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)