Acta Pharmacol Sin 2003 Apr; 23 (4): 342-381
Proceedings
Proceedings of the 12th China and Japan Joint Meeting on Pharmacology (CJP-2002)
and the 8th International Symposium on Biopeptides Medical Sciences (BMS-2002)
2002 October 27-30, Shanghai, China
Organizers: LEE Wen-Hsiung, HIGASHINO Hideaki, YUAN Wen-Jun
IL-01 Significance of double-blind, placebo-controlled clinical studies
on herbal (Kampo) medicines
Shuji TAKAORI
Professor Emeritus, Kyoto University, Kyoto 606-8501, Japan
To elucidate the efficacy of Choto-san (Diao-Teng-San)
in comparison with a placebo, a double-blind study was
carried out in 139 patients suffering from vascular
dementia. Choto-san extract glanules (7.5 g/d) and a
placebo, were given 3 times a day for 12 weeks, respectively. Informed consent was obtained from the
patients and/or their families. Choto-san was
statistically superior to the placebo in global improvement
rating, utility rating, and global improvement ratings of
subjective symptoms, psychiatric symptoms and disturbance in daily living activities. Such items as
spontaneity of conversation, lack of facial expression,
decline in simple mathematical ability, nocturnal delirium,
sleep disturbance, hallucination or delusion, and putting
on and taking off clothes were significantly improved
at one or more evaluation points in those taking
Choto-san compared to those taking the placebo. However,
no significant difference between the Choto-san and
placebo groups was observed in the global improvement rating of neurological symptoms at any of the
evaluation points. There was no significant difference
between the two groups in terms of the overall safety
rating. These results suggest that Choto-san has a
favorable effect on vascular dementia and can be
recommended as a treatment for this condition. Double-blind,
placebo-controlled studies are significant to evaluate the
efficacy and safety of herbal medicines.
IL-02 Molecular physiology of volume-regulated
chloride channels in heart
Dayue DUAN
Center of Biomedical Research Excellence, Department of
Pharmacology, School of Medicine University of Nevada, Reno,
Nevada 89557, USA
Changes in cell volume are known to regulate chloride (Cl-) channels
in many cell types. To date at least two types of volume-regulated chloride
channels have been described in cardiovascular system: the outward rectifier
Cl- channel (ORCC or Clvol) and the Cl- inward rectifier
(Clir). Clvol in the heart is characterized by activation by hypotonic cell
swelling (Clswell), outward rectification, greater permeability to iodide than
chloride, slow inactivation at positive potentials, and an intermediate (about
50 pS) unitary conductance. Basally active Cl- channels (Clb), which
share many properties of Clvol, have also been observed under isotonic conditions
in cardiac and endothelial cells. The single channel underlying both Clb and
Clswell is the same ORCC. The gene encoding Clvol in these cells may be ClC-3.
However, the differences in pharmacology and modulation of Clvol in various
cell types and species also point to the possibility of a heterogeneous family
of multiple volume-regulated Cl- channels. Clir has been recently
discovered in mammalian cardiac cells in our laboratory. Clir may be encoded
by ClC-2, another member of the ClC family. In hypertrophied heart Clvol is
persistently activated. The molecular expression and functional distribution
of volume-regulated Cl- channels (ClC-2 and ClC-3) in cardiac cells,
vascular smoot muscle cells and endothelial cells strongly implicate important
roles of these channels in the regulation of cardiovascular function. The most
obvious function of the volume-regulated Cl- channels may be their
contributions to regulatory volume decrease (RVD). Activation of the volume-regulated
Cl- channels may cause a loss of intracellular Cl- ions
and water follows, effectively reducing cell volume to normal levels. In the
heart activation of Cl- currents can cause important changes in action
potential characteristics, which may increase the dispersion of electrophysiological
properties and provide the substrate for the occurrence of arrhythmias under
conditions that cause cell swelling. Activation of ClC-3 channels may mainly
cause a shortening of APD due to its outward rectification, and activation of
the inward rectifier ClC-2 channels may normally play a much more prominent
role in the pacemaker activity of SA or AV nodal cells in the heart. A combination
of multiple approaches, including integrated physiology, pharmacology, electrophysiology,
and functional genomics and proteomics, will be necessary for defining the function
of the volume-regulated Cl- channels in the heart. (Supported by
AHA Grant-in-Aid #9950153N, NIH P20 RR15581, and HL63914)
IL-03 Gene therapy strategy for vascular
dysfunction in low renin hypertension
Alex F CHEN
Director, Laboratory of Molecular Vascular Biology,
Department of Pharmacology and Toxicology and the Neuroscience
Program, College of Human Medicine, Michigan State University,
East Lansing, Mi 48824-1317, USA
Although hypertension is a key risk factor for
atherosclerosis and stoke, the underlying celluar and
molecular mechanisms remain to be delineated. One of the
earliest events in hypertension is endothelial dysfunction.
Decreased nitric oxide (NO) and/or increased oxidative
stress (eg, superoxide anions) are causative factors for
such an event in the blood vessel wall. Consequently, a
dysfunctional endothelium promotes vasoconstriction,
platelet aggregation, adhesion molecule-induced
leukocyte adhesion, and smooth muscle cell proliferation. Our
research interests are in vascular biology, pharmacology,
and gene therapy of diseased animal models, including
hypertension, diabetes, and ischemic stroke. Some of
our most recent findings of gene transfer of endothelial
NO synthase (eNOS) and manganese superoxide dismutase (MnSOD) on endothelin-1 (ET-1)-induced
vascular dysfunction related to oxidative stress in
deoxycorticosterone-acetate (DOCA)-salt hypertensive rats
will be presented. Our ultimate goal is to translate basic
research to clinical arena for gene therapy of
cardiovascular disease.
IL-04 Delaying brain mitochondrial decay and
aging with mitochondrial antioxidants and metabolites
LIU Jian-Kang, Bruce N AMES
Division of Biochemistry and Molecular Biology, University of
California, Berkeley, CA 94720; Children's Hospital Oakland
Research Institute, Oakland, CA 94609, Canada
Mitochondria decay with age due to the oxidation of lipids, proteins, RNA and
DNA. Some of this decay can be reversed in aged animals by feeding the mitochondrial
metabolites acetylcarnitine and lipoic acid. In this presentation, we summarize
our recent studies on the effects of these mitochondrial metabolites and mitochondrial
antioxidants (a-phenyl-N-t-butyl nitrone and N-t-butyl hydroxylamine)
on the age-associated mitochondrial decay of the brain of old rats, neuronal
cells, and human diploid fibroblast cells. In the studies with old rats, these
mitochondrial metabolites and antioxidants improve the age-associated decline
of ambulatory activity and memory, partially restore mitochondrial structure
and function, inhibit the age-associated increase of oxidative damage to lipids,
proteins, and nucleic acids, elevate the levels of antioxidants, and restore
the activity and substrate binding affinity of a key mitochondrial enzyme, carnitine
acetyltrasferase. These mitochondrial metabolites and antioxidants protect neuronal
cells from neurotoxin- and oxidant-induced toxicity, oxidative damage, delay
the normal senescence of human diploid fibroblast cells, and inhibit oxidant-induced
senescence acceleration. These results suggest a plausible mechanism: with age
increased, oxidative damage to proteins and lipid membranes, particularly in
mitochondria, causes a deformation of structure of enzymes, with a consequent
decrease of enzyme activity as well as substrate binding affinity for their
substrates; an increased level of substrate restores the velocity of the reaction
and restores mitochondrial function, thus delaying mitochondrial decay and aging.
This loss of activity due to coenzyme or substrate binding appears to be true
for a number of other enzymes as well, including mitochondrial complex III and
IV. In conclusion, dietary supplements of mitochondrial antioxidants/metabolites
as well as mirconutrients may be effective in delaying mitochondrial decay,
thus delaying aging and preventing age-associated diseases.
IL-05 Changeover from bridging to global
development strategy in clinical drug development
---
significance of ethnic similarity in pharmacokinetics
KANADA Heihachiro
International Clinical Research Organization for Medicine
(InCROM) 4-12-11 kasuga, Suita-Shi, Osaka 565-0853, Japan
Since 1992, ICH had been scientifically evaluating the ethnic factors upon
medicine's effects and considering, how to facilitate the use of the foreign
clinical data. In August 1998, the guidelines originated in ICH. Agreement was
issued in Japan while it provides the prerequisites for accepting foreign clinical
data as documents to be reviewed for approval of the new drug application. Consequently,
Bridging strategy has been taking an important part at each pharmaceutical company
in drug development strategy. Based upon this guideline, Bridging-studies were
conducted and those data were submitted together with foreign clinical data,
and, in result, seven new medicines had been approved accepting foreign clinical
data (Review report by PMDEC, August 2002). According to the report, the dose-response
studies as confirmatory studies at the late phase II or phase III were conducted
generally as bridging studies. Several concrete examples are going to be shown
in this seminar. Currently, drug development strategy has been changing over
from Bridging in order to utilize existing foreign clinical data to Global considering
simultaneous development. Namely, in the trends of conduct of international
simultaneous clinical trials through globalization and speeding up of drug development
thus, the strategy has been initiating simultaneous development at the early
stage in drug development. Nevertheless, the both strategies basically necessitate
pharmacokinetic data showing similar profile among different ethnics in order
to approve the Bridging. Although ICH guidelines have been developed by the
three core regions such as Japan, USA, and EU, Bridging of clinical data between
East Asian ethnics could be performed after showing similarities in pharmacokinetic
profiles, because these ethnic groups bear many historical linkages. From this
viewpoint, we started to investigate similarity in physiological factors and
to compare pharmacokinetic profile between East Asian (ie, Chinese, Korean,
and Japanese). In this seminar, the results from two studies are presented:
1) Comparison of pharmacological parameters among four ethnic groups (Study
of acarbose effect under sucrosel loading in Chinese, Korean, Japanese, and
Caucasian). 2) Comparison of physiological factors influencing pharmacokinetics
among East Asians (study of the gastric emptying in Chinese, Korean, and Japanese).
The variety of methods is available to compare ethnic diversities utilizing
pharmacokinetic studies, which comprise a basis for Bridging, and/or other supportive
clinical pharmacological studies. For instance, a method using one site with
one protocol or, that is to say, the studies among ethnic groups. Thus, this
design could be applicable to many situations in the clinical pharmacological
studies including parmacokinetic studies as far as future global development
is concerned. Presently, however, considering to pharmaceutical market in China
and Korea, that are quickly developing economically and scientifically, global
development that deals with ethnically similar East Asia, as one region is a
future subject in drug develop-ment. Finally, as an established CRO, InCROM
has one medical institution specialized in clinical trials in UK and three similar
medical institutions in the home, where clinical trials including the Bridging-studies
are performed.
IL-06 In vitro differentiation of embryonic stem
cells to cardiomyocytes lacking the sarcoplasmic reticulum ryanodine receptor
YANG Huang-Tian, Kenneth R BOHELER1
Laboratory of Molecular Cardiology, Health Science Center, CAS/SIBS &
SCA S and SSMU, Shanghai 200025, China;
1Laboratory of Cardiovascular Science, NIH/NIA, Baltimore, MD
21224, USA
AIM: Contraction of cardiac muscle is controlled through the process
of Ca2+ release. A key component of this release process is the type
2 ryanodine receptor (RyR2). This study is to elucidate the functional properties
of RyR2 in early differential cardiac myocytes by gene targeting of mouse embryonic
stem (ES) cells. METHODS: Using homologous recombination and Crerecombinase-loxP
system, the R1 ES cells were targeted to insert loxP sites flanking an upstream
exon. A plasmid expressing Cre recombinase was transfected into the ES cells
to create RyR2 knockout ES cells (RyR2-/-). WT ES cells RyR2-/-
cell lines were cultivated in suspension to differentiate into cardiac
myocytes. The RyR2+/+ and RyR2-/- ES cell lines were tested
for their ability to differentiate into cardiac myocytes and were measured for
Ca2+ signaling. RESULTS: RyR2+/+ and RyR2-/-
cells both differentiated into cardiomyocytes. Contracting cell clusters were
isolated for single cell analyses. Fluorometric Ca2+ transient measurements
showed that RyR2+/+ cardiomyocytes, but not RyR2-/-, had
Ca2+ transients evoked by caffeine (20 
mol/L),
an activator of RyR2. Ryanodine (10 mol/L),
an inhibitor of the RyR2, did not inhibit spontaneous Ca2+ transients
in the RyR2-/- cardiomyocytes, indicating that these cells lacked
a functional RyR2. Studies with carbachol (10 mol/L)
demonstrated that both atrial and ventricular cells were present. CONCLUSION:
Loss of RyR2 does not prevent differentiation of cardiomyocytes; embryonic
lethality at 10 dpc is due to subsequent functional defects associated with
loss of RyR2; and these genetically modified ES cells represent a unique model
system to study the function of RyR in excitation-contraction coupling.
IL-07 Some characteristics of SHRSP and
M-SHRSP
Tsuneyuki SUZUKI
Department of Pathology, Kinki University School of Medicine,
Osaka, Japan
In 1974, using SHR substrains for selective inbreeding,
Okamoto et al established a colony of SHRSP with
severe hypertension. In 1981, Okamoto et al using these
SHRSP, developed a colony of M-SHRSP with very severe hypertension at a very early age. These strains
are maintained in our laboratory of Kinki University.
This report is designed to elucidate some
characteristics of male SHRSP and M-SHRSP.
Cerebrospinal fluid pressure Cerebrospinal fluid
pressure was measured by the cisternal pancture method,
and brain parenchyma water content and brain volume
were also measured. When SHRSP and M-SHRSP blood pressure reached 210 mmHg, their cerebrospinal fluid
pressure also began to rise; the correlation was positive.
However, no cerebrospinal fluid pressure increase was
found for normotensive rats. Positive correlation was
found between blood pressure and brain parenchyma water content, between brain parenchyma water
content and brain volume, between the former and
cerebrospinal fluid pressure, and between brain volume and
cerebrospinal fluid pressure. These results appear to
suggest that severe hypertension increases cerebral
blood vessel hyperpermeability and brain edema.
Plasma rennin concentration The changes in plasma
rennin concentration (PRC) due to development of
hypertension were studied by using plasma rennin RIA
kit. There were extraordinary increases in PRC for
both SHRSP and M-SHRSP (blood pressure over 250 mmHg) and PRC levels were positively correlated with
blood pressure; however PRC values remained normal
even for these strains as long as their blood pressures
remained below 210 mmHg. These results suggest that
the increased PRC in SHRSP and M-SHRSP with high blood pressure levels is a result of this extraordinary
rise in blood pressure (above 210 mmHg); while, in
turn, the increased PRC further aggrevates the hypertension.
Plasma aldosterone concentration The changes in
plasma aldosterone concentration (PAC) were studied
by using plasma aldosterone RIA kit and compared with
the changes of PRC. PAC increased significantly in
M-SHRSP over that in WKY from 10 weeks of age (blood
pressure over 230 mmHg). At 12 weeks PAC values were about six times those of similarly aged WKY. In
SHRSP the PAC increased more slowly and reached to
only 2.5 times of the WKY values at 25 weeks.
Similarly to the increase in PAC, significant increases in PRC
for M-SHRSP took place at 10 weeks. SHRSP PRC increases occured much earlier than the PAC increases,
being seen at 12 weeks. There were positive
correlations between blood pressure and PAC, between blood
pressure and PRC, and between PRC and PAC in mature M-SHRSP with blood pressures over 210 mmHg.
Based upon previous reports and these findings on SHRSP and M-SHRSP characteristics, it may be
concluded that symptoms and progress in hypertension
of SHRSP and M-SHRSP are very similar to those in human hypertension and malignant hypertension.
S-01 Nitric oxide metabolite (NOX) concentrations
in serum of hospital workers and patients consulting doctors at a hospital
HIGASHINO Hideaki1,2, MUKAI
Hidenori2, MIYA Hirohisa2, MIYA
Yoshihisa2
1Department of Pharmacology, Kinki University School
of Medicine, Osaka-Sayama 589-8511; 2Department of
Internal Medicine, Higashi-Yodogawa Hospital, Osaka
589-8511, Japan
AIM: Nitric oxide produced through eNOS, nNOS and
iNOS must reflect the internal body activities, and
therefore we may be able to guess the occasional
pathophysiological situations. METHODS: From this
concept, we measured the concentrations of nitric
oxide metabolites (NOX) in the serum of 456 patients aged
14 to 96 years consulting doctors at a hospital and 80
healthy hospital workers at rest with each permission.
The values were analyzed in relation to diseases, complications, genders, and ages.
RESULTS: 1) NOX levels in serum increased with age in female with a
positive correlation, but not in male. 2) Although, overall,
there was no relationship between NOX and blood
pressure levels, a positive correlation was found only in
60-70 years old male. 3) Higher levels of NOX were found
in diabetes, renal dysfunction, hyperlipemia,
myocardial infarction, acute enteritis, helicobacter pyroripositive
gastritis, type C hepatitis, rheumatoid arthritis, and
cancers groups than in a healthy group. 4) Apoplexy,
hyperuricemia, osteoporosis, respiratory diseases, and
thyroiditis did not show a higher NOX level. CONCLUSION:
1) NOX levels in serum increase with age. 2) Progress of atherosclerosis will stimulate the
production of NO, since NOX levels were higher in elder and male patients with life-style diseases. 3)
Several cytokines stimulating NO production through iNOS
will relate to the diseases such as acute enteritis,
gastritis caused by Helicobacter pylori, rheumatoid arthritis,
and cancers. 4) A measurement of NOX in the patients
will be useful for understanding of causes, status, and
prognosis.
S-02 Restoration of impaired arterial baroreflex:
a new strategy for cardiovascular diseases
SU Ding-Feng
Department of Pharmacology, Second Military Medical
University, Shanghai 200433, China
Arterial baroreflex (ABR) is an important regulatory
mechanism in cardiovascular activities. It was reported
that ABR function related to the sudden death after acute
myocardial infarction. The present lecture summarizes
the works completed in our department in the past10
years.
ABR function predicted end organ damage in
hypertension It was found that both ABR-BP and
ABR-HP very closely related to end organ damage in SHR.
ABR dysfunction was not the cause of hypertension but it predicted the end organ damage in hypertension.
Interruption of ABR induced severe organ damages
Interruption of ABR was completed by sinoaortic
denervation (SAD). It was found that there were sever
organ damages in SAD rats, such as vascular remodeling,
myocardial remodeling, renal damages, etc.
Mechanisms underlying for organ damage in SAD
rats A series of observations were done in SAD rats.
BPV is increased in SAD rats, irregular tissue perfusion
may contribute to organ damage. The activations of
tissue renin angiotensin system and inflammatory
cellular factors and myocardial apoptosis may play also an
important role in this procession. These were evidenced
by the works with RT-PCR and microsatellite DNA-PCR and RAPD PCR,
etc.
Ketanserin improved the impaired ABR function in
SHR Ketanserin enhanced both ABR-HP and ABR-BP in
SHR. Compared with prazosin and ritanserin, it was found
that the effect of ketanserin on ABR function was
centrally and mediated by 5-HT2A receptors.
Perspective The possibility to develop a strain of rats
possessing spontaneous ABR deficiency will be mentioned.
S-03 Pharmacokinetics of ibudilast sustained
realease capsules in healthy volunteers
ZHANG Cai-Li, LOU Jian-Shi, ZHANG Jing-Ling, JIAO Jian-Jie, WEN Xiao-Na ,
XIAO Xiang-Qian
Division of Clinical Pharmacology, Tianjin Medical University,
Tianjin 300070, China
AIM: To study the pharmacokinetics of ibudilast sustained release capsules
in Chinese healthy volunteers. METHODS: Tirty-two male volunteers, age
18-24 (21±2) a, body weight 55-81 (65±7.3) kg, height 168-184 (171±5)
cm were recruited. They were divided into two groups randomely. 1) Single dosage
groups: 5,10, or 20 mg ibudilast sustained release capsules given orally to
the fasted volunteers. 2) Multiple dosage groups: 10 mg capsule Q12 h, for 8
consecutive days. The drug was provided by Tianjin Zhong wei Pharmaceutical
Company Ltd. Blood samples for pharmacokinetic analyses were collected pre-dose
and then at 0.17, 0.33, 0.67, 1, 2, 3, 4, 6, 8, 12, 24, 30, 36, and 48 h after
dosing for single dosage groups. For multiple dosage group, on d 5, d 6, and
d 7 pre-dose and at 4 h after dosing and on d 8 the times for blood collection
were the same as that of single dosage groups. HPLC was used to determine the
blood concentration of ibudilast. RESULTS: The pharmacokinetic parameters
of 5 mg and 10 mg groups were: Tmax (1.6±1.2) h and (1.6±0.8)
h, Cmax (17±5) g/L
and (25±7) g/L, AUC (103±24)
g·L-1·h and
(144±65) g·L-1·h,
T1/2 (5.4±2.1) h and (4.4±2.2) h, respectively.
For 20 mg group: Tmax (3.3±1.6) h, Cmax
(56±22)g/L, AUC (623±185)
g·L-1·h,
T1/2 (31±15) h. Following multiple dosing of 10 mg, steady
state Cmax was (45±18) g/L
and steady state Cmin was (19±9) g/L,
the peak to trough fluctuation index (DF) was 85 %±9 %, and AUCss
(768±340) g·L-1·h.
Following the last dosing of 10 mg, the pharmacokinetic parameters were: Tmax
(2.7±1.6) h, Cmax (48±21) g/L,
AUC (658±278) g·L-1·h,
and T1/2 (33±13) h. CONCLUSION: After multiple
dosing of 10 mg ibudilast sustained release capsule, the peak to trough blood
concentration fluctuation keeps stable, and the recommended dosage is 10 mg
twice daily.
S-04 Gastric oxidative stress and hemorrhagic
ulcer in Salmonella typhimurium-infected rats
HUNG Chen-Road
Department of Pharmacology, College of Medicine, National
Cheng-Kung University, Tainan 70101, Taiwan, China
AIM: The infection of Salmonella typhimurium (S typhimurium)
may lead to various organ diseases. This research was to propose that the infection
of S typhimurium caused gastric oxidative stress and disruption of gastric
mucosal barriers, and thus resulted in gastric hemorrhagic ulcer in rats.
METHODS: Male pathogen-free Wistar rats were deprived of food for 24 h.
Single injection of live culture of S typhimurium (1×106-1×109
CFU/rat in 1.0 mL of sterilized phosphate buffer saline (PBS) was challenged
iv to rat 5 h after withdrawal of food. Age-matched control rats received PBS
only. After gastric surgery and vagotomy, rat stomachs were irrigated for 3
h with either normal saline or a simulated rat gastric juice containing HCl
100 mmol/L, pepsin 600 mg/L, and NaCl 54 mmol/L. Gastric parameters, including
mucosal glutathione and lipid peroxide generation as well as luminal hemoglobin
content and mucosal damage were determined. RESULTS: The production
of gastric hemorrhagic ulcer and various gatric parmeters was dependent on the
concentration of S typhimurium injected. The maximal response was obtained
at the concentration of 1×109 CFU/rat. The presence of luminal
gastric juice produced gastric hemorrhagic damage. High relation of ulcer formation
to mucosal lipid peroxide generation and glutathione levels was also observed
in those S typhimurium-infected rats. This exacerbation of ulcerogenic
parameters was dose-dependently inhibited by pretreatment of ofloxacin. Pretreatment
of antioxidants, such as reduced glutathione, allopurinol and dimethylsulfoxide,
also caused amelioration of gastric hemorrhagic damage in S typhimurium-infected
rats. CONCLUSION: Infection of S typhimurium may produce gastric
hemorrhage and mucosal ulceration via aggravation of gastric oxidative stress
that can be effectively ameliorated by antioxidants or antibiotics.
S-05 Effects of prousion on scavenging free
radicals
PAN Jia-Hu, HASHINMOTO Masakazu1, JIN Jian,
CHANG Jian-Jie, NIE Jin
Department of Pharmacology, School of Pharmacy, Fudan
University, Shanghai 200032, China; 1Japan Health Promotion
Association, 7-17-801 Nihonbashi Yokoyama-cho, Chuou-ku
Tokyo 103-0003, Japan
AIM: To detect the effects of prousion on hydroxyl free radicals and
superoxide free radicals in vitro. METHODS: Prousion, natural
porous ore material with some biological functions found in Japan, Japan Patent
Application Number: BFC4j (B2002-52397). Prousion-A, B, C (P-A, P-B, P-C), offered
by Dr M Hashimoto, were the powders mixed of these porous ore in different proportions
(grey powder, d 
3 m).
The effects of prousion on hydroxyl free radical and superoxide free radicals
were determined by the assays of deoxyribose and xanthine oxidase, respectively.
Their direct effects on these free radicals were also performed with the Electron
spin resonance (ESR). RESULTS: 1) IC50 for P-A, P-B, and
P-C on scavenging hydroxyl free radicals by the assay of deoxyribose was 0.2209,
0.2962, and 0.2183 g/L, respectively, the order for their actions was C>A>B.
The 95 % confidence limit was 0.0909-0.5367, 0.1302-0.6741 g/L, and 0.0895-0.5322
g/L, respectively. 2) The inhibition produced by P-A, P-B, and P-C at the concentration
of 1.4 mg/L on hydroxide free radicals detected by ESR was 35.24 %, 36.92 %,
and 55.14 %, respec-tively. This action was in a dose-dependent manner and the
order for their actions was C>B
A,
confirming the results obtained by the assay of deoxyribose. 3) IC50
for P-A, P-B, and P-C on scavenging superoxide free radicals by the assay of
xanthine oxidase was 0.4025, 0.3854, and 0.3626 g/L, respectively, the order
was C>B>A. The 95 % confidence limit was 0.2065-0.7847, 0.2041-0.7276,
and 0.1823-0.7213 g/L, respectively. 4) The inhibition by P-A, P-B, and P-C
at the concentration of 0.34 mg/L on superoxide free radicals detected by ESR
was 5.99 %, 9.15 %, and 9.15 %, respectively, also confirming the results of
xanthine oxidase assay. CONCLUSION: Prousion has the properties scavenging
hydroxide and superoxide free radicals.
S-06 Steroids modulate placental CRH gene
transcriptional activity through cAMP regulatory
element
NI Xin, HOU Yue, Richard NICHOLSON1, Roger
SMITH1
Department of Physiology, Second Military Medical University,
Shanghai 200433, China; 1Mothers and babies Research Center,
Endocrine Unit, John Hunter Hospital, NSW 2310, Australia
AIM: To explore the regulation of corticotropin-releasing hormone (CRH)
gene expression by steroid hormones in placenta and its molecular mechanism.
METHODS: Human primary cultured placental trophoblasts were transfected
with CRH-luciferase reporter genes and treated with estrogen, progesterone,
and their antagonists. RESULTS: Estradiol (E2) inhibited basal and cAMP-stimulated
CRH promoter activity. A pure estrogen antagonist ICI182, 780 not only blocked
repression of CRH gene expression by E2, but up-regulated CRH promoter activity.
This effect appeared to occur specifically through ER
-mediated
mechanism, as similar effects were found in the ER
over-expression trophoblasts. Through deletion and mutagenesis analyses we found
that estradiol inhibition of the CRH gene required cAMP regulatory element (CRE).
Progesterone (P4) treatment resulted in a decrease in CRH promoter activity.
Addition of Ru486 or inhibition of endogenous P4 production by placental cells,
led to an increase of promoter activity. It was also found that the repression
of CRH gene expression by P4 also required CRE. This CRE sequence conferred
E2 and P4 inhibitions upon a heterologous promoter (rabbit 
-globin).
CONCLUSION: Estrogen and progesterone play inhibitory roles in the regulation
of CRH gene transcription in human placental trophoblasts. These effects occurred
through CRE of CRH gene.
S-07 Soluble protein of a novel costimulatory
molecule, Dectin-2, breaks ultraviolet B-induced immune tolerance in contact hypersensitivity in
mice
ARAGANE Yoshinori, MAEDA Akira, YAMAZAKI Fumie, UENO Kengo, SAKANO Fumiaki, TEZUKA
Tadashi, ARIIZUMI Kiyoshi1
Department of Dermatology, Kinki University School of Medicine,
Osakasayama 589-8511, Japan; 1Department of Dermatology,
Southwestern Medical Center, University of Texas, Dallas,
Texas 77030-0708, USA
AIM: One of hazardous adverse effects of ultraviolet B light (UV) exposure
to skin may be best exemplified with its immunosuppressive property on contact
hypersensitivity (CHS) and the subsequent induction of tolerance. Bearing in
mind thoughts that artificial break of impaired cutaneous immune response may
lead to regain host's immune competence, we focused on a novel costimulatory
molecule, Dectin (Dec)-2, shown to be selectively expressed on antigen presenting
cells, such as epidermal Langerhans cells (LC). METHODS & RESULTS:
Mice exposed to low doses of UV and sensitized thereafter with hapten, dinitrofluorobenzene
(DNFB) through the exposed skin area showed markedly ameliorated CHS upon resensitization
and rechallenge to the same animals. When soluble Dec-2 (sol-Dec2), an inhibitor
of cognate Dec-2, was injected iv before resensitization, following rechallenge
led to full occurrence of CHS, indicating that sol-Dec2 breaks UV-induced tolerance.
This is the first demonstration that functional neutralization of costimulatory
molecule restores impaired cutaneous immune response. To further exploit mechanisms
underlying this phenomenon, T-cells obtained from the UV-tolerized animals were
cocultured in vitro with epidermal cell suspension (consisting of 5 %-10
% LC) derived from naive animals in the presence of water soluble DNFB analogue,
DNBS, resulting in apoptosis of LC as demonstrated by FACS analysis using an
antibody directed against murine major histocompatibility antigen class II (Ia)
and annexin V. Interestingly, sol-Dec2 suppressed apoptosis of LC by those T-cells,
indicating that sol-Dec2 blocks LC apoptosis via interference with Dec-2-mediated
signals transduced from the T-cells to LC. CONCLU-SION: The present study
demonstrates for the first time that sol-Dec2 prevents apoptosis of LC by hapten
specific T-cells, thereby leading to break of UV-induced immune tolerance.
S-08 Carrageenan inflammation increases
sensitivity of vanilloid receptor-1 without changes in its
expression level in periphery
ZHANG Hong-Wei, ANDOH Tsugunobu, NOJIMA Hiroshi, KURAISHI Yasushi
Department of Applied Pharmacology, Faculty of
Pharmaceutical Sciences, Toyama Medical & Pharmaceutical University,
Toyama 930-01, Japan
AIM: Vanilloid receptor-1 (VR1) is a ligand-gated non-selective cation
channel expressed predominantly by primary sensory neurons. It functions as
a transducer of painful thermal and chemical stimuli in vivo. Carrageenan
treatment has been shown to induce axonal flow of VR1 mRNA from the dorsal root
ganglia to the dorsal horn and to increase the sensitivity of VR1 in the latter
region. The present study was conducted to determine whether changes in VR1
expression in the periphery would be involved in the carrageenan-induced thermal
hyperalgesia. METHODS: Carrageenan (1 mg/site) was unilaterally injected
into the plantar region of the hind paw of rats. The activity of the saphenous
nerve and VR1 expression were examined 6 h after carrageenan, when thermal hyperalgesia
peaked. RESULTS: Intraplantar pretreatment with the VR1 antagonist capsazepine
(0.3 and 0.6 mg/site) attenuated thermal hyperalgesia induced by carrageenan.
The saphenous nerve in the inflamed paw displayed sensitization to capsaicin
(1-10 g/site) and this enhanced responsiveness
was significantly inhibited by pretreatment with capazepine (10 g/site).
There was a significant increase in VR1 mRNA in the inflamed skin and a decreased
trend in the ipsilateral dorsal root ganglia (L4-L6). There were no significant
increases in VR1 protein and the distribution of VR1-positive fibers in the
inflamed skin. CONCLUSION: The results support the idea that VR1 receptors
are involved in inflammation-induced thermal hyperalgesia. Increase in VR1 sensitivity
rather than in VR1 translation in the periphery may chiefly contribute to this
hyperalgesia.
S-09 Dectin-2 works as a costimulatory molecule
that induces production of interleukin-10
ARAGANE Yoshinori, MAEDA Akira, YAMAZAKI Fumie, UENO Kengo, SAKANO Fumiaki, TEZUKA
Tadashi, ARIIZUMI Kiyoshi1
Department of Dermatology, Kinki University School of Medicine,
Osakasayama 589-8511, Japan; 1Department of Dermatology,
Southwestern Medical Center, University of Texas, Dallas, Texas
77030-0708, USA
AIM: Using subtractive cDNA cloning technology, we currently isolated
dectin-2 (Dec-2) that selectively is expressed on bone marrow-derived epidermal
residents, Langerhans cells (LC). LC, with their capacity to potently operate
antigen presentation, may maintain host's homeostasis by eliminating invasion
of bacteria, viruses or industrial and environmental compounds. Keeping in mind
costimulatory function of its homologue Dec-1, we were interested to explore
whether Dec-2 also serves as a costimulatory molecule. METHODS: To first
assess co-stimulatory function of Dec-2, naïve splenic T-cells were obtained
from mice, which were cultured in vitro on plastic dishes coated with
serial amounts of a monoclonal antibody directed against CD3 (
CD3) (0, 0.1, 0.3, 1, or 3 mg/L) for 48 h and T-cell growth monitored by an
MTT assay. RESULTS: Consequently, it was found that T-cells significantly
proliferated on plate-bound CD3
in a concentration higher than 1 mg/L, while those less than 0.3 mg/L turned
out to be suboptimal. Intriguingly, when plates were coated with both graded
amounts of CD3 and soluble Dec-2
10 mg/L, significantly augmented proliferation of T-cells were detected even
with suboptimal concentrations of
CD3, indicating that Dec-2 works as a costimulatory molecule. To further exploit
impacts of Dec-2 on T-cells, the supernatants of the above culture were tested
for cytokine production by ELISA assays, demonstrating that CD3-induced
production of interleukin (IL)-10 was markedly augmented by plate-bound sol-Dec2,
which, however failed to do so for IL-4 or interferon-
.
CONCLUSION: Dec2 possesses costimulatory functions on T-cells, leading
to selective production of immunosuppressive type cytokine, IL-10.
S-10 Effect of bio-catalyzer on macrophage
function in ODUS/Odu II. In vitro experiments
SHINOHARA Mitsuko, OHURA Kiyoshi
Department of Pharmacology, Osaka Dental University, Hirakata
573-1121, Japan
AIM: Bio-catalyzer (Bio-normalizer: BN, Sun-O
International Inc, Japan), a product of yeast fermentation of
Carica papaya Linn, is sold as a natural food in Japan. On the
11th China-Japan Joint Meeting on Pharmacology, we
reported that 2 % BN administered to rats with naturally
occurring gingivitis rat (ODUS/Odu) in
vitro. In this study, we measured the influence of BN on macrophages in
ODUS/Odu. METHODS: We prepared BN at final
concentrations of 0.1 g/L, 1
g/L, 10 g/L, 0.1 mg/L, 1 mg/L, and 10 mg/L in RPMI
medium 1640. Macrophages were collected 4 d after ip injection
of 1 % oyster glycogen. We measured chemotaxis, phagocytosis, and superoxide anion production of rat
macrophages. RESULTS: Macrophage chemotaxis with
BN in 1:20 ZAS was significantly suppressed compared
with the control, except at concentrations of 0.1
g/L-0.1 mg/L. It was dose-dependently suppressed in
1:20 ZAS. Macrophage phagocytosis at all
concentrations of BN was significantly suppressed
(P<0.01) after 2 d compared with the controls. Superoxide anion
production by rat macrophages in the presence of stimulated PMA
was significantly different after 3 h, except BN 0.1
g/L. CONCLUSION: The above findings suggested
that BN affects immune function.
S-11 Effect of AT1-receptor antagonist on
microvascular injury in cerbral cortex of stroke-prone SHR
ITO Hiroyuki, TAKEMORI Kumiko, ISHIDA Hiroyuki,
SUZUKI Tsuneyuki
Department of Pathology, Kinki University School of Medicine,
Osaka-sayama, Osaka 589-8511, Japan
AIM: To elucidate the possible involvement of
angiotensin II (AII) in the pathogenesis of hypertensive cerbral
injury, the effects of AT1-receptor antagonist on adhesion
molecule expression in leukocytes and cerbral microvessels
were investigated using stroke-prone SHR (SHRSP).
METHODS: Male SHRSP at 19 weeks of age were treated
with AT1-receptor antagonist (TCV-116, Takeda, 1
mg/kg/day, po) for 4 weeks. Furthermore, 5-week-old rats
were treated similarly for 5 weeks and maintained for
further 17 weeks. At the end of experiment, pathological
findings of the brain were examined and the expression of
following molecules was investigated: adhesion molecules
(Mac-1 in leukocyte and ICAM-1 in endothelial cell),
BBB-associated molecules (Glut-1 and Aquaporin-4). Plasma
AII concentration and brain ACE activity were also
investigated. RESULTS: Blood pressure level was not
too much different between two groups. Nonetheless,
brain weight in the TCV group was significantly lighter
and the incidence of cerebral lesion was low as compared
to the control. Both adhesion molecule and
BBB-associated molecule expression were significantly lower in the
TCV group. Similar results were also obtained in the
experiment of early and short-term treatment. The plasma
AII and brain ACE activity were lower than in the control
and plasma NO metabolite content was also lower in the
TCV group. CONCLUSION: AT1-receptor antagonist
was effective for prevention of hypertensive cerebral
damage in SHRSP with pressure-independent manner, even in
early and short-term treatment. This could be due to the
inhibition of leukocyte-endothelial cell adhesion and
subsequent BBB dysfunction by NO radical. Thus, AII may
be closely involved in the pathogenesis of hypertensive
end-organ damage.
O-01 Effect of CA4-3, a polysacchride isolated from
Liuwei Dihuang decoction, on immune responses
FU Yan-Rong, ZHANG Yong-Xiang
Beijing Institute of Pharmacology and Toxicology, Beijing 100850,
China
AIM: To study the immunopharmacological effect of
CA4-3, an acidic polysaccharide extracted from Luiwei
Dihuang decoction, and the possible mechanisms
underlying its effects. METHODS:
3H-TdR incorporation method was employed to assess the effect of
CA4-3 on lymphoctye proliferation. IgG production was
determined by enzyme-linked immunosorbant assay (ELISA).
T-cell subsets were analyzed by florescent activated
cell sorting (FACS). Electrophoretic mobility shift
assay (EMSA) was used to evaluate the binding activity
of nuclear factor-kappa B (NF-
B) and B cell
specific activating protein (BSAP). RESULTS:
CA4-3 significantly enhanced the spontaneous proliferation of
splenocytes of normal mice when given both in
vitro and in vivo. Spontanoeous and LPS-induced IgG
production by splenocyte was also promoted by CA4-3
(10, 100 mg/L) in vitro. After oral administration of
CA4-3 (19, 38 mg/kg), the percentage of double
positive thymocyte
(CD4+CD8
-) was significantly reduced while that of single positive thymocyte
(CD4+CD8
- and CD8+CD4
-) was significantly increased. However,
CA4-3 showed no influence when it was co-cultured with
thymocytes. CA4-3 (100 mg/L) in vitro significantly
facilitated the binding activities of
NF-B and BSAP. CONCLUSION: CA4-3 potentiated immune
function. The major direct target cell of CA4-3 is
B-cell, and its effect on T-cell seems to be mediated by
other ways. The mechanism underlying its immunopotentiating effect is related to the increased
binding activity of NF-B and BSAP.
O-02 Effects of Cobalt-60 radiation on
synthesis of adrenomedullin and endothelin in rat
vascular smooth muscle cells
ZHONG Guang-Zhen, CHEN Feng-Rong, BU
Ding-Fang1, QI Yong-Fen2, NIU
Da-Di2, WANG Shu-Heng2, PANG
Yong-Zheng2, TANG Chao-Shu2
Department of Cardiovascular Disease, the Third Hospital,
Peking University; 1Institute of Cardiovascular Disease Research,
Peking University First Hospital, Beijing 100034;
2Institute of Cardiovascular Research, Peking University, Beijing100083,
China
AIM: To study the effects of Cobalt-60
radiation on gene expression and secretion of adrenomedullin
and endothelin in cultured rat vascular smooth muscle
cells. METHODS: Rat vascular smooth muscle cells
were cultured in Dulbecco's modified Eagle's medium
containing 10 % fetal bovine serum and were radiated
with Cobalt-60 radiation at doses of 1, 14, and
25Gy. mRNA levels of adrenomedullin and endothelin
in the cells were studied by reverse-transcriptative
competitive polymerase chain reaction. Secretion of
adrenomedullin and endothelin by the cells was studied
by radioimmunoassay. RESULTS: Secretion of
adrenomedullin increased by 270 % and 233 % with 14
and 25Gy radiation, and adrenomedullin mRNA level increased by 82.4 % and 101 %, respectively.
Secretion of endothelin decreased by 27.3 % and 58.0 % in
vascular smooth muscle cells with 14 and 25Gy
radiation respectively. Endothelin mRNA level decreased by
47.1 % and 40.2 % in smooth muscle cells with 14 and
25Gy radiation, respectively. CONCLUSION:
Cobalt-60 radiation can increase adrenomedullin and
decrease endothelin both at gene transcription and
protein levels.
O-03 Effect of mouse IL-12 plasmid on airway
inflammation and cytokines of asthmatic model in BALB/c mice.
LI He-Quan, SHAO Chuan-Sen, XIE Qiang-Min, BIAN
Ru-Lian
Medical School of Zhejiang University, Hangzhou 310031, China
AIM: To investigate the effect of mouse IL-12 gene
expressive plasmid (mIL-12 plasmid) on the airway
inflammation and the cytokines level in asthmatic mouse
and to analyze the probable mechanism.
METHODS: A mouse asthma model was established by sensitiation
with ovalbumin (OVA). The animals were divided into
six groups including asthmatic model group (sensitized
with OVA plus challenging with OVA by aerosol), model
control group (sensitized with OVA plus aerosolizing
with normal saline), mIL-12 plasmid preventive group,
mIL-12 plasmid treatment group, empty plasmid preventive group, and empty plasmid group. The EOS
count and concentration of IL-4, IL-5, and
IFN- in the mice bronchoalveolar lavage fluids (BALF) were
detected. RESULTS: The EOS count and
concentration of IL-4, IL-5, and IFN- from the model
control group were
(0.01±0.03)×108/L, (24±4),
(63±10), and (725±59) ng/L, respectively, those from
the mIL-12 plasmid preventive group were
(0.06±0.04)×108/L, (43±13), (66±14), and (626±60) ng/L, and
those from the mIL-12 plasmid treatment group were
(0.11±0.12)×108/L, (38±14), (33±6), and (661±40) ng/
L, showing significant differences compared with the
asthmatic model group
[(3.0±1.2)×108/L, (122±45),
(126±34), and (435±49) ng/L] (P<0.01). The EOS
count and concentration of IL-4, IL-5, and
IFN- in BALF from the mIL-12 plasmid preventive group
and the mIL-12 plasmid treatment group also showed
significantly differences as compared with those from
the empty plasmid preventive group
[(2.1±0.9)×108/L, (110±24), (112±11), and (464±51) ng/L] and the empty
treatment plasmid group
[(2.1±0.9)×108/L, (88±17),
(107±6), and (481±64) ng/L] (P<0.01).
CONCLUSION: mIL-12 plasmid can significantly
inhibit the airway inflammation, and the probable
mechanism may be balancing Th1/Th2 cell subsets.
O-04 Effects of endothelin-1 mRNA antisense
oligodeoxynucleotides on ischemic arrhythmia in isolated rat
hearts1
QIU Jing-Wei, LIN Li, YANG Ling, PAN Xiu-Jie, REN
An-Jing, CHEN Hong, YUAN Wen-Jun2
Department of Physiology, Second Military Medical University,
Shanghai 200433, China
AIM: Endothelin-1 (ET-1) level in plasma is incresead in patients with
acute myocardial ischemia. Our previous experiments showed that ET-1 mRNA antisense
oligodeoxynucleotides (AS-ODN) ameliorated acute ischemic arrhythmia in intact
rats. The present study was to determine whether ET-1 AS-ODN also could suppress
the acute ischemic arrhythmia caused by coronary occlusion in isolated rat hearts.
To illustrate the involvement of cardiac ET-1 in ischemic arrhythmia, the effects
of ET-1 mRNA AS-ODN on acute ischemic arrhythmia were observed. METHODS:
ET-1 AS-ODN was injected iv to rats 2 h before heart isolation, and then the
ischemic arrhythmia caused by occlusion of the left anterior descending (LAD)
coronary artery was observed in isolated perfused heart. RESULTS: (1)
In normal perfused rat hearts, a small number of ventricular ectopic beats (VEB)
were observed, and neither ventricular tachycardia (VT) nor heart arrest occurred.
(2) LAD occlusion elicited various ischemic arrhythmias in isolated hearts.
No differences in the incidences and numbers of the arrhythmias were found between
isolated hearts which had received normal saline (NS) and those which had received
ET-1 ODN (sense-ODN and scrambled-ODN) in vivo. VT occurred in all hearts,
and the percentage of heart arrest was 33.3 % in NS group, 12.5 % in sense-ODN
group, and 25 % in scrambled ODN group. (3) ET-1 AS-ODN dose-dependently decreased
the incidence of VT, the arrhythmia score, the numbers of single VEB and VT,
and no heart arrest was observed in the hearts pretreated with ET-1 AS-ODN.
CONCLUSION: Endogenous ET-1 does play an important role in the genesis of
acute ischemic arrhythmia in isolated rat hearts.
1 Project was supported by the National Natural Science
Foundation of China (No 30070306).
2 Correspondence to Prof YUAN Wen-Jun.
O-05 Losartan protects neonatal rat cardiac myocytes from advanced glycation end-products-
induced apoptosis
ZENG Ping, XU Ding-Li, LI Zheng, LAI Wen-Yan,
REN Hao
Department of Cardiology, Nanfang Hospital, The First Military
Medical University, Guangzhou 510515, China
AIM: To investigate effects of advanced glycation end-products (AGE)
on cell cycle distribution and apoptosis in neonatal rat cardiac myocytes (CM);
to study whether losartan, an angiotensin-converting enzyme inhibitor anent,
protects CM from AGE-induced apoptosis and affects the distribution of cell
cycle. METHODS: CM primarily cultured for 3-5 d were exposed to vehicle
(control), AGE-BSA (50 and100 mg/L) in the presence or absence of losartan (10-5
mmol/L) for 12, 24, and 48 h. Serum-starved cells used in cell cycle distribution
analysis were cultured with 1 % fetal bovine serum for 24 h before analyzing.
Cells were harvested; Flow cytometer (FC) was used to detect the magnitude of
apoptosis and cell cycle distribution. RESULTS: No significant differences
were observed on cell cycle distribution in CM after treatment with AGE and/or
losartan. The average values of apoptosis in the groups of control, AGE (50
and 100 mg/L) were 3.9±0.3, 6.7±1.0, 8.6±0.9 at 12 h, 6.0±0.4,
10.3±0.5, 12.9±1.5 at 24 h, and 8.8±0.7, 14.5±0.8, 18.5±0.8
at 48 h, respectively. The average values in the groups of AGE (50 mg/L)+losartan
(10-5 mmol/L), AGE (100 mg/L)+losartan (10-5 mmol/L) were
4.8±0.3, 6.5±0.4 at 12 h, 8.6±1.3, 10.2±1.8 at 24 h, and
12.9±2.2, 16±4 at 48 h, respectively. CONCLUSION: AGE significantly
increases the magnitude of apoptosis in CM in a time- and dose-dependent manner.
Losartan inhibit sAGE induced apoptosis in CM.
O-06 Effect of interleukin-2 on contraction and
intracellular pH in isolated ventricular myocytes
CAO Chun-Mei, XIA Qiang, SHEN Yue-Liang, YE Zhi-Guo, CHEN Jun-Zhu
Department of Physiology, Zhejiang University School of Medicine,
Hangzhou 310031, China
AIM: Cytokines are low molecular weight proteins that
act as communication signals between cells of the
immune system and as systemic mediators of the host's
response to infection. Recent observations suggest that
proinflammatory cytokines can modulate cardiovascular functions, but direct myocardial effects of cytokines
remain to be elucidated. The aim of the present study
is to determine the effects of interleukin-2 (IL-2), one
of cytokines, on contractility and intracellular pH
(pHi) in isolated rat ventricular myocytes and underlying
mechanisms. METHODS: Video tracking system and
spectrofluorometry were employed to measure the
contractility and intracellular pH in enzymatically isolated
ventricular myocytes, where BCECF/AM was used as intracellular pH indicator.
RESULTS: IL-2 (2.5-200 kU/L) depressed the contraction in a dose-dependent
manner. IL-2 decreased ±dL/dtmax, dL, and
end-diastolic cell length. Inactive IL-2 at 200 kU/L did not
significantly decrease ±dL/dtmax, dL, or cell length.
Pretreatment with naloxone (10 nmol/L), a universal opioid
receptor antagonist, or nor-BNI (10 nmol/L), a specific
opioid receptor antagonist, abolished the
inhibitory effect of IL-2 on contraction; naltrindole (1
mol / L), a specific 
-opioid receptor
antagonist, had no effect. IL-2 (200 kU/L) decreased
the pHi of cardiaomyocytes, which could be attenuated
by nor-BNI (10 nmol/L). CONCLUSION: It is
concluded that the decreased contraction and
pHi of ventricular myocytes by IL-2 is mediated via the cardiac
opioid receptor.
O-07 Inhibitory effect of DL111-IT of
angiogenesis in vivo and in vitro
ZHOU Hui-Jun, LI Jun, WU Guo-Dong
Department of Pharmacology, College of Pharmaceutical
Sciences, Zhejiang University, Hangzhou 310031, China
AIM: To investigate the ability of DL111-IT to inhibit
angiogenesis in experimental models in vivo and
in vitro. METHODS: The chorioallantoic membrane (CAM) of
the chick embryo was used to measure angiogenesis.
The rabbit cornea was operated to assess neovascular
growth. The human umbilical vein endothelial cell
(HUVEC) was used to determine cytotoxicity by MTT.
RESULTS: DL111-IT can induce a dose-dependent
antiangiogenic reaction in the CAM. The counts of
blood vessels in the CAM given DL111-IT 500, 250, 10, and 0
g/embryo were 0, 23, 84, 162, and 197, respectively; Meanwhile
ID50 of the chick embryos (n=10) was 0.039 mg/embryo (0.013-0.100).
In the rabbit model of neovascularization induced in
the cornea by suturing DL111-IT at 3, 1, 0.3, and 0
mg/eye per 3 d the length of the blood vessels were 0/1,
1-2/2, 1-4/3, and 10.06±1.39/18 (mm/total number vessels,
n=4). When HUVEC was grown in vitro
and subjected to increasing concentrations of DL111-IT, inhibition of
HUVEC cells cultured was observed and
IC50 of the HUVEC cell was 15.86, 11.41, and 0.94 mg/L in 24, 48, and 72 h.
CONCLUSION: DL111-IT exerts inhibitory effects on
angiogenesis both in vivo and in
vitro.
O-08 Antimetastatic activity of Liuwei Dihuang
decoction, a typical traditional Chinese medicinal
prescription
QI Chun-Hui, WANG Yan, ZHAO Xiu-Nan, ZHANG Yong-Xiang, SHEN
Bei-Fen1
Beijing Institute of Pharmacology and Toxicology,
1Beijing Institute of Basic Medical Science, Beijing 100850, China
AIM: To evaluate the antimetastatic activity of Liuwei
Dihuang decoction (LW), a typical traditional Chinese
medicinal prescription for nourishing "Kidney-Yin", and
to explore its possible mechanisms. METHODS:
An experimental lung metastatic model of B16 melanoma
and spontaneous lung metastatic model of mouse lung
adenocarcinoma (MLA795) were employed, and
3H-TdR incorporation, ELISA, FACS (florescent activated cell
sorting), EMSA (electrophoretic mobility shift assay)
and gelatinase activity assay were used in the experiments.
RESULTS: Oral administration of LW (5 and 10 g/kg for 14 d) reduced the organ colonization
of the lungs in both metastatic models and modulated
the balance of humoral and cellular immune responses
disturbed in tumor-bearing mice. LW was showed to
be effective in promoting the activity of NK and LAK in
MLA795 model mice. In addition, the mRNA expression and gelatinase activity of matrix
metalloproteinase-9 (MMP-9) were significantly inhibited and AP-1 signal
transduction pathway was partially blocked after oral
treatment with LW. CONCLUSION: These results
suggest that LW possesses an antimetastatic effect in
experimental lung metastasis mice, which is likely to be
reached by modulating the balance of immune function
to intensify immune surveillant function, and inhibiting
the gelatinase activity of MMP-9 and the signaling
pathway mediated by AP-1.
O-09 Effect of anoxia-fast and anti-tireness of
Fumaria medicated wine
ZHANG Jian, ZHANG Ji, YANG Yong-Li1, GUO
Shou-Jun1
Department of Pharmacology, Lanzhou Medical College, Lanzhou
730000; 1College of life Science, Northwest Normal University,
Lanzhou 730070, China
AIM: To study the anoxia-fast and anti-tireness effect
of Fumaria medicated wine. METHODS: The
anoxia-fast effect of the medicated wine was investigated by
applying the pathological model of asphyxiant anoxia
under normal atmospheric pressure in mice, the model
of heart muscle anoxia caused by isoprenaline, and the
model of brain anoxia in decapitated mice. The
anti-tireness effect of the wine was studied by applying the
experiment of mice swimming. RESULTS: In the
experiments of above pathological models of anoxia
Fumaria medicated wine increased the utilization rate
of blood oxygen in mouse body, decreased the speed
of oxygen consumption in mouse organism, and remarkably prolonged the mouse swimming time.
CONCLUSION: Fumaria medicated wine is a
composite pharmaceutical preparation which mainly
consists of the extract of Corydalis adunca Maxim (a
natural Tibetan medicine in Gansu) supplemented by
Cuscuta chinensis Lam. And Morinada
officinalis How according to the medicallit erature, dehydrocory-daline, DHC
has effects of dilating coronary blood vessels,
inhibiting aggregation of thrombocytes, improving
micro-circulation, and increasing the anoxia-fast ability of mice.
Morind offcinalis How stimulates
hypothalamic-pituitary-adrenal axis, HPA promotes releasing
adrenocortical hormones, Cuscuta chinensis Lam, besides above
actions, increases immunoreaction of organism and
resists decrepitude. The results of this study tally with
the reports in medical literature, showing that fumaria
medicated wine has anoxia-fast and anti-tireness actions.
Therefore, this study provides a scientific basis for
developing tonic beverages from Fumaria medicated wine.
O-10 Effect of hyperhomocysteinemia on calcified
vessel
YANG Ying, TANG Chao-Shu1, LI Chun-Yue
Department of Physiology, Inner Mongolia Medical College
Huhhot 010059; 1The Cardiovascular Institute, the First
Hospital of Peking University, Beijing 100034, China
AIM: To observe the effect of hyperhomocysteinemia on calcified vessel
and explore the regulatory mechanism of vascular calcification. METHODS:
Rats were subjected to VD3 plus nicotin to develop the calcified
vessel model, and at the same time fed with high methionine diet to induce hyperhomocysteinemia.
The animals were divided into four groups: (1) control, where no specific agent
was used; (2) Hcy treated-group, where rats were fed high methionine diet; (3)
VDN group, where rats were fed with nicotin plus VD3 im injection;
(4) VDN+Hcy group, where rats were fed with nicotin plus VD3 im injection+high
methionine diet. All animals were treated with above agents for 6 week.
RESULTS: Calcium deposition was increased by 9.09-fold and 12.21-fold in
VDN group and VDN+Hcy group compared with control group, and VDN+Hcy group was
increased by 34.29 % compared with that of VDN group (P<0.01). Plasma
MDA content and conjugated diene content in VDN+Hcy group increased by 18.75
% and 2.89-fold (P<0.01) respectively, aortic osteocalcin level and
alkaline phosphatase activity in VDN group increased by 2.81-fold and 45.57
% compared with control group. However, aortic osteocalcin level and alkaline
phosphatase activity in VDN+Hcy had no significant change compared with control
(P>0.05). Tissue HE stain showed that the vascular endothelium was
damaged in Hcy, VDN, and VDN+Hcy groups, and Von Kossa stain showed medium calcium
deposition in VDN and VDN+Hcy group. CONCLUSION: Hyperhomocysteinemia
accelerates vascular calcifica-tion, and its mechanism needs further to be explored.
P-01 Caveolin-1/ERK1/2 signal transduction
pathway mediates probucol regulation on vascular
remodeling after percutaneous transluminal angioplasty in rabbits
LIAO Duan-Fang, YANG Yun-Bo
Institute of Pharmacy & Pharmacology, Nanhua University,
Hengyang 421001, China
AIM: To investigate the correlation between
preventive effects of probucol on restenosis and its improving
vascular remodeling by inhibition of ERK1/2 activation.
METHODS: New Zealand rabbit thoracic aorta
atherosclerosis was induced by 3.5 F balloon catheter
injury following a 4-week feeding with high cholesterol
diet, and percutaneous translumanal angioplasty (PTA)
was performed with 3.5F balloon catheter. After 2 weeks
of PTA, the bore and diameter of aorta, intimal elastic
lamina (IEL), extral elastic lamina (EEL), neointimal area
(NEA), medial area (MA), and NEA/MA ratio were measured. The relaxation and contraction responses
of rabbit aortic rings to acetylcholine, serotonin,
norepinephrine, and potassium chloride were measured
by bioassay. ERK1/2 activity was determined by
Western blot using phospho-ERK1/2 antibody, and the
expression of MKP-1 and caveolin-1 was determined by
Western blot using MKP-1 or caveolin-1 antibody, respectively.
RESULTS: Probucol treatment for 5 weeks significantly improved the restenosis of aorta as
shown by increasing bore, diameter, lumen area, IEL,
EEL of restenotic aorta and decreasing NEA, NEA/MA.
In addition, probucol could protect the relaxation
responsibility of restenotic vessel to acetylcholine and
decrease the contraction responsibility to serotonin,
norepinephrine, and potassium chloride. Western blot
analysis showed that probucol markedly inhibited the
ERK1/2 activity by enhancing the expression of caveolin-1
and MKP-1 of restenotic vessel wall. Furthermore,
probucol also showed to enhance nitric oxide level of
serum and inhibit expression of c-myc and PCNA.
CONCLUSION: Probucol prevented restenosis by
improving vascular remodeling after angioplasty. The
regulating effect of probucol on remodeling is related to
enhancement of expression of caveolin-1 and MKP-1 and
therefore to inhibition of ERK1/2 activity.
P-02 Effect of long term cigarette smoke on
hemodynamics in spontaneously hypertensive rats ---
histological evaluation of endothelin receptors
NAKASHIMA Toshikatsu1, OHNO
Norikazu1, TANAKA Takeshi1,
KUBO Kaoru2, NAKATANI
Toshiaki1, KITA Taizo1,3,
YONETANI Yukio1, KONISHI Noboru1
1Department of Pharmacology;
2Institute of Laboratory Animals;
3Department of 2nd Pathology, Nara Medical University,
Kashihara, Nara 634-8521, Japan
AIM: Although cigarette smoke is a risk factor for
coronary heart disease, the relationship between
cardiovascular changes associated with hypertension and tobacco
smoking remains unknown. We investigated the effects of chronic cigarette smoke on cardiovascular
function using mature male spontaneously hypertensive rat
(SHR/NCrj) and normotensive Wistar Kyoto rat (WKY/NCrj).
METHODS: SHR and WKY were exposed to smoke (30 cigarettes for 20
min·d-1) in a chamber, 5 d each week for 8 weeks using the Hamburg II smoking
machine. Both blood pressure (BP) and heart rate (HR)
were measured once each week by the tail cuff method.
The hemodynamics from 15 min to 50 min after cigarette smoke were measured on Thursday every week,
from 125 min to 165 min taken at 2 and 24 h after last
cigarette smoke. The density of endothelin (ET)
receptor was evaluated by immunohistochemistry.
RESULTS: In SHR, an early response of HR after
cigarette smoke, but at 24 h, HR and systolic BP (SBP) were
lowered. This change was well phenomenon for SHR under hypertensive and tachycardiac state. In WKY, an
early response of HR after cigarette smoke increased
significantly, and at 24 h recovered to the basal state.
The density of ET A receptor in adrenal medulla of no
smoke SHR was weaker compared to no smoke WKY, but the density was increased by cigarette smoke. In
WKY, the density did not change. CONCLUSION:
It is said that ET A receptor in adrenal medulla modulates
the synthesis of catecholamine. We speculate that this
catecolamine modulation system through ET A receptor induced the well condition for smoke SHR.
P-03 Preventive effect of ET-1 pretreatment on
hypoxia-induced injury in cultured neonatal rat
cardiomyocytes
PAN Yan-Xia 1,2, LIN Li 1, YUAN Wen-Jun
1
1Department of Physiology, Second Military Medical University,
Shanghai 200433; 2Department of Physiology, Fujian Medical
University, Fuzhou 350004, China
AIM: To observe the effects of ET-1 pretreatment on
hypoxia-induced injury and changes in intracellular free
calcium concentration
([Ca2+]i) in cultured neonatal rat
cardiomyocytes. METHODS: The supernatant lactate
dehydrogenase (LDH), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were
determined in the cultured cardiomyocytes subjected to
hypoxia induced by incubation in a 3 %
O2-5 % CO2 hypoxic atmosphere for 12 h at 37 ºC with or without
ET-1 pretreatment. [Ca2+]i were measured with
Ca2+-sensitive fluorescent probe fluo-3/AM under laser
scanning confocal microscope. Fluorescence intensity
emitted from fluo-3/AM-loaded cells reflected the
concentration of [Ca2+]i. The hypoxia model used in
[Ca2+]i measurement was estabilshed by constant perfusing
cardiomyocytes for 30 min with hypoxic DMEM solution containing
Na2S2O4 1mmol/L and equilibrated with
95% N2-5 % CO2 . Pretreatment with ET-1 consisted
of three cycles of ET-1 perfusion (5 min for each)
followed by ET-1-free DMEM solution (10 min for each)
prior to hypoxia. RESULTS: (1) Under pretreatment
with ET-1 0.01-1 nmol/L, LDH release and supernatant
MDA content were decreased, but SOD activity was enhanced dose-dependently, as compared with the
hypoxia group (P<0.01). (2) The spontaneous calcium
transient in cultured cardiomyocytes was terminated in
30 s, and then [Ca2+]i was increased markedly after
perfusion with hypoxic solution. (3) ET-1 0.01-1
nmol/L increased the frequency of
[Ca2+]i transient in cultured cardiomyocytes in a dose-dependent manner .
The termination of [Ca2+]i transient and the elevation
of [Ca2+]i caused by hypoxia were postponed under
pretreatment with ET 0.01-1 nmol/L. CONCLUSION:
Pretreatment with ET-1 0.01-1 nmol/L attenuated
hypoxia-induced injury partially due to improving SOD
activity, decreasing in MDA production, and inhibiting
[Ca2+]i elevation. ET-1 pretreatment possibly protected
rather than injured cultured neonatal rat cardiomyocytes
under the condition of hypoxia.
P-04 Animal models of rhinorrhea induced by
carbamylcholine
PAN Jia-Hu, CHANG Jian-Jie , NIE Jing
Department of Pharmacology, School of Pharmacy, Fudan
University, Shanghai 200032, China
AIM: To set up the rabbit and rat models of rhinorrhea induced by carbamylcholine
(Car), a parasympathomimetic agent. METHODS: The rhinorrhea animal models
induced by Car were produced on rabbit and rat. The revised method of filter
paper strips was used to collect the nasal mucosal secretion and monitor the
nasal response. Ipratropium bromide (IB), a typical anticholinergic agent, was
used to check the effects on hypersecretion induced by Car. RESULTS: 1)
Nasal secretion was obviously increased by Car in a dose-dependent manner. Car
86 g /kg and 30-min challenge were
adopted for the rabbit model, and Car 0.64 mg/kg and 30-min challenge were used
for the rat model. 2) The rabbit nasal secretary kinetics induced by Car was
in a prolonged pattern, quite different from the quick pattern induced by methacholine.
The rabbit nasal secretion induced by Car was increased slowly and reached the
peak with the amount of (54±32) mg per 2 min at about the 13th min, and
then decreased to near the basic level 30 min later. The rat nasal secretary
kinetics induced by Car was a little different from that of rabbits, in which
it was increased quickly, reached the peak at about the 7th minute with the
amount of (8.2±2.7) mg per 2 min, and went back to the basic level after
31 min. 3) IB effectively inhibited the hypersecretion on both rabbit and rat
models challenged by Car in a dose-dependent manner. CONCLUSION: The
rabbit and rat rhinorrhea models were successfully produced by Car. Their nasal
secretary kinetics proved to be the prolonged patterns and the nasal hypersecretion
induced by Car was obviously inhibited by ipratropium bromide.
P-05 Effects of prousion on rabbit models with high
plasma lipid
PAN Jia-Hu, HASHIMOTO Masakazu1, CHANG
Jian-Jie, NIE Jin, JIN Jian
Department of Pharmacology, School of Pharmacy, Fudan
University, Shanghai 200032, China; 1Japan Health Promotion
Association, 7-17-801 Nihonbashi Yokoyama-cho, Chuou-ku,
Tokyo 103-0003, Japan
AIM: To check the toxicity of prousion-B and detect
its effects on the rabbit models with high plasma lipid.
METHODS: Prousion-B (P-B): one kind of prousion
powder (grey powder, d3
m) made of natural porous ore found in Japan, Japan Patent
Application Number: BFC4j (B2002-52397), offered by Dr
M Hashimoto. 1) The acute toxicity of P-B suspended
with 0.2 % CMC was performed on the mice of Kunming
species. 2) The rabbit models with high plasma lipid
were used to determine the effects of P-B in
vivo, ie, the rabbits were divided into 6 groups: blank control,
model, positive control (nicotinic acid), P-B in different
dosage (0.1, 0.2, and 0.4 g/kg). The rabbits were given
ig with the above drugs once a day at the same time
with high lipid food for 6 weeks. 3) The blood lipid
levels were checked in 2 weeks and the blood samples
were taken for the examination of some enzyme
activities reflecting the heart and liver functions.
RESULTS: 1) The dosage of P-B in the largest suspension
concentration (3 g/kg) did not induce death of the mice,
showing that the toxicity of P-B was low. 2) Although there
was no significant difference between the rabbit groups
given with P-B and the control in statistics, P-B showed
a tendency to decrease blood total cholesterol (TC),
low-density lipoprotein (LDL), and apolipoprotein B
(apB) levels in the rabbits. 3) P-B obviously inhibited
the increase of total creatine kinase (TCK) activity and
glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) levels induced by high
plasma lipids in the rabbit models, reflecting that P-B
had certain protective functions on the rabbit model with
high plasma lipid. 4) P-B showed no obvious effects
on kidney function, blood electrolyte levels, and
erythrocyte index in rabbit models. CONCLUSION:
Prousion-B had certain protective functions on the
rabbit model with high plasma lipid.
P-06 Role of the caudal ventrolateral medulla
I1-imidazoline receptors in controlling the
cardiovascular activities
WANG Wei-Zhong, YUAN Wen-Jun, BEI Jie
Department of Physiology, Second Military Medical University,
Shanghai 200433, China
AIM: Many papers showed that the I1-imidazoline receptors
within the center nerve system played an important role in controlling the cardiovascular
activities. The purpose of the present study was to explore the possible role
of I1-imidazoline receptors within the caudal ventrolateral medulla
(CVLM) in regulation of cardiovascular activities in anesthetized rats.
METHODS and RESULTS: Unilateral microinjection of 2 nmol of idazoxan, a
mixed antagonist of I1-imidazoline receptors and 2-adrenoceptors,
into the CVLM significantly (P<0.01) decreased blood pressure (BP),
heart rate (HR), and the firing rate of presympathetic neurons in the rostral
ventrolateral medulla (RVLM). Moreover, the CVLM unilateral injection of idazoxan
significantly (P<0.01) reduced the inhibitory responses of the ipsilateral
RVLM presympathetic neurons evoked by stimulation of aortic nerve and elevation
of BP, and partially inhibited the neuronal cardiac cycle-related rhythm. Depressor
responses evoked by aortic nerve stimulation were significantly (P<0.01)
attenuated 10 and 20 min after bilateral microinjection of idazoxan 2 nmol (each
side) into the CVLM. However, injection of 500 pmol of yohimbine, a selective
2-adrenoceptor antagonist,
into the CVLM did not affect the resting cardiovascular activities and baroreceptor
reflex. CONCLUSION: the CVLM I1-imidazoline receptors played
an important role in tonic and reflex control of peripheral cardiovascular activities.
P-07 Stimulating endothelin synthesis and
secretion of rat aorta by rat urotensin II
ZHANG Yong-Gang, WEI Rui-Hong1, CAO
Jun2, BU Ding-Fang, PANG Yong-Zheng, TANG Chao-Shu
Institute of Cardiovascular Research, the First Hospital, Peking
University, Beijng 100034; 1Department of Internal Medicine,
Affiliated Hospital of Handan Medical School, Handan 056002;
2Department of Pathophysiology, Ningxia Medical College,
Yinchuan 750004, China
AIM: To investigate the effect and signal transduction pathways of urotensin
II (UII) on endothelin production in cultured aortic tissues of rat. METHODS:
Aortic slices were incubated with different concentration of UII. Contents
of endothelin (ET) both in medium and tissues were measured with radioimmunal
assay. Different inhibitors were added to the medium to study the roles of different
signal transduction pathways in the stimulating effect of UII on production
of ET. RESULTS: UII significantly stimulated ET secretion (ET in medium)
and production (ET both in medium and tissues) from rat aortic slices. After
3-h incubation, ET production (including secretion) were increased significantly
in 10 nmol/L of UII group than control group (pg/mg, wet weight: 4.5±0.7
vs 1.83±0.20, P<0.01). After 6 h incubation, it was shown
that UII induced ET secretion and production in a time and concentration-dependent
manner, which were increased by 59.2 %, 108.0 %, 159.6 %, and 178.0 % in secretion
(P<0.01), and 40.6 %, 68.4 %, 103.1 %, 105.7 % in production (P<0.01)
induced by UII 0.1 nmol/L-0.1 mol/L,
respec-tively. Furthermore, the effects of UII on ET-induction were inhibited
by 34.1 %, 24.5 %, 32.2 %, 32.1 %, and 27.6 % in secretion (P<0.01)
and by 33.5 %, 31.5 %, 25.8 %, 28.0 %, and 32.5 % in production (P<0.01)
when incubation with nicardipine, H7, PD98059, cyclosposphamide,
or actinomycin D, which are inhibitors of calcium channel, PKC, MAPK, protein
synthesis, and mRNA production, respectively. CONCLUSION: UII stimulated
ET synthesis and release from rat aortic tissues, and this effect might be mediated
by Ca2+, PKC, and MAPK signal transduction pathways. It was proposed
that UII might play importent roles in cardiovascular homeostasis and pathology
by cross-talking with other vasoactive peptides (such as ET), which deserves
further investigation.
P-08 Blocking effect of Stauntonae saponin on
nerve conduction
YE Wen-Bo, ZHANG Hui-Qi, JIN Rong-Hua
College of Life and Environment Sciences, Shanghai Teachers
University, Shanghai 200234, China
AIM: To assess the effects of Stauntoniae saponin (SS) on nerve conduction.
METHODS: SS was extracted from Stauntonia chinensis DC, a medicinal
herb used for relieving the pain. After extraction, 0.3 % SS was prepared and
applied on the saphenous nerve of SD rats. Following the application, the amplitude
of the compound action potentials was compared with its control, and the nerve
was taken for surveying by transmission electron microscope. RESULTS: SS
functioned to demyelinate nerve and to block nerve conduction. Sixty minutes
after application, the myelin sheath and the axon membrane of nerve fibers were
adsorbed with many SS, and the amplitude of A component of the compound action
potentials dropped to 0.22±0.28 (n=5, P<0.01 vs control).
CONCLUSION: Stauntoniae saponin may have effects on destruction of nerve
structure and blockage of nerve conduction, resulting from its appetency with
axon membrane and myelin sheaths of nerve fibers.
P-09 Role of RVLM angiotensin II receptors in
cardiovascular activities of rats
PAN Yan-Xia1,2, WANG
Wei-Zhong1, DAI Xiu-Zhong2, BAI
Jie3, YUAN Wen-Jun1
1Department of Physiology, Second Military Medical University,
Shanghai 200433; 2Department of Physiology, Fujian Medical
University, Fuzhou 350004; 3Department of Physiology, Ningxia
Medical College, Yinchuan 750004, China
AIM: To investigate the role of the angiotensin II (Ang
II) receptors within the rostral ventrolateral medulla
(RVLM), a pressor cardiovascular centre, in the tonic
cardiovascular activities and the arterial baroreceptor
reflex of rats. METHODS: The effects of Ang II and
Ang II receptor antagonist bilaterally microinjected into
the RVLM on blood pressure (BP), heart rate (HR), and
arterial baroreceptor reflex were observed in
urethane-anesthetized SD rats. RESULTS: Bilateral
microinjection of Ang II (100 pmol in 100 nL for each side) into
the RVLM increased both BP and HR significantly
(P<0.01, n=6). In contrast, bilateral RVLM
microinjection of [Sar1, Thr8] Ang II (10, 100, and 1000 pmol in
100 nL for each side, n=5 for each dose), an Ang II
receptor antagonist, decreased both BP and HR
dose-dependently (P<0.01). Besides, [Sar1, Thr8] Ang II
abolished the effects of Ang II on BP and HR. In
regard to the arterial baroreceptor reflex, the decrease
of BP evoked by electrical stimulation of the aortic
nerve was not altered by bilateral RVLM microinjection of either Ang II (100 pmol in 100 nL for each
side, n=6, P>0.05) or [Sar1, Thr8] Ang II (100 pmol
in 100 nL for each side, n=6, P>0.05).
CONCLUSION: The RVLM Ang II receptors were involved in
maintaining the tonic excitatory cardiovascular activities, while
not in mediating the arterial baroreceptor reflex.
P-10 Concentrations of PAMP in blood and tissues
of rats with sepsis shock
JIANG Wei, REN Yong-Sheng, JIANG Hong-Feng, SHAO Yi, CAI Da-Yong, TANG Chao-Shu
Institute of Cardiovascular Diseases, the First Hospital, Health
Sciences Center, Peking university, Beijing 100083, China
AIM: To study the role of proadrenomedullin
N-terminal 20 peptide (PAMP) in sepsis shock. METHODS:
We employed the method described by IH Chaudry
et al to get animal model of polymicrobial sepsis by cecal
ligation and puncture (CLP) to study the role of PAMP
in sepsis shock. Twenty-four male Sprague Dawley rats (250-330 g) were divided into three groups (ie, 10
and 20 h after CLP or sham-operation, 8 rats each group), 10 h after CLP represents the early,
hyperdynamic stage of sepsis (ES) and 20 h after CLP
represents the late, hypodynamic stage of sepsis (LS). At
10 or 20 h after CLP or sham operation, the blood was
drawn from the left common carotid artery for blood
PAMP assay, and the heart and thoracic aorta were also
harvested for tissue PAMP assay. The levels of PAMP
were measured by a radioimmunoassay (RIA) kit special for the rat PAMP from Phoenix Co
(USA). RESULTS: We found that the concentrations of PAMP
in blood of sepsis rats were (86±11) pmol/L (early shock,
n=7) and (109±26) pmol/L (late shock,
n=7) pmol/L, respectively, and were higher
(P<0.01) than that of the control [(58±10) pmol/L,
n=7]. Furthermore, the PAMP in blood of late shock was 26.60 % more than that of
the early shock but without significant statistical
differences. The contents of PAMP in heart of sepsis rats were (1.06±0.18) (early shock,
n=6) and (0.91±0.11) ng per g protein (late shock,
n=6), respec-tively, and the contents of PAMP in
early shock were higher (P<0.05) than that of the control [(0.84±0.13)
ng per g protein, n=6]. The contents of PAMP in aorta of
sepsis rats were (2.2±0.8) (early shock,
n=6) and (2.3±0.9) ng per g protein (late shock,
n=6) respectively, and compared with that of the control [(2.5±0.9) ng
per g protein, n=6)], both decreased little but there were
no significant differences in statistics. CONCLUSION:
PAMP increases significantly in blood of the sepsis
shock rats and in heart only of early shock. The change
of PAMP in sepsis may contribute to the occurrence of
the hyperdynamic response and its transition to the
hypodynamic phase of sepsis. Further study needs to be
done to learn how PAMP is involved in the procedure
of sepsis shock.
P-11 Roles of different peptide fragments derived
from proadrenomedullin in the regulation of vascular tone in isolated rat aorta
REN Yong-Sheng1,2, DONG
Xian-Hong1, LI Ju-Xiang1, ZHONG
Guang-Zhen1, WU
Sheng-Ying1,2, TANG Chao-shu1
1Department of Physiology and Pathophysiology, Peking
University, Beijing 100083; 2Department of Physiology, Yunyang
Medical College, Shiyan 442000, China
AIM: The effects of different peptides derived from
proadrenomedullin, adrenomedullin (ADM),
proadreno-medullin N-terminal 20 peptide (PAMP), and
adreno-tensin (ADT), individual or in combination, on vascular
tone were investigated in isolated rat aorta. Their
effects on cAMP accumulation and nitric oxide synthesis
were also evaluated in cultured rat aorta. RESULTS:
ADM 1-100 nmol/L induced dose-dependent relaxation
in aorta precontracted with phenylephrine. PAMP or
ADT had no effect on the aortic ring precontracted with
phenylephrine. In the presence of PAMP, ADM 10 nmol/L, no longer induced relaxant responses.
Coadministration of ADM with ADT decreased the vascular tone, not different from ADM alone. Incubation
of aortia with ADM (1-100 nmol/L) for 2 h caused a
significant increase in nitrite generation. This effect of
adrenomedullin was significantly abrogated in the
presence of PAMP or ADT. ADM (10 nmol/L)-induced nitrite production by vascular tissue was significantly
decreased by PAMP (10 nmol/L) or ADT (10 nmol/L).
ADM (1-100 nmol/L) concentration-dependently increased the cAMP levels of the incubated aortic tissue.
PAMP (10 nmol/L) and ADT (10 nmol/L) were more potent than the same concentration of ADM in
stimulating cAMP accumulation. Co-administration of ADM
with PAMP or ADT, further increased cAMP as compared with ADM alone, but the effect of PAMP and
ADT on ADM was not additive or synergistic but an antagonist one.
CONCLUSION: The relaxing effect of ADM on aorta was antagonized by PAMP, whereas
ADT had no effect on the ADM-induced relaxant effect.
The effect of PAMP on ADM induced vasorelaxation, probably through an NO-dependent pathway.
P-12 Protective effect of MK-801, an NMDA antagonist, on noise-induced hearing loss
DIAO Ming-Fang, ZHANG Yan-Min, LIU Hai-Ying, GAO Wen-Yuan
Department of Physiology, Second Military Medical University, Shanghai 200433,
China
AIM: To investigate whether MK-801, an NMDA
antagonist, plays a role in transient threshold loss (TTS)
or permanent threshold loss (PTS) arising from noise
exposure. METHODS: The Guinea pigs were randomly
assigned to four groups. Group I was treated with
MK-801 (0.5 mg/kg, ip) just before and immediately
after noise trauma (4-kHz tone at 110 dB SPL for 3 h),
Group II was treated with physiologic saline (0.5
mg/kg, ip) by the same way as Group I. Group III and
Group IV were noise-exposed (4-kHz tone at 115 dB SPL for 5 h) and treated with MK-801 (0.5 mg/kg, ip)
and physiologic saline (0.5 mg/kg, ip), respectively by
the same way as Group I. ABR of Group I and II was
recorded before and immediately 7 d after noise exposure.
ABR of Group III and IV were recorded before and 7
d and 21 d after noise exposure. The spiral ganglion neurons were analyzed by morphological
methods. RESULTS: The threshold shifts of both Group
I and II immediately after noise trauma were 24-63
dB(2-8 kHz), and 7 d after noise exposure the threshold of
most animals recovered to the level of threshold
measured before exposure. Seven days post exposure, the
threshold shifts of Group III and IV were 5-17 dB and
6-33 dB (2-10 kHz), respectively (P<0.05,
t-test) , and 3 weeks after noise exposure , the threshold shifts were
6-20 dB for Group III, and 15-30 dB for Group IV
(P<0.05, t-test). The spiral ganglion neurons were
protected in Group III compared with Group IV
(P<0.05, t-test). CONCLUSION:
MK-801 protects against PTS, not TTS.
P-13 A functional and histological study of chronic
intravenous admistration of vancomycin on the inner ear of guinea pig
LIU Hai-Ying, GAO Wen-Yuan, DIAO Ming-Fang, ZHANG Yan-min
Department of Physiology, Second Military Medical University,
Shanghai 200433, China
AIM: To observe the chronic ototoxic effects on guinea pigs treated
with daily single iv injection of different doses of vancomycin (V) for 14 d.
METHODS: Guinea pigs were divided into three groups: V 216 mg/kg, iv,
once per day for 14 d (group A); V 108 mg/kg, iv, once per day for 14 d (group
B); and saline control (group C). The duration of post-rotatory nystagmus and
the thresholds of auditory brainstem response (ABR) were measured before and
after treatment. The threshold shifts were calculated afterwards. For histological
examination the cochleas were dissected for hair cell counting, scaning electron
microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: (1)
In intra-groups and intra-individuals, there was no significant difference in
the duration of post-rotatory nystagmus before treatment and 14 or 28 d after
administration. (2) For saline control, there was almost no ABR threshold shifts
produced after click and trapezoide stimuli 14 or 28 d after administration.
There was only an average of 0.3 to 1.7 dB threshold shifts produced after click
and 2, 4, 6, and 8 kHz tone stimuli after 14-d V (108 mg/kg) treatment, with
no deterioration 2 weeks later. In group A (216 mg/kg), the mean hearing loss
of 4 and 6 kHz tone 14 d postadministration, ranging from 5.0 to 5.7 dB, was
significantly different from those of the saline control, but not after click
and 2, 8 kHz trapezoid stimuli. There was no significant difference between
group A and C 28 d after administration. (3) Histological examination showed
that there was 1 %-4 % outer hair cell loss in group A and B, not significantly
different from those of the saline control. There was no inner hair cell loss
in all groups. The results of SEM and TEM showed that no change of ultramicronic
structure of hair cells occurred after V treatment. CONCLUSION: The
admistration of a single daily dose, corresponding 16 and 8 times of clinical
dosage of V for 14 d, respectively, has no effects on vestibular function, and
only has a mild and transient hearing damage on guinea pigs.
P-14 Propofol restrains release of catecholamine
during ischemia and reperfusion in isolated working hearts in rats
WANG Zhong, XU Mei-Ying, DENG Xiao-Ming,
YUAN Wen-Jun, CHEN Hong
Department of Anesthesiology, Changhai Hospital, Shanghai
200433, China
AIM: To investigate the protective effect of propofol
on the heart with ischemia-reperfusion in isolated
working heart preparation. METHODS: Rat hearts were
isolated and perfused with oxygenized Krebs-Henseleit solution. After 15-min
equilibration, all rat hearts were subjected to global ischemia for 25 min followed
by 30-min reperfusion. Propofol was administered in perfusates 10, 50, and 100
mol/L before ischemia and during
30-min reperfusion. Coronary flow (CF), cardiac output (CO), heart rate (HR)
and product of left ventricular developed pressure (LVDP) and heart rate (LVDP×HR)
were detected at 5-min intervals before and after ischemia. Coronary effluent
was sampled for CK and catecholamine determination. RESULTS: During
reperfusion, the heart function of propofol-treated group recovered significantly
better than that of control (P<0.05). The value of CK release, the
concentrations in norepinephrine and epinephrine of propofol-treated groups
decreased significantly compared to control groups (P<0.05), while
the concentration of dopamine was not significantly changed. CONCLUSION:
Propofol protects isolated rat heart from ischemia-reperfusion injury and
restrains release of cardiac catecholamine following ischemia-reperfusion injury.
P-15 Role of electroacupuncture on pressor
response to angiotensin-(1-7) in rostral ventrolateral
medulla in rats
WANG Jin, SHEN Lin-Lin, CAO Yin-Xiang, ZHU
Da-Nian
Department of Physiology and Pathophysiology, Shanghai Medical Center of
Fudan University, Shanghai 200032, China
AIM: To assess the role of electroacupuncture on pressor response to
angiotensin-(1-7) [Ang-(1-7)] in the rostral ventrolateral medulla (RVLM) in
rats. METHODS: Rats were subjected to electroacupuncture (EA), microinjection,
and microdialysis in the RVLM combined with high performance liquid chromatography
(HPLC)-fluorescent detection. RESULTS: Unilateral microinjection of Ang-(1-7)
into the RVLM of anesthetized rats caused an increase in blood pressure (BP)
[from (12.5±0.5) to (15.6±0.5) kPa] accompanied by an increased
release of excitatory amino acid (EAA) glutamate [from (70±12) to (152±17)
pmol/20 L]. In contrast, microinjection
of Ang779, a selective antagonist of Ang-(1-7) receptor, into the RVLM caused
a decrease in BP [from (12.7±0.3) to (10.1±0. 4) kPa] accompanied
by a deceased release of EAA glutamate [from (68±14) to (33±4) pmol/20L]
as well as an increased release of inhibitory amino acid (IAA) glycine (Gly)
[from (98±8) to (165±11) pmol/20 L],
taurine (Tau) [from (87±12) to (142±18) pmol/20 L],
-aminobutyric acid (GABA) [from
(92±6) to (146±16) pmol/20 mL]. After EA stimulation at bilateral
Zusanli (St36) (4 Hz and 20 Hz alternately, 0.5 ms pulse, 4 mA for 20 min),
Ang-(1-7) or Ang779 was administered into the RVLM, again. EA obviously reduced
the pressor effect of Ang-(1-7) [from (12.62±0.26) to (13.25±0.21)
kPa] as well as the increment of EAA Glu release [from (79±10) to (85±12)
pmol/20 L]. EA also clearly attenuated
the depressor effect of Ang 779 [from (12.6±0.3) to (12.02±0.28) kPa]
as well as the decrement of EAA Glu release [from (81±18) to (78±17)
pmol/20 L] and the increment of IAA
release Gly [from (113±10) to (114±8) pmol/20 L],
GABA [from (87±5) to (91±12) pmol/20 L].
CONCLU-SION: EA may attenuate the pressor effect of Ang-(1-7) and depressor
effect of Ang779 through regulating corresponding amino acid neurotransmitters
release in the RVLM.
P-16 Effect of angiotensin-(1-7) in amino acid
neurotransmitter mediated blood pressure regulation
of rat caudal ventrolateral medulla
WANG Jin, SHEN Lin-Lin, CAO Yin-Xiang, ZHU Da-Nian
Department of Physiology and Pathophysiology, Shanghai
Medical Center of Fudan University, Shanghai 200032, China
AIM: To investigate the linkage between angiotensin-(1-7) [Ang-(1-7)]
and the release of amino acid neurotransmitters in the caudal ventrolateral
medulla (CVLM). METHODS: Rats were subjected to microinjection and microdialysis
in CVLM combined with high performance liquid chromatography (HPLC)-fluorescent
detection. RESULTS: Unilateral microinjection of Ang-(1-7) into the
CVLM of anesthetized rats with urethane and chloralose caused an decrease in
mean arterial pressure (MAP) [from (12.85±0.27) to (10.6 ±0.4) kPa]
accompanied by an increased release of excitatory amino acid glutamate [from
(11.0±1.4) to (21±3) pmol/20 L]
and a decreased release of inhibitory amino acid taurine [from (97±8) to
(59±7) pmol/20 L]. In contrast,
microinjection of Ang779, a selective antagonist of Ang-(1-7) receptor, caused
an increase in MAP [from (12.75±0.27) to (14.9±0.4) kPa] accompanied
by a decreased release of glutamate [from (12.0±1.2) to (5.0±0.7)
pmol/20 L] and an increased release
of taurine [from (98±6) to (140±6) pmol/20 L].
CONCLUSION: The depressor effect of Ang-(1-7) in the CVLM may be partially
due to an increased release of glutamate and a decreased release of taurine.
P-17 Effects of A3243G point mutation on
amino-acylation of human mitochondrial tRNALeu(UUR)
WANG Zhen-Cheng, WANG Xue-Min, JIAO Bing-Hua
Department of Biochemistry and Molecular Biology, Second
Military Medical University, Shanghai 200433,
China
AIM: To search for the pathogenetic mechanism of human mitochondrial
A3243G mutated tRNALeu(UUR) gene, which causes the MELAS encephalomyopathy,
maternally inherited diabetes, or chronic progressive external ophlthalmoplegia.
METHODS: The wild-type and mutant-type human mt tRNALeu(UUR)
genes were synthesized and transcribed in vivo. The kinetic parameters
of mtLeuRS expressed in E coli were determined with wild-type and mutant-type
human mt tRNALeu(UUR), respectively. RESULTS: The Kcat
and Km values of wild tRNALeu(UUR) were 0.24 S-1
and 9.1 mol/L while those of mutant
tRNALeu(UUR) were 0.22 S-1 and 13.3 mol/L.
The Kcat/Km value of wild tRNALeu(UUR)
was about 2-fold higher than that of mutant tRNALeu(UUR). CONCLUSION:
Human mitochondrial tRNALeu(UUR) gene A3243G point mutant can
remarkably reduce its aminoacylation activity, suggesting that it would be one
of the mechanisms that the mutation can produce such clinical phenotypes.
P-18 Effect of nucleotide modification on aminoacylation of human mitochondrial
tRNALeu(UUR)
WANG Zhen-Cheng, WANG Xue-Min , JIAO Bing-Hua
Department of Biochemistry and Molecular Biology, Second
Military Medical University, Shanghai 200433,
China
AIM: To assess the effect of nucleotide modification on aminoacylation
of human mitochondrial tRNALeu(UUR). METHODS: The human mitochondrial
tRNALeu(UUR) genes were transcribed in vitro with T7 RNA polymerase
and the tRNALeu(UUR) without modificated nucleotide was purified
and recovered by Urea-PAGE. The modified tRNALeu(UUR) was purified
from the cellular total tRNA of E coli JM109 which has recombinant plasmid
containg wild tRNALeu(UUR) gene. The kinetic parameters of human
mitochondrial leucyl-tRNA synthetase (mtLeuRS) were determined with two kinds
of tRNALeu(UUR). RESULTS: The Kcat and
Km values of tRNALeu(UUR) transcribed in vitro
were 11.67 mol/L and 0.17 S-1
while those of tRNALeu(UUR) expressed in E coli were 9.1 mol/L
and 0.24 S-1. The Kcat/Km values
between two kinds of tRNA were not significantly different. CONCLUSION:
Nucleotide modification may be not essential to the aminoacylation of human
mitochondrial tRNALeu(UUR).
P-19 Detection of mitochondrial DNA deletion by a
modified PCR method
WANG Zhen-Cheng, Zhu Ke-Jun, WANG Xue-Min
, JIAO Bing-Hua
Department of Biochemistry and Molecular Biology, Second
Military Medical University, Shanghai 200433, China
AIM: To develop a simple and efficient method to
detect small populations of mitochondrial DNA deletion.
METHODS: Using peripheral blood cell DNA, which
was obtained from a victim who was accidently exposed to a
60Co radiation source 11 years ago, as template, PCR was performed and multiple products
including many artifacts were generated. These PCR
products were fractionated on agarose gel and gel-purified. The real products derived from deleted mtDNA
were authenticated by comparing the products of the
secondary nested PCR. RESULTS: A new mtDNA
deletion, which spans 889 bp from nt 11688 to nt 12576,
was detected in the peripheral blood cells of the
victim. CONCLUSION: The new PCR-based method was
more efficient to detect small populations of mtDNA
deletion than other routine methods. MtDNA deletion
was found in the victim, suggesting the relationship
between the deletion and phenotypes of the disease.
P-20 Effect of thiopental, propofol, and midazolom
on arrhythmias during ischemia-reperfusion in isolated-perfused rat heart
WU Jing-Xiang, XU Mei-Ying, CHEN Hong1
Department of Anethesiology, Changhai Hospital;
1Department of Pharmacology, Second Militoary Medical University,
Shanghai 200433, China
AIM: To assess the effect of three widely used iv anesthetics on the
arrhythmias during ischemia-reperfusion in isolated-perfused rat heart and the
roles of the activity of cardiac sodium pump and the level of endogenous digitalis-like
factor (EDLF) in their anti-arrhythmias effect. METHODS: Thiopental
50 mol/L, propofol 25 mol/L,
and midazolom 1.5 mol/L were used
in isolated working rat heart subjected to 30-min warm globle ischemia followed
by 30-min reperfusion. Arrhythmia score (AS) was recorded. Activity of cardiac
sodium pump was estimated using phosphorus measurement. The level of tissue
EDLF was measued by radioimmunoassay. RESULTS: AS of treatment groups
was lower vs control (thiolpental 1.1±0.7, propofol 0.9±0.8,
and midazolam 1.0±0.8 vs control 2.9±0.9), while the activity
of sodium pump and the level of EDLF were higher compared with control. Negative
correlation had been observed between the AS and sodium pump activity. The level
of EDLF and sodium pump activity were negatively correlated in control but positive
correlated in the treatment. The EDLF and AS were positive correlated in control
but non-correlated in the treatment. CONCLUSION: The three anesthetics
have an anti-arrhythmias effect on ischemia reperfused rat heart and the effect
may be related to the sodium pump protection. Whether the protective effect
is through decreasing EDLF level needs to be further investigated.
P-21 Construction of recombinant adenovirus
vector encoding NF-B RNA aptamer
YOU Xing-Ji,YANG Sheng-Sheng, CHAI Zai-Long, ZHANG Zhi-Zhen, MAO Ji-Fang, JIAO Bing-Hua
Department of Biochemistry and Molecular Biology, Department
of Basic Medicine, Second Military University, Shanghai
200433, China
AIM: Cloning NF-B (nuclear factor-kappa B)
RNA aptamer cDNA and construction of recombinant Adenovirus vector of pAdeasy/aptamer cDNA for
further investigation on its biological activities with inhibi
tion on gene expression regulated by
NF-B. METHODS: The NF-
B RNA aptamer cDNA was cloned into pGEM-7Z vector by the procedure of
annealing and ligation of six synthetic oligonucleotide
fragments, then inserted into pShuttle-CMV vector. The
direction and sequence of insert of
NF-B aptamer cDNA were confirmed by DNA sequence
analysis. Following the co-transformation of plasmid
DNA of pAdeasy with pShuttle-CMV/aptamer cDNA, the recombinant vector of pAdeasy/aptamer cDNA was
obtained. RESULTS: The digested recombinant DNA
of pAdeasy/aptamer cDNA with Pac I showed 4.5 kb and 30 kb fragments in agarose gel, which indicated
that aptamer cDNA fragment was successfully recombined into adenovirus vector.
CONCLUSION: The production of the recombinant adenovirus vector
encoding NF-B RNA aptamer could provide the
foundation for investigation of its activity and a gene
therapy method for treatment of acute and chronic
inflammation caused by NF-B over-activity in
clinic.
P-22 5-lipoxygenase increases resistance of HeLa
cells to apoptosis
ZHOU Bin , ZHANG Qin , QUAN Hua, CHENG Min-He , CHEN Xin-Sheng , YIN Ming
Department of Pharmacology, School of Pharmacy, Second
Military Medical University, Shanghai 200433, China
AIM: To investigate whether the expression of 5-lipoxygenase increased
their survival in HeLa cells to apoptosis induced by staurosporine. METHODS:
A full-length human 5-lipoxygenase cDNA was cloned into the expression vector
pBI-G. HeLa cells were transfected with FuGENE 6. Stable HeLa cell clones expressing
5-lipoxygenae were selected with hygromycin B 0.2 g/L. Cell viabilities were
assessed by MTT assay. Apoptosis was analyzed using a FACSCalibur with APO-BRDU
kit. RESULTS: Stable HeLa cell clones expressing 5-lipoxygenase or vector
alone were isolated after 4 weeks in the presence of hygromycin B 0.2 g/L. Under
the normal culture conditions, expression of 5-lipo-xygenase at the levels of
the line used in the present study did not seem to affect HeLa cell survival
or growth. Staurosporine (0.1 mol/L)
treatment for 24 h induced apoptosis. Apoptosis in untransfected cells and vector-transfected
cells (about 40 %) was greater than that in cell lines expressing 5-lipoxygenase
(about 15 %). The level of apoptosis induced by staurosporine in 5-lipoxygenase
expressing cells was higher after treatment with 5-lipoxygenase inhibitor AA861
(5 mol/L) and MK886 (5 mol/L)
than control. CONCLUSION: The expression of 5-lipoxygenase can increase
the resistance of HeLa cells to apoptosis induced by staurosporine.
P-23 Effects of angelica on Ox-LDL-induced ET-1
release from human vascular endothelial cells
YAN Xiao-Hong, OU YANG Jing-Ping , YAN Ying-Fang,
CUI Yie-Jian, SHUN Gui-Hong, HU Jian-Ming
Department of Physiology, Medical College of Wuhan University,
Wuhan 430071, China
AIM: To investigate the effects of angelica on ET-1
release induced by oxidized low-density lipoprotein
(Ox-LDL) from vascular endothelial cells. METHODS:
After human umbilical vein endothelial cells (HUVEC) were
incubated with Ox-LDL (0.1 g/L), Ox-LDL+Angelica
(0.2 g/L), Ox-LDL+Angelica (2 g/L), and Ox-LDL+angelica
(20 g/L) for 24 h in vitro, ET-1 release content from
vascular endothelial cells were determined by
radioimmunoassay. RESULTS: HUVEC were incubated
with Ox-LDL (0.1 g/L) for 24 h, the release content of
ET-I from vascular endothelial cells significantly increased
(P<0.05). Angelica (0.2 g/L) had no effect on
Ox-LDL-induced release content of ET-1, angelica (2 and 20 g/L)
had inhibitory effects on Ox-LDL-induced release
content of ET-1, and increasing concentrations of
angelica produced a concentration-dependent inhibition tendency
of Ox-LDL-induced release content of ET-1, when the
concentration of angelica was 20 g/L, the inhibitory
effect was most pronounced. CONCLUTION: Angelica
has suppressive effects on Ox-LDL-induced ET-1 release from cultured HUVEC.
P-24 Opioid receptor antagonists modulate
Ca2+-activated K+ channels in mesenteric arterial smooth
muscle cells of rats in hemorrhagic shock
KAI Li1,2, WANG
Zhong-Feng3, HU De-Yao1, SHI
Yu-Liang 3, LIU Liang-Ming1
1Institute of Surgery, Daping Hospital; 2Department
of Pharmacology, Third Military Medical University, Chongqing 400038; 3Institute
of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy
of Sciences, Shanghai 200031, China
AIM: Previous studies have indicated a significant
enhancement of activity of large-conductance
Ca2+-activated K+ channel
(BKCa) in mesentric arterial vascular smooth muscle cells isolated from rats in the vascular
hyporesponsive stage of hemorrhagic shock. In the
present study, the effect of opioid receptor antagonist
on BKCa activity in the vascular smooth muscle cells of
rats in the hyporesponse stage of hemorrhagic shock
was investigated. METHODS: Inside-out
configuration of the patch-clamp technique was used.
RESULTS: naloxone (10 mol/L) down-regulated the
activity of BKCa by reducing open probability
(Po) and open frequency of the channels. The reduction of
Po resulted from a decrease of mean open time and an
increase of the slow closed time constant. Naltrindole
and nor-binaltorphimine (100 nmol/L) had the similar
effect to that of naloxone, but no significant effect of
-funaltrexamine (100 nmol/L) on the activity of
the channels was found. CONCLUSION: These
results suggest that - and
- opioid receptors, but
not-receptors, may be involved in the regulation of
BKCa in the vascular hyporesponse stage, and that inhibition of
BKCa may be one of the mechanisms of the opioid receptor antagonists in
improving the response of resistance arteries to
vasoactive stimulants during the decompensatory stage of
hemorrhagic shock.
P-25 Protective effect of baicalin on primary
cultures of neonatal rat cardiomyocytes deprived of
oxygen and glucose
LIU Hua, WU Xiao-Dong, YAN Qian, XU De-Yi, JIA Hong-Bin
Department of Pharmacology, School of Basic Medical Sciences,
Southeast University, Nanjing 210009, China
AIM: To investigate the protective effect of baicalin on oxygen and
glucose deprivation in neonatal rat cardiomyocytes. METHODS: Cardiomyocytes
isolated from Sprague-Dawley rats (aging 2-3 d) hearts were grown in DMEM culture
medium containing 15 % fetal bovine serum. The cell density was adjusted to
1×108 cell·L-1, and 1 mL of the suspension was
pippeted into each well of 24-well culture plates. The cells were incubated
at 37 ºC in 5 % CO2 atmosphere for 3 d. The cultured rat cardiomyocytes
were divided into different groups (control, oxygen and glucose deprivation
(model), and therapeutic groups with baicalin 0.1, 1, and 10 g·L-1).
The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the supernatant
of cultured cardiomyocytes were measured by bioassay method. The struction of
cell was observed. RESULTS: Compared with the control group, SOD activity
was lower, and MDA production was higher in oxygen and glucose deprivation group
(P<0.01). The struction of cell was damaged seriously. After treatment
with baicalin in three groups, SOD activities were higher, and MDA production
was less than model group (P<0.01). The struction of cell was also
protected by baicalin. CONCLUSION: Baicalin has a protective effect
on oxygen and glucose deprivation in neonatal rat cardiomyocytes.
P-26 Effect of GbE on levels of excitatory amino
acids in extracellullar fluid of rat hippocampus
following cerebral ischemia
YU Ren-Luan
Department of Pharmacology, Beijing Military Medical College,
Beijing 100071, China
AIM: To investigate the change of EAA level of rat
hippocampus and to discuss the protective effect of
Ginkgo biloba extract (GbE) during ischemic
reperfusion. METHODS: In this study, male adult SD
rats (250-300 g) were used. Ischemic reperfusion rat
models were established by Pulsinelli's "Four-vessel
occlusion"method. The experiment used the
intracerebral microdialysis technique to investigate the effect of
GbE on extracellular EAA levels, ischemic (30
min)-reperfusion (30 min) in the rat hippocampus .The
microdialysates were analyzed by high performance
liquid chromatography with fluorescent detection.
RESULTS: Excitatory amino acid concentration of
saline group in the hippocampal dialysate was significantly
higher than that of sham group (P<0.01); excitatory
amino acid content of GbE groups (three doses)
decreased dramatically in a dose-dependent manner in
microdialysates. CONCLUSION: GbE reduced release
of EAA by cerebral cells in case of ischemia.
P-27 Effect of ACG on immunologic function in
tumor-bearing mice
YE Jia, JANG Yi-Ming, LI Chang-Ling
Department of Pharmacology, School of Pharmaceutical Sciences,
Beijing University, Beijing 100083, China
AIM: To investigate the antitumor effect of ACG and the possible mechanisms
involving immunologic function in tumor-bearing mice. METHODS: Mice
were inoculated with tumor cells (S180 or Lewis lung cancer). The therapeutic
mice were treated with ACG (50 g·kg-1·d-1, po)
for 21 d to observe the rate of tumor inhibition, and to determine the activity
of IL-2 and the subsets of T-cells, with normal saline (NS) as control.
RESULTS: 1) In mice inoculated with S180 tumor cells, the tumor weight
of mice receiving ACG was (1.1±0.5) g, significantly lighter than that
of NS control (1.6 g±0.5 g, P<0.05), with the rate of tumor inhibition
32.7 %. Similarly, in C57BL mice inoculated with Lewis lung cancer cells, the
tumor weight of mice receiving ACG was (0.9±0.5) g, significantly lighter
than that of NS (1.6 g±0.7 g, P<0.05), with the rate of tumor
inhibition 42.77 %. 2) Inoculation of S180 or Lewis lung sarcoma cells resulted
in impairment of immunologic function, including decrease of IL-2 activity and
abnormal ratio of T-cells (Th/Ts<1). In the presence of ACG, the activity
of IL-2 was enhanced (55 kU/L±16 kU/L vs 31 kU/L ±9 kU/L, P<0.01),
and the percent of Th cells was raised (23 %±6 % vs 17 %±6
%, P<0.05). The abnormal Th/Ts ratio was rectified to 1.6±0.6,
close to the normal (1.7±0.5). CONCLU-SION: ACG inhibits the growth
of tumor in tumor-bearing mice, which is associated with increasing IL-2 activity
and normalizing T-cell subgroups. It indicates that ACG exerted an antitumor
activity through a mechanism involving the enhancement of immunologic function.
P-28 Interaction between endogenous carbon
monoxide and nitric oxide in pathogenesis of
myocardial injury of stress-induced hypertension in rats
CUI Hao-Jun, SHAO Min
Department of Physiology, Inner Mongolia Medical College, Huhhot
010059, China
AIM: To assess the interaction between endogenous carbon monoxide (CO)
and nitric oxide (NO) in pathogenesis of myocardial injury of stress-induced
hypertention in rats. METHODS: Stress-induced hypertension (SIH) was
affected by electric shock on hind paw of rat and noise stimulus to establish
conditioned reflex. The electrocardiograms (ECG) were observed, malondialdehyde
(MDA), superoxide dismutase (SOD), and NO levels in serum and myocardial tissue
were measured, and at the same time the HbCO formation with blood and myocardial
tissue were tested and myocardial tissue structures were observd. RESULTS:
The rate of SIH group was not different from that of control group, ST
segment was significantly taller than normal group, and QT interval was longer
than normal group (P<0.05). After treatment with L-Arginine
(L-Arg), the above changes were reversed (P<0.05). In SIH group,
serum and myocardial tissue MDA content increased significantly, but SOD and
NO contents decreased in SIH group (P<0.01). The HbCO formation in
blood and tissue was elevated (P<0.01). These changes could be reversed
by L-Arg (P<0.01). Microscopic observation showed that degree
of injury to myocardial structure in L-Arg treated group was slighter
than control. CONCLUSION: Endogenous NOS and nitric oxide systems are
inhibited, and the heme oxygenase (HO)/carban monoxide (CO) systems compensated
up-regulation. L-Arg attenuated myocardial injury in SIH rats. Both
endogenous CO and NO were involved in the pathogenesis of myocardial injury
of stress-induced hypertension in rats, and they could compensate each other.
P-29 Protective effect of resveratrol on acute
experimental spinal cord injury in rats
YANG Ying-Bao1,2, PIAO Ying-Jie2
1Department of Pharmacology;
2Department of Anatomy, First Military Medical University, Guangzhou 510515, China
AIM: To study the effects of resveratral (Res) on oxygen free radical
level and Ca2+, Mg2+-ATPase activities in rats after spinal
cord injury (SCI). methods: The weight-dropping method was used to produce
the experimental spinal cord injury in adult rats. Res and methylprednisolone
(MPSS) were given ip by the bolus injections of 50 and 100 mg/kg, respectively,
immediately after SCI. And then the effects of Res on malondialdehyde (MDA)
content, reactive oxygen species (ROS) level, superoxide dismutase (SOD) activity,
Ca2+, Mg2+-ATPase activities in local injured spinal cord
were determined at 1, 24, and 48 h after SCI compared with MPSS. Results:
Res significantly inhibited increase of the MDA content and ROS level and prevented
reduction of SOD activity in the injured spinal cord tissue in comparison with
the SCI model at the doses of 50 mg/kg and 100 mg/kg, and the most remarkable
effects were induced by Res 100 mg/kg at 48 h with the inhibition rate over
52 %, 40 %, and 58 %. At the same time, Res 100 mg/kg obviously augmented Ca2+-ATPase,
Mg2+-ATPase, and Ca2+, Mg2+-ATPase activities
at all time points after SCI in rats with the greatest improving rates over
71 %, 180 %, and 120 % at 1, 48 , and 1 h after SCI, respectively. The effects
of Res were greater than those of MPSS. conclusion: Res may reduce oxygen
free radicals and then effectively protect the spinal cord through improving
the Ca2+, Mg2+-ATPase systems in SCI, suggesting that
Res may have a good potent therapeutic effect on SCI.
P-30 Effects of Tiao-xin-fang on long-term
potentiation in rat hippocampal slices
WAN Qin, ZHANG Yong-Xiang
Beijing Institute of Pharmacology and Toxicology, Beijing 100850,
China
AIM: To investigate the effects of Tiao-Xin-Fang (TXF), a traditional
Chinese medicinal prescription, on long-term potentiation in rat hippocampal
slices. METHODS: Population spikes (PS) were evoked by extracellular
microelectrode recording technique in CA1 area of rat hippocampal slices, and
tetanic stimulation (100 Hz, 100 pulse) was used to induce long-term potentiation
(LTP). LTP was thought to be successfully induced when the amplitude of PS was
increased more than 20 % above the baseline for at least 30 min. RESULTS:
Under normal conditions, the successful rate of evoking LTP was about 60
% and after tetanic stimulation the PS amplitude increased significantly while
PS latent period decreased. In vitro application of TXF (7.45 g/L) had
little effect on either the sharp or amplitude of PS, indicating that
there was no marked change in basal synaptic transmission. But after tetanus
the LTP-inducing rate and the PS amplitude were significantly enhanced compared
with the control (P<0.01). Pre-incubation of hippocampal slices with
-amyloid (0.2 mol/L)
or corticosterone (2 mol/L) significantly
attenuated both LTP inducting rate and PS amplitude, suggesting that either
of them had an inhibitory effect on LTP generation. TXF (7.45 g/L) co-application
with -amyloid or corticosterone
obviously antagonized their inhibitory effects, and both LTP-inducing rate and
PS amplitude improved. CONCLUSION: TXF enhanced the LTP-inducing rate
and the PS amplitude after tetanic stimulation in normal rat hippocampal slices
and also antagonized the inhibitory effects of -amyloid
and corticosterone on LTP generation, which may be one of the mechanisms of
TXF in improving the impaired learning and memory function of patients with
Alzheimer's disease.
P-31 Pharmacokinetics and distribution of penehyclidine hydrochloride raceme and its four
optical isomers in rabbits and mice
XUE Ming1,2, RUAN
Jin-Xiu1, YUAN Shu-Lan1, ZHANG
Zhen-Qin1, QIAO Jian-Zhong1
1Institute of Pharmacology and Toxicology, Academy of Military
Medical Sciences, Beijing 100850; 2Department of Pharmacology,
Capital University of Medical Sciences, Beijing 100054, China
AIM: To investigate and compare the pharmacokinetics and distribution
of penehyclidine hydrochloride (PH) raceme, a novel anti-cholinerigic drug invented
by Institute of Pharmacology and Toxicology AMMS, and its four optical isomers
in rabbits and mice. METHODS: Blood and tissure concentrations of PH
raceme and its four optical isomers were determined by gas chromatography-mass
spectrometry with selected ion monitoring (GC-MS/SIM) PH (m/z 175) and PH-D5
(m/z 180) as internal standard. RESULTS: The concentration-time profile
of PH raceme and its four optical isomers were all best fitted to the first
order absorption two-compartment open model after in a single dose (0.1 mg/kg)
in rabbits and mice. Differences were found in the absorption, the distribution
and the elimination between PH raceme and its four isomers. The main pharmacokinetic
parameters of PH for rabbits and mice were as follows: t1/2
0.12 and 0.23 h, t1/2
8.4 and 3.3 h, t1/1Ka 0.0066 and 0.013 h, Tmax
0.024 and 0.067 h, Cmax 30.2 and 18.7 g/L,
AUC 107.6 and 50.4 g·L-1·h.
There were some differences in blood drug concentration of PH raceme and the
four optical isomers after dosing the PH and the four isomers. The distribution
in mice showed that the tissue concentration of R-2 isomer had a high level
which R-2 isomer had a great affinity to mAChr. And the isomer with S-configuration
in mice brain eliminated fast and the isomer with R-configuration eliminated
slowly. CONCLUSION: There was a relationship between the pharmacodynamics
and the pharacokinetics for R-configuration at carbon-3 of quiniuclidinol
ring to the chiral drug. This finding provides important information for developing
a novel chiral drug for clinical use.
P-32 Protective effect of Danbiqing granule on acute
cholangitis
LIN Xiu-Zhen, GONG Yan-Ling, WANG Hong-Bo
Departmen of Pharmacology, Tianjin Medical University, Tianjin
300070, China
AIM: To investigate the protective effect of Danbiqing
granule on experimental bacterial cholangitis, and the
mechanisms of its anti-inflammatory effect.
METHODS: Rabbits models of acute bacterial
cholangitis were established successfully. All rabbits were
divided into 6 groups, and serum PLA2, NO, and plasma
TXB2,
6-keto-PGF1 levels were detected.
RESULTS: Compared with control group, serum
PLA2, NO, and plasma TXB2 levels increased significantly in
model group (P<0.05). There was a slight increase in
plasma 6-keto-PGF1 level, though not
statistically significant (P>0.05). Serum NO,
PLA2, and plasma TXB2 levels decreased in Danbiqing groups in different
degrees (P<0.05), while there was no significant change
in plasma 6-keto-PGF1 level
(P>0.05). CONCLUSION: Danbiqing granule exerts an
anti-inflammatory effect by inhibiting the over production of
inflammatory factor and lipid mediator.
P-33 Effects of E2020 on learning and memory
behaviors and brain monoamine and amino acid neurotransmitters in senescence-accelerated
mice
ZHOU Wen-Xia, ZHANG Yong-Xiang , Cheng Jun-Ping
Beijing Institute of Pharmacology and Toxicology, Beijing
100850, China
AIM: To study the effects of E2020, a cholinesterase
inhibitor, which has been used clinically in the
treatment of Alzheimer's disease (AD), on central learning
and memory behaviors and brain monoamine and amino
acid neurotransmitters in senescence-accelerated mice
(SAM). METHODS: Computer-controlled step-down
test of passive avoidance performance and water maze
test, a spatial memory task, were employed to evaluate
the learning and memory ability of SAM. Reverse-phase
high performance liquid chromatography and electrochemical detection (HPLC-EC) was used to determine
the concentrations of monoamines including
norepinephrine (NE), dopamine (DA), serotonin (5-HT),
3,4-dihydroxyphenylacetic acid (DOPAC), homovanilic acid
(HVA), and 5-hydroxy-indoleacetic acid (5-HIAA), as
well as those of amino acid neurotransmitters including
aspartate (Asp), glutamate (Glu), glutamine (Gln),
glycine (Gly), taurine (Tau), and -aminobutyric acid
(GABA) in the cerebral cortex, hippocampus, and hypothalamus.
RESULTS: The learning and memory ability in step-down test of 5-month-old SAM-prone/8
(SAMP8), a senescence-accelerated sub-strain of SAM,
was significantly declined compared with the age-matched SAM-resitance/1 (SAMR1), a
senescence-resistant sub-strain of SAM. The spatial memory of
SAMP8 tested by water maze was also found to deteriorate compared to that of SAMR1. Meanwhile, the
levels of monoamines including NE in cerebral cortex
and hippocampus, 5-HT and 5-HIAA in hippocampus
of SAMP8 were significantly increased. In addition,
the levels of Glu and Gln in hippocampus and hypothalamus were also obviously increased, while Asp in
hippocampus decreased and other neurotransmitters showed no obvious change in the three brain regions.
Chronic oral administration of E2020 (1 mg/kg, ig, for
8 weeks) significantly ameliorated the spatial memory
ability in SAMP8, but showed no significant improving
effect on learning and memory behaviors in step-down
test. The elevated levels of NE in hippocampus and
Glu in hippocampus and hypothalamus in SAMP8 were significantly decreased after E2020 treatment.
CONCLUTION: Chronic oral administration of E2020
improved the spatial memory in SAMP8, suggesting that
E2020 ameliorates age-related deterioration of learning
and memory function. The preliminary results suggest
that restoration of the levels of brain monoamine and
amino acid neurotransmitters may be another way,
besides inhibiting cholinesterase, for E2020 to achieve the
improving effect on the learning and memory function
in AD.
P-34 Bi-directional regulation of deep peroneal
nerve inputs on pressor and depressor effects induced by area postrema excitation in rats
ZHOU Su-Ya, ZHU Yi-Ping, XU Jin-Fang, XIA Qiang
Department of Physiology, Zhejiang University School of Medicine,
Hangzhou 310031, China
AIM: To investigate the regulation of deep peroneal nerve
(DPN) inputs on cardiovascular effects caused by area
postrema (AP) excitation in rats and to analyze its
mechanism. METHODS: The mean arterial blood
pressure (MAP) and heart rate (HR) in anesthetized
Sprague-Dawley rats were monitored. Electric stimulation was
used to excite neurons and nerve fibers in AP and DPN.
RESULTS: High frequency (80 Hz) electric stimulation
on AP caused pressor effect while low frequency (20
Hz) stimulation caused depressor effect. The changes
of MAP were (3.45±0.25) kPa (P<0.05) and (_3.1±0.6)
kPa (P<0.01), respectively. Both high and low frequency
stimulation on AP decreased HR. The pressor or depressor effects and bradycardia induced by AP
excitation were inhibited by electric stimulation of DPN
(P<0.01). The percentages of inhibitory rate were 73.6 %,
97.6 % (P<0.01, P<0.05) and 73.8 %, 69.4 %
(P<0.01, P<0.05), respectively. The regulatory effects of DPN
inputs on AP excitation were blocked by intravenous
naloxone (1.5 mg/kg, P<0.01). But scopolamine (0.25
mg/kg) did not change the effect of DPN inputs on AP
excitation (P>0.05). CONCLUSION:
The DPN inputs regulated bi-directionally the pressor and depressor
effects caused by AP excitation, in which opioid peptide,
not acetylchloine, may be involved.
P-35 Role of chloroquine in protecting mice from
challenge with bacterial DNA
ZHOU Hong, ZHENG Jiang, LU Yong-Ling, LUO Ping
Department of Pharmacology, School of Medicine;
1Medical Research Center, Southwestern Hospital,
Third Military Medical University, Chongqing 400038, China
AIM: To investigate the role of chloroquine in protecting mice from
challenge with bacterial DNA (bDNA). METHODS: First, mice were injected
with chloroquine (20 mg/kg) by tail vein, and then 30 mg/kg weight bDNA and
CT DNA (Calf DNA, as control DNA) were injected at once. Mice mortality within
7 d was observed. Human monocyte cell line, THP-1 was cultivated in 48-well
plates in vitro. After adding chloroquine (1 g/L) into the plates for
4 h, bDNA and CT DNA 10 mg/L were added, too. Cells were cultivated
in 5 % CO2 incubator at 37 ºC for 20 h. Then the supernant was
obtained to test the concentration of TNF-
and IL-6. TLR9 expressions on THP-1 cell surface induced by bDNA for 4 h were
detected by flow cytometry when cells were pre-cultured with chloroquine
(1.0 g/L) or not. RESULTS: All mice (10/10) died within seven days
after bDNA injection, while only 4/10 mice injected with chloroquine died. bDNA
induced strong release of TNF-
and IL-6, and high expression of TLR9 on the cell surface. But when pretreated
with chloroquine, cells stimulated by bDNA released a small quantity of cytokines,
and TLR9 expression decreased substantially. CONCLUSION: Chloroquine
had the ability to protect mice challenged by bDNA, and this protective action
was associated with lower cytokines release. So chloroquine may be a very effective
agent that can be used clinically to treat sepsis or systemic inflammatory response
syndrome induced by bacteria.
P-36 Regulation of wound neutrophil function by
W11-a12
CHEN Xiao-Hong1, CHENG Tiao-Min, SU Yong-Ping,
RAN Xin-Ze
Institute of Combined Injury of CPLA,
1Department of Pharmacology, Third Military Medical University, Chongqing
400038, China
AIM: To explore the mechanism of W11-a12, a polyalcohol
compound extracted from Periplaneta American, in enhancing wound healing in
rats, especially in improving the initiation of repair. METHODS: Rats
were wounded on back. Wound exudate neutrophils were collected from polyvinyl
alcohol sponges implanted in incisions. RESULTS: The supernatant of
wound neutrophil was able to promote the proliferation and migration of cultured
normal fibroblasts (FB) as well as vascular endothelial cells (VEC). After cultured
confluent monolayer FB and VEC were wounded with a needle, the wound closure
of FB and VEC was evidently promoted by addition of wound neutrophil supernatant.
Results also showed that the neutrophil supernatant improved 3H-proline
incorporation into FB. Administration of W11-a12 (10 mg/wound,
24-48 h) at the wound site significantly elevated all the above functions of
wound neutrophil. Simultaneously, W11-a12 exhibited weakening
the inhibition of proliferation of cultured VEC and FB by cocultured wound neutrophil.
CONCLUSION: Our study provided evidence for the first time that wound
neutrophil directly contributed to the initiation of healing and the formation
of primary granulation tissue. And W11-a12 regulated functions
of wound neutrophils, which might occupy a potential position in enhancing initiation
of wound healing by W11-a12.
P-37 Characteristic of heart and thorax aorta in
diabetic cardiomyopathy rat
ZHANG Chun-hong1,2, ZANG
Weijin1, YU Xiao-Jiang1, HU
Hao1, SUN Qiang, LV Jun1
1Division of Cardiovascular Physiology and Pharmacology, Xi'an
JiaoTong University Medical College, Xi'an 710061; 2Division of Endocrinology,
the Second Hospital, Xi'an JiaoTong University, Xi'an 710004, China
AIM: To study the characteristics of the heart and the reactivity of
the thorax aorta in diabetic cardiomyopathy rat. METHODS: Tirty Sprague-Dawley
rats were divided into normal group (n=8), diabetic cardiomyopathy model group
(n=12), and insulin treatment group (n=10). After 12 weeks, the hearts were
removed and the ultrastructure was observed by electron microscopy. And isometric
contractile response and relaxation response to norepinephrine and phentolamine
were observed in the thorax aorta isolated from diabetic cardiomyopathy rats.
RESULTS: (1) The ultrastructures of the cardiac muscles in normal group
and insulin group were almost the same. There was a decrease in the number of
the myofibrils in model group, where the muscle silk was ruptured; the hyperplasia
of the mitochondrion was obvious at the part of the cardiac muscle; the sarcoplasmic
reticulum became dilated, and the necrosis of cardiac myofibrils appeared at
last. (2) The maximal contractile responses to norepinephrine varied among the
arteries in an order of diabetic cardiomyopathy group> normal group
insulin
group. But the EC50 values of norepinephrine in all groups were almost
the same. The relaxation responses to phentolamine were not significantly different
between the three groups, and EC50 values of phentolamine were almost
the same, too. CONCLUSION: In the presence of diabetic cardio-myopathy,
the reactivity in some big vessels has already changed, such as the contractile
responses. The change may be related to hyperglycemia. Treatment with insulin
is beneficial to diabetic cardiomyopathy in the heart and some vessels.
P-38 Designing anti-mast cell degranulation
compounds
SUN Ren-Shan, CHENG Tian-Min, HAO Fei
Department of Dermatology, Southwest Hospital, Third Military
Medical University, Chongqing 400038, China
AIM: To design some compounds which possess the
capacity to inhibit mast cell degranulation. METHODS:
A set of isoquinoline compounds of anti-mast cell
degranulation was investigated by 3D-QSAR. Their pharmacophore model was identified by DISCO
program. According to the pharmacophore model,
3D-QSAR models for the activity and toxicity were built by
CoMFA. RESULTS: The crossvalidated Rcv2 and
non-crossvalidated R2 (correlation coefficient) for the
structure-activity and structure-toxicity models were 0.654
and 0.662, 0.990 and 0.983, respectively. The
confidence levels of all these models were all over 99
% by referring statistics F table. These models showed
relevant and high predictability. CONCLUSION:
New bioactive lead compounds were designed on the basis
of these models. Their predicted activities are higher
and predicted toxicity is lower than the known compounds. The synthesis of these new compounds
is in progress.
P-39 Signal transduction of TNF-alpha induced
cardioprotection in hypoxic/reoxygenation
FU Chen, XIA Qiang, CAO Chun-Mei, JIANG Hui-Di
Department of Physiology, Zhejiang University School of Medicine,
Hangzhou 310031, China
AIM: Tumor necrosis factor-
(TNF-) could reduce myocardial ischemia/reperfusion injury by
activating manganese superoxide dismutase (MnSOD).
However, the mechanism of the MnSOD activation by
TNF- is poorly understood. So in the present
study we tried to determine the role of reactive oxygen
species and mitochondrial ATP-sensitive potassium
(KATP) channels in mediating this effect in cultured
cardiomyocytes during simulated hypoxia/reoxygenation
(H/R). METHODS: Neonatal rat ventricular myocytes
were pretreated with TNF- or diazoxide for 12
h and then subjected to continuous hypoxia for 12 h,
followed by reoxygenation for 6 h. In another series of
experiments, before H/R, the myocytes were first
pretreated with mitochondrial antioxidant N-acetylcysteine
(NAC), mitochondrial electron transport inhibitors
antimycin A, specific nitric oxide synthase inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME) or mitochondrial selective
KATP channel inhibitor 5-hydroxydecanoic
(5-HD) for 3 h, followed by 12 h of
TNF- (100 kU/L) pretreatment. The MnSOD activity of the cell
was measured after hypoxia/reoxygenation. Cell injury
was assessed in terms of lactic dehydrogenase (LDH)
release. RESULTS: TNF- (10, 50, 100, or
500 kU/L) significantly increased the MnSOD activity
and reduced LDH release of neonatal rat ventricular
myocytes. Diazoxide (50 mol/L), a selective
opener of the mitochondrial KATP channel, decreased the
LDH release but did not elevate MnSOD activity of the
myocytes. Pretreatment with NAC (50
mol/L), antimycin A (50 mol/L),
L-NAME (100 mol/L), or 5-HD
(100mol/L) attenuated the increase of MnSOD activity and reduction of
LDH level induced by TNF-. CONCLUSION:
TNF- induces cardioprotection via oxygen
radical signals and activation of mitochondrial
KATP channels. Through this intracellular signaling pathway,
TNF- induces MnSOD activity and attenuates LDH
release in cultured cardiomyocytes.
P-40 Effects of huperzine A and E2020 on learning
and memory related gene expression in hippocampus of senescence-accelerated mice
ZHOU Wen-Xia, ZHANG Yong-Xiang
Beijing Institute of Pharmacology and Toxicology, Beijing 100850,
China
AIM: To study the effects of huperzine A and E2020,
two cholinesterase inhibitors, which are being used in
the treatment of Alzheimer's disease (AD), on learning
and memory related gene expression in hippocampus of senescence-accelerated mice (SAM).
METHODS: Hippocampi were dissected and the total RNA was
isolated from the tissue. The expression of the genes
including mineralocorticoid receptor (MR), presenilin 1
and 2 (PS-1 and PS-2), tau, amyloid precursor protein
(APP), apoprotein E (apoE) and bcl-2 in the
hippocampus of SAM were determined by reverse transcription
polymerase chain reaction (RT-PCR). The effect of
huperzine A and E2020 on hippocampal gene expressions were then studied.
RESULTS: The expression levels of hippocampal genes including MR, tau, PS-2,
and APP in 5-month-old SAM-prone/8 (SAMP8), a senescence-accelerated substrain of SAM, were
significantly elevated, but the expression of apoE was
decreased, compared with those in age-matched SAM-resitance/1 (SAMR1), a senescence resistant sub-strain
of SAM. While in the 12-month-old SAMP8, the expression levels of the genes of MR and tau were
significantly elevated, but that of bcl-2 was decreased. Oral
administration of huperzine A (0.05 mg/kg, ig, for 8
weeks) corrected the disturbed gene expressions of tau
and PS-2 in 5-month-old SAMP8, and MR and PS-2 in 12-month-old SAMP8. While E2020 (1 mg/kg, ig, for
8 weeks) restored the abnormal expressions of MR, tau and APP in 5-month-old SAMP8, and MR, PS-1,
and PS-2 in 12-month-old SAMP8. CONCLUSION:
Continuous oral administration of huperzine A or E2020
modulated the expressions of learning and memory
related genes in hippocampus in 5- and 12-month-old
SAMP8, which might be one of the mechanisms of Huperzine A and E2020 in improving the impaired
learning and memory function of AD patients.
P-41 Effect of Liuwei Dihuang decoction on the
balance of the hypothalamus-pituitary-ovary axis of senescence-accelerated mice
MA Yuan, ZHOU Wen-Xia, CHENG Jun-Ping, ZHANG Yong-Xiang
Beijing Institute of Pharmacology and Toxicology, Beijing 100850,
China
AIM: To study the modulating effect of Liuwei Dihuang
decoction (LW) on the balance of the hypothalamuspituitary-ovary (HPO) axis of senescence-accelerated
mice (SAM). METHODS: Radioimmunoassay (RIA)
was used to test the concentrations of serum estradiol,
hypothalamic -endorphin (-EP), and
substance P (SP). Western blot was used to quantify
the level of luteinizing hormone (LH) in the pituitary
and the levels of estrogen receptor
(ER) expressed in pituitary and ovary.
RESULTS: The oestrus cycle and diestrus of 9-month-old SAM-Prone/8
(SAMP8), a senescence-accelerated sub-strain of SAM,
were significantly prolonged compared with the
age-matched SAM-resitance/1 (SAMR1), a
senescence-resistant sub-strain of SAM. The concentration of
serum estradiol was significantly decreased, while the level
of pituitary LH was significantly elevated in SAMP8 in
comparison with age-matched SAMR1. The contents of
-EP and SP in hypothalamus, as well as the
levels of ER in both pituitary and ovary were
significantly decreased in SAMP8 compared with SAMR1. Chronic oral administration of LW
significantly shortened the diestrus of SAMP8, but showed
little effect on the concentration of serum estradiol.
Treatment with LW at the doses of 5, 10, and 15 g/kg
significantly decreased the pituitary LH level of SAMP8.
The levels of hypothalamic -EP and ovary
ER expression were both significantly restored
after LW treatment. CONCLUSION: Chronic oral
administration of LW improved or corrected the
imbalance of HPO axis in 9-month-old SAMP8. Modulation
of the levels of hypothalamic -EP and ovary
ER expression may contribute to the effect of LW
on HPO axis disturbed in SAMP8.
P-42 Effect of Liuwei Dihuang decoction on
hypothalamus-pituitary-ovary axis in hanging
stress-loaded female mice
MA Yuan, ZHOU Wen-Xia, CHENG Jun-Ping, ZHANG Yong-Xiang
Beijing Institute of Pharmacology and Toxicology, Beijing 100850,
China
AIM: To study the effect of Liuwei Dihuang (LW) decoction on stress-induced
imbalance of hypothalamus-pituitary-ovary (HPO) axis in female mice. METHODS:
Mice were subjected to hanging stress every day (30 min for the first 3
d and with 30 min increasing for every other 3 d). The total duration of stress
was 25 d. Radioimmunoassay (RIA) was used to test the concentrations of hypothalamic
gonadotropin-releasing hormone (GnRH), -endorphin
(-EP), substance P (SP), and serum
estradiol, and Western blot was employed to quantify the levels of luteinizing
hormone (LH) of the pituitary and estrogen receptor
(ER) expressed in both pituitary
and ovary. High-performance liquid chromatography connected with an electrochemical
detector (HPLC-ECD) was used to determine the content of monoamine neurotransmitters
in hypothalamus. RESULTS: The body weights of stress-loaded mice were
markedly decreased and the estrus cycle, as well as the diestrus was significantly
prolonged. Meanwhile, the level of pituitary LH in stress-loaded mice was significantly
decreased, and that of hypothalamic GnRH tended to be decreased, while the levels
of serum estradiol and the contents of hypothalamic-EP,
SP, and monoamine neurotransmitters were significantly increased. Oral administration
of LW showed little effect on the oestrus cycle in stressed mice, but it showed
significant improving effects on the balances of hypothalamic GnRH, pituitary
LH and abnormal ovary ER expression
disturbed by stress. The concentrations of serum estradiol, hypothalamic SP
and monoamine neurotransmitters of stress-loaded mice were significantly restored
after LW administration. CONCLUSION: Hanging stress disturbed the balance
of HPO axis in mice and oral administration of LW showed significant improving
or restoring effect on it in hanging stress-loaded mice. These effects could
be achieved by decreasing the concentrations of hypothalamic SP and monoamine
neurotransmitters and promoting the expression of ER
in the ovary.
P-43 Effect of serum from Liuwei Dihuang
decoction-treated rats on synaptic function of cultured
hippocampal neurons
YANG Sheng, ZHANG Yong-Xiang
Beijing Institute of Pharmacology and Toxicology, Beijing 100850,
China
AIM: To investigate the synaptic mechanism
underlying the cognition-enhancing effect of Liuwei Dihuang
decoction (LW). METHODS: Whole-cell recording
technique was used to observe the spontaneous
synaptic activity and paired-pulse depression (PPD) of rat
hippocampal neurons cultured with the serum from LWtreated rats, termed LW-containing serum (LWCS). PPD
was a simple form of use-dependent synaptic plasticity
and was measured in response to paired stimulation.
PPD indicated some presynaptic mechanism of synaptic function.
RESULTS: After hippocampal neurons were treated with LWCS for 48 h, the frequency of
spontaneous action current (sAC) and miniature
excitatory postsynaptic current (mEPSC) of neurons were
significantly increased, but the amplitude of mEPSC
remained unchanged. The degree of PPD in LWCS-treated neurons measured at 1-s interval was
significantly decreased compared with control.
CONCLU-SION: LWCS enhances synaptic function of the
primary cultured hippocampal neurons, possibly by
affecting the presynaptic site of the neurons, which may
contribute to the cognition-enhancing effect of LW.
P-44 Preliminary protective mechanism of
3,6-dimethamidodibenzopyriodonium gluconate on myocardial ischemia-reperfusion injury in rats
LI Xi-Kuan, GOU Wei1, SHI Xiao-lian, LIU Jun-Tian,
SUN Xiao-Ming1, ZHAO Jin1, BIAN
Wei-Guo2
Department of Pharmacology, Medical College of Xi-an Jiaotong
University; 1Function Center Medical College of Xi-an Jaotong
University; 298(7) Grade of Clinical Medicine of Xi-an Jiaotong
University, Xi-an 710061, China
AIM: To study the mechanism of
3,6-dimethamidodi-benzopyriodonium gluconate (IHC-93) on
myocardial ischemia-reperfusion injury. METHODS:
The modle was established by ligating and unting the
descending anterior branch of left coronary artery.
RESULTS: IHC-93 at the doses of 0.25 and 0.5
mg/kg increased superoxide dismutase (SOD) activity in the myocardium, decreased malondialdehyde
(MDA) content in serum and myocardium. IHC-93, at the doses of 0.125, 0.25, and 0.5 mg/kg increased
6-keto-PGF1 and decreased
TXB2 contet in serum. CONCLUSION:
The preliminary mechanisms of IHC-93 are possibly related to the inhibition
of lipid peroxidation, increase of
PGI2, and decrease of
TXB2.
P-45 Effects of astragalus polysaccharide injectable powder (APS-P) on the
proliferation and mobilization of murine hematopoietic stem cells
WENG Ling, LIU Xue-Ying, LIU Yang, ZHANG Ying, ZHAO Lin-Ai, Karen LEU
Pharmagenesis Incorporation, Polo Building, 6
Guan-Hua-Xi-Li, Chaoyang District, Beijing 100020, China
AIM: Astragalus polysaccharide injectable powder
(APS-P), developed by Pharmagenesis, has been approved by the SDA of China as a Chinese New Drug
Class II. The present study was undertaken to evaluate
the APS-P on hematopoietic activities in normal or
cyclophosphamide (CyP)-treated C57BL/6 mice. METHODS:
Normal or cyclophosphamide-treated C57BL/6 mice were injected APS-P daily for 2, 3, 5, 7,
or 9 d, and then bone marrow, peripheral blood and
spleen cells were collected on d 3, 4, 6, 8, or 10. Cells
were stained with APC-Sca-1, PerCP-c-kit and
PE-Lineage (CD3, CD4, CD8, Tr119, Mac-1, CD11b, and
Gr-1) for flow cytometry analysis. Lin-c-kit
+Sca-1+ (surface phenotypes of hematopoietic stem cells) cells were
numerated and compared between different groups.
RESULTS: APS-P treatment increased the number of
Lin-c-kit+sca-1+ cells in bone marrow, spleen and
peripheral blood in normal mice and also helped their
recovery after CyP treatment. CONCLUSION: The
increase of
Lin-c-kit+Sca-1+ cells in bone marrow could
be due to stem cell proliferation, while the increase in
spleen or peripheral blood might be due to the
mobilization of bone marrow stem cells. These results suggest
the possibility of clinical application of APS-P in
autologous or allogeneic peripheral stem cell
transplantation in addition to preventing and/or treating
chemotherapy-associated myelosuppression.
P-46 Astragalus polysaccharide accelerated the
recovery of granulocytes and macrophage bone marrow progenitors (GM-CFC) in irradiated or
5-FU-treated mice
Stacie A DALRYMPLE, Voltaire PAJE, Geoff SPENCER, Karen LEU
Pharmagenesis Incorporation, Polo Building, 6
Guan-Hua-Xi-Li, Chaoyang District, Beijing 100020,
China
AIM: A purified extract, Astragalus polysaccharide (APS), has
been isolated from Astragalus membranaceus var momgholicus (AM),
a medicinal plant documented to have immunostimulating and hematopoietic activities.
The objective of this study is to study the efficacy of Astragalus polysaccharide
in accelerating the recovery of bone marrow granulocytes and macrophage progenitors
(GM-CFC) in sublethally irradiated mice or 5-fluorouracil-treated mice.
METHODS: Female BALB/c mice were sublethally irradiated (X-ray, 450 cGy)
and injected subcutaneously daily with APS for 7 d. Another group of mice were
treated with 5-FU (150 mg/kg) and then given APS for 3 d. Bone marrow cells
were harvested and cultured for GM-CFC, in a complete methylcellulose medium
with pokeweed mitogen spleen cell conditioned medium as a source of colony stimulating
factors. Colonies of greater than 50 cells were scored after 6-7 d of incubation
at 37 ºC. RESULTS: Sc administration of APS significantly accelerated
the recovery of GM-CFC in irradiated or 5-FU-treated mice in a dose-dependent
manner. Furthermore, APS was as effective as G-CSF (100 g/kg)
in promoting the GM-CFC recovery. CONCLU-SION: APS provided an alternative
to G-CSF therapy for the treatment of neutropenia.
P-47 Vasoactive effects of atropine on blood vessel
in rabbit ear
LIU Shu-Qin, ZANG Wei-Jin, LI Zeng-Li, YU Xiao-Jiang, CHENG Liang
Department of Pharmacology, Medical College, Xi-an JiaoTong
University, Xi-an 710061, China
AIM: To investigate the vasoactive effects of atropine and the involved
mechanism. MTHODS: (1) Great auricular nerve (1.5 cm-length) in the
left lateral ear root of rabbit was cut off under general anesthesia. All the
denervated rabbits were raised for 14 d and were administered with reserpine
25 mg/kg for 2 d (ip, qd). Rabbits were then administered with saline 0.5 mL/kg,
atropine 0.2 mg/kg (0.5 g/L) or chlorpromazine 2.0 mg/kg (im). (2) Ears of 10
denervated rabbits were isolated and perfused with locke's solution bubbled
with 95 % O2+5 % CO2 through dorsalis auricular arteria
intubation tube under constant perfusion pressure. Saline, chlorpromazine, atropine,
acetylcholine, acetylcholine+atropine, noradrenaline, or noradrenaline +atropine
were added into Locke's solution, and the maximal and minimal effluent flows
under each drug treatment were recorded. RESULTS: (1) The rabbit ear
blood vessel was net-dilated 1 min into atropine administration. Chlorpromazine
net-dilated ear blood vessel 5 min into administration, more fiercely than atropine.
(2) As compared with saline, chlorpromazine 1 mol/L
or acetylcholine 0.25 mol/L significantly
increased the effluent of isolated ears from denervated rabbits. However, atropine
(3 mol/L, 30 mol/L,
0.3 mmol/L) did not increase the effluent (P>0.05). Acetylcholine
0.25 mol/L failed to increase the
effluent when co-administered with atropine 3mol/L,
but not 0.3mol/L. Noradrenaline (0.1mol/L)
decreased the effluent very significantly (P<0.01), while this disease
weakened in the presence of atropine (30 mol/L,
0.3 mmol/L). CONCLUSION: The present study supported a direct blood
vessel-dilating effect of chlorpromazine, but not atropine. It is suggested
that atropine dilates blood vessel contracted by -adrenoceptor
activation.
P-48 Forskolin protects cultured cerebellar
granule neurons against diphenylhydantoin-mediated
apoptosis by elevating [cAMP]i in a
[Ca2+]i-independent way
ZHAO Ling-Zhi, HUANG Yi-Jun, SU Xing-Wen,
JIANG Wei-Jian, ZHANG Hai-Xin, QIU Peng-Xin,
YAN Guang-Mei
Department of Pharmacology, Sun Yat-Sen University of Medical
School, Sun Yat-Sen University, Guangzhou 510080, China
AIM: To study the effect of forskolin on apoptosis of cultured cerebellar
granule neurons (CGN) induced by diphenylhydantoin (DPH) and its putative mechanisms.
METHODS: Cerebellar granule neurons culture; FDA stain, hochest 33258
stain and agar gel electrophoresis were used to test morphological and biological
characters of apoptotic cells. RESULTS: Forskolin (2-20 mol/L),
an activator of AC, dose-dependently protected CGN against DPH (100 mol/L)-induced
neuronal apoptosis as assessed by morphological inspection (FDA stain), chromatin
condensation (Hochest stain) and DNA fragmentation. This neuroprotective effect
could not be blocked by LY294002 (the inhibitor of PI3K) and PD98059 (the inhibitor
of MEK1). It was abolished by specific membrane-permeable inhibitor (Rp-cAMP)
and specific PKA inhibitor (H89), and mimicked by potent membrane-permeable
activator (Sp-cAMP) and cyclic AMP-specific phosphodiesterase inhibitor (rolipram).
Activation of cAMP synthesis by intracellular Ca2+ was thought to
be the main mode of cAMP generation in the brain and decreasing [Ca2+]i
was considered an important effector for DPH to induce CGN to death. However,
the survival effect of forskolin was unaffected by inhibitors of ryanodine-sensitive
Ca2+ release (dantrolene) and inhibitor of IP3 sensitive
Ca2+ release (2APB), nor was it inhibited by L-type channel blockers
(nifedipine and nimodipine), N-methyl-K-aspartate (NMDA) receptor blocker
(MK801), and inhibitor of CaMKII (KN93). CONCLUSION: Forskolin protected
cerebellar granule neurons from apoptosis mediated by diphenylhydantoin
in a PI3K, and MEK1-independent manner. It is likely to elevate cyclic AMP content
by a mechanism which does not require increasing [Ca2+]i.
P-49 Protective effect of midazolam on injuried
cultured PC12 cells
ZHANG Tao-Tao, JIANG Wei-Jian, Ma
Jue1, QIU Peng-Xin, YAN Guang-Mei
Department of Pharmacology, Medical School of Sun Yat-sen
University, Guangzhou 510080; 1Department of Anesthesiology,
Guangdong Provincial People's Hospital, Guangzhou
510080,China
AIM: There are different opinions of the role of brain protection induced
by iv anesthetics. This study is to investigate the role of midazolam on cultured
PC12 cells injuried by hydrogen peroxide in vitro. METHODS: PC12
cells were prepared to observe morphology of neurons with phase-contrast microscope;
neuronal viability was assessed by FDA staining and MTT method; Hoechst 33258
staining was used to reveal the nuclear morphology of PC12 cells; DNA fragmentation
was detected by agrose gel electrophoresis. RESULTS: PC12 cells grown
nearly confluent, then were exposed to different treatments for 18 h. Apoptosis
of hydroperoxide (150 mol/L)-induced
PC12 cells was blocked by midazolam in a dose-dependent manner at a range of
50-150 mol/L; neuronal viability
increased from 21 %±5 % to 73 %±6 % (P<0.01) compared with
control. This effect was reversed by LY294002 20 mol/L,
the specific inhibitor of PI3K (neuronal viability 14 %±5 %, there was
no statistical difference compared with control). In addition, with increased
concentrations (200-300 mol/L), midazolam
induced apoptosis of PC12 cells in a dose-dependent manner. In hydroperoxide
group, midazolam plus hydroperoxide plus LY294002 group, and high-dose midazolam
group, Hoechst 33258 nuclear staining showed nuclear condensation. Agrose gel
electrophoresis showed DNA "ladder"; whereas in midazolam (150 mol/L)
plus hydroperoxide group, the nuclear feature was normal and there was no DNA
" ladder". CONCLU-SION: At a range of 0-150 mol/L,
midazolam protected PC12 cells against hydroperoxide-induced apoptosis, and
this role was PI3K/Akt signaling pathway-dependent. Beyond 200 mol/L,
midazolam itself caused apoptosis of PC12 cell.
P-50 Rifampine does not exacerbate
isoniazid-induced short term hepatotoxicity in rats
YUE Jiang, PENG Ren-Xiu
Department of Pharmacology, Medical College of Wuhan
University, Wuhan 430071, China
AIM: To investigate the effect of rifampine (RMF) on short term hepatotoxicity
induced by isoniazid (INH) young male Wistar rats. METHODS: Rats were
either treated with INH alone (100 mg/kg, ip), or co-administered with RMF (100
mg/kg, ig) for 21 d. Liver injury was assessed by serum ALT, AST, and alkaline
phosphatase (ALP) and histopathological examinations. Aniline hydroxylase (ANH)
activity was measured as the enzyme marker of CYP2E1 that was involved in hepatotoxicity
induced by small organic molecules such as APAP and CCl4. And CYP
2E1 mRNA expression was determined by semi-quantitative RT-PCR. RESULTS:
Serum ALT and AST were significantly higher in INH group than in the control
[(42±6) vs (32±7) U/L and (135±16) vs (126±11)
U/L, respectively] whereas there were no differences between RMF-INH group [(43±10)
and (140±7) U/L, respectively] and INH group (P>0.05). Meanwhile,
serum ALP in INH group (291 U/L±24 U/L) increased by 11 % compared with
the control, but no significant change was observed in RMF-INH group (311 U/L±34
U/L) as compared with INH group. Furthermore, ANH activity in INH group increased
454 % compared with the control [(0.37±0.05) mol
· g-1 · min-1], and increased by 32 % compared
with RMF-INH group. There were no significant differences in CYP2E1 mRNA expression
between the control, INH and RMF-INH groups. Moreover, histopathological examinations
showed that the slices in the two groups exhibited an equally mild degree of
hepatic injury, characterized by hydropic degeneration and spotty necrosis.
CONCLUSION: RMF does not significantly modify the metabolism of INH.
Obviously, the results of the present study suggest that RMF does not exacerbate
INH-induced hepatotoxicity in young rats pretreated for 21 d. ANH activity was
not paralleled with the level of hepatic impairment. In addition, the mechanism
of CYP2E1 induction by INH is not due to activation of mRNA transcription. The
interaction of INH and RMF during chronic administration remains to be further
studied.
P-51 Metabolic characteristics of nicotine
C-oxidation in Chinese liver microsomes in vitro
XIA Xue-Yan, PENG Ren-Xiu, WANG Hui, YANG Jing,
KONG Rui
Department of Pharmacology, Medical College of Wuhan
University, Wuhan 430071, China
AIM: To acquaint with the kinetic characteristics of nicotine C-oxidation
catalyzed by cytochrome P450 2A6(CYP2A6). Some clinical drugs were used to explore
their inhibitory effects on the major pathway of nicotine metabolism. METHODS:
Human liver microsomes were prepared by the method of Rance. The main metabolite
of nicotine C-oxidase, cotinine, was measured by a reversed phase assay
of HPLC. The kinetic constant of nicotine C-oxidase was determined. CYP2A6
specific inhibitors and some clinical drugs were employed to observe their effects
on nicotine C-oxidase in vitro. The expression of liver CYP2A6
mRNA was measured by semi-quantitative RT-PCR. RESULTS: The cotinine
formation depended on microsomal protein content, reaction time, and substrate
concentration. The Km and Vmax values of
nicotine C-oxidase were ranged from 8.6-63.6 mol/L
and 0.06-0.68 mol·min-1·g-1
, their variations were 7.4-fold and 11.3-fold, respectively. The formation
of cotinine was markedly inhibited (>50 %) by coumarin (0.1-100 mol/L)
and pilocarpine (0.1-100 mol/L),
the Ki value (IC50) was 9.15 and 8.12 mol/L,
respectively. Ketoconazole (0.1-100 mol/L)
also inhibited nicotine C-oxidase activity, and Ki value
was 66.24 mol/L. Rifampin had a slightly
inhibitory effect on nicotine metabolism, while -naphthoflavone
and diethyldithio carbamic (DDC) had no obvious effects. A significant correlation
was observed between CYP2A6 mRNA expression and nicotine C-oxidase oxidase activity.
CONCLUSION: CYP2A6 is the principlal human nicotine C-oxidase
and the determinant factor in smoking behavior. Various studies suggest that
the individual variability of CYP2A6 is large (>40-fold). Compared with our
data, the kinetics of nicotine C-oxidase also exhibited a considerable
individual variation; moreover, the expression of CYP2A6 mRNA varied in human
liver (26-fold). However, in the nicotine C-oxidation of our study, the
average value of Km (26.4 mol/L)
and Vmax (0.28 mol·min-1·g-1
) was similar to the report in whites. It is known that nicotine and cumarin
both are the typical substrate of CYP2A6. In our published data, rifampin (2.5-500
mol/L) obviously inhibited cumarin
7-hydroxylation (IC50=38.8 mol/L).
In contrast, its effect on cotinine formation was weak. Another commonly used
drug, ketoconazole, also presented an inhibitory effect on nicotine C-oxidase.
P-52 Effects of interleukin-6 on the development
of autoimmune diabetes in nod mice
XIANG Ming, Ferdinando NICOLETTI1
College of Pharmacy, Tongji Medical University, Wuhan 430030,
China; 1Institute of Microbiology, University of Milan, Italy
AIM: To investigate the anti-inflammatory properties of IL-6 on the
development of IDDM (insulin-dependent diabetes mellitus) in NOD mice. METHODS:
IL-6 was administered exogenously in female NOD mice (spontaneous model)
and cyclophosphamide-treated NOD mice (accelerated model). During the study
period, the mice were evaluated for development of diabetes once a week by glycosuria.
At the end of experiment, 6 euglycosuria mice from each group were sacrificed
for histoimmunological analysis, and the production of IL-1,
TNF-, and IFN-
from splenic lymphoid cells was detected. RESULTS: The incidence of
IDDM in spontaneous model (20 %) was significantly reduced 8 weeks after treatment.
TNF- (P<0.05) and IFN-
(P<0.01) were decreased obviously, but no variation was found in the
production of IL-1. CONCLUTION:
IL-6 protected rodent models against IDDM, which was accompanied by a dramatic
reduction of TNF- and IFN-
production. It was suggest that in hibiting the secretion of these cytokines
might be a pivotal mechanism by which IL-6 exerts its anti-inflammatory properties.
P-53 In vitro 22a- and 24-hydroxylation of 18
a-glycyrrhetic acid is mediated by CYP3A1/2 and
2C9/10 in rat liver microsomes
YANG Jing, PENG Ren-Xiu , KONG Rui
Department of Pharmacology, Wuhan University Medical School,
Wuhan 430071, China
AIM: To identify the cytochrome P450 isoforms involved in 22a-hydroxylation
and 24-hydroxylation of 18-glycyrrhetic
acid (GA) in rat liver. METHODS: The metabolism of GA to two of major
metabolites, 22a-hydro-GA and 24-hydro-GA was studied in rat liver microsomes
by a reversed phase HPLC assay. Selective chemical inhibitors to various cytochrome
P450 isoforms were employed to conduct inhibition experiments. The relationship
between the formation rate of 22a-hydro-GA and erythromycin N-demethylase
activities was analyzed. RESULTS: The formation of metabolites of GA
depended on incubation time (10-40 min), substrate concentration (25-200 mol/L)
and microsome protein concentration (0.25-1.0 g/L). The kinetic behaviors of
22a-hydroxylation and 24-hydroxylation of GA were described well by a single-enzyme
Michaelis-Menten equation [Km was (30±9) and (70±18)
mol/L, Vmax was
(7.9±1.4) and (3.4±1.0)mol·min-1
·g-1, respectively]. Inhibition experiments showed that troleandomycin
(0.025-0.1 mmol/L) and erythromycin (0.25-1.0 mmol/L) as potent CYP3A1/2 inhibitors,
reduced 22a-hydroxylation in a dose-dependent manner (the maximum inhibitory
rate was 59.6 % and 44.7 %, respectively), while sulfaphenazole, an inhibitor
towards 2C9/10, did not display significant inhibition. 22a-hydroxylation of
GA correlated well with erythromycin N-demethylase activities (r=
0.864, P<0.01, n=10). Sulfaphenazole (0.025-0.1 mmol/L) as
a potent CYP2C9/10 inhibitor, reduced 24-hydroxylation of GA in a dose-dependent
manner, the maxium inhibitory rate was 69.5 %, while troleandomycin, an inhibitor
of CYP3A1/2 did not display significant inhibition. The 24-hydroxylation of
GA did not correlate with erythromycin N-demethylase activities (r=0.310,
P>0.05, n=10). CONCLUSION: The 22a-hydroxylation and 24-hydroxylation
of GA in rat liver microsomes are mediated by CYP3A1/2 and CYP2C9/10.
P-54 Cytoprotective effect of
18-glycyrrhizic acid on acetaminophen-induced acute injury of
hepatocytes
YANG Jing, PENG Ren-Xiu , KONG Rui
Department of Pharmacology, Wuhan University Medical School,
Wuhan 430071, China
AIM: To study cytoprotective effect and molecular mechnisms of18-glycyrrhizic
acid (GL) on acetaminophen-induced acute injury of hepatocytes. METHODS:
Rat hepatocytes in sandwich cultures were randomly allocated into control
group, model control group, GL group, and GL plus L-NAME group. Hepatocytes
were exposed to acetaminophen (AAP 20 mmol/L) for 12 h to induce acute injury
of hepatocytes. In the pretreatment groups, L-NAME and/or GL were given
before administration of AAP. The activities of alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) in medium were measured by enzyme assays.
CYP2E1 expression in hepatocytes was determined by enzyme assays and semiquanti-tative
reverse transcriptase-polymerase chain reaction (RT-PCR). AAP concentration
in medium was assayed by HPLC. The contents of cGMP and cAMP in hepatocytes
were measured by radioimmunoassay. Nuclear factor kappa-B (NF-B)
activation was determined by sandwich ELISA. RESULTS: (1) After
administration of acetaminophen (20 mmol/L) to the medium, the leakage of ALT
and AST increased significantly to a stable level, serving as a good hepatocyte
injury model. (2) When hepatocytes were pretreated with GL of different concentrations
(50-200 mg/L) for 48 h prior to acetaminophen exposure, the elevated leakage
of ALT and AST induced by acetaminophen was dose-dependently reduced. Levels
of transaminases almost returned to normal, and in the meantime CYP2E1 (Aniline
hydroxylase, ANH) activity was decreased by 1.2 %, 40.0 %, and 57.7 %, respectively
and mRNA expression was inhibited in a dose (50 -200 mg/L)-dependent manner,
the maximum inhibitory rate was 61.2 %. AAP concentration in medium pretreated
with GL (100-200 mg/L) was higher than that in medium treated with AAP
alone. In addition, after administration of GL remarkable increase of glutathione
content was observed. (3) L-NAME almost reversed this effect. GL could
enhance the intracellular cGMP content and suppress NF-B
activation, but had no effect on cAMP content. CONCLUSION: The decrease
of elevated transaminase levels by GL is mediated in part by inhibition of CYP2E1
expression, which was regulated at the mRNA levels by suppressing NF-B
activation via NO-cGMP pathways.
P-55 Effects of clearing-heat herb on content of
NT in rat hypothalamus
XU Gang
Department of Pharmacology, Branch Campus of the First
Military Medical University, Guangzhou 510315, China
AIM: Clinical practice has shown that clearing-heat
herb is effective in treating infective and inflammatory
fever. We selected typical clearing-heat herb
Honeysuckle Flower and Forsythia Fruit to form a
clearing-heat prescription to analyze its antipyretic mechanism.
The objective of this experiment is to observe effects
of the clearing-heat prescription on content of neurotensin (NT) in rat hypothalamus and analyze the
relationship between NT and the antipyretic mechanism
of clearing-heat herb. METHODS: Rat fever models
were by infecting yeast solution hypodermically, and
pouring the set up medicine down the throat of the
animals. The content of NT in rat hypothalamus was
examined by radioimmunoassay (RIA). RESULTS:
The body temperature and content of NT in rat
hypothalamus of model group was higher than those of control
group. The body temperature of therapeutic group was
lower than that of model group, but the former content
of NT in hypothalamus was higher than the latter.
CONCLUSION: Clearing-heat herb has an antipyretic
action, and its antipyretic mechanism is connected with
change of NT in hypothalamus. Rising of NT of model
group shows that NT participated in the process of
fever. Going up of NT of therapeutic group is relevant
to antipyretic action of clearing-heat herb because NT
is an important antipyretic medium that takes part in
negative regulation of body temperature. NT could
prevent temperature from going up too much. So
reinforcing negative regulation of temperature and
suppressing positive regulation of temperature by increasing the
content of NT in temperature regulation center may be
one of the antipyretic mechanisms of clearing-heat herb.
P-56 Effects of cryptoporus volvatus ferment
substance on leukotriene production from
polymorphonuclear leukocytes in rats in vitro
JIN Sai-Hong, XIE Qiang-Min, CHEN Ji-Qiang, BIAN
Ru-Lian
Zhejiang Respiratory Drug Research Laboratory of the State Drug
Administration of China, Medical School of Zhejiang University,
Hangzhou 310031, China
AIM: To study inhibitory effects of cryptoporus
volvatus ferment substance (CVFS) on leukotriene
production in polymorphonuclear leukocytes in rats
in vitro. METHODS: Polymorphonuclear leukocytes (PMN)
were stimulated by calcimycin in rats in
vitro to produce leukotriene (LT)
B4, C4, and D4. The
concentrations of LTB4, C4, and
D4 were measured by reversed-phase high-pressure liquid chromatography (HPLC).
RESULTS: CVFS 0.025, 0.1, and 0.4 g/L
decreased LTB4, C4, and
D4 production from PMN in a
concentration-dependent manner. LTB4 of CVFS 0.025, 0.1, and
0.4 g/L groups was (148±46), (93±33), and
(43±9) ng per 107 cells, respectively, significantly lower than that
of control group (215±101) ng per 107
cells (P<0.05). LTC4 of CVFS 0.025, 0.1, and 0.4 g/L groups was
(176±37), (132±65), and (106±29) ng per
107 cells, respectively, significantly lower than that of control
group (514±310) ng per 107 cells
(P<0.05). LTD4 of CVFS 0.025, 0.1, and 0.4 g/L groups was
(257±70), (227±78) and (157±59) ng per
107 cells, respectively, significantly lower than that of control group
(514±249) ng per 107 cells
(P<0.05). CONCLUSION: Supression
of leukotrienes release may be a CVFS effect
mechanism of anti-inflammation and anti-asthma.
P-57 Application of vascular smooth muscle strips
in study of sulfonylurea receptors
LI Jun, HE Hua-Mei , ZHANG Le-Zhi
Department of Pharmacology, Third Military Medical University,
Chongqing 400038, China
AIM: To assess the application of vascular smooth
muscle strips in study of sulfonylurea receptors (SUR).
METHODS: Comparatively study the binding
characteristics of [3H] glibenclamide
([3H] Gli) to aortic smooth
muscle strips (ASMS) and to aortic smooth muscle cell
membranes (ASMM) under different experimental conditions.
RESUILTS: After incubation at 25ºC in
HEPES buffer (pH 7.40) for 60 min, the specific
binding of [3H]Gli 3 nmol/L to ASMM in rats was only
(0.5±0.2) pmol/g protein, accounting for 8.8 % of total binding. Variation
of the buffer (Tris or PBS), variation of the incubation conditions (time, temperature,
and pH) addition of ATP 1 mmol/L, preservation of the phosphorylation state
of the preparation with the presence of NaF 50 mmol/L and MgATP 0.1 mmol/L,
inhibition of membrane proteolysis by the addition of phenylmethylsulfonyl fluoride
, bestatin and pepstatin 1 mol/L
each into Ca2+-free HEPES buffer(replacing CaCl2 5 mmol/L
with egtazic acid 1mmol/L) produced no alteration in the specific binding of
[3H]Gli to ASMM. As compared with the results from ASMM, after incubation
at 25 ºC in HEPES buffer with a pH range of 6.0 to 8.5 for 60 min, specific
binding of [3H]Gli 3 nmol/L to ASMS was 16.8-19.2 pmol/g wet weight,
accounting for 56 %-62 % of total binding. A good linear correlation (r=0.993,
P<0.01) between specific binding and wet weight of ASMS over a range
of wet weight from 2 to 8 mg was obtained. Specific binding increased with temparature
across the range of 4 to 25 ºC, but not in the range of 25-37 ºC.
Furthermore, coefficient of variation for specific bound of [3H]Gli
to ASMS was lower than that to ASMM. CONCLUSION: Studying SUR of aortic
smooth muscles replacing ASMM with ASMS is a convenient, rapid, and well-repeatable
method. Not only can it economize animals but also can increase the specific
binding due to preventing physiological properties and environments of SUR from
being destroyed by homogenizing and centrifuging when ASMM are prepared.
P-58 A new animal model for Tourette syndrome
ZHANG Ru-Yi, LI lin, ZUO Ping-Ping1, YE Cui-Fei, AI
Hou-Xi
Department of Pharmacology, Xuan-wu Hospital of Capital
University of Medical Sciences, Beijing Key Laboratory for Brain
Aging, Beijing 100053; 1Department of Pharmacology, Research
Institute of Basic Medicine of Chinese Academy of Medical
Sciences, Beijing 100005, China
AIM: To develop a rat model for Tourette syndrome
(TS) which mimics the behavior and the underlying pathophysiology of clinical patients, and may be used
as the first pharmacological paradigm in China.
METHODS: A TS rat model was developed by peritoneal injection of iminodipropionitrile.
Stereotyped behaviors of model rats were recorded, the open field test was used
to test the excitability of the central nervous system of model rats, and climbing
times were also recorded to evaluate muscle strength of model rats. High performance
liquid chromatography was used to detect the content of dopamine and homovanillic
acid in striatum of model rats. Radioactive ligand combination test was used
to detect the activity of dopamine receptor type II in striatum of model rats.
RESULTS: TS model rats exhibited abnormal rotation, choreatic head and
neck movement and tic-like jerk of the head, decreased excitability of the central
nervous system and decreased muscle strength. There was no change in the content
of dopamine in striatum, but the content of homovanillic acid increased. The
activity of dopamine receptor type II in striatum increased markedly. CONCLUSION:
The first animal model for TS in China was developed successfully. The model
rats can mimic most of the changes in behavior, content of neurotrans-mitters,
and activity of dopamine receptor type II in TS patients.
P-59 Effect of Congsheng capsule on intracellular
Ca2+ concentration and membrane integrality of
neurons after cerebral ischemia
ZHAO Ling, XU Qiu-Ping, LI Lin
Department of Pharmacology, Xuan-Wu Hospital of Capital
University of Medical Sciences, Beijing Key Laboratory for Brain
Aging, Beijing 100053, China
AIM: To investigate the effect of Congsheng capsule (CSC) on intracellular
Ca2+ concentration increase and membrane integrality of cultured
neurons caused by cerebral ischemia. METHODS: Ca2+ concentration
in intra-synaptosomes and neurons was measured using double wavelength fluorescence
spectrophotometer. Synaptosomes and neurons were loaded with fluorescent dye
Fura-2/AM. Drug serum was used to study the action of CSC on neuronal damage
during hypoxia (3 h) /reoxygenation (0, 3, 6, and18 h) which was reflected by
the release of lactate dehydrogenase (LDH) from cells into culture medium.
RESULTS: CSC 3 and 9 g/kg decreased intra-synaptosomal Ca2+ concentration
increase induced by repeated cerebral ischemia and reperfusion combined with
caudal bloodletting in mice. The serum preparation of Congsheng capsule inhibited
the increased level of [Ca2+]i caused by hypoxia/hypoglycemia
for 1 h or glutamate 200 mol/L for
30 min. The serum preparation of CSC (2, 4, and 8 g/kg) attenuated neuronal
damage during hypoxia (3 h)/reoxygenation (0, 3, 6, and18 h) by decreasing LDH
release. CONCLUSION: The protective effect of Congsheng capsule on cerebral
ischemia may be achieved out by inhibiting increased level of [Ca2+]i
induced by cerebral ischemic damage, protecting membrane integrality after cerebral
ischemic damage.
P-60 Effect of serum from Liuwei Dihuang
decoction-treated rats on long-term potentiation in
hippocampal slices from senescence accelerated mice
ZHANG He-Ping , ZHANG Yong-Xiang
Beijing Institute of Pharmacology and Toxicology, Beijing 100850,
China
AIM: To investigate age-related changes of long-term
potentiation (LTP) in hippocampal slices from
senescence-accelerated mice (SAM) and the effect of the
serum from Liuwei Dihuang decoction (LW)-treated rats.
METHODS: Extracellular microelectrode
recording technique slices. The serum was isolated from the
rats treated with LW by oral administration, termed
LW-containing serum (LW-CS). RESULTS: With the
advancing of age, population spike (PS) amplitude was
gradually decreasing in both SAM-prone/8 (SAMP8), a
senescence accelerated prone substrain of SAM, and
SAM-prone/1 (SAMR1), a senescence resistant substrain of SAM. The amplitude of PS was
significantly reduced in both 6 and 12-month-old SAMP8
compared with that in age-matched SAMR1, however, they showed no differences compared with
2-month-old mice in both strains. When the hippocampal slices
from 6-month-old SAMP8 were incubated with LW-CS from LW-treated rats for 30 min, the amplitude of
PS evoked by tetanus was significantly potentiated.
CONCLUSION: SAMP8 showed age-related decline
of hippocampal LTP induction evoked by tetanus,
suggesting that the hippocampal synaptic plasticity is
reduced with aging in SAMP8. LW-CS facilitated the
induction of hippocampal LTP, suggesting that the
improving effect of LW on learning and memory in SAMP8
was related to the amelioration of synaptic plasticity of
the hippocampus.
P-61 Research of JMS inhibitory effect on Duck HBV in vivo
ZHAO Yan-Ling, HUANG Ying, ZHANG Xin-Quan, CAI Guang-Ming, XIAO Xiao-He, HE
Cheng-Shan,
LI Zhuang1
Department of Pharmacy, 302 Hospital of CPLA, Beijing
100039; 1Institute of Medicinal Biotechnology, Chinese Academy of
Medical Sciences, Beijing 100050, China
AIM: To investigate the inhibitory effect of JMS, an
active constitute from Potentilla anserine
L, on duck hepatitis B vxirus DNA (DHB VDNA) and duck
hepatitis B surface antigen (DHBsAg). METHODS:
DHBV- infected ducklings were treated with JMS 0.6, 0.3, and
0.15 g/kg (ig, bid) for 21 d and dynamics of DHBVDNA
was tested in liver, kidney, pancreas, and spleen.
Supercoiled DNA (SC DNA) extracted from liver was
detected by Southern blot hybridization. RESULTS:
(1) JMS 0.3 and 0.6 g/kg significantly inhibited serum
DHBsAg; (2) JMS 0.6 and 0.3 g/kg inhibited serum DHBV DNA from 14 to 21d, JMS 0.6 g/kg inhibited
serum DHBV DNA earlier even 7 d after administration
of JMS. (3) JMS 0.3 and 0.6 g/kg obviously inhibited
DHBV DNA in liver and kidney, but no inhibition was
found in spleen and pancreas. (4) The replication of
DHBV DNA in liver of ducklings treated with JMS 0.6 g/kg for 21 d showed that JMS apparently inhibited
the synthesis of linear DNA (L DNA), relaxed circular
DNA (RC DNA) and SC DNA. CONCLUSION: JMS inhibited DHBV DNA replication in DHBV-infected
ducks, showing its potential anti-hepatitis B effect for
future clinical use.
P-62 HCY-2 gene expression in ventricular
myocardium during progression of heart failure rat
model induced by myocardial infarction
LI Zheng, XU Ding-Li, ZENG Ping, LAI Wen-Yan,
REN Hao
Department of Cardiology, Nanfang Hospital, the First Military
Medical University, Guangzhou 510515, China
AIM: HCY-2 gene, which was recently identified by
homocysteine stimulation, was closely concerned with
growth, differentiation, and apoptosis of vascular
smooth muscle cells. The purpose of this study is to
investigate the role of HCY-2 gene expression in
ventricular myocardium during progression of heart failure
induced by myocardial infarction. METHODS:
Male Sprague-Dawley rats (200-300 g) underwent either a
left coronary artery ligation, a model of chronic heart
failure (CHF), or control group (sham-operated rats).
The expression of HCY-2 gene was assessed by Western blot on 3 d (acute myocardial infarction stage), 3
weeks (ventricular hypertrophy stage), and 6 weeks
(heart failure stage) after operation. RESULTS:
The HCY-2 gene was increased 25 % (1.36±0.07
vs
1.09±0.08, P<0.01), 24 %
(1.40±0.09 vs 1.14±0.06, P<0.01), and 48 %
(1.62±0.10 vs 1.09±0.07, P<0.01)
in CHF rats on 3 d, 3 weeks, and 6 weeks as compared
with control group respectively. The densitometry value
of HCY-2 of rat CHF model on 3 weeks did not obviously increased as compared with 3-d group of CHF
model (P>0.05). However, the densitometry value of
HCY-2 in 6-week group of CHF model significantly
increased 15 % (P<0.05) as compared with 3-week group
of CHF model. There was no significant difference in
control rats on 3 d, 3 weeks, and 6 weeks
(P>0.05). CONCLUSION: There was increased HCY-2 gene
expression in the progression of heart failure. HCY-2 gene
may be an important target gene in heart failure
treatment and prevention.
P-63 HSP70 is involved in protection of thermal
preconditioning from apoptosis in cerebellar granule neurons induced by repolarization
CHEN Li-Jun, SHU Xing-Wen, QIU Peng-Xin, HUANG Yi-Jun, YAN Guang-Mei
Department of Pharmacology, Zhongshan Medical College, Sun
Yat-Sen University, Guangzhou 510089, China
AIM: To explore the possibility that expressions of different heat shock
proteins were specifically involved in the protection against apoptosis of cerebellar
granule neurons induced by repolarization. METHODS: Western blot was
used to detect expressions of HSP27, HSP70, and HSP90 induced by thermal preconditioning
(TP) in cerebellar granule neurons; RT-PCR (reverse transcription-polymerase
chain reaction) to detect the expression level of HSP70 mRNA; Western blot to
detect the effect of HSP70 antisense oligodeoxynucleotides on expression of
HSP70. RESULTS: There was no expression of HSP27 in cerebellar granule
neurons with TP at 44 ºC. Expression of HSP90 was obvious in cerebellar
granule neurons both without and with TP at 44 ºC for different time. TP
at 44 ºC induced specifically and obviously expression of HSP70 in cerebellar
granule neurons, and the longer the TP, the more obvious the expression of HSP70
was. Furthermore, the expression of HSP70 mRNA increased while duration of TP
was longer.HSP70 antisense oligodeoxynucleotides (10 mol/L
) inhibited expression of HSP70 in cerebellar granule neurons with TP at 44
ºC, and at the same time, abolished protection of TP from apoptosis in
cerebellar granule neurons induced by repolarization. CONCLUSION: HSP70
is involved in protection of thermal preconditioning from apoptosis in cerebellar
granule neurons induced by repolarization.
P-64 Inhibitory effect of nicotinic acid on platelet
aggregation in rabbit
ZHU Min-Heng, WU Yue
Department of Pharmacology, Pharmaceutical College, Shihezi
University, Shihezi 832002, China
AIM: To investigate the effect of nicotinic acid (NA)
on rabbit platelet aggregation in vivo and
in vitro. METHODS: Platelet aggregation in rabbit blood induced
by collagen, adenosine diphosphate (ADP), and
arachidonic acid (AA) was measured by turbidimetry.
RESULTS: NA (2, 3, 4, and 6 g/L) evidently inhibited
abnormal increase of platelet aggregation induced by
ADP and collagen in vitro, and the same effects were
observed in NA (200, 300, 400, and 500 mg/kg) in
vivo. Both in vivo and in vitro, NA selectively inhibited
the platelet aggregation induced by ADP and collagen,
but not by AA. Aspirin significantly inhibited abnormal
increase of platelet aggregation induced by collagen, ADP
and AA, both in vivo and in vitro.
CONCLUSION: NA inhibited platelet aggregation.
P-65 Expression of the gene for thrombin-like
enzyme from Agkistrodon acutus venom in High
FiveTM insect cell
LIANG Xiu-Xia, CHEN Jia-Shu, QIU Peng-Xin, YAN Guang-Mei
Department of Pharmacology, Zhongshan Medical College, Sun
Yat-Sen University, Guangzhou 510080, China
AIM: To express the gene for thrombin-like enzyme
(TLE) in insect cells using a baculovirus expression
system. METHODS: The cDNA of TLE encoding DNA
fragment was inserted into baculovirus transfer vector
pMelBac, and then High five cells were transfected with
wild Bac-N-Blue baculovirus DNA to produce the recombinant baculovirus by homologous recombination.
The recombinant virus was identified by PCR assay and electromicroscopic observation.
RESULTS: The High five cells infected with the recombinant baculovirus
containing TLE cDNA fragment expressed biologically
active rTLE, which was secreted into the supernatant.
The level reached the highest 72 h after infection with
recombinant baculovirus. SDS-PAGE showed the expressed TLE degraded
A chain of bovine and human fibrinogen, which was similar to natural
TLE. CONCLUSION: TLE can be expressed and secreted
into the supernatant in High five insect cells. The rTLE
has the activity of degrading the A chain of
fibrinogen.
P-66 Pharmacological study of tricin for hepatic encephalopthy
XU Dong-Hui, TANG Xu, MEI Xue-Ting, ZHANG Chang-Hua, XU Shi-Bo
Department of Pharmacy, Zhongshan University, Guangzhou 510275, China
AIM: High blood ammonia is the main reason for hepatic coma in hepatic
encephalopthy. Sodium glutamate is used clinically to reduce blood ammonia,
but it has some side effects. The present study aimed to assess the effects
of tricin (5,4'-dihydroxy-3',5'-dimethoxy-7-(-D-glucopyranosyloxy)-flavone)
on blood ammonia in a rat hepatic coma model. METHODS: Rats were injected
with CH3COONH4 5.5 mmol/kg and 0.1 % CHCl3
(sc) to establish a high blood ammonia model. Tricin was ip injected to observe
the effects on blood ammonia and duration of hepatic coma. RESULTS: Tricin
(20, 60, and 180 mg/kg, ip) reduced the high level of blood ammonia and the
duration of hepatic coma caused by CH3COONH4 and CHCl3.
CONCLUSION: Tricin potentially serves the curative of hepatic coma in
hepatic encephalopthy.
Host: Association of CJP/JCP Club International and
BMS Club International
Supported by: Golden Castle Science Fund
Shanghai Society for Physiological Sciences
Shanghai Society for Pharmacology