Acute restraint stress induces endothelial dysfunction: role of vasoconstrictor prostanoids and oxidative stress

Mental stress has been linked to increased morbidity and mortality in coronary artery disease and to atherosclerosis progression. Experimental studies have suggested that damage to the endothelium may be an important mechanism. Endothelial function was studied in 10 healthy men (aged 50. 4+/-9.6 years) and in 8 non-insulin-dependent diabetic men (aged 52. 0+/-7.2 years). Brachial artery flow-mediated dilation (FMD, endothelium dependent) and response to 50 microg of sublingual glyceryl trinitrate (GTN, endothelium independent) were measured noninvasively by use of high-resolution ultrasound before and after (30, 90, and 240 minutes) a standardized mental stress test. The same protocol without mental stress was repeated on a separate occasion in the healthy men. In healthy subjects, FMD (5.0+/-2.1%) was significantly (P:<0.01) reduced at 30 and 90 minutes after mental stress (2.8+/-2.3% and 2.3+/-2.4%, respectively) and returned toward normal after 4 hours (4.1+/-2.0%). Mental stress had no effect on the response to GTN. In the repeated studies without mental stress, FMD did not change. The diabetic subjects had lower FMD than did the control subjects (3.0+/-1.5% versus 5.0+/-2.1%, respectively; P:=0.02) but showed no changes in FMD (2.7+/-1.1% after 30 minutes, 2.8+/-1.9% after 90 minutes, and 3.1+/-2.3% after 240 minutes) or GTN responses after mental stress. These findings suggest that brief episodes of mental stress, similar to those encountered in everyday life, may cause transient (up to 4 hours) endothelial dysfunction in healthy young individuals. This might represent a mechanistic link between mental stress and atherogenesis.

Superoxide (O-2) and nitric oxide (NO) act to kill invading microbes in phagocytes. In macrophages NO is synthesized by inducible nitric oxide synthase (iNOS, NOS 2) from L-arginine (L-Arg) and oxygen; however, O-2 was thought to be produced mainly by NADPH oxidase. Electron paramagnetic resonance (EPR) spin trapping experiments performed in murine macrophages demonstrate a novel pathway of O-2 generation. It was observed that depletion of cytosolic L-Arg triggers O-2 generation from iNOS. This iNOS-mediated O-2 generation was blocked by the NOS inhibitor N-nitro-L-arginine methyl ester or by L-Arg, but not by the noninhibitory enantiomer N-nitro-D-arginine methyl ester. In L-Arg-depleted macrophages iNOS generates both O-2 and NO that interact to form the potent oxidant peroxynitrite (ONOO-), which was detected by luminol luminescence and whose formation was blocked by superoxide dismutase, urate, or L-Arg. This iNOS-derived ONOO- resulted in nitrotyrosine formation, and this was inhibited by iNOS blockade. iNOS-mediated O-2 and ONOO- increased the antibacterial activity of macrophages. Thus, with reduced L-Arg availability iNOS produces O-2 and ONOO- that modulate macrophage function. Due to the existence of L-Arg depletion in inflammation, iNOS-mediated O-2 and ONOO- may occur and contribute to cytostatic/cytotoxic actions of macrophages.

Previous studies from our laboratory have demonstrated that the mitochondrial protein manganese superoxide dismutase is inactivated, tyrosine nitrated, and present as higher molecular mass species during human renal allograft rejection. To elucidate mechanisms whereby tyrosine modifications might result in loss of enzymatic activity and altered structure, the effects of specific biological oxidants on recombinant human manganese superoxide dismutase in vitro have been evaluated. Hydrogen peroxide or nitric oxide had no effect on enzymatic activity, tyrosine modification, or electrophoretic mobility. Exposure to either hypochlorous acid or tetranitromethane (pH 6) inhibited (approximately 50%) enzymatic activity and induced the formation of dityrosine and higher mass species. Treatment with tetranitromethane (pH 8) inhibited enzymatic activity 67% and induced the formation of nitrotyrosine. In contrast, peroxynitrite completely inhibited enzymatic activity and induced formation of both nitrotyrosine and dityrosine along with higher molecular mass species. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that only three (Y34, Y45, and Y193) of the nine total tyrosine residues in manganese superoxide dismutase were nitrated by peroxynitrite. Inspection of X-ray crystallographic data suggested that neighboring glutamate residues associated with two of these tyrosines may promote targeted nitration by peroxynitrite. Tyr34, which is present in the active site, appeared to be the most susceptible residue to peroxynitrite-mediated nitration. Collectively, these observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.