SELECTIVE SEQUESTRATION OF NITRIC-OXIDE BY SUBCELLULAR COMPONENTS OF VASCULAR SMOOTH-MUSCLE AND PLATELETS - RELATIONSHIP TO NITRIC-OXIDE STIMULATION OF THE SOLUBLE GUANYLYL CYCLASE

Sequestration of nitric oxide (NO) by subcellular fractions isolated f rom bovine pulmonary arterial medial layer (BPA) and rabbit platelets (RP) was studied utilizing a novel chemiluminescence - headspace gas t echnique. Sequestration in all fractions was similarly rapid (5 min) a nd remained constant for at least 30 min. When incubated with 108 pmol of NO, the BPA mitochondrial, microsomal, and nuclear fractions seque stered 22.8 +/- 1.9, 20.5 +/- 2.2 and 15.2 +/- 3.6% of the NO, respec tively (n = 14). However, significantly more of the 108 pmol of NO, 36 .8 +/- 2.8 and 32.9 +/- 3.6%, respectively, was sequestered by the BPA homogenate (about 2 mg protein/ml) and BPA cytosolic fraction (about 1 mg protein/ml) (n = 19). Also, RP cytosolic fraction (about 3 mg pro tein/ml) sequestered a greater amount of NO than any BPA fraction when incubated with 108 pmol of NO (83.0 +/- 1.0%; n = 3). Analysis of the binding data obtained for the BPA homogenate and cytosolic fraction w as consistent with the existence of two binding sites, one site with a K-d of approximately 100 nM and another with a K-d of approximately 1 mu M. Both the BPA homogenate fraction and the cytosolic fraction as well as the RP cytosolic fraction were shown to have soluble guanylyl cyclase activity. The nitrovasodilator sodium nitroprusside (SNP) caus ed a concentration-dependent increase in the activity of this enzyme i n all these fractions. Maximum stimulations caused by 1 mM SNP in BPA homogenate fraction, BPA cytosolic fraction, and RP cytosolic fraction were equivalent to 2-, 4- and 3-fold increases in catalytic activity, respectively. No effect of SNP was observed in BPA mitochondrial, mic rosomal, or nuclear fraction. Prior incubation of BPA and RP cytosolic fractions with authentic NO significantly stimulated the soluble guan ylyl cyclase activity. In both the BPA and RP cytosolic fractions, max imal stimulation brought about by prior incubation with authentic NO w as equivalent to approximately 60% of that caused by 100 mu M SNP. Thu s, incubation of subcellular fractions from two nitrovasodilator-sensi tive tissues with authentic NO resulted in significant sequestration o f the free radical in these fractions and a concentration-dependent ac tivation of the soluble guanylyl cyclase. In conclusion, the chemilumi nescence - headspace gas technique is a suitable method for the study of NO sequestration in subcellular fractions of various tissues. Also, this study demonstrates that NO is sequestered preferentially by subc ellular fractions of BPA and RP that contain soluble guanylyl cyclase activity, and that the sequestration of NO in these fractions stimulat es the catalytic activity of this enzyme.