Vanadate-induced activation of cytosolic phospholipase A2alpha in L929 cells: Roles of tyrosine kinase, protein kinase C, and extracellular signal-regulated kinase

Orthovanadate (Na3VO4), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of alpha-type cytosolic phospholipase A2 (cPLA2alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na3VO4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA2alpha, were used along with a clone of C12 cells that are stably expressing cPLA2alpha (C12-cPLA2alpha cells). In the presence of a Ca2+ ionophore (10 microM A23187), 5 and 10mM Na3VO4 synergistically stimulated AA release from L929 and C12-cPLA2alpha cells, and to a much lesser extent from control C12 cells. The release of AA by Na3VO4/A23187 was inhibited by a selective cPLA2alpha inhibitor (3 microM pyrrophenone). The release of AA by Na3VO4/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 microM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 microM U0126). The phosphorylation of ERK1/2 was stimulated by Na3VO4, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na3VO4 stimulates AA release largely via cPLA2alpha activation in Ca2+-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na3VO4-induced AA release from L929 cells.     

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.     

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.     

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.     

Mechanisms Of Endothelin-1-Induced Potentiation Of Noradrenaline Response In Rat Mesenteric Artery

1. Subthreshold concentrations of endothelin (ET)-1 enhance the contractile responses to noradrenaline (NA). We investigated possible mechanisms underlying the ET-1-induced enhancement of vasoconstrictor responses to NA in rat perfused mesenteric arteries. 2. Perfusion of arteries with subpressor dose of ET-1 (3 x 10-10 mol/L) significantly potentiated the pressor responses to NA (10-6, 3 x 10-6 and 10-5 mol/L) and this action of ET-1 was endothelium independent. 3. The protein kinase C (PKC) inhibitors staurosporine (10-8 mol/L) and calphostin C (10-7 mol/L) markedly attenuated the ET-1-induced enhancement of NA responses. Vasoconstrictor responses to NA were potentiated when vessels were perfused with phorbol 12-myristate 13-acetate (10-8 mol/L). 4. The potentiating effect of ET-1 was efficiently suppressed by Y-27632 (10-6 mol/L), a selective Rho-kinase inhibitor. In the presence of both staurosporine and Y-27632, contractile responses to NA alone were decreased markedly and ET-1-induced potentiation was abolished. 5. Both staurosporine and Y-27632 decreased contractile responses to NA in arteries of deoxycorticosterone acetate (DOCA)-salt hypertensive rats to levels observed in normotensive control animals. 6. These findings suggest that ET-1-mediated potentiation of responses to NA occurs through activation of either PKC or Rho-kinase. This mechanism seems to contribute to the enhanced vasoconstrictor responces to NA observed in DOCA-salt hypertensive rats, in which the responses to NA are enhanced tonically by endogenous vascular ET-1.     

Stimulation of catecholamine biosynthesis via the protein kinase C pathway by endothelin-1 in PC12 rat pheochromocytoma cells

It has been reported that endothelins (ETs) stimulate catecholamine release from chromaffin cells. However, it is not known whether ETs also affect catecholamine biosynthesis. Thus, using a rat pheochromocytoma cell line, PC12, we examined the effects of ETs on catecholamine biosynthesis. The mRNA level and activity of tyrosine hydroxylase (TH), a rate-limiting enzyme in catecholamine biosynthesis, were increased significantly by endothelin-1 (ET-1) (100nM). These stimulatory effects were inhibited completely by a blocker for the A-type endothelin receptor, BQ-123 [cyclo(D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl)] (1 microM), but not by a blocker for the B-type endothelin receptor, BQ-788 (N-cis 2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine (1 microM). Also, Ro-32-0432 (3-[8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido-[1,2-a]indol-10-yl]-4-(1-methyl-3-indolyl)-H-pyrrole-2,5-dione hydrochloride) (100nM), a protein kinase C inhibitor, completely inhibited ET-1-induced increases in TH activity and mRNA level. Furthermore, ET-1 (100nM) significantly stimulated protein kinase C activity, as well as inositol 1,4,5-triphosphate production; these stimulatory effects were abolished by BQ-123 but not by BQ-788. Moreover, ET-1 (100nM) significantly increased both the TH-protein level and the intracellular catecholamine content. By contrast to ET-1, endothelin-3 did not affect catecholamine synthesis. These results indicate that ET-1, but not ET-3, stimulates catecholamine synthesis through the PKC pathway in PC12 cells. Also, the use of selective ET receptor antagonists suggests that the effects of ET-1 on catecholamine biosynthesis are mediated through ET(A).     

Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.     

Involvement of mitogen-activated protein kinases and protein kinase C in cadmium-induced prostaglandin E2 production in primary mouse osteoblastic cells

We previously reported that cadmium (Cd) induced prostaglandin E₂ (PGE₂) biosynthesis through the activation of cytosolic phospholipase A₂ (cPLA₂) and induction of cyclooxygenase 2 (COX-2) in primary mouse osteoblastic cells. In the present study, we further investigated the mechanism of PGE₂ production by Cd focusing on the main mitogen-activated protein kinase (MAPK) subfamilies that mediate prostaglandin synthesis, extracellular signal-regulated kinase (ERK1/2 MAPK), c-jun-amino-terminal kinase (JNK MAPK) and p38 MAPK, and protein kinase C (PKC) which is activated by Cd in several kinds of cells. Cd at 2 μM and above stimulated PGE₂ production in osteoblastic cells and its production was inhibited by the kinase-specific inhibitors PD98059, SB203580, curcumin, and calphostin C. Calphostin C also inhibited the production of PGE₂ by phorbol 12-myristate 13-acetate (PMA), which is a potent activator of PKC. PD98059 inhibited PGE₂ production stimulated by PMA as well as Cd, indicating that activation of PKC by ERK1/2 MAPK was necessary for Cd-stimulated PGE₂ production. Moreover, Cd stimulated the phosphorylation of these three MAPKs, and inhibition of the phosphorylation of ERK1/2 MAPK by calphostin C was also observed. On the other hand, Cd was found to phosphorylate cPLA₂ and the phosphorylation was inhibited by PD98059, indicating that cPLA₂ was activated by Cd through ERK1/2 MAPK and released arachidonic acid (AA), a substrate of COX-2, from membranous phospholipids. From these results, it was suggested that activation of each of the ERK1/2, p38, and JNK MAPK cascades in addition to that of PKC and cPLA₂ played an important role in the Cd-stimulated biosynthesis of PGE₂ in mouse osteoblastic cells.     

CysLT1 signal transduction in differentiated U937 cells involves the activation of the small GTP-binding protein Ras

We investigated the signal transduction pathway(s) of leukotriene D(4) (LTD(4)) in the human promonocytic U937 cells, a cell line known to constitutively express CysLT(1) receptors. Herein, we demonstrate that LTD(4) specifically acts on a CysLT(1) receptor to dose-dependently increase (three to five-fold over basal) RasGTP through a G(i/o) protein. In fact, while cytosolic Ca(2+) ([Ca(2+)](i)) increase was only partially sensitive to pertussis toxin (PTx), Ras activation was almost completely inhibited by the same toxin. Furthermore, the phospholipase C (PLC) inhibitor U73122 completely inhibited both [Ca(2+)](i) and RasGTP increase, suggesting that in these cells PLC is the point of convergence for both PTx insensitive and sensitive pathways leading to [Ca(2+)](i) release and Ras activation. Indeed, chelating intracellular Ca(2+) strongly (>70%) prevented LTD(4)-induced Ras activation, indicating that this ion plays an essential role for CysLT(1)-induced downstream signaling in differentiated U937 (dU937) cells. In addition, while Src did not appear to be substantially involved in CysLT(1)-induced signaling, genistein was able to partially inhibit LTD(4)-induced [Ca(2+)](i) transient ( approximately 34%) and almost completely prevented Ras activation (>90%), suggesting a potential role for other Ca(2+)-dependent tyrosine kinases in LTD(4)-induced signaling. Finally, agonist-induced CysLT(1) stimulation was followed by a specific extracellular regulated kinase (ERK) 1/2 phosphorylation, an event with a pharmacological profile similar to that of Ras activation, partially ( approximately 40%) sensitive to Clostridium sordellii lethal toxin and totally blocked by PTx. In conclusion, LTD(4)-induced CysLT(1) receptor activation in dU937 cells leads to Ras activation and ERK phosphorylation mostly through a PTx-sensitive G(i/o) protein, PLC, and Ca(2+)-dependent tyrosine kinase(s).     

Arachidonic acid release mediated by OX1 orexin receptors

Background and purpose:

We have previously shown that lipid mediators, produced by phospholipase D and C, are generated in OX₁ orexin receptor signalling with high potency, and presumably mediate some of the physiological responses to orexin. In this study, we investigated whether the ubiquitous phospholipase A₂ (PLA₂) signalling system is also involved in orexin receptor signalling.

Experimental approach:

Recombinant Chinese hamster ovary-K1 cells, expressing human OX₁ receptors, were used as a model system. Arachidonic acid (AA) release was measured from ³H-AA-labelled cells. Ca²⁺ signalling was assessed using single-cell imaging.

Key results:

Orexins strongly stimulated [³H]-AA release (maximally 4.4-fold). Orexin-A was somewhat more potent than orexin-B (pEC₅₀= 8.90 and 8.38 respectively). The concentration–response curves appeared biphasic. The release was fully inhibited by the potent cPLA₂ and iPLA₂ inhibitor, methyl arachidonyl fluorophosphonate, whereas the iPLA₂ inhibitors, R- and S-bromoenol lactone, caused only a partial inhibition. The response was also fully dependent on Ca²⁺ influx, and the inhibitor studies suggested involvement of the receptor-operated influx pathway. The receptor-operated pathway, on the other hand, was partially dependent on PLA₂ activity. The extracellular signal-regulated kinase, but not protein kinase C, were involved in the PLA₂ activation at low orexin concentrations.

Conclusions and implications:

Activation of OX₁ orexin receptors induced a strong, high-potency AA release, possibly via multiple PLA₂ species, and this response may be important for the receptor-operated Ca²⁺ influx. The response coincided with other high-potency lipid messenger responses, and may interact with these signals.     

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.     

Non-dioxin-like polychlorinated biphenyls induce a release of arachidonic acid in liver epithelial cells: a partial role of cytosolic phospholipase A(2) and extracellular signal-regulated kinases 1/2 signalling

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) have been shown to act as tumor promoters in liver; however, the exact mechanisms of their action are still only partially understood. One of the interesting effects of NDL-PCBs is the acute inhibition of gap junctional intercellular communication (GJIC), an effect, which has been often found to be associated with tumor promotion. As previous studies have suggested that NDL-PCB-induced disruption of lipid signalling pathways might correspond with GJIC inhibition, we investigated effects of PCBs on the release of arachidonic acid (AA) in the rat liver epithelial WB-F344 cell line, a well-established model of liver progenitor cells. We found that both 2,2',4,4'-tetrachlorobiphenyl (PCB 47) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), but not the dioxin-like, non-ortho-substituted, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), induce a massive release of AA. The AA release, induced by PCB 153, was partially inhibited by extracellular signal-regulated kinases 1/2 (ERK1/2) signalling inhibitor, U0126, and by cytosolic phospholipase A(2) (cPLA(2)) inhibitor, AACOCF(3). Although PCB 153 induced both ERK1/2 and p38 activation, the specific p38 kinase inhibitor, SB203580, had no effect on AA release. Inhibitors of other phospholipases, including phosphatidylcholine-specific phospholipase C or phosphatidylinositol-specific phospholipase C, were also without effect. Taken together, our findings suggest that the AA release, induced by non-dioxin-like PCBs in liver progenitor cell line, is partially mediated by cytosolic PLA(2) and regulated by ERK1/2 kinases. Our results suggest that more attention should be paid to cell signalling pathways regulated by AA or eicosanoids after PCB exposure, which might be involved in their toxic effects.     

Inhibitory effect of glutamate release from rat cerebrocortical nerve terminals by α2 adrenoceptor agonist dexmedetomidine

The present study examined the effect of dexmedetomidine, an α₂ adrenoceptor agonist, on endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes). We also explored the possible mechanism that triggers dexmedetomidine to act. Dexmedetomidine dose-dependently inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine. Presynaptic α_2A adrenoceptors were involved in this release inhibition, with the α_2A antagonist (but not by the α_2B/C antagonist) blocking the dexmedetomidine-mediated inhibition. The effect of dexmedetomidine on the evoked glutamate release was prevented by the chelating extracellular Ca²⁺ ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate did not have any effect on the action of dexmedetomidine. Dexmedetomidine decreased the degree of depolarization-induced increase in the intrasynaptosomal Ca²⁺ levels, but did not affect the synaptosomal membrane potential. The inhibitory effect of dexmedetomidine on evoked glutamate release was abolished by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but was insensitive to the endoplasmic reticulum ryanodine receptors or mitochondrial Na⁺/Ca²⁺ exchange. In addition, the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors prevented dexmedetomidine from inhibiting glutamate release. Further, western blotting showed that dexmedetomidine decreased the 4-aminopyridine-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase 1 and 2 and synapsin I, the main presynaptic target of mitogen-activated protein kinase. Thus, we concluded that dexmedetomidine acts at α_2A adrenoceptors present on cerebrocortical nerve terminals inhibit the release of glutamate. We further concluded that this effect is linked to the suppression of voltage-dependent Ca²⁺ channels and mitogen-activated protein kinase activity.     

Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

Acute flunitrazepam (1 μM) exposure for 1 h reduced GABA_A receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABA_A receptor protein expression, that appears to be dependent on protein kinase C activity.     

Activation of osteoblastic functions by a mediator of pain, bradykinin

We investigated the effects of bradykinin (BK) on the production of interleukin (IL)-6 and prostaglandin PGE(2), whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as the signal transduction systems involved, in human osteoblasts (SaM-1 cells). BK receptors B1 (B1R) and B2 (B2R) were expressed in SaM-1 and osteosarcoma (SaOS-2, HOS, and MG-63) cells. Treatment of SaM-1 cells with BK increased the synthesis of both IL-6 and PGE(2) and the increase in both was blocked by HOE140 (B2R antagonist), but not by Des-Arg(9)-[Leu(8)]-BK (B1R antagonist). U-73122, a phospholipase C (PLC) inhibitor, suppressed BK-induced IL-6 and PGE(2) synthesis in SaM-1 cells. In addition, BK caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)]i), which was inhibited by pretreatment with HOE140 or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) blocker. Furthermore, both SB203580 (an inhibitor of p38 mitogen-activated protein kinase [MAPK]) and PD98059 (an inhibitor of MEK, upstream of ERK) attenuated the BK-induced IL-6 and PGE(2) synthesis. BK treatment resulted in the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK)1/2, and 2-APB could suppress BK-induced phosphorylation of ERK1/2. These findings suggest that BK increased both IL-6 and PGE(2) synthesis in osteoblastic cells via B2R and that PLC, IP(3)-induced [Ca(2+)]i, MEK, and MAPKs were involved in the signal transduction in these cells.     

Inhibition of the MEK/ERK pathway has no effect on agonist-induced aggregation of human platelets

The activation of human platelets by a variety of agonists is accompanied by the phosphorylation of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein (MAP) kinases. However, the role(s) of, and the substrate(s) for, these enzymes in platelet function remain unclear. Studies on ERKs in platelets have relied on pharmacological tools, including an inhibitor of ERK activation, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene]. In the present study, the effects of U0126 and its "inactive" analogue, U0125 [1,4-diamino-2,3-dicyano-1,4-bis(phenylthio)butadiene], on human platelet aggregation and MAP kinase activity were examined. Several agonists with a variety of signaling pathways were studied including thrombin, a thromboxane analogue, arachidonic acid, collagen, calcium ionophores, and the phorbol ester phorbol myristate acetate (PMA). U0126, at concentrations consistent with inhibition of the isolated enzyme, inhibited ERK phosphorylation, and therefore MEK activation, in response to each agonist. Under such conditions, U0126 did not affect the phosphorylation of a second MAP kinase, p38(MAPK); however, platelet aggregation was also unaffected. Higher concentrations of U0126, and of U0125, inhibited platelet aggregation in response to collagen and PMA with no effect on that induced by the other agonists. These results dissociate ERK activation from platelet aggregation, suggesting an alternative role for ERKs in platelet function. In addition, the effects of higher concentrations of U0126 are likely due to an action on protein kinase C, likely unrelated to ERK inhibition, suggesting that the inhibitor concentration is crucial to the interpretation of such studies.     

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.     

NOX4/Src regulates ANP secretion through activating ERK1/2 and Akt/GATA4 signaling in beating rat hypoxic atria

Nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are the major enzymatic source of reactive oxygen species (ROS). NOX2 and NOX4 are expressed in the heart but its role in hypoxia-induced atrial natriuretic peptide (ANP) secretion is unclear. This study investigated the effect of NOX on ANP secretion induced by hypoxia in isolated beating rat atria. The results showed that hypoxia significantly upregulated NOX4 but not NOX2 expression, which was completely abolished by endothelin-1 (ET-1) type A and B receptor antagonists BQ123 (0.3 µM) and BQ788 (0.3 µM). ET-1-upregulated NOX4 expression was also blocked by antagonists of secreted phospholipase A2 (sPLA2; varespladib, 5.0 µM) and cytosolic PLA2 (cPLA2; CAY10650, 120.0 nM), and ET-1-induced cPLA2 expression was inhibited by varespladib under normoxia. Moreover, hypoxia-increased ANP secretion was evidently attenuated by the NOX4 antagonist GLX351322 (35.0 µM) and inhibitor of ROS N-Acetyl-D-cysteine (NAC, 15.0 mM), and hypoxia-increased production of ROS was blocked by GLX351322. In addition, hypoxia markedly upregulated Src expression, which was blocked by ET receptors, NOX4, and ROS antagonists. ET-1-increased Src expression was also inhibited by NAC under normoxia. Furthermore, hypoxia-activated extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) were completely abolished by Src inhibitor 1 (1.0 µM), and hypoxia-increased GATA4 was inhibited by the ERK1/2 and Akt antagonists PD98059 (10.0 µM) and LY294002 (10.0 µM), respectively. However, hypoxia-induced ANP secretion was substantially inhibited by Src inhibitor. These results indicate that NOX4/Src modulated by ET-1 regulates ANP secretion by activating ERK1/2 and Akt/GATA4 signaling in isolated beating rat hypoxic atria.     

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.