B. Balestra

Prejunctional muscarinic inhibitory control of acetylcholine release in the human isolated detrusor: involvement of the M4 receptor subtype.

Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. In detrusor strips preincubated with [3H]‐choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]‐ACh (76%) and [3H]‐choline (24%). Electrically‐evoked [3H]‐ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release. The effects of various subtype‐preferring muscarinic receptor antagonists were evaluated on [3H]‐ACh release and muscle contraction. The rank potency (−log EC50) orders at pre‐ and postjunctional level were: atropine 4‐diphenyl‐acetoxy‐N‐piperidine (4‐DAMP)>mamba toxin 3 (MT‐3)>tripitramine>para‐fluorohexahydrosiladiphenidol (pF‐HHSiD)methoctraminepirenzepine>tripinamide, and atropine4‐DAMP>pF‐HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT‐3, respectively. The comparison of pre‐ and post‐junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.

Role of agonist-dependent receptor internalization in the regulation of μ opioid receptors

Organotypic cultures and ileal neuromuscular preparations were used to determine (i) whether endogenous release of opioids by electrical stimulation induces mu receptor endocytosis, and (ii) whether and under which conditions ligand-induced mu receptor endocytosis influences the responsiveness of neurons expressing native mu receptors. In longitudinal muscle-myenteric plexus preparations, electrical stimulation at 20 Hz induced a prominent endocytosis of mu receptors in enteric neurons, indicating endogenous release of opioids. A similar massive endocytosis was triggered by exogenous application of the mu receptor agonist, [D-Ala(2),MePhe(4), Gly-ol(5)] enkephalin, whereas exogenous application of morphine was ineffective. [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin and morphine induced a concentration-dependent inhibition of neurogenic cholinergic twitch contractions to electrical stimulation at 0.1 Hz. beta-Chlornaltrexamine shifted to the right the inhibitory curve of both agonists with a concentration-dependent reduction of the maximum agonist response, which is consistent with the existence of spare mu opioid receptors. Under these conditions, the induction of mu receptor endocytosis by exogenously applied [D-Ala(2), MePhe(4),Gly-ol(5)] enkephalin diminished the inhibitory effect of this agonist on twitch contractions and tritiated acetylcholine release. In contrast, there was no reduction of the inhibitory effect of morphine, which failed to induce mu receptor endocytosis, on neurogenic cholinergic response. These results provide the first evidence for the occurrence of mu receptor endocytosis in neurons by endogenously released opioids and show that agonist-dependent mu receptor endocytosis could serve as a mechanism to regulate mu opioid receptor responsiveness to ligand stimulation when the opioid receptor reserve is reduced.

Functional and neurochemical changes of the gastrointestinal tract in a rodent model of Parkinson's disease.

Patients with Parkinsons disease develop motor disturbances often accompanied by peripheral autonomic dysfunctions, including gastrointestinal disorders, such as dysphagia, gastric stasis and constipation. While the mechanisms subserving enteric autonomic dysfunctions are not clearly understood, they may involve the enteric dopaminergic and/or nitrergic systems. In the present study, we demonstrate that rats with unilateral 6-hydroxydopamine lesion of nigrostriatal dopaminergic neurons develop a marked inhibition of propulsive activity compared to sham-operated controls, as indicated by a 60% reduction of daily fecal output at the 4th week of observation. Immunohistochemical data revealed that 6-hydroxydopamine treatment did not affect the total number of HuC/D-positive myenteric neurons in both the proximal and distal segments of ileum and colon. Conversely, in the distal ileum and proximal colon the number of nitrergic neurons was significantly reduced. These results suggest that a disturbed distal gut transit, reminiscent of constipation in the clinical setting, may occur as a consequence of a reduced propulsive motility, likely due to an impairment of a nitric oxide-mediated descending inhibition during peristalsis.

Colonic mucosal mediators from patients with irritable bowel syndrome excite enteric cholinergic motor neurons.

Background  Mediators released in the mucosal milieu have been suggested to be involved in visceral hypersensitivity and abdominal pain in patients with irritable bowel syndrome (IBS). However, their impact on myenteric neurons remains unsettled.

5-Hydroxytryptamine receptors that facilitate excitatory neuromuscular transmission in the guinea-pig isolated detrusor muscle.

1 In isolated detrusor strips from the guinea‐pig urinary bladder, contractile responses to electrical field stimulation were mostly mediated by neurally released acetylcholine (ACh) and adenosine 5′‐triphosphate (ATP). 2 5‐Hydroxytryptamine (5‐HT) produced a concentration‐dependent increase in the amplitude of stimulated detrusor strip contractions. The 5‐HT concentration‐response curve showed a biphasic profile: the high potency phase was obtained at sub‐micromolar concentrations (10–300 nM), while the low potency phase in the range 1–30 μm. The maximum response of the first phase was 30% of the total 5‐HT response. 3 Like 5‐HT, the 5‐HT3 receptor agonist, 2‐methyl‐5‐hydroxytryptamine (2‐methyl‐5‐HT: 0.3–100 μm), the 5‐HT2 receptor agonist, (±)‐l‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI: 30 nM‐3 μm) and the 5‐HT4 receptor agonist, 5‐methoxytryptamine (5‐MeOT: 0.1–30 μm) potentiated, though with lower potency, detrusor contractions. The resulting concentration‐response curves were monophasic in nature. 2‐Methyl‐5‐HT had a maximum effect comparable to that of 5‐HT. By contrast, the maximal effects of DOI and 5‐MeOT were only 20% and 30% of that elicited by 30 μm 5‐HT, respectively. 4 The 5‐HT3 receptor antagonist, granisetron (0.3 μm) had no effect on the high potency phase, but caused a rightward parallel shift of the low potency phase of the 5‐HT curve (pKB = 7.3). Granisetron (0.3 μm) antagonized with comparable affinity (pKB= 7.1) 5‐HT‐induced responses after pharmacological isolation of 5‐HT3 receptors with the 5‐HT1/5‐HT2 receptor antagonist, methiothepin (0.3 μm) and the 5‐HT4 receptor antagonist, GR 125487 (30 nM). Granisetron (0.1, 0.3 and 1 μm) competitively antagonized the potentiating effect of 2‐methyl‐5‐HT with an estimated pA2 of 7.3. 5 Methiothepin (0.3 μm) and the 5‐HT2A receptor antagonist, ketanserin (0.3 μm) produced a slight inhibition of the first phase of the 5‐HT curve. In the presence of ketanserin, an equimolar concentration of methiothepin was ineffective in further reducing the effect of 5‐HT. Similarly, the 5‐HT4 receptor antagonist, GR 125487 (30 nM) slightly inhibited the first phase of the 5‐HT curve. Conversely, this phase was suppressed when detrusor strips were coincubated with ketanserin (or methiothepin) and GR 125487. 6 In a separate set of experiments, the interactions of 5‐HT with either the purinergic or cholinergic components of excitatory neuromuscular transmission were investigated. In the presence of hyoscine (1 μm), 5‐HT was mostly effective at sub‐micromolar concentrations, while in the presence of the P2‐purinoceptor antagonist, suramin (300 μm), 5‐HT‐induced potentiation was mainly obtained with micromolar concentrations. 7 Thus, in electrically stimulated detrusor strips from guinea‐pig, 5‐HT potentiated excitatory neuromuscular transmission by activating at least three separate neural 5‐HT receptors. These include the 5‐HT2A and 5‐HT4 receptors, which mediate the 5‐HT high potency phase mainly by activation of purinergic transmission. On the other hand, the potentiating effect caused by micromolar concentrations of 5‐HT mostly involves cholinergic transmission and is mediated by the 5‐HT3 receptors.

Two subtypes of enteric non-opioid σ receptors in guinea-pig cholinergic motor neurons

Abstract In the longitudinal muscle-myenteric plexus preparation (LMMP) of the guinea-pig ileum, the non-opioid σ receptors agonists, 1,3-di-ortho-tolylguanidine (DTG) and (+)N-allyl-N-normetazocine [(+)SKF 10,047], had opposite effects on nerve-mediated cholinergic contractions caused by electrical field stimulation. DTG (0.1–10 μM) inhibited and (+)SKF 10,047 (0.1–10 μM) markedly enhanced these contractile responses. Both effects were evaluated in the presence (0.5 or 1 μM) of the putative antagonists at central σ sites: haloperidol, rimcazole, BMY 14802 and dextromethorphan. Haloperidol and dextromethorphan were ineffective. Rimcazole antagonized the effect of both DTG and (+)SKF 10.047. BMY 14802 antagonized the (+)SKF 10,047-mediated excitatory response only. These results suggest that two σ receptor subtypes are present in enteric cholinergic motor neurons innervating the longitudinal coat. Rimcazole and BMY 14802 may provide useful tools for the characterization of peripheral non-opioid σ receptors.

Role of galanin receptor 1 in peristaltic activity in the guinea pig ileum

Galanin effects are mediated by distinct receptors, galanin receptor 1 (GAL-R1), GAL-R2 and GAL-R3. Here, we analyzed 1) the role of GAL-R1 in cholinergic transmission and peristalsis in the guinea-pig ileum using longitudinal muscle-myenteric plexus preparations and intact segments of the ileum in organ bath, and 2) the distribution of GAL-R1 immunoreactivity in the myenteric plexus with immunohistochemistry and confocal microscopy. Galanin inhibited electrically stimulated contractions of longitudinal muscle-myenteric plexus preparations with a biphasic curve. Desensitization with 1 microM galanin suppressed the high potency phase of the curve, whereas the GAL-R1 antagonist, RWJ-57408 (1 microM), inhibited the low potency phase. Galanin (3 microM) reduced the longitudinal muscle contraction and the peak pressure, and decreased the compliance of the circular muscle. All these effects were antagonized by RWJ-57408 (1 or 10 microM). RWJ-57408 (10 microM) per se did not affect peristalsis parameters in normal conditions, nor when peristalsis efficiency was reduced by partial nicotinic transmission blockade with hexamethonium. In the myenteric plexus, GAL-R1 immunoreactivity was localized to neurons and to fibers projecting within the plexus and to the muscle. GAL-R1 was expressed mostly by cholinergic neurons and by some neurons containing vasoactive intestinal polypeptide or nitric oxide synthase. This study indicates that galanin inhibits cholinergic transmission to the longitudinal muscle via two separate receptors; GAL-R1 mediates the low potency phase. The reduced peristalsis efficiency could be explained by inhibition of the cholinergic drive, whereas the decreased compliance is probably due to inhibition of descending neurons and/or to the activation of an excitatory muscular receptor. Endogenous galanin does not appear to affect neuronal pathways subserving peristalsis in physiologic conditions via GAL-R1.

5-HT4 receptors contribute to the motor stimulating effect of levosulpiride in the guinea-pig gastrointestinal tract.

BACKGROUND The dopamine D2 receptor antagonist levosulpiride is a substituted benzamide derivative, whose gastrokinetic properties are exploited clinically for the management of functional dyspepsia. However, for other benzamide derivatives, such as cisapride and mosapride, agonism towards serotonin 5-HT4 receptors is considered the main mechanism leading to gastrointestinal prokinesia. AIMS To assess whether levosulpiride is able to activate 5-HT4 receptors in the guinea-pig isolated gastrointestinal tract. MATERIALS AND METHODS Circular muscle strips from gastric antrum, and colonic longitudinal muscle strips were used to detect electrically stimulated neurogenic contractions. The effect of levosulpiride was assessed in the absence and presence of GR125487, a selective 5-HT4 receptor antagonist. Furthermore, potential interaction of levosulpiride with 5-HT3 receptors and tissue cholinesterases was assessed in unstimulated ileal longitudinal muscle-myenteric plexus preparations. RESULTS Antral and colonic strip contractions were cholinergic/tachykinergic in nature. Micromolar concentrations of levosulpiride potentiated submaximal responses, through a mechanism competitively antagonized by GR125487 (pKB=9.4). In LMMPs, levosulpiride slightly affected contractions caused by the 5-HT, receptor agonist 2-methyl-5-HT, and had no effect on contractions to exogenous acetylcholine. CONCLUSIONS Our results indicate that levosulpiride acts as a moderate agonist at the 5-HT4 receptor. This property, together with antagonism at D2 receptors, may contribute to its gastrointestinal prokinetic effect.

Response of colonic motility to dopaminergic stimulation is subverted in rats with nigrostriatal lesion: relevance to gastrointestinal dysfunctions in Parkinson's disease

Constipation is extremely common in patients with Parkinsons disease (PD) and has been described in PD animal models. In this study, we investigated whether a PD‐like degeneration of dopaminergic neurons of the substantia nigra can influence peristalsis in colonic segments of rats by impacting on enteric dopaminergic transmission.

5-HT3 receptor involvement in descending reflex relaxation in the rabbit isolated distal colon

In whole segments of rabbit distal colon with mucosa removed, descending reflex relaxations of the circular muscle (descending inhibition) elicited by inflating (0.1-1 ml) an intraluminal balloon, were partially antagonized by 100 microM hexamethonium and the 5-HT3 receptor antagonist, ondansetron (3 microM), and abolished by 1 microM tetrodotoxin. The inhibitory effects of hexamethonium and ondansetron were additive. Conversely, hexamethonium (100 microM) and ondansetron (3 microM) failed to reduce electrically induced non-adrenergic non-cholinergic (NANC) relaxations of colonic circular muscle. It is concluded that interneuronally released acetylcholine and 5-hydroxytryptamine (5-HT) activate descending inhibitory pathways supplying the circular coat, via nicotinic and 5-HT3 receptors, respectively. This evidence suggests a functional involvement of 5-hydroxytryptaminergic transmission in the descending inhibition of rabbit colon.