Facilitation Of Acetylcholine Release Research Articles

Synaptotagmin-7 facilitates acetylcholine release in splanchnic nerve-chromaffin cell synapses during nerve activity

Disturbances that threaten homeostasis elicit activation of the sympathetic nervous system (SNS) and the adrenal medulla. The effectors discharge as a unit to drive global and immediate changes in whole-body physiology. Descending sympathetic information is conveyed to the adrenal medulla via preganglionic splanchnic fibers. These fibers pass into the gland and synapse onto chromaffin cells, which synthesize, store, and secrete catecholamines and vasoactive peptides. While the importance of the sympatho-adrenal branch of the autonomic nervous system has been appreciated for many decades, the mechanisms underlying transmission between presynaptic splanchnic neurons and postsynaptic chromaffin cells have remained obscure. In contrast to chromaffin cells, which have enjoyed sustained attention as a model system for exocytosis, even the Ca2+ sensors that are expressed within splanchnic terminals have not yet been identified. This study shows that a ubiquitous Ca2+-binding protein, synaptotagmin-7 (Syt7), is expressed within the fibers that innervate the adrenal medulla, and that its absence can alter synaptic transmission in the preganglionic terminals of chromaffin cells. The prevailing impact in synapses that lack Syt7 is a decrease in synaptic strength and neuronal short-term plasticity. Evoked excitatory postsynaptic currents (EPSCs) in Syt7 KO preganglionic terminals are smaller in amplitude than in wild-type synapses stimulated in an identical manner. Splanchnic inputs also display robust short-term presynaptic facilitation, which is compromised in the absence of Syt7. These data reveal, for the first time, a role for any synaptotagmin at the splanchnic-chromaffin cell synapse. They also suggest that Syt7 has actions at synaptic terminals that are conserved across central and peripheral branches of the nervous system.

Lesions of the nucleus basalis magnocellularis (Meynert) induce enhanced somatosensory responses and tactile hypersensitivity in rats

We used the immunotoxin 192 immunoglobulin G-saporin to produce a selective cholinergic lesion in the nucleus basalis of Meynert (NBM) of rats and investigated whether the NBM lesion led to tactile hypersensitivity in the forepaw. The paw mechanical threshold test showed that the lesioned rats had a decreased threshold compared to the control. Surprisingly, there was a significant positive correlation between mechanical threshold and survival rate of NBM cholinergic neurons. Furthermore, using local field potential (LFP) recordings and voltage-sensitive dye (VSD) imaging, we found that the forepaw-evoked response in the primary somatosensory cortex (S1) was significantly enhanced in both amplitude and spatial extent in the NBM-lesioned rats. The neurophysiological measures of S1 response, such as LFP amplitude and maximal activated cortical area depicted by VSD, were also correlated with withdrawal behavior. Additional pharmacological experiments demonstrated that forepaw-evoked responses were increased in naive rats by blocking S1 cholinergic receptors with mecamylamine and scopolamine, while the response decreased in NBM-lesioned rats with the cholinergic agonist carbachol. In addition, NBM burst stimulation, which facilitates acetylcholine release in the S1, suppressed subsequent sensory responses to forepaw stimulation. Taken together, these results suggest that neuronal loss in the NBM diminishes acetylcholine actions in the S1, thereby enhancing the cortical representation of sensory stimuli, which may in turn lead to behavioral hypersensitivity.

Sex differences in the cardiac cholinergic response to adrenalin-induced myocardial necrosis and light desynchronosis

The aim was to identify sex differences in the cardiac response to cholinergic stimuli in the development of myocardial necrosis and light desynchronosis.Materials and methods. Experiments were conducted in 144 albino rats (72 males, 72 females). Myocardial necrosis was induced by adrenaline (0.5 mg/kg, intramuscularly).The intensity of bradycardia that occurred as a response to intravenous acetylcholine injection and electrical stimulation of vagus nerve was measured. The animals were divided into 4 groups: 1 – rats that were in a light balance (day/night); 2 – rats exposed to light deprivation for 10 days; 3 – rats exposed to permanent lighting for 10 days; 4 – animals exposed to permanent lighting for 10 days and injected with melatonin (5 mg/kg) intraperitoneally 1 hour before the necrosis modeling.Results. The development of myocardial necrosis when occurring in light deprivation was characterized by an increased responsiveness of the rat heart to cholinergic stimulation due to an increase in both cholinoreceptors sensitivity and release of acetylcholine from the vagus nerve terminals in females, and due to only an increased cholinoreceptors sensitivity in males. The development of myocardial necrosis in rats exposed to permanent lighting was characterized by significantly higher sensitivity of cholinergic structures of the heart than that in light balance, especially in females. Melatonin injection in the development of myocardial necrosis contributed to the heart cholinergic structures response to the stimulation, which was close to that observed in conditions of light balance, though it remained somewhat higher: due to the facilitation of acetylcholine release from vagus nerve in females, and it was combined with a higher sensitivity of cholinoreceptors in males.Conclusions. The development of myocardial necrosis in light desynchronosis (light deprivation or permanent lighting) causes an increased heart response to cholinergic stimulation in rats. Such effects are stronger, especially in females in conditions of permanent lighting. The injection of melatonin in rats with myocardial necrosis and permanent lighting results in an approximation (but not restoration) of the heart sensitivity to cholinergic stimuli parameters to those that are observed in light balance. However, the heart response to cholinergic stimulation remains higher, especially in males.

Synergy between 5-HT4 receptor stimulation and phosphodiesterase 4 inhibition in facilitating acetylcholine release in human large intestinal circular muscle.

Gastroprokinetic properties of 5-HT4 receptor agonists, such as prucalopride, are attributed to activation of 5-HT4 receptors on cholinergic nerves innervating smooth muscle in the gastrointestinal smooth muscle layer, increasing acetylcholine release and muscle contraction. In porcine stomach and colon, phosphodiesterase (PDE) 4 has been shown to control the signaling pathway of these 5-HT4 receptors. The aim of this study was to investigate the PDE-mediated control of these 5-HT4 receptors in human large intestine. Circular smooth muscle strips were prepared from human large intestine; after incubation with [³H]-choline, electrically induced tritium outflow was determined as a measure for acetylcholine release. The influence of PDE inhibition on the facilitating effect of prucalopride on electrically induced acetylcholine release was studied. The non-selective PDE inhibitor IBMX enhanced the facilitating effect of prucalopride on electrically induced acetylcholine release. The selective inhibitors vinpocetine (PDE1), EHNA (PDE2) and cilostamide (PDE3) did not influence, while rolipram and roflumilast (PDE4) enhanced the prucalopride-induced facilitation to the same extent as IBMX. In human large intestinal circular muscle, the intracellular pathway of 5-HT4 receptors facilitating cholinergic neurotransmission to large intestinal circular smooth muscle is controlled by PDE4. If the synergy between 5-HT4 receptor agonism and PDE4 inhibition is confirmed in a functional assay with electrically induced cholinergic contractions of human large intestinal circular smooth muscle strips, combination of a selective 5-HT4 receptor agonist with a selective PDE4 inhibitor might enhance the in vivo prokinetic effect of the 5-HT4 receptor agonist in the large intestine.

Synergistic effect between 5-HT4 receptor agonist and phosphodiesterase 4-inhibitor in releasing acetylcholine in pig gastric circular muscle in vitro

5-HT4 receptor agonists have a gastroprokinetic effect by facilitating acetylcholine release from cholinergic nerves innervating gastrointestinal smooth muscle. The role of phosphodiesterase (PDE) 4 in the signal transduction pathway of the 5-HT4 receptors located on the cholinergic neurons towards the circular muscle layer in pig stomach was investigated by analysis of acetylcholine release. Circular muscle strips were prepared from pig proximal stomach and tritium outflow, induced by electrical field stimulation, was studied as a marker for acetylcholine release after incubation with [3H]-choline. The PDE4-inhibitor roflumilast concentration-dependently (0.1–1µM) enhanced the facilitating effect of a submaximally effective concentration of the 5-HT4 receptor agonist prucalopride (0.01µM) on electrically induced acetylcholine release. Roflumilast (0.3µM) enhanced acetylcholine release per se but in the combined presence of roflumilast and prucalopride, acetylcholine release was enhanced more than the sum of the effect of the 2 compounds alone. The 5-HT4 receptor agonist velusetrag concentration-dependently (0.01–0.1µM) enhanced acetylcholine release; the effect of the minimally effective concentration (0.01µM) was significantly enhanced by 1µM of the PDE4-inhibitor rolipram, again to a level higher than the sum of the effect of the 2 compounds alone. The synergistic effect between 5-HT4 receptor agonists and PDE4-inhibitors demonstrates that the intracellular pathway of the 5-HT4 receptors located on cholinergic neurons towards pig gastric circular muscle is controlled by PDE4. Combining a 5-HT4 receptor agonist with a PDE4-inhibitor might thus enhance its gastroprokinetic effect.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

Selective 5-HT4 receptor agonists such as prucalopride are used as human prokinetics, since activation of 5-HT4 receptors on intestinal cholinergic neurons facilitates acetylcholine release. 5-HT4 receptors, linked to adenylyl cyclase, act via generation of cAMP. None of the 4 in vitro studies on 5-HT in horses provided evidence for neuronal 5-HT4 receptors, but none used the protocol as described in human studies [1-4]. To investigate whether functional 5-HT4 receptors are present in the equine small intestine. In vitro organ bath set up, applying electrical field stimulation (EFS) in longitudinal and circular smooth muscle strips. Results were similar in both muscle layers. In the presence of 0.3 mmol/l NG-Nitro-L-arginine methyl ester and 0.3 μmol/l apamine, excluding effects of the inhibitory transmitters NO and ATP, EFS induced voltage-dependent on-contractions; these were neurogenic as they were abolished by 3 μmol/l tetrodotoxin. At a voltage inducing 50% of the maximal amplitude, the submaximal EFS-induced contractions were cholinergic as atropine (1 μmol/l) abolished them. Prucalopride (0.3 μmol/l) did not increase the amplitude of these submaximal EFS-induced contractions. Even in the presence of the nonselective phosphodiesterase inhibitor IBMX, previously shown to enhance the effect of neuronal 5-HT4 receptors by inhibiting breakdown of their 2nd messenger cAMP [5], prucalopride (3 μmol/l) had no influence. Also 5-HT (10 μmol/l), a full agonist at 5-HT4 receptors, tested in the presence of methysergide and granisetron to exclude interaction with other 5-HT receptor subtypes, did not enhance EFS-induced submaximal contractions. There is no evidence for presence of 5-HT4 receptors on the cholinergic neurons of the equine small intestine. These results question the application of 5-HT4 prokinetic drugs in horses. Ethical animal research: Research ethics committee oversight not currently required by this conference: the study was performed on material collected at an abattoir. Sources of funding: None. Competing interests: None declared.

Procognitive effect of AC-3933 in aged mice, and synergistic effect of combination with donepezil in scopolamine-treated mice

We have previously reported that AC-3933, a newly developed benzodiazepine receptor partial inverse agonist, facilitates acetylcholine release in the hippocampus and ameliorates scopolamine-induced memory deficits in rats. To further confirm the procognitive effect of AC-3933, we assessed in this study the beneficial effects of this compound in aged mice using the Y-maze and object recognition tests. In addition, we investigated the synergistic effect of AC-3933 and donepezil, a cholinesterase inhibitor, on scopolamine-induced memory impairment in mice. In aged mice, oral administration of AC-3933 at doses of 0.05–0.1mg/kg and 0.05mg/kg significantly improved spatial working memory and episodic memory, respectively. In scopolamine-treated mice, both AC-3933 and donepezil significantly ameliorated memory deficits in the Y-maze test at doses of 0.3–3mg/kg and 10–15mg/kg, respectively. The beneficial effect of AC-3933, but not that of donepezil, on scopolamine-induced memory impairment was antagonized by flumazenil, a benzodiazepine receptor antagonist, indicating that the procognitive action of AC-3933 arises via a mechanism different from that of donepezil. Co-administration of donepezil at the suboptimal dose of 3mg/kg with AC-3933 at doses of 0.1–1mg/kg significantly ameliorated scopolamine-induced memory impairment, suggesting that AC-3933 potentiates the effect of donepezil on memory impairment induced by cholinergic hypofunction. These findings indicate that AC-3933 not only has good potential as a cognitive enhancer by itself, but also is useful as a concomitant drug for the treatment of Alzheimer׳s disease.

P.5.c.002 Nanoparticles as disease-modifying mediators for brain therapy: focus on Huntington's disease

Optimized-SopungSunkiwon (OSS) is a multi-herbal formula that contains six medicinal herbs from SopungSunkiwon, a traditional medicine used for neurodegenerative disorders. In this study, we investigated the anti-amnesic effects of OSS in a dementia model. Acetylcholinesterase (AChE) inhibition assay was performed to investigate the cholinergic antagonistic effect of OSS. In addition, a step-through passive-avoidance test was performed with scopolamine-induced memory impairment in mice, and immunohistochemistry was analyzed to investigate synaptic formation with synaptic proteins. OSS inhibited AChE activity, resulting in significant improvement of memory functions. In the passive-avoidance test, the latency time of OSS-treated mice was significantly longer than that of either the control or scopolamine-treated group. In the immunohistochemical analysis, synaptic proteins such as synaptophysin and PSD-95 were significantly increased in OSS-treated mice. These results demonstrate that OSS may affect impairment and enhancement of memory and increase synaptophysin and PSD-95 facilitating acetylcholine release and synaptic growth.

Angiotensin AT1–Receptor Blockers Enhance Cardiac Responses to Parasympathetic Nerve Stimulation via Presynaptic AT1 Receptors in Pithed Rats

In the present study, we investigated the effects of angiotensin AT1-receptor blockers, KT3-671 and losartan, on the cardiac vagal neurotransmission in pithed rats. The bradycardia induced by vagal nerve stimulation (VNS, at 5 Hz) was potentiated significantly and dose-dependently by KT3-671 and also losartan. This enhancement effect of KT3-671 (10 mg/kg) was slightly potent than that of losartan (10 mg/kg). On the other hand, an angiotensin AT2-receptor blocker, PD123319 (10 mg/kg), did not affect VNS-induced bradycardia. KT3-671 and losartan did not affect the exogenous acetylcholine-evoked bradycardia. Intravenous infusion of AngII (100 ng/kg per min) attenuated the VNS-induced bradycardia. This inhibitory effect of AngII on bradycardia was restored by both KT3-671 and losartan. These results suggest that endogenous AngII can have a tonic inhibitory effect on cardiac vagal transmission by stimulating the presynaptic AT1 receptors not AT2 receptors. Suppression of this mechanism by the AT1-receptor blockers causes the facilitation of acetylcholine release from vagal nerve endings. This acceleratory effect of AT1-receptor blockers on cardiac vagal neurotransmission may contribute to the lack of reflex tachycardia following hypotension.

P21 Endogenous H2S produced by CSE selectively facilitates acetylcholine release from synaptic terminals of central origin in mouse superior mesenteric ganglion

Abdominal sympathetic prevertebral ganglia in mammals play a major physiological role in regulating motility, absorption and secretion in the gastrointestinal tract and its accessory organs. Hydrogen sulfide (H2S) has emerged as an important messenger molecule in the central nervous system and enteric nervous system. We designed experiments to test the hypotheses that prevertebral ganglia have the enzymatic machinery to generate H2S, that H2S is continuously generated and released in the prevertebral ganglia and that H2S modulates fast cholinergic nicotinic excitatory synaptic input (F-EPSP). Methods The superior mesenteric ganglion (SMG) and attached splanchnic and colonic nerve trunks were dissected from adult SJL/J mice. The use of mouse tissue was approved by the Institutional Animal Care and Use Committee. Sharp glass microelectrodes were used for intracellular recordings and bi-polar stimulating electrodes were used to evoke F-EPSPs. Immunostaining was performed with antibodies for cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) and with vesicular acetylcholine transporter to identify preganglionic cholinergic terminals. Since the amount of tissue in mouse prevertebral ganglia was less than the amount required for the assay method (gas chromatography), we used guinea pig prevertebral ganglia to measure endogenous H2S generation. Results Immunoreactivity of CSE was found in neurons and glia cells. There was no colocalization of CSE with immunoreactivity of vesicular acetylcholine transporter. Immunoreactivity of CBS was only found in some glia cells. There was no colocalization between immunoreactivity of CBS and immunoreactivity of vesicular acetylcholine transporter. Endogenous H2S was generated and released from homogenized guinea pig sympathetic prevertebral ganglia at a rate of 2.52 ± 0.52 pmol/min/mg tissue weight, suggesting that H2S is generated in intact prevertebral ganglia. To determine whether the F-EPSPs are potentiated by endogenously released H2S in mouse SMG, we tested the effect of inhibiting H2S break-down using stigmatellin, a specific sulfide quinone reductase inhibitor. Stigmatellin (1 μM) significantly (p dl -propargylglycine (PAG) (1 μM) on F-EPSPs evoked by splanchnic and colonic nerve stimulation in the presence of PAG. The amplitude and area of F-EPSPs evoked by splanchnic nerve stimulation were significantly (p 0.05) effect on F-EPSPs evoked by colonic nerve stimulation (30.2 ± 6.1 mV and 808 ± 235 ms · mV with PAG vs. 26.9 ± 7.6 mV and 855 ± 241 ms · mV before PAG; n = 5). Conclusions H2S is enzymatically generated and released in the mouse prevertebral ganglia where it potentiates fast cholinergic synaptic input in a pathway specific manner.

The facilitating effect of prucalopride on cholinergic neurotransmission in pig gastric circular muscle is regulated by phosphodiesterase 4

The influence of the selective 5-HT4 receptor agonist prucalopride on acetylcholine release from cholinergic nerve endings innervating pig gastric circular muscle and the possible regulation of this effect by phosphodiesterases (PDEs) was investigated, as PDEs have been shown to control the response to 5-HT4 receptor activation in pig left atrium. Circular muscle strips were prepared from pig proximal stomach and either submaximal cholinergic contractions or tritium outflow after incubation with [3H]-choline, induced by electrical field stimulation, were studied. Prucalopride concentration-dependently increased the amplitude of submaximal cholinergic contractions and of acetylcholine release induced by electrical field stimulation. The effect of the highest concentration tested (0.3 μM) on cholinergic contractions was antagonized by the selective 5-HT4 receptor antagonist GR113808 but not by granisetron or methysergide; the antagonism of prucalopride by GR113808 was confirmed in the release assay. The non-selective PDE-inhibitor 3-isobutyl-methyl-xanthine (IBMX) concentration-dependently reduced the amplitude of the cholinergic contractions; 3 μM IBMX reduced the cholinergic contractions maximally by 16% but it enhanced the facilitating effect of prucalopride from 51 to 83%. IBMX (10 μM) induced and enhanced the facilitating effect of prucalopride on electrically induced acetylcholine release. The selective inhibitors vinpocetine (PDE1), EHNA (PDE2) and cilostamide (PDE3) did not influence the effect of prucalopride on acetylcholine release but the PDE4-inhibitor rolipram (1 μM) enhanced the facilitating effect of prucalopride to the same extent as IBMX. These results demonstrate that 5-HT4 receptors are present on the cholinergic nerves towards the pig gastric circular muscle, facilitating acetylcholine release; the intracellular transduction pathway of this facilitation is regulated by PDE4. Combination of a 5-HT4 receptor agonist with selective inhibition of the PDE involved in this regulation of transmitter release might enhance the prokinetic effect of the 5-HT4 receptor agonist.

Dopamine–Galanin Receptor Heteromers Modulate Cholinergic Neurotransmission in the Rat Ventral Hippocampus

Previous studies have shown that dopamine and galanin modulate cholinergic transmission in the hippocampus, but little is known about the mechanisms involved and their possible interactions. By using resonance energy transfer techniques in transfected mammalian cells, we demonstrated the existence of heteromers between the dopamine D(1)-like receptors (D(1) and D(5)) and galanin Gal(1), but not Gal(2) receptors. Within the D(1)-Gal(1) and D(5)-Gal(1) receptor heteromers, dopamine receptor activation potentiated and dopamine receptor blockade counteracted MAPK activation induced by stimulation of Gal(1) receptors, whereas Gal(1) receptor activation or blockade did not modify D(1)-like receptor-mediated MAPK activation. Ability of a D(1)-like receptor antagonist to block galanin-induced MAPK activation (cross-antagonism) was used as a "biochemical fingerprint" of D(1)-like-Gal(1) receptor heteromers, allowing their identification in the rat ventral hippocampus. The functional role of D(1)-like-Gal receptor heteromers was demonstrated in synaptosomes from rat ventral hippocampus, where galanin facilitated acetylcholine release, but only with costimulation of D(1)-like receptors. Electrophysiological experiments in rat ventral hippocampal slices showed that these receptor interactions modulate hippocampal synaptic transmission. Thus, a D(1)-like receptor agonist that was ineffective when administered alone turned an inhibitory effect of galanin into an excitatory effect, an interaction that required cholinergic neurotransmission. Altogether, our results strongly suggest that D(1)-like-Gal(1) receptor heteromers act as processors that integrate signals of two different neurotransmitters, dopamine and galanin, to modulate hippocampal cholinergic neurotransmission.

Effects of optimized-SopungSunkiwon on memory impairment and enhancement

Optimized-SopungSunkiwon (OSS) is a multi-herbal formula that contains six medicinal herbs from SopungSunkiwon, a traditional medicine used for neurodegenerative disorders. In this study, we investigated the anti-amnesic effects of OSS in a dementia model. Acetylcholinesterase (AChE) inhibition assay was performed to investigate the cholinergic antagonistic effect of OSS. In addition, a step-through passive-avoidance test was performed with scopolamine-induced memory impairment in mice, and immunohistochemistry was analyzed to investigate synaptic formation with synaptic proteins. OSS inhibited AChE activity, resulting in significant improvement of memory functions. In the passive-avoidance test, the latency time of OSS-treated mice was significantly longer than that of either the control or scopolamine-treated group. In the immunohistochemical analysis, synaptic proteins such as synaptophysin and PSD-95 were significantly increased in OSS-treated mice. These results demonstrate that OSS may affect impairment and enhancement of memory and increase synaptophysin and PSD-95 facilitating acetylcholine release and synaptic growth.

Influence of 5-HT4receptor activation on acetylcholine release in human large intestine with endometriosis

The 5-HT(4) receptor agonist prucalopride enhances large intestinal contractility by facilitating acetylcholine release through activation of 5-HT(4) receptors on cholinergic nerves and is effective in patients with constipation. Patients with intestinal endometriosis can present with constipation. We investigated in vitro whether large intestinal endometriotic infiltration influences contractility and facilitation of acetylcholine release by prucalopride. Sigmoid colon or rectum circular muscle strips were obtained at the level of an endometriotic nodule with infiltration of the Auerbach plexus, and at a macroscopically healthy site at least 5 cm cranially from the nodule, in patients undergoing laparoscopic colorectal resection because of symptomatic bowel endometriosis. Responses to muscarinic receptor stimulation and to electrical field stimulation (EFS), and the facilitating effect of prucalopride on acetylcholine release were evaluated. The EC50 and E(max) of the contractile responses to the muscarinic receptor agonist carbachol did not differ between healthy and lesioned strips. EFS-induced on-contractions were not different between the healthy and lesioned strips, while the non-nitrergic relaxant responses induced by EFS were decreased in the lesioned strips. The facilitating effect of prucalopride on acetylcholine release in healthy strips was similar to that reported before in macroscopically healthy colon tissue of patients with colon cancer; in lesioned strips, the effect of prucalopride was fully maintained in 6/8 patients and absent in two. Large intestinal endometriosis does not lead to a systematic interference with the cholinergic facilitating effect of prucalopride.

The effect of mosapride citrate on constipation in patients with diabetes

Objective Constipation is one of the most common gastrointestinal complications suspected to degrade the quality of life in diabetic patients. Mosapride citrate is a novel selective 5-HT4 receptor agonist, which enhances gastric emptying and motility in the upper gastrointestinal tract facilitating acetylcholine release from the enteric cholinergic neurons. Since the receptors of 5-HT4 have been recently found in the lower intestine including the rectum in humans, we tested if mosapride improves constipation in diabetic patients. Patients and methods Diabetic patients with constipation were treated with mosapride citrate (15 mg/day, n = 20) or domperidone (30 mg/day, n = 12) as controls for 8 weeks. Bowel frequency was monitored and gastrointestinal symptoms were evaluated by Gastrointestinal Symptoms Rating Scale (GSRS). Results Administration of mosapride resulted in increased bowel frequency after 4 and 8 weeks of administration, while no change was seen in the control group. Mosapride increased bowel frequency in even patients with diabetic polyneuropathy. Mosapride improved reflux and constipation evaluated by GSRS. As previously reported, glycemic control also improved by mosapride in these patients. Conclusions Administration of mosapride increased bowel frequency and ameliorated symptoms of reflux and constipation possibly stimulating the lower intestine. Given that mosapride has those beneficial aspects, it may be a useful prokinetic agent in treating diabetic patients with constipation.

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

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 M(2) 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 M(3) receptors activation, as predicted by blockade with J104129 (a selective M(3) antagonist), 4-DAMP and atropine, whereas the selective M(2) 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 ( approximately 60%) evoked adenosine outflow (5 Hz, 3000 pulses). Oxotremorine (300 micromol 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 M(2) and facilitatory M(3) autoreceptors. While the M(2) inhibition is prevalent during brief stimulation periods, muscarinic M(3) facilitation is highlighted during sustained nerve activity as it depends on extracellular adenosine accumulation leading to activation of facilitatory A(2A) receptors.

P4.20 On the role of adenosine (A 2A) tonus and cross talk with NK 1 and M 3 receptors regulating acetylcholine release from myenteric neurons of the rat ileum

Gastrointestinal motility relies on the release of acetylcholine (ACh), purines and tachykinins (e.g. substance P) from excitatory enteric neurons. Tachykinins acting via NK1 and NK3 receptors facilitate cholinergic neurotransmission in the enteric nervous system. Recently, we showed that muscarinic M3 receptors facilitate ACh release indirectly by increasing adenosine outflow from stimulated myenteric neurons leading to the activation of excitatory A2A receptors [1]. It is well established that adenosine A2A receptors play a widespread role in synaptic plasticity via a sophisticated interaction with receptors for other neurotransmitters/neuromodulators. In the myenteric plexus, endogenous adenosine may arise from the catabolism of released ATP through the ectonucleotidase pathway together with the release of the nucleoside per se via the equilibrative transport system [2]. Preliminary accounts from our lab, suggest that CAP-sensitive tachykinergic neurons are an important source of purines in the rat myenteric plexus. These findings prompted us to investigate the cross talk between facilitatory A2A, M3 and NK1 receptors in rat longitudinal muscle-myenteric plexus preparations, focusing our attention on the role of tachykinergic nerve fibres which can be selectively eliminated (>95%) by neonatal capsaicin administration (CAP, 50 mg/kg, SC).

Cross talk between facilitatory adenosine A2A, Tachykinin NK1 and Muscarinic M3 receptors in the rat Mynteric plexus: on the role of Capsaicin-sensitive Tachykinergic neurons

Event Abstract Back to Event Cross talk between facilitatory adenosine A2A, Tachykinin NK1 and Muscarinic M3 receptors in the rat Mynteric plexus: on the role of Capsaicin-sensitive Tachykinergic neurons I. Silva1*, C. Vieira1, F. Ferreirinha1, M. Duarte-Araujo1 and P. Correia- De-Sa1 1 Instituto de Ciencias Biomedicas Abel Salazar - Universidade do Porto (ICBAS-UP), Laboratorio de Farmacologia e Neurobiologia, Unidade Multidisciplinar de Investigacao Biomedica (UMIB), Portugal Gastrointestinal motility relies on the release of acetylcholine (ACh), purines and tachykinins (e.g. substance P) from excitatory enteric neurons. Tachykinins (TK) acting via NK1 and NK3 receptors facilitate cholinergic neurotransmission in the enteric nervous system. Recently, we showed that muscarinic M3 receptors facilitate ACh release indirectly by increasing adenosine outflow from stimulated myenteric neurons leading to the activation of excitatory A2A receptors (Vieira et al., 2009, Neurogastroenterol. Motil. in press). These findings prompted us to investigate the cross talk between facilitatory A2A, M3 and NK1 receptors in the rat myenteric plexus, focusing our attention on the role of tachykinergic nerve fibres which can be selectively eliminated (>95%) by neonatal capsaicin administration (CAP, 50 mg/Kg, SC).The muscarinic agonist, oxotremorine (Oxo, 300 μM) and the selective adenosine A2A agonist, CGS21680C (CGS, 3 nM), increased evoked (5 Hz, 200 pulses) [3H]-ACh release from myenteric neurons respectively by 34±4% (n=5) and 53±10% (n=5) in controls (C) but were devoid of effects in CAP animals. Selective activation of NK1 receptors with Sar9,Met(O2)11-Substance P (s,m-SP, 300 nM) restored CGS facilitation (46±17%, n=6) to control levels in CAP rats. Cross talk between A2A and NK1 receptors was evaluated using selective antagonists of NK1 (L732128, 20 nM) and A2A (ZM241385, 50 nM) receptors. Pretreatment with s,m-SP did not restore Oxo facilitation (3±7%, n=4) in CAP animals. Incubation of C with the M3 antagonist (J104129, 6 nM) attenuated CGS (3±5%, n=5)-induced facilitation of [3H]-ACh release, but (like in CAP rats) its effect was partially recovered (23±2%, n=3) in the presence of s,m-SP.Data suggest that facilitation of [3H]-ACh release by A2A receptors requires tonic activation of NK1 receptors by endogenously released TK from CAP-sensitive myenteric neurons. Confocal microscopy studies show that A2A receptors co-localize with cholinergic nerve fibres (labelled with VAChT), but not with CAP-sensitive neurons exhibiting substance P immunoreactivity. Data also indicate that endogenous release of TK (as well as adenosine, see Vieira et al., 2009) may be tightly controlled by M3 receptors. In support of this hypothesis, we showed that CAP-sensitive tachykinergic neurons might be an important source of purines in the rat myenteric plexus (see, Marques et al., 2009, this meeting).Work supported by FCT (FEDER funding, PTDC/CVT/74462/2006) Conference: 11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009. Presentation Type: Poster Presentation Topic: Neuronal Communication Citation: Silva I, Vieira C, Ferreirinha F, Duarte-Araujo M and Correia- De-Sa P (2009). Cross talk between facilitatory adenosine A2A, Tachykinin NK1 and Muscarinic M3 receptors in the rat Mynteric plexus: on the role of Capsaicin-sensitive Tachykinergic neurons. Front. Neurosci. Conference Abstract: 11th Meeting of the Portuguese Society for Neuroscience. doi: 10.3389/conf.neuro.01.2009.11.149 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Aug 2009; Published Online: 12 Aug 2009. * Correspondence: I. Silva, Instituto de Ciencias Biomedicas Abel Salazar - Universidade do Porto (ICBAS-UP), Laboratorio de Farmacologia e Neurobiologia, Unidade Multidisciplinar de Investigacao Biomedica (UMIB), Porto, Portugal, isabelsds@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers I. Silva C. Vieira F. Ferreirinha M. Duarte-Araujo P. Correia- De-Sa Google I. Silva C. Vieira F. Ferreirinha M. Duarte-Araujo P. Correia- De-Sa Google Scholar I. Silva C. Vieira F. Ferreirinha M. Duarte-Araujo P. Correia- De-Sa PubMed I. Silva C. Vieira F. Ferreirinha M. Duarte-Araujo P. Correia- De-Sa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Tachykinin regulation of cholinergic transmission in the limbic/prefrontal territory of the rat dorsal striatum: implication of new neurokinine 1‐sensitive receptor binding site and interaction with enkephalin/mu opioid receptor transmission

The tachykinin neurokinin 1 receptors (NK(1)Rs) regulation of acetylcholine release and its interaction with the enkephalin/mu opioid receptors (MORs) transmission was investigated in the limbic/prefrontal (PF) territory of the dorsal striatum. Using double immunohistochemistry, we first showed that in this territory, cholinergic interneurons contain tachykinin NK(1)Rs and co-express MORs in the last part of the light period (afternoon). In slices of the striatal limbic/PF territory, following suppression of the dopaminergic inhibitory control of acetylcholine release, application of the tachykinin NK(1)R antagonist, SSR240600, markedly reduced the NMDA-induced acetylcholine release in the morning but not in the afternoon when the enkephalin/MOR regulation is operational. In the afternoon, the NK(1)R antagonist response required the suppression of the enkephalin/MOR inhibitory control of acetylcholine release by betafunaltrexamine. The pharmacological profile of the tachykinin NK(1)R regulation tested by application of the receptor agonists [[Pro(9)]substance P, neurokinin A, neuropeptide K, and substance P(6-11)] and antagonists (SSR240600, GR205171, GR82334, and RP67580) indicated that the subtype of tachykinin NK(1)R implicated are the new NK(1)-sensitive receptor binding site. Therefore, in the limbic/PF territory of the dorsal striatum, endogenous tachykinin facilitates acetylcholine release via a tachykinin NK(1)R subtype. In the afternoon, the tachykinin/NK(1)R and the enkephalin/MOR transmissions interact to control cholinergic transmission.

Dissociation between M1‐facilitation of acetylcholine release and crosstalk with A2A‐ and M2‐receptors on rat motoneurons

AbstractAcetylcholine (ACh) facilitates its own release acting at muscarinic M1 receptors on rat motoneurons. While the M1 positive feedback mechanism is operative there is a concomitant suppression of the ability of muscarinic M2‐inhibitory and adenosine A2A‐facilitatory receptors to control [3H]ACh release. We aimed at investigating whether M1‐occlusion of M2 and A2A receptors function could result from interplay at second messengers level. Drugs blocking the IP3 pathway, like LiCl and 2‐aminoethoxydiphenylborane (2‐APB), but not the selective PKC inhibitor, chelerythrine, attenuated M1 facilitation by McN‐A‐343. PKC activation with phorbol 12‐myristate 13‐acetate mimicked the ability of McN‐A‐343 to suppress M2‐inhibition and A2A‐facilitation of [3H]ACh release caused by oxotremorine and CGS 21680C, respectively. Co‐application of chelerythrine together with McN‐A‐343 restored oxotremorine‐inhibition and CGS 21680C‐facilitation, but this was not observed when the M1 agonist was applied together with LiCl and 2‐APB. McN‐A‐343 also masked facilitation of [3H]ACh release caused by stimulators of the cyclic AMP pathway, forskolin and rolipram. Data suggest that M1‐facilitation of ACh release results mainly from activation of the IP3 pathway. This mechanism can be dissociated from the way M1 receptor operates suppression of neuromodulation through M2‐inhibitory and A2A‐facilitatory receptors, which might involve secondary PKC activation.