Abstract
Nitric oxide (NO) is synthesized by a number of cells from a guanidino nitrogen atom of L-arginine by the action of either constitutive or inducible NO synthases, both of which form citrulline as a co-product. We have determined the source of the oxygen in both NO and in citrulline formed by the constitutive NO synthase from the vascular endothelium and brain and by the inducible NO synthase from the murine macrophage cell line J774. All these enzymes incorporate molecular oxygen both into NO and into citrulline. Furthermore, activated J774 cells form NO from omega-hydroxyl-L-arginine, confirming the proposal that this compound is an intermediate in the biosynthesis of NO.
Highlights
The demonstration of the synthesis of nitric oxide (NO)' from a guanidino nitrogen atom of L-arginine by vascular endothelial cells [1]and activated macrophages [2] led to the identification of the L-arginine/NO pathway in many other charide and interferon-y andby vascular endothelial cells and brain cytosol in the presence of lSOzor H2lsO.we have attempted to elucidate the mechanism of NO synthesis by both constitutive andinducible NO synthases
The use of plasma spray mass spectrometry allowed the determination of the pseudomolecular ion of citrulline with high sensitivity and specificity.Nitric oxide formation by cells was analyzed as its reaction product with morpholine, nitrosomorpholine, by GCMS since this method allowed the accurate identification of nitrosomorpholine at a specific retention time relative to the internalstandard
The murine monocytic leukemia cell line 5774, in which the Ca2+-independent NO synthase had been induced by lipopolysaccharide and interferon-y, synthesized NO and citrulline from L-arginine
Summary
Morpholine in an atmosphere of air resulted in the formation of a product (92.3 & 3.0% l60, n = 4) with an m / z 117 at the relative retentiontime ( t R ) of authentic nitrosomorpholine (Fig. M ) .This was changed by 2 atomic mass units to m/z. When 5774 cells were incubatedin H2180 or H2160 the only product observed had anm/z 117, and thiswas >92% enriched in I 6 0 equivalent volumes of dichloromethane prior to the final dry down in either case. The formation of this product was inhibited by and analysis.
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