Protein kinase C activation increases the rate and magnitude of agonist-induced delta-opioid receptor down-regulation in NG108-15 cells.
Protein kinase C (PKC) activation was examined for its role in delta-opioid receptor down-regulation in the neuroblastoma X glioma hybrid cell line NG108-15. Incubation of NG108-15 cells for 2 hr at 37 degrees with up to 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), a phorbol ester that activates PKC, had no effect on opioid binding to membranes prepared from these cells. However, as little as 3 nM PMA incubated with an opioid agonist and NG108-15 cells potentiated the decrease and the rate of decrease of opioid binding, compared with agonist alone. Scatchard analysis of [3H][D-Ala2,D-Leu5]enkephalin (DADLE) binding revealed that NG108-15 cells incubated for 3 hr with 1 nM DADLE and 30 nM PMA displayed a > 50% reduction in the number of [3H]DADLE binding sites with no affinity change at the remaining sites, compared with cells treated with DADLE alone. The antagonist naloxone blocked both DADLE-induced and PMA-enhanced DADLE-induced down-regulation. The agonists morphine and cyclazocine, which alone were unable to induce delta receptor down-regulation, did so in the presence of PMA. The PKC inhibitor staurosporine and down-regulation of PKC by chronic PMA treatment blocked PMA potentiation of DADLE-induced down-regulation, but not "normal" DADLE-induced down-regulation. The enhancement of down-regulation by PMA was unaffected by either metabolic inhibitor or incubations at 20 degrees, conditions that blocked down-regulation by DADLE alone. NG108-15 cells incubated with [3H]DADLE and PMA retained more [3H]DADLE than cells incubated with [3H]DADLE alone, suggesting that PMA enhanced receptor internalization instead of merely inhibiting membrane binding. The diacylglycerol 1-oleoyl-2-acetyl-glycerol and bradykinin substituted for PMA but not carbachol, indicating that PKC activated physiologically may play a role in delta receptor down-regulation.
Gucker
S
S
Department of Pharmacology, University of Rochester, School of Medicine and Dentistry, New York 14642.
Bidlack
J M
JM
D000470:Alkaloids / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D015536:Down-Regulation; D016308:Enkephalin, Leucine-2-Alanine / Q000378:metabolism; D004789:Enzyme Activation; D005048:Etorphine / Q000494:pharmacology; D066298:In Vitro Techniques; D009270:Naloxone / Q000494:pharmacology; D009294:Narcotics / Q000494:pharmacology; D009474:Neurons / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D009294:Narcotics; D017465:Receptors, Opioid, delta; D009270:Naloxone; D005048:Etorphine; D016308:Enkephalin, Leucine-2-Alanine; D002738:Chloroquine; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate