Stimulation of phospholipase D via alpha1-adrenergic receptors in Madin-Darby canine kidney cells is independent of PKCalpha and -epsilon activation.

We have demonstrated previously that protein kinase Calpha (PKCalpha) 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 alpha1-adrenergic receptor subtype, promoted transient translocation of PKCalpha and more prolonged translocation of PKCepsilon 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 alpha1-adrenergic antagonist prazosin. D609, an inhibitor of phosphatidylcholine hydrolysis, blocked translocation of PKCalpha and PKCepsilon but did not inhibit PLD activation. Unlike results with PMA, or with the P2 purinergic receptor agonist ATP, epinephrine-stimulated PLD activity was not inhibited in MDCK-D1 cells in which PKCalpha expression is attenuated by an antisense cDNA construct or in cells in which PKC activity was inhibited by 1 microM 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 alpha1-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.