Mode of ethanol administration influences the severity of hemorrhage-induced lactic acidemia in conscious guinea pigs.

Mechanisms that limit metabolic acidemia during shock are limited by ethanol (EtOH). This may be due to (1) loss of respiratory compensation, (2) a greater fall in cardiac output, (3) altered removal of plasma lactate by the liver, and (4) alterations in central nervous system orchestration of compensatory responses. We have previously shown that loss of metabolic compensation during hemorrhage is correlated with plasma EtOH concentrations. The present study determines if the mode of ethanol administration influences compensation during hemorrhage. Male guinea pigs were administered EtOH (1 g/kg, 30% wt/vol) via intraperitoneal (IP) or intragastric (IG) routes. After 30 minutes, 60% of the estimated blood volume was removed. Animals remained in shock for 30 minutes were resuscitated with lactated Ringer solution and monitored for 3 hours. Plasma EtOH levels were similar in the 2 groups at the initiation of, and during, hemorrhage and resuscitation. Animals given EtOH IP exhibited more severe acidemia. The mode of EtOH administration may affect hepatic ethanol and lactate metabolism, thus exacerbating acidemia. An altered central nervous system response may impact compensatory responses during shock. Our results indicate that the "history" of the EtOH episode may be an important determinant in the compensation for hemorrhage and resuscitation.

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 microM 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 microM) or monobromotrimethylammoniobimane (20 microM), which covalently label the reduced total or extracellular-reduced thiols, respectively. H2O2 or the conditioned buffer predominantly 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 microM). 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 predominantly intracellular and dependent activation on the p38 MAP kinase. Thus coronary metabolic dilation appears to be mediated by redox-dependent signals.

Knockout mice reveal that the angiotensin II type 1B receptor links to smooth muscle contraction.

BACKGROUND: Rodents express two isoforms of the angiotensin II type 1 (AT(1)) receptor: AT(1A) and AT(1B). It is unclear which receptor subtype mediates contraction in response to angiotensin II in various arteries. We tested the hypothesis that the AT(1B) receptor is the predominant receptor that mediates contraction in the abdominal aorta in response to angiotensin II. METHODS: Isometric tension responses to angiotensin II were determined in abdominal aortic rings obtained from male wild-type and AT(1B) receptor knockout mice. The rings were suspended in an organ bath of a wire myograph and contractions to angiotensin II and other vasoconstrictors were determined. RESULTS: Angiotensin II contracted aortic segments from wild-type mice; however, this response was virtually absent in rings obtained from AT(1B) receptor knockout mice. Contractions in response to K(+) and U46619 (thromboxane A(2) mimetic) were not different between rings obtained from wild-type and AT(1B) receptor knockout mice. CONCLUSIONS: Reduced angiotensin II contraction is not related to a generalized decrease in smooth muscle function, rather it is specifically due to genetic ablation of the AT(1B) receptor. Our data support the concept that AT(1B) receptors couple to contraction in the mouse abdominal aorta, a function that parallels the single known AT(1) receptor in human vascular smooth muscle.

Hydrogen peroxide: a feed-forward dilator that couples myocardial metabolism to coronary blood flow.

OBJECTIVE: We tested the hypothesis that hydrogen peroxide (H2O2), the dismutated product of superoxide (O2*-), couples myocardial oxygen consumption to coronary blood flow. Accordingly, we measured O2*- and H2O2 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 H2O2. METHODS AND RESULTS: The production of O2*- is coupled to oxidative metabolism because inhibition of complex I (rotenone) or III (antimycin) enhanced the production of O2*- during pacing by about 50% and 400%, respectively; whereas uncoupling oxidative phosphorylation by decreasing the protonmotive force with carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) decreased pacing-induced O2*- production. The inhibitor of cytosolic NAD(P)H oxidase assembly, apocynin, did not affect O2*- production by pacing. Aliquots of buffer from paced myocytes produced vasodilation of isolated arterioles (peak response 67+/-8% percent of maximal dilation) that was significantly reduced by catalase (5+/-0.5%, P<0.05) or the antagonist of Kv channels, 4-aminopyridine (18+/-4%, P<0.05). In intact animals, tissue concentrations of H2O2 are proportionate to myocardial oxygen consumption and directly correlated to coronary blood flow. Intracoronary infusion of catalase reduced tissue levels of H2O2 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 pO2. CONCLUSIONS: Taken together, our results demonstrate that O2*- is produced in proportion to cardiac metabolism, which leads to the production of the vasoactive reactive oxygen species, H2O2. Our results further suggest that the production of H2O2 in proportion to metabolism couples coronary blood flow to myocardial oxygen consumption.

H2O2-induced redox-sensitive coronary vasodilation is mediated by 4-aminopyridine-sensitive K+ channels.

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.

Reduced functional expression of K(+) channels in vascular smooth muscle cells from rats made hypertensive with N{omega}-nitro-L-arginine.

Smooth muscle membrane potential is determined, in part, by K(+) channels. In the companion paper to this article, we demonstrated that superior mesenteric arteries from rats made hypertensive with N(omega)-nitro-l-arginine (l-NNA) are depolarized and express less K(+) channel protein compared with those from normotensive rats. In the present study, we used patch-clamp techniques to test the hypothesis that l-NNA-induced hypertension reduces the functional expression of K(+) channels in smooth muscle. In whole cell experiments using a Ca(2+)-free pipette solution, current at 0 mV, largely due to voltage-dependent K(+) (K(V)) channels, was reduced approximately 60% by hypertension (2.7 +/- 0.4 vs. 1.1 +/- 0.2 pA/pF). Current at +100 mV with 300 nM free Ca(2+), largely due to large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels, was reduced approximately 40% by hypertension (181 +/- 24 vs. 101 +/- 28 pA/pF). Current blocked by 3 mM 4-aminopyridine, an inhibitor of many K(V) channel types, was reduced approximately 50% by hypertension (1.0 +/- 0.4 vs. 0.5 +/- 0.2 pA/pF). Current blocked by 1 mM tetraethylammonium, an inhibitor of BK(Ca) channels, was reduced approximately 40% by hypertension (86 +/- 14 vs. 53 +/- 19 pA/pF). Differences in BK(Ca) current magnitude are not attributable to changes in single-channel conductance or Ca(2+)/voltage sensitivity. The data support the hypothesis that l-NNA-induced hypertension reduces K(+) current in vascular smooth muscle. Reduced molecular and functional expression of K(+) channels may partly explain the depolarization and augmented contractile sensitivity of smooth muscle from l-NNA-treated rats.

Leptin receptors are expressed in coronary arteries, and hyperleptinemia causes significant coronary endothelial dysfunction.

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.

C-reactive protein does not relax vascular smooth muscle: effects mediated by sodium azide in commercially available preparations.

C-reactive protein (CRP), an acute-phase protein and newly recognized indicator of cardiovascular risk, may have direct actions on the vascular wall. Previous studies suggest that CRP is a vasodilator that activates smooth muscle K(+) channels. We examined the reported vasoactive properties of CRP and further explored its mechanisms of action. CRP decreased blood pressure in rats and increased coronary flow in open-chest dogs at a constant coronary perfusion pressure. CRP relaxed rat aortic rings and mesenteric small arteries that were contracted with phenylephrine. Relaxation was not affected by endothelial denudation or inhibition of nitric oxide (NO) synthase but was blocked by inhibition of soluble guanylate cyclase or K(+) channels. CRP solutions remained effective, i.e., elicited vasodilation, even after boiling or enzymatic digestion, which suggests the presence of a nonprotein contaminant. Sodium azide (NaN(3), 0.1%) is the preservative used for commercially available CRP and a potential source of NO. NaN(3) elicited the same cardiovascular effects as CRP preparations at equal concentrations, and its actions were blocked by inhibition of guanylate cyclase and K(+) channels. NaN(3)-free CRP, prepared by gel-filtration centrifugation and confirmed by electrophoresis, had no effect on vascular tone. Inhibition of vascular smooth muscle catalase with 3-amino-1,2,4-triazole completely prevented the effects of NaN(3) and NaN(3)-containing CRP solutions. We demonstrate that the acute vasoactive properties of commercially available CRP preparations are attributable to NaN(3) (and subsequent production of NO by catalase); therefore, this study suggests a reappraisal of the acute role of CRP in regulating vascular tone.

Ethanol intoxication and lactated Ringer's resuscitation prolong hemorrhage-induced lactic acidosis.

Ethanol (EtOH) blunts the respiratory and metabolic compensation during hemorrhage, resulting in a more severe lactic acidemia. We hypothesized that lactated Ringer's (LR) resuscitation may exacerbate this lactic acidemia. Male guinea pigs were implanted with arterial and venous catheters. Two days after catheter placement, conscious animals were injected intraperitoneally with 1 g/kg EtOH, 0.3 g/kg EtOH, or an equal volume of water 30 min before hemorrhage (60% of estimated blood volume). After 30 min of hemorrhagic shock, animals were resuscitated with isotonic saline (S) or LR at 1 mL/min (three times shed blood volume). Mean arterial blood pressure (MABP) was not affected by pretreatment with either dose of EtOH, but was significantly decreased by hemorrhage in all groups. Both S and LR resuscitation slightly increased MABP, but neither restored it to prehemorrhage values. Blood lactate levels increased in all groups during hemorrhage and remained elevated for 3 h in animals pretreated with 1 g/kg EtOH. In the group pretreated with 0.3 g/kg EtOH, pH decreased during shock but returned to prehemorrhage levels during the resuscitation period. Resuscitation with S returned pH to prehemorrhage values in animals pretreated with 1.0 g/kg EtOH. Resuscitation with LR did not exacerbate, but did prolong, the lactic acidemia after shock in animals pretreated with 1.0 g/kg EtOH. Administration of additional lactate during intoxication and hypovolemia for hemodynamic stabilization before blood transfusion may exacerbate a metabolic stress.