AJP - Lung Cellular and Molecular Physiology, Vol 267, Issue 6 815-L822, Copyright © 1994 by American Physiological Society
Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle
K. M. Mohazzab and M. S. Wolin
Department of Physiology, New York Medical College, Valhalla 10595.
Sources of superoxide anion (O2-.) production in calf pulmonary artery
smooth muscle homogenate and subcellular fractions were examined in this
study by measurement of the chemiluminescence produced by the reaction of
O2-. with 50 microM lucigenin, because recent evidence suggests that
endogenously produced reactive O2 species appear to mediate certain
vascular responses. In the homogenate fraction, an NADH (0.1 mM)-dependent
oxidoreductase activity was the major detected source of chemiluminescence.
NADPH (0.1 mM) produced only 3% of the O2-. observed with NADH.
Quantitation of certain other potential sources of O2-. (under optimized
conditions), including xanthine oxidase (0.1 mM hypoxanthine), mitochondria
(5 mM succinate + 30 microM antimycin), cyclooxygenase/lipoxygenase (1
microM arachidonic acid + 0.1 mM NADPH), or autooxidation (0.1 mg/ml
superoxide dismutase), resulted in the detection of minimal amounts (<
3% of NADH) of chemiluminescence. Estimation of mitochondrial O2-.
production from tissue respiration rates suggests that lucigenin is a poor
detector of intramitochondrial O2-.. These observations were confirmed by
examination of chemiluminescence produced by subcellular fractions, where
the major activity detected was an NADH oxidoreductase, which fractionated
in a manner closely matching the activity of the microsomal marker enzyme
rotenone-insensitive NADH-cytochrome c reductase. Because this NADH
oxidoreductase appears to be a major vascular smooth muscle-derived source
of O2-. production, this system has the potential to be an important
endogenous source for the generation of vasoactive reactive O2 species.
