endothelin-1 and herbimycin

Chronic hypokalemia increases NHE3 activity in OKP cells. The aim of the present study was to determine whether an autocrine mechanism is involved in this activation.. After incubation of OKP cells in normal-K(+) and low-K(+) media for 24 h, the potassium concentration in the low-K(+) media was adjusted to a normal level. These conditioned media were then used as the normal-K(+) and low-K(+) supernatants. Other OKP cells were incubated in these normal-K(+) and low-K(+) supernatants and the mechanism of Na(+)/H(+) antiporter activation was examined.. The EIPA-resistant Na(+)/H(+) antiporter activity of OKP cells increased after 4 h incubation in the low-K(+) supernatant, and the amount of NHE3 protein increased at 24 h. Since both BQ788 and saralasin blocked this antiporter activation, the supernatant concentration of endothelin I (ET-I) and angiotensin II (Ang-II) were measured. The ET-I concentration was reduced, but the Ang-II concentration remained unchanged. There was a significant association between a reduction in the ET-I concentration and an increase in Na(+)/H(+) antiporter activity, but only when Ang-II was present in the supernatant.. An autocrine mechanism is involved in the activation of NHE3 in OKP cells. Both ET-I and Ang-II play a role in this activation.

Topics: Acidosis; Amiloride; Angiotensin II; Animals; Autocrine Communication; Benzoquinones; Cell Line; Culture Media, Conditioned; Dose-Response Relationship, Drug; Endothelin B Receptor Antagonists; Endothelin-1; Hydrogen-Ion Concentration; Kidney; Lactams, Macrocyclic; Oligopeptides; Opossums; Piperidines; Potassium; Protein-Tyrosine Kinases; Quinones; Receptor, Endothelin B; Rifabutin; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers; Time Factors

Small-artery responses to vasoconstrictor agonists are important for vascular function. To investigate the signaling pathways involved in contraction, we studied the activation and regulation of p38 mitogen-activated protein kinases (p38MAPKs) and heat shock protein (HSP) kinase by endothelin and noradrenaline in rat mesenteric arteries. Both vasoconstrictors activated p38alpha and/or p38beta but not p38gamma or p38delta, leading to increased HSP kinase activity. p38MAPK activation by noradrenaline was maximum between 2 and 10 minutes and was wholly dependent on calcium influx but insensitive to the tyrosine kinase inhibitor herbimycin A. In contrast, endothelin induced a biphasic response, with activation at 2 and 10 minutes. The early activity was wholly dependent on calcium influx and inhibited by herbimycin A. The later activity was only 50% calcium dependent, was insensitive to herbimycin A, but was 50% inhibited by genistein, a nonselective tyrosine kinase inhibitor. With both agonists, p38MAPK activity returned to basal by 30 minutes. SB203580, a p38MAPK inhibitor, blocked agonist-induced HSP kinase activity, and herbimycin A inhibited activation by endothelin but not by noradrenaline. In addition, SB203580 inhibited noradrenaline-induced contraction but had little effect on contraction to endothelin. These data show that vasoconstrictors use different upstream activators of p38MAPK in vascular tissue and that the p38MAPK pathway is selectively implicated in the contractile response to noradrenaline in small arteries.

Topics: Animals; Arteries; Benzoquinones; Calcium; Endothelin-1; Enzyme Activation; Female; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Lactams, Macrocyclic; Mesenteric Arteries; Mitogen-Activated Protein Kinases; Norepinephrine; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; Vasoconstriction

Upregulation of endothelin-1 (ET-1) synthesis in venous bypass grafts in response to arterial levels of blood pressure may play a major role in graft failure. To investigate this hypothesis, isolated segments of the rabbit jugular vein were perfused at physiological (0 to 5 mm Hg) and nonphysiological (20 mm Hg) levels of intraluminal pressure. As judged by reverse transcription-polymerase chain reaction analysis (mRNA level), neither endothelin-converting enzyme nor endothelin A receptor expression appeared to be pressure sensitive. In contrast, there was a profound and time-dependent increase in endothelial prepro-ET-1 mRNA and intravascular ET-1 abundance (by ELISA) as well as in smooth muscle endothelin B receptor mRNA and functional protein (by superfusion bioassay) on raising the perfusion pressure from 5 to 20 mm Hg, but not from 0 to 5 mm Hg, for up to 12 hours. Video microscopy analysis revealed that the segments were distended by 75% at 5 mm Hg and near maximally at 20 mm Hg compared with the resting diameter at 0 to 1 mm Hg. Treatment of the segments with actinomycin D (1 micromol/L), the specific protein kinase C inhibitor, Ro 31-8220 (0.1 micromol/L), or the c-Src family-specific tyrosine kinase inhibitor, herbimycin A (0.1 micromol/L), demonstrated that the pressure-induced expression of these gene products occurs at the level of transcription and requires activation of protein kinase C, but not c-Src. In venous bypass grafts such deformation-induced changes in gene expression may contribute not only to acute graft failure through ET-1-induced vasospasm but also to endothelin A receptor- and/or endothelin B receptor-mediated smooth muscle cell hyperplasia and graft occlusion.

Topics: Animals; Aspartic Acid Endopeptidases; Base Sequence; Benzoquinones; Dactinomycin; DNA Primers; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Enzyme Inhibitors; In Vitro Techniques; Jugular Veins; Lactams, Macrocyclic; Male; Metalloendopeptidases; Pressure; Protein Kinase Inhibitors; Protein Kinases; Protein Precursors; Quinones; Rabbits; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Rifabutin; RNA, Messenger; Stress, Mechanical; Up-Regulation

Ets-1 is a transcription factor that activates expression of matrix-degrading proteinases such as collagenase and stromelysin. To study the control of ets-1 gene expression in rat vascular smooth muscle cells (VSMC), cells were exposed to factors known to regulate VSMC migration and proliferation. Platelet-derived growth factor-BB (PDGF-BB), endothelin-1 (ET-1), and phorbol 12-myristate 13-acetate (PMA) induced a dose-dependent expression of ets-1 mRNA. These effects were abrogated by inhibition of protein kinase C (PKC) by H-7 or chronic PMA treatment. Ets-1 mRNA was superinduced by PDGF-BB and ET-1 in the presence of cycloheximide. The chelation of intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester and the depletion of endoplasmic reticulum intracellular Ca2+ concentration ([Ca2+]i) by thapsigargin inhibited PDGF-BB- and ET-1-induced ets-1 mRNA, whereas ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid had no effect. However, [Ca2+]i release alone was not sufficient to increase ets-1 mRNA. Forskolin blocked ET-1-, PDGF-BB-, and PMA-induced ets-1 mRNA, as well as inositol phosphate formation, consistent with an effect through impairment of PKC activation. Inhibitors of ets-1 gene expression, such as H-7 and herbimycin A, inhibited the ET-1 induction of collagenase I mRNA. We propose that ets-1 may be an important element in the orchestration of matrix proteinase expression and of vascular remodeling after arterial injury.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Becaplermin; Benzoquinones; Calcium; Cells, Cultured; Collagenases; Culture Media, Serum-Free; Cyclic AMP; Cycloheximide; Endoplasmic Reticulum; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Extracellular Matrix; Gene Expression Regulation; Lactams, Macrocyclic; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; Protein Kinase C; Protein Synthesis Inhibitors; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Proto-Oncogene Proteins c-sis; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; RNA, Messenger; Transcription Factors; Transcription, Genetic

The effect of the macrophage- and T-lymphocyte-derived cytokine oncostatin M (OSM) on endothelin-1 (ET-1) production in cultured human umbilical cord vein endothelial cells (HUVEC) was studied. OSM (2.5-10 ng/ml) stimulated ET-1 production and the expression of preproendothelin-1 mRNA. The stimulatory effect of OSM was reversed by anti-interleukin (IL)-6 IgG (33 microg/ml). IL-6 (10 ng/ml) was shown to stimulate ET-1 production. The tyrosine kinase inhibitors herbimycin (250-500 ng/ml) and genistein (1-4 microg/ml) suppressed basal ET-1 production and reversed the stimulatory effect of OSM, whereas daidzein (1-8 microg/ml), a less active analog of genistein, had no effect on basal ET-1 production and only partly reversed the stimulatory effect of OSM. The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibited ET-1 production. Downregulation of protein kinase C (PKC) with PMA (1 microM) preincubation potentiated OSM-induced ET-1 production. In summary, OSM stimulated ET-1 production in cultured HUVEC. The stimulation was probably mediated by IL-6. Furthermore, the present data suggest that tyrosine kinase activation was involved in ET-1 stimulation and that PKC activation leads to suppression of basal and OSM-stimulated ET-1 production.

Topics: Benzoquinones; Cell Division; Cell Survival; Cells, Cultured; Drug Interactions; Endothelin-1; Endothelins; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation; Genistein; Growth Inhibitors; Humans; Isoflavones; Lactams, Macrocyclic; Oncostatin M; Peptides; Protein Precursors; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Transcription, Genetic; Umbilical Veins

Endothelin (ET-1), a contractor and mitogen in the vasculature, enhanced cAMP production (t1/2, 2.2 min; EC50, 89 +/- 6.3 nM) and stimulated activity of the cAMP-dependent protein kinase (PKA) in pig coronary arteries. These responses were blunted by the protein tyrosine kinase (PTK) inhibitors genistein and herbimycin-A, but not by inhibitors of protein kinase C or cyclooxygenase. In contrast, forskolin-stimulated cAMP production was unaffected by PTK inhibition. Immunoblot analysis revealed that ET-1 induced a concentration-dependent protein tyrosine (PT) phosphorylation. Sarafotoxin-c, a selective ETB receptor agonist, had no effect on either cAMP levels or PT phosphorylation. Moreover, pervanadate (PV), a potent inhibitor of PT phosphatases, enhanced both cAMP formation and PT phosphorylation, both of which were blocked by PTK inhibitors. The effects of ET-1 and PV were not additive, suggesting a similar mode of activation, whereas responses to ET-1 and forskolin were synergistic. These findings indicate that AC and PKA are activatable via a nonreceptor PTK-dependent pathway downstream from the G-protein-linked ETA receptor. Because cAMP is a dilator and antimitogen in smooth muscle, stimulation of AC activity may be a negative feedback mechanism regulating ET-1-induced vasoconstriction and/or mitogenesis.

Topics: Adenylyl Cyclases; Animals; Benzoquinones; Colforsin; Coronary Vessels; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Genistein; GTP-Binding Proteins; In Vitro Techniques; Kinetics; Lactams, Macrocyclic; Muscle, Smooth, Vascular; Peptides; Protein-Tyrosine Kinases; Quinones; Receptor, Endothelin A; Receptors, Endothelin; Rifabutin; Signal Transduction; Swine; Vanadates; Viper Venoms

Endothelin-1 (ET-1) is known to induce the contraction and proliferation of glomerular mesangial cells. ET-1 has been shown to activate p42 and p44 mitogen-activated protein kinases (MAPKs), also known as extracellular signal regulated kinases (ERKs), through both protein kinase C (PKC) and protein tyrosine kinase (PTK)-dependent pathways. However, an involvement of c-Jun NH2-terminal kinase (JNK), one of members of the MAPK family, in ET-1 signaling in mesangial cells has not yet been elucidated. To clarify this point, we examined whether ET-1 could activate JNK and the mechanism of activation in cultured mesangial cells. ET-1 enhanced the activities of JNK in a dose-dependent (10(-8) M maximum) and time-dependent manner, with a peak at 15 minutes. ET-1-induced activation of JNK was blocked by BQ-123, an antagonist for the ETA receptor. The depletion of PKC by prolonged treatment with phorbol 12,13 dibutyrate or the inhibition of PKC by GF 109203X failed to inhibit ET-1-induced activation of JNK. In contrast, ET-1-induced activation of JNK was significantly reduced by calcium chelation (with BAPTA/AM and EGTA). In addition, ionomycin, a calcium ionophore, and thapsigargin, an intracellular calcium-rising agent, were able to induce the activation of JNK. ET-1-induced activation of JNK was also inhibited by PTK inhibitors (herbimycin A and genistein). Furthermore, ET-1 increased the DNA-binding activity of AP-1 containing c-Jun and c-Fos proteins. These results indicate that ET-1 is able to activate JNK in glomerular mesangial cells through PKC-independent and PTK-dependent pathways and intracellular calcium is necessary to the activation of JNK.

Topics: Animals; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; DNA; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Genistein; Glomerular Mesangium; Isoflavones; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; Mitogen-Activated Protein Kinases; Peptides, Cyclic; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Transcription Factor AP-1

Endothelin 1 (ET-1) is produced in ovarian cancer cell lines and has been shown to act through ET(A) receptors as an autocrine growth factor to promote tumor cell proliferation in vitro. In OVCA 433 cells, the efficacy of ET-1 as a stimulus of [3H]thymidine incorporation was equivalent to that of epidermal growth factor. ET-1 also stimulated the rapid expression of c-fos, an action mediated by ET(A) receptors. The mitogenic action of ET-1 was not mediated by a pertussis toxin-sensitive G protein. An analysis of the effects of inhibition and depletion of protein kinase C (PKC) on mitogenic responses demonstrated that PKC was necessary but not sufficient for maximal stimulation by ET-1. In quiescent OVCA 433 cells, ET-1-induced stimulation of [3H]thymidine incorporation was prevented by two structurally distinct inhibitors of tyrosine kinase, herbimycin A and genistein. These results indicate that both PKC and protein tyrosine kinase participate in ET-1-stimulated mitogenic signaling. ET-1 rapidly stimulated tyrosine phosphorylation of several cellular proteins, among which p125FAK and p42 mitogen-activated protein kinase were identified. The additivity between the potent mitogenic actions of ET-1 and epidermal growth factor is consistent with the independence of their signal transduction pathways in ovarian cancer cells. These findings also indicate that intracellular signaling between the ET(A) receptor and a yet unidentified tyrosine kinase is involved in the mitogenic response to ET-1.

Topics: Benzoquinones; Blotting, Northern; Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion Molecules; Cell Division; DNA, Neoplasm; Endothelin-1; Enzyme Inhibitors; Epidermal Growth Factor; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation; Genes, fos; Genistein; Humans; Immunoblotting; Indoles; Isoflavones; Lactams, Macrocyclic; Maleimides; Ovarian Neoplasms; Pertussis Toxin; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

Mediators including the neuropeptide endothelin-1 (ET-1), which are released in response to injury, modulate the expression of cell adhesion molecules on leukocytes and endothelial cells. The mechanisms underlying this process are not clear. In this study we investigated the effect of endothelin-1 on the expression of tyrosine phosphorylated proteins in human blood monocytes. Endothelin-1 caused an increase in tyrosine phosphorylated proteins in monocytes in a time-dependent and dose-dependent manner, the Mr 60, 80 and 110 kDa proteins being the most prominent. This effect was blocked by pre-incubating the monocytes with the selective tyrosine kinase inhibitors genistein or herbimycin A. Endothelin-1 induced upregulation of tyrosine phosphorylated proteins appears to be mediated by the ETA receptor. Unlike our previously reported studies in endothelial cells, immunoprecipitation with anti-src or anti-JAK antibodies followed by immunoblotting with PY20 in human blood monocytes revealed that these proteins of Mr 60, 80 and 110 kDa were not related to src or JAK kinases. These findings suggest that ET- exerts its effect on monocytes by a pathway involving tyrosine kinases other than src or JAK kinases.

Topics: Adult; Antibodies, Blocking; Benzoquinones; Blotting, Western; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Female; Genistein; Humans; In Vitro Techniques; Isoflavones; Janus Kinase 1; Lactams, Macrocyclic; Male; Monocytes; Phosphorylation; Precipitin Tests; Protein-Tyrosine Kinases; Proteins; Quinones; Receptors, Endothelin; Rifabutin; src-Family Kinases; Time Factors; Tyrosine

To investigate the novel interaction between endothelin-1 (ET-1) and cellular protein tyrosine kinases (PTK), we asked whether Ca2+ influx links ET-1 receptors to PTK activation. In glomerular mesangial cells, ET-1 stimulated a biphasic increase in PTK activity in anti-phosphotyrosine immunoprecipitates that temporally correlated with increased tyrosine phosphorylation of cellular proteins. ET-1 increased tyrosine phosphorylation of proteins in the cytosol and in a puncture distribution consistent with focal adhesions. Addition of ionomycin to increase Ca2+ influx stimulated PTK activity, and inhibition of extracellular Ca2+ influx blocked PTK activation by ET-1. ET-1 increased autophosphorylation of pp60c-src, which was mimicked by addition of ionomycin and inhibited by chelation of extracellular Ca2+. In addition, a selective PTK inhibitor blocked induction of c-fos mRNA by ionomycin, suggesting that Ca(2+)-stimulated PTKs contribute to a signaling pathway regulating immediate early gene expression. Taken together, these results demonstrate that ET-1 stimulates nonreceptor PTK activity, including pp60c-src, by activating Ca2+ channels and subsequent influx of extracellular Ca2+.

Topics: Animals; Benzoquinones; Calcium; Calcium Channels; Endothelin-1; Enzyme Activation; Ionomycin; Lactams, Macrocyclic; Male; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-fos; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; RNA, Messenger; src-Family Kinases