The role of reactive oxygen species in the endothelium derived hyperpolarising factor response

EDHF is the endothelium-dependent but nitric oxide (NO) and prostacyclin (PGI2) -independent vasodilatation pathway that is the dominant relaxation mechanism in the microcirculation, and has been shown to require IKCa and SKCa channel activation and also functional myoendothelial gap junctions. Reactive oxygen species (ROS) have been implicated in the response, but their precise role remains uncertain. To investigate this, the cremaster muscle circulation of freshly killed rats was perfused with a Krebs buffer solution containing albumin (1 g.l-1) and either FITC-albumin (2 g.l-1) to measure changes in diameter, or Fura-PE3AM (10 μM) for 60 minutes to selectively load the endothelium, allowing endothelial [Ca2+]i to be recorded. The preparation was placed on the stage of an intravital microscope to measure vessel diameter, and endothelial [Ca2+]i was estimated from the 360/380 nm excitation ratio, emission at > 510 nm. The preparation was superfused with phenylephrine (30 μM), L-NAME (300 μM) and indomethacin (3 μM) to evoke arteriolar constriction while preventing the synthesis of NO and PGI2. Carbachol (10 μM) caused a 74.5 ± 2.3% (n = 65) relaxation and the 360/380 nm ratio increased by 25.6 ± 1.6% (n = 69). Both responses were substantially reduced by a ROS scavenging combination of superoxide dismutase and catalase (100 U.ml-1 each). The possibility that ROS are important for Ca2+ release from stores was examined by applying carbachol in Ca2+-free solution containing EGTA. Carbachol application then resulted in a transient [Ca2+]i increase that was reduced by SOD and catalase. The CYP 2C9 inhibitor sulfaphenazole (10 μM) reduced relaxation and the endothelial [Ca2+]i increase, as did the phospholipase C blocker U73122. These and other data to be presented suggest that ROS produced by arachidonic acid metabolism via CYP 2C9 promote EDHF mediated relaxation mainly by enhancing the inositol trisphosphate-mediated release of Ca2+ from endothelial stores.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2013

Author dc:creator

• Chidgey, James

Advisors dc:contributor.advisor

• Aaronson, Philip Irving
• Fraser, Paul Anthony

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/fbc0bac4-9a82-4156-bf12-64e705df631f

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/fbc0bac4-9a82-4156-bf12-64e705df631f