Calcium and Protein Kinase C (PKC)-Related Kinase Mediate α1A-Adrenergic Receptor-Stimulated Activation of Phospholipase D in Rat-1 Cells, Independent of PKC

• Published in: The Journal of pharmacology and experimental therapeutics 2002-12, Vol.303 (3), p.1206-1215
• Main Authors: Parmentier, Jean-Hugues, Ahmed, Aftab, Ruan, Ying, Gandhi, Gautam K., Saeed, Abdelwahab E., Malik, Kafait U.
• Format: Article
• Language: English
• ISSN: 0022-3565
• DOI: 10.1124/jpet.102.041384

A previous study conducted in rat-1 cells expressing α1A-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 α1A-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 α1A-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.