Mitogen-activated protein kinases regulate COX-2 and mucosal recovery in ischemic-injured porcine ileum

Mitogen-activated protein kinase (MAPK) pathways transduce signals from a diverse array of extracellular stimuli. The three primary MAPK-signaling pathways are the extracellular regulated kinases (ERK1/2), p38 MAPK, and c-Jun NH(2)-terminal kinase (JNK). Previous research in our laboratory has shown that COX-2-elaborated prostanoids participate in recovery of mucosal barrier function in ischemic-injured porcine ileum. Because COX-2 expression is regulated in part by MAPKs, we postulated that MAPK pathways would play an integral role in recovery of injured mucosa. Porcine mucosa was subjected to 45 min of ischemia, after which tissues were mounted in Ussing chambers, and transepithelial electrical resistance (TER) was monitored as an index of recovery of barrier function. Treatment of tissues with the p38 MAPK inhibitor SB-203580 (0.1 mM) or the ERK1/2 inhibitor PD-98059 (0.1 mM) abolished recovery. Western blot analysis revealed that SB-203580 inhibited upregulation of COX-2 that was observed in untreated ischemic-injured mucosa, whereas PD-98059 had no effect on COX-2 expression. Inhibition of TER recovery by SB-203580 or PD-98059 was overcome by administration of exogenous prostaglandin E(2) (1 microM). The JNK inhibitor SP-600125 (0.1 mM) significantly increased TER and resulted in COX-2 upregulation. COX-2 expression appears to be positively and negatively regulated by the p38 MAPK and the JNK pathways, respectively. Alternatively, ERK1/2 appear to be involved in COX-2-independent reparative events that remain to be defined.     

MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone. p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-linked p38 MAPK.     

PKC-dependent delayed metabolic preconditioning is independent of transient MAPK activation

In this study we used an in vitro model of delayed preconditioning to investigate activation of mitogen-activated protein kinases (MAPKs) and their potential role in protection. Neonatal rat cardiomyocytes were preconditioned using a buffer containing glycolytic inhibitors and low pH (minimal metabolic preconditioning; MMPC) consisting of modified Krebs buffer, 10 mM 2-deoxyglucose, and 20 mM lactate, pH 6.8, for 2 h followed by 24 h of simulated reperfusion before lethal simulated ischemia (LSI). MAPK activation during the MMPC protocol was determined using phospho-specific antisera and the effect on protection determined following LSI. Rapid, transient phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 MAPK was observed during each of the MMPC, reperfusion, and LSI phases; an effect blocked by MAPK inhibitors PD-98059 and SB-203580, respectively, but not by the protein kinase C (PKC) inhibitor Ro31-8220. However, although MMPC was blocked by Ro31-8220, treatment with the MAPK inhibitors during the preconditioning protocol did not block delayed protection conferred by MMPC. Thus the data suggest that, in this model of delayed preconditioning, protection appears to be PKC dependent but independent of ERK1/2 or p38 MAPK activation.     

Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells

Rhythmic strain stimulates Caco-2 proliferation. We asked whether mitogen-activated protein kinase (MAPK) activation mediates strain mitogenicity and characterized upstream signals regulating MAPK. Caco-2 cells were subjected to strain on collagen I-precoated membranes or antibodies to integrin subunits. Twenty-four hours of cyclic strain increased cell numbers compared with static conditions. MAPK-extracellular signal-regulated kinase (ERK) kinase inhibition (20 microM PD-98059) blocked strain mitogenicity. p38 Inhibition (10 microM SB-202190) did not. Strain rapidly and time-dependently activated focal adhesion kinase (FAK), paxillin, ERK1 and 2, and p38 on collagen. c-Jun NH(2)-terminal kinase (JNK)1 and 2 exhibited delayed activation. Similar activation occurred when Caco-2 cells were subjected to strain on a substrate of functional antibody to the alpha2-, alpha3-, alpha6-, or beta1-integrin subunits but not on a substrate of functional antibody to the alpha5-subunit. FAK inhibition by FAK397 transfection blocked ERK2 and JNK1 activation by in vitro kinase assays, but pharmacological protein kinase C inhibition did not block ERK1 or 2 activation by strain. Strain-induced ERK signals mediate strain's mitogenic effects and may require integrins and FAK activation.     

Inhibition of p38 reduces myocardial infarction injury in the mouse but not pig after ischemia-reperfusion

The MAPK family member p38 is activated in the heart after ischemia-reperfusion (I/R) injury. However, the cardioprotective vs. proapoptotic effects associated with p38 activation in the heart after I/R injury remain unresolved. Another issue to consider is that the majority of past studies have employed the rodent as a model for assessing p38's role in cardiac injury vs. protection, while the potential regulatory role in a large animal model is even more uncertain. Here we performed a parallel study in the mouse and pig to directly compare the extent of cardiac injury after I/R at baseline or with the selective p38 inhibitor SB-239063. Infusion of SB-239063 5 min before ischemia in the mouse prevented ischemia-induced p38 activation, resulting in a 25% reduction of infarct size compared with vehicle-treated animals (27.9 +/- 2.9% vs. 37.5 +/- 2.7%). In the pig, SB-239063 similarly inhibited myocardial p38 activation, but there was no corresponding effect on the degree of infarction injury (43.6 +/- 4.0% vs. 41.4 +/- 4.3%). These data suggest a difference in myocardial responsiveness to I/R between the small animal mouse model and the large animal pig model, such that p38 activation in the mouse contributes to acute cellular injury and death, while the same activation in pig has no causative effect on these parameters.     

Vitamin C-induced activation of phospholipase D in lung microvascular endothelial cells: regulation by MAP kinases

Our earlier studies have shown that vitamin C at pharmacological doses (mM) induces loss of redox-dependent viability in bovine lung microvascular endothelial cells (BLMVECs) that is mediated by oxidative stress. Therefore, here, we investigated the vitamin C-induced activation of the lipid signaling enzyme, phospholipase D (PLD) in BLMVECs. Monolayer cultures of BLMVECs were treated with vitamin C (0-10 mM) for different time periods (0-2 h) and the activity of PLD was determined. Vitamin C induced activation of PLD in BLMVECs in a time- and dose-dependent fashion that was significantly attenuated by antioxidants, p38 mitogen-activated protein kinase (p38 MAPK)-specific inhibitor (SB203580), extracellular signal-regulated protein kinase (ERK)-specific inhibitor (PD98059), and transient transfection of cells with dominant-negative (DN)-p38 MAPK and DN-ERK1/ERK2. Vitamin C also induced phosphorylation and enhanced the activities of p38 MAPK and ERK in BLMVECs in a time-dependent fashion. It was also evident that vitamin C induced translocation of PLD(1) and PLD(2), association of p38 MAPK and ERK with PLD(1) and PLD(2), threonine phosphorylation of PLD(1) and PLD(2) and SB203580- and PD98059-inhibitable threonine phosphorylation of PLD(1) in BLMVECs. Transient transfection of BLMVECs with DN-p38 MAPK and DN-ERK1/ERK2 resulted in marked attenuation of vitamin C-induced phosphorylation of threonine in PLD(1) and PLD(2). We, for the first time, showed that vitamin C at pharmacological doses, activated PLD in the lung microvascular ECs through oxidative stress and MAPK activation.     

Characterization of protein kinase pathways responsible for Ca2+ sensitization in rat ileal longitudinal smooth muscle

We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca2+ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 microM microcystin induced LC20 diphosphorylation and contraction of beta-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF-109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 muM, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca2+ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle.     

Evidence for the role of p38 MAP kinase in hypoxia-induced pulmonary vasoconstriction

Mitogen-activated protein (MAP) kinases regulate smooth muscle cell contraction. Hypoxia contracts pulmonary arteries by mechanisms that are incompletely understood. We hypothesized that hypoxic contraction of pulmonary arteries involves activation of the MAP kinases. To test this hypothesis, we studied the effects of SB-202190, a p38 MAP kinase inhibitor, PD-98059 and UO-126, two structurally different MEKK inhibitors, and anisomycin, a stimulator of p38 MAP kinase on acute hypoxia-induced contraction in rat conduit pulmonary artery rings precontracted with phenylephrine or KCl. Hypoxia induced a transient contraction, followed by a relaxation, and then a slowly developing sustained contraction. Hypoxia also significantly increased phosphorylation of p38 MAP kinase. SB-202190 did not affect the transient phase but abrogated the sustained phase of hypoxic contraction, whereas anisomycin enhanced both phases of contraction. SB-202190 also attenuated and anisomycin enhanced the phenylephrine-induced contraction. In contrast, PD-98059 and UO-126 had minimal effects on either hypoxic or phenylephrine-induced contraction. None of the treatments modified KCl-induced contraction. We conclude that p38, but not the ERK1/ERK2 MAP kinase pathway, mediates the sustained phase of hypoxic contraction in isolated rat pulmonary arteries.     

Degradation of alpha-actin filaments in venous smooth muscle cells in response to mechanical stretch

Mechanical stretch has been shown to induce the degradation of alpha-actin filaments in smooth muscle cells (SMC) of experimental vein grafts. Here, we investigate the possible role of ERK1/2 and p38 MAPK in regulating this process using an ex vivo venous culture model that simulates an experimental vein graft. An exposure of a vein to arterial pressure induced a significant increase in the medial circumferential strain, which induced rapid alpha-actin filament disruption, followed by degradation. The percentage of SMC alpha-actin filament coverage was reduced significantly under arterial pressure (91 +/- 1%, 43 +/- 13%, 51 +/- 5%, 28 +/- 3%, and 19 +/- 5% at 1, 6, 12, 24, and 48 h, respectively), whereas it did not change significantly in specimens under venous pressure at theses times. The degradation of SMC alpha-actin filaments paralleled an increase in the relative activity of caspase 3 (3.0 +/- 0.7- and 1.7 +/- 0.4-fold increase relative to the control level at 6 and 12 h, respectively) and a decrease in SMC density (from the control level of 1,368 +/- 66 cells/mm(2) at time 0 to 1,205 +/- 90, 783 +/- 129, 845 +/- 61, 637 +/- 55, and 432 +/- 125 cells/mm(2) at 1, 6, 12, 24, and 48 h of exposure to arterial pressure, respectively). Treatment with a p38 MAPK inhibitor (SB-203580) significantly reduced the stretch-induced activation of caspase 3 at 6 h (from 3.0 +/- 0.7- to 2.2 +/- 0.3-fold) in conjunction with a significant rescue of alpha-actin filament degradation (from 43 +/- 13% to 69 +/- 15%) at the same time. Treatment with an inhibitor for the ERK1/2 activator (PD-98059), however, did not induce a significant change in the activity of caspase 3 or the percentage of SMC alpha-actin filament coverage. These results suggest that p38 MAPK and caspase 3 may mediate stretch-dependent degradation of alpha-actin filaments in vascular SMCs.     

Reactive nitrogen species control apoptosis and autophagy in K562 cells: implication of TAp73α induction in controlling autophagy

The biological outcome of nitric oxide (NO) and reactive nitrogen species (RNS) in regulating pro survival and pro death autophagic pathways still demand further investigation. In the present study, we investigated the effect of nitrosative stress in K562 cells using NO donor compound DETA-NONOate, peroxynitrite, and SIN-1. Exposure to NO, peroxynitrite, and SIN-1 caused decrease in K562 cell survival. NO induced autophagy but not apoptosis or necrosis in K562 cells. In contrast, peroxynitrite and SIN-1 treatment induced apoptosis in K562 cells. Surprisingly, inhibition of autophagic response using 3-methyladenine led to the induction of apoptosis in K562 cells. Increase in 5’adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was only observed in the presence of NO donor indicated that AMPK was crucial to induce autophagy in K562 cells. We for the first time discovered a novel role of p73 in autophagy induction under nitrosative stress in K562 cells. TAp73α was only induced upon exposure to NO but not in the presence of peroxynitrite. Reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio remained unaltered upon NO exposure. Our data suggest a complex network of interaction and cross regulations between NO and p73. These data open a new path for therapies based on the abilities of RNS to induce autophagy-mediated cell death.     

Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-beta

Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway, but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-beta is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-beta1 mRNA and protein to a greater extent than nonasthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-beta1 stimulated the growth of nonconfluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-beta1 on ASM cells was inhibited by selective inhibitors of TGF-beta receptor I kinase (SD-208), phosphatidylinositol 3-kinase (PI3K, LY-294002), ERK (PD-98059), JNK (SP-600125), and NF-kappaB (AS-602868). On the other hand, p38 MAPK inhibitor (SB-203580) augmented TGF-beta1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector-expressing Smad4, Smad7, or dominant-negative Smad3 and found no involvement of these Smads in TGF-beta1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-beta1-induced proliferation. Our findings suggest that TGF-beta1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK, and JNK, whereas p38 MAPK is a negative regulator. NF-kappaB is also involved in the TGF-beta1 mitogenic signaling, but Smad pathway does not appear important.     

T3 increases Na-K-ATPase activity via a MAPK/ERK1/2-dependent pathway in rat adult alveolar epithelial cells

Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3K-dependent pathway. In these cells, dopamine and beta-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase alpha(1)-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway.     

Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes

Mitogen-activated protein kinases (MAPKs) play different regulatory roles in signaling oxidative stress-induced apoptosis in cardiac ventricular myocytes. The regulation and functional role of cross-talk between p38 MAPK and extracellular signal-regulated kinase (ERK) pathways were investigated in cardiac ventricular myocytes in the present study. We demonstrated that inhibition of p38 MAPK with SB-203580 and SB-239063 enhanced H(2)O(2)-stimulated ERK phosphorylation, whereas preactivation of p38 MAPK with sodium arsenite reduced H(2)O(2)-stimulated ERK phosphorylation. In addition, pretreatment of cells with the protein phosphatase 2A (PP2A) inhibitors okadaic acid and fostriecin increased basal and H(2)O(2)-stimulated ERK phosphorylation. We also found that PP2A coimmunoprecipitated with ERK and MAPK/ERK (MEK) in cardiac ventricular myocytes, and H(2)O(2) increased the ERK-associated PP2A activity that was blocked by inhibition of p38 MAPK. Finally, H(2)O(2)-induced apoptosis was attenuated by p38 MAPK or PP2A inhibition, whereas it was enhanced by MEK inhibition. Thus the present study demonstrated that p38 MAPK activation decreases H(2)O(2)-induced ERK activation through a PP2A-dependent mechanism in cardiac ventricular myocytes. This represents a novel cellular mechanism that allows for interaction of two opposing MAPK pathways and fine modulation of apoptosis during oxidative stress.     

Actin reorganization and morphological changes in human neutrophils stimulated by TNF,GM-CSF,and G-CSF: the role of MAP kinases

Stimulation of human neutrophils with tumor necrosis factor- (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) resulted in decreased fluorescence intensity of FITC-phalloidin (actin depolymerization) and morphological changes. Cytokine-induced actin depolymerization was dependent on the concentration of cytokines used as stimuli. The maximal changes were detected at 10 min after stimulation with TNF or GM-CSF and at 20 min after stimulation with G-CSF. Cytokine-induced actin depolymerization was sustained for at least 30 min after stimulation. In contrast, N-formyl-methionyl-leucyl-phenylalanine (FMLP) rapidly (within 45 s) induced an increase in the fluorescence intensity of FITC-phalloidin (actin polymerization) and morphological changes. TNF- and GM-CSF-induced actin depolymerization and morphological changes, but not FMLP-induced responses, were partially inhibited by either PD-98059, an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase, or SB-203580, an inhibitor of p38 MAPK, and were almost completely abolished by these inhibitors in combination. G-CSF-induced responses were almost completely abolished by PD-98059 and were unaffected by SB-203580. These findings are consistent with the ability of these cytokines to activate the distinct MAPK subtype cascade in human neutrophils. Phosphorylated ERK and p38 MAPK were not colocalized with F-actin in neutrophils stimulated by cytokines or FMLP. Furthermore, FMLP-induced polarization and actin polymerization were prevented by cytokine pretreatment. These findings suggest that TNF, GM-CSF, and G-CSF induce actin depolymerization and morphological changes through activation of ERK and/or p38 MAPK and that cytokine-induced actin reorganization may be partly responsible for the inhibitory effect of these cytokines on neutrophil chemotaxis. neutrophil; actin reorganization; cytokines; mitogen-activated protein kinase; tumor necrosis factor-; granulocyte-macrophage colony stimulating factor     

Two distinct mechanisms of nitric oxide-mediated neuronal cell death show thiol dependency

To better understand the mechanism(s) underlying nitricoxide (· NO)-mediated toxicity, in the presence and absenceof concomitant oxidant exposure, postmitotic terminally differentiatedNT2N cells, which are incapable of producing · NO, wereexposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1).Exposure to SIN-1, which generated peroxynitrite in the range of25-750 nM/min, produced a concentration- and time-dependentdelayed cell death. In contrast, a critical threshold concentration(>440 nM/min) was required for · NO to produce significantcell injury. Examination of cells by electron microscopy shows alargely necrotic injury after peroxynitrite exposure but mainlyapoptotic-like morphology after · NO exposure. Cellularlevels of reduced thiols correlated with cell death, and pretreatmentwith N-acetylcysteine (NAC) fully protected from cell death ineither PAPA/NO or SIN-1 exposure. NAC given within the first 3 hposttreatment further delayed cell death and increased theintracellular thiol level in SIN-1 but not · NO-exposedcells. Cell injury from · NO was independent of cGMP,caspases, and superoxide or peroxynitrite formation. Overall, exposureof non-· NO-producing cells to · NO orperoxynitrite results in delayed cell death, which, although occurringby different mechanisms, appears to be mediated by the loss ofintracellular redox balance.

     

Lysophosphatidic acid enhances interleukin-13 gene expression and promoter activity in T cells

Airway smooth muscle cells (ASMC) are a source of inflammatory chemokines that may propagate airway inflammatory responses. We investigated the production of the CXC chemokine growth-related oncogene protein-alpha (GRO-alpha) from ASMC induced by cytokines and the role of MAPK and NF-kappaB pathways. ASMC were cultured from human airways, grown to confluence, and exposed to cytokines IL-1beta and TNF-alpha after growth arrest. GRO-alpha release, measured by ELISA, was increased by >50-fold after IL-1beta (0.1 ng/ml) or 5-fold after TNF-alpha (1 ng/ml) in a dose- and time-dependent manner. GRO-alpha release was not affected by the T helper type 2 cytokines IL-4, IL-10, and IL-13. IL-1beta and TNF-alpha also induced GRO-alpha mRNA expression. Supernatants from IL-1beta-stimulated ASMC were chemotactic for neutrophils; this effect was inhibited by anti-GRO-alpha blocking antibody. AS-602868, an inhibitor of IKK-2, and PD-98059, an inhibitor of ERK, inhibited GRO-alpha release and mRNA expression, whereas SP-600125, an inhibitor of JNK, reduced GRO-alpha release without effect on mRNA expression. SB-203580, an inhibitor of p38 MAPK, had no effect. AS-602868 but not PD-98059 or SP-600125 inhibited p65 DNA-binding induced by IL-1beta and TNF-alpha. By chromatin immunoprecipitation assay, IL-1beta and TNF-alpha enhanced p65 binding to the GRO-alpha promoter, which was inhibited by AS-602868. IL-1beta- and TNF-alpha-stimulated expression of GRO-alpha from ASMC is regulated by independent pathways involving NF-kappaB activation and ERK and JNK pathways. GRO-alpha released from ASMC participates in neutrophil chemotaxis.     

SIN-1-induced cytotoxicity in mixed cortical cell culture: peroxynitrite-dependent and -independent induction of excitotoxic cell death

3-Morpholinosyndnomine (SIN-1) has been reported to be a peroxynitrite (OONO(-)) donor because it produces both nitric oxide (NO) and superoxide (O(2)(-).) upon decomposition in aqueous solution. However, SIN-1 can decompose to primarily NO in the presence of electron acceptors, including those found in biological tissues, making it necessary to determine the release product(s) formed in any given biological system. In a mixed cortical cell culture system, SIN-1 caused a concentration-dependent increase in cortical cell injury with a parallel increase in the release of cellular proteins containing 3-nitrotyrosine into the culture medium. The increase in 3-nitrotyrosine immunoreactivity, a footprint of OONO(-) production, was specific for SIN-1 as exposure to neurotoxic concentrations of an NO donor (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (DETA/NO), or NMDA did not result in the nitration of protein tyrosine residues. Both SIN-1-induced injury and 3-nitrotyrosine staining were prevented by the addition of either 5,10,15,20-Tetrakis (4-sulfonatophenyl) prophyrinato iron (III) [FeTPPS], an OONO(-) decomposition catalyst, or uric acid, an OONO(-) scavenger. Removal of NO alone was sufficient to inhibit the formation of OONO(-) from SIN-1 as well as its cytotoxicity. Removal of O(2)(-). and the subsequently formed H(2)O(2) by superoxide dismutase (SOD) plus catalase likewise prevented the nitration of protein-bound tyrosine but actually enhanced the cytotoxicity of SIN-1, indicating that cortical cells can cope with the oxidative but not the nitrosative stress generated. Finally, neural injury induced by SIN-1 in unadulterated cortical cells was prevented by antagonism of AMPA/kainate receptors, while blockade of the NMDA receptor was without effect. In contrast, activation of both NMDA and non-NMDA receptors contributed to the SIN-1-mediated neurotoxicity when cultures were exposed in the presence of SOD plus catalase. Thus, whether SIN-1 initiates neural cell death in an OONO(-)-dependent or -independent manner is determined by the antioxidant status of the cells. Further, the mode of excitotoxicity by which injury progresses is determined by the NO-related species generated.