Perfluorinated carboxylic acids inhibit cyclooxygenase pathway more potently than 12-lipoxygenase pathway in rat platelets

Two perfluorinated carboxylic acids (PFCAs), pentadecafluorooctanoic acid (PDFOA) and heptadecafluorononanoic acid (HDFNA), were investigated for potential modulatory effects on the cyclooxygenase (COX) and 12-lipoxygenase (LOX) metabolisms in rat platelets. Both PDFOA and HDFNA dose-dependently inhibited the formation of a COX metabolite, 12-HHT, without any effect on that of a LOX metabolite, 12-HETE, at concentrations ranging from 10 to 100 μM. These two PFCAs up to 100 μM did not affect platelet membrane integrity, and COX-1 and -2 protein expression levels in Caco-2 cells. These results suggest that PDFOA and HDFNA have the potential to modify platelet function by inhibiting the COX pathway at activity level, but not at protein level.     

Increase in 12-lipoxygenase activity in platelets of spontaneously hypertensive rats

12-Lipoxygenase activity in platelets of spontaneously hypertensive rats was investigated. Enzyme activity was measured in the absence and the presence of reduced glutathione. In both assay conditions, 12-lipoxygenase activity in platelets of spontaneously hypertensive rats was significantly higher than that in platelets of normotensive rats. Since 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, has been reported to be a potent chemoattractant for aortic smooth muscle cells, increase in biosynthesis of 12-HETE in platelets of spontaneously hypertensive rats might contribute to the explanation of pathogenesis of vascular disorder commonly found in hypertension patients.     

Albumin modifies the metabolism of hydroxyeicosatetraenoic acids via 12-lipoxygenase in human platelets

12-Lipoxygenase and cyclooxygenase 1 are the dominating enzymes that metabolize arachidonic acid in human platelets. In addition to the conversion of arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, 12-lipoxygenase can also utilize 5(S)-hydroxyeicosatetraenoic acid and 15(S)-hydroxyeicosatetraenoic acid to form 5(S), 12(S)-dihydroxyeicosatetraenoic acid and 14(R), 15(S)-dihydroxyeicosatetraenoic acid, respectively. Furthermore, 15(S)-hydroxyeicosatetraenoic acid works as an inhibitor for 12-lipoxygenase. In the present paper we have studied the influence of albumin on the in vitro metabolism of 5 - and 15 -hydroxyeicosatetraenoic acids, and 5,15 -dihydroxyeicosatetraenoic acid by the platelet 12-lipoxygenase. The presence of albumin reduced the formation of 5(S),12(S)- dihydroxyeicosatetraenoic acid from 5(S)-hydroxyeicosatetraenoic acid, however, it had no effect on the 12(S)-hydroxyeicosatetraenoic acid production from endogenous arachidonic acid. In contrast, when 15(S)-hydroxyeicosatetraenoic acid was incubated with activated platelets, the formation of 14(R), 15(S)- dihydroxyeicosatetraenoic acid was stimulated by the presence of albumin. Furthermore, albumin reduced the inhibitory action 15(S)-hydroxyeicosatetraenoic acid had on 12(S)-hydroxyeicosatetraenoic acid formation from endogenous arachidonic acid. However, addition of exogenous arachidonic acid (20 microm) to the incubations inverted the effects of albumin on the conversion of 15(S)-hydroxyeicosatetraenoic acid to 14(R),15(S)- dihydroxyeicosatetraenoic acid and the production of 12(S)-hydroxyeicosatetraenoic acid in these incubations. Based on the Scatchard equation, the estimates of the binding constants to albumin were 1.8 x 10(5) for 15 -HETE, 1.4 x 10(5) for 12-HETE, and 0.9 x 10(5) for 5 -HETE respectively. These results suggest an important role of albumin for the regulation of the availability of substrates for platelet 12-lipoxygenase.     

12/15-lipoxygenase inhibitors in diabetic nephropathy in the rat

The 12/15-lipoxygenase (12/15-LO) pathway is activated in diabetes mellitus (DM), increasing 12(S)-hydroxyeicosatetraenoic acid (12-HETE). We showed that a 12-LO inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC) inhibited 12/15-LO activity in vivo and assessed the efficacy of another 12/15-LO inhibitor, N-benzyl-N-hydroxy-5-phenylpentamidine (BHPP), to diminish urinary 12-HETE and ameliorate diabetic nephropathy (DN) over 4 months. Rats studied were control (C, n=8), DM (n=6), and rats injected with BHPP (C+BHPP, n=4) and (DM+BHPP, n=5). BHPP 3 mg/kg/day decreased urinary (U) 12-HETE/creatinine (cr) by 30-50% after one injection and after 1 week of daily injections in DM rats. U 12-HETE/cr excretion increased paradoxically in controls given BHPP. There was a highly significant relationship between U 12-HETE/cr excretion and U alb/cr (r=0.79, P<10(-5)), demonstrating that renal 12/15-LO pathway activation is associated with albuminuria. BHPP did not inhibit glomerular collagen synthesis or improve histology. More sustained 12-LO inhibition may improve albuminuria in DN.     

Nitric oxide triggers the toxicity due to glutathione depletion in midbrain cultures through 12-lipoxygenase

Glutathione (GSH) depletion is the earliest biochemical alteration shown to date in brains of Parkinson's disease patients. However, data from animal models show that GSH depletion by itself is not sufficient to induce nigral degeneration. We have previously shown that non-toxic inhibition of GSH synthesis with l-buthionine-(S,R)-sulfoximine in primary midbrain cultures transforms a nitric oxide (NO) neurotrophic effect, selective for dopamine neurons, into a toxic effect with participation of guanylate cyclase (GC) and cGMP-dependent protein kinase (PKG) (Canals, S., Casarejos, M. J., de Bernardo, S., Rodríguez-Martín, E., and Mena, M. A. (2001) J. Neurochem. 79, 1183-1195). Here we demonstrate that arachidonic acid (AA) metabolism through the 12-lipoxygenase (12-LOX) pathway is also central for this GSH-NO interaction. LOX inhibitors (nordihydroguaiaretic acid and baicalein), but not cyclooxygenase (indomethacin) or epoxygenase (clotrimazole) ones, prevent cell death in the culture, even when added 10 h after NO treatment. Furthermore, the addition of AA to GSH-depleted cultures precipitates a cell death process that is indistinguishable from that initiated by NO in its morphology, time course, and 12-LOX, GC, and PKG dependence. The first AA metabolite through the 12-LOX enzyme, 12-hydroperoxyeicosatetraenoic acid, induces cell death in the culture, and its toxicity is greatly enhanced by GSH depletion. In addition we show that if GSH synthesis inhibition persists for up to 4 days without any additional treatment, it will induce a cell death process that also depends on 12-LOX, GC, and PKG activation. In this study, therefore, we show that the signaling pathway AA/12-LOX/12-HPETE/GC/PKG may be important in several pathologies in which GSH decrease has been documented, such as Parkinson's disease. The potentiating effect of NO over such a signaling pathway may be of relevance as part of the cascade of events leading to and sustaining nerve cell death.     

Studies on the glutathione S-transferase of human platelets

The glutathione S-transferases of human platelets have been compared with those of erythrocytes. Although wide variations in activity were found, in individual subjects, the activity in these cell types was significantly correlated. The enzymes demonstrated similar isoelectric points and electrophoretic mobilities and it appears that the platelet enzyme is also a product of the GST3 locus. There was no correlation between platelet enzyme activity and plasma concentrations of retinol and cholesterol, but in men, the relationship between activity and carotene was significant. It is suggested that GST3 isoenzyme activity depends on vitamin A.     

Regulation of cyclooxygenase and 12-lipoxygenase catalysis by phospholipid hydroperoxide glutathione peroxidase in A431 cells

Regulation of arachidonate metabolism in human epidermoid carcinoma A431 cells by phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cytosolic glutathione peroxidase (GPx1) was studied. In order to study the effect of reduced glutathione (GSH) on the catalysis regulation of these oxygenation enzymes, diethyl maleate was used to deplete the intracellular GSH. In the presence of 13-hydroperoxyoctadecadienoic acid, the enzymatic catalysis of cyclooxygenase and 12-lipoxygenase was significantly increased in the GSH-depleted cells. In terms of the inhibitory effect on 12-lipoxygenase, PHGPx was more sensitive to GSH concentrations than GPx1. Inhibition of PHGPx activity by the treatment of cells with antisense oligonucleotide of PHGPx mRNA increased the enzymatic catalysis of both cyclooxygenase and 12-lipoxygenase. In conclusion, the results indicate that catalysis of cyclooxygenase and 12-lipoxygenase in A431 cells was regulated by redox-reaction, and PHGPx seems to play an important role in the controlling of these reactions.     

Oxidation of reduced glutathione by subcellular fractions of rat liver

1. A new method was used to diminish the autoxidation of GSH. 2. The oxidation of GSH by liver homogenates was studied with regard to concentration of homogenate, concentration of GSH, time, pH and anaerobiosis. 3. GSH was oxidized by recombinations of the supernatant with microsomes and with mitochondria. Each fraction alone caused little oxidation. 4. Proteins in the supernatant were required to obtain the effect, and low-molecular-weight compounds in the same fraction increased its effect. 5. GSH diminished the formation of malonaldehyde in homogenates. 6. GSH prevented a stimulating effect of the supernatant on the formation of malonaldehyde in microsomes and in mitochondria. 7. The malonaldehyde formation in microsomes together with the supernatant did not start until the concentration of endogenous low-molecular-weight thiols had decreased to a low level. 8. It is suggested that part of the oxidation of GSH in homogenates is coupled to a mechanism that counteracts the peroxidation of membrane lipids.     

Measurement of reduced and oxidized glutathione in cultures of adult rat hepatocytes

A reversed-phase ion-exchange high-performance liquid chroamtographic technique, suitable for the separate measurement of reduced (GSH) and oxidized (GSSG) glutathione in cultures of adult rat hepatocytes, is described. A commercially available Nucleosil 120-7NH₂ column was used. A complete run took ca. 22 min. The retention times for GSH and GSSG were 10.6 and 12.7 min, respectively, providing a resolution coefficient of 1.4. The coefficients of variation for GSH and GSSG were ca. 5 and 25%, respectively, for freshly isolated hepatocytes, and 16 and 15%, respectively, for 24-h cultured hepatocytes. The detector response was linear as a function of GSH and GSSG concentration and the hepatocytes concentration studied. Addition of up to 1.5 mg/ml bovine serum albumin to the culture medium had no effect on the linearity. The recovery for standards, ranging from 0 to 150 nmol of GSH or GSSG per millilitre in the presence of hepatocytes, was 98% for GSH and 80% for GSSG. The detection limit of the method was between 0.5 and 1.0 nmol of GSH and GSSG per millilitre. In cultured rat hepatocytes, the GSH content increased during the first 24 h of culture, followed by a slow decrease. After six days of culture, the GSH content was less than 50% of the value found for freshly isolated hepatocytes. GSSG was present in cultured rat hepatocytes in only small amounts and becomes unmeasurable after four days of culture.     

Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism stimulates cell growth and metastasis of various cancer cells and the 12-LO metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], enhances proliferation of aortic smooth muscle cells (SMCs). However, pulmonary vascular effects of 12-LO have not been previously studied. We sought evidence for a role of 12-LO and 12(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that 12-LO gene and protein expression is elevated in lung homogenates of rats exposed to chronic hypoxia. Immunohistochemical staining with a 12-LO antibody revealed intense staining in endothelial cells of large pulmonary arteries, SMCs (and possibly endothelial cells) of medium and small-size pulmonary arteries and in alveolar walls of hypoxic lungs. 12-LO protein expression was increased in hypoxic cultured rat pulmonary artery SMCs. 12(S)-HETE at concentrations as low as 10(-5) microM stimulated proliferation of pulmonary artery SMCs. 12(S)-HETE induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 12(S)-HETE-stimulated SMC proliferation was blocked by the MEK inhibitor PD-98059, but not by the p38 MAPK inhibitor SB-202190. Hypoxia (3%)-stimulated pulmonary artery SMC proliferation was blocked by both U0126, a MEK inhibitor, and baicalein, an inhibitor of 12-LO. We conclude that 12-LO and its product, 12(S)-HETE, are important intermediates in hypoxia-induced pulmonary artery SMC proliferation and may participate in hypoxia-induced pulmonary hypertension.     

Functional characteristic of PC12 cells with reduced microsomal glutathione transferase 1

Microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activities and is active in biotransformation of xenobiotics and in defense against oxidative stress. To assess MGST1 role in the development and functioning of PC12 cells, we constructed a cell line with reduced MGST1 (PC12_M). Real-time PCR and immunoblot assays showed MGST1 expression lowered to 60 % and immunocytochemical analyses demonstrated an altered concentration and distribution of the enzyme. PC12_M cells revealed a larger tendency to grow in clusters, weaker adhesion, irregular shape of bodies, short neurite outgrowth and higher percentage of necrotic cells (34 %). The total GSTs activity determined with non-specific substrate CDNB (1-chloro-2,4-dinitrobenzene) decreased by 15-20 %, whereas that with DCNB (2,4-dichloro-1-nitrobenzene), a substrate more specific for cytosolic GSTs, was similar to the one in control cells. This suggests that reduction of MGST1 cannot be compensated by other glutathione transferases. In PC12_M cells the total glutathione content was higher by 15-20 %, whereas the GSSG/GSH ratio was lower than in control cells. Moreover, the laminin-dependent migration rate was much faster in control cells than in PC12_M, suggesting some alterations in the metastatic potential of the line with suppressed MGST1. The amount of MAP kinases (p38, JNK, ERK1/2) was elevated in PC12_M cells but their phosphorylation level declined. Microarray analysis showed changed expression of several genes, which may be linked with differentiation and necrosis of PC12_M cells. Our data suggest that MGST1 could be an important regulator of PC12 cells development and might have significant effects on cell growth and proliferation, probably through altered expression of genes with different biological function.     

Glutathione disulfide enhances the reduced glutathione inhibition of lipid peroxidation in rat liver microsomes

Experiments were undertaken to examine the effects of reduced (GSH) and oxidized (GSSG) glutathione on lipid peroxidation of rat liver microsomes. Dependence on microsomal alpha-tocopherol was shown for the GSH inhibition of lipid peroxidation. However, when GSH (5 mM) and GSSG (2.5 mM) were combined in the assay system, inhibition of lipid peroxidation was enhanced markedly over that with GSH alone in microsomes containing alpha-tocopherol. Surprisingly, the synergistic inhibitory effect of GSH and GSSG was also observed for microsomes that were deficient in alpha-tocopherol. These data suggest that there may be more than one factor responsible for the glutathione-dependent inhibition of lipid peroxidation. The first is dependent upon microsomal alpha-tocopherol and likely requires GSH for alpha-tocopherol regeneration from the alpha-tocopheroxyl radical during lipid peroxidation. The second factor appears to be independent of alpha-tocopherol and may involve the reduction of lipid hydroperoxides to their corresponding alcohols. One, or possibly both, of these factors may be activated by GSSG through thiol/disulfide exchange with a protein sulfhydryl moiety.     

Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase

Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways.     

Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species

The effect of enzymatically generated reduced oxygen metabolites on the activity of hepatic microsomal glutathione S-transferase activity was studied to explore possible physiological regulatory mechanisms of the enzyme. Noradrenaline and the microsomal cytochrome P-450-dependent monooxygenase system were used to generate reduced oxygen species. When noradrenaline (greater than 0.1 mM) was incubated with rat liver microsomes in phosphate buffer (pH 7.4), an increase in microsomal glutathione S-transferase activity was observed, and this activation was potentiated in the presence of a NADPH-generating system; the glutathione S-transferase activity was increased to 180% of the control with 1 mM noradrenaline and to 400% with both noradrenaline and NADPH. Superoxide dismutase and catalase inhibited partially the noradrenaline-dependent activation of the enzyme. In the presence of dithiothreitol and glutathione, the activation of the glutathione S-transferase by noradrenaline, with or without NADPH, was not observed. In addition, the activation of glutathione S-transferase activity by noradrenaline and glutathione disulfide was not additive when both compounds were incubated together. These results indicate that the microsomal glutathione S-transferase is activated by reduced oxygen species, such as superoxide anion and hydrogen peroxide. Thus, metabolic processes that generate high concentrations of reduced oxygen species may activate the microsomal glutathione S-transferase, presumably by the oxidation of the sulfhydryl group of the enzyme, and this increased catalytic activity may help protect cells from oxidant-induced damage.     

Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma

The distribution of the glutathionyl moiety between reduced and oxidized forms in rat plasma was markedly different than that for the cysteinyl moiety. Most of the glutathionyl moiety was present as mixed disulfides with cysteine and protein whereas most of the cysteinyl moiety was present as cystine. Seventy percent of total glutathione equivalents was bound to proteins in disulfide linkage. The distribution of glutathione equivalents in the acid-soluble fraction was 28.0% as glutathione, 9.5% as glutathione disulfide, and 62.6% as the mixed disulfide with the cysteinyl moiety. In contrast, 23% of total cysteine equivalents was protein-bound. The distribution of cysteine equivalents in the acid-soluble fraction was 5.9% as cysteine, 83.1% as cystine, and 10.8% as the mixed disulfide with the glutathionyl moiety. A first-order decline in glutathione occurred upon in vitro incubation of plasma and was due to increased formation of mixed disulfides of glutathione with cysteine and protein. This indicates that plasma thiols and disulfides are not at equilibrium, but are in a steady-state maintained in part by transport of these compounds between tissues during the inter-organ phase of their metabolism. The large amounts of protein-bound glutathione and cysteine provide substantial buffering which must be considered in analysis of transient changes in glutathione and cysteine. In addition, this buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins.     

The content of reduced glutathione in rat liver after poisoning with white phosphorus

The content of hepatic GSH was evaluated in rats after poisoning with white phosphorus. In addition, liver damage following the administration of the hepatotoxin was assessed by determining hepatic triglyceride accumulation. Experiments in parallel were carried out in an attempt to evaluate the enhanced susceptibility of hepatic tissue to peroxidative decomposition of unsaturated lipids 'in vitro', as measured by the production of TBA-reacting substances. Our data indicate that only in the early stage of intoxication is it possible to detect a slight decrease of GSH content in the liver, while during the subsequent stages the concentration of GSH was unaffected. At 6 hours of intoxication the level of hepatic triglycerides was significantly increased. Pretreatment with GSH was followed by an amelioration of fatty infiltration, but the content of hepatic GSH was unchanged. The production of TBA-reacting products was found enhanced only at 6 hours of intoxication. These results are discussed in relation to the role of lipid peroxidation in liver injury by white phosphorus.     

Characterization of 12-L-hydroperoxyeicosa-5,8,10,14-tetraenoic acid peroxidase in platelets by monoclonal antibody against glutathione peroxidase

In the present investigation, 12-L-hydroxyeicosa-5,8,14-tetraenoic acid (12-HPETE) peroxidase in the platelet 12-lipoxygenase pathway was characterized by using a monoclonal antibody to erythrocyte glutathione peroxidase. Pure glutathione peroxidase was used for the immunization of mice. Monoclonal antibody directed against the erythrocyte glutathione peroxidase was obtained from hybridomas, following fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with the enzyme. The subclass of monoclonal antibody was immunoglobulin M with kappa-light chain. Enzyme activity assays using cumene hydroperoxide and [1-14C]12-HPETE as substrates were employed. The monoclonal antibody reacted with glutathione peroxidase in the cumene hydroperoxide assay. In order to see whether platelet 12-HPETE peroxidase reacts with the monoclonal antibody, platelet cytosol and glutathione peroxidase were incubated with the monoclonal antibody and the antibody was precipitated by goat anti-mouse immunoglobulin M. The activities of platelet 12-HPETE peroxidase and glutathione peroxidase remaining were then assayed by using [1-14C]12-HPETE as substrate. The ability of glutathione peroxidase to transform 12-HPETE to 12-HETE was removed by the monoclonal antibody; however, the activity of platelet cytosol was not removed by the antibody. The results indicated that the antigenic specificity of 12-HPETE peroxidase in the platelet 12-lipoxygenase pathway is different from that of erythrocyte glutathione peroxidase.     

Central and peripheral monitoring of the cellular redox state after reduced glutathione administration in the rat

In this study we measured reduced glutathione as DTNB reactive material in different brain areas as well as in liver and kidney of rat, before and after exogenous administration of GSH. Treatment with GSH produced an increase in DTNB positive material as well as a decrease of lipoperoxidation, in central and peripheral organs of rat, suggesting the possibility of an exogenous modulation of redox balance in mammalian cells.     

Effects of silencing leukocyte-type 12/15-lipoxygenase using short interfering RNAs

The leukocyte-type 12/15-lipoxygenase (12/15-LO) has been implicated in the pathogenesis of atherosclerosis, hypertension, and diabetes. 12/15-LO and its products are associated with LDL oxidation, cellular growth, migration, adhesion, and inflammatory gene expression in monocytes/macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs). Our objective, therefore, was to develop novel expression vectors for short interfering RNAs (siRNAs) targeting 12/15-LO to evaluate its functional relevance in macrophages and VSMCs. We used a PCR-based approach to rapidly identify effective siRNA target sites on mouse 12/15-LO and initially tested their efficacy on a fusion construct of 12/15-LO cDNA and enhanced green fluorescent protein. We then cloned these U6 promoter+siRNA PCR products into plasmid vectors [short hairpin siRNAs (shRNAs)] to knockdown endogenous 12/15-LO expression in mouse macrophages and also rat and mouse VSMCs. Furthermore, the functional effects of shRNA-mediated 12/15-LO knockdown were noted by the reduced oxidant stress and chemokine [monocyte chemoattractant protein-1 (MCP-1)] expression in a differentiated mouse monocytic cell line as well as by the reduced cellular adhesion and fibronectin expression in VMSCs. Knocking down 12/15-LO expression also reduced the expression of inflammatory genes, MCP-1, vascular cell adhesion molecule-1, and interleukin-6 in VSMCs. Our results illustrate the functional relevance of 12/15-LO activation in macrophages and VSMCs and its relationship to oxidant stress and inflammation.