Activation of PLC- 1 by Gi/o-coupled receptor agonists

Murthy, Karnam S., Huiping Zhou, Jiean Huang, and Srinivas N. Pentyala. Activation of PLC1 by Gi/o-coupled receptor agonists. Am J Physiol Cell Physiol 287: C1679–C1687, 2004; doi:10.1152/ ajpcell.00257.2004.—The mechanism of phospholipase (PLC)activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca stimulated an eightfold increase in PLC1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three Gi/o-coupled receptor agonists (somatostatin, -opioid agonist [D-Pen,D-Pen]enkephalin, and A1 agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC1(E341R/D343R; 65–76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca influx and was not observed in the absence of extracellular Ca , but was partly inhibited by nifedipine (16–30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca channels. Treatment of the cells with a Gq/13-coupled receptor agonist, CCK-8, caused only transient, PLC1-mediated PI hydrolysis. Unlike Gi/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or G 13 minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine ( 25%) and strongly inhibited by SKF-96365 ( 75%) and in cells expressing PLC1(E341R/D343R). Agonist-independent Ca release or Ca influx via voltage-gated Ca channels stimulated only moderate PI hydrolysis (2to 3-fold), which was abolished by PLC1 antibody or nifedipine. We conclude that PLC1 is activated by Gi/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca influx via store-operated Ca channels.