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Increased oxidative stress is a known cause of cardiac dysfunction in animals and patients with diabetes, but the sources of reactive oxygen species [e.g., superoxide anion ([O.sup.-.sub.2])] and the mechanisms underlying [O.sup.-.sub.2] production in diabetic hearts are not clearly understood. Our aim was to determine whether NADPH oxidase (Nox) is a source of [O.sup.-.sub.2] and whether glucose-6-phosphate dehydrogenase (G6PD)-derived NADPH plays a role in augmenting [O.sup.-.sub.2] generation in diabetes. We assessed cardiac function, Nox and G6PD activities, NADPH levels, and the activities of antioxidant enzymes in heart homogenates from young (9-11 wk old) Zucker lean and obese (fa/fa) rats. We found that myocardial G6PD activity was significantly higher in fa/fa than in lean rats, whereas superoxide dismutase and glutathione peroxidase activities were decreased (P < 0.05). [O.sup.-.sub.2] levels were elevated (70-90%; P < 0.05) in the diabetic heart, and this elevation was blocked by the Nox inhibitor gp-[91.sup.ds-tat] (50 [micro]M) or by the mitochondrial respiratory chain inhibitors antimycin (10 [micro]M) and rotenone (50 [micro]M). Inhibition of G6PD by 6-aminonicotinamide (5 mM) and dihydroepiandrosterone (100 [micro]M) also reduced (P < 0.05) [O.sup.-.sub.2] production. Notably, the activities of Nox and G6PD in the fa/fa rat heart were inhibited by chelerythrine, a protein kinase C inhibitor. Although we detected no changes in stroke volume, cardiac output, or ejection fraction, left ventricular diameter was slightly increased during diastole and systole, and left ventricular posterior wall thickness was decreased during systole (P < 0.05) in Zucker fa/fa rats. Our findings suggest that in a model of severe hyperlipidema and hyperglycemia Nox-derived [O.sup.-.sub.2] generation in the myocardium is fueled by elevated levels of G6PD-derived NADPH. Similar mechanisms were found to activate [O.sup.-.sub.2] production and induce endothelial dysfunction in aorta. Thus G6PD may be a useful therapeutic target for treating the cardiovascular disease associated with type 2 diabetes, if second-generation drugs specifically reducing the activity of G6PD to near normal levels are developed. pentose phosphate pathway; protein kinase C; adenylate cyclase; cardiac function

Protein kinase C (PKC) stimulation of NAD(P)H oxidases (Nox) is an important component of multiple vascular disease processes; however, the relationship between oxidase activation and the regulation of vascular smooth muscle contraction by PKC remains poorly understood. Therefore, we examined the signaling cascade of PKC-elicited Nox activation and the role of superoxide and hydrogen peroxide in mediating PKC-induced vascular contraction. Endothelium-denuded bovine coronary arteries showed a PKC-dependent basal production of lucigenin (5 [micro]M)-detected Nox oxidase-derived superoxide, which was stimulated fourfold by PKC activation with 10 [micro]M phorbol 12,13-dibutyrate (PDBu). PDBu appeared to increase superoxide generation by Nox2 through both [p47.sup.phox] and peroxide-dependent Src activation mechanisms based on the actions of inhibitors, properties of Src phosphorylation, and the loss of responses in aorta from mice deficient in Nox2 and [p47.sup.phox]. The actions of inhibitors of contractile regulating mechanisms, scavengers of superoxide and peroxide, and responses in knockout mouse aortas suggest that a major component of the contraction elicited by PDBu appeared to be mediated through peroxide derived from Nox2 activation stimulating force generation through Rho kinase and calmodulin kinase-II mechanisms. Superoxide generated by PDBu also attenuated relaxation to nitroglycerin. Peroxide-derived from Nox2 activation by PKC appeared to be a major contributor to the thromboxane [A.sub.2] receptor agonist U46619 (100 nM)-elicited contraction of coronary arteries. Thus a [p47.sup.phox] and Src kinase activation of peroxide production by Nox2 appears to be an important contributor to vascular contractile mechanisms mediated through activation of PKC. hydrogen peroxide; NADPH oxidase; protein kinase C; Src kinases; coronary artery

When recovering from heart failure (HF), the myocardium displays a marked plasticity and can regain normal gene expression and function; however, recovery of substrate oxidation capacity has not been explored. We tested whether cardiac functional recovery is matched by normalization of energy substrate utilization during post-HF recovery. HF was induced in dogs by pacing the left ventricle (LV) at 210-240 beats/min for 4 wk. Tachycardia was discontinued, and the heart was allowed to recover. An additional group was studied in HF, and healthy dogs served as controls (n = 8/group). Cardiac free fatty acids (FFAs) and glucose oxidation were measured with [[sup.3]H]oleate and [[sup.14]C]glucose. At 10 days of recovery, hemodynamic parameters returned to control values; however, the contractile response to dobutamine remained depressed, LV end-diastolic volume was 28% higher than control, and the heart mass-to-body mass ratio was increased (9.8 [+ or -] 0.4 vs. 7.5 [+ or -] 0.2 g/kg, P < 0.05). HF increased glucose oxidation (76.8 [+ or -] 19.7 nmol x [min.sup.-1] x [g.sup.-1]) and decreased FFA oxidation (20.7 [+ or -] 6.4 nmol x [min.sup.-1] x [g.sup.-1]), compared with normal dogs (24.5 [+ or -] 6.3 and 51.7 [+ or -] 9.6 nmol x [min.sup.-1] x [g.sup.-1], respectively), and reversed to normal values at 10 days of recovery (25.4 [+ or -] 6.0 and 46.6 [+ or -] 6.7 nmol x [min.sup.-1] x [g.sup.-1], respectively). However, similar to HF, the recovered dogs failed to increase glucose and fatty acid uptake in response to pacing stress. The activity of myocardial citrate synthase and aconitase was significantly decreased during recovery compared with that in control dogs (58 and 27% lower, respectively, P < 0.05), indicating a persistent reduction in mitochondrial oxidative capacity. In conclusion, cardiac energy substrate utilization is normalized in the early stage of post-HF recovery at baseline, but not under stress conditions. dilated cardiomyopathy; fatty acids; glucose; tachypacing

Mitochondrial biogenesis is involved in the control of cell metabolism, signal transduction, and regulation of mitochondrial reactive oxygen species (ROS) production. Despite the central role of mitochondria in cellular aging and endothelial physiology, there are no studies extant investigating agerelated alterations in mitochondrial biogenesis in blood vessels. Electronmicroscopy and confocal microscopy (en face Mitotracker staining) revealed that in aortas of F344 rats, a decline in mitochondrial biogenesis occurs with aging. In aged vessels, the expression of the mitochondrial biogenesis factors (including mitochondrial transcription factor A and peroxisome proliferator-activated receptor-[gamma]/coactivator-1) was decreased. The vascular expression of complex I, III, and IV significantly declined with age, whereas aging did not alter the expression of complex II and V. Cytochrome c oxidase (COX) expression/activity exhibited the greatest age-related decline, which was associated with increased mitochondrial ROS production in the aged vessels. In cultured coronary arterial endothelial cells, a partial knockdown of COX significantly increased mitochondrial ROS production. In conclusion, vascular aging is characterized by a decline in mitochondrial mass in the endothelial cells and an altered expression of components of the mitochondrial electron transport chain likely due to a dysregulation of mitochondrial biogenesis factors. We posit that impaired mitochondrial biogenesis and downregulation of COX may contribute to the increased mitochondrial oxidative stress in aged endothelial cells. vascular senescence

This study examines in endothelium-denuded bovine pulmonary arteries the effects of increasing heme oxygenase-I (HO-1) activity on relaxation and soluble guanylate cyclase (sGC) activation by nitric oxide (NO). A 24-h organ culture with 0.1 mM cobalt chloride (COC12) or 30 [micro]M Co-protoporphyrin IX was developed as a method of increasing HO-1 expression. These treatments increased HO-1 expression and HO activity by approximately two- to fourfold and lowered heme levels by 40-45%. Induction of HO-1 was associated with an attenuation of pulmonary arterial relaxation to the NO-donor spermine-NONOate. The presence of a HO-1 inhibitor 30 [micro]M chromium mesoporphyrin during the 24-h organ culture (but not acute treatment with this agent) reversed the attenuation of relaxation to NO seen in arteries co-cultured with agents that increased HO-1. Relaxation to isoproterenol, which is thought to be mediated through cAMP, was not altered in arteries with increased HO-1. Inducers of HO-1 did not appear to alter basal sGC activity in arterial homogenates or expression of the [[beta].sub.1]-subunit of sGC. However, the increase in activity seen in the presence of 1 [micro]M spermine-NONOate was attenuated in homogenates obtained from arteries with increased HO-1. Since arteries with increased HO-I had decreased levels of superoxide detected by the chemiluminescence of 5 [micro]M lucigenin, superoxide did not appear to be mediating the attenuation of relaxation to NO. These data suggest that increasing HO-1 activity depletes heme, and this is associated with an attenuation of pulmonary artery relaxation and sGC activation responses to NO. cGMP; cobalt protoporphyrin; chromium mesoporphyrin; superoxide

Cholesterol is a primary constituent of the plasmalemma, including the lipid rafts/caveolae, where various G protein-coupled receptors colocalize with signaling proteins and channels. By manipulating cholesterol in rabbit and rat ventricular myocytes using methyl-[beta]-cyclodextrin (M[beta]CD), we studied the role of cholesterol in the modulation of L-type [Ca.sup.2+] currents ([I.sub.Ca,L]). M[beta]CD was mainly dialyzed from BAPTA-containing pipette solution during whole cell clamp. In rabbit myocytes dialyzed with 30 mM M[beta]CD for 10 min, a positive shift in membrane potential at half-maximal activation ([V.sub.0.5]) from -8 to -2 mV developed and was associated with an increase in current density at positive potentials (42% at +20 mV vs. time-matched controls). Isoproterenol (ISO) increased [I.sub.Ca,L] approximately threefold and caused a negative shift in [V.sub.0.5] in control cells, but it did not increase [I.sub.Ca,L]. in M[beta]CD-treated myocytes, nor did it shift [V.sub.0.5]. The effect of M[beta]CD (10 or 30 mM) was concentration dependent: 30 mM M[beta]CD suppressed the ISO-induced increase in [I.sub.Ca,L] more effectively than 10 mM M[beta]CD. M[beta]CD dialysis also abolished the increase in [I.sub.Ca,L] elicited by forskolin or dibutyryl cAMP, but not that elicited by (-)BAY K 8644. External application of M[beta]CD-cholesterol complex to rat myocytes attenuated the M[beta]CD-mediated inhibition of the ISO-induced increase of [I.sub.Ca,L]. Biochemical analysis confirmed that the myocytes' cholesterol content was diminished by M[beta]CD and increased by M[beta]CD-cholesterol complex. Cholesterol thus appears to contribute to the regulation of basal [I.sub.Ca,L] and [beta]-adrenergic cAMP/ PKA-mediated increases in [I.sub.Ca,L]. We suggest that cholesterol affects the structural coupling between L-type [Ca.sup.2+] channels and adjacent regulatory proteins. lipid raft; adenosine 3',5'-cyclic monophosphate; protein kinase A; phosphorylation

Protoporphyrin IX is an activator of soluble guanylate cyclase (sGC), but its role as an endogenous regulator of vascular function through cGMP has not been previously reported. In this study we examined whether the heme precursor [delta]-aminolevulinic acid (ALA) could regulate vascular force through promoting protoporphyrin IX-elicited activation of sGC. Exposure of endothelium-denuded bovine pulmonary arteries (BPA) in organoid culture to increasing concentrations of the heme precursor ALA caused a concentration-dependent increase in BPA epifluorescence, consistent with increased tissue protoporphyrin IX levels, associated with decreased force generation to increasing concentrations of serotonin. The force-depressing actions of 0.1 mM ALA were associated with increased cGMP-associated vasodilator-stimulated phosphoprotein (VASP) phosphorylation and increased sGC activity in homogenates of BPA cultured with ALA. Increasing iron availability with 0.1 mM FeSO4 inhibited the decrease in contraction to serotonin and increase in sGC activity caused by ALA, associated with decreased protoporphyrin IX and increased heme. Chelating endogenous iron with 0.1 mM deferoxamine increased the detection of protoporphyrin IX and force depressing activity of 10 [micro]M ALA. The inhibition of sGC activation with the heme oxidant 10 [micro]M 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) attenuated the force depressing actions of an NO donor without altering the actions of ALA. Thus control of endogenous formation of protoporphyrin IX from ALA by the availability of iron is potentially a novel physiological mechanism of controlling vascular function through regulating the activity of sGC. guanosine 3',5'-cyclic monophosphate; heme metabolism; iron; vasodilation doi: 10.1152/ajplung.00482.2005

Hypoxia relaxes endothelium-denuded bovine coronary arteries (BCA) through mechanisms that do not appear to involve reactive oxygen species, prostaglandins, or nitric oxide. Because of similarities in the relaxation of BCA to hypoxia ([Po.sub.2] = 8-10 Torr) and inhibitors of the pentose phosphate pathway (PPP) including 6-aminonicotinamide and epiandrosterone, we measured NADPH and NADP and found that hypoxia caused NADPH oxidation (decreased NADPH/NADP). The relaxation to hypoxia was similar to previously reported properties of relaxation to PPP inhibitors in that both responses were associated with glutathione oxidation and depressed intracellular calcium release and calcium influx-mediated contractile responses. Inhibitors of potassium channels had minimal effects on these relaxation responses. Relaxation to hypoxia and PPP inhibitors were attenuated by a thiol reductant (3 mM dithiothreitol) and by eliciting contraction with an activator of protein kinase C (phorbol 12,13-dibutyrate). In the presence of contraction to U-46619. relaxation to hypoxia and PPP inhibitors were attenuated by the sarco(endo)plasmic reticulum [Ca.sup.2+]ATPase pump inhibitor 200 [micro]M cyclopiazonic acid and by 10 mM pyruvate. Hypoxia decreased BCA levels of glucose-6-phosphate but not ATP. Pyruvate prevented the hypoxia-elicited decrease in glucose-6-phosphate and glutathione oxidation, and it increased NADPH levels under hypoxia to levels observed under normoxia. Thus hypoxia causes a metabolic stress on the PPP that promotes BCA relaxation through processes controlled by lowering the levels of cytosolic NADPH. calcium; oxygen sensor; pentose phosphate pathway; pyruvate; sarco(endo)plasmic reticulum calcium-adenosinetriphosphatase pump

The mechanisms through which thiol oxidation and cellular redox influence the regulation of soluble guanylate cyclase (sGC) are poorly understood. This study investigated whether promoting thiol oxidation via inhibition of NADPH generation by the pentose phosphate pathway (PPP) with 1 mM 6-aminonicotinamide (6-AN) or the thiol oxidant diamide (1 mM) alters sGC activity and cGMP-associated relaxation to nitric oxide (NO) donors [S-nitroso-N-acetylpenicillamine (SNAP) and spermine-NONOate]. Diamide and 6-AN inhibited NO-elicited relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and stimulation of sGC activity in BPA homogenates. Treatment of BPA with the thiol reductant DTT (1 mM) reversed inhibition of NO-mediated relaxation and sGC stimulation by 6-AN. The increase in cGMP protein kinase-associated phosphorylation of vasodilator-stimulated phosphoprotein on [Ser.sup.239] elicited by 10 [micro]M SNAP was also inhibited by diamide. Activation of sGC by SNAP was attenuated by low micromolar concentrations of GSSG in concentrated, but not dilute, homogenates of BPA, suggesting that an enzymatic process contributes to the actions of GSSG. Relaxation to agents that function through cAMP (forskolin and isoproterenol) was not altered by inhibition of the pentose phosphate pathway or diamide. Thus a thiol oxidation mechanism controlled by the regulation of thiol redox by NADPH generated via the pentose phosphate pathway appears to inhibit sGC activation and cGMP-mediated relaxation by NO in a manner consistent with its function as an important physiological redox-mediated regulator of vascular function. cGMP; glutathione redox; NADPH redox; nitrovasodilators

Vascular smooth muscle (VSM) derived from pulmonary arteries generally contract to hypoxia, whereas VSM from systemic arteries usually relax, indicating the presence of basic oxygen-sensing mechanisms in VSM that are adapted to the environment from which they are derived. This review considers how fundamental processes associated with the generation of reactive oxygen species (ROS) by oxidase enzymes, the metabolic control of cytosolic NADH, NADPH and glutathione redox systems, and mitochondrial function interact with signaling systems regulating vascular force in a manner that is potentially adapted to be involved in [Po.sub.2] sensing. Evidence for opposing hypotheses of hypoxia, either decreasing or increasing mitochondrial ROS, is considered together with the [Po.sub.2] dependence of ROS production by Nox oxidases as sensors potentially contributing to hypoxic pulmonary vasoconstriction. Processes through which ROS and NAD(P)H redox changes potentially control interactive signaling systems, including soluble guanylate cyclase, potassium channels, and intracellular calcium are discussed together with the data supporting their regulation by redox in responses to hypoxia. Evidence for hypothesized potential differences between systemic and pulmonary arteries originating from properties of mitochondrial ROS generation and the redox sensitivity of potassium channels is compared with a new hypothesis in which differences in the control of cytosolic NADPH redox by the pentose phosphate pathway results in increased NADPH and Nox oxidase-derived ROS in pulmonary arteries, whereas lower levels of glucose-6-phosphate dehydrogenase in coronary arteries may permit hypoxia to activate a vasodilator mechanism controlled by oxidation of cytosolic NADPH. hydrogen peroxide; hypoxia; Nox oxidase; mitochondria; pentose phosphate pathway

Because systems controlled by basal NAD(P)H oxidase activity appear to contribute to differences in responses of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to hypoxia, we characterized the Nox oxidases activities present in these vascular segments and how cytosolic NAD(P)H redox systems could be controlling oxidase activity. BPA generated ~60-80% more lucigenin (5 [micro]M) chemiluminescence detectable superoxide than BCA. Apocynin (10 [micro]M), a NAD(P)H oxidase inhibitor, and 6-aminonicotinamide (1 mM), a pentose phosphate inhibitor (PPP), both attenuated (approximately by 50-70%) superoxide detected in BPA and BCA. There was no significant difference in the expression of Nox2 or Nox4 mRNA or protein detected by Western blot analysis. NADPH and NADH increased superoxide in homogenates and isolated microsomal membrane fractions in a manner consistent with BPA and BCA having similar levels of oxidase activity. BPA had 4.2-fold higher levels of NADPH than BCA. The activity and protein levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting PPP enzyme generating cytosolic NADPH, were 1.5-fold higher in BPA than BCA. Thus BPA differ from BCA in that they have higher levels of G6PD activity, NADPH, and superoxide. Because both arteries have similar levels of Nox expression and activity, elevated levels of cytosolic NADPH may contribute to increased superoxide in BPA. glucose-6-phosphate dehydrogenase; lactate; NAD(P)H oxidase; Nox2; Nox4; pentose phosphate pathway.

Pentose phosphate pathway (PPP) inhibitors, 6-aminonicotinamide (6-AN) and epiandrosterone (Epi), were employed to examine whether changes in NADP(H) redox regulates contractile force in endothelium-removed bovine coronary arteries (BCAs). 6-AN (0.01-5 mM) or Epi (1-500 [micro]M) elicited dose-dependent relaxation in BCAs contracted with 30 mM KCl, 0.1 [micro]M U-44619, and endothelin-1 but not with phorbol 12,13-dibutyrate, a protein kinase C activator that causes [Ca.sup.2+]-independent contraction. Relaxation to PPP inhibition was associated with oxidation of NADPH and glutathione (GSH). Relaxation to 6-AN was not mediated by [H.sub.2][O.sub.2], because it was not altered by hypoxia or the peroxide scavenger ebselen (100 [micro]M). The thiol reductant DTT (3 mM) attenuated the relaxation to 6-AN and Epi by 30-40%. Inhibition of glycolysis of mitochondrial electron transport did not elicit relaxation in BCAs contracted with 30 mM KCl, suggesting these pathways may not be involved in relaxation elicited by PPP inhibition. High doses of [K.sup.+] channel blockers [e.g., TEA (10 mM) and 4-aminopyridine (10 mM)] only partially inhibited the relaxation to 6-AN. On the basis of changes in the fura-2 fluorescence ratio, 6-AN and Epi appeared to markedly reduce intracellular [Ca.sup.2+]. Thus PPP inhibition oxidizes NADPH and GSH and appears to activate a novel coordination of redox-controlled relaxing mechanisms in BCAs mediated primarily through decreasing intracellular C[a.sup.2+] calcium; redox signaling; vasodilator mechanisms