Negative regulation of Ca2+ influx during P2Y2 purinergic receptor activation is mediated by Gβγ-subunits

We have previously reported that P2Y2 purinoceptors and muscarinic M3 receptors trigger Ca2+ responses in HT-29 cells that differ in their timecourse, the Ca2+ response to P2Y2 receptor activation being marked by a more rapid decline of intracellular Ca2+ concentration ([Ca2+]i) after the peak response and that this rapid decline of [Ca2+]i was slowed in cells expressing heterologous β-adrenergic receptor kinase (βARK). In the present study, we demonstrate that, during P2Y2 receptor activation, βARK expression increases the rate of Gd3+-sensitive Mn2+ influx, a measure of the rate of store-operated Ca2+ entry from the extracellular space, during P2Y2 activation and that this effect of βARK is mimicked by exogenous α-subunits of Gq, G11 and Gi2. The effect of βARK on the rate of Mn2+ influx is thus attributable to its ability to scavenge G protein βγ-subunits released during activation of P2Y2 receptor. We further find that the effect of βARK on the rate of Mn2+ influx during P2Y2 receptor activation can be overcome by arachidonic acid. In addition, the UTP-induced Mn2+ influx rate was significantly increased by inhibitors of phospholipase A2 (PLA2) and an siRNA directed against PLA2β, but not by an siRNA directed against PLA2α or by inhibitors of arachidonic acid metabolism. These findings provide evidence for the existence of a P2Y2 receptor-activated signalling system that acts in parallel with depletion of intracellular Ca2+ stores to inhibit Ca2+ influx across the cell membrane. This signalling process is mediated via Gβγ and involves PLA2β and arachidonic acid.