DIFFERENTIAL REGULATION OF PHOSPHOLIPASE-C AND PHOSPHOLIPASE-D BY PHORBOL ESTERS AND THE PHYSIOLOGICAL ACTIVATORS CARBACHOL AND GLUTAMATE IN ASTROCYTES FROM CHICKEN-EMBRYO CEREBRUM AND CEREBELLUM

Primary astrocytic cultures derived from day-15 chick embryo (E15) cer ebral hemispheres (CH) or cerebellum (CB) express a calcium/phospholip id-dependent isoform as the major protein kinase C (PKC-alpha/beta). P KC was activated (translocation of activity from cytosol to membrane) following stimulation with carbachol, so we tested for activation of p hospholipase C (PLC) as the source of diacylglycerol released from pol yphosphoinositide (PIP2) hydrolysis. Carbachol activated PLC (inositol phosphate release) 4-fold in a time- and dose-dependent manner in cor tical (CH) astrocytes, but there was no activation of PLC in astrocyte s from cerebellum (CB). Pirenzepine, but not gallamine, attenuated bot h carbachol-induced PKC translocation and PIP2 hydrolysis in E15CH ast rocytes, arguing for contribution of M(1) subtype. The phorbol ester T PA completely inhibited PIP2 hydrolysis, both basal and carbachol-stim ulated, and elicited a stronger, but shorter (10 min) activation of PK C than that observed with carbachol. We investigated phospholipase D ( PLD) activation as an alternate source of diacylglycerol in astrocytes , since the ratio of PLC to PKC activation by carbachol was lower in a strocytes than observed in neurons. We observed a dramatic (10-fold) t ime- and dose-dependent activation of PLD by TPA in CH and a 3-fold in crease in CB. The duration of TPA-dependent PLD activation correlated well with increased cell proliferation and changes in astrocytic pheno type markers. Carbachol-stimulated PLD activation was observed in CH b ut not in CB astrocytes, being mostly dependent on the M(3) receptor s ubtype in the former. In contrast, glutamate elicited a greater PLD ac tivation in CB astrocytes than in CH astrocytes. TPA activation of PLD was totally blocked by staurosporine (PKC inhibitor) and genistein (a tyrosine kinase inhibitor) in cerebellar (CB) astrocytes; however, to tal inhibition of TPA-dependent PLD activation was only achieved in co rtical (CH) astrocytes after addition of EGTA. Thapsigargin activated PLD in both populations, further emphasizing the PLD activation depend ency on [Ca2+](i). Taken together with our previous observations that TPA induces proliferation, cytoskeleton changes, and decreases of glut amine synthetase activity, these data suggest that phospholipase D is a differential but important participant in the regulation of the sign alling of mitosis and differentiation in astrocytes during their devel opment.