cAMP catalyzing phosphodiesterases control cholinergic muscular activity but their inhibition does not enhance 5-HT4 receptor-mediated facilitation of cholinergic contractions in the murine gastrointestinal tract

Background: As the signal transduction of 5-HT4 receptors on cholinergic neurons innervating smooth muscle is controlled by phosphodiesterase (PDE) 4 in porcine stomach and colon, and human large intestine, the in vivo gastroprokinetic effects of a 5-HT4 receptor agonist might be enhanced by combination with a selective PDE4 inhibitor. The presence of 5-HT4 receptors on cholinergic neurons towards murine gastrointestinal circular muscle was recently shown. If the control of this receptor pathway by PDE4 is also present in mice, this might be a good model for in vivo testing of the combination therapy. Therefore this study investigates the role of cAMP catalyzing PDEs in smooth muscle cell activity and in the intraneuronal signal transduction of the 5-HT4 receptors in the gastrointestinal tract of C57Bl/6J mice. Methods: In circular smooth muscle strips from murine fundus, jejunum, and colon, submaximal cholinergic contractions were induced by either electrical field stimulation (EFS) or by carbachol (muscarinic receptor agonist). The influence of the PDE inhibitors IBMX (non-selective), vinpocetine (PDE1), EHNA (PDE2), cilostamide (PDE3), and rolipram (PDE4) was tested on these contractions and on the facilitating effect of a submaximal concentration of prucalopride (5-HT4 receptor agonist) on EFS-induced contractions. Results: In the three gastrointestinal regions, IBMX and cilostamide concentrationdependently decreased carbachol- as well as EFS-induced contractions. Some inhibitory effect was also observed with rolipram. In the fundus a non-significant trend for an enhancement of the facilitating effect of prucalopride on EFS-induced contractions was observed with IBMX, but none of the selective PDE inhibitors enhanced the facilitating effect of prucalopride in fundus, jejunum or colon. Conclusion: In analogy with the porcine gastrointestinal tract, in murine fundus, jejunum, and colon circular smooth muscle PDE3 is the main regulator of the cAMP turnover, with some contribution of PDE4. In contrast to the porcine gastrointestinal tract, the in vitro facilitation of electrically induced cholinergic contractions by 5-HT4 receptor stimulation could not be enhanced by specific PDE inhibition. The C57Bl/6J murine model is thus not suitable for in vivo testing of a 5-HT4 receptor agonist combined with a selective PDE4 inhibitor

Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium

Our objective was to investigate the role of phosphodiesterase (PDE)3 and PDE4 and cGMP in the control of cAMP metabolism and of phosphorylation of troponin I (TnI) and phospholamban (PLB) when 5-HT4 receptors are activated in pig left atrium. Electrically paced porcine left atrial muscles, mounted in organ baths, received stimulators of particulate guanylyl cyclase (pGC) or soluble guanylyl cyclase (sGC) and/or specific PDE inhibitors followed by 5-HT or the 5-HT4 receptor agonist prucalopride. Muscles were freeze-clamped at different moments of exposure to measure phosphorylation of the cAMP/protein kinase A targets TnI and PLB by immunoblotting and cAMP levels by enzyme immunoassay. Corresponding with the functional results, 5-HT only transiently increased cAMP content, but caused a less quickly declining phosphorylation of PLB and did not significantly change TnI phosphorylation. Under combined PDE3 and PDE4 inhibition, the 5-HT-induced increase in cAMP levels and PLB phosphorylation was enhanced and sustained, and TnI phosphorylation was now also increased. Responses to prucalopride per se and the influence thereupon of PDE3 and PDE4 inhibition were similar except that responses were generally smaller. Stimulation of pGC together with PDE4 inhibition increased 5-HT-induced PLB phosphorylation compared to 5-HT alone, consistent with functional responses. sGC stimulation hastened the fade of inotropic responses to 5-HT, while cAMP levels were not altered. PDE3 and PDE4 control the cAMP response to 5-HT4 receptor activation, causing a dampening of downstream signalling. Stimulation of pGC is able to enhance inotropic responses to 5-HT by increasing cAMP levels, while sGC stimulation decreases contraction to 5-HT cAMP independently

Identifiabilité structurelle et identification de systèmes couplés par les sorties

International audienceCet article s'intéressè a l'identification des syst emes de grande taille qui peuvent etre décomposés en une collection de sous-syst emes couplés par les sorties. Il est d'abord montré que si le syst eme global est structurellement identifiable, alors tous les sous-syst emes le sont egalement, en considérant les sorties comme de nouvelles entrées. Cette propriété est ensuite utilisée pour proposer une procédure d'identification décentralisée. L'efficacité de l'approche pro-posée est illustrée sur un exemple académique. Mots-clés— Identifiabilité structurelle, Identification décen-tralisée, Syst emes de grande taille. I. Introduction Les syst emes technologiques de grande taille tels que les syst emes de transport, les syst emes electriques, les syst emes de bâtiments. . .sont omniprésents dans notre vie moderne. La grande taille de ces syst emes a conduit au développement de diverses techniques pour réduire la complexité de leur etude. Une approche possible est de considérer le grand syst eme comme une collection de nom-breux sous-syst emes plus simples. L'identification [1] est un point crucial pour l'´ elaboration d'une stratégie de contrôle basée sur un mod ele. D'un point de vue pratique, leprobì eme de la taille des syst emes se posé egalement pour l'identification paramétrique. Pour simplifier l'identification des syst emes de grande taille, de nombreux auteurs ont essayé d'exploiter leur structure. Dans [5], le syst eme global est hiérarchisé et une méthode itérative est proposée pour l'identification. Dans [7], les auteurs s' intéressent aux syst emes circulants [3], et ils exploitent leurs propriétés pour définir une procédure originale d'identification. Une autre technique exploite la propriété de découplage en boucle fermée afin de définir une collection d'observateurs décentralisés pour les syst emes non linéaires interconnectés [12]. Dans cet article, nous supposons que le syst eme global est structurellement identifiable. L'identifiabilité structu-relle est une propriété importante quand il faut evaluer les param etres du syst eme, car elle garantit l'unicité des param etres [15]. Dans [13], l'auteur se concentre sur les syst emes compartimentaux pour etudier l'identifiabi-lité structurelle du syst eme global, et dans [4], l'identi-fiabilité pratique peut etre vérifiée pour des conditions suffisantes sur les signaux d'interaction entre les sous-syst emes. Dans ce document, on consid ere un ensemble de syst emes linéaires couplés par leurs sorties. Un exemple est représenté sur la figure 1. De nombreux processus peuvent etre modélisés sous cette forme, c'est le cas par exemple des syst emes thermiques dans les bâtiments [8]