THE INHIBITION OF PHOSPHOINOSITIDE SYNTHESIS AND MUSCARINIC-RECEPTOR-MEDIATED PHOSPHOLIPASE-C ACTIVITY BY LI+ AS SECONDARY, SELECTIVE, CONSEQUENCES OF INOSITOL DEPLETION IN 1321N1 CELLS

Conditions are described for culture of 1321N1 cells under which cellu lar inositol is decreased from similar to 20 mM to < 0.5 mM but phosph oinositide concentrations are unaffected. The effects of the muscarini c-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinos itide turnover in these or in inositol-replete cells was examined afte r steady-state [H-3]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar ini tial (0-15 min) rates of phospholipase C (PLC) activity, in the presen ce of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [H-3 ]PtdIns concentrations declined dramatically only in depleted cells. I n inositol-depleted cells, carbachol alone evoked increased concentrat ions of [H-3]inositol, [H-3]InsP(1), [H-3]InsP(2), [H-3]InsP(3) and [H -3]InsP(4), which were largely sustained over 90 min, and concentratio ns of [3H]PtdIns, [H-3]PtdInsP and [H-3]PtdInsP(2) were decreased only to similar to 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was preven ted and, although accumulation of [H-3]InsP(1), [H-3]InsP(2) and [H-3] InsP(3) was initially (0-30 min) potentiated, rates of accumulation of [H-3]InsP(1) and concentrations of [H-3]polyphosphates later (> 30-60 min) declined, and concentrations of [H-3]PtdIns, [H-3]PtdInsP and [H -3]PtdInsP(2) were decreased respectively to similar to 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, s timulated PLC activity was decreased by similar to 70% compared with t he initial rate in depleted cells. This decreased PLC activity was ref lected by changes in the stimulated concentrations of [H-3]Ins(1,3,4)P -3 but not of [H-3]Ins(1,4,5)P-3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimula ted accumulation of [H-3]inositol 1:2cyclic,4,5-trisphosphate. These d ata suggest that receptor-mediated PLC activity is selectively impaire d by Li+ as a secondary consequence of inositol monophosphatase inhibi tion in cells which are highly dependent on inositol re-cycling, but i mply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not rea dily attributed to decreased PtdInsP(2) availability. The potential fo r complex regulation of PLC and PtdIns synthase is discussed.