Proceedings of The Physiological Society

University of Manchester (2010) Proc Physiol Soc 19, PC184

Poster Communications

Reactive oxygen species (ROS) from cytochrome P450 mediate the endothelial [Ca2+]i increase during endothelial derived hyperpolarising factor (EDHF) responses.

J. G. Chidgey1,2, P. I. Aaronson1, P. A. Fraser2

1. Asthma, Allergy and Lung Biology Division, King's College London, London, United Kingdom. 2. Cardiovascular Division, King's College London, London, United Kingdom.


The nitric oxide and prostacyclin-independent vasodilatation following carbachol administration 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. The cremaster muscle circulation of freshly killed Wistar 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 with Fura-PE3 AM (10 µM) 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 360/380 nm excitation ratio, emission > 510 nm, using an extended ISIS camera. The significance of changes was assessed by paired t test. The preparation was routinely superfused with phenylephrine (30 µM), L-NAME (300 µM) and indomethacin (3 µM). This caused a 31.1% ± 2.1, n = 44 (mean ± se) constriction of the maximum diameter. Relaxation was measured as the % decrease in constriction. Addition of carbachol (10 µM) resulted in 72.6% ± 2.4, n = 55 relaxation and the 360/380 nm ratio increased by 24.7% ± 1.5, n = 62. Apamin (500 µM) and TRAM-34 (10 µM) resulted in a reduced relaxation (from 79.4% ± 1.0 to 14.7% ± 1.7, n = 9; p < 0.001) showing that EDHF is the dominant relaxation pathway in the cremaster microcirculation. 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 (100 U.ml-1 each; relaxation from 74.4% ± 10.9 to 42.3% ± 15.4, ratio from 19.1% ± 3.5 to 5.5% ± 2.1, n = 6; p < 0.05). The cytochrome P450 inhibitors 6-(2-propargyloxyphenyl) hexanoic acid (10 µM) and 17-octadecynoic acid (50 µM) reduced relaxation (from 65.3% ± 7.7 to 18.8% ± 5.5 and from 62.9% ± 8.8 to 21.9% ± 3.2, n = 4; p <0 .01) and the endothelial [Ca2+]i increase (from 22.2% ± 2.2 to 9.1% ± 1.0 and from 21.5% ± 2.7 to 8.1% ± 2.1, n = 4; p < 0.01), as did the selective cytosolic phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone (3 µM; relaxation from 68.7% ± 3.7 to 17.1% ± 4.4, ratio from 27.6% ± 6.0 to 5.9% ± 0.4, n = 5; p < 0.05) (1). The epoxyeicosatrienoic acids antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 µM) did not significantly affect the EDHF response (relaxation from 73.0% ± 7.1 to 72.7% ± 6.1, ratio from 24.% ± 4.1 to 21.7% ± 3.2, n = 4; p > 0.05) (2). These data suggest that ROS produced by arachidonic acid metabolism via cytochrome P450 play an important role in EDHF mediated relaxation by increasing endothelial [Ca2+]i.

Where applicable, experiments conform with Society ethical requirements