ALTERED LEVELS OF PHOSPHATIDYLINOSITOL PHOSPHOLIPASE-C ACTIVITY IN RAT-LIVER CELLS IN RESPONSE TO INSULIN, EPINEPHRINE, ACETYLCHOLINE AND BACTERIAL PHOSPHOLIPASE-C

Rat liver cells were homogenized and subsequently fractionated by a simplified method based on microfiltration, which proved to give a high recovery of membrane-bound phosphatidylinositol phospholipase C (PI-PLC) activity. The effects of insulin, acetylcholine (AC). epinephrine (EN) and bacterial phopsholipase C (bPLC) on the PI-PLC activity were studied after in vitro treatment of isolated membranes or after in situ application in rat liver. A dose-dependent increase of membranous PI-PLC (up to 3-fold) and corresponding 36 to 72% decline of the cytosolic activity were established upon treatment with supraphysiological doses of insulin or with bPLC, respectively. AC induced a biphasic response with a maximal stimulation in the micromolar range. On the other hand EN promoted a slight but significant dose-dependent inhibition of PI-PLC in both cytosol and membranes. Sodium fluoride was also a potent inhibitor of the membrane-associated PI-PLC with an EC50 value of about 5 mM. The combined assay with NaF and EN revealed no additivity between their inhibitory effects, suggesting a common step in the mechanism(s) of inhibition caused by the two agents. The stimulatory effects of insulin and AC were partially reduced by soluble cytosolic factors, which still remain to be identified. When insulin and AC were applied in combination in the presence of cytosol, this resulted in a 56% inhibition of PI-PLC below the control level.