Shahabi N A, Sharp B M
Endocrine-Neuroscience Research Laboratory, Minneapolis Medical Research Foundation, Minnesota.
J Pharmacol Exp Ther. 1993 Jan;264(1):276-81.
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)
据报道，蛋白激酶-C（PKC）的激活可改变多种受体-配体相互作用，包括肿瘤坏死因子α与免疫细胞的相互作用。因此，我们研究了佛波酯对β-内啡肽与前单核细胞样U937细胞系上纳洛酮抗性受体结合的影响。将完整的U937细胞与佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA，100 nM）在22℃孵育30分钟后，125I-β-内啡肽的特异性结合最大程度降低了约40%。只有PMA（10 - 150 nM），而非无生物学活性的佛波醇4α-佛波醇12,13-十二烷酸酯，会引起这种快速的、剂量依赖性的下调。PMA不干扰放射受体分析，与细胞膜孵育时也不会诱导下调。Scatchard分析显示，PMA显著降低了受体数量和平衡解离常数（对照组为10,640个受体/细胞，Kd = 2.9±0.1 nM；150 nM PMA处理组为4,868个受体/细胞，Kd = 1.5±0.7 nM）。用PKC抑制剂N-（2-氨乙基）-5-异喹啉磺酰胺或1-（5-异喹啉磺酰基）-2-甲基哌嗪预孵育细胞可消除PMA的作用。下调是可逆的；从培养基中去除100 nM PMA后，3小时部分恢复结合，24小时完全恢复。在22℃下，未观察到125I-β-内啡肽的内化，且PMA对此无影响。（摘要截短于250字）
