In the accompanying paper [Khare et al., Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G993-G1004, 1999], activation of protein kinase C- (PKC-) was shown to be involved in the stimulation of phospholipase D (PLD) by 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 12-O-tetradecanoylphorbol 13-acetate (TPA) in Caco-2 cells. Monomeric or heterotrimeric G proteins, as well as pp60^c-src have been implicated in PLD activation. We therefore determined whether these signal transduction elements were involved in PLD stimulation by 1,25(OH)₂D₃ or TPA. Treatment with C3 transferase, which inhibits members of the Rho family of monomeric G proteins, markedly diminished the ability of 1,25(OH)₂D₃, but not TPA, to stimulate PLD. Brefeldin A, an inhibitor of ADP-ribosylation factor proteins, did not, however, significantly reduce the stimulation of PLD by either of these agents. Moreover, 1,25(OH)₂D₃, but not TPA, activated pp60^c-src and treatment with PP1, a specific inhibitor of the pp60^c-src family, blocked the ability of 1,25(OH)₂D₃ to activate PLD. Pretreatment of cells with pertussis toxin (PTx) markedly reduced the stimulation of PLD by either agonist. PTx, moreover, inhibited the stimulation of pp60^c-src and PKC- by 1,25(OH)₂D₃. PTx did not, however, block the membrane translocation of RhoA induced by 1,25(OH)₂D₃ or inhibit the stimulation of PKC- by TPA. These findings, taken together with those of the accompanying paper, indicate that although 1,25(OH)₂D₃ and TPA each activate PLD in Caco-2 cells in part via PKC-, their stimulation of PLD differs in a number of important aspects, including the requirement for pp60^c-src and RhoA in the activation of PLD by 1,25(OH)₂D₃, but not TPA. Moreover, the requirement for different signal transduction elements by 1,25(OH)₂D₃ and TPA to induce the stimulation of PLD may potentially underlie differences in the physiological effects of these agents in Caco-2 cells.

calcitriol; phorbol esters; pertussis toxin; G proteins; signal transduction

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