Cytosolic phospholipase C activity: II. Relationship to concanavalin A-induced phosphatidylinositol-turnover in splenocytes.
We have described in the first paper the coupling between cytosolic Gi alpha and cytosolic PLC activity in a cell free preparation. In order to establish the functional significance of the cytosolic Gi alpha coupled soluble PLC, we examined the effects of DEX, NaF, and trifluoperizine (TFP) on concanavalin A (Con A)-induced PI-turnover in intact splenocytes and, in parallel, on soluble PLC activity in cytosol preparations. Cytosolic PLC activity was measured with [3H]PI and [3H]PIP2 as substrates. 1) The Con A-induced increase (2-4 fold) in PI-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 Gi alpha immunoreactivity 3-6-fold as expected if cytosolic Gi alpha was coupled to soluble PLC activation. 2) DEX (10(-7) M), administered 6 h prior to Con A administration, inhibited the Con A-induced increase in PI-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 Gi alpha immunoreactivity. 3) We have demonstrated in the first paper that NaF and TFP 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 was similar to PLC gamma, we measured PI-turnover induced by Con A or NaVO3 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 (50 nM), but the NaVO3-induced PI-turnover was increased over 2-fold by PMA (50 nM), 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.
Akompong
T
T
Laboratory of Neuroendocrinology, Rockefeller University, New York, New York 10021.
Spencer
R L
RL
McEwen
B S
BS
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D003208:Concanavalin A / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008954:Models, Biological; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D012969:Sodium Fluoride / Q000494:pharmacology; D012995:Solubility; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D010738:Type C Phospholipases / Q000096:biosynthesis / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D010716:Phosphatidylinositols; D003208:Concanavalin A; D014268:Trifluoperazine; D014638:Vanadates; D003907:Dexamethasone; D012969:Sodium Fluoride; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D013755:Tetradecanoylphorbol Acetate