The Mechanism Underlying The Excitatory Effect Of Oxytocin And Vasopressin On The Gastric Motility In Male Rats

Partâ… :The mechanism underlying the excitory effect of oxytocin on gastric motility in male ratsObjective:Oxytocin(OT) is a neurohypopHysial hormone that is produced in hypothalamic supraoptic nucleus and paraventricular nucleus,and exerts complicated and variable effect on gastrointestinal motility depending on the part of the gut and the animal species tested.The mechanism involved in OT effect on the contraction of the gut is unknown.Monstein et al.reported that oxytocin receptor(OTR) mRNA was expressed in the human gut.As far as we know,however,there is no report about the cell type that expressed OTR in gut.The mechanism about the effect of OT on the gut is unknown.The aim of this study is to investigate the effect of OT on gastric motility and involved mechanism and to localize OTR in the stomach in rats.Methods:Recording of the gastric motility in vivoThe rat was anesthetized by amobarbital sodium(56 mg/kg).The trachea and external jugular vein and the right femoral artery of the rat were cannulated.In order to monitor the gastric pressure,a plastic ballonet was inserted into the stomach. Normal saline(1 ml) was infused into the ballonet to maintain the pressure in the ballonet at about 30 mmHg.The stomach was initially equilibrated for 60 min before performing experiments.Some animals were administered with devazepide,the CCK₁ receptor antagonist,10 min before OT injection.Recording of the contraction of muscle stripsAfter the rat was sacrificed,the stomach was put in 4℃krebs solution and then cut along the lesser curvature.Four kinds of full thickness muscle strips from the stomach(4 mm wide,10 mm long) were prepared.They are muscle strips parallel to the long axis of the circular muscle layer of gastric body,parallel to the long axis of the longitudinal muscle layer of gastric body,parallel to the circular muscle of gastric antrum,and canalis pyloricus.The mucosa layer was carefully removed.The muscle strips were suspended in tissue chambers containing 5 ml Krebs solution(37℃).The strips were administrated with OT after equilibration for 60 min with flushing every 15 min.Measurement of the length of the isolated cells of smooth muscleThe gastric body was removed after the rat was sacrificed.The mucosa layer was stripped with fine scissors,and the muscle sheet was cut into strips(2 mm long,2 mm wide).The strips were then incubated for two successive 45-min periods in 8 ml Hepes-Ringer solution containing 0.1%collagenaseâ…¡and 0.01%soybean trypsin inhibitor at 31℃.After incubation with enzyme,the muscle strips were washed 3 times with enzyme-free Hepes-Ringer solution,and then oscillated in 5 ml Hepes-Ringer solution for 30 min at 31℃.The cells were obtained by filtration through 500-μm Nylon mesh.Only one cell in a dish was observed.Western blot analysisThe gastric body homogenates without the mucosa layer were electropHoresed (12%SDS-PAGE) and transferred to nitrocellulose membranes.Membranes were blocked for 1 hr at room temperature in blocking buffer(5%nonfat dry milk,TTBS), washed in TTBS(0.1%Tween 20,50 mM Tris,and 150 mM NaCl),and incubated overnight with goat anti-OTR polyclonal antibody(1:400,sc 8102;Santa Cruz).After washing three times,membranes were incubated for 1 hr at room temperature with HRP-conjugated secondary antibodies(1:20000).Finally,immunoreactive proteins were detected by ECL plus.ImmunohistochemistrySpecimens of the gastric body were sectioned(10μm thickness) in a cryostat. Activity of endogenous peroxidase was blocked with 3%hydrogen peroxide.After three rinses in PBS,5%normal rabbit serum was applied for 15 min,and then the sections were incubated with primary goat anti-OTR polyclonal antibody(diluted 1:100) overnight in a humid chamber at 4℃.After the sections were washed,they were incubated with biotinylated rabbit anti-goat serum(for 30 min at room temperature).The sections were next washed and then treated with HRP -labeled streptavidin- complex for 30 min at room temperature.After several rinses, peroxidase was revealed by a 3,3'-diaminobenzidine tetrahydrochloride substrate kit.Statistic AnalysisIn the in vivo studies,the peak amplitudes of gastric pHasic contraction were measured.Mean amplitude for 3-min period before application of OT was taken as the baseline.The peak amplitude after OT treatment was normalized by subtracting the baseline,In the experiments on muscle strips,the average tension for 3-min period before treatment with OT was taken as the baseline,The average tension after each OT treatment was normalized to a standardized ratio R.The value are presented as mean±SEM.Differences between groups were evaluated by One way ANOVA and Students t test,P<0.05 was considered to be significantly different.Results:1 The effect of exogenous OT on gastric motility showed a bipHasic pattern.OT induced a transient decrease and subsequent increase on intragastric pressure.The peak amplitude of pHasic contraction decreased by 3.04±0.94 mmHg at 1 min and increased by 4.16±2.76 and 4.84±3.00 mmHg at 13 and 15 min,respectively. Pretreatment with devazepide,a CCK₁ receptor antagonist,blocked the transient inhibitory effect of OT on gastric motility.2 OT concentration-dependently excited the contraction of circular and longitudinal muscle strips of gastric body,circular muscle strips of gastric antrum and pyloric spHincter.The average tension reached their highest level at 1^st or 3^rd min.At third minute following treatment of four doses of OT(10^-9 M-10^-6 M),R of the circular muscle strips of gastric body increased from 1(baseline) to 1.18±0.09,2.13±0.32 (P<0.05 vs.control),3.92±0.55(P<0.05 vs.control) and 5.04+0.61(P <0.05 vs.control ),respectively;R of the longitudinal muscle strips of gastric body increased to 1.04±0.02,1.13±0.08,1.39±0.14(P＜0.05 vs.control) and 2.20±0.22(P＜0.05 vs.control).At the first minute following treatment of three doses of OT(10^-8 M-10^-6 M),R of the pyloric spHincter increased to 1.33±0.10(P<0.05 vs.control ),1.48±0.17 (P<0.05 vs.control) and 1.53±0.16(P<0.05 vs.control ) respectively;R of the circular muscle strips of gastric antrum increased to 1.03±0.02(P＜0.05 vs.control),1.31±0.13(P<0.05 vs.control) and 1.52±0.26(P<0.05 vs.control ) respectively.3 Atosiban(10^-6 M),a competitive antagonist of oxytocin receptors,inhibited the spontaneous contraction of the circular muscle strips of gastric body.Pretreatment with atosiban(10^-6 M) partially blocked the excitatory effect of OT at 10^-8 - 10^-7 M on the contractions of the circular muscle strips.Pretreatment with TTX(10^-5 M),the specific blocker of voltage-gated Na⁺ channels in the nerve fiber,and atropine had no apparent effect.4 Administration of OT(10^-8 M-10^-6 M) induced contraction of the cells,and the length of the cells decreased.The contractile response to OT(10^-6 M) was completely abolished by pretreatment of atosiban(10^-6 M) and 2-APB,the antagonist of IP₃ receptor.5 Immunoreactive bands of appropriate molecular mass for OTR were identified in female rat uterus,male rat stomach smooth muscle and colon.OTR immunoreactivity was expressed on gastric smooth muscle cells.Both longitudinal and circular muscles expressed OTR.Conclusions:OT exerted a tonic excitatory effect on the gastric motility.OTR expressed on the smooth muscle mediated the excitatory effect of OT on gastric motility in rats.The transit inhibitory effect of OT on gastric motility in vivo was mediated by the release of CCK. Partâ…¡The effect of vasopressin on gastric motility in male ratsObjective:Vasopressin(VP) is another neurohypopHysial hormone.Although the molecular structure of VP differs from that of OT by only two amino acids(80%homology), they have clearly divergent pHysiological activities.VP is involved in the regulation of osmotic balance and cardiovascular function.There were data shown VP influenced the gastrointestinal motility.Monstein et al.have reported vasopressin receptor(VPR) mRNA was expressed in the human gut.As far as we know,however,there is no report about the cell type that expressed VPR in gut.The aim of this study is to localize VPR in stomach and to investigate the effect of VP on gastric motility in rats.Methods:Recording of the contraction of muscle stripsThe method followed that of partâ… .ImmunohistochemistrySpecimens of the gastric body were sectioned(10μm thickness) in a cryostat. Activity of endogenous peroxidase was blocked with 3%hydrogen peroxide.After three rinses in PBS,5%normal rabbit serum was applied for 15 min.Then the sections were incubated with primary goat anti-V_1a receptor polyclonal antibody (diluted 1:100,Santa Cruz) overnight in a humid chamber at 4℃.After the sections were washed,they were incubated with polymer peroxidase-anti-goat serum for 30 min at room temperature.After several rinses,peroxidase was revealed by a DAB tetrahydrochloride substrate kit.Double labeling immunodetectionColocalization of V_1a receptor and neuronal nuclei(NeuN) was performed on paraffin sections(4μm).After deparaffinization and hydration,sections were preincubated with 5%rabbit serum for 1h and then in a 1:100 dilution of anti-V_1a receptor polyclonal antibody and a 1:100 dilution of the anti-NeuN monoclonal antibody overnight at 4℃.After washing with PBS,the sections were incubated at room temperature for 1h with secondary antibody as follows:TRITC (rhodamine)-conjugated rabbit anti-goat IgG(1:50),FITC(fluorescein)-conjugated rabbit anti-mouse IgG(1:50).The immunoactivity were captured by using a fluorescence microscope.Results:1 VP(10^-9 - 10^-7 M) concentration-dependently excited the contraction of circular muscle strips of gastric body.The average tension of the muscle strips bagan to increased 1 min following VP administration,reached the highest level at 3^rd min.The excitatory effect of VP(10^-9 M) on the tension of gastric muscle strips was maintained more than 15 min.2 V-1880(10^-7 M),a potent V₁ receptor antagonist,inhibited the spontaneous contraction of the circular muscle strips.3 Pretreatment with V-1880(10^-7 M) and atropine(10^-6 M) partially blocked the excitatory effect of VP(10^-7 M) on the contraction of the muscle strips.Pretreatment with TTX(10^-6 M) completely blocked the excitatory effect of VP(10^-7 M). Pretreatment of hexamethonium(10^-5 M),the blocker of ganglia nicotinic receptors, did not influence it.4 V_1a receptors were expressed in the neurons of the myenteric plexus.5 Both V_1a and NeuN were located in the myenteric plexus of the gastric stomach. After overlapping the two pHotos,it is clearly that V_1a was expressed on the neurons of the myenteric plexus.Conclusions:The mechanism of VP increasing the contraction of the stomach differs from that of OT.V_1a receptor in myenteric nerves mediated the excitatory effect of VP on circular muscle strips of gastric body in vitro in rats.