Calmodulin-dependent Nitric Oxide Synthase Research Articles

Bradykinin Stimulates Endothelial Nitric Oxide(NO) Production by Calcium/Calmodulin-Dependent NO Synthase.

The cellular mechanism by which bradykinin induces nitric oxide (NO) formation was studied in cultured bovine endothelial cells. The basal release of nitrate/nitrite (NOx) from endothelial cells increased linearly during one-min incubation and reached plateau levels thereafter. Bradykinin dose-dependently and similarly stimulated NOx release and cyclic GMP production, both of which were abolished by NG-monomethyl L-arginine. D-Arg-[Hyp3, Thio5′8, D-Phe7]-bradykinin (B2-antagonist), but not Leu8-des Arg9-bradykinin (B1-antagonist), inhibited bradykinin-induced production of both NOx and cyclic GMP. Removal of extracellular Ca2+ by EGTA failed to affect bradykinin-induced NOx and cyclic GMP production, whereas intracellular Ca2+ release inhibitor (TMB-8) completely abolished NOx and cyclic GMP production stimulated by bradykinin. A relatively selective calmodulin inhibitor (W-7) blocked bradykinin-induced production of NOx and cyclic GMP. Our data suggest that bradykinin stimulates B2 receptor-mediated NO formation via activation of Ca2+/calmodulin-dependent constitutive NO synthase, and that NO may function as an autocrine/paracrine factor in endothelial cells. (Hypertens Res 1993; 16: 131-137)

Identification of inducible calmodulin-dependent nitric oxide synthase in the liver of rats.

A calmodulin-dependent nitric oxide synthase was significantly induced in the liver of rats treated intravenously with heat-killed Propionibacterium acnes and 5 days later with Escherichia coli lipopolysaccharide. The apparent calmodulin-dependent and -independent isozymes were separated by Mono Q column chromatography after their partial purification by 2',5'-ADP-agarose affinity chromatography. Both enzymes had a molecular weight of 125,000 as determined by SDS-polyacrylamide gel electrophoresis and required NADPH, tetrahydrobiopterin, and dithiothreitol as cofactors. Their activities were completely inhibited by the specific nitric oxide synthase inhibitors NG-monomethyl-L-arginine and N omega-nitro-L-arginine at 80 and 800 microM, respectively. The peptide maps of these two isozymes with lysylendopeptidase and their reverse-phase column chromatographic profiles were indistinguishable. In the presence of bovine calmodulin, the purified calmodulin-dependent isozyme behaved as a calmodulin-independent isozyme on Mono Q column chromatography. The purified calmodulin-independent isozyme was converted to a calmodulin-dependent isozyme by EDTA and EGTA. Calmodulin blot analysis using 125I-calmodulin showed that the two isozymes bound calmodulin equally efficiently.

Induction of Ca 2+/calmodulin-dependent NO synthase in various organs of rats by Propionibacterium acnes and lipopolysaccharide treatment

Ca 2+/calmodulin-dependent nitric oxide synthase was found to be induced during rat liver necrosis caused by administration of Propionibacterium acnes and E. coli lipopolysaccharide to rats. Examination of the specific induction of C a2+/calmodulin-dependent NO synthase showed that the enzyme was induced in the lung, spleen and colon as well as the liver. Northern blot analysis revealed that the induction occurred at the transcriptional level.

Identification and characterization of a calmodulin-dependent nitric oxide synthase from GH3 pituitary cells.

A nitric oxide synthase activity stimulated more than 30-fold by the concurrent presence of Ca2+ and calmodulin (CaM), and inhibited by trifluoperazine (50 microM), has been identified in extracts of GH3 pituitary cells. The CaM-dependent nitric oxide synthase of the crude extract was stimulated more than 9-fold by (6R)-5,6,7,8-tetrahydro-L-biopterin with half-maximal stimulation occurring at a concentration of 300 nM. Fractionation of the extract on DEAE-cellulose enhanced nitric oxide synthase specific activity up to 300-fold and provided a preparation which on Western blot analysis possessed a 152 kDa protein which cross-reacted with antibodies to homogeneous bovine brain nitric oxide synthase. The DEAE-cellulose-purified enzyme exhibited apparent Km values of 4.3 microM, 0.4 microM, 0.3 microM and 4 nM for L-arginine, NADPH, Ca2+ and CaM respectively. The CaM-dependent nitric oxide synthase of GH3 extract bound to 2',5'-ADP-agarose and was eluted by NADPH with a 500-fold increased specific activity. Citrulline formation by the ADP-agarose-purified enzyme was inhibited by NG-nitro-L-arginine, NG-methyl-L-arginine and Nitro Blue Tetrazolium with apparent Ki values of 0.2, 1.8 and 7 microM respectively. The ADP-agarose-purified enzyme displayed cytochrome c reductase activity which was stimulated more than 18-fold by the concurrent presence of Ca2+ and CaM and inhibited by trifluoperazine. NG-Nitro-L-arginine and NG-methyl-L-arginine did not inhibit the cytochrome c reductase activity.

Clinical relevance of endothelium‐derived relaxing factor (EDRF).

1. In addition to metabolic and neurohumoral factors endothelium-derived autacoids like the nitric oxide radical NO and prostacyclin are effective regulators of vascular tone and thus tissue perfusion. NO is produced in endothelial cells from L-arginine by a Ca2+/calmodulin-dependent enzyme NO synthase. In addition, the NO radical is ultimately cleaved from all nitrovasodilators and resembles their vasoactive and antiaggregatory principle, which is used under pathological conditions as substitution therapy for impaired endothelial function and autacoid production. Impaired endothelium-dependent vasomotor control has been documented in hypercholesterolaemia, atheromatosis, diabetes, hypertension, and in reperfusion damage. L-arginine supplementation is effective in a few instances.

Purification of a Ca 2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum : Cofactor-role of tetrahydrobiopterin

L-Arginine-derived nitric oxide acts as an inter- and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine-inducible enzyme in macrophages and a constitutive, Ca 2+-regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'-ADP-Sepharose. The enzyme was identified as an NO synthase with a specific NO-chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO-sensitive detection system. The purified NO synthase was, besides Ca 2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.