DIFFERENCES IN THE EFFECTS OF VASOPRESSIN AND OXYTOCIN ON FELINE GASTRIC CORPUS MOTILITY: SELECTIVE ACTION OF VASOPRESSIN ON LONGITUDINAL MUSCLE

Pharmacological Research, Vol. 37, No. 5, 1998

DIFFERENCES IN THE EFFECTS OF VASOPRESSIN AND OXYTOCIN ON FELINE GASTRIC CORPUS MOTILITY: SELECTIVE ACTION OF VASOPRESSIN ON LONGITUDINAL MUSCLE ˇ ´ SLOBODAN JANKOVIC ´ U and DUSAN ˇ GORDANA MIRCIC, BELESLIN Accepted 5 March 1998

The effect of vasopressin and oxytocin on the contractile activity of preparations isolated from the feline gastric corpus wall was investigated. Vasopressin Ž1.5= 10y9 ]2.1= 10y7 M., but not oxytocin, evoked concentration-dependent tonic contractions only of longitudinal muscle strips. At the same time, vasopressin Ž1.5= 10y9 ]2.1= 10y7 M. potentiated the magnitude of amplitudes, but not the frequency, of spontaneous contractions. Both the vasopressin V1 receptor antagonist dŽCH 2 .5-ŽMe. 2-Tyr-AVP and the predominantly vasopressin V2 receptor antagonist dŽCH 2 .5 , D-Ile 2 , Ile 4-AVP, the non-selective muscarinic receptor antagonist, atropine, the predominantly selective muscarinic M1 receptor antagonist, pirenzepine, the predominantly selective muscarinic M2 antagonist, methoctramine, the predominantly selective muscarinic M3 receptor antagonist, para-fluoro-hexahydrosiladifenidol, and the calcium channel blocker, nifedipine, but not the ganglion blocking agent, mecamylamine, depressed or blocked the tonic contractions induced by vasopressin. Among the antagonists, only atropine and nifedipine inhibited the spontaneous contractions. On the other hand, the anticholinesterase, physostigmine, potentiated both the vasopressin-induced tonic and spontaneous contractions. With regard to the receptors, the vasopressin-induced tonic contractions are mediated at least in part through vasopressin V1 and V2 receptors, non-selective muscarinic and selective muscarinic M 1 , M 2 and M 3 receptors. The increase in amplitudes of spontaneous contractions is mediated only via-nonselective muscarinic receptors. Vasopressin receptors appear to be located mostly pre-synaptically, although the direct effect of vasopressin on post-synaptic receptors cannot be excluded. The pA 2 values suggests rather V1a than V1b vasopressin receptor subtype involvement in tonic contractions vasopressin had produced. The tonic as well as spontaneous contractions are calcium-dependent. In addition, these results point to the existence of non-selective muscarinic receptors, which participate in the regulation of both tonic and spontaneous contractions, while muscarinic M 1 , M 2 and M 3 receptors subserve only the tonic contractions. Q 1998 The Italian Pharmacological Society KEY

WORDS:

stomach, contractions, vasopressin receptors, muscarinic receptors.

INTRODUCTION Two neurohypophyseal hormones, oxytocin and vasopressin, exert considerable activity in the central nervous system w1x, in vascular smooth muscle w2, 3x, in myometrium w4x and in some extravascular and extrauterine smooth muscles w5]10x. Prior investigations focused mainly on the central nervous system, vascular smooth muscle and uterus. Indeed, both excitatory and inhibitory effects of these hormones on gastrointestinal motility have been reported, depending on the specific segment of the gut, animal species, or even on experimental conditions w11]15x. Immunohistochemical examinations have shown the presence of cells Žcrypt cells of the stomach and U

Corresponding author.

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small intestine . containing vasopressin and vasopressin-associated human neurophysin, with capability of local vasopressin synthesis w16x. In addition, available experimental data suggest that functional oxytocin w17x and vasopressin w18x receptors exist in the vagal dorsal motor nucleus, and that oxytocin releasing axons from the hypothalamic paraventricular nucleus exhibit tonic inhibitory effects on gastric motility and secretion by increasing the excitability of the central vagal nerves in nucleus tractus solitarii and in vagal dorsal motor nucleus w19, 20x. However, interactions between the intrinsic cholinergic system within the wall of the gastrointestinal tract and the neurohypophyseal hormones have not yet been demonstrated. In the light of these observations, the aim of this study was to: Ž1. examine the effects of vasopressin and oxytocin on the contractile activity of the prepaQ1998 The Italian Pharmacological Society

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rations isolated from the feline gastric corpus wall; Ž2. determine the possible mechanism of action of these hormones; and Ž3. define the types of receptors mediating the effects of oxytocin and vasopressin. Moreover, the effects of oxytocin and vasopressin on the isolated preparations of the gastric corpus of pregnant cats were investigated.

MATERIALS AND METHODS The strips of gastric wall were obtained from 40 cats Žfour pregnant. of both sex, weighing between 1.5 and 4.0 kg, after a 24-h fasting period. The cats were anaesthetized with thiopentone sodium Ž30 mg kgy1 i.p.. and exsanguinated by a sectioning of the carotid arteries. Full wall-thickness longitudinal and circular strips Ž25 mm= 6 mm. were resected from the anterior side of gastric corpus, starting 5 mm distal from the oesophageal]gastric junction.

Preparation of longitudinal and circular smooth muscle strips Mucosa was removed from the gastric wall strips by a sharp dissection. For the purpose of studying the response of longitudinal smooth muscle layer, four incisions through whole thickness Žeach 1 mm apart from the other. were made parallel to the longer axis of the longitudinal strip interrupting the circular and oblique smooth muscle layers. For the purpose of studying the response of circular smooth muscle layer, four incisions through whole thickness Žeach 1 mm apart from the other. were made parallel to the longer axis of the circular strip interrupting the longitudinal and oblique smooth muscle layers. The smooth muscle strips were suspended into the 20-ml isolated organ bath filled with Tyrode solution ŽNaCl 137 mM, KCl 2.68 mM, CaCl 2 1.8 mM, MgCl 2 1.0 mM, NaH 2 PO4 0.417 mM, NaHCO3 11.9 mM and glucose 5.5 mM per litre of solution.. One end of the strips was attached for the holder in the bath bottom and the other to an isotonic recording system. The nutrient solution was kept at 378C, and bubbled slowly Žto avoid foaming. with 100% O 2 . The strips were loaded with 1.0 g. After mounting and loading, the strips were allowed to equilibrate for approx. 45 min. During this period the organ baths were washed with fresh Ž378C. Tyrode solution every 15 min.

Experimental procedure After the equilibration period, concentration-response curves were obtained by cumulative addition of vasopressin Ž1.5= 10y9 ]2.1= 10y7 M. to the strips. Increasing concentrations of vasopressin were added when the previous concentration had produced its equilibrium response. Experiments fol-

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lowed a multiple curve design. If the first and second concentration-response curves were not superimposable, but differed by more than 10%, the data from a particular tissue were rejected. The following protocol was used: Ž1. concentration-response curve with vasopressin Žused as tissue control., followed by three washes, addition of the antagonist and a 20-min equilibration period; and Ž2. concentration-response curve with vasopressin. In all experiments, one longitudinal and one circular isolated preparation from the same animal were run in parallel but without antagonist, in order to check for any time-dependent changes in sensitivity to the agonists.

Statistical analysis The contraction induced by each concentration of vasopressin was expressed as a percentage of the maximum contraction induced by vasopressin and used for construction of concentration-response curves. Concentration-response relationship was determined by linear regression calculated according to the method of least squares w21x. The concentration of vasopressin eliciting 50% of its own maximum response ŽEC 50 . and its confidence limits Ž1.96 = SE. were determined graphically for each curve by linear interpolation w22x. The pA 2 values Žylog molar concentration of antagonist reducing the agonist response by a factor of two. for p-fluorohexahydro-sila-difenidol ŽpFHHSiD., pirenzepine, atropine, methoctramine, w dŽCH 2 .5-Tyr-ŽMe. 2 x-AVP and w dŽCH 2 .5 , D-Ile 2 , Ile 4 x-AVP were determined from a Schild plot w23, 24x using vasopressin as the antagonist. The concentration ratios Žthe ratio between the EC 50 value for vasopressin in the presence and absence of an antagonist. at different antagonist concentrations for the different vasopressinrantagonist pairs were calculated for each experiment. Thus, the mean values of concentration ratios for an vasopressinrantagonist pair were plotted in a Schild diagram using regression analysis, and pA 2 was obtained from the intercept of the regression line on the abscissa w23x. The significance of the Schild plot linearity was tested by analysis of variance w22x. The difference of the slope to unity was verified by Student’s t-test and was considered not significant if P) 0.05. The results are expressed as means " 95% confidence intervals; n refers to the number of cats from whom gastric strips were taken. Two-way analysis of variance Žtwo-way ANOVA. was used when two blocks of groups were analysed Že.g. responses on different concentrations of vasopressin in presence and in absence of an antagonist.. Statistical differences between two means were determined by Student’s t-test for paired or unpaired observations where appropriate. A value of P- 0.05 was considered to be statistically significant w25x.

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Drugs The drugs used in these experiments were: oxytocin ŽSandoz, Germany.; 8-L-lysine-vasopressin ŽSandoz, Germany.; nifedipine ŽZdravlje, FR Yugoslavia.; physostigmine-salicylate ŽSigma Chemical Co., USA.; mecamylamine hydrochloride ŽSigma Chemical Co., USA.; p-fluoro-hexahydro-sila-difenidol ŽpFHHSiD. ŽResearch Biochemicals Inc., USA.; pirenzepine dihydrochloride ŽBoeh. Ingelheim Pharma GmbH, Austria.; atropine sulphate ŽSanofarma, FR Yugoslavia.; methoctramine tetrahydrochloride ŽDipartimento di Scienze Farmaceutiche, Universita Degli Studi di Bologna, Italy.; w1-Ž bmercapto-b , b-cyclopentamethylene-propionic acid.2- Ž O -m e th yl . tyrosin e x a rgin in e -va sopre ssin Žw dŽCH 2 .5 , O ŽMe. 2 Tyrx-AVP. ŽBachem, Switzerland.; and w1-Ž b-mercapto-b , b-cyclopentamethylen e-propion ic acid . , 2- D -isoleu cin e, 4- D isoleucinexarginine-vasopressin Žw dŽCH 2 .5 , D-Ile 2 , Ile 4 x-AVP. ŽBachem, Switzerland.. All drugs were prepared immediately before the experiment and stored on 48C until used. All agents were dissolved in distilled water and diluted to the desired concentration with buffer. Previous experiments had shown that the solvents used had no effect on the preparations. All drugs were added directly to the bath in a volume of 200 m l and the concentrations given are the calculated final concentrations in the isolated bath solution.

RESULTS

Effects of ¨ asopressin on gastric corpus wall strips from non-pregnant cats

The 8-L-lysine-vasopressin Ž1.5= 10y9 ]2.1= 10y7 M. produced concentration-dependent tonic contractions of the longitudinal smooth muscle strips isolated from feline gastric corpus Ž r s 0.99; P0.01. wFigs 1ŽA. and 2ŽA.x. The EC 50 for 8-L-lysinevasopressin was 1.53" 0.02= 10y8 M. At the same time, the amplitudes of spontaneous contractions were increased in a concentration-dependent manner Ž r s 0.98; P- 0.01; EC 50 s 1.06" 0.01= 10y8 M. wFigs 1ŽA. and 2ŽB.x. However, frequency Žnot shown. of the spontaneous contractions was unaffected by 8-L-lysine-vasopressin Ž1.5= 10y9 ]1.2= 10y6 M.. On the other hand, neither the tone nor the spontaneous activity of the circular smooth muscle preparations isolated from the gastric corpus Žnot shown. were affected by 8-L-lysine-vasopressin Ž1.5 = 10y9 ]1.2= 10y6 M..

Effects of oxytocin on gastric corpus wall strips from non-pregnant cats

Oxytocin added into the organ bath Ž1.5 = 10y9 ]1.2= 10y6 M. did not affect the tone or the spontaneous activity of the isolated longitudinal and

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circular smooth muscle preparations Žnot shown. from the feline stomach corpus Ž n s 5..

Effects of 8- L -lysine-¨ asopressin and oxytocin on the gastric corpus wall strips from pregnant cats

Both 8-L-lysine-vasopressin Ž1.5 = 10y9 ]1.2 = 10 M. and oxytocin Ž1.5= 10y9 ]1.2= 10y6 M. had no significant effect on the tone Ž P) 0.05. as well as on the spontaneous contractions Žnot shown. of the isolated longitudinal and circular smooth muscle preparations of gastric corpus taken from pregnant cats Ž n s 4.. y6

Vasopressin receptor antagonists In the longitudinal smooth muscle strips both the predominantly selective vasopressin V1 receptor antagonist w d Ž CH 2 . 5 -O Ž M e . 2 -Tyr x -AVP Ž 1.9 = 10y8 ]1.7= 10y7 M. and the predominantly selective vasopressin V2 antagonist w dŽCH 2 .5 , D-Ile 2 , Ile 4 xAVP Ž1.5= 10y8 ]1.4= 10y7 M. produced significant shifts to the right in a concentration-dependent manner Ž P- 0.05 for both antagonists studied. of the concentration-response curve of tonic contractions for vasopressin wFig. 1ŽB. and ŽC., respectively, and Fig. 2x. The data from the experiments with vasopressin receptor antagonists were analysed as described by Arunlakshana and Schild w23x. Both antagonists yielded straight lines with similar slopes, but different pA 2 values ŽTable I.. The predominantly selective vasopressin V1 receptor blocker w dŽCH 2 . 5-O ŽMe. 2-Tyrx-AVP Ž1.9 = 10y8 ]1.7= 10y7 M. and the predominantly selective vasopressin V2 receptor blocker w dŽCH 2 .5 , D-Ile 2 , Ile 4 x-AVP Ž1.5= 10y8 ]1.4= 10y7 M. did not affect significantly Ž P) 0.05 for both antagonist studied. the increase in amplitude wFig. 1ŽB. and ŽC., respectively, and Fig. 2x and the frequency Žnot shown. of spontaneous activity of the concentration-response curves for vasopressin.

Non-specific receptor antagonists 3.5.1. Muscarinic receptor antagonists The effects of atropine Ž9.6= 10y1 0 ]9.6= 10y8 M., a non-selective antagonist of muscarinic M receptors, pirenzepine Ž1.6= 10y8 ]1.6= 10y6 M., a predominantly selective antagonist of muscarinic M 1 receptors, methoctramine Ž2.7= 10y8 ]2.7= 10y7 M., a predominantly selective antagonist of muscarinic M 2 receptors, and pFHHSiD Ž5.2= 10y8 ]5.2= 10y7 M., a predominantly selective antagonist of muscarinic M 3 receptors, were concentration-dependent and they induced a significant shift to the right Ž P- 0.05 for all antagonist studied. of the concentration-response curves for tonic contractions of vasopressin wFig. 1ŽD., Figs 3 and 4x. The data from the experiments with muscarinic receptor antagonists were analysed as described by Arunlakshana and Schild w23x. The experiments with atropine, pirenzepine,

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Fig. 1. Experimental recordings of ŽA. the changes in the activity of the longitudinal smooth muscle strips isolated from feline gastric corpus induced by 8-L-lysine-vasopressin Žconcentrations achieved in isolated organ bath at marked points: 1.5= 10y9 M, 9.0= 10y9 M, 3.2= 10y8 M, 6.2= 10y8 M, 1.2= 10y7 M and 2.1= 10y7 M, respectively.; ŽB. the antagonistic effect of V1 receptor antagonist w dŽCH 2 .5-O ŽMe. 2-Tyrx-AVP Ž5.8= 10y8 M.: left}control effect of 8-L-lysine-vasopressin Žconcentrations achieved in isolated organ bath at marked points: 1.5= 10y9 M, 9.0= 10y9 M, 3.2= 10y8 M, 6.2= 10y8 M, 1.2= 10y7 M and 2.1= 10y7 M, respectively.; right}effect of 8-L-lysine-vasopressin Žconcentrations achieved in isolated organ bath at marked points: 1.5= 10y9 M, 9.0= 10y9 M, 3.2= 10y8 M, 6.2= 10y8 M, 1.2= 10y7 M, 2.1= 10y7 M, 5.1= 10y7 M and 1.3= 10y6 M, respectively. in the presence of the V1 receptor antagonist; ŽC. the antagonistic effect of V2 antagonist w dŽCH 2 .5 , D-Ile 2 , Ile 4 x-AVP Ž4.7= 10y8 M.: left}control effect of 8-L-lysine-vasopressin Žconcentrations achieved in isolated organ bath at marked points: 1.5= 10y9 M, 9.0= 10y9 M, 3.2= 10y8 M, 6.2= 10y8 M, 1.2= 10y7 M and 2.1= 10y7 M, respectively.; right}effect of 8-L-lysine-vasopressin Žconcentrations achieved in isolated organ bath at marked points: 1.5= 10y9 M, 9.0= 10y9 M, 3.2= 10y8 M, 6.2= 10y8 M, 1.2= 10y7 M, 2.1= 10y7 M, 5.1= 10y7 M and 1.3= 10y6 M, respectively. in the presence of the V2 receptor antagonist; and ŽD. the antagonistic effect of atropine Ž9.6= 10y9 M.: left}control effect of 8-L-lysine-vasopressin Žconcentrations achieved in isolated organ bath at marked points: 1.5= 10y9 M, 9.0= 10y9 M, 3.2= 10y8 M, 6.2= 10y8 M, 1.2= 10y7 M and 2.1= 10y7 M, respectively.; right}effect of 8-L-lysine-vasopressin Žconcentrations achieved in isolated organ bath at marked points: 1.5= 10y9 M, 9.0= 10y9 M, 3.2= 10y8 M, 6.2= 10y8 M, 1.2= 10y7 M, 2.1= 10y7 M and 5.1= 10y7 M, respectively. in the presence of the atropine.

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Fig. 2. The effects of predominantly selective V1 receptor antagonist w dŽCH 2 .5-O ŽMe. 2-Tyrx-AVP and the predominantly selective vasopressin V2 antagonist w dŽCH 2 .5 , D-Ile 2 , Ile 4 x-AVP on tonic contractions and increase in amplitude of spontaneous contractions produced by vasopressin in longitudinal strips of feline gastric corpus. ŽA. Concentration-response curves of tonic contractions for vasopressin in absence ŽB. and presence of 1.9= 10y8 M Žv., 5.8= 10y8 M Ž'. and 1.7= 10y7 M Ž%. w dŽCH 2 .5-O ŽMe. 2-Tyrx-AVP. ŽB. Concentration-response curves of increase in amplitude for vasopressin in absence ŽB. and presence of 1.7= 10y7 M Žv. w dŽCH 2 .5-O ŽMe. 2-Tyrx-AVP. ŽC. Concentration-response curves of tonic contractions for vasopressin in absence ŽB. and presence of 1.5= 10y8 M Žv., 4.7= 10y8 M Ž'. and 1.4= 10y7 M Ž%. w dŽCH 2 .5 , D-Ile 2 , Ile 4 x-AVP. ŽD. Concentration-response curves of increase in amplitude for vasopressin in absence ŽB. and presence of 1.4= 10y7 M Žv. w dŽCH 2 .5 , D-Ile 2 , Ile 4 x-AVP. Each point represents the mean response Ž n s 6]12.. Confidence limits are avoided for the sake of clarity and do not exceed 15% of mean value for the each point. Responses are expressed as a percentage of the maximal tonic contraction or maximal increase in amplitude of spontaneous contractions produced by vasopressin in the same experiment.

methoctramine and pFHHSiD yielded straight lines Žfor all antagonists studied. with various slopes and pA 2 values. Similar pA 2 values were obtained for pirenzepine, methoctramine and pFHHSiD ŽTable I.. However, the pA 2 value of atropine, a non-selective muscarinic receptor antagonist, differed Ž P-

0.05. from those of the muscarinic receptor subtypes antagonists ŽTable I.. Atropine Ž9.6 = 10y1 0 ]9.6 = 10y8 M., but not pirenzepine Ž1.6= 10y6 M., methoctramine Ž2.7= 10y7 M. and pFHHSiD Ž5.2= 10y7 M., induced significant shift to the right in a concentration-depen-

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Table I PA 2 values and Schild regression slopes of the antagonists used in the study. The results are expressed as means" 95% confidence limits Type of the response Tonic contractions Tonic contractions Tonic contractions Tonic contractions Tonic contractions Tonic contractions Increase in amplitude

Antagonist

pA2

Slope

w dŽCH2 .5-O ŽMe. -Tyrx-AVP w dŽCH2 .5 , D-Ile2 , Ile4 x-AVP Atropine Pirenzepine Methoctramine pFHHSiD Atropine

8.84" 0.01 6.30" 0.25 9.83" 0.69 7.58" 0.12 7.79" 0.96 7.37" 0.58 10.35" 1.03

0.86" 0.08 0.83" 0.06 0.88" 0.11 0.82" 0.06 1.13" 0.13 1.11" 0.15 0.98" 0.21

2

dent manner Ž P- 0.05. of the increase in amplitudes of spontaneous contractions elicited by vasopressin wFig. 1ŽD., Figs 3 and 4x. The slopes of the Schild regressions for tonic contractions and for increase in amplitudes were not significantly different from unity ŽTable I.. The pA 2 values were not significantly different for tonic contractions and the increase in amplitudes ŽTable I.. A calcium channel blocker The calcium channel blocker, nifedipine Ž1.9= 10y7 M. shifted to the right and depressed significantly Ž P- 0.01. the maximum response of concentration-response curves for tonic contractions and for the increase in amplitudes of the longitudinal muscle strips induced by vasopressin ŽFig. 5.. A ganglion blocking agent The ganglion blocking agent, mecamylamine Ž1.6= 10y5 M. did not significantly affect the concentration-response curves for tonic contractions and the increase in amplitudes of the longitudinal muscle strips produced by vasopressin Žnot shown..

An acetylcholinesterase inhibitor The acetylcholinesterase inhibitor, physostigmine Ž1.6= 10y8 M. significantly shifted to the left the concentration-response curves for tonic contractions and the increase in amplitudes of spontaneous activity of longitudinal muscle strips caused by vasopressin ŽFig. 5.. At the same time, physostigmine enhanced the maximum responses of tonic contractions and the increase in amplitudes of concentration-response curves for vasopressin ŽFig. 5..

DISCUSSION The results of these experiments confirm and extend our previous observations w15x that vasopressin, but not oxytocin, produced concentration-dependent tonic contractions only on longitudinal muscle strips isolated from feline gastric corpus. Concentrations of vasopressin eliciting the contractions were similar to concentrations necessary for contracting isolated blood vessels in ¨ itro. At the same time vasopressin increased the amplitudes, but not the frequency, of

spontaneous contractions. Selective effects of neuropeptides and acetylcholine mediating contraction of the longitudinal of circular muscle layers in the intestine of various species have already been described. For instance, the opioid peptides met-enkephalin, leu-enkephalin, beta-endorphin and dynorphin contracted the circular muscles but none had any affect on the longitudinal muscle layer of the guinea-pig and human jejunum. On the other hand, acetylcholine, the octapeptide of cholecystokinin and substance P were capable of mediating contraction on both longitudinal and circular muscle layers of the guinea-pig and human jejunum w26x. It follows, therefore, that the selective effects of neuropeptides obtained in the present and previous experiments w26x reflects, most probably, the functional integration of longitudinal and circular muscles in the complex regulation of motor activity of various parts of gastrointestinal tract. The present experiments further revealed that neither vasopressin not oxytocin affected the contractions of the gastric corpus wall strips isolated from the pregnant cats. It is well known that oxytocin receptors are up-regulated by estrogens and down-regulated by progesterone in many tissues, while vasopressin receptors are much less regulated by steroid hormones w27x. Moreover, it has been suggested that the anatomical and histological changes in the uterine artery during pregnancy are estrogen-dependent w28x, and that estrogens reduced contractions of human myometrial arteries in response to vasopressin w29x. If this had been the mechanism of failure of vasopressin to contract the longitudinal muscle layer of the pregnant feline gastric corpus, this would also support the concept that estrogens, apart from oxytocin, affect the contractile effect of vasopressin as well. It is well known that vasopressin induces contraction of vascular smooth muscle w30]33x and myometrium w34, 27x through vasopressin V1 receptor activation. However, there is evidence that the vasocontractile effect of vasopressin is also mediated by vasopressin V2 receptors w35x. The findings of the present experiments showed that both the vasopressin V1 receptor-preferring antagonist dŽCH 2 .5O ŽMe. 2-Tyr-AVP w36x and the predominantly vaso-

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Fig. 3. The effects of atropine and pirenzepine on tonic contractions and increase of amplitude of spontaneous contractions produced by vasopressin in longitudinal gastric strips. ŽA. Concentration-response curves of tonic contractions for vasopressin in the absence ŽB. and presence of 9.6= 10y10 M Žv., 9.6= 10y9 M Ž'. and 9.6= 10y8 M Ž%. atropine. ŽB. Concentration-response curves of increase in amplitude for vasopressin in the absence ŽB. and presence of 9.6= 10y10 M Žv., 9.6= 10y9 M Ž'. and 9.6= 10y8 M Ž%. atropine. ŽC. Concentration-response curves of tonic contractions for vasopressin in the absence ŽB. and presence of 1.6= 10y8 M Žv., 1.6= 10y7 M Ž'. and 1.6= 10y6 M Ž%. pirenzepine. ŽD. Concentration-response curves of increase in amplitude for vasopressin in the absence ŽB. and presence of 1.6= 10y6 M Žv. pirenzepine. Each point represents the mean response Ž n s 6]12.. Confidence limits are avoided for the sake of clarity and do not exceed 15% of mean value for the each point. Responses are expressed as a percentage of the maximal tonic contraction or maximal increase in amplitude of spontaneous contractions produced by vasopressin in the same experiment.

pressin V2 antagonist dŽCH 2 .5 , D-Ile 2 , Ile 4-AVP w37x inhibited the tonic contraction, but not the amplitudes of spontaneous contractions of the longitudinal muscle layers of feline gastric corpus caused by vasopressin. The pA 2 value for the dŽCH 2 .5 , D-Ile 2 , Ile 4-AVP obtained in our study Ž6.30" 0.25. was

significantly lower than the value reported for vasopressin V2 receptors Ž8.12" 0.12. w37]39x and correspond to the value obtained for V1 subtypes of vasopressin receptors Ž6.65" 0.25. w38, 40x. The pA 2 value obtained in the present experiments Ž8.84" 0.15. for dŽCH 2 .5-O ŽMe. 2-Tyr-AVP was somewhat

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Fig. 4. The effects of predominantly selective muscarinic M 2 receptor antagonist methoctramine and the predominantly selective muscarinic M 3 antagonist pFHHSiD on tonic contractions and increase in amplitude of spontaneous contractions produced by vasopressin in longitudinal strips of feline gastric corpus. ŽA. Concentration-response curves of tonic contractions for vasopressin in absence ŽB. and presence of 2.7= 10y8 M Žv., 9.0= 10y8 M Ž'. and 2.7= 10y7 M Ž%. methoctramine. ŽB. Concentration-response curves of increase in amplitude for vasopressin in absence ŽB. and presence of 2.7= 10y7 M Žv. methoctramine. ŽC. Concentration-response curves of tonic contractions for vasopressin in absence ŽB. and presence of 5.2= 10y8 M Žv., 1.7= 10y7 M Ž'. and 5.2= 10y7 M Ž%. pFHHSiD. ŽD. Concentration-response curves of increase in amplitude for vasopressin in absence ŽB. and presence of 5.2= 10y7 M Žv. pFHHSiD. Each point represents the mean response Ž n s 6]12.. Confidence limits are avoided for the sake of clarity and do not exceed 15% of mean value for the each point. Responses are expressed as a percentage of the maximal tonic contraction or maximal increase in amplitude of spontaneous contractions produced by vasopressin in the same experiment.

lower than the values obtained for vasopressin V1 receptors in canine femoral artery ŽpA 2 s 9.5. w41x, rabbit submucosal arterioles Ž10.15" 0.15. w42x, and in human platelets ŽpA 2 s 9.21. w43x, but it was similar to the values obtained for vasopressin V1

receptors in guinea-pig and human submucosal arterioles Ž8.75" 0.25. w42x and in human and guinea-pig uterine arteries Ž8.55" 0.12. w44x. The reason for this variation on d Ž CH 2 . 5 -O ŽMe . 2 -Tyr-AVP probably excludes the role of V1b vasopressin recep-

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Fig. 5. The effects of acetylcholinesterase inhibitor physostigmine and calcium channel blocker nifedipine on tonic contractions and increase in amplitude of spontaneous contractions produced by vasopressin in longitudinal strips of feline gastric corpus. ŽA. Concentration-response curves of tonic contractions for vasopressin in absence ŽB. and presence of 1.6= 10y8 M Žv. physostigmine. ŽB. Concentration-response curves of increase in amplitude for vasopressin in absence ŽB. and presence of 1.6= 10y8 M Žv. physostigmine. ŽC. Concentration-response curves of tonic contractions for vasopressin in absence ŽB. and presence of 1.9= 10y7 M Žv. nifedipine. ŽD. Concentration-response curves of increase in amplitude for vasopressin in absence ŽB. and presence of 1.9= 10y7 M Žv. nifedipine. Each point represents the mean response Ž n s 6]12.. Confidence limits are avoided for the sake of clarity and do not exceed 15% of mean value for the each point. Responses are expressed as a percentage of the maximal tonic contraction or maximal increase in amplitude of spontaneous contractions produced by vasopressin in the same experiment.

tors, and in addition with the failure of oxytocin to provoke any changes in feline gastric corpus motility, suggests that vasopressin V1a receptor subtype mediated its contractile effect. The failure of the vasopressin antagonists used in our study to affect the increase in amplitude of

spontaneous contractions produced by vasopressin itself suggests less affinity of vasopressin antagonists used in the study for population of vasopressin receptors involved in control of spontaneous contractions. Whether this means that different subtype of vasopressin receptors is involved in control of spon-

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taneous activity or not could be answered only after testing a variety of type- and subtype-selective vasopressin antagonists. The muscarinic M 1 , M 2 and M 3 receptor subtypes are present in the gastrointestinal tract w45]50x. The M 1 and M 2 receptor subtypes are mainly localised in the nervous elements, while the M 2 and M 3 receptor subtypes in the smooth muscle w45]47x. In the present experiments both non-selective and predominantly selective muscarinic receptor antagonists depressed and blocked the tonic contractions of feline gastric corpus caused by vasopressin. It is, therefore, reasonable to suppose that vasopressin releases acetylcholine, whereas the muscarinic receptor antagonists in turn block the contractile effect of acetylcholine. The non-selective muscarinic antagonist, atropine was the most potent. Of predominantly selective muscarinic antagonist the most active were the muscarinic M 1 receptor subtype antagonists. This finding points to the nervous component of vasopressin action. The nervous elements activated by vasopressin should be post-ganglionic Žor ‘end’. since the ganglion blocking agent, mecamylamine did not affect the tonic contractions induced by vasopressin. This interpretation strengthens the potentiation of tonic contractions of vasopressin by the acetylcholinesterase inhibitor physostigmine. Since it is believed that muscarinic M 2 receptors inhibit the transmitter release w45, 46x, then the contractile response of vasopressin most likely is mediated through other muscarinic receptor subtypes localised in the smooth muscle. For instance, in the upper gastrointestinal tract smooth muscles muscarinic M 2 and M 3 receptor subtypes have been found w47x. It could be, therefore, supposed that vasopressin may release acetylcholine from nerve endings which in turn contracts the longitudinal smooth muscle of gastric corpus. However, the direct smooth muscle contracting action of vasopressin can not be excluded. An interesting finding of the present experiments is that of all used antagonists only the non-selective muscarinic receptor antagonist, atropine, blocked the increase in amplitudes of spontaneous contractions caused by vasopressin. On the other hand, the predominantly selective M 1 muscarinic antagonist, pirenzepine, M 2 muscarinic antagonist methoctramine as well as M 3 muscarinic antagonist pFHHSiD failed to block the potentiation of spontaneous contractions produced by vasopressin. As already pointed out, both the non-selective and the predominantly selective muscarinic receptor antagonists depressed or blocked the tonic contractions caused by vasopressin. It appears, therefore, that the tonic contractions and the spontaneous contractions produced by released acetylcholine are mediated through muscarinic receptors coupled with different transduction systems. There were no significant differences between pA 2 values of atropine for tonic

ŽpA 2 s 9.83" 0.69. and spontaneous Ž10.35" 1.03. contractions. The pA 2 values for the predominantly selective M 1 , M 2 and M 3 muscarinic receptor antagonists were significantly lower than those for atropine. Thus, the existence of a muscarinic receptor population coupled to different transduction system and controlling spontaneous contractions seems likely. It has been postulated that muscarinic receptor activation can regulate one post-synaptic transduction mechanisms within the same tissue w51]53x. However, it is not yet known with certainty whether one muscarinic receptor is linked to more than one transduction mechanisms or various transduction mechanisms are coupled to different muscarinic receptor subtypes in a distinct tissue. In the circular muscle of guinea-pig stomach, for example, the different transduction mechanisms have different sensitivities to the antagonists and a muscarinic M 3 receptor activates three transduction mechanisms with different efficacies w53x. In the present experiments it was observed that atropine, a non-selective muscarinic receptor antagonist, is much more potent than the predominantly selective muscarinic receptor subtype antagonists. In addition, of all muscarinic receptor antagonists used, only atropine blocked the amplitudes of spontaneous contractions. Thus, the results of our study suggest that muscarinic receptors involved in control of spontaneous contractions link to separate transduction mechanism. In support of this interpretation are the observations of Ashkenazi et al. w26x and Parekh and Brading w53x that muscarinic M 2 and M 3 receptor subtypes link to several different transduction mechanisms. Furthermore, in the presence of nifedipine both the tonic contractions and contractions of spontaneous activity were inhibited. This might suggests that the muscarinic receptors in longitudinal muscle of feline stomach are coupled by transduction mechanisms to voltage-gated Žnifedipine-sensitive. Ca2q channels. On the contrary, in circular muscle of the guinea-pig gastric fundus a nifedipine-insensitive calcium influx operates w54x.

ACKNOWLEDGEMENTS This work was partially financed by Ministry of Science and Technology of Republic of Serbia. The authors also wish to thank to Prof. Dr Carlo Melchiorre, who kindly supplied them with methoctramine, free of charge.

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