R-type calcium channels in myenteric neurons of guinea pig small intestine

Currents carried by L-, N-, and P/Q-type calcium channels do not account for the total calcium current in myenteric neurons. This study identified all calcium channels expressed by guinea pig small intestinal myenteric neurons maintained in primary culture. Calcium currents were recorded using whole cell techniques. Depolarizations (holding potential = -70 mV) elicited inward currents that were blocked by CdCl(2) (100 microM). Combined application of nifedipine (blocks L-type channels), Omega-conotoxin GVIA (blocks N-type channels), and Omega-agatoxin IVA (blocks P/Q-type channels) inhibited calcium currents by 56%. Subsequent addition of the R-type calcium channel antagonists, NiCl(2) (50 microM) or SNX-482 (0.1 microM), abolished the residual calcium current. NiCl(2) or SNX-482 alone inhibited calcium currents by 46%. The activation threshold for R-type calcium currents was -30 mV, the half-activation voltage was -5.2 +/- 5 mV, and the voltage sensitivity was 17 +/- 3 mV. R-type currents activated fully in 10 ms at 10 mV. R-type calcium currents inactivated in 1 s at 10 mV, and they inactivated (voltage sensitivity of 16 +/- 1 mV) with a half-inactivation voltage of -76 +/- 5 mV. These studies have accounted for all of the calcium channels in myenteric neurons. The data indicate that R-type calcium channels make the largest contribution to the total calcium current in myenteric neurons. The relatively positive half-activation voltage and rapid activation kinetics suggest that R-type channels could contribute to calcium entry during somal action potentials or during action potential-induced neurotransmitter release.     

Experimental colitis modulates the functional properties of NMDA receptors in dorsal root ganglia neurons

N-methyl-D-aspartate (NMDA) receptors (NMDARs) on spinal afferent neurons regulate the peripheral and central release of neuropeptides involved in the development of hyperalgesia. We examined the effect of experimental colitis on the molecular and functional properties of NMDARs on these neurons. Lumbosacral dorsal root ganglia (DRG) were collected from adult rats 5 days after the induction of colitis for whole cell patch-clamp recording, Western blot analysis, and quantitative RT-PCR. Compared with neurons from control rats, those taken from animals with colitis had a threefold higher density of NMDA currents in both retrograde-labeled, colon-specific, and unlabeled DRG neurons. Increased current densities were not observed in DRG neurons taken from thoracic spinal levels. There was no significant change in NMDA or glycine affinity or in voltage-dependent Mg2+ inhibition; however, there was a 10-fold decrease in sensitivity to the NR2B subunit-selective antagonist ifenprodil. Quantitative RT-PCR and Western blot analysis indicated a 28% increase in the expression of NR2B with little or no change in the other three NR2 subunits. The addition of the Src family tyrosine kinase inhibitor PP2 (10 microM) decreased NMDAR currents in neurons from colitis but not control rats. Conversely, pretreatment of DRG neurons from control animals with 100 microM sodium orthovanadate increased NMDAR currents and decreased ifenprodil sensitivity to levels similar to those observed in neurons from animals with colitis. In conclusion, colonic inflammation upregulates the activity of NMDARs in all DRG neurons within ganglia innervating this tissue through mechanisms involving increased expression and persistent tyrosine phosphorylation.     

Urocortin 3 expression at baseline and during inflammation in the colon: Corticotropin releasing factor receptors cross-talk

Urocortins (Ucn1-3), members of the corticotropin-releasing factor (CRF) family of neuropeptides, are emerging as potent immunomodulators. Localized, cellular expression of Ucn1 and Ucn2, but not Ucn3, has been demonstrated during inflammation. Here, we investigated the role of Ucn3 in a rat model of Crohn's colitis and the relative contribution of CRF receptors (CRF₁ and CRF₂) in regulating Ucn3 expression at baseline and during inflammation. Ucn3 mRNA and peptide were ubiquitously expressed throughout the GI tract in naïve rats. Ucn3 immunoreactivity was seen in epithelial cells and myenteric neurons. On day 1 of colitis, Ucn3 mRNA levels decreased by 80% and did not recover to baseline even by day 9. Next, we ascertained pro- or anti-inflammatory actions of Ucn3 during colitis. Surprisingly, unlike observed anti-inflammatory actions of Ucn1, exogenous Ucn3 did not alter histopathological outcomes during colitis and neither did it alter levels of pro-inflammatory cytokines IL-6 and TNF-α. At baseline, colon-specific knockdown of CRF₁, but not CRF₂ decreased Ucn3 mRNA by 78%, whereas during colitis, Ucn3 mRNA levels increased after CRF₁ knockdown. In cultured cells, co-expression of CRF₁ + CRF₂ attenuated Ucn3-stimulated intracellular Ca²⁺ peak by 48% as compared to cells expressing CRF₂ alone. Phosphorylation of p38 kinase increased by 250% during colitis and was significantly attenuated after Ucn3 administration. Thus, our results suggest that a balanced and coordinated expression of CRF receptors is required for proper regulation of Ucn3 at baseline and during inflammation.     

Neuronal Ca2+ sensor-1/frequenin functions in an autocrine pathway regulating Ca2+ channels in bovine adrenal chromaffin cells

NCS-1/frequenin belongs to a family of EF-hand-containing Ca(2+) sensors expressed mainly in neurons. Overexpression of NCS-1/frequenin has been shown to stimulate neurotransmitter release but little else is known of its cellular roles. We have constructed an EF-hand mutant, NCS-1(E120Q), as a likely dominant inhibitor of cellular NCS-1 function. Recombinant NCS-1(E120Q) showed an impaired Ca(2+)-dependent conformational change but could still bind to cellular proteins. Transient expression of this mutant, but not NCS-1, in bovine adrenal chromaffin cells increased non-L-type Ca(2+) channel currents. Cells expressing NCS-1(E120Q) no longer responded effectively to the removal of autocrine purinergic/opioid inhibition of Ca(2+) currents but still showed voltage-dependent facilitation. These data are consistent with the existence of both voltage-dependent and voltage-independent pathways for Ca(2+) channel inhibition in chromaffin cells. Our results suggest a novel function for NCS-1 specific for the voltage-independent autocrine pathway that negatively regulates non-L-type Ca(2+) channels in chromaffin cells.     

TH and NPY in sympathetic neurovascular cultures: role of LIF and NT-3

The sympathetic nervous system is an important determinant of vascular function. The effects of the sympathetic nervous system are mediated via release of neurotransmitters and neuropeptides from postganglionic sympathetic neurons. The present study tests the hypothesis that vascular smooth muscle cells (VSM) maintain adrenergic neurotransmitter/neuropeptide expression in the postganglionic sympathetic neurons that innervate them. The effects of rat aortic and tail artery VSM (AVSM and TAVSM, respectively) on neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were assessed in cultures of dissociated sympathetic neurons. AVSM decreased TH (39 +/- 12% of control) but did not affect NPY. TAVSM decreased TH (76 +/- 10% of control) but increased NPY (153 +/- 20% of control). VSM expressed leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3), which are known to modulate NPY and TH expression. Sympathetic neurons innervating blood vessels expressed LIF and NT-3 receptors. Inhibition of LIF inhibited the effect of AVSM on TH. Inhibition of neurotrophin-3 (NT-3) decreased TH and NPY in neurons grown in the presence of TAVSM. These data suggest that vascular-derived LIF decreases TH and vascular-derived NT-3 increases or maintains NPY and TH expression in postganglionic sympathetic neurons. NPY and TH in vascular sympathetic nerves are likely to modulate NPY and/or norepinephrine release from these nerves and are thus likely to affect blood flow and blood pressure. The present studies suggest a novel mechanism whereby VSM would modulate sympathetic control of vascular function.     

Dextran sodium sulfate-induced colitis reveals nicotinic modulation of ion transport via iNOS-derived NO

In normal colon, ACh elicits a luminally directed Cl- efflux from enterocytes via activation of muscarinic receptors. In contrast, in the murine model of dextran sodium sulfate (DSS)-induced colitis, an inhibitory cholinergic ion transport event due to nicotinic receptor activation has been identified. The absence of nicotinic receptors on enteric epithelia and the ability of nitric oxide (NO) to modulate ion transport led us to hypothesize that NO mediated the cholinergic nicotinic receptor-induced changes in ion transport. Midportions of colon from control and DSS-treated mice were examined for inducible NO synthase (iNOS) expression by RT-PCR and immunofluorescence or mounted in Ussing chambers for assessment of cholinergic-evoked changes in ion transport (i.e., short-circuit current) with or without pretreatment with pharmacological inhibitors of NO production. iNOS mRNA and protein levels were increased throughout the tissue from DSS-treated mice and, notably, in the myenteric plexus, where the majority of iNOS immunoreactivity colocalized with the enteric glial cell marker glial fibrillary acidic protein. The drop in short-circuit current evoked by the cholinomimetic carbachol in tissue from DSS-treated mice was prevented by selective inhibitors of iNOS activity [N6-(1-iminoethyl)-lysine HCl and N-[3-(aminomethyl)benzyl]acetamidine] or an NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or by removal of the myenteric plexus. Thus, in this model of colitis, a "switch" occurs from muscarinic to nicotinic receptor-dominated control of cholinergic ion transport. The data indicate a novel pathway involving activation of nicotinic receptors on myenteric neurons, resulting in release of NO from neurons or enteric glia and, ultimately, a dampening of stimulated epithelial Cl- secretion that would reduce secretory diarrhea.     

Platelet-activating factor in the enteric nervous system of the guinea pig small intestine

Platelet-activating factor (PAF) is a proinflammatory mediator that may influence neuronal activity in the enteric nervous system (ENS). Electrophysiology, immunofluorescence, Western blot analysis, and RT-PCR were used to study the action of PAF and the expression of PAF receptor (PAFR) in the ENS. PAFR immunoreactivity (IR) was expressed by 6.9% of the neurons in the myenteric plexus and 14.5% of the neurons in the submucosal plexus in all segments of the guinea pig intestinal tract as determined by double staining with anti-human neuronal protein antibody. PAFR IR was found in 6.1% of the neurons with IR for calbindin, 35.8% of the neurons with IR for neuropeptide Y (NPY), 30.6% of the neurons with IR for choline acetyltransferase (ChAT), and 1.96% of the neurons with IR for vasoactive intestinal peptide (VIP) in the submucosal plexus. PAFR IR was also found in 1.5% of the neurons with IR for calbindin, 51.1% of the neurons with IR for NPY, and 32.9% of the neurons with IR for ChAT in the myenteric plexus. In the submucosal plexus, exposure to PAF (200-600 nM) evoked depolarizing responses (8.2 +/- 3.8 mV) in 12.4% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.5% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology, whereas in the myenteric preparations, depolarizing responses were elicited by a similar concentration of PAF in 9.5% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.0% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology. The results suggest that subgroups of secreto- and musculomotor neurons in the submucosal and myenteric plexuses express PAFR. Coexpression of PAFR IR with ChAT IR in the myenteric plexus and ChAT IR and VIP IR in the submucosal plexus suggests that PAF, after release in the inflamed bowel, might act to elevate the excitability of submucosal secretomotor and myenteric musculomotor neurons. Enhanced excitability of motor neurons might lead to a state of neurogenic secretory diarrhea.     

Calcium permeability changes and neurotransmitter release in cultured brain neurons. II. Temporal analysis of neurotransmitter release

The coupling between depolarization-induced calcium entry and neurotransmitter release was studied in rat brain neurons in culture. The endogenous dopamine content of the cells was determined by high performance liquid chromatography utilizing electrochemical detection. The amount of dopamine in unstimulated cells was found to be about 16 ng/mg of protein. Depolarization of the neurons by elevated K+ caused a Ca2+-dependent release of dopamine from the cells. Following 1 min of depolarization, the cellular dopamine content and the amount of [3H]dopamine in cells preloaded with the radioactive transmitter were reduced by 35%. The release of [3H]dopamine by the neurons was measured at 1.5-6-s intervals by a novel rapid dipping technique. Depolarization in the presence of Ca2+ (1.8 mM) enhanced the rate of neurotransmitter release by 90-fold (0.072 +/- 0.003 s-1) over the basal release in the presence of Ca2+. The evoked release consisted of a major rapidly terminating phase (t1/2 = 9.6 s) which comprised about 40% of the neurotransmitter content of the cells and a subsequent slower efflux (t1/2 = 575 s) which was observed during following prolonged depolarization. Predepolarization of the cells in the absence of extracellular Ca2+ did not affect the kinetics of the evoked release. The fast evoked release could be re-elicited in the cells after 20 min "rest" in reference low K+ buffer. The effects of varying the extracellular Ca2+ concentrations on the kinetic parameters of the evoked release were measured. The amount of neurotransmitter released during the fast kinetic phase was very sensitive to the external Ca2+ (from 0% in the absence of Ca2+ to 40% of the neurotransmitter content at Ca2+ 0.3 mM). The rate constant of the fast release did not depend on the extracellular Ca2+, whereas the rate constant of the slow release increased from 0.0004 +/- 0.0001 s-1 at 0.4 mM Ca2+ to 0.0012 +/- 0.0002 s-1 at 0.8 mM Ca2+. The fast evoked release was inhibited by verapamil in a concentration-dependent manner. By contrast, verapamil enhanced the basal and the slow release independent of the presence of Ca2+. Both fast and slow phases of the evoked release were blocked by Co2+. Addition of Co2+ within the first 6 s after the onset of depolarization inhibited the fast release but failed to do so when added later on.(ABSTRACT TRUNCATED AT 400 WORDS)     

Presynaptic nicotinic acetylcholine receptors in the myenteric plexus of guinea pig intestine

Presynaptic nicotinic acetylcholine receptors (nAChRs) were studied in myenteric plexus preparations from guinea pig ileum using intracellular electrophysiological methods. Microapplication of nicotine (1 mM) caused a biphasic depolarization in all AH neurons (n = 30) and in 36 of 49 S neurons. Cytisine (1 mM) caused fast depolarizations in S neurons and no response in AH neurons. Mecamylamine (10 microM) blocked all responses caused by nicotine and cytisine. TTX (0.3 microM) blocked slow excitatory synaptic potentials in S and AH neurons but had no effect on fast depolarizations caused by nicotine. Nicotine-induced slow depolarizations were reduced by TTX in two of twelve AH neurons (79% inhibition) and four of nine S neurons (90+/-12% inhibition). Slow nicotine-induced depolarizations in the remaining neurons were TTX resistant. TTX-resistant slow depolarizations were inhibited after neurokinin receptor 3 desensitization caused by senktide (0.1 microM); senktide desensitization inhibited the slow nicotine-induced depolarization by 81+/-5% and 63+/-15% in AH and S neurons, respectively. A low-calcium and high-magnesium solution blocked nicotine-induced slow depolarizations in AH neurons. In conclusion, presynaptic nAChRs mediate the release of substance P and/or neurokinin A to cause slow depolarizations of myenteric neurons.     

Spatial organization of neurons in the dorsal motor nucleus of the vagus synapsing with intragastric cholinergic and nitric oxide/VIP neurons in the rat

The dorsal motor nucleus of the vagus (DMV) contains preganglionic neurons that control gastric motility and secretion. Stimulation of different parts of the DMV results in a decrease or an increase in gastric motor activities, suggesting a spatial organization of vagal preganglionic neurons in the DMV. Little is known about how these preganglionic neurons in the DMV synapse with different groups of intragastric motor neurons to mediate contraction or relaxation of the stomach. We used pharmacological and immunohistochemical methods to characterize intragastric neural pathways involved in mediating gastric contraction and relaxation in rats. Microinjections of L-glutamate (L-Glu) into the rostral or caudal DMV produced gastric contraction and relaxation, respectively, in a dose-related manner. Intravenous infusion of hexamethonium blocked these actions, suggesting mediation via preganglionic cholinergic pathways. Atropine inhibited gastric contraction by 85.5 +/- 4.5%. Gastric relaxation was reduced by intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 52.5 +/- 11.9%) or VIP antagonist (56.3 +/- 14.9%). Combined administration of L-NAME and VIP antagonist inhibited gastric relaxation evoked by L-Glu (87.8 +/- 4.3%). Immunohistochemical studies demonstrated choline acetyltransferase immunoreactivity in response to L-Glu microinjection into the rostral DMV in 88% of c-Fos-positive intragastric myenteric neurons. Microinjection of L-Glu into the caudal DMV evoked expression of nitric oxide (NO) synthase and VIP immunoreactivity in 81 and 39%, respectively, of all c-Fos-positive intragastric myenteric neurons. These data indicate spatial organization of the DMV. Depending on the location, microinjection of L-Glu into the DMV may stimulate intragastric myenteric cholinergic neurons or NO/VIP neurons to mediate gastric contraction and relaxation.     

Characterisation of the interaction of the C-terminus of the dopamine D2 receptor with neuronal calcium sensor-1

NCS-1 is a member of the neuronal calcium sensor (NCS) family of EF-hand Ca(2+) binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca(2+) channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1 via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca(2+) and D2 peptide. The data show that NCS-1 binds D2 peptide with a K(d) of ～14.3 μM and stoichiometry of peptide binding to NCS-1 of 2:1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer.     

5-Hydroxytryptophan activates colonic myenteric neurons and propulsive motor function through 5-HT4 receptors in conscious mice

Serotonin [5-hydroxytryptamine (5-HT)] acts as a modulator of colonic motility and secretion. We characterized the action of the 5-HT precursor 5-hydroxytryptophan (5-HTP) on colonic myenteric neurons and propulsive motor activity in conscious mice. Fos immunoreactivity (IR), used as a marker of neuronal activation, was monitored in longitudinal muscle/myenteric plexus whole mount preparations of the distal colon 90 min after an intraperitoneal injection of 5-HTP. Double staining of Fos IR with peripheral choline acetyltransferase (pChAT) IR or NADPH-diaphorase activity was performed. The injection of 5-HTP (0.5, 1, 5, or 10 mg/kg ip) increased fecal pellet output and fluid content in a dose-related manner, with a peak response observed within the first 15 min postinjection. 5-HTP (0.5-10 mg/kg) dose dependently increased Fos expression in myenteric neurons, with a maximal response of 9.9 +/- 1.0 cells/ganglion [P < 0.05 vs. vehicle-treated mice (2.3 +/- 0.6 cells/ganglion)]. There was a positive correlation between Fos expression and fecal output. Of Fos-positive ganglionic cells, 40 +/- 4% were also pChAT positive and 21 +/- 5% were NADPH-diaphorase positive in response to 5-HTP, respectively. 5-HTP-induced defecation and Fos expression were completely prevented by pretreatment with the selective 5-HT4 antagonist RS-39604. These results show that 5-HTP injected peripherally increases Fos expression in different populations of cholinergic and nitrergic myenteric neurons in the distal colon and stimulates propulsive colonic motor function through 5-HT4 receptors in conscious mice. These findings suggest an important role of activation of colonic myenteric neurons in the 5-HT4 receptor-mediated colonic propulsive motor response.     

Neuronal calcium sensor-1 facilitates neuronal exocytosis through phosphatidylinositol 4-kinase

This work tested the theory that neuronal calcium sensor-1 (NCS-1) has effects on neurotransmitter release beyond its actions on membrane channels. We used nerve-ending preparations where membrane channels are bypassed through membrane permeabilization made by mechanical disruption or streptolysin-O. Nerve ending NCS-1 and phosphatidylinositol 4-kinase (PI4K) are largely or entirely particulate, so their concentrations in nerve endings remain constant after breaching the membrane. Exogenous, myristoylated NCS-1 stimulated nerve ending phosphatidylinositol 4-phosphate [PI(4)P] synthesis, but non-myristoylated-NCS-1 did not. The N-terminal peptide of NCS-1 interfered with PI(4)P synthesis, and with spontaneous and Ca(2+)-evoked release of both [(3)H]-norepinephrine (NA) and [(14)C]-glutamate (glu) in a concentration-dependent manner. An antibody raised against the N-terminal of NCS-1 inhibited perforated nerve ending PI(4)P synthesis, but the C-terminal antibody had no effects. Antibodies against the N- and C-termini of NCS-1 caused significant increases in mini/spontaneous/stimulation-independent release of [(3)H]-NA from perforated nerve endings, but had no effect on [(14)C]-glu release. These results support the idea that NCS-1 facilitates nerve ending neurotransmitter release and phosphoinositide production via PI4K and localizes these effects to the N-terminal of NCS-1. Combined with previous work on the regulation of channels by NCS-1, the data are consistent with the hypothesis that a NCS-1-PI4K (NP, neuropotentiator) complex may serve as an essential linker between lipid and protein metabolism to regulate membrane traffic and co-ordinate it with ion fluxes and plasticity in the nerve ending.     

Neuroplasticity in the smooth muscle of the myenterically and extrinsically denervated rat jejunum

The objective of this study was to examine the effects of two different denervation procedures on the distribution of nerve fibers and neurotransmitter levels in the rat jejunum. Extrinsic nerves were eliminated by crushing the mesenteric pedicle to a segment of jejunum. The myenteric plexus and extrinsic nerves were eliminated by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). The effects of these two denervation procedures were evaluated at 15 and 45 days. The level of norepinephrine in whole segments of jejunum was initially reduced by more than 76% after both denervation procedures, but by 45 days the level of norepinephrine was the same as in control tissue. Tyrosine hydroxylase (nor-adrenergic nerve marker) immunostaining was absent at 15 days, but returned by 45 days. However, the pattern of noradrenergic innervating axons was altered in the segment deprived of myenteric neurons. Immunohistochemical studies showed protein gene product 9.5 (PGP 9.5)-immunoreactive fibers in whole-mount preparations of the circular smooth muscle in the absence of the myenteric plexus and extrinsic nerves. At 45 days, the number of nerve fibers in the circular smooth muscle increased. Vasoactive intestinal polypeptide (VIP)-immunoreactive fibers, a subset of the PGP 9.5 nerve fibers, were present in the circular smooth muscle at both time points examined. Choline acetyltransferase (CAT) activity and VIP and leucine enkephalin levels were measured in separated smooth muscle and submucosa-musosal layers of the denervated jejunum. VIP and leucine-enkephalin levels were no different from control in tissue that was extrinsically denervated alone. However, the levels of these peptides were elevated two-fold in the smooth muscle 15 and 45 days after myenteric and extrinsic denervation. In the submucosa-mucosa, VIP and leucine enkephalin levels also were elevated two-fold at 15 days, but comparable to control at 45 days. CAT activity was equal to control in the smooth muscle but elevated two-fold in the submucosa-mucosa at both times. These results provide evidence for innervation of the circular smooth muscle by the submucosal plexus. Moreover, these nerve fibers originating from the submucosal plexus proliferate in the absence of the myenteric plexus. Furthermore, the myenteric neurons appear to be essential for normal innervation of the smooth muscle by the sympathetic nerve fibers. It is speculated that the sprouting of the submucosal plexus induced by myenteric plexus ablation is mediated by increased production of trophic factors in the hyperplastic smooth muscle.     

Distribution of intrinsic nerve cell bodies and axons which take up aromatic amines and their precursors in the small intestine of the guinea-pig

Summary The sites of uptake, decarboxylation and retention of 1-dopa and the uptake and retention of dopamine and 6-hydroxytryptamine in the small intestine of the guinea-pig have been localised histochemically with a fluorescence technique for arylethylamines. In segments of ileum from untreated guinea-pigs only noradrenergic axons are fluorescent; these axons were eliminated by surgical denervation (crushing nerves running to the intestine through the mesentery) or by chemical denervation with 6-hydroxydopamine. In denervated segments of ileum, cell bodies and processes of intrinsic neurons become fluorescent after the injection of 1-dopa, dopamine or 6-hydroxytryptamine and the inhibition of monoamine oxidase, as do cells of Brunner's glands and Paneth cells. About 11% of the nerve cell bodies in the submucous plexus and 0.4% of those in the myenteric plexus become fluorescent. Varicose intrinsic axons which take up amines are found amongst the nerve cell bodies of the myenteric and submucous plexuses. They also ramify in the principal connections of the plexuses, in the tertiary strands of the myenteric plexus, in the deep muscular plexus and contribute sparse supplies of axons to arterioles in the submucosa and to the lamina propria of the mucosa. The axons are resistant to the degenerative actions of 6-hydroxydopamine.It is suggested that the intrinsic amine handling axons are more likely to utilise an indolamine related to 5-hydroxytryptamine than they are to utilise a catecholamine as a neurotransmitter.     

Morphological and Functional Alterations of the Myenteric Plexus in Rats with TNBS-Induced Colitis

The 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced model of experimental colitis was used to investigate the time-course of alterations in enteric neurotransmission and/or smooth muscle function that occur in chronic inflammation. Myenteric plexus morphology (immunocytochemical markers), functional integrity of cholinergic neurons (³H-choline uptake, acetylcholine release and contractile response to electrical field stimulation) and smooth muscle integrity (contractile response to exogenous acetylcholine) were determined 2, 7, 15, and 30 days after TNBS treatment. In TNBS-treated rats extensive ulcerations of the mucosa and/or the submucosa and increase in colonic weights were accompanied by significant reduction in ³H-choline uptake, acetylcholine release and contractile response to stimulation of enteric nerves. These changes were maximal 7 and 15 days after TNBS treatment. Immunocytochemical marker (PGP 9.5, SNAP 25, synaptophysin and S100 protein) expression was absent in necrotic areas of colons removed 7 days post-injury and partially reduced in colons removed 15 days after TNBS treatment. By contrast, the contractile response to exogenous acetylcholine was significantly increased after 7 days in both inflamed and uninflamed regions and returned to control values by day 30. Likewise, an almost complete recovery of neural cholinergic function and of myenteric plexus morphology was observed 30 days after TNBS treatment. These data suggest that TNBS-induced colitis is associated with progressive and selective alterations in myenteric plexus structure and function, with consequent reduction of cholinergic neurotransmission and abnormality in colonic contractility. The reversibility of myenteric plexus disruption is a clear indication of neuronal plasticity within enteric nervous system as an adaptative mechanism against inflammatory challenges.     

Down-regulation of voltage-gated Ca2+ channels by neuronal calcium sensor-1 is beta subunit-specific

Neuronal Ca(2+) sensor protein-1 (NCS-1) is a member of the Ca(2+) binding protein family, with three functional Ca(2+) binding EF-hands and an N-terminal myristoylation site. NCS-1 is expressed in brain and heart during embryonic and postnatal development. In neurons, NCS-1 facilitates neurotransmitter release, but both inhibition and facilitation of the Ca(2+) current amplitude have been reported. In heart, NCS-1 co-immunoprecipitates with K(+) channels and modulates their activity, but the potential effects of NCS-1 on cardiac Ca(2+) channels have not been investigated. To directly assess the effect of NCS-1 on the various types of Ca(2+) channels we have co-expressed NCS-1 in Xenopus oocytes, with Ca(V)1.2, Ca(V)2.1, and Ca(V)2.2 Ca(2+) channels, using various subunit combinations. The major effect of NCS-1 was to decrease Ca(2+) current amplitude, recorded with the three different types of alpha(1) subunit. When expressed with Ca(V)2.1, the depression of Ca(2+) current amplitude induced by NCS-1 was dependent upon the identity of the beta subunit expressed, with no block recorded without beta subunit or with the beta(3) subunit. Current-voltage and inactivation curves were also slightly modified and displayed a different specificity toward the beta subunits. Taken together, these data suggest that NCS-1 is able to modulate cardiac and neuronal voltage-gated Ca(2+) channels in a beta subunit specific manner.     

Agonists of cannabinoid receptor 1 and 2 inhibit experimental colitis induced by oil of mustard and by dextran sulfate sodium

Oil of mustard (OM) is a potent neuronal activator that is known to elicit visceral hyperalgesia when given intracolonically, but the full extent to which OM is also proinflammatory in the gastrointestinal tract is not known. We have previously shown that male CD-1 mice given a single administration of 0.5% OM develop a severe colitis that is maximum at day 3 and that gradually lessens until essentially absent by day 14. OM-induced neuronal stimulation is reported to be reduced by cannabinoid agonists, and cannabinoid receptor 1 (CB1R)-/- mice have exacerbated experimental colitis. Therefore, we examined the role of cannabinoids in this OM-induced 3-day model of colitis in CD-1 mice and in a 7-day dextran sulfate sodium (DSS) colitis model in BALB/c mice. In OM colitis, the CB1R-selective agonist ACEA and the CB2R-selective agonist JWH-133 reduced (P < 0.05) colon weight gain (means +/- SE; 82 +/- 13% and 47 +/- 15% inhibition, respectively), colon shrinkage (98 +/- 24% and 42 +/- 12%, respectively), colon inflammatory damage score (49 +/- 11% and 40 +/- 12%, respectively), and diarrhea (58 +/- 12% and 43 +/- 11%, respectively). Histological damage was similarly reduced by these treatments. Likewise, CBR agonists attenuated DSS colitis, albeit at higher doses; ACEA at 10 mg/kg, twice daily, inhibited (P < 0.05) macroscopic and microscopic scores (46 +/- 9% and 63 +/- 7%, respectively); whereas 20 mg/kg, twice daily, of JWH-133 was required to diminish (P < 0.05) macroscopic and microscopic scores (29 +/- 7% and 43 +/- 5%, respectively). CB1R and CB2R immunostaining of colon sections revealed that CB1R in enteric neurons was more intense in colitic vs. control mice; however, CB1R was also increased in the endothelial layer in OM colitis only. CB2R immunostaining was more marked in infiltrated immune cells in OM colitis. These findings validate the OM colitis model with respect to the DSS model and provide strong support to the emerging idea that cannabinoid receptor activation mediates protective mechanisms in experimental colitis. The demonstration of CB1R agonist effects in colitis support the neurogenic nature of the OM-induced colitis model and reinforce the importance of neuronal activation in intestinal inflammation.     

5-HT4 receptor activation facilitates recovery from synaptic rundown and increases transmitter release from single varicosities of myenteric neurons

5-HT(4) receptor agonists facilitate synaptic transmission in the enteric nervous system, and these drugs are used to treat constipation. In the present study, we investigated the effects of the 5-HT(4) receptor agonist, renzapride, on rundown and recovery of fast excitatory postsynaptic potentials (fEPSPs) during and after trains of stimulation and on transmitter release from individual myenteric neuronal varicosities. Intracellular electrophysiological methods were used to record fEPSPs from neurons in longitudinal muscle myenteric plexus preparations of guinea pig ileum in vitro. During trains of supramaximal electrical stimulation (10 Hz, 2 s), fEPSP amplitude declined (time constant = 0.6 +/- 0.1 s) from 17 +/- 2 mV to 0.7 +/- 0.3 mV. Renzapride (0.1 microM) did not change the time constant for fEPSP rundown, but it decreased the time constant for recovery of fEPSP amplitude after the stimulus train from 7 +/- 2 s to 1.6 +/- 0.2 s (P < 0.05). 5-HT (0.1 microM) also increased fEPSPs and facilitated recovery from rundown. The adenylate cyclase activator, forskolin (1 muM), mimicked the actions of renzapride and 5-HT, whereas H-89, a protein kinase A (PKA) inhibitor, blocked the effects of renzapride. We used nicotinic acetylcholine receptor containing outside-out patches obtained from myenteric neurons maintained in primary culture to detect acetylcholine release from single varicosities. Renzapride (0.1 microM) increased release probability twofold. We conclude that 5-HT(4) receptors activate the adenylyl cyclase-PKA pathway to increase acetylcholine release from single varicosities and to accelerate recovery from synaptic rundown. These responses may contribute to the prokinetic actions of 5-HT(4) receptor agonists.