Abstract
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)
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|