Cesura A M, Bertocci B, Da Prada M
Department of Pharmacology, University of Milan, Italy.
Eur J Pharmacol. 1990 Sep 4;186(1):95-104. doi: 10.1016/0014-2999(90)94064-5.
The carrier for 5-hydroxytryptamine (5-HT) of the 5-HT storage organelles of blood platelets was characterized by [3H]dihydrotetrabenazine binding and [125I]azidoiodokentanserin photoaffinity labeling. [3H]Dihydrotetrabenazine bound with high affinity to membrane preparations from different animal species. The [3H]dihydrotetrabenazine Bmax value was about 10-fold higher in rabbit (9.4 +/- 1.3 pmol/mg protein) than in human, rat and guinea-pig preparations (Bmax values = 1.1 +/- 0.2, 1.2 +/- 0.1 and 0.52 +/- 0.06 pmol/mg protein, respectively). After rabbit platelet subcellular fractionation, [3H]dihydrotetrabenazine binding was highly enriched in the fraction corresponding to pure 5-HT organelles, whereas ligand binding was much lower in the other subcellular fractions. Conversely, [3H]paroxetine binding sites were more concentrated in the lower density fractions, with no binding to the 5-HT granules. In competition experiments, [3H]dihydrotetrabenazine binding to human platelet membranes and rabbit platelet 5-HT organelles was markedly inhibited by the benzo[a]quinolizine derivatives, tetrabenazine and Ro 4-1284, and by ketanserin. In isolated rabbit platelet 5-HT organelles, reserpine showed a relatively high IC50 (930 nM), but the presence of ATP increased its potency about 10-fold. Paroxetine, methysergide and carrier substrates had little or no effect. After photoaffinity labeling of rabbit 5-HT granules with [125I]azidoiodoketanserin, the radioactivity was incorporated into several polypeptides. The presence of Ro 4-1284, reserpine and ketanserin prevented the labeling of a polypeptide of 85 kDa. The data obtained suggest that this protein represents a component of the granular carrier which binds [3H]dihydrotetrabenazine.
通过[3H]二氢丁苯那嗪结合和[125I]叠氮碘酮色林光亲和标记对血小板5-羟色胺（5-HT）储存细胞器的5-HT载体进行了表征。[3H]二氢丁苯那嗪与来自不同动物物种的膜制剂具有高亲和力结合。[3H]二氢丁苯那嗪的Bmax值在兔（9.4±1.3 pmol/mg蛋白质）中比在人、大鼠和豚鼠制剂中高约10倍（Bmax值分别为1.1±0.2、1.2±0.1和0.52±0.06 pmol/mg蛋白质）。兔血小板亚细胞分级分离后，[3H]二氢丁苯那嗪结合在对应于纯5-HT细胞器的级分中高度富集，而配体结合在其他亚细胞级分中则低得多。相反，[3H]帕罗西汀结合位点在较低密度级分中更集中，与5-HT颗粒无结合。在竞争实验中，[3H]二氢丁苯那嗪与人血小板膜和兔血小板5-HT细胞器的结合被苯并[a]喹嗪衍生物、丁苯那嗪和Ro 4-1284以及酮色林显著抑制。在分离的兔血小板5-HT细胞器中，利血平显示出相对较高的IC50（930 nM），但ATP的存在使其效力增加约10倍。帕罗西汀、甲基麦角新碱和载体底物几乎没有影响或没有影响。用[125I]叠氮碘酮色林对兔5-HT颗粒进行光亲和标记后，放射性被掺入几种多肽中。Ro 4-1284、利血平和酮色林的存在阻止了85 kDa多肽的标记。获得的数据表明，该蛋白质代表结合[3H]二氢丁苯那嗪的颗粒载体的一个组分。
