Activation of PLC-δ1 by Gi/o-coupled receptor agonists
The mechanism of phospholipase (PLC)-δ activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca2+ stimulated an eightfold increase in PLC-δ1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three Gi/o-coupled receptor agonists (somatostatin, δ-opioid agonist [D-Pen2,D-Pen5]enkephalin, and A1 agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-δ1(E341R/D343R; 65–76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca2+ influx and was not observed in the absence of extracellular Ca2+, but was partly inhibited by nifedipine (16–30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca2+ channels. Treatment of the cells with a Gq/13-coupled receptor agonist, CCK-8, caused only transient, PLC-β1-mediated PI hydrolysis. Unlike Gi/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-δ1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or Gα13 minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-δ1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca2+ 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 PLC-δ1(E341R/D343R). Agonist-independent Ca2+ release or Ca2+ influx via voltage-gated Ca2+ channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-δ1 antibody or nifedipine. We conclude that PLC-δ1 is activated by Gi/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca2+ influx via store-operated Ca2+ channels.