Activation of phospholipase D by guanosine 5'[gamma-thio]triphosphate and AlF4- in bovine corneal epithelial cells.

We have investigated the regulation of phospholipase D (PLD) by guanine nucleotides and AlF4- in bovine corneal epithelial cells (BCEC) prelabeled with [3H]myristic acid. In the presence of ethanol, AlF4- increased the production of [3H]PA and [3H]PET indicating activation of PLD in these cells. The effects of AlF4- were time- and dose-dependent. Addition of guanosine 5[gamma-thio]triphosphate (GTP gamma S), 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 gamma S-induced activation of PLD. Direct addition of GTP gamma S 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 GTP gamma S in the microsomal fraction was absolutely dependent on the presence of Ca2+ > 0.5 microM. Addition of Ca2+ (10-100 microM) alone dose-dependently stimulated the PLD activity. Treatment of the microsomal fraction with phorbol esters had no effect on the ability of GTP gamma S 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.