RECEPTOR-MEDIATED PHOSPHOLIPASE-D ACTIVITY IN PRIMARY ASTROGLIAL CULTURES

Phospholipase D, an enzyme involved in signal transduction cascades, c atalyses the formation of phosphatidic acid and, when ethanol is prese nt, the formation of phosphatidylethanol. In the present study we demo nstrate that stimulation of muscarinic acetylcholine receptors as well as P2-purinergic receptors induces activation of phospholipase D in p rimary cultures of astroglial cells. Both the hydrolysis and the trans phosphatidylation reactions were stimulated by receptor agonists. Carb achol and ATP induced a rapid increase in the amount of H-3!phosphati dic acid in astroglial cells prelabelled with H-3!oleic acid. When et hanol (150 mM) was present, phosphatidylethanol was formed. Furthermor e, the receptor-mediated increase in the concentration of phosphatidic acid was inhibited by ethanol, indicating that the phosphatidic acid production was indeed mediated by phospholipase D. The formation of ph osphatidylethanol was concentration dependent, with a half-maximal eff ective concentration of 5 x 10(-5) M for carbachol and 10(-5) M for AT P. The carbachol-induced response was almost completely inhibited by a tropine, indicating activation of phospholipase D via muscarinic recep tors. The purinergic response is most probably mediated via P2-recepto rs since ADP was almost as efficient as ATP in inducing phosphatidylet hanol formation, whereas AMP was significantly less potent. We conclud e that astroglial cells in primary culture display muscarinic and puri nergic receptors coupled to phospholipase D. The relationship to cell function needs to be further investigated. (C) 1993 Wiley-Liss, Inc.