Redox-dependent coronary metabolic dilation

We have observed that hydrogen peroxide (H₂O₂), the dismutated product of superoxide, is a coronary metabolic dilator and couples myocardial oxygen consumption to coronary blood flow. Because the chemical activity of H₂O₂ favors its role as an oxidant, and thiol groups are susceptible to oxidation, we hypothesized that coronary metabolic dilation occurs via a redox mechanism involving thiol oxidation. To test this hypothesis, we studied the mechanisms of dilation of isolated coronary arterioles to metabolites released by metabolically active (paced at 400 min) isolated cardiac myocytes and directly compared these responses with authentic H₂O₂. Studies were performed under control conditions and using interventions designed to reduce oxidized thiols [0.1 μM dithiothreitol (DTT) and 10 mM N-acetyl-L-cysteine (NAC)]. Aliquots of the conditioned buffer from paced myocytes produced vasodilation of isolated arterioles (peak response, 71% ± 6% of maximal dilation), whereas H₂O₂ produced complete dilation (92% ± 7%). Dilation to either the conditioned buffer or to H₂O₂ was significantly reduced by the administration of either NAC or DTT. The location of the thiols oxidized by the conditioned buffer or of H₂O₂ was determined by the administration of the fluorochromes monochlorobimane (20 μM) or monobromotrimethylammoniobimane (20 μM), which covalently label the reduced total or extracellular-reduced thiols, respectively. H₂O₂ or the conditioned buffer predominately oxidized intracellular thiols since the fluorescent signal from monochlorobimane was reduced more than that of monobromotrimethylammoniobimane. To determine whether one of the intracellular targets of thiol oxidation that leads to dilation is the redox-sensitive kinase p38 mitogen-activated protein (MAP) kinase, we evaluated dilation following the administration of the p38 inhibitor SB-203580 (10 μM). The inhibition of p38 attenuated dilation to either H₂O₂ or to the conditioned buffer from stimulated myocytes by a similar degree, but SB-203580 did not attenuate dilation to nitroprusside. Western blot analysis for the activated form of p38 (phospho-p38) in the isolated aortae revealed robust activation of this enzyme by H₂O₂. Taken together, our results show that an active component of cardiac metabolic dilation, like that of H ₂O₂, produces dilation by the oxidation of thiols, which are predominately intracellular and dependent activation on the p38 MAP kinase. Thus coronary metabolic dilation appears to be mediated by redox-dependent signals.