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 vasc ular smooth muscle cells cultured on microcarrier beads and stimulated for 20 h with interleukin la was investigated. Unlike the unstable re laxation elicited by authentic nitric oxide (NO) in a cascade superfus ion bioassay system, the effluate from vascular smooth muscle cells in duced a stable relaxation that was susceptible to inhibition by oxyhem oglobin. Three putative endogenous NO carriers mimicked this stable re laxing effect: S-nitroso-L-cysteine, low molecular weight dinitrosyl-i ron complexes (DNICs), and the adduct of N-G-hydroxy-L-arginine (HOArg ) with NO, Inactivation of S-nitroso L-cysteine by Hg2+ ions or trappi ng of DMICs with agarose-bound bovine serum albumin abolished their re laxing effects, whereas that of the vascular smooth muscle cell efflua te 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 i nstantaneously generated upon reaction of HOArg with authentic NO unde r bioassay conditions, Its pharmacological profile was indistinguishab le from that of the vascular smooth muscle cell effluate, as judged by comparative bioassay with different vascular and nonvascular smooth m uscle preparations, Moreover, up to 100 nM HOArg was detected in the e ffluate from interleukin 1 beta-stimulated vascular smooth muscle cell s, suggesting that sufficient amounts of HOArg are released from these cells to spontaneously generate the HOArg-NO adduct. This intercellul ar NO carrier probably accounts for the stable L-arginine-derived rela xing factor released from cytokine-stimulated vascular smooth muscle t ells and also from other NO-producing cells, such as macrophages and n eutrophils.