Muscarinic M3 facilitation of acetylcholine release from rat myenteric neurons depends on adenosine outflow leading to activation of excitatory A2A receptors

Abstract Acetylcholine (ACh) is a major excitatory neurotransmitter in the myenteric plexus, and it regulates its own release acting via muscarinic autoreceptors. Adenosine released from stimulated myenteric neurons modulates ACh release preferentially via facilitatory A_2A receptors. In this study, we investigated how muscarinic and adenosine receptors interplay to regulate ACh from the longitudinal muscle–myenteric plexus of the rat ileum. Blockade of the muscarinic M₂ receptor with 11‐[[2‐1[(diethylamino) methyl‐1‐piperidinyl]‐ acetyl]]‐5,11‐dihydro‐6H‐pyrido [2,3‐b][1,4] benzodiazepine‐6‐one (AF‐DX 116), 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP) and atropine facilitated [³H]ACh release evoked by short stimulation trains (5 Hz, 200 pulses). Prolonging stimulus train length (>750 pulses) shifted muscarinic autoinhibition towards facilitatory M₃ receptors activation, as predicted by blockade with J104129 (a selective M₃ antagonist), 4‐DAMP and atropine, whereas the selective M₂ antagonist, AF‐DX 116, was without of effect. Blockade of A_2A receptors with ZM 241385, inhibition of adenosine transport with dipyridamole, and inhibition of ecto‐5′‐nucleotidase with concanavalin A, all attenuated release inhibition caused by 4‐DAMP. J104129 and 4‐DAMP, but not AF‐DX 116, decreased (～60%) evoked adenosine outflow (5 Hz, 3000 pulses). Oxotremorine (300 μmol L^?1) facilitated the release of [³H]ACh (34 ± 4%, n = 5) and adenosine (57 ± 3%, n = 6) from stimulated myenteric neurons. 4‐DAMP, dipyridamole and concanavalin A prevented oxotremorine‐induced facilitation. ZM 241385 blocked oxotremorine facilitation of [³H]ACh release, but kept adenosine outflow unchanged. Thus, ACh modulates its own release from myenteric neurons by activating inhibitory M₂ and facilitatory M₃ autoreceptors. While the M₂ inhibition is prevalent during brief stimulation periods, muscarinic M₃ facilitation is highlighted during sustained nerve activity as it depends on extracellular adenosine accumulation leading to activation of facilitatory A_2A receptors.     

Signalling mechanism coupled to 5-hydroxytryptamine4 receptor-mediated facilitation of fast synaptic transmission in the guinea-pig ileum myenteric plexus

5-hydroxytryptamine (HT)4 receptor agonists stimulate gastrointestinal motility partly by facilitating acetylcholine release from myenteric neurones. However, the signalling mechanisms that couple 5-HT4 receptor activation to increased transmitter release in the myenteric plexus are unknown. We used conventional intracellular electrophysiological methods to record fast excitatory postsynaptic potentials (fEPSPs) from neurones in the guinea-pig ileum myenteric plexus preparation. The substituted benzamide, renzapride, acted at 5-HT4 receptors to facilitate fEPSPs. This response was mimicked by forskolin, an activator of adenylate cyclase. Facilitation of fEPSPs by renzapride and forskolin was not blocked by treating tissues with pertussis toxin (PTX) (2 h, 2 microg mL-1). Facilitation of fEPSPs caused by renzapride was blocked by the non-selective protein kinase inhibitors, staurosporine (1 micromol L-1) and H-8 (30 micromol L-1) and by the selective protein kinase A (PKA) inhibitor, H-89 (10 micromol L-1). These data indicate that 5-HT4 receptors act via a PTX-resistant mechanism to activate PKA. Protein kinase A activation leads to an increase in transmitter release from myenteric nerve terminals and a facilitation of fast excitatory synaptic transmission.     

Dynamics of fast synaptic excitation during trains of stimulation in myenteric neurons of guinea-pig ileum

Fast excitatory postsynaptic potentials (fEPSPs) occur in bursts in the myenteric plexus during evoked motor reflexes in the guinea-pig ileum in vitro. This study used electrophysiological methods to study fEPSPs during stimulus trains to mimic bursts of synaptic activity in vitro. The amplitude of fEPSPs or fast excitatory postsynaptic currents (EPSCs) declined (rundown) during stimulus trains at frequencies of 0.5, 5, 10 and 20 Hz. At 0.5 Hz, fEPSP or fEPSC amplitude declined by 50% after the first stimulus but remained constant for the remainder of the train. At 5, 10 and 20 Hz, synaptic responses ran down completely with time constants of 0.35, 0.21 and 0.11 s, respectively. Recovery from rundown occurred with a time constant of 7 s. Mecamylamine, a nicotinic cholinergic receptor antagonist, or PPADS, a P2X receptor antagonist, reduced fEPSP amplitude, but they had no effect on rundown. Responses caused by trains of ionophoretically applied ATP or ACh (to mimic fEPSPs) did not rundown. Blockade of presynaptic inhibitory muscarinic, adenosine A1, opioid, alpha2-adrenergic and 5-HT1A receptors or pertussis toxin (PTX) treatment did not alter rundown. Antidromic action potentials followed a 10-Hz stimulus train. Iberiotoxin (100 nM), a blocker of large conductance calcium activated K+ (BK) channels, did not alter rundown. These data suggest that synaptic rundown is not due to: (a) action potential failure; (b) nicotinic or P2X receptor desensitization; (c) presynaptic inhibition mediated by pertussis-toxin sensitive G-proteins, or (d) BK channel activation. Synaptic rundown is likely due to depletion of a readily releasable pool (RRP) of neurotransmitter.     

A novel calcium-sensitive potassium conductance is coupled to P2X3 subunit containing receptors in myenteric neurons of guinea pig ileum

This study characterized P2X receptors in guinea pig ileum myenteric S neurons (n = 124) in vitro using electrophysiological methods. ATP or alpha,beta-methylene ATP (alpha,beta-mATP), an agonist at P2X(1) and P2X(3) subunit containing receptors, depolarized 103 neurons (85%). Pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (10 micromol L(-1)) blocked ATP- and alpha,beta-mATP-induced depolarizations. ATP-induced depolarizations and fast excitatory postsynaptic potentials (fEPSPs) were reduced by trinitrophenyl-ATP (10 micromol L(-1)), an antagonist that can block P2X(3) receptors. Ivermectin (10 micromol L(-1)), a modulator of P2X(4) and P2X(4/6) receptors, had no effect on alpha,beta-mATP-induced depolarizations. In 58% of neurons, the alpha,beta-mATP induced-depolarization was followed by an afterhyperpolarization (AHP) (P2X-AHP). Under voltage clamp, alpha,beta-mATP induced an inward current followed by an outward current which reversed polarity at 0 and -80 mV respectively. The P2X-AHP was reduced in low extracellular Ca(2+) solutions. Blockers of large, intermediate and small conductance Ca(2+)-activated K(+) channels or voltage-gated K(+) channels did not inhibit the P2X-AHP. Half of the neurons exhibiting the P2X-AHP contained nitric oxide synthase (NOS)-immunoreactivity (ir). In summary, NOS-ir S neurons express P2X(3) subunit containing P2X receptors. P2X receptors couple to activation of a Ca(2+)-activated K(+) conductance that mediates an AHP. As P2X receptors contribute to fEPSPs, the P2X-AHP may modulate S neuron excitability during purinergic synaptic transmission.     

Effect of the stable thromboxane derivative, carbocyclic thromboxane A2, on membrane potential of rat myenteric neurones in culture

The effects of carbocyclic thromboxane A(2) (cTXA(2); 10(-6) mol L(-1)) on membrane potential and cytosolic Ca(2+) concentration were measured with the whole-cell patch-clamp or the fura-2 method, respectively, at rat myenteric ganglia. cTXA(2) caused a hyperpolarization of myenteric neurones from -19.3 +/- 2.5 to -29.3 +/- 2.3 mV. In addition, the eicosanoid potentiated the carbachol-induced depolarization from 4.2 +/- 1.0 mV under control conditions to 11.1 +/- 1.1 mV in the presence of the cTXA(2) (n = 9). The hyperpolarization was abolished by internal application of CsCl (140 mmol L(-1)), a non-selective blocker of K(+) channels, or EGTA (11 mmol L(-1)in the pipette solution), a chelator of intracellular Ca(2+). A similar inhibition was observed in the presence of charybdotoxin (10(-7) mol L(-1)). Fura-2 imaging experiments revealed a cTXA(2)-evoked increase in the intracellular Ca(2+) concentration as indicated by a rise in the fura-2 ratio signal. This response was mediated by a release of Ca(2+) from intracellular stores as sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase blockade with cyclopiazonic acid (5 x 10(-5) mol L(-1)) completely abolished the response to cTXA(2). A similar inhibition was observed after blockade of phospholipase C with U-73122 (10(-5) mol L(-1)). These results suggest an activation of Ca(2+)-activated K(+) channels by cTXA(2) after stimulation of phospholipase C.     

Prostaglandin E2 (PGE2)-evolted chloride secretion in guinea-pig colon is mediated by nerve-dependent and nerve-independent mechanisms

Abstract Conventional flux chamber and intracellular recording methods were used to investigate the effect of prostaglandin E₂ on ion transport and on electrical behaviour of submucosal neurones in guinea-pig colon. In flux chamber experiments, prostaglandin E₂ evoked a dose-dependent increase in short-circuit current. The response was reduced by serosal addition of bumetanide, tetrodotoxin or atropine, but not hexamethonium or piroxicam. This indicates that the response to prostaglandin E₂ was mediated in part by activation of chloride secretion via submucosal neurons. Application of prostaglandin E₂ to submucosal neurones evoked a depolarization of the membrane potential associated with an enhanced spike discharge which was frequently triggered by fEPSPs. The depolarizing response to prostaglandin E₂ was not affected by tetrodotoxin, indicative of a direct effect of prostaglandin E₂ on the impaled neurones. However, the increased spike activity was synaptically driven suggesting an additional activation of other cells. Prostaglandin E₂ had no excitatory or inhibitory effect on cholinergic fast excitatory postsynaptic potentials. The study suggests that prostaglandin E₂ may function as a neuromodulator to evoke nerve-mediated chloride secretion through activation of submucosal neurones. The results further indicate that prostaglandin E₂ may influence mucosal function by altering electrical behaviour of submucosal neurones leading to spread of excitation throughout the plexus.     

In vivo labelling of the adenosine A2A receptor in mouse brain using the selective antagonist [3H]SCH 58261

The selective A2A receptor antagonist [3H]SCH 58261 was injected intravenously in mice and the radioactivity accumulating in various brain regions was determined by tissue sampling. Radioactivity levels in regions of interest such as the striatum were highest 15 min after injection and quickly declined thereafter (30 min and 1 h postinjection) in a time-dependent manner. The amount of labelling was ranked as follows: striatum (4.6 +/- 0.3 fmol/mg protein) > cortex > hippocampus > pons = hypothalamus > cerebellum (0.5 +/- 0.05 fmol/mg protein). Specific labelling of the A2A receptor occurred in striatum and cortex because significantly less radioactivity accumulated in these areas from adenosine A2A receptor knockout mice as compared to wild-type littermates. In control outbred CD1 mice, a striatum-to-cerebellum ratio of 7.6 +/- 0.6 was found. At 30 min postinjection, the nonselective adenosine receptor antagonist caffeine reduced the radioactivity due to [3H]SCH 58261 in the striatum by 32% at 1 mg/kg i.p. and by 66% at the stimulant dose of 6.25 mg/kg i.p. Radioactivity in the striatum was lowered, respectively, by 66 and 86% 30 min after injection of 3 or 10 mg/kg i.p. doses of unlabelled SCH 58261. The present results indicate that [3H]SCH 58261 directly labels striatal A2A receptors in vivo. Thus [3H]SCH 58261 is an excellent tool for studying brain distribution and A2A receptor occupancy of various compounds ranging from xanthines, such as caffeine, to other A2A antagonists.     

Blockade of A2a Adenosine Receptors Positively Modulates Turning Behaviour and c-Fos Expression Induced by D1 Agonists in Dopamine-denervated Rats

In rats with unilateral 6-hydroxydopamine lesions of the dopaminergic nigrostriatal pathway, administration of the A_2a adenosine antagonist SCH 58261 alone did not induce any motor asymmetry but strongly potentiated the contralateral turning behaviour induced by the dopamine D₁ agonist SKF 38393. SCH 58261 also increased the number of Fos-like positive nuclei induced by SKF 38393 in the 6-hydroxydopamine-lesioned striatum. Intense potentiation of D₁-dependent turning behaviour and c-Fos expression was also observed after administration of the A_2a/A₁ antagonist CGS 15943. Administration of the A₁ adenosine receptor antagonist DPCPX induced a small potentiation of D₁-mediated contralateral turning while c-Fos expression induced by SKF 38393 was not modified. The results suggest that endogenous adenosine acting on A_2a receptors can exert an inhibitory influence on the functional expression of D₁-mediated responses in dopamine-denervated rats, and propose new possible therapeutic approaches in the treatment of Parkinson's disease.     

α2-Adrenoceptors couple to inhibition of R-type calcium currents in myenteric neurons

Alpha2-adrenoceptors inhibit Ca2+ influx through voltage-gated Ca2+ channels throughout the nervous system and Ca2+ channel function is modulated following activation of some G-protein coupled receptors. We studied the specific Ca2+ channel inhibited following alpha2-adrenoceptor activation in guinea-pig small intestinal myenteric neurons. Ca2+ currents (I(Ca2+)) were studied using whole-cell patch-clamp techniques. Changes in intracellular Ca2+ (delta[Ca2+]i) in nerve cell bodies and varicosities were studied using digital imaging where Ca2+ influx was evoked by KCl (60 mmol L(-1)) depolarization. The alpha2-adrenoceptor agonist, UK 14 304 (0.01-1 micromol L(-1)) inhibited I(Ca2+) and delta[Ca2+]i; maximum inhibition of I(Ca2+) was 40%. UK 14 304 did not affect I(Ca2+) in the presence of SNX-482 or NiCl2 (R-type Ca2+ channel antagonists). UK 14 304 inhibited I(Ca2+) in the presence of nifedipine, omega-agatoxin IVA or omega-conotoxin, inhibitors of L-, P/Q- and N-type Ca2+ channels. UK 14 304 induced inhibition of I(Ca2+) was blocked by pertussis toxin pretreatment (1 microg mL(-1) for 2 h). Alpha2-adrenoceptors couple to inhibition of R-type Ca2+ channels via a pertussis toxin-sensitive pathway in myenteric neurons. R-type channels may be a target for the inhibitory actions of noradrenaline released from sympathetic nerves on to myenteric neurons.     

Laser microdissection as a new tool to investigate site‐specific gene expression in enteric ganglia of the human intestine

Background Myenteric ganglia are key‐structures for the control of intestinal motility and their mRNA expression profiles might be altered under pathological conditions. A drawback of conventional RT‐PCR from full‐thickness specimens is that gene expression analysis is based on heterogeneously composed tissues. To overcome this problem, laser microdissection combined with real‐time RT‐PCR can be used to detect and quantify low levels of gene expression in isolated enteric ganglia. Methods Fresh unfixed full‐thickness specimens of sigmoid colon were obtained from patients (n = 8) with diseases unrelated to intestinal motility disorders. 10 μm cryo‐sections were mounted on membrane‐coated slides and ultra‐rapidly stained with toluidine blue. Myenteric ganglia were isolated by laser microdissection and catapulting for mRNA isolation. Real‐time RT‐PCR was performed for selected growth factors, neurotransmitter receptors and specific cell type markers. Key Results Collection of 0.5 mm² of ganglionic tissue was sufficient to obtain positive RT‐PCR results. Collection of 4 mm² resulted in ct‐values allowing a reliable quantitative comparison of gene expression levels. mRNA analysis revealed that neurotrophic growth factor, neurotrophin‐3, serotonin receptor 3A, PGP 9.5 and S100β are specifically expressed in myenteric ganglia of the human colon. Conclusions & Inferences Laser microdissection combined with real‐time RT‐PCR is a novel technique to reliably detect and quantify site‐specific expression of low‐abundance mRNAs (e.g. growth factors, neurotransmitter receptors) related to the human enteric nervous system. This technical approach expands the spectrum of available tools to characterize enteric neuropathologies underlying human gastrointestinal motility disorders at the molecular biological level.     

Effects of modulators of Ca2+-activated, intermediate-conductance potassium channels on motility of the rat small intestine, in vivo

The movements of the intestine shift between different motor patterns, including between propulsion and mixing, but there is little information concerning mechanisms that may lead to changes in the patterns of motility. We have investigated the influence on intestinal motility of drugs that affect the after-hyperpolarization potential (AHP) of intrinsic primary afferent neurons (IPANs). The current of the AHP is carried by the intermediate conductance, calcium-activated, potassium (IK) channel. In anaesthetized rats, the IK channel blocker, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (0.05-1 mg kg(-1), i.v.) disrupted the regular propulsive pressure waves that occur in the small intestine and reduced propulsion of the contents (after 1 mg kg(-1), the fluid propelled was <25% of control). If the propulsion in the intestine was regular, the IK channel opener, 5,6-dichloro-1-ethyl-2-benzimidazolinone (DC-EBIO, 0.1 mg kg(-1) h(-1)) had no effect. DC-EBIO (0.1 mg kg(-1) h(-1)) restored propulsive activity after the nitric oxide synthase inhibitor, Nomega-nitro-l-arginine had changed motility to a mixing pattern. We suggest that the AHP determines the synchrony of action potential firing in synaptically coupled IPANs, and that this synchrony influences the patterns of firing of muscle motor neurons, and hence the pattern of contraction of the muscle and whether the pattern is predominantly propulsive or predominantly mixing.     

Evidence for functional NK1-tachykinin receptors on motor neurones supplying the circular muscle of guinea-pig small and large intestine

The guinea-pig intestine was investigated to determine which neurones are excited via NK1 receptors. The specific NK1 receptor agonists [Sar9, Met(O2)11]-SP and septide contracted the circular muscle of all regions via a tetrodotoxin (TTX)-insensitive mechanism. In the proximal colon, they also evoked a TTX-sensitive relaxation; in the distal colon, the contractions were larger when nerve impulses were blocked with TTX, indicating that the agonists excited inhibitory motor neurones. In the duodenum and ileum, TTX reduced agonist-evoked contractions indicating that excitatory motor neurones were activated. In the presence of indomethacin, TTX enhanced contractions of ileal circular muscle evoked by these agonists suggesting that NK1 receptors were on inhibitory motor neurones. Blockade of nitric oxide synthase (NOS) enhanced NK1 receptor agonist evoked contractions of duodenal circular muscle, indicating that the agonists excite inhibitory motor neurones in duodenum. Neurones immunoreactive for NK1 receptors were studied in the duodenum and distal colon. As reported previously for the ileum, 1 some neurones were immunoreactive for NOS and had Dogiel type I morphology; features characteristic of inhibitory motor neurones. In conclusion, there are functional NK1 receptors on excitatory and inhibitory motor neurones in the guinea-pig small intestine and on inhibitory motor neurones in the colon.     

Differential release of β‐NAD+ and ATP upon activation of enteric motor neurons in primate and murine colons

Background The purinergic component of enteric inhibitory neurotransmission is important for normal motility in the gastrointestinal (GI) tract. Controversies exist about the purine(s) responsible for inhibitory responses in GI muscles: ATP has been assumed to be the purinergic neurotransmitter released from enteric inhibitory motor neurons; however, recent studies demonstrate that β‐nicotinamide adenine dinucleotide (β‐NAD⁺) and ADP‐ribose mimic the inhibitory neurotransmitter better than ATP in primate and murine colons. The study was designed to clarify the sources of purines in colons of Cynomolgus monkeys and C57BL/6 mice. Methods High‐performance liquid chromatography with fluorescence detection was used to analyze purines released by stimulation of nicotinic acetylcholine receptors (nAChR) and serotonergic 5‐HT₃ receptors (5‐HT₃R), known to be present on cell bodies and dendrites of neurons within the myenteric plexus. Key Results Nicotinic acetylcholine receptor or 5‐HT₃R agonists increased overflow of ATP and β‐NAD⁺ from tunica muscularis of monkey and murine colon. The agonists did not release purines from circular muscles of monkey colon lacking myenteric ganglia. Agonist‐evoked overflow of β‐NAD⁺, but not ATP, was inhibited by tetrodotoxin (0.5 μmol L^?1) or ω‐conotoxin GVIA (50 nmol L^?1), suggesting that β‐NAD⁺ release requires nerve action potentials and junctional mechanisms known to be critical for neurotransmission. ATP was likely released from nerve cell bodies in myenteric ganglia and not from nerve terminals of motor neurons. Conclusions & Inferences These results support the conclusion that ATP is not a motor neurotransmitter in the colon and are consistent with the hypothesis that β‐NAD⁺, or its metabolites, serve as the purinergic inhibitory neurotransmitter.     

Phospholipase A2 and Somatostatin Release are Activated in Response to N-Methyl-D-Aspartate Receptor Stimulation in Hypothalamic Neurons in Primary Culture

We have recently shown that glutamate primarily induces somatostatin release in hypothalamic neurons through N-methyl-D-aspartate (NMDA)-type receptor sites. Here we report that glutamate and NMDA also stimulate the release of [³H]arachidonic acid in a dose-dependent manner. The NMDA-induced effects (arachidonic acid release and somatostatin secretion) were both inhibited by MK-801, an NMDA receptor-type antagonist, or mepacrine, a phospholipase A₂ inhibitor. In addition, mepacrine was able to inhibit A23187-stimulated arachidonic acid release and somatostatin secretion. p-Bromophenacylbromide, another phospholipase A₂ inhibitor, also blocked NMDA-induced secretion of somatostatin. However, responses to NMDA were unaffected by H7 (inhibitor of protein kinase C), nordihydroguaiaretic acid or indomethacin (inhibitors of lipoxygenase and cyclooxygenase). Melittin, a phospholipase A₂ activator, was found to stimulate both responses, but omission of extracellular Ca²⁺ from the incubation media strongly reduced melittin-induced somatostatin release. Six-h pertussis toxin pretreatment did not significantly reduce the action of NMDA on either of the two parameters studied. High-performance liquid chromatography analysis of [₃H]metabolites released in the medium after NMDA stimulation revealed that [₃H]arachidonic acid was the only detectable metabolite. External addition of arachidonic acid increased the release of somatostatin, whereas E² and F²α prostaglandins had no effect. Our results show a close correlation between arachidonic acid release and somatostatin secretion, the two parameters we investigated.     

FlCRhR/cyclic AMP signaling in myenteric ganglia and calbindin-D28 intrinsic primary afferent neurons involves adenylyl cyclases I, III and IV

The aims of this study were to improve insight into cAMP signaling in myenteric neurons and glia and identify the adenylyl cyclase (AC) isoforms expressed in myenteric ganglia of the guinea-pig small intestine. An increase in the intracellular cAMP levels was measured indirectly by an increase in the 520 nm/580 nm fluorescence emission ratio of the protein kinase A fluorosensor FlCRhR. Forskolin or pituitary adenylyl cyclase activating peptide caused an increase in cAMP levels in cell somas and neurites and elicited a slow EPSP-like response in myenteric AH/Type 2 neurons, whereas the inactive form of forskolin was without these effects. Glia displayed similar cAMP responses. Immunoblot analysis showed that AC I, III and IV were present in myenteric ganglia, with AC I being detected as two bands of 160 kDa and 185 kDa, AC III as two bands near 220 kDa, and AC IV as two bands of greater than 220 kDa. Pretreatment with N-ethylmaleimide and N-glycosidase F revealed an AC IV band at 115 kDa. Preabsorption with specific blocking peptides prevented detection of AC I or AC IV immunoreactive proteins. In ganglia which expressed strong AC IV immunoreactivity, no immunoreactive bands were detected for AC II, AC V/VI, AC VII or AC VIII. The amount of AC isoforms expressed in myenteric ganglia followed the order of AC IVIII>I. Immunofluorescent labeling studies revealed that AC I, AC III and AC IV were variably expressed in myenteric neurons and glia of the duodenum, jejunum and ileum. In the guinea-pig ileum, AC I, III and IV immunoreactivities were respectively present in 26%, 58% and 89% of calbindin-D₂₈—colabeled myenteric neurons. These findings suggest that (1) AC I, AC III and AC IV variably contribute to cAMP signaling in myenteric ganglia, (2) AC I, AC III and AC IV may be differentially expressed in distinct subsets of calbindin-D₂₈ neurons which may represent intrinsic primary afferent myenteric neurons. Our study also provides direct evidence for activation of cAMP-dependent protein kinase.     

R‐type Ca2+ channels contribute to fast synaptic excitation and action potentials in subsets of myenteric neurons in the guinea pig intestine

Background R‐type Ca²⁺ channels are expressed by myenteric neurons in the guinea pig ileum but the specific function of these channels is unknown. Methods In the present study, we used intracellular electrophysiological techniques to determine the function of R‐type Ca²⁺ channels in myenteric neurons in the acutely isolated longitudinal muscle‐myenteric plexus. We used immunohistochemical methods to localize the Ca_V2.3 subunit of the R‐type Ca²⁺ channel in myenteric neurons. We also studied the effects of the non‐selective Ca²⁺ channel antagonist, CdCl₂ (100 μmol L^?1), the R‐type Ca²⁺ channel blockers NiCl₂ (50 μmol L^?1) and SNX‐482 (0.1 μmol L^?1), and the N‐type Ca²⁺ channel blocker ω‐conotoxin GVIA (CTX 0.1 μmol L^?1) on action potentials and fast and slow excitatory postsynaptic potentials (fEPSPs and sEPSPs) in S and AH neurons in vitro. Key Results Ca_V2.3 co‐localized with calretinin and calbindin in myenteric neurons. NiCl₂ and SNX‐482 reduced the duration and amplitude of action potentials in AH but not S neurons. NiCl₂ inhibited the afterhyperpolarization in AH neurons. ω‐conotoxin GVIA, but not NiCl₂, blocked sEPSPs in AH neurons. NiCl₂ and SNX‐482 inhibited cholinergic, but not cholinergic/purinergic, fEPSPs in S neurons. Conclusions and Inferences These data show that R‐type Ca²⁺ channels contribute to action potentials, but not slow synaptic transmission, in AH neurons. R‐type Ca²⁺ channels contribute to release of acetylcholine as the mediator of fEPSPs in some S neurons. These data indicate that R‐type Ca²⁺ channels may be a target for drugs that selectively modulate activity of AH neurons or could alter fast synaptic excitation in specific pathways in the myenteric plexus.