CMP ACTIVATES REVERSAL OF PHOSPHATIDYLINOSITOL SYNTHASE AND BASE-EXCHANGE BY DISTINCT MECHANISMS IN RAT PITUITARY GH3 CELLS

CMP is known to activate phosphatidylinositol (PtdIns)/inositol (Ins) base exchange and has been reported to activate reversal of PtdIns synthase also. Because it is possible that PtdIns synthase acting in the reverse direction, followed by re-incorporation of ambient Ins, could be responsible for base-exchange activity, we characterized these processes in rat pituitary GH3 cells. In permeabilized GH3 cells prelabelled with [H-3]Ins and incubated in buffer with LiCl but without added Ins, CMP stimulated rapid accumulation of [H-3]Ins and decreases in [H-3]PtdIns; the K(m) for CMP was 1.7 mM. CDP and CTP were less effective, whereas 2'-CMP, 3'-CMP, other nucleoside monophosphates and cytidine did not influence this process. In permeabilized cells prelabelled to isotopic equilibrium with [H-3]Ins and [P-32]P(i), CMP stimulated decreases in both the P-32 and H-3 labelling of PtdIns, but did not increase that of [P-32]phosphatidic acid. These findings demonstrate that in the absence of added Ins the effect of CMP is not via activation of base exchange nor via a phospholipase D, but by reversal of PtdIns synthase. In permeabilized cells prelabelled with [H-3]Ins and [P-32]P(i), unlabelled Ins inhibited loss of P-32 labelling of PtdIns caused by CMP while markedly stimulating loss of H-3 labelling of PtdIns and release of [H-3]Ins. These data demonstrate that Ins inhibits reversal of PtdIns synthase, but stimulates base exchange. We conclude that in GH3 cells reversal of PtdIns synthase and PtdIns/Ins base exchange are both stimulated by CMP, but are distinct processes.