Quantitative imaging mass spectrometry of renal sulfatides: validation by classical mass spectrometric methods

Owing to its capability of discriminating subtle mass-altering structural differences such as double bonds or elongated acyl chains, MALDI-based imaging MS (IMS) has emerged as a powerful technique for analysis of lipid distribution in tissue at moderate spatial resolution of about 50 μm. However, it is still unknown if MS¹-signals and ion intensity images correlate with the corresponding apparent lipid concentrations. Analyzing renal sulfated glycosphingolipids, sulfatides, we validate for the first time IMS-signal identities using corresponding sulfatide-deficient kidneys. To evaluate the extent of signal quenching effects interfering with lipid quantification, we surgically dissected the three major renal regions (papillae, medulla, and cortex) and systematically compared MALDI IMS of renal sulfatides with quantitative analyses of corresponding lipid extracts by on-target MALDI TOF-MS and by ultra-performance LC-ESI-(triple-quadrupole)tandem MS. Our results demonstrate a generally strong correlation (R² > 0.9) between the local relative sulfatide signal intensity in MALDI IMS and absolute sulfatide quantities determined by the other two methods. However, high concentrations of sulfatides in the papillae and medulla result in an up to 4-fold signal suppression. In conclusion, our study suggests that MALDI IMS is useful for semi-quantitative dissection of relative local changes of sulfatides and possibly other lipids in tissue.     

Involvement of MAPK activation in chemokine or COX-2 productions by Toxoplasma gondii

This experiment focused on MAPK activation in host cell invasion and replication of T. gondii, as well as the expression of CC chemokines, MCP-1 and MIP-1 alpha , and enzyme, COX-2/prostaglandin E2 (PGE2) in infected cells via western blot, [3H]-uracil incorporation assay, ELISA and RT-PCR. The phosphorylation of ERK1/2 and p38 in infected HeLa cells was detected at 1 hr and/or 6 hr postinfection (PI). Tachyzoite proliferation was reduced by p38 or JNK MAPK inhibitors. MCP-1 secretion was enhanced in infected peritoneal macrophages at 6 hr PI. MIP-1 alpha mRNA was increased in macrophages at 18 hr PI. MCP-1 and MIP-1 alpha were reduced after treatment with inhibitors of ERK1/2 and JNK MAPKs. COX-2 mRNA gradually increased in infected RAW 264.7 cells and the secretion of COX-2 peaked at 6 hr PI. The inhibitor of JNK suppressed COX-2 expression. PGE2 from infected RAW 264.7 cells was increased and synthesis was suppressed by PD98059, SB203580, and SP600125. In this study, the activation of p38, JNK and/or ERK1/2 MAPKs occurred during the invasion and proliferation of T. gondii tachyzoites in HeLa cells. Also, increased secretion and expression of MCP-1, MIP-1 alpha , COX-2 and PGE2 were detected in infected macrophages, and appeared to occur via MAPK signaling pathways.     

NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.     

Down-regulation of HIV-1 infection by inhibition of the MAPK signaling pathway

The human immunodeficiency virus type 1 (HIV-1) can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase (MAPK) signal pathway can positively regulate the replication of HIV-1, but exactly how each MAPK pathway affects HIV-1 infection and replication is not understood. In this study, we used the Extracellular signal-regulated kinase (ERK) pathway inhibitor, PD98059, the Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, and the p38 pathway inhibitor, SB203580, to investigate the roles of these pathways in HIV-1 replication. We found that application of PD98059 results in a strong VSV-G pseudotyped HIV-1_NL4-3 luciferase reporter virus and HIV-1_NL4-3 virus inhibition activity. In addition, SB203580 and SP600125 also elicited marked VSV-G pseudotyped HIV-1_NL4-3 luciferase reporter virus inhibition activity but no HIV-1_NL4-3 virus inhibition activity. We also found that SB203580 and SP600125 can enhance the HIV-1 inhibition activity of PD98059 when cells were treated with all three MAPK pathway inhibitors in combination. Finally, we show that HIV-1 virus inhibition activity of the MAPK pathway inhibitors was the result of the negative regulation of HIV-1 LTR promoter activity.     

Vanadium salts stimulate mitogen-activated protein (MAP) kinases and ribosomal S6 kinases

Effect of several vanadium salts, sodium orthovanadate, vanadyl sulfate and sodium metavanadate on protein tyrosine phosphorylation and serine/threonine kinases in chinese hamster ovary (CHO) cells overexpressing a normal human insulin receptor was examined. All the compounds stimulated protein tyrosine phosphorylation of two major proteins with molecular masses of 42 kDa (p42) and 44 kDa (p44). The phosphorylation of p42 and p44 was associated with an activation of mitogen activated protein (MAP) kinase as well as increased protein tyrosine phosphorylation of p42^mapk and p44^mapk. Vanadinm salts also activated the 90 kDa ribosomal s6 kinase (p90^rsk) and 70 kDa ribosomal s6 kinase (p70^s6k). Among the three vanadium salts tested, vanadyl sulfate appeared to be slightly more potent than others in stimulating MAP kinases and p70^s6k activity. It is suggested that vanadium-induced activation of MAP kinases and ribosomal s6 kinases may be one of the mechanisms by which insulin like effects of this trace element are mediated.Abbreviations eIF-4 eukaryotic protein synthesis initiation factor-4 - GRB-2 growth factor receptor bound protein-2 - GSK-3 Glycogen Synthase Kinase-3 - IRS-1 insulin receptor substrate-1 - ISPK insulin stimulated protein kinase - MAPK mitogen activated protein kinase, also known as - ERK extracellular signal regulated kinase - MAPKK mitogen activated protein kinase kinase, also known as-MEK, MAPK or ERK kinase - PHAS-1 phosphorylated heat and acid stable protein regulated by insulin - PI3K phosphatidyl inositol 3-kinase - PP1-G protein phosphatase-glycogen bound form - PTK protein tyrosine kinase - PTPase protein tyrosine phosphatase - rsk ribosomal s6 kinases - shc src homology domain containing protein - SOS son of sevenless     

Renal sulfatides: sphingoid base-dependent localization and region-specific compensation of CerS2-dysfunction

Mammalian kidneys are rich in sulfatides. Papillary sulfatides, especially, contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their cer­amide (Cer) anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound’s function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and Cer synthase 2 (CerS)2-deficient mice by imaging MS (IMS) and by LC-MS² (in controls for the cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS² analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within the renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in the medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. CerS2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH, as previously observed in sulfatide-depleted kidneys. The reasons may be the almost unchanged C22-sulfatide levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

The mitogen-activated protein kinase pathway contributes to vanadate toxicity in vascular smooth muscle cells

Vanadate has been considered in the treatment of diabetes because of its insulin-like effects. However, it has severe toxic effects in both animal and man. In cultured cells, vanadate can either cause death or be growth stimulatory, depending on the cell type and growth conditions. Here, we report that in baboon aortic smooth muscle cells (SMCs), vanadate induced p42/p44 mitogen-activated protein kinase (MAPK) activity. This effect was abolished in the presence of the specific MAPK kinase (MAPKK) inhibitor PD098059. Although activation of p42/p44^MAPK/MAPKK is generally thought to be necessary for proliferation, in SMCs, vanadate did not promote DNA synthesis and inhibited thymidine incorporation stimulated by platelet-derived growth factor (PDGF)-BB in a dose dependent fashion (IC₅₀: 30 M). Prolonged exposure to vanadate exerted cytotoxic effects. Cells retracted, rounded up and detached from the substratum. These vanadate-induced morphological changes were blocked in the presence of PD098059. The addition of PDGF-BB further activated p42/p44^MAPK/MAPKK in the presence of vanadate and substantially increased vanadate toxicity. We conclude from these observations that activation of the p42/p44^MAPK/MAPKK signalling module contributes to the cytotoxic effects induced by vanadate.     

Growth suppression of Leydig TM3 cells mediated by aryl hydrocarbon receptor

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental toxicity in reproductive organs. To elucidate the function of AhR, we generated stable transformants of TM3 cells overexpressing wild-type aryl hydrocarbon receptor (AhR) or its mutants which carried mutations in nuclear localization signal or nuclear export signal. In the presence of 3-methylcholanthrene (MC), proliferation of the cells transfected with wild-type AhR was completely suppressed, whereas cells expressing AhR mutants proliferated in a manner equivalent to control TM3 cells, suggesting AhR-dependent growth inhibition. The suppression was associated with up-regulation of cyclin-dependent kinase inhibitor p21^Cip1, which was abolished by pretreatment with actinomycin D. A p38 MAPK specific inhibitor, SB203580, blocked the increase of p21^Cip1 mRNA in response to MC. Treatment with indigo, another AhR ligand, failed to increase of p21^Cip1 mRNA, although up-regulation of mRNA for CYP1A1 was observed. These data suggest AhR in Leydig cells mediates growth inhibition by inducing p21^Cip1.     

Cadmium induces vascular permeability via activation of the p38 MAPK pathway

The vasculature of various organs is a targeted by the environmental toxin, cadmium (Cd). However, mechanisms leading to pathological conditions are poorly understood. In the present study, we examined the effect of cadmium chloride (CdCl₂) on human umbilical vein endothelial cells (HUVECs). At 4 μM, CdCl₂ induced a hyper-permeability defect in HUVECs, but not the inhibition of cell growth up to 24 h. This effect of CdCl₂ was dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. The p38 MAPK inhibitor SB203850 suppressed the CdCl₂-induced alteration in trans-endothelial electrical resistance in HUVEC monolayers, a model measurement of vascular endothelial barrier integrity. SB203850 also inhibited the Cd-induced membrane dissociation of vascular endothelial (VE) cadherin and β-catenin, the important components of the adherens junctional complex. In addition, SB203850 reduces the Cd-induced expression and secretion of tumor necrosis factor α (TNF-α). Taken together, our findings suggest that Cd induces vascular hyper-permeability and disruption of endothelial barrier integrity through stimulation of p38 MAPK signaling.     

Mitochondrial Ca uptake is inhibited by a concerted action of p38 MAPK and protein kinase D

Angiotensin II elicits cytosolic Ca²⁺ signal that is transferred into the mitochondria. Previously we found in H295R cells that this signal transfer is enhanced by both the inhibition of p38 MAPK and a novel isoform of PKC [G. Szanda, P. Koncz, A. Rajki, A. Spät, Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca²⁺ uptake, Cell Calcium 43 (2008) 250–259]. Now we report that simultaneous activation of these protein kinases (by TNFα and PMA + an inhibitor of the conventional PKC isoforms, respectively) attenuates the transfer of cytosolic Ca²⁺ signal, elicited by depolarisation or store-operated Ca²⁺ influx, into the mitochondria. The Ca²⁺ uptake enhancing effect of the p38 MAPK inhibitor SB202190 is due to the inhibition of p38 MAPK and not to a direct mitochondrial action. Protein kinases reduce mitochondrial [Ca²⁺] by inhibiting the uptake mechanism. The threshold of mitochondrial Ca²⁺ uptake may depend on the activity of p38 MAPK. The silencing of protein kinase D (PKD) also results in enhanced transfer of Ca²⁺ signal from the cytosol into the mitochondria. Our data indicate that Ca²⁺ mobilising agonists, through the simultaneous activation of p38 MAPK, a novel PKC isoform and PKD, exert a negative feed-forward action on mitochondrial Ca²⁺ uptake, thus reducing the risk of Ca²⁺ overload.     

Mechanism of induction of muscle protein loss by hyperglycaemia

Treatment of murine myotubes with high glucose concentrations (10 and 25 mM) stimulated protein degradation through the ubiquitin-proteasome pathway, and also caused activation (autophosphorylation) of PKR (double-stranded-RNA-dependent protein kinase) and eIF2α (eukaryotic initiation factor 2α). Phosphorylation of PKR and eIF2α was also seen in the gastrocnemius muscle of diabetic ob/ob mice. High glucose levels also inhibited protein synthesis. The effect of glucose on protein synthesis and degradation was not seen in myotubes transfected with a catalytically inactive variant (PKRΔ6). High glucose also induced an increased activity of both caspase-3 and -8, which led to activation of PKR, since this was completely attenuated by the specific caspase inhibitors. Activation of PKR also led to activation of p38MAPK (mitogen activated protein kinase), leading to ROS (reactive oxygen species) formation, since this was attenuated by the specific p38MAPK inhibitor SB203580. ROS formation was important in protein degradation, since it was completely attenuated by the antioxidant butylated hydroxytoluene. These results suggest that high glucose induces muscle atrophy through the caspase-3/-8 induced activation of PKR, leading to phosphorylation of eIF2α and depression of protein synthesis, together with PKR-mediated ROS production, through p38MAPK and increased protein degradation.     

Inhibitors of the tyrosine kinase signaling cascade attenuated thrombin-induced guinea pig airway smooth muscle cell proliferation

Airway remodeling is one of the major hallmarks of asthma. The present study examined the effects of tyrosine kinase inhibitors on thrombin-induced guinea pig ASM cell proliferation, in comparison with inhibitors of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K). The ASM cells expressed smooth muscle alpha-actin and myosin, and responded to thrombin by increasing cytosolic Ca(+2). Thrombin (1-10 U/ml) induced [(3)H]thymidine incorporation into ASM cells. Tyrphostin 47, a broad-spectrum tyrosine kinase inhibitor, PP2, a Src-specific inhibitor, and piceatannol, a Syk-selective inhibitor, significantly attenuated thrombin-induced [(3)H]thymidine incorporation. In addition, the tyrosine kinase inhibitors significantly reduced thrombin-induced cyclin D(1) expression in ASM cells. PD098059 and U0126, two MAPK kinase inhibitors, and LY294002, a PI3K inhibitor, significantly blocked thrombin-induced [(3)H]thymidine incorporation and cyclin D(1) expression in ASM cells. Our data show that inhibitors of Src and, probably Syk, can modulate thrombin-induced ASM cell proliferation, which may have therapeutic potential for asthma.     

Immediate prostaglandin E2 synthesis in rat 3Y1 fibroblasts following vasopressin V1a receptor stimulation

Arginine vasopressin (AVP) induces immediate prostaglandin E(2) (PGE(2)) production in rat 3Y1 fibroblasts. Judging from effects of several inhibitors, cytosolic phospholipase A(2)alpha (cPLA(2)alpha) and cyclooxygenase-1 (COX-1) were mainly involved in this reaction. The antagonist of vasopressin receptor V1a, and not that of V2, inhibited the AVP-induced PGE(2) synthesis, indicating that AVP activates cPLA(2)alpha through V1a receptor. Treatment of 3Y1 cells with AVP resulted in transient activation of p44/42 mitogen-activated protein kinase (MAPK) and cPLA(2)alpha, and phosphatidylinositol 3-kinase (PI3K) inhibitor blocked not only AVP-induced PGE(2) synthesis but also MAPK activation, suggesting that PI3K is involved in the AVP-induced MAPK and cPLA(2)alpha activation, which initiates the production of PGE(2). These results suggest that PGE(2) generated by the stimulation of AVP probably modulates the physiological effects of AVP.     

Sphingosine 1-phosphate induces cyclooxygenase-2 via Ca2+-dependent, but MAPK-independent mechanism in rat vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on prostaglandin I(2) (PGI(2)) production and cyclooxygenase (COX) expression in cultured rat vascular smooth muscle cells (VSMCs) were investigated. S1P stimulated PGI(2) production in a concentration-dependent manner, which was completely suppressed by NS-398, a selective COX-2 inhibitor, as determined by radioimmunoassay. S1P stimulated COX-2 protein and mRNA expressions in a concentration- and time-dependent manner, while it had no effect on COX-1 expression. S1P(2) and S1P(3) receptors mRNA were abundantly expressed in rat VSMCs. Suramin, an antagonist of S1P(3) receptor, almost completely inhibited S1P-induced COX-2 expression. Pretreatment of VSMCs with pertussis toxin (PTX) partially, but significantly inhibited S1P-induced PGI(2) production and COX-2 expression. S1P also activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). However, neither PD 98059, a selective inhibitor of ERK activation, nor SB 203580, a selective inhibitor of p38 MAPK, had a significant inhibitory effect on S1P-induced COX-2 expression, suggesting that the MAPK activation does not play main roles in S1P-induced COX-2 induction. S1P-induced COX-2 expression was inhibited by PP2, an inhibitor of Src-family tyrosine kinase, Ca(2+) depletion, and GF 109203X, an inhibitor of protein kinase C (PKC). These results suggest that S1P stimulates COX-2 induction in rat VSMCs through mechanisms involving Ca(2+)-dependent PKC and Src-family tyrosine kinase activation via S1P(3) receptor coupled to PTX-sensitive and -insensitive G proteins.     

Enhancement of (−)-epigallocatechin-3-gallate and theaflavin-3-3′-digallate induced apoptosis by ascorbic acid in human lung adenocarcinoma SPC-A-1 cells and esophageal carcinoma Eca-109 cells via MAPK pathways

Tea polyphenols (−)-epigallocatechin-3-gallate (EGCG) and theaflavin-3-3′-digallate (TF₃) are two prospective compounds in cancer prevention and treatment. Ascorbic acid (Vc) is essential to a healthy diet as well as being a highly effective antioxidant. In this work, the effects of the combination of EGCG or TF₃ with Vc on the apoptosis and caspases-3/9 activities in human lung adenocarcinoma SPC-A-1 cells and esophageal carcinoma Eca-109 cells were determined. Furthermore, the role of mitogen-activated protein kinases (MAPK) pathways in the apoptosis induced by TF₃ or EGCG together with Vc were studied using three MAPK inhibitors (ERK inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580). Our results showed that Vc could enhance the EGCG and TF₃ induced apoptosis in SPC-A-1 and Eca-109 cells, and this effect involved the activation of caspase-3 and 9. EGCG, TF₃ and Vc could activate MAPK pathways respectively, and each compound activated different MAPK subfamilies in different cells. This may explain the enhancement of EGCG and TF₃ induced apoptosis by Vc in SPC-A-1 and Eca-109 cells, and will ultimately aid the design of more effective anti-cancer treatments.     

Inhibition of p38 MAP kinase activity up-regulates multiple MAP kinase pathways and potentiates 1,25-dihydroxyvitamin D(3)-induced differentiation of human leukemia HL60 cells

Differentiation therapy for neoplastic diseases has potential for supplementing existing treatment modalities but its implementation has been slow. One of the reasons is the lack of full understanding of the complexities of cellular pathways through which signals for differentiation lead to cell maturation. This was addressed in this study using HL60 cells, a well-established model of differentiation of neoplastic cells. SB 203580 and SB 202190, specific inhibitors of a signaling protein p38 MAP kinase, were found to markedly accelerate monocytic differentiation of HL60 cells induced by low concentrations of 1,25-dihydroxyvitamin D(3) (1,25D(3)). Surprisingly, inhibition of p38 activity resulted in sustained enhancement of p38 phosphorylation and of its in vitro activity in the absence of the inhibitor, indicating up-regulation of the upstream components of the p38 pathway. In addition, SB 203580 or SB 202190 treatment of HL60 cells resulted in a prolonged activation of the JNK and, to a lesser extent, the ERK pathways. The data are consistent with the hypothesis that in HL60 cells an interruption of a negative feedback loop from a p38 target activates a common regulator of multiple MAPK pathways. The possibility also exists that JNK and/or ERK pathways amplify a differentiation signal provided by 1,25D(3).     

Dual role of the p38 MAPK/cPLA2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists

p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase A₂ (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.