Yoo H, Fallgren B, Lindahl A, Wahlestedt C
Department of Neurology and Neuroscience, Cornell University Medical College, New York, NY 10021.
Eur J Pharmacol. 1994 Jun 15;268(1):55-63. doi: 10.1016/0922-4106(94)90119-8.
Alfa-trinositol (or D-myo-inositol 1,2,6-trisphosphate) was recently found to, e.g., inhibit agonist-induced vasoconstriction and display antiinflammatory properties. However, its mechanism of action is unknown, although effects on Ca2+ fluxes, perhaps by interfering with endogenous inositol phosphate(s), have been suggested. Here we describe the existence of specific [3H]alpha-trinositol binding sites and compare these with binding sites for naturally occurring inositol phosphates. For this purpose we developed a tritiated analog of alpha-trinositol and used it in a centrifugation binding assay on extensively washed membranes from rat tissues. The degree of specific [3H] alpha-trinositol binding was markedly increased as a result of the many wash steps, indicating the existence of endogenous binding inhibitor(s). A single population of [3H] alpha-trinositol binding sites, displaying a KD of 159 nM and a Bmax of 71 pmol/mg protein, was present in cardiac membranes assayed at pH 7.4. Similar binding site densities were detected also in liver > lung > brain. The relative density of [3H] alpha-trinositol sites in cardiac membranes was 8-fold higher than [3H]Ins(1,4,5)P3 but 2-fold and 4-fold lower than [3H]Ins(1,3,4,5)P4 and [3H]InsP6 binding sites, respectively. Competition binding studies indicated the ability of Ins(1,3,4,5)P4 and InsP6, but not Ins(1,4,5)P3, to potently displace [3H] alpha-trinositol binding. Conversely, unlabelled alpha-trinositol showed relatively low potency vs. [3H]InsP6, but the novel inositol phosphate was virtually equipotent with Ins(1,3,4,5)P4 in inhibiting [3H]Ins(1,3,4,5)P4 binding. Finally, analyses of binding at different pH and ionic conditions revealed differences between alpha-trinositol and the three other previously studied inositol phosphates, although distinct similarities between alpha-trinositol and Ins(1,3,4,5)P4 were again observed.
α-三磷酸肌醇（或D-肌醇1,2,6-三磷酸）最近被发现，例如，可抑制激动剂诱导的血管收缩并具有抗炎特性。然而，其作用机制尚不清楚，尽管有人提出它可能通过干扰内源性肌醇磷酸对Ca2+通量产生影响。在此，我们描述了特异性[3H]α-三磷酸肌醇结合位点的存在，并将其与天然存在的肌醇磷酸的结合位点进行比较。为此，我们开发了一种α-三磷酸肌醇的氚代类似物，并将其用于对大鼠组织经大量洗涤后的膜进行离心结合测定。由于多次洗涤步骤，特异性[3H]α-三磷酸肌醇结合程度显著增加，表明存在内源性结合抑制剂。在pH 7.4条件下测定的心脏膜中存在单一群体的[3H]α-三磷酸肌醇结合位点，其KD为159 nM，Bmax为71 pmol/mg蛋白。在肝脏>肺>脑中也检测到了类似的结合位点密度。心脏膜中[3H]α-三磷酸肌醇位点的相对密度比[3H]肌醇-1,4,5-三磷酸高8倍，但分别比[3H]肌醇-1,3,4,5-四磷酸和[3H]肌醇六磷酸结合位点低2倍和4倍。竞争结合研究表明，肌醇-1,3,4,5-四磷酸和肌醇六磷酸能够有效取代[3H]α-三磷酸肌醇结合，而肌醇-1,4,5-三磷酸则不能。相反，未标记的α-三磷酸肌醇对[3H]肌醇六磷酸的效力相对较低，但这种新型肌醇磷酸在抑制[3H]肌醇-1,3,4,5-四磷酸结合方面与肌醇-1,3,4,5-四磷酸几乎等效。最后，在不同pH和离子条件下的结合分析揭示了α-三磷酸肌醇与其他三种先前研究的肌醇磷酸之间的差异，但再次观察到α-三磷酸肌醇与肌醇-1,3,4,5-四磷酸之间存在明显的相似性。
