ACTIVATION OF PHOSPHOLIPASE-C BY G-PROTEIN BY SUBUNITS IN DDTF(1)MF-2 CELLS

Adenosine A(1) receptors directly stimulate inositol phospholipid hydrolysis and Ca2+ mobilization through a pertussis toxin sensitive mechanism in DDT(1)MF-2 cells. In the present study we have investigated whether G protein beta gamma subunits (G beta gamma) are capable of stimulating phospholipase C in DDT(1)MF-2 cell membrane preparations using lipid vesicles containing [H-3]phosphatidylinositol 4,5-bisphosphate. DDT(1)MF-2 cell membrane and soluble fractions were found to contain phospholipase C activity which was stimulated by increases in free Ca2+ ion concentration. G beta gamma purified from bovine retinal transducin produced significant increases in phospholipase C activity in DDT(1)MF-2 cell membranes. G beta gamma-dependent activation of phospholipase C, while virtually absent in the presence of low Ca2+ ion concentrations, increased markedly with increasing free Ca2+ ion concentration. These data suggest that membrane bound phospholipase C in DDT(1)MF-2 cells is sensitive to Ca2+, and may be stimulated conditionally by G beta gamma subunits, i.e. G beta gamma subunits activate the enzyme only in the presence of Ca2+. G beta gamma subunits also stimulated soluble phospholipase C in DDT(1)MF-2 cells. These findings support the hypothesis that G(i) beta gamma subunits are involved in adenosine A(1) receptor stimulated phospholipase C/Ca2+ signalling in DDT(1)MF-2 cells.