Endothelial nitric oxide generating enzyme(s) in the bovine aorta: subcellular location and metabolic characterization

Abstract

The metabolic production of nitric oxide (NO) from bovine endothelial homogenates and subcellular fractions was examined. NO was quantified using a sensitive and specific ozone redox-chemiluminescence detector. Endogenously produced NO was detected in the headspace gas of bovine vascular endothelial homogenates supplemented with superoxide dismutase and an NADPH regenerating system, which had been incubated at 0 degrees C for 3 hr. An identical system incubated at 37 degrees C for 3 hr did not produce NO. Both superoxide dismutase and an NADPH regenerating system were required for observing endogenous NO production from endothelial homogenates. Among the various endothelial subcellular fractions tested, only the 1000 x g supernatant manifested significant generation of endogenous NO. Addition of 1.4 mM L-arginine (but not D-arginine, L-citrulline or L-lysine) resulted in significant enhancement of NO production from the 15,500 and 210,000 x g pellet fractions. L-arginine did not stimulate NO production from either homogenates or subcellular fractions of coronary vascular smooth muscle cells. Exogenously added calcium or magnesium was not required for NO generation in the 210,000 x g pellet stimulated with L-arginine, whereas L-canavanine (1.4 mM) and SKF 525-A (0.1 mM) significantly inhibited NO production with this preparation. Analysis of the enzyme marker data from various subcellular fractions suggests that the endothelial NO-generating enzyme system is membrane-bound and might be associated with the plasma membrane.