Hydrogen peroxide-induced arachidonic acid release in L929 cells; roles of Src, protein kinase C and cytosolic phospholipase A2alpha

Hydrogen peroxide (H(2)O(2)) stimulates the release of arachidonic acid from cells, but the signaling mechanism(s) involved remains to be elucidated. We investigated the roles of alpha-type cytosolic phospholipase A(2) (cPLA(2)alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of arachidonic acid from L929 cells (a murine fibroblast cell line), C12 cells (a variant of L929 that lacks cPLA(2)alpha) and a stable clone of C12 cells expressing cPLA(2)alpha (C12-cPLA(2)alpha cells). In the presence of 10 muM A23187, 100 nM phorbol myristate acetate (PMA) and 1 mM H(2)O(2) synergistically stimulated arachidonic acid release from L929 cells and C12-cPLA(2)alpha cells, and to a much lesser extent from C12 cells. The reagents alone and co-treatment with PMA and H(2)O(2) without A23187 had marginal effects. No arachidonic acid was released by PMA/A23187 or H(2)O(2)/A23187 in CaCl(2)-free buffer and the release was inhibited by a selective cPLA(2)alpha inhibitor (3 microM pyrrophenone). Addition of 10 microM H(2)O(2), which did not stimulate arachidonic acid release with A23187, enhanced the response to PMA/A23187. The release induced by PMA/A23187 and by H(2)O(2)/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X) and in PKC-depleted cells, and by a Src inhibitor (2 microM PP2). The phosphorylation of extracellular signal-regulated kinase 1/2 induced by PMA/A23187 and H(2)O(2)/A23187 was significantly decreased by inhibitors of PKC and Src. These findings suggest that H(2)O(2) with Ca(2+) stimulates arachidonic acid release via cPLA(2)alpha in a Src- and PKC-dependent manner in L929 cells. The role of cross-talk between Src and PKC in arachidonic acid release is discussed.     

Mechanisms of the influence of magnolol on eicosanoid metabolism in neutrophils

We have demonstrated that magnolol suppressed thromboxane B2 (TXB2) and leukotriene B4 (LTB4) formation in A23187-stimulated rat neutrophils. Maximum inhibition was obtained with about 10 microM magnolol. Magnolol was more effective in the inhibition of cyclooxygenase (COX) activity than in the inhibition of 5-lipoxygenase (5-LO) activity as assessed by means of enzyme activity determination in vitro and COX and 5-LO metabolic capacity analyses in vivo. Magnolol alone stimulated cytosolic phospholipase A2 (cPLA2) phosphorylation and the translocation of 5-LO and cPLA2 to the membrane, and evoked arachidonic acid (AA) release. Recruitment of both 5-LO and cPLA2 to the membranes was suppressed by EGTA. Arachidonyl trifluoromethyl ketone (AACOCF3), a PLA2 inhibitor, bromoenol lactone (BEL), a Ca2+-independent PLA2 (iPLA2) inhibitor, and EGTA suppressed the magnolol-induced AA release. However, none of the follows affected magnolol-induced AA-release: 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), a p38 mitogen-activated protein kinase (MAPK) inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), a MAPK kinase (MEK) inhibitor, or 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X), a protein kinase C (PKC) inhibitor. In addition, magnolol at 30 microM did not stimulate the p38 MAPK and extracellular signal-regulated kinase 2 (ERK2) enzyme activities. These results indicated that magnolol inhibits the formation of prostaglandins and leukotrienes in A23187-stimulated rat neutrophils, probably through a direct blockade of COX and 5-LO activities. The stimulatory effects of magnolol at high concentration on the membrane association of 5-LO and cPLA2 are attributable to the elevation of [Ca2+]i, and on the AA release is likely via activation of cPLA2 and iPLA2.     

Release of arachidonic acid induced by tumor necrosis factor-alpha in the presence of caspase inhibition: evidence for a cytosolic phospholipase A2alpha-independent pathway

Stimulation of L929 cells with tumor necrosis factor-alpha (TNFalpha) caused cell death accompanied by a release of arachidonic acid (AA). Although the inhibition of caspases has been shown to cause necrosis in TNFalpha-treated L929 cells, its role in the TNFalpha-induced release of AA has not been elucidated. The release of AA is tightly regulated by phospholipase A(2) (PLA(2)). To find out the mechanisms underlying the TNFalpha-induced release of AA, we investigated the relationship between TNFalpha stimulation and PLA(2) regulation with and without zVAD, an inhibitor of caspases. In the present study, we found that treatment with TNFalpha and zVAD stimulated release of AA and cell death in C12 cells (a variant of L929 cells lacking alpha type of cytosolic PLA(2) (cPLA(2)alpha)). Stimulation with TNFalpha/zVAD also caused the release of AA from L929-cPLA(2)alpha-siRNA cells. Treatment with pyrrophenone (a selective inhibitor of cPLA(2)alpha) completely inhibited the TNFalpha-induced release of AA, but only partially inhibited the TNFalpha/zVAD-induced response in L929 cells. The TNFalpha/zVAD-induced release of AA from C12 and L929-cPLA(2)alpha-siRNA cells was pyrrophenone-insensitive, but inhibited by treatment with butylated hydroxyanisole (BHA, an antioxidant). Treatment with dithiothreitol, which inactivates secretory PLA(2) activity, decreased the amount of AA released by TNFalpha/zVAD. TNFalpha/zVAD appears to stimulate release of AA from C12 cells in a cPLA(2)alpha-independent, BHA-sensitive manner. The possible roles of secretory PLA(2) and reactive oxygen species from different pools in the release of AA and cell death were discussed.     

Ceramide-1-phosphate activates cytosolic phospholipase A2alpha directly and by PKC pathway

Ceramide-1-phosphate (C1P), a novel bioactive sphingolipid, is implicated in the vital cellular processes such as cell proliferation and inflammation. The role of C1P on activity of cytosolic phospholipase A2alpha (cPLA2alpha), a key enzyme for the release of arachidonic acid (AA) and prostanoids, has not been well elucidated. In this study, we investigated the effect of C1P on the release of AA from L929 cells and a variant, which lacks cPLA2alpha expression, C12 cells. C1P at 30 microM alone induced AA release from L929 cells without an increase in intracellular Ca2+ concentration. C1P-induced AA release was marginal in C12 cells, and treatment with an intracellular Ca2+ chelator (BAPTA-AM) or an inhibitor of cPLA2alpha (2 microM pyrrophenone) decreased C1P-induced AA release in L929 cells. C1P increased the enzymatic activity of cPLA2alpha over two-fold in the presence of Ca2+. C1P triggered the translocation of cPLA2alpha and its C2 domain from the cytosol to the perinuclear region in CHO-K1 cells. Interestingly, C1P at 10 microM synergistically enhanced ionomycin-induced AA release from L929 cells. The AA release induced by C1P with and without ionomycin decreased by treatment with protein kinase C (PKC) inhibitor (10 microM GF109203X) and in the PKC-depleted cells. C1P at 10 microM stimulated the translocation of PKC (alpha and delta) from the soluble to the membrane fractions. We propose that C1P stimulates AA release via two mechanisms; direct activation of cPLA2alpha, and the PKC-dependent pathway.     

Expression of cytosolic phospholipase A2 alpha in murine C12 cells, a variant of L929 cells, induces arachidonic acid release in response to phorbol myristate acetate and Ca2+ ionophores, but not to tumor necrosis factor-alpha

Tumor necrosis factor-alpha (TNFalpha)-induced cell death is regulated through the release of arachidonic acid (AA) by group IVA cytosolic phospholipase A2 (cPLA2alpha) in the murine fibroblast cell line L929. However, the signaling pathway by which TNFalpha activates cPLA2alpha remained to be solved. We examined AA release in L929 cells, in a variant of L929 (C12 cells) lacking cPLA2alpha, and in C12 cells transfected with cPLA2alpha expression vectors. In transient and stable clones of C12 cells expressing cPLA2alpha, Ca2+ ionophore A23187 and phorbol myristate acetate (PMA) stimulated AA release within 90 min, although no response to TNFalpha was observed within 6 h. These results suggest that C12 cells may lack the components necessary for TNFalpha-induced AA release, in addition to cPLA2alpha. PMA is known to stimulate AA release via phosphorylation of Ser505 in cPLA2alpha by activating extracellular signal-regulated kinases (ERK1/2). However, PMA-induced AA release from C12 cells expressing mutant cPLA2alpha S505A (mutation of Ser505 to Ala), which is not phosphorylated by ERK1/2, was similar to that from L929 cells and C12 cells expressing wild-type cPLA2alpha. The role of Ser505 phosphorylation in AA release induced by PMA is also discussed.     

Up-regulation of cytosolic phospholipase A2alpha expression by N,N-diethyldithiocarbamate in PC12 cells; involvement of reactive oxygen species and nitric oxide

Disulfiram (an alcohol-aversive drug) and related compounds are known to provoke several side effects involving behavioral and neurological complications. N,N-diethyldithiocarbamate (DDC) is considered as one of the main toxic species of disulfiram and acts as an inhibitor of superoxide dismutase. Since arachidonic acid (AA) formation is regulated by reactive oxygen species (ROS) and related to toxicity in neuronal cells, we investigated the effects of DDC on AA release and expression of the alpha type of cytosolic phospholipase A(2) (cPLA(2)alpha) in PC12 cells. Treatment with 80-120 microM DDC that causes a moderate increase in ROS levels without cell toxicity stimulated cPLA(2)alpha mRNA and its protein expression. The expression was mediated by extracellular-signal-regulated kinase (ERK1/2), one of the mitogen-activated protein kinases. Treatment with N(G) nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase, 1 mM) and oxy-hemoglobin (a scavenger of nitric oxide, 2 mg/mL) abolished the DDC-induced responses (ERK1/2 phosphorylation and cPLA(2)alpha expression). We also showed DDC-induced up-regulation of the mRNA expression of lipocortin 1, an inhibitor of PLA(2). Furthermore, DDC treatment of the cells enhanced Ca(2+)-ionophore-induced AA release in 30 min, although the effect was limited. Changes in AA metabolism in DDC-treated cells may have a potential role in mediating neurotoxic actions of disulfiram. In this study, we show the first to demonstrate the up-regulation of cPLA(2)alpha expression by DDC treatment in neuronal cells.     

Effects of synthetic sphingosine-1-phosphate analogs on arachidonic acid metabolism and cell death

Sphingolipid metabolites such as sphingosine regulate cell functions including cell death and arachidonic acid (AA) metabolism. D-erythro-C18-Sphingosine-1-phosphate (D-e-S1P), a sphingolipid metabolite, acts as an intracellular messenger in addition to being an endogenous ligand of some cell surface receptors. The development of S1P analogs may be useful for studying and/or regulating S1P-mediated cellular responses. In the present study, we found that several synthetic S1P analogs at pharmacological concentrations stimulated AA metabolism and cell death in PC12 cells. D-erythro-N,O,O-Trimethyl-C18-S1P (D-e-TM-S1P), L-threo-O,O-dimethyl-C18-S1P (L-t-DM-S1P) and L-threo-O,O-dimethyl-3O-benzyl-C18-S1P (L-t-DMBn-S1P) at 100 microM stimulated [(3)H]AA release from the prelabeled PC12 cells. L-t-DMBn-S1P at 20 microM increased prostanoid formation in PC12 cells. L-t-DMBn-S1P-induced AA release was inhibited by D-e-sphingosine, but not by the tested PLA(2) inhibitors. L-t-DMBn-S1P did not stimulate the activity of cytosolic phospholipase A(2alpha) (cPLA(2alpha)) in vitro and the translocation of cPLA(2alpha) in the cells, and caused AA release from the cells lacking cPLA(2alpha). These findings suggest that L-t-DMBn-S1P stimulated AA release in a cPLA(2alpha)-independent manner. In contrast, D-e-S1P and D-erythro-N-monomethyl-C18-S1P caused cell death without AA release in PC12 cells, and the effects of D-e-TM-S1P, L-t-DM-S1P and L-t-DMBn-S1P on cell death were limited. Synthetic S1P analogs may be useful tools for studying AA metabolism and cell death in cells.     

Inhibition of arachidonic acid release and cytosolic phospholipase A2 alpha activity by D-erythro-sphingosine

Sphingolipid metabolites such as sphingosine 1-phosphate (S1P) and ceramide can mediate many cellular events including apoptosis, stress responses and growth arrest. Although ceramide stimulates arachidonic acid metabolism in several cells, the effects of sphingosine and its endogenous analogs have not been established. We investigated the effects of D-erythro-sphingosine and its metabolites on arachidonic acid release in the two cells and on the activity of cytosolic phospholipase A2alpha. C2-Ceramide (N-acetyl-D-erythro-sphingosine, 100 microM) alone stimulated [3H]arachidonic acid release and enhanced the ionomycin-induced release from the prelabeled PC12 cells and L929 cells. In contrast, exogenous addition of D-erythro-sphingosine inhibited the responses in a concentration-dependent manner in the two cell lines. D-erythro-sphingosine, D-erythro-N,N-dimethylsphingosine (D-erythro-DMS) and D-erythro-dihydrosphingosine (D-erythro-DHS) significantly inhibited mastoparan-, but not Na3VO4-, stimulated arachidonic acid release in PC12 cells. D-erythro-S1P and DL-threo-DHS showed no effect on the responses. Production of prostaglandin F2alpha was also enhanced by C2-ceramide (20 microM) and suppressed by D-erythro-sphingosine (10 microM) in PC12 cells. An in vitro study revealed that D-erythro-sphingosine, D-erythro-DMS and D-erythro-DHS directly inhibited cytosolic phospholipase A2alpha activity. These findings suggest that ceramide and D-erythro-analogs of sphingosine have opposite effects on phospholipase A2 activity and thus regulate arachidonic acid release from cells.     

Endothelin-1-induced arachidonic acid release by cytosolic phospholipase A2 activation in rat vascular smooth muscle via extracellular signal-regulated kinases pathway

The present study investigates whether endothelin-1 (ET-1), like noradrenaline (NA), stimulates the release of arachidonic acid (AA) via cytosolic phospholipase A2 (cPLA2) in rat tail artery. In tail artery segments labelled with [3H]AA, ET-1-induced AA release in a concentration-dependent manner with an EC50 of 1.3 nM. The effect of ET-1 was inhibited by bosentan and was insensitive to BQ788, suggesting the involvement of ETA receptor. The stimulation of AA release induced by ET-1 was prevented by arachydonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2 and not by RHC80267, a diacylglycerol lipase inhibitor. Furthermore, PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK) cascade and calphostin C, a protein kinase C (PKC) inhibitor, prevented the stimulation of AA release induced by ET-1 and NA. Immunoblotting of the cytosolic fraction of rat tail arteries stimulated with ET-1 or NA showed an increase in extracellular signal-regulated kinases (ERKs) phosphorylation and this effect was abolished by calphostin C treatment. These findings show that in rat tail artery ET-1 and NA induce a sequential activation of protein kinase C and extracellular signal-regulated kinases that results in stimulation of AA release via cPLA2 activation. This may represent a general pathway by which G-proteins coupled receptors stimulate AA release and its metabolites in vascular smooth muscle.     

Role of cytosolic phospholipase A2 in the enhancement of alpha2-adrenoceptor-mediated vasoconstriction by the thromboxane-mimetic U46619 in the porcine isolated ear artery: comparison with vasopressin-enhanced responses

Pre-contraction with the thromboxane-mimetic U46619 enhances the subsequent alpha(2)-adrenoceptor-mediated vasoconstriction in the porcine ear artery through an enhanced activation of ERK-MAP kinase. In this study we determined the role of cPLA(2) in this enhanced response, and determined whether vasopressin is also able to enhance alpha(2)-adrenoceptor-mediated vasoconstriction through the same pathway. The cPLA(2) inhibitors AACOCF3 (50 microM) and MAFP (50 microM) both inhibited the U46619-enhanced alpha(2)-adrenoceptor response, but had no effect on the direct alpha(2)-adrenoceptor response. AACOCF3 also inhibited the enhanced ERK activation associated with the enhanced alpha(2)-adrenoceptor-mediated vasoconstriction. Pre-contraction with arachidonic acid mimicked the effect of U46619 by enhancing the contractile response to the alpha(2)-adrenoceptor agonist UK14304 (1 microM) and enhancing the alpha(2)-adrenoceptor-mediated ERK activation. Pre-contraction with vasopressin also enhanced the contractile response to UK14304, but neither PD98059 (50 microM) nor AACOCF3 (50 microM) had any effect this vasopressin-enhanced response, indicating that neither the ERK pathway, nor cPLA(2) are involved in vasopressin-enhanced responses. The alpha(2)-adrenceptor-stimulated activation of ERK was also unaffected by pre-contraction with vasopressin. On the other hand, inhibition of PKCzeta inhibited the enhanced alpha(2)-adrenoceptor contraction after pre-contraction with both U46619 and vasopressin. This study demonstrates that alpha(2)-adrenoceptor-mediated vasoconstriction can be enhanced through two different pathways-one dependent upon the enhanced activation of ERK-MAP kinase through activation of cPLA(2), and the other through a different, ERK/cPLA(2)-independent pathway.     

Oxidative stress and alpha1-adrenoceptor-mediated stimulation of the Cl-/HCO3- exchanger in immortalized SHR proximal tubular epithelial cells

BACKGROUND AND PURPOSE: This study evaluated the signalling coupled to the alpha1-adrenoceptor-induced stimulation of the Cl-/HCO3- exchanger in hypertension. EXPERIMENTAL APPROACH: The Na+ -independent HCO3- transport system activity was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rat (SHR) and their normotensive control (Wistar Kyoto rat; WKY). KEY RESULTS: Noradrenaline increased Cl-/HCO3- exchanger activity with EC50 values of 0.6 and 5.3 microM in SHR and WKY cells, respectively. These effects were abolished by prazosin, but not by yohimbine. Phenylephrine increased Cl-/HCO3- exchanger activity in SHR and WKY cells (EC50 of 2.6 and 4.9 microM, respectively). Phenylephrine-mediated increase in Cl-/HCO3- exchanger activity in WKY and SHR cells was inhibited by protein kinase C (PKC), MAPK/ERK kinase (MEK) and p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. The expression of alpha1A- and alpha1B-adrenoceptors was identical in WKY and SHR cells. SHR cells generated more H2O2 than WKY cells. In SHR cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H2O2 and abolished their hypersensitivity to phenylephrine, but failed to affect basal Cl-/HCO3- exchanger activity. H2O2-dependent stimulation of Cl-/HCO3- exchange activity was significantly higher in SHR than in WKY cells. CONCLUSIONS AND IMPLICATIONS: Differences between WKY and SHR cells on their sensitivity to alpha1-adrenoceptor stimulation did not correlate with the abundance of alpha1A- and alpha1B-adrenoceptors and may be related to the increased generation of H2O2, which may amplify the response downstream of alpha1-adrenoceptor activation.     

Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons

BACKGROUND AND PURPOSE: Stimulation of astrocytes by the alpha(2)-adrenoceptor agonist dexmedetomidine, a neuroprotective drug, transactivates epidermal growth factor (EGF) receptors. The present study investigates signal pathways leading to release of an EGF receptor ligand and those activated during EGF receptor stimulation, and the response of neurons to dexmedetomidine and to astrocyte-conditioned medium. EXPERIMENTAL APPROACH: Phosphorylation of ERK(1/2) was determined by western blotting and immunocytochemistry, and phosphorylation of EGF receptors by immunoprecipitation and western blotting. mRNA expression of fos family was measured by RT-PCR. KEY RESULTS: Pertussis toxin (0.2 microg ml(-1)) an inhibitor of betagamma subunit dissociation from Galpha(i) protein, and GF 109203X (500 nM), a protein kinase C inhibitor, abolished ERK(1/2) phosphorylation. PP1 (10 microM), inhibiting Src kinase and GM 6001 (10 microM), an inhibitor of Zn-dependent metalloproteinase, abolished ERK(1/2) phosphorylation by dexmedetomidine (50 nM), but not that by EGF (10 ng ml(-1)), showing Src kinase and metalloproteinase activation during the first stage only; AG 1478 (1 microM), an inhibitor of the EGF receptor tyrosine kinase, abolished ERK(1/2) phosphorylation. Dexmedetomidine-induced EGF receptor phosphorylation was prevented by AG 1478, GM 6001, PP1 and GF 109203X and its induction of cfos and fosB by AG 1478 and by U0126 (10 microM), an inhibitor of ERK phosphorylation, indicating downstream effects of ERK(1/2) phosphorylation. EGF and conditioned medium from dexmedetomidine-treated astrocytes, but not dexmedetomidine itself, induced ERK phosphorylation in primary cultures of cerebellar neurons. CONCLUSIONS AND IMPLICATIONS: Dexmedetomidine-induced transactivation pathways were delineated. Its paracrine effect on neurons may account for its neuroprotective effects.     

Extracellular ATP activates ERK1/ERK2 via a metabotropic P2Y1 receptor in a Ca2+ independent manner in differentiated human skeletal muscle cells

ATP is released at the neuromuscular junction to regulate development and proliferation. The sequential expression of P2X and P2Y receptors has been correlated to these effects in many species and cell lines. We have therefore investigated ATP mediated signalling in differentiated primary human skeletal muscle cells. ATP was capable to trigger Ca2+ transients in these cells via P2Y receptors which were not attributable to Ca2+ influx via P2X receptors. Instead, ATP propagated the formation of inositol phosphate (IP) with an EC50 of 21.3 microM. The Ca2+ transient provoked by ATP was abrogated roughly 75% by the phospholipase C (PLC) inhibitor, U73122. Interestingly, the ryanodine sensitive Ca2+ pool was not involved in ATP triggered Ca2+ release. On mRNA level and by a pharmacological approach we confirmed the presence of the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Substantially, ATP activated IP formation via a P2Y1 receptor. In addition, ATP elicited extracellular signal regulated kinase (ERK)1/2 phosphorylation in a time and concentration dependent manner, again mainly via P2Y1 receptors. The ATP mediated ERK1/2 phosphorylation was strictly dependent on phospholipase C and PI3 kinase activity. Importantly, ATP mediated ERK1/2 phosphorylation was Ca2+ independent. This observation was corroborated by the finding that conventional protein kinase C inhibitors did not suppress ATP triggered ERK1/2 phosphorylation. Taken together, these observations highlight the importance of ATP as a co-neurotransmitter at the neuromuscular junction via dual signalling, i.e. IP3 receptor mediated Ca2+ transients and Ca2+ insensitive phosphorylation of ERK1/2.     

Novel effect of carvedilol on Ca2+ movement in renal tubular cells

The effect of carvedilol on intracellular free Ca(2+) levels ([Ca(2+)](i)) has not been explored previously. This study was aimed to examine the effect of carvedilol on Ca(2+) handling in renal tubular cells. Madin-Darby canine kidney cells were used as a model for renal tubular cells and fura-2 was used as a fluorescent Ca(2+) probe. Carvedilol increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 5 microM. Extracellular Ca(2+) removal partly inhibited the [Ca(2+)](i) signals. Carvedilol-induced Ca(2+) influx was verified by measuring Mn(2+)-induced quench of fura-2 fluorescence. Carvedilol-induced store Ca(2+) release was reduced by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) but not with 5 microM ryanodine or 2 microM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). Carvedilol (30 microM)-induced Ca(2+) release was not affected by inhibiting phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-l)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; 2 microM), but was potentiated by increasing cAMP levels or inhibiting protein kinase C. The carvedilol-induced Ca(2+) mobilization was not significantly sequestered by the endoplasmic reticulum or mitochondria. This study shows that carvedilol increased [Ca(2+)](i) in renal tubular cells by causing Ca(2+) release from the endoplasmic reticulum and other unknown stores in an inositol-1,4,5-trisphosphate-independent manner, and by inducing Ca(2+) influx. The Ca(2+) release was modulated by cAMP and protein kinase C.     

Mechanisms for prostaglandin E2 formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.     

Enhancement of peripheral benzodiazepine receptor ligand-induced apoptosis and cell cycle arrest of esophageal cancer cells by simultaneous inhibition of MAPK/ERK kinase

Specific ligands of the peripheral benzodiazepine receptor (PBR) activate pro-apoptotic and anti-proliferative signaling pathways. Previously, we found that PBR ligands activated the p38 mitogen-activated protein kinase (MAPK) pathway in esophageal cancer cells, and that the activation of p38MAPK contributed to tumor cell apoptosis and cell cycle arrest. Here, we report that PBR ligands also activate the pro-survival MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in esophageal cancer cells, which might compromise the efficacy of PBR ligands. Hence, a combination treatment of PBR ligands and MEK inhibitors, which are emerging as promising anticancer agents, was pursued to determine whether this treatment could lead to enhanced apoptosis and cell cycle arrest. Using Western blotting we demonstrated a time- and dose-dependent phosphorylation of ERK1/2 in response to PBR ligands. Apoptosis was investigated by assessment of mitochondrial alterations and caspase-3 activity. Cell cycle arrest was measured by flow cytometric analysis of stained isolated nuclei. The inhibition of MEK/ERK with a pharmacologic inhibitor, 2'-amino-3'-methoxyflavone (PD 98059), resulted in a synergistic enhancement of PBR-ligand-induced growth inhibition, apoptosis and cell cycle arrest. Specifity of the pharmacologic inhibitor was confirmed by the use of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 0126), a second MEK/ERK inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U 0124), a structural analogue of it which does not display any affinity to MEK. Enhanced pro-apoptotic and anti-proliferative effects were observed both in KYSE-140 esophageal squamous cancer and OE-33 adenocarcinoma cells, suggesting that this effect was not cell-type specific. In addition, the PBR-mediated overexpression of the stress response gene (growth arrest and DNA-damage-inducible gene gadd153) was synergistically enhanced by MEK inhibition. This is the first report of enhanced PBR-ligand-mediated apoptosis and cell cycle arrest by simultaneous MEK inhibition, suggesting a new anticancer strategy.     

Activated human hydroxy-carboxylic acid receptor-3 signals to MAP kinase cascades via the PLC-dependent PKC and MMP-mediated EGFR pathways

BACKGROUND AND PURPOSE 3-Hydroxy-octanoate, recently identified as a ligand for, the orphan GPCR, HCA(3), is of particular interest given its ability to treat lipid disorders and atherosclerosis. Here we demonstrate the pathway of HCA(3)-mediated activation of ERK1/2. EXPERIMENTAL APPROACH Using CHO-K1 cells stably expressing HCA(3) receptors and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA(3) receptors, HCA(3)-mediated activation of ERK1/2 was measured by Western blot. KEY RESULTS HCA(3)-mediated activation of ERK1/2 was rapid, peaking at 5 min, and was Pertussis toxin sensitive. Our data, obtained by time course analyses in combination with different kinase inhibitors, demonstrated that on agonist stimulation, HCA(3) receptors evoked ERK1/2 activation via two distinct pathways, the PLC/PKC pathway at early time points (≤ 2 min) and the MMP/ epidermal growth factor receptor (EGFR) transactivation pathway with a maximum response at 5 min. Furthermore, our present results also indicated that the βγ-subunits of the G(i) protein play a critical role in HCA(3)-activated ERK1/2 phosphorylation, whereas β-arrestins and Src were not required for ERK1/2 activation. CONCLUSIONS AND IMPLICATIONS We have described the molecular mechanisms underlying the coupling of human HCA(3) receptors to the ERK1/2 MAP kinase pathway in CHO-K1 and A431 cells, which implicate the G(i) protein-initiated, PLC/PKC -and platelet-derived growth factor receptor/EGFR transactivation-dependent pathways. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the HCA(3)-mediated activation of ERK1/2.