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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

A promising therapeutic approach to diminish pathological inflammation is to inhibit the increased production and/or biological activity of proinflammatory cytokines (e.g., TNF-[alpha], IL-6). The production of proinflammatory cytokines is controlled at the gene level by the activity of transcription factors, such as NF-[kappa]B. Phosphatidylinositol 3-kinase (PI3K), a lipid kinase, is known to induce the activation of NF-[kappa]B. Given this, we hypothesized that inhibitors of PI3K activation would demonstrate anti-inflammatory potential. Accordingly, we studied the effects of a preferential p110[alpha]/[gamma] PI3K inhibitor (compound 8C; PIK-75) in inflammation-based assays. Mechanism-based assays utilizing human cells revealed that PIK-75-mediated inhibition of PI3K activation is associated with dramatic suppression of downstream signaling events, including AKT phosphorylation, IKK activation, and NF-[kappa]B transcription. Cell-based assays revealed that PIK-75 potently and dose dependently inhibits in vitro and in vivo production of TNF-[alpha] and IL-6, diminishes the induced expression of human endothelial cell adhesion molecules (E-selectin, ICAM-1, and VCAM-1), and blocks human monocyte-endothelial cell adhesion. Most importantly, PIK-75, when administered orally in a therapeutic regimen, significantly suppresses the macroscopic and histological abnormalities associated with dextran sulfate sodium-induced murine colitis. The efficacy of PIK-75 in attenuating experimental inflammation is mediated, at least in part, due to the downregulation of pertinent inflammatory mediators in the colon. Collectively, these results provide first evidence that PIK-75 possesses anti-inflammatory potential. Given that PIK-75 is known to exhibit anti-cancer activity, the findings from this study thus reinforce the cross-therapeutic functionality of potential drugs. phosphatidylinositol 3-kinase; nuclear factor-[kappa]B; cytokines; colitis doi: 10.1152/ajpcell.00461.2009.

Infusion of ANG II in hyperlipidemic mice augments atherosclerosis and causes formation of abdominal aortic aneurysms (AAAs). The purpose of this study was to define the contribution of ANG II-induced hypertension to these vascular pathologies. Male apolipoprotein E (apoE)- and LDL receptor (LDLr)-deficient mice were infused with ANG II (1,000 ng x [kg.sup.-1] x [min.sup.-1]) or norepinephrine (NE; 5.6 mg x [kg.sup.-1] x [day.sup.-1]) for 28 days. Infusion of ANG II or NE increased mean arterial pressure (MAP; ANG II, 133 [+ or -] 2.8; NE, 129 [+ or -] 13 mmHg) to a similar extent compared with baseline blood pressures (MAP, 107 [+ or -] 2 mmHg). Abdominal aortic width increased in both apoE-deficient (apo[E.sup.-/-]) or LDLr-deficient (LD[Lr.sup.-/-]) mice infused with ANG II (apo[E.sup.-/-]: 1.4 [+ or -] 0.1; LD[Lr.sup.-/-]: 1.6 [+ or -] 0.2 mm). In contrast, NE did not change diameters of abdominal aortas (apo[E.sup.-/-]: 0.91 [+ or -] 0.03; LD[Lr.sup.-/-]: 0.87 [+ or -] 0.02 mm). Similarly, atherosclerotic lesions in aortic arches were much greater in mice infused with ANG II compared with NE. At a subpressor infusion rate of ANG II (500 ng x [kg.sup.-1] x [min.sup.-1]), AAAs developed in 50% of apo[E.sup.-/-] mice. Alternatively, administration of hydralazine (250 mg/l) to ANG II-infused apo[E.sup.-/-] mice (1,000 ng x [kg.sup.-1] x [min.sup.-1]) lowered systolic blood pressure (day 28: ANG II, 157 [+ or -] 6; ANG II/hydralazine, 135 [+ or -] 6 mmHg) but did not prevent AAA formation or atherosclerosis. These results demonstrate that infusion of ANG II to hyperlipidemic mice induces AAAs and augments atherosclerosis independent of increased blood pressure. aneurysms; hypertension; vascular lesions

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

Ulcerative colitis is an autoimmune-inflammatory disease characterized by increased proliferation of colonic epithelial cells, dysregulation of signal transduction pathways, elevated mucosal T cell activation, increased production of proinflammatory cytokines, and enhanced leukocyte infiltration into colonic interstitium. Several compounds that possess antiproliferative properties and/or inhibit cytokine production exhibit a therapeutic effect in murine models of colitis. Mammalian target of rapamycin (mTOR), a protein kinase regulating cell proliferation, is implicated in colon carcinogenesis. In this study, we report that a novel haloacyl aminopyridine-based molecule (P2281) is a mTOR inhibitor and is efficacious in a murine model of human colitis. In vitro studies using Western blot analysis and cell-based ELISA assays showed that P2281 inhibits mTOR activity in colon cancer cells. In vitro and in vivo assays of proinflammatory cytokine production revealed that P2281 diminishes induced IFN-[gamma] production but not TNF-[alpha] production, indicating preferential inhibitory effects of P2281 on T cell function. In the dextran sulfate sodium (DSS) model of colitis, 1) macroscopic colon observations demonstrated that P2281 significantly inhibited DSS-induced weight loss, improved rectal bleeding index, decreased disease activity index, and reversed DSS-induced shortening of the colon; 2) histological analyses of colonic tissues revealed that P2281 distinctly attenuated DSS-induced edema, prominently diminished the leukocyte infiltration in the colonic mucosa, and resulted in protection against DSS-induced crypt damage; and 3) Western blot analysis showed that P2281 blocks DSS-induced activation of mTOR. Collectively, these results provide direct evidence that P2281, a novel mTOR inhibitor, suppresses DSS-induced colitis by inhibiting T cell function and is a potential therapeutic for colitis. Given that compounds with anticancer activity show promising anti-inflammatory efficacy, our findings reinforce the cross-therapeutic functionality of potential drugs. colon; inflammation; T cells; IFN-[gamma]; mammalian target of rapamycin; dextran sulfate sodium

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

Adipose tissue expresses components of the renin-angiotensin system (RAS). Angiotensin converting enzyme (ACE2), a new component of the RAS, catabolizes the vasoconstrictor peptide ANG II to form the vasodilator angiotensin 1-7 [ANG-(1-7)]. We examined whether adipocytes express ACE2 and its regulation by manipulation of the RAS and by high-fat (HF) feeding. ACE2 mRNA expression increased (threefold) during differentiation of 3T3-L1 adipocytes and was not regulated by manipulation of the RAS. Male C57BL/6 mice were fed low- (LF) or high-fat (HF) diets for 1 wk or 4 mo. At 1 wk of HF feeding, adipose expression of angiotensinogen (twofold) and ACE2 (threefold) increased, but systemic angiotensin peptide concentrations and blood pressure were not altered. At 4 mo of HF feeding, adipose mRNA expression of angiotensinogen (twofold) and ACE2 (threefold) continued to be elevated, and liver angiotensinogen expression increased (twofold). However, adipose tissue from HF mice did not exhibit elevated ACE2 protein or activity. Increased expression of ADAM17, a protease responsible for ACE2 shedding, coincided with reductions in ACE2 activity in 3T3-L1 adipocytes, and an ADAM17 inhibitor decreased media ACE2 activity. Moreover, ADAM17 mRNA expression was increased in adipose tissue from 4-mo HF-fed mice, and plasma ACE2 activity increased. However, HF mice exhibited marked increases in plasma angiotensin peptide concentrations (LF: 2,141 [+ or -] 253; HF: 6,829 [+ or -] 1,075 pg/ml) and elevated blood pressure. These results demonstrate that adipocytes express ACE2 that is dysregulated in HF-fed mice with elevated blood pressure compared with LF controls. angiotensin I-converting enzyme; angiotensin

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

We aimed to determine 1) the mechanism(s) that enables glucose-6-phosphate dehydrogenase (G6PD) to regulate serum response factor (SRF)- and myocardin (MYOCD)-driven smooth muscle cell (SMC)-restricted gene expression, a process that aids in the differentiation of SMCs, and 2) whether G6PD-mediated metabolic reprogramming contributes to the pathogenesis of vascular diseases in metabolic syndrome (MetS). Inhibition of G6PD activity increased (>30%) expression of SMC-restricted genes and concurrently decreased (40%) the growth of human and rat SMCs ex vivo. Expression of SMC-restricted genes decreased (>100-fold) across successive passages in primary cultures of SMCs isolated from mouse aorta. G6PD inhibition increased Myh11 (47%) while decreasing (>50%) Sca-1, a stem cell marker, in cells passaged seven times. Similarly, CRISPR-Cas9-mediated expression of the loss-of-function Mediterranean variant of G6PD (S188F; G6PDS188F) in rats promoted transcription of SMC-restricted genes. G6PD knockdown or inhibition decreased (48.5%) histone deacetylase (HDAC) activity, enriched (by 3-fold) H3K27ac on the Myocd promoter, and increased Myocd and Myh11 expression. Interestingly, G6PD activity was significantly higher in aortas from JCR rats with MetS than control Sprague-Dawley (SD) rats. Treating JCR rats with epiandrosterone (30 mg/kg/day), a G6PD inhibitor, increased expression of SMC-restricted genes, suppressed Serpine1 and Epha4, and reduced blood pressure. Moreover, feeding SD control (littermates) and G6PDS188F rats a high-fat diet for 4mo increased Serpine1 and Epha4 expression and mean arterial pressure in SD but not G6PDS188F rats. Our findings demonstrate that G6PD downregulates transcription of SMC-restricted genes through HDAC-dependent deacetylation and potentially augments the severity of vascular diseases associated with MetS. [ABSTRACT FROM AUTHOR]

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

Nitric oxide (NO) donors generally relax vascular preparations through cGMP-mediated mechanisms. Relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and coronary arteries to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) is almost eliminated by inhibition of soluble guanylate cyclase activation with 10 [micro]M 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), whereas only a modest inhibition of relaxation is observed under hypoxia (P[O.sub.2] = 8-10 Torr). This effect of hypoxia is independent of the contractile agent used and is also observed with NO gas. ODQ eliminated SNAP-induced increases in cGMP under hypoxia in BPA. cGMP-independent relaxation of BPA to SNAP was not attenuated by inhibition of [K.sup.+] channels (10 mM tetraethylammonium), myosin light chain phosphatase (0.5 [micro]M microcystin-LR), or adenylate cyclase (4 [micro]M 2',5'-dideoxyadenosine). SNAP relaxed BPA contracted with serotonin under [Ca.sup.2+]-free conditions in the presence of hypoxia and ODQ, and contraction to [Ca.sup.2+] readdition was also attenuated. The sarcoplasmic reticulum [Ca.sup.2+]-reuptake inhibitor cyclopiazonic acid (0.2 mM) attenuated SNAp-mediated relaxation of BPA in the presence of ODQ. Thus hypoxic conditions appear to promote a cGMP-independent relaxation of BPA to NO by enhancing sarcoplasmic reticulum [Ca.sup.2+] reuptake. calcium; guanylate cyclase; nitrovasodilator

Pyridine nucleotides, such as NADPH and NADH, are emerging as critical players in the regulation of heart and vascular function. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is the primary source and regulator of cellular NADPH. In the current study, we have identified two isoforms of G6PD (slow and fast migrating) and functionally characterized the slow migrating isoform of G6PD (G6PD545) in bovine and human arteries. We found that G6PD545 is eluted in the caveolae fraction of vascular smooth muscle (VSM) and has a higher maximum rate of reaction (Vmax: 1.65-fold) than its fast migrating isoform (G6PD515). Interestingly, caveolae G6PD forms a complex with the pore-forming α1C-subunit of the L-type Ca2+ channel, Cav1.2, as demonstrated by a proximity ligation assay in fixed VSMCs. Additionally, Förster resonance energy transfer (FRET) analysis of HEK293-17T cells cotransfected with red fluorescent protein (RFP)-tagged G6PD545 (C-G6PD545) and green fluorescent protein (GFP)-tagged Cav1.2-(Cav1.2-GFP) demonstrated strong FRET signals as compared with cells cotransfected with Cav1.2-GFP and C-G6PD515. Furthermore, L-type Ca2+ channel conductance was larger and the voltage-independent component of availability (c1) was augmented in C-G6PD545 and Cav1.2-GFP cotransfectants compared with those expressing Cav1.2-GFP alone. Surprisingly, epiandrosterone, a G6PD inhibitor, disrupted the G6PD-Cav1.2 complex, also decreasing the amplitude of L-type Ca2+ currents and window currents, thereby reducing the availability of the c1 component. Moreover, overexpression of adeno-G6PD545-GFP augmented the KCl-induced contraction in coronary arteries compared with control. To determine whether overexpression of G6PD had any clinical implication, we investigated its activity in arteries from patients and rats with metabolic syndrome and found that G6PD activity was high in this disease condition. Interestingly, epiandrosterone treatment reduced elevated mean arterial blood pressure and peripheral vascular resistance in metabolic syndrome rats, suggesting that the increased activity of G6PD augmented vascular contraction and blood pressure in the metabolic syndrome. These data suggest that the novel G6PD-Cav1.2 interaction, in the caveolae fraction, reduces intrinsic voltage-dependent inactivation of the channel and contributes to regulate VSM L-type Ca2+ channel function and Ca2+ signaling, thereby playing a significant role in modulating vascular function in physiological/pathophysiological conditions. NEW & NOTEWORTHY In this study we have identified a novel isozyme of glucose-6-phosphate dehydrogenase (G6PD), a metabolic enzyme, that interacts with and contributes to regulate smooth muscle cell l-type Ca2+ ion channel function, which plays a crucial role in vascular function in physiology and pathophysiology. Furthermore, we demonstrate that expression and activity of this novel G6PD isoform are increased in arteries of individuals with metabolic syndrome and in inhibition of G6PD activity in rats of metabolic syndrome reduced blood pressure. [ABSTRACT FROM AUTHOR]

The effect of metabolic alterations in NADH oxidase-derived O2- on NO-elicited bovine pulmonary artery (BPA) relaxation and soluble guanylate cyclase (sGC) was investigated. Lactate and pyruvate were used to control NADH-dependent O2- production in BPA. Results indicated that cytosolic NADH redox and Cu/Zn superoxide dismutase play important roles in regulating the inhibitory effects of O2- derived from NADH oxidase and sGC activity.

Rat hearts were analyzed after O2 perfusion to determine the effects of nitroglycerin (NTG) on the cardiac production and release of prostaglandin F2alpha (PGF2alpha) and thromboxane A2 (TxA2). O2 perfusion of rat hearts enhanced the release of PGF2alpha and TxA2 which augmented vasoconstriction indicating the role of prostaglandins in vasoconstriction during coronary perfusion pressure. Furthermore, the activity of NO was inactived by O2 perfusion by regulating prostaglandin synthesis.

Pulmonary hypertension (PH) is a multicellular and progressive disease with a high mortality rate. Among many cell types, hematopoietic stem cells (HSCs) are incriminated in the pathogenesis of PH. However, our understanding of the mechanisms that increase HSCs in blood and lungs of hypertensive animals or patients and the role played by HSCs in the pathogenesis of PH remains elusive. Studies suggest that glycolysis is critical for the survival and growth of HSCs. In various cell types from hypertensive lungs of animals and patients, glycolysis and the glucose-6-phosphate dehydrogenase (G6PD) activity are increased. Herein, we demonstrated in mice that chronic hypoxia increased HSCs (CD34+, CD117+, CD133+, CD34+/CD117+, and CD34+/CD133+) in bone marrow and blood and around hypertensive pulmonary arteries in a time-dependent manner. Intriguingly, we found fewer CD133+ cells in the bone marrow of C57BL/6 mice compared with Sv129J mice, and C57BL mice developed less severe chronic hypoxia-elicited PH and heart failure than Sv129J mice. Similarly, the numbers of CD34+ and CD117+ cells in blood of patients with pulmonary arterial hypertension (PAH) were higher (>3-fold) compared with healthy individuals. By allogeneic bone marrow transplantation, we found that GFP+ bone marrow cells infiltrated the lungs and accumulated around the pulmonary arteries in lungs of hypoxic mice, and these cells contributed to increased α-adrenergic receptor-mediated contraction of the pulmonary artery cultured in hypoxia. Inhibition of G6PD activity with (3β,5α)-3,21-dihydroxypregnan-20-one, a novel and potent G6PD inhibitor, decreased HSCs in bone marrow, blood, and lungs of hypoxic mice and reduced α-agonist-induced contraction of the pulmonary artery and established hypoxia-induced PH. We did not observe CD133+ cells around the pulmonary arteries in the lungs of chronically hypoxic G6PD-deficient mice. Furthermore, knockdown of G6PD and inhibition of G6PD activity: 1) downregulated canonical and noncanonical Wnt and Fzd receptors genes; 2) upregulated Bmpr1a; 3) decreased Cxcl12, and 4) reduced HSC (CD117+ and CD133+) numbers. In all, our findings demonstrate unexpected function for bone marrow-derived HSCs in augmenting α-adrenergic receptor-mediated contraction of pulmonary arteries and remodeling of pulmonary arteries that contribute to increase pulmonary vascular resistance in PAH patients and hypoxic mice and suggest that G6PD, by regulating expression of genes in the WNT and BMPR signaling, contributed to increase and release of HSCs from the bone marrow in response to hypoxic stimuli. [ABSTRACT FROM AUTHOR]

Children surviving cancer and chemotherapy are at risk for adverse health events including heart failure that may be delayed by years. Although the early effects of doxorubicin-induced cardiotoxicity may be attributed to a direct effect on the cardiomyocytes, the mechanisms underlying the delayed or late effects (8–20 yr) are unknown. The goal of this project was to develop a model of late-onset doxorubicin-induced cardiotoxicity to better delineate the underlying pathophysiology responsible. The underlying hypothesis was that doxorubicin-induced “late-onset cardiotoxicity” was the result of mitochondrial dysfunction leading to cell failure and death. Wistar rats, 3–4 wk of age, were randomly assigned to vehicle or doxorubicin injection groups (1–45 mg/kg). Cardiovascular function was unaltered at the lower dosages (1–15 kg/mg), but beginning at 6 mo after injection significant cardiac degradation was observed in the 45 mg/kg group. Doxorubicin significantly increased myocardial mitochondrial DNA (mtDNA) damage. In contrast, in isolated c-kit left ventricular (LV) cells, doxorubicin treatment did not increase mtDNA damage. Biomarkers of senescence within the LV were significantly increased, suggesting accelerated aging of the LV. Doxorubicin also significantly increased LV histamine content suggestive of mast cell activation. With the use of flow cytometry, a significant expansion of the c-kit and stage-specific embryonic antigen 1 cell populations within the LV were concomitant with significant decreases in the circulating peripheral blood population of these cells. These results are consistent with the concept that doxorubicin induced significant damage to the cardiomyocyte population and that although the heart attempted to compensate it eventually succumbed to an inability for self-repair. [ABSTRACT FROM AUTHOR]