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

Abstract

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.

Introduction

The highly selective 5-HT₄ receptor agonist prucalopride accelerates colonic transit and increases stool frequency in healthy volunteers (Emmanuel et al., 1998; Bouras et al., 1999) and is efficient in patients with severe chronic constipation resistant to laxative treatment (Camilleri et al., 2008; Tack et al., 2009). The main mechanism for these effects of prucalopride is thought to be interaction with facilitatory 5-HT₄ receptors on cholinergic nerve endings towards colonic smooth muscle, which have been shown in different species also at other levels of the gastrointestinal tract (Gershon and Tack, 2007). In human colon, these receptors were shown to be present on cholinergic nerve endings towards the longitudinal (Prins et al., 2000) and circular muscle (Leclere et al., 2005; Cellek et al., 2006). Untill it was withdrawn because of the potential of cardiac dysrhythmias, the non-selective 5-HT₄ receptor agonist cisapride was worldwide used for treatment of gastro-esophageal reflux and for increasing gastric emptying in gastroparesis, and less so for constipation. Facilitatory 5-HT₄ receptors are indeed also present on cholinergic nerve endings in human stomach circular muscle (Leclere and Lefebvre, 2002) similar to those described in the stomach of other species such as guinea pig (Takada et al., 1999) and dog (Prins et al., 2001a); prucalopride indeed accelerates gastric emptying in man (Bouras et al., 2001). In pig, which is a good model for the study of gastrointestinal issues in view of similar morphology and physiology of the gastrointestinal tract (Miller and Ullrey, 1987), we have shown before the presence of 5-HT₄ receptors on cholinergic neurons controlling longitudinal muscle but did not investigate circular muscle. The first aim of this study was therefore to investigate whether 5-HT₄ receptors also facilitate acetylcholine release from cholinergic neurons innervating the circularly oriented muscle in porcine stomach.

When comparing the effect of prucalopride on electrically induced cholinergic contractions in porcine proximal stomach longitudinal muscle with that in electrically paced porcine left atrial pectinate muscles, a clear and sustained enhancement of the cholinergic contractions in proximal stomach was observed but the positive inotropic effect of prucalopride was transient and very weak and only became prominent and sustained upon phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methyl-xanthine (IBMX), illustrating a regulatory role of PDEs on 5-HT₄ receptor function in atrium (De Maeyer et al., 2006a, De Maeyer et al., 2006b), which has since been shown to be related to PDE3 and PDE4 activity (Galindo-Tovar et al., 2009). The robust and sustained effect of prucalopride in porcine stomach makes it unclear whether regulatory PDEs are involved. We did not test IBMX versus prucalopride in porcine stomach (De Maeyer et al., 2006a) but a recent detailed immunohistochemical study of PDE2A distribution in different species including dog, monkey and man, showed that the strongest immunoreactivity for PDE2A outside the central nervous system was present in enteric ganglia from the stomach to the colon (Stephenson et al., 2009). The second aim of this study was therefore to investigate whether PDEs control the effect of 5-HT₄ receptor stimulation on cholinergic neurotransmission in pig gastric circular muscle.