Negative regulation of Ca(2+) influx during P2Y(2) purinergic receptor activation is mediated by Gbetagamma-subunits.
We have previously reported that P2Y(2) purinoceptors and muscarinic M(3) receptors trigger Ca(2+) responses in HT-29 cells that differ in their timecourse, the Ca(2+) response to P2Y(2) receptor activation being marked by a more rapid decline of intracellular Ca(2+) concentration ([Ca(2+)](i)) after the peak response and that this rapid decline of [Ca(2+)](i) was slowed in cells expressing heterologous beta-adrenergic receptor kinase (betaARK). In the present study, we demonstrate that, during P2Y(2) receptor activation, betaARK expression increases the rate of Gd(3+)-sensitive Mn(2+) influx, a measure of the rate of store-operated Ca(2+) entry from the extracellular space, during P2Y(2) activation and that this effect of betaARK is mimicked by exogenous alpha-subunits of G(q), G(11) and G(i2). The effect of betaARK on the rate of Mn(2+) influx is thus attributable to its ability to scavenge G protein betagamma-subunits released during activation of P2Y(2) receptor. We further find that the effect of betaARK on the rate of Mn(2+) influx during P2Y(2) receptor activation can be overcome by arachidonic acid. In addition, the UTP-induced Mn(2+) influx rate was significantly increased by inhibitors of phospholipase A(2) (PLA(2)) and an siRNA directed against PLA(2)beta, but not by an siRNA directed against PLA(2)alpha or by inhibitors of arachidonic acid metabolism. These findings provide evidence for the existence of a P2Y(2) receptor-activated signalling system that acts in parallel with depletion of intracellular Ca(2+) stores to inhibit Ca(2+) influx across the cell membrane. This signalling process is mediated via Gbetagamma and involves PLA(2)beta and arachidonic acid.
Hu
Haibi
H
The University of Sydney, New South Wales, Australia.
O'Mullane
Lauren M
LM
Cummins
Michelle M
MM
Campbell
Craig R
CR
Hosoda
Yutaka
Y
Poronnik
Philip
P
Dinudom
Anuwat
A
Cook
David I
DI
D000255:Adenosine Triphosphate / Q000378:metabolism; D016718:Arachidonic Acid / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D025461:Feedback, Physiological; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D054513:Group IV Phospholipases A2 / Q000235:genetics / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D008345:Manganese / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D043587:Receptor, Muscarinic M3 / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D051552:beta-Adrenergic Receptor Kinases / Q000235:genetics / Q000378:metabolism
D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; C546385:P2RY2 protein, human; D034741:RNA, Small Interfering; D043587:Receptor, Muscarinic M3; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D016718:Arachidonic Acid; D008345:Manganese; D000255:Adenosine Triphosphate; D051552:beta-Adrenergic Receptor Kinases; D054513:Group IV Phospholipases A2; D002118:Calcium