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 Hideaki^1,2, 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: T_max (1.6±1.2) h and (1.6±0.8) h, C_max (17±5) g/L and (25±7) g/L, AUC (103±24) g·L^-1·h and (144±65) g·L^-1·h, T_1/2 (5.4±2.1) h and (4.4±2.2) h, respectively. For 20 mg group: T_max (3.3±1.6) h, C_max (56±22)g/L, AUC (623±185) g·L^-1·h, T_1/2 (31±15) h. Following multiple dosing of 10 mg, steady state C_max was (45±18) g/L and steady state C_min was (19±9) g/L, the peak to trough fluctuation index (DF) was 85 %±9 %, and AUC_ss (768±340) g·L^-1·h. Following the last dosing of 10 mg, the pharmacokinetic parameters were: T_max (2.7±1.6) h, C_max (48±21) g/L, AUC (658±278) g·L^-1·h, and T_1/2 (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>BA, 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 (pH_i) 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/dt_max, dL, and end-diastolic cell length. Inactive IL-2 at 200 kU/L did not significantly decrease ±dL/dt_max, 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 pH_i of cardiaomyocytes, which could be attenuated by nor-BNI (10 nmol/L). CONCLUSION: It is concluded that the decreased contraction and pH_i 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 Taizo^1,3, 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-Xia^1,2, 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-Sheng^1,2, DONG Xian-Hong¹, LI Ju-Xiang¹, ZHONG Guang-Zhen¹, WU Sheng-Ying^1,2, 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 tRNA^Leu(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 tRNA^Leu(UUR) gene, which causes the MELAS encephalomyopathy, maternally inherited diabetes, or chronic progressive external ophlthalmoplegia. METHODS: The wild-type and mutant-type human mt tRNA^Leu(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 tRNA^Leu(UUR), respectively. RESULTS: The K_cat and K_m values of wild tRNA^Leu(UUR) were 0.24 S^-1 and 9.1 mol/L while those of mutant tRNA^Leu(UUR) were 0.22 S^-1 and 13.3 mol/L. The K_cat/K_m value of wild tRNA^Leu(UUR) was about 2-fold higher than that of mutant tRNA^Leu(UUR). CONCLUSION: Human mitochondrial tRNA^Leu(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 tRNA^Leu(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 tRNA^Leu(UUR). METHODS: The human mitochondrial tRNA^Leu(UUR) genes were transcribed in vitro with T7 RNA polymerase and the tRNA^Leu(UUR) without modificated nucleotide was purified and recovered by Urea-PAGE. The modified tRNA^Leu(UUR) was purified from the cellular total tRNA of E coli JM109 which has recombinant plasmid containg wild tRNA^Leu(UUR) gene. The kinetic parameters of human mitochondrial leucyl-tRNA synthetase (mtLeuRS) were determined with two kinds of tRNA^Leu(UUR). RESULTS: The K_cat and K_m values of tRNA^Leu(UUR) transcribed in vitro were 11.67 mol/L and 0.17 S^-1 while those of tRNA^Leu(UUR) expressed in E coli were 9.1 mol/L and 0.24 S^-1. The K_cat/K_m values between two kinds of tRNA were not significantly different. CONCLUSION: Nucleotide modification may be not essential to the aminoacylation of human mitochondrial tRNA^Leu(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 Li^1,2, 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 (BK_Ca) 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 BK_Ca 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 BK_Ca by reducing open probability (P_o) and open frequency of the channels. The reduction of P_o 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 BK_Ca in the vascular hyporesponse stage, and that inhibition of BK_Ca 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-Bao^1,2, 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 Ming^1,2, 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: t_1/2 0.12 and 0.23 h, t_1/2 8.4 and 3.3 h, t_1/1Ka 0.0066 and 0.013 h, T_max 0.024 and 0.067 h, C_max 30.2 and 18.7 g/L, AUC 107.6 and 50.4 g·L^-1·h. T