Obesity is associated with marked increases in plasma leptin concentration, and hyperleptinemia is an independent risk factor for coronary artery disease. As a result, the purpose of this investigation was to test the following hypotheses: 1) leptin receptors are expressed in coronary endothelial cells; and 2) hyperleptinemia induces coronary endothelial dysfunction. RT-PCR analysis revealed that the leptin receptor gene is expressed in canine coronary arteries and human coronary endothelium. Furthermore, immunocytochemistry demonstrated that the long-form leptin receptor protein (ObRb) is present in human coronary endothelium. The functional effects of leptin were determined using pressurized coronary arterioles (<130 microm) isolated from Wistar rats, Zucker rats, and mongrel dogs. Leptin induced pharmacological vasodilation that was abolished by denudation and the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and was absent in obese Zucker rats. Intracoronary leptin dose-response experiments were conducted in anesthetized dogs. Normal and obese concentrations of leptin (0.1-3.0 microg/min ic) did not significantly change coronary blood flow or myocardial oxygen consumption; however, obese concentrations of leptin significantly attenuated the dilation to graded intracoronary doses of acetylcholine (0.3-30.0 microg/min). Additional experiments were performed in canine coronary rings, and relaxation to acetylcholine (6.25 nmol/l-6.25 micromol/l) was significantly attenuated by obese concentrations of leptin (625 pmol/l) but not by physiological concentrations of leptin (250 pmol/l). The major findings of this investigation were as follows: 1) the ObRb is present in coronary arteries and coupled to pharmacological, nitric oxide-dependent vasodilation; and 2) hyperleptinemia produces significant coronary endothelial dysfunction.
Hydrogen peroxide (H(2)O(2)) is a proposed endothelium-derived hyperpolarizing factor and metabolic vasodilator of the coronary circulation, but its mechanisms of action on vascular smooth muscle remain unclear. Voltage-dependent K(+) (K(V)) channels sensitive to 4-aminopyridine (4-AP) contain redox-sensitive thiol groups and may mediate coronary vasodilation to H(2)O(2). This hypothesis was tested by studying the effect of H(2)O(2) on coronary blood flow, isometric tension of arteries, and arteriolar diameter in the presence of K(+) channel antagonists. Infusing H(2)O(2) into the left anterior descending artery of anesthetized dogs increased coronary blood flow in a dose-dependent manner. H(2)O(2) relaxed left circumflex rings contracted with 1 muM U46619, a thromboxane A(2) mimetic, and dilated coronary arterioles pressurized to 60 cmH(2)O. Denuding the endothelium of coronary arteries and arterioles did not affect the ability of H(2)O(2) to cause vasodilation, suggesting a direct smooth muscle mechanism. Arterial and arteriolar relaxation by H(2)O(2) was reversed by 1 mM dithiothreitol, a thiol reductant. H(2)O(2)-induced relaxation was abolished in rings contracted with 60 mM K(+) and by 10 mM tetraethylammonium, a nonselective inhibitor of K(+) channels, and 3 mM 4-AP. Dilation of arterioles by H(2)O(2) was antagonized by 0.3 mM 4-AP but not 100 nM iberiotoxin, an inhibitor of Ca(2+)-activated K(+) channels. H(2)O(2)-induced increases in coronary blood flow were abolished by 3 mM 4-AP. Our data indicate H(2)O(2) increases coronary blood flow by acting directly on vascular smooth muscle. Furthermore, we suggest 4-AP-sensitive K(+) channels, or regulating proteins, serve as redox-sensitive elements controlling coronary blood flow.
Objective-We tested the hypothesis that hydrogen peroxide (H 2 O 2 ), the dismutated product of superoxide (O 2 ⅐Ϫ ), couples myocardial oxygen consumption to coronary blood flow. Accordingly, we measured O 2 ⅐Ϫ and H 2 O 2 production by isolated cardiac myocytes, determined the role of mitochondrial electron transport in the production of these species, and determined the vasoactive properties of the produced H 2 O 2 . Methods and Results-The production of O 2⅐Ϫ is coupled to oxidative metabolism because inhibition of complex I (rotenone) or III (antimycin) enhanced the production of O 2 ⅐Ϫ during pacing by about 50% and 400%, respectively; whereas uncoupling oxidative phosphorylation by decreasing the protonmotive force with carbonylcyanide-ptrifluoromethoxyphenyl-hydrazone (FCCP) decreased pacing-induced O 2 ⅐Ϫ production. The inhibitor of cytosolic NAD(P)H oxidase assembly, apocynin, did not affect O 2 ⅐Ϫ production by pacing. Aliquots of buffer from paced myocytes produced vasodilation of isolated arterioles (peak response 67Ϯ8% percent of maximal dilation) that was significantly reduced by catalase (5Ϯ0.5%, PϽ0.05) or the antagonist of Kv channels, 4-aminopyridine (18Ϯ4%, PϽ0.05). In intact animals, tissue concentrations of H 2 O 2 are proportionate to myocardial oxygen consumption and directly correlated to coronary blood flow. Intracoronary infusion of catalase reduced tissue levels of H 2 O 2 by 30%, and reduced coronary flow by 26%. Intracoronary administration of 4-aminopyridine also shifted the relationship between myocardial oxygen consumption and coronary blood flow or coronary sinus pO 2 . Conclusions-Taken together, our results demonstrate that O 2⅐Ϫ is produced in proportion to cardiac metabolism, which leads to the production of the vasoactive reactive oxygen species, H 2 O 2 . Our results further suggest that the production of H 2 O 2 in proportion to metabolism couples coronary blood flow to myocardial oxygen consumption. Key Words: reactive oxygen species Ⅲ coronary circulation Ⅲ vasodilation Ⅲ microcirculation T he coupling of blood flow to metabolism is the most important vasomotor adjustment for the regulation of oxygen delivery to metabolically active organ systems. This matching, termed metabolic dilation, or metabolic or active hyperemia, is critical to ensure adequate oxygen delivery for aerobic metabolism and adequate organ function. 1 Although the factor or factors responsible for the coupling of flow to metabolism have been actively pursued for decades, no metabolite has been casually linked to the process of metabolic hyperemia or has withstood critical evaluation. 1-3 Most investigations have pursued the idea that the metabolic regulation of flow is a negative feedback pathway, in which an imbalance between oxygen supply (delivered via flow) and oxygen demands, ie, demands exceed supply, results in the production of a metabolic dilator. The adenosine hypothesis was such a scheme, in which oxygen demands, in excess of supply would increase the production of adenosine through hydro...
We have observed that hydrogen peroxide (H 2O2), the dismutated product of superoxide, is a coronary metabolic dilator and couples myocardial oxygen consumption to coronary blood flow. Because the chemical activity of H 2O2 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 M 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% Ϯ 6% of maximal dilation), whereas H 2O2 produced complete dilation (92% Ϯ 7%). Dilation to either the conditioned buffer or to H 2O2 was significantly reduced by the administration of either NAC or DTT. The location of the thiols oxidized by the conditioned buffer or of H 2O2 was determined by the administration of the fluorochromes monochlorobimane (20 M) or monobromotrimethylammoniobimane (20 M), which covalently label the reduced total or extracellular-reduced thiols, respectively. H 2O2 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 redoxsensitive kinase p38 mitogen-activated protein (MAP) kinase, we evaluated dilation following the administration of the p38 inhibitor SB-203580 (10 M). 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 H2O2, 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. coronary circulation; coronary microcirculation; reactive oxygen species; vasodilatation THE OXIDATION OF THIOL GROUPS is involved in many biological processes. Thiol oxidation induces protein conformation changes by converting the free thiols (-SH) into sulfenic acids (SO Ϫ ), sulfinic acids (SOO Ϫ ), sulfonic acids (SOOO Ϫ ), and disulfide bridges (S-S). Thiol oxidation is involved in many cellular processes, e.g., p38 mitogen-activating protein (MAP) kinase activation (2, 23), inhibition of p56 (lck) tyrosine kinase ...
We conclude that hyperbaric oxygen limits infarct size in the reperfused rabbit heart and that the effect can be achieved when hyperbaric oxygen is begun at reperfusion.
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