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PubMed1986

Quantitative use of weak bases for estimation of cellular pH gradients.

An improvement in the usual procedure for estimating cellular H+ gradients by distribution of weak bases is described, which involves evaluation of and correction for the permeability of the ionic form of the sensor. In the case of methylamine, unidirectional uptake (influx) of the methylammonium ion is calculated by comparing the total influx of [14C]methylamine with the influx of the highly permeant ion [14C]tetraphenyl-phosphonium ([14C]TPP+) for two experimental situations in which the membrane potential differs. Comparison of the potential-dependent changes in unidirectional influx of methylamine and TPP+ allows calculation of the magnitude of influx for the methylammonium ion. This value can then be used to determine the error in the H+ gradient as estimated from the steady-state distribution of methylamine across the plasma membrane. By using ATP-depleted isolated small intestine cells from the chick as the test system, and imposed H+ gradients of defined magnitude, it can be shown that the observed error matches the calculated error.

Acid-Base Equilibrium↗
PubMed1987

Inhibition and activation of Na+-Ca2+ exchange activity by quinacrine.

Quinacrine either inhibited or stimulated Na-Ca exchange in cardiac sarcolemmal vesicles, depending on the experimental conditions. When present in the assay medium for Na-Ca exchange, quinacrine inhibited both Nai-dependent Ca2+ uptake (Ki = 50 microM) and Nao-dependent Ca2+ efflux. Quinacrine's inhibition of Ca2+ efflux was attenuated by high concentrations of Na+. Quinacrine also blocked Na-Na and Ca-Ca exchange activities in the vesicles. The inhibitory effects of quinacrine on Na-Ca exchange activity are qualitatively similar to those reported previously for amiloride derivatives. When Na-loaded vesicles were preincubated with quinacrine and then assayed for Na-Ca exchange in a quinacrine-free medium, stimulation of exchange activity was observed. This stimulation was reversible on the removal of bound quinacrine and involved in a reduction in the apparent Km for extravesicular Ca2+. Stimulation of exchange activity under these conditions was also observed with the lipophilic cation tetraphenylphosphonium. Since Ca2+, quinacrine and tetraphenylphosphonium all bind strongly to sarcolemmal membranes, it is suggested that the observed stimulation of exchange activity involves a local electrostatic effect of the bound cations in accelerating a rate-limiting step in the reaction mechanism for Na-Ca exchange.

Animals↗
PubMed1987

Dependence of L-arginine accumulation on membrane potential in cultured human fibroblasts.

The cell-to-medium distribution ratios at steady state of L-arginine (RArg) and of the lipid-soluble cation tetraphenylphosphonium (RTPP) were studied as a function of the membrane potential (Em) in adult human fibroblasts. The relationship between RArg and Em was qualitatively similar to that of RTPP and Em. Quantitatively, RArg and RTPP differed in that 1) RTPP was much greater than RArg when Em was near zero, indicating a significant binding component in the uptake of TPP+ but not of L-arginine, and 2) after a correction for binding when Em is near zero, RTPP was still greater than RArg so that RT/F . ln RTPP exceeded RT/F . ln RArg by 10-25 mV. The pattern of the redistribution of accumulated TPP+ and arginine after an alteration of Em was identical. In null-point experiments, the external [K+] for which there were no changes in cellular TPP+ or L-arginine in the presence of high valinomycin (the null points) were very similar for the two probes. Em calculated from the null-point measurements (-70(-)-80 mV) was also very similar to RT/F . ln RArg and thus smaller than RT/F.ln RTPP. It was concluded that 1) there was an additional TPP+ binding as cellular [TPP] rose in response to more negative membrane potentials, 2) the transport system for L-arginine in these cells (system y+) operates as a facilitated diffusion system driven by the membrane potential, and 3) in some circumstances, L-arginine could be employed as a probe of Em.

Arginine↗
PubMed1987

SITS-sensitive Cl- conductance pathway in chick intestinal cells.

The unidirectional influx of 36Cl- into isolated chick epithelial cells is 30% inhibited by 300 microM SITS. Characteristics of the SITS-sensitive flux pathway were examined in terms of sensitivity to changes in membrane potential and intracellular pH. Potential dependence was evaluated using unidirectional influx of [14C]tetraphenylphosphonium ([14C]-TPP+) as a qualitative sensor of diffusion potentials created by experimentally imposed gradients of Cl-. Steady-state distribution of [14C]methylamine ([14C]MA) was used to examine for Cl(-)-dependent changes in intracellular pH. Imposed Na+ gradients, but not Cl- gradients, induce changes in [14C]MA distribution. SITS does not alter the [14C]MA distribution observed in cells with imposed gradients of Na+ and Cl-. Both results suggest that inhibition of Cl(-)-OH- exchange system is not the basis for the SITS effect on Cl- influx. However, if relative permeabilities for ion pairs via conductance pathways are compared, it can be shown that SITS causes a marked reduction of Pcl relative to either PNa or PK. SITS also inhibits electrically induced influx of [14C]TPP+ or [14C] alpha-methylglucoside driven by imposed Cl- gradients. Conversely, electrically driven Cl- influx can be blocked by SITS. These observations are all consistent with a SITS-sensitive Cl- conductance pathway associated with the plasma membrane of chick intestinal cells. No Cl(-)-OH- exchange capability can be detected for chick intestinal cells.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1989

Influx of L-arginine is an indicator of membrane potential in human fibroblasts.

The net influx of L-arginine (JARG) was employed as an indicator of the membrane potential in human fibroblasts. Cell depolarization, obtained by increasing [K+]out, decreased both JARG and the net influx of the lipid soluble cation tetraphenylphosphonium (JTPP), a probe of membrane potential. JTPP, but not JARG, was influenced by the mitochondrial potential and exhibited a component dependent on intracellular and/or extracellular binding. JARG was sensitive to changes in the membrane potential induced by Na+-dependent transport of L-proline or by the activity of Na+-K+-ATPase. In the presence of 50 microM valinomycin, JARG was markedly influenced by the distribution ratio of K+ in a range of [K+]out from 1.5 to 100 mM. In this range of [K+]out, membrane potential (Em) varied from -90 to -23 mV, and calibration of JARG vs. the membrane potential yielded a linear relationship. These results indicate the following: 1) that the net influx of TPP+ is not a reliable indicator of membrane potential in cultured human fibroblasts; 2) that in the same cells the net influx of L-arginine can be employed as an index of membrane potential; 3) that in a range of Em from -23 to -90 mV the activity of system y+ (the membrane agency devoted to L-arginine transport in cultured human fibroblasts) exhibits no saturation of potential-dependent activation of transport.

Arginine↗
PubMed1991

Sodium-dependent succinate transport by isolated chick intestinal cells.

Isolated chick intestinal epithelial cells take up succinate by a Na(+)-coupled transport system similar in some characteristics to those described for renal epithelium. The transport system exhibits a hyperbolic dependence on succinate concentration but a sigmoidal dependence on Na+ concentration. Best nonlinear fit of the Na+ dependence data to the Hill equation indicates a Michaelis constant for half-maximal transport rate (Km) for Na+ of approximately 20 mM, a maximal transport rate (Vmax) of 1.1 nmol succinate.min-1.mg protein-1, and a Hill coefficient of 2.5. Nearly equivalent fit was obtained with trial Hill coefficients down to 2.0. The data for succinate dependence indicated a Km of 25 microM and Vmax of 1.05 nmol.min-1.mg protein-1. The kinetic parameters indicate a higher affinity, lower capacity system than for succinate transport in the renal brush-border system. Thiocyanate-induced diffusion potentials cause no change in Na(+)-dependent succinate influx despite pronounced effects on the influx of tetraphenylphosphonium and on Na(+)-dependent alpha-methylglucoside (AMG) and alanine uptake. Several other dicarboxylic and tricarboxylic metabolic intermediates (but not the dicarboxylic amino acids) compete with succinate for uptake via the transport system. The data are consistent with the likelihood that these cells have a succinate transport system with a 2Na+:1succinate stoichiometry per transport cycle. The system catalyzes no net charge transfer and is therefore different from the potential-responsive succinate transporter described for renal tissue.

Alanine↗
PubMed1994

Na(+)-coupled alanine transport in LLC-PK1 cells.

Transport of alanine (Ala) was characterized in LLC-PK1 renal epithelial cells. Transport capability for Ala falls by 75% in postconfluent cultures, while Na(+)-coupled alpha-methylglucoside (AMG) transport rises more than fourfold during the same interval. The kinetics of Ala transport were characterized in ATP-depleted cells to allow experimental imposition of changes in Na+ gradient and control of membrane potential across the plasma membrane. At 100 microM Ala and 135 mM Na+, > 98% of the unidirectional Ala influx is dependent on the presence of Na+ in cells from postconfluent cultures. Li+ is only 1% as effective as Na+, and other monovalent cations are ineffective in supporting Ala uptake. alpha-(Methylamino)isobutyric acid (MeAIB; 5 mM) causes only a small inhibition (approximately 10%) of 100 microM Ala influx. The low selectivity for Li+; low sensitivity to competition by MeAIB or aminoisobutyric acid; pronounced inhibition by serine, homoserine, cysteine, homocysteine and threonine; moderate inhibition by valine, isoleucine, proline and histidine; and lack of inhibition by lysine, arginine, and aspartate are more consistent with those characteristics reported for entry via the ASC amino acid transport system rather than those associated with the A system. Alanine influx exhibits a hyperbolic relationship with increasing Ala or Na+ concentration. Kinetic analysis suggests a single transport pathway with a Michaelis constant (Km) for alanine of 380 microM (when Na+ is 135 mM), apparent Km for Na+ of 32 mM (with 100 microM Ala), and a maximum velocity of 7 nmol.min-1.mg cell protein-1. An interior-negative diffusion potential induces a similar enhancement of [14C]alanine or [14C]tetraphenylphosphonium influx (approximately 40%). In contrast, AMG influx is enhanced by a factor of 2.2 under the same conditions. AMG uptake also shows a sigmoidal relationship with Na+ concentration. Hill coefficients are 1.56 for AMG and 1.0 for alanine. Direct measurement of Na(+)-Ala coupling stoichiometry yields a value of 1.01 +/- 0.07. Under the same conditions, Na(+)-AMG coupling stoichiometry is 2.1 +/- 0.25. The difference in coupling stoichiometries provides an explanation for differences in intensity of interaction between Na(+)-coupled transport systems for sugars and amino acids.

Alanine↗
PubMed1999

NADPH oxidase inhibition does not interfere with low PO2 transduction in rat and rabbit CB chemoreceptor cells.

The aim of the present work was to elucidate the role of NADPH oxidase in hypoxia sensing and transduction in the carotid body (CB) chemoreceptor cells. We have studied the effects of several inhibitors of NADPH oxidase on the normoxic and hypoxia-induced release of [3H]catecholamines (CA) in an in vitro preparation of intact CB of the rat and rabbit whose CA deposits have been labeled by prior incubation with the natural precursor [3H]tyrosine. It was found that diphenyleneiodonium (DPI; 0.2-25 microM), an inhibitor of NADPH oxidase, caused a dose-dependent release of [3H]CA from normoxic CB chemoreceptor cells. Contrary to hypoxia, DPI-evoked release was only partially Ca2+ dependent. Concentrations of DPI reported to produce full inhibition of NADPH oxidase in the rat CB did not prevent the hypoxic release response in the rat and rabbit CB chemoreceptor cells, as stimulation with hypoxia in the presence of DPI elicited a response equaling the sum of that produced by DPI and hypoxia applied separately. Neopterin (3-300 microM) and phenylarsine oxide (0.5-2 microM), other inhibitors of NADPH oxidase, did not promote release of [3H]CA in normoxic conditions or affect the response elicited by hypoxia. On the basis of effects of neopterin and phenylarsine oxide, it is concluded that NADPH oxidase does not appear to play a role in oxygen sensing or transduction in the rat and rabbit CB chemoreceptor cells in vitro and, in the context of the present study, that DPI effects are not related to NADPH oxidase inhibition.

Animals↗
PubMed2004

PKC-delta-dependent activation of oxidative stress in adipocytes of obese and insulin-resistant mice: role for NADPH oxidase.

Oxidative stress is thought to be one of the causative factors contributing to insulin resistance and type 2 diabetes. Previously, we showed that reactive oxygen species (ROS) production is significantly increased in adipocytes from high-fat diet-induced obese and insulin-resistant mice (HF). ROS production was also associated with the increased activity of PKC-delta. In the present studies, we hypothesized that PKC-delta contributes to ROS generation and determined their intracellular source. NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) reduced ROS levels by 50% in HF adipocytes, and inhibitors of NO synthase (L-NAME, 1 mM), xanthine oxidase (allopurinol, 100 microM), AGE formation (aminoguanidine, 10 microM), or the mitochondrial uncoupler (FCCP, 10 microM) had no effect. Rottlerin, a selective PKC-delta inhibitor, suppressed ROS levels by approximately 50%. However, neither GO-6976 nor LY-333531, effective inhibitors toward conventional PKC or PKC-beta, respectively, significantly altered ROS levels in HF adipocytes. Subsequently, adenoviral-mediated expression of wild-type PKC-delta or its dominant negative mutant (DN-PKC-delta) in HF adipocytes resulted in either a twofold increase in ROS levels or their suppression by 20%, respectively. In addition, both ROS levels and PKC-delta activity were sharply reduced by glucose depletion. Taken together, these results suggest that PKC-delta is responsible for elevated intracellular ROS production in HF adipocytes, and this is mediated by high glucose and NADPH oxidase.

Adipocytes↗
PubMed2004

NADPH oxidase-derived oxidant stress is critical for neutrophil cytotoxicity during endotoxemia.

Neutrophils can cause liver injury during endotoxemia through generation of reactive oxygen species. However, the enzymatic source of the oxidant stress and the nature of the oxidants generated remain unclear. Therefore, we investigated the involvement of NADPH oxidase in the pathophysiology by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) in the galactosamine/endotoxin (700 mg/kg Gal:100 microg/kg ET) model of liver injury. In addition, we measured chlorotyrosine as indicator for hypochlorous acid formation by myeloperoxidase. Gal/ET treatment of male C3HeB/FeJ mice resulted in sinusoidal neutrophil accumulation and parenchymal cell apoptosis (14 +/- 3% of cells) at 6 h. At 7 h, 35% of neutrophils had transmigrated. The number of apoptotic cells increased to 25 +/- 2%, and the overall number of dead cells was 48 +/- 3%; many of them showed the characteristic morphology of necrosis. Hepatocytes, which colocalized with extravasated neutrophils, stained positive for chlorotyrosine and 4-hydroxynonenal (4-HNE) protein adducts. In contrast, animals pretreated with DPI (2.5 mg/kg) were protected against liver injury at 7 h (necrosis = 20 +/- 2%). These livers showed little chlorotyrosine or 4-HNE staining, but apoptosis and neutrophil accumulation and extravasation remained unaffected. However, DPI-treated animals showed serious liver injury at 9 h due to sustained apoptosis. The results indicate that NADPH oxidase is responsible for the neutrophil-derived oxidant stress, which includes formation of hypochlorous acid by myeloperoxidase. Thus NADPH oxidase could be a promising therapeutic target to prevent neutrophil-mediated liver injury. However, the long-term benefit of this approach needs to be investigated in models relevant for human liver disease.

Animals↗
PubMed2001

Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcohol-induced liver injury in the rat.

The oxidant source in alcohol-induced liver disease remains unclear. NADPH oxidase (mainly in liver Kupffer cells and infiltrating neutrophils) could be a potential free radical source. We aimed to determine if NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI) affects nuclear factor-kappaB (NF-kappaB) activation, liver tumor necrosis factor-alpha (TNF-alpha) mRNA expression, and early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g. kg(-1). day(-1)) continuously for up to 4 wk, using the Tsukamoto-French intragastric enteral feeding protocol. DPI or saline vehicle was administered by subcutaneous injection for 4 wk. Mean urine ethanol concentrations were similar between the ethanol- and ethanol plus DPI-treated groups. Enteral ethanol feeding caused severe fat accumulation, mild inflammation, and necrosis in the liver (pathology score, 4.3 +/- 0.3). In contrast, DPI significantly blunted these changes (pathology score, 0.8 +/- 0.4). Enteral ethanol administration for 4 wk also significantly increased free radical adduct formation, NF-kappaB activity, and TNF-alpha expression in the liver. DPI almost completely blunted these parameters. These results indicate that DPI prevents early alcohol-induced liver injury, most likely by inhibiting free radical formation via NADPH oxidase, thereby preventing NF-kappaB activation and TNF-alpha mRNA expression in the liver.

Animals↗
PubMed2005

Hypoxic reperfusion of the ischemic heart and oxygen radical generation.

Postischemic myocardial contractile dysfunction is in part mediated by the burst of reactive oxygen species (ROS), which occurs with the reintroduction of oxygen. We hypothesized that tissue oxygen tension modulates this ROS burst at reperfusion. After 20 min of global ischemia, isolated rat hearts were reperfused with temperature-controlled (37.4 degrees C) Krebs-Henseleit buffer saturated with one of three different O2 concentrations (95, 20, or 2%) for the first 5 min of reperfusion and then changed to 95% O2. Additional hearts were loaded with 1) allopurinol (1 mM), a xanthine oxidase inhibitor, 2) diphenyleneiodonium (DPI; 1 microM), an NAD(P)H oxidase inhibitor, or 3) Tiron (10 mM), a superoxide scavenger, and were then reperfused with either 95 or 2% O2 for the first 5 min. ROS production and tissue oxygen tension were quantitated using electron paramagnetic resonance spectroscopy. Tissue oxygen tension was significantly higher in the 95% O2 group. However, the largest radical burst occurred in the 2% O2 reperfusion group (P < 0.001). Recovery of left ventricular (LV) contractile function and aconitase activity during reperfusion were inversely related to the burst of radical production and were significantly higher in hearts initially reperfused with 95% O2 (P < 0.001). Allopurinol, DPI, and Tiron reduced the burst of radical formation in the 2% O2 reperfusion groups (P < 0.05). Hypoxic reperfusion generates an increased ROS burst originating from multiple pathways. Recovery of LV function during reperfusion is inversely related to this oxygen radical burst, highlighting the importance of myocardial oxygen tension during initial reperfusion.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed2002

Gene transfer of Cu/Zn SOD to cerebral vessels prevents FPI-induced CBF autoregulatory dysfunction.

The goal of this study was to determine whether gene transfer of human copper-zinc (Cu/Zn) superoxide dismutase (SOD) has preventive effects on cerebral blood flow (CBF) autoregulatory dysfunction after fluid percussion injury (FPI). Rats subjected to FPI (2-2.5 atm) exhibited enhanced activity of reduced NADP (NADPH) oxidase in the cerebral vasculature. In line with these findings, the rats showed not only reduced vasodilation of the pial artery in response to calcitonin gene-related peptide and levcromakalim but also impaired autoregulatory vasodilation in response to acute hypotension. The FPI-induced hemodynamic alterations were significantly prevented by pretreatment with diphenyleneiodonium (10 micromol/l), an NAD(P)H oxidase inhibitor. Intracisternal application of recombinant adenovirus (100 microl of 1 x 10(10) pfu/ml)-encoding human Cu/Zn SOD 3 days before FPI prevented the impairment of vasodilation to hypotension and vasorelaxants, resulting in the restoration of CBF autoregulation. Our findings demonstrate that FPI-induced impairment of CBF autoregulation is closely related with NAD(P)H oxidase-derived superoxide anion, and these alterations can be prevented by the recombinant adenovirus-mediated transfer of human Cu/Zn SOD gene to the cerebral vasculature.

Adenoviridae↗
PubMed2006

Insulin-induced activation of hypoxia-inducible factor-1 requires generation of reactive oxygen species by NADPH oxidase.

Hypoxia-inducible factor (HIF)-1 activation in response to hypoxia requires mitochondrial generation of reactive oxygen species (ROS). In contrast, the requirement of ROS for HIF-1 activation by growth factors like insulin remains unexplored. To explore that, insulin-sensitive hepatic cell HepG2 or cardiac muscle cell H9c2 cells were pretreated with NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) or apocynin and HIF-1 activation was tested by electrophoretic mobility shift and reporter gene assay. Antioxidants DPI or apocynin completely blocked insulin-stimulated HIF-1 activation. The restoration of HIF-1 activation by H(2)O(2) in DPI-pretreated cells not only confirmed the role of ROS but also identified H(2)O(2) as the responsible ROS. The role of NADPH oxidase was further confirmed by greater stimulation of HIF-1 during simultaneous treatment of suboptimal concentration of insulin along with NADPH but not by NADH. The role of oxidant generated by insulin is found to inhibit the protein tyrosine phosphatase as suggested by the following observations. First, tyrosine phosphatase-specific inhibitor sodium vanadate compensates DPI-inhibited HIF-1 activity. Second, sodium vanadate stimulates HIF-1 activation with suboptimal concentration of insulin. Third, DPI and pyrrolidene dithiocarbamate (PDTC) blocks insulin-receptor tyrosine kinase activation. The activity of phosphatidylinositol 3-kinase as evidenced by Akt phosphorylation, involved in HIF-1 activation, is also dependent on ROS generation by insulin. Finally, DPI pretreatment blocked insulin-stimulated expression of genes like VEGF, GLUT1, and ceruloplasmin. Overall, our data provide strong evidence for the essential role of NADPH oxidase-generated ROS in insulin-stimulated activation of HIF-1.

Acetophenones↗
PubMed2004

17beta-estradiol inhibits cyclic strain-induced endothelin-1 gene expression within vascular endothelial cells.

It has been well documented previously that 17beta-estradiol (E2) exerts a protective effect on cardiovascular tissue. The possible role of E2 in the regulation of endothelin (ET)-1 production has been previously reported, although the complex mechanisms by which E2 inhibits ET-1 expression are not completely understood. The aims of this study were to examine whether E2 was able to alter strain-induced ET-1 gene expression and also to identify the putative underlying signaling pathways that exist within endothelial cells. For cultured endothelial cells, E2 (1-100 nM), but not 17alpha-estradiol, inhibited the level of strain-induced ET-1 gene expression and also peptide secretion. This inhibitory effect elicited by E2 was able to be prevented by the coincubation of endothelial cells with the estrogen receptor antagonist ICI-182,780 (1 microM). E2 also inhibited strain-enhanced NADPH oxidase activity and intracellular reactive oxygen species (ROS) generation as measured by the redox-sensitive fluorescent dye 2',7'-dichlorofluorescin diacetate and the level of extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, the presence of E2 and antioxidants such as N-acetylcysteine and diphenylene iodonium were able to elicit a decrease in the level of strain-induced ET-1 secretion, ET-1 promoter activity, ET-1 mRNA, ERK phosphorylation, and activator protein-1 binding activity. In summary, we demonstrated, for the first time, that E2 inhibits strain-induced ET-1 gene expression, partially by interfering with the ERK pathway via the attenuation of strain-induced ROS generation. Thus this study delivers important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of estrogen on the cardiovascular system.

Acetylcysteine↗
PubMed2005

The direct physiological effects of mitoK(ATP) opening on heart mitochondria.

The mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoK(ATP) opening that should be observable at the level of the mitochondrion. MitoK(ATP) opening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+ influx, caused either by mitoK(ATP) opening or valinomycin, and K+ efflux caused by the mitochondrial K+/H+ antiporter. We show that increasing K+ influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+ cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+ influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+ influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoK(ATP) openers and blockers with those of valinomycin, we show that four independent assays of mitoK(ATP) activity yield quantitatively identical results for mitoK(ATP)-mediated K+ transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+ channel and establish the physiological consequences of mitoK(ATP) opening for mitochondria.

Adenosine Triphosphate↗
PubMed2006

Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase.

Reactive oxygen species (ROS) play a central role in the pathogenesis of many cardiovascular diseases, such as atherosclerosis and hypertension. Endothelial NADPH oxidase is the major source of intracellular ROS. The present study investigated the role of endothelial NADPH oxidase-derived ROS in angiopoietin-1 (Ang-1)-induced angiogenesis. Exposure of porcine coronary artery endothelial cells (PCAECs) to Ang-1 (250 ng/ml) for periods up to 30 min led to a transient and dose-dependent increase in intracellular ROS. Thirty minutes of pretreatment with the NADPH oxidase inhibitors diphenylene iodinium (DPI, 10 microM) and apocynin (200 microM) suppressed Ang-1-stimulated ROS. Pretreatment with either DPI or apocynin also significantly attenuated Ang-1-induced Akt and p44/42 MAPK phosphorylation. In addition, inhibition of NADPH oxidase significantly suppressed Ang-1-induced endothelial cell migration and sprouting from endothelial spheroids. Using mouse heart microvascular endothelial cells from wild-type (WT) mice and mice deficient in the p47(phox) component of NADPH oxidase (p47(phox-/-)), we found that although Ang-1 stimulated intracellular ROS, Akt and p42/44 MAPK phosphorylation, and cell migration in WT cells, the responses were strikingly suppressed in cells from the p47(phox-/-) mice. Furthermore, exposure of aortic rings from p47(phox-/-) mice to Ang-1 demonstrated fewer vessel sprouts than WT mice. Inhibition of the Tie-2 receptor inhibited Ang-1-induced endothelial migration and vessel sprouting. Together, our data strongly suggest that endothelial NADPH oxidase-derived ROS play a critical role in Ang-1-induced angiogenesis.

Acetophenones↗
PubMed2006

Inhibition of cardiac contractility by 5-hydroxydecanoate and tetraphenylphosphonium ion: a possible role of mitoKATP in response to inotropic stress.

This study investigates the role of the mitochondrial ATP-sensitive K+ channel (mitoKATP) in response to positive inotropic stress. In Langendorff-perfused rat hearts, inotropy was induced by increasing perfusate calcium to 4 mM, by adding 80 microM ouabain or 0.25 microM dobutamine. Each of these treatments resulted in a sustained increase in rate-pressure product (RPP) of approximately 60%. Inhibition of mitoKATP by perfusion of 5-hydroxydecanoate (5-HD) or tetraphenylphosphonium before induction of inotropic stress resulted in a marked attenuation of RPP. Inhibition of mitoKATP after induction of stress caused the inability of the heart to maintain a high-work state. In human atrial fibers, the increase in contractility induced by dobutamine was inhibited 60% by 5-HD. In permeabilized fibers from the Langendorff-perfused rat hearts, inhibition of mitoKATP resulted, in all cases, in an alteration of adenine nucleotide compartmentation, as reflected by a 60% decrease in the half-saturation constant for ADP [K1/2 (ADP)]. We conclude that opening of cardiac mitoKATP is essential for an appropriate response to positive inotropic stress and propose that its involvement proceeds through the prevention of stress-induced decrease in mitochondrial matrix volume. These results indicate a physiological role for mitoKATP in inotropy and, by extension, in heart failure.

Animals↗