Differential actions of urocortins on neurons of the myenteric division of the enteric nervous system in guinea pig distal colon

Background and purpose:

Urocortins (Ucns) 1, 2 and 3 are corticotropin-releasing factor (CRF)-related neuropeptides and may be involved in neural regulation of colonic motor functions. Nevertheless, details of the neural mechanism of action for Ucns have been unclear. We have, here, tested the hypothesis that Ucns act in the enteric nervous system (ENS) to influence colonic motor behaviour.

Experimental approach:

We used intracellular recording with ‘sharp’ microelectrodes, followed by intraneuronal injection of biocytin, and immunohistochemical localization of CRF₁ and CRF₂ receptors in guinea pig colonic tissue.

Key results:

Application of Ucn1 depolarized membrane potentials and elevated excitability in 58% of AH-type and 60% of S-type colonic myenteric neurons. In most of the neurons tested, depolarizing responses evoked by Ucn-1 were suppressed by the CRF₁ receptor antagonist NBI 27914, but were unaffected by the CRF₂ receptor antagonist antisauvagine-30. The selective CRF₂ receptor agonists, Ucn2 and Ucn3, evoked depolarizing responses in 12 and 8% of the AH-type myenteric neurons, respectively, and had no effect on S-type neurons. Antisauvagine-30, but not NBI 27914, suppressed these Ucn2- and Ucn3-evoked responses. Immunohistochemical staining identified CRF₁ as the predominant CRF receptor subtype expressed by ganglion cell somas, while CRF₂-immunoreactive neuronal somas were sparse. Ucns did not affect excitatory synaptic transmission in the ENS.

Conclusions and implications:

The results suggest that Ucns act as neuromodulators to influence myenteric neuronal excitability. The excitatory action of Ucn1 in myenteric neurons was primarily at CRF₁ receptors, and the excitatory action of Ucn2 and Ucn3 was at CRF₂ receptors.     

Pharmacological characterization of canine bradykinin receptors in prostatic culture and in isolated prostate

The objective of this study was to characterize pharmacologically bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, BK) receptors in the canine prostate. Primary cultures of canine prostate stromal (PS) and epithelial cells (PE) were established and then characterized using cell-specific antibodies (actin, vimentin and cytokeratin). Cultured cells were assayed for BK receptors using fluorometric imaging plate reader assays. In addition, isolated strips of the canine prostate were studied for BK-induced isometric contraction. PS cells were labeled only with anti-actin and -vimentin antibodies, while the anti-cytokeratin antibodies labeled only the PE cells. In cultured prostate cells, the BK receptor 2 (B2)-preferring agonist BK induced mobilization of intracellular Ca(2+) in a concentration-dependent manner with potencies (log[EC(50)]mid R:PE, pEC(50)) of 8.72+/-0.12 in PS and 8.75+/-0.06 in PE cells. In contrast, the BK receptor 1 (B1)-selective agonist [des-Arg(9)]BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) did not elicit any significant effect (pEC(50)<5) on Ca(2+) responses. BK agonism (10 nm) was inhibited by HOE-140 (D-arginyl-L-arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahhydro-3-isoquinolinecarbonyl-L-(2a,3b,7ab)-octahydro-1H-indole-2-carbonyl-L-arginine), a B2-selective antagonist, with a log[IC(50)] (pIC(50)) of 8.11+/-0.19 and 9.23+/-0.20 in PS and PE cells, respectively. [des-Arg(10)]HOE-140 (d-arginyl-l-arginlyl-l-prolyl-trans-4-hydroxy-l-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-(2a, 3b,7ab)-octahydro-1H-indole-2-carbonyl), a B1-selective antagonist, displayed weak antagonism with pIC(50) values of 4.87+/-0.23 and 6.38+/-0.16 in PS and PE cells, respectively. Isolated tissue strips of the canine prostate contracted to BK (10 microm) but not to [des-Arg(9)]BK (10 microm). BK-induced contractility was attenuated by HOE-140 (1 microm). In conclusion, canine prostates express functional B2 receptors, with no apparent B1 receptor subtypes.     

Mediation by B1 and B2 receptors of vasodepressor responses to intravenously administered kinins in anaesthetized dogs.

1. Vasodepressor responses to intravenous (i.v.) injection of bradykinin (BK) and des-Arg9-BK, a selective B1 kinin receptor agonist, were characterized following i.v. pretreatment with selective B1 ([Leu8]-des-Arg9-BK) and B2 (Hoe 140) kinin receptor antagonists in anaesthetized dogs. 2. Des-Arg9-BK (0.05-3.3 nmol kg-1) produced dose-dependent decreases in mean arterial blood pressure with a ED50 0.4 nmol kg-1. The vasodepressor effects evoked by des-Arg9-BK (0.6 nmol kg-1) and BK (0.2 nmol kg-1) were greater after i.v. and i.a. injections, respectively. 3. The vasodepressor response to BK (0.6 nmol kg-1) but not to des-Arg9-BK (0.6 nmol kg-1) was significantly (P < 0.001) blocked by pretreatment with the B2 receptor antagonist, Hoe 140. 4. The vasodepressor response to des-Arg9-BK (0.6 nmol kg-1) but not to BK (0.6 nmol kg-1) was significantly (P < 0.001) reduced by pretreatment with the selective B1 receptor antagonist, [Leu8]-des-Arg9-BK. Although both B1 and B2 receptor antagonists caused a transient fall in blood pressure, their inhibitory action was unlikely to be related to a desensitization mechanism. 5. Inhibition of prostaglandin synthesis with indomethacin prevented the vasodepressor response induced by arachidonic acid (1 mg kg-1, i.v.) but not that to BK or des-Arg9-BK (0.6 nmol kg-1). 6. These results suggest, firstly, that the vasodepressor responses to i.v. BK and des-Arg9-BK are mediated by the activation of B2 and B1 receptors, respectively; secondly, that prostaglandins are not involved in the vasodepressor responses to kinins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of bradykinin on bradykinin-B2 receptor in rat aortic vascular smooth muscle cells and the involved signal transduction pathways

The aim of this paper is to study the effect of bradykinin (BK) on bradykinin-B2 receptor as well as the possible involved signal transduction pathways in cultured rat aortic vascular smooth muscle cells (VSMCs). Rat aortic VSMCs were cultured. Cells after 4–6 passages were used in the experiment. VSMCs were incubated with BK, BK + B2 receptor inhibitor (HOE-140), BK + MEK inhibitor (PD98059), BK + mitogen-activated protein kinase (MAPK) inhibitor (apigenin), BK + phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), and BK + Akt inhibitor to evaluate the expression of B2 receptor and phosphorylation of signaling molecules MAPK, Akt, and PI3K by Western blot. (1) BK markedly up-regulated the expression of B2 receptor in VSMC. (2) Apigenin, PD98059, Akt inhibitor, and LY294002 inhibited upregulation of B2 receptor induced by BK. (3) Signal transduction pathways of MAPK and PI3K were involved in the up-regulation of B2 receptor by BK mediation. Results suggest that bradykinin can up-regulate the expression of B2 receptor in VSMCs.     

The mechanism of action of narcotic analgesics in the guinea-pig ileum.

1. Intracellular recordings were made from neurones in the myenteric plexus of the guinea-pig ileum. Single myenteric ganglia were maintained in vitro and drugs were applied by adding them to the perfusing solution. 2. Narcotic analgesics hyperpolarized the membrane of a proportion of neurones in the myenteric plexus. 3. The membrane hyperpolarization was sometimes associated with a decrease in input resistance. These effects reduced the excitability of myenteric neurones. 4. The effects of narcotics occurred at low concentrations (10 nM to 1 micrometer), were stereospecific and were reversed by naloxone. 5. It is proposed that the morphine-sensitive neurones may be the cholinergic efferents to the muscle layers. By hyperpolarizing these neurones, morphine may prevent their excitation by electric field stimulation. This may explain why narcotic analgesics reduce the output of acetylcholine and the contractile response of this preparation when it is excited by field stimulation.     

Effect of a kinin B2 receptor antagonist on LPS- and cytokine-induced neutrophil migration in rats

1 This study examines the involvement of kinins in neutrophil migration into rat subcutaneous air pouches triggered by lipopolysaccharide (LPS), as well as the putative roles played by kinin B(1) and B(2) receptors, tumour necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and selectins in this response. 2 LPS (5 ng to 10 micro g cavity(-1)) injected into the 6-day-old pouch induced a dose- and time-dependent neutrophil migration which peaked between 4 and 6 h, and was maximal following the dose of 100 ng cavity(-1) (saline: 0.46+/-0.1; LPS: 43+/-3.70 x 10(6) cells cavity(-1) at 6 h). 3 Bradykinin (BK) (600 nmol) injected into the pouch of saline-treated rats induced only modest neutrophil migration (0.73+/-0.16 x 10(6) cells cavity(-1)). A more robust response to BK (3.2+/-0.25 x 10(6) cells cavity(-1)) was seen in animals pretreated with captopril, but this was still smaller than the responses to IL-1beta or TNF-alpha (15 pmol: 23+/-2.2 x 10(6) and 75 pmol: 29.5+/-2 x 10(6) cells cavity(-1), respectively). Nevertheless, the B(1) agonist des-Arg(9)-BK (600 nmol) failed to induce neutrophil migration. 4 HOE-140 (1 and 2 mg kg(-1)), a B(2) receptor antagonist, reduced LPS-induced neutrophil migration. HOE-140 also reduced the neutrophil migration induced by BK, but had no effect on the migration promoted by IL-1beta or TNF-alpha. des-Arg(9)-[Leu(8)]-BK, B(1) receptor antagonist was ineffective in changing neutrophil migration caused by any of these stimuli. 5 Neutrophil migration induced by LPS or BK was reduced by interleukin-1 receptor antagonist (IL-1ra) (1 mg kg(-1)), sheep anti-rat TNF serum (anti-TNF serum) (0.3 ml cavity(-1)), and the nonspecific selectin inhibitor fucoidin (10 mg kg(-1)). 6 TNF-alpha levels in the pouch fluid were increased by LPS or BK injection, peaking at 0.5-1 h and gradually declining thereafter up to 6 h. IL-1beta levels increased steadily throughout the 6 h period. HOE-140 markedly inhibited the rise in IL-1beta and TNF-alpha levels in pouch fluid triggered by both stimuli. 7 These results indicate that BK participates importantly in selectin-dependent neutrophil migration into the air pouch triggered by LPS in the rat, by stimulating B(2) receptors coupled to synthesis/release of TNF-alpha and IL-1beta.     

Pharmacological characterization of RMP-7, a novel bradykinin agonist in smooth muscle

RMP-7 is a bradykinin (BK) agonist designed to be resistant to kininases such as angiotensin-converting enzyme (ACE). Pharmacological assays were performed with RMP-7 in isolated guinea-pig ileum and rat mesenteric artery. RMP-7 induced contractile responses in the guinea-pig ileum, where the apparent affinity of the peptide (pD2) was significantly lower than that determined for BK (7.3 +/- 0.07 vs. 8.3 +/- 0.05, respectively). HOE-140 blocked this effect indicating that B2 receptor was involved. Captopril (1 microM) had no potentiating effect on RMP-7 but increased pD2 value determined for BK (8.8 +/- 0.1), confirming a high resistance of RMP-7 to the ACE. In rat mesenteric artery, RMP-7 induced endothelium-dependent relaxation (7.8 +/- 0.4), with a higher affinity than that of BK which induced vasodilatation only in the presence of 1 microM captopril (6.9 +/- 0.36). Nevertheless, the maximum effect induced by RMP-7 was lower than that of BK in contrast to that observed in guinea-pig ileum although B2 receptor was involved in both cases. We concluded that: RMP-7 is greatly resistant to the ACE and that the receptor sites activated by RMP-7 and BK show important differences in vascular and non-vascular preparations probably due to the different sensitivity of the B2 receptor to RMP-7.     

Localization of endothelin ETB receptors on the myenteric plexus of guinea-pig ileum and the receptor-mediated release of acetylcholine.

1. The type of endothelin (ET) receptor located on the myenteric neurones of guinea-pig ileum was determined by receptor autoradiography and function of the receptor was examined by release experiments of acetylcholine (ACh) from the longitudinal muscle myenteric plexus (LM-MP) preparations. 2. Specific [125I]-ET-1 binding sites were distributed in muscle layers, myenteric and submucous plexuses, and mucosa layers. High-grain densities were detected in both myenteric and submucous plexuses. 3. Binding in the myenteric plexus was abolished by incubation with either IRL 1620 (endothelin ETB receptor agonist) or BQ 788 (endothelin ETB receptor antagonist), but not with BQ 123 (endothelin ETA receptor antagonist). The [125I]-IRL 1620 binding sites were evident in the myenteric plexus. Thus, the endothelin receptor located on the myenteric neurones is of the ETB type. 4. ET-1 (10(-10)-3 x 10(-8) M) and ET-3 (10(-10)-3 x 10(-8) M) evoked 3H outflow from LM-MP preparations of ileum preloaded with [3H]-choline, in a concentration-dependent manner. There was no significant difference between maximum amounts of ET-1-evoked and ET-3-evoked 3H outflow. 5. ET-1 and ET-3 evoked outflow of 3H was BQ 788-sensitive, but BQ 123-insensitive. Both evoked outflows of 3H were Ca(2+)-dependent and tetrodotoxin-sensitive. 6. These results indicate that the endothelin ETB receptor is located on the enteric cholinergic neurones and that stimulation evokes the release of ACh.     

Implication of the bradykinin receptors in antigen-induced pulmonary inflammation in mice

1. The involvement of bradykinin (BK) receptors in the allergic inflammation associated with airway hyper-reactivity (AHR) was evaluated by means of the selective bradykinin B(1) receptor (BKB(1)-R) antagonists R-715 (Ac-Lys-[D-betaNal(7), Ile(8)]desArg(9)-BK) and R-954 (Ac-Orn[Oic(2), alpha-MePhe(5), D-betaNal(7), Ile(8)]desArg(9)-BK) or the selective bradykinin B(2) receptor (BKB(2)-R) antagonist HOE-140 (D-Arg(0)-Hyp(3)-Thi(5)-D-Tic(7)-Oic(8)-BK). Cellular migration and AHR were examined 24 h after the second ovalbumin (OA) challenge. 2. R-715 (10-500 microg kg(-1)) and R-954 (1-100 microg kg(-1)) injected intravenously (i.v.), 5 min prior to aerosol OA challenges, decreased by approximately 50% the induced lung eosinophilia in OA-sensitized mice but did not reduce AHR. 3. HOE-140 (1 microg kg(-1)) administered in the same manner, decreased mononuclear cell and eosinophil infiltration in the bronchoalveolar lavage fluid (BALF) of OA-sensitized mice. Moreover, treatment of OA-sensitized mice with HOE-140 (100 microg kg(-1)) completely abolished the AHR to carbachol. 4. The BKB(1)-R agonist desArg(9)-BK (DBK; 10-1000 microg kg(-1)) administered intratrachealy to normal mice had no effect on the basal cell counts recovered in BALF nor on the plasma extravasation, while the BKB(2)-R selective agonist BK (20 microg kg(-1)) stimulated mononuclear cell migration, neutrophilia and plasma extravasation in normal mouse lungs. Such effects were inhibited by HOE-140 (10 microg kg(-1)). 5. Our results suggest that the airway inflammatory response induced by antigen challenge in mice is mediated by stimulation of both BKB(1)-R and BKB(2)-R.     

Suppression of nicotinic synaptic transmission by adenosine in myenteric ganglia of the guinea-pig gastric antrum.

Conventional intracellular recording techniques were used to investigate actions of adenosine on nicotinic cholinergic transmission in myenteric neurons of the gastric antrum. Adenosine or the more potent derivatives, 5'-N-ethylcarboxamidoadenosine (NECA), 5'-N-cyclopropylcarboxamidoadenosine, 1-deaza-2-chloro-N6-cyclopentyladenosine or N6-cyclopentyladenosine reversibly and dose dependently inhibited the fast excitatory postsynaptic potentials (fast EPSPs) in 60% of the gastric neurons. Neither adenosine nor NECA affected excitatory responses to the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodine. The EC50 concentration for inhibition of the fast excitatory postsynaptic potential (EPSP) by adenosine was 55 microM NECA was a more potent inhibitor than adenosine. The specific adenosine receptor antagonists 1,3-dipropyl-8-p-sulfophenyl xanthine or 1,3-dipropyl-8-(cyclopentyl) xanthine blocked the inhibitory effects of adenosine or NECA. Fast EPSPs were enhanced by superfusion of the antagonists alone, suggestive of ongoing inhibition of nicotinic transmission by endogenous adenosine. The antagonists had no effect on resting membrane properties, excitability or antidromic action potentials. In neurons with suppression of fast EPSPs, adenosine did not suppress all cholinergic inputs to the same neuron. The results suggest that adenosine inhibits nicotinic transmission by interacting with presynaptic P1 adenosine receptors located at cholinergic release sites.     

Mianserin blocks alpha 2 adrenoceptors in submucous neurones of the guinea-pig caecum

Intracellular recordings were made from submucous plexus neurones of the guinea-pig caecum in vitro. The peak amplitude of the adrenergic inhibitory postsynaptic potential (IPSP) was depressed by mianserin in a dose-dependent manner (300 nM-100 microM). This was due to a direct blockade of postsynaptic alpha 2 adrenoceptors. The nicotinic excitatory postsynaptic potential (EPSP) and the non-cholinergic EPSP were not affected by mianserin (100 microM). The presynaptic inhibition of the release of acetylcholine, mediated by presynaptic alpha 2 receptors, was also blocked by mianserin (30 microM). The results suggest that mianserin antagonizes both pre- and post-synaptic alpha 2 adrenoceptors in enteric plexus neurones.     

Neuronal 5-HT receptors in the periphery

5-Hydroxytryptamine (5-HT) induces responses in neurones from all branches of the mammalian peripheral nervous system. Responses may be excitatory or inhibitory and are mediated through at least four distinct receptor sites.One receptor mediates excitation in motoneurones and preganglionic sympathetic neurones and can be designated a D (or possibly 5-HT₂) receptor since “classical” antagonists such as methysergide, metergoline or cinanserin are potent and selective antagonists at this site. A second receptor mediating neuronal excitation can be positively identified on the basis of susceptibility to blockade by small concentrations of 1αH,3α,5αH-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222) and the weak or negligible affinity, relative to 5-HT, of certain agonists such as 5-methoxytryptamine. Such sites mediate depolarization of sympathetic and parasympathetic neurones and excitation of both the cell bodies and terminals of primary afferent fibres. A third receptor, mediating neuronal excitation, is the classical M-receptor of Gaddum and Picarelli, at this stage clearly identified only on postganglionic parasympathetic neurones of the guinea-pig myenteric plexus. These sites can be differentiated from other excitatory 5-HT receptors since MDL 72222 is neither potent nor selective as an antagonist and 5-methoxytryptamine approaches the potency of 5-HT as an agonist. (3α-Homotropanyl)-1-methyl-5-fluoro-indole-3-carboxylic acid is a potent, surmountable antagonist of 5-HT at the M-receptor of the ileum, but is non-selective.Neuronal inhibitory responses have been observed using electrophysiological techniques or by monitoring the decrease in depolarization-evoked release of transmitter in enteric, parasympathetic and sympathetic neurones. Largely negative results, using selective agonists and antagonists, allow the receptor(s) mediating inhibition to be clearly differentiated from the three neuronal excitatory receptors for 5-HT. Comparison of relative potencies of agonists suggests similarities with the 5-HT₁ recognition site of the central nervous system; no selective antagonist has yet emerged to permit their positive identification.     

Bradykinin B2 receptor evoked K+ permeability increase mediates relaxation in the rat duodenum.

We have investigated the receptors and associated coupling mechanisms that mediate the smooth muscle relaxant response to bradykinin (BK) in the rat duodenum in vitro. Relaxation in response to BK seems due to a direct action on the longitudinal smooth muscle since effects were demonstrable in the presence of ibuprofen, mepyramine, atropine, guanethidine (all 1 microM), hexamethonium (10 microM) and TTX (0.3 microM). Receptors involved are of the B2 subtype since agonists and antagonists active at B1 receptors were essentially inactive, and the B2 receptor antagonist Lys,Lys-[Hyp3,Thi5,8,D-Phe7]BK was a potent competitive antagonist of BK-induced relaxation (pKB of 7.2 +/- 0.1). The activity of both BK and the antagonist were unchanged by the presence of peptidase inhibitors including the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (mergetpa, 10 microM), which prevents conversion of BK analogues to des-Arg9-B1-active products. In high-K+ solution, BK (0.1-10 microM) produced concentration-related increases in 86Rb efflux. Both this permeability increase in high-K+ solution, and the relaxant responses in Krebs solution, were inhibited by low concentrations (10-100 nM) of apamin, as well as the B2 receptor antagonist Lys,Lys-[Hyp3,Thi5,8,D-Phe7]BK (1 microM). These results are compatable with the proposal that BK-evoked relaxation of the rat duodenum is mediated via a subset of B2 receptors for which the antagonist Lys,Lys-[Hyp3,Thi5,8,D-Phe7]BK has a high affinity, and results from stabilisation of the smooth muscle membrane through the opening of apamin-sensitive 86Rb-permeable calcium-activated K+ channels.     

Opioid inhibition of synaptic transmission in the guinea-pig myenteric plexus.

Intracellular recordings were made from neurones in the myenteric plexus of the guinea-pig ileum. Presynaptic nerves were excited by a focal stimulating electrode on an interganglionic strand. Fast excitatory postsynaptic potentials (e.p.s.ps) were depressed in amplitude by morphine and [Met5]enkephalin in the concentration range of 1 nM-1 microM. Nicotinic depolarizations evoked by exogenously applied acetylcholine (ACh) were not affected by these opioids. Hyperpolarization of the presynaptic fibres probably contributed to the depression of the fast e.p.s.p. because fast e.p.s.ps evoked by low stimulus voltages were more depressed than those evoked by high stimulus voltages and fast e.p.s.ps resulting from activation of a single presynaptic fibre were blocked in a non-graded manner. Opioids depressed the slow e.p.s.p. in those neurones in which they did not change the resting membrane potential. The slow e.p.s.p. was increased in amplitude in those neurones hyperpolarized by opioids. Depolarizations resulting from application of barium, substance P or ACh were also enhanced by opioids. Equivalent circuit models in which opioids increase, and substance P or ACh decrease, the same potassium conductance could account for this enhancement. The actions of opioids were prevented or reversed by naloxone (1 nM-1 microM). It is concluded that morphine and enkephalin inhibit the release of ACh and a non-cholinergic transmitter from fibres of the myenteric plexus, and that this may involve a hyperpolarization of presynaptic fibres. Additionally, opioids can interact postsynaptically with other substances which affect membrane potassium conductances.     

The effects of calcium concentration on the inhibition of cholinergic neurotransmission in the myenteric plexus of guinea-pig ileum by adenine nucleotides.

1 Adenosine and the adenine nucleotides AMP, ADP, ATP, cyclic AMP, NAD, NADP and NADH produced a dose-related inhibition of the contractile response of guinea-pig ileum longitudinal muscle-myenteric plexus strips to low frequencies (less than 1 Hz) of electrical field stimulation. 2 These compounds inhibited hexamethonium-sensitive contractions induced by nicotine but did not alter the responses to exogenous acetylcholine, and the acetylcholine output from the myenteric plexus was inhibited by the adenyl compounds. These findings indicate that adenine derivatives act at a presynaptic site on postganglionic cholinergic neurones. 3 The degree of inhibition produced by adenine compounds was inversely related to the calcium concentration of the bath fluid over a range of calcium concentrations (1 to 5 mM) that had no effect on the responses of the muscle to exogenous acetylcholine. 4 The inhibition produced by adenine derivatives was antagonized by theophylline and augmented by dipyridamole. Both of these interactions were sensitive to, and synergistic with, alterations of the concentration of calcium in the bath fluid. 5 The results suggest that adenine compounds inhibit acetylcholine release from the myenteric plexus by diminishing the availability of intracellular calcium ions required for neurotransmitter release.     

Involvement of enteric neurones in the response of guinea-pig ileum preparations to metoclopramide.

The role of myenteric neurones in mediating the stimulant effects of metoclopramide in vitro in the guinea-pig ileum has been investigated using the non-ionic surfactant Triton X-100. Histological examination of the ileum 30 days after application of Triton X-100 to the serosal surface demonstrated a marked reduction in the number of ganglion cells and nerve elements in the myenteric plexus. Longitudinal muscle-myenteric plexus (LM-MP) preparations from Triton X-100-treated animals were unresponsive to dimethylphenylpiperazinium and responded poorly or not at all to electrical field stimulation. Metoclopramide (30 microM) elicited small contractions in LM-MP preparations from control and sham-operated animals but failed to contract Triton X-100-treated tissues. However, tissues responded in a similar manner to exogenous acetylcholine (ACh). These results demonstrate the importance of a prejunctional site of action for metoclopramide in this tissue and suggest that contractile responses to the drug are mediated indirectly, probably by increased release of ACh from myenteric neurones.     

Cellular distribution of vanilloid VR1 receptor immunoreactivity in the guinea-pig myenteric plexus

Recent investigations suggest that vanilloid receptor-1 (VR1) immunoreactivity occurs in the intestine. We have determined and quantified this immunoreactivity in the myenteric plexus with respect to cholinergic and neurofilament protein-positive neurones. Guinea-pig and rat preparations were dual-labelled with specific antibodies raised in rabbit or goat against vanilloid receptor-1 and against other neurochemical markers. In the rat ileum, both vanilloid receptor antibodies were co-distributed, whereas in the guinea-pig ileum and colon, tertiary fibres were also detected with the goat antibody. In the guinea-pig, all vanilloid receptor-1-immunoreactive cell bodies were choline acetyltransferase-immunopositive (100%) and showed some immunoreactivity to neurofilament proteins (NFP-200 kDa (79%) or triplet (10.8%)) or calretinin. Immunoreactive fibres in the secondary plexus co-localised with calcitonin gene-related peptide (CGRP) and with substance P, calretinin and synapsin I in the tertiary plexus. Subpopulations of cholinergic neurones including sensory, interneuronal and secretory neurones express vanilloid receptor-1. Co-localisation with substance P and calretinin in fibres suggests that vanilloid receptor-1 may be expressed by excitatory motor neurones. The association of vanilloid receptors with calcitonin gene-related peptide and synaptic protein in fibres implies a role for vanilloid receptors in neurotransmitter/neuropeptide release. Although it is likely that at least some of the vanilloid receptor-bearing fibres originate in immunopositive myenteric soma, the origin of all these fibres cannot be identified in the present study.     

The differential contribution of endogenous prostaglandins to the release of acetylcholine from the myenteric plexus of the guinea-pig ileum.

1. Prostaglandin E (PGE) may be essential for maintaining the sensitivity of the myenteric plexus of guinea-pig ileum to nicotine. The contributions of prostaglandins to nervous activity evoked by different stimuli have now been investigated by measuring the amount of acetylcholine (ACh) released from the myenteric plexus of the guinea-pig ileum. 2. The amount of ACh released in response to dimethylphenylpiperazinium (DMPP) or substance P was depressed to about 40% of control by 2.8 microM indomethacin (Ind), whereas the release of ACh induced by 5-hydroxytryptamine (5-HT) was not affected. The inhibitory effects of Ind were overcome by 14.3 nM PGE2. 3. Mepacrine 5 microM, an inhibitor of phospholipase A2, depressed the release of ACh in response to DMPP and substance P to the same extent as Ind. These inhibitory effects of mepacrine were overcome by arachidonic acid (10 microM), but not by arachidonic acid plus Ind. The release of ACh evoked by 5-HT or electrical field stimulation (EFS) was also inhibited to about 60% of control by mepacrine but these inhibitions were overcome by arachidonic acid (10 microM) either in the absence or the presence of Ind. 4. The results suggest that endogenous prostaglandins and arachidonic acid contribute to the maintenance of the excitability of the myenteric plexus by DMPP and substance P. By contrast, the release of ACh induced by 5-HT and EFS may be regulated by arachidonic acid and not by prostaglandins.     

Interactions between serotonin and cisapride on myenteric neurons

Intracellular recording methods were used to investigate the interactions between serotonin (5-HT) and cisapride on myenteric neurons of guinea-pig small intestine. Serotonin had three actions on the neurons. One was a slowly rising depolarization associated with increased input resistance and discharge of spikes that lasted six or more times longer than the duration of the 5-HT application. The second action was a transient depolarization associated with decreased input resistance and brief discharge of spikes. This response desensitized quickly and could be evoked only at intervals of 2 to 3 min. The third action of 5-HT was presynaptic inhibition of acetylcholine release at nicotinic synapses. Cisapride reduced or abolished both the prolonged and transient responses to 5-HT. The threshold concentration for reduction of the responses was 0.1 microM and the responses were abolished at 1.0 to 10 microM. Cisapride suppressed stimulus-evoked slow excitatory postsynaptic potentials (EPSPs) in the same cells for which cisapride blocked the prolonged responses to 5-HT. There were no effects of cisapride on resting electrical behavior or spike generation. Cisapride reduced the amplitude of fast cholinergic EPSPs, suggesting that it behaved as an agonist at the presynaptic serotonergic receptors.     

The action of neurotensin on single myenteric neurones.

The action of neurotensin was studied on single myenteric neurones within ganglia of the myenteric plexus isolated from the guinea-pig ileum. Drugs were applied by adding them to the perfusing Krebs solution. Extracellular recording with glass suction electrodes indicated that neurotensin (100 pM-300 nM) caused a dose-dependent excitation of about 50% of myenteric neurones; the remaining neurones were unaffected. This effect persisted in calcium-free solutions. Intracellular recording showed that a similar proportion of Type 1 myenteric neurones were depolarized by neurotensin: this was associated with an increase in membrane resistance. Type 2 cells were either depolarized or hyperpolarized by neurotensin. The depolarization persisted in calcium-free solutions. The hyperpolarization disappeared in calcium-free solutions, suggesting either that the potential change itself is calcium-dependent or that it was due to release by neurotensin of a hyperpolarizing substance.