Nitric oxide induces acetylcholine-mediated contractions in the guinea-pig small intestine

In longitudinal muscle/myenteric plexus preparations of the guinea-pig ileum, exogenous nitric oxide (NO) induced concentration-dependent relaxations. In tissues at basal tone, NO (3 × 10^–6 M) induced a moderate relaxation followed by a pronounced contraction, consisting of a quick and sustained component. Tetrodotoxin (5 × 10^–7 M) abolished both phases of the contraction. Atropine (5 × 10^–7 M) abolished the quick component and reduced the sustained component of the contraction; the latter was further suppressed by the selective NK₁ receptor antagonist CP 96,345. Hexamethonium (5 × 10^–5 M) failed to affect the contractile response to NO. It is concluded that administration of exogenous NO in the guinea-pig ileum can lead to activation cholinergic and to a lesser degree tachykininergic neurones. Correspondence to: L. Barthó at the above address     

Increase by NO synthase inhibitors of acetylcholine release from guinea-pig myenteric plexus

The effects of nitric oxide (NO) synthase inhibitors on the electrically evoked release of [³H]acetylcho-line were studied in guinea-pig myenteric plexus preparations preincubated with [³H]choline. N^G-monomethyl-l-arginine (EC₅₀ 5.3 mol l^–1) and N^G-nitro-l-arginine (EC₅₀ 1.3 mol l^–1) concentration-dependently increased the evoked release of [³H]acetylcholine without affecting the basal outflow. The facilitatory effect of N^G-monomethyl-l-arginine was prevented by l-arginine but not by d-arginine. The results suggest that endogenous NO inhibits the depolarisation-evoked release of acetylcholine. Correspondence to: H. Kilbinger at the above address     

Evidence that the contractile response of the guinea-pig ileum to capsaicin is due to substance P release

Summary The mechanism of action of capsaicin on the guinea-pig isolated ileum was studied. Capsaicin (1.5×10^–6mol l^–1) produced a large contractile response of the ileum which exhibited marked tachyphylaxis. The response was reduced by treatment of preparations with atropine, 5×10^–6mol l^–1, and by substance P-autodesensitization, and was abolished by a combination of these treatments. It was concluded that capsaicin released both substance P and acetylcholine from the guinea-pig ileum. Evidence for a cholinergic component in the action of high concentrations of substance P was also obtained and therefore it is proposed that capsaicin releases substance P from neurones in the ileum, and the released substance P in turn stimulates cholinergic neurones to release acetylcholine, as well as producing direct muscle contraction.     

Differential role of tachykinin NK3 receptors on cholinergic excitatory neurotransmission in the mouse stomach and small intestine

Background and purpose:

Tachykinin NK₃ receptors are widely expressed in the mouse gastrointestinal tract but their functional role in enteric neuromuscular transmission remains unstudied in this species. We investigated the involvement of NK₃ receptors in cholinergic neurotransmission in the mouse stomach and small intestine.

Experimental approach:

Muscle strips of the mouse gastric fundus and ileum were mounted in organ baths for tension recordings. Effects of NK₃ agonists and antagonists were studied on contractions to EFS of enteric nerves and to carbachol.

Key results:

EFS induced frequency-dependent tetrodotoxin-sensitive contractions, which were abolished by atropine. The cholinergic contractions to EFS in the stomach were enhanced by the NK₃ antagonist SR142801, but not affected by the NK₃ agonist senktide or neurokinin B. The cholinergic contractions to EFS in the small intestine were not affected by SR142801, but dose-dependently inhibited by senktide and neurokinin B. This inhibitory effect was prevented by SR142801 but not by hexamethonium. SR142801, senktide or neurokinin B did not induce any response per se in the stomach and small intestine and did not affect contractions to carbachol.

Conclusions and implications:

NK₃ receptors modulate cholinergic neurotransmission differently in the mouse stomach and small intestine. Blockade of NK₃ receptors enhanced cholinergic transmission in the stomach but not in the intestine. Activation of NK₃ receptors inhibited cholinergic transmission in the small intestine but not in the stomach. This indicates a physiological role for NK₃ receptors in mouse stomach contractility and a pathophysiological role in mouse intestinal contractility.     

Prejunctional M1 and postjunctional M3 muscarinic receptors in the circular muscle of the guinea-pig ileum

The effects of subtype-selective muscarinic receptor antagonists on electrically evoked release of acetylcholine and muscle contraction were compared in circular muscle preparations of the guinea-pig ileum. Incubation of the preparation with [³H]choline resulted in the formation of [³H]acetylcholine. Electrical stimulation caused the release of [³H]acetylcholine which was abolished by tetrodotoxin and omission of calcium from the medium. 5-Hydroxytryptamine (10 M) and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (300 M) did not change acetylcholine release. The muscarinic antagonists pirenzepine (M1 selective), AF-DX 116 (M2 selective) and hexahydrosiladifenidol (M3 selective) caused concentration-dependent increases in the evoked release of acetylcholine, and inhibitions of the circular muscle contraction. The postjunctional affinity constants (pA₂ values) obtained for hexahydrosiladifenidol (8.06), pirenzepine (6.95) and AF-DX 116 (6.60) identified the muscular receptor as an M3 subtype. Pirenzepine was more potent in facilitating the evoked release than hexahydrosiladifenidol and AF-DX 116. These findings suggest that the release of acetylcholine in the circular muscle is inhibited by M1 muscarinic autoreceptors whereas muscle contraction is mediated by M3 receptors.     

Sensitization of the smooth muscle by prostaglandin E1 contributes to reversal of drug-induced inhibition of the guinea-pig ileum

Summary Prostaglandin (PG)E₁ reverses drug-induced inhibition of the electrically stimulated guineapig ileum. The effect of PGE₁ is in part related to a sensitizing action on the smooth muscle and in part to an increased acetylcholine release from the myenteric plexus.     

NK1- and NK3-receptor mediated inhibition of 5-hydroxytryptamine release from the vascularly perfused small intestine of the guinea-pig

The effects of tachykinins on the spontaneous release of 5-hydroxytryptamine (5-HT) from the enterochromaffin cells into the portal circulation was investigated in vitro using the vascularly perfused isolated guinea-pig small intestine. 5-HT was determined by HPLC with electrochemical detection. Test substances were applied intraarterially. Substance P (SP) caused a concentration-dependent decrease in 5-HT outflow with an EC50 of 50pmol/l. Similarly, the selective NK1 receptor agonist SP methyl ester (1nmol/l) significantly inhibited 5-HT outflow (to 51±3%). When tetrodotoxin (1μmol/l) was added to the arterial perfusion medium, the inhibition by SP of 5-HT outflow was not affected. The selective NK1 receptor antagonist CP 99994 [(+)-(2S,3S)-3-(2-methoxyben-zylamino)-2-phenylpiperidine] (0.1μmol/l) prevented the inhibitory effect of SP (0.1μmol/l). Neither GR 94800 (PhCO-Ala-Ala-DTrp-Phe-DPro-Pro-NleNH₂) (0.1μmol/l) nor SR 142801 [(S)-(N)-(1-(3-(1-benzoyl-3-(3, 4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenyl-piperidin-4-yl)-N-methylacetamide] (10nmol/l), which are selective NK2 and NK3 receptor antagonists, changed the SP-mediated inhibition. The selektive NK3 receptor agonist senktide (10nmol/l) also decreased the 5-HT outflow (to 57±5%). This inhibition was prevented by SR 142801 (10nmol/l) and by tetrodotoxin. CP 99994 (0.1μmol/l) significantly antagonized the senktide-mediated inhibition of 5-HT outflow. The outflow of 5-HT was unaffected when CP 99994, GR 94800 or SR 142801 alone were added to the perfusion medium. It is concluded that the release of 5-HT from enterochromaffin cells is directly inhibited by NK1 receptors, and indirectly by neuronal NK3 receptors whose stimulation leads to the release of SP. Received: 11 June 1997 / Accepted: 28 July 1997     

Endomorphin-1 and endomorphin-2 activate µ-opioid receptors in myenteric neurons of the guinea-pig small intestine

The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine and human brain bind with high affinity and selectivity to central μ-opioid receptors. In the digestive tract, a comprehensive pharmacological analysis of the receptors involved in endomorphin action has not been reported. In this study, we analyzed the effects of endomorphin-1 and endomorphin-2 on longitudinal muscle-myenteric plexus preparations (LMMPs) from the guinea-pig ileum. Both peptides (30 pM–1 μM) inhibited (–log EC₅₀ values: 8.61 and 8.59, respectively) the amplitude of electrically-induced twitch contractions in a concentration-dependent fashion, up to its abolition. Conversely, in unstimulated LMMPs, they failed to affect contractions to applied acetylcholine (100 nM). In stimulated LMMPs, the highly selective μ-opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP), caused a concentration-dependent (30 nM–1 μM), parallel rightward shift of endomorphin-1 and endomorphin-2 inhibitory curves, without depression of their maximum. Following Schild analysis, calculated pA ₂ values were 7.81 and 7.85, respectively, with slopes not different from unity. Concentration-response curves to both peptides were not affected by 30 nM naltrindole (a selective δ-receptor antagonist) or 30 nM nor-binaltorphimine (a selective κ-receptor antagonist). These results demonstrate that endomorphins selectively activate μ-opioid receptors located on excitatory myenteric plexus neurons, and that they act as full agonists. Received: 6 August 1998 / Accepted: 5 October 1998     

Effects of cromakalim on acetylcholine release and smooth muscle contraction in guinea-pig small intestine

Summary The effects of the potassium channel opener cromakalim on smooth muscle contraction and ³H-acetylcholine release were studied simultaneously in guinea-pig longitudinal muscle myenteric plexus preparations which had been preincubated with ³H-choline. Cromakalim (10 mol/1) inhibited more markedly the smooth muscle contractions caused by the release of endogenous acetylcholine (via electrical stimulation or via activation of nicotine- and 5-HT₃-receptors) than contractions induced by pilocarpine. Cromakalim (10 ~mol/1) did not affect the release of ³Hacetylcholine evoked by electrical stimulation or by stimulation of nicotine- and 5-HT₃-receptors. In contrast, the release of ³H-acetylcholine caused by stimulation of M₁-receptors was concentration-dependently reduced by cromakalim (1–100 gmol/1). The results suggest that the relaxant effect of cromakalim on smooth muscle contraction is not caused by a reduction of acetylcholine release from myenteric neurones. An opening of cromakalim-sensitive potassium channels may be involved in the inhibition of the M₁-receptor mediated acetylcholine release.This work was supported by the Deutsche Forschungsgemeinschaft (Ki 210/6-3). Send offprint requests to H. Schwörer at the above address     

Indirect evidence for the inhibition of enteric substance P neurones by opiate agonists but not by capsaicin

There is good evidence indicating that hyoscine-resistant contractions of the guinea-pig ileum evoked by stimulation of the intramural nerves are mediated by substance P (SP). In the present experiments, non-cholinergic neurogenic ileum contractions to field stimulation (100 imp., 5-50 Hz) were inhibited by the opiate agonists morphine and [D-Met2,Pro5]enkephalinamide (10(-6) M) in a naloxone-reversible manner. Neither morphine nor naloxone influenced the musculodirect contracting effect of histamine. Capsaicin, a drug that has been shown to deplete SP from primary afferent neurones, exerted no long-lasting effect on non-cholinergic contractions to field stimulation. Repeated administration of long trains of stimuli (900 impulses) resulted in a progressive decrease of the contractions evoked. Addition of naloxone (3 X 10(-7) M) restored the original height of the responses. The above inhibitory action of opiate agonists and of repeated long-train stimulation was 3-6 times greater at 5 Hz than at 50 Hz. It is concluded that opiate agonists inhibit the release of SP from intramural neurones of the guinea-pit ileum. The decrease in responses to repeated long-train stimulations is mediated, at least in part, by the release of endogenous opioid substance(s).     

Morphine leads to contraction of the ileal circular muscle via inhibition of the nitrergic pathway in mice

Morphine inhibits small intestinal transit in mice, although few mu-opioid receptors are present in the ileum. The present study focused on the action of morphine in the isolated mouse ileum to reveal the mechanism by which morphine inhibits mouse small intestinal transit. In the isolated circular muscle, morphine caused tonic contraction. This contraction was potently inhibited by naloxone and the mu-opioid receptor antagonist cyprodime. Moreover, the response was almost completely inhibited by tetrodotoxin and N(G)-nitro-L-arginine, but only moderately inhibited by atropine and indomethacin. In the isolated longitudinal muscle, morphine caused no or only slight contractions. Furthermore, electrically induced contraction was dose-dependently depressed by morphine, an effect that was not reversed by naloxone. These findings indicate that 1) morphine-induced circular muscle contraction occurs in the mouse ileum, 2) the contraction occurs through mu-opioid receptors mainly by inhibiting the release of nitric oxide from nitrergic nerves, although cholinergic nerves are at least partly involved in this contractile mechanism, and 3) inhibition of descending relaxation of peristalsis by morphine may slow small intestinal transit.     

The effects of metoclopramide on acetylcholine release and on smooth muscle response in the isolated guinea-pig ileum

Summary The effects of metoclopramide on smooth muscle contraction and on release of acetylcholine were studied in the guinea-pig myenteric plexus longitudinal muscle preparation. Acetylcholine was determined either as endogenous acetylcholine, or as labelled transmitter from strips preloaded with ³H-choline.Metoclopramide caused an increase in resting tension of longitudinal muscle as well as an increase in resting output of either endogenous or labelled acetylcholine. Tetrodotoxin abolished the metoclopramide-evoked increase in transmitter release. The increase in smooth muscle tension was clearly related to the increase in resting output. The effects of metoclopramide on both longitudinal muscle contraction and resting release of labelled acetylcholine were prevented in the presence of a concentration of 5-hydroxytryptamine (5-HT) that desensitized 5-HT receptors. This suggests that metoclopramide stimulates neuronal 5-HT receptors and, thereby, facilitates acetylcholine release.Metoclopramide augmented the twitch-like contractions induced by field stimulation at 0.1 Hz. Contractions elicited at 1 Hz were only slightly enhanced. Similarly, metoclopramide facilitated only the release of labelled acetylcholine evoked by electrical stimulation at 0.1 Hz, but not that at 1 Hz. The facilitatory effetts of metoclopramide on twitch height and evoked release could not be attributed to a blockade of presynaptic inhibitory -adrenoceptors, dopamine or muscarine receptors.     

Target sites for the inhibition of prostacyclin effect in guinea-pig ileum

Summary In the guinea-pig terminal ileum a maximally effective concentration of prostacyclin (PGI₂) (1 ol/l) induced contractions that were partially resistant to tetrodotoxin (TTX) 0.1 mol/l, to low temperature (20°C) and to atropine (30 nmol/l). Half maximum contractions evoked by PGI₂ (20 nmol/l) were abolished by TTX and by low temperature, which did not modify the response to exogenous acetylcholine (ACh), as well as by atropine. Procaine (5–500 ol/l) caused a concentration-dependent inhibition of contractions induced by PGI₂ (20 nmol/l and 1 mol/l) and by equieffective concentrations of ACh (20 nmol/l and 0.4 ol/l, respectively). The order of magnitude for this inhibition was ACh 20 nmol/l = PGI₂ 20 nmol/l > PGI₂1 mol/l > ACh 0.4 mol/l. In preparations exposed to TTX or to low temperature procaine (50 mol/l) did not affect the residual response to PGI₂ (1 mol/l). Quercetin (1 and 5 ol/l) inhibited the effect of PGI₂ and, at higher concentrations, it also caused partial depression of the responses to ACh. Quercetin did not alter TTX-resistant and low temperature-resistant contractions induced by PGI₂ 1 mol/l. Carbonyl cyanide-trifluoromethoxyphenyl hydrazone (FCCP) (0.1–1 ol/l) reduced the effect of PGI₂ and of ACh to approximately the same extent and inhibited the residual response to PGI₂ 1 mol/l in preparations treated with TTX or expressed to low temperature. The present results show that PGI₂, besides acting on cholinergic neurons, also exerts a direct effect on smooth muscle cells and FCCP can be used to block this effect. In contrast procaine and quercetin selectively inhibit the ACh-mediated component of PGI₂ action. Send offprint requests to R. M. Gaion     

Opioid mu and kappa receptors on axons of cholinergic excitatory motor neurons supplying the circular muscle of guinea-pig ileum

Summary In preparations of guinea-pig ileum comprising the circular muscle and the axonal processes of myenteric neurons, electrical stimulation evoked contractions of the circular muscle which were abolished by tetrodotoxin and by hyoscine, indicating that they resulted from action potential-mediated release of acetylcholine. The selective mu opioid agonist, (d-Ala²-N-Me-Phe⁴-Gly⁵-ol)-enkephalin (DAGO), and the selective kappa opioid agonist, trans-(±)-3,4-dichloro-N-(2-(I-pyrrolidinyl) cyclohexyl) benzeneacetamide, U-50488H, caused concentration-dependent and naloxone-reversible inhibitions of nerve-mediated contractions. The experiments indicate that opioid mu and kappa receptors are present on the axonal processes of cholinergic excitatory motor neurons supplying the circular muscle of the guinea-pig ileum.Send offprint requests to S. Johnson at the above address     

Muscarinic inhibition of acetylcholine release from a novel in vitro preparation of the guinea-pig trachea

Summary An isolated preparation of the guinea-pig trachea is described which allows the simultaneous measurement of acetylcholine release and smooth muscle contraction. Incubation of the epithelium-free preparation with [³H]choline resulted in the formation of [³H]acetylcholine. Electrical stimulation caused the release of [³H]acetylcholine and a contractile response. Tetrodotoxin and omission of calcium from the medium abolished both the evoked release and contractions.The muscarinic agonists oxotremorine, carbachol and pilocarpine concentration-dependently inhibited the electrically evoked acetylcholine release and contracted the tracheal smooth muscle. Pre- and postsynaptic EC₅₀ values for a given agonist were not different. Atropine (100 nmol/l) significantly faciliated the evoked acetylcholine release. A concentration of 10 nmol/l atropine did not change the evoked release but antagonized the inhibitory effect of oxotremorine. It is concluded that presynaptic muscarine autoreceptors inhibit the release of acetylcholine from parasympathetic nerves of the guinea-pig trachea.Send offprint requests to G. D'Agostino at the above address     

The biphasic response of the isolated guinea-pig ileum by bradykinin

The isolated guinea-pig ileum, challenged by agonist, was used to study the effect of bradykinin. In the presence of acetylcholine producing approximately 60% of maximum contraction, bradykinin caused relaxation followed by contraction. The biphasic response to bradykinin was also found in the presence of histamine, eledoisin, angiotensin, prostaglandin F_2α and transmural electrical stimulation. The conditions for bradykinin-induced relaxation were not found after treatment by bradykinin, and potassium or barium chloride. Under conditions where bradykinin produced a biphasic response, acetylcholine, histamine, eledoisin, angiotensin, prostaglandin F_2α and lysine-vasopressin only contracted the ileum, while adrenaline, noradrenaline, oxytocin, calcium and magnesium chloride only relaxed. On increasing the percentage of maximum contraction with acetylcholine, an inverse relationship with relaxation by bradykinin was found. Tachyphylaxis was not present with the bradykinin-induced relaxation. The relaxing effect of bradykinin is more likely to be due to a direct action on the muscle cell membrane than to a release of a mediator or to blockade of a receptor mediating contraction.     

D-Met2, Pro5]enkephalinamide and dynorphin-(1-13) inhibit the cholinergic contraction induced in the guinea-pig ileum by substance P

In the isolated guinea-pig ileum, the sustained phase of the longitudinal contractile response to substance P is, unlike the initial peak response, mediated by stimulation of cholinergic neurons. This cholinergically mediated response to substance P was inhibited by the specific substance P antagonist, [D-Pro2,D-Trp7,9]substance P, which suggests that substance P stimulates enteric cholinergic neurons through a specific site of action. [D-Met2,Pro5]enkephalinamide and dynorphin-(1-13) also decreased whereas naloxone increased the sustained response to substance P. The results indicate that the enteric cholinergic neurons, which are stimulated by substance P, are also under the control of enkephalin and/or dynorphin neurons.     

Effect of SR 142801 on nitric oxide-dependent and independent responses to tachykinin NK3 receptor agonists in isolated guinea-pig colon

We have determined the ability of the novel nonpeptide tachykinin (TK) NK₃ receptor antagonist, SR 142801, [(S) -(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide] in inhibiting the nitric oxide (NO)-independent prejunctional inhibition of cholinergic twitches and the NO-dependent relaxation produced by the NK₃ receptor selective agonist, senktide, in the circular muscle of the guinea-pig proximal colon. Under moderate load (10 mN) and isometric recording of mechanical activity, single pulse electrical field stimulation (EFS) produced atropine- and tetrodotoxin-sensitive twitch contractions of mucosa-free circular muscle strips from the guinea-pig proximal colon. In the presence of NK₁ and NK₂ receptor antagonists (SR 140333 0.01 M and GR 94800 0.1 M, respectively) the NK₃ receptor selective agonist, senktide (EC₅₀ 33 pM) and the NK₃ receptor preferring natural TK, neurokinin B (NKB, EC₅₀ 13 pM) produced a concentration-dependent slowly developing inhibition of cholinergic twitches. Senktide (1 nM) did not affect the contractile response to acetylcholine (1 gM) indicating that depression of evoked twitches occurs prejunctionally. The inhibitory effect of senktide was 'unaffected when evoked in the presence of the cyclooxygenase inhibitor (S)-ketoprofen (10 M), guanethidine (10 M), naloxone (0.3 M), the GABA_B receptor antagonist 2-hydroxysaclofen (10 M) or the combined application of the adenosine A₁ and A₂ receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (10 M) and 3,7-dimethyl-l-propargylxanthine (30 M) respectively.In the presence of NK₁ and NK₂ receptor antagonists, the NO-synthase inhibitor l-nitroarginine (L-NOARG 30-100 M) did not affect twitch inhibition induced by senktide (EC₅₀ 33 pM). The response to NKB (EC₅₀ 95 pM) was slightly reduced by L-NOARG, yet the bulk of the inhibitory effect of both agonists on cholinergic twitches was substantially independent of NO generation. SR 142801 (0.1–0.3 M) produced a moderate rightward shift of the concentration-response curve to senktide without depression of the Emax to the agonist, yielding an apparent pK_B value of 7.65.Under low resting tone (3 mN) and isotonic recording of mechanical activity, mucosa-free circular muscle strips from the guinea-pig proximal colon gained a high intrinsic tone suitable for testing the response to relaxant agents. In the presence of atropine (1 M), guanethidine (3 M), SR 140333 (0.01 [M) and GR 94800 (0.1 LM), senktide (EC₅₀ 50 pM) produced a concentration-dependent relaxation of the strips, which was blocked by L-NOARG. SR 142801 (0.01–0.1 M) produced a large rightward shift of the L-NOARG-sensitive concentration-response curve to senktide yielding an apparent pKB value of 8.62. Under isometric recording condition, SR 142801 (0.1 M) did not affect twitch inhibition produced by 3 nM clonidine. Under isotonic recording condition, SR 142801 did not affect the L-NOARG-sensitive relaxation produced by EFS.The present results indicate that NK₃ receptor stimulation produces a NO-dependent relaxation of the guinea-pig colon and a substantially NO-independent prejunctional inhibition of cholinergic twitches. The variable affinities of SR 142801 in antagonizing various senktide-induced neuromodulatory effects in the guinea-pig intestine suggest a possible intraspecies heterogeneity of NK₃ receptors in the enteric nervous system.     

Cisplatin increases the release of 5-hydroxytryptamine (5-HT) from the isolated vascularly perfused small intestine of the guinea-pig: Involvement of 5-HT3 receptors

Summary Isolated segments of the guinea-pig small intestine were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent determined by high pressure liquid chromatography with electrochemical detection. Release of acetylcholine from isolated superfused intestinal segments was determined as outflow of [³H]radioactivity from preparations preincubated with [³H]choline. Cisplatin (3 M) increased the outflow of 5-HT and 5-HIAA by about 90%. At 30 and 100 M cisplatin decreased the outflow of 5-HT and its metabolite by 40%–50%. The stimulatory effect of cisplatin was consistently observed only when the bicarbonate-phosphate buffer of the Tyrode's solution was replaced by HEPES-buffer. The stimulatory effect of cisplatin was abolished in the absence of extracellular calcium or presence of tetrodotoxin (1 M). The stimulatory effect of cisplatin was also prevented by hexamethonium (100 M) or scopolamine (100 nM). The 5-HT₃ receptor antagonists ondansetron and ICS 205-930 in concentrations as low as 1 pM also abolished the stimulatory effect of cisplatin. The 5-HT₃ receptor antagonist MDL 72222 prevented the stimulatory effect of cisplatin only at a concentration of 1 M. None of the 5-HT₃ receptor antagonists alone significantly altered the outflow of 5-HT and 5-HIAA.Cisplatin (3 M) enhanced the outflow of [³H]radioactivity from intestinal segments and caused longitudinal muscle contractions that were abolished by 100 nM scopolamine.In conclusion, cisplatin, at concentrations which occur during anti-cancer therapy in humans and induce emesis, increases the release of 5-HT from the enterochromaffin cells of the small intestine of the guinea-pig. This effect of cisplatin is mediated by a cascade of events which involves release of acetylcholine and stimulation of 5-HT₃ receptors. Send offprint requests to H. Schwörer at his present address     

Involvement of substance P in the excitatory action of GABAA agonists on cholinergic neurons in the guinea-pig ileum

Summary 1. The possible involvement of substance P (SP) in cholinergic contractions induced by GABA_A agonists in the guinea-pig ileum was further investigated. 2. Responses evoked by 3-aminopropane sulphonic acid (3-APS) or muscimol consisted of a rapid phasic contraction followed in 70% of preparations by a tonic contraction, usually smaller in amplitude but considerably longer in duration. Phasic and tonic components were sensitive to bicuculline, neurogenic (cholinergic) in nature and susceptible to desensitization. 3. Capsaicin (0.2 M) pretreatment and SP receptor desensitization caused by 3 different priming SP concentrations (10 nM, 30 nM, 100 nM), depressed both components of the 3-APS-induced response, the magnitude of antagonism being greater for tonic contractions. Similar findings were obtained by using 10 M (d-Pro⁴, d-Trp^7,9)SP-(4–11), even though the degree of antagonism caused by this SP antagonist was consistently lower. 4. These results indicate that depression of SP receptor function achieved by three different procedures decreases cholinergic contractile responses to GABA_A agonists in the guinea-pig ileum. This provides further support for the hypothesis that GABA_A receptor activation evokes both direct and indirect stimulation of enteric cholinergic neurons and that SP and/or a related peptide play an important role in mediating the indirect component of the cholinergic response. Send offprint requests to M. Tonini at the above address