Shah C M, Locke I C, Chowdrey H S, Gordge M P
Department of Biomedical Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, U.K.
Biochem Soc Trans. 2003 Dec;31(Pt 6):1450-2. doi: 10.1042/bst0311450.
RSNOs (S-nitrosothiols) regulate platelet and megakaryocyte function, and may act in vivo as a nitric oxide reservoir. There is a discrepancy between the spontaneous rate of NO release from different RSNO compounds and their pharmacological effects, implying that target cells may mediate biological activity either by metabolism of RSNOs or by displaying cell surface receptors. In the present study, we sought evidence for rapid cell-mediated metabolism of RSNOs. Exposure of platelets to GSNO (S-nitrosoglutathione) for as little as 5 s inhibited thrombin-induced platelet aggregation by >95%; however, AlbSNO (S-nitrosoalbumin) was much less effective over these short time periods. Incubation of 1 microM GSNO or AlbSNO with platelets and megakaryocytes resulted in a 25-34% loss of RSNO recoverable from the supernatant (P <0.02) within 30 s. This rapid cell-mediated RSNO decay did not progress further over 5 min, and could not be accounted for by release of free NO. The gamma-glutamyl transpeptidase inhibitor acivicin (100 microM) partially decreased GSNO decay, whereas the membrane-impermeable thiol-blocking agent 5,5'-dithiobis-(2-nitrobenzoic acid) (100 microM) completely blocked cell-mediated GSNO decay and partially blocked AlbSNO decay. Our results highlight differences between high- and low-molecular-mass RSNOs with regard to their rapid metabolism/uptake and subsequent cellular responses, and indicate a critical role for extracellular thiols in RSNO metabolism by platelets and megakaryocytes.
S-亚硝基硫醇（RSNOs）调节血小板和巨核细胞功能，并且可能在体内作为一氧化氮的储存库发挥作用。不同RSNO化合物的一氧化氮自发释放速率与其药理作用之间存在差异，这意味着靶细胞可能通过RSNOs的代谢或通过展示细胞表面受体来介导生物活性。在本研究中，我们寻找RSNOs细胞介导的快速代谢的证据。血小板暴露于GSNO（S-亚硝基谷胱甘肽）仅5秒就可抑制凝血酶诱导的血小板聚集达95%以上；然而，在这些短时间内，AlbSNO（S-亚硝基白蛋白）的效果要差得多。将1微摩尔的GSNO或AlbSNO与血小板和巨核细胞一起孵育，导致30秒内上清液中可回收的RSNO损失25%-34%（P<0.02）。这种快速的细胞介导的RSNO衰变在5分钟内没有进一步进展，并且不能用游离一氧化氮的释放来解释。γ-谷氨酰转肽酶抑制剂阿西维辛（100微摩尔）部分降低了GSNO的衰变，而膜不可渗透的硫醇阻断剂5,5'-二硫代双（2-硝基苯甲酸）（100微摩尔）完全阻断了细胞介导的GSNO衰变，并部分阻断了AlbSNO的衰变。我们的结果突出了高分子量和低分子量RSNOs在快速代谢/摄取及其随后的细胞反应方面的差异，并表明细胞外硫醇在血小板和巨核细胞对RSNO的代谢中起关键作用。
