Regulation of phospholipase D activity in neuroblastoma cells

Abstract

The regulation of phospholipase D was studied in human neuroblastoma cells using phosphatidylethanol as a marker of the enzyme activity. Carbachol induced phospholipase D activity in SH-SY5Y cells. Muscarinic antagonists inhibited the response with potencies suggesting that muscarinic M₁ receptors are responsible for the activation. In permeabilized SH-SY5Y cells, both the carbachol- and GTPγS-induced Peth formation was inhibited by GDPβS, indicating that both responses are mediated via a G-protein. The protein kinase C inhibitors, bisindolylmaleimide and staurosporine significantly inhibited the carbachol-induced Peth formation whereas H7 had no effect. Thus, the cholinergic activation of phospholipase D in SH-SY5Y cells is probably mediated via a direct receptor-G-protein coupling but an involvement of protein kinase C cannot be excluded. Calmidazolium, a calmodulin antagonist, induced an increase in phosphatidylethanol formation in both SH-SY5Y and IMR-32 cells. This effect was inhibited by genistein and tyrphostin, indicating a tyrosine kinase dependent pathway for phospholipase D activation in neuroblastoma cells.