Nitric oxide-mediated activation of extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of newborn piglets

Previous studies have shown that mitogen-activated protein kinases, such as extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK), mediate signal transduction from cell surface receptors to the nucleus and phosphorylate anti-apoptotic proteins thereby regulating programmed cell death. The present study tests the hypotheses that hypoxia activates ERK and JNK in neuronal nuclei of newborn piglets and the hypoxia-induced activation of ERK and JNK is mediated by nitric oxide (NO). Activated ERK and JNK were assessed by determining phosphorylated ERK and JNK using immunoblotting in six normoxic (Nx) and 10 hypoxic (Hx) and five N-nitro-L-arginine (a NOS inhibitor, 40 mg/kg,) -pretreated hypoxic (N-nitro-L-arginine [NNLA]-Hx) 3-5 day old piglets. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Cortical neuronal nuclei were isolated and the nuclear protein was analyzed for activated ERK and JNK using anti-phosphorylated ERK and JNK antibodies. Protein bands were detected using enhanced chemiluminescence method and analyzed by imaging densitometry. Protein density was expressed as absorbance ODxmm(2). ATP levels were 4.57+/-0.45 micromoles/g brain in the Nx group, 1.29+/-0.23 micromoles/g brain in the Hx group (P<0.05 vs Nx) and 1.50+/-0.14 micromoles/g brain in the NNLA-Hx group (P<0.05 vs Nx). PCr levels were 3.77+/-0.36 micromoles/g brain in the Nx group, 0.77+/-0.13 micromoles/g brain in the hypoxic group (P<0.05) and 1.02+/-0.24 in the NNLA-Hx group (P<0.05 vs Nx). Density of phosphorylated ERK protein was 170.5+/-53.7 ODxmm(2) in the Nx group as compared with 419.6+/-63.9 ODxmm(2) in the hypoxic group (P<0.001 vs Nx) and 270.0+/-28.7 in the NNLA-Hx group (P<0.002 vs Hx). Density of phosphorylated JNK protein was 172.8+/-42.8 ODxmm(2) in the normoxic group as compared with 364.6+/-60.1 ODxmm(2) in the Hx group (P<0.002) and 254.8+/-24.8 in the NNLA-Hx group (P<0.002 vs Hx). The data demonstrate increased phosphorylation of ERK and JNK during hypoxia indicating that hypoxia results in activation of ERK and JNK in neuronal nuclei of newborn piglets. The administration of NNLA, a NOS inhibitor, prevented the hypoxia-induced phosphorylation of ERK and JNK indicating that the hypoxia-induced activation of ERK and JNK in the cerebral cortical nuclei of newborn piglets is NO-mediated     

Low concentrations of nitric oxide (NO) induced cell death in PC12 cells through activation of p38 mitogen-activated protein kinase (p38 MAPK) but not via extracellular signal-regulated kinases (ERK1/2) or c-Jun N-terminal protein kinase (JNK)

Nitric oxide (NO), a highly reactive gaseous molecule, has been previously reported to induce apoptosis-like cell death even at a low concentration in PC12 cells. In this study, we examined NO-induced activation of members of the mitogen-activated protein kinase (MAPK) family, i.e., p38 MAPK, extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal protein kinase (JNK). Following the exposure of PC12 cells to an NO donor, (+)-(E)-4-ethyl-2-[hydroxyimino]-5-nitro-3-hexenamide (NOR3; 100 muM), the phosphorylation level of p38 MAPK increased time dependently from 2 to 6 h, but that of both ERK1/2 and JNK did not. Treatment with a p38 MAPK inhibitor SB203580 partially blocked the NOR3-induced cell death. Neither PD98059, U0126 (inhibitors of ERK1/2) nor SP600125 (a specific inhibitor of JNK) treatments had any significant effect on the NOR3-induced cell death. These findings suggest that the activation of a p38 MAPK pathway, but not that of ERK1/2 or JNK, plays an essential role in the apoptosis-like cell death induced by low concentrations of NO.     

c-Jun N-terminal kinase and p38 mitogen-activated protein kinase mediate double-strand RNA-induced inducible nitric oxide synthase expression in microglial cells

Double-stranded RNA (dsRNA) has been implicated as a potential immune stimulant in activating microglia, which can cause chronic neurodegeneration. In this study, we examined the involvement of different types of mitogen-activated protein kinases (MAPKs) in the induction of inducible nitric oxide synthase (iNOS) by dsRNA in microglial cells. Nitric oxide production was increased after exposure of microglia to 50 μg/mL dsRNA. Levels of dsRNA-induced nitrite production in a line of immortalized murine microglia (BV2) and in primary cultures of murine microglia were decreased by inhibition of JNK or p38 MAPK, but were increased by inhibition of extracellular signal-regulated kinase. Similar results were shown in the levels of dsRNA-induced iNOS gene expression in BV2 cells. Phosphorylation levels of p38 MAPK were increased, depending on p38 MAPK inhibitor concentrations, while activation levels of MAPKAPK2, a known p38 substrate, were inhibited. Thus, it is likely that SB203580 inhibited the kinase activity of p38 MAPK, resulting in the loss of a feedback inhibition regulatory loop of p38 MAPK in BV2 cells. These findings suggest that dsRNA stimulated iNOS expression via MAPK signaling pathways, including JNK and p38 MAPK.     

Mechanism of Ca2+-influx and Ca2+/calmodulin-dependent protein kinase IV activity during in utero hypoxia in cerebral cortical neuronal nuclei of the guinea pig fetus at term

Previously we showed that following hypoxia there is an increase in nuclear Ca(2+)-influx and Ca(2+)/calmodulin-dependent protein kinase IV activity (CaMK IV) in the cerebral cortex of term guinea pig fetus. The present study tests the hypothesis that clonidine administration will prevent hypoxia-induced increased neuronal nuclear Ca(2+)-influx and increased CaMK IV activity, by blocking high-affinity Ca(2+)-ATPase. Studies were conducted in 18 pregnant guinea pigs at term, normoxia (Nx, n=6), hypoxia (Hx, n=6) and clonidine with Hx (Hx+Clo, n=6). The pregnant guinea pig was exposed to a decreased FiO(2) of 0.07 for 60 min. Clonidine, an imidazoline inhibitor of high-affinity Ca(2+)-ATPase, was administered 12.5 microg/kg IP 30 min prior to hypoxia. Hypoxia was determined biochemically by ATP and phosphocreatine (PCr) levels. Nuclei were isolated and ATP-dependent (45)Ca(2+)-influx was determined. CaMK IV activity was determined by (33)P-incorporation into syntide 2 for 2 min at 37 degrees C in a medium containing 50mM HEPES (pH 7.5), 2mM DTT, 40muM syntide 2, 0.2mM (33)P-ATP, 10mM magnesium acetate, 5 microM PKI 5-24, 2 microM PKC 19-36 inhibitor peptides, 1 microM microcystine LR, 200 microM sodium orthovanadate and either 1mM EGTA (for CaMK IV-independent activity) or 0.8mM CaCl(2) and 1mM calmodulin (for total activity). ATP (mumoles/gbrain) values were significantly different in the Nx (4.62+/-0.2), Hx (1.65+/-0.2, p<0.05 vs. Nx), and Hx+Clo (1.92+/-0.6, p<0.05 vs. Nx). PCr (mumoles/g brain) values in the Nx (3.9+/-0.1), Hx (1.10+/-0.3, p<0.05 vs. Nx), and Hx+Clo (1.14+/-0.3, p<0.05 vs. Nx). There was a significant difference between nuclear Ca(2+)-influx (pmoles/mg protein/min) in Nx (3.98+/-0.4), Hx (10.38+/-0.7, p<0.05 vs. Nx), and Hx+Clo (7.35+/-0.9, p<0.05 vs. Nx, p<0.05 vs. Hx), and CaM KIV (pmoles/mg protein/min) in Nx (1314.00+/-195.4), Hx (2315.14+/-148.5, p<0.05 vs. Nx), and Hx+Clo (1686.75+/-154.3, p<0.05 vs. Nx, p<0.05 vs. Hx). We conclude that the mechanism of hypoxia-induced increased nuclear Ca(2+)-influx is mediated by high-affinity Ca(2+)-ATPase and that CaMK IV activity is nuclear Ca(2+)-influx-dependent. We speculate that hypoxia-induced alteration of high-affinity Ca(2+)-ATPase is a key step that triggers nuclear Ca(2+)-influx, leading to CREB protein-mediated increased expression of apoptotic proteins and hypoxic neuronal death.     

Differential regulation of mast cell cytokines by both dexamethasone and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580

Activated mast cells generate multiple cytokines but it is not known if these can be differentially regulated by pharmacological agents. We report here that the glucocorticoid dexamethasone (DEX) preferentially inhibited Ag-induced expression of IL-4 and IL-6 mRNA relative to TNF-alpha mRNA in RBL-2H3 cells. Likewise, the drug more readily inhibited release of IL-4 than TNF-alpha protein. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), enhanced Ag-induced TNF-alpha mRNA expression without affecting IL-4 or IL-6 mRNA. At the protein level, SB203580 exerted little effect on TNF-alpha release but inhibited IL-4 release; notably, the ratio of TNF-alpha : IL-4 increased markedly with the concentration of SB203580, confirming the differential regulation of these cytokines. PD98059, an inhibitor of MAPK kinase (MEK), a component of the p44/42 MAPK pathway, partially inhibited Ag-induced expression of mRNA for all three cytokines while cyclosporin A inhibited Ag-induced IL-4 and IL-6 mRNA more readily than TNF-alpha mRNA. Ag activation of the cells led to phosphorylation of p38 and p44/42 MAPK but this was not influenced by DEX. In conclusion, mast cell cytokines can be differentially regulated pre- and post-translationally by DEX and SB203580 but there does not appear to be a direct mechanistic link between the actions of these two drugs.     

Ketamine reduces inducible superoxide generation in human neutrophils in vitro by modulating the p38 mitogen-activated protein kinase (MAPK)-mediated pathway

Many cellular stresses and inflammatory stimuli can activate p38 mitogen-activated protein kinase (MAPK), a serine/threonine kinase in the MAPK family. The different stimuli act via different receptors or signalling pathways to induce phosphorylation of the cytosolic protein p47^phox, one subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Formyl–methionyl–leucyl–phenylalanine (fMLP) has been shown to induce the p38 MAPK phosphorylation during the respiratory burst in human neutrophils. Here, we show that treatment with S(+)-ketamine or R(-)-ketamine at different concentrations (50, 100, 200, 400 µM) reduced fMLP-induced superoxide anion generation and p47^phox phosphorylation in neutrophils in a concentration-dependent manner (y = −0·093x + 93·35 for S(+)-ketamine and y = −0·0982x + 95·603 for R(-)-ketamine, respectively). While treatment with 50 µM ketamine inhibited fMLP-induced superoxide generation by 10%, treatment with 400 µM S(+)-ketamine and R(-)-ketamine reduced fMLP-induced superoxide generation to 60·5 ± 8·3% and 60·0 ± 8·5%, respectively, compared with that in neutrophils treated with fMLP alone. Furthermore, treatment with ketamine down-regulated both fMLP-induced p47^phox and isoproterenol-induced p38 MAPK phosphorylation and superoxide production. Interestingly, treatment with SB203580, the p38 MAPK inhibitor, also mitigated fMLP-induced superoxide anion generation and p38 MAPK and p47^phox phosphorylation as well as apoptosis in a concentration-dependent fashion in neutrophils. Therefore, ketamine racemes inhibited fMLP-induced superoxide anion generation and p47^phox phosphorylation by modulating fMLP-mediated p38 MAPK activation in neutrophils.