endothelin-1 and cobaltous-chloride

Topics: Cell Hypoxia; Cell Line; Cell Survival; Cobalt; Diterpenes; Endothelial Cells; Endothelin-1; Heme Oxygenase-1; Humans; Hydroxylation; Hypoxia-Inducible Factor 1, alpha Subunit; NF-E2-Related Factor 2; p38 Mitogen-Activated Protein Kinases; Prolyl Hydroxylases; Reactive Oxygen Species; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction

Fetal striatal transplantation has emerged as a new therapeutic strategy in Huntington's disease (HD). Hypoxia is one of the microenvironmental stress conditions to which fetal tissue is exposed as soon as it is isolated and transplanted into the diseased host brain. Mechanisms that support neuroblast survival and replenishment of damaged cells within the HD brain in the hypoxic condition have yet to be fully elucidated. This study is aimed at investigating the molecular pathways associated with the hypoxic condition in human fetal striatal neuroblasts (human striatal precursor (HSP) cells), using the hypoxia-mimetic agent cobalt chloride (CoCl2). We analyzed the effect of CoCl2 on HSP cell proliferation and on the expression of hypoxia-related proteins, such as hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Moreover, we evaluated fibroblast growth factor 2 (FGF2; 50ng/ml) and endothelin-1 (ET-1; 100nM) proliferative/survival effects in HSP cells in normoxic and hypoxic conditions. Dose-response experiments using increasing concentrations of CoCl2 (50-750μM) showed that the HSP cell growth was unaffected after 24h, while it increased at 48h, with the maximal effect observed at 400μM. In contrast, cell survival was impaired at 72h. Hypoxic conditions determined HIF-1α protein accumulation and increased gene and protein expression of VEGF, while FGF2 and ET-1 significantly stimulated HSP cell proliferation both in normoxic and hypoxic conditions, thus counteracting the apoptotic CoCl2 effect at 72h. The incubation with selective receptor (FGFR1, endothelin receptor A (ETA) and endothelin receptor B (ETB)) inhibitors abolished the FGF2 and ET-1 neuroprotective effect. In particular, ET-1 stimulated HSP cell survival through ETA in normoxic conditions and through ETB during hypoxia. Accordingly, ETA expression was down-regulated, while ETB expression was up-regulated by CoCl2 treatment. Overall, our results support the idea that HSP cells possess the machinery for their adaptation to hypoxic conditions and that neurotrophic factors, such as FGF2 and ET-1, may sustain neurogenesis and long-term survival through complex receptor-mediated mechanisms.

Topics: Cell Hypoxia; Cell Proliferation; Cell Survival; Cells, Cultured; Central Nervous System Agents; Cobalt; Corpus Striatum; Dose-Response Relationship, Drug; Endothelin-1; Fetal Stem Cells; Fibroblast Growth Factor 2; Fibroblast Growth Factors; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Nerve Tissue Proteins; Neural Stem Cells; Oxidoreductases Acting on CH-CH Group Donors; Receptor, Endothelin A; Receptor, Endothelin B; Receptor, Fibroblast Growth Factor, Type 1; RNA, Messenger; Vascular Endothelial Growth Factor A

Tumour hypoxia, being widespread in solid tumours, is related to an increased risk of invasion and metastasis as well as to resistance to chemotherapy and radiotherapy. Hypoxia-inducible factor-1alpha (HIF-1alpha) has emerged as the key regulator of the cellular response to hypoxia. In primary breast cancers, HIF-1alpha is overexpressed, and high levels of HIF-1alpha predict for early relapse and increased metastasis. The endothelin (ET) axis, comprising the peptides ET-1, -2, -3 and their receptors A (ETAR) and B (ETBR), is another regulatory system of major relevance in human breast cancer. However, little is known about the interaction of HIF-1alpha and the ET axis in breast carcinomas. Therefore, we analysed expression of HIF-1alpha and the ET axis in 600 breast cancer tissue samples by immunohistochemistry, observing a significant correlation between expression of HIF-1alpha and ET-1 (P<0.001). In vitro, hypoxia was found to double ET-1 secretion of MCF-7 breast cancer cells (203.5% of controls; P<0.001), thereby promoting an invasive phenotype. Of note, real-time PCR analysis revealed that the increase of ET-1 was not due to enhanced transcription of the ET-1 gene. In invasion assays, breast cancer cell invasiveness was strongly increased by hypoxia (150.0% of controls; P=0.007). Most important, this increase was completely inhibited by the selective ETAR antagonist atrasentan. In conclusion, we provide evidence for a relevant interaction between hypoxia and the ET axis in breast cancer cells. Our data suggest that tumour hypoxia induces breast carcinoma invasiveness by releasing intracellularly stored ET-1. However, induction of invasiveness may be inhibited by selective ETAR antagonism, thus emphasising the promising status of the ET axis as a therapeutic target in breast cancer.

Topics: Antineoplastic Agents; Aspartic Acid Endopeptidases; Atrasentan; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cobalt; Endothelin A Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Staging; Pyrrolidines; Receptor, Endothelin A; Time Factors; Up-Regulation

We investigated whether endothelin-2/vasoactive intestinal contractor (ET-2/VIC) gene expression, upregulated by hypoxia in cancer cells, was associated with differentiation in neuronal cells. RT-PCR analysis, morphological observations, and immunostaining revealed that CoCl2, a hypoxic mimetic agent, at 200 microM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced neurite outgrowth in PC-12 rat pheochromocytoma cells. These effects induced by 200 microM CoCl2 were completely inhibited by the antioxidant N-acetyl cysteine at 20 mM. In addition, CoCl2 increased the level of intracellular reactive oxygen species (ROS) at an early stage. Furthermore, interleukin (IL)-6 gene expression was upregulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by ROS may be associated with neuronal differentiation through the regulation of IL-6. When the cells were treated with 500 microM CoCl2 for 24 hr, however, ET-2/VIC gene expression disappeared, IL-6 gene expression was downregulated, and necrosis was subsequently induced in the PC-12 cells.

Topics: Animals; Antimutagenic Agents; Antioxidants; Cell Differentiation; Cell Division; Cobalt; Endothelin-1; Endothelin-2; Gene Expression; Intercellular Signaling Peptides and Proteins; Interleukin-6; Neurites; PC12 Cells; Peptides; Rats; Reactive Oxygen Species

Hypoxia-inducible factor (HIF)-1alpha is an important transcriptional factor that activates the gene expression of glycolytic enzymes, which are activated as compensation for impaired beta-oxidation of fatty acid in the failing heart. We reported that cardiac endothelin (ET)-1 expression is markedly increased in heart failure. The mechanism, however, is unknown. Because we found an HIF-1alpha binding site in the 5'-promoter region of the ET-1 gene, we hypothesized that HIF-1alpha is involved in this mechanism.. In rat cardiomyocytes, luciferase assay and electrophoretic mobility shift assay showed that HIF-1alpha transcriptionally activates ET-1 gene expression by direct interaction with the predicted DNA binding site in the 5'-promoter region. HIF-1alpha mRNA and ET-1 mRNA in the failing heart increased during the aggravation of heart failure in vivo in animal models, ie, rats with myocardial infarction and hamsters with cardiomyopathy. In cultured cardiomyocytes treated with a mitochondrial inhibitor, HIF-1alpha mRNA and ET-1 mRNA were markedly increased with activated glycolysis, and antisense oligonucleotide for HIF-1alpha largely inhibited the increased gene expression of ET-1.. The present study revealed a novel molecular mechanism of upregulation of myocardial ET-1 in heart failure, indicating that induction of HIF-1alpha to stimulate glycolysis as an adaptation in heart failure against impaired energy metabolism alternatively causes an elevation of cardiac ET-1 gene expression as a maladaptation.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Cells, Cultured; Cobalt; Cricetinae; DNA-Binding Proteins; DNA, Antisense; Endothelin-1; Energy Metabolism; Gene Expression Regulation; Hemodynamics; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Luciferases; Mitochondria; Myocardial Infarction; Myocardium; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Transcription Factors

We investigated whether impairment of myocardial energy metabolism attenuates cardiac function and increases cardiac endothelin-1 (ET-1) gene expression in rats. Three weeks after commencing administration of cobalt chloride (CoCl2), an inhibitor of mitochondrial function, the peak positive first derivative of left ventricular (LV) pressure, an indicator of myocardial contractility, was significantly decreased in the CoCl2-treated rats. LV end-diastolic pressure and right ventricular systolic pressure were increased in the CoCl2-treated rats. Echocardiography showed that fractional shortening was significantly decreased in the CoCl2-treated rats. Myocardial expressions of acyl-CoA synthase mRNA, an enzyme involved in fatty acid utilization, was markedly decreased in the CoCl2-treated rats. Under such conditions, myocardial expression of preproendothelin-1 mRNA and atrial natriuretic peptide (ANP) mRNA, molecular markers of heart failure, was markedly increased in the CoCl2 rats. In conclusion, the data suggest that impairment of myocardial energy metabolism causes hemodynamic abnormality and increases molecular markers of heart failure (ET-1, ANP mRNA). These data suggest that myocardial energy metabolism is one of the factors involved in the upregulation of ET-1 gene expression in the failing heart.

Topics: Animals; Atrial Natriuretic Factor; Cobalt; Endothelin-1; Endothelins; Energy Metabolism; Heart Failure; Male; Myocardium; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger

We evaluated the contribution of cytochrome P-450 (CYP450)-dependent arachidonic acid (AA) metabolites and prostanoids to the renal hemodynamic and tubular effects of endothelin-1 (ET-1) in anesthetized rats. Either ET-1 (0.3, 1.0, and 3 pmol.kg-1.min-1) or vehicle was infused intravenously during two to three 30-min clearance experimental periods. Only high-dose ET-1 increased mean arterial pressure: control, 75 +/- 3 mmHg vs. experimental, 84 +/- 4 mmHg. A dose-dependent diuretic-natriuretic response to ET-1 occurred despite progressive declines in glomerular filtration rate (GFR) and renal blood flow. In the face of a 36% reduction in GFR in response to the highest dose of ET-1, urinary sodium excretion (UNaV) increased threefold from 0.57 +/- 0.11 to 1.6 +/- 0.10 mumol.100 g-1.min-1. Indomethacin (5 mg/kg) decreased basal GFR from 1.2 +/- 0.3 ml.100 g-1.min-1 to 0.8 +/- 0.1 ml.100 g-1.min-1 and potentiated the GFR lowering action of ET-1 associated with reductions in UNaV and urine volume. Cobalt chloride (CoCl2) and dibromododec-11-enoic acid (DBDD), which diminish CYP450-dependent AA metabolism through different mechanisms, were used to identify CYP450 products mediating the renal functional actions of ET-1. DBDD (12.5 micrograms/min) reduced urinary excretion of 20-hydroxyeicosatetraenoic acid from 3.4 +/- 0.9 (control) to 1.1 +/- 0.6 ng/h and abolished the negative effects of ET-1 on GFR while decreasing the diuretic-natriuretic action of ET-1. Similar effects were produced by CoCl2. Clotrimazole, an inhibitor of epoxygenase activity, was without effect on ET-1-induced renal functional changes. Thus the capacity of ET-1 to enhance prostaglandin production was primarily expressed in terms of positive effects on renal hemodynamics. In contrast, CYP450 products promoted sodium excretion despite negative effects on renal hemodynamics.

Topics: Anesthesia, General; Animals; Clotrimazole; Cobalt; Cytochrome P-450 Enzyme System; Eicosanoids; Endothelin-1; Fatty Acids, Monounsaturated; Glomerular Filtration Rate; Indomethacin; Kidney; Male; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Renal Circulation; Vascular Resistance

In the rat isolated perfused kidney, 5,8,11,14-eicosatetraynoic acid, an inhibitor of all pathways of arachidonic acid (AA) metabolism, diminished endothelin-1 (ET-1)- and angiotensin II (ANG II)-induced renal vasoconstriction by approximately 60-70%. We then examined the individual contribution of each oxygenase, cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P-450 (CYP) to the vasoconstrictor effects of ET-1 and ANG II. Inhibition of COX with indomethacin reduced by 30-40% the vasoconstrictor responses to ET-1 and ANG II. Inhibition of 12-LOX with baicalein and 5- and 12-LOX with 5,8,11-eicosatriynoic acid attenuated ANG II-induced renal vasoconstriction by approximately 40-60% but did not affect responses to ET-1. In contrast, 12,12-dibromododec-11-enoic acid (DBDD), an inhibitor of the CYP omega/omega 1-hydroxylase pathway, diminished ET-1-induced renal vasoconstriction by 30-40%, an effect reproduced by depletion of CYP enzymes with CoCl2. Neither DBDD nor CoCl2 affected renal vasoconstriction elicited by ANG II. ET-1 increased efflux of 19- and 20-hydroxyeicosatetraenoic acid, an effect reduced by DBDD. Thus products of the COX and CYP pathways contribute to the renal vasoconstrictor response to ET-1, whereas COX- and LOX-derived eicosanoids contribute to the response to ANG II, accounting for > or = 80% of the vasoactivity of the peptides.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Angiotensin II; Animals; Arachidonic Acids; Cobalt; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme System; Endothelin-1; Enzyme Inhibitors; Flavanones; Flavonoids; Indomethacin; Lipoxygenase; Lipoxygenase Inhibitors; Male; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Renal Circulation; Vasoconstriction