An improved capillary electrophoresis method for measuring tissue metabolites associated with cellular energy state.
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An improved method for the measurement of tissue metabolites associated with cellular energetic state by capillary electrophoresis is described. This method allows 17 compounds present in a mixture of standards to be determined simultaneously within 43 min with good reproducibility. ATP, ADP, AMP, UTP, IMP, inosine, hypoxanthine, creatine, phosphocreatine, UDP-galactose, NAD and NADH were detected in samples of either rat heart tissue or rat neonatal cardiomyocytes. This method can detect compounds at concentrations of 5 microm in samples. Recoveries for ATP and phosphocreatine added to cardiomyocyte samples were 99.4 +/- 2.1% and 103.1 +/- 3.3%, respectively (mean +/- SEM, n = 3). Our method has been comprehensively validated and is capable of measuring a wider range of tissue metabolites important in assessing cellular energy status than existing methods. (+info)
Effects of high glucose on the hypoxic isolated guinea pig heart: interactions with ATP-dependent K+ channels?
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The effect of perfusion with elevated glucose concentrations on hypoxic myocardium was investigated in isolated Langendorff guinea pig hearts. For that purpose, mechanical (heart rate, systolic peak pressure and coronary flow) and electrophysiological (monophasic action potential duration=MAP, ectopic beats) data were evaluated. At the end of the experiments the hearts were examined histologically after trypan blue vital staining for quantification of irreversible myocardial damage. In the absence of insulin moderate glucose elevation (from 5 to 15 mM) exerted beneficial effects on hypoxic hearts: the depressed contraction was improved, the action potential shortening partly reversed and the percentage of irreversibly damaged myocytes diminished. Glucose did not have any effect on heart rate and arrhythmias under hypoxia or reperfusion. A contribution of cardiac ATP-dependent K+ channels to the effects of glucose could be excluded by further experiments. Thus, blocking these channels with high glibenclamide concentrations did not affect the action of glucose on MAP and contraction. To some degree the glucose effect on MAP, but not on systolic pressure, was also observable under normoxic conditions. (+info)
Gene delivery of Kir6.2/SUR2A in conjunction with pinacidil handles intracellular Ca2+ homeostasis under metabolic stress.
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Metabolic injury is a complex process affecting various tissues, with intracellular Ca2+ loading recognized as a common precipitating event leading to cell death. We have recently observed that cells overexpressing recombinant ATP-sensitive K+ (KATP) channel subunits may acquire resistance against metabolic stress. To examine whether, under metabolic challenge, intracellular Ca2+ homeostasis can be maintained by an activator of channel proteins, we delivered Kir6.2 and SUR2A genes, which encode KATP channel subunits, into a somatic cell line lacking native KATP channels. Hypoxia-reoxygenation was simulated by application and removal of the mitochondrial poison 2,4 dinitrophenol. Under such metabolic stress, Ca2+ loading was induced by Ca2+ influx during hypoxia and release of Ca2+ from intracellular stores during reoxygenation. Delivery of Kir6.2/SUR2A genes, in conjunction with the KATP channel activator pinacidil, prevented intracellular Ca2+ loading irrespective of whether the channel opener was applied throughout the duration of hypoxia-reoxygenation or transiently during the hypoxic or reoxygenation stage. In all stages of injury, the effect of pinacidil was inhibited by the selective antagonist of KATP channel, 5-hydroxydecanoate. The present study provides evidence that combined use of gene delivery and pharmacological targeting of recombinant proteins can handle intracellular Ca2+ homeostasis under hypoxia-reoxygenation irrespective of the stage of the metabolic insult. (+info)
Hypoxia-mediated regulation of gene expression in mammalian cells.
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The molecular mechanism underlying oxygen sensing in mammalian cells has been extensively investigated in the areas of glucose transport, glycolysis, erythropoiesis, angiogenesis and catecholamine metabolism. Expression of functionally operative representative proteins in these specific areas, such as the glucose transporter 1, glycolytic enzymes, erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase are all induced by hypoxia. Recent studies demonstrated that both transcriptional activation and post-transcriptional mechanisms are important to the hypoxia-mediated regulation of gene expression. In this article, the cis-acting elements and trans-acting factors involved in the transcriptional activation of gene expression will be reviewed. In addition, the mechanisms of post-transcriptional mRNA stabilization will also be addressed. We will discuss whether these two processes of regulation of hypoxia-responsive genes are mechanistically linked and co-operative in nature. (+info)
Role of endothelial nitric oxide synthase in endothelial cell migration.
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Endothelium-derived nitric oxide (NO) and its precursor L-arginine have been implied to promote angiogenesis, but little is known about the precise mechanism. The inhibition of endogenous NO formation by Nomega-nitro-L-arginine methyl ester (L-NAME) (1 mmol/L) but not its inactive enantiomer D-NAME (1 mmol/L) inhibited endothelial cell sprouting from the scratched edge of the cultured bovine aortic endothelial cell monolayer. Inhibition of endogenous NO release by L-NAME was confirmed by amperometric measurement using an NO-specific electrode. In the modified Boyden chamber, L-NAME (1 mmol/L) significantly inhibited endothelial cell migration, whereas L-NAME did not affect endothelial DNA synthesis as assessed by analysis of [3H]thymidine incorporation. We then examined alteration of endothelial cell adhesion molecule expression after the inhibition of NO by L-NAME in cultured human umbilical vein endothelial cells. In both normoxic and hypoxic conditions, L-NAME (1 mmol/L) inhibited surface expression of integrin alphavbeta3, which is an important integrin facilitating endothelial cell survival and angiogenesis. However, L-NAME did not affect the expression of platelet endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, vascular endothelial adhesion molecule-1, gap junction protein connexin 43, and VE-cadherin, which have been reported to potentially affect angiogenesis. In summary, inhibition of endothelial NO synthase by L-NAME attenuated endothelial cell migration but not proliferation in vitro. Furthermore, endogenous endothelium-derived NO maintains the functional expression of integrin alphavbeta3, a mediator for endothelial migration, survival, and angiogenesis. Endothelium-derived NO, thus, may play an important role in mediating angiogenesis by supporting endothelial cell migration, at least partly, via an integrin-dependent mechanism. (+info)
Defective vascularization of HIF-1alpha-null embryos is not associated with VEGF deficiency but with mesenchymal cell death.
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Hypoxia-inducible factor 1 (HIF-1) is a dimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits that plays an essential role in mammalian O2 homeostasis. In Hif1a-/- knockout mice, complete deficiency of HIF-1alpha resulted in cardiac and vascular malformations and embryonic lethality at E10.5. Between E8. 75 and E9.25 striking vascular regression and abnormal remodeling occurred in the cephalic region concomitant with marked mesenchymal cell death. Similar vascular defects were observed in HIF-1alpha- and VEGF-deficient embryos and VEGF mRNA expression was not induced by hypoxia in Hif1a-/- embryonic stem cells. Surprisingly, Hif1a-/- embryos demonstrated increased VEGF mRNA expression compared to wild-type embryos. In tissue culture cells, VEGF mRNA expression was induced by glucose deprivation independent of HIF-1alpha, providing a mechanism for increased VEGF mRNA expression in Hif1a-/- embryos, in which absence of adequate tissue perfusion resulted in both O2 and glucose deprivation. Rather than being associated with VEGF deficiency, the vascular defects in Hif1a-/- embryos were spatially correlated with cell death, the onset of which preceded vascular regression. (+info)
Myocyte-dependent regulation of endothelial cell syndecan-4 expression. Role of TNF-alpha.
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Syndecan-4 is a unique member of the syndecan gene family that has the ability to bind and activate protein kinase C-alpha. Whereas increased syndecan-4 levels have been noted in ischemic hearts, little is known regarding the regulation of its expression. To investigate the role of cardiac myocytes in induction of syndecan-4 expression, human endothelial cells (ECV304) were exposed to a medium conditioned by primary mouse cardiac myocytes or H9c2 cells. The medium conditioned by hypoxic but not normal myocytes was able to induce syndecan-4 expression in ECV cells. Western analysis of the conditioned medium demonstrated an increased presence of tumor necrosis factor-alpha (TNF-alpha) in the medium conditioned by hypoxic but not normal myoblasts. Primary cardiac myocytes collected from the wild type C57/129 but not the homozygous TNF-alpha-/- knockout mice were able to induce syndecan-4 expression in ECV cells when cultured under hypoxic conditions. In vitro studies demonstrated that TNF-alpha induced endothelial cell syndecan-4 expression by both increasing syndecan-4 gene expression in an NF-kappaB-dependent manner and by prolonging syndecan-4 mRNA half-life. We conclude that TNF-alpha is the principal factor produced by the ischemic myocytes that is responsible for induction of endothelial cell syndecan-4 expression and that this requires both transcriptional and posttranscriptional mechanisms. (+info)
Hypoxia-associated induction of early growth response-1 gene expression.
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The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298-8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 approximately 12 torr) displayed increased Egr-1 expression because of de novo biosynthesis, with a approximately 10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to -424/-65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning -424/-375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation of Egr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-betaII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediated Egr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1beta and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition. (+info)