Characterisation of P2Y receptor-mediated contractile signalling and its regulation by G protein coupled receptor kinases and arrestin proteins in a rat bladder smooth muscle

ATP released from parasympathetic nerves can mediated bladder contraction, can activate purinergic P2Y/Gq/11-coupled G protein-coupled receptors (GPCR) expressed on detrusor (bladder) smooth muscle cells (DSMC). P2Y/Gq/11 signalling activates phospholipase C (PLC) and increases intracellular calcium concentrations to induce contraction. DSMC contractile GPCR activity is tightly regulated to prevent inappropriate contraction/incontinence. Additionally, GPCRs activity is regulated by G protein-coupled receptor kinase (GRK) and arrestin proteins, it is likely that they play a similar role in DSMC, and may help to maintain continence. Combining confocal imaging, calcium-sensitive dyes and selective P2X and P2Y receptor agonists/antagonists, showed that after 3-4 days in culture DSMC calcium signals were mediated by P2Y1 and P2Y2, but not P2X, P2Y4 or P2Y6 receptors. Repeated agonist additions indicated a desensitization of P2Y1 and P2Y2 activated phospholipase C (PLC)/Ca2+ signals, which was restored when the washout period between agonist challenges was increased. Transfection of DSMC with dominant-negative, catalytically inactive GRK mutants, which block endogenous GRK function, showed that P2Y1 and P2Y2 receptor stimulated calcium signalling was selectively regulated by GRK3 and GRK2, respectively. Furthermore, desensitization of P2Y1 and P2Y2 receptor PLC/Ca2+ was attenuated following RNAi-mediated knockdown of arrestin2 or arrestin3, suggesting both arrestins were able to regulate P2Y1/2 receptor signalling. To mimic the effects of obstructive bladder, DSMC were mechanically stretched which resulted in increased GRK2, and decreased GRK3/5/6 expression. These data show that DSMC express functional P2Y1/P2Y2 receptors which mediate purinergic agonist PLC/Ca2+ signalling, implying roles for P2Y1 and P2Y2 in bladder contraction and voiding. Furthermore, P2Y1 and P2Y2 receptor are selectively regulated by GRK and arrestin proteins, which suggests that GRK and arrestin proteins play an important role in the regulation of bladder tone. Furthermore, since GRK expression following mechanical stretch this may in turn affect GPCR signalling and produce dysregulation of DSMC contraction observed during incontinence.