Real time measurement of nitric oxide produced ex vivo by luminol-H2O2 chemiluminescence method.
Endothelium-derived relaxing factor, identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase. A newly developed aqua-chemiluminescence system was used to measure the amount of NO release from the isolated, perfused rat kidney concomitantly with the pressure changes (Kikuchi, K., Nagano, T., Hayakawa, H., Hirata, Y., and Hirobe, M. (1993) Anal. Chem. 65, 1794-1799). In normotensive rats, basal NO release was estimated to be 86 +/- 6 fmol min-1 (g of kidney weight)-1, and endothelium-dependent vasodilators increased NO release with a concomitant pressure reduction. Pretreatment of the kidney with detergents to damage the vascular endothelium diminished the responses of NO and perfusion pressure to acetylcholine, while infusion of authentic NO solution caused an increase in the chemiluminescence and a concomitant decrease in the pressure. The half-life of NO in O2-saturated Krebs-Henseleit buffer was determined as 6.41 s, and the decay constant (Kd) was 0.108 s-1. The measuring system involves a lag time of 15 s after passage of the solution through the kidney, so that the measured value is 13.0% of the true luminal NO concentration in the kidney, up to 400 pM. These observations enabled us to quantitate the degree to which NO released from the rat kidney changes perfusion pressure. Acetylcholine-induced vasodilation and NO release were markedly attenuated in deoxycorticosterone acetate-salt hypertensive rats, in which the endothelium is known to be markedly damaged. Thus, this assay system may make it possible to evaluate the extent of injury to the endothelium in conditions involving vascular damage.
Kikuchi
K
K
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Nagano
T
T
Hayakawa
H
H
Hirata
Y
Y
Hirobe
M
M
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D003900:Desoxycorticosterone; D006973:Hypertension / Q000503:physiopathology*; D007668:Kidney / Q000378:metabolism*; D008163:Luminescent Measurements; D008165:Luminol; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D009569:Nitric Oxide; D003900:Desoxycorticosterone; D008165:Luminol; D000109:Acetylcholine