Oliva D, Pocchiari F, Allievi L, Rovati G E, Nicosia S
Institute of Pharmacological Sciences, University of Milan, Italy.
Pharmacol Res. 1992 Sep;26(2):187-99. doi: 10.1016/s1043-6618(05)80132-x.
Here we report the identification of binding sites for 3H-ketanserin in human platelet membranes. At 4 degrees C, 3H-ketanserin binding is saturable (Bmax = 0.58 pmol/mg protein), rapid (equilibrium being attained within 20 min) and reversible. The kinetics of the association and dissociation curves are consistent with the existence of a single class of binding sites, as confirmed also by computer-assisted analysis of the saturation curve. Specific binding is increased by Ca2+ and Mg2+. 3H-ketanserin binding is inhibited by serotonin (Ki = 48.5 microM), unlabeled ketanserin (Ki = 3-15 nM), as well as by another antiserotonergic drug, methysergide (Ki = 32.6 microM). However, other selective 5-HT2 ligands, such as ritanserin, spiperone and cyproheptadine fail to interact with 3H-ketanserin binding. On the contrary, tetrabenzine, a monoamine depleting agent, when preincubated at 30 degrees C, did inhibit the specific binding completely. 3H-ketanserin specific binding is inhibited in a dose-dependent fashion by some calcium blocking agents, with different potencies: verapamil (Ki = 2.25 microM), diltiazem (Ki = 139 microM) and SIM6080, a new Ca(2+)-antagonist related to the phenylalkylamines (Ki = 5.22 microM). Flunarizine inhibited 3H-ketanserin specific binding only at relatively high concentrations (IC50 greater than 100 microM), while nitrendipine did not show any inhibitory effect up to 20 microM. The present evidence indicates that all the sites labeled by 3H-ketanserin at 4 degrees C might be coincident with the monoamino transporter identified in other systems, and that they might play a role in the modulation of platelet aggregation exerted by some calcium blocking agents.
在此，我们报告了在人血小板膜中3H-酮色林结合位点的鉴定情况。在4℃时，3H-酮色林结合具有饱和性（Bmax = 0.58 pmol/mg蛋白质）、快速性（20分钟内达到平衡）且可逆。结合和解离曲线的动力学与单一类结合位点的存在相一致，这也通过饱和曲线的计算机辅助分析得到证实。特异性结合可被Ca2+和Mg2+增强。3H-酮色林结合受到5-羟色胺（Ki = 48.5 microM）、未标记的酮色林（Ki = 3 - 15 nM）以及另一种抗5-羟色胺能药物麦角异胺（Ki = 32.6 microM）的抑制。然而，其他选择性5-HT2配体，如利坦色林、螺哌隆和赛庚啶，未能与3H-酮色林结合相互作用。相反，单胺耗竭剂丁苯那嗪在30℃预孵育时，确实能完全抑制特异性结合。一些钙阻滞剂以剂量依赖方式抑制3H-酮色林特异性结合，效力不同：维拉帕米（Ki = 2.25 microM）、地尔硫卓（Ki = 139 microM）以及与苯烷基胺相关的新型Ca(2+)拮抗剂SIM6080（Ki = 5.22 microM）。氟桂利嗪仅在相对高浓度时（IC50大于100 microM）抑制3H-酮色林特异性结合，而尼群地平在高达20 microM时未显示任何抑制作用。目前的证据表明，在4℃时被3H-酮色林标记的所有位点可能与在其他系统中鉴定出的单胺转运体一致，并且它们可能在某些钙阻滞剂对血小板聚集的调节中发挥作用。
