Intact Bovine Aortic Endothelial Cells Research Articles

Mitochondrial Targeted Coenzyme Q, Superoxide, and Fuel Selectivity in Endothelial Cells

BackgroundPreviously, we reported that the “antioxidant” compound “mitoQ” (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates.Methods and ResultsTo further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity.ConclusionsIn summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over fatty acid oxidation at the intact cell level.

Molecular Mechanisms of Angiotensin II–Mediated Mitochondrial Dysfunction

Mitochondrial dysfunction is a prominent feature of most cardiovascular diseases. Angiotensin (Ang) II is an important stimulus for atherogenesis and hypertension; however, its effects on mitochondrial function remain unknown. We hypothesized that Ang II could induce mitochondrial oxidative damage that in turn might decrease endothelial nitric oxide (NO.) bioavailability and promote vascular oxidative stress. The effect of Ang II on mitochondrial ROS, mitochondrial respiration, membrane potential, glutathione, and endothelial NO. was studied in isolated mitochondria and intact bovine aortic endothelial cells using electron spin resonance, dihydroethidium high-performance liquid chromatography -based assay, Amplex Red and cationic dye fluorescence. Ang II significantly increased mitochondrial H2O2 production. This increase was blocked by preincubation of intact cells with apocynin (NADPH oxidase inhibitor), uric acid (scavenger of peroxynitrite), chelerythrine (protein kinase C inhibitor), N(G)-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor), 5-hydroxydecanoate (mitochondrial ATP-sensitive potassium channels inhibitor), or glibenclamide. Depletion of p22(phox) subunit of NADPH oxidase with small interfering RNA also inhibited Ang II-mediated mitochondrial ROS production. Ang II depleted mitochondrial glutathione, increased state 4 and decreased state 3 respirations, and diminished mitochondrial respiratory control ratio. These responses were attenuated by apocynin, 5-hydroxydecanoate, and glibenclamide. In addition, 5-hydroxydecanoate prevented the Ang II-induced decrease in endothelial NO. and mitochondrial membrane potential. Therefore, Ang II induces mitochondrial dysfunction via a protein kinase C-dependent pathway by activating the endothelial cell NADPH oxidase and formation of peroxynitrite. Furthermore, mitochondrial dysfunction in response to Ang II modulates endothelial NO. and generation, which in turn has ramifications for development of endothelial dysfunction.

Genistein Activates the 3′,5′-Cyclic Adenosine Monophosphate Signaling Pathway in Vascular Endothelial Cells and Protects Endothelial Barrier Function

The soy phytoestrogen, genistein, has an array of biological actions, including weak estrogenic effects, inhibition of tyrosine kinase, and cellular antioxidant activity. Recent studies showed that genistein may improve vascular function, but the mechanism is unclear. We show that genistein stimulates intracellular cAMP accumulation in intact bovine aortic endothelial cells and human umbilical vein endothelial cells over an incubation period of 30 min. Increases in intracellular cAMP are evoked by as low as 10 nm genistein but not by estrogen. These increases in cAMP may result primarily from enhanced adenylate cyclase activity by a mechanism that does not involve genomic actions or estrogen receptors. The cAMP induced by genistein activates cAMP-dependent protein kinase (PKA) in bovine aortic endothelial cells. The activation of PKA phosphorylates and activates cAMP response element-binding protein, leading to up-regulation of cAMP response element-containing gene expression. In addition, activation of PKA protects thrombin-induced endothelial monolayer permeability, a novel cardioprotective effect of genistein mediated by the cAMP/PKA cascade. These findings demonstrate that a nongenomic action of genistein leads to activation of the cAMP/PKA signaling system to protect the vascular barrier function and alter the expression of cAMP-regulated genes, thereby providing a novel mechanism underlying some of the cardiovascular protective effects proposed for soy phytoestrogens.

Chronic exposure to high glucose impairs bradykinin-stimulated nitric oxide production by interfering with the phospholipase-C-implicated signalling pathway in endothelial cells: evidence for the involvement of protein kinase C

Overwhelming evidence indicates that endothelial cell dysfunction in diabetes is characterised by diminished endothelium-dependent relaxation, but the matter of the underlying molecular mechanism remains unclear. As nitric oxide (NO) production from the endothelium is the major player in endothelium-mediated vascular relaxation, we investigated the effects of high glucose on NO production, and the possible alterations of signalling pathways implicated in this scenario. NO production and intracellular Ca(2+) levels ([Ca(2+)](i)) were assessed using the fluorescent probes 4,5-diaminofluorescein diacetate and fura-2 respectively. Exposure of cultured bovine aortic endothelial cells to high glucose for 5 or 10 days significantly reduced NO production induced by bradykinin (but not by Ca(2+) ionophore) in a time- and dose-dependent manner. This was probably due to an attenuation in bradykinin-induced elevations of [Ca(2+)](i) under these conditions, since a close correlation between [Ca(2+)](i) increases and NO generation was observed in intact bovine aortic endothelial cells. Both bradykinin-promoted intracellular Ca(2+) mobilisation and extracellular Ca(2+) entry were affected. Moreover, bradykinin-induced formation of Ins(1,4,5)P(3), a phospholipase C product leading to increases in [Ca(2+)](i), was also inhibited following high glucose culture. This abnormality was not attributable to a decrease in inositol phospholipids, but possibly to a reduction in the number of bradykinin receptors. The alterations in NO production, the increases in [Ca(2+)](i), and the bradykinin receptor number due to high glucose could be largely reversed by protein kinase C inhibitors and D: -alpha-tocopherol (antioxidant). Chronic exposure to high glucose reduces NO generation in endothelial cells, probably by impairing phospholipase-C-mediated Ca(2+) signalling due to excess protein kinase C activation. This defect in NO release may contribute to the diminished endothelium-dependent relaxation and thus to the development of cardiovascular diseases in diabetes.

Genistein acutely stimulates nitric oxide synthesis in vascular endothelial cells by a cyclic adenosine 5'-monophosphate-dependent mechanism.

Genistein may improve vascular function, but the mechanism of this effect is unclear. We tested the hypothesis that genistein directly regulates vascular function through stimulation of endothelial nitric oxide synthesis. Genistein activated endothelial nitric oxide synthase (eNOS) in intact bovine aortic endothelial cells and human umbilical vein endothelial cells over an incubation period of 10 min. The maximal eNOS activity was at 1 microm genistein. Consistent with this activation pattern, 1 microm genistein maximally stimulated the phosphorylation of eNOS at serine 1179 at 10 min of incubation. The rapid activation of eNOS by genistein was not dependent on RNA transcription or new protein synthesis and was not blocked by a specific estrogen receptor antagonist. In addition, inhibition of MAPK or phosphatidylinositol 3-OH kinase/Akt kinase had no affect on eNOS activation by genistein. Furthermore, the genistein effect on eNOS was also independent of tyrosine kinase inhibition. However, inhibition of cAMP-dependent kinase [protein kinase A (PKA)] by H89 completely abolished the genistein-stimulated eNOS activation and phosphorylation, suggesting that genistein acted through a PKA-dependent pathway. These findings demonstrated that genistein had direct nongenomic effects on eNOS activity in vascular endothelial cells, leading to eNOS activation and nitric oxide synthesis. These effects were mediated by PKA and were unrelated to an estrogenic effect. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for soy phytoestrogens.

Dehydroepiandrosterone stimulates nitric oxide release in vascular endothelial cells: evidence for a cell surface receptor

Dehydroepiandrosterone (DHEA) improves vascular function, but the mechanism of this effect is unclear. Since nitric oxide (NO) regulates vascular function, we hypothesized that DHEA affects the vasculature by increasing endothelial NO production. Physiological concentrations of DHEA stimulated NO release from intact bovine aortic endothelial cells (BAEC) within 5 min. This effect was mediated by activation of endothelial nitric oxide synthase (eNOS) in BAEC and human umbilical vein endothelial cells (HUVEC). Dehydroepiandrosterone increased cyclic GMP (cGMP) levels in BAEC, consistent with its effect on NO production. Albumin-conjugated DHEA also stimulated NO release, suggesting that DHEA stimulates eNOS by a plasma membrane-initiated signal. Tamoxifen blocked estrogen-stimulated NO release from BAEC, but did not inhibit the DHEA effect. Pertussis toxin abolished the acute effect of DHEA on NO release. Dehydroepiandrosterone had no effect on intracellular calcium fluxes. However, inhibition of tyrosine kinases or the mitogen-activated protein (MAP) kinase kinase (MEK) blocked NO release and cGMP production in response to DHEA. These findings demonstrate that physiological concentrations of DHEA acutely increase NO release from intact vascular endothelial cells, by a plasma membrane-initiated mechanism. This action of DHEA is mediated by a steroid-specific, G-protein coupled receptor, which activates eNOS in both bovine and human cells. The release of NO is independent of intracellular calcium mobilization, but depends on tyrosine- and MAP kinases. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for DHEA.

Dehydroepiandrosterone stimulates nitric oxide release in vascular endothelial cells: evidence for a cell surface receptor

Dehydroepiandrosterone (DHEA) improves vascular function, but the mechanism of this effect is unclear. Since nitric oxide (NO) regulates vascular function, we hypothesized that DHEA affects the vasculature by increasing endothelial NO production. Physiological concentrations of DHEA stimulated NO release from intact bovine aortic endothelial cells (BAEC) within 5 min. This effect was mediated by activation of endothelial nitric oxide synthase (eNOS) in BAEC and human umbilical vein endothelial cells (HUVEC). Dehydroepiandrosterone increased cyclic GMP (cGMP) levels in BAEC, consistent with its effect on NO production. Albumin-conjugated DHEA also stimulated NO release, suggesting that DHEA stimulates eNOS by a plasma membrane-initiated signal. Tamoxifen blocked estrogen-stimulated NO release from BAEC, but did not inhibit the DHEA effect. Pertussis toxin abolished the acute effect of DHEA on NO release. Dehydroepiandrosterone had no effect on intracellular calcium fluxes. However, inhibition of tyrosine kinases or the mitogen-activated protein (MAP) kinase kinase (MEK) blocked NO release and cGMP production in response to DHEA. These findings demonstrate that physiological concentrations of DHEA acutely increase NO release from intact vascular endothelial cells, by a plasma membrane-initiated mechanism. This action of DHEA is mediated by a steroid-specific, G-protein coupled receptor, which activates eNOS in both bovine and human cells. The release of NO is independent of intracellular calcium mobilization, but depends on tyrosine- and MAP kinases. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for DHEA.

Regulation of cyclooxygenase activity by metamizol

The ability of metamizol to inhibit cyclooxygenase-1 and cyclooxygenase-2 activities has been evaluated using different cyclooxygenase sources. Metamizol inhibited purified cyclooxygenase-1 and cyclooxygenase-2 with an IC 50 of about 150 μg/ml. A similar IC 50 value for cyclooxygenase-2 was obtained in lipopolysaccharide-activated broken murine macrophages. Consistent with these findings, molecular models of the complexes between cyclooxygenase-1 or cyclooxygenase-2 with 4-methylaminoantipyrine, the major active derivative of metamizol, suggested a common binding mode to both isoforms. In intact cells, however, the inhibition profiles were markedly different. The IC 50 values of metamizol for cyclooxygenase-1 in intact bovine aortic endothelial cells (BAEC) cells and human platelets were 1730±150 μg/ml and 486±56 μg/ml, respectively. Inhibition of cyclooxygenase-2 activity in murine macrophages and primary human leukocytes activated by lipopolysaccharide yielded IC 50 values of 12±1.8 μg/ml and 21±2.9 μg/ml, respectively. These data indicate that the IC 50 values obtained with purified enzymes or disrupted cells cannot always be extrapolated to the cyclooxygenase inhibitory activity of nonsteroidal antiinflammatory drugs (NSAIDs) in intact cells. The data presented here also indicate that cyclooxygenase-2 inhibition could play an important role in the pharmacological effects of metamizol.

Regulation of nitric oxide synthesis by oxygen in vascular endothelial cells.

Vascular endothelial cells synthesize nitric oxide (NO) in response to agonists that elevate cytosolic free Ca2+ concentrations. Once activated, NO synthase (NOS) requires arginine, NADPH, and O2 as cosubstrates. In this study, we investigated the role of O2 in regulating endothelial NOS activity in intact bovine aortic endothelial cells by measuring the rate of nitrite (NO2-) and nitrate (NO3-) production after conversion of NO2- to S-nitrosoglutathione before analysis or after reduction of NO2- and NO3- to NO using acidic vanadium chloride. The basal rate of NO2- production was 6.5 +/- 0.8 pmol.min-1.mg protein-1. Thapsigargin (TG, 1 microM) elevated free cytosolic Ca2+ concentration and increased the rate of NO2- synthesis. At maximal concentrations of TG, the rate of stimulated NO2- production was linear for at least 20 min and was eightfold higher than the basal rate (53.5 +/- 1.8 pmol.min-1.mg protein-1). Incubation of cells in gas mixtures chosen to produce PO2 values in the physiological range led to a progressive fall in the rate of TG-stimulated NO2- production, as O2 concentrations were reduced from that of room air. The half-maximal effective concentration for NO2- production by intact cells was found to occur at 38 Torr. PO2 values higher than that of room air did not lead to a change in the rate of TG-stimulated NO2- production. To confirm that measurement of NO2- accurately reflects total NO production, both NO2- plus NO3- were measured in buffer samples from cells incubated in either room air or N2. The sum of these NO oxidation products was inhibited similarly by hypoxia. These findings suggest that O2 is an important determinant of NOS activity in hypoxic tissues or in vascular beds such as the pulmonary arterial or fetal circulation where PO2 values in the range of 40 Torr are encountered normally.

Evidence that most high-affinity ATP binding sites on aortic endothelial cells and membranes do not correspond to P 2 receptors

It has recently been demonstrated that two types of ATP receptors, the P 2 y and P 2 u receptors, are coexpressed on bovine aortic endothelial cells. The aim of the present study was to characterize directly P 2 y and P 2 u subtypes on intact bovine aortic endothelial cells and on membranes prepared from these cells using adenosine 5′-0-(3-thio[ 35S]triphosphate) ([ 35S]ATPγS). [ α- 32P]ATP and [ α- 32P]UTP as radioligands. [ 35S]ATPγS binding to bovine aortic endothelial cell membranes was saturable and apparently involved a single class of high-affinity binding sites ( K d : 14 ± 11 nM; B max 1.6 ± 0.7 pmol/mg protein; mean ± S.D.). A similar class of high-affinity binding sites was identified with [ α- 32P]ATP ( K d : 14 ± 9 nM; B max: 1.7 ± 1.1 pmol/mg protein; mean ± S.D.). Competition experiments showed that only one third of these sites bound 2-methylthio-ATP (2-MeSATP) with high affinity ( K i : 21 ± 5 and 14 ± 10 nM, mean ± S.D., for [ 35S]ATPγS and [ α- 32P]ATP, respectively) and might therefore represent the P 2 y receptors. UTP did not compete with [ 35S]ATPγS or [ α- 32P]ATP for binding at the remaining sites, indicating that they are not the P 2 u receptors. No high-affinity UTP binding sites could be detected using [ α- 32P]UTP. [ 35S]ATPγS binding to intact bovine aortic endothelial cells was competed by ATPγS ( K d : 1.0 ± 0.5 gmM; mean ± S.D.), but not by 2-MeSATP and UTP, indicating that these binding sites are neither the P 2 y nor the P 2 u receptors.

Inhibition of endothelial nitric oxide synthase activity by protein kinase C.

Nitric oxide (NO) is an important molecular messenger accounting for endothelium-derived relaxing factor. Recently, NO synthase (NOS) from cultured endothelial cells has been purified and molecularly cloned. To evaluate the effect of phosphorylation by protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) on endothelial constitutive NOS catalytic activity, we incubated purified endothelial NOS with PKC or PKA. Endothelial NOS was stoichiometrically phosphorylated by PKC and PKA. In intact bovine aortic endothelial cells (BAECs), NOS was phosphorylated by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). NOS activity measured by the conversion of [3H]arginine to [3H]citrulline in homogenates of BAECs treated with TPA or phorbol 12,13-dibutyrate was reduced by 30%, whereas dibutylyl cyclic AMP did not affect NOS activity. Moreover, we measured NO release from cultured BAECs by a chemiluminescence method to examine the effect of PKC and PKA on endothelial NOS activity. In cultured BAECs, ATP gamma S and A23187 induced NO release in time- and dose-dependent manners. Phorbol esters such as TPA and phorbol 12,13-dibutyrate dose dependently inhibited NO release stimulated by A23187 as well as ATP gamma S. Reduction of NO release by TPA was almost completely prevented by pretreatment with staurosporine, an inhibitor of PKC. NO release by A23187 was increased in PKC-downregulated BAECs. In contrast, dibutylyl cyclic AMP or 8-bromo cyclic GMP had no effect on NO release from BAECs induced by A23187 or ATP gamma S. These results indicate that phosphorylation of NOS by PKC is associated with a reduction of its catalytic activity in vascular endothelial cells.

Effects of phosphoramidon in endothelial cell cultures on the endogenous synthesis of endothelin-1 and on conversion of exogenous big endothelin-1 to endothelin-1.

Several studies have shown that phosphoramidon (PHOS) reduces the release of endothelin-1 (ET-1) from cultured endothelial cells. Moreover, the main endothelin-converting enzyme (ECE) activity in these cells is a membrane-bound metallopeptidase that is also inhibited by PHOS. We have investigated further the role of the PHOS-sensitive ECE in the conversion of big ET-1 to ET-1. ET-1 was measured using a radioimmunoassay specific for the C-terminal ET[16-21] sequence. The effect of PHOS on the production of ET-1 from endogenous precursors was determined using cultured bovine aortic endothelial cells (BAECs) and the human endothelial cell line (EA.hy 926). The concentrations of ET-1 accumulating in the medium over 24 h from BAECs were lowered by PHOS (-27% 10 microM, -76% 100 microM). In contrast, with EA.hy 926 cells, the same concentrations of PHOS increased by five- to sixfold the amount of ET-1 present in the medium after 24-h incubation. In other experiments, incubation of big ET-1 (1 microM) with intact BAECs or EA.hy 926 cells resulted in the generation of ET-1, and with both cell types this was inhibited by PHOS (IC50: BAECs = 6.4 microM; EA.hy 926 = 0.26 microM). These results are consistent with both cell types having a PHOS-sensitive ECE that is readily accessible to exogenous big ET-1 and is therefore probably located on the plasma membrane. Furthermore, another intracellular ECE may play a part in the endogenous intracellular formation of ET-1 in EA.hy 926 cells.

Identification and partial characterization of an adenosine(5′)tetraphospho(5′)adenosine hydrolase on intact bovine aortic endothelial cells

The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.