Reactive oxygen species facilitate the EDH response in arterioles by potentiating intracellular endothelial Ca(2+) release

There is abundant evidence that H(2)O(2) can act as an endothelium-derived hyperpolarizing factor in the resistance vasculature. However, whilst scavenging H(2)O(2) 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 H(2)O(2). We have examined the role of H(2)O(2) in EDH-dependent vasodilatation by simultaneously measuring vascular diameter and changes in endothelial cell (EC) [Ca(2+)](i) during the application of H(2)O(2) or carbachol, which triggers EDH. Carbachol (10 µM) induced dilatation of phenylephrine-preconstricted rat cremaster arterioles was largely (73%) preserved in the presence of indomethacin (3 µM) and l-NAME (300 µM). This residual NO- and prostacyclin-independent dilatation was reduced by 89% upon addition of apamin (0.5 µM) and TRAM-34 (10 µM), and by 74% when an extracellular ROS scavenging mixture of SOD and catalase (S&C; 100 U ml(−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 1 µM H(2)O(2) was present in the bath. H(2)O(2) -induced dilatation, which occurred only at concentrations ≥100 µM, 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 H(2)O(2), but not by carbachol. Inhibiting PLC, PLA(2) or CYP450 2C9 each greatly reduced the carbachol-induced increase in EC [Ca(2+)](i) and vasodilatation, but adding 10 µM H(2)O(2) during PLA(2) 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, H(2)O(2) promotes EDH by acting within EC to enhance Ca(2+) release.