Differential regulation of phospholipases C and 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) cerebral hemispheres (CH) or cerebellum (CB) express a calcium/phospholipid-dependent isoform as the major protein kinase C (PKC-alpha/beta). PKC was activated (translocation of activity from cytosol to membrane) following stimulation with carbachol, so we tested for activation of phospholipase C (PLC) as the source of diacylglycerol released from polyphosphoinositide (PIP2) hydrolysis. Carbachol activated PLC (inositol phosphate release) 4-fold in a time- and dose-dependent manner in cortical (CH) astrocytes, but there was no activation of PLC in astrocytes from cerebellum (CB). Pirenzepine, but not gallamine, attenuated both carbachol-induced PKC translocation and PIP2 hydrolysis in E15CH astrocytes, arguing for contribution of M1 subtype. The phorbol ester TPA completely inhibited PIP2 hydrolysis, both basal and carbachol-stimulated, and elicited a stronger, but shorter (10 min) activation of PKC 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 astrocytes than observed in neurons. We observed a dramatic (10-fold) time- and dose-dependent activation of PLD by TPA in CH and a 3-fold increase in CB. The duration of TPA-dependent PLD activation correlated well with increased cell proliferation and changes in astrocytic phenotype markers. Carbachol-stimulated PLD activation was observed in CH but not in CB astrocytes, being mostly dependent on the M3 receptor subtype in the former. In contrast, glutamate elicited a greater PLD activation 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, total inhibition of TPA-dependent PLD activation was only achieved in cortical (CH) astrocytes after addition of EGTA. Thapsigargin activated PLD in both populations, further emphasizing the PLD activation dependency on [Ca2+]i. Taken together with our previous observations that TPA induces proliferation, cytoskeleton changes, and decreases of glutamine synthetase activity, these data suggest that phospholipase D is a differential but important participant in the regulation of the signalling of mitosis and differentiation in astrocytes during their development.
Mangoura
D
D
Department of Pediatrics MC5058, University of Chicago Medical School, IL 60637, USA.
Sogos
V
V
Pelletiere
C
C
Dawson
G
G
D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology*; D002478:Cells, Cultured / Q000166:cytology / Q000502:physiology; D002531:Cerebellum / Q000166:cytology; D002540:Cerebral Cortex / Q000166:cytology; D002642:Chick Embryo; D018698:Glutamic Acid / Q000494:pharmacology*; D009024:Morphogenesis / Q000502:physiology; D018721:Muscarinic Agonists / Q000494:pharmacology*; D010703:Phorbol Esters / Q000494:pharmacology*; D010739:Phospholipase D / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D010738:Type C Phospholipases / Q000502:physiology*
D002273:Carcinogens; D018721:Muscarinic Agonists; D010703:Phorbol Esters; D018698:Glutamic Acid; D002217:Carbachol; D010738:Type C Phospholipases; D010739:Phospholipase D; D002118:Calcium