Leysen J E, Eens A, Gommeren W, van Gompel P, Wynants J, Janssen P A
Department of Biochemical Pharmacology, Janssen Research Foundation, Beerse, Belgium.
J Pharmacol Exp Ther. 1988 Jan;244(1):310-21.
In mammalian striatal tissue and cat platelets, [3H]ketanserin labels besides serotonin-S2 receptors nonserotonergic saturable binding sites. The sites have been distinguished and characterized in [3H]ketanserin binding assays by selective inhibition with tetrabenazine (Ki = 4 nM), a monoamine depleting agent. In rats, the nonserotonergic ketanserin sites were enriched in the striatum (KD = 12.4 +/- 0.3 nM, maximal number of binding sites = 53.2 +/- 11.8 fmol/mg of tissue at pH 7.7, 37 degrees C) and nucleus accumbens. The sites were decreased by 65 to 78% after 6-hydroxydopamine lesions, suggesting an association with dopaminergic nerve terminals. In in vitro superfusion experiments using [3H]dopamine, [3H]norepinephrine and [3H]serotonin loaded rat brain tissue and [3H]serotonin loaded human platelets, 5 min superfusion with 10(-6) M ketanserin, tetrabenazine and reserpine caused instantaneously a marked increase in tritium efflux. The effect was attenuated by the monoamine oxidase inhibitor, pargyline, in brain slices but not in platelets. High-performance liquid chromatography analysis of endogenous catecholamines, serotonin and metabolites in superfusates from striatal slices revealed that stimulation with these drugs provoked mainly release of 3,4-dihydroxybenzeneacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid. Potencies of a series of ketanserin derivatives, benzoquinolizine derivatives and a variety of drugs affecting neurotransmission were assessed in the in vitro release test using [3H]dopamine loaded striatal slices, and in [3H]ketanserin binding assays to nonserotonergic sites in the striatum and to serotonin-S2 receptors in brain tissue. Activities of drugs in the release test correlated strongly with their binding affinities for nonserotonergic ketanserin sites (rs = 0.83, n = 30, P less than .001). High potency in the latter two tests was confined to few close structural congeners of ketanserin and tetrabenazine. Distinct structural activity relationships for interaction with nonserotonergic ketanserin sites and serotonin-S2 receptors were found. It was concluded that nonserotonergic ketanserin sites mediate release of oxidated metabolites of biogenic amines from nerve endings and of serotonin from platelets. Hence release of biogenic amine metabolites or of cytoplasmic amines is probably not a mere diffusion process but involves specific membranous molecules. Unlike tetrabenazine, ketanserin caused no obvious depletion of central catecholamine and indoleamine stores. Implications of these findings for the mechanism of action of the drugs are discussed.
在哺乳动物的纹状体组织和猫的血小板中，[3H]酮色林除了标记5-羟色胺-S2受体外，还标记非5-羟色胺能的可饱和结合位点。在[3H]酮色林结合试验中，通过用单胺耗竭剂丁苯那嗪（Ki = 4 nM）进行选择性抑制，已对这些位点进行了区分和表征。在大鼠中，非5-羟色胺能的酮色林位点在纹状体（KD = 12.4±0.3 nM，在pH 7.7、37℃时最大结合位点数 = 53.2±11.8 fmol/mg组织）和伏隔核中富集。6-羟基多巴胺损伤后，这些位点减少了65%至78%，表明与多巴胺能神经末梢有关。在使用[3H]多巴胺、[3H]去甲肾上腺素和[3H]5-羟色胺加载的大鼠脑组织以及[3H]5-羟色胺加载的人血小板进行的体外灌流实验中，用10(-6) M酮色林、丁苯那嗪和利血平进行5分钟灌流会立即导致氚外流显著增加。单胺氧化酶抑制剂帕吉林在脑片中减弱了这种作用，但在血小板中没有。对纹状体切片灌流液中内源性儿茶酚胺、5-羟色胺及其代谢产物的高效液相色谱分析表明，用这些药物刺激主要引起3,4-二羟基苯乙酸、高香草酸和5-羟基吲哚乙酸的释放。在使用[3H]多巴胺加载的纹状体切片的体外释放试验以及对纹状体中非5-羟色胺能位点和脑组织中5-羟色胺-S2受体的[3H]酮色林结合试验中，评估了一系列酮色林衍生物、苯并喹嗪衍生物和各种影响神经传递的药物的效能。药物在释放试验中的活性与其对非5-羟色胺能酮色林位点的结合亲和力密切相关（rs = 0.83，n = 30，P <. .001）。后两种试验中的高效能仅限于酮色林和丁苯那嗪的少数紧密结构类似物。发现了与非5-羟色胺能酮色林位点和5-羟色胺-S2受体相互作用的明显构效关系。得出的结论是，非5-羟色胺能酮色林位点介导生物胺氧化代谢产物从神经末梢的释放以及5-羟色胺从血小板的释放。因此，生物胺代谢产物或细胞质胺的释放可能不仅仅是一个扩散过程，而是涉及特定的膜分子。与丁苯那嗪不同，酮色林不会导致中枢儿茶酚胺和吲哚胺储备明显耗竭。讨论了这些发现对药物作用机制的意义。
