Myenteric Plexus-longitudinal Muscle Strips Research Articles

Interaction of αβ-Methylene ATP with the Cholinergic Twitch Response in the Guinea-Pig Ileum

In this study, direct effects of the P2X purinoceptor agonist αβ-methylene ATP (αβ-meATP) and effects on the cholinergic twitch response of the electrically stimulated longitudinal muscle-myenteric plexus (LMMP) strip of the guinea-pig ileum, were investigated. αβ-meATP (1, 3, and 10 µmol/l) induced short-lasting contractions on its own, followed by an inhibition of the twitch response during its presence in the organ bath. The inhibitor of small conductance Ca²⁺-activated K⁺ (SK) channels, apamin (100 nmol/l), prevented the inhibitory effect of αβ-meATP on the twitch response, whereas tetraethylammonium (300 µmol/l), a blocker of voltage-gated K⁺ channels and an inhibitor at nicotinic acetylcholine receptors, augmented the inhibitory effect of αβ-meATP on the twitch response. It is concluded, that there is a functional interaction between P2X receptors and nicotinic receptors in the LMMP strip, and that a major part of the excitatory input to the cholinergic motor neuron evoking the twitch response is purinergic and not nicotinergic.

Nicotine-Induced Modulation of the Cholinergic Twitch Response in the Ileum of Guinea Pig

In the present study, the direct drug effects of nicotine and its effects on the cholinergic twitch responses of the electrically stimulated longitudinal muscle-myenteric plexus strip from the ileum of guinea pig were investigated. Nicotine dose-dependently (0.3-10 µmol/l) evoked the well-known contractile responses on its own. Whereas the interposed twitch responses remained present without a change in height at 1 µmol/l nicotine, a nicotine concentration of 3 µmol/l slightly and a concentration of 10 µmol/l markedly diminished the twitch during their presence. After the washout of 1-10 µmol/l nicotine, the height of the twitch response was also temporarily and significantly reduced by 30-77%. The P2X purinoceptor agonist αβ-methylene ATP (1-10 µmol/l) dose-dependently induced contractions on its own and reduced the twitch response during its presence in the organ bath; however, it did not diminish the twitch responses after washout of the drug as nicotine did. The P2X antagonist pyridoxalphosphate-6-azophenyl-2′-4′-disulphonic acid, the NMDA channel blocker MK-801 and the inhibitor of small conductance Ca²⁺-activated K⁺ (SK) channels apamin reduced the contractile effect of 1 µmol/l nicotine. Apamin also significantly prevented the ‘post-nicotine inhibition of the twitch' following the washout of 1-3 µmol/l nicotine. As a conclusion, we provide evidence for a functional interaction between nicotinic receptors and the P2X receptors in the ileum of the guinea pig. The ‘post-nicotine inhibition of the twitch' is not due to nicotinic acetylcholine receptor desensitization or transmitter depletion, but most probably the secondary effects of nicotine on SK channels determine the reduced cholinergic motor neuron excitability.

Non-synaptic Cholinergic Modulation of Neurogenic Twitches of the Guinea-pig Ileum

The effect of cholinergic and anticholinergic compounds on conduction of neuronal excitation has been studied in myenteric plexus-longitudinal muscle strips from the guinea-pig ileum. A preparation in a special triple bath was drawn through two rubber membranes dividing the strip into three segments. Neurogenic stimulation of the oral segment set up nerve action potentials propagating aborally across the middle segment (10 mm) so that the aboral segment might be also invaded, eventually. Drugs were added to the middle segment to affect neuronal propagation (non-synaptic effects) which was monitored by twitch height of the aboral segment. The application of acetylcholine to the middle segment augmented aboral twitches. The effects of nicotine, pilocarpine and oxotremorine were selectively blocked by (+)-tubocurarine, pirenzepine and atropine, respectively. The effect of acetylcholine was suppressed by pirenzepine and atropine and mimicked by doubling of KCl concentration. The effect of acetylcholine may be thus explained by the facilitated propagation of nerve action potentials in partially depolarized cholinergic terminals via stimulation of muscarinic receptors. The adenylate cyclase system is not directly involved in the mechanism of muscarinic facilitation of neuronal propagation in the terminals; however, it may participate in the modulation of a final common effector mechanism.

Activation of a myenteric 5-hydroxytryptamine-like receptor by metoclopramide.

Cholinergically mediated contractions were evoked by electrical field stimulation (EFS) of guinea-pig distal ileum longitudinal muscle-myenteric plexus strips. Metoclopramide, 0.1-100 microM, dose-dependently increased the contractions, probably by increasing acetylcholine (ACh) release; contractions evoked by exogenous ACh in the presence of tetrodotoxin were not increased by metoclopramide. 5-Hydroxytryptamine (5-HT) 0.3-30 nM similarly increased the height of the EFS-evoked contractions, although the maximum increase was less than for metoclopramide; higher concentrations of 5-HT (3 and 30 microM) had no effects or caused inhibition. The compound, ICS 205-930, 0.001-1 microM, had no effect on the EFS-evoked contractions and caused inhibition at higher concentrations. Preincubation of the tissues with 5-HT, 0.3-30 nM, did not affect the increase in EFS-evoked contractions caused by metoclopramide, 1 or 100 microM, whereas 5-HT, 3 and 30 microM, prevented the response caused by metoclopramide 1 microM, but not 100 microM. ICS 205-930, 0.1 microM, had no effect on the increase in EFS-evoked contractions caused by metoclopramide 1 or 100 microM. The drug, at least in low concentrations, may therefore increase cholinergically mediated contractions of guinea-pig ileum by stimulating 5-HT-like receptors within the myenteric plexus, which differ from those antagonized by ICS 205-930.

Inhibition of calcium/calmodulin‐dependent protein kinase kinase (CaMKK) and calcium/calmodulin‐dependent protein kinase II (CaMKII) reduces colonic longitudinal muscle contraction by inhibition of myosin light chain kinase (MLCK)

Contraction of longitudinal muscle by a muscarinic agonist is regulated by the opposing actions of MLCK and myosin light chain phosphatase (MLCP) upon the phosphorylation level of the 20-KDa subunit of myosin light chain. However, the signaling pathways leading to alterations of MLCK and MLCP activity are poorly understood. Aim To determine the role of CaMKK and CaMKII in the regulation of colonic longitudinal muscle contraction. Methods Longitudinal muscle-myenteric plexus strips were prepared from rat colon for measurement of isometric contraction in organ chambers or used to prepare tissue lysates for immunokinase assay. Strips were exposed to CaMKK inhibitor STO-609, or CaMKII inhibitor KN-62, and carbachol (CCh; 0.01–100 uM) induced contraction or MLCK activity was measured and compared to control. Results CCh-induced contraction in colonic longitudinal muscle was significantly attenuated by both STO-609 (22–42% inhibition) and KN-62 (42–49% inhibition). Consistent with their effect on contraction, STO-609 and KN-62 significantly reduced MLCK activity (68 and 64% inhibition, respectively) as measured by immunokinase assay. Conclusion Contraction of longitudinal muscle from rat colon in response to muscarinic receptor stimulation is mediated partly through activation of CaMKK and CaMKII. Supported by NIDDKD.

Analgesic activity and pharmacological characterization of N-[1-phenylpyrazol-3-yl]- N-[1-(2-phenethyl)-4-piperidyl] propenamide, a new opioid agonist acting peripherally

We previously reported the synthesis of three new opioid agonists as well as their in vitro and in vivo activity [Girón, R., Abalo, R., Goicoechea, C., Martín, M.I., Callado, L.F., Cano, C., Goya, P., Jagerovic, N. 2002. Synthesis and opioid activity of new fentanyl analogs. Life Sci. 71, 1023–1034]. One of them, N-[1-phenylpyrazol-3-yl]- N-[1-(2-phenethyl)-4-piperidyl)] propenamide (IQMF-4) , showed an interesting antinociceptive activity. Intraperitoneally (i.p.) administered, it was as effective as fentanyl or morphine, being less potent than fentanyl but more so than morphine. The aim of the present work was to evaluate its antinociceptive effect by different routes of administration, using the hot plate test, and to investigate possible side effects, such as tolerance and withdrawal, in vitro, using the myenteric plexus-longitudinal muscle strip preparation from guinea pig ileum, and in vivo, using the hot plate test. IQMF-4 was more potent than morphine when administered per os (p.o.), but less potent when administered intracerebroventricularly (i.c.v.). By both routes, fentanyl is more potent that IQMF-4. When IQMF-4 was administered i.p., naloxone methiodide, a peripherally acting antagonist, was able to completely block its antinociceptive effect, whereas, after i.c.v. administration, the blockade was only partial. An interesting feature of the new compound is that it induces tolerance in vitro but not in vivo. Moreover, though i n vitro withdrawal was not different from fentanyl or morphine, in vivo withdrawal symptoms were significantly less frequent in mice treated with IQMF-4 than in those treated with morphine or fentanyl. Although more assays are required, these results show that IQMF-4 appears to be a potent analgesic compound with an interesting peripheral component, and reduced ability to induce dependence.

Stimulus-evoked opioid inhibition in guinea-pig longitudinal muscle-myenteric plexus strip is modulated by NMDA receptors

Longitudinal muscle-myenteric plexus (LMMP) strips of the guinea-pig ileum were used to investigate the stimulus-evoked endogenous opioid inhibition and its modulation by ionotropic glutamate receptors. Regular cholinergic twitch responses evoked by a short 3-s-field stimulation in intervals of 80 s were found reduced after an interposed period of prolonged 40-s-field stimulation. In the presence of a peptidase-inhibitor-cocktail, the cholinergic twitch response following the period of prolonged stimulation was even further reduced as compared to normal Tyrode solution without peptidase inhibitors. In both instances, the impairment of the cholinergic twitch response was completely abolished by naloxone thus demonstrating its opioidergic nature. This endogenous inhibitory opioid effect was significantly mitigated by the NMDA-receptor antagonist MK-801, but not by the AMPA/kainate receptor antagonist CNQX. These results demonstrate by functional experiments that there is a significant opioid-mediated inhibition in guinea-pig LMMP preparations evoked by a prolonged electrical stimulation, and that an NMDA antagonist can mitigate the opioid inhibition.

Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3- 4- 5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]- N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by μ selective antagonists (naloxone and/or CTOP) but not by the δ selective antagonist naltrindole, thus indicating that the compound acted as a μ opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the μ-receptor.

Blockade by pertussis toxin of the opioid effect on guinea pig ileum. Contractility and electrophysiological neuronal recording

The aim of the present study was to analyse the correlation between functional and electrophysiological effects of the opioids in guinea pig ileum. Preparations of guinea pig ileum myenteric plexus-longitudinal muscle strips were used to compare the effect of two opioids, morphine (a μ-agonist) and U-50,488H (a κ-agonist) on the electrically-induced contractile response and the excitatory postsynaptic potentials exhibited by the myenteric neurones when the internodal strands are electrically stimulated. Both opioids dose-dependently inhibited the contractile responses and the fast excitatory postsynaptic potentials (fEPSPs) in S neurones but did not modify the amplitude of the slow postsynaptic potentials. Pertussis toxin pretreatment was able to antagonise the effects of both morphine and U-50,448H. From our results it can be suggested that the effect of the opioids on guinea pig ileum involves the inhibition of neuronal fEPSPs.

Evidence for a neurotransmitter role of glutamate in guinea pig myenteric plexus neurons

Longitudinal muscle-myenteric plexus strips of the guinea pig ileum were used to investigate a possible transmitter role of glutamate (Glu) in the enteric nervous system. Glu was released from this nerve muscle preparation by high K +-depolarization in a Ca 2+-dependent manner, by electrical field stimulation and by the ganglionic stimulant dimethylphenylpiperazinium which indicates its neural origin. Contractions of the longitudinal muscle evoked by electrical stimulation of the myenteric nerves or by Glu, were significantly reduced by the N-methyl- d-aspartate (NMDA)-receptor antagonist FR115427 (9 and 18 μM), whereas contractions induced by histamine were unaffected. The results show that the amino acid Glu is likely to play an excitatory neurotransmitter role via NMDA receptors in the myenteric plexus of the guinea pig.

Effect of butanedione monoxime on the contractility of guinea pig ileum and on the electrophysiological activity of myenteric S-type neurones

2,3-Butanedione monoxime (BDM) has demonstrated protective effects on isolated cardiac tissues, and on smooth muscle but its mechanism of action is not fully understood. To simultaneously study the effect of BDM on muscle contractility and on neuronal activity, the effect of BDM was tested in the contractile force of myenteric plexus-longitudinal muscle strips and in electrophysiological activity of myenteric S-type neurones of guinea pig ileum. BDM reduces, in a dose-dependent manner, the force of the spontaneous motility and the contractions induced by acetylcholine, bethanechol and electrical stimulation. The same BDM concentrations depolarize the neuronal membrane and reduce the rate of evoked firing. The effect of BDM can be attributed to a direct effect on the smooth muscle and to modifications of the neuronal activity.

Effects of mercuric chloride and methyl mercury on cholinergic neuromuscular transmission in the guinea-pig ileum.

The effects of mercuric chloride (HgCl2) and methyl mercury (MeHg) were examined on basal mechanical activity and electrically-induced neurogenic cholinergic contractions (twitch contractions) in longitudinal muscle-myenteric plexus strips from guinea-pig distal ileum. Both compounds at 0.33 microM slightly enhanced the amplitude of twitch contractions in approximately 50% preparations. This effect was probably due to facilitation of acetylcholine (ACh) release since 0.1 and 1 microM mercurials increased electrically-evoked tritium outflow from [3H]choline preloaded muscle layer with attached myenteric plexus. Conversely, higher mercury concentrations inhibited twitch contractions (HgCl2 IC50 = 21.3 +/- 6.4 microM; MeHg IC50 = 45.1 +/- 5.5 microM), as well as contractions to exogenous ACh (0.1 microM) in resting preparations, and concomitantly increased the basal tone. The former effects possibly reflected an antimuscarinic activity of mercury, while the latter was related to alterations of calcium homeostasis in the effector cells. Indeed, the effect of HgCl2 on basal tone was antagonized by the Ca2+ entry blocker nifedipine (3, 10, 30 nM), indicating Hg-induced facilitation of Ca2+ influx through voltage-dependent channels. On the whole, our results suggest that cholinergic neuromuscular transmission and Ca(2+)-dependent mechanisms underlying smooth muscle contractility are targets for mercury toxicity in the intestine.

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine.

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed. 2. NO (10(-7) M) induced only contraction while 10(-6) M NO induced contraction followed by relaxation. Methylene blue (up to 10(-4) M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10(-6) M NO. Administration of 8-bromo-cyclic GMP (10(-6)-10(-4) M) only induced relaxation. 3. Sodium nitroprusside (SNP; 10(-7)-10(-5) M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3':5'-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10(-8) M NO and 10(-6) M SNP; it was increased by a factor of 2 during contraction with 10(-7) M NO, and by a factor of 12 during relaxation with 3 x 10(-6) M NO. 4. The NO-induced contractions were not affected by ryanodine (3 x 10(-5) M) but were concentration-dependently reduced by nifedipine (10(-8)-10(-7) M) and apamin (3 x 10(-9)-3 x 10(-8) M). 5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca2+ influx through L-type Ca2+ channels, that might be activated in response to the closure of Ca(2+)-dependent K+ channels.

Modulation of kappa-opioid receptor mediated tolerance in the guinea-pig ileum by chronic co-administration of dihydropyridines.

The influence of the L-type Ca2+ channel modulators nimodipine (a Ca2+ blocker) and BAY K 8644 (a Ca2+ activator) on the expression of tolerance to the inhibitory effects of kappa- and mu-opioid agonists in the guinea-pig ileum from guinea-pigs rendered tolerant to the kappa-opioid receptor agonist U-50,488H was investigated. Tolerance to U-50,488H was induced by its administration (15 mg/kg twice a day) for 4 days. Control groups received saline at the same time schedule. Chronic infusion of guinea-pigs with nimodipine (2 micrograms/microliters/h for 7 days) or BAY K 8644 (0.5 microgram/microliters/h for 7 days), did not cause any modification of the height of contractions induced by electrical stimulation of the myenteric plexus-longitudinal muscle (MPLM) preparation from naïve guinea-pigs. The Ca2+ antagonist nimodipine increases the potency of U-50,488H (selective kappa agonist) to reduce the amplitude of neurogenic contractions of the MPLM strip in naïve animals, whereas the Ca2+ activator BAY K 8644 induced the opposite effect. However, the effect of DAMGO (selective mu agonist) was not modified in guinea-pigs infused with nimodipine or BAY K 8644. Tolerance to the inhibitory effects of both U-50,488H and DAMGO was observed following administration of U-50,488H for 4 days and was revealed as a rightward shift of the concentration-response curves for the two agonists. Chronic infusion of guinea-pigs with nimodipine concurrently with chronic U-50,488H, markedly attenuated the expression of selective tolerance to U-50,488H as well as the cross-tolerance between U-50,488H and DAMGO. By the contrary, the magnitude of tolerance to U-50,488H and to DAMGO was enhanced by concomitant infusion of BAY K 8644. The results suggest that, in the GPI, kappa-opioid receptor may be functionally linked to the dihydropyridine sensitive Ca2+ channel: The blockade of the channel increased whereas its activation reduced the potency of U-50,488H. In chronic experiments, nimodipine prevented the expression of tolerance to U-50,488H and the cross-tolerance between U-50,488H and DAMGO, whereas BAY K 8644 produced the opposite effect. These results suggest that, in the GPI, selective tolerance to kappa-agonist as well as cross-tolerance between kappa- and mu-opioid agonists would involve activation of L-type Ca2+ channels, which could indicate that intracellular Ca2+ may be the final common pathway through which myenteric neurons adapt to the chronic opioid exposure.

Effect of chronic administration of dihydropyridine Ca 2+ channel ligands on sufentanil-induced tolerance to μ- and κ-opioid agonists in the guinea pig ileum myenteric plexus

The present investigation was aimed at elucidating if the entry of Ca 2+ plays a role in the development of tolerance to μ- and κ-opioid agonists in the guinea pig ileum myenteric plexus. For this purpose, the influence of the L-type Ca 2+ channel modulators nimodipine (Ca 2+ blocker) and Bay K 8644 (Ca 2+ activator) on the expression of tolerance to the inhibitory effects of μ- and κ-opioid agonists in the ileum of guinea pigs rendered tolerant to sufentanil was investigated. Chronic perfusion of guinea pigs with nimodipine (2 μg/μl/h for 7 days) or Bay K 8644 (0.5 μg/μl/h for 7 days) did not cause any modification of the height of contractions induced by electrical stimulation of the myenteric plexus-longitudinal muscle (MPLM) strip from naive animals. Tolerance to sufentanil (a selective μ-agonist) was induced by s.c. implantation of osmotic minipumps for 7 days, which deliver at 2 μg/μl/h. Control groups received saline. Tolerance to sufentanil as well as to U-50,488H (selective κ-agonist) was observed following chronic treatment with sufentanil and was revealed as a rightward shift of the concentration-response curves. Chronic perfusion of guinea pigs with the Ca 2+ antagonist nimodipine concurrently with chronic sufentanil, markedly blocked the expression of tolerance to sufentanil, as well as the cross-tolerance between sufentanil and U-50,488H. On the contrary, when guinea pigs were perfused with the Ca 2+ agonist Bay K 8644 concurrently with sufentanil, it enhanced the magnitude of tolerance to both sufentanil and U-50,488H. These results suggest that, in guinea pig ileum, chronic exposure to opioids may involve the activation of L-type Ca 2+ channel, which would indicate that intracellular Ca 2+ may be one of the final pathways through which myenteric neurons adapt to the chronic opioid exposure.

Nitric oxide causes contraction in the rat isolated small intestine

The effect of authentic nitric oxide (NO) was investigated on rat ileal myenteric plexus-longitudinal muscle strips in the presence of atropine. NO (10 −10 to 3 × 10 −6M) caused a quick, transient contraction of the smooth muscle, with an optimum concentration of 10 −7M, as well as a slower contraction with a higher optimum concentration. In preparations sub-maximally pre-contracted with 5-hydroxytryptamine, NO (10 −7 to 3 × 10 −6M) also caused a quick relaxant response. These responses to NO (10 −6M) were not influenced by tetrodotoxin, indicating a direct effect on the smooth muscle.

Studies on the mechanism of the contractile action of rat calcitonin gene-related peptide and of capsaicin on the guinea-pig lleum: Effect of hCGRP (8–37) and CGRP tachyphylaxis

Both the neuropeptide calcitonin gene-related peptide (CGRP) and the sensory stimulant drug capsaicin exert excitatory as well as inhibitory actions in the guinea-pig ileum. It has been known that their excitatory effects are due to activation of myenteric neurons. In the present study it has been established that a fairly high concentration (3 μM) of the CGRP antagonist human alpha CGRP (8–37) [hCGRP (8–37)], which has been known to reverse the inhibitory effects of rCGRP, failed to inhibit longitudinal contractions of ileal longitudinal muscle-myenteric plexus strips due to rat (r) CGRP. Instead, a slight enhancement of the contractions was observed, probably due to the inhibition of the relaxant effect. Tachyphylaxis to rCGRP was able to markedly reduce the contractile action of rCGRP but did not influence contractions due to capsaicin (1 μM). It is concluded that myenteric neuronal excitation due to rCGRP involves receptors not sensitive to hCGRP (8–37). Moreover, neuronal excitation by capsaicin does not seem to involve a CGRP-like mediator.

In vitro study of the interaction of salmon calcitonin with ?, ? and ? opioid agonists

A possible interaction of salmon-calcitonin with opioid systems was studied in isolated tissues. Neurogenic contractions were elicited by electrical stimulation in guinea-pig ileum myenteric plexus-longitudinal muscle strips, rabbit vas deferens and mouse vas deferens. Bremazocine inhibited neurogenic contractions in all three tissues (presumably through kappa-receptors) [D-Pen2, D-Pen5]enkephalin and [Met5]enkephalin inhibited contractions in mouse vas deferens (presumably through delta-receptors), and [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) inhibited contractions in guinea-pig ileum and mouse vas deferens (presumably through mu-receptors). All inhibitory effects were concentration-dependent. Salmon-calcitonin 0.1 IU/ml increased the effect of bremazocine in guinea-pig ileum and rabbit vas deferens and also increased the effects of [D-Pen2, D-Pen5]enkephalin and [Met5]enkephalin in mouse vas deferens. In contrast, salmon-calcitonin up to 0.4 IU/ml did not change the effect of bremazocine in mouse vas deferens and the effect of DAMGO in guinea-pig ileum and mouse vas deferens. It is concluded that salmon-calcitonin enhances agonist effects at opioid kappa- and delta- but not at opioid mu-receptors. The level of this interaction remains to be elucidated. The interaction may be the basis of the analgesic effect of salmon-calcitonin in vivo.

Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus

The effects of three different opioid agonists on contractions and [3H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine3 (5-HT3) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu (mu)-opioid receptor agonist (D-Ala2,NMe-Phe4,Gly-ol]-enkephalin (DAMGO; 1 nM-100 nM) and the selective kappa (kappa)-opioid receptor agonist U50488 (10 nM-1 microM) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC50 estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM-51 nM. The selective delta (delta)-opioid receptor agonist [D-Pen2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 microM). 3H-overflow from LMMP preparations preincubated with [3H]-choline was measured as an indicator of [3H]-ACh release. DAMGO (1 nM-100 nM) and U50488 (10 nM-1 microM) inhibited the increases in release of [3H]-ACh evoked by 5-HT (10 microM) and by senktide (10 nM) in a concentration-dependent manner. IC50 estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [3H]-ACh release and lay in the range 6 nM-23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

An attempt to localize the site of action of different agents within cholinergic motor neurones of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum by the triple bath method

The site of action of cholinergic, adrenergic, peptidergic and opioid agents was studied in myenteric plexus-longitudinal muscle strips from the guinea pig ileum. A preparation in a special triple bath was drawn through two rubber membranes, dividing the strip into three segments. Neurogenic stimulation of the oral segment, set up nerve action potentials also in the neurones projecting axons up to the aboral segment. These axons, turning into varicose nerve terminals, conducted action potentials aborally across the middle segment, that was up to 10 mm wide. Finally, the nerve terminals, extending into the aboral segment, might be also invaded triggering twitches. Agents were added, either to the oral segment, to affect the genesis and spread of action potentials in the proximal parts of cholinergic neurones (cell bodies, axon hillocks, initial segments and axon preterminals) or they were added to the middle segment to affect propagation of action potentials in varicose nerve terminals. As a result, the amplitude of aboral twitches reflected their effects at each site, quantitatively. Noradrenaline and ethylketocyclazocine were more effective at the site of varicose nerve terminals, whereas substance P, acetylcholine and oxotremorine were more effective at the proximal parts; pilocarpine and nicotine were effective at both sites. Changes in membrane polarization might be the final common effect in the mechanism of action of all the stimulatory agents used.