endothelin-1 and pyrazolanthrone

The sole FDA-approved drug treatment for ischemic stroke is tissue-type plasminogen activator (tPA). However, upregulation of JNK mitogen-activated protein kinase (MAPK) and endothelin 1 (ET-1) by tPA after stroke contributes to impaired cerebrovascular autoregulation. Wild-type (wt) tPA can bind to the lipoprotein-related receptor (LRP), which mediates vasodilation, or NMDA receptors (NMDA-Rs), exacerbating vasoconstriction. Elevations in IL-6, a marker of inflammation that accompanies stroke, are reported to be an adverse prognostic factor. We hypothesized that IL-6 released into CSF after stroke by wt-tPA through activation of NMDA-Rs and upregulation of ET-1 and JNK contribute to impairment of cerebrovascular autoregulation and increased histopathology. Results show that IL-6 was increased post stroke in pigs, which was increased further by wt-tPA. Co-administration of the IL-6 antagonist LMT-28 with wt-tPA prevented impairment of cerebrovascular autoregulation and necrosis of hippocampal cells. wt-tPA co-administered with the JNK inhibitor SP 600125 and the ET-1 antagonist BQ 123 blocked stroke-induced elevation of IL-6. Co-administration of LMT-28 with wt-tPA blocked the augmentation of JNK and ET-1 post stroke. In conclusion, IL-6 released after stroke, which is enhanced by wt-tPA through activation of NMDA-Rs and upregulation of ET-1 and JNK, impairs cerebrovascular autoregulation and increases histopathology. Strategies that promote fibrinolysis while limiting activation of NMDA-Rs and upregulation of IL-6 may improve the benefit/risk ratio compared to wt-tPA in treatment of stroke.

Topics: Animals; Anthracenes; Cerebral Cortex; Disease Models, Animal; Endothelin-1; Hippocampus; Homeostasis; Interleukin-6; Necrosis; Oxazolidinones; Random Allocation; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Stroke; Swine; Tissue Plasminogen Activator; Up-Regulation

Endothelin-1 (ET-1) is a 21-amino acid peptide with multifunctional regulation. Initial research indicated that ET-1 is related to the inflammatory pathogenesis of periodontitis and involved in the regulation of cytokines, but the mechanisms involved remain unclear. The primary aim of this study is to investigate how ET-1 affects proinflammatory cytokine expression in human periodontal ligament (hPDL) cells.. hPDL cells were obtained from both healthy (H)- and periodontitis (P)-affected periodontal tissues. H-hPDL and P-hPDL cells were treated with ET-1 (1, 10, and 100 nM) for 12, 24, and 48 hours. The untreated cells served as a control. To confirm the specificity of the ET-1 effects, 100 nM of the specific endothelin A (ETA) receptor antagonist BQ123 and 100 nM of the specific ETB receptor antagonist BQ788, as negative control, were used. To examine the signaling pathways and molecular mechanisms involved in ET-1-mediated cytokine expression, H-hPDL and P-hPDL cells were pretreated with specific inhibitors for extracellular signal-regulated kinase (ERK1/2) (PD98059), c-Jun N-terminal kinase (SP600125), and p38 kinase (SB203580) for 1 hour before 100 nM ET-1 stimulation. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 messenger RNA (mRNA) and protein levels were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.. ET-1 dose- and time-dependently induced the production of proinflammatory cytokines TNF-α, IL-1β, and IL-6 by H-hPDL and P-hPDL cells at both mRNA and protein levels. However, ETA and ETB receptor antagonists inhibited the stimulatory effects of ET-1 on inflammatory cytokine expression in H-hPDL and P-hPDL cells. Furthermore, inhibitors of the mitogen-activated protein kinases (MAPKs) significantly reduced ET-1-stimulated TNF-α, IL-1β, and IL-6 expression in H-hPDL and P-hPDL cells.. ET-1 may be involved in the inflammatory process of periodontitis, at least in part, by stimulating proinflammatory cytokine production via the MAPK pathway in hPDL cells.

Topics: Adult; Anthracenes; Calcium-Calmodulin-Dependent Protein Kinases; Cell Culture Techniques; Cells, Cultured; Cytokines; Dose-Response Relationship, Drug; Endothelin B Receptor Antagonists; Endothelin Receptor Antagonists; Endothelin-1; Female; Flavonoids; Humans; Imidazoles; Interleukin-1beta; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Peptides, Cyclic; Periodontal Ligament; Periodontitis; Piperidines; Pyridines; Time Factors; Tumor Necrosis Factor-alpha

Resistin and endothelin (ET)-1 have been reported to inhibit adipogenesis and regulate adipocyte insulin resistance, respectively. Although both hormones interact with each other, the exact signaling pathway of ET-1 to act on resistin gene expression is still unknown. Using 3T3-L1 adipocytes, we investigated the signaling pathways involved in ET-1-stimulated resistin gene expression. The up-regulation of resistin mRNA expression by ET-1 depends on concentration and timing. The concentration of ET-1 that increased resistin mRNA levels by 100%-250% was approximately 100 nM for a range of 0.25-12 hours of treatment. Treatment with actinomycin D blocked ET-1-increased resistin mRNA levels, suggesting that the effect of ET-1 requires new mRNA synthesis. Treatment with an inhibitor of the ET type-A receptor, such as N-[1-Formyl-N-[N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophyl]-D-tryptophan (BQ610), but not with the ET type-B receptor antagonist N-[(cis-2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine (BQ788), blocked ET-1, increased the levels of resistin mRNA, and phosphorylated levels of downstream signaling molecules, such as ERK1/2, c-Jun N-terminal kinases (JNKs), protein kinase B (AKT), and signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment of specific inhibitors of either ERK1/2 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene [U0126] and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one [PD98059], two inhibitors of MEK1), JNKs (SP600125), phosphatidylinositol 3-kinase/AKT (LY294002 and Wortmannin), or Janus kinase 2 (JAK2)/STAT3 ((E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide, AG490) prevented ET-1-increased levels of resistin mRNA and reduced the ET-1-stimulated phosphorylation of ERK1/2, JNKs, AKT, and STAT3, respectively. However, the p38 kinase antagonist 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580) did not alter the effect of ET-1. These results imply that ET type-A receptor, ERK1/2, JNKs, AKT, and JAK2, but not ET type-B receptor or p38, are necessary for the ET-1 stimulation of resistin gene expression. In vivo observations that ET-1 increased resistin mRNA and protein levels in sc and epididymal adipose tissues support the in vitro findings.

Topics: 3T3-L1 Cells; Adipose Tissue; Androstadienes; Animals; Anthracenes; Butadienes; Chromones; Dactinomycin; Endothelin-1; Flavonoids; Gene Expression Profiling; Gene Expression Regulation; Male; MAP Kinase Kinase 4; Mice; Mice, Inbred C57BL; Morpholines; Nitriles; Oligopeptides; Piperidines; Proto-Oncogene Proteins c-akt; Resistin; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Wortmannin

The cardiac L-type Ca(2+) channel current (I(Ca,L)) plays an important role in controlling both cardiac excitability and excitation-contraction coupling and is involved in the electrical remodeling during postnatal heart development and cardiac hypertrophy. However, the possible role of endothelin-1 (ET-1) in the electrical remodeling of postnatal and diseased hearts remains unclear. Therefore, the present study was designed to investigate the transcriptional regulation of I(Ca,L) mediated by ET-1 in neonatal rat ventricular myocytes using the whole-cell patch-clamp technique, quantitative RT-PCR and Western blotting. Furthermore, we determined whether the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway is involved. ET-1 increased I(Ca,L) density without altering its voltage dependence of activation and inactivation. In line with the absence of functional changes, ET-1 increased L-type Ca(2+) channel pore-forming α1C-subunit mRNA and protein levels without affecting the mRNA expression of auxiliary β- and α2/δ-subunits. Furthermore, an actinomycin D chase experiment revealed that ET-1 did not alter α1C-subunit mRNA stability. These effects of ET-1 were inhibited by the ETA receptor antagonist BQ-123 but not the ETB receptor antagonist BQ-788. Moreover, the effects of ET-1 on I(Ca,L) and α1C-subunit expression were abolished by the ERK1/2 inhibitor (PD98059) but not by the p38 MAPK inhibitor (SB203580) or the c-Jun N-terminal kinase inhibitor (SP600125). These findings indicate that ET-1 increased the transcription of L-type Ca(2+) channel in cardiomyocytes via activation of ERK1/2 through the ETA receptor, which may contribute to the electrical remodeling of heart during postnatal development and cardiac hypertrophy.

Topics: Animals; Animals, Newborn; Anthracenes; Blotting, Western; Cells, Cultured; Endothelin-1; Enzyme Inhibitors; Flavonoids; Imidazoles; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Oligopeptides; Patch-Clamp Techniques; Peptides, Cyclic; Piperidines; Pyridines; Rats; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in solid organ transplant recipients. Endothelin-1 (ET-1) is implicated in the pathogenesis of atherosclerosis and is one of the potential therapeutic targets. This study was conducted to evaluate the effect of mycophenolic acid (MPA), an immunosuppressant for the transplant recipients, on tumor necrosis factor-alpha (TNF-alpha)-induced ET-1 production in aortic endothelial cells.. In cultured human aortic endothelial cells, TNF-alpha increased ET-1 through AP-1 and NF-kappaB, whereas MPA attenuated it by reducing both AP-1 and NF-kappaB DNA-binding activities. TNF-alpha increased ET-1 via c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), but not extracellular signal-regulated kinase. N-acetylcysteine that downregulated TNF-alpha-induced reactive oxygen species (ROS) inhibited JNK activation, but not p38 MAPK. N-acetylcysteine, SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated TNF-alpha-induced DNA-binding activities of both AP-1 and NF-kappaB. MPA inhibited JNK and p38 MAPK activations as well as ROS generation. N-acetylcysteine, SP600125, SB203580 and MPA had no effect on either TNF-alpha-induced IkappaBalpha degradation or p65 nuclear translocation, but attenuated p65 Ser276 phosphorylation.. MPA attenuated TNF-alpha-induced ET-1 production through inhibitions of ROS-dependent JNK and ROS-independent p38 MAPK that regulated NF-kappaB as well as AP-1. These findings suggest that MPA could have an effect of amelioration of atherosclerosis.

Topics: Acetylcysteine; Anthracenes; Cells, Cultured; Endothelin-1; Endothelium, Vascular; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Mycophenolic Acid; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Pyridines; Reactive Oxygen Species; Transcription Factor AP-1; Tumor Necrosis Factor-alpha

We previously showed that endothelin-1 (ET-1) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that protein kinase C (PKC)-dependent p44/p42 mitogen-activated protein (MAP) kinase plays a part in the IL-6 synthesis. In the present study, we investigated the effect of (-)-epigallocatechin gallate (EGCG), one of the major flavonoids containing in green tea, on ET-1-induced IL-6 synthesis in osteoblasts and the underlying mechanism. EGCG significantly reduced the synthesis of IL-6 stimulated by ET-1 in MC3T3-E1 cells as well primary cultured mouse osteoblasts. SB203580, a specific inhibitor of p38 MAP kinase, but not SP600125, a specific SAPK/JNK inhibitor, suppressed ET-1-stimulated IL-6 synthesis. ET-1-induced phosphorylation of p38 MAP kinase was not affected by EGCG. On the other hand, EGCG suppressed the phosphorylation of p44/p42 MAP kinase induced by ET-1. Both the IL-6 synthesis and the phosphorylation of p44/p42 MAP kinase stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of PKC, were markedly suppressed by EGCG. The phosphorylation of MEK1/2 and Raf-1 induced by ET-1 or TPA were also inhibited by EGCG. These results strongly suggest that EGCG inhibits ET-1-stimulated synthesis of IL-6 via suppression of p44/p42 MAP kinase pathway in osteoblasts, and the inhibitory effect is exerted at a point between PKC and Raf-1 in the ET-1 signaling cascade.

Topics: Animals; Anthracenes; Catechin; Cells, Cultured; Endothelin-1; Enzyme Activation; Flavonoids; Imidazoles; Interleukin-6; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Osteoblasts; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-raf; Pyridines; Tetradecanoylphorbol Acetate

In the early development of the central nervous system, neural progenitor cells divide in an asymmetric manner and migrate along the radial glia cells. The radial migration is an important process for the proper lamination of the cerebral cortex. Recently, a new mode of the radial migration was found at the intermediate zone where the neural progenitor cells become multipolar and reduce the migration rate. However, the regulatory signals for the radial migration are unknown. Using the migration assay in vitro, we examined how neural progenitor cell migration is regulated. Neural progenitor cells derived from embryonic mouse telencephalon migrated on laminin-coated dishes. Endothelin (ET)-1 inhibited the neural progenitor cell migration. This ET-1 effect was blocked by BQ788, a specific inhibitor of the ETB receptor, and by the expression of a carboxyl-terminal peptide of Galpha q but not Galpha i. The expression of constitutively active mutant of Galpha q, Galpha qR183C, inhibited the migration of neural progenitor cells. Moreover, the inhibitory effect of ET-1 was suppressed by the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the expression of the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of the JNK pathway. Using the slice culture system of embryonic brain, we demonstrated that ET-1 and the constitutively active mutant of Galpha q caused the retention of the neural progenitor cells in the intermediate zone and JNK-binding domain of JNK-interacting protein-1 abrogated the effect of ET-1. These results indicated that G protein-coupled receptor signaling negatively regulates neural progenitor cell migration through Gq and JNK.

Topics: Animals; Anthracenes; Cell Movement; Cerebral Cortex; Endothelin-1; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gq-G11; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Inbred ICR; Multipotent Stem Cells; Mutation; Neurons; Receptor, Endothelin B; Receptors, G-Protein-Coupled; Signal Transduction; Tissue Culture Techniques

We have reported that endothelin-1 (ET-1) activates p38 mitogen-activated protein (MAP) kinase through protein kinase C in osteoblast-like MC3T3-E1 cells, and that p38 MAP kinase plays a role in the ET-1-induced heat shock protein 27 (HSP27). Recently, we found that stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is activated by ET-1 in these cells. In the present study, we have investigated the involvement of SAPK/JNK in ET-1-induced HSP27 in MC3T3-E1 cells.. The concentration of HSP27 in soluble extracts of the cells, the expression of mRNA for HSP27, and the phosphorylation of SAPK/JNK were determined by an enzyme immunoassay, Northern blot analysis, and Western blot analysis respectively.. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced ET-1-stimulated HSP27 accumulation. The inhibitory effect of SP600125 was dose dependent in the range between 1 and 50 microM. SP600125 reduced the ET-1-increased level of HSP27 mRNA. Calphostin C and Go 6976, inhibitors of protein kinase C, reduced the ET-1-induced phosphorylation of SAPK/JNK. 12-O-Tetradecanoylphorbol-13-acetate, a direct activator of protein kinase C, induced SAPK/JNK phosphorylation, which was suppressed by SP600125. A combination of SP600125 and p38 MAP kinase inhibitor such as SB203580 and PD169316 additively reduced the ET-1-stimulated accumulation of HSP27.. These results strongly suggest that JNK plays a part in ET-1-induced HSP27 in addition to p38 MAP kinase in osteoblasts.

Topics: Animals; Anthracenes; Blotting, Northern; Blotting, Western; Carbazoles; Endothelin-1; Enzyme Activators; Enzyme Inhibitors; Gene Expression Regulation; Heat-Shock Proteins; Immunoenzyme Techniques; Indoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular Chaperones; Naphthalenes; Neoplasm Proteins; Osteoblasts; p38 Mitogen-Activated Protein Kinases; RNA, Messenger; Tetradecanoylphorbol Acetate

We previously reported that endothelin-1 (ET-1) activates both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that not p44/p42 MAP kinase but p38 MAP kinase participates in the ET-1-induced vascular endothelial growth factor (VEGF) synthesis. In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) in ET-1-induced VEGF synthesis in these cells. ET-1 significantly induced the phosphorylation of JNK in a dose-dependent manner in the range between 0.1 and 100 nM. SP600125, an inhibitor of JNK, markedly reduced the ET-1-induced VEGF synthesis. A combination of SP600125 and SB203580 additively reduced the ET-1-stimulated VEGF synthesis. SP600125 suppressed the ET-1-induced phosphorylation of JNK, while having no effect on the phosphorylation of p38 MAP kinase elicited by ET-1. SB203580, an inhibitor of p38 MAP kinase, hardly affected the ET-1-induced phosphorylation of JNK. These results strongly suggest that JNK plays a role in ET-1-induced VEGF synthesis in addition to p38 MAP kinase in osteoblasts.

Topics: Animals; Anthracenes; Bone and Bones; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Growth Factors; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Imidazoles; Intercellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Lymphokines; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Osteoblasts; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Time Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors