Sequestration of nitric oxide (NO) by subcellular fractions isolated from bovine pulmonary arterial medial layer (BPA) and rabbit platelets (RP) was studied utilizing a novel chemiluminescence – headspace gas technique. Sequestration in all fractions was similarly rapid (5 min) and remained constant for at least 30 min. When incubated with 108 pmol of NO, the BPA mitochondrial, microsomal, and nuclear fractions sequestered 22.8 ± 1.9, 20.5 ± 2.2 and 15.2 ± 3.6% of the NO, respectively (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 protein/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 was consistent with the existence of two binding sites, one site with a Kd of approximately 100 nM and another with a Kd of approximately 1 μM. 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) caused a concentration-dependent increase in the activity of this enzyme in 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, microsomal, or nuclear fraction. Prior incubation of BPA and RP cytosolic fractions with authentic NO significantly stimulated the soluble guanyiyl cyclase activity. In both the BPA and RP cytosolic fractions, maximal stimulation brought about by prior incubation with authentic NO was equivalent to approximately 60% of that caused by 100 μM SNP. Thus, incubation of subcellular fractions from two nitrovasodilator-sensitive tissues with authentic NO resulted in significant sequestration of the free radical in these fractions and a concentration-dependent activation of the soluble guanylyl cyclase. In conclusion, the chemiluminescence – 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 subcellular fractions of BPA and RP that contain soluble guanylyl cyclase activity, and that the sequestration of NO in these fractions stimulates the catalytic activity of this enzyme.Key words: nitric oxide, vascular smooth muscle, platelets, soluble guanylyl cyclase.
Fresh look on old substances – new class of NO-independent regulators of soluble guanylyl cyclase activity and function
