We studied how 5-HT(4) receptor-mediated inotropic responses are regulated at the level of cAMP in porcine left atrium. We used selective phosphodiesterase (PDE) inhibitors to assess which PDE subtypes are responsible for the fade with time of inotropic responses to 5-HT(4) receptor activation with 5-HT and the 5-HT(4) receptor agonist prucalopride. A possible cross-talk via PDEs between cGMP and 5-HT(4) receptor-induced cAMP signalling was evaluated. Electrically paced left atrial pectinate muscles from young male pigs (15-25 kg) were studied in vitro. Simultaneous inhibition of PDE3 plus PDE4 subtypes was necessary to increase the amplitude and completely prevent the fade of the inotropic response to 5-HT and prucalopride. When responses to 5-HT or prucalopride had faded 1 h after addition, the nonspecific PDE-inhibitor IBMX still fully recovered inotropic responses. Stimulation of particulate guanylyl cyclase, together with PDE2 and PDE4 inhibition, delayed the fade of the response to 5-HT, while stimulation of soluble guanylyl cyclase independently of PDEs accelerated the fade of the response to 5-HT. In conclusion, both PDE3 and PDE4 subtypes are responsible for the suppression and the fade of the inotropic response to 5-HT and prucalopride. Signalling through the 5-HT(4) receptor remains fully active for at least 90 min with PDEs continuously regulating the response. cGMP levels, elevated by activation of particulate guanylyl cyclase under PDE2 inhibition, can indirectly enhance 5-HT(4) receptor-mediated signalling, at least when also PDE4 is inhibited, presumably through inhibition of PDE3. Elevation of cGMP generated by soluble guanylyl cyclase attenuates responses to 5-HT independently of PDEs.

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-HT 4 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-HT 4 receptor agonist prucalopride. Muscles were freezeclamped 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-HT 4 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.

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