Abstract

A previous study conducted in rat-1 cells expressing α_1A-adrenergic receptors showed that phenylephrine (PHE) stimulates phospholipase D (PLD) activity. This study was conducted to determine the contribution of protein kinase C (PKC) to PHE-induced PLD activation in these cells. PKC inhibitors bisindolylmaleimide (BIM) I and Ro 31-8220, but not Gö 6976 or a pseudosubstrate peptide inhibitor of PKCα, decreased PLD activity and arachidonic acid release elicited by PHE. However, antisense oligonucleotides directed against PKC α, δ, ε, and η reduced PKC isoform levels by about 80% but failed to alter PHE-induced PLD activation, indicating that these PKC isoforms are not involved in PLD activation elicited by α_1A-adrenergic receptor stimulation. Ectopic expression of a kinase-deficient mutant of the PKC-related kinase PKN significantly attenuated PHE-induced PLD activation. On the other hand, BIM I and Ro 31-8220 blocked PHE-mediated increase in intracellular Ca²⁺ but Gö 6976 and the peptide inhibitor did not. In the absence of extracellular Ca²⁺, PHE failed to increase PLD activity. These results indicate that α_1A-adrenergic receptor-stimulated PLD activation is mediated by a mechanism independent of PKCα, δ, ε, and η, but dependent on a PKC-related kinase, PKN. Moreover, PKC inhibitors BIM I and Ro 31-8220 block PHE-induced PLD activity by inhibiting calcium signal. Caution should be used in interpreting the data obtained with PKC inhibitors in vivo.