PT - JOURNAL ARTICLE AU - E A Kowaluk AU - P Seth AU - H L Fung TI - Metabolic activation of sodium nitroprusside to nitric oxide in vascular smooth muscle. DP - 1992 Sep 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 916--922 VI - 262 IP - 3 4099 - http://jpet.aspetjournals.org/content/262/3/916.short 4100 - http://jpet.aspetjournals.org/content/262/3/916.full SO - J Pharmacol Exp Ther1992 Sep 01; 262 AB - Sodium nitroprusside (SNP) is thought to exert its vasodilating activity, at least in part, by vascular activation to nitric oxide (NO), but the activation mechanism has not been delineated. This study has examined the potential for vascular metabolism of SNP to NO in bovine coronary arterial smooth muscle subcellular fractions using a sensitive and specific redox-chemiluminescence assay for NO. SNP was readily metabolized to NO in subcellular fractions, and the dominant site of metabolism appeared to be located in the membrane fractions. NO-generating activity was significantly enhanced by, but did not absolutely require, the addition of a NADPH-regenerating system, NADPH per se, NADH or cysteine. A correlation analysis of NO-generating activity (in the presence of a NADPH-regenerating system) with marker enzyme activities indicated that the SNP-directed NO-generating activity was primarily membrane-associated. Radiation inactivation target-size analysis revealed that the microsomal SNP-directed NO-generating activity was relatively insensitive to inactivation by radiation exposure, suggesting that the functioning catalytic unit might be quite small. A molecular weight of 5 to 11 kDa was estimated. NO-generating activity could be solubilized from the crude microsomes with 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulfonate, and the solubilized extract was subjected to gel filtration chromatography. NO-generating activity was eluted in two peaks: one peak corresponding to an approximate molecular weight of 4 kDa, thus confirming the existence of a small molecular weight NO-generating activity, and a second activity peak corresponding to a molecular weight of 112 to 169 kDa, the functional significance of which is unclear at present.(ABSTRACT TRUNCATED AT 250 WORDS)