Conditions required for the measurement of nitric oxide synthase activity in a myenteric plexus/smooth muscle preparation from the rat ileum.
Nitric oxide synthase (NOS) activity was measured, by the conversion of arginine to citrulline, in a preparation from the rat ileum consisting of the myenteric plexus and smooth muscle layers. A variety of incubating media were used in order to establish the optimal conditions required for the assay. NOS activity was present in the soluble fraction and was Ca(2+)- and calmodulin-dependent, characteristic of neuronal NOS. Exogenous Ca2+ was required for activity to be detectable but NOS activity progressively decreased with Ca2+ concentrations above 1.25 mM. Activity varied with arginine concentration, reaching saturation at 6 microM, and required the addition of the co-substrate NADPH. Endogenous levels of co-factors in the crude soluble fraction were not sufficient to maintain NOS activity. Omission of flavin adenine dinucleotide and tetrahydrobiopterin from the incubation medium reduced activity by 90%, and both co-factors had to be present for maximal activity to occur. These results emphasize the need to control assay conditions when measuring NOS activity in crude preparations from peripheral tissue.
Lincoln
J
J
Department of Anatomy and Developmental Biology, University College London, UK.
Messersmith
W A
WA
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007082:Ileum / Q000201:enzymology; D066298:In Vitro Techniques; D009130:Muscle, Smooth / Q000201:enzymology*; D009197:Myenteric Plexus / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000032:analysis* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D000975:Antioxidants; D002147:Calmodulin; D005182:Flavin-Adenine Dinucleotide; D001708:Biopterins; D002956:Citrulline; D009249:NADP; D005486:Flavin Mononucleotide; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin; D002118:Calcium