Contribution of phospholipase C-beta3 phosphorylation to the rapid attenuation of opioid-activated phosphoinositide response.
Activation of the delta-opioid receptor in NG108-15 neuroblastoma X glioma hybrid cells results in a transient increase at the intracellular level of inositol-1,4,5-triphosphate [Ins(1,4,5)P3]. This time course in the transient increase in the Ins(1,4,5)P3 level is distinctly different from that observed in the homologous opioid receptor desensitization as measured by the inhibition of adenylyl cyclase activity. One probable mechanism for this rapid loss in Ins(1,4,5)P3 response is the feedback regulation of the phospholipase C activity. Regulation by protein phosphorylation was suggested by the observations that the opioid-mediated response was potentiated by calphostin C, an inhibitor of protein kinase C (PKC), and was abolished by either phorbol-12-myristate-13-acetate, a PKC activator, or calyculin A, a protein phosphatase1/2A inhibitor. The direct phosphorylation of phospholipase C was demonstrated by immunoprecipitation of PLC-beta3 from metabolically labeled NG108-15 cells challenged with the delta-selective agonist [D-Pen2, D-Pen5]enkephalin (DPDPE). A time- and DPDPE concentration-dependent and naloxone-reversible increase in the PLC-beta3 phosphorylation can be demonstrated. This PLC-beta3 phosphorylation was mainly due to PKC activation because pretreatment of NG108-15 cells with calphostin C could block the DPDPE effect. Activation of the PLC-beta3 by DPDPE was one of the prerequisites for agonist-mediated PLC-beta3 phosphorylation because the aminosteroid phospholipase C inhibitor U73122 could block the DPDPE effect. In addition to DPDPE, lysophosphatidic acid (LPA) stimulated the PLC-beta3 phosphorylation, but bradykinin did not. Furthermore, the LPA- and DPDPE-mediated PLC-beta3 phosphorylation was additive and was much less than that observed with phorbol-12-myristate-13-acetate. The effect of DPDPE was specific to PLC-beta3; the betagamma-insensitive phospholipase C-beta1 was not phosphorylated in the presence of either DPDPE or LPA. These results indicate that although PKC phosphorylation of PLC-beta3 is not obligatory for the opioid receptor desensitization, it seems to play a significant facilatory role in the mechanisms allowing desensitization of opioid-activated phospholipase C response before that of adenylyl cyclase inhibition.[1]References
- Contribution of phospholipase C-beta3 phosphorylation to the rapid attenuation of opioid-activated phosphoinositide response. Strassheim, D., Law, P.Y., Loh, H.H. Mol. Pharmacol. (1998) [Pubmed]
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