Purinergic agonist and G protein stimulation of phospholipase D in rat liver plasma membranes. Independence from phospholipase C activation.

Hormonal regulation of phospholipase D (PLD) was studied in isolated rat liver plasma membranes. Purinergic agents and a submaximal concentration of guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S), a non-hydrolyzable analog of GTP, synergistically stimulate phosphatidylethanol formation, a measure of PLD activity. The rank order of efficacy for stimulation of PLD activity in the presence of 0.2 microM GTP gamma S was beta, gamma-methylene-ATP > adenosine 5'-0-(3-thiotriphosphate) = ATP = ADP = 2-methylthio-ATP > alpha, beta-methylene-ATP = UTP. This pattern of activation does not conform to the series at known P2 receptors. GTP gamma S stimulated PLD activity in a dose-dependent manner, and the GTP gamma S dose-response curve for phosphatidylethanol formation was shifted to the left by an analog of ATP. Activation of PLD by purinergic agents in the presence of GTP gamma S supports the involvement of a purinergic receptor of the P2 class and a GTP-binding protein. Purinergic agents competitively inhibited [35S]adenosine 5'-0-(3-thiotriphosphate) binding to plasma membranes in the rank order adenosine 5'-0'(3-thiotriphosphate) > ATP > alpha,beta-methylene-ATP = UTP >> beta, gamma-methylene-ATP = ADP. Stimulation of phosphoinositide phospholipase C (PI-PLC) by purinergic agents, as measured by release of radioactivity from endogenously myo[3H]inositol-labeled plasma membranes, occurred in the order alpha, beta-methylene-ATP >> 2-methylthio-ATP. Beta, gamma-methylene-ATP had little effect on PI-PLC activity. Different dose-response relationships for agonist-stimulation of PI-PLC and PLD indicate that activation of PI-PLC is not involved in stimulation of PLD in rat liver plasma membranes, and suggest that purinergic activation of PLD occurs via a pathway involving a G protein and a heretofore uncharacterized P2 receptor.