Localization of 12-hydroxyeicosatetraenoic acid in endothelial cells

Bovine aortic endothelial cells take up 12-hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product formed from arachidonic acid. The uptake of [3H]12-HETE reached a maximum in 2 to 4 h. At this time, from 75 to 80% of the incorporated radioactivity was contained in phospholipids, about 85% of the esterified radioactivity remained in the form of 12-HETE, and at least 90% of the phospholipid radioactivity was present in the sn-2-position. Subcellular fractionation on Percoll and sucrose gradients demonstrated that 65 to 74% of the radioactivity was present in membranes enriched in NADPH-cytochrome c reductase and UDP-galactosyl transferase. The specific radioactivity relative to protein of these intracellular membranes was 2.9-times higher than in a plasma membrane fraction enriched in 5'-nucleotidase. A similar intracellular localization was observed when [3H]5-HETE or [3H]arachidonic acid were taken up. The 12-HETE was contained primarily in the choline glycerophospholipids of the microsomal membranes. After incorporation, [3H]12-HETE was removed from the cell lipids much more rapidly than [3H]arachidonic acid, and 80% of the radioactivity released into the medium during the first hour remained as 12-HETE. Because it accumulates in microsomal membranes, 12-HETE uptake may perturb certain intracellular processes and thereby lead to endothelial dysfunction. The relatively rapid removal of the newly incorporated 12-HETE may be an important protective mechanism that prevents excessive accumulation and more extensive endothelial damage.     

Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells

Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides.     

Disparate effects of 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid in vascular endothelial and smooth muscle cells and in cardiomyocytes

The expression and activity of the arachidonic acid-metabolizing enzyme leukocyte-type 12-lipoxygenase (12-LO) are augmented in cultured vascular endothelial and smooth muscle cells exposed to high glucose concentrations and in blood vessels of diabetic animals. The product of this enzyme, 12-hydroxyeicosatetraenoic acid (12-HETE), evokes two types of interactions in these cells: on one hand it acts as a pro-inflammatory factor that contributes to the initiation and progression of atherosclerotic lesions. Yet on the other, it protects the same cells against deleterious effects of high levels of intracellular glucose by downregulating the glucose transport system in the cells. In addition, it has been shown that 12-LO and 12-HETE support insulin-dependent glucose transporter-4 translocation to the plasma membrane by maintaining intact actin fiber network in the cardiomyocytes. Here we focus on the disparate cellular interactions by which 12-LO and 12-HETE affect the glucose transport system in vascular endothelial and smooth muscle cells and in cardiomyocytes.     

Arachidonic acid, 12- and 15-hydroxyeicosatetraenoic acids, eicosapentaenoic acid, and phospholipase A2 induce starfish oocyte maturation

In starfish oocyte maturation (meiosis reinitiation) is induced by the natural hormone 1-methyladenine (1-Me-Ade). This paper shows that arachidonic acid (AA) induces oocyte maturation at concentrations above 0.5 microM. This maturation shares many characteristics with 1-MeAde-induced maturation: same kinetics, same required contact time, same stimulations of protein phosphorylation and sodium influx. Although calcium facilitates the AA-induced but not the 1-MeAde-induced maturation, AA, like 1-MeAde, does not stimulate the uptake of calcium. Calcium does not facilitate the uptake of AA by oocytes. Out of 36 different fatty acids (saturated and unsaturated), only eicosatetraenoic (AA) and eicosapentaenoic acids were found to mimic 1-MeAde. Calcium-dependent phospholipases A2 from bee venom and Naja venom also induce maturation (0.1-1 unit/ml) when added externally to the oocytes. Phospholipase A2 inhibitors (quinacrine, bromophenacylbromide) block maturation; inhibition is reversed by increasing the 1-MeAde concentration and only occurs during the hormone-dependent period. AA is usually metabolized through oxidation by cyclooxygenase or lipoxygenase. Cyclooxygenase inhibitors (acetylsalicylic acid, indomethacin, tolazoline) do not block maturation; prostaglandins E2, D2, F2 alpha, I2, and thromboxane B2 do not induce meiosis reinitiation. On the other hand, lipoxygenase inhibitors (quercetin, butylated hydroxytoluene, and eicosatetraynoic acid) block 1-MeAde-induced maturation; although leukotrienes (A4, B4, C4, D4, E4) have no effects on oocytes, two other lipoxygenase products, 12- and 15-hydroxyeicosatetraenoic acids (and their corresponding hydroperoxy-) induce oocyte maturation (around 1 microM). The possible mode of action of the fatty acids inducing oocyte maturation is discussed.     

Quantitative measurement of 5-, 12-, and 15-hydroxyeicosatetraenoic acid together with 12-hydroxyheptadecatrienoic acid by stable isotope dilution gas chromatography-negative ion chemical ionization-mass spectrometry

A stable isotope dilution gas chromatography-negative ion chemical ionization-mass spectrometry assay for simultaneous quantitative measurement of 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, and 12-hydroxyheptadecatrienoic acid in one single GC/MS run was established. 18O2-Labeled analogs as internal standards, prepared according to conventional procedures, were found to be useful for this application. A sample processing and derivatization sequence providing highly purified compounds with a recovery of 42.7% was elaborated. The detection limit was in the femtomole range.     

Involvement of protein kinase C in angiotensin II-mediated release of 12-hydroxyeicosatetraenoic acid in bovine adrenal glomerulosa cells.

The present study was conducted to determine whether protein kinase C was involved in angiotensin II-mediated release of 12-hydroxyeicosatetraenoic acid (12-HETE) from bovine adrenal glomerulosa cells. Activators of protein kinase C, 12-O-tetradecanoylphorbol 4-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG), significantly increased release of 12-HETE. The effect of OAG was potentiated by BAYK8644, a stimulator of calcium entry. Sphingosine, H-7 and staurosporine, which inhibited the activity of protein kinase C in vitro, almost completely blocked 12-HETE release induced by TPA. These agents also significantly reduced angiotensin II-mediated 12-HETE release. When time course of the liberation of 12-HETE was measured, angiotensin II elicited sustained release of 12-HETE, which was inhibited by staurosporine. These results indicate that angiotensin II induces sustained release of 12-HETE, a feed forward regulator of aldosterone secretion, and that protein kinase C may be involved in this process.     

A re-evaluation of the mitogenic effect of serotonin on vascular endothelial cells

Serotonin is an extracellular mediator recognized by seven different types of receptors, thus giving rise to pleiotropic intracellular responses. One of these responses is the activation of proliferation for a number of cell types. The induction of proliferation of otherwise quiescent endothelial cells is a key step of angiogenesis. Previously published work concerning the effect of serotonin on endothelial cell proliferation is controversial. The present work is aimed to re-evaluate the mitogenic role of serotonin on endothelial cells, since a pro-angiogenic role for serotonin could be hypothesized if its mitogenic potential on these cells were confirmed. By using three different types of endothelial cells and three experimental approaches, we demonstrate that serotonin cannot be considered a general mitogen for endothelial cells.     

Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions.

Cell surface ecto-nucleotidases are considered the major effector system for inactivation of extracellular adenine nucleotides, whereas the alternative possibility of ATP synthesis has received little attention. Using a TLC assay, we investigated the main exchange activities of 3H-labeled adenine nucleotides on the cultured human umbilical vein endothelial cells. Stepwise nucleotide degradation to adenosine occurred when a particular nucleotide was present alone, whereas combined cell treatment with ATP and either [3H]AMP or [3H]ADP caused unexpected phosphorylation of 3H-nucleotides via the backward reactions AMP --> ADP --> ATP. The following two groups of nucleotide-converting ecto-enzymes were identified based on inhibition and substrate specificity studies: 1) ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase; 2) ecto-nucleotide kinases, adenylate kinase, and nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase possessed the highest activity, as revealed by comparative kinetic analysis, and was capable of using both adenine and nonadenine nucleotides as phosphate donors and acceptors. The transphosphorylation mechanism was confirmed by direct transfer of the gamma-phosphate from [gamma-32P]ATP to AMP or nucleoside diphosphates and by measurement of extracellular ATP synthesis using luciferin-luciferase luminometry. The data demonstrate the coexistence of opposite, ATP-consuming and ATP-generating, pathways on the cell surface and provide a novel mechanism for regulating the duration and magnitude of purinergic signaling in the vasculature.     

Characterization of a novel chemokine-containing storage granule in endothelial cells: evidence for preferential exocytosis mediated by protein kinase A and diacylglycerol

We have recently shown that several proinflammatory chemokines can be stored in secretory granules of endothelial cells (ECs). Subsequent regulated exocytosis of such chemokines may then enable rapid recruitment of leukocytes to inflammatory sites. Although IL-8/CXCL8 and eotaxin-3/CCL26 are sorted to the rod-shaped Weibel-Palade body (WPB), we found that GROalpha/CXCL1 and MCP-1/CCL2 reside in small granules that, similarly to the WPB, respond to secretagogue stimuli. In the present study, we report that GROalpha and MCP-1 colocalized in 50- to 100-nm granules, which occur throughout the cytoplasm and at the cell cortex. Immunofluorescence confocal microscopy revealed no colocalization with multimerin or tissue plasminogen activator, i.e., proteins that are released from small granules of ECs by regulated exocytosis. Moreover, the GROalpha/MCP-1-containing granules were Rab27-negative, contrasting the Rab27-positive, WPB. The secretagogues PMA, histamine, and forskolin triggered distinct dose and time-dependent responses of GROalpha release. Furthermore, GROalpha release was more sensitive than IL-8 release to inhibitors and activators of PKA and PKC but not to an activator of Epac, a cAMP-regulated GTPase exchange factor, indicating that GROalpha release is regulated by molecular adaptors different from those regulating exocytosis of the WPB. On the basis of these findings, we designated the GROalpha/MCP-1-containing compartment the type 2 granule of regulated secretion in ECs, considering the WPB the type 1 compartment. In conclusion, we propose that the GROalpha/MCP-1-containing type 2 granule shows preferential responsiveness to important mediators of EC activation, pointing to the existence of selective agonists that would allow differential release of selected chemokines.     

The priming effect of 12(S)-hydroxyeicosatetraenoic acid on lymphocyte phospholipase D involves specific binding sites.

We have previously shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE)-enrichment primed human peripheral blood mononuclear cells for phospholipase D activation by mitogens. Given that 12(S)-HETE-enriched cells stimulated with concanavalin A released free 12(S)-HETE in the extracellular medium, and that the priming effect of 12(S)-HETE on phospholipase D was suppressed by the non-permeant drug, suramin, we hypothesized an extracellular mechanism for 12(S)-HETE-induced PLD activation. Using [3H]12(S)-HETE as a ligand and a rapid filtration technique, we have pointed out the presence of specific low-affinity 12(S)-HETE binding sites on intact human mononuclear cells and lymphocytes. [3H]12(S)-HETE binding was efficiently displaced by other monohydroxylated and n-3 fatty acids but not by oleate and arachidonate, and was also significantly inhibited by suramin and pertussis toxin. Furthermore, 12(S)-HETE-induced PLD activation was strongly inhibited by pertussis toxin and genistein, but was not PKC-dependent. In addition, 12(S)-HETE also potentiated the ConA-induced tyrosine phosphorylation of a 46-50 kDa protein, which was inhibited by genistein. Collectively, these results suggest that 12(S)-HETE binding sites on human lymphocytes may be coupled to phospholipase D through pertussis toxin sensitive G-proteins and tyrosine kinases.     

Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

Retinal progenitor cells （RPCs） are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid （RA） is a powerful growth/differentiation factor that generally causes growth inhibition, differentiation and/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affecting RPC proliferation. The enhanced cell survival was accompanied by a significant upregulation of the expression of protein kinase A （PKA） and several protein kinase C （PKC） isoforms. Treatment of cells grown in RA-free media with 8-bromoadenosine3＇,5＇-cyclic monophosphate, a known activator of PKA, resulted in an anti-apoptotic effect similar to that caused by RA; whereas the PKA inhibitor N-[2-（p-bromocinnamylamino）ethyl]-5-isoquinolinesul- fonamide dihydrochloride led to a significant （-32%） increase in apoptosis. In contrast, treatment of RPCs with any of two PKC selective inhibitors, 2,2＇,3,3＇,4,4＇-hexahydroxy-1,1 ＇-biphenyl-6,6＇-dimethanol dimethyl ether and bisindolylmaleimide XI, led to diminished apoptosis; while a PKC activator, phorbol 12-myristate 13-acetate, increased apoptosis. These and other data suggest that the effect of RA on RPC survival is mostly due to the increased anti-apoptotic activity elicited by PKA, which might in turn be antagonized by PKC. Such a mechanism is a new example of tight regulation of important biological processes triggered by RA. Although the detailed mechanisms remain to be elucidated, we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changed expression of p53 or bcl-2, and appears to be independent of 15-amyloid, Fas ligand, TNF-α, ganglioside GM1 and ceramide C 16-induced apoptotic pathways.     

The inhibition of arachidonic acid mobilization in human platelets by R59 022, a diacylglycerol kinase inhibitor

R59 022 (6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1- piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one) has been suggested as an inhibitor of diacylglycerol kinase in erythrocyte membranes and intact platelets. In the present study, we have investigated the effects of this drug on arachidonic acid mobilization occurring in response to thrombin in intact human platelets. Our results indicate that release of arachidonic acid from membrane phospholipids such as phosphatidylcholine and phosphatidylinositol was severely impaired by R59 022 and the extent of inhibition amounted to 77% and 84%, respectively, as compared to controls. This resulted in a dramatic decrease in the accumulation of free arachidonic acid (labeled/unlabeled) and the percent inhibition of free arachidonic acid accumulation amounted to 80-90% as compared to controls. Furthermore, the drug caused a significant accumulation of thrombin-induced diacylglycerol (labeled) without affecting the formation of labeled phosphatidic acid (PA). We found no significant changes in the radioactivity of either phosphatidylethanolamine or phosphatidylserine following stimulation with thrombin in the presence or absence of R59 022. We conclude that the observed inhibition of thrombin-induced arachidonic acid mobilization by R59 022 may be due to its effects on the activities of diacylglycerol lipase/phospholipase A2. In addition, the failure of further stimulation of thrombin-induced PA by R59 022 may indicate that PA-specific phospholipase A2 is either not involved in the release of arachidonic acid or is not a major source for arachidonic acid release in thrombin-stimulated human platelets. These findings may prove to be important when this drug is used as a selective inhibitor of diacylglycerol kinase.     

(-)-Epicatechin elevates nitric oxide in endothelial cells via inhibition of NADPH oxidase

Dietary (-)-epicatechin is known to improve bioactivity of (*)NO in arterial endothelium of humans, but the mode of action is unclear. We used the fluorophore 4,5-diaminofluorescein diacetate to visualize the (*)NO level in living human umbilical vein endothelial cells (HUVEC). Untreated cells showed only a weak signal, whereas pretreatment with (-)-epicatechin (10 microM) or apocynin (100 microM) elevated the (*)NO level. The effects were more pronounced when the cells were treated with angiotensin II with or without preloading of the cells with (*)NO via PAPA-NONOate. While (-)-epicatechin scavenged O2(*-), its O-methylated metabolites prevented O2(*-) generation through inhibition of endothelial NADPH oxidase activity, even more strongly than apocynin. From the effect of 3,5-dinitrocatechol, an inhibitor of catechol-O-methyltransferase (COMT), on HUVEC it is concluded that (-)-epicatechin serves as 'prodrug' for conversion to apocynin-like NADPH oxidase inhibitors. These data indicate an (*)NO-preserving effect of (-)-epicatechin via suppression of O2(*-)-mediated loss of (*)NO.     

Antiproliferative effect of nitric oxide on rat glomerular mesangial cells via inhibition of mitogen-activated protein kinase.

The effect of nitric oxide (NO) donors and lipopolysaccharide (LPS) on the proliferation of rat glomerular mesangial cells was characterized. Exogenous application of a NO donor inhibited serum-induced proliferation in a time- and dose-dependent manner. S-Nitrosoglutathione (GSNO) also increased cGMP generation and arachidonic acid release, but it did not cause any measurable increase in the cytosolic Ca2+ concentration. Chelation of cytosolic Ca2+ or inhibition of mitogen-activated protein kinase (MAPK) kinase had an inhibitory effect on proliferation, but neither enhanced the antiproliferative effect of GSNO. In contrast, inhibition of guanylate cyclase or phospholipase A2 had no effect on proliferation, but partially reversed GSNO-induced antiproliferation by approximately 98 and 65%, respectively. GSNO did not cause cell death. Incubation of cells with LPS induced endogenous NO generation and had an antiproliferative effect. LPS-induced antiproliferation was reversed completely by inhibition of nitric oxide synthase and partially by inhibition of guanylate cyclase or phospholipase A2. GSNO or LPS inhibited serum-induced MAPK activation, and both effects were partially reversed by inhibition of guanylate cyclase or phospholipase A2. Inclusion of 8-bromo-cGMP or arachidonic acid in the growth medium resulted in a similar antiproliferative effect. In conclusion, in rat glomerular mesangial cells, MAPK inhibition and an antiproliferative effect could be induced by either an increase in the cellular concentration of NO or exposure of the cells to LPS. Part of the effect of NO was attributable to the increased cellular cGMP generation and arachidonic acid release.     

Differential regulation of phosphatidylcholine biosynthesis by 12-O-tetradecanoylphorbol-13-acetate and diacylglycerol in NG108-15 neuroblastoma x glioma hybrid cells

12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor promoter and potent activator of protein kinase C, stimulates [3H]choline incorporation into phosphatidylcholine (PtdCho) in NG108-15 cells (Liscovitch, M., Freese, A., Blusztajn, J. K. and Wurtman, R. J. (1986) J. Neurochem. 47, 1936-1941). In the present study we demonstrate that two cell-permeant diacylglycerols, sn-1-oleoyl-2-acetylglycerol and sn-1,2-dioctanoylglycerol, also stimulate [3H]choline incorporation into PtdCho. However, the effect of diacylglycerol is additional to that produced by a maximally effective concentration of TPA (0.5 microM), suggesting that the two agents may not act via the same mechanism. In addition, the protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (at 200 microM) inhibits the action of TPA by 59% while not affecting that of diacylglycerol. Finally, preincubation of the cells with TPA (0.1 microM) for 24 h reduces protein kinase C activity in the cells and completely abolishes the effect of additional TPA on choline incorporation. In contrast, diacylglycerol-induced stimulation of PtdCho biosynthesis was not inhibited in the cells that were desensitized to TPA. These results suggest that the effect of the two cell-permeant diacylglycerols on PtdCho biosynthesis either is not mediated by protein kinase C activation, or, is mediated by a TPA-insensitive isoenzyme of protein kinase C.     

The expression of COX-2 in VEGF-treated endothelial cells is mediated through protein tyrosine kinase

Cyclooxygenase (COX), existing as the COX-1 and COX-2 isoforms, converts arachidonic acid to prostaglandin H2, which is then further metabolized to various prostaglandins. Vascular endothelial growth factor (VEGF) has been shown to play important roles in inflammation and is upregulated by the prostaglandin E series through COX-2 in several cell types. Here, we have investigated the effects of VEGF on the COX isoform expressed in human umbilical vein endothelial cells (HUVEC). The signalling mechanism of the COX isoform expressed in endothelial cells activated with VEGF will be also investigated using the tyrosine kinase inhibitor, genistein, and protein kinase C inhibitor, staurosporine. The activity of COX-2 was assessed by measuring the production of 6-keto-prostaglandin F1alpha in the presence of exogenous arachidonic acids (10 microM, 10 min) by enzyme immunoassay. The expression of COX isoform protein was detected by immunoblot using specific antibodies. Untreated HUVEC contained no COX-2 protein. In HUVEC treated with VEGF (0.01-50 ng/ml), COX-2 protein, but not COX-1, and COX activity were increased in a dose-dependent manner. Interestingly, the increased COX-2 protein and activity in response to VEGF (10 ng/ml) was inhibited by the tyrosine kinase inhibitor, genistein (0.05-5 microg/ml), but not by the protein kinase C inhibitor, staurosporine (0.1-10 ng/ml). Thus, the induction of COX-2 by VEGF in endothelial cells was mediated through protein tyrosine kinase, and the uses of specific COX-2 inhibitors in these conditions, in which VEGF was involved, might have a role.     

The effects of ox-LDL in human atherosclerosis may be mediated in part via the toll-like receptor 4 pathway

Toll-like receptor 4 (TLR4) may provide a potential pathophysiological link between lipids and infection/inflammation and atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) makes it more atherogenic than its native form. The purpose of the study was to investigate the relationship between the effects of ox-LDL in human atherosclerosis and the expression of TLR4. We studied the relationship between TLR4 and ox-LDL, pro and con, using both real-time quantitative RT-PCR and RNA interference technology through in vitro cell culture. Nuclear factor kappa B (NF-κ B) activity and the concentrations of monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were detected by ELISA. The results showed that the expression of TLR4 increased in response to ox-LDL. Simultaneously, NF-κ B relative activity and the concentrations of MCP-1 and IL-8 in cell supernatant were upregulated by ox-LDL in a dose-dependent manner. TLR4 expression was inhibited by small interference RNA(siRNA) plasmid expression vectors; NF-κ B activity and the secretions of MCP-1 and IL-8 in response to ox-LDL were significantly lower in the group whereinTLR4 expression has been inhibited than that in the group wherein TLR4 expression has not been inhibited. We suggest that the atherogenic effects of ox-LDL could be mediated in part via the TLR4 pathway. Furthermore, inhibition of TLR4 expression may downregulate the NF-κ B activity and secretions of MCP-1 and IL-8 in monocytes due to oxidized LDL, resulting in the alleviation of the progress of atherosclerosis.     

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide

Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant-pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosan-induced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed.     

Viability inhibition effect of gambogic acid combined with cisplatin on osteosarcoma cells via mitochondria-independent apoptotic pathway

We previously demonstrated that gambogic acid (GA) is a promising chemotherapeutic compound for human osteosarcoma treatment. The aim of this study was to detect whether the combination of lower-dose GA (0.3 mg/L) and cisplatin (CDDP) (1 mg/L) could perform a synergistic effect on inhibiting tumor in four osteosarcoma cell lines. Our results showed that the combination between GA at lower dose and CDDP significantly exerts a synergistic effect on inhibiting the cellular viability in MG63, HOS, and U2OS cells. In contrast, an antagonistic character was detected in SAOS2 cells exposed to the combined use of lower-dose GA (0.3 mg/L) and CDDP (1 mg/L). Then, analysis of cell cycle showed the combination of both drugs significantly induced the G₂/M phase arrest, without any difference relative to GA treatment alone, in MG63 cells. Flow-cytometric analysis of cell apoptosis displayed that the apoptotic rate in the combination group is higher than that in GA treatment alone in MG63, HOS, and U2OS cells. The combined use of both drugs had no effect on mitochondrial membrane potential, but promoted the apoptosis-inducing function through triggering of CDDP in the three cell lines. By measurement of mitochondrial membrane potential, the activity of caspase-3 and the expressions of caspase-8 and caspase-9, it was showed that the apoptosis-promoting effect of the combined use of both drugs could be dependent on the death receptor apoptosis pathway, not dependent on the mitochondria apoptosis mechanism. This research, for the first time, demonstrates that GA could increase the chemotherapeutic effect of CDDP in human osteosarcoma treatment through inducing the cell cycle arrest and promoting cell apoptosis.     

NAD(P)H-dependent reduction of 12-ketoeicosatetraenoic acid to 12(R)- and 12(S)-hydroxyeicosatetraenoic acid by rat liver microsomes

The possibility that 12-keto-5,8,10,14 eicosatetraenoic acid (12-KETE) could be used as substrate by reductase(s) to generate 12-hydroxyeicosatetraenoic acid (12-HETE) was investigated using rat liver microsomes as a source of enzyme activity. Microsomes catalyzed the time-dependent reduction of 12-KETE to 12-HETE in a reaction that required NAD(P)H. The maximal specific activity of 12-HETE formation was 1.7 nmol/min/mg of protein in the presence of NADH. The reaction could not be detected in the absence of cofactor or by using heat inactivated microsomes. The identity of the 12-HETE product was established by U.V. spectroscopy and co-elution with 12-HETE in two different systems of RP-HPLC. Resolution of the methyl esters of reaction products by chromatography on chiral columns also indicated that the reduction of 12-KETE with either NADPH or NADH generated a mixture of 12(S)- and 12(R)-HETE in a ratio of about 2:1. The results demonstrate the presence of a 12-KETE reductase activity in rat liver microsomes which can form both the R and S isomers of 12-HETE.