Reactive oxygen species facilitate the EDH response in arterioles by potentiating intracellular endothelial Ca(2+) release.
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.
King's College London, Faculty of Life Sciences and Medicine, Division of Asthma, Allergy & Lung Biology, London, United Kingdom.
James
J
Chidgey
King's College London, Faculty of Life Sciences and Medicine, Cardiovascular Division, London, United Kingdom.
Paul A
PA
Fraser
King's College London, Faculty of Life Sciences and Medicine, Division of Asthma, Allergy & Lung Biology, London, United Kingdom. Electronic address: philip.aaronson@kcl.ac.uk.
Philip I
PI
Aaronson
D000818:Animals; D001160:Arterioles / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D001685:Biological Factors / Q000378:metabolism*; D020013:Calcium Signaling; D002217:Carbachol / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008297:Male; D008564:Membrane Potentials; D018721:Muscarinic Agonists / Q000494:pharmacology; D017208:Rats, Wistar; D014664:Vasodilation
D001685:Biological Factors; D018721:Muscarinic Agonists; C067410:endothelium-dependent hyperpolarization factor; D002217:Carbachol; D006861:Hydrogen Peroxide
Arteriole; Ca(2+); Endothelium dependent hyperpolarization; H(2)O(2); Reactive oxygen species; Vasodilation