Differential effects of p38 MAP kinase inhibitors SB203580 and SB202190 on growth and migration of human MDA-MB-231 cancer cell line

p38 mitogen-activated protein kinase (MAPK) belongs to the MAPK superfamily, phosphorylating serine and/or threonine residues of the target proteins. The activation of p38 MAPK leads to cell growth, differentiation, inflammation, survival or apoptosis. In this study, we tested the effect of two highly specific and potent inhibitors of p38 MAPK (namely, SB203580 and SB202190) on human breast cancer cell line MDA-MB-231 to elucidate the controversial role of p38 MAPK on cell proliferation and/or cell migration/metastasis further. It was determined that the IC₅₀ value of SB203580 was 85.1 µM, while that of SB202190 was 46.6 µM, suggesting that SB202190 is slightly more effective than SB203580. To verify the effect of each inhibitor on cell proliferation and cytotoxicity, the cells were treated with various doses of SB203580 and SB202190 and examined using iCELLigence system. No significant effect of 1 and 5 µM of both inhibitors were seen on cell proliferation as compared to the DMSO-treated control cells for up to 96 h. On the other hand, both SB203580 and SB202190 significantly prevented cell proliferation at a concentration of 50 µM. SB202190 was again more effective than SB203580. Afterwards, we tested the effect of each inhibitor on cell migration using wound assay. Both SB203580 and SB202190 significantly reduced cell migration in a time-dependent manner at a concentration of 50 µM. However, interestingly it was observed that a low and noncytotoxic dose of 5 µM of SB203580 and SB202190 also did cause significant cell migration inhibition at 48 h of the treatment, corroborating the fact that p38 MAPK pathway has a critical role in cell migration/metastasis. Then, we tested whether each p38 MAPK inhibitor has any effect on cell adhesion during a treatment period of 3 h using iCELLigence system. A concentration of only 50 µM of SB202190 reduced cell adhesion for about 1.5 h (p < 0.001); after that period of time, cell adhesion in 50 µM SB202190-treated cells returned to the level of the control cells. To determine the mechanism of growth and cell migration inhibitory effects of p38 MAPK inhibitors, the activation/inactivation of various proteins and enzymes was subsequently analyzed by PathScan^® Intracellular Signaling Array kit. The ERK1/2 phosphorylation level was not modified by low concentrations (1 or 5 µM) of SB202190 and SB203580; while a high concentration (50 µM) of both inhibitors caused significant reductions in the ERK1/2 phosphorylation. In addition, it was determined that both p38 MAPK inhibitors caused significant increases on the Ser15 phosphorylation of mutant p53 in MDA-MB-231 under these experimental conditions; while SB202190 was more potent than SB203580.     

SB203580 Modulates p38 MAPK Signaling and Dengue Virus-Induced Liver Injury by Reducing MAPKAPK2, HSP27, and ATF2 Phosphorylation

Dengue virus (DENV) infection causes organ injuries, and the liver is one of the most important sites of DENV infection, where viral replication generates a high viral load. The molecular mechanism of DENV-induced liver injury is still under investigation. The mitogen activated protein kinases (MAPKs), including p38 MAPK, have roles in the hepatic cell apoptosis induced by DENV. However, the in vivo role of p38 MAPK in DENV-induced liver injury is not fully understood. In this study, we investigated the role of SB203580, a p38 MAPK inhibitor, in a mouse model of DENV infection. Both the hematological parameters, leucopenia and thrombocytopenia, were improved by SB203580 treatment and liver transaminases and histopathology were also improved. We used a real-time PCR microarray to profile the expression of apoptosis-related genes. Tumor necrosis factor α, caspase 9, caspase 8, and caspase 3 proteins were significantly lower in the SB203580-treated DENV-infected mice than that in the infected control mice. Increased expressions of cytokines including TNF-α, IL-6 and IL-10, and chemokines including RANTES and IP-10 in DENV infection were reduced by SB203580 treatment. DENV infection induced the phosphorylation of p38MAPK, and its downstream signals including MAPKAPK2, HSP27 and ATF-2. SB203580 treatment did not decrease the phosphorylation of p38 MAPK, but it significantly reduced the phosphorylation of MAPKAPK2, HSP27, and ATF2. Therefore, SB203580 modulates the downstream signals to p38 MAPK and reduces DENV-induced liver injury.     

Reduced Parasite Motility and Micronemal Protein Secretion by a p38 MAPK Inhibitor Leads to a Severe Impairment of Cell Invasion by the Apicomplexan Parasite Eimeria tenella

E. tenella infection is associated with a severe intestinal disease leading to high economic losses in poultry industry. Mitogen activated protein kinases (MAPKs) are implicated in early response to infection and are divided in three pathways: p38, extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). Our objective was to determine the importance of these kinases on cell invasion by E. tenella. We evaluated the effect of specific inhibitors (ERK: PD98059, JNKII: SP600125, p38 MAPK: SB203580) on the invasion of epithelial cells. Incubation of SP600125 and SB203580 with epithelial cells and parasites significantly inhibited cell invasion with the highest degree of inhibition (90%) for SB203580. Silencing of the host p38α MAPK expression by siRNA led to only 20% decrease in cell invasion. In addition, when mammalian epithelial cells were pre-treated with SB203580, and washed prior infection, a 30% decrease in cell invasion was observed. This decrease was overcome when a p38 MAPK activator, anisomycin was added during infection. This suggests an active but limited role of the host p38 MAPK in this process. We next determined whether SB203580 has a direct effect on the parasite. Indeed, parasite motility and secretion of micronemal proteins (EtMIC1, 2, 3 and 5) that are involved in cell invasion were both decreased in the presence of the inhibitor. After chasing the inhibitor, parasite motility and secretion of micronemal proteins were restored and subsequently cell invasion. SB203580 inhibits cell invasion by acting partly on the host cell and mainly on the parasite.     

Tri-substituted imidazole analogues of SB203580 as inducers for cardiomyogenesis of human embryonic stem cells

The p38α mitogen-activated protein kinase (MAPK) inhibitor SB203580 had been reported to enhance the cardiomyogenesis of human embryonic stem cells (hESCs). To investigate if tri-substituted imidazole analogues of SB203580 are equally effective inducers for cardiomyogenesis of hESCs, and if there is a correlation between p38α MAPK inhibition and cardiomyogenesis, we designed and synthesized a series of novel tri-substituted imidazoles with a range of p38α MAPK inhibitory activities. Our studies demonstrated that suitably designed analogues of SB203580 can also be inducers of cardiomyogenesis in hESCs and that cell growth is affected by changes in the imidazole structures.     

Pyridinylimidazole compound SB 203580 inhibits the activity but not the activation of p38 mitogen-activated protein kinase.

p38 MAPK is a Ser/Thr protein kinase activated by various inflammatory cytokines and a variety of stress stimuli. It is involved in many physiological processes, including the production of inflammatory cytokines. We have previously reported the design and synthesis of a series of pyridinylimidazole compounds that are selective inhibitors of p38 MAPK. These compounds, exemplified by SB 203580, are exceptionally effective in cell-based assays, including the inhibition of inflammatory cytokine production. SB 203580 is widely used as a tool to dissect the role of p38 MAPK in various physiological processes. It has previously been established that SB 203580 acts primarily to block the catalytic activity of p38 MAPK. However, it has been suggested that in cells, the compounds could also inhibit p38 MAPK activation by virtue of their ability to bind to the inactive enzyme. We undertook careful studies to definitively demonstrate that treatment with SB 203580 had no effect on Thr(180) and Tyr(182) phosphorylation, and hence activation of p38 in vivo. SB 203580, however, potently inhibited the activity of p38 MAPK as demonstrated by the inhibition of the activation of MAPKAP K2, a specific physiological substrate of p38 MAPK. This was observed regardless of stimuli or cell type. Identical results were obtained when the p38 MAPK cascade was partially reconstituted in vitro. Thus, our data clearly indicate that SB 203580 specifically inhibits the activity of p38 MAPK but not its activation by upstream MAPKK.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

Evidence for the Involvement of p38 MAPK Activation in Barnacle Larval Settlement

The barnacle Balanus ( = Amphibalanus) amphitrite is a major marine fouling animal. Understanding the molecular mechanism of larval settlement in this species is critical for anti-fouling research. In this study, we cloned one isoform of p38 MAPK (Bar-p38 MAPK) from this species, which shares the significant characteristic of containing a TGY motif with other species such as yeast, Drosophila and humans. The activation of p38 MAPK was detected by an antibody that recognizes the conserved dual phosphorylation sites of TGY. The results showed that phospho-p38 MAPK (pp38 MAPK) was more highly expressed at the cyprid stage, particularly in aged cyprids, in comparison to other stages, including the nauplius and juvenile stages. Immunostaining showed that Bar-p38 MAPK and pp38 MAPK were mainly located at the cyprid antennules, and especially the third and fourth segments, which are responsible for substratum exploration during settlement. The expression and localization patterns of Bar-p38 MAPK suggest its involvement in larval settlement. This postulation was also supported by the larval settlement bioassay with the p38 MAPK inhibitor SB203580. Behavioral analysis by live imaging revealed that the larvae were still capable of exploring the surface of the substratum after SB203580 treatment. This shows that the effect of p38 MAPK on larval settlement might be by regulating the secretion of permanent proteinaceous substances. Furthermore, the level of pp38 MAPK dramatically decreased after full settlement, suggesting that Bar-p38 MAPK maybe plays a role in larval settlement rather than metamorphosis. Finally, we found that Bar-p38 MAPK was highly activated when larvae confronted extracts of adult barnacle containing settlement cues, whereas larvae pre-treated with SB203580 failed to respond to the crude adult extracts.     

Antiproliferative effect of IL-1 is mediated by p38 mitogen-activated protein kinase in human melanoma cell A375.

The role of p38 mitogen-activated protein kinase (MAPK) in IL-1-induced growth inhibition was investigated using IL-1-sensitive human melanoma A375-C2-1 cells and IL-1-resistant A375-R8 cells. In both cells, p38 MAPK was activated by IL-1. A selective inhibitor for p38 MAPK, SB203580, almost completely recovered the IL-1-induced growth inhibition in A375-C2-1 cells. IL-1-induced IL-6 production was also suppressed by SB203580. However, the reversal effect of SB203580 was not due to the suppression of IL-6 production because the SB203580 effect was still observed in the presence of exogenous IL-6. Down-regulation of ornithine decarboxylase (ODC) activity as well as its protein level has been shown to be essential for IL-1-induced growth inhibition. SB203580 also reversed the IL-1-induced down-regulation of ODC activity and intracellular polyamine levels without affecting ODC mRNA levels in A375-C2-1 cells. In IL-1-resistant R8 cells, however, IL-1 only slightly suppressed ODC activity. In A375-C2-1 cells, the mRNA expression level of antizyme (AZ), a regulatory factor of ODC activity, has been shown to be up-regulated by IL-1. IL-1-induced up-regulation of AZ mRNA level was not affected by SB203580. These findings demonstrate that p38 MAPK plays an important role in IL-1-induced growth inhibition in A375 cells through down-regulating ODC activity without affecting the level of ODC mRNA and AZ mRNA. In IL-1-resistant A375-R8 cells, IL-1 signaling pathway is deficient between p38 MAPK activation and down-regulation of ODC activity.     

Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB203580

SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imi dazole) is widely used as a specific inhibitor of p38 mitogen-activated protein kinase (MAPK). Here, we report that SB203580 activates the serine/threonine kinase Raf-1 in quiescent smooth muscle cells in a dose-dependent fashion. The concentrations of SB203580 required lie above those necessary to inhibit p38 MAPK and we were unable to detect basal levels of active p38 MAPK. SB203580 does not directly activate Raf-1 in vitro, and fails to activate Ras, MEK, and ERK in intact cells. In vitro, however, SB203580-stimulated Raf-1 activates MEK1 in a coupled assay. We conclude that activation of Raf-1 by SB203580 is not mediated by an inhibition of p38 MAPK, is Ras-independent, and is uncoupled from MEK/ERK signaling.     

Time course of the MAPK and PI3-kinase response within 24 h of skeletal muscle overload.

Knowledge of the molecular mechanisms by which skeletal muscle hypertrophies in response to increased mechanical loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. To gain insight into potential early signaling mechanisms associated with skeletal muscle hypertrophy, the temporal pattern of mitogen-activated protein kinase (MAPK) phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activity during the first 24 h of muscle overload was determined in the rat slow-twitch soleus and fast-twitch plantaris muscles after ablation of the gastrocnemius muscle. p38alpha MAPK phosphorylation was elevated for the entire 24-h overload period in both muscles. In contrast, Erk 2 and p54 JNK phosphorylation were transiently increased by overload, returning to the levels of sham-operated controls by 24 h. PI3-kinase activity was increased by muscle overload only at 12 h of overload and only in the plantaris muscle. In summary, sustained elevation of p38alpha MAPK phosphorylation occurred early in response to muscle overload, identifying this pathway as a potential candidate for mediating early hypertrophic signals in response to skeletal muscle overload.     

Inhibition of nucleoside transport by p38 MAPK inhibitors

While investigating the ability of p38 MAPK to regulate cytarabine (Ara C)-dependent differentiation of erythroleukemia K562 cells, we observed effects that indicated that the imidazoline class of p38 MAPK inhibitors prevented nucleoside transport. Incubation of K562 cells with SB203580, SB203580-iodo, or SB202474, an analogue of SB203580 that does not inhibit p38 MAPK activity, inhibited the uptake of [3H]Ara C or [3H]uridine and the differentiation of K562 cells. Consistent with the effects of these compounds on the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (ENT1), incubation with SB203580 or SB203580-iodo eliminated the binding of [3H]NBMPR to K562 cells or membranes isolated from human erythrocytes. Furthermore, using a uridine-dependent cell type (G9c), we observed that SB203580 or SB203580-iodo efficiently inhibited the salvage synthesis of pyrimidine nucleotides in vivo. Thus these studies demonstrate that the NBMPR-sensitive equilibrative nucleoside transporters are novel and unexpected targets for the p38 MAPK inhibitors at concentrations typically used to inhibit protein kinases.     

The role of RIP2 in p38 MAPK activation in the stressed heart

The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinase-dead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.     

Falcarindiol inhibits LPS-induced inflammation via attenuating MAPK and JAK-STAT signaling pathways in murine macrophage RAW 264.7 cells

The purpose of the study was to investigate the mechanism of total flavone of Desmodium styracifolium (TFDS) in regulating the formation of urinary calculi. Protein levels of KIM-1, LC3-II, p-p38 were measured by Western blot. The effect of different COM concentrations, different TFDS concentrations, SB203580 (specific inhibitor of p38/MAPK), and overexpression of KIM-1 on cell viability were detected by WST-1 assay. The apoptotic cells and FITC positive cells were detected by flow cytometry. HK-2 cell viability decreased with the increase of COM concentration, and the protein levels of KIM-1, LC3-II, and p-p38 increased with the time. Blocking the p38/MAPK pathway or co-cultured with TFDS inhibited the effects of COM on apoptosis and autophagy of HK-2 cells. In addition, blocking the p38/MAPK pathway inhibited the expression of KIM-1. In COM-induced cells, after treated with SB203580, overexpression of KIM-1 could reverse the protection effect of SB203580 on COM-induced cell damage and the inhibition of SB203580 on COM-induced excessive autophagy, suggesting p38/MAPK regulated KIM-1 to regulate COM-induced cell apoptosis and autophagy. Finally, we proved that TFDS inhibited p38/MAPK pathway. And the protection effect of COM-induced cell injury increased with the increase of TFDS concentration, and the adhesion between COM and cells decreased with the increase of TFDS concentration. With the increase of the concentration of TFDS, p38/MAPK pathway was gradually inhibited, and KIM-1 and autophagy related proteins were decreased. TFDS inhibited HK-2 cell apoptosis and autophagy by regulating KIM-1 via p38/MAPK pathway.     

p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression

The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38alpha/gamma-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression.     

Modulatory effect of interleukin-1α on expression of structural matrix proteins,MMPs and TIMPs in human cardiac myofibroblasts: Role of p38 MAP kinase

The proinflammatory cytokine interleukin-1 (IL-1) elicits catabolic effects on the myocardial extracellular matrix (ECM) early after myocardial infarction but there is little understanding of its direct effects on cardiac myofibroblasts (CMF), or the role of p38 mitogen-activated protein kinase (MAPK). We used a focused RT-PCR microarray to investigate the effects of IL-1α on expression of 41 ECM genes in CMF cultured from different patients, and explored regulation by p38 MAPK.IL-1α (10 ng/ml, 6 h) had minimal effect on mRNA expression of structural ECM proteins, including collagens, laminins, fibronectin and vitronectin. However, it induced marked increases in expression of specific ECM proteases, including matrix metalloproteinases MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (gelatinase-B) and MMP-10 (stromelysin-2). Conversely, IL-1α reduced mRNA and protein expression of ADAMTS1, a metalloproteinase that suppresses neovascularization. IL-1α increased expression of TIMP-1 slightly, but not TIMP-2. Data for MMP-1, MMP-2, MMP-3, MMP-9, MMP-10 and ADAMTS1 were confirmed by quantitative real-time RT-PCR. Tumor necrosis factor-alpha (TNFα), another important myocardial proinflammatory cytokine, did not alter expression of these metalloproteinases. IL-1α strongly activated the p38 MAPK pathway in human CMF. Pharmacological inhibitors of p38-α/β (SB203580) or p38-α/β/γ/δ (BIRB-0796) reduced MMP-3 and ADAMTS1 mRNA expression, but neither inhibitor affected MMP-9 levels. MMP-1 and MMP-10 expression were inhibited by BIRB-0796 but not SB203580, suggesting roles for p38-γ/δ.In summary, IL-1α induces a distinct pattern of ECM protein and protease expression in human CMF, in part regulated by distinct p38 MAPK subtypes, affirming the key role of IL-1α and CMF in post-infarction cardiac remodeling.     

Inhibition of p38 MAPK facilitates ex vivo expansion of skin epithelial progenitor cells

Ex vivo expansion of skin epithelial stem cells has long attracted great interest because of the potential utilization in transplantation and gene therapy. The use of cultured stem or progenitor cells was limited by the lack of applicable culturing system with both satisfactory expansion efficacy and well suppressed differentiation ex vivo. The p38 mitogen-activated protein kinase (MAPK) pathways are responsible for cell growth and differentiation process. We investigated the function of p38 inhibitor SB203580 in the ex vivo expansion of skin epithelial progenitor cells by comparing media with or without addition of this inhibitor. Our results showed that the culturing medium with murine 3T3 feeder layers added with 10 μM SB203580 was more effective in promoting clonal growth of human skin epithelial progenitors or stem cells than the conventional medium without SB203580. The clone initial day in cells treated with 10 μM SB203580 came 2 d earlier with higher colony formation efficiency. The skin epithelial progenitor cells treated with 10 μM SB203580 formed clones that were uniformly smaller in size, longer in sustained proliferation, shorter in clone doubling time, higher in S-phase cells percentage, and lower in levels of differentiation markers such as K10 along with higher levels of stem-cell-associated markers such as p63, K15, and ABCG2 than those cultured in the conventional medium. Collectively, these results indicate that the p38 MAPK pathways inhibitor SB203580 can be used as a culture medium additive that helps to achieve more effective ex vivo expansion of skin epithelial progenitor cells.     

Stimulation of p38 MAP kinase reduces acidosis and Na(+) overload in preconditioned hepatocytes

Ischemic preconditioning has been shown to improve liver resistance to hypoxia/reperfusion damage. A signal pathway involving A(2A)-adenosine receptor, G(i)-proteins, protein kinase C and p38 MAP kinase is responsible for the development of hypoxic preconditioning in hepatocytes. However, the coupling of this signal pathway with the mechanisms responsible for cytoprotection is still unknown. We have observed that stimulation of A(2A)-adenosine receptors or of p38 MAPK by CGS21680 or anisomycin, respectively, appreciably reduced intracellular acidosis and Na(+) accumulation developing during hypoxia. These effects were reverted by p38 MAPK inhibitor SB203580 as well as by blocking vacuolar proton ATPase with bafilomycin A(1). SB203580 and bafilomycin A(1) also abolished the cytoprotective action exerted by both CGS21680 and anisomycin. We propose that the stimulation of p38 MAPK by preconditioning might increase hepatocyte resistance to hypoxia by activating proton extrusion through vacuolar proton ATPase, thus limiting Na(+) overload promoted by Na(+)-dependent acid buffering systems.