The 5-HT4 receptor agonist prucalopride does not facilitate cholinergic neurotransmission in circular and longitudinal smooth muscle preparations of equine mid-jejunum.

BACKGROUND: Postoperative ileus (POI) remains an important cause of death in horses. The recently developed selective 5-HT4 receptor agonists such as prucalopride target 5-HT4 receptors on myenteric cholinergic neurons to enhance acetylcholine release and GI motility. No clearcut in vitro evaluation whether highly selective 5-HT4 receptor agonists enhance submaximal cholinergic neurotransmission towards the muscle layer has been performed in horses. OBJECTIVES: To identify functional 5-HT4 receptors in equine jejunum. STUDY DESIGN: In vitro experimental study. METHODS: Circular and longitudinal smooth muscle strips (mid-jejunum) were mounted in organ baths between 2 platinum electrodes allowing electrical field stimulation (EFS). To delineate the conditions to obtain purely cholinergic responses, voltage-response curves were studied. To investigate the influence of prucalopride and 5-HT, submaximal cholinergic contractions at a single voltage were induced. RESULTS: In circular and longitudinal strips, EFS induced voltage-dependent neurogenic on-contractions when the bathing medium contained a NO-synthesis inhibitor and apamin to prevent inhibitory responses to NO and ATP. Contractions at a voltage inducing 50% of maximal amplitude were cholinergic, as they were blocked by atropine. These contractions were not influenced by prucalopride (up to 3µM), even in the presence of the phosphodiesterase inhibitor isobutyl-methyl-xanthine to inhibit breakdown of the second messenger of 5-HT4 receptors, cAMP. Also the full 5-HT4 receptor agonist 5-HT did not influence the EFS-induced submaximal cholinergic contractions. Moreover, prucalopride did not influence muscle tone continuously enhanced with KCl. CONCLUSIONS: There are no functional 5-HT4 receptors on myenteric cholinergic neurons nor muscular 5-HT4 receptors in equine jejunum.

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 [(3)H]-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.

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

The influence of the selective 5-HT(4) 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-HT(4) 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 [(3)H]-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-HT(4) 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-HT(4) 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-HT(4) receptor agonist with selective inhibition of the PDE involved in this regulation of transmitter release might enhance the prokinetic effect of the 5-HT(4) receptor agonist.

Mechanisms of action of hydrogen sulfide in relaxation of mouse distal colonic smooth muscle.

Hydrogen sulfide (H(2)S) has been suggested as a gaseous neuromodulator in mammals. The aim of this study was to examine the influence of H(2)S on contractility in mouse distal colon. The effect of sodium hydrogen sulfide (NaHS; H(2)S donor) on prostaglandin F(2alpha) (PGF(2alpha))-contracted circular muscle strips of mouse distal colon was investigated. In addition, tension and cytosolic calcium concentration ([Ca(2+)](cyt)) in the mouse distal colon strips were measured simultaneously in the presence of NaHS. NaHS caused concentration-dependent relaxation of the pre-contracted mouse distal colon strips. The NaHS-induced relaxation was not influenced by the K(+) channels blockers glibenclamide, apamin, charybdotoxin, barium chloride and 4-aminopyridine. The relaxation by NaHS was also not influenced by the nitric oxide inhibitor L-NAME, by the soluble guanylate cyclase respectively adenylate cyclase inhibitors ODQ and SQ 22536, by the nerve blockers capsazepine, omega-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The initiation of the NaHS-induced relaxation was accompanied by an increase in [Ca(2+)](cyt), but once the relaxation was maximal and sustained, no change in [Ca(2+)](cyt) was measured. This calcium desensitization is not related to the best known calcium desensitizing mechanism as the myosin light chain phosphatase (MLCP) inhibitor calyculin-A and the Rho-kinase inhibitor Y-27632 had no influence. We conclude that NaHS caused concentration-dependent relaxations in mouse distal colon not involving the major known K(+) channels and without a change in [Ca(2+)](cyt). This calcium desensitization is not related to inhibition of Rho-kinase or activation of MLCP.

Jejunal cholinergic, nitrergic, and soluble guanylate cyclase activity in postoperative ileus.

BACKGROUND: In animal models of postoperative ileus (POI), inflammation of the intestine plays an important role in the pathogenesis of POI. Changes in alpha(2)-adrenoceptors and nitrergic regulation have been proposed to be implicated. The aim of our study was to investigate the presynaptic alpha(2)-receptor-mediated control of cholinergic nerve activity, the nitrergic nerve activity, and the possible role of soluble guanylate cyclase (sGC) during the inflammatory phase of POI. METHODS: Ileus was induced by anesthesia and manipulation of the rat jejunum. Rats were treated with the sGC inhibitors methylene blue or ODQ; nonoperated animals served as controls. After 24 h, plasma and jejunal tissue were collected for biochemical assays, nitric oxide synthase-1 (NOS-1)-immunohistochemistry, acetylcholine (Ach)-release experiments, and muscle tension experiments. RESULTS: In all operated animal groups, myeloperoxidase activity was significantly increased, which indicates initiation of an inflammatory response. The alpha(2)-adrenoceptor agonist UK14,304 reduced electrically induced Ach-release similarly in operated and nonoperated animals. In strips of operated animals, electrically induced nitrergic relaxations were decreased, whereas relaxations induced by exogenous nitric oxide (NO) remained unchanged compared with control. The number of myenteric neurons and the percentage of NOS-1-positive neurons were not influenced. Plasmatic cyclic guanosine monophosphate (cGMP) levels were decreased in all operated groups, whereas jejunal cGMP levels were unchanged compared with nonoperated controls; treatment with sGC inhibitors did not reduce plasmatic cGMP levels. CONCLUSIONS: This study demonstrates that presynaptic alpha(2)-receptor mediated control of intestinal cholinergic nerve activity is unchanged during manipulation-induced inflammation. However, this inflammation induces impaired nitrergic neurotransmission related to decreased NOS-1 activity in the nitrergic nerves.