CHARACTERIZATION OF THE STABLE L-ARGININE-DERIVED RELAXING FACTOR RELEASED FROM CYTOKINE-STIMULATED VASCULAR SMOOTH-MUSCLE CELLS AS AN N-G-HYDROXY-L-ARGININE-NITRIC OXIDE ADDUCT

The nature of an L-arginine-derived relaxing factor released from vascular smooth muscle cells cultured on microcarrier beads and stimulated for 20 h with interleukin la was investigated. Unlike the unstable relaxation elicited by authentic nitric oxide (NO) in a cascade superfusion bioassay system, the effluate from vascular smooth muscle cells induced a stable relaxation that was susceptible to inhibition by oxyhemoglobin. Three putative endogenous NO carriers mimicked this stable relaxing effect: S-nitroso-L-cysteine, low molecular weight dinitrosyl-iron complexes (DNICs), and the adduct of N-G-hydroxy-L-arginine (HOArg) with NO, Inactivation of S-nitroso L-cysteine by Hg2+ ions or trapping of DMICs with agarose-bound bovine serum albumin abolished their relaxing effects, whereas that of the vascular smooth muscle cell effluate remained unaffected. In addition, neither S-nitrosothiols nor DNICs were detectable in the effluate from these cells, as judged by UV and electron spin resonance (ESR) spectroscopy, The HOArg-NO adduct was instantaneously generated upon reaction of HOArg with authentic NO under bioassay conditions, Its pharmacological profile was indistinguishable from that of the vascular smooth muscle cell effluate, as judged by comparative bioassay with different vascular and nonvascular smooth muscle preparations, Moreover, up to 100 nM HOArg was detected in the effluate from interleukin 1 beta-stimulated vascular smooth muscle cells, suggesting that sufficient amounts of HOArg are released from these cells to spontaneously generate the HOArg-NO adduct. This intercellular NO carrier probably accounts for the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle tells and also from other NO-producing cells, such as macrophages and neutrophils.