EXPRESSION OF A FUNCTIONAL NEUTROPHIL-TYPE NADPH OXIDASE IN CULTURED RAT CORONARY MICROVASCULAR ENDOTHELIAL-CELLS

Objectives: The production of reactive oxygen species (e.g., superoxid e) by endothelial cells is relevant to tissue injury during ischemia-r eperfusion, and may also play a role in intracellular signaling pathwa ys. However, the molecular identities of the enzymes responsible for e ndothelial superoxide production are poorly defined, although xanthine oxidase, NADH/NADPH oxidoreductases and nitric oxide synthase are amo ng proteins suggested to contribute. Recent studies suggest that an NA DH/NADPH oxidase similar to that found in neutrophils is an important source of superoxide in vascular smooth muscle. Methods: We investigat ed whether a phagocyte-type NADH/NADPH oxidase complex is present in r at cultured coronary microvascular endothelial cells. The expression o f NADPH oxidase components was studied by RT-PCR and Western blot anal yses, while functional activity was assessed by measurement of superox ide production by lucigenin-enhanced chemiluminescence. Results: The m ajor component of the pha,oocyte-type NADH/NADPH oxidase complex, a cy tochrome b558 heterodimer, was shown to be present both at mRNA and pr otein levels, using oligonucleotide primers designed from published ne utrophil and vascular smooth muscle sequences and anti-neutrophil anti bodies respectively. Functional activity of the enzyme was also confir med by NADPH-evoked superoxide production in cell homogenates, which w as inhibited either by the superoxide chelator Tiron or by diphenylene iodonium, an inhibitor of the oxidase. Conclusions: A functional phago cyte-type NADPH oxidase is expressed in coronary microvascular endothe lial cells. where it may contribute to the physiological and/or pathop hysiological effects of reactive oxygen species. These data, together with reports of the presence of a similar oxidase in other non-phagocy tic cell types, suggest that this enzyme complex is widely expressed i n many tissues where it may subserve signaling and other functions. (C ) 1998 Elsevier Science B.V.