Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.12.2 (MEK)
 18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study investigated the effects of C7 and C9 aliphatic (n-heptane, n-nonane), naphthenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) and aromatic (toluene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat brain synaptosome fraction. Methyl mercury (MeHg) was included as a positive control. Exposure of the synaptosomes to the hydrocarbons produced a concentration-dependent linear increase in the formation of the fluorescence of 2',7'-dichlorofluorescein (DCF) as a measure of the production of ROS and RNS. Formation of RNS was demonstrated by preincubation of the synaptosome fraction with the neuronal nitric oxide synthase (nNOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME), which reduced the MeHg and TMCH-stimulated fluorescence by 51% and 65%, respectively. The naphthenic hydrocarbon TMCH showed the strongest potential for ROS and RNS formation in rat brain synaptosomes, followed by TMB, toluene, n-nonane, n-heptane, and methylcyclohexane, respectively. TMCH was selected for mechanistic studies of the formation of ROS. Both MeHg and TMCH induced an increase in intracellular calcium concentration [Ca(2+)]i as measured with Fura-2. Blockade of voltage-dependent Ca(2+) channels with lanthanum prior to stimulation with MeHg and TMCH led to a reduction in the ROS/RNS formation of 72% and 70%, respectively. Furthermore, addition of cyclosporin A (CSA), a blocker of the mitochondrial permeability transition pore (MTP), lowered both the MeHg and TMCH-elevated DCF fluorescence by 72% and 59%. Preincubation of the synaptosome fraction with the protein tyrosine kinase inhibitor genistein lowered the MeHg and TMCH-stimulated fluorescence by 85% and 91%, respectively. Addition of the extracellular signal-regulated protein kinase (MEK)-1 and -2 inhibitor U0126 reduced the fluorescence stimulated by MeHg and TMCH by 62% and 63%. Furthermore, the protein kinase C inhibitor bisindolylmaleimide reduced the fluorescence stimulated by MeHg and TMCH by 52% and 56%. The compound 1-(6-[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122), which inhibits phospholipase C, was shown to decrease the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, respectively. The phospholipase A2 (PLA2) inhibitor 7,7-dimethyl eicosadienoic acid (DEDA) reduced fluorescence in response to MeHg and TMCH by 49% and 54%. Simultaneous addition of L-NAME, CSA, and DEDA to the synaptosome fraction totally abolished the DCF fluorescence. In conclusion, C7 and C9 aliphatic, naphthenic, and aromatic hydrocarbons stimulated formation of ROS and RNS in rat brain synaptosomes. The naphthenic hydrocarbon TMCH stimulated formation of ROS and RNS in the synaptosomes through Ca(2+)-dependent activation of PLA2 and nNOS, and through increased transition permeability of the MTP. Exposure of humans to the naphthenic hydrocarbon TMCH may stimulate formation of free radicals in the brain, which may be a key factor leading to neurotoxicity.
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PMID:The effect of aliphatic, naphthenic, and aromatic hydrocarbons on production of reactive oxygen species and reactive nitrogen species in rat brain synaptosome fraction: the involvement of calcium, nitric oxide synthase, mitochondria, and phospholipase A. 1137 3

Basic fibroblast growth factor (bFGF) is an important angiogenic factor produced by hearts subjected to ischemia. However, the direct effects of bFGF on myocardial cells are unknown. Primary cultured cardiac myocytes from neonatal rats were stimulated with lipopolysaccharide (LPS), a potent inducer of inducible nitric oxide synthase (iNOS), in the presence or the absence of bFGF. LPS induced the expression of iNOS in cardiac myocytes, demonstrated at both mRNA and protein levels. We showed that LPS activated the apoptotic pathway, evidenced by TUNEL staining, DNA ladder formation, and morphologic features. LPS-induced apoptosis was blocked by the administration of L-NAME, an inhibitor of NOS. This indicates that LPS induces apoptosis via an iNOS-dependent pathway. Administration of bFGF completely inhibited myocardial cell apoptosis induced by hydrogen peroxide or acidic medium as well as LPS. To determine signaling pathways for this inhibitory effect, we utilized PD098059, an MEK-1-specific inhibitor. PD098059 blocked bFGF-induced activation of ERK (extracellularly responsive kinase)-1/2 and neutralized the apoptotic inhibitory effect of bFGF. These findings demonstrate that LPS induces myocardial cell apoptosis in an iNOS-dependent manner. The results also suggest that bFGF is a protective factor against myocardial cell apoptosis and that this protection requires the MEK-1-ERK pathway.
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PMID:Basic fibroblast growth factor protects cardiac myocytes from iNOS-mediated apoptosis. 1180 11

TRH has been reported to possess several neurophysiological actions in the brain. To gain insights into the molecular mechanisms underlying these effects, particularly in the cerebellum, we attempted to clone a cDNA that was regulated by TRH using TRH knockout mice and subtractive cDNA analysis. Over 100 clones obtained by subtractive hybridization analysis between the wild-type and TRH-1-cerebellum were analyzed. Four clones among them were identical and cdc2-related kinase (PFTAIRE protein kinase 1 (PFTK1)) cDNA, which was previously reported to be expressed only in the brain and testis. PFTK1 mRNA levels in the euthyroid TRH-1- cerebellum supplemented with thyroid hormone were significantly decreased compared with those in the wild-type. Induction of PFTK1 mRNA by TRH was also observed in a time- and dose-dependent manner in human medulloblastoma-derived HTB-185 cells that expressed TRH receptor subtype I mRNA. In addition, treatment of 8-Br-cGMP significantly increased PFTK1 mRNA levels, and a specific inhibitor of cGMP production, ODQ, completely blocked TRH-induced expression of PFTK1 mRNA. Furthermore, induction of PFrK1 mRNA by TRH was significantly inhibited by a NOS specific inhibitor, L-NAME, but not by a MEK inhibitor, PD98059 or a calcium channel inhibitor, nimodipine. These findings demonstrated, for the first time, a novel pathway between a neuropeptide and a cell cycle related peptide in the brain, and PFTK1 may be a key regulator for TRH action in t he cerebellum through t he NO-cGMP pathway.
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PMID:A novel TRH-PFTAIRE protein kinase 1 pathway in the cerebellum: subtractive hybridization analysis of TRH-deficient mice. 1207 16

Adenosine is released from the myocardium, endothelial cells, and skeletal muscle in ischemia and is an important regulator of coronary blood flow. We have already shown that acute (2 min) activation of A2a purinoceptors stimulates NO production in human fetal umbilical vein endothelial cells (1) and now report a key role for p42/p44 mitogen-activated protein kinases (p42/p44MAPK) in the regulation of the l-arginine-nitric oxide (NO) signaling pathway. Expression of mRNA for the A2a-, A2b-, and A3-adenosine receptor subtypes was abundant whereas A1-adenosine receptor mRNA levels were negligible. Activation of A2a purinoceptors by adenosine (10 microM) or the A2a receptor agonist CGS21680 (100 nM) resulted in an increase in l-arginine transport and NO release that was not mediated by changes in intracellular Ca2+, pH, or cAMP. Stimulation of endothelial cells with adenosine was associated with a membrane hyperpolarization and phosphorylation of p42/p44MAPK. l-NAME abolished the adenosine-induced hyperpolarization and stimulation of l-arginine transport whereas sodium nitroprusside activated an outward potassium current. Genistein (10 microM) and PD98059 (10 microM), an inhibitor of MAPK kinase 1/2 (MEK1/2), inhibited adenosine-stimulated l-arginine transport, NO production, and phosphorylation of p42/p44MAPK. We found no evidence for activation of eNOS via the serine/threonine kinase Akt/PKB (protein kinase B) in adenosine-stimulated cells. Our results provide the first evidence that adenosine stimulates the endothelial cell l-arginine-NO pathway in a Ca2+-insensitive manner involving p42/p44MAPK, with release of NO leading to a membrane hyperpolarization and activation of l-arginine transport.
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PMID:Early activation of the p42/p44MAPK pathway mediates adenosine-induced nitric oxide production in human endothelial cells: a novel calcium-insensitive mechanism. 1237 81

We tested the hypothesis that steady laminar shear stress activates the glucocorticoid receptor (GR) and its transcriptional signaling pathway in an effort to investigate the potential involvement of GR in shear stress-induced antiatherosclerosis actions in the vasculature. In both bovine aortic endothelial cells (BAECs) and NIH3T3 cells expressing GFP-GR chimeric protein, wall shear stress of 10 or 25 dynes/cm2 caused a marked nuclear localization of GFP-GR within 1 hour to an extent comparable to induction with 25 micromol/L dexamethasone. The shear mediated nuclear localization of GFP-GR was significantly reduced by 25 micromol/L of the MEK1 inhibitor (PD098059) or the PI 3-kinase inhibitor (LY294002). Also, Western blots demonstrated translocation of endogenous GR into nucleus of sheared BAECs. Promoter construct studies using glucocorticoid response element (GRE)-driven expression of secreted alkaline phosphatase (SEAP) indicated that BAECs exposed to shear stress of 10 and 25 dynes/cm2 for 8 hours produced >9-fold more SEAP (n=6; P<0.005) than control cells, a level comparable to that observed with dexamethasone. Shear stress enhanced SEAP expression at 6 hours was reduced 50% (n=5; P<0.005) by MEK1/2 or PI 3-kinase inhibitors, but not by the NO inhibitor, L-NAME. Finally, in human internal mammary artery, endothelial GR is found to be highly nuclear localized. We report a new shear responsive transcriptional element, GRE. The finding that hemodynamic forces can be as potent as high dose glucocorticoid steroid in activating GR and GRE-regulated expression correlates with the atheroprotective responses of endothelial cells to unidirectional arterial shear stress.
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PMID:Shear stress causes nuclear localization of endothelial glucocorticoid receptor and expression from the GRE promoter. 1259 39

Reactive oxygen species (superoxide anion, hydrogen peroxide, and nitric oxide) are involved in human sperm capacitation and associated tyrosine (Tyr) phosphorylation through a cAMP- and protein kinase A-mediated pathway. Recently, we evidenced the double phosphorylation of the threonine-glutamine-Tyr motif (P-Thr-Glu-Tyr-P) in human sperm proteins of 80 and 105 kDa during capacitation. The objective of the present study was to investigate the role of reactive oxygen species in the regulation of this process and to immunolocalize the P-Thr-Glu-Tyr-P motif in human spermatozoa. Superoxide dismutase and catalase did not prevent, and exogenous addition of superoxide anion or hydrogen peroxide did not trigger, the increase in P-Thr-Glu-Tyr-P related to sperm capacitation. However, l-NAME (a competitive inhibitor of l-arginine for nitric oxide synthase) prevented, and a nitric oxide donor promoted, the increase in P-Thr-Glu-Tyr-P related to sperm capacitation. In addition, l-arginine reversed the inhibitory effect of l-NAME on capacitation and the associated increase of P-Thr-Glu-Tyr-P. Therefore, the regulation of P-Thr-Glu-Tyr-P is specific to nitric oxide and not to superoxide anion or hydrogen peroxide. The nitric oxide-mediated increase of P-Thr-Glu-Tyr-P involved protein Tyr kinase, MEK or MEK-like kinase, and protein kinase C but not protein kinase A. The P-Thr-Glu-Tyr-P motif was immunolocalized to the principal piece region of spermatozoa. In conclusion, nitric oxide regulates the level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation. These data evidence, to our knowledge for the first time, a specific role for nitric oxide in signal transduction events leading to sperm capacitation.
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PMID:Nitric oxide regulates the phosphorylation of the threonine-glutamine-tyrosine motif in proteins of human spermatozoa during capacitation. 1260 10

Hepatocyte growth factor (HGF) is a potent mitogen for vascular endothelial cells (EC); however, signal transduction pathways for HGF-stimulated EC growth remain unclear. In the present study we investigated the role of Src family kinases and nitric oxide (NO) in HGF-stimulated EC growth. Human umbilical vein endothelial cells (HUVEC) were stimulated with HGF and NO was measured by an NOx analyzing HPLC system. Activation of ERK1/2 and p38 MAPK was assessed by Western blot. NO production in HUVEC increased 1.8-fold by HGF. A Src family kinases inhibitor PP1 inhibited HGF-stimulated NO production by 71%. HUVEC growth increased 1.9-fold in cell number by HGF. PP1 and Nitro-L-arginine methylester (L-NAME) inhibited HGF-stimulated HUVEC growth by 51 and by 71%. ERK1/2 and p38 MAPK were phosphorylated by HGF and a MEK inhibitor PD98059 and a p38 MAPK inhibitor SB203580 inhibited HGF-stimulated HUVEC growth by 66% and by 58%; however, HGF-induced phosphorylation of ERK1/2 and p38 MAPK was not inhibited by L-NAME, indicating that NO is not an upstream activator of ERK1/2 and p38 MAPK. These findings demonstrated that Src family kinases regulate HGF-stimulated NO production in HUVEC and that HGF stimulates HUVEC growth through NO-dependent and NO-independent pathways.
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PMID:Src family kinases and nitric oxide production are required for hepatocyte growth factor-stimulated endothelial cell growth. 1261 72

An elevated extracellular concentration of D-glucose (i.e. hyperglycaemia) inhibits cell proliferation and incorporation of the endogenous nucleoside thymidine into DNA in human umbilical vein endothelial cells (HUVECs). Cells in their log-phase of growth (3.7 +/- 0.3 days, n = 27) incubated for 30 min with 25 mM D-glucose, but not with equimolar concentrations of L-glucose or D-mannitol, exhibited reduced [3H]thymidine incorporation and cell growth rate, with no change in cell viability (> 98 %), total DNA, protein content or cell volume. Incubation with D-glucose activated protein kinase C (PKC), endothelial NO synthase (eNOS), p42 and p44 mitogen-activated protein kinases (p42/44(mapk)), but inhibited superoxide dismutase (SOD). Incubation with D-glucose also increased cGMP and cAMP levels. The effect of D-glucose was blocked by the PKC inhibitor calphostin C, the MAP kinase kinase 1/2 (MEK1/2) inhibitor PD-98059, the eNOS inhibitor L-NAME, the protein kinase G (PKG) inhibitor KT-5823 and the protein kinase A (PKA) inhibitor KT-5720. In the presence of 5 mM D-glucose, [3H]thymidine incorporation and cell growth were reduced by the PKC activator phorbol 12-myristate 13-acetate (PMA), the NO donor S-nitroso-N-acetyl-L,D-penicillamine (SNAP), dibutyryl cGMP, dibutyryl cAMP and the Ca2+ ionophore A-23187. The effect of A-23187 was blocked by calphostin C and PD-98059. D-Glucose-dependent inhibition of thymidine incorporation and cell proliferation is associated with increased PKC, eNOS, and MEK1/2, but decreased SOD activity, and higher intracellular levels of cGMP, cAMP and Ca2+ in HUVECs. These are cellular mechanisms which may reduce endothelial cell growth in pathological conditions such as in diabetes mellitus or hyperglycaemia.
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PMID:Hyperglycaemia inhibits thymidine incorporation and cell growth via protein kinase C, mitogen-activated protein kinases and nitric oxide in human umbilical vein endothelium. 1262 26

Chronic inhibition of nitric oxide (NO) synthesis induces cardiac remodeling independent of systemic hemodynamic changes in rats. We examined whether long-acting dihydropyridine calcium channel blockers block myocardial remodeling and whether the activation of 70-kDa S6 kinase (p70S6K) and extracellular signal-regulated kinase (ERK) are involved. Ten groups of Wistar-Kyoto rats underwent 8 weeks of drug treatment consisting of a combination of NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME), an inactive isomer (D-NAME), amlodipine (1 or 3 mg/kg per day), or benidipine (3 or 10 mg/kg per day). In other groups, L-NAME was also used in combination with a p70S6K inhibitor (rapamycin), a MEK inhibitor (PD98059), and hydralazine. Systolic blood pressure (SBP), heart rate, and left ventricular weight (LVW) were measured, together with histological examinations and kinase assay. L-NAME increased SBP and LVW (1048+/-22 versus 780+/-18 mg, P<0.01) compared with the control, showing a significant increase in cross-sectional area of cardiomyocytes after 8 weeks. Amlodipine, benidipine, or hydralazine equally attenuated the increase in SBP induced by L-NAME. However, both amlodipine and benidipine but not hydralazine attenuated the increase in LVW by L-NAME (789+/-27, 825+/-20 mg, P<0.01, and 1118+/-29 mg, NS, respectively), also confirmed by histological analysis. L-NAME caused a 2.2-fold/1.8-fold increase in p70S6K/ERK activity in myocardium compared with the control, both of which were attenuated by both amlodipine and benidipine but not hydralazine. Both rapamycin and PD98059 attenuated cardiac hypertrophy in this model. Thus, long-acting dihydropyridine calcium channel blockers inhibited cardiac hypertrophy induced by chronic inhibition of NO synthesis by inhibiting both p70S6K and ERK in vivo.
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PMID:Long-acting Ca2+ blockers prevent myocardial remodeling induced by chronic NO inhibition in rats. 1262 37

Using clonal derivatives of spontaneous mammary tumours in C3H/HeJ mice, we had earlier shown that tumour-derived nitric oxide (NO), resulting from endothelial type (e) NO synthase (NOS) expression by tumour cells, promoted tumour growth and metastasis by multiple mechanisms: stimulation of tumour cell invasiveness, migration and angiogenesis. Our present study examined the signaling mechanisms underlying NO-mediated promotion of tumour cell migration in a highly metastatic and high eNOS-expressing C3H/HeJ mammary tumour cell line, C3L5. C3L5 cell migration was reduced in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor) in a concentration-dependent manner and restored in the additional presence of excess L-arginine (NOS substrate), confirming a migration-promoting role of endogenous NO. Migratory capacity of C3L5 cells was reduced after treatment with the guanylate cyclase (GC) inhibitor 1-H-[1,2,4]oxadiaxolo[4,3-a]quinolalin-1-one (ODQ) and restored in the additional presence of 8-bromoguanosine 3'5'-cyclic monophosphate (8-Br cGMP, cGMP analogue), demonstrating a pivotal role for GC in C3L5 cell migration. Mitogen-activated protein kinase kinase (MAPKK; MEK) inhibitor, UO126, blocked migration, demonstrating MEK involvement in C3L5 cell migration. Furthermore, both ODQ and UO126 blocked migration-restoring effects of L-arginine in L-NAME-treated cells, indicating that GC and MAPK pathways are required for endogenous NO-mediated migratory responses. Similarly, L-NAME reduced and additional treatment with excess L-arginine or sodium nitroprusside (SNP, NO donor) stimulated phosphorylation of extracellular signal-regulated kinases (ERK(1/2)), demonstrating a role for endogenous and exogenous NO in ERK(1/2) activation. ODQ inhibited ERK(1/2) activation, whereas 8-Br cGMP stimulated ERK(1/2) phosphorylation in L-NAME-treated cells, indicating that cGMP is a downstream effector of NOS for ERK(1/2) activation. Finally, both ODQ and UO126 blocked the capacity of L-arginine to restore ERK(1/2) phosphorylation in L-NAME-treated cells, demonstrating that GC and MEK are both required for endogenous NO-mediated MAPK activation. Together, these results indicate sequential activation of NOS, GC and MAPK pathways in mediating signals for C3L5 cell migration, an essential step in invasion and metastasis. Since NOS activity is positively associated with human breast cancer progression, the present results are relevant for development of therapeutic modalities for this disease.
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PMID:Nitric oxide-mediated promotion of mammary tumour cell migration requires sequential activation of nitric oxide synthase, guanylate cyclase and mitogen-activated protein kinase. 1284 43


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