Abstract
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) [Ca2+]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 [Ca2+]i increase by 74%. When applied in Ca2+-free external medium, carbachol caused a transient increase in EC [Ca2+]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 [Ca2+]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 Fe2+ chelators to block the formation of ∙OH. A cell permeant chelator was able to inhibit EC Ca2+ store release, but cell impermeant chelators reduced both the vasodilatation and EC Ca2+ 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 Ca2+ release.
Highlights
Endothelium-dependent hyperpolarization (EDH) plays an important role in the vasodilatation of small arteries and arterioles induced by diverse stimuli including locally released factors, blood flow, and low pH [1]
We speculate that arteries/arterioles may fall into three categories with regard to the involvement of H2O2 in EDH
As evidenced by the lack of effect of catalase, H2O2 is probably not involved in this response [4], such a role has not been exhaustively examined for every stimulus which triggers EDH in these preparations
Summary
Endothelium-dependent hyperpolarization (EDH) plays an important role in the vasodilatation of small arteries and arterioles induced by diverse stimuli including locally released factors, blood flow, and low pH [1]. EDH was initially viewed as resulting from the release of a diffusible endothelial factor, termed EDHF [2], but is thought to involve the transmission of endothelial cell hyperpolarization to the underlying vascular smooth muscle cells (VSMCs) via myoendothelial gap junctions [3]; the relative importance of each mechanism is thought to vary between different arteries/arterioles and depends on the degree of pre-existing vascular excitation [4].
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