Release-modulating acetylcholine receptors in cholinergic neurones of the guinea-pig ileum.

1 Twitch responses of the guinea-pig ileum to electrical transmural stimulation (0.2 Hz) were smaller after a dose of acetylcholine (ACh) than before it. The magnitude of the post-ACh inhibition of twitch was dose-dependent. 2 The post-ACh inhibition of twitch could not be explained in terms of post-junctional desensitization and was not modified by guanethidine, thymoxamine, propranolol or naloxone. Inhibition of twitch also followed high frequency stimulation (10 Hz) but this inhibition, unlike that following ACh, was partially antagonized by naloxone. 3 Hexamethonium (C6) in concentrations known to block contractions to nicotine, potentiated the post-ACh inhibition of twitch and modified the pattern of recovery. An initial rapid phase followed by a slower phase was converted by C6 to an initial slow phase followed by a more rapid rate of recovery. 4 The C6-sensitive (nicotinic) component of twitch recovery after ACh was also dose-dependent and contributed greatly to the rapid recovery during the first minute after ACh washout, whereas during the same period the C6-resistant inhibition remained relatively constant; thereafter both components declined. The C6-resistant inhibition was considered to be due to the activation of prejunctional muscarinic receptors. 5 5-Hydroxytryptamine (5-HT) and nicotine also caused inhibition of twitch but the pattern of response differed from those due to ACh, the maximum inhibition usually being produced 1 min after recommencing stimulation. High doses of 5-HT produced inhibitory responses similar to those following ACh, whereas nicotine produced a characteristic triphasic pattern of response. 6 It is concluded that ACh acts on at least two prejunctional receptors subserving a modulatory role on transmitter release, a nicotinic receptor whose activation enhances ACh output and a muscarinic receptor whose activation leads to an inhibition of transmitter secretion.     

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.     

Involvement of neurokinins in the non-cholinergic response to activation of 5-HT3 and 5-HT4 receptors in guinea-pig ileum.

1. The effects of vasopressin and deamino-8-D-arginine vasopressin (DDAVP, desmopressin) were studied in artery rings (0.8-1 mm in external diameter) obtained from portions of human omentum during the course of abdominal operations (27 patients). 2. In arterial rings under resting tension, vasopressin produced concentration-dependent, endothelium-independent contractions with an EC50 of 0.59 +/- 0.12 nM. The V1 antagonist d(CH2)5Tyr(Me)AVP (1 microM) and the mixed V1-V2 antagonist desGly-d(CH2)5D-Tyr(Et)ValAVP (0.01 microM) displaced the control curve to vasopressin to the right in a parallel manner without differences in the maximal responses. In the presence of indomethacin (1 microM) the contractile response to vasopressin was significantly increased (P < 0.01). 3. In precontracted arterial rings, previously treated with the V1 antagonist, d(CH2)5Tyr(Me)AVP (1 microM), vasopressin produced endothelium-dependent relaxation. This relaxation was reduced significantly (P < 0.05) by indomethacin (1 microM) and unaffected by the V1-V2 receptor antagonist desGly-d(CH2)5D-Tyr(Et)ValAVP (1 microM) or by NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mM). 4. The selective V2 receptor agonist, DDAVP, caused endothelium-independent, concentration-dependent relaxations in precontracted arterial rings that were inhibited by the mixed V1-V2 receptor antagonist, but not by the V1 receptor antagonist or by pretreatment with indomethacin or L-NAME. 5. Results from this study suggest that vasopressin is primarily a constrictor of human mesenteric arteries by V1 receptor stimulation; vasopressin causes dilatation only during V1 receptor blockade. The relaxation appears to be mediated by the release of vasodilator prostaglandins from the endothelial cell layer and is independent of V2 receptor stimulation or release of nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

A facilitatory effect of bicuculline on the enteric neurones in the guinea-pig isolated colon.

Changes in the efficiency of the peristaltic reflex, acetylcholine (ACh) output and motor responses to transmural and periarterial nerve stimulation produced by bicuculline and gamma-aminobutyric acid (GABA) receptor desensitization were investigated in the guinea-pig isolated colon. Bicuculline, at concentrations unable to affect spontaneous colonic motility and lacking anticholinesterase activity, produced a dose-dependent increase of both the efficiency of the peristaltic reflex and the stimulated ACh output. Such effects could not be observed in GABA-desensitized preparations. A frequency-dependent potentiation of the cholinergic excitatory and non-adrenergic non-cholinergic (NANC) inhibitory responses to transmural stimulation was also observed in the presence of bicuculline. Conversely bicuculline exhibited an inhibitory effect on the relaxation induced by periarterial nerve stimulation. Acute GABA-desensitization was unable to affect the contractile responses to transmural stimulation, the ACh output and the efficiency of the peristaltic reflex. On the contrary, desensitization was able to mimic the effects of bicuculline on the inhibitory responses to both transmural and periarterial nerve stimulation. Our results are consistent with a significant role played by an intrinsic GABAergic pathway in the modulation of both cholinergic excitatory and NANC inhibitory neurones. The hypothesis is advanced that a feed-back modulation carried out through bicuculline-sensitive GABAergic synapses could operate during the propagation of peristaltic motor activity.     

Myenteric 5-HT-containing neurones activate the descending cholinergic excitatory pathway to the circular muscle of guinea-pig ileum.

1. Participation of myenteric 5-hydroxytryptamine (5-HT)-containing neurones in the ascending and descending pathways of the guinea-pig isolated ileum was investigated in a new preparation. Transmural electrical stimulation of the longitudinal muscle-myenteric plexus (LM-MP) portion of the preparation caused ascending and descending contractions of circular or longitudinal muscle in the attached, intact segments situated orally or anally to the point of stimulation. 2. All contractions of LM-MP stimulation were abolished by tetrodotoxin (0.2 microM). The ascending and descending contractions of circular muscles were also abolished by atropine and inhibited to about 50% by hexamethonium. They were not affected by desensitization to substance P (SP) or by the SP antagonist, (D-Pro2,D-Trp7,9)-substance P. The contractions of longitudinal muscles were inhibited by about 45% by hexamethonium and abolished by a combination of atropine with SP desensitization or the SP antagonist, (D-Pro2,D-Trp7,9)-substance P. 3. Desensitization to 5-HT, ICS 205-930 (1 microM) or cocaine (1 microM) reduced the descending contraction of circular muscle by 80-90%, without significantly affecting the ascending contraction. Methysergide (0.2 microM) failed to alter either contraction. 4. 5-HT desensitization, ICS 205-930 and cocaine only partially reduced the descending contraction of longitudinal muscle. A similar reduction of the ascending contraction (20-30%) was also observed. Methysergide had no effects on either contraction. 5. Contractions of either circular or longitudinal muscle produced by field stimulation of the intact segment were not significantly affected by any of the 5-HT receptor antagonists tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of mesaconitine-induced contractile response in guinea-pig ileum

Mesaconitine (MA) caused contractions of the guinea-pig isolated ileum in a dose-dependent manner (10-8-10-5 g ml-1), slightly potentiated the contractile response to acetylcholine (ACh) and histamine and enhanced responses to electrical stimulation. Repeated application of MA (10-5 g ml-1) produced tachyphylaxis. Atropine blocked contractions to MA (3 X 10-M g ml-1), but only partially those to MA (10-5 G ML-1). Morphine, strychnine and hemicholinium-3, but not hexamethonium, also inhibited MA-induced contractions. Contractions produced by both doses of MA were abolished by cocaine, tetrodotoxin, or noradrenaline, or by previous cooling of the ileum. The atropine-resistant contractions produced by MA (10-5 g ml-1) were blocked by indomethacin. MA (3 X 10-7-10-5 g ml-1) elicited a dose-dependent release of ACh from the isolated ileum which was blocked by treatment with tetrodotoxin or cocaine, or exclusion of calcium ions from the bath. It is concluded that the contractions induced by lower doses of MA are brought about by the release of ACh from the postganglionic cholinergic nerve and that the contractions by higher doses could also be mediated by release of prostaglandins from the ileum.     

Substance P mediates atropine-sensitive response of guinea-pig ileum to serotonin

The action of serotonin (5HT) was investigated on guinea-pig isolated ileum. Low concentrations of 5HT (10(-8) - 2.5 x 10(-7) mol x 1(-1)) produced contractile responses which were abolished by atropine and by methionine enkephalin but not by methysergide. Higher concentrations of 5HT (5 x 10(-7) - 5 x 10(-6) mol x 1(-1)) produced contractions which were inhibited by methysergide. The atropine-sensitive response was also abolished following desensitization of preparations to substance P and by the substance P antagonist [D-Pro2,D-Phe7,D-Trp9]SP. It was concluded that the atropine-sensitive response to 5HT is mediated by substance P.     

P2x-purinoceptors of myenteric neurones from the guinea-pig ileum and their unusual pharmacological properties.

1. Whole-cell and outside-out patch clamp recordings were used to characterize the physiological and pharmacological properties of the P2x-purinoceptors of myenteric neurones from the guinea-pig ileum. 2. Adenosine 5'-triphosphate (ATP) and analogues (1-3000 microM) evoked a rapid inward current in > 90% of all recorded neurones. The reversal potential of this current was dependent on the extracellular sodium concentration, at +14 +/- 1.9, 0 +/- 1.6 and -12 +/- 1 mV for 166, 83 and 42 mM of sodium, respectively. The fast activation and inactivation of this current occurred even when guanosine 5'-triphosphate (GTP) was omitted from the pipette solution or substituted with an equimolar concentration of guanosine 5'-o-[2-thiotriphosphate] (GTP-gamma-S). Single channel currents were observed when these outside-out membrane patches were exposed to ATP (10-30 microM). These channels have a unitary conductance of about 17 picosiemens. 3. The rank-order of potency of the agonists used to induce the whole-cell currents was: ATP-gamma-S = ATP = 2-methylthio-ATP (2-Me-S-ATP) > > alpha, beta-methylene ATP = beta, gamma-methylene ATP; adenosine and uridine 5'-triphosphate (UTP) (up to 1 mM) were inactive. 4. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1-30 microM) antagonized the effects of ATP (1 mM) with an IC50 of 4 microM. alpha, beta-Methylene ATP (100 microM) did not affect the ATP (30 microM)-induced current. Cibacron Blue 3GA increased the ATP activated cationic current whereas Basilen Blue E-3G had a very weak antagonistic effect (IC50 > or = 3 mM). Suramin potentiated the currents induced by ATP through a mechanism that was independent of its inhibitory effect on ectonucleotidase activity, as suramin also potentiated the effect of alpha, beta-methylene ATP (an ATP analogue that is resistant to nucleotidases). 5. In conclusion, the myenteric P2x-purinoceptor shares some properties with other purinoceptors in particular with the P2x4- and P2x6-purinoceptors. This receptor has also some unusual pharmacological properties suggesting that myenteric neurones express a novel subtype of P2x-purinoceptors. The properties of this receptor, however, might be a result of the combination of two or more of the homomeric purinoceptors so far characterized.     

The properties of the clonidine withdrawal response of guinea-pig isolated ileum.

The dependence-inducing effects of clonidine were investigated on the guinea-pig isolated ileum. Clonidine produced relaxation of the ileum with a threshold concentration between 0.01 and 0.1 mumol 1(-1). Washout of clonidine did not induce a withdrawal contraction. Following 2 min contact of the ileum with clonidine, 1 mumol 1(-1), addition of phentolamine, 5 mumol 1(-1), induced a contracture. The phentolamine-precipitated withdrawal contracture did not increase in height with a longer period of contact (32 min) of the ileum with clonidine. The phentolamine-precipitated withdrawal contracture following 2 min contact of ileum with clonidine was abolished by atropine, 5 mumol 1(-1), and substance P (SP) antagonists, (D-Pro2,D-Phe7, D-Trp9)-SP and spantide, 10 mumol 1)-1). [Met5]enkephalin, 1 mumol 1(-1), abolished the withdrawal response to clonidine and clonidine, 1 mumol 1(-1), abolished the withdrawal response to [Met5]enkephalin. Following 2 min contact of the ileum with noradrenaline, 5 mumol 1(-1), washout or addition of phentolamine or yohimbine, 5 mumol 1(-1), also induced a withdrawal response. The noradrenaline washout withdrawal response was abolished by atropine, 5 mumol 1(-1), and spantide, 10 mumol 1(-1). Since clonidine dependence may be induced as rapidly as opiate dependence in the ileum and the pharmacology of the withdrawal responses is similar, it is suggested that they both induce the same post-receptor neuronal feedback disturbance in which substance P neurones play a major role.     

The effect of muscarine on cholinoceptive neurones subserving the peristaltic reflex of guinea-pig isolated ileum.

Muscarine, added serosally to the guinea-pig isolated ileum. contracted the longitudinal and the circular muscles and stimulated peristaltic activity during a continuously raised intraluminal pressure. This effect was long-lasting and dose-dependent. Muscarine acting serosally also antagonized the inhibition of peristalsis caused by serosal application of ganglionic blocking agents (hexamethonium, tetraethylammonium, nicotine, dimethylphenylpiperazinium or tetramethylammonium), (+)tubocurarine, gallamine, atropine, hyoscine, morphine or morphine-like drugs (pethidine and methadone). noradrenaline, adrenaline, dopamine, 5-hydroxytryptamine, histamine, calcium and magnesium. This antagonism of the blocking agents by muscarine was complete for the circular muscle, but only partial for the longitudinal muscle. The results support the view that the cholinoceptive sites within the ganglia of the myenteric plexus subserving the peristaltic reflex have properties of both nicotinic and muscarinic receptors. The long-lasting effect of muscarine suggests that the mechanism activated by these receptors differs from conventional synaptic transmission. It is possible that the cholinoceptive neurones sensitive to muscarine behave as a “biological generator”, and when activated produce long-lasting peristaltic activity.     

Effect of metoclopramide on peristalsis and antiperistalsis in isolated frog stomach and guinea-pig ileum

The action of metoclopramide was studied on peristaltic as well as antiperistaltic activity in frog stomach and guinea-pig ileum. It was observed that metoclopramide enhances normal peristaltic activity by acting on dopamine-receptors whereas it inhibits antiperistalsis by some unknown mechanism. It is, therefore, inferred that inhibition of antiperistaltic activity might be an additional mechanism for its antiemetic effect. It also explains the pharmacological basis for its use in oesophagitis due to gastro-oesophageal reflux.     

Actions of the muscle relaxant chandonium iodide on guinea-pig ileum and vas deferens preparations.

The effects of the newly synthesized neuromuscular blocking agent, chandonium iodide (17a-methyl-3beta-pyrrolidino-17a-aza-D-homo-5-androstene dimethiodide) have been investigated on guinea-pig isolated ileum and vas deferens preparations. On the ileum, chandonium (0-1-10-0 mug ml(-1); 1-6 X 10(-7) M-1-6 X 10(-5) M) had weak muscarinic receptor blocking action (pA2 is 5-7), but no antihistamine properties at the concentration tested. No evidence for anticholinesterase actions was found. On the vas deferens, chandonium (10-50 mug ml(-1); 1-6-8-1 X 10(-5) M) potentiated responses to exogenous noradrenaline; responses to electrical stimulation were potentiated only in the presence of 50 mug ml(-1) chandonium. No adrenoceptor or adrenergic neuron blockade was found. The results provide evidence that chandonium acts selectively at acetylcholine receptors and that it is more active as a nicotinic receptor antagonist than as a muscarinic receptor antagonist.     

Mechanism of mesaconitine-induced contractile response in guinea-pig ileum*

Mesaconitine (MA) caused contractions of the guinea-pig isolated ileum in a dose-dependent manner (10^?8 ?10^?5 g ml^?1), slightly potentiated the contractile response to acetylcholine (ACh) and histamine and enhanced responses to electrical stimulation. Repeated application of MA (10^?5 g ml^?1) produced tachyphylaxis. Atropine blocked contractions to MA (3 times 10^?7 g ml^?1), but only partially those to MA (10^?5 g ml^?1). Morphine, strychnine and hemicholinium-3, but not hexamethonium, also inhibited MA-induced contractions. Contractions produced by both doses of MA were abolished by cocaine, tetrodotoxin, or noradrenaline, or by previous cooling of the ileum. The atropine-resistant contractions produced by MA (10^?5 g ml^?1) were blocked by indomethacin. MA (3 times 10^?7 ?10^?5 g ml^?1) elicited a dose-dependent release of ACh from the isolated ileum which was blocked by treatment with tetrodotoxin or cocaine, or exclusion of calcium ions from the bath. It is concluded that the contractions induced by lower doses of MA are brought about by the release of ACh from the postganglionic cholinergic nerve and that the contractions by higher doses could also be mediated by release of prostaglandins from the ileum.     

Pharmacological evidence for the presence of cholecystokinin-containing neurones in the mesenteric nerves supplying the guinea-pig ileum

In guanethidine-treated, capsaicin-desensitized segments of guinea-pig isolated ileum electrical stimulation of mesenteric nerves resulted in contractions reaching 10 to 30% of the maximal longitudinal spasm. These responses were abolished by tetrodotoxin or atropine, and were reduced by half by the selective cholecystokinin antagonist lorglumide. It is concluded that neuronal processes whose endings release cholecystokinin-like material within the gut wall run in the mesenteric nerves and contribute to the capsaicin-resistant contractile response by releasing endogenous acetylcholine.     

Involvement of SRS-A in the Schultz-Dale response of the guinea-pig small intestine.

1 The anaphylactic reaction of the guinea-pig ileum, the so called Schultz-Dale reaction, shows a biphasic response: a short rapid contraction followed by a partial relaxation and a slow contractile response. 2 Dose-response curves with ovalbumin as an antigen were obtained for the quick and slow contraction of this anaphylactic reaction. 3 Mepyramine (1 microgram/ml) blocked the rapid first contraction, but failed to abolish the slow one in about 50% of the animals studied. 4 The SRS-A antagonist, FPL 55712, significantly depressed the slow sustained contraction during the Schultz-Dale reaction. Disodiumcromoglycate was without effect on both phases when it was added 5 min before addition of the antigen. However, when added simultaneously with the antigen it produced a 30% suppression of the slow phase in the highest concentration used.     

The effects of ruthenium red on the response of guinea-pig ileum to capsaicin

Ruthenium red has recently been found to inhibit the effects of capsaicin on peripheral terminals of sensory neurones. Thus the effects of ruthenium red on the responses of the guinea-pig isolated ileum to capsaicin, acetylcholine (ACh), substance P (SP) and nicotine were investigated. Ruthenium red, 5 mumol/l, abolished responses to capsaicin 1.5 mumol/l and nicotine 2 mumol/l, and shifted the concentration-response lines to ACh and SP to the right. Pretreatment of ileum preparations with ruthenium red, 12.5 mumol/l for 2 min, prevented desensitization of ileum responses to capsaicin tested 30 min later. Tetrodotoxin, 1 mumol/l, abolished the response to capsaicin on control preparations and those pretreated with atropine, 5 mumol/l, ruthenium red, 12.5 mumol/l or spantide, 10 mumol/l. It is proposed that capsaicin acts via a specific receptor coupled to a receptor-operated membrane calcium channel, and that ruthenium red binds irreversibly to the calcium channel part of the complex but reversibly to some other site which prevents the action or binding of capsaicin at its specific receptor.     

Effects of cannabinoid receptor ligands on electrophysiological properties of myenteric neurones of the guinea-pig ileum

1. The effect of cannabinoid receptor agonists was studied in guinea-pig myenteric neurones in vitro by use of conventional intracellular recording techniques.
2. Exposure of myenteric neurones of the S-cell type to the cannabinoid receptor agonists WIN 55,212-2 (100 nM) and CP 55,940 (100 nM) reversibly and significantly depressed the amplitude of fast excitatory synaptic potentials (fast e.p.s.ps) by 46% and 37%, respectively.
3. The depressant effect of WIN 55,212-2 and CP 55,940 on fast e.p.s.p. amplitude (expressed as the area above the amplitude-time curve (mVs)) was significantly greater than that of the vehicle, Tween 80, which had no detectable effect.
4. The inhibitory effect of WIN 55,212-2 appeared to be concentration-dependent over the range 1–100 nM. WIN 55,212-3, its (−)-enantiomer (100 nM), was inactive.
5. The cannabinoid CB₁ receptor antagonist, SR141716A (1 μM), reversed the inhibitory effects of WIN 55,212-2 on fast e.p.s.ps in 38% of neurones tested (3/8) and acetylcholine (ACh)-induced depolarizations in 42% of neurones tested (5/12).
6. When tested on its own, SR141716A (1 μM) caused a 40–50% reduction in the amplitude of fast e.p.s.ps (n=9).
7. WIN 55,212-2 reversibly depressed the amplitude of the slow e.p.s.p. and, in 2 out of 7 neurones, this effect was reversed by SR141716A (1 μM).
8. It is concluded that cannabinoid-induced inhibition of fast cholinergic synaptic transmission occurred by reversible activation of both presynaptic and postsynaptic CB₁ receptors and that slow excitatory synaptic transmission can also be reversibly depressed by cannabinoids. Furthermore, it would seem that subpopulations of myenteric S-neurones and their synapsing cholinergic and non-cholinergic, non-adrenergic terminals are not endowed with cannabinoid receptors.

     

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.