Studies of nitric oxide over the past two decades have highlighted the fundamental importance of gaseous signaling molecules in biology and medicine. The physiological role of other gases such as carbon monoxide and hydrogen sulfide (H 2 S) is now receiving increasing attention. Here we show that H 2 S is physiologically generated by cystathionine γ-lyase (CSE) and that genetic deletion of this enzyme in mice markedly reduces H 2 S levels in the serum, heart, aorta, and other tissues. Mutant mice lacking CSE display pronounced hypertension and diminished endothelium-dependent vasorelaxation. CSE is physiologically activated by calcium-calmodulin, which is a mechanism for H 2 S formation in response to vascular activation. These findings provide direct evidence that H 2 S is a physiologic vasodilator and regulator of blood pressure.Nitric oxide (NO) and carbon monoxide (CO) are established physiologic messenger molecules, and NO has an important role as an endothelial cell-derived relaxing factor (EDRF) and regulator of blood pressure (1,2). Indirect evidence has implicated another endogenous gasotransmitter, hydrogen sulfide (H 2 S), in similar functions (3-7). H 2 S can be produced by cystathionine γ-lyase (CSE) or cystathionine β-synthase (CBS) (3,4), but definitive evidence for either of these enzymes in the physiologic formation of H 2 S is lacking.To investigate the role of H 2 S as a physiologic vasorelaxant and determinant of blood pressure, we generated mice with a targeted deletion of the gene encoding CSE (8) (fig. S1, A to C). The homozygous (CSE −/− ) and heterozygous (CSE −/+ ) mutant mice were viable, fertile, and indistinguishable from their control wild-type littermates (CSE +/+ ) in terms of growth pattern.
Nitric oxide mediates vascular relaxing effects of endothelial cells, cytotoxic actions of macrophages and neutrophils, and influences of excitatory amino acids on cerebellar cyclic GMP. Its enzymatic formation from arginine by a soluble enzyme associated with stoichiometric production of citrulline requires NADPH and Ca2 . We show that nitric oxide synthetase activity requires calmodulin. Utilizing a 2',5'-ADP affinity column eluted with NADPH, we have purified nitric oxide synthetase 6000-fold to homogeneity from rat cerebellum. The purified enzyme migrates as a single 150-kDa band on SDS/PAGE, and the native enzyme appears to be a monomer.Endothelium-derived relaxing factor, a labile substance formed by endothelial cells, which mediates vasodilation, has been shown to be identical to nitric oxide (NO) (1-3). In addition to relaxing blood vessels, NO has multiple messenger functions as it has been demonstrated in macrophages (4) and in brain tissue (5-7). NO appears responsible for the cytotoxic effects of macrophages and neutrophils (8). We have obtained direct evidence for NO as a messenger for the influences of the excitatory amino acid glutamate on cGMP in the cerebellum (7). We showed a striking enhancement by glutamate and other excitatory amino acids of the conversion of arginine to NO and the associated formation of citrulline. Moreover we observed that NV-monomethyl-L-arginine (MeArg), an inhibitor ofthe enzymatic conversion of arginine to NO, inhibits glutamate-elicited cGMP formation, an influence selectively reversed by excess arginine.Evidence that NO mediates functions of tissues as diverse as the brain, endothelium, and blood cells suggests a widespread role for NO as a messenger molecule. Localizing NO formation at a cellular level throughout the body would be greatly facilitated by immunohistochemical identification of NO synthetase, the NO-forming enzyme. The mechanism of conversion of arginine to NO, presently unclear (9) 220C, assays were terminated with 2 ml of 20 mM Hepes, pH 5.5/2 mM EDTA, and were applied to 1-ml columns of Dowex AG50WX-8 (Na+ form), which were eluted with 2 ml of water.[3H]Citrulline was quantified by liquid scintillation spectroscopy of the 4-ml flow-through. Purification of NO Synthetase. Eighteen rat cerebella were homogenized in 100 ml of ice-cold buffer A [50 mM Tris HCl, pH 7.4/1 mM EDTA/antipain (10 mg/liter)/leupeptin (10 mg/liter)/soybean trypsin inhibitor (10 mg/liter)/pepstatin (10 mg/liter)/chymostatin (10 mg/liter)/phenylmethylsulfonyl fluoride (100 mg/liter)], and all subsequent procedures were carried out at 4°C. The homogenate was centrifuged at 20,000 x g for 15 min, and the supernatant was loaded at 2 ml/min onto a 20-ml column of diethylaminoethyl (DEAE) equilibrated with buffer A. The column was washed with 50 ml of buffer A and eluted with a 100-ml linear gradient of 0-400 mM NaCl in buffer A. Fractions (2.5 ml) were assayed for enzyme activity. Fractions containing the first peak of activity from the DEAE column were pooled and added to ...
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