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Introduction
Natriuretic peptides (NP) are a polypeptide family with
many bioactivities and physiologically function as hormones
or neurotransmitters[1]. Up to now, NP include the atrial
natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type
natriuretic peptide (CNP) and dendroaspis natriuretic
peptide (DNP), Micrurus natriuretic peptide, and ventricular
natriuretic peptide. They are distributed all over the body and
they have natriuretic-diuretic[2] and
vasorelaxation[3] functions designed to lower blood
pressure[4] and control electrolyte
homeostasis[5]. Since immunoactivity of NP was
found in the stomach and small intestines of rats, the
investigation of the relationship between NP and the
gastrointestinal function was
preluded[6]. The studies about the
physiological functions of NP have mainly focused on gastric
absorption[7], secretion[8], and intestinal
motility[9], but there are few of NP studies about the regulation of gastric smooth
muscle motility.
Our previous study[10] indicated that DNP inhibited
gastric motility in the gastric antrum of guinea pigs. DNP, a 38
amino acid peptide, was isolated from the venom of the green
mamba. It has structural and functional similarities to other
members of the NP family. Studies about the physiological
function of DNP have mainly focused on the cardiovascular
system[11], urinary
system[12], and genitals[13]. Study about
the relationship between DNP and gastrointestinal functions
are few. Kim et al[14] demonstrated that the DNP system
presents in the rat colon and regulates colonic motility as a
local regulator. It is the first report to investigate the
relationship between DNP and gastrointestinal functions.
Up to now, there is no report of the relationship between
DNP and gastric motility. The aim of present study was to
determine whether the NP receptor (NPR) is present in the
stomach of guinea pigs and to investigate the effect of DNP
on the gastric motility of guinea pigs and its mechanism.
Materials and methods
Strip preparation EWG/B guinea pigs of either sex
(weighing 300±50 g) and bred by the Experimental Animal
Center of Dalian Medical University(Dalian, China) were
euthanized by a lethal intravenous dose of pentobarbital
sodium (50 mg/kg).The abdomen of each rat was opened
along the midline, and the stomach was removed and placed
in pre-oxygenated Tyrode's solution at room temperature.
The mucous layer was removed and strips (approximately
2.0 mm×15.0 mm) of gastric antral circular muscles were
prepared. The longer axis of the stomach was cut parallel to
the circular muscle fibers. The muscle strips were placed in
a chamber. One end of the strip was fixed on the lid of the
chamber through a glass claw; the other end was attached to
an isometric force transducer (TD-112S, Nihon
Kohden-Kogyo Japan) to record contractions. The chamber (2 mL
volume) was constantly perfused with pre-oxygenated
Tyrode's solution at 1 mL/min. The temperature was
maintained at 37.0±0.5 °C by a water bath thermostat (WC/09-05,
Chongqing, China). The muscle strips were allowed to
incubate for at least 40 min before the experiments were started.
Autoradiograph of the NPR
method[15] The iodinated DNP was prepared by the modified chloramine-T method,
divided, and kept at -70 °C until use. The iodinated DNP was
repurified by HPLC, and the specific activity was
approximately 2000 Ci/mmol. The anti-DNP antibody was purchased
from Peninsula Laboratories (San Carlos, Californai, USA).
The lyophilized samples were reconstituted with phosphate
buffer (pH 7.4) containing 50 mmol/L NaCl, 0.1% bovine
serum albumin (BSA), 0.1% Triton X-100, and 0.01% sodium
azide. Synthetic DNP was used as a standard. After
incubation with the anti-DNP antibody for 24 h at 4 °C,
approximately 15 000 cpm of 125I-DNP was added, and the samples
were incubated again for an additional 24 h at 4 °C. When
125I-DNP bonded with its receptor thoroughly, the tissue was
covered by photographic emulsion. At the site of the
radioactive material, the radioactive emission acts on the silver
halide in the emulsion. Subsequent development and
fixation turned the radiated silver halide into black grains.
Cell preparation and electrophysiological recording
Guinea pigs of either sex weighing 250_350 g were euthanized
by a lethal intravenous injection of pentobarbital sodium (50
mg/kg). The antral part of the stomach was rapidly cut, then
the mucosal layer was separated from the muscle layers. The
longitudinal layer of muscle was then dissected from the
other muscle layers using fine scissors and then cut into
small segments (1 mm×4 mm). These segments were kept in
modified Kraft_Bruhe (K_B) medium at 4 °C for 15 min. Then
they were incubated at 36 °C in 4 mL of digestion medium
(Ca-free physiologic salt solution [Ca-free PSS]) containing
0.1% collagenase II, 0.1% dithioerythritol, 0.15% trypsin
inhibitor, and 0.2% BSA for 25_35 min. The softened muscle
segments were transferred into the modified K_B medium,
and the single cells were dispersed by gentle trituration with
a wide-bore, fire-polished glass pipette. The isolated gastric
myocytes were kept in modified K_B medium at 4 °C until
they were ready for use.
Isolated cells were transferred to a 0.1 mL chamber on the
stage of an inverted microscope (IX-70 Olympus, Tokyo,
Japan) and allowed to settle for 10_15 min. The cells were
continuously perfused with a isosmotic PSS at a rate of
0.9_1.0 mL/min. An 8-channel perfusion system (L/M-sps-8, List
Electronics, Berlin, Germany) was used to exchange the
solution. The calcium-activated potassium currents
(IK[ca]) was recorded using the conventional whole cell patch-clamp
technique[16,17]. Patch-clamp pipettes were manufactured
from borosilicate glass capillaries (GC 150T-7.5, Clark
Electro-medical Instruments, London , UK) using a 2-stage puller
(PP-83, Narishige, Tokyo, Japan). The resistance of the patch
pipette was 3_5 MW¸ when filled with pipette solution.
Liquid junction potentials were canceled prior to the seal
forma-tion. Whole-cell currents were recorded with an Axopatch
1-D patch-clamp amplifier (Axon Instruments, Foster City
California, USA), and data were filtered at 1 KHz. Command
pulses, data acquisition, and storage were applied by using
the IBM-compatible, 486-grade computer and pCLAMP 6.02
software (Axon Foster City, California, USA). Spontaneous
transient outward currents were recorded simultaneously by
an EPC-10-HEAKA amplifier (HEAKA Instruments, Berlin,
Germany). All experiments were performed at room
temperature (20_25 °C).
Drugs and solutions Tyrode's solution containing (in
mmol/L) NaCl 147, KCl 4,
MgCl2·6H2O 1.05,
CaCl2 ·2H2O 0.42,
Na2PO4·2H2O 1.81, and 5.5 mmol/L glucose was used.
Ca2+-free PSS containing (in mmol/L) NaCl 134.8, KCl 4.5, glucose
5, and N-(2-hydroxyethyl) piperazine-N-(2-ethanesulphonic
acid) (HEPES) 10 was adjusted to pH 7.4 with Tris
(hydroxy-methyl) aminomethane. Modified K_B solution containing
(in mmol/L) L-glutamate 50, KCl 50, taurine 20,
KH2PO4 20,
MgCl2·6H2O 3, glucose 10, HEPES 10, and egtazic acid 0.5
was adjusted to pH 7.40 with KOH. PSS containing (in
mmol/L) NaCl 134.8, KCl 4.5,
MgCl2·6H2O 1,
CaCl2 ·2H2O 2, glucose 5, HEPES 10, and sucrose 110 was adjusted to pH
7.4 with Tris. In order to abolish delayed rectifier potassium
currents (IK[V]), external solution contained 4-aminopyridine
10 mmol/L, a selective inhibiter of
IK(V). The pipette solution recording
IK(ca) contained (in mmol/L) potassium-aspartic acid
110, Mg-ATP 5, HEPES 5,
MgCl2·6H2O 1.0, KCl 20, egtazic
acid 0.1, di-tris-creatine phosphate 2.5, and
disodium-creatine phosphate 2.5; its pH was adjusted to 7.3 with KOH.
Tetraethylammonium (TEA), DNP, LY83583, and zaprinast
were made up as stock solutions. All chemicals in this
experiment were purchased from Sigma (St Louis, MO, USA).
Data analysis All data was expressed as mean±SD.
Statistical significance was evaluated by a
t-test. Differences were considered to be significant when a
P-value was less than 0.05.
Results
Distribution of the NPR in the stomach wall of guinea
pigs Using the radioautograph technique, the distribution
of the NPR in the different regions of the stomach of guinea
pigs was detected. The NPR existed both in the
mucosal layer and muscle layer, and the distribution order of the NPR
in density was: antrum>body>fundus in the muscle layer
(n=6, Figure 1)
Effect of DNP on spontaneous contraction in antral
circular smooth muscles of guinea pigs The spontaneous
contraction usually appeared approximately 40 min after
incubating the muscle strips in Tyrode's solution. The effects of
10 nmol/L DNP on spontaneous contraction in gastric antral
circular smooth muscles of guinea pigs were observed.
After administering DNP, the spontaneous contraction was
significantly inhibited. (n=6, Figure 2). Different
concentrations of DNP obviously inhibited spontaneous contraction
in a dose-dependent manner, and the inhibition percentages
was 35%±7%, 54%±8%, 78%±13%, and 94%±6% at 1, 10,
100, and 1000 nmol/L respectively
(n=6, Figure 3). High concentration (1000 nmol/L) of DNP completely inhibited
spontaneous contraction and shifted down the base line.
Effect of LY83583 and zaprinast on DNP-induced
inhibition To further investigate the mechanism by which DNP
inhibited gastric motility in guinea pigs, the effect of DNP on
gastric motility was observed in the condition of
administering LY83583, an inhibitor of guanylate cyclase, and zaprinast
as a phosphoesterase inhibitor to change the production of
cGMP. LY83583 (10 nmol/L) markedly diminished the
inhibitory effect of DNP on spontaneous contraction, but could
not completely abolish the inhibitory effect
(n=8, Figure 4). Zaprinast (100 nmol/L) potentiated the inhibitory effect of
DNP on spontaneous contraction (n=8, Figure 5).
Effect of DNP on spontaneous contraction in the
presence of TEA It is well known that nitric oxide (NO) and NP
are cGMP generation systems in the body. Our previous
studies demonstrated[16,17] that sodium nitroprusside, a NO
donor, inhibited the spontaneous contraction of gastric
smooth muscles via increasing
IK(ca). So in the present study,
the effect of TEA, a non-selective potassium channel blocker,
on the DNP-induced inhibitory effect on gastric motility was
observed. After the muscle strips were pretreated with TEA
(10 mmol/L), the inhibitory effect of DNP (10 nmol/L) on
spontaneous contraction was significantly diminished
(n=6,
Figure 6).
Effect of DNP on
IK(ca) Under the conventional whole cell
patch-clamp mode, the membrane potential was clamped at
-60 mV, and the IK(ca) was elicited by step voltage command
pulse from -40 mV to 100 mV for 400 ms with a 20 mV
increment at 10 s intervals. 10 nmol/L DNP
(n=6, Figure 7) markedly increased the
IK(ca), and the increasing amplitude was
62.31%±3.22% at 60 mV.
Discussion
Our present study clearly shows that the NPR existed in
the stomach of guinea pigs and its density was largest in the
antrum. DNP, a new member of the NP family, inhibited the
motility of gastric antral smooth muscles in a
dose-dependent manner. The DNP-induced inhibitory effect was
diminished by LY83583, an inhibitor of guanylate cyclase, and
was potentiated by zaprinast, a phosphoesterase inhibitor.
TEA, a non-selective potassium channel blocker, also
suppressed the DNP-induced inhibitory effect. DNP increased
IK(ca) in a dose-dependent manner in the gastric circular
myocytes of guinea pigs.
The NPR was extensively distributed in many tissues
and systems; for example, in the rat
brain[18], in the kidney of the Japanese eel
(Anguilla japonica)[19], in the guinea pig
cecum[20], and in the rat heart
[21]. In the present study, we found that the NPR also exists in the stomach of guinea pigs
and the density is largest in the gastric antrum. There are
many reports about ANP, BNP, and CNP, however, studies
about DNP are few. The studies about DNP have focused
on the cardiovascular system. Ha et
al[22]suggested that DNP protects postischemic myocardial injury by an
increased ratio of Bcl-2 to the Bax protein after
ischemia-reperfusion. Ha et al[23]
demonstrated that DNP induces the apoptosis of cardiac muscle cells. Kim
et al[14] demonstrated that the DNP system presents in the rat colon and regulates
colonic motility as a local regulator. Kim et
al's report was the first report about the relationship between DNP and
gastrointestinal functions. In present study, DNP significantly
inhibited the spontaneous contraction of gastric antrum in
guinea pigs.
NP are similar to NO, which is a cGMP generation system
in the living body. Their physiological functions are very
important in life science. Ha et
al[23] demonstrated that DNP may induce the apoptosis of muscle cells by the cGMP
pathway. However, Singh et al[11]
reported that vasodilatation in human mammary glands was predominantly mediated
via the direct activation of smooth muscle NPR-A. It is
probable that there is a direct and indirect activation of NPR to
exert the physiological function of DNP. Up to now, there
are only a few studies investigating the mechanism of DNP
action, so the key point of this study was to determine the
relationship between DNP-induced inhibition and cGMP in
gastric antral circular smooth muscles. In present study, the
DNP-induced inhibition was diminished by LY83583, an
inhibitor of guanylate cyclase, and was potentiated by
zaprinast, a phosphoesterase inhibitor. Our study indicated
that the DNP-induced inhibitory effect on the spontaneous
contraction of gastric antral smooth muscles was mediated
by cGMP in guinea pigs.
In our present study, TEA weakened the effect of the
DNP-induced inhibition on spontaneous contraction. It is
well known that the potassium channel has a direct
relationship to the relaxation of smooth muscles. There are 2 kinds
of potassium currents, IK(ca) and delayed rectified potassium
currents in the gastric antral smooth muscle cells of guinea
pigs[24,25]. The present study demonstrated that DNP
increased IK(ca) in a dose-dependent manner. It could be
concluded that the potassium channel participated in the
DNP-induced relaxation. Many previous studies also reported
that the potassium channel is involved in the NP-induced
regulation of many physiological functions. Van der Zander
et al[25] demonstrated that NO and
IK(ca) regulated the effect of the BNP-induced relaxation on vascular smooth muscles.
Up to now, there is no report about the relationship between
DNP and ion channels. Our present study indicates that
DNP inhibits gastric motility by activating the
IK(ca).
In summary, our study confirms that the NPR system
exists in the stomach of guinea pigs, and the density of the
NPR is hightest in the antrum. DNP inhibits gastric motility
of circular smooth muscles. The DNP-induced inhibition
enhances cGMP production, and IK(ca)
may be involved in this process.
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