Activation of protein kinase C rapidly down-regulates naloxone-resistant receptors for beta-endorphin on U937 cells.
Activation of protein kinase-C (PKC) has been reported to modify a variety of receptor-ligand interactions, including that of tumor necrosis factor alpha with immune cells. Thus, we studied the effect of phorbol esters on the binding of beta-endorphin to naloxone-resistant receptors on the promonocyte-like U937 cell line. After incubating intact U937 cells with phorbol 12-myristate 13-acetate (PMA, 100 nM) at 22 degrees C for 30 min, the specific binding of 125I-beta-endorphin was maximally reduced by approximately 40%. Only PMA (10-150 nM), and not the biologically inactive phorbol, 4 alpha-phorbol 12,13-didecanoate, caused this rapid, dose-dependent down-regulation. PMA did not interfere with the radioreceptor assay nor did it induce down-regulation when incubated with cell membrane. Scatchard analysis revealed that PMA significantly reduced both the number of receptors and Kd (10,640 receptors/cell and Kd = 2.9 +/- 0.1 nM for control vs. 4,868 receptors/cell and Kd = 1.5 +/- 0.7 nM for 150 nM PMA). The effect of PMA was abolished by preincubating cells with the inhibitors of PKC, N-(2-aminoethyl)-5 isoquinolinesulfonamide or 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine. Down-regulation was reversible; removing 100 nM PMA from the media partially restored binding by 3 h and completely by 24 h. At 22 degrees C, internalization of 125I-beta-endorphin was not observed, and this was not altered by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)
Endocrine-Neuroscience Research Laboratory, Minneapolis Medical Research Foundation, Minnesota.
N A
NA
Shahabi
B M
BM
Sharp
D015536:Down-Regulation / Q000502:physiology*; D004351:Drug Resistance; D004789:Enzyme Activation; D006801:Humans; D007457:Iodine Radioisotopes; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D007948:Leukemia, Monocytic, Acute / Q000201:enzymology / Q000378:metabolism; D009270:Naloxone / Q000494:pharmacology*; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011957:Receptors, Opioid / Q000378:metabolism / Q000502:physiology*; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000648:ultrastructure; D001615:beta-Endorphin / Q000378:metabolism*
D007457:Iodine Radioisotopes; D007546:Isoquinolines; D010703:Phorbol Esters; D011957:Receptors, Opioid; D013449:Sulfonamides; C008876:phorbol-12,13-didecanoate; D009270:Naloxone; D001615:beta-Endorphin; C044388:N-(2-aminoethyl)-5-isoquinolinesulfonamide; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate