Chidgey James, Fraser Paul A, Aaronson Philip I
King's College London, Faculty of Life Sciences and Medicine, Division of Asthma, Allergy & Lung Biology, London, United Kingdom.
King's College London, Faculty of Life Sciences and Medicine, Cardiovascular Division, London, United Kingdom.
Free Radic Biol Med. 2016 Aug;97:274-284. doi: 10.1016/j.freeradbiomed.2016.06.010. Epub 2016 Jun 16.
There is abundant evidence that H2O2 can act as an endothelium-derived hyperpolarizing factor in the resistance vasculature. However, whilst scavenging H2O2 can abolish endothelial dependent hyperpolarization (EDH) and the associated vascular relaxation in some arteries, EDH-dependent vasorelaxation can often be mimicked only by using relatively high concentrations of H2O2. We have examined the role of H2O2 in EDH-dependent vasodilatation by simultaneously measuring vascular diameter and changes in endothelial cell (EC) [Ca(2+)]i during the application of H2O2 or carbachol, which triggers EDH. Carbachol (10µM) induced dilatation of phenylephrine-preconstricted rat cremaster arterioles was largely (73%) preserved in the presence of indomethacin (3µM) and l-NAME (300µM). This residual NO- and prostacyclin-independent dilatation was reduced by 89% upon addition of apamin (0.5µM) and TRAM-34 (10µM), and by 74% when an extracellular ROS scavenging mixture of SOD and catalase (S&C; 100Uml(-1) each) was present. S&C also reduced the carbachol-induced EC [Ca(2+)]i increase by 74%. When applied in Ca(2+)-free external medium, carbachol caused a transient increase in EC [Ca(2+)]i. This was reduced by catalase, and was enhanced when 1µM H2O2 was present in the bath. H2O2 -induced dilatation, which occurred only at concentrations ≥100µM, was reduced by a blocking antibody to TRPM2, which had no effect on carbachol-induced responses. Similarly, iberotoxin and Rp-8bromo cGMP reduced the vasodilatation induced by H2O2, but not by carbachol. Inhibiting PLC, PLA2 or CYP450 2C9 each greatly reduced the carbachol-induced increase in EC [Ca(2+)]i and vasodilatation, but adding 10µM H2O2 during PLA2 or CYP450 2C9 inhibition completely restored both responses. The nature of the effective ROS species was investigated by using Fe(2+) chelators to block the formation of ∙OH. A cell permeant chelator was able to inhibit EC Ca(2+) store release, but cell impermeant chelators reduced both the vasodilatation and EC Ca(2+) influx, implying that ∙OH is required for these responses. The results indicate that rather than mediating EDH by acting directly on smooth muscle, H2O2 promotes EDH by acting within EC to enhance Ca(2+) release.
有充分证据表明，H2O2可作为阻力血管中内皮源性超极化因子。然而，虽然清除H2O2可消除某些动脉中的内皮依赖性超极化（EDH）及相关血管舒张，但EDH依赖性血管舒张通常仅在使用相对高浓度的H2O2时才能模拟。我们通过在应用H2O2或卡巴胆碱（触发EDH）期间同时测量血管直径和内皮细胞（EC）[Ca(2+)]i的变化，研究了H2O2在EDH依赖性血管舒张中的作用。在吲哚美辛（3µM）和L-NAME（300µM）存在的情况下，卡巴胆碱（10µM）诱导的苯肾上腺素预收缩大鼠提睾肌小动脉舒张在很大程度上（73%）得以保留。加入蜂毒明肽（0.5µM）和TRAM-34（10µM）后，这种不依赖一氧化氮和前列环素的残余舒张减少了89%，而当存在超氧化物歧化酶和过氧化氢酶的细胞外活性氧清除混合物（S&C；各100Uml(-1)）时，减少了74%。S&C还使卡巴胆碱诱导的EC [Ca(2+)]i增加减少了74%。当在无钙的细胞外培养基中应用时，卡巴胆碱导致EC [Ca(2+)]i短暂增加。这被过氧化氢酶减少，而当浴液中存在1µM H2O2时则增强。仅在浓度≥100µM时发生的H2O2诱导的舒张被TRPM2阻断抗体减少，该抗体对卡巴胆碱诱导的反应无影响。同样，iberotoxin和Rp-8溴环鸟苷酸减少了H2O2诱导的血管舒张，但不影响卡巴胆碱诱导的反应。抑制磷脂酶C、磷脂酶A2或细胞色素P450 2C9均大大减少了卡巴胆碱诱导的EC [Ca(2+)]i增加和血管舒张，但在抑制磷脂酶A2或细胞色素P450 2C9期间加入10µM H2O2可完全恢复这两种反应。通过使用亚铁螯合剂阻断∙OH的形成来研究有效活性氧物质的性质。一种细胞渗透性螯合剂能够抑制EC钙库释放，但细胞非渗透性螯合剂减少了血管舒张和EC钙内流，这意味着这些反应需要∙OH。结果表明，H2O2不是通过直接作用于平滑肌来介导EDH，而是通过在EC内起作用以增强钙释放来促进EDH。
