Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC227

Poster Communications

Reactive oxygen species (ROS) from cytochrome P450 2C9 mediate Ca2+ release from endothelial stores during endothelial derived hyperpolarising factor (EDHF) responses.

J. Chidgey1, P. I. Aaronson1, P. A. Fraser1

1. King's College London, London, United Kingdom.


EDHF is the endothelium-dependent but nitric oxide and prostacyclin-independent vasodilatation pathway and has been characterised as requiring KCa3.1 and KCa2.3 channel activation as well as functional myoendothelial gap junctions. H2O2 has also been implicated in this EDHF response, but its role has yet to be elucidated. To investigate this the cremaster muscle circulation of freshly killed Wistar rats was perfused with a Krebs buffer solution containing albumin (1 g.l-1) and Fura-PE3 AM (10µM) for 60 minutes, which led to selective loading of the Ca2+ indicator into the endothelium. The preparation was placed on the modified stage of an intravital microscope to measure vessel diameter, and endothelial [Ca2+]i was estimated from the 360/380nm excitation ratio, emission > 510nm, using an extended ISIS camera. The significance of changes was assessed by paired t test. The preparation was routinely superfused with phenylephrine (30µM), L-NAME (300 µM) and indomethacin (3µM). This caused a 36.8 ± 3.2%, n=54 (mean ± SEM) constriction of the maximum diameter. Relaxation was measured as the % decrease in constriction. Addition of carbachol (10µM) resulted in 74.5 ± 2.3%, n=65 relaxation and the 360/380 nm ratio increased by 25.6 ± 1.6%, n=69. The EDHF mediated relaxation and endothelial [Ca2+]i increase in response to carbachol was substantially reduced by including a ROS scavenging combination of superoxide dismutase and catalase (100U.ml-1 each; relaxation from 74.9 ± 8.9% to 37.5 ± 13.5%, ratio from 19.3 ± 3.3% to 5.3 ± 1.7%, n=6; p<0.05). The possibility that ROS are important for Ca2+ release from stores was examined by applying EGTA in Ca2+-free solutions. Carbachol application then resulted in transient Fura ratio increase that was reduced by SOD and catalase (from 20.5 ± 2.8% to 10.4 ± 1.5% n=5; p<0.01). The phospholipase C inhibitor U73122 (3µM) reduced the EDHF response to carbachol (relaxation 74.9 ± 2.8% to 10.6 ± 2.4%, ratio 22.9 ± 2.5% to 3.2 ± 0.6%, n=5; p<0.05). The KCa3.1 and KCa2.3 channel activator NS309 (10µM) caused a comparable relaxation to carbachol (65.6 ± 2.2%, n=5) that was not blocked by SOD and catalase (relaxation 64.3 ± 2.0%, n=5) suggesting the ROS work before this step in the pathway. The selective CYP 2C9 inhibitor sulfaphenazole (10µM) reduced relaxation (from 80.2±3.6% to 23.7 ± 2.6%, n=5; p<0.05) and the endothelial [Ca2+]i increase (from 23.1 ± 2.3% to 8.6 ± 1.2%, n=5; p<0.05). In the Ca2+-free preparation sulfaphenazole also inhibited the Ca2+ release from stores (ratio from 17.0 ± 2.0% to 7.7 ± 0.8%, n=5; p<0.05). These data suggest that ROS produced by arachidonic acid metabolism via CYP 2C9 promote EDHF mediated relaxation mainly by enhancing the release of Ca2+ from endothelial stores.

Where applicable, experiments conform with Society ethical requirements