Platelet regulation by NO/cGMP signaling and NAD(P)H oxidase-generated ROS

Platelets play a crucial role in the physiology of primary hemostasis and pathophysiological processes such as arterial thrombosis. Accumulating evidence suggests a key regulatory role of both NO and reactive oxygen species (ROS) in platelets. While the inhibitory role of NO/cGMP signaling in both murine and human platelets is well established, recent data suggest that intracellular ROS generation is involved in platelet activation. Thrombin-induced intracellular ROS production was inhibited by NAD(P)H oxidase inhibitors (DPI and apocynin), cyclooxygenase inhibitor (acetylsalicylic acid), and superoxide scavengers (tiron and MnTMPyP). Furthermore, thrombin (Trap6)-induced platelet aggregation and thrombus formation on collagen under high shear was inhibited by NAD(P)H oxidase inhibitors (DPI and apocynin), whereas secretion and platelet shape change were not affected. Inhibition of alphaIIbbeta3 activation by NAD(P)H oxidase inhibitors and superoxide scavengers was independent of NO/cGMP signaling demonstrating a direct role of platelet NAD(P)H oxidase-generated ROS for integrin alphaIIbbeta3 activation.     

Upregulation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase activity increases oxidative stress in failing human heart

BackgroundWe previously found that higher NADPH levels produced by glucose-6-phosphate dehydrogenase (G6PD) can enhance myocardial superoxide generation by NAD(P)H oxidase in a dog model of dilated cardiomyopathy. Therefore, we tested whether G6PD activity is elevated and enhances NADPH level and increases NAD(P)H oxidase-derived superoxide production in the myocardium from patients with heart failure from ischemic cardiomyopathy.Methods and ResultsSurgical discards of left ventricle were collected from 8 congestive heart failure patients undergoing surgical ventricular restoration procedures, whereas control left ventricle tissue was obtained from 5 normal donor hearts deemed not suitable for transplantation. Biochemical assays were performed in tissue homogenates. We found that superoxide and hydrogen peroxide were elevated, respectively, by 9- and 3-fold in failing versus normal hearts (P < .05). The NAD(P)H oxidase inhibitors gp91^ds-tat, apocynin, and diphenyleneiodonium, significantly inhibited superoxide generation by approximately 75%, 89%, and 91%, respectively. Superoxide production by NAD(P)H oxidase increased 10- and 3-fold by adding NADPH (100 μmol/L) and NADH (100 μmol/L), respectively, in a DPI- and gp91^ds-tat–inhibitable manner. Interestingly, chelerythrine, a PKC inhibitor, and PP2, a Src kinase family inhibitor, reduced G6PD activity (0.29 ± 0.04 nM·min·mg protein) by 50% and 51% and these inhibitors also decreased myocardial superoxide by 99% and 79%, respectively. Furthermore, 6-aminonicotinamide, a G6PD inhibitor, decreased myocardial superoxide production by 71%.ConclusionsThese data suggest that high NAD(P)H oxidase, fueled by G6PD-derived NADPH, generates most of the superoxide in failing hearts of patients with ischemic cardiomyopathy. In addition, PKC-Src kinase signaling pathways seem to coordinate the activation of both G6PD and NAD(P)H oxidase in human cardiac muscle.     

Genetic and pharmacological analyses of Syk function in alphaIIbbeta3 signaling in platelets

Agonists induce inside-out alphaIIbbeta3 signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in alphaIIbbeta3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of alphaIIbbeta3 by adenine diphosphate (ADP) +/- epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in alphaIIbbeta3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits alphaIIbbeta3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.     

Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling

Fibrinogen binding to integrin alphaIIbbeta3 mediates platelet aggregation and requires agonist-induced "inside-out" signals that increase alphaIIbbeta3 affinity. Agonist regulation of alphaIIbbeta3 also takes place in megakaryocytes, the bone marrow cells from which platelets are derived. To facilitate mechanistic studies of inside-out signaling, we describe here the generation of megakaryocytes in quantity from murine embryonic stem (ES) cells. Coculture of ES cells for 8-12 days with OP9 stromal cells in the presence of thrombopoietin, IL-6, and IL-11 resulted in the development of large, polyploid megakaryocytes that produced proplatelets. These cells expressed alphaIIbbeta3 and platelet glycoprotein Ibalpha but were devoid of hematopoietic stem cell, erythrocyte, and leukocyte markers. Mature megakaryocytes, but not megakaryocyte progenitors, specifically bound fibrinogen by way of alphaIIbbeta3 in response to platelet agonists. Retrovirus-mediated expression of the reporter gene, green fluorescent protein, in ES cell-derived megakaryocytes did not affect viability or alphaIIbbeta3 function. On the other hand, retroviral expression of CalDAG-GEFI, a Rap1 exchange factor identified by megakaryocyte gene profiling as a candidate integrin regulator, enhanced agonist-induced activation of Rap1b and fibrinogen binding to alphaIIbbeta3 (P < 0.01). These results establish that ES cells are a ready source of mature megakaryocytes for integrin studies and other biological applications, and they implicate CalDAG-GEFI in inside-out signaling to alphaIIbbeta3.     

NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment

Platelets, although not phagocytotic, have been suggested to release O. Since O-producing reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases can be specifically activated by certain agonists and are found in several nonphagocytotic tissues, we investigated whether such an enzyme is the source of platelet-derived O. We further studied which agonists cause platelet O release and whether platelet-derived O influences thrombus formation in vitro. Collagen, but not adenosine 5'-diphosphate (ADP) or thrombin, increased O formation in washed human platelets. This was a reduced nicotinamide adenine dinucleotide (NADH)-dependent process, as shown in platelet lysates. Consistent with a role of a platelet, NAD(P)H oxidase expression of its subunits p47(phox) and p67(phox) and inhibition of platelet O formation by diphenylene-iodoniumchloride (DPI) and by the specific peptide-antagonist gp91ds-tat were observed. Whereas platelet-derived O did not influence initial aggregation, platelet recruitment to a preformed thrombus following collagen stimulation was significantly attenuated by superoxide dismutase (SOD) or DPI. It was also inhibited when ADP released during aggregation was cleaved by the ectonucleotidase apyrase. ADP in supernatants of collagen-activated platelets was decreased in the presence of SOD, resulting in lower ADP concentrations available for recruitment of further platelets. Exogenous O increased ADP- concentrations in supernatants of collagen-stimulated platelets and induced irreversible aggregation when platelets were stimulated with otherwise subthreshold concentrations of ADP. These results strongly suggest that collagen activation induces NAD(P)H oxidase-dependent O release in platelets, which in turn enhances availability of released ADP, resulting in increased platelet recruitment.     

ADAP is required for normal alphaIIbbeta3 activation by VWF/GP Ib-IX-V and other agonists

Interaction between von Willebrand factor (VWF) and platelet GP Ib-IX-V is required for hemostasis, in part because intracellular signals from VWF/GP Ib-IX-V activate the ligand-binding function of integrin alphaIIbbeta3. Because they also induce tyrosine phosphorylation of the ADAP adapter, we investigated ADAP's role in GP Ib-IX-V signal transduction. Fibrinogen or ligand-mimetic POW-2 Fab binding to alphaIIbbeta3 was stimulated by adhesion of ADAP+/+ murine platelets to dimeric VWF A1A2 but was significantly reduced in ADAP-/- platelets (P<.01). alphaIIbbeta3 activation by ADP or a Par4 thrombin receptor agonist was also decreased in ADAP-/- platelets. ADAP stabilized the expression of another adapter, SKAP-HOM, via interaction with the latter's SH3 domain. However, no abnormalities in alphaIIbbeta3 activation were observed in SKAP-HOM-/- platelets, which express normal ADAP levels, further implicating ADAP as a modulator of alphaIIbbeta3 function. Under shear flow conditions over a combined surface of VWF A1A2 and fibronectin to test interactions involving GP Ib-IX-V and alphaIIbbeta3, respectively, ADAP-/- platelets displayed reduced alphaIIbbeta3-dependent stable adhesion. Furthermore, ADAP-/- mice demonstrated increased rebleeding from tail wounds. These studies establish ADAP as a component of inside-out signaling pathways that couple GP Ib-IX-V and other platelet agonist receptors to alphaIIbbeta3 activation.     

Acute pressor effect of central angiotensin II is mediated by NAD(P)H-oxidase-dependent production of superoxide in the hypothalamic cardiovascular regulatory nuclei

BACKGROUND: Centrally applied angiotensin II (Ang II) increases sympathetic nervous activity and mean arterial blood pressure (MAP), but the mediation of these effects is not fully understood. OBJECTIVE: To test the hypothesis that central effects of Ang II are mediated by reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H]-oxidase-dependent production of superoxide in the hypothalamus. METHODS: Under isoflurane anesthesia, male Sprague-Dawley rats were given an intracerebroventricular infusion of either artificial cerebrospinal fluid or apocynin (4 microg/kg per min), a selective inhibitor for NAD(P)H oxidase, for 30 min, followed by Ang II (20 ng) or carbachol (200 ng), while MAP and heart rate were measured at the femoral artery. At the end of the experiments, hydroethidine, a superoxide-sensitive fluorescent dye, was infused intravenously for 10 min, and superoxide production was assessed in the vasoregulatory hypothalamic nuclei using confocal microscopy. RESULTS: Ang II elicited a rapid 11 +/- 2-mmHg increase in MAP and a 16 +/- 2-beats/min decrease in heart rate. Apocynin abolished these effects of Ang II in a specific manner, as carbachol-induced increases in MAP were unaffected by the inhibition of NAD(P)H oxidase (MAP increased by 9 +/- 2 and 8 +/- 1 mmHg in the absence and presence of apocynin, respectively). In response to Ang II, apocynin-sensitive production of superoxide increased significantly in the nuclei of the anterior hypothalamus, in the subfornical organ, and in the paraventricular nucleus of the hypothalamus. CONCLUSION: These findings demonstrate that acute pressor responses of central Ang II are mediated by NAD(P)H-oxidase-dependent production of superoxide in the hypothalamus.     

UV-C irradiation disrupts platelet surface disulfide bonds and activates the platelet integrin alphaIIbbeta3

UV-C irradiation has been shown to be effective for pathogen reduction in platelet concentrates, but preliminary work indicated that UV-C irradiation of platelets can induce platelet aggregation. In this study, the mechanism underlying this phenomenon was investigated. Irradiation of platelets with UV-C light (1500 J/m(2)) caused platelet aggregation, which was dependent on integrin alphaIIbbeta3 activation (GPIIb/IIIa). This activation occurred despite treatment with several signal transduction inhibitors known to block platelet activation. UV-C also induced activation of recombinant alphaIIbbeta3 in Chinese hamster ovary (CHO) cells, an environment in which physiologic agonists fail to activate. Activation of alphaIIbbeta3 requires talin binding to the beta3 tail, yet alphaIIbbeta3-Delta724 (lacking the talin binding site) was activated by UV-C irradiation, excluding a requirement for talin binding. The UV-C effect appears to be general in that beta(1) and beta(2) integrins are also activated by UV-C. To explain these findings, we investigated the possibility of UV-C-induced photolysis of disulfide bonds, in analogy with the activating effect of reducing agents on integrins. Indeed, UV-C induced a marked increase in free thiol groups in platelet surface proteins including alphaIIbbeta3. Thus, UV-C appears to activate alphaIIbbeta3 not by affecting intracellular signal transduction, but by reduction of disulfide bonds regulating integrin conformation.     

Serotonin 5-HT(2B) receptor blockade prevents reactive oxygen species-induced cardiac hypertrophy in mice

We established previously that 5-HT(2B) receptors are involved in cardiac hypertrophy through the regulation of hypertrophic cytokines in cardiac fibroblasts. Moreover, the generation of reactive oxygen species and tumor necrosis factor-alpha through the activation of reduced nicotinamide-adenine dinucleotide phosphate [NAD(P)H] oxidase has been implicated in cardiac hypertrophy. In this study, we investigated whether 5-HT(2B) receptors could be involved in the development of cardiac hypertrophy associated with superoxide anion production. Therefore, we measured the effects of serotonergic 5-HT(2B) receptor blockade on left-ventricular superoxide anion generation in 2 established pharmacological models of cardiac hypertrophy, ie, angiotensin II and isoproterenol infusions in mice. Angiotensin II infusion for 14 days increased superoxide anion concentration (+32%), NAD(P)H oxidase maximal activity (+84%), and p47(phox) NAD(P)H oxidase subunit expression in the left ventricle together with hypertension (+37 mm Hg) and cardiac hypertrophy (+17% for heart weight:body weight). The 5-HT(2B) receptor blockade by a selective antagonist (SB215505) prevented the increase in cardiac superoxide generation and hypertrophy. Similarly, infusion for 5 days of isoproterenol increased left-ventricular NAD(P)H oxidase activity (+48%) and cardiac hypertrophy (+31%) that were prevented by the 5-HT(2B) receptor blockade. Finally, in the primary culture of left-ventricular cardiac fibroblasts, angiotensin II and isoproterenol stimulated NAD(P)H oxidase activity. This activation was prevented by SB215505. These findings suggest that the 5-HT(2B) receptor may represent a new target to reduce cardiac hypertrophy and oxidative stress. Its blockade affects both angiotensin II and beta-adrenergic trophic responses without significant hemodynamic alteration.     

Generation of reactive oxygen species in blood platelets

The generation of superoxide anion radicals (O . - 2 ) and the other reactive oxygen species (ROS) was estimated by means of cytochrome c reduction and chemiluminescence, as well in resting blood platelets and in platelets stimulated by thrombin in the presence or absence of some inhibitors of pathways involved in platelet activation. We used allopurinol (xanthine oxidase inhibitor), wortmannin (PI 3-kinase inhibitor) and staurosporine (protein kinase C inhibitor). To determine the involvement of the glutathione in ROS generation, we used L-buthionine sulfoximine (BSO) which blocks GSH synthesis. Our results confirmed that thrombin stimulates the production of ROS concomitant with metabolism of arachidonate and production of malonyldialdehyde (MDA) in blood platelets ( P < 0.05) and showed that, in the presence of inhibitors, the generation of ROS in platelets (resting and stimulated) was reduced. This indicates that xanthine oxidase, PI 3-kinase or protein kinase C take part in the formation of ROS in blood platelets. Moreover, adhesion of platelets to fibrinogen and secretion of adenine nucleotides from platelets after wortmannin and staurosporine action was also inhibited. BSO not only decreased GSH level, but also reduced the amount of ROS; a correlation between the depletion of GSH and the decrease of ROS was observed ( R = -0.987; P < 0.02). It is concluded that in blood platelets, ROS are produced in the receptor-mediated signaling pathways and platelet activation (arachidonic acid metabolism, the glutathione cycle, metabolism of phosphoinositoides and due to xanthine oxidase). Our results support the importance of ROS in platelet function.     

Generation of reactive oxygen species in blood platelets

The generation of superoxide anion radicals (O2*-) and the other reactive oxygen species (ROS) was estimated by means of cytochrome c reduction and chemiluminescence, as well in resting blood platelets and in platelets stimulated by thrombin in the presence or absence of some inhibitors of pathways involved in platelet activation. We used allopurinol (xanthine oxidase inhibitor), wortmannin (PI 3-kinase inhibitor) and staurosporine (protein kinase C inhibitor). To determine the involvement of the glutathione in ROS generation, we used L-buthionine sulfoximine (BSO) which blocks GSH synthesis. Our results confirmed that thrombin stimulates the production of ROS concomitant with metabolism of arachidonate and production of malonyldialdehyde (MDA) in blood platelets (P < 0.05) and showed that, in the presence of inhibitors, the generation of ROS in platelets (resting and stimulated) was reduced. This indicates that xanthine oxidase, PI 3-kinase or protein kinase C take part in the formation of ROS in blood platelets. Moreover, adhesion of platelets to fibrinogen and secretion of adenine nucleotides from platelets after wortmannin and staurosporine action was also inhibited. BSO not only decreased GSH level, but also reduced the amount of ROS; a correlation between the depletion of GSH and the decrease of ROS was observed (R = -0.987; P < 0.02). It is concluded that in blood platelets, ROS are produced in the receptor-mediated signaling pathways and platelet activation (arachidonic acid metabolism, the glutathione cycle, metabolism of phosphoinositoides and due to xanthine oxidase). Our results support the importance of ROS in platelet function.     

GPIIb/IIIa antagonists and other anti-integrins

Platelet aggregation involves the binding of adhesive proteins (fibrinogen, von Willebrand factor) to the alphaIIbbeta3 integrin, which assumes a high-affinity state for adhesive proteins during platelet activation. The occupied integrin sends signals back into the platelet, and the bound adhesive protein forms the bridges linking platelets together. Anti-integrin therapy is designed to inhibit this process in arterial thrombosis. Abciximab, mouse-human chimeric Fab fragments, blocks platelet aggregation and provides proven clinical benefit in acute situations such as in patients with unstable angina undergoing angioplasty or stenting. Eptifibatide and tirofiban are small molecular mass inhibitors also in current use. In contrast, oral inhibitors of alphaIIbbeta3 have proved disappointing, provoking increased mortality without assuring an adequate blockade of alphaIIbbeta3. The problems of using anti-integrin therapy are discussed in this article as are ways of improving its efficacity. Final thoughts provide ideas for a new generation of inhibitors.     

ADMA impairs nitric oxide-mediated arteriolar function due to increased superoxide production by angiotensin II-NAD(P)H oxidase pathway

Asymmetrical dimethylarginine (ADMA) is thought to be an endogenous regulator of arteriolar tone by inhibiting NO synthase. However, our previous studies showed that, in isolated arterioles, ADMA induced superoxide production as well. Thus, the mechanisms by which ADMA affects arteriolar tone remain obscure. We hypothesized that ADMA, by activating NAD(P)H oxidase, increases superoxide production, interfering with NO mediation of flow-induced dilation. In the presence of indomethacin, isolated arterioles from rat gracilis muscle ( approximately 160 microm at 80 mm Hg) were incubated with ADMA (10(-4) mol/L), which elicited significant constriction (from 162+/-4 to 143+/-4 microm) and eliminated the dilations to increases in intraluminal flow (from a maximum 31+/-2% to 3+/-1%; P<0.05). In the presence of ADMA, superoxide dismutase plus catalase restored dilations to flow (from a maximum 3+/-1% to 28+/-2%). Endothelial denudation or incubation of arterioles with the NAD(P)H oxidase inhibitor apocynin or the angiotensin-converting enzyme inhibitor quinapril inhibited ADMA-induced constriction. In addition, apocynin, quinapril, or the angiotensin type 1 receptor blocker losartan restored flow-induced dilations reduced by ADMA. Furthermore, inhibition of NO synthase abolished the "superoxide dismutase/catalase-restored" flow-induced dilation in the presence of ADMA. ADMA-induced increased production of superoxide, assessed by dihydroethidium fluorescence, was inhibited by apocynin, quinapril, or losartan. We suggest that ADMA activates the local renin-angiotensin system, and the angiotensin II released activates NAD(P)H oxidase; superoxide produced interferes with the bioavailability of NO, resulting in diminished flow-induced dilation, a mechanism that may contribute to the development of arteriolar dysfunction and increased tone associated with elevated ADMA levels.     

Platelet-mediated alterations in cardiac cellular electrophysiology

Recent clinical and experimental evidence indicates that platelet activation contributes to the arrhythmogenic effects of myocardial ischaemia, but little is known about the electrophysiological effects produced by controlled platelet activation under conditions of normal perfusion and how these might relate to effects during ischaemia. To investigate this, we studied changes in cardiac cellular electrophysiology and arrhythmogenesis during infusion of platelets [10(8)/ml] in isolated, perfused guinea-pig hearts during normal perfusion and global myocardial ischaemia. Hearts were studied in four groups: group A (n = 4) receiving frozen/thawed (activated) platelets; group B (n = 4) receiving normal platelets in the presence of 10(-9) M platelet activating factor (PAF); group C (n = 9) receiving buffer only during normal perfusion and myocardial ischaemia; group D (n = 9) receiving platelets during normal perfusion and myocardial ischaemia. Infusion of platelets (group D) had no effects during normal perfusion, but activated platelets (group A) decreased action potential duration (APD) from 165 +/- 1 ms to 138 +/- 5 ms (mean +/- SE) at 15 min of normal perfusion (P less than 0.02) and produced ventricular fibrillation (VF) in 3/4 at 21 +/- 1 min. Infusion of platelets in the presence of PAF (group B) produced similar reductions of APD during normal perfusion and VF in 2/4. During ischaemia, platelets (group D) increased the incidence of VF (100% vs 56% group C, P less than 0.05) and enhanced the ischaemia-induced reductions in APD (107 +/- 3 ms vs 121 +/- 5 ms (group C) P less than 0.05 at 15 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calreticulin-independent regulation of the platelet integrin alphaIIbbeta3 by the KVGFFKR alphaIIb-cytoplasmic motif

The platelet integrin alphaIIbbeta3 alters conformation in response to platelet activation and ligand binding, although the molecular mechanisms involved are not known. We previously showed that a lipid modified peptide, corresponding to the membrane proximal 989KVGFFKR995 portion of the alphaIIb cytoplasmic tail, independently activates platelet alphaIIbbeta3. Calreticulin (CRT) is a potential integrin regulatory protein based on its interaction with the highly conserved alpha-integrin sequence KxGFFKR. We therefore examined the possible interaction of calreticulin and alphaIIbbeta3 in human platelets. We demonstrate that calreticulin in platelets is localised to the granulomere. In contrast, the known integrin-binding protein talin accumulates at the periphery of spreading platelets and colocalises with alphaIIbbeta3 during the process of adhesion. An interaction between calreticulin and alphaIIbbeta3 could not be demonstrated using co-immunoprecipitation techniques under various platelet activation states, even in the presence of covalent chemical crosslinkers. Thus, calreticulin does not functionally interact with the major integrin in human platelets. In order to identify proteins that interact with the integrin KVGFFKR motif we then used a peptide 'pull-down' assay from platelet lysates with biotinylated peptides and demonstrate that only the alphaIIb and beta3 subunits selectively and individually interact with this sequence. This interaction is divalent cation-dependent, has high-affinity, and occurs both with purified alphaIIbbeta3 complex and with electroeluted alpha and beta subunits. Thus, our data show that the conserved integrin KVGFFKR domain interacts primarily with the alpha and beta cytoplasmic tails and not with CRT in human platelets.     

Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca2+ channel function during acute hypoxia

OBJECTIVE: The role of NAD(P)H oxidase in regulating cellular production of reactive oxygen species (ROS) and the L-type Ca2+ channel during acute hypoxia was examined in adult ventricular myocytes from guinea pig. METHODS: The fluorescent indicator dihydroethidium (DHE) was used to detect superoxide and the response of the L-type Ca2+ channel to beta-adrenergic receptor stimulation was used as a functional reporter since hypoxia increases the sensitivity of the L-type Ca2+ channel (I(Ca-L)) to isoproterenol (Iso). RESULTS: Hypoxia caused a 41.2+/-5.2% decrease in the rate of the DHE signal (n=21; p<0.01). Of the classical NAD(P)H oxidase inhibitors, DPI but not apocynin mimicked the effect of hypoxia on the sensitivity of I(Ca-L) to Iso. However, the potent NAD(P)H oxidase agonist angiotensin II had no effect on cellular superoxide or the sensitivity of I(Ca-L) to Iso. Although DPI inhibits NAD(P)H oxidase, it also decreased superoxide in isolated mitochondria in a concentration-dependent manner. Partial inhibition of mitochondrial function with nanomolar concentrations of FCCP or myxothiazol mimicked the effect of hypoxia on cellular superoxide and the sensitivity of I(Ca-L) to Iso. In addition, hypoxia caused a 69.3+/-0.8% decrease in superoxide in isolated mitochondria (n=4; p<0.01), providing direct evidence for a role for the mitochondria. CONCLUSIONS: Our data suggest that mitochondria appear to be involved in oxygen sensing, regulation of cellular ROS, and the function of I(Ca-L) during acute hypoxia in cardiac myocytes and NAD(P)H oxidase does not appear to contribute substantially.     

Angiotensin II and oxidative stress in Dahl Salt-sensitive rat with heart failure

Reactive oxygen species have an important pathogenic role in organ damage. We investigated the role of oxidative stress via nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the kidney of the Dahl salt-sensitive (DS) rats with heart failure (DSHF). Eleven-week-old DS rats fed an 8%-NaCl diet received either vehicle or imidapril (1 mg/kg per day) for 7 weeks. The renal expression of the NAD(P)H oxidase p47phox and endothelial NO synthase were evaluated. In DSHF rats, associated with increased renal angiotensin II, mRNA and protein expression of NAD(P)H oxidase p47phox were enhanced with an increase in renal lipid peroxidation production (0.33+/-0.03 versus 0.22+/-0.01 nmol/mg protein, P<0.05) and urinary excretion of hydrogen peroxide (26.9+/-6.6 versus 9.5+/-2.1 U/mg creatinine, P<0.01) compared with levels in Dahl salt-resistant rats. The endothelial NO synthase expression was decreased in the kidney. Treatment with imidapril reduced renal angiotensin II and NAD(P)H oxidase expression and the oxidative products (kidney lipid peroxidation product: 0.16+/-0.02, P<0.001; urinary hydrogen peroxide: 3.1+/-0.2, P<0.01 versus DSHF rats). Imidapril significantly decreased albuminuria and reduced glomerulosclerosis without changes in the blood pressure. In conclusion, DSHF rats showed increased oxidative stress in the kidney via NAD(P)H oxidase. Blockade of local angiotensin II with subpressor dose of imidapril inhibited NAD(P)H oxidase and prevented renal damage.