PHOSPHOLIPASE-D ACTIVITY IN PC12 CELLS - EFFECTS OF OVEREXPRESSION OFALPHA(2A)-ADRENERGIC RECEPTORS

PC12 neuronal cells express a membrane phospholipase D (PLD) activity that is detected at similar levels in undifferentiated or differentiat ed cells. The regulation of this activity by agonists was explored, Me mbrane phospholipase D activity was increased by treatment of cells wi th the phorbol ester phorbol 12-myristate 13-acetate (PMA) or with ner ve growth factor, The ability of PMA to activate PLD was confirmed in intact PC12 cells. Basal activity of PLD in membranes was reduced in R G20, a PC12 cell line overexpressing the human alpha(2A)-adrenergic re ceptor. PMA did not increase PLD activity in RG20 cells, as assessed b oth in membrane preparations and in intact cells, Cyclic AMP levels di d not regulate phospholipase D activity in either cell type. However, incubation of RG20 cells with the alpha(2)-adrenergic antagonist rauwo lscine or with pertussis toxin increased membrane PLD activity and res tored activation of PLD by PMA. These data suggest that the effects of the overexpressed alpha(2A)-adrenergic receptor on PLD activity are m ediated by precoupling of the receptor to the heterotrimeric GTP bindi ng protein, G(i), but are independent of adenylate cyclase regulation. The results of this study suggest that membrane phospholipase D activ ity can be negatively regulated via G(i) in PC12 cells.