Cytosolic phospholipase C activity: II. Relationship to concanavalin A-induced phosphatidylinositol-turnover in splenocytes

We have described in the first paper the coupling betweencytosolic Giα and cytosolic PLC activity in a cell free preparation. In order to establish the functional significance of the cytosolic Giα coupled soluble PLC, we examined the effects of Dex, NaF, and trifluopeerizine (TEP) on concanavalin A(Con A)‐induced PI‐turnover in intact slenocytes and, in parallel, on soluble PLC activity in cytosol preparations. Vytosolic PLC activity was measured with [3H]PIP and [3H]PIP2 as substrates. (1) The con A‐induced increase (2–4 fold) in Pl‐turnover in intact splenocytes was paralleled by an 1.2–5‐fold increase in soluble PLC activity in vitro. Con A administration also increased cytosolic Giα immunoreactivity 3–6‐fold as expected if cytosolic Giα was coupled to soluble PLC activation. (2) DEX (10−7 M), administered 6 h prior to Con A administration inbited the Con A‐induced increase in Pl‐turnover in intact splenocytes. This was paralleled by DEX inhibition of the Con A‐induced increase in soluble PLC activity measured in vitro and cytosolic Giα imunoreactivity. (3) We have demonstrated in the first paper that NaF and TEP inhibited soluble PLC activity. Here we show that NaF and TFP inhibited the Con A‐induced increase in PI‐turnover extending the similarities between soluble PLC activity and Con A‐ Stimulated PLC Activity in intact splenocytes. (4) In order to examine Whether or not the Con A‐induced PLC activity and Con A‐stimulated PLC activity in intact splenocytes. (4) In order to examine Whether or not the Con A‐induced PLC was similar to PLCγ, we measured PI‐turnover induced by Con A or BaVO3 in combination with DEX and PMA. Whereas the Con A‐induced PI‐turnover was significantly inhibited (40–60%) by DEX, the NaVO3 ‐induced PI‐turnover was not affected by DEX. The Con A‐induced PI‐turnover was not affected by PMA (50nM), But the NaVO3‐induced Pi‐turnover was increased over 2‐fold PMA (50nM), suggesting that the Con A‐induced PLC in intact splenocytes is different from NaVO3‐induced PLC. Based on these results a model for the sequential activation of substrate‐specific PLCs in splenocyte by mitogen is presented.