Opioid peptides activate phospholipase D and protein kinase C-ε in chicken embryo neuron cultures

Abstract

The μ-opioid peptide morphiceptin stimulated a Ca2+-independent protein kinase C (PKC-ε) that is expressed both in embryonic day 6 chicken telencephalon and in derived neuronal cultures. This activation was seen as a 2-fold increase in the activity and level of cytosolic PKC-ε and as a transient increase in membrane-associated PKC-ε following morphiceptin treatment. Morphiceptin did not activate phospholipase C-mediated phosphatidylinositol hydrolysis but did transiently activate (2- to 3-fold) phospholipase D (PLD), as measured by phosphatidylethanol formation in neuron cultures derived from embryonic day 6 or day 7 cerebral hemispheres. This PLD activation could provide an alternative source of diacylglycerol for the activation of PKC-ε and was naloxone-reversible and at least partially blocked by the tyrosine kinase inhibitor herbimycin A. Addition of phorbol 12-myristate 13-acetate stimulated both PLD and PKC-ε activities to a greater extent than opioids. The phorbol ester and insulin stimulation of PLD was also blocked by herbimycin. Both morphiceptin (in a naloxone-reversible manner) and phorbol ester increased phosphorylation of similar cytosolic proteins in intact cells, demonstrating a functional role for the PKC-ε activation by opioids. This is evidence that opioid receptors are transiently coupled to tyrosine kinase, PLD and PKC-ε activation and, by implication, to neuronal cell growth during brain morphogenesis.