Activation of arachidonate release and cytosolic phospholipase A2 via extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in macrophages stimulated by bacteria or zymosan

The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A2 (cPLA2). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA2 in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA2 activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA2 activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA2 activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA2 and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages.     

Extracellular-regulated kinase activation regulates replication of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> intracellularly in primary human monocytes

Mycobacterium avium-intracellulare (MAI) is a ubiquitous environmental pathogen that causes disseminated infection in immunocompromised patients, such as those with human immunodeficiency virus, interleukin-12 deficiency, or interferon-γ receptor mutation. Colony morphotypes are associated with MAI pathogenicity. Our previous studies have reported that smooth-transparent (SmT) morphotypes are more virulent and induce less cytokine (interleukin-1β and tumor necrosis factor-α) production by human monocytes than the smooth-domed (SmD) morphotypes. Mitogen-activated protein (MAP) kinases such as extracellular-regulated kinase (ERK) are activated by the phagocytosis of particle antigens in macrophages, and this ERK activation subsequently influences cytokine expression and the control of intracellular pathogen growth. The influence of MAP kinase activation on MAI replication in human monocytes was examined. Peripheral blood monocytes isolated from healthy subjects by Ficoll-Hypaque sedimentation were infected with virulent SmT or avirulent SmD MAI without or with MAP kinase inhibitors. MAP kinase activities were determined by in vitro kinase assay, intracellular MAI growth by CFU assay, and cytokines by enzyme-linked immunosorbent assay. MAI infection induced ERK and p38 activation. Inhibition of ERK by PD98059, but not p38, significantly increased intracellular MAI growth. Tumor necrosis factor-α release and interleukin-1β production in response to MAI were reduced by MAP kinase inhibition. p38 inhibition tended to reduce cytokine production more substantially. These data suggest that ERK activation limits intra-monocytic MAI replication and enhances monocytic cytokine release, whereas p38 activation influences only cytokine release. The effect of MAP kinases on MAI growth might thus be mediated by the modulation of cytokine production.     

The differential interaction of p38 MAP kinase and tumor necrosis factor-alpha in human alveolar macrophages and monocytes induced by Mycobacterium tuberculois

Cellular signaling by TNF-alpha is mediated through activation of mitogen activated protein (MAP) kinases. In particular, p38 MAP kinase is activated in mononuclear phagocytes and may be important in sustaining TNF-alpha activity. Here, we compared the activation and mutual regulation of p38 MAP kinase and TNF-alpha by MTB in human alveolar macrophages (AM) and blood monocytes (MN). AM and autologous MN were prepared, and stimulated by MTB at 1:1 (bacteria/cell). MAP kinase activation was assessed by immunoprecipitation and kinase activity. TNF-alpha mRNA was assessed by real-time RT-PCR, and TNF-alpha immunoreactivity was assessed by ELISA. MTB-induced p38MAP kinase rapidly in AM as compared to MN, and inhibition of p38 MAP kinase by SB203580 reduced both TNF-alpha mRNA and protein. Activation of ERK (1/2) by MTB followed similar kinetics in both AM and MN. TNF-alpha produced by MTB sustained p38 MAP kinase activation in MN only. These data suggest that interaction of resident pulmonary macrophages and the more immature MN with MTB differ with regard to both p38 MAP kinase activation and TNF-alpha expression.     

Toll-like receptor 9-mediated cytosolic phospholipase A2 activation regulates expression of inducible nitric oxide synthase

Although CpG containing DNA is an important regulator of innate immune responses via toll-like receptor 9 (TLR9), excessive activation of this receptor is detrimental to the host. Here, we show that cytosolic phospholipase A₂ (cPLA₂) activation is important for TLR9-mediated inducible nitric oxide synthase (iNOS) expression. Activation of TLR9 signaling by CpG induces iNOS expression and NO production. Inhibition of TLR9 blocked the iNOS expression and NO production. The CpG also stimulates cPLA₂-hydrolyzed arachidonic acid (AA) release. Inhibition of cPLA₂ activity by inhibitor attenuated the iNOS expression by CpG response. Additionally, knockdown of cPLA₂ protein by miRNA also suppressed the CpG-induced iNOS expression. Furthermore, the CpG rapidly phosphorylates three MAPKs and Akt. A potent inhibitor for p38 MAPK or Akt blocked the CpG-induced AA release and iNOS expression. These results suggest that TLR9 activation stimulates cPLA₂ activity via p38 or Akt pathways and mediates iNOS expression.     

Expression of phospholipases A2 in primary human lung macrophages: Role of cytosolic phospholipase A2-α in arachidonic acid release and platelet activating factor synthesis

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA₂) and secreted phospholipases A₂ (sPLA₂) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA₂s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA₂ (iPLA₂). Two structurally-divergent inhibitors of group IV cPLA₂ completely block arachidonic acid release by macrophages in response to non-physiological (Ca²⁺ ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA₂s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA₂s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA₂ inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA₂-alpha, iPLA₂ and several sPLA₂s. Cytosolic PLA₂-alpha is the major enzyme responsible for lipid mediator production in human macrophages.     

Hyperforin induces Ca-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A2 activation through select phospholipid interactions

Here, we investigated the modulation of cytosolic phospholipase A₂ (cPLA₂)-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10 µM) versus unstimulated cells, which was blocked by cPLA₂α-inhibition, and induced translocation of cPLA₂ to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca²⁺ by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA₂ at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA₂ phosphorylation. However, neither AA release nor translocation of cPLA₂ was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA₂ in the presence of Ca²⁺. However, when Ca²⁺ was omitted, hyperforin caused a prominent increase in cPLA₂ activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional ¹H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA₂ activation by enabling its access towards select lipid membranes independent of Ca²⁺ ions. Such Ca²⁺- and phosphorylation-independent mechanism of cPLA₂ activation may apply also to other membrane-interfering molecules.     

Phosphatidylinositol 3-kinase in zymosan- and bacteria-induced signalling to mobilisation of arachidonic acid in macrophages

Stimulation of mouse peritoneal macrophages with zymosan or bacteria results in activation of 85-kDa cytosolic phospholipase A(2) (cPLA(2)) and release of arachidonate. We have investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the signalling leading to activation of cPLA(2) and release of arachidonate in response to zymosan and the bacterium Prevotella intermedia. The specific PtdIns 3-kinase inhibitor wortmannin completely inhibited zymosan- and bacteria-induced release of arachidonate with an IC(50) value of 10-20 nM. Wortmannin also completely inhibited the zymosan-induced activation of cPLA(2), while the cPLA(2) activation by bacteria was partially inhibited by about 50%. Further experiments showed that zymosan-induced activation of extracellular signal-regulated kinase was inhibited, and bacteria-induced activation of the kinase strongly reduced, in the presence of wortmannin. Also zymosan-induced activation of p38 mitogen-activated protein kinase was inhibited by wortmannin, while p38 activation induced by bacteria was not. The zymosan- and bacteria-induced activation of phospholipase C, as determined by the generation of inositol phosphates, was also inhibited by wortmannin. Moreover, zymosan caused activation of PtdIns 3-kinase, which was totally inhibited by wortmannin. In contrast to zymosan and bacteria, arachidonate release induced by calcium ionophore alone, or further amplified by phorbol ester, was not sensitive to wortmannin. These results suggest that PtdIns 3-kinase constitutes a critical component in the zymosan- and bacteria-induced signalling leading to release of arachidonate and that PtdIns 3-kinase is positioned upstream of phospholipase C in this pathway.     

Tryptase Activation of Immortalized Human Urothelial Cell Mitogen-Activated Protein Kinase

The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA₂ activation. Incubation with the membrane phospholipid-derived PLA₂ hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA₂ activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS.     

Activation of phospholipase A2 and MAP kinases by oxidized low-density lipoproteins in immortalized GP8.39 endothelial cells

In immortalized rat brain endothelial cells (GP8.39), we have previously shown that oxidized LDL (oxLDL), after 24-h treatment, stimulates arachidonic acid release and phosphatidylcholine hydrolysis by activation of cytosolic phospholipase A₂ (cPLA₂). A putative role for MAPKs in this process has emerged. Here, we studied the contribution of Ca²⁺-independent phospholipase A₂ (iPLA₂), and the role of the MAP kinase family as well as both cPLA₂ and iPLA₂ mRNA expression by RT-PCR in oxLDL toxicity to GP8.39 cells in vitro. The activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH₂-terminal kinase (JNK) was assessed with Western blotting and kinase activity assays. iPLA₂ activity, which was found as a membrane-associated enzyme, was more stimulated by oxLDL compared with native LDL. The phosphorylation of ERK1/2, p38 and JNKs was also significantly enhanced in a dose-dependent manner. PD98059, an ERK inhibitor, SB203580, a p38 inhibitor, and SP600125, an JNK inhibitor, abolished the stimulation of all three members of the MAPK family by oxLDL. Confocal microscopy analysis and subcellular fractionation confirmed either an increase in phosphorylated form of ERKs, p38 and JNKs, or their nuclear translocation upon activation. A strong inhibition of MAPK activation was also observed when endothelial cells were treated with GF109203X, a PKC inhibitor, indicating the important role of both PKC and all three MAPKs in mediating the maximal oxLDL response. Finally, compared with samples untreated or treated with native LDL, treatment with oxLDL (100 μM hydroperoxides) for 24 h significantly increased the levels of constitutively expressed iPLA₂ protein (by 5.1-fold) and mRNA (by 3.1-fold), as well as cPLA₂ protein (by 4.4-fold) and mRNA (by 1.5-fold). Together, these data link the stimulation of PKC–ERK–p38–JNK pathways and PLA₂ activity by oxLDL to the prooxidant mechanism of the lipoprotein complex, which may initially stimulate the endothelial cell reaction against noxious stimuli as well as metabolic repair, such as during inflammation and atherosclerosis.     

The involvement of a P38-like MAP kinase in ABA-induced and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-mediated stomatal closure in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

SB203580 is a specific inhibitor of p38 mitogen-activated protein (MAP) kinase and has been widely used to investigate the physiological roles of p38 in animal and yeast cells. Here by using an epidermal strip bioassay, laser-scanning confocal microscopy and whole-cell patch clamp analysis, we assess the effects of pyridinyl imidazoles-like SB203580 on the H₂O₂ signaling in guard cells of Vicia faba L. The results indicated that SB203580 blocks H₂O₂- or ABA-induced stomatal closure, ABA-induced H₂O₂ generation, and decrease in K⁺ fluxing across plasma membrane of Vicia guard cells by application of ABA and H₂O₂, whereas its analog SB202474 had no effect on these events. Thus, these results suggest that activation of p38-like MAP kinase modulates guard cell ROS signaling in response to stress.     

A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin-stimulated proliferation of vascular smooth muscle cells

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA₂) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA₂ are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA₂ and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA₂ by 1.5-fold (p < 0.01), which was blocked by the cPLA₂ inhibitor MAFP (10 μM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 μM; 15 min) and ERK1/2 inhibitor PD98059 (20 μM; 15 min) abolished the INS-mediated increase in cPLA₂ phosphorylation by 59% (p < 0.001), and by 75% (p < 0.001), respectively. Further, inhibition of cPLA₂ with cPLA₂ inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% (p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% (p < 0.001). The effect of INS on VSMCs proliferation was significantly (p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA₂ and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA₂ activity.     

Effects of prostaglandin D2 on Na-dependent phosphate transport activity and its intracellular signaling mechanism in osteoblast-like cells

Inorganic phosphate (Pi) transport probably represents an important function of bone-forming cells in relation to extracellular matrix mineralization. In the present study, we investigated the effect of prostaglandin D₂ (PGD₂) on Pi transport activity and its intracellular signaling mechanism in MC3T3-E1 osteoblast-like cells. PGD₂ stimulated Na-dependent Pi uptake time- and dose-dependently in MC3T3-E1 cells during their proliferative phase. A protein kinase C (PKC) inhibitor calphostin C partially suppressed the stimulatory effect of PGD₂ on Pi uptake. The selective inhibitors of mitogen-activated protein (MAP) kinase pathways such as ERK, p38 and Jun kinases suppressed PGD₂-induced Pi uptake. The inhibitors of phosphatidylinositol (PI) 3-kinase and S₆ kinase reduced this effect of PGD₂, while Akt kinase inhibitor did not. These results suggest that PGD₂ stimulates Na-dependent Pi transport activity in the phase of proliferation of osteoblasts. The mechanisms responsible for this effect are activation of PKC, MAP kinases, PI 3-kinase and S₆ kinase.     

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.     

Regulation of MAPK pathways in response to purinergic stimulation of adult rat cardiac myocytes

We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA₂ translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA₂ may be a downstream target of MSK1.     

Phospholipase C-γ2 via p38 and ERK1/2 MAP kinase mediates diperoxovanadate-asparagine induced human platelet aggregation and sCD40L release

Abstract

Objective

Redox imbalance either inside platelets or in their immediate surroundings prove detrimental to their physiologic functions during haemostasis. This study was therefore aimed to assess the effect of peroxide radicals on platelet functions and underlying signalling mechanisms using asparagine-conjugated diperoxovanadate (DPV-Asn).

Methods

Platelet aggregation, ATP secretion, TxB₂ release, intra-platelet calcium mobilization, protein tyrosine phosphorylation, GPIIbIIIa activation by PAC1 labelling and sCD40L release (enzyme-linked immunosorbent assay) was monitored using various concentrations of DPV-Asn. Cell viability was assessed by Annexin V labelling, MTT assay, LDH leakage and mitochondrial membrane potential by JC-1.

Results

Platelet aggregation induced by DPV-Asn was chiefly regulated by dense granule secretion, thromboxane A₂ (TxA₂) generation, intra-platelet [Ca²⁺] influx, GPIIbIIIa activation and sCD40L release, which were significantly reduced in presence of U73122 (PLC inhibitor), aspirin (COX), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor). This was further corroborated by enhanced tyrosine phosphorylation of numerous platelet proteins including PLC-γ2, which apparently played a central role in transducing peroxide signals to regulate [Ca²⁺] influx and phosphorylation of p38 and ERK1/2 MAP kinase.

Discussion

Peroxide radicals critically regulate the thrombo-inflammatory functions of platelets via the PLCγ2-p38-ERK1/2-TxA₂ pathway, which closely resembles the clinical scenario of various pathologies like hyperglycemia and atherosclerosis during which oxidative stress disrupts platelet functions.     

(S)-tetrahydroisoquinoline alkaloid inhibits LPS-induced arachidonic acid release through downregulation of cPLA2 expression

Sepsis, a systemic inflammatory response syndrome, remains a potentially lethal condition. (S)-1-α-Naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) is noted as a drug candidate for sepsis. Many studies have demonstrated its significant anti-inflammatory effects. Here we first examined whether CKD712 inhibits lipopolysaccharide (LPS)-induced arachidonic acid (AA) release in the RAW 264.7 mouse monocyte cell line, and subsequently, its inhibitory mechanisms. CKD712 reversed LPS-associated morphological changes in the RAW 264.7 cells, and inhibited LPS-induced release of AA in a concentrationdependent manner. The inhibition was apparently due to the diminished expression of a cytosolic form of phospholipase A₂ (cPLA₂) by CKD712, resulting from reduced NF-κB activation. Furthermore, CKD712 inhibited the activation of ERK1/2 and SAP/JNK, but not of p38 MAPK. CKD712 had no effect on the activity or phosphorylation of cPLA₂ and on calcium influx. Our results collectively suggest that CKD712 inhibits LPS-induced AA release through the inhibition of a MAPKs/NF-κB pathway leading to reduced cPLA₂ expression in RAW 264.7 cells.     

Coupling of M(2) muscarinic receptors to ERK MAP kinases and caldesmon phosphorylation in colonic smooth muscle

Coupling of M(2) and M(3) muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M(3) receptors were selectively inactivated by N, N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard (40 nM). ACh elicited activation of extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 MAP kinases in control muscles and increased phosphorylation of caldesmon (Ser(789)), a putative downstream target of MAP kinases. Alkylation of M(3) receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 microM AF-DX 116 completely prevented activation of ERK and p38 MAP kinase and prevented caldesmon phosphorylation. Caldesmon phosphorylation was blocked by the MAP kinase/ERK kinase inhibitor PD-98509 but not by the p38 MAP kinase inhibitor SB-203580. These results indicate that colonic smooth muscle M(2) receptors are coupled to ERK and p38 MAP kinases. Activation of ERK, but not p38 MAP kinases, results in phosphorylation of caldesmon in vivo, which is a novel function for M(2) receptor activation in smooth muscle.     

Involvement of ERK in NMDA receptor-independent cortical neurotoxicity of hydrogen sulfide

Hydrogen sulfide (H₂S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H₂S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypan blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c, leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H₂S involves activation of the MEK/ERK pathway and some apoptotic mechanisms.