Transforming growth factor-beta stimulates articular chondrocyte cell growth through p44/42 MAP kinase (ERK) activation

Transforming growth factor-beta1 (TGF-beta1) stimulates articular chondrocyte cell proliferation and extracellular matrix formation. We reported previously that immediate and transient expression of c-fos mRNA through protein kinase C activation is required for the mitogenic effect of TGF-beta1 on cultured rat articular chondrocytes (CRAC). In gel kinase assays using myelin basic protein (MBP) showed that total cell lysates from cells treated with TGF-beta1 caused rapid phosphorylation of MBP, which suggests the involvement of mitogen-activated protein kinase (MAPK) activation. To identify specific MAPK pathways activated by TGF-beta1, we performed in vitro kinase assays using specific substrates. TGF-beta1 induced a rapid activation of extracellular signal regulated kinase (ERK) with a peak at 5 min, which decreased to basal levels within 240 min after TGF-beta1 stimulation. In contrast, the c-jun N-terminal kinase activity increased only about 2.5-fold after 240 min of stimulation and p38 MAPK activity did not change significantly. ERK activation by TGF-beta1 was also confirmed by in vivo phosphorylation assays of Elk1. However, a specific MEK1 inhibitor, PD98059, significantly decreased TGF-beta1 induced Elk1 phosphorylation in a dose-dependent manner. Furthermore, PD98059 reduced the TGF-beta1-induced cell growth by 40%. These results indicate that TGF-beta1 specifically activates MEK1 and subsequent ERK pathways in CRAC, and that the activation of this MAPK pathway plays a role in the mitogenic response to TGF-beta1.     

Transforming growth Factor-beta1 induces phenotypic modulation of human lung fibroblasts to myofibroblast through a c-Jun-NH2-terminal kinase-dependent pathway

Myofibroblasts play an important role in the fibrogenic process of pulmonary fibrosis. Transforming growth factor (TGF)-beta is well known to induce the phenotypic modulation of fibroblasts to myofibroblasts; however, the intracellular signal regulating induction of the myofibroblastic phenotype of human lung fibroblasts (HLF) has not been determined. In the present study, we examined the role of the mitogen-activated protein kinase (MAPK) superfamily in inducing the phenotypic modulation of HLF to myofibroblasts characterized by alpha-smooth-muscle actin expression, in order to clarify this issue. The results showed that: (1) TGF-beta1 caused the phenotypic modulation of HLF to myofibroblasts in a dose- and a time-dependent manner; (2) TGF-beta1 induced increases in c-Jun-NH2- terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (Erk) phosphorylation and activity; (3) the inhibitors CEP-1347, SB 203580, and PD 98059 attenuated TGF-beta1-induced JNK, p38 MAPK, and Erk activity, respectively; and (4) CEP-1347, but not SB 203580 or PD 98059, attenuated the TGF-beta1-induced phenotypic modulation of HLF to myofibroblasts in a dose-dependent manner. These results indicate that TGF-beta1 is capable of inducing the myofibroblastic phenotype of HLF, and that JNK regulates the phenotypic modulation of TGF-beta1-stimulated HLF to myofibroblasts.     

Differential roles of extracellular signal-regulated kinase 1/2 and p38MAPK in mechanical load-induced procollagen alpha1(I) gene expression in cardiac fibroblasts

OBJECTIVE AND METHODS: We have previously demonstrated that mechanical loading of cardiac fibroblasts leads to increased synthesis and gene expression of the extracellular matrix protein collagen. We hypothesised that the upregulation of procollagen gene expression in cardiac fibroblasts, in response to cyclic mechanical load, is mediated by one or more members of the MAP kinase family. To test this hypothesis, the effect of mechanical load on the activation of extracellular signal-regulated kinase (ERK) 1/2, p46/54JNK, and p38MAPK was examined in rat cardiac fibroblasts. RESULTS: Peak phosphorylation of ERK 1/2, p38MAPK kinases, and p46/54JNK was observed following 10-20 min of continuous cyclic mechanical load. Mechanical load significantly increased procollagen alpha1(I) mRNA levels up to twofold above static controls after 24 h. This increase was completely abolished by the MEK 1/2 inhibitor U0126, with no effect on basal levels. In contrast, SB203580, a specific inhibitor of p38MAPK, enhanced both basal and stretch-stimulated levels of procollagen mRNA. Consistent with this finding, selective activation of the p38MAPK signalling pathway by expression of MKK6(Glu), a constitutive activator of p38MAPK, significantly reduced procollagen alpha1(I) promoter activity. SB203580-dependent increase in procollagen alpha1(I) was accompanied by ERK 1/2 activation, and inhibition of this pathway completely prevented SB203580-induced procollagen alpha1(I) expression. CONCLUSIONS: These results suggest that mechanical load-induced procollagen alpha1(I) gene expression requires ERK 1/2 activation and that the p38MAPK pathway negatively regulates gene expression in cardiac fibroblasts. These pathways are likely to be key in events leading to matrix deposition during heart growth and remodelling induced by mechanical load.     

Role of MAP kinases and their cross-talk in TGF-beta1-induced apoptosis in FaO rat hepatoma cell line

Transforming growth factor (TGF) beta1 is a potent inducer of apoptosis in the liver. During TGF-beta1-induced apoptosis, 3 mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38 kinase) showed simultaneously sustained activation in FaO rat hepatoma cells. TGF-beta1-induced apoptosis was markedly enhanced when ERK activation was selectively inhibited by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. In contrast, both interfering with p38 activity by overexpression of the dominant negative (DN) MKK6 mutant and inhibition of the JNK pathway by overexpression of the DN SEK1 mutant resulted in suppression of mitochondrial cytochrome c release, abrogating TGF-beta1-induced apoptosis. In addition, antiapoptotic Bcl-2 blocked mitochondrial cytochrome c release, suppressing TGF-beta1-induced activation of JNK and p38. Inhibition of ERK activity enhanced TGF-beta1-induced p38 and JNK activation. However, inhibition of the JNK pathway suppressed p38 but induced transient ERK activation. Similarly, interfering with the p38 pathway also attenuated JNK activation but generated transient ERK activation in response to TGF-beta1. These results indicate that disrupting one MAP kinase pathway affects the TGF-beta1-induced activation of other MAP kinases, suggesting cross-talk among MAP kinase pathways. In conclusion, we propose that the balance and integration of MAP kinase signaling may regulate commitment to TGF-beta1-induced apoptosis modulating the release of cytochrome c from mitochondria.     

Atrial natriuretic peptide inhibits transforming growth factor beta-induced Smad signaling and myofibroblast transformation in mouse cardiac fibroblasts

This study tested the hypothesis that activation of atrial natriuretic peptide (ANP)/cGMP/protein kinase G signaling inhibits transforming growth factor (TGF)-beta1-induced extracellular matrix expression in cardiac fibroblasts and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Left ventricular hypertrophy and fibrosis, collagen deposition, and myofibroblast transformation of cardiac fibroblasts in response to pressure overload by transverse aortic constriction were exaggerated in ANP-null mice compared with wild-type controls. ANP and cGMP inhibited TGF-beta1-induced myofibroblast transformation, proliferation, collagen synthesis, and plasminogen activator inhibitor-1 expression in cardiac fibroblasts isolated from wild-type mice. Following pretreatment with cGMP, TGF-beta1 induced phosphorylation of Smad3, but the resultant pSmad3 could not be translocated to the nucleus. pSmad3 that had been phosphorylated with recombinant protein kinase G-1alpha was analyzed by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ion trap tandem mass spectrometry. The analysis revealed phosphorylation of Ser309 and Thr388 residues, sites distinct from the C-terminal Ser423/425 residues that are phosphorylated by TGF-beta receptor kinase and are critical for the nuclear translocation and down-stream signaling of pSmad3. These results suggest that phosphorylation of Smad3 by protein kinase G is a potential molecular mechanism by which activation of ANP/cGMP/protein kinase G signaling disrupts TGF-beta1-induced nuclear translocation of pSmad3 and downstream events, including myofibroblast transformation, proliferation, and expression of extracellular matrix molecules in cardiac fibroblasts. We postulate that this process contributes to the antifibrogenic effects of the natriuretic peptide in heart.     

Epigallocatechin-3-gallate suppresses TNF-α -induced production of MMP-1 and -3 in rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) synovial fibroblasts produce matrix metaloproteinases (MMPs), which destruct cartilage and bone in RA joint. Tumor necrosis factor-α (TNF-α) is one of the most important mediator leading to MMP production in RA synovial fibroblasts. Here we show that epigallocatechin-3-Gallate (EGCG) suppresses TNF-α-induced production of MMP-1 and MMP-3 in RA synovial fibroblasts, which was accompanied by inhibition of mitogen activated protein kinase (MAPK) and activator protein-1 (AP-1) pathways. EGCG treatment resulted in dose-dependent inhibition of TNF-α-induced production of MMP-1 and MMP-3 at the protein and mRNA levels in RA synovial fibroblast. EGCG treatment also inhibited TNF-α-induced phosphorylation of MAPKs, such as ERK1/2, p38, JNK. Electrophoretic mobility shift assay revealed that EGCG inhibits binding of AP-1 proteins to its response elements in synovial fibroblast treated. Thus, EGCG may play a role in regulating inflammation and bone destruction in RA patients.     

Resveratrol inhibits transforming growth factor-β-induced proliferation and differentiation of ex vivo human lung fibroblasts into myofibroblasts through ERK/Akt inhibition and PTEN restoration

The authors investigated the role of resveratrol (RV), a natural poliphenolic molecule with several biological activities, in transforming growth factor-β (TGF-β)-induced proliferation and differentiation of ex vivo human pulmonary fibroblasts into myofibroblasts. The effects of RV treatment were evaluated by analyzing TGF-β-induced α-smooth muscle actin (α-SMA) expression and collagen production, as well as cell proliferation of both normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. Results demonstrate that RV inhibits TGF-β-induced cell proliferation of both normal and pathological lung fibroblasts, attenuates α-SMA expression at both the mRNA and protein levels, and also inhibits intracellular collagen deposition. In order to understand the molecular mechanisms, the authors also investigated the effects of RV treatment on signaling pathways involved in TGF-β-induced fibrosis. The authors show that RV inhibited TGF-β-induced phosphorylation of both extracellular signal-regulated kinases (ERK1/2) and the serine/threonine kinase, Akt. Moreover, RV treatment blocked the TGF-β-induced decrease in phosphatase and tensin homolog (PTEN) expression levels.     

气道上皮损伤对上皮下成纤维细胞转分化的影响及其在支气管哮喘气道高反应发生中的地位

目的探讨损伤气道上皮对上皮下成纤维细胞活化、转分化为肌纤维母细胞的影响及其在支气管哮喘(简称哮喘)气道高反应中的地位。方法原代培养的人气道上皮下成纤维细胞与经脂多糖(LPS)处理后给予机械划伤的人气道上皮细胞(16HBE)共培养,分别加入内皮素(ET)受体A拮抗剂BQ123、转化生长因子β1(TGF-β1)中和抗体及丝裂原活化蛋白激酶(MAPKs)信号通路特异性抑制剂,应用免疫组化、免疫印迹以及5-溴-2′-脱氧尿苷(BrdU)掺入等方法,检测上皮下成纤维细胞α平滑肌肌动蛋白(α-SMA)的表达与细胞增殖情况;将细胞转染反义内皮素转换酶(anti-ECE)mRNA表达载体并运用酶谱分析法,研究ET-1与基质金属蛋白酶(MMPs)的相互作用。结果损伤上皮诱导并增强了上皮下成纤维细胞α-SMA表达及细胞增殖,同时诱导了上皮下成纤维细胞p38丝裂原活化蛋白激酶(p38MAPK)及细胞外信号调节激酶(ERK1/2)信号通路的活化;BQ123、TGF-β1中和抗体及p38MAPK、ERK1/2抑制剂均分别抑制或部分抑制了α-SMA的表达。与对照(转染空载体)及转染anti-ECEmRNA表达载体上皮细胞比较,转染空载体的上皮细胞损伤后上清液中ET-1分泌[机械划伤+LPS刺激的pEGFPN2转染16HBE组为(15·00±0·86)pg/ml;机械划伤+LPS刺激的anti-ECE转染16HBE组为(7·57±0·94)pg/ml]增加(P均<0·01)。结论损伤上皮通过释放ET-1和(或)TGF-β1诱导了上皮下成纤维细胞向肌纤维母细胞转分化并促进了细胞的增殖,这一过程涉及了MAPKs家族的p38MAPK与ERK1/2信号通路的激活。     

Pigment epithelium-derived factor in idiopathic pulmonary fibrosis: a role in aberrant angiogenesis

Pigment epithelium-derived factor (PEDF) is a 50-kD protein with angiostatic and neurotrophic activities that regulates vascular development within the eye. PEDF expression was increased in the lungs of patients with idiopathic pulmonary fibrosis (IPF) based on microarray analyses. Angiogenesis has been implicated in the pathogenesis of fibrotic lung diseases, we therefore hypothesized that regional abnormalities in vascularization occur in IPF as a result of an imbalance between PEDF and vascular endothelial growth factor. We demonstrated that vascular density is regionally decreased in IPF within the fibroblastic foci, and that within these areas PEDF was increased, whereas vascular endothelial growth factor was decreased. PEDF colocalized with the fibrogenic cytokine, transforming growth factor (TGF)-beta 1, particularly within the fibrotic interstitium and the fibroblastic focus, and prominently within the epithelium directly overlying the fibroblastic focus. This suggested that TGF-beta 1 might regulate PEDF expression. Using 3T3-L1 fibroblasts and human lung fibroblasts, we showed that PEDF was indeed a TGF-beta 1 target gene. Collectively, our findings implicate PEDF as a regulator of pulmonary angiogenesis and an important mediator in IPF.     

Direct effects of high glucose and insulin on protein synthesis in cultured cardiac myocytes and DNA and collagen synthesis in cardiac fibroblasts

The present study examined the direct effects of high glucose and insulin on protein synthesis in cardiac myocytes and DNA and collagen synthesis in cardiac fibroblasts. Cultured rat cardiac myocytes and fibroblasts were grown in media containing normal glucose, high glucose, or osmotic control, and incubated with or without insulin. In cardiac myocytes, high glucose had no effect, but insulin increased protein synthesis and atrial natriuretic peptide (ANP) secretion and gene expression. The extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor and the protein kinase C (PKC) inhibitor blocked insulin-induced protein synthesis. In cardiac fibroblasts, high glucose and osmotic control media increased DNA synthesis. Collagen synthesis and fibronectin and transforming growth factor-beta1 (TGF-beta1) mRNA expression were stimulated by high glucose, but not by osmotic control. Insulin increased DNA and collagen synthesis in fibroblasts, and the insulin-induced increase in DNA synthesis was blocked by the phosphatidylinositol 3 kinase (PI3K) inhibitor. Our findings suggest that cardiomyocyte protein synthesis is mainly regulated by insulin rather than high glucose and both high glucose and insulin contribute to fibroblast DNA and collagen synthesis. High glucose accelerates fibroblast DNA synthesis and collagen synthesis, and fibronectin and TGF-beta1 mRNA expression, dependent or independent of osmotic stress. Insulin regulates myocyte protein synthesis and fibroblast DNA synthesis through different intracellular mechanisms.     

软脂酸通过丝裂原活化蛋白激酶通路促进血管内皮细胞凋亡

目的探讨软脂酸(PA)诱导的血管内皮细胞凋亡中丝裂原活化蛋白激酶(MAPK)通路的作用。方法将人脐静脉内皮细胞(HUVEC)分对照组、PA组、MAPK通路干预组[分别先用p38抑制剂SB203580、氨基末端激酶(JNK)抑制剂PD98059、细胞外信号调节激酶(ERK)抑制剂SP600125干预]再分为PA+SB组、PA+PD组、PA+SP组。流式细胞仪检测细胞凋亡率;Western blot法检测caspase-3、磷酸化p38、JNK和ERK1/2表达水平;分光光度法检测caspase-3的活性。结果与对照组比较,PA组、PA+SB组、PA+PD组、PA+SP组HUVEC凋亡及caspase-3表达和活性明显增加,PA组磷酸化p38MAPK表达明显增加(P<0.05)。与PA组比较,PA+SB组HUVEC细胞凋亡率、caspase-3表达和活性明显降低(P<0.05);而PA+PD组和PA+SP组HUVEC凋亡率、caspase-3表达和活性无明显变化(P>0.05)。结论 PA通过p38MAPK通路促进内皮细胞凋亡。     

Growth arrest- and DNA-damage-inducible 45β gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1β-induced apoptosis in insulin-producing INS-1E cells

Aims/hypothesis IL-1β is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1β activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1β-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45β (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study was to investigate if Gadd45b prevents IL-1β-induced beta cell MAPK signalling and apoptosis.Materials Rat insulinoma INS-1E cells and mouse beta-TC3 cells stably expressing Gadd45b were generated. The effects of Gadd45b expression on signalling by JNK, ERK and p38 were assessed by Western blotting and kinase assays. Apoptosis rate was measured by terminal deoxynucleotidyl-mediated dUTP-biotin nick end-labelling (TUNEL) and an ELISA designed to detect apoptotic nucleosomes. Expression of endogenous Gadd45b mRNA was measured by RT-PCR.Results In INS-1E and beta-TC3 cells, expression of Gadd45b inhibited IL-1β-induced activation of JNK and ERK, but augmented IL-1β-mediated p38 activity. IL-1β-induced nitric oxide production and decreases in insulin content and secretion were reduced by GADD45β. IL-1β-induced apoptosis was reduced by GADD45β by up to 77%. Although IL-1β stimulated the time-dependent induction of endogenous Gadd45b in INS-1E cells and rat islets, expression levels were lower in these cells than in IL-1β-exposed NIH-3T3 and 3DO T cells.Conclusions/interpretation Inadequate induction of Gadd45b, which encodes a novel beta cell JNK and ERK inhibitor, may in part explain the pro-apoptotic response of beta cells to IL-1β.     

Salicylate inhibition of extracellular signal-regulated kinases and inducible nitric oxide synthase

The expression of inducible nitric oxide synthase (iNOS) is a characteristic response to inflammation and can be inhibited with sodium salicylate. We used the cytokine-induced iNOS induction in cardiac fibroblasts as a model system in which to test the hypothesis that effects on mitogen-activated protein kinases (MAPKs) may explain the mechanism by which salicylate exerts its anti-inflammatory effects. Tumor necrosis factor-alpha (TNF-alpha) alone can induce extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase activity in a rapid and transient manner, whereas interferon-gamma (IFN-gamma) can induce only ERK. The inhibition of either the ERK pathway or p38 MAPK activity with selective inhibitors blocked cytokine-induced iNOS protein and nitrite production. Salicylate treatment inhibited iNOS expression induced by TNF-alpha and IFN-gamma and attenuated the phosphorylation of ERK by TNF-alpha and IFN-gamma either alone or in combination. Salicylate had no obvious effect on the activation of p38 MAPK or c-Jun N-terminal kinase. The results showed that salicylate inhibited the phosphorylation of ERK and iNOS expression induced by cytokines in a dose-dependent manner and suggested that salicylate exerts its anti-inflammatory action in part through inhibition of the ERK pathway and iNOS induction.     

The role of protein kinase B and mitogen-activated protein kinase in epidermal growth factor and tumor necrosis factor alpha-mediated rat hepatocyte survival and apoptosis

Perturbation of hepatocyte growth regulation is associated with a number of liver diseases such as fibrosis and cancer. These diseases are mediated by a network of growth factors and cytokines that regulate the induction of hepatocyte proliferation and apoptosis. In this study, we have investigated the role of signaling pathways activated by tumor necrosis factor alpha (TNF-alpha) and epidermal growth factor (EGF) in the regulation of apoptosis induced by transforming growth factor beta(1) (TGF-beta(1)), because this physiological factor is believed to regulate spontaneous apoptosis in the liver. We show that pretreatment with (10 ng/mL) EGF or (25 ng/mL) TNF-alpha can suppress TGF-beta(1)-induced apoptosis by 73% and 50%, respectively, in isolated rat hepatocytes. However, suppression of TGF-beta(1)-induced apoptosis by EGF and TNF-alpha occurs via different protein kinase signaling pathways. Using specific inhibitors, we show that suppression of apoptosis by EGF is dependent on activation of phosphoinositide 3-kinase (PI 3-kinase) and the extracellular signal regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathways, but not p38 MAP kinase. In contrast, suppression of TGF-beta(1)-induced apoptosis by TNF-alpha does not require PI 3-kinase and protein kinase B (PKB or Akt)-mediated pathways, but is dependent on ERK and p38 MAP kinase activity. These data contribute to our understanding of the intracellular survival signals that play a role in normal liver homeostasis and in diverse pathological conditions.     

Hypertension accelerates diabetic nephropathy in Wistar fatty rats,a model of type 2 diabetes mellitus,via mitogen-activated protein kinase cascades and transforming growth factor-beta1

Although it is known that diabetic nephropathy is accelerated by hypertension, the mechanisms involved in this process are not clear. In this study we aimed to clarify these mechanisms using male Wistar fatty rats (WFR) as a type 2 diabetic model and male Wistar lean rats (WLR) as a control. Each group was fed a normal or high sodium diet from the age of 6 to 14 weeks. We determined the blood pressure and urinary albumin excretion (UAE). At the end of the study, the expressions of mitogen-activated protein kinases (MAPK) and transforming growth factor-beta1 (TGF-beta1) were examined in the isolated glomeruli by Western blot analysis, and the number of glomerular lesions was determined by conventional histology. High sodium load caused hypertension and a marked increase in UAE in the WFR but not in the WLR. Glomerular volume was increased in the hypertensive WFR. There was no difference among the four groups in the expression of c-Jun-NH2-terminal kinase (JNK). In contrast, the expressions of extracellular signal-regulated kinase 1/2 (ERK1/2) and its upstream regulator, MAPK/ERK kinase 1 (MEK1), were augmented in the hypertensive WFR. Expression of p38 MAPK was increased in the normotensive WFR, and further enhanced in the hypertensive WFR. Moreover, administration of high sodium load to WFR augmented the expression of TGF-beta1. In conclusion, systemic hypertension in WFR accelerates the diabetic nephropathy in type 2 diabetes via MEK-ERK and p38 MAPK cascades. TGF-beta1 is also involved in this mechanism.     

Regulation of sarcolemmal Na(+)/H(+) exchange by hydrogen peroxide in adult rat ventricular myocytes

OBJECTIVE: To characterise the effects of exogenous H(2)O(2) on sarcolemmal Na(+)/H(+) exchanger (NHE) activity and determine the roles of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC) in observed effects. METHODS: Sarcolemmal H(+) efflux rate (J(H)) was determined by microepifluorescence at a pH(i) of 6.70 in adult rat ventricular myocytes, after two consecutive acid pulses in HCO(3)(-)-free medium; before the second pulse, cells (n=7-10/group) were exposed to H(2)O(2) or vehicle and the change in J(H) (DeltaJ(H)) was used to quantify the change in NHE activity. ERK and p38 MAPK activities were determined by immunoblotting with phosphospecific antibodies. RESULTS: Relative to control, DeltaJ(H) was increased by a 10-min exposure to 100, but not 1 or 10 microM H(2)O(2) (1000 microM was not tolerated); 3 or 6 min exposure to 100 microM H(2)O(2) was without effect. ERK and p38 MAPK activities were both increased by 100 microM H(2)O(2) (peak at 6 min); the ERK kinase inhibitor PD98059 (10 microM), but not the p38 MAPK inhibitor SB203580 (1 microM), inhibited the H(2)O(2)-induced increase in DeltaJ(H). H(2)O(2)-induced ERK activation was inhibited not only by PD98059 (10 microM), but also by the non-selective tyrosine kinase inhibitor genistein (3-100 microM), the EGF receptor kinase inhibitor AG1478 (3-300 nM) and the Src family kinase inhibitor PP2 (0.1-10 microM). The PKC inhibitors GF109203X (0.3-10 microM) and chelerythrine (1-30 microM) were without effect on ERK activation, although the former abolished the H(2)O(2)-induced increase in DeltaJ(H). CONCLUSIONS: Our data demonstrate that, in adult rat ventricular myocytes, (i) hydrogen peroxide stimulates sarcolemmal NHE activity, (ii) this response requires activation of ERK and PKC, but not p38 MAPK, (iii) ERK activation occurs through tyrosine kinase-mediated, but PKC-independent, mechanisms