Arachidonic acid enhances TPA-induced differentiation in human leukemia HL-60 cells via reactive oxygen species-dependent ERK activation

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of differentiation in human leukemia cells; however, the effects of arachidonic acid (AA) on TPA-induced differentiation are still unclear. In the present study, we investigated the contribution of AA to TPA-induced differentiation of human leukemia HL-60 cells. We found that treatment of HL-60 cells with TPA resulted in increases in cell attachment and nitroblue tetrazolium (NBT)-positive cells, which were significantly enhanced by the addition of AA. Stimulation of TPA-induced intracellular reactive oxygen species (ROS) production by AA was detected in HL-60 cells via a DCHF-DA analysis, and the addition of the antioxidant, N-acetyl-cysteine (NAC), was able to reduce TPA+AA-induced differentiation in accordance with suppression of intracellular peroxide elevation by TPA+AA. Furthermore, activation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by TPA+AA was identified in HL-60 cells, and the ERK inhibitor, PD98059, but not the JNK inhibitor, SP600125, inhibited TPA+AA-induced NBT-positive cells. Suppression of TPA+AA-induced ERK protein phosphorylation by PD98059 and NAC was detected, and AA enhanced ERK protein phosphorylation by TPA was in HL-60 cells. AA clearly increased TPA-induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression, which was inhibited by NAC and PD98059 addition. Eicosapentaenoic acid (EPA) as well as AA showed increased intracellular peroxide production and differentiation of HL-60 cells elicited by TPA. Evidence of AA potentiation of differentiation by TPA in human leukemia cells HL-60 via activation of ROS-dependent ERK protein phosphorylation was first demonstrated herein.     

ras-transformation of MDCK cells alters responses to phorbol ester without altering responses to bradykinin

The results of studies to evaluate the hypothesis that the 21 kDa GTP-binding protein derived from the ras oncogene is involved in regulation and coupling of hormone receptors to phospholipase activity have thus far been inconsistent. We therefore examined the effect of H-ras transformation on basal, tumor-promoting phorbol ester (TPA)-stimulated, and bradykinin-mediated phospholipid hydrolysis in Madin Darby canine kidney cells (MDCK) by comparing H-ras-transformed MDCK cells (MDCK-RAS) to two non-transformed strains of MDCK cells (MDCK-D1 and MDCK-ATCC). In unstimulated MDCK-RAS, diacylglycerol (DAG), inositol phosphate accumulation, and choline phosphate release were increased while arachidonic acid and arachidonic acid metabolite (AA) release was not increased, suggesting that ras transformation increased phospholipase C activity. Protein kinase C (PK-C) activity was decreased, and specific binding of [3H]phorbol ester was reduced in MDCK-RAS relative to the non-transformed MDCK cells suggesting that elevated DAG may activate and thereby down-regulate PK-C. Consistent with this finding in MDCK-RAS, TPA-stimulated AA release and subsequent prostaglandin E2 production were decreased, while TPA-stimulated choline phosphate release was increased. Bradykinin receptor-stimulated phospholipid hydrolysis in MDCK-RAS was similar to that of non-transformed cells, suggesting that the ras-derived protein does not directly couple bradykinin receptors to phospholipases in MDCK cells. However, the ability of TPA-treatment to inhibit bradykinin-stimulated phosphoinositide hydrolysis and enhance bradykinin-stimulated AA release was attenuated in MDCK-RAS. Additionally, in MDCK-RAS the conversion of arachidonic acid to prostaglandin E2 was substantially reduced. We conclude that ras transformation of MDCK cells increases DAG levels, thereby activating and, in turn, down-regulating PK-C and certain responses to TPA. Since activation of PK-C may result in a variety of effects on signal transduction pathways, we propose that increased DAG and altered PK-C levels associated with ras transformation may account for the inconsistent effects previously observed in studies evaluating the effect of ras transformation on phospholipases and other signal transduction systems.     

Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells. Evidence that bradykinin mediates noninterdependent activation of phospholipases A2 and C

Many types of peptide hormone and neurotransmitter receptors mediate hydrolysis of phosphoinositides (PI) and arachidonic acid and arachidonic acid metabolite (AA) release, but the relation between these responses is not clearly defined. We have characterized bradykinin (BK)-mediated AA release and PI hydrolysis in clonal Madin-Darby canine kidney cells (MDCK-D1). Both responses occurred over a similar dose range in response to the B1 and B2 receptor agonist, BK, but not in response to the B1 receptor-selective agonist des-Arg-BK. To test whether AA release occurs via a mechanism which is sequential to and dependent upon PI hydrolysis, we used the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates protein kinase C. TPA treatment blocked BK-mediated PI hydrolysis in MDCK-D1 cells, while at the same time and at similar concentrations enhancing BK-mediated AA release. Thus, TPA treatment dissociated BK-mediated AA release from PI hydrolysis. In addition, treatment of MDCK-D1 cells with neomycin blocked BK-mediated hydrolysis of phosphatidylinositol bisphosphate without reducing BK-mediated AA release. BK treatment increased formation of lysophospholipids with a time course in accord with BK-mediated AA release, indicating that at least part of the BK-mediated AA release was likely derived from activation of phospholipase A2. BK-mediated lysophospholipid production was enhanced by pretreatment with TPA, suggesting that the mechanism of AA release before and after treatment with TPA was the same. BK-mediated AA release and lysophospholipid production was dependent on the presence of extracellular calcium, while the enhanced responses to BK in the presence of TPA were not dependent on the presence of extracellular calcium. TPA treatment also enhanced AA release and lysophospholipid production in response to the calcium ionophore A23187. From these data we propose that BK, acting at B2 receptors, promotes AA release in MDCK cells via a mechanism which is 1) independent of polyphosphoinositide hydrolysis by phospholipase C, 2) dependent upon influx of extracellular calcium and activation of phospholipase A2, and 3) enhanced by activation of protein kinase C.     

Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis

Many stimulators of prostaglandin production are thought to activate the Ca2+- and phospholipid-dependent protein kinase first described by Nishizuka and his colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. In this paper we report evidence that the activation of protein kinase C caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) is involved in the increased prostaglandin production induced by 12-O-tetradecanoylphorbol-13-acetate in Madin-Darby canine kidney (MDCK) cells. We have shown that TPA activates protein kinase C in MDCK cells with similar dose response curve as observed for TPA induction of arachidonic acid release in MDCK cells. Activation of protein kinase C was associated with increased phosphorylation of proteins of 40,000 and 48,000 daltons. We used two compounds (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-(5-isoquinolinesulfonyl)piperazine) known to inhibit protein kinase C by different mechanisms to further examine if activation of protein kinase C was involved in the increased synthesis of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C partially purified from MDCK cells and that ET-18-OMe inhibited the phosphorylation of proteins by protein kinase C in the intact cells. Addition of either compound during or after TPA treatment decreased both release of arachidonic acid from phospholipids and prostaglandin synthesis. Release of [3H]arachidonic acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinolinesulfonyl)piperazine addition. However, arachidonic acid release stimulated by A23187 is not blocked by Et-18-OMe. When assayed in vitro, treatment of cells with Et-18-OMe did not prevent the enhanced conversion of arachidonic acid into prostaglandins induced by pretreatment of cells with TPA. Our results suggest that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase C by TPA.     

Protein kinase C activation amplifies prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin F2 alpha (PGF2 alpha) stimulated arachidonic acid (AA) release in a dose-dependent manner in the range between 1 nM and 10 microM. 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2 alpha in a dose-dependent manner. 4 alpha-phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2 alpha-induced AA release. 1-oleoyl-2-acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2 alpha. H-7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2 alpha-induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2 alpha-induced AA release, while it suppressed the amplification by OAG of PGF2 alpha-induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol trisphosphate stimulated by PGF2 alpha. Under the same condition, PGF2 alpha indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2 alpha-induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2 alpha-induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like cells.     

Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid release and acetylcholine-induced vascular relaxation in rabbit aorta

ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A2 (PLA2), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [14C]AA and stimulated with methacholine (10(-5) M). Free [14C]AA was released by methacholine. Methacholine decreased the [14C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [14C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta.     

Cytosolic phospholipase A2 is an effector of Jak/STAT signaling and is involved in platelet-derived growth factor BB-induced growth in vascular smooth muscle cells

Platelet-derived growth factor-BB (PDGF-BB) is a potent mitogen and chemoattractant for vascular smooth muscle cells (VSMC). To understand its mitogenic and chemotactic signaling events, we studied the role of cytosolic phospholipase A(2) (cPLA(2)) and the Jak/STAT pathway. PDGF-BB induced the expression and activity of cPLA(2) in a time-dependent manner in VSMC. Arachidonyl trifluoromethyl ketone, a potent and specific inhibitor of cPLA(2), significantly reduced PDGF-BB-induced arachidonic acid release and DNA synthesis. PDGF-BB stimulated tyrosine phosphorylation of Jak-2 in a time-dependent manner. In addition, PDGF-BB activated STAT-3 as determined by its tyrosine phosphorylation, DNA-binding activity, and reporter gene expression, and these responses were suppressed by AG490, a selective inhibitor of Jak-2. AG490 and a dominant-negative mutant of STAT-3 also attenuated PDGF-BB-induced expression of cPLA(2,) arachidonic acid release, and DNA synthesis in VSMC. Together, these results suggest that induction of expression of cPLA(2) and arachidonic acid release are involved in VSMC growth in response to PDGF-BB and that these events are mediated by Jak-2-dependent STAT-3 activation.     

TLR-4 mediated group IVA phospholipase A2 activation is phosphatidic acid phosphohydrolase 1 and protein kinase C dependent

Group IVA phospholipase A₂ (GIVA PLA₂) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA₂. We also studied the effect of PAP-1 and PKC on Ca^+ 2 induced and synergy enhanced GIVA PLA₂ activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo₂-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca^+ 2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA₂ synergistic activation by stimulation with Kdo₂-Lipid A and ATP.     

Stimulation and inhibition of neoplastic cell growth by tumor promoter-treated macrophages

Macrophages treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent inflammatory and tumor-promoting agent, can have the diametrically opposed functions of contact-mediated tumor cytotoxicity and release of soluble clonal proliferation factor(s) for tumor cells. In vitro TPA treatment of macrophages at 1.0 ng/ml induced prostaglandin E₂ release and morphological changes analogous to cell activation. In addition, conditioned medium from macrophages pulsed with TPA enhanced M109 carcinoma colony formation in vitro. Although macrophages were not rendered tumoricidal by TPA in vitro, cytotoxic macrophages were recovered from mice following ip treatment with TPA at 1–100 μg/kg. This indicated an indirect pathway for the activation of macrophages by TPA. The very weak tumor promoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate lacked effects on macrophages at all doses tested. The possibility that macrophage secretions (e.g., prostaglandin E₂, angiogenesis-stimulating factor(s), and clonal proliferation factor(s) for carcinogen-triggered cells) may be involved in the tumor promotion process is discussed.     

Effect of 1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-acether) on leukocytes I. Analysis of the in vitro migration of human neutrophils

The effect of synthetic 1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-acether) and of 1-O-octadecyl-sn-glycero-3-phosphocholine (lyso-PAF-acether) on human neutrophil migration was studied in modified Boyden chambers, with the following results: (1) By checker-board analysis and deactivation experiments, the factors are chemokinetic at low (10^?8 M) and chemotactic at higher concentrations (10^?6 M), with lyso-PAF-acether being less potent at all concentrations. (2) Cross-deactivation occurs between the two PAF compounds, but not with two other chemotactic factors, suggesting a specific, common receptor for the PAFs on the neutrophil membrane. (3) Other chemotactic substances may act as potentiating or additive factors to the PAF compounds. (4) Inhibition of arachidonic acid turnover during chemotaxis by compound BW 755 C enhances leukocyte chemotaxis towards the PAF compounds and towards other chemotactic factors. The data suggest that PAF and its lyso-derivate may contribute in a unique and potent fashion to leukocyte accumulation at inflammatory sites.     

Dual effect of the phorbol ester TPA on arachidonic acid release from HeLa cells

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induces release of arachidonic acid (AA) from HeLa cells with a maximum at 2-3 h. Subsequently the extracellular level of AA decreases. Cycloheximide (CH, 10(-5) M does not influence the release of AA, however, it causes the AA level to remain elevated. In the presence of TPA and CH (i) re-uptake of AA is not altered, (ii) re-incorporation of AA into phosphatidylinositol (and phosphatidylethanolamine) is largely increased, and (iii) the level of lysophosphatidylinositol is elevated. The latter two phenomena can be prevented by fluocinolone acetonide (10(-8) M), i.e. by inhibition of phospholipase A2 (PLA2). These data point to a continuously elevated PLA2 activity in the presence of TPA and CH. The phorbol ester appears to induce a proteinaceous principle which diminishes PLA2 activity.     

Hydrolysis of novel diacylglycerol and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [³H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, ever crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-¹⁴C]palmitoyl-2-acetyl-sn-glycerol and [1-¹⁴C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-tinies the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[³H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Role of arachidonic acid release in the G2 delay induced by tumor promoter TPA in HeLa cells

The tumor promoter TPA2 (12-O-tetradecanoylphorbol-13-acetate) has been shown to exhibit a radiomimetic activity on the cell cycle of HeLa cells (V. Kinzel, J. Richards, and M. St?hr (1980) Science 210, 429). The response includes a delay of cells in G2 phase. The relation between TPA-induced release of arachidonic acid (AA) and the inhibition in G2 phase was studied. Exogenous AA (greater than 10(-4) M; in presence of 10% serum) is shown to delay HeLa cells in G2 and to enhance the effectiveness of TPA in this respect. The inhibition of the TPA-induced AA liberation by fluocinolone acetonide, however, does not influence the TPA-effected G2 delay. The diacylglycerols 1,2-dioctanoyl-glycerol and 1-oleoyl-2-acetylglycerol delay HeLa cells in G2 but without major stimulation of AA liberation. On the basis of the data it is concluded that AA released from HeLa cells due to the action of TPA is not involved in the TPA-induced delay of cells in G2 phase.     

Epidermal growth factor stimulates prostaglandin biosynthesis by canine kidney (MDCK) cells.

Serum and/or arachidonic acid stimulated prostaglandin production by dog kidney (MDCK) cells. Epidermal growth factor (EGF) at concentrations of 10^?9 to 10^?10 M stimulated the biosynthesis of prostaglandins by MDCK cells but not that by human fibroblasts (D-550), mouse fibroblasts (3T3), transformed mouse fibroblasts (MC5-5), and rabbit aorta endothelial cells (CLO). EGF also stimulated the release of radioactivity from MDCK cells radioactively labelled with [³H]arachidonic acid.     

Protein kinase C activation inhibits stress-induced synthesis of heat shock protein 27 in osteoblast-like cells: Function of arachidonic acid

Exposure of osteoblast-like MC3T3-E1 cells to sodium arsenite (arsenite) increased the level of heat shock protein 27 (hsp27). The effect of arsenite was dose-dependent in the range of 50 to 200 μM. Arsenite also stimulated arachidonic acid release dose-dependently in the range between 50 and 200 μM in these cells. Both indomethacin, an inhibitor of cyclooxygenase, and nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly enhanced the arsenite-induced accumulation of hsp27. Melittin, an activator of phospholipase A₂, significantly enhanced the arsenite-induced accumulation of hsp27. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, inhibited the arsenite-induced accumulation of hsp27. In contrast, 4α-phorbol 12, 13-didecanoate (4α-PDD), a PKC-nonactivating phorbol ester, had little effect. TPA suppressed the arsenite-induced arachidonic acid release, but 4α-PDD had little effect. Arsenite no longer affected cAMP accumulation, inositol phosphates formation nor the formation of choline and phosphocholine in these cells. These results suggest that the response to stress of hsp27 is coupled with the metabolic activity of the arachidonic acid cascade, and the activation of PKC inhibits the induction of hsp27 through the suppression of arachidonic acid release in osteoblast-like cells. © 1996 Wiley-Liss, Inc.     

Bradykinin-stimulated arachidonic acid release from MDCK cells is not protein kinase C dependent

Bradykinin (BK)-induced release of arachidonic acid (AA) fromMadin-Darby canine kidney (MDCK) D1 cells was investigated. Phorbol12-myristate 13-acetate (PMA) caused a synergistic increase in BK- andA-23187-induced release of AA but alone had no effect on this release.Inhibition of protein kinase C (PKC) with bisindolmaleimide I (BIS)abolished the synergistic effects of PMA but did not affect AA releasecaused by BK or A-23187 alone. Downregulation of PKC with 100 nM PMAresulted in a reduction of AA release induced by BK or A-23187addition, which corresponded to a decrease in cytoplasmic phospholipaseA₂(cPLA₂) activity as measured incell extracts. Although Western blotting revealed no differences in cPLA₂ expression as a result ofPMA treatment, phosphorylation of the enzyme, as assessed byphosphoserine content, was significantly reduced in PKC-depleted cells.These results imply that, with PKC downregulation, subsequent BKstimulation results in aCa²⁺-dependent translocation of aless phosphorylated, less active form ofcPLA₂. Any stimulation of PKC byBK addition did not appear as a significant event in onset reponsesleading to AA release. On the other hand, inhibition of themitogen-activated protein kinase (MAPK) cascade with the MAPK kinaseinhibitor, PD-98059, significantly decreased BK-induced release of AA,a finding that, with our other results, points to the existence of aPKC-independent route for stimulation of MAPK and the propagation ofonset responses.

     

Differential crosstalk between P2X7 and arachidonic acid in activation of mitogen-activated protein kinases

Accumulating evidence indicates that astroglial syncytium plays key role in normal and pathological brain functions. Astrocytes both in vitro and in situ respond to extracellular adenine-based nucleotides via the activation of P2 receptors. Massive release of ATP from neurons and glial cells occurs as a result of pathological conditions of the brain leading to neuroinflammation and involving P2X7 receptors. In this study, we investigated whether P2X7 stimulation on cultured cortical astrocytes promoted a differential activation of mitogen-activated protein kinases (MAPKs), and whether the second messenger arachidonic acid (AA), which is also a key modulator of neuroinflammation, affected the P2X7-mediated MAPK phosphorylation. The results show that the synthetic P2X7 receptor agonist 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP), induced a concentration-dependent phosphorylation of MAPK ERK1/2, JNK and p38. Stimulation of ERK1/2, JNK and p38 phosphorylation was also obtained by pathophysiological levels of extracellularly applied AA. Interestingly, a robust potentiation of ERK1/2 phosphorylation was elicited by co-application of BzATP and AA, whereas no differences were observed in JNK or p38 phosphosignals. The kinases activation showed a differential dependence on the presence of extracellular Ca²⁺. The potentiation of BzATP-mediated ERK1/2 phosphorylation was also observed in human embryonic kidney cells (HEK293) stably transfected with rat P2X7, but not in HEK cells expressing truncated P2X7 receptor lacking the full cytoplasmic carboxy-terminal or in those carrying the structurally related rat P2X2. AA and BzATP synergism in ERK1/2 activation was abolished by cyclo-oxygenase and lipoxygenase pathway inhibitors.The result that ERK1/2-mediated transduction pathway is synergistically modulated by ATP and AA signalling depicts possible novel pharmacological targets for interfering with pathological activation of astroglial cells.     

Arachidonic acid stimulates interleukin-6 release from rat peritoneal macrophages in vitro: Evidence for a prostacyclin-dependent mechanism

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (Mø) in vitro. AA (0.5–16 μM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 μM AA generating a peak of IL-6 release (3-5-fold). AA (0.5–16 μM) also induced an increasing release of the AA metabolite thromboxane B₂ (TXB₂). The AA-induced release of IL-6 occurred within 1–2 h of incubation, whereas TXB₂ concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 μM and 40.0 μM) effectively blocked the generation of TXB₂, but increased prostacyclin (PGI₂) generation and potentiated the release of IL-6. In addition, PGI₂, as well as the PGI₂ agonists iloprost and cicaprost, stimulated IL-6 release from Mø by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI₂ (but not TXA₂) is involved in AA-induced IL-6 release from peritoneal Mø.     

Oxysterol activation of arachidonic acid release and protaglindin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase activity

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca²⁺ level. Since both Ca²⁺ variations and protein C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxyterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC₅₀ (4 × 10⁻⁹ M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE₂ synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3β,25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca²⁺ ion fluxes, therefore oxysterols could affed earlier events triggered by serum growth factor binding to their cell membrane receptors.