Parent C A, Lagarde M, Venton D L, Le Breton G C
Department of Pharmacology, University of Illinois, Chicago 60612.
J Biol Chem. 1992 Apr 5;267(10):6541-7.
We previously demonstrated that nonesterified as well as esterified eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) inhibit U46619-induced platelet aggregation and [3H]U46619 specific binding to washed human platelets. It was also demonstrated that esterification of these fatty acids resulted in a decrease in the affinity of [3H]U46619 for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. In order to investigate the specificity of this inhibition, the effects of 20:5n-3 and 22:6n-3 on the function and binding of the platelet alpha 2-adrenergic receptor were studied. It was found that neither 20:5n-3 nor 22:6n-3 (nonesterified or esterified) altered epinephrine-induced aggregation or [3H]yohimbine specific binding. Moreover, Scatchard analysis revealed that esterification with either 20:5n-3 or 22:6n-3 did not alter the dissociation constant for [3H]yohimbine binding. Modulation of the TXA2/PGH2 receptor by 20:5n-3 and 22:6n-3 was next evaluated using CHAPS- and digitonin-solubilized platelet membranes. [3H]SQ29,548 dissociation constants of 26.5 nM and 20.8 nM were measured for CHAPS and digitonin-solubilized membranes, respectively. Competitive binding experiments in these solubilized preparations revealed that 20:5n-3 or 22:6n-3 blocked [3H] SQ29,548 binding with IC50 values in the range of 6-15 microM, while concentrations of these fatty acids of up to 100 microM showed no effect on [3H]yohimbine binding. On the other hand, the IC50 values for inhibition of [3H] SQ29,548 binding by linoleic acid (18:2n-6) and gamma-linolenic acid (18:3n-6) were in the range of 150 microM. Furthermore, 18:2n-6 and 18:3n-6 showed similar inhibitory effects on [3H]yohimbine binding. Finally, competition binding studies performed in a partially purified TXA2/PGH2 receptor preparation also demonstrated inhibition of [3H]SQ29,548 binding by 20:5n-3 and 22:6n-3. Collectively, these findings support the notion that 20:5n-3 and 22:6n-3 can selectively and directly modulate TXA2/PGH2 receptor function, and that this mechanism of action may contribute to the antiplatelet activity associated with diets rich in these fatty acids.
我们之前证明，未酯化以及酯化的二十碳五烯酸（20:5n-3）和二十二碳六烯酸（22:6n-3）可抑制U46619诱导的血小板聚集以及[3H]U46619与洗涤后的人血小板的特异性结合。还证明这些脂肪酸的酯化导致[3H]U46619对血栓素A2/前列腺素H2（TXA2/PGH2）受体的亲和力降低。为了研究这种抑制的特异性，研究了20:5n-3和22:6n-3对血小板α2-肾上腺素能受体功能和结合的影响。发现20:5n-3和22:6n-3（未酯化或酯化）均未改变肾上腺素诱导的聚集或[3H]育亨宾特异性结合。此外，Scatchard分析表明，用20:5n-3或22:6n-3酯化不会改变[3H]育亨宾结合的解离常数。接下来使用CHAPS和洋地黄皂苷增溶的血小板膜评估20:5n-3和22:6n-3对TXA2/PGH2受体的调节作用。对于CHAPS和洋地黄皂苷增溶的膜，分别测得[3H]SQ29,548的解离常数为26.5 nM和20.8 nM。在这些增溶制剂中的竞争性结合实验表明，20:5n-3或22:6n-3以6-15 microM范围内的IC50值阻断[3H] SQ29,548结合，而这些脂肪酸浓度高达100 microM时对[3H]育亨宾结合无影响。另一方面，亚油酸（18:2n-6）和γ-亚麻酸（18:3n-6）抑制[3H] SQ29,548结合的IC50值在150 microM范围内。此外，18:2n-6和18:3n-6对[3H]育亨宾结合表现出相似的抑制作用。最后，在部分纯化的TXA2/PGH2受体制剂中进行的竞争结合研究也证明20:5n-3和22:6n-3可抑制[3H]SQ29,548结合。总的来说，这些发现支持以下观点，即20:5n-3和22:6n-3可以选择性地直接调节TXA2/PGH2受体功能，并且这种作用机制可能有助于富含这些脂肪酸的饮食相关的抗血小板活性。
