Activation of phospholipase D by guanosine 5′[γ-thio]triphosphate and AlF−4 in bovine corneal epithelial cells

We have investigated the regulation of phospholipase D (PLD) by guanine nucleotides and AlF−4 in bovine corneal epithelial cells (BCEC) prelabeled with [3H]myristic acid. In the presence of ethanol, AlF−4 increased the production of [3H]PA and [3H]PET indicating activation of PLD in these cells. The effects of AlF−4 were time-and dose-dependent. Addition of guanosine 5[γ-thio]triphosphate (GTPγS), to streptolysin O-permeabilized cells also resulted in increased accumulation of [3H]PA and [3H]PEt. Other guanine and adenine nucleotides were ineffective, and guanosine thiodiphosphate inhibited the GTP-γS-induced activation of PLD. Direct addition of GTγ7S to microsomal fraction prepared from [3H]myristate-labeled BCEC resulted in increased formation of [3H]PEt in a time-and dose-dependent manner. The activation of PLD by GTPγS in the microsomal fraction was absolutely dependent on the presence of Ca2+ >0.5 μM. Addition of Ca2+ (10-100 μM) alone dose-dependently stimulated the PLD activity. Treatment of the microsomal fraction with phorbol esters had no effect on the ability of GTPγS to stimulate PLD. Addition of isoproterenol to BCEC resulted in several-fold stimulation of cAMP, but it had no effect on basal or PDBu-induced stimulation of PLD. Taken together, the data suggest that a GTP-binding protein is involved in regulation of PLD in BCEC, and that maximal stimulation of PLD probably results from an interaction between Ca2+, PKC and G-protein in BCEC.