Safa A R, Glover C J, Sewell J L, Meyers M B, Biedler J L, Felsted R L
J Biol Chem. 1987 Jun 5;262(16):7884-8.
A radioactive photoactive dihydropyridine calcium channel blocker, [3H]azidopine, was used to photoaffinity label plasma membranes of multidrug-resistant Chinese hamster lung cells selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic fluorograms revealed the presence of an intensely radiolabeled 150-180-kDa doublet in the membranes from drug-resistant but not from the drug-sensitive parental (DC-3F) cells. A similar radiolabeled doublet was barely detected in a drug-sensitive partial revertant (DC-3F/ADX-U) cell line. The 150-180-kDa doublet exhibited a specific half-maximal saturable photolabeling at 1.07 X 10(-7) M [3H]azidopine. The dihydropyridine binding specificity was established by competitive blocking of specific photolabeling with nonradioactive azidopine as well as with nonphotoactive calcium channel blockers nimodipine, nitrendipine, and nifedipine. In addition, [3H]azidopine photolabeling was blocked by verapamil and diltiazem but was stimulated by excess prenylamine and bepridil suggesting a cross-specificity for up to four different classes of calcium channel blockers. The 150-180-kDa calcium channel blocker acceptor co-electrophoresed exactly with the 150-180-kDa surface membrane glycoprotein (gp150-180 or P-glycoprotein) Vinca alkaloid acceptor from multidrug-resistant cells and was immunoprecipitated by polyclonal antibody recognizing gp150-180. [3H]Azidopine photolabeling of the 150-180-kDa component in the presence of excess vinblastine was reduced over 90%, confirming the identity or close relationship of the calcium channel blocker acceptor and the gp150-180 Vinca alkaloid acceptor. The [3H]azidopine photolabeling of gp150-180 also was reduced by excess actinomycin D, adriamycin, or colchicine, demonstrating a broad gp150-180 drug recognition capacity. The ability of gp150-180 to recognize multiple natural product cytotoxic drugs as well as calcium channel blockers suggests a direct function for gp150-180 in the multidrug resistance phenomenon and a role in the circumvention of that resistance by calcium channel blockers.
一种放射性光活性二氢吡啶钙通道阻滞剂[3H]叠氮平，被用于对选择出的对长春新碱（DC - 3F/VCRd - 5L）或放线菌素D（DC - 3F/ADX）具有耐药性的多药耐药中国仓鼠肺细胞的质膜进行光亲和标记。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳荧光图谱显示，在耐药细胞而非药物敏感的亲本（DC - 3F）细胞的膜中存在一条强烈放射性标记的150 - 180 kDa双峰。在药物敏感的部分回复突变体（DC - 3F/ADX - U）细胞系中几乎检测不到类似的放射性标记双峰。150 - 180 kDa双峰在1.07×10(-7) M [3H]叠氮平浓度下表现出特异性的半数最大饱和光标记。通过用非放射性叠氮平以及非光活性钙通道阻滞剂尼莫地平、尼群地平和硝苯地平竞争性阻断特异性光标记，确定了二氢吡啶结合特异性。此外，[3H]叠氮平光标记被维拉帕米和地尔硫䓬阻断，但被过量的普尼拉明和苄普地尔刺激，这表明对多达四类不同的钙通道阻滞剂存在交叉特异性。150 - 180 kDa钙通道阻滞剂受体与多药耐药细胞中的150 - 180 kDa表面膜糖蛋白（gp150 - 180或P - 糖蛋白）长春花生物碱受体精确共电泳，并被识别gp150 - 180的多克隆抗体免疫沉淀。在过量长春碱存在下，[3H]叠氮平对1
