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 cellular inositol is decreased from similar to 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [H-3]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [H-3]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations 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 concentrations 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 prevented 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+, stimulated PLC activity was decreased by similar to 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected 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 stimulated accumulation of [H-3]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively impaired by Li+ as a secondary consequence of inositol monophosphatase inhibition in cells which are highly dependent on inositol re-cycling, but imply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not readily attributed to decreased PtdInsP(2) availability. The potential for complex regulation of PLC and PtdIns synthase is discussed.