Drímal J
Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava.
Gen Physiol Biophys. 1994 Apr;13(2):137-48.
The low affinity binding sites identified in crude membranes from different excitable tissues with the dihydropyridine (DHP) calcium (Ca2+) channel ligands have confused researches in the field of Ca2+ channels as they can represent low affinity state(s) of the DHP receptor, or they can be labelled with DHP-type Ca2+ channel ligands. The aim of this communication was to provide more evidence for the existence of separate DHP binding sites on the surface of cultured green monkey renal cells (GMRC). The saturation ligand binding experiments with [3H]-nitrendipine (NTP) and photoaffinity labelling studies with (-)-[3H]-azidopine (AZI) were performed in order to identify and further characterize the DHP receptor on cultured GMRC. Specific high affinity sites identified on GMRC with [3H]-NTP (Bmax = 0.78 +/- 0.03 pmol/mg protein and KD = 0.06 +/- 0.1 nmol/l in native cells) and photolabelled with AZI represent DHP receptor on L-type Ca2+ channels. The low affinity binding sites photolabelled with AZI on GMRC (9.84 +/- 2.4 pmol/mg protein and KD = 3.21 +/- 1.25 nmol/l in native cells) were significantly increased after preincubation of GMRC with low concentrations of DHPs nitrendipine and nisoldipine. Preincubation of GMRC with Ca2+ channel agonist (-)BAYK 8644 significantly reduced specific photolabelling with AZI on GMRC and increased low affinity labelling. Preincubation of (+)BAYK 8644 was without any effect. Niguldipine (DHP with the voluminous substituent on the port side of the DHP ring) partially inhibited specific photolabelling with AZI on GMRC and also partially reduced the maximal number of low affinity binding sites labelled with AZI. Our results support the hypothesis of separate subsites in the region of DHP receptor of GMRC and the existence of the "marginal" photolabelling of specific DHP binding sites identified on Ca2+ channels.
在不同可兴奋组织的粗制膜中，用二氢吡啶（DHP）钙（Ca2+）通道配体鉴定出的低亲和力结合位点，一直困扰着Ca2+通道领域的研究人员，因为它们可能代表DHP受体的低亲和力状态，或者它们可以被DHP型Ca2+通道配体标记。本通讯的目的是为培养的绿猴肾细胞（GMRC）表面存在独立的DHP结合位点提供更多证据。为了鉴定并进一步表征培养的GMRC上的DHP受体，进行了用[3H]-尼群地平（NTP）的饱和配体结合实验以及用（-）-[3H]-叠氮平（AZI）的光亲和标记研究。在GMRC上用[3H]-NTP鉴定出的特异性高亲和力位点（天然细胞中Bmax = 0.78 +/- 0.03 pmol/mg蛋白，KD = 0.06 +/- 0.1 nmol/l），并用AZI进行光标记，代表L型Ca2+通道上的DHP受体。GMRC上用AZI光标记的低亲和力结合位点（天然细胞中为9.84 +/- 2.4 pmol/mg蛋白，KD = 3.21 +/- 1.25 nmol/l），在GMRC用低浓度的DHP尼群地平和尼索地平预孵育后显著增加。GMRC用Ca2+通道激动剂（-）BAYK 8644预孵育，显著降低了GMRC上用AZI的特异性光标记，并增加了低亲和力标记。（+）BAYK 8644预孵育没有任何影响。尼鲁地平（DHP环左侧带有大量取代基的DHP）部分抑制了GMRC上用AZI的特异性光标记，也部分减少了用AZI标记的低亲和力结合位点的最大数量。我们的结果支持GMRC的DHP受体区域存在独立亚位点的假设，以及Ca2+通道上鉴定出的特异性DHP结合位点存在“边缘”光标记的假设。
