u-0126 and Disease-Models--Animal

Excessive secretion of airway mucus may be an important pathological factor of air pollution-induced acute asthma attacks. Treatment of airway mucus hypersecretion improves asthma aggravated by air pollutants. Qufeng Xuanbi Formula (QFXBF) has been used to treat asthma for more than 30 years. However, whether QFXBF inhibits asthmatic mucus secretion exacerbated by air pollutants has not yet been established.. This study aimed to evaluate the effect of QFXBF on airway mucus secretion and the mechanism of action in an air pollutant benzo[a]pyrene (BaP)-induced mouse model of aggravated asthma.. Ovalbumin (OVA) and BaP co-exposure were used to establish the aggravated asthma model. The average enhanced pause (Penh), serum OVA-specific IgE, and changes in lung histopathology were determined. 16HBE cells exposed to BaP, treatment with QFXBF, arylhydrocarbon receptor (AhR) signal antagonist SR1, reactive oxygen species (ROS) antagonist NAC, or extracellular signal-regulated kinase (ERK1/2) signal antagonist U0126 were established to investigate the effect of QFXBF on BaP-induced mucus secretion and its target. The mRNA and protein expression levels of MUC5AC in the lung tissue and 16HBE cells were examined. We also studied the effect of QFXBF on ROS production. Finally, the protein expression of AhR, phospho-extracellular signal-regulated kinases (p-ERK1/2), and ERK1/2 in 16HBE cells and lung tissues was determined by western blotting.. Administration of QFXBF significantly alleviated the pathological symptoms, including Penh, serum OVA-specific IgE, and changes in lung histopathology in a BaP-induced mouse model of aggravated asthma. QFXBF inhibited MUC5AC expression in asthmatic mice and 16HBE cells exposed to BaP. ROS production, AhR expression, and ERK1/2 phosphorylation were significantly increased in BaP-induced asthmatic mice and 16HBE cells. Signaling pathway inhibitors StemRegenin 1 (SR1), NAC, and U0126 significantly inhibitedBaP-induced MUC5AC expression in 16HBE cells. SR1 reversed Bap-induced ROS production and ERK activation, and NAC inhibited Bap-induced ERK activation. In addition, QFXBF regulated AhR signaling, inhibited ROS production, reversed ERK activation, and downregulated mucus secretion to improve asthma aggravated by air pollutant BaP.. QFXBF can ameliorate mucus secretion in BaP-induced aggravated asthmatic mice and 16HBE cells, and the specific mechanism may be related to the inhibition of the AhR/ROS/ERK signaling pathway.

Topics: Air Pollutants; Animals; Asthma; Benzo(a)pyrene; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Immunoglobulin E; Lung; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mucus; Ovalbumin; Reactive Oxygen Species

Topics: Animals; Butadienes; Cell Line; Cell Proliferation; Cell Survival; Chromones; Cytokines; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Endometriosis; Epigenesis, Genetic; Female; Gene Expression Regulation; Humans; MAP Kinase Signaling System; Mice; Morpholines; Nitriles; Proto-Oncogene Proteins c-akt; Receptors, Progesterone

Peripheral nerve injuries (PNIs) often result in persistent neuropathic pain, seriously affecting quality of life. Existing therapeutic interventions for PNI-induced neuropathic pain are far from satisfactory. Extracellular signal-regulated kinases (ERKs) and p38 have been found to participate in triggering and maintaining PNI-induced neuropathic pain. However, ERK and p38 also contribute to axonal regeneration and motor function recovery after PNI, making it difficult to inhibit ERK and p38 for therapeutic purposes. In this study, we simultaneously characterized neuropathic pain and motor function recovery in a mouse sciatic nerve crush injury model to identify the time window for therapeutic interventions. We further demonstrated that delayed delivery of a combination of ERK and p38 inhibitors at three weeks after PNI could significantly alleviate PNI-induced neuropathic pain without affecting motor function recovery. Additionally, the combined use of these two inhibitors could suppress pain markedly better than either inhibitor alone, possibly reducing the required dose of each inhibitor and alleviating the side effects and risks of the inhibitors when used individually.

Topics: Animals; Axons; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Imidazoles; Male; Mice, Inbred C57BL; Nerve Regeneration; Neuralgia; Nitriles; p38 Mitogen-Activated Protein Kinases; Peripheral Nerve Injuries; Pyridines; Recovery of Function; Sciatic Nerve; Treatment Outcome

To study the possible mechanism of ghrelin in heart failure and how it works.. In vitro results demonstrated that ghrelin alleviates cardiac function and reduces myocardial fibrosis in rats with heart failure. Moreover, ghrelin intervention increased PTEN expression level and reduced ERK, c-jun, and c-Fos expression level; in vivo experiments demonstrated that ghrelin intervention reduces mast memory expression and increases cardiomyocyte surface area, PTEN expression level, ERK, c-jun, c-Fos expression level, and cell surface area, while ERK blockade suppresses mast gene expression and reduces cell surface area.. In vitro experimental results prove that we have successfully constructed a rat model related to heart failure, and ghrelin can alleviate the heart function of heart failure rats and reduce myocardial fibrosis. In addition, ghrelin is closely related to the decrease of the expression levels of ERK, c-jun, and c-Fos, but it can also increase the expression of PTEN in the rat model; in vivo experiments proved that we successfully constructed an in vitro cardiac hypertrophy model, and the intervention of ghrelin would reduce the expression of hypertrophic memory and increase the surface area of cardiomyocytes, increase the expression level of PTEN, and reduce the expression levels of ERK, c-jun, and c-Fos, while the blockade of PTEN will increase the expression of hypertrophy genes and increase the cell surface area, while the blockade of ERK will increase the expression of hypertrophic genes, which in turn will make the cell surface area reducing.. Ghrelin inhibits the phosphorylation and nuclear entry of ERK by activating PTEN, thereby controlling the transcription of hypertrophic genes, improving myocardial hypertrophy, and enhancing cardiac function.

Topics: Animals; Butadienes; Cell Enlargement; Cell Line; Computational Biology; Disease Models, Animal; Female; Fibrosis; Gene Expression; Ghrelin; Heart Failure; MAP Kinase Signaling System; Mast Cells; Myocytes, Cardiac; Nitriles; Phenanthrenes; PTEN Phosphohydrolase; Rats; Rats, Sprague-Dawley

Osteoarthritis (OA) is a chronic, age‑related osteoarthropathy that causes a considerable decline in quality of life, as well as economic losses due to its high incidence and poor prognosis. Mitogen‑activated protein kinases (MAPKs) regulate multiple cellular processes, including proliferation, differentiation and apoptosis, in certain diseases, such as cancer, diabetes and Alzheimer's disease. The present study aimed to investigate the regulatory role of the MAPK signaling pathway in early‑stage OA. A rabbit model of early‑stage OA was induced by treatment with the enzyme papain. U0126 [an extracellular signal‑regulated kinase (ERK) inhibitor], SP600125 [a Jun NH2‑terminal kinase (JNK) inhibitor] and SB203580 (a p38 inhibitor) were administered to the rabbits via intra‑articular injection. The severity of OA was assessed by histological examination using H&E, toluidine blue and safranin‑O/fast green staining, as well by analyzing the glycosaminoglycan (GAG) content and determining the OA Research Society International (OARSI) score. Western blotting was used to detect the protein expression levels of matrix metalloproteinase‑3 (MMP3), ERK, phosphorylated (p)‑ERK, p38, p‑p38, JNK, p‑JNK, Beclin1, UNC‑51‑like kinase 1 (ULK1) and microtubule‑associated protein 1 light chain 3 (LC3)II/I. U0126, SP600125 or SB203580 treatment significantly decreased the OARSI scores and significantly increased the GAG levels in the cartilaginous tissues of OA model rabbits. These results indicated that the MAPK inhibitors reduced the severity of OA‑induced injury at the early stage. Western blotting results demonstrated that MAPK inhibition significantly decreased the protein expression levels of MMP3 in OA cartilage. The protective effect of MAPK inhibitors in OA was mediated via the activation of autophagy, as demonstrated by the increased protein expression levels of LC3II/I, ULK1 and Beclin1. Overall, the data indicated that MAPK inhibitors may exert a protective effect against OA by restoring compromised autophagy. Furthermore, the present study suggested that MAPK inhibitors may represent a potential pharmacological strategy for treating OA in the future.

Topics: Animals; Anthracenes; Autophagy; Butadienes; Disease Models, Animal; Imidazoles; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Osteoarthritis; Protein Kinase Inhibitors; Pyridines; Rabbits; Severity of Illness Index

Depression is a mental and emotional disorder that has made an opening great burden to the society. Paeoniflorin showed remarkable antidepressant-like effects in multiple animal models with depressive disorders. However, the molecule of paeoniflorin on depression is less studied. This study aims to explore the effect and the molecular mechanism of paeoniflorin on depression in a chronic restraint stress (CRS) mice model. CRS model of C57BL/6 J mice was set up. Sucrose preference test (SPT), tail suspension test (TST), open field test (OFT) and forced swimming test (FST) were used to assess depression symptoms. Immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were implemented to detect the expression changes of the proteins involved in extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Results showed that paeoniflorin treatment decreased the degree of depression in the CRS mice. Further analysis showed that the expression of ERK1/2 proteins was significantly downregulated, while paeoniflorin could elevate the expression of ERK1/2 proteins in CRS mice. Finally, it showed that inhibiting signaling ERK1/2 pathway could aggravate the depressive behavior when treatment with ERK-specific inhibitor U0126, while the condition could be partially relieved when treated with paeoniflorin. In conclusion, the present study demonstrated that paeoniflorin attenuated chronic stress-induced depression-like behavior in mice by affecting the ERK1/2 pathway. These findings provided the basis for the molecular mechanism of paeoniflorin on the effect of depression, which support paeoniflorin might act as an important drug in the treatment of depression.

Topics: Animals; Behavior, Animal; Butadienes; Cell Count; Chronic Disease; Depression; Disease Models, Animal; Gene Expression Regulation; Glucosides; Hippocampus; Male; MAP Kinase Signaling System; Mice, Inbred C57BL; Monoterpenes; Neurons; Nitriles; Stress, Psychological

Spontaneously hypertensive rats (SHRs) have increased daily or induced sodium intake compared to normotensive rats. In normotensive rats, angiotensin II (ANG II)-induced sodium intake is blocked by the inactivation of p42/44 mitogen-activated protein kinase, also known as extracellular signal-regulated protein kinase1/2 (ERK1/2). Here we investigated if inhibition of ERK1/2 pathway centrally would change sodium appetite and intracerebroventricular (icv) ANG II-induced pressor response in SHRs. SHRs (280-330 g, n = 07-14/group) with stainless steel cannulas implanted in the lateral ventricle (LV) were used. Water and 0.3 M NaCl intake was induced by the treatment with the diuretic furosemide + captopril (angiotensin converting enzyme blocker) subcutaneously or 24 h of water deprivation (WD) followed by 2 h of partial rehydration with only water (PR). The blockade of ERK1/2 activation with icv injections of U0126 (MEK1/2 inhibitor, 2 mM; 2 μl) reduced 0.3 M NaCl intake induced by furosemide + captopril (5.0 ± 1.0, vs. vehicle: 7.3 ± 0.7 mL/120 min) or WD-PR (4.6 ± 1.3, vs. vehicle: 10.3 ± 1.4 mL/120 min). PEP7 (selective inhibitor of AT1 receptor-mediated ERK1/2 activation, 2 nmol/2 μL) icv also reduced WD-PR-induced 0.3 M NaCl (2.8 ± 0.7, vs. vehicle: 6.8 ± 1.4 mL/120 min). WD-PR-induced water intake was also reduced by U0126 or PEP7. In addition, U0126 or PEP7 icv reduced the pressor response to icv ANG II. Therefore, the present results suggest that central AT1 receptor-mediated ERK1/2 activation is part of the mechanisms involved in sodium appetite and ANG II-induced pressor response in SHRs.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Appetite; Butadienes; Captopril; Disease Models, Animal; Furosemide; Humans; Hypertension; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Rats; Rats, Inbred SHR; Receptor, Angiotensin, Type 1; Sodium

In acute ischemic stroke, the only FDA-approved drug; recombinant tissue plasminogen activator (rt-PA) is limited by restricted time-window due to an enhanced risk of hemorrhagic transformation which is thought to be caused by metalloproteinase (MMP). In experimental stroke inhibitors of the mitogen-activated protein kinase kinase extracellular signal-regulated kinase kinase (MEK) 1/2 pathways reduce the MMPs. This study evaluated whether a MEK1/2 inhibitor in combination with rt-PA can prevent the detrimental effects of delayed rt-PA therapy in stroke. Thromboembolic stroke was induced in C57 black/6J mice and the MEK1/2 inhibitor U0126 was administrated 3.5 h and rt-PA 4 h post stroke-onset. Treatment with rt-PA demonstrated enhanced MMP-9 protein levels and hemorrhagic transformation which was prevented when U0126 was given in conjunction with rt-PA. By blocking the MMP-9 with U0126 the safety of rt-PA administration was improved and demonstrates a promising adjuvant strategy to reduce the harmful effects of delayed rt-PA treatment in acute ischemic stroke.

Topics: Animals; Butadienes; Disease Models, Animal; Drug Discovery; Drug Therapy, Combination; Drug-Related Side Effects and Adverse Reactions; Hemorrhage; Humans; Ischemic Stroke; Male; Matrix Metalloproteinase 9; Mice; Mitogen-Activated Protein Kinase Kinases; Nitriles; Signal Transduction; Tissue Plasminogen Activator; Treatment Outcome

Topics: Acetylation; Anacardic Acids; Animals; Butadienes; Cardiomegaly; Cell Survival; Disease Models, Animal; Female; Histone Acetyltransferases; Histones; Male; MAP Kinase Signaling System; MEF2 Transcription Factors; Mice; Myocytes, Cardiac; Nitriles; p300-CBP Transcription Factors; Phenylephrine; Signal Transduction

This study focused on the relationship between extracellular-regulated kinase (ERK) and obesity-induced increases in neuropathic pain. We fed rats a high-fat diet to establish the obesity model, and rats were given surgery to establish the chronic compression of the dorsal root ganglia (CCD) model. U0126 was applied to inhibit ERK, and metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) was applied to cause AMP-activated protein kinase (AMPK) activation. Paw withdrawal mechanical threshold (PWMT) were calculated to indicate the level of neuropathic pain. The data indicated that compared with normal CCD rats, the PWMT of obese CCD rats were decreased, accompanied with an increase of ERK phosphorylation, NAD(P)H oxidase 4 (NOX4) protein expression, oxidative stress and inflammatory level in the L4 to L5 spinal cord and dorsal root ganglia (DRG). Administration of U0126 could partially elevate the PWMT and reduce the protein expression of NOX4 and the above pathological changes in obese CCD rats.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Apoptosis; Butadienes; Diet, High-Fat; Disease Models, Animal; Enzyme Inhibitors; Ganglia, Spinal; Hypoglycemic Agents; Inflammation; Male; MAP Kinase Signaling System; Metformin; NADPH Oxidase 4; Neuralgia; Nitriles; Obesity; Oxidative Stress; Pain Threshold; Phosphorylation; Rats, Wistar; Ribonucleotides; Spinal Cord

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Topics: A549 Cells; Acrylates; Aged; Animals; Antigens, CD; Bleomycin; Butadienes; Cadherins; Calpain; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation; Humans; Lung; Male; Mice; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Protease Inhibitors; Protein Kinase Inhibitors; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta1

Obstructive sleep apnea syndrome (OSAS) is a common and complex disorder that is associated with liver injury. Moreover, previous studies have revealed that chronic intermittent hypoxia (CIH) is associated with the development of non‑alcoholic fatty liver disease and hepatic fibrosis. However, the underlying molecular mechanisms remain largely unknown. The present study aimed to investigate whether chronic intermittent hypoxia induced hepatic fibrosis, in addition to determining its underlying mechanisms, in CIH model rats using immunohistochemistry, western blotting and reverse transcription‑quantitative PCR. The present results suggested that CIH caused hepatic fibrosis and increased the expression levels of interleukin (IL)‑1β, IL‑8, monocyte chemotactic‑1, tumor necrosis factor‑α, intercellular adhesion molecule‑1 and vascular cell adhesion molecule‑1 in the liver; these conditions could be reversed by Toll‑like receptor 4 (TLR4) short hairpin RNA lentivirus treatment. Moreover, immunohistochemistry and western blotting results indicated that TLR4 and NF‑κB expression levels were significantly increased in the CIH and CIH‑TLR4 empty vector lentivirus group. However, protein expression levels of TLR4, NF‑κB, inhibitor of NF‑κB and phosphorylated‑mitogen‑activated protein kinase (MAPK)‑1 in the hypoxia/reoxygenation group were significantly higher compared with the control group (P<0.05), and these results were reversed by the MAPK inhibitor U0126 in vitro. Collectively, the present preliminary results suggested that inflammation and the TLR4/NF‑κB/MAPK signaling pathway may be involved in CIH‑induced liver fibrosis.

Topics: Animals; Butadienes; Cell Line; Disease Models, Animal; Enzyme Inhibitors; Gene Silencing; Hepatic Stellate Cells; Hypoxia; Inflammation; Liver Cirrhosis; Male; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Rats; Rats, Sprague-Dawley; Signal Transduction; Sleep Apnea, Obstructive; Toll-Like Receptor 4

Vascular pathophysiological changes after haemorrhagic stroke, such as phenotypic modulation of the cerebral arteries and cerebral vasospasms, are associated with delayed cerebral ischemia (DCI) and poor outcome. The only currently approved drug treatment shown to reduce the risk of DCI and improve neurologic outcome after aneurysmal subarachnoid haemorrhage (SAH) is nimodipine, a dihydropyridine L-type voltage-gated Ca2+ channel blocker. MEK1/2 mediated transcriptional upregulation of contractile receptors, including endothelin-1 (ET-1) receptors, has previously been shown to be a factor in the pathology of SAH. The aim of the study was to compare intrathecal and subcutaneous treatment regimens of nimodipine and intrathecal treatment regimens of U0126, a MEK1/2 inhibitor, in a single injection experimental rat SAH model with post 48 h endpoints consisting of wire myography of cerebral arteries, flow cytometry of cerebral arterial tissue and behavioural evaluation. Following ET-1 concentration-response curves, U0126 exposed arteries had a significantly lower ET-1max than vehicle arteries. Arteries from both the intrathecal- and subcutaneous nimodipine treated animals had significantly higher ET-1max contractions than the U0126 arteries. Furthermore, Ca2+ concentration response curves (precontracted with ET-1 and in the presence of nimodipine) showed that nimodipine treatment could result in larger nimodipine insensitive contractions compared to U0126. Flow cytometry showed decreased protein expression of the ETB receptor in U0126 treated cerebral vascular smooth muscle cells compared to vehicle. Only U0126 treatment lowered ET-1max contractions and ETB receptor levels, as well as decreased the contractions involving nimodipine-insensitive Ca2+ channels, when compared to both intrathecal and subcutaneous nimodipine treatment. This indicate that targeting gene expression might be a better strategy than blocking specific receptors or ion channels in future treatments of SAH.

Topics: Animals; Butadienes; Calcium Channel Blockers; Cerebral Arteries; Disease Models, Animal; Male; Mitogen-Activated Protein Kinase Kinases; Muscle Contraction; Muscle, Smooth, Vascular; Nimodipine; Nitriles; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Subarachnoid Hemorrhage; Up-Regulation; Vasoconstriction

Irisin, a myokine, is cleaved from the extracellular portion of fibronectin domain-containing 5 protein in skeletal muscle and myocardium and secreted into circulation as a hormone during exercise. Irisin has been found to exert protective effects against lung and heart injuries. However, whether irisin influences myocardial infarction (MI) remains unclear. In this study we investigated the therapeutic effects of irisin in an acute MI model and its underlying mechanisms. Adult C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery and treated with irisin for 2 weeks after MI. Cardiac function was assessed using echocardiography. We found that irisin administration significantly alleviated MI-induced cardiac dysfunction and ventricular dilation at 4 weeks post-MI. Irisin significantly reduced infarct size and fibrosis in post-MI hearts. Irisin administration significantly increased angiogenesis in the infarct border zone and decreased cardiomyocyte apoptosis, but did not influence cardiomyocyte proliferation. In human umbilical vein endothelial cells (HUVEC), irisin significantly increased the phosphorylation of ERK, and promoted the migration of HUVEC detected in wound-healing and transwell chamber migration assay. The effects of irisin were blocked by the ERK inhibitor U0126. In conclusion, irisin improves cardiac function and reduces infarct size in post-MI mouse heart. The therapeutic effect is associated with its pro-angiogenic function through activating ERK signaling pathway.

Topics: Animals; Apoptosis; Butadienes; Cell Movement; Disease Models, Animal; Fibronectins; Human Umbilical Vein Endothelial Cells; Humans; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Neovascularization, Pathologic; Nitriles; Recombinant Proteins

Painful diabetic neuropathy (PDN) is a severely debilitating chronic pain syndrome. Spinal chemokine CXCL13 and its receptor CXCR5 were recently demonstrated to play a pivotal role in the pathogenesis of chronic pain induced by peripheral tissue inflammation or nerve injury. In this study we investigated whether CXCL13/CXCR5 mediates PDN and the underlying spinal mechanisms. We used the db/db type 2 diabetes mice, which showed obvious hyperglycemia and obese, long-term mechanical allodynia, and increased expression of CXCL13, CXCR5 as well as pro-inflammatory cytokines TNF-α and IL-6 in the spinal cord. Furthermore, in the spinal cord of db/db mice there is significantly increased gliosis and upregulated phosphorylation of cell signaling kinases, including pERK, pAKT and pSTAT3. Mechanical allodynia and upregulated pERK, pAKT and pSTAT3 as well as production of TNF-α and IL-6 were all attenuated by the noncompetitive NMDA receptor antagonist MK-801. If spinal giving U0126 (a selective MEK inhibitor) or AG490 (a Janus kinase (JAK)-STAT inhibitor) to db/db mice, both of them can decrease the mechanical allodynia, but only inhibit pERK (by U0126) or pSTAT3 (by AG490) respectively. Acute administration of CXCL13 in C57BL/6J mice resulted in exacerbated thermal hyperalgesia and mechanical allodynia, activation of the pERK, pAKT and pSTAT3 pathways and increased production of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6), which were all attenuated by knocking out of Cxcr5. In all, our work showed that chemokine CXCL13 and its receptor CXCR5 in spinal cord contribute to the pathogenesis of PDN and may help develop potential novel therapeutic approaches for patients afflicted with PDN.

Topics: Animals; Butadienes; Chemokine CXCL13; Cytokines; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Disease Models, Animal; Dizocilpine Maleate; Hyperalgesia; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Nitriles; Proto-Oncogene Proteins c-akt; Receptors, CXCR5; Signal Transduction; Spinal Cord; STAT3 Transcription Factor; Tyrphostins

Hypertrophic cardiomyopathy occurs along with pathological phenomena such as cardiac hypertrophy, myocardial fibrosis and cardiomyocyte activity. However, few of the specific molecular mechanisms underlying this pathological condition have been mentioned.. All target proteins and markers expression in the study was verified by PCR and western bloting. H9c2 cell morphology and behavior were analyzed using immunofluorescent and proliferation assays, respectively. And, the CTGF protein secreted in cell culture medium was detected by ELISA.. We found that high expression of CTGF and low expression of EGFR were regulated by ERK1/2 signaling pathway during the cardiac hypertrophy induced by Ang-II stimulation. CTGF interacted with EGFR, and the interaction is reduced with the stimulation of Ang-II. ERK1/2 serves as the center of signal control during the cardiac hypertrophy.. The ERK1/2 cooperates with GPCR and EGFR signaling, and promotes the occurrence and development of cardiac hypertrophy by regulating the expression and binding states of CTGF and EGFR. The study revealed a regulation model based on ERK1/2, suggesting that ERK1/2 signaling pathway may be an important control link for mitigation of hypertrophic cardiomyopathy treatment.

Topics: Angiotensin II; Animals; Butadienes; Cardiomegaly; Cardiomyopathy, Hypertrophic; Cell Enlargement; Cell Line; Connective Tissue Growth Factor; Disease Models, Animal; ErbB Receptors; Heart Ventricles; MAP Kinase Signaling System; Myocytes, Cardiac; Nitriles; Phosphorylation; Rats; Receptors, G-Protein-Coupled; Signal Transduction

The specific mechanisms underlying cyclin-dependent kinase 5 (Cdk5)-mediated neuropathic pain at the spinal cord level remain elusive. The aim of the present study was to explore the role of crosstalk between Cdk5/p35 and extracellular signal-regulated kinase 1/2 (ERK1/2) signalling in mediating spinal astrocyte activity via the PPARγ pathway in a rat model of chronic constriction injury (CCI). Here, we quantified pain behaviour after CCI; detected the localization of p35, Cdk5, phosphorylated ERK1/2 (pERK1/2), phosphorylated peroxisome proliferator-activated receptor γ (pPPARγ), neuronal nuclei (a neuronal marker), glial fibrillary acidic protein (GFAP, an activated astrocyte marker) and ionized calcium binding adaptor molecule 1 (a microglial marker) in the dorsal horn using immunofluorescence; measured the protein levels of Cdk5, p35, pERK1/2, pPPARγ and GFAP using western blot analysis; and gauged the enzyme activity of Cdk5/p35 kinase using a Cdk5/p35 kinase activity assay kit. Tumour necrosis factor-α, interleukin (IL)-1β and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). Ligation of the right sciatic nerve induced mechanical allodynia; thermal hyperalgesia; and the time-dependent upregulation of p35, pERK1/2 and GFAP and downregulation of pPPARγ. p35 colocalized with Cdk5, pERK1/2, pPPARγ, neurons and astrocytes but not microglia. Meanwhile, intrathecal injection of the Cdk5 inhibitor roscovitine, the mitogen-activated ERK kinase (MEK) inhibitor U0126 and the PPARγ agonist pioglitazone prevented or reversed behavioural allodynia, increased pPPARγ expression, inhibited astrocyte activation and alleviated proinflammatory cytokine (tumour necrosis factor-α, IL-1β, and IL-6) release from activated astrocytes. Furthermore, crosstalk between the Cdk5/p35 and ERK1/2 pathways was observed with CCI. Blockade of either Cdk5/p35 or ERK1/2 inhibited Cdk5 activity. These findings indicate that spinal crosstalk between the Cdk5/p35 and ERK1/2 pathways mediates astrocyte activity via the PPARγ pathway in CCI rats and that targeting this crosstalk could be an effective strategy to attenuate CCI and astrocyte-derived neuroinflammation.

Topics: Animals; Astrocytes; Butadienes; Constriction, Pathologic; Cyclin-Dependent Kinase 5; Disease Models, Animal; Enzyme Inhibitors; Male; MAP Kinase Signaling System; Nitriles; Phosphotransferases; PPAR gamma; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Roscovitine; Sciatic Neuropathy; Spinal Cord

The aim was to evaluate the beneficial effect of early mitogen-activated protein kinase (MEK)1/2 inhibition administered at a clinical relevant time-point using the transient middle cerebral artery occlusion model and a dedicated rodent magnetic resonance imaging system (9.4T) to monitor cerebrovascular changes non-invasively for 2 weeks.. Transient middle cerebral artery occlusion was induced in male rats for two hours followed by reperfusion. The specific MEK1/2 inhibitor U0126 was administered ip at 6 and 24 hours post-reperfusion. Neurological functions were evaluated by 6- and 28-point tests. 9.4 T magnetic resonance imaging was used to monitor morphological infarct changes at day 2, 8 and 14 after stroke and to evaluate cerebral perfusion at day 14. Immunohistochemistry evaluation of Ki67 was performed 14 days post-stroke.. U0126 improved long-term behavioural outcome and significantly reduced infarct size. In addition, cerebral perfusion in U0126-treated animals was improved compared to the vehicle group. Immunohistochemistry showed a significant increase in Ki67. Early MEK1/2 inhibition improves long-term functional outcome, promotes recovery processes after stroke and most importantly provides a realistic time window for therapy.

Topics: Animals; Brain; Butadienes; Cerebrovascular Circulation; Disease Models, Animal; Infarction, Middle Cerebral Artery; Ki-67 Antigen; Magnetic Resonance Imaging; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Protein Kinase Inhibitors; Rats, Wistar; Recovery of Function; Time Factors

Sevoflurane has been shown to stimulate or depress memory in adult rats; however, the cellular mechanism of this bidirectional effect has not been fully investigated.. We used an intra-hippocampal microinfusion of U0126 to suppress ERK activation. Male SD rats were randomly assigned to four groups: Sham, 0.11%SEV, 0.3%SEV and 0.3%+U0126. They received bilateral injections of U0126 or saline. Rats were anesthetized, and Inhibitory Avoidance (IA) training was performed immediately after anesthesia. The memory retention latency was observed 24 h later. In another experiment, the hippocampus was removed 45 min after IA training to assess ARC expression, the synapsin 1 protein levels and the phosphorylation level of ERK.. Treatment with 0.11%SEV led to rapid phosphorylation of ERK, while 0.3%SEV inhibited phosphorylation; the latter change was reversed by the microinfusion of U0126 in the hippocampus. The memory latency result had similar tendencies. The local infusion of U0126 abolished the 0.3%SEV-induced memory impairment and ERK inhibition. Selective upregulations of ARC and synapsin 1 proteins were observed in the 0.3%SEV group compared with the 0.11%SEV group.. The results indicate that different doses of sevoflurane trigger synaptic plasticity-related cytoskeleton proteins through the ERK signaling pathway. This novel modulation by inhalational agents may help to reduce their side-effects on memory function.

Topics: AIDS-Related Complex; Anesthetics, Inhalation; Animals; Butadienes; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Escape Reaction; Hippocampus; Learning Disabilities; Male; MAP Kinase Signaling System; Memory Disorders; Methyl Ethers; Nitriles; Rats; Rats, Sprague-Dawley; Sevoflurane; Synapsins

The pineal hormone melatonin influences the secretion of insulin by pancreatic islets via the G‑protein‑coupled melatonin receptors 1 and 2 that are expressed in pancreatic β‑cells. Genome‑wide association studies indicate that melatonin receptor 1B (MTNR1B) single nucleotide polymorphisms are tightly associated with type 2 diabetes mellitus (T2DM). However, the underlying mechanism is unclear. Raf‑1 serves a critical role in the mitogen‑activated protein kinase (MAPK) pathways in β‑cell survival and proliferation and, therefore, may be involved in the mechanism by which melatonin impacts on T2DM through MTNR1B. In the present study, the mRNA expression of the two mouse insulin genes Ins1 and Ins2 was investigated in MIN6 cells treated with different concentrations of melatonin, and insulin secretion was detected under the same conditions. Following the overexpression or silencing of MTNR1B, the activities of components of the MAPK signaling pathway, including Raf‑1 and ERK, were evaluated. The impact of MTNR1B knockdown on the melatonin‑regulated insulin gene expression and insulin secretion were also investigated. The results demonstrated that exogenous melatonin inhibited the expression of insulin mRNA in the MIN6 cells. Insulin secretion by the MIN6 cells, however, was not affected by melatonin. The MAPK signaling pathway was inhibited in MIN6 cells by treatment with melatonin or the overexpression of MTNR1B. The knockdown of MTNR1B totally attenuated the regulating effect of melatonin on insulin gene expression. Additionally, the inductive effect of melatonin on the expression of insulin mRNA was attenuated when the activities of Raf‑1 or ERK were blocked using the chemical inhibitors GW5074 and U0126, respectively. It may be concluded that melatonin exerts an inhibitory effect on insulin transcription via MTNR1B and the downstream MAPK signaling pathway.

Topics: Animals; Butadienes; Diabetes Mellitus, Type 2; Disease Models, Animal; Gene Expression Regulation; Humans; Indoles; Insulin; Islets of Langerhans; MAP Kinase Signaling System; Melatonin; Mice; Nitriles; Phenols; Proto-Oncogene Proteins c-raf; Receptor, Melatonin, MT2

This study aims to highlight some of the genes that are differentially regulated by ERK1/2 signaling in TGFβ-induced EMT in lens, and their potential contribution to this pathological process.. Rat lens epithelial explants were cultured with or without TGFβ over a 3-day-culture period to induce EMT, in the presence or absence of UO126 (ERK1/2 signaling inhibitor), both prior to TGFβ-treatment, or 24 or 48 hours after TGFβ treatment. Smad2/3-nuclear immunolabeling was used to indicate active TGFβ signaling, and quantitative RT-PCR was used to analyze changes in the different treatment groups in expression of the following representative genes: TGFβ signaling (Smad7, Smurf1, and Rnf111), epithelial markers (Pax6, Cdh1, Zeb1, and Zeb2), cell survival/death regulators (Bcl2, Bax, and Bad) and lens mesenchymal markers (Mmp9, Fn1, and Col1a1), over the 3 days of culture.. ERK1/2 was found to regulate the expression of Smurf1, Smad7, Rnf11, Cdh1, Pax6, Zeb1, Bcl2, Bax, and Bad genes in lens cells. TGFβ signaling was evident by nuclear localization of Smad2/3 and this was effectively blocked by pre-treatment with UO126, but not by post-treatment with this ERK1/2 signaling inhibitor. TGFβ induced the expression of its signaling partners (Smad7, Smurf1, and Rnf111), as well as lens mesenchymal genes (Mmp9, Fn1, and Col1a1), consistent with its role in inducing an EMT. These TGFβ-responsive signaling genes, as well as the mesenchymal markers, were all positively regulated by ERK1/2-activity. The expression levels of the lens epithelial genes we examined, and genes that were associated with cell death/survival, were not directly impacted by TGFβ.. TGFβ-mediated ERK1/2 signaling positively modulates the expression of mesenchymal genes in lens epithelial explants undergoing EMT, in addition to regulating TGFβ-mediated regulatory genes. Independent of TGFβ, ERK1/2 activity can also regulate the expression of endogenous lens epithelial genes, highlighting its potential key role in regulation of both normal and pathological lens cellular processes.

Topics: Animals; Butadienes; Cataract; Cells, Cultured; Disease Models, Animal; Epithelial-Mesenchymal Transition; Gene Expression; Lens, Crystalline; MAP Kinase Signaling System; Nitriles; Rats; Rats, Wistar; Signal Transduction; Transforming Growth Factor beta2

Growth retardation is a constant feature of Noonan syndrome (NS) but its physiopathology remains poorly understood. We previously reported that hyperactive NS-causing SHP2 mutants impair the systemic production of insulin-like growth factor 1 (IGF1) through hyperactivation of the RAS/extracellular signal-regulated kinases (ERK) signalling pathway. Besides endocrine defects, a direct effect of these mutants on growth plate has not been explored, although recent studies have revealed an important physiological role for SHP2 in endochondral bone growth. We demonstrated that growth plate length was reduced in NS mice, mostly due to a shortening of the hypertrophic zone and to a lesser extent of the proliferating zone. These histological features were correlated with decreased expression of early chondrocyte differentiation markers, and with reduced alkaline phosphatase staining and activity, in NS murine primary chondrocytes. Although IGF1 treatment improved growth of NS mice, it did not fully reverse growth plate abnormalities, notably the decreased hypertrophic zone. In contrast, we documented a role of RAS/ERK hyperactivation at the growth plate level since 1) NS-causing SHP2 mutants enhance RAS/ERK activation in chondrocytes in vivo (NS mice) and in vitro (ATDC5 cells) and 2) inhibition of RAS/ERK hyperactivation by U0126 treatment alleviated growth plate abnormalities and enhanced chondrocyte differentiation. Similar effects were obtained by chronic treatment of NS mice with statins. In conclusion, we demonstrated that hyperactive NS-causing SHP2 mutants impair chondrocyte differentiation during endochondral bone growth through a local hyperactivation of the RAS/ERK signalling pathway, and that statin treatment may be a possible therapeutic approach in NS.

Topics: Animals; Butadienes; Cell Differentiation; Cell Proliferation; Chondrocytes; Disease Models, Animal; Growth Plate; Humans; Insulin-Like Growth Factor I; MAP Kinase Signaling System; Nitriles; Noonan Syndrome; Protein Tyrosine Phosphatase, Non-Receptor Type 11

Moderate hypothermia (MH) used as treatment for neurological diseases has a protective effect; however, its mechanism remains unclear. Neuronal autophagy is a fundamental pathological process of early brain injury in subarachnoid hemorrhage (SAH). We found that moderate activation of autophagy can reduce nerve cells damage. In this study, We found that MH can moderately increase the level of autophagy in nerve cells and improve the neurological function in rats. This type of autophagy activation is dependent on extracellular signal-regulated kinase (ERK) signaling pathways. The level of neuronal autophagy was down-regulated significantly by using U0126, an ERK signaling pathway inhibitor. In summary, these results suggest that MH can moderately activate neuronal autophagy through ERK signaling pathway, reduce nerve cell death, and produce neuroprotective effects.

Topics: Animals; Autophagy; Brain Edema; Brain Injuries; Butadienes; CA1 Region, Hippocampal; Disease Models, Animal; Hypothermia, Induced; Male; MAP Kinase Signaling System; Neurons; Neuroprotection; Nitriles; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage

Striatal-enriched phosphatase 61 (STEP61) is a member of intracellular protein tyrosine phosphatases, which is involved in the regulation of synaptic plasticity and a line of neuropsychiatric disorders. This protein tyrosine phosphatase is also abundant in pain-related spinal cord dorsal horn neurons. However, whether and how this tyrosine phosphatase modulates the nociceptive plasticity and behavioral hypersensitivity remain largely unknown. The present study recorded the long-term potentiation (LTP) of primary afferent C fiber-evoked field potentials in vivo in superficial dorsal horn of rats, and tested the possible role of STEP61 in spinal LTP. We found that LTP induction significantly increased STEP61 phosphorylation at Ser221 residue, a key molecular event that has been shown to impair the phosphatase activity. The STEP61 hypoactivity allowed for the activation of three substrates, GluN2B subunit-containing N-methyl-d-aspartate-subtype glutamate receptors, Src-family protein tyrosine kinase member Fyn and extracellular signal-regulated kinase 1/2, through which the thresholds for LTP induction were noticeably decreased. To reinstate STEP61 activity, we overexpressed wild-type STEP61 [STEP61(WT)] in spinal dorsal horn, finding that STEP61(WT) completely blunted LTP induction. Behavioral tests showed that LTP blockade by STEP61(WT) correlated with a long-lasting alleviation of thermal hypersensitivity and mechanical allodynia induced by chronic constriction injury of sciatic nerves. These data implicated that STEP61 exerted a negative control over spinal nociceptive plasticity, which might have therapeutic benefit in pathological pain.

Topics: Afferent Pathways; Animals; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Green Fluorescent Proteins; Hyperalgesia; Long-Term Potentiation; Male; Nerve Fibers; Neuralgia; Nitriles; Pain Measurement; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-fyn; Rats; Rats, Sprague-Dawley; Sensory Receptor Cells; Spinal Cord Dorsal Horn; Transduction, Genetic

Topics: Animals; Butadienes; Disease Models, Animal; Dynamins; Enzyme Inhibitors; Male; Mitochondria; Mitochondrial Dynamics; Mitogen-Activated Protein Kinase 12; Muscarinic Agonists; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Nitriles; Phosphorylation; Pilocarpine; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Signal Transduction; Status Epilepticus; TRPC Cation Channels

Short and long acting NMDA receptor (NMDAR) antagonists exert their antidepressant-like effects by activating signaling pathways involved in the synthesis of synaptic proteins and formation of new synaptic connections in the prefrontal cortex (PFC) of rats. The blockade of the ERK pathway abolishes ketamine and Ro 25-6981 antidepressant potency. However, the role of ERK in the antidepressant-like activity of short acting NMDAR antagonists is still unclear. More puzzling is the fact that the precise role of ERK in the short and long lasting effects of long-acting NMDAR antagonists is unknown. In this study, we show that zinc, (Zn) a short-acting NMDAR antagonist evokes only transient ERK activation, which is observed 7 min after its administration in the PFC of rats. In contrast to Zn, the long acting NMDAR antagonist Ro 25-6981 produces persistent ERK activation lasting up to 24 h. Pretreatment with the MAPK/ERK inhibitor (U0126) totally abolished Zn and Ro 25-6981 antidepressant-like activities in the forced swim test in rats. However, when U0126 is administered 15 min after Zn or Ro 25-6981 both compounds maintain their short-lasting antidepressant-like activity. On the other hand, posttreatment with U0126 significantly attenuated the long lasting antidepressant-like activity of Ro 25-6981. These results indicate that the activation of ERK is crucial for the short- and long lasting antidepressant-like activity observed in the FST in rats.

Topics: Animals; Antidepressive Agents; Aspartic Acid; Butadienes; Depressive Disorder; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Male; Motor Activity; Nitriles; Organometallic Compounds; Phenols; Phosphorylation; Piperidines; Prefrontal Cortex; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Synaptosomes; Time Factors; Zinc Compounds

Myocardial infarction is the leading cause of death worldwide. Restoration of blood flow rescues myocardium but also causes ischemia-reperfusion injury. Here, we show that in a mouse model of chronic neuropathic pain, ischemia-reperfusion injury following myocardial infarction is reduced, and this cardioprotection is induced via an anterior nucleus of paraventricular thalamus (PVA)-dependent parasympathetic pathway. Pharmacological inhibition of extracellular signal-regulated kinase activation in the PVA abolishes neuropathic pain-induced cardioprotection, whereas activation of PVA neurons pharmacologically, or optogenetic stimulation, is sufficient to induce cardioprotection. Furthermore, neuropathic injury and optogenetic stimulation of PVA neurons reduce the heart rate. These results suggest that the parasympathetic nerve is responsible for this unexpected cardioprotective effect of chronic neuropathic pain in mice.Various forms of preconditioning can prevent ischemic-reperfusion injury after myocardial infarction. Here, the authors show that in mice, the presence of chronic neuropathic pain can have a cardioprotective effect, and that this is dependent on neural activation in the paraventricular thalamus.

Topics: Animals; Butadienes; Chronic Pain; Disease Models, Animal; Enzyme Inhibitors; Ganglionic Blockers; Heart Rate; Hexamethonium; Lidocaine; Male; Mice, Inbred C57BL; Midline Thalamic Nuclei; Myocardial Infarction; Myocardial Reperfusion Injury; Neuralgia; Nitriles; Optogenetics

Extracellular signal-regulated kinase (ERK) 1/2 is activated during acute phase of stroke and contributes to stroke pathology. We have found that acute treatment with MEK1/2 inhibitors decreases infarct size and neurological deficits 2 days after experimental stroke. However, it is not known whether benefits of this inhibition persist long-term. Therefore, the aim of this study was to assess neurological function, infarct size and recovery processes 14 days after stroke in male rats to determine long-term outcome following acute treatment with the MEK1/2 inhibitor U0126.. Transient middle cerebral artery occlusion was induced in male rats. U0126 or vehicle was given at 0 and 24 h of reperfusion. Neurological function was assessed by staircase, 6-point and 28-point neuroscore tests up to 14 days after induction of stroke. At day 14, infarct volumes were determined and recovery processes were evaluated by measuring protein expression of the tyrosine kinase receptor Tie-2 and nestin. Levels of p-ERK1/2 protein were determined.. Acute treatment with U0126 significantly improved long-term functional recovery, reduced infarct size, and enhanced Tie-2 and nestin protein expression at 14 days post-stroke. There was no residual blockade of p-ERK1/2 at this time point.. It is demonstrated that benefits of early treatment with U0126 persist beyond subacute phase of ischaemic stroke in male rats. Prevention of ERK1/2 activation in the acute phase results in improved long-term functional outcome and enhances later-stage recovery processes. These results expand our understanding of the benefits and promise of using MEK1/2 inhibitors in stroke recovery.

Topics: Animals; Blotting, Western; Brain; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Image Processing, Computer-Assisted; Immunohistochemistry; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Rats; Rats, Wistar; Recovery of Function; Stroke

Aortic valve disease (AVD) is one of the leading causes of cardiovascular mortality. Abnormal expression of hyaluronan (HA) and its synthesizing/degrading enzymes have been observed during latent AVD however, the mechanism of impaired HA homeostasis prior to and after the onset of AVD remains unexplored. Transforming growth factor beta (TGFβ) pathway defects and biomechanical dysfunction are hallmarks of AVD, however their association with altered HA regulation is understudied. Expression of HA homeostatic markers was evaluated in diseased human aortic valves and TGFβ1-cultured porcine aortic valve tissues using histology, immunohistochemistry and Western blotting. Further, porcine valve interstitial cell cultures were stretched (using Flexcell) and simultaneously treated with exogenous TGFβ1±inhibitors for activated Smad2/3 (SB431542) and ERK1/2 (U0126) pathways, and differential HA regulation was assessed using qRT-PCR. Pathological heavy chain HA together with abnormal regional expression of the enzymes HAS2, HYAL1, KIAA1199, TSG6 and IαI was demonstrated in calcified valve tissues identifying the collapse of HA homeostatic machinery during human AVD. Heightened TSG6 activity likely preceded the end-stage of disease, with the existence of a transitional, pre-calcific phase characterized by HA dysregulation. TGFβ1 elicited a fibrotic remodeling response in porcine aortic valves similar to human disease pathology, with increased collagen and HYAL to HAS ratio, and site-specific abnormalities in the expression of CD44 and RHAMM receptors. Further in these porcine valves, expression of HAS2 and HYAL1 was found to be differentially regulated by the Smad2/3 and ERK1/2 pathways, and CD44 expression was highly responsive to biomechanical strain. Leveraging the regulatory pathways that control both HA maintenance in normal valves and early postnatal dysregulation of HA homeostasis during disease may identify new mechanistic insight into AVD pathogenesis.

Topics: Adolescent; Aged; Animals; Aortic Valve; Benzamides; Butadienes; Cell Adhesion Molecules; Cells, Cultured; Dioxoles; Disease Models, Animal; Gene Regulatory Networks; Heart Valve Diseases; Homeostasis; Humans; Hyaluronic Acid; Middle Aged; Nitriles; Swine; Transforming Growth Factor beta1; Young Adult

We investigated the mechanisms underlying the suppression of the rewarding effects of opioids using the femur bone cancer (FBC) mouse model. The rewarding and antinociceptive effects of subcutaneously administered morphine and oxycodone in the FBC model mice were assessed using the conditioned place preference test and the von-Frey test. In FBC mice, antinociceptive doses of morphine (30 mg/kg) and oxycodone (5 mg/kg) did not produce the rewarding effects but excessive doses of morphine (300 mg/kg) and oxycodone (100 mg/kg) did. Western blot analyses revealed a transient and significant increase in phosphorylated-extracellular regulated kinase (p-ERK) levels in ventral tegmental area (VTA) 5 min after the administration of morphine in sham-group. Interestingly, in FBC group, a regular dose of morphine did not increase p-ERK levels but a high dose of morphine caused an increase in p-ERK level 5 min after administration. The rewarding effects of a regular dose of and a high dose of morphine in the sham-operation and FBC model, respectively, were significantly inhibited by the MEK inhibitor. The suppression of p-ERK might result in resistance to these rewarding effects under the conditions of bone cancer.

Topics: Analgesics; Animals; Butadienes; Conditioning, Psychological; Disease Models, Animal; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Mice; Morphine; Nitriles; Oxycodone; Phosphorylation; Radioligand Assay; Receptors, Opioid, mu; Reward; Up-Regulation; Ventral Tegmental Area

Amyloid-β (Aβ) produces neurotoxicity in the brain and causes neuronal death, but the endogenous defense mechanism that is activated on Aβ insult is less well known. Here we found that acute Aβ increases the expression of PIAS1 and Mcl-1 via activation of MAPK/ERK, and Aβ induction of PIAS1 enhances HDAC1 SUMOylation in rat hippocampus. Knockdown of PIAS1 decreases endogenous HDAC1 SUMOylation and blocks Aβ induction of Mcl-1. Sumoylated HDAC1 reduces it association with CREB, increases CREB binding to the Mcl-1 promoter and mediates Aβ induction of Mcl-1 expression. Transduction of SUMO-modified lenti-HDAC1 vector to the hippocampus of APP/PS1 mice rescues spatial learning and memory deficit and long-term potentiation impairment in APP/PS1 mice. It also reduces the amount of amyloid plaque and the number of apoptotic cells in CA1 area of APP/PS1 mice. Meanwhile, HDAC1 SUMOylation decreases HDAC1 binding to the neprilysin promoter. These results together reveal an important role of HDAC1 SUMOylation as a naturally occurring defense mechanism protecting against Aβ toxicity and provide an alternative therapeutic strategy against AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Apoptosis; Butadienes; Disease Models, Animal; Epigenesis, Genetic; Gene Expression; HEK293 Cells; Hippocampus; Histone Deacetylase 1; Humans; Male; Mice; Mice, Transgenic; Neuroprotection; Nitriles; Protein Binding; Protein Inhibitors of Activated STAT; Rats; Rats, Sprague-Dawley; RNA Interference; Sumoylation

Sensory mismatch between actual motion information and anticipated sensory patterns (internal model) is the etiology of motion sickness (MS). Some evidence supports that hippocampus might involve the neural storage of the "internal model". This study established an "internal model" acquisition-retention behavioral model using a repeated habituation rotation training protocol. We tried to identify the hippocampal subregion involved in "internal model" retention using chemical lesion methods. Hippocampal kinases (CaMK, CaMKIV, CREB and ERK1/2) phosphorylation in the target subregion was assayed and the effects of kinase inhibitors (KN93 or U0126) on "internal model" retention were investigated. The activities of potential kinases (CaMKII and CREB) were also examined in otoliths deficit het/het mice. In habituated rats, CA1 lesion reproduced MS-related behavioral responses on "internal model" retention day. Habituation training increased CaMKII and CREB activity but had no effect on CaMKIV and ERK1/2 activity in the CA1, while inhibition of CaMKII but not ERK1/2 impaired "internal model" retention. In het/het mice, CaMKII and CREB were not activated in the CA1 on the retention day. These results suggested that CaMKII/CREB pathway might potentially contribute to the storage of the "internal model" in the hippocampal CA1 after motion sickness induced by vestibular stimulation.

Topics: Animals; Benzylamines; Butadienes; CA1 Region, Hippocampal; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinase Type 4; CREB-Binding Protein; Disease Models, Animal; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Motion Sickness; Nitriles; Otolithic Membrane; Phosphorylation; Rats; Rats, Sprague-Dawley; Rotation; Signal Transduction; Sulfonamides

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

Topics: Animals; Antibodies, Neutralizing; Apoproteins; beta-Thalassemia; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 6; Butadienes; Cytokines; Disease Models, Animal; Gene Expression Regulation; Hepatocytes; Hepcidins; Humans; Liver; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle Proteins; Nitriles; Phosphorylation; RNA, Messenger; Signal Transduction; Smad Proteins; Transferrin

Trimethyltin (TMT) toxicity causes histopathological damage in the hippocampus and induces seizure behaviors in mice. The lesions and symptoms recover spontaneously over time; however, little is known about the precise mechanisms underlying this recovery from TMT toxicity. We investigated changes in the brain-derived neurotrophic factor/extracellular signal-regulated kinases (BDNF/ERK) signaling pathways in the mouse hippocampus following TMT toxicity. Mice (7 weeks old, C57BL/6) administered TMT (2.6 mg/kg intraperitoneally) showed acute and severe neurodegeneration with increased TUNEL-positive cells in the dentate gyrus (DG) of the hippocampus. The mRNA and protein levels of BDNF in the hippocampus were elevated by TMT treatment. Immunohistochemical analysis showed that TMT treatment markedly increased phosphorylated ERK1/2 expression in the mouse hippocampus 1-4 days after TMT treatment, although the intensity of ERK immunoreactivity in mossy fiber decreased at 1-8 days post-treatment. In addition, ERK-immunopositive cells were localized predominantly in doublecortin-positive immature progenitor neurons in the DG. In primary cultured immature hippocampal neurons (4 days in vitro), BDNF treatment alleviated TMT-induced neurotoxicity, via activation of the ERK signaling pathway. Thus, we suggest that BDNF/ERK signaling pathways may be associated with cell differentiation and survival of immature progenitor neurons, and will eventually lead to spontaneous recovery in TMT-induced hippocampal neurodegeneration.

Topics: Animals; Brain-Derived Neurotrophic Factor; Butadienes; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Flavonoids; Hippocampus; Hydro-Lyases; Male; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neurons; Neurotoxicity Syndromes; Nitriles; Recovery of Function; Seizures; Time Factors; Trimethyltin Compounds

To explore the role of IL-33 in asthmatic airway remodeling.. Male BALB/c mice were randomly divided into 3 groups: a control group, an ovalbumin (OVA) group, and an anti-IL-33 antibody combined with OVA group. The airway remodeling features in mice were observed by HE staining. In addition, the expressions of IL-33, alpha smooth muscle actin (α-SMA), and type 1 collagen (Col1) in the airway of mice were detected by immunohistochemistry and Western blotting. Finally, Western blotting was used to determine the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen- and stress-activated protein kinase 1 (MSK1) in the lungs of mice. In vitro, human lung fibroblasts (HLF-1) were pretreated with the ERK1/2 inhibitor U0126 or the MSK1 inhibitor H89 respectively, and then treated with the human recombinant IL-33 (rIL-33). Then real-time quantitative PCR and Western blotting were used to test the expressions of α-SMA and Col1. Immunofluorescence cytochemistry and Western blotting were also used to observe the phosphorylation of ERK1/2 and MSK1 in HLF-1 cells.. The pre-treatment with the ERK1/2 inhibitor U0126 or anti-IL-33 antibody significantly abolished the OVA-induced airway remodeling, increased expressions of IL-33, α-SMA, Col1, and phosphorylation of ERK1/2 and MSK1 in the airway of mice. In vitro, the increased expressions of α-SMA and Col1 and the phosphorylation of ERK1/2 and MSK1 induced by rIL-33 in HLF-1 cells were markedly inhibited by the pre-treatment with U0126 or H89.. IL-33 promotes airway remodeling in asthmatic mice via the ERK1/2-MSK1 signaling pathway.

Topics: Airway Remodeling; Animals; Asthma; Butadienes; Disease Models, Animal; Humans; Interleukin-33; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Nitriles; Ribosomal Protein S6 Kinases, 90-kDa

Thrombolysis with recombinant tissue plasminogen activator (rtPA) is the most effective drug treatment for acute ischemic stroke within 4.5h after symptom onset. However, the use of rtPA may increase the risk of hemorrhagic transformation (HT), particularly when it is administered after the first 4.5h. However, no effective treatments are available to reduce the HT risk. Disruption of the blood-brain barrier (BBB) is central to the genesis of HT. Connexin43 (Cx43)-mediated gap junction intercellular communication (GJIC) has been demonstrated to regulate the integrity of the BBB in ischemia. We investigated the effect of Cx43 on BBB permeability during rtPA-induced HT. Spontaneously hypertensive rats (SHRs) underwent a 1.5-h middle cerebral artery occlusion and were treated with rtPA at 4.5h. The rats were sacrificed at 24h, and their brains were evaluated for BBB permeability and the expression of tight junction (TJ) proteins and Cx43. We examined whether the effects were Cx43 dependent using multiple Cx43 inhibitors. Phosphorylated Cx43 (p-Cx43) but not total Cx43 protein expression was increased after rtPA treatment. Delayed rtPA administration induced significant HT and BBB disruption. These effects were attenuated by inhibitors that blocked GJIC and Cx43 phosphorylation and expression but not Cx43 redistribution. Additionally, rtPA administration upregulated p-Cx43 expression in hypoxia/reoxygenation (H/R)-exposed brain endothelial cells. These effects were suppressed by the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126. We suggest that rtPA-associated hemorrhage due to an alteration in the integrity of the BBB is highly associated with an increase in p-Cx43 resulting from the activation of the PI3K and ERK pathways.

Topics: Animals; Blood-Brain Barrier; Butadienes; Capillary Permeability; Cells, Cultured; Cerebral Hemorrhage; Chromones; Connexin 43; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Fibrinolytic Agents; Infarction, Middle Cerebral Artery; Male; Morpholines; Nitriles; Phosphorylation; Rats, Inbred SHR; Thrombolytic Therapy; Tissue Plasminogen Activator

Less disruption of the blood-brain barrier (BBB) after severe ischemic stroke is one of the beneficial outcomes of ischemic preconditioning (IP). However, the effect of IP on tight junctions (TJs), which regulate paracellular permeability of the BBB, is not well understood. In the present study, we examined IP-induced changes in TJs before and after middle cerebral artery occlusion (MCAO) in mice, and the association between changes in TJs and tolerance to a subsequent insult. After IP, we found decreased levels of transmembrane TJ proteins occludin and claudin-5, and widened gaps of TJs with perivascular swelling at the ultrastructural level in the brain. An inflammatory response was also observed. These changes were reversed by inhibition of extracellular signal-regulated kinase1/2 (ERK1/2) via the specific ERK1/2 inhibitor U0126. After MCAO, reduced brain edema and inflammatory responses were associated with altered levels of angiogenic factors and cytokines in preconditioned brains. Pretreatment with U0126 reversed the neuroprotective effects of IP against MCAO. These findings suggest that ERK1/2 activation has a pivotal role in IP-induced changes in TJs and inflammatory response, which serve to protect against BBB breakdown and inflammation after ischemic stroke.

Topics: Animals; Brain Edema; Brain Infarction; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Infarction, Middle Cerebral Artery; Ischemic Preconditioning, Myocardial; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle Strength; Neurologic Examination; Nitriles; Tight Junctions; Time Factors; Vascular Endothelial Growth Factor A

Cerebral vasospasm and late cerebral ischemia (LCI) remain leading causes of mortality in patients experiencing a subarachnoid hemorrhage (SAH). This occurs typically 3 to 4 days after the initial bleeding and peaks at 5 to 7 days. The underlying pathophysiology is still poorly understood. Because SAH is associated with elevated levels of endothelin-1 (ET-1), focus has been on counteracting endothelin receptor activation with receptor antagonists like clazosentan, however, with poor outcome in clinical trials. We hypothesize that inhibition of intracellular transcription signaling will be an effective approach to prevent LCI. Here, we compare the effects of clazosentan versus the MEK1/2 blocker U0126 in a rat model of SAH. Although clazosentan directly inhibits the contractile responses in vivo to ET-1, it did not prevent SAH-induced upregulation of ET receptors in cerebral arteries and did not show a beneficial effect on neurologic outcome. U0126 had no vasomotor effect by itself but counteracts SAH-induced receptor upregulation in cerebral arteries and improved outcome after SAH. We suggest that because SAH induces elevated expression of several contractile receptor subtypes, it is not sufficient to block only one of these (ET receptors) but inhibition of transcriptional MEK1/2-mediated upregulation of several contractile receptors may be a viable way towards alleviating LCI.

Topics: Animals; Brain Ischemia; Butadienes; Cerebral Arteries; Dioxanes; Disease Models, Animal; Endothelin-1; Enzyme Inhibitors; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Pyridines; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptors, Endothelin; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles; Up-Regulation

Sex differences are well known in cerebral ischemia and may impact the effect of stroke treatments. In male rats, the MEK1/2 inhibitor U0126 reduces ischemia-induced endothelin type B (ETB) receptor upregulation, infarct size and improves acute neurologic function after experimental stroke. However, responses to this treatment in females and long-term effects on outcome are not known. Initial experiments used in vitro organ culture of cerebral arteries, confirming ERK1/2 activation and increased ETB receptor-mediated vasoconstriction in female cerebral arteries. Transient middle cerebral artery occlusion (tMCAO, 120 minutes) was induced in female Wistar rats, with U0126 (30 mg/kg intraperitoneally) or vehicle administered at 0 and 24 hours of reperfusion, or with no treatment. Infarct volumes were determined and neurologic function was assessed by 6-point and 28-point neuroscores. ETB receptor-mediated contraction was studied with myograph and protein expression with immunohistochemistry. In vitro organ culture and tMCAO resulted in vascular ETB receptor upregulation and activation of ERK1/2 that was prevented by U0126. Although no effect on infarct size, U0126 improved the long-term neurologic function after experimental stroke in female rats. In conclusion, early prevention of the ERK1/2 activation and ETB receptor-mediated vasoconstriction in the cerebral vasculature after ischemic stroke in female rats improves the long-term neurologic outcome.

Topics: Animals; Butadienes; Cerebrovascular Circulation; Disease Models, Animal; Enzyme Inhibitors; Female; Immunohistochemistry; MAP Kinase Signaling System; Neuroprotective Agents; Nitriles; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Recovery of Function; Stroke; Vasoconstriction

Focal adhesion kinase (FAK) is one of the nonreceptor protein tyrosine kinases critical for the dynamic regulation of cell adhesion structures. Recent studies have demonstrated that FAK is also localized at excitatory glutamatergic synapses and is involved in long-term modification of synaptic strength. However, whether FAK is engaged in nociceptive processing in the spinal dorsal horn remains unresolved. The current study shows that intraplantar injection of complete Freund's adjuvant (CFA) in mice significantly increases FAK autophosphorylation at Tyr397, indicating a close correlation of FAK activation with inflammatory pain. FAK activation depended on the activity of N-methyl-D-aspartate-subtype glutamate receptor (NMDAR) and metabotropic glutamate receptor (mGluR) because pharmacological inhibition of NMDAR or group I mGluR totally abolished FAK phosphorylation induced by CFA. The active FAK operated to stimulate extracellular signal-regulated kinase1/2 (ERK1/2), which boosted the protein expression of GluN2B subunit-containing NMDAR at the synaptosomal membrane fraction. Inhibition of FAK activity by spinal expression of a kinase-dead FAK(Y397F) mutant repressed ERK1/2 hyperactivity and reduced the synaptic concentration of NMDAR in CFA-injected mice. Electrophysiological recording demonstrated that intracellular loading of specific anti-FAK antibody significantly reduced the amplitudes of NMDAR-mediated excitatory postsynaptic currents on lamina II neurons from inflamed mice but not from naive mice. Behavioral tests showed that spinal expression of FAK(Y397F) generated a long-lasting alleviation of CFA-induced mechanical allodynia and thermal hyperalgesia. These data indicate that FAK might exaggerate NMDAR-mediated synaptic transmission in the spinal dorsal horn to sensitize nociceptive behaviors.

Topics: Animals; Butadienes; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Focal Adhesion Kinase 1; Freund's Adjuvant; Male; MAP Kinase Signaling System; Mice; Mice, Inbred Strains; Mutation; Neurons; Neurotransmitter Agents; Nitriles; Patch-Clamp Techniques; Peripheral Nervous System Diseases; Phosphorylation; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Spinal Cord Dorsal Horn; Subcellular Fractions; Synaptic Potentials; Transduction, Genetic

Protease-activated receptor type 2 (PAR2) is known to play an important role in inflammatory, visceral, and cancer-evoked pain based on studies using PAR2 knockout (PAR2(-/-)) mice. We have tested the hypothesis that specific activation of PAR2 is sufficient to induce a chronic pain state through extracellular signal-regulated kinase (ERK) signaling to protein synthesis machinery. We have further tested whether the maintenance of this chronic pain state involves a brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (trkB)/atypical protein kinase C (aPKC) signaling axis. We observed that intraplantar injection of the novel highly specific PAR2 agonist, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at), evokes a long-lasting acute mechanical hypersensitivity (median effective dose ∼12 pmoles), facial grimacing, and causes robust hyperalgesic priming as revealed by a subsequent mechanical hypersensitivity and facial grimacing to prostaglandin E2 (PGE2) injection. The promechanical hypersensitivity effect of 2-at is completely absent in PAR2(-/-) mice as is hyperalgesic priming. Intraplantar injection of the upstream ERK inhibitor, U0126, and the eukaryotic initiation factor (eIF) 4F complex inhibitor, 4EGI-1, prevented the development of acute mechanical hypersensitivity and hyperalgesic priming after 2-at injection. Systemic injection of the trkB antagonist ANA-12 similarly inhibited PAR2-mediated mechanical hypersensitivity, grimacing, and hyperalgesic priming. Inhibition of aPKC (intrathecal delivery of ZIP) or trkB (systemic administration of ANA-12) after the resolution of 2-at-induced mechanical hypersensitivity reversed the maintenance of hyperalgesic priming. Hence, PAR2 activation is sufficient to induce neuronal plasticity leading to a chronic pain state, the maintenance of which is dependent on a BDNF/trkB/aPKC signaling axis.

Topics: Animals; Azepines; Behavior, Animal; Benzamides; Brain-Derived Neurotrophic Factor; Butadienes; Chronic Pain; Dinoprostone; Disease Models, Animal; Facial Expression; Hydrazones; Hyperalgesia; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Nitriles; Protein Kinase C; Receptor, PAR-2; Receptor, trkB; Signal Transduction; Thiazoles

Activation of liver X receptor (LXR) inhibits atherosclerosis but induces hypertriglyceridemia. In vitro, it has been shown that mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor synergizes LXR ligand-induced macrophage ABCA1 expression and cholesterol efflux. In this study, we determined whether MEK1/2 (U0126) and LXR ligand (T0901317) can have a synergistic effect on the reduction of atherosclerosis while eliminating LXR ligand-induced fatty livers and hypertriglyceridemia. We also set out to identify the cellular mechanisms of the actions.. Wild-type mice were used to determine the effect of U0126 on a high-fat diet or high-fat diet plus T0901317-induced transient dyslipidemia and liver injury. ApoE deficient (apoE(-/-)) mice or mice with advanced lesions were used to determine the effect of the combination of T0901317 and U0126 on atherosclerosis and hypertriglyceridemia. We found that U0126 protected animals against T0901317-induced transient or long-term hepatic lipid accumulation, liver injury, and hypertriglyceridemia. Meanwhile, the combination of T0901317 and U0126 inhibited the development of atherosclerosis in a synergistic manner and reduced advanced lesions. Mechanistically, in addition to synergistic induction of macrophage ABCA1 expression, the combination of U0126 and T0901317 maintained arterial wall integrity, inhibited macrophage accumulation in aortas and formation of macrophages/foam cells, and activated reverse cholesterol transport. The inhibition of T0901317-induced lipid accumulation by the combined U0126 might be attributed to inactivation of lipogenesis and activation of lipolysis/fatty acid oxidation pathways.. Our study suggests that the combination of mitogen-activated protein kinase kinase 1/2 inhibitor and LXR ligand can function as a novel therapy to synergistically reduce atherosclerosis while eliminating LXR-induced deleterious effects.

Topics: Animals; Aorta; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Butadienes; Chemical and Drug Induced Liver Injury; Cholesterol; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Fatty Liver; Female; Foam Cells; Hep G2 Cells; Humans; Hydrocarbons, Fluorinated; Hypertriglyceridemia; Liver; Liver X Receptors; Male; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Orphan Nuclear Receptors; Protein Kinase Inhibitors; Signal Transduction; Sulfonamides

Microglia accumulation plays detrimental roles in the pathology of germinal matrix hemorrhage (GMH) in the immature preterm brain. However, the underlying mechanisms remain poorly defined. Here, we investigated the effects of a cannabinoid receptor 2 (CB2R) agonist on microglia proliferation and the possible involvement of the mitogen-activated protein kinase (MAPK) family pathway in a collagenase-induced GMH rat model and in thrombin-induced rat microglia cells. We demonstrated that activation of CB2R played a key role in attenuating brain edema, neuronal degeneration, microglial accumulation and the phosphorylated extracellular signal-regulated kinase (p-ERK) protein level 24 h following GMH. In vitro, Western blot analysis and immunostaining indicated that ERK and P38 phosphorylation levels in microglia stimulated by thrombin were decreased after JWH-133 (CB2R selective agonist) treatment in a concentration-dependent manner. Microglia proliferation (EDU + microglia) and inflammatory and oxidative stress responses were attenuated by UO126 (ERK pathway inhibitor) 24 h after thrombin stimulation, an activity that was prevented by AM630 (CB2R selective antagonist). Overall, these findings suggest that activation of the endocannabinoid system might attenuate inflammation-induced secondary brain injury after GMH in rats by reducing microglia accumulation through a mechanism involving ERK dephosphorylation. Enhancing CB2R activation is a potential treatment to slow down the course of GMH in preterm newborns.

Topics: Animals; Brain; Brain Edema; Butadienes; Cannabinoids; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Indoles; Intracranial Hemorrhages; Male; Microglia; Nerve Degeneration; Neuroimmunomodulation; Nitriles; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Random Allocation; Receptor, Cannabinoid, CB2; Thrombin

Chronic stress is a potent risk factor for depression, but the mechanism by which stress causes depression is not fully understood. To investigate the molecular mechanism underlying stress-induced depression, C57BL/6 inbred mice were treated with repeated restraint to induce lasting depressive behavioral changes. Behavioral states of individual animals were evaluated using the forced swim test, which measures psychomotor withdrawals, and the U-field test, which measures sociability. From these behavioral analyses, individual mice that showed depression-like behaviors in both psychomotor withdrawal and sociability tests, and individuals that showed a resiliency to stress-induced depression in both tests were selected. Among the neuropeptides expressed in the amygdala, thyrotropin-releasing hormone (TRH) was identified as being persistently up-regulated in the basolateral amygdala (BLA) in individuals exhibiting severe depressive behaviors in the two behavior tests, but not in individuals displaying a stress resiliency. Activation of TRH receptors by local injection of TRH in the BLA in normal mice produced depressive behaviors, mimicking chronic stress effects, whereas siRNA-mediated suppression of either TRH or TRHR1 in the BLA completely blocked stress-induced depressive symptoms. The TRHR1 agonist, taltirelin, injection in the BLA increased the level of p-ERK, which mimicked the increased p-ERK level in the BLA that was induced by treatment with repeated stress. Stereotaxic injection of U0126, a potent inhibitor of the ERK pathway, within the BLA blocked stress-induced behavioral depression. These results suggest that repeated stress produces lasting depression-like behaviors via the up-regulation of TRH and TRH receptors in the BLA.

Topics: Animals; Basolateral Nuclear Complex; Butadienes; Chronic Disease; Depressive Disorder; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Male; Mice, Inbred C57BL; Nitriles; Psychotropic Drugs; Receptors, Thyrotropin-Releasing Hormone; Restraint, Physical; RNA, Small Interfering; Stress, Psychological; Thyrotropin-Releasing Hormone

Quetiapine (QUE) and repetitive transcranial magnetic stimulation (rTMS) have been considered to be possible monotherapies for depression or adjunctive therapies for the treatment of the resistant depression, but the underlying mechanisms remain unclear. The present study aimed to assess the effects of combined QUE and rTMS treatment on depressive-like behaviors, hippocampal proliferation, and the in vivo and in vitro expressions of phosphorylated extracellular signal-regulated protein kinase (pERK1/2) and brain-derived neurotrophic factor (BDNF) in male Sprague-Dawley rats. The administration of QUE and rTMS was determined not only to reverse the depressive-like behaviors of rats exposed to chronic unpredictable stress (CUS) but also to restore the protein expressions of pERK1/2 and BDNF and cell proliferation in the hippocampus. Additionally, QUE and rTMS promoted the proliferation and increased the expression of pERK1/2 and BDNF in hippocampal-derived neural stem cells (NSCs), and these effects were abolished by U0126. Taken together, these results suggest that the antidepressive-like effects of QUE and rTMS might be related to the activation of the BDNF/ERK signaling pathway and the up-regulation of cell proliferation in the hippocampus.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Brain-Derived Neurotrophic Factor; Butadienes; Cell Proliferation; Cell Survival; Cells, Cultured; Depression; Disease Models, Animal; Hippocampus; Male; MAP Kinase Signaling System; Neural Stem Cells; Nitriles; Quetiapine Fumarate; Rats; Transcranial Magnetic Stimulation

Diabetic neuropathy comprises dying back of nerve endings that reflects impairment in axonal plasticity and regenerative nerve growth. Metabolic changes in diabetes can lead to a dysregulation of hormonal mediators, such as cytokines, that may constrain distal nerve fiber growth. Interleukin-17 (IL-17A), a proinflammatory and neurotropic cytokine produced by T-cells, was significantly reduced in sciatic nerve of streptozotocin (STZ)-diabetic rats. Thus we studied the effect of IL-17A on the phenotype of sensory neurons derived from age matched control or type 1 diabetic rats. The aims were to determine the ability of IL-17A to enhance neurite outgrowth in cultured sensory neurons, investigate the signaling pathways activated by IL-17A, study the role of mitochondria and mechanistically link to neurite outgrowth.. IL-17A (10 ng/ml; p<0.05) significantly and dose-dependently increased total neurite outgrowth in cultures of adult dorsal root ganglia (DRG) sensory neurons derived from both control and streptozotocin (STZ)-diabetic rats. This enhancement was mediated by IL-17A-dependent activation of extracellular-regulated protein kinase (ERK) and phosphoinositide-3 kinase (PI-3K) signal transduction pathways. Pharmacological blockade of one of these activated pathways triggered complete inhibition of neurite outgrowth. IL-17A augmented mitochondrial bioenergetic function of sensory neurons derived from control or diabetic rats and this was also mediated via ERK or PI-3K. IL-17A-dependent elevation of bioenergetic function was associated with augmented expression of proteins of the mitochondrial electron transport system complexes.. IL-17A enhanced axonal plasticity through activation of ERK and PI-3K pathways and was associated with augmented mitochondrial bioenergetic function in sensory neurons.

Topics: Animals; Antibiotics, Antineoplastic; Butadienes; Cells, Cultured; Chromones; Diabetes Mellitus, Experimental; Disease Models, Animal; Electron Transport Complex IV; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Ganglia, Spinal; Interleukin-17; Male; MAP Kinase Signaling System; Mitochondria; Morpholines; Multienzyme Complexes; Neurites; Nitriles; Rats; Rats, Sprague-Dawley; Sensory Receptor Cells; Streptozocin

2-CL-IB-MECA, (A3 adenosine receptor agonist)(A3AR) mediated cardioprotection is well documented although the associated intracellular signalling pathways remain unclear. Here we demonstrate a role of the pro-survival signalling pathways MEK1/2-ERK1/2 and PI3K/AKT and their effect on modifying Caspase-3 activity in A3AR mediated cardioprotection.. Isolated perfused rat hearts or primary adult rat cardiac myocytes were subjected to ischaemia/hypoxia and reperfusion/reoxygenation, respectively. 2-CL-IB-MECA (1 nM) was administered at the onset of reperfusion/reoxygenation in the presence and absence of either the PI3K inhibitor Wortmannin (5 nM) or MEK1/2 inhibitor UO126 (10 μM). Heart tissues were harvested for assessment of p-ERK1/2(Thr202/Tyr204) or p-AKT (Ser-473) status or underwent infarct size assessment. Cardiac myocytes underwent flow-cytometric analysis for apoptosis, necrosis, cleaved-caspase 3/p-BAD (Ser-112 and Ser-136) activity post-reoxygenation.. 2-CL-IB-MECA significantly reduced infarct size compared to non-treated controls, where co-administration with either of the kinase inhibitors abolished the infarct sparing effects. Administration of 2-CL-IB-MECA at reperfusion significantly upregulated the status of p-ERK1/2 and p-AKT compared to time matched controls in a UO126 and Wortmannin sensitive manner respectively. 2-CL-IB-MECA when administered throughout reoxygenation significantly reduced apoptosis, necrosis, cleaved-caspase 3 activity and increased p-BAD (Ser-112) and p-BAD (Ser-136) activity in myocytes subjected to hypoxia/reoxygenation injury. The cytoprotective effect was abolished by co-administration with the kinase inhibitors Wortmannin and/or UO126.. We have described the molecular mechanisms associated with A3AR mediated cardioprotection indicating a role for the pro-survival signalling pathways that decrease caspase-3 activity. These observations provide novel insight into the pharmacological effects of A3ARs in ameliorating myocardial ischaemia/reperfusion injury.

Topics: Adenosine; Adenosine A3 Receptor Agonists; Androstadienes; Animals; Apoptosis; Butadienes; Cardiotonic Agents; Caspase 3; Disease Models, Animal; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitriles; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A3; Signal Transduction; Wortmannin

Rosmarinic acid (RA) is an important component of Chinese herbal medicine treatments and has been demonstrated to exert therapeutic effects in mood disorders. The present study was designed to assess the effects of RA on post-traumatic stress disorder (PTSD)-like symptoms, hippocampal cell proliferation and phosphorylation extracellular regulated protein kinases (pERK1/2) expression. We found that administration of RA (10mg/kg) alleviated PTSD-like symptoms in rats exposed to an enhanced single prolonged stress (ESPS) paradigm and restored hippocampal proliferation and pERK1/2 expression. Interestingly, the effects of RA were inhibited by the blockage of the ERK signaling. These data support the use of RA for treating PTSD and indicate that the ERK1/2 signaling cascade may play a critical role in the therapeutic efficacy of RA in treating such conditions.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bromodeoxyuridine; Butadienes; Cell Proliferation; Cell Survival; Cells, Cultured; Cinnamates; Depsides; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Freezing Reaction, Cataleptic; Hippocampus; Male; MAP Kinase Signaling System; Maze Learning; Motor Activity; Neural Stem Cells; Nitriles; Rats; Rats, Sprague-Dawley; Rosmarinic Acid; Stress Disorders, Post-Traumatic

Hyperbaric oxygen (HBO) is emerging as a therapy for brain ischemia, although its benefits are still debated. The present study aimed to investigate the effect of HBO on brain damage in a rat model of transient focal cerebral ischemia and its underlying mechanism of action. Male Wistar rats, which had suffered 1.5h of transient middle cerebral artery occlusion (tMCAO) and had a Longa's neuron score of 3, were given pure oxygen at 3.0 atm absolute, for 60 min after the third hour of reperfusion. After 24h of reperfusion, rat brains were removed and studied. 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin and eosin staining revealed that the infarct ratio in the HBO group increased remarkably when compared with the MCAO group. Up-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation was detected in the HBO group because of reactive oxygen species (ROS) generation. Autophagy appeared to be obstructed in the HBO group. Administration of the ERK1/2 inhibitor U0126 decreased the infarct ratio and improved protein clearance by autophagy in the HBO group. Collectively, these results suggest that HBO enlarges the area of brain damage via reactive oxygen species-induced activation of ERK1/2, which interrupts autophagy flux.

Topics: Animals; Autophagy; Brain; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Hyperbaric Oxygenation; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Rats; Rats, Wistar; Reactive Oxygen Species; Up-Regulation

Despite accumulating evidence of the clinical effectiveness of acupuncture, its mechanism remains largely unclear. We assume that molecular signaling around the acupuncture needled area is essential for initiating the effect of acupuncture. To determine possible bio-candidates involved in the mechanisms of acupuncture and investigate the role of such bio-candidates in the analgesic effects of acupuncture, we conducted 2 stepwise experiments. First, a genome-wide microarray of the isolated skin layer at the GB34-equivalent acupoint of C57BL/6 mice 1 hour after acupuncture found that a total of 236 genes had changed and that extracellular signal-regulated kinase (ERK) activation was the most prominent bio-candidate. Second, in mouse pain models using formalin and complete Freund adjuvant, we found that acupuncture attenuated the nociceptive behavior and the mechanical allodynia; these effects were blocked when ERK cascade was interrupted by the mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) inhibitor U0126 (.8 μg/μL). Based on these results, we suggest that ERK phosphorylation following acupuncture needling is a biochemical hallmark initiating the effect of acupuncture including analgesia.. This article presents the novel evidence of the local molecular signaling in acupuncture analgesia by demonstrating that ERK activation in the skin layer contributes to the analgesic effect of acupuncture in a mouse pain model. This work improves our understanding of the scientific basis underlying acupuncture analgesia.

Topics: Acupuncture Analgesia; Animals; Blotting, Western; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Formaldehyde; Freund's Adjuvant; Gene Expression; Male; MAP Kinase Signaling System; Mice, Inbred C57BL; Nitriles; Oligonucleotide Array Sequence Analysis; Pain; Pain Management; Pain Threshold; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Skin Physiological Phenomena; Time Factors

The aim of this study was to investigate the effect of electroacupuncture (EA) on cell proliferation and its molecular mechanisms. Sixty rats were randomly divided into 5 groups: sham operation control (SC), ischemia control (IC), EA, EA and DMSO injection (ED), EA and U0126 injection (EU). All the groups, with the exception of SC, underwent middle cerebral artery occlusion (MCAO), and DMSO or U0126 was injected into the rat in the ED or EU group 30 min prior to MCAO. Cell proliferation was evaluated by proliferating cell nuclear antigen (PCNA) immunostaining. The changes of cell cycle proteins (cyclin D1, CDK4, cyclin E, CDK2, p21 and p27) and the ERK1/2 pathway activation were examined by RT-PCR and western blot analysis. The results showed that the positive cell numbers of PCNA immunostaining in the EA and ED groups were more than those in the IC group (P<0.05). The mRNA and protein levels of p21 or p27 were obviously increased, however, the mRNA and protein levels of cyclin D1, CDK4, cyclin E and CDK2 were reduced in the IC and EU groups. The findings suggested that EA activates the ERK1/2 signaling pathway to protect brain injury during cerebral ischemia. However, this positive effect of EA can be blocked by U0126.

Topics: Animals; Butadienes; Cell Proliferation; Disease Models, Animal; Electroacupuncture; Infarction, Middle Cerebral Artery; Male; MAP Kinase Signaling System; Neurons; Nitriles; Rats; Rats, Sprague-Dawley

Itch, chronic itch in particular, can have a significant negative impact on an individual's quality of life. However, the molecular mechanisms underlying itch processing in the central nervous system remain largely unknown.. We report here that activation of ERK signaling in the spinal cord is required for itch sensation. ERK activation, as revealed by anti-phosphorylated ERK1/2 immunostaining, is observed in the spinal dorsal horn of mice treated with intradermal injections of histamine and compound 48/80 but not chloroquine or SLIGRL-NH2, indicating that ERK activation only occurs in histamine-dependent acute itch. In addition, ERK activation is also observed in 2, 4-dinitrofluorobenzene (DNFB)-induced itch. Consistently, intrathecal administration of the ERK phosphorylation inhibitor U0126 dramatically reduces the scratching behaviors induced by histamine and DNFB, but not by chloroquine. Furthermore, administration of the histamine receptor H1 antagonist chlorpheniramine decreases the scratching behaviors and ERK activation induced by histamine, but has no effect on DNFB-induced itch responses. Finally, the patch-clamp recording shows that in histamine-, chloroquine- and DNFB-treated mice the spontaneous excitatory postsynaptic current (sEPSC) of dorsal horn neurons is increased, and the decrease of action potential threshold is largely prevented by bathing of U0126 in histamine- and DNFB-treated mice but not those treated with chloroquine.. Our results demonstrate a critical role for ERK activation in itch sensation at the spinal level.

Topics: Animals; Butadienes; Chloroquine; Dinitrofluorobenzene; Disease Models, Animal; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Histamine; Male; MAP Kinase Signaling System; Mice; Mice, Inbred ICR; Neuroglia; Nitriles; Oligopeptides; Phosphorylation; Posterior Horn Cells; Proto-Oncogene Proteins c-fos; Pruritus; Receptors, Histamine H1; Sensation; Spinal Cord; Time Factors

Stressful stimuli can exacerbate persistent pain disorder. However, the underlying mechanism is still unknown. Here, to reveal the underlying mechanism for stressful stimuli-induced hyperalgesia in chronic pain, we investigated the effect of extracellular signal-regulated kinase1/2 (ERK1/2) activation on pain hypersensitivity using single-prolonged stress (SPS) model, complete Freund's adjuvant (CFA) model and SPS + CFA model. The experimental results revealed significantly reduced paw withdrawal threshold in the SPS, CFA, and SPS + CFA group compared with the control group. However, the increased phosphorylation of ERK1/2 in the medial prefrontal cortex (mPFC) was observed in the SPS- or SPS + CFA-exposed group but not the CFA group compared with control group. There was also a significant increase in mPFC ERK1/2 phosphorylation and mechanical allodynia after SPS + CFA treatment compared to SPS or CFA treatment alone. Furthermore, inhibiting ERK1/2 phosphorylation by microinjection of U0126, a MAPK kinase (MEK) inhibitor, into the mPFC attenuated SPS + CFA- and SPS- but not CFA-induced mechanical allodynia, anxiety-like behavior, and cognitive impairments. These results suggest that the activation of ERK1/2 in the mPFC may contribute to the process of stress-induced cognitive and emotional disorders, leading to an increase in pain sensitivity.

Topics: Animals; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hyperalgesia; MAP Kinase Signaling System; Nitriles; Phosphorylation; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Stress Disorders, Post-Traumatic; Stress, Psychological

Castration-resistant prostate cancer is an incurable heterogeneous disease that is characterized by a complex multistep process involving different cellular and biochemical changes brought on by genetic and epigenetic alterations. These changes lead to the activation or overexpression of key survival pathways that also serve as potential therapeutic targets. Despite promising preclinical results, molecular targeted therapies aimed at such signaling pathways have so far been dismal. In the present study, we used a PTEN-deficient mouse model of prostate cancer to show that plasticity in castration-resistant tumors promotes therapeutic escape. Unlike castration-naïve tumors which depend on androgen receptor and PI3K/AKT signal activation for growth and survival, castration-resistant tumors undergo phenotypic plasticity leading to increased intratumoral heterogeneity. These tumors attain highly heterogeneous phenotypes that are characterized by cancer cells relying on alternate signal transduction pathways for growth and survival, such as mitogen-activated protein kinase and janus kinase/signal transducer and activator of transcription, and losing their dependence on PI3K signaling. These features thus enabled castration-resistant tumors to become insensitive to the therapeutic effects of PI3K/AKT targeted therapy. Overall, our findings provide evidence that androgen deprivation drives phenotypic plasticity in prostate cancer cells and implicate it as a crucial contributor to therapeutic resistance in castration-resistant prostate cancer. Therefore, incorporating intratumoral heterogeneity in a dynamic tumor model as a part of preclinical efficacy determination could improve prediction for response and provide better rationale for the development of more effective therapies.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Butadienes; Carcinogenesis; Cell Proliferation; Combined Modality Therapy; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Everolimus; Humans; Male; Mice; Mice, Transgenic; Molecular Targeted Therapy; Nitriles; Orchiectomy; Phenotype; Prostate; Prostatic Neoplasms, Castration-Resistant; PTEN Phosphohydrolase; Receptors, Androgen; Sirolimus

Neuropathic pain is clinically challenging because it is resistant to alleviation by morphine. The nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways may be involved in the development of neuropathic pain. The aim of our study was to examine the influence of a chronic, intrathecal administration of parthenolide (PTL, inhibitor of NF-κB) and U0126 (inhibitor of MEK1/2) on nociception and morphine effectiveness in a rat model of neuropathy.. The chronic constriction injury of the sciatic nerve in Wistar rats was performed. PTL and U0126 were injected chronic intrathecally and morphine was injected once at day 7. To evaluate allodynia and hyperalgesia, the von Frey and cold plate tests were used, respectively. The experiments were carried out according to IASP rules. Using qRT-PCR we analyzed mRNAs of μ-(mor), δ-(dor) and κ-(kor)-opioid receptors in the lumbar spinal cord after drugs administration.. The administration of PTL and U0126 decreased allodynia and hyperalgesia and significantly potentiated morphine effect. The mor, dor and kor mRNAs were down-regulated 7 days after injury in the ipsilateral spinal cord. The PTL and U0126 significantly up-regulated the mRNA levels of all opioid receptors. The levels of mor and dor mRNAs were much higher compared to those in naïve, but only the kor levels returned to control values.. These results indicate that the inhibition of the NF-κB pathway has better analgesic effects. Both inhibitors similarly potentiate morphine analgesia, which parallels the up-regulation of both mor and dor mRNAs expression spinal levels of the model of neuropathy.

Topics: Analgesics; Analgesics, Opioid; Animals; Butadienes; Disease Models, Animal; Drug Synergism; Hyperalgesia; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Morphine; Neuralgia; NF-kappa B; Nitriles; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Sciatic Nerve; Sesquiterpenes; Signal Transduction

To examine the possible involvement and regulatory mechanisms of extracellular signal-regulated kinase (ERK) pathway in the temporomandibular joint (TMJ) of rats subjected to chronic sleep deprivation (CSD).. Rats were subjected to CSD using the modified multiple platform method (MMPM). The serum levels of corticosterone (CORT) and adrenocorticotropic hormone (ACTH) were tested and histomorphology and ultrastructure of the TMJ were observed. The ERK and phospho-ERK (p-ERK) expression levels were detected by Western blot analysis, and the MMP-1, MMP-3, and MMP-13 expression levels were detected by real-time quantitative polymerase chain reaction (PCR) and Western blotting.. The elevated serum CORT and ACTH levels confirmed that the rats were under CSD stress. Hematoxylin and eosin (HE) staining and scanning electron microscopy (SEM) showed pathological alterations in the TMJ following CSD; furthermore, the p-ERK was activated and the mRNA and protein expression levels of MMP-1, MMP-3, and MMP-13 were upregulated after CSD. In the rats administered with the selective ERK inhibitor U0126, decreased tissue destruction was observed. Phospho-ERK activation was visibly blocked and the MMP-1, MMP-3, and MMP-13 mRNA and protein levels were lower than the corresponding levels in the CSD without U0126 group.. These findings indicate that CSD activates the ERK pathway and upregulates the MMP-1, MMP-3, and MMP-13 mRNA and protein levels in the TMJ of rats. Thus, CSD induces ERK pathway activation and causes pathological alterations in the TMJ. ERK may be associated with TMJ destruction by promoting the expression of MMPs.

Topics: Adrenocorticotropic Hormone; Animals; Butadienes; Cartilage, Articular; Corticosterone; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; Nitriles; Rats; Sleep Deprivation; Temporomandibular Joint

Detection of external irritants by head nociceptor neurons has deep evolutionary roots. Irritant-induced aversive behavior is a popular pain model in laboratory animals. It is used widely in the formalin model, where formaldehyde is injected into the rodent paw, eliciting quantifiable nocifensive behavior that has a direct, tissue-injury-evoked phase, and a subsequent tonic phase caused by neural maladaptation. The formalin model has elucidated many antipain compounds and pain-modulating signaling pathways. We have adopted this model to trigeminally innervated territories in mice. In addition, we examined the involvement of TRPV4 channels in formalin-evoked trigeminal pain behavior because TRPV4 is abundantly expressed in trigeminal ganglion (TG) sensory neurons, and because we have recently defined TRPV4's role in response to airborne irritants and in a model for temporomandibular joint pain. We found TRPV4 to be important for trigeminal nocifensive behavior evoked by formalin whisker pad injections. This conclusion is supported by studies with Trpv4(-/-) mice and TRPV4-specific antagonists. Our results imply TRPV4 in MEK-ERK activation in TG sensory neurons. Furthermore, cellular studies in primary TG neurons and in heterologous TRPV4-expressing cells suggest that TRPV4 can be activated directly by formalin to gate Ca(2+). Using TRPA1-blocker and Trpa1(-/-) mice, we found that both TRP channels co-contribute to the formalin trigeminal pain response. These results imply TRPV4 as an important signaling molecule in irritation-evoked trigeminal pain. TRPV4-antagonistic therapies can therefore be envisioned as novel analgesics, possibly for specific targeting of trigeminal pain disorders, such as migraine, headaches, temporomandibular joint, facial, and dental pain, and irritation of trigeminally innervated surface epithelia.

Topics: Animals; Butadienes; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fixatives; Formaldehyde; Keratinocytes; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morpholines; Neurons; Nitriles; Pain; Pyrroles; Trigeminal Ganglion; TRPV Cation Channels; Ubiquitin Thiolesterase; Vibrissae

Cooling following cardiac arrest can improve survival significantly. However, delays in achieving target temperature may decrease the overall benefits of cooling. Here, we test whether lipid emulsion, a clinically approved drug reported to exert cardioprotection, can rescue heart contractility in the setting of delayed cooling in stunned mouse cardiomyocytes.. Cell culture study.. Academic research laboratory.. Cardiomyocytes isolated from 1- to 2-day-old C57BL6 mice.. Cardiomyocytes were exposed to 30 minutes of ischemia followed by 90 minutes of reperfusion and 10 minutes of isoproterenol with nine interventions: 1) no additional treatment; 2) intraischemic cooling at 32 °C initiated 10 minutes prior to reperfusion; 3) delayed cooling started 20 minutes after reperfusion; 4) lipid emulsion + delayed cooling; 5) lipid emulsion (0.25%) administered at reperfusion; 6) lipid emulsion + intraischemic cooling; 7) delayed lipid emulsion; 8) lipid emulsion + delayed cooling + Akt inhibitor (API-2, 10 µM); and 9) lipid emulsion + delayed cooling + Erk inhibitor (U0126, 10 µM). Inhibitors were given to cells 1 hour prior to ischemia.. Contractility was recorded by real-time phase-contrast imaging and analyzed with pulse image velocimetry in MATLAB (Mathworks, Natick, MA). Ischemia diminished cell contraction. The cardioprotective effect of cooling was diminished when delayed but was rescued by lipid emulsion. Further, lipid emulsion on its own improved recovery of the contractility to a greater extent as intraischemic cooling. However, cotreatment of lipid emulsion and intraischemic cooling did not further improve the recovery compared to either treatment alone. Furthermore, Akt and Erk inhibitors blocked lipid emulsion-induced protection.. Lipid emulsion improved contractility and rescued contractility in the context of delayed cooling. This protective effect required Akt and Erk signaling. Lipid emulsion might serve as a treatment or adjunct to cooling in ameliorating myocardial ischemia/reperfusion injury.

Topics: Animals; Butadienes; Cardiotonic Agents; Chlorpropamide; Disease Models, Animal; Hypothermia, Induced; Ischemia; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Muscle Contraction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitriles; Proto-Oncogene Proteins c-akt; Time Factors

Corticosteroids are regularly used to treat autoimmune diseases, such as bullous pemphigoid (BP). In BP, autoantibodies bind to type XVII collagen (COL17), located at the dermal-epidermal junction. A crucial role of neutrophils in experimental BP has been established. Specifically, reactive oxygen species and proteolytic granule enzymes mediate tissue injury. Therefore, we investigated the effects of methylprednisolone (MP) on neutrophils, which are likely to be affected by topical treatment. First, MP inhibited dermal-epidermal separation ex vivo in cryosections of the human skin induced by co-incubation of BP autoantibodies with neutrophils from healthy volunteers. Next, MP inhibited neutrophil activation in vitro induced by immune complexes (ICs) of COL17 and autoantibodies. This neutrophil activation was associated with phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and Akt. In turn, inhibition of ERK1/2, p38 MAPK, or Akt phosphorylation inhibited neutrophil activation by IC in vitro and dermal-epidermal separation ex vivo. In addition, we observed an increase of p38 MAPK phosphorylation in dermal infiltrates of BP patients. Treatment of mice with either MP or inhibitors of p38-MAPK or ERK1/2 phosphorylation impaired induction of autoantibody- or irritant-induced neutrophil-dependent inflammation. We here identify the inhibition of Akt, ERK1/2, and p38 MAPK phosphorylation as molecular mechanisms to promote MP's therapeutic effects.

Topics: Adult; Animals; Antigen-Antibody Complex; Autoantibodies; Butadienes; Dermis; Disease Models, Animal; Enzyme Inhibitors; Epidermal Cells; Epidermis; Epidermolysis Bullosa Acquisita; Glucocorticoids; Humans; Imidazoles; MAP Kinase Signaling System; Methylprednisolone; Mice; Mice, Inbred C57BL; Neutrophils; Nitriles; Pemphigoid, Bullous; Pyridines; Reactive Oxygen Species; Respiratory Burst

Extracellular signal‑regulated kinase (ERK) 1/2 in the spinal cord has been implicated in the development of neuropathic pain and inflammatory pain. However, a limited number of studies have investigated the role of spinal ERK in incisional pain. The present study aimed to determine the role of ERK in the spinal cord in incisional pain. Incisional pain was established in rats by a unilateral hind paw incision. ERK1/2 expression was analyzed by immunohistochemistry. Hypersensitivity to pain was evaluated by measuring the paw withdrawal threshold using the von Frey test. The mitogen‑activated protein kinase kinase (MEK) inhibitor, U0126, was administered 20 min prior to or 10 min following the incision by intrathecal or intraperitoneal injection. Phosphorylated ERK1/2 in the ipsilateral L4‑5 spinal superficial dorsal horn was activated 1 min following the incision, reached its peak level at 5 min and then returned to the basal level 20 min following the incision. Pretreatment, but not post‑treatment with U0126 markedly attenuated the pain hypersensitivity induced by the incision. Therefore, the present study indicates that the transient activation of spinal ERK1/2 contributes to the initiation of pain hypersensitivity following surgical incision.

Topics: Animals; Butadienes; Disease Models, Animal; Enzyme Activation; Hyperalgesia; Hypersensitivity; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Nitriles; Pain, Postoperative; Phosphorylation; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Skin; Spinal Cord

Spinal muscular atrophy (SMA), a recessive neurodegenerative disease, is characterized by the selective loss of spinal motor neurons. No available therapy exists for SMA, which represents one of the leading genetic causes of death in childhood. SMA is caused by a mutation of the survival-of-motor-neuron 1 (SMN1) gene, leading to a quantitative defect in the survival-motor-neuron (SMN) protein expression. All patients retain one or more copies of the SMN2 gene, which modulates the disease severity by producing a small amount of stable SMN protein. We reported recently that NMDA receptor activation, directly in the spinal cord, significantly enhanced the transcription rate of the SMN2 genes in a mouse model of very severe SMA (referred as type 1) by a mechanism that involved AKT/CREB pathway activation. Here, we provide the first compelling evidence for a competition between the MEK/ERK/Elk-1 and the phosphatidylinositol 3-kinase/AKT/CREB signaling pathways for SMN2 gene regulation in the spinal cord of type 1 SMA-like mice. The inhibition of the MEK/ERK/Elk-1 pathway promotes the AKT/CREB pathway activation, leading to (1) an enhanced SMN expression in the spinal cord of SMA-like mice and in human SMA myotubes and (2) a 2.8-fold lifespan extension in SMA-like mice. Furthermore, we identified a crosstalk between ERK and AKT signaling pathways that involves the calcium-dependent modulation of CaMKII activity. Together, all these data open new perspectives to the therapeutic strategy for SMA patients.

Topics: Animals; Animals, Newborn; Butadienes; Calcium; Cell Survival; Cells, Cultured; Chromatin Immunoprecipitation; Coculture Techniques; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Exploratory Behavior; Extracellular Signal-Regulated MAP Kinases; Female; Ganglia, Spinal; Humans; Male; Mice; Mice, Knockout; Motor Neurons; Muscle Cells; Muscular Atrophy, Spinal; N-Methylaspartate; Nitriles; Signal Transduction; Stem Cells; Survival of Motor Neuron 2 Protein

Upregulation of vasoconstrictor receptors in cerebral arteries, including endothelin B (ETB) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors, has been suggested to contribute to delayed cerebral ischemia, a feared complication after subarachnoid hemorrhage (SAH). This receptor upregulation has been shown to be mediated by intracellular signalling via the mitogen activated protein kinase kinase (MEK1/2)--extracellular regulated kinase 1/2 (ERK1/2) pathway. However, it is not known what event(s) that trigger MEK-ERK1/2 activation and vasoconstrictor receptor upregulation after SAH.We hypothesise that the drop in cerebral blood flow (CBF) and wall tension experienced by cerebral arteries in acute SAH is a key triggering event. We here investigate the importance of the duration of this acute CBF drop in a rat SAH model in which a fixed amount of blood is injected into the prechiasmatic cistern either at a high rate resulting in a short acute CBF drop or at a slower rate resulting in a prolonged acute CBF drop.. We demonstrate that the duration of the acute CBF drop is determining for a) degree of early ERK1/2 activation in cerebral arteries, b) delayed upregulation of vasoconstrictor receptors in cerebral arteries and c) delayed CBF reduction, neurological deficits and mortality. Moreover, treatment with an inhibitor of MEK-ERK1/2 signalling during an early time window from 6 to 24 h after SAH was sufficient to completely prevent delayed vasoconstrictor receptor upregulation and improve neurological outcome several days after the SAH.. Our findings suggest a series of events where 1) the acute CBF drop triggers early MEK-ERK1/2 activation, which 2) triggers the transcriptional upregulation of vasoconstrictor receptors in cerebral arteries during the following days, where 3) the resulting enhanced cerebrovascular contractility contribute to delayed cerebral ischemia.

Topics: Analysis of Variance; Animals; Antipyrine; Area Under Curve; Blood Pressure; Brain Ischemia; Butadienes; Carbon Isotopes; Cerebral Arteries; Cerebrovascular Circulation; Disease Models, Animal; Enzyme Inhibitors; Laser-Doppler Flowmetry; Male; MAP Kinase Signaling System; Motor Activity; Nervous System Diseases; Nitriles; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Receptor, Serotonin, 5-HT1B; Signal Transduction; Subarachnoid Hemorrhage; Up-Regulation

In this study, we aimed to compare the effects of low- and high-quality cardiopulmonary resuscitation (CPR) on cardioprotection by induced hypothermia (IH) at 34 °C and examine whether extracellular signal-regulated kinase or endothelial nitric oxide synthase mediates this cardioprotection. Left ventricle infarct sizes were evaluated in six groups of rat hearts (n = 6) following Langendorff perfusion and triphenyltetrazolium chloride staining. Controls underwent 30 min of global ischemia at 37 °C, followed by 10 min of simulated low- or high-quality CPR reperfusion and 90 min of reperfusion at 75 mmHg. The IH groups underwent IH at 34 °C during reperfusion. The U0126 group received U0126 (60 μM)-an extracellular signal-regulated kinase inhibitor-during reperfusion at 34 °C. The L-NIO (N-(1-iminoethyl)-L-ornithine dihydrochloride) group received L-NIO (2 μM)-an endothelial nitric oxide synthase inhibitor-5 min before global ischemia at 37 °C to the end of reperfusion at 34 °C. Infarct size did not significantly differ between the control and IH groups receiving low-quality CPR. However, IH with high-quality CPR reduced the infarct size from 47.2% ± 10.2% to 26.0% ± 9.4% (P = 0.005). U0126 reversed the IH-induced cardioprotection (45.9% ± 9.4%, P = 0.010), whereas L-NIO had no significant effect. Cardiopulmonary resuscitation quality affects IH-induced cardioprotection. Extracellular signal-regulated kinase may mediate IH-induced cardioprotection.

Topics: Animals; Butadienes; Cardiopulmonary Resuscitation; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hemodynamics; Hypothermia, Induced; In Vitro Techniques; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Nitric Oxide Synthase Type III; Nitriles; Ornithine; Rats; Rats, Sprague-Dawley

Tungsten carbide-cobalt (WC-Co) nanoparticle composites have wide applications because of their hardness and toughness. WC-Co was classified as "probably carcinogenic" to humans by the International Agency for Research on Cancer (IARC) in 2003. It is believed that the toxicity and carcinogenesis of WC-Co is associated with particle size. Recent studies demonstrated that the tumor suppressor gene programmed cell death 4 (PDCD4) and its upstream regulator miR-21 have been considered as oncogenes for novel cancer prevention or anticancer therapies. The present study examined the effects of WC-Co nanoparticles on miR-21-PDCD4 signaling in a mouse epidermal cell line (JB6 P+). The results showed that (i) exposure of JB6 cells to WC-Co stimulated a increase of miR-21 generation; (ii) WC-Co also caused inhibition of PDCD4, a tumor suppressor protein and downstream target of miR-21, expression in JB6 cells; (iii) inhibition of ERKs with ERK inhibitor U0126 significantly reversed WC-Cominus;induced PDCD4 inhibition, but inhibition of p38 with p38 inhibitor SB203580 did not; and (iv) ROS scavengers, N-acetyl-L-cysteine and catalase, blocked the inhibitory effect of WC-Co on PDCD4 expression, while superoxide dismutase promoted the inhibitory effect. These findings demonstrate that WC-Co nanoparticles induce miR-21 generation, but inhibit PDCD4 production, which may be mediated through ROS, especially endogenous H2O2, and ERK pathways. Unraveling the complex mechanisms associated with these events may provide insights into the initiation and progression of WC-Co-induced carcinogenesis.

Topics: Animals; Apoptosis Regulatory Proteins; Butadienes; Cell Line; Cell Transformation, Neoplastic; Cobalt; Disease Models, Animal; Enzyme Inhibitors; Epidermis; Hydrogen Peroxide; Imidazoles; In Vitro Techniques; MAP Kinase Signaling System; Mice; MicroRNAs; Mitogen-Activated Protein Kinase Kinases; Nanoparticles; Nitriles; Occupational Exposure; Pyridines; Reactive Oxygen Species; RNA-Binding Proteins; Signal Transduction; Tungsten Compounds

In the present study, we examined the effects of intracerebroventricular administration of extracellular signal-regulated protein kinase- (ERK) and p38-specific inhibitors, U0126 and PD169316, respectively, on apoptosis induced by amyloid beta (Aβ) in rats. To investigate the effects of these compounds, we evaluated intracellular signalling pathways of apoptosis, as well as inflammatory and antioxidant pathways, 7 and 20 days after Aβ injection. We found that caspase-3 and Bax/Bcl-2 ratio, two hallmarks of apoptosis, were significantly decreased in the rats pre-treated with U0126 and PD169316, 7 days after Aβ injection. This observation was in agreement with the results of immunostaining analysis of the hippocampus that showed decreased levels of terminal transferase dUTP nick end labelling positive cells in the hippocampus of U0126 and PD169316 pre-treated rats, compared with the Aβ-injected group. We also chased the changes in the levels of calpain-2 and caspase-12, two ER factors, in the Aβ-injected and treatment groups. Decreased levels of calpain-2 and caspase-12 in U0126 and PD169316 pre-treated rats confirmed the protective effects of these inhibitors. Furthermore, we studied the effect of two stress-sensing transcription factors, nuclear-related factor-2 (Nrf2) and nuclear factor-кB (NF-кB), in Aβ-injected as wells as U0126 and PD169316 pre-treated rats. U0126 and PD169316 activated Nrf2 and suppressed NF-кB pathways, 7 days after Aβ injection. These antioxidant and inflammatory pathways restored to the vehicle level within 20 days. Taken together, our findings reinforce and extend the notion of the potential neuroprotective role of ERK and/or p38 inhibitors against the neuronal toxicity induced by Aβ.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Apoptosis; Blotting, Western; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hippocampus; Imidazoles; In Situ Nick-End Labeling; Injections, Intraventricular; Male; MAP Kinase Signaling System; NF-E2-Related Factor 2; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Wistar

L-glutamine (Gln) is a nonessential amino acid that is the most abundant amino acid in plasma. Gln has been reported to have an anti-inflammatory activity that involves deactivation of mitogen-activated protein kinases (MAPKs) in a MAPK phosphatase (MKP)-1-dependent manner. This study investigated the role of Gln in the inhibition of DNFB-induced allergic contact dermatitis (CD) in the ears of mice, and specifically the involvement of Gln in p38 MAPK inhibition. Topical application of Gln or the p38 inhibitor, SB202190, suppressed DNFB-induced CD. Gln application inhibited DNFB-induced p38 phosphorylation. Western blot analysis revealed that Gln application resulted in early phosphorylation and protein induction of MKP-1. MKP-1 small interfering RNA (siRNA), but not control siRNA, abrogated Gln-mediated early phosphorylation, protein induction of MKP-1, deactivation of p38, and Gln-mediated suppression of CD. The extracellular signal-regulated kinase (ERK) inhibitor, U0126, blocked Gln-induced MKP-1 phosphorylation and protein induction, as well as Gln suppression of CD. These results suggest that Gln suppresses DNFB-induced CD via deactivation of p38 MAPK through the early induction of MKP-1, the negative regulator of p38, in an ERK-dependent manner.

Topics: Animals; Butadienes; Cell Line; Cells, Cultured; Dermatitis, Contact; Dinitrofluorobenzene; Disease Models, Animal; Dual Specificity Phosphatase 1; Enzyme Inhibitors; Female; Glutamine; Humans; Keratinocytes; MAP Kinase Kinase 4; Mice; Mice, Inbred C57BL; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; RNA, Small Interfering

Bacterial clearance is one of the most important beneficial consequences of the innate immune response. Chemokines are important mediators controlling leukocyte trafficking and activation, whereas reactive oxygen and nitrogen species are effectors in bacterial killing. In the present work, we used in vivo and in vitro models of infections to study the role of monocyte chemoattractant protein 1 (MCP-1)/CCL2 and nitric oxide (NO) in the bacterial clearance in sepsis. Our results show that MCP-1/CCL2 and NO levels are increased in the peritoneal cavity of mice 6 h after sepsis induced by cecal ligation and puncture. Pretreatment with anti-MCP-1/CCL2 monoclonal antibodies increased the number of colony-forming units (CFUs) recovered in the peritoneal lavage fluid. Moreover, CFU counts were increased in the peritoneal fluid of CCR2 mice subjected to cecal ligation and puncture. In vitro stimulation of peritoneal macrophages with recombinant MCP-1/CCL2 reduced CFU counts in the supernatant after challenge with Escherichia coli. Conversely, treatment with anti-MCP-1/CCL2 increased CFU counts under the same experimental condition. Stimulation of cultured macrophages with MCP-1/CCL2 and interferon had a synergistic effect on NO production. Macrophages from CCL2 mice showed a consistent decrease in NO production when compared with wild-type controls after stimulation with LPS + interferon. Finally, we showed incubation of macrophages with E. coli, and the ERK inhibitor U0126 increased CFU numbers and decreased intracellular levels of NO. In conclusion, we demonstrated for the first time that MCP-1/CCL2 has a crucial role in the clearance of bacteria by mechanisms involving increased expression of inducible NO synthase and production of NO by ERK signaling pathways.

Topics: Animals; Ascitic Fluid; Bacterial Infections; Butadienes; Cells, Cultured; Chemokine CCL2; Colony Count, Microbial; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Macrophage Activation; Macrophages, Peritoneal; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitriles; Phagocytosis; Recombinant Proteins; Sepsis

Peripheral amylin inhibits eating via the area postrema (AP). Because amylin activates the extracellular-signal regulated kinase 1/2 (ERK) pathway in some tissues, and because ERK1/2 phosphorylation (pERK) leads to acute neuronal responses, we postulated that it may be involved in amylin's eating inhibitory effect. Amylin-induced ERK phosphorylation (pERK) was investigated by immunohistochemistry in brain sections containing the AP. pERK-positive AP neurons were double-stained for the calcitonin 1a/b receptor, which is part of the functional amylin-receptor. AP sections were also phenotyped using dopamine-β-hydroxylase (DBH) as a marker of noradrenergic neurons. The effect of fourth ventricular administration of the ERK cascade blocker U0126 on amylin's eating inhibitory action was tested in feeding trials. The number of pERK-positive neurons in the AP was highest ∼10-15 min after amylin treatment; the effect appeared to be dose-dependent (5-20 μg/kg amylin). A portion of pERK-positive neurons in the AP carried the amylin-receptor and 22% of the pERK-positive neurons were noradrenergic. Pretreatment of rats with U0126 decreased the number of pERK-positive neurons in the AP after amylin injection. U0126 also attenuated the ability of amylin to reduce eating, at least when the animals had been fasted 24 h prior to the feeding trial. Overall, our results suggest that amylin directly stimulates pERK in AP neurons in a time- and dose-dependent manner. Part of the AP neurons displaying pERK were noradrenergic. At least under fasting conditions, pERK was shown to be a necessary part in the signaling cascade mediating amylin's anorectic effect.

Topics: Animals; Anorexia; Appetite Regulation; Area Postrema; Butadienes; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fourth Ventricle; Islet Amyloid Polypeptide; Male; MAP Kinase Signaling System; Nitriles; Rats; Rats, Sprague-Dawley; Receptors, Islet Amyloid Polypeptide; Time Factors

IL-6 has been shown to play a major role in collagen up-regulation process during cardiac hypertrophy, although the precise mechanism is still not known. In this study we have analyzed the mechanism by which IL-6 modulates cardiac hypertrophy. For the in vitro model, IL-6-treated cultured cardiac fibroblasts were used, whereas the in vivo cardiac hypertrophy model was generated by renal artery ligation in adult male Wistar rats (Rattus norvegicus). During induction of hypertrophy, increased phosphorylation of STAT1, STAT3, MAPK, and ERK proteins was observed both in vitro and in vivo. Treatment of fibroblasts with specific inhibitors for STAT1 (fludarabine, 50 μM), STAT3 (S31-201, 10 μM), p38 MAPK (SB203580, 10 μM), and ERK1/2 (U0126, 10 μM) resulted in down-regulation of IL-6-induced phosphorylation of specific proteins; however, only S31-201 and SB203580 inhibited collagen biosynthesis. In ligated rats in vivo, only STAT3 inhibitors resulted in significant decrease in collagen synthesis and hypertrophy markers such as atrial natriuretic factor and β-myosin heavy chain. In addition, decreased heart weight to body weight ratio and improved cardiac function as measured by echocardiography was evident in animals treated with STAT3 inhibitor or siRNA. Compared with IL-6 neutralization, more pronounced down-regulation of collagen synthesis and regression of hypertrophy was observed with STAT3 inhibition, suggesting that STAT3 is the major downstream signaling molecule and a potential therapeutic target for cardiac hypertrophy.

Topics: Animals; Antineoplastic Agents; Butadienes; Cardiomegaly; Cells, Cultured; Collagen; Disease Models, Animal; Down-Regulation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Humans; Imidazoles; Interleukin-6; Male; MAP Kinase Signaling System; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Rats; Rats, Wistar; STAT1 Transcription Factor; STAT3 Transcription Factor; Vidarabine

Despite increased understanding of the fundamental biology regulating cardiomyocyte hypertrophy and heart failure, it has been challenging to find novel chemical or genetic modifiers of these pathways. Traditional cell-based methods do not model the complexity of an intact cardiovascular system and mammalian models are not readily adaptable to chemical or genetic screens. Our objective was to create an in vivo model suitable for chemical and genetic screens for hypertrophy and heart failure modifiers.. Using the developing zebrafish, we established that the cardiac natriuretic peptide genes (nppa and nppb), known markers of cardiomyocyte hypertrophy and heart failure, were induced in the embryonic heart by pathological cardiac stimuli. This pathological induction was distinct from the developmental regulation of these genes. We created a luciferase-based transgenic reporter line that accurately modelled the pathological induction patterns of the zebrafish nppb gene. Utilizing this reporter line, we were able to show remarkable conservation of pharmacological responses between the larval zebrafish heart and adult mammalian models.. By performing a focused screen of chemical agents, we were able to show a distinct response of a genetic model of hypertrophic cardiomyopathy to the histone deacetylase inhibitor, Trichostatin A, and the mitogen-activated protein kinase kinase 1/2 inhibitor, U0126. We believe this in vivo reporter line will offer a unique approach to the identification of novel chemical or genetic regulators of myocardial hypertrophy and heart failure.

Topics: Animals; Animals, Genetically Modified; Butadienes; Cardiomyopathy, Hypertrophic; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation, Developmental; Genes, Reporter; Genetic Markers; Genetic Testing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Luciferases; Mammals; Natriuretic Peptides; Nitriles; Signal Transduction; Zebrafish

Endothelin ET(B) receptors mediate under normal physiological conditions vasorelaxation in coronary arteries. However, vasocontractile ET(B) receptors appear in coronary arteries of ischemic heart disease patients. Interestingly, organ culture of isolated coronary arteries also induces upregulation of vasocontractile ET(B) receptors. This study examines the early time course and mechanism behind upregulation of contractile ET(B) receptors in isolated rat coronary arteries during short-term organ culture.. Coronary artery segments were mounted in wire-myographs and incubated in physiological saline solution. Contractions were measured after exposure to the specific ET(B) receptor agonist Sarafotoxin 6c (S6c) and the endogenous agonists endothelin-1 and endothelin-3. Protein localization and levels of ET(B) and phosphorylated-extracellular-signal-regulated-kinase-1/2 (ERK1/2) were examined by immunohistochemistry.. Fresh arteries showed negligible vasoconstriction to S6c. However, incubation for only 4 and 7h increased S6c contractions two- and seven-fold, respectively. Furthermore, 7h incubation enhanced vasocontractile responses to endothelin-3 and increased ET(B) receptor density in vascular smooth muscle cells. ERK1/2 was activated rapidly after start of incubation. Moreover, incubation with either the transcriptional inhibitor actinomycin D or the mitogen-activated-protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 attenuated contractile ET(B) receptor upregulation. U0126 attenuated ET(B) receptor protein levels after 24 h of incubation.. Coronary arteries rapidly upregulate vasocontractile ET(B) receptors during organ culture via transcriptional mechanisms and MEK-ERK1/2 signalling. This model may mimic the mechanisms seen in ischemic conditions. Furthermore, these findings have important experimental implications in tissue bath experiments lasting for more than 4h.

Topics: Animals; Butadienes; Coronary Vessels; Dactinomycin; Disease Models, Animal; Endothelin-1; Endothelin-3; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Ischemia; Nitriles; Organ Culture Techniques; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Time Factors; Transcription, Genetic; Up-Regulation; Vasoconstriction; Vasoconstrictor Agents; Viper Venoms

Subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. It is suggested that the associated inflammation is mediated through activation of the mitogen-activated protein kinase (MAPK) pathway which plays a crucial role in the pathogenesis of delayed cerebral ischemia after SAH. The aim of this study was first to investigate the timecourse of altered expression of proinflammatory cytokines and matrix metalloproteinase in the cerebral arteries walls following SAH. Secondly, we investigated whether administration of a specific mitogen-activated protein kinase kinase (MEK)1/2 inhibitor, U0126, given at 6 h after SAH prevents activation of the MEK/extracellular signal-regulated kinase 1/2 pathway and the upregulation of cerebrovascular inflammatory mediators and improves neurological function.. SAH was induced in rats by injection of 250 μl of autologous blood into basal cisterns. U0126 was given intracisternally using two treatment regimens: (A) treatments at 6, 12, 24 and 36 h after SAH and experiments terminated at 48 h after SAH, or (B) treatments at 6, 12, and 24 h after SAH and terminated at 72 h after SAH. Cerebral arteries were harvested and interleukin (IL)-6, IL-1β, tumor necrosis factor α (TNF)α, matrix metalloproteinase (MMP)-9 and phosphorylated ERK1/2 (pERK1/2) levels investigated by immunohistochemistry. Early activation of pERK1/2 was measured by western blot. Functional neurological outcome after SAH was also analyzed.. Expression levels of IL-1β, IL-6, MMP-9 and pERK1/2 proteins were elevated over time with an early increase at around 6 h and a late peak at 48 to 72 h post-SAH in cerebral arteries. Enhanced expression of TNFα in cerebral arteries started at 24 h and increased until 96 h. In addition, SAH induced sensorimotor and spontaneous behavior deficits in the animals. Treatment with U0126 starting at 6 h after SAH prevented activation of MEK-ERK1/2 signaling. Further, U0126 significantly decreased the upregulation of inflammation proteins at 48 and 72 h following SAH and improved neurological function. We found no differences between treatment regimens A and B.. These results show that SAH induces early activation of the MEK-ERK1/2 pathway in cerebral artery walls, which is associated with upregulation of proinflammatory cytokines and MMP-9. Inhibition of the MEK-ERK1/2 pathway by U0126 starting at 6 h post-SAH prevented upregulation of cytokines and MMP-9 in cerebral vessels, and improved neurological outcome.

Topics: Animals; Astrocytes; Blood Transfusion, Autologous; Butadienes; Cerebral Arteries; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Exploratory Behavior; Extracellular Signal-Regulated MAP Kinases; Glial Fibrillary Acidic Protein; Male; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinases; Motor Activity; Nervous System Diseases; Nitriles; Rats; Rats, Sprague-Dawley; Signal Transduction; Statistics, Nonparametric; Subarachnoid Hemorrhage; Time Factors; Tumor Necrosis Factor-alpha

As with other herpesviruses, human cytomegalovirus (hCMV) has the ability to establish lifelong persistence and latent infection following primary exposure, salivary glands (SMGs) being the primary site of both. In the immunocompromised patient, hCMV is a common cause of opportunistic infections, and subsequent morbidity and mortality. Elucidating the molecular pathogenesis of CMV-induced disease is critical to the development of more effective and safer drug therapies. In the present study, we used a novel mouse postnatal SMG organ culture model of mCMV-induced dysplasia to investigate a candidate signaling network suggested by our prior studies (COX-2/AREG/EGFR/ERK). The objective was to employ small molecule inhibitors to target several key steps in the autocrine loop, and in this way ameliorate pathology. Our results indicate that upregulation of ERK phosphorylation is necessary for initial mCMV-induced pathogenesis, and that ErbB receptor family phosphorylation and downstream signaling are highly relevant targets for drug discovery.

Topics: Amphiregulin; Animals; Animals, Newborn; Blotting, Western; Butadienes; Cell Proliferation; Cells, Cultured; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Diclofenac; Disease Models, Animal; EGF Family of Proteins; Enzyme Inhibitors; ErbB Receptors; Female; Glycoproteins; Herpesviridae Infections; Host-Pathogen Interactions; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 3; Muromegalovirus; Nitriles; Organ Culture Techniques; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Submandibular Gland

The emergence of the 2009 H1N1 pandemic swine influenza A virus is a good example of how this viral infection can impact health systems around the world in a very short time. The continuous zoonotic circulation and reassortment potential of influenza A viruses (IAV) in nature represents an enormous public health threat to humans. Beside vaccination antivirals are needed to efficiently control spreading of the disease. In the present work we investigated whether the MEK inhibitor U0126, targeting the intracellular Raf/MEK/ERK signaling pathway, is able to suppress propagation of the 2009 pandemic IV H1N1v (v=variant) as well as highly pathogenic avian influenza viruses (HPAIV) in cell culture and also in vivo in the mouse lung. U0126 showed antiviral activity in cell culture against all tested IAV strains including oseltamivir resistant variants. Furthermore, we were able to demonstrate that treatment of mice with U0126 via the aerosol route led to (i) inhibition of MEK activation in the lung (ii) reduction of progeny IAV titers compared to untreated controls (iii) protection of IAV infected mice against a 100× lethal viral challenge. Moreover, no adverse effects of U0126 were found in cell culture or in the mouse. Thus, we conclude that U0126, by inhibiting the cellular target MEK, has an antiviral potential not only in vitro in cell culture, but also in vivo in the mouse model.

Topics: Animals; Antiviral Agents; Butadienes; Cell Line; Cell Survival; Disease Models, Animal; Dogs; Enzyme Inhibitors; Female; Humans; Influenza A virus; Lung; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Mitogen-Activated Protein Kinase Kinases; Nitriles; Orthomyxoviridae Infections; Survival Analysis

Post-myocardial infarction ventricular remodeling is associated with the expression of a variety of factors including S100B that can potentially modulate myocyte apoptosis.. This study was undertaken to investigate the expression and function of S100B and its receptor, the receptor for advanced glycation end products (RAGE) in both postinfarction myocardium and in a rat neonatal myocyte culture model.. In a rat model of myocardial infarction following coronary artery ligation, we demonstrate in periinfarct myocytes, upregulation of RAGE, induction of S100B, and release into plasma with consequent myocyte apoptosis. Using a coimmunoprecipitation strategy, we demonstrate a direct interaction between S100B and RAGE. In rat neonatal cardiac myocyte cultures, S100B at concentrations > or = 50 nmol/L induced myocyte apoptosis, as evidenced by increased terminal DNA fragmentation, TUNEL, cytochrome c release from mitochondria to cytoplasm, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p53, increased expression and activity of proapoptotic caspase-3, and decreased expression of antiapoptotic Bcl-2. Transfection of a full-length cDNA of RAGE or a dominant-negative mutant of RAGE resulted in increased or attenuated S100B-induced myocyte apoptosis, respectively. Inhibition of ERK1/2 by U0126/PD-98059 or overexpression of a dominant negative p53 comparably inhibited S100B-induced myocyte apoptosis.. These results suggest that interaction of RAGE and its ligand S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53 signaling. This receptor-mediated mechanism is uniquely amenable to therapeutic intervention.

Topics: Animals; Apoptosis; Butadienes; Caspase 3; Cell Line; Cytochromes c; Cytosol; Disease Models, Animal; DNA Fragmentation; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation; Humans; Mitochondria, Heart; Mitogen-Activated Protein Kinase 3; Muscle Proteins; Myocardial Infarction; Myocytes, Cardiac; Nerve Growth Factors; Nitriles; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptor for Advanced Glycation End Products; Receptors, Immunologic; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Signal Transduction; Tumor Suppressor Protein p53; Ventricular Remodeling

Cerebral ischemia from middle cerebral artery wall (MCA) occlusion results in increased expression of cerebrovascular endothelin and angiotensin receptors and activation of the mitogen-activated protein kinase (MAPK) pathway, as well as reduced local cerebral blood flow and increased levels of pro-inflammatory mediators in the infarct region. In this study, we hypothesised that inhibition of the cerebrovascular inflammatory reaction with a specific MEK1/2 inhibitor (U0126) to block transcription or a combined receptor blockade would reduce infarct size and improve neurological score.. Rats were subjected to a 2-hours middle cerebral artery occlusion (MCAO) followed by reperfusion for 48 hours. Two groups of treated animals were studied; (i) one group received intraperitoneal administration of a specific MEK1/2 inhibitor (U0126) starting at 0, 6, or 12 hours after the occlusion, and (ii) a second group received two specific receptor antagonists (a combination of the angiotensin AT1 receptor inhibitor Candesartan and the endothelin ETA receptor antagonist ZD1611), given immediately after occlusion. The middle cerebral arteries, microvessels and brain tissue were harvested; and the expressions of tumor necrosis factor-alpha (TNF-alpha), interleukin-1ss (IL-1ss), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and phosphorylated ERK1/2, p38 and JNK were analysed using immunohistochemistry.. We observed an infarct volume of 25 +/- 2% of total brain volume, and reduced neurological function 2 days after MCAO followed by 48 hours of recirculation. Immunohistochemistry revealed enhanced expression of TNF-alpha, IL-1ss, IL-6 and iNOS, as well as elevated levels of phosphorylated ERK1/2 in smooth muscle cells of ischemic MCA and in associated intracerebral microvessels. U0126, given intraperitoneal at zero or 6 hours after the ischemic event, but not at 12 hours, reduced the infarct volume (11.7 +/- 2% and 15 +/- 3%, respectively), normalized pERK1/2, and prevented elevation of the expressions of TNF-alpha IL-1ss, IL-6 and iNOS. Combined inhibition of angiotensin AT1 and endothelin ETA receptors decreased the volume of brain damaged (12.3 +/- 3; P < 0.05) but only slightly reduced MCAO-induced enhanced expression of iNOS and cytokines. The present study shows elevated microvascular expression of TNF-alpha, IL-1ss, IL-6 and iNOS following focal ischemia, and shows that this expression is transcriptionally regulated via the MEK/ERK pathway.

Topics: Animals; Brain; Butadienes; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Infarction, Middle Cerebral Artery; Male; MAP Kinase Kinase Kinases; Neurologic Examination; Nitric Oxide Synthase Type II; Nitriles; Pyrazines; Rats; Rats, Wistar; Signal Transduction; Sulfonamides; Time Factors

Exercise preconditioning has been shown to reduce neuronal damage in ischemic/reperfusion (I/R) injury. ERK1/2 signaling in injury has been thought to modulate neuroprotection. In this study, we investigated the effects of ERK1/2 activation on the expression and activity of MMP-9 and downstream neuronal apoptosis. Adult male Sprague-Dawley rats were subjected to 30min of exercise on a treadmill for 3 weeks. Stroke was induced by a 2-h middle cerebral artery (MCA) occlusion using an intraluminal filament. Apoptotic protein caspase-3 and neuronal apoptosis in cortex and striatum was determined by Western blot at 24h reperfusion and TUNEL staining at 48h reperfusion in 5 I/R injury groups: no treatment, MMP-9 inhibitor (doxycycline), pre-ischemic exercise, exercised animals undergone ERK1/2 inhibition (U0126), and dual inhibition of ERK1/2 and MMP-9 in exercised ischemic rats. Cerebral MMP-9 expression in ischemic rats with different treatment was determined at 6, 12 and 24h reperfusion by real-time PCR for mRNA, Western blot for protein and zymography for enzyme activity. Exercise preconditioning significantly (p<0.05) reduced apoptosis determined by caspase-3 and TUNEL. In non-exercised rats, doxycycline treatment had significant (p<0.05) reductions in apoptosis after I/R injury. The dual ERK1/2-MMP-9 inhibited exercised animals had significantly (p<0.05) reduced neuronal apoptosis that was similar to that seen in exercised ischemic rats. MMP-9 expression in I/R injury was significantly (p<0.05) reduced in the exercised animals as compared to non-exercised controls. When ERK1/2 was inhibited, the reduced MMP-9 expression was reversed to the level seen in the non-exercised controls. This study has suggested that exercise-induced neuroprotection in I/R injury may be mediated by MMP-9 and ERK1/2 expression, leading to a reduction in neuronal apoptosis.

Topics: Analysis of Variance; Animals; Apoptosis; Brain; Butadienes; Caspase 3; Disease Models, Animal; Doxycycline; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Physical Conditioning, Animal; Rats; Rats, Sprague-Dawley; Reperfusion; Time Factors

Amyloid β (Aβ) peptide plays a central role in neuronal apoptosis, promoting oxidative stress, lipid peroxidation, caspase pathway activation and neuronal loss. Our previous study has shown that bone marrow-derived mesenchymal stem cells (BM-MSCs) reduce Aβ deposition when transplanted into acutely-induced Alzheimer's disease (AD) mice brain. However, the impact of reduced Aβ deposition on memory impairment and apoptosis by BM-MSCs has not yet been investigated. Therefore, the aim of the present study was to investigate the neuroprotective mechanism of BM-MSCs in vitro and in vivo. We found that BM-MSCs attenuated Aβ-induced apoptotic cell death in primary cultured hippocampal neurons by activation of the cell survival signaling pathway. These anti-apoptotic effects of BM-MSCs were also observed in an acutely-induced AD mice model produced by injecting Aβ intrahippocampally. In addition, BM-MSCs diminished Aβ -induced oxidative stress and spatial memory impairment in the in vivo model. These findings lead us to hypothesize that BM-MSCs ameliorate Aβ -induced neurotoxicity and cognitive decline by inhibiting apoptotic cell death and oxidative stress in the hippocampus. These findings provide support for a potentially beneficial role for BM-MSCs in the treatment of AD.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Bone Marrow Transplantation; Butadienes; Cells, Cultured; CREB-Binding Protein; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Ethidium; Extracellular Signal-Regulated MAP Kinases; Hippocampus; In Situ Nick-End Labeling; Maze Learning; Memory Disorders; Mesenchymal Stem Cell Transplantation; Mice; Mice, Inbred C57BL; Neurons; Nitriles; Oxidative Stress; Phosphorylation; Reaction Time; Statistics, Nonparametric; Superoxides

Two pathways that have been shown to mediate cerebral ischemic damage are the MEK/ERK cascade and the pro-apoptotic deltaPKC pathway. We investigated the relationship between these pathways in a rat model of focal ischemia by observing and modifying the activation state of each pathway. The ERK1/2 inhibitor, U0126, injected at ischemia onset, attenuated the increase in phosphorylated ERK1/2 (P-ERK1/2) after reperfusion. The deltaPKC inhibitor, deltaV1-1, delivered at reperfusion, did not significantly change P-ERK1/2 levels. In contrast, the deltaPKC activator, psi deltaRACK, injected at reperfusion, reduced ERK1/2 phosphorylation measured 4 h after reperfusion. Additionally, U0126 pretreatment at ischemia onset reduced infarct size compared with vehicle, but U0126 injected at the onset of reperfusion had no protection. Finally, combination of U0126 injection at ischemia onset plus deltaV1-1 injection at reperfusion further reduced infarct size, while combination of U0126 delivered at ischemia onset with psi deltaRACK injected at reperfusion increased infarct size compared with U0126 alone. In conclusion, we find that inhibiting both the MEK/ERK and the deltaPKC pathways offers greater protection than either alone, indicating they likely act independently.

Topics: Animals; Brain Ischemia; Butadienes; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Enzyme Inhibitors; Male; Mitogen-Activated Protein Kinase 3; Neuroprotective Agents; Nitriles; Phosphorylation; Protein Kinase C-delta; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Stroke; Up-Regulation

Endothelin (ET)-1 is a likely candidate for a key role in diabetic vascular complications. However, no abnormalities in the vascular responsiveness to ET-1 have been identified in the chronic stage of type 2 diabetes. Our goal was to look for abnormalities in the roles played by ET receptors (ET(A) and ET(B)) in the mesenteric artery of the type 2 diabetic Goto-Kakizaki (GK) rat and to identify the molecular mechanisms involved. Using mesenteric arteries from later-stage (32-38 wk old) individuals, we compared the ET-1-induced contraction and the relaxation induced by the selective ET(B) receptor agonist IRL1620 between GK rats and control Wistar rats. Mesenteric artery ERK activity and the protein expressions for ET receptors and MEK were also measured. In GK rats (vs. age-matched Wistar rats), we found as follows. 1) The ET-1-induced contraction was greater and was attenuated by BQ-123 (ET(A) antagonist) but not by BQ-788 (ET(B) antagonist). In the controls, BQ-788 augmented this contraction. 2) Both the relaxation and nitric oxide (NO) production induced by IRL1620 were reduced. 3) ET-1-induced contraction was enhanced by N(G)-nitro-l-arginine (l-NNA; NO synthase inhibitor) but suppressed by sodium nitroprusside (NO donor). 4) The enhanced ET-1-induced contraction was reduced by MEK/ERK pathway inhibitors (PD-98059 or U0126). 5) ET-1-stimulated ERK activation was increased, as were the ET(A) and MEK1/2 protein expressions. 6) Mesenteric ET-1 content was increased. These results suggest that upregulation of ET(A), a defect in ET(B)-mediated NO signaling, and activation of the MEK/ERK pathway together represent a likely mechanism mediating the hyperreactivity to ET-1 examined in this study.

Topics: Angiotensin II; Animals; Arginine Vasopressin; Butadienes; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Endothelin-1; Endothelins; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Male; MAP Kinase Kinase Kinases; Mesenteric Arteries; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitriles; Nitroarginine; Nitroprusside; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Piperidines; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Signal Transduction; Vasoconstriction; Vasodilation

The pathogenesis of chronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation associated with lung neutrophilia and elevated levels of pro-inflammatory mediators in the bronchoalveolar lavage fluid or sputum of patients. Recent findings revealed that mitogen-activated protein kinase (MAPK) signaling cascade is involved in the inflammatory response of lung injury. In the present study we could elucidate the role of extracellular signal-related MAPK in the murine model of LPS-induced acute lung injury by using U0126, a specific inhibitor of MEK1/2, upstream kinases of ERK. Phosphorylation of ERK was inhibited by U0126 in vivo as well as in vitro. In freshly isolated human peripheral blood mononuclear cells U0126 dose-dependently blocked the release of IL-2 and TNF-alpha. For in vivo studies mice were exposed to aerosolized LPS to induce an acute lung injury mimicking some aspects of COPD. This led to a recruitment of neutrophils to the lung and to the release of pro-inflammatory cytokines into bronchoalveolar lavage. Pretreatment of mice with U0126 significantly reduced lung neutrophilia and diminished levels of TNF-alpha and chemotactic MIP-2 and KC in bronchoalveolar fluid. U0126 also decreased albumin levels in BAL fluid, a marker of vascular leakage. Histological examination of lung tissues revealed that ERK MAPK inhibition using U0126 efficiently attenuated LPS-induced pulmonary inflammatory responses. These data suggest that ERK signaling plays an important role in acute lung injury and pharmacologic inhibition of ERK provides a promising new therapeutic strategy for lung inflammatory diseases and in particular COPD.

Topics: Animals; Butadienes; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Humans; Inflammation; Leukocytes, Mononuclear; Lipopolysaccharides; Lung Injury; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Nitriles

Hindlimb unweighting (HLU) of rats is a model used to mimic the cephalic fluid shift potentially involved in the orthostatic intolerance experienced by astronauts. Certain arteries in these rats exhibit a decreased contractile response to adrenergic agonists. It was shown previously that this may be caused by changes in thick filament regulation (Summers et al., Vascul Pharmacol 48: 208-214, 2008). In the present study, it was hypothesized that HLU also modifies thin filament regulation by effects on p38(MAPK) and ERK. Abdominal aorta rings from 20-day HLU rats and untreated controls were subjected to phenylephrine and phorbol 12,13-dibutyrate (PDBU) concentration response curves in the presence and absence of two inhibitors: the p38(MAPK) inhibitor SB-203580 and the MEK inhibitor U-0126. SB-203580 decreased control sensitivity to both agonists, but HLU sensitivity was not significantly affected. U-0126, which blocks enzymes immediately upstream of ERK, affected sensitivity to both agonists equally between control and HLU. Western blot analysis revealed no change in total levels of p38(MAPK) and its downstream target heat shock protein 27 but did reveal a decrease in phosphorylated levels of both after stimulation with PDBU and phenylephrine after HLU treatment. Neither total ERK nor phosphorylated levels after stimulation were affected by HLU. Total levels of caldesmon, a molecule downstream of both pathways, were decreased, but phosphorylated levels after stimulation were decreased by roughly twice as much. The results of this study demonstrate that HLU downregulates p38(MAPK), but not ERK, signaling. In turn, this may decrease actin availability for contraction.

Topics: Adrenergic alpha-Agonists; Animals; Aorta, Abdominal; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Hindlimb Suspension; HSP27 Heat-Shock Proteins; Imidazoles; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nitriles; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Phorbol 12,13-Dibutyrate; Pyridines; Rats; Rats, Wistar; Signal Transduction

Cocaine-induced changes in glutamatergic synaptic transmission in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) play a key role in cocaine behavioral effects. Activation of ionotropic glutamate receptor NMDA receptor (NMDAR) in the VTA is critical for the development of cocaine psychomotor sensitization. However, the role of NMDAR in the NAc, a brain area critical for the expression of cocaine psychomotor sensitization, remains to be explored. Here, we show that repeated noncontingent cocaine injections increased NAc NMDAR subunits, NR1, NR2A, and NR2B 21 d, but not 1 d, after withdrawal from cocaine. These changes were associated with an increase in the GluR1 subunit of the AMPA receptor. We also found a time-dependent increase in extracellular signal-regulated kinase (ERK) activity which correlated with the increased expression of NMDAR subunits. Furthermore, the increase in GluR1 and ERK activity was blocked after inhibition of NR2B-containing NMDAR during the development of cocaine psychomotor sensitization or when the MEK (mitogen-activated protein/ERK kinase) inhibitor was microinjected into the NAc 21 d after withdrawal from cocaine. Together, these results suggest that the development of cocaine psychomotor sensitization triggers a delayed increase in the expression of NMDAR subunits in the NAc, which in turn enhances the activity of ERK. Enhanced ERK activity drives the increased expression of the GluR1 subunits, which increases the excitability of NAc neurons after prolonged withdrawal from cocaine and results in enduring expression of psychomotor sensitization.

Topics: Analysis of Variance; Animals; Butadienes; Cocaine; Cocaine-Related Disorders; Disease Models, Animal; Dopamine Uptake Inhibitors; Drug Administration Schedule; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Locomotion; Male; Nitriles; Nucleus Accumbens; Piperidines; Psychomotor Performance; Rats; Receptors, N-Methyl-D-Aspartate; Subcellular Fractions; Time Factors

Cerebral ischemia is usually characterized by a reduction in local blood flow and metabolism and by disruption of the blood-brain barrier in the infarct region. The formation of oedema and opening of the blood-brain barrier in stroke is associated with enhanced expression of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1).. Here, we found an infarct volume of 24.8 +/- 2% and a reduced neurological function after two hours of middle cerebral artery occlusion (MCAO), followed by 48 hours of recirculation in rat. Immunocytochemistry and confocal microscopy revealed enhanced expression of MMP-9, TIMP-1, and phosphorylated ERK1/2 in the smooth muscle cells of the ischemic MCA and associated intracerebral microvessels. The specific MEK1/2 inhibitor U0126, given intraperitoneal zero or 6 hours after the ischemic event, reduced the infarct volume significantly (11.8 +/- 2% and 14.6 +/- 3%, respectively; P < 0.05), improved neurological function, normalized expression of phosphorylated ERK1/2, and reduced expression of MMP-9 and TIMP-1 in the vessel walls. Administration of U0126 12 hours after MCAO did not alter the expression of MMP-9. Immunocytochemistry showed no overlap in expression between MMP-9/TIMP-1 and the astrocyte/glial cell marker GFAP in the vessel walls.. These data are the first to show that the elevated vascular expression of MMP-9 and TIMP-1, associated with breakdown of the blood-brain barrier following focal ischemia, are transcriptionally regulated via the MEK/ERK pathway.

Topics: Actins; Animals; Astrocytes; Brain Infarction; Butadienes; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Infarction, Middle Cerebral Artery; Male; MAP Kinase Kinase Kinases; Matrix Metalloproteinase 1; Matrix Metalloproteinase 9; Microvessels; Muscle, Smooth; Neurologic Examination; Nitriles; Rats; Rats, Wistar; Signal Transduction; Tetrazolium Salts; Tissue Inhibitor of Metalloproteinase-1

Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is a potent hematopoietic cytokine. In the present study, we examined whether GM-CSF is neuroprotective in retinal ganglion cells (RGCs). First, we studied the expression of GM-CSF and the GM-CSF-alpha-receptor in rat and human retina and in RGC-5 cells. Then, RGC-5 cells were incubated with apoptosis-inducing agents (e.g., staurosporine, glutamate and NOR3). The cell death was assessed by Live-Death-Assays and apoptosis-related-proteins were examined by immunoblotting. In addition, the expression of phosphorylated ERK1/2-pathway-proteins after incubation with GM-CSF and after inhibiting MEK1/2 with U0126 was analyzed. To assess the in vivo-effect, first staurosporine or GM-CSF plus staurosporine was injected into the vitreous body of Sprague-Dawley rats. In a second axotomy model the optic nerve was cut and GM-CSF was injected into the vitreous body. In both models, the RGCs were labeled retrogradely with either Fluoro-Gold or 4-Di-10-Asp and counted. As a first result, we identified GM-CSF and the GM-CSF-alpha-receptor in rat and human retina as well as in RGC-5 cells. Then, in the RGC-5 cells GM-CSF counteracts induced cell death in a dose-and time-dependent manner. With respect to apoptosis, Western blot analysis revealed a decreased Bad-expression and an increased Bcl-2-expression after co-incubation with GM-CSF. Concerning signaling pathways, incubation with GM-CSF activates the ERK1/2 pathway, whereas inhibition of MEK1/2 with U0126 strongly decreased the phosphorylation downstream in the ERK1/2 pathway, and the antiapoptotic activity of GM-CSF in vitro. Like in vitro, GM-CSF counteracts the staurosporine-induced cell death in vivo and protects RGCs from axotomy-induced degeneration. Our data suggest that GM-CSF might be a novel therapeutic agent in neuropathic disease of the eye.

Topics: Adult; Aged; Animals; Apoptosis; bcl-Associated Death Protein; Blotting, Western; Butadienes; Cells, Cultured; Disease Models, Animal; Glaucoma; Glutamic Acid; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydroxylamines; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroanatomical Tract-Tracing Techniques; Nitriles; Nitro Compounds; Optic Nerve Injuries; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Retinal Ganglion Cells; Staurosporine

The protective effect of sevoflurane preconditioning against spinal cord ischemia/reperfusion (I/R) is unclear. We designed this study to investigate whether sevoflurane preconditioning could induce rapid ischemic tolerance to the spinal cord in a rabbit model of transient spinal cord ischemia and how the role of extracellular signal-regulated kinase (ERK) is involved.. To test whether preconditioning with sevoflurane induces rapid ischemic tolerance, New Zealand White male rabbits were randomly assigned to three groups. Animals in the Sev group received preconditioning with 3.7% sevoflurane (1.0 minimum alveolar anesthetic concentration) in 96% oxygen for 30 min, whereas animals in the O(2) group serving as controls inhaled only 96% oxygen for 30 min. The Sham group received the same anesthesia and surgical preparation but no preconditioning or spinal cord I/R. To evaluate the role of ERK activation in sevoflurane preconditioning, rabbits were randomly assigned to four groups. U0126, an ERK inhibitor, was administered IV 20 min before the beginning of preconditioning in the U0126 + O(2) and U0126 + Sev groups. Dimethylsulfoxide was administered IV at the same time in the vehicle + O(2) and vehicle + Sev groups. At 1 h after preconditioning, the animals were subjected to spinal cord I/R induced by infrarenal aorta occlusion. All animals were assessed at 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested for histopathological examination, TUNEL staining, and Western blot of phosphor-ERK1/2.. The animals in the Sev group had higher neurological scores and more normal motor neurons than those in the O(2) group (P < 0.01 for each comparison). Compared with vehicle + Sev group, the U0126 + Sev group had worse neurological outcomes, fewer viable neurons, more apoptotic neurons, and significantly decreased ERK1/2 phosphorylation (P

Topics: Anesthetics, Inhalation; Animals; Aorta, Abdominal; Apoptosis; Butadienes; Cell Survival; Constriction; Disease Models, Animal; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Hemodynamics; Male; Methyl Ethers; Motor Neurons; Neurologic Examination; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Rabbits; Reperfusion Injury; Sevoflurane; Spinal Cord; Spinal Cord Ischemia; Time Factors

The mitogen-activated protein kinase family plays an important role in several types of pain. However, the detailed role of phosphorylated extracellular signal-regulated kinase (pERK) in the region of injured peripheral nerve is poorly understood. In this study, we investigated whether pERK in injured sciatic nerve contributes to neuropathic pain induced by partial sciatic nerve ligation (PSL) in mice.. Mice received PSL; pERK1/2 (p44/42) in sciatic nerve was measured by both Western blotting and immunohistochemistry. U0126 (an ERK kinase inhibitor) was injected twice, an intraneural injection (20 nmol/2 microL) 30 min before PSL, and a perineural injection (20 nmol/10 microL) on Day 1 after PSL. Thermal hyperalgesia and tactile allodynia induced by PSL were evaluated by the thermal paw withdrawal test and the von Frey test, respectively.. As measured by Western blotting, in sham-operated mice, the levels of pERK1/2 in sciatic nerve were constant and the same as those in naive mice across Days 1-14. In PSL-operated mice, a significant increase in pERK1/2 was observed on Day 1 after PSL and persisted until Day 3. As measured by immunohistochemistry, immunoreactivity of pERK1/2 in PSL-operated sciatic nerve was markedly increased in comparison with that in sham-operated sciatic nerve on Day 1 after PSL. In the sciatic nerve on Day 1 after PSL, as indicated by double immunostaining, the increased immunoreactivity of pERK1/2 was colocalized with glial fibrillary acidic protein (GFAP), a marker of Schwann cells, but not F4/80, a marker of macrophages. PSL-induced thermal hyperalgesia was significantly attenuated by treatment with U0126 on Days 3, 7, and 14 after PSL. The PSL-induced tactile allodynia was also significantly attenuated by treatment with U0126 on Days 7 and 14 after PSL.. Activation of ERK in Schwann cells of the injured peripheral nervous system may play an important role in the development of neuropathic pain. Our results suggest that pERK itself and ERK-related mediators are potential therapeutic targets for the treatment of neuropathic pain.

Topics: Analgesics; Animals; Behavior, Animal; Butadienes; Disease Models, Animal; Enzyme Activation; Hyperalgesia; Ligation; Male; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Pain Measurement; Pain Threshold; Phosphorylation; Protein Kinase Inhibitors; Schwann Cells; Sciatic Nerve; Sciatic Neuropathy; Sciatica; Time Factors

Plasticity in the spinal dorsal horn is thought to underlie, at least in part, pain behavior after peripheral nerve injury. Homer1 proteins play an important role in synaptic plasticity through an activity-dependent remodeling of the postsynaptic density (PSD). In this study, we examined the early consequences of the loose ligation of the sciatic nerve on the levels of Homer1a and Homer1b/c proteins in the PSD of spinal dorsal horn neurons.. Male rats were randomly assigned to control, sham-operated, or ligated groups. Four hours after sciatic exposure or ligation, the animals were anesthetized and killed. Dorsal horn ipsilateral and contralateral quadrants were homogenized and centrifuged to obtain a PSD-containing LP1 fraction. Homer1 isoforms were identified in Western immunoblots. In some animals, Homer1 small interfering RNA (siRNA), nontarget siRNA, MK-801, or U01026 was injected intrathecally before surgery to assess the effects of this treatment on the levels of Homer1 isoforms and on 2 signs of injury-associated pain behavior, a shift in weight-bearing distribution and thermal hyperalgesia.. In ligated animals, the protein levels of Homer1a increased and those of Homer1b/c decreased in the ipsilateral LP1 fraction of the spinal dorsal horn. In contrast, no changes were detected in the contralateral LP1 fraction of ligated animals or the ipsilateral or contralateral LP1 fraction of sham-operated animals. Intrathecal injections of Homer1 siRNA, but not nontarget siRNA, 2 h before the ligation prevented the accumulation of Homer1a and loss of Homer1b/c in the ipsilateral LP1 fraction. The same pretreatment with Homer1 siRNA also alleviated both a shift in weight-bearing behavior and thermal hyperalgesia in the ligated animals. Intrathecal injections of MK-801 or U0126 15 min before the ligation similarly prevented the injury-associated changes in Homer1 protein levels and the behavioral signs of pain.. The ligation-associated changes in the protein levels of Homer1a and Homer1b/c in the ipsilateral PSD of spinal dorsal horn neurons may be an important early reflection of the injury-associated plasticity that in time leads to the development of persistent pain.

Topics: Animals; Behavior, Animal; Butadienes; Carrier Proteins; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Homer Scaffolding Proteins; Injections, Spinal; Ligation; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuralgia; Neuronal Plasticity; Nitriles; Posterior Horn Cells; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; RNA Interference; RNA, Small Interfering; Sciatic Nerve; Sciatic Neuropathy; Time Factors

The laterocapsular division of the central nucleus of the amygdala (CeLC) has emerged as an important site of pain-related plasticity and pain modulation. Glutamate and neuropeptide receptors in the CeLC contribute to synaptic and behavioral changes in the arthritis pain model, but the intracellular signaling pathways remain to be determined. This study addressed the role of PKA, PKC, and ERK in the CeLC. Adult male Sprague-Dawley rats were used in all experiments. Whole-cell patch-clamp recordings of CeLC neurons were made in brain slices from normal rats and from rats with a kaolin/carrageenan-induced monoarthritis in the knee (6 h postinduction). Membrane-permeable inhibitors of PKA (KT5720, 1 microM; cAMPS-Rp, 10 microM) and ERK (U0126, 1 microM) activation inhibited synaptic plasticity in slices from arthritic rats but had no effect on normal transmission in control slices. A PKC inhibitor (GF109203x, 1 microM) and an inactive structural analogue of U0126 (U0124, 1 microM) had no effect. The NMDA receptor-mediated synaptic component was inhibited by KT5720 or U0126; their combined application had additive effects. U0126 did not inhibit synaptic facilitation by forskolin-induced PKA-activation. Administration of KT5720 (100 microM, concentration in microdialysis probe) or U0126 (100 microM) into the CeLC, but not striatum (placement control), inhibited audible and ultrasonic vocalizations and spinal reflexes of arthritic rats but had no effect in normal animals. GF109203x (100 microM) and U0124 (100 microM) did not affect pain behavior. The data suggest that in the amygdala PKA and ERK, but not PKC, contribute to pain-related synaptic facilitation and behavior by increasing NMDA receptor function through independent signaling pathways.

Topics: Amygdala; Animals; Arthritis; Behavior; Butadienes; Carbazoles; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Indoles; Male; Maleimides; Neuronal Plasticity; Neurons; Nitriles; Pain; Protein Kinase C; Protein Kinase Inhibitors; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Thionucleotides

Polycystic kidney disease (ADPKD) results from failure of the kidney to properly maintain three-dimensional structure after loss of either polycystin-1 or -2. Mice with kidney selective inactivation of Pkd1 during embryogenesis develop profound renal cystic disease and die from renal failure within 3 weeks of birth. In this model, cysts form exclusively from cells in which Cre recombinase is active, but the apparent pace of cyst expansion varies by segment and cell type. Intercalated cells do not participate in cyst expansion despite the presence of cilia up to at least postnatal day 21. Cystic segments show a persistent increase in proliferation as determined by bromodeoxyuridine (BrdU) incorporation; however, the absolute proliferative index is dependent on the underlying proliferative potential of kidney tubule cells. Components of the extracellular regulated kinase (MAPK/ERK) pathway from Ras through MEK1/2 and ERK1/2 to the effector P90(RSK) are activated in both perinatal Pkd1 and adult Pkd2 ortholgous gene disease models. The pattern of MAPK/ERK activation is focal and does not correlate with the pattern of active proliferation identified by BrdU uptake. The possibility of a causal relationship between ERK1/2 activation and cyst cell proliferation was assessed in vivo in the acute perinatal Pkd1 model of ADPKD using MEK1/2 inhibitor U0126. U0126 treatment had no effect on progression of cyst formation in this model at doses sufficient to reduce phospho-ERK1/2 in cystic kidneys. Cysts in ADPKD exhibit both increased proliferation and activation of MAPK/ERK, but cyst growth is not prevented by inhibition of ERK1/2 activation.

Topics: Animals; Apoptosis; Butadienes; Cell Proliferation; Cysts; Disease Models, Animal; Enzyme Activation; Kidney; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mice; Mice, Mutant Strains; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Polycystic Kidney, Autosomal Dominant; Protein Kinase Inhibitors; TRPP Cation Channels

Inhibition of cytokines offers modest protection from injury in animal models of lung ischemia-reperfusion. Improved strategies would selectively inhibit the transcriptional activation response to oxidative stress. Mitogen-activated protein kinases (p38, c-jun N-terminal kinase, extracellular signal-regulated kinase) have been shown to be activated after oxidative stress and in animal models of acute inflammatory lung injury. We hypothesized that mitogen-activated protein kinase inhibition would block downstream transcriptional activation, providing robust protection from lung ischemia-reperfusion injury.. Experimental rats received inhibitors of p38, c-jun kinase, or extracellular signal-regulated kinase before in situ left lung ischemia-reperfusion. Immunohistochemistry localized cellular sites of mitogen-activated protein kinase activation. Several markers of lung injury were assessed. Enzyme-linked immunosorbent assay measured soluble cytokine and chemokine contents. Western blotting assessed mitogen-activated protein kinase phosphorylation. Electromobility shift assays measured transcription factor nuclear translocation.. Immunohistochemistry localized p38 and c-jun kinase activations in positive controls to alveolar macrophages. Extracellular signal-regulated kinase was activated in endothelial and epithelial cells. Animals treated with p38 or c-jun kinase inhibitor demonstrated significant reductions in transcription factor activation and markers of lung injury. Extracellular signal-regulated kinase inhibition was not protective. Western blotting confirmed inhibitor specificity.. Inhibition of p38 and c-jun kinase provided significant protection from injury. The alveolar macrophage appears to be the key coordinator of injury in response to oxidative stress. Therapeutically targeting specific cell population (macrophage) responses to oxidative stress has the potential benefit of reducing lung reperfusion injury severity while leaving host immune responses intact.

Topics: Animals; Anthracenes; Blotting, Western; Bronchoalveolar Lavage Fluid; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Female; Immunohistochemistry; Inflammation Mediators; Lung Diseases; Male; Mitogen-Activated Protein Kinases; Nitriles; Oxidative Stress; Peroxidase; Phosphorylation; Probability; Random Allocation; Rats; Rats, Sprague-Dawley; Reference Values; Reperfusion Injury; Sensitivity and Specificity

Changes in the properties of visceral sensory neurons contribute to the development of gastrointestinal pain. However, little is known about the molecules involved in mechanosensation from the gastrointestinal tract. We investigated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinase cascade, in dorsal root ganglion (DRG) and nodose ganglion (NG) neurons by noxious gastric distention (GD) and its involvement in acute visceral pain in rats.. Electromyographic responses to gastric balloon distention through gastrostomy were recorded from the acromiotrapezius muscle in rats after splanchnic nerve resection or vagotomy and in control rats. We then examined the phosphorylated-ERK1/2 (p-ERK1/2) labeling in the DRG and NG after GD using immunohistochemistry.. Gastric distention induced p-ERK1/2 in DRG and NG neurons with a peak at 2 minutes after stimulation. We found a stimulus intensity-dependent increase in the number of activated neurons, and this activation corresponded well with the incidence of the visceromotor response. Most of these p-ERK1/2-labeled neurons were small- and medium-sized neurons that coexpressed transient receptor potential vanilloid 1 ion channel and acid-sensing ion channel 3. Splanchnic nerve resection, but not vagotomy, affected the visceromotor response, and attenuated the ERK1/2 activation in DRG neurons produced by GD. Furthermore, intrathecal administration of the mitogen-activated protein kinase kinase 1/2 inhibitor, U0126, altered the response to noxious GD.. The activation of ERK1/2 pathways in DRG neurons by noxious GD may be correlated with functional activity, and may be involved in acute visceral pain.

Topics: Abdominal Pain; Acid Sensing Ion Channels; Acute Disease; Animals; Butadienes; Catheterization; Disease Models, Animal; Electromyography; Enzyme Activation; Enzyme Inhibitors; Ganglia, Spinal; Gastric Emptying; Immunohistochemistry; Male; Membrane Proteins; Mitogen-Activated Protein Kinase 3; Nerve Tissue Proteins; Neurofilament Proteins; Neurons, Afferent; Nitriles; Nodose Ganglion; Physical Stimulation; Rats; Rats, Sprague-Dawley; Sodium Channels; Stomach; TRPV Cation Channels

Cerebral ischaemia is associated with elevated levels of endothelin B (ETB) receptors in the ipsilateral middle cerebral artery (MCA). This up-regulation of ET receptors occurs via de novo transcription involving mitogen-activated protein kinases (MAPK). The aim of this study was to examine the effect of inhibition of the MAP kinase/ERK kinase (MEK)1/2 on ET receptor alteration, brain damage, and neurology in experimental cerebral ischaemia. Transient middle cerebral artery occlusion (MCAO) was induced in male Wistar rats by the intraluminal filament technique. The animals received 100 mg/kg intraperitoneally of the MEK1/2 inhibitor U0126 or vehicle in conjunction with the occlusion. After 24 h, the rats were decapitated and the brains removed. The middle cerebral arteries were dissected out and examined with myographs or immunohistochemistry. The ischaemic areas of the brains were compared. After the MCAO, the contractile responses of the ETA and ETB receptors were augmented in the ipsilateral MCA. U0126 decreased this alteration in ET receptor response. Furthermore, treatment with U0126 significantly decreased the brain damage and improved neurological scores. Immunohistochemistry showed that there were lower protein levels of phosphorylated extracellular signal-regulated kinases (ERK)1/2 and phosphorylated transcription factor Elk-1 in the U0126-treated rats compared to control. The results show that treatment with the MEK1/2 inhibitor U0126 in ischaemic stroke decreases brain damage, neurological symptoms, and ET receptor alteration. The vascular effects of U0126 provide new perspective on possible mechanisms of actions of MAPK inhibition in cerebral ischaemia.

Topics: Animals; Butadienes; Cerebral Infarction; Disease Models, Animal; Enzyme Inhibitors; Functional Laterality; Gene Expression Regulation; Infarction, Middle Cerebral Artery; Male; Mitogen-Activated Protein Kinases; Muscle Contraction; Neurologic Examination; Nitriles; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B

Patients with neurofibromatosis type 1 (NF1), resulting from neurofibromin gene mutations, frequently suffer from deficits in learning and spatial memory. Mice heterozygous for functional deletion of the NF1 gene (NF1(+/-) mice) also exhibit compromised spatial learning, and deficits in early-stage hippocampal long-term potentiation (LTP). Neurofibromin is a multifunctional protein which acts in part as an inhibitory constraint on Ras signalling, and the deficits in early-stage LTP and spatial learning have been linked to Ras hyperactivation. However, the downstream targets of Ras hyperactivation that lead to cognitive disruption are unknown. The levels of activity of signalling molecules potentially downstream of Ras were therefore studied in NF1(+/-) mice. Elevated phospho-ERK (pERK) levels were observed in the hippocampi from NF1(+/-) mice, while phospho-Akt/PKB (pAkt) and phospho-eIF4E (peIF4E) levels were unchanged relative to wild-type mice. Hippocampal levels of phospho-CREB (pCREB) were also increased, suggesting potential changes in late-phase LTP in NF1(+/-) mice. Indeed, LTP was found to be impaired for at least 4 h following induction in NF1(+/-) mice, linking neurofibromin function with the long-term maintenance of LTP. Remarkably, U0126, an inhibitor of ERK activation, at doses which reduced the hyperactive pERK levels in NF1(+/-) mice to the levels observed in control mice, caused a reduction in the deficits in early-phase LTP and completely rescued the long-term LTP deficits. In contrast to the abundant evidence that reductions in ERK activity lead to impaired plasticity, these data indicate that ERK hyperactivation in a partial model of type 1 neurofibromatosis leads to deficits in long-lasting hippocampal plasticity.

Topics: Animals; Butadienes; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Electric Stimulation; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Hippocampus; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurofibromatosis 1; Neuronal Plasticity; Nitriles; Phosphorylation; Recovery of Function

Plasminogen (Plg) activator inhibitor-1 (PAI-1) is an important fibrosis-promoting molecule. Whether this effect can be attributed to PAI-1's activity as an inhibitor of plasmin generation is debated. This study was designed to investigate the role of Plg in renal fibrosis using in vivo and in vitro approaches. Plg-deficient (Plg-/-) and wild-type (Plg+/+) C57BL/6 mice were subjected to unilateral ureteral obstruction or sham surgery (n = 8/group; sham, days 3, 7, 14, and 21). Plg deficiency was confirmed by the absence of Plg mRNA, protein, and plasmin activity. After 21 d of unilateral ureteral obstruction, total kidney collagen was significantly reduced by 35% in the Plg-/- mice. Epithelial-to-mesenchymal transition (EMT), as typified by tubular loss of E-cadherin and acquisition of alpha-smooth muscle actin, was also significantly reduced in Plg-/- mice, 76% and 50%, respectively. Attenuation of EMT and fibrosis severity in the Plg-/- mice was associated with significantly lower levels of phosphorylated extracellular signal-regulated kinase (ERK) and active TGF-beta. In vitro, addition of plasmin (20 microg/ml) to cultures of murine tubular epithelial cells initiated ERK phosphorylation within minutes, followed by phenotypic transition to fibroblast-specific protein-1+, alpha-smooth muscle actin+, fibronectin-producing fibroblast-like cells. Both plasmin-induced ERK activation and EMT were significantly blocked in vitro by the protease-activated receptor-1 (PAR-1) silencing RNA; by pepducin, a specific anti-PAR-1 signaling peptide; and by the ERK kinase inhibitor UO126. Plasmin-induced ERK phosphorylation was enhanced in PAR-1-overexpressing tubular cells. These findings support important profibrotic roles for plasmin that include PAR-1-dependent ERK signaling and EMT induction.

Topics: Actins; Animals; Butadienes; Cadherins; Cell Movement; Collagen; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Fibrinolysin; Fibrosis; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitriles; Phosphorylation; Plasminogen Activator Inhibitor 1; Receptor, PAR-1; Signal Transduction; Transforming Growth Factor beta; Ureteral Obstruction

Arteries from hypertensive animals and humans have increased spontaneous tone. Increased superoxide anion (superoxide) contributes to elevated blood pressure (BP) and spontaneous tone in hypertension. The association between the extracellular signaling-regulated kinase 1/2 (ERK1/2)-mitogen-activated protein kinase (MAPK) signaling pathway and generation of superoxide and spontaneous tone in isolated aorta was studied in angiotensin II (ANG II)-infused hypertensive (HT) rats. Systolic BP, phosphorylation of ERK, aortic superoxide formation, and aortic spontaneous tone were compared in sham normotensive and HT rats. Infusion of ANG II (0.5 mg x kg(-1) x day(-1) for 6 days) significantly elevated the systolic BP (P<0.01). The phosphorylation of ERK1/2 vs. total ERK1/2 in thoracic aorta was enhanced, and superoxide was increased in the HT vs. the sham group (P<0.01). Spontaneous tone developed in the HT group, but not in the normotensive group. MAPK/ERK1/2 (MEK1/2)-ERK1/2 signaling pathway inhibitors, PD-98059 (10 micromol/l), and U-0126 (10 micromol/l), significantly reduced the phosphorylation of ERK1/2, superoxide generation (P<0.01), and spontaneous tone (P<0.01) in HT. These findings suggest that ANG II infusion induces the production of superoxide and spontaneous tone and that both are dependent on ERK-MAPK activation. In endothelium-denuded aorta, however, MEK1/2 inhibitors did not inhibit the spontaneous tone, even though they significantly reduced superoxide generation similar to endothelium-intact aorta. These data suggest that inhibition of ERK1/2 signaling pathway, via PD-98059 or U-0126, may regulate spontaneous tone in an endothelium-dependent manner. In conclusion, these findings support the importance of the ERK1/2 signaling pathway in modulating vascular oxidative stress and subsequently mediating spontaneous tone in HT.

Topics: Angiotensin II; Animals; Aorta, Thoracic; Blood Pressure; Butadienes; Disease Models, Animal; Endothelium, Vascular; Enzyme Activation; Flavonoids; Hypertension; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Oxidative Stress; Phosphorylation; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Superoxides; Vasoconstriction

Ischemic spinal cord injury is a serious complication of aortic surgery. Although the extracellular signal-regulated kinases 1 and 2 are generally regarded as related to cell proliferation and survival, increasing evidence suggests that the role of the extracellular signal-regulated kinase pathway in ischemia/reperfusion injury is much more sophisticated.. Spinal cord ischemia in rats was induced by occluding the thoracic descending aorta with a balloon catheter introduced through a femoral artery, accompanied by concomitant exsanguination. Rats in the control group were given dimethyl sulfoxide (vehicle) before undergoing spinal cord ischemia/reperfusion injury. In the U0126-treated group, rats were pretreated with a specific inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinases 1 and 2, U0126, to inhibit extracellular signal-regulated kinases 1 and 2 phosphorylation. The sham-operated rats underwent aortic catheterization without occlusion. Parameters, including neurologic performance, neuronal survival, inflammatory cell infiltration, and interleukin-1beta production in the spinal cords, were compared between groups.. Early extracellular signal-regulated kinases 1 and 2 phosphorylation was observed after injury in the control group, followed by abundant microglial accumulation in the infarct area and increased interleukin-1beta expression. In the U0126 group, U0126 treatment completely blocked extracellular signal-regulated kinases 1 and 2 phosphorylation. Microglial activation and spinal cord interleukin-1beta levels were significantly reduced. Neuronal survival and functional performance were improved.. The mitogen-activated protein kinase/extracellular signal-regulated kinase pathway may play a noxious role in spinal cord ischemia/reperfusion injury by participating in inflammatory reactions and cytokine production. Targeting this pathway may be of potential value in terms of therapeutic intervention.

Topics: Animals; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Interleukin-1beta; Male; Microglia; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Spinal Cord Ischemia

The epithelial sodium channel (ENaC) is expressed in a variety of tissues, including the renal collecting duct, where it constitutes the rate-limiting step for sodium reabsorption. Liddle's syndrome is caused by gain-of-function mutations in the beta and gamma subunits of ENaC, resulting in enhanced Na reabsorption and hypertension. Epidermal growth factor (EGF) causes acute inhibition of Na absorption in collecting duct principal cells via an extracellular signal-regulated kinase (ERK)-dependent mechanism. In experiments with primary cultures of collecting duct cells derived from a mouse model of Liddle's disease (beta-ENaC truncation), it was found that EGF inhibited short-circuit current (Isc) by 24 +/- 5% in wild-type cells but only by 6 +/- 3% in homozygous mutant cells. In order to elucidate the role of specific regions of the beta-ENaC C terminus, Madin-Darby canine kidney (MDCK) cell lines that express beta-ENaC with mutation of the PY motif (P616L), the ERK phosphorylation site (T613A), and C terminus truncation (R564stop) were created using the Phoenix retroviral system. All three mutants exhibited significant attenuation of the EGF-induced inhibition of sodium current. In MDCK cells with wild-type beta-ENaC, EGF-induced inhibition of Isc (<30 min) was fully reversed by exposure to an ERK kinase inhibitor and occurred with no change in ENaC surface expression, indicative of an effect on channel open probability (P(o)). At later times (>30 min), EGF-induced inhibition of Isc was not reversed by an ERK kinase inhibitor and was accompanied by a decrease in ENaC surface expression. Our results are consistent with an ERK-mediated decrease in ENaC open probability and enhanced retrieval of sodium channels from the apical membrane.

Topics: Amiloride; Amino Acid Motifs; Animals; Butadienes; Cell Line; Disease Models, Animal; Dogs; Down-Regulation; Epidermal Growth Factor; Epithelial Sodium Channel Blockers; Epithelial Sodium Channels; Hypertension; Kidney; Membrane Potentials; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Protein Structure, Tertiary; Protein Transport; Sodium; Sodium Channel Blockers; Syndrome; Transfection

Pluripotent embryonic stem (ES) cell therapy may be an attractive source for postinfarction myocardial repair and regeneration. However, the specific stimuli and signal pathways that may control ES cell-mediated cardiomyogenesis remains to be completely defined. The aim of the present study was to investigate (1) the effect and underlying signal transduction pathways of leukemia inhibitory factor (LIF) and bone-morphogenic protein-2 (BMP-2)-induced mouse ES cell (mES-D3 line) differentiation into cardiomyocytes (CMC) and (2) the efficacy of CMC precommitted mES cells for functional and anatomical cardiac repair in surgically-induced mouse acute myocardial infarction (AMI) model. Various doses of LIF and BMP-2 and their inhibitors or blocking antibodies were tested for mES differentiation to CMC in vitro. CMC differentiation was assessed by mRNA and protein expression of CMC-specific markers, Connexin-43, CTI, CTT, Mef2c, Tbx5, Nkx2.5, GATA-4, and alphaMHC. LIF and BMP-2 synergistically induced the expression of CMC markers as early as 2 to 4 days in culture. Signaling studies identified STAT3 and MAP kinase (ERK1/2) as specific signaling components of LIF+BMP-2-mediated CMC differentiation. Inhibition of either STAT3 or MAPK activation by specific inhibitors drastically suppressed LIF+BMP-2-mediated CMC differentiation. Moreover, in mouse AMI, transplantation of lentivirus-GFP-transduced, LIF+BMP-2 precommitted mES cells, improved post-MI left ventricular functions, and enhanced capillary density. Transplanted cells engrafted in myocardium and differentiated into CMC and endothelial cells. Our data suggest that LIF and BMP-2 may synergistically enhance CMC differentiation of transplanted stem cells. Thus augmentation of LIF/BMP-2 downstream signaling components or cell type specific precommitment may facilitate the effects of ES cell-based therapies for post-MI myocardial repair and regeneration.

Topics: Animals; Biomarkers; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Butadienes; Capillaries; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Drug Synergism; Embryonic Stem Cells; Enzyme Inhibitors; Leukemia Inhibitory Factor; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Myocardial Infarction; Myocytes, Cardiac; Nitriles; Phosphorylation; Serine; Signal Transduction; STAT3 Transcription Factor; Stem Cell Transplantation; Transforming Growth Factor beta; Triterpenes; Tyrosine

HIV encephalitis (HIVE) is a neurodegenerative disease seen in approximately one in four terminally infected patients. Macaques infected with the simian immunodeficiency virus develop encephalitis (SIVE) very similar to the human disease. Neurodegeneration in both these conditions occurs from the effects of toxic viral proteins and neurotoxins derived from activated brain macrophages. Activated macrophages in the brain of macaques with SIVE can be labeled in vivo using positron emission tomography (PET) using PK11195, a ligand that binds the peripheral benzodiazepine receptor (PBR). However, the functional significance and mechanisms mediating increased PK11195 binding in activated brain macrophages are not known. Using post mortem tissues from macaques with SIVE and macrophages cell cultures activated with lipopolysaccharide (LPS), we show that [(3)H](R)-PK11195 binding is increased in activated macrophages. Increased [(3)H](R)-PK11195 binding in LPS-activated macrophages was reversed by pharmacologically inhibiting class III phosphatidylinositol-3 kinase (PI3-kinase), but was not altered by inhibiting the mitogen-activated protein kinase (MAP-kinase) pathway. Our results suggest that activated macrophages in lentiviral encephalitis show increased [(3)H](R)-PK11195 binding in a PI3-kinase-dependent fashion which may help elucidate the function of PBR in activated brain macrophages in HIVE and other neuroinflammatory diseases.

Topics: Analysis of Variance; Animals; Antineoplastic Agents; Autoradiography; Binding, Competitive; Butadienes; Chromones; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalitis; Enzyme Inhibitors; HIV; Isoquinolines; Lipopolysaccharides; Macaca; Macrophage Activation; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Positron-Emission Tomography; Simian Immunodeficiency Virus; Tritium

To study role of external signal regulated kinase (ERK) and transforming growth factor beta(1) (TGF-beta1) in asthma airway remodeling and to explore the regulation of glucocorticoids on ERK, TGF-beta1, and airway remodeling.. Thirty SD rats were randomly divided into 3 equal groups: control group; asthma group, undergoing intra-peritoneal injection of ovalbumin (OVA) on days 1 and 8 and inhalation of OVA every other day for 8 weeks since day 15 to establish chronic asthma models; dexamethasone (DM) intervention group, undergoing intra-peritoneal injection of DM 30 min before every inhalation instigation; and control group, receiving normal saline instead of DM. 1 - 2 hours after the last instigation the left lungs were taken out. The total bronchial wall thickness (Wat) and smooth muscle thickness (Wam) were measured by image analysis system. Phosphorylated ERK (P-ERK) was detected by immunohistochemistry. 1 - 2 hours after the last instigation blood samples were collected from the femoral artery. The concentration of transforming growth factor (TGF)-beta1 in the serum was measured by sandwich ELISA. Rat airway epithelial cells were cultured, stimulated with platelet-derived growth factor-BB (PDGF-BB, 1, 10, 25, or 50 microg/L), U0126 (specific inhibitor of phosphorylation of ERK), or budesonide (BUD). Western blotting was used to detect the P-ERK level. The level of TGF-beta1 in the cell culture supernatant was detected by sandwich ELISA.. The Wat and Wam of the asthma group was significantly higher than those of the control group (both P < 0.01), and the Wat and Wam of the DM group were both significantly lower than those of the asthma group (both P < 0.01). The mean optical density of P-ERK and concentration of TGF-beta1 in the serum of the asthma group were 31.1 +/- 2.2 and 28.1 +/- 7.4 microg/L respectively, both significantly higher than those of the control group (12.8 +/- 2.4 and 13.6 +/- 2.7 microg/L respectively, both P < 0.01), and the mean optical density of P-ERK and concentration of TGF-beta1 in the serum of the DM group were 18.7 +/- 3.1 and 15.0 +/- 3.2 microg/L respectively, both significantly lower than those asthma group (both P < 0.01). In the PDGF-BB (25 microg/L) stimulated cells marked phosphorylation of ERK occurred 15 min later, the level of P-ERK remained high up to 8 hour later, and the maximal activation occurred at the period of 2 h - 4 h later, 6.5 +/- 0.4 times that of the control value (P < 0.01). The phosphorylation levels of ERK depended on the concentration of PDGF-BB and the maximal level phosphorylation was detected with the concentration of PDGF-BB of 50 microg/L, which was 4.1 +/- 0.3 times that of the control value (P < 0.01). U0126 and BUD inhibited the phosphorylation of ERK in the cells stimulated by PDGF-BB of the concentration of 25 microg/L. there was no difference in the level of TGF-beta1 in the cell culture supernatant among different groups.. Phosphorylation of ERK and TGF-beta1 have an important role in asthma airway remodeling; PDGF-BB does not induce normal rat airway epithelial cells to product or release TGF-beta1 by phosphorylation of ERK. Glucocorticoids can inhibit phosphorylation of ERK.

Topics: Animals; Asthma; Becaplermin; Bronchi; Butadienes; Dexamethasone; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Glucocorticoids; Injections, Intraperitoneal; Lung; Nitriles; Phosphorylation; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Random Allocation; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta1

The aim of this study was to determine whether inhibition of the extracellular-regulated kinase signaling pathway decreases acute amphetamine-induced behavioral activity and neuropeptide gene expression in the rat striatum. Western blotting revealed that extracellular-regulated kinase1/2 phosphorylation was highly induced in the rat striatum 15 min after an acute amphetamine (2.5 mg/kg, i.p.) injection without altering the total amount of extracellular-regulated kinase protein. In a separate experiment, the systemic injection of SL327, a selective inhibitor of extracellular regulated kinase kinase that crosses the blood-brain barrier, 1 h prior to amphetamine administration decreased amphetamine-induced vertical and horizontal activity. Quantitative in situ hybridization histochemistry showed that SL327 abolished the high levels of preproenkephalin and preprodynorphin mRNA induced by amphetamine in the striatum with no alteration of their basal levels. In another set of experiments, the hyperlocomotor activity induced by amphetamine was reduced by pretreatment with intra-striatal infusion of U0126. U0126 also blocked the amphetamine-induced increases in phospho-extracellular-regulated kinase and preproenkephalin and preprodynorphin gene expression in the striatum. These data indicate that activation of the extracellular-regulated kinase cascade contributes to the behavioral effects and changes in striatal neuropeptide gene expression induced by acute amphetamine.

Topics: Aminoacetonitrile; Amphetamine; Amphetamine-Related Disorders; Animals; Butadienes; Corpus Striatum; Disease Models, Animal; Dynorphins; Enkephalins; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Male; MAP Kinase Signaling System; Motor Activity; Neuropeptides; Nitriles; Phosphorylation; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation

Pain during inflammatory joint diseases is enhanced by the generation of hypersensitivity in nociceptive neurons in the peripheral nervous system. To explore the signaling mechanisms of mechanical hypersensitivity during joint inflammation, experimental arthritis was induced by injection of complete Freund's adjuvant (CFA) into the synovial cavity of rat knee joints. As a pain index, the struggle threshold of the knee extension angle was measured. In rats with arthritis, the phosphorylation of extracellular signal-regulated kinase (ERK), induced by passive joint movement, increased significantly in dorsal root ganglion (DRG) neurons innervating the knee joint compared to the naïve rats that received the same movement. The intrathecal injection of a MEK inhibitor, U0126, reduced the phosphorylation of ERK in DRG neurons and alleviated the struggle behavior elicited by the passive movement of the joint. In addition, the injection of U0126 into the joint also reduced the struggle behavior. These findings indicate that the ERK signaling is activated in both cell bodies in DRG neurons and peripheral nerve fibers and may be involved in the mechanical sensitivity of the inflamed joint. Furthermore, the phosphorylated ERK-positive neurons co-expressed the P2X3 receptor, and the injection of TNP-ATP, which antagonizes P2X receptors, into the inflamed joint reduced the phosphorylated ERK and the struggle behavior. Thus, it is suggested that the activation of the P2X3 receptor is involved in the phosphorylation of ERK in DRG neurons and the mechanical hypersensitivity of the inflamed knee joint.

Topics: Adenosine Triphosphate; Animals; Arthritis, Experimental; Axonal Transport; Butadienes; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Freund's Adjuvant; Ganglia, Spinal; Hyperalgesia; Injections, Intra-Articular; Injections, Spinal; Male; Neurons, Afferent; Nitriles; Osteoarthritis, Knee; Pain; Phosphorylation; Protein Processing, Post-Translational; Purinergic P2 Receptor Antagonists; Range of Motion, Articular; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Signal Transduction; Stifle; Stress, Mechanical

Thalidomide has gained renewed interest as a cancer therapeutic due to its potential antiangiogenic effects. The thalidomide analogues CPS11 and CPS49 are active in preclinical angiogenesis assays and xenograft model systems, but the biochemical basis for these observations is unclear.. To address this question, we assessed the toxicity of these thalidomide analogues in cancer cells, endothelial cells, and genetically modified cells using assays that measure apoptotic and nonapoptotic cell death. Phosphospecific and native antibodies were used in immunoblotting and immunohistochemical experiments to assess the activation states of kinases that control cellular survival in vitro and in vivo.. CPS49 predominantly induced nonapoptotic cell death in lung cancer cells, prostate cancer cells, and endothelial cells in a dose-dependent manner, whereas CPS11 was not cytotoxic. CPS49 did not inhibit kinases that promote survival, such as Akt or extracellular signal-regulated kinase, but rather rapidly activated the stress kinase p38 pathway in both cancer cells and endothelial cells. CPS49 activated p38 in tumor xenografts. Using p38alpha-/- cells or an inhibitor of p38, we show that the presence and activation of p38alpha is important for cytotoxicity in all cell types examined.. Our studies identify a unifying mechanism of action for cytotoxicity of the tetraflourinated thalidomide analogue, CPS49, and suggest that activation of p38 could serve as a biomarker in clinical trials with CPS49.

Topics: Animals; Butadienes; Cell Death; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Endothelial Cells; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; Mice; Mice, SCID; Mitogen-Activated Protein Kinase 14; Neoplasms; Nitriles; Pyridines; Structure-Activity Relationship; Thalidomide; Xenograft Model Antitumor Assays

Glucagon-like peptide 1 (GLP-1), a gut incretin hormone that stimulates insulin secretion, also activates antiapoptotic signaling pathways such as phosphoinositide 3-kinase and mitogen-activated protein kinase in pancreatic and insulinoma cells. Since these kinases have been shown to protect against myocardial injury, we hypothesized that GLP-1 could directly protect the heart against such injury via these prosurvival signaling pathways. Both isolated perfused rat heart and whole animal models of ischemia/reperfusion were used, with infarct size measured as the end point of injury. In both studies, GLP-1 added before ischemia demonstrated a significant reduction in infarction compared with the valine pyrrolidide (an inhibitor of its breakdown) or saline groups. This protection was abolished in the in vitro hearts by the GLP-1 receptor antagonist exendin (9-39), the cAMP inhibitor Rp-cAMP, the PI3kinase inhibitor LY294002, and the p42/44 mitogen-activated protein kinase inhibitor UO126. Western blot analysis demonstrated the phosphorylation of the proapoptotic peptide BAD in the GLP-1-treated groups. We show for the first time that GLP-1 protects against myocardial infarction in the isolated and intact rat heart. This protection appears to involve activating multiple prosurvival kinases. This finding may represent a new therapeutic potential for this class of drug currently undergoing clinical trials in the treatment of type 2 diabetes.

Topics: Animals; Blood Pressure; Butadienes; Chromones; Disease Models, Animal; Enzyme Inhibitors; Glucagon; Glucagon-Like Peptide 1; Heart Rate; In Vitro Techniques; Insulin; Insulin Secretion; Male; MAP Kinase Signaling System; Morpholines; Myocardial Infarction; Myocardial Reperfusion Injury; Nitriles; Peptide Fragments; Phosphoinositide-3 Kinase Inhibitors; Protein Precursors; Rats; Rats, Sprague-Dawley; Time Factors

Using a rat model of craving and relapse, we have previously found time-dependent increases in cue-induced cocaine seeking over the first months of withdrawal from cocaine, suggesting that drug craving incubates over time. Here, we explored the role of the amygdala extracellular signal-regulated kinase (ERK) signaling pathway in this incubation. Cocaine seeking induced by exposure to cocaine cues was substantially higher after 30 withdrawal days than after 1 withdrawal day. Exposure to these cues increased ERK phosphorylation in the central, but not the basolateral, amygdala after 30 d, but not 1 d, of withdrawal. After 30 d of withdrawal from cocaine, inhibition of central, but not basolateral, amygdala ERK phosphorylation decreased cocaine seeking. After 1 d of withdrawal, stimulation of central amygdala ERK phosphorylation increased cocaine seeking. Results suggest that the incubation of cocaine craving is mediated by time-dependent increases in the responsiveness of the central amygdala ERK pathway to cocaine cues.

Topics: Amygdala; Anesthetics, Local; Animals; Behavior, Addictive; Behavior, Animal; Blotting, Western; Butadienes; Cocaine; Cocaine-Related Disorders; Conditioning, Operant; Cues; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Extinction, Psychological; Extracellular Signal-Regulated MAP Kinases; Food; Injections, Intravenous; Male; N-Methylaspartate; Nitriles; Phosphorylation; Rats; Rats, Long-Evans; Reinforcement, Psychology; Self Administration; Signal Transduction; Substance Withdrawal Syndrome; Time Factors

We have studied the involvement of the thrombin receptor [protease-activated receptor-1 (PAR-1)] in astrogliosis, because extravasation of PAR-1 activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders. PAR1-/- animals show a reduced astrocytic response to cortical stab wound, suggesting that PAR-1 activation plays a key role in astrogliosis associated with glial scar formation after brain injury. This interpretation is supported by the finding that the selective activation of PAR-1 in vivo induces astrogliosis. The mechanisms by which PAR-1 stimulates glial proliferation appear to be related to the ability of PAR-1 receptor signaling to induce sustained extracellular receptor kinase (ERK) activation. In contrast to the transient activation of ERK by cytokines and growth factors, PAR-1 stimulation induces a sustained ERK activation through its coupling to multiple G-protein-linked signaling pathways, including Rho kinase. This sustained ERK activation appears to regulate astrocytic cyclin D1 levels and astrocyte proliferation in vitro and in vivo. We propose that this PAR-1-mediated mechanism underlying astrocyte proliferation will operate whenever there is sufficient injury-induced blood-brain barrier breakdown to allow extravasation of PAR-1 activators.

Topics: Amides; Analysis of Variance; Animals; Animals, Newborn; Astrocytes; Blotting, Northern; Blotting, Western; Brain Injuries; Bromodeoxyuridine; Butadienes; Cell Count; Cell Movement; Cell Proliferation; Cells, Cultured; Coculture Techniques; Colforsin; Cyclin D1; Disease Models, Animal; Drug Interactions; Enzyme Inhibitors; Functional Laterality; Glial Fibrillary Acidic Protein; Gliosis; Immunohistochemistry; Male; MAP Kinase Kinase Kinases; Mice; Mice, Knockout; Microglia; Nitriles; Oligopeptides; Pyridines; Receptor, PAR-1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thrombin; Time Factors

It has been proposed that mitogen-activated protein kinase (MAPK) pathways may play a role in the regulation of pro-inflammatory cytokines, such as interlukine-1, during cerebral ischemia. Our previous study showed that extracellular-signal-regulated kinases 1 and 2 (ERK 1/2) were activated during focal cerebral ischemia in mice [J. Cereb. Blood Flow Metab. 20 (2000) 1320]. However, the effect of ERK 1/2 activation in focal cerebral ischemia is still unclear. In this study we reported that in vivo phospho-ERK 1/2 expression increased following 30 min of middle cerebral artery occlusion (MCAO) in the mouse brain in both the ischemic core and perifocal regions. Western blot analysis and immunohistochemistry demonstrated that pro-treatment with 1,4-diamino-2,3-dicyano-1,4-bis butadiene (U0126) [J. Biol. Chem. 273 (1998) 18623] could significantly inhibit mouse brain phospho-MEK 1/2 and phospho-ERK 1/2 expression after 1-2 h of MCAO (p<0.05). Compared to the control group of mice, brain infarct volume was significantly decreased after 24 h of MCAO in the U0126-treated mice (27+/-6 vs. 46+/-9 mm(2), p<0.05). Inhibition of the MEK/ERK 1/2 pathway also prevented downstream kinase Elk-1 phosphorylation, and further reduced cytokine IL-1beta mRNA, but not TNFalpha, IL-1alpha, or chemokine MIP-1alpha mRNA expression. Our data demonstrates that in vivo the close linking of MEK 1/2, ERK 1/2, Elk-1, and IL-1 mRNA expression in the cerebral ischemia animals suggests that ERK 1/2 pathway activation is important in pro-inflammatory cytokine IL-1beta signaling, which induces an inflammatory response and exacerbates ischemic brain injury. Inhibiting the ERK 1/2 pathway may therefore provide a novel approach for the reduction of ischemia-induced IL-1beta overexpression.

Topics: Animals; Blotting, Western; Brain; Brain Ischemia; Butadienes; Chemokine CCL3; Chemokine CCL4; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Immunohistochemistry; Infarction, Middle Cerebral Artery; Interleukin-1; Macrophage Inflammatory Proteins; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Time Factors; Tumor Necrosis Factor-alpha

The extracellular signal-regulated kinase pathway of the mitogen-activated protein kinase signal transduction cascade has been implicated in the neuronal and endothelial dysfunction witnessed following cerebral ischemia-reperfusion injury. Extracellular signal-regulated kinase is activated by mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2. We evaluated the ability of a mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2-specific inhibitor (U0126) to block extracellular signal-regulated kinase activation and mitigate ischemic neuronal damage in a model of deep hypothermic circulatory arrest.. Piglets underwent normal flow cardiopulmonary bypass (control, n = 4), deep hypothermic circulatory arrest (n = 6), and deep hypothermic circulatory arrest with U0126 (n = 5) at 20 degrees C for 60 minutes. The deep hypothermic circulatory arrest with U0126 group was given 200 microg/kg of U0126 45 minutes prior to initiation of bypass followed by 100 microg/kg at reperfusion. Following 24 hours of post-cardiopulmonary bypass recovery, brains were harvested. Eleven distinct cortical regions were evaluated for neuronal damage using hematoxylin and eosin staining. A section of ischemic cortex was further evaluated by immunohistochemistry with rabbit polyclonal antibody against phosphorylated extracellular signal-regulated kinase 1/2.. The deep hypothermic circulatory arrest and deep hypothermic circulatory arrest with U0126 groups displayed diffuse ischemic changes. However, the deep hypothermic circulatory arrest with U0126 group possessed significantly lower neuronal damage scores in the right frontal watershed zone of cerebral cortex, basal ganglia, and thalamus (P < or =.05) and an overall trend toward neuroprotection versus the deep hypothermic circulatory arrest group. This neuroprotection was accompanied by nearly complete blockade of phosphorylated extracellular signal-regulated kinase in the cerebral vascular endothelium.. In this experimental model of deep hypothermic circulatory arrest, U0126 blocked extracellular signal-regulated kinase activation and provided a significant neuroprotective effect. These results support targeting of the extracellular signal-regulated kinase pathway for inhibition as a novel therapeutic approach to mitigate neuronal damage following deep hypothermic circulatory arrest.

Topics: Animals; Animals, Newborn; Brain Ischemia; Butadienes; Cardiopulmonary Bypass; Cerebrovascular Circulation; Disease Models, Animal; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Heart Arrest, Induced; Hypothermia, Induced; Immunohistochemistry; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Models, Cardiovascular; Neurons; Nitriles; Postoperative Complications; Signal Transduction; Swine; Treatment Outcome

Extracellular signal-regulated protein kinase (ERK) is a mitogen-activated protein kinase (MAPK) that mediates several cellular responses to mitogenic and differentiation signals, and activation of ERK in dorsal horn neurons by noxious stimulation is known to contribute to pain hypersensitivity. In order to elucidate the pathophysiological mechanisms of the cauda equina syndrome, secondary to spinal canal stenosis, we evaluated walking dysfunction triggered by forced exercise and activation of ERK in the dorsal horn using a rat model of neuropathic intermittent claudication. Rats in the lumbar canal stenosis (LCS) group showed a shorter running distance from 1 to 14 days after surgery. Two minutes after running on the treadmill apparatus, phosphorylation of ERK was induced in neurons in the superficial laminae in the LCS group but not in the sham group, whereas there was no change in the deeper laminae. Intrathecal administration of the MAPK kinase inhibitor, U0126, 30 min before running, clearly increased the running distance, whereas there was no significant change in the vehicle control group 3 days after surgery. In addition, a prostaglandin E1 analog, OP-1206 alpha-CD, administered orally, improved the walking dysfunction, and further, inhibited activation of ERK following running 7 days after surgery. These findings suggest that intermittent claudication triggered by forced walking might affect the phosphorylation of ERK in the superficial laminae, possibly via transient (partial) ischemia of the spinal cord. ERK activation in the dorsal horn neurons may be involved in the transient pain in the neuropathic intermittent claudication model.

Topics: Alprostadil; Analysis of Variance; Animals; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Immunohistochemistry; Intermittent Claudication; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Spinal Stenosis; Walking

Transcription factor Ets-1 has been reported to regulate angiogenesis in vascular endothelial cells. Here, we investigated a mechanism that may regulate the expression of Ets-1 in vascular endothelial growth factor (VEGF)- and hypoxia-induced retinal neovascularization and that may have potential to inhibit ocular neovascular diseases. VEGF and hypoxia increased Ets-1 expression in cultured bovine retinal endothelial cells. The VEGF-induced mRNA increase of Ets-1 was suppressed by a tyrosine kinase inhibitor (genistein), by inhibitors of MEK (mitogen-activated protein and extracellular signal-regulated kinase kinase) (PD98059 and UO126), and by inhibitors of protein kinase C (GF109203X, staurosporine, and Gö6976). Dominant-negative Ets-1 inhibited VEGF-induced cell proliferation, tube formation, and the expression of neuropilin-1 and angiopoietin-2. In a mouse model of proliferative retinopathy, Ets-1 mRNA was up-regulated. Intravitreal injection of dominant-negative Ets-1 suppressed retinal angiogenesis in a mouse model of proliferative retinopathy. In conclusion, VEGF induces Ets-1 expression in bovine retinal endothelial cells and its expression is protein kinase C/ERK pathway-dependent. Ets-1 up-regulation is involved in the development of retinal neovascularization, and inhibition of Ets-1 may be beneficial in the treatment of ischemic ocular diseases.

Topics: Adenoviridae; Angiopoietin-2; Animals; Blotting, Northern; Blotting, Western; Butadienes; Carbazoles; Cattle; Cell Division; Disease Models, Animal; DNA; Enzyme Inhibitors; Flavonoids; Genes, Dominant; Humans; Hypoxia; Indoles; Maleimides; Mice; Models, Biological; Neovascularization, Pathologic; Neuropilin-1; Nitriles; Phosphorylation; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Reperfusion Injury; Retina; RNA, Messenger; Staurosporine; Time Factors; Transcription Factors; Up-Regulation; Vascular Endothelial Growth Factor A

Mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in the activation of inflammatory cells. Recent findings revealed that the activity of p42/44 MAPK (also known as extracellular signal-regulated kinase (ERK)) in the lungs was significantly higher in asthmatic mice than in normal controls. We hypothesized that inhibition of ERK activity may have anti-inflammatory effects in allergic asthma. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of VCAM-1 expression, and airway hyperresponsiveness. Intraperitoneal administration of U0126, a specific MAPK/ERK kinase inhibitor, significantly (p < 0.05) inhibited OVA-induced increases in total cell counts, eosinophil counts, and IL-4, IL-5, IL-13, and eotaxin levels recovered in bronchoalveolar lavage fluid in a dose-dependent manner. U0126 also substantially (p < 0.05) reduced the serum levels of total IgE and OVA-specific IgE and IgG1. Histological studies show that U0126 dramatically inhibited OVA-induced lung tissue eosinophilia, airway mucus production, and expression of VCAM-1 in lung tissues. In addition, U0126 significantly (p < 0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine in a dose-dependent manner. Western blot analysis of whole lung lysates shows that U0126 markedly attenuated OVA-induced tyrosine phosphorylation of ERK1/2. Taken together, our findings implicate that inhibition of ERK signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.

Topics: Animals; Anti-Inflammatory Agents; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Butadienes; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Eosinophils; Immunoglobulin G; Male; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Mucus; Nitriles; Ovalbumin; Phosphorylation; Vascular Cell Adhesion Molecule-1

We have investigated the role of spinal extracellular signaling-regulated kinase-1 and -2 (ERK1/2) in a model of visceral pain and hyperalgesia induced by intracolonic instillation of irritants in adult mice. Instillation of either capsaicin or mustard oil induced a significant activation of lumbosacral spinal ERK1/2, measured by immunoblot, with a peak 2.4-fold increase over control levels between 45 and 90 min post-treatment. Intracolonic saline did not produce significant activation of lumbosacral spinal ERK1/2, and none of the treatments evoked ERK1/2 activation in thoracic or cervical spinal cord. These studies suggested a preferential nuclear localization, which was explored by subcellular fractionation. Both mustard oil and capsaicin produced a redistribution of phosphorylated ERK1/2 from cytosol into the nucleus that was statistically significant at 45 min after treatment. Spinal ERK1/2 activation with capsaicin treatment correlated with the development of prolonged referred hyperalgesia. The upstream inhibitor of ERK phosphorylation, U0126 (100-400 microg/kg, i.v., 10 min pre-capsaicin), dose-dependently inhibited referred hyperalgesia 3-6 h after capsaicin. Treatment with U0126 did not affect spontaneous pain behavior or colon inflammation. Our data show that ERK activation plays a specific role in maintaining prolonged referred (secondary) hyperalgesia in visceral pain. The time course and subcellular localization of the effects observed suggest that ERK is involved in transcriptional events underlying the maintenance of secondary hyperalgesia.

Topics: Alkaloids; Animals; Behavior, Animal; Butadienes; Cytosol; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hyperalgesia; Immunoblotting; Male; Mice; Mice, Inbred Strains; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mustard Plant; Nitriles; Pain; Pain Measurement; Physical Stimulation; Plant Extracts; Plant Oils; Reaction Time; Spinal Cord; Time Factors; Visceral Afferents

Hyperplasia of middle-ear mucosa (MEM) during otitis media (OM) is thought to be partially mediated by the actions of growth factors and their receptors. The intracellular pathway leading from the small G-protein Ras to the extracellular regulated kinases (Erks) often links growth factor stimulation to cellular proliferation. This study assessed whether this pathway is involved in MEM hyperplasia during bacterial OM via the activation of Erk1/Erk2 in MEM of an in vivo rat bacterial OM model. Activation was maximal at 1 and 6 h and at 1 week after introduction of bacteria into the middle ear. Additionally, an in vitro model of rat MEM in bacterial OM was treated with farnesyl transferase inhibitor 277 or the Mek inhibitor U0126. MEM explants treated with either inhibitor demonstrated significant suppression of bacterially induced growth. These data support a role for Ras and Erk signaling in MEM hyperplasia during bacterial OM.

Topics: Animals; Butadienes; Disease Models, Animal; Ear, Middle; Enzyme Inhibitors; Haemophilus Infections; Haemophilus influenzae; Hyperplasia; In Vitro Techniques; Male; Methionine; Mitogen-Activated Protein Kinases; Mucous Membrane; Nitriles; Otitis Media; ras Proteins; Rats; Signal Transduction; Time Factors

Activation of ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinase in dorsal horn neurons of the spinal cord by peripheral noxious stimulation contributes to short-term pain hypersensitivity. We investigated ERK activation by peripheral inflammation and its involvement in regulating gene expression in the spinal cord and in contributing to inflammatory pain hypersensitivity. Injection of complete Freund's adjuvant (CFA) into a hindpaw produced a persistent inflammation and a sustained ERK activation in neurons in the superficial layers (laminae I-IIo) of the dorsal horn. CFA also induced an upregulation of prodynorphin and neurokinin-1 (NK-1) in dorsal horn neurons, which was suppressed by intrathecal delivery of the MEK (MAP kinase kinase) inhibitor U0126. CFA-induced phospho-ERK primarily colocalized with prodynorphin and NK-1 in superficial dorsal horn neurons. Although intrathecal injection of U0126 did not affect basal pain sensitivity, it did attenuate both the establishment and maintenance of persistent inflammatory heat and mechanical hypersensitivity. Activation of the ERK pathway in a subset of nociceptive spinal neurons contributes, therefore, to persistent pain hypersensitivity, possibly via transcriptional regulation of genes, such as prodynorphin and NK-1.

Topics: Animals; Butadienes; Disease Models, Animal; Enkephalins; Enzyme Activation; Enzyme Inhibitors; Freund's Adjuvant; Hindlimb; Hyperalgesia; Inflammation; Injections, Spinal; Male; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Pain; Posterior Horn Cells; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Substance P; Up-Regulation

1. IL-13 is an important mediator in inflammatory diseases such as asthma. IL-13 is mainly produced by T cells. However, signalling pathways leading to induction of this cytokine are not well-characterized. We analysed the regulation of IL-13 in human peripheral blood mononuclear cells and CD4(+) T cells. 2. Cyclosporine (CsA) and FK-506 inhibited IL-13 synthesis, when cells were stimulated by TPA/ionomycin. However, stimulation by alpha-CD3/alpha-CD28 led to an enhanced IL-13 synthesis. 3. NF-kappa B inhibitor N-tosyl-L-lysine chloromethylketone (TLCK) inhibited IL-13 synthesis more effectively after TPA/ionomycin stimulation. After alpha-CD3/alpha-CD28 stimulation, only 300 microM TLCK inhibited IL-13 synthesis. Dexamethasone inhibited IL-13 equally effective after alpha-CD3/alpha-CD28 and TPA/ionomycin stimulation. 4. p38 MAPK inhibitor SB203580 inhibited IL-13 synthesis only partially. MEK inhibitor U0126 inhibited TPA/ionomycin induced IL-13 synthesis very effectively, whereas alpha-CD3/alpha-CD28 stimulated IL-13 induction was resistant to this drug. 5. These results were confirmed in purified CD4(+) T cells. In difference to PBMCs alpha-CD3/alpha-CD28 stimulated IL-13 synthesis was effectively inhibited by CsA, FK-506 and U0126. 6. Therefore U0126 was tested in an animal model of allergic asthma. We could demonstrate for the first time that inhibition of the MEK - ERK cascade is a therapeutic option for asthma. Intraperitoneal administration of 10 mg kg(-1) U0126 reduced lung eosinophilia in ovalbumin-challenged Brown Norway rats by 44%. 7. These results demonstrate that different signalling pathways are involved in regulating IL-13 synthesis in primary human T cells. Characterizing highly potent inhibitors of IL-13 synthesis can be exploited to identify new drugs to treat immunological diseases such as asthma.

Topics: Animals; Anti-Asthmatic Agents; Asthma; Butadienes; Calcium Signaling; CD4-Positive T-Lymphocytes; Cells, Cultured; Cyclosporine; Disease Models, Animal; DNA-Binding Proteins; Enzyme Inhibitors; Humans; Inhibitory Concentration 50; Injections, Intraperitoneal; Interleukin-13; Leukocytes, Mononuclear; Lymphocytes; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; NF-kappa B; NFATC Transcription Factors; Nitriles; Nuclear Proteins; Pulmonary Eosinophilia; Rats; Rats, Inbred BN; RNA, Messenger; Tacrolimus; Time Factors; Transcription Factors

It has been suggested that mitogen-activated protein kinase (MAPK) is involved in cerebral vasospasm after subarachnoid hemorrhage. The present study was undertaken to explore the inhibitory effect of U0126, a novel MAPK inhibitor, in the contraction of the rabbit basilar artery by 3 spasmogens: hemolysate, oxyhemoglobin, and bloody cerebrospinal fluid (CSF) from patients with vasospasm.. The contraction and relaxation of rabbit basilar arteries were measured by isometric tension. MAPK immunoprecipitation was assessed by Western blot analysis.. (1) Pretreatment of the rabbit basilar arteries with U0126 reduced contractions to hemolysate, oxyhemoglobin, or bloody CSF applied subsequently. (2) In the absence of endothelial cells, U0126 produced an inhibitory effect similar to the contractions induced by hemolysate, oxyhemoglobin, or bloody CSF. (3) U0126 relaxed the sustained contraction induced by hemolysate, oxyhemoglobin, or bloody CSF. (4) Hemolysate, oxyhemoglobin, and bloody CSF enhanced MAPK immunoprecipitation. (5) U0126 reduced MAPK immunoprecipitation induced by hemolysate, oxyhemoglobin, and bloody CSF. (6) Hemolysate, oxyhemoglobin, and bloody CSF significantly increased MAPK activity in the rabbit basilar artery. (7) U0126 abolished the effect of hemolysate, oxyhemoglobin, or bloody CSF on MAPK activation.. This study demonstrated a role of MAPK in the contraction of rabbit basilar arteries by hemolysate, oxyhemoglobin, and bloody CSF. MAPK inhibitor U0126 may be useful in the treatment of cerebral vasospasm.

Topics: Animals; Basilar Artery; Blotting, Western; Butadienes; Cerebrospinal Fluid; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Female; Hemoglobins; Hemolysis; In Vitro Techniques; Isometric Contraction; Male; Mitogen-Activated Protein Kinases; Nitriles; Oxyhemoglobins; Precipitin Tests; Rabbits; Vasodilator Agents; Vasospasm, Intracranial

Mitogen-activated protein kinase (MAPK) may be involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage. This study was conducted to investigate the ability of the MAPK inhibitors PD-98059 and U-0126 to reverse vasospasm in a double-hemorrhage model in dogs.. Twenty-two adult mongrel dogs of either sex, each weighing 18 to 24 kg, were divided randomly into four groups: control SAH (four dogs), vehicle- (dimethyl sulfoxide, six dogs), PD-98059- (six dogs), and U-0126-treated groups (six dogs). The double-hemorrhage model was created by an autologous blood injection into the cisterna magna on Days 0 and 2. An intracisternal injection of MAPK inhibitors was administered once per day on Days 3 through 6. Cerebral angiography was performed on Days 0 and 7 before the animals were killed. Western blot analysis was used to study the effects of hemorrhage and drug treatment on the MAPK immunoprecipitation. Severe vasospasm developed in the dogs in the control and vehicle-treated groups (basilar artery [BA] diameter reduction 46.6 +/- 5.5% and 49.3 +/- 4.6%, respectively). In the PD-98059-treated group, most of the dogs developed mild vasospasm (18.9 +/- 6.2%). In the U-0126-treated group, severe vasospasm was observed despite treatment (39.6 +/- 6.4%). The PD-98059 but not the U-0126 abolished MAPK immunoprecipitation in the spastic BAs. However, treatment with either PD-98059 or U-0126 improved the clinical scores of the dogs.. The present study is the first in which the effects of MAPK inhibitors on vasospasm have been investigated in vivo. The authors demonstrate that MAPK may play a role in vasospasm and that PD-98059 is a potential candidate for the treatment of cerebral vasospasm.

Topics: Animals; Butadienes; Cerebral Angiography; Cerebral Arteries; Disease Models, Animal; Dogs; Flavonoids; Mitogen-Activated Protein Kinases; Nitriles; Subarachnoid Hemorrhage; Vasodilator Agents; Vasospasm, Intracranial