Tachykinin receptors in the guinea-pig isolated bronchi.

The aim of the study was to assess which tachykinin receptors mediate the contractile response in the guinea-pig isolated bronchi. Experiments with natural tachykinins and receptor-selective tachykinin agonists were performed in the absence or presence of peptidase inhibitors and in bronchi pretreated with phenoxybenzamine. Both NK-1 (substance P, substance P methylester and septide) and NK-2 (neurokinin A, [beta-Ala8]neurokinin A-(4-10) and MDL 28,564) receptor agonists produced concentration-dependent contraction. NK-3 agonists (senktide and [MePhe7]neurokinin B) were active only at high concentrations. Phenoxybenzamine pretreatment reduced the maximal response to NK-1 agonists and produced a rightward shift of the curve to NK-2 agonists, without depression of the maximum. Five tachykinin antagonists selective for the NK-1 (L 668,169) or the NK-2 (MEN 10,207, MEN 10,376, L 659,877 and R 396) receptor were tested against substance P methylester and [beta-Ala8]neurokinin A-(4-10). The results indicated that these receptor-selective antagonists maintain their characteristic even when tested in a multireceptor assay such as the guinea-pig bronchus. The rank order of potency of NK-2 antagonists against [beta-Ala8]neurokinin A-(4-10) was MEN 10,207 = MEN 10,376 greater than L 659,877 much greater than R 396. This pattern, with the observation of the full agonist activity of MDL 28,564, indicates that in addition to NK-1 receptors, NK-2 receptors also are present in the guinea-pig bronchi and belong to the same subtype (NK-2A) as present in the rabbit pulmonary artery.     

Tachykinin NK1 and NK2 receptor antagonists and atropine-resistant ascending excitatory reflex to the circular muscle of the guinea-pig ileum.

1. The aim of this study was to investigate the effect of various antagonists, selective for the tachykinin NK1 or NK2 receptor, on the atropine-resistant ascending excitatory reflex (AER) to the circular muscle of the guinea-pig ileum elicited by radial stretch (balloon distension) or electrical field stimulation. 2. Submaximal and maximal atropine- (1 microM) resistant AER elicited by balloon distension averaged about 40-50% and 70-90% of maximal circular spasm to 80 mM KCl, respectively. The NK1 receptor antagonist, (+/)-CP 96,345 (1 microM) inhibited both maximal and submaximal AER. FK 888 (1-3 microM) inhibited submaximal AER only. RP 67,580 (1 microM) was ineffective. The NK2 receptor antagonist, GR 94,800, inhibited both maximal and submaximal AER at all concentrations tested (0.1-3.0 microM), while SR 48,968 was effective only at 1.0 microM. The NK2 receptor antagonists, MEN 10,376 and MEN 10,573 inhibited both submaximal and maximal AER at 10 and 1.0 microM, respectively. 3. In other experiments, an NK1 receptor antagonist, (+/-)-CP 96,345 or FK 888 (1.0 microM in each case) was administered first and the effect of GR 94,800 (1.0 microM) on the residual AER response was determined; or GR 94,800 was administered first and the effect of (+/-)-CP 96,345 or FK 888 was determined. The results of these experiments indicated an additive effect produced by the combined treatment with NK1 and NK2 receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tachykinin NK1 but not NK2 receptors mediate non-cholinergic excitatory junction potentials in the circular muscle of guinea-pig colon.

1. The effect of tachykinin NK1 and NK2 receptor antagonists on noncholinergic excitatory junction potentials (e.j.ps) evoked by electric field stimulation (EFS) in the circular muscle of the guinea-pig proximal colon was investigated by means of a sucrose-gap technique. 2. In the presence of 1 microM atropine, submaximal EFS (10 Hz, 20-30 V, 0.5 ms pulse width, 1 s train duration) evoked an inhibitory junction potential (i.j.p.) followed by e.j.p. with superimposed action potentials (APs) and contraction. Addition of either NG-nitro-L-arginine (L-NOARG, 0.1 mM) or apamin (0.1 microM) inhibited the evoked i.j.p. and the combined administration of the two agents almost abolished it. In the presence of both L-NOARG and apamin, an atropine-resistant e.j.p. was the only electrical response evoked by EFS in 50% of cases and a small i.j.p. (10% of original amplitude) followed by e.j.p. was evident in the remainder. 3. In the presence of L-NOARG and apamin, the tachykinin NK1 receptor antagonists, (+/-)-CP 96,345 and GR 82,334 (10 nM-3 microM) concentration-dependently inhibited the atropine-resistant e.j.p. and accompanying contraction evoked by EFS. EC50 values were: 0.77 microM (e.j.p. inhibition) and 0.22 microM (inhibition of contraction) for (+/-)-CP 96,345; 0.61 microM (e.j.p. inhibition) and 0.20 microM (inhibition of contraction) for GR 82,334. The tachykinin NK2 receptor antagonists, MEN 10,376 (up to 3 microM) and SR 48,968 (up to 1 microM) had no effect on the atropine-resistant e.j.p. MEN 10,376 (3 microM) but not SR 48,968 produced a slight inhibition of the evoked contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Tachykininergic transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of NK2 receptors.

1. The effect of newly developed, receptor-selective tachykinin antagonists (GR 71,251 for NK1 receptors, MEN 10,376 and L 659,877 for NK2 receptors) on noncholinergic transmission to the circular muscle of the guinea-pig ileum has been investigated. 2. In circular muscle strips of the ileum, electrical field stimulation in the presence of atropine (2 microM) and apamin (0.1 microM) evoked a complex motor response. The tonic primary contraction in this response was reduced by GR 71,251 (10 microM) and MEN 10,376 (3-10 microM) but not by L 659,877 (up to 10 microM). The presence of apamin was necessary in this experimental arrangement to unmask an atropine-resistant primary contraction, sensitive to tachykinin antagonists. The motor response was abolished by tetrodotoxin. 3. In circular strips of the ileum GR 71,251 (10 microM) inhibited the tonic contraction produced by [Sar9] substance P sulphone, a selective NK1 receptor agonist but not that produced by [beta Ala8] neurokinin A (4-10), a selective NK2 receptor agonist. By contrast, MEN 10,376 antagonized the effect of the NK2 agonist while leaving the response to the NK1 agonist unaffected. 4. In whole segments of the ileum, distension of the gut wall by an intraluminal balloon placed at about 1 cm from the point of recording of mechanical activity of the circular muscle produced atropine-sensitive phasic contractions (ascending enteric reflex). In the presence of atropine (2 microM), a noncholinergic response was elicited, which required larger volumes of distension that the cholinergic one. The atropine-resistant ascending enteric reflex was enhanced by apamin (0.1 microM) and abolished by tetrodotoxin, either in the presence or absence of apamin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galanin suppresses neurally evoked contractions of circular muscle in the guinea-pig ileum

The effect of galanin, a 29 amino-acid peptide, on intestinal smooth muscles was studied in guinea-pig ileum. Galanin did not affect the basal activity of longitudinal or circular muscles. Galanin decreased neurally evoked circular muscle contractions in a dose-dependent manner, but failed to affect neurally evoked longitudinal muscle contractions. Galanin also decreased neurally evoked circular muscle contractions in the presence of atropine. The neurally evoked phasic contractions were blocked by TTX. These findings indicate that galanin has an inhibitory role in circular muscle contractions via myenteric neurons in the guinea-pig ileum.     

Opioid dependence in myenteric neurons innervating the circular muscle of guinea-pig ileum

Summary Guinea-pigs were treated with morphine for 6–8 days by subcutaneous implantation of pellets, each containing a mixture of morphine base (120 mg) and morphine hydrochloride (35 mg). Each guinea-pig received a single pellet. Mechanical activity of the circular muscle was recorded in vitro in preparations comprising the circular muscle and myenteric plexus. Exposure to morphine was maintained by addition of 1 M morphine to the organ baths. After 90 min, morphine was withdrawn, either by repeatedly washing tissues in morphine-free Krebs' solution , or by addition of naloxone to reduce the occupancy of the opioid receptors by morphine. Withdrawal of morphine resulted in markedly enhanced contractile activity compared with that in circular muscle-myenteric plexus preparations from untreated control guinea-pigs. The withdrawal contractions were abolished by tetrodotoxin (600 nM) and greatly reduced by hyoscine (1 M), indicating that they resulted from action potential discharge in myenteric neurons that release acetylcholine onto the circular muscle. Activation of the cholinergic excitatory motor neurons was not secondary to synaptic activation by cholinergic interneurons, because hexamethonium (100 M) did not affect withdrawal contractions. The withdrawal response may therefore arise in the cholinergic excitatory motor neurons themselves, or in neurons that activate them via noncholinergic mechanisms. Send offprint requests to S. Johnson at the above address     

Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea-pig ileum and proximal colon.

1. The aim of this study was the pharmacological characterization of tachykinin NK1 and NK2 receptors mediating contraction in the circular muscle of the guinea-pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [Sar9]SP sulphone, [Glp6,Pro9]SP(6-11) (septide) and [beta Ala8]NKA(4-10) was investigated. The affinities of various peptide and nonpeptide antagonists for the NK1 and NK2 receptor was estimated by use of receptor selective agonists. 2. The natural agonists, SP and NKA, produced concentration-dependent contraction in both preparations. EC50 values were 100 pM and 5 nM for SP, 1.2 nM and 19 nM for NKA in the ileum and colon, respectively. The action of SP and NKA was not significantly modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). 3. Synthetic NK1 and NK2 receptor agonists produced concentration-dependent contraction of the circular muscle of the ileum and proximal colon. EC50 values were 83 pM, 36 pM and 10 nM in the ileum, 8 nM, 0.7 nM and 12 nM in the colon for [Sar9]SP sulphone, septide and [beta Ala8]NKA-(4-10), respectively. The pseudopeptide derivative of NKA(4-10), MDL 28,564 behaved as a full or near-to-full agonist in both preparations, its EC50s being 474 nM and 55 nM in the ileum and colon, respectively. 4. Nifedipine (1 microM) abolished the response to septide and [Sar9]SP sulphone in the ileum and produced a rightward shift and large depression of the response in the colon. The response to [beta Ala8]NKA(4-10) was abolished in the ileum and largely unaffected in the colon. 5. The NK1 receptor antagonists, (+/-)-CP 96,34, FK 888 and GR 82,334 competitively antagonized the response to septide and [Sar9]SP sulphone in both preparations without affecting that to [beta Ala8]NKA(4-10). In general, the NK1 receptor antagonists were significantly more potent toward septide than [Sar9]SP sulphone in both preparations. 6. The NK2 receptor antagonists, GR 94,800 and SR 48,968 selectively antagonized the response to [beta Ala8]NKA(4-10) without affecting that to [Sar9]SP sulphone or septide in the ileum and colon. SR 48,968 produced noncompetitive antagonism of the response to the NK2 receptor agonist in the ileum and competitive antagonism in the colon. 7. MEN 10,376 and the cyclic pseudopeptide MEN 10,573 antagonized in a competitive manner the response to [beta Ala8]NKA(4-10) in the ileum and colon.(ABSTRACT TRUNCATED AT 400 WORDS)     

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     

Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid CB1 receptors

1. The effect of cannabinoid drugs has been investigated on cholinergic and non-adrenergic non-cholinergic (NANC) contractile responses to the circular smooth muscle of guinea-pig ileum elicited by electrical field stimulation (EFS).
2. The cannabinoid receptor agonist WIN 55,212-2 (1–1000 nM) and the putative endogenous ligand anandamide (0.1–100 μM) both produced a concentration-dependent inhibition of the cholinergic (9–57% and 1–51% inhibition) and NANC (9–55% and 2–57% inhibition) contractile responses. WIN 55,212-2 and anandamide did not modify the contractions produced by exogenous acetylcholine or substance P.
3. Apamin (30 nM), a blocker of Ca²⁺-activated K⁺ channels, reduced the inhibitory effect of WIN 55,212-2 on cholinergic, but not NANC, contractile response. N^G-nitro-L-arginine methyl ester (100 μM), an inhibitor of nitric oxide synthase, or naloxone (1 μM), an opioid receptors antagonist, did not modify the inhibitory effect of WIN 55,212-2 on both cholinergic and NANC contractions.
4. The inhibitory effects of WIN 55,212-2 and anandamide on both cholinergic and NANC contractile response was competitively antagonized by the cannabinoid CB₁ receptor antagonist SR 141716A (10–1000 nM).
5. In absence of other drugs, SR 141716A (1–1000 nM) enhanced cholinergic (1–45% increase) and NANC (2–38% increase) contractile responses elicited by electrical stimulation, but did not modify the contractions produced by acetylcholine or substance P.
6. It is concluded that activation of prejunctional cannabinoid CB₁ receptors produces inhibition of cholinergic and NANC excitatory responses in the guinea-pig circular muscle. The inhibition of cholinergic (but not NANC) transmission involves activation of apamin-sensitive K⁺ channels. In addition, an endogenous cannabinoid ligand could inhibit cholinergic and NANC transmission in the guinea-pig ileal circular muscle.

     

The action of drugs on the circular muscle strip from the guinea-pig isolated ileum

The circular muscle strip is a new preparation for examining the action of drugs on the circular muscle of the guinea-pig isolated intestine. The preparation differed from the longitudinal muscle in that it was insensitive to drugs which act on autonomic effector tissues but, after inhibition of cholinesterase, it responded readily to choline esters, 5-hydroxytryptamine, histamine and nicotine. This behaviour necessitated the treatment of each strip with the anticholinesterase NN-diisopropylphosphodiamidic fluoride (mipafox) before each experiment. The contractions of the strip by 5-hydroxytryptamine, histamine and nicotine were abolished by procaine, botulinum toxin (Type A), morphine and hemicholinium, whilst the actions of acetylcholine and methacholine were unaffected. Contractions of the strip in response to each of the drugs were abolished by atropine and hyoscine. The action of nicotine was specifically antagonized by hexamethonium, that of 5-hydroxytryptamine by desensitization of the tissue to 5-hydroxytryptamine, and that of histamine either by desensitization of the tissue to histamine or by mepyramine. It is postulated that 5-hydroxytryptamine, histamine and nicotine stimulate specific receptor sites within the intramural nerve plexuses of the guinea-pig isolated ileum. Finally, botulinum toxin (Type A), morphine or hemicholinum, acting on the neuronal elements of the intramural plexuses, depressed the contractions of the circular muscle strip due to histamine or nicotine more readily than those due to 5-hydroxytryptamine.     

Characterization of kappa-opioid receptors in the guinea-pig ileum

Equilibrium binding saturation studies with [3H]bremazocine, under mu- and delta-suppressed conditions and [3H]U69593 have demonstrated that both radioligands bind with high affinity to an apparently homogeneous population of binding sites in the guinea-pig ileum longitudinal muscle-myenteric plexus preparation. In competition studies, the absolute affinities and slopes of the inhibition curves for several unlabelled ligands against [3H]bremazocine were not significantly different to those against [3H]U69593 and were consistent with binding to the kappa-opioid binding site. In the intestinal layers underlying the longitudinal muscle-myenteric plexus [3H]bremazocine, under kappa-selective conditions, recognized both a high and low affinity site. In contrast, [3H]U69593 bound to a homogeneous population of binding sites. The [3H]U69593 binding site and the [3H]bremazocine high affinity site demonstrated comparable characteristics to the single, kappa site identified in the longitudinal muscle layer. The nature of the low affinity site was not investigated due to difficulties associated with low specific binding, and its significance therefore remains to be investigated.     

Effects of adenosine on contractile response of circular muscle in electrically stimulated guinea-pig ileum

The action of adenosine on the electrically induced mechanical response of circular muscle in isolated guinea-pig ileum has been investigated. Electrical stimulation (0.1 Hz) elicited the twitch response, which was completely abolished by tetrodotoxin (0.2 microM), morphine (1 microM) and atropine (0.1 microM). Adenosine (0.1-100 microM) markedly depressed the twitch response in a concentration-dependent manner, and the concentration-depression curve for adenosine was significantly shifted to the right in the presence of theophylline (30 microM). On the other hand, the contractile responses induced by acetylcholine (1-300 microM) were not effected by adenosine at all. The present investigation suggests that the twitch response is mediated through acetylcholine released from the intramural cholinergic nerves supplying the circular muscle of guinea-pig ileum, and adenosine has an inhibitory effect on the cholinergic transmission, probably via P1-purinoceptors.     

Tachykinin receptors in the guinea-pig isolated oesophagus: a complex system

1. The tachykinin receptors mediating contraction of isolated longitudinal strips of the guinea-pig oesophageal body were characterized with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) as well as the analogues, [Sar⁹,Met(O₂)¹¹]SP, [Nle¹⁰]NKA(4–10) and [MePhe⁷]NKB, selective for NK₁, NK₂ and NK₃, receptors, respectively. Experiments were performed both in the absence and presence of a cocktail of peptidase inhibitors, captopril (1 μM), thiorphan (1 μM) and amastatin (20 μM), in order to determine whether membrane bound proteases are important in the metabolism of tachykinins in this preparation.
2. All agonists produced concentration-dependent contractile effects. The presence of the peptidase inhibitors shifted the concentration-response curves of SP, [Nle¹⁰]NKA(4–10) and [MePhe⁷]NKB significantly leftwards and the concentration-response curve of NKB was shifted significantly rightwards. However, the EC₅₀ values were significantly different only for [Nle¹⁰]NKA(4–10) and NKB.
3. In the presence of the peptidase inhibitors, the EC₅₀ values of the selective agonists, [MePhe⁷]NKB (0.6 nM) and [Nle¹⁰]NKA(4–10) (66 nM) indicated the presence of both tachykinin NK₃ and NK₂ receptors. [MePhe⁷]NKB produced less than 50% of the maximal response obtained with the other agonists. Since [Sar⁹,Met(O₂)¹¹]SP produced a small response in the nanomolar concentration range in about 30% of the preparations tested, it is possible that some NK₁ receptors were also present.
4. Assuming competitive antagonism, the NK₂-selective antagonist SR 48,968 (30 nM) gave apparent pK_B values of 8.13 and 8.65 for [Nle¹⁰]NKA(4–10) in the absence and presence of peptidase inhibitors, respectively, supporting the presence of NK₂ receptors.
5. The NK₃-selective antagonist SR 142,801 (0.1 μM), suppressed responses to low (0.1–10 nM) concentrations of [MePhe⁷]NKB. These contractile responses to [MePhe⁷]NKB were also abolished by atropine (0.6 μM) suggesting that this response was mediated via cholinergic nerves.
6. It is concluded that the guinea-pig oesophagus is a complex system which has both NK₂ and NK₃ receptors and possibly some NK₁ receptors as well.

     

Some pharmacological properties of the circular and longitudinal muscle strips from the guinea-pig isolated ileum

A circular and a longitudinal muscle strip were prepared from adjacent parts of a guinea-pig ileum and a direct pharmacological comparison made under identical conditions. The longitudinal preparation was sensitive to acetylcholine, methacholine, carbachol, 5-hydroxytryptamine, histamine and nicotine, while the circular preparation was insensitive to 5-hydroxytryptamine, histamine and nicotine, and responded to the choline esters only in high concentrations. Incubation of the preparations with the anticholinesterase, mipafox (NN-diisopropylphosphodiamidic fluoride), sensitized both preparations to the action of acetylcholine; potentiation of the contraction of the longitudinal muscle was 16-times; that of the circular one 4,000-times. The longitudinal muscle was more sensitive than the circular muscle to acetylcholine whether both were treated with mipafox or not. Bradykinin and substance P both stimulated the longitudinal but not the circular muscle, an effect not modified after mipafox. Hyoscine antagonized the responses of the circular muscle strip, treated with mipafox, to acetylcholine and to histamine, but on the longitudinal muscle strip the response to histamine was not affected, the response to acetylcholine being competitively antagonized. Morphine, in the same concentrations on both circular and longitudinal muscle strips, antagonized the stimulant actions of nicotine and to a lesser extent of 5-hydroxytryptamine, but the responses to histamine on the longitudinal muscle strip were not antagonized by morphine which was in contrast to its action on the circular muscle strip. These observations showed that the main differences in the responses of the circular and longitudinal muscle of the guinea-pig ileum to drugs were in the intrinsic properties of the smooth muscle cells. In addition cholinesterase may protect the circular muscle cells. Finally the circular muscle strip preparation proved to be a useful tool to study the action of drugs on the nervous plexuses of the ileum of the guinea-pig.     

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.     

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)     

Electrophysiological and mechanical characteristics of histamine receptors in smooth muscle cells of the guinea-pig ileum

To elucidate the role of the histamine receptor in functions related to intestinal motility, we investigated the effects of histamine and its antagonists on electrical and mechanical activities in longitudinal and circular layers of the terminal region of the guinea-pig ileum. Histamine hyperpolarized the membrane in the circular smooth muscle cells by increasing the permeability of K+ and it transiently inhibited generation of the spike while resting tone was elevated. Cimetidine (CIM) inhibited the hyperpolarization and relaxation induced by histamine while mepyramine (MEP) inhibited the contraction but did not affect the histamine-induced hyperpolarization. In the presence of CIM, histamine did not depolarize the membrane but did lower the threshold potential required for generation of the spike potential, increased the appearance of the spike and enhanced the phasic contraction. Histamine, in 20 mM K+ solution, hyperpolarized the membrane and produced a biphasic response, an initial relaxation and a subsequently generated contraction, in a concentration-dependent manner. In the longitudinal smooth muscle cells, histamine depolarized the membrane, and enhanced both generation of the spike and the contraction. MEP (0.1 microM) but not CIM (1 microM) blocked the histamine-induced responses. CIM at a higher concentration (10 microM) enhanced the histamine-induced contraction, while histamine did not relax the tissue precontracted by 20 mM K+. These results indicate that the circular muscle cells have both H1 and H2 receptors while the longitudinal muscle cells have the H1 receptor. The excitatory responses induced by activation of the H1 receptor in smooth muscle cells differ in these layers.     

Role of kappa opioid receptors in modulating cholinergic twitches in the circular muscle of guinea-pig colon.

1. Single pulse electrical field stimulation (EFS, 0.5 ms pulse width, 60 V at a frequency of 0.05 Hz) induced twitch contractions of mucosa-free circular muscle strips from the guinea-pig proximal colon which were abolished by atropine (0.3 microM), tetrodotoxin (0.3 microM) or omega-conotoxin GVIA (0.1 microM). 2. Various opioid receptor agonist concentration-dependently inhibited twitches with the following rank order of potency (EC50 values in brackets): U 50488 (0.31 nM) > dermorphin (4.3 nM) = dynorphin A (1-13) (6.2 nM) > [D-Ala2, N-MePhe4, Gly5-ol]-enkephalin (DAMGO, 33.5 nM) = [D-Ala2, D-Leu5]-enkephalin (DADLE, 60 nM) > [D-Pen2, D-Pen2, D-Pen5]-enkepahlin (DPDPE, 1144 nM). 3. Peptidase inhibitors (captopril, thiorphan and bestatin, 1 microM each) did not modify the amplitude of twitches. In the presence of peptidase inhibitors the concentration-response curve to dynorphin A (1-13) was displaced to the left to yield an EC50 of 0.35 nM, comparable to that of the selective kappa receptor agonist, U50488. The curves to the other opioid receptor agonist were unaffected by peptidase inhibitors. 4. DPDPE, DADLE, dermorphin and DAMGO consistently induced a concentration-unrelated transient increase in basal tone and a small and transient facilitation of twitches before development of their inhibitory effect. These transient excitatory effects were not observed upon application of dynorphin A (1-13) or U 50488. The contraction produced by DPDPE (30 nM) was largely inhibited (> 80%) by 1 microM atropine. 5. Twitches suppression induced by dynorphin A (1-13) (30 nM) was partly reversed (46 +/- 8%, n = 6) by naloxone (0.3 microM). The potent and selective kappa opioid receptor antagonist nor-binaltorphimine (Nor-BNI, 3-100 nM)) did not affect the amplitude of twitches and potently antagonized (pKB 9.83 +/- 0.09, n = 10) the inhibitory effect of dynorphin. 6. Naloxone (1-300 nM) concentration-dependently depressed the cholinergic twitches: this depressant effect was largely counteracted in the presence of apamin (0.1 microM) and NG-nitro-L-arginine (30 microM) which potentiated cholinergic twitches on their own. 7. Dynorphin A (1-13) (10 nM, n = 6) did not affect the contractile response to exogenous acetylcholine (1 microM), indicating that depression of evoked twitches occurs prejunctionally. 8. We conclude that multiple opioid receptors modulate cholinergic twitches in the circular muscle of guinea-pig proximal colon. While mu and delta opioid receptor agonists produced mixed excitatory and inhibitory effects, kappa opioid receptors, activated by sub-nanomolar concentrations of dynorphin A (1-13), mediate a powerful and pure prejunctional inhibition of acetylcholine release.