Abstract

We have demonstrated previously that protein kinase C_α(PKC_α) plays a key role in regulating phospholipase D (PLD) activation by nucleotides and the phorbol ester phorbol-12-myristate-13-acetate in Madin-Darby canine kidney (MDCK-D1) cells. In the current work, we investigated PLD activation in MDCK-D1 cells triggered by the adrenergic receptor agonist epinephrine and its mechanism of activation. Epinephrine, acting through the α₁-adrenergic receptor subtype, promoted transient translocation of PKC_α and more prolonged translocation of PKC_ε to the membrane fraction, indicating activation of these two isoforms. In addition, epinephrine promoted activation of PLD, as shown by a sustained accumulation of phosphatidylethanol. All of these events were blocked by pretreatment of cells with the α₁-adrenergic antagonist prazosin. D609, an inhibitor of phosphatidylcholine hydrolysis, blocked translocation of PKC_α and PKC_ε but did not inhibit PLD activation. Unlike results with PMA, or with the P₂purinergic receptor agonist ATP, epinephrine-stimulated PLD activity was not inhibited in MDCK-D1 cells in which PKC_αexpression is attenuated by an antisense cDNA construct or in cells in which PKC activity was inhibited by 1 μm GF 109203X. However, PLD activation by epinephrine was abolished by concomitant incubation of cells with the calcium chelator EGTA. These data, together with previous results, are consistent with the hypothesis that in MDCK-D1 cells, epinephrine acting on α₁-adrenergic receptors, promotes a rapid increase in cytosolic Ca²⁺ that promotes activation of PLD through an as-yet poorly defined mechanism. The data demonstrate that different types of G protein-linked receptors that activate PLD can mediate this activation in either a PKC activation-dependent or -independent manner within a single cell type.