Inhibition of mitochondrial creatine kinase activity by D-2-hydroxyglutaric acid in cerebellum of young rats

D-2-Hydroxyglutaric aciduria (DHGA) is a neurometabolic disorder biochemically characterized by tissue accumulation and excretion of high amounts of D-2-hydroxyglutaric acid (DGA). Although the affected patients have predominantly severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In previous studies we have demonstrated that DGA, at concentrations as low as 0.25 mM, significantly decreased creatine kinase activity and other parameters of energy metabolism in cerebral cortex of young rats. In the present study, we investigated the effect of DGA (0.25-5 mM) on total creatine kinase (tCK) activity, as well as on CK activity in cytosolic (Cy-CK) and mitochondrial (Mi-CK) preparations from cerebellum of 30-day-old Wistar rats in order to test whether the inhibitory effect of DGA on CK was tissue specific. We verified that tCK (22% inhibition) and Mi-CK (40% inhibition) activities were moderately inhibited by DGA at concentrations of 2.5 mM and higher, in contrast to Cy-CK, which was not affected by the acid. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by preincubation of the homogenates with reduced glutathione, suggesting that the inhibition of CK activity by DGA is possibly mediated by modification of essential thiol groups of the enzyme. Our present results therefore demonstrate a relatively weak inhibitory effect of DGA on cerebellum Mi-CK activity, as compared to that provoked in cerebral cortex, and may possibly be related to the neuropathology of DHGA, characterized by cerebral cortex abnormalities.     

Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro

D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.     

Reticulocyte lipoxygenase, ingensin, and ATP-dependent proteolysis

Lipoxygenase purified from rabbit reticulocyte lysate has a molecular mass of 68 kDa on SDS gel and a pI of 5.97. Lipoxygenase is inhibited by nordihydroguaiaretic acid (NDGA), 3-amino-1-(m-(trifluoromethyl)phenyl)-2-pyrazoline (BW755C), 5,8,11,14-eicosatetraynoic acid (ETYA), salicylhydroxamate (SHAM) or hemin. Metal ions or nucleotides do not affect its activity. The addition of certain of these inhibitors to the reticulocyte extract also inhibited the ATP-dependent proteolysis of casein, one of the distinct characteristics of reticulocytes. No clear correlation between lipoxygenase activity and ATP-dependent proteolysis could be detected. Hemin and NDGA inhibited both processes, but the concentrations necessary for inhibition were quite different. SHAM completely inhibited lipoxygenase, but not proteolysis. o-Phenanthroline inhibited ATP-dependent proteolysis, but had no effect on lipoxygenase activity. We have also purified a high-molecular-mass protease, ingensin, from reticulocyte extract. This protease accounted for more than 90% of the casein-degrading activity in reticulocyte extract. NDGA inhibited ingensin at the same concentrations required for inhibition of ATP-dependent proteolysis. These results suggest that lipoxygenase is not indispensable for the ATP-dependent proteolysis and the novel high-molecular-mass protease, ingensin, may be involved in the process.     

Omega 6-oxygenation of 6, 9, 12-octadecatrienoic acid in human platelets

[1-14C]6, 9, 12-Octadecatrienoic acid was incubated with suspensions of human platelets. Three monohydroxy acids were isolated, i.e. 10LS-hydroxy-6, 8-pentadecadienoic acid, 10LS-hydroxy-6, 8, 12-octadecatrienoic acid, and 13LS-hydroxy-6, 9, 11-octadecatrienoic acid. Aspirin (0.5 mM) and indomethacin (10 microM) completely inhibited formation of the first mentioned compound whereas 5, 8, 11, 14-eicosatetraynoic acid (34 microM) inhibited formation of all three compounds. Isolation of 13LS-hydroxy-6, 9, 11-octadecatrienoic acid demonstrates that human platelets possess a lipoxygenase activity catalyzing omega 6-oxygenation of suitable poly-unsaturated fatty acids.     

The effect of selected arachidonic acid metabolites on natural killer cell activity

The effect of arachidonic acid (AA) metabolites of lipoxygenase(s) was evaluated on natural killer (NK) cell activity in Fischer F344 rat splenic lymphocytes and compared with prostaglandin E2 (PGE2), a known inhibitor of NK cell lytic activity. It was observed that 5(S),12(S)-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid (5(S),12(S)-diHETE, EZEZ) inhibited NK cell activity to a degree comparable to the inhibitory effects of PGE2. This compound maximally inhibited NK cell activity at concentrations of 10(-6) and 10(-8) M. PGE2 and 5(S),12(S)-diHETE (EZEZ) inhibited NK activity to an identical degree at all concentrations and effector:target (E:T) cell ratios tested. Of the other lipoxygenase pathway metabolites screened, 8(S),15(S)-all trans-diHETE and 8(S),15(S)-diHETE (EZEZ) also inhibited NK activity, but only at 10(-6) M and a 50:1 E:T cell ratio. These findings provide further evidence that the lipoxygenase and cyclooxygenase pathways produce metabolites which can modulate NK cell function, and that 5(S),12(S)-diHETE (EZEZ), which has not been previously tested for effects on NK cells, may have a significant immunoregulatory role.     

Purification and characterization of a glycolic acid (GA) oxidase active toward diglycolic acid (DGA) produced by DGA-utilizing Rhodococcus sp. No. 432

Diglycolic acid (DGA) oxidizing activity was found in crude extracts of Rhodococcus sp. no. 432 grown in DGA. Glycolic acid (GA) oxidase was purified approximately 80 times by treatment with streptomycin sulfate, precipitation with (NH₄)₂SO₄, chromatographies with DEAE-cellulose, DEAE-Toyopearl and Butyl-Toyopearl, and gel filtration on Toyopearl HW-55. The purified GA oxidase was almost homogeneous on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The purity was calculated to be more than 95%. The molecular weight of the enzyme, which appeared to consist of three identical units, was 158,000. Each subunit of GA oxidase included one molecule of FAD as a cofactor. The isoelectric point of the enzyme was around 5.3. GA oxidase was stable below 30°C and at the pH range of 6.0–8.5. The optimum pH and temperature were around 7.5 and 40°C, respectively. Oxygen, cytochrome c, ferricyanide and 2,6-dichlorophenol indophenol (DCIP) acted as an electron acceptor. The activity of GA oxidase was strongly inhibited by potassium cyanide, quinine, quinacrine, monoiodoacetate, 1,4-benzoquinone and some heavy metal ions. GA oxidase also had activity in DGA, GA, glyoxylic acid (GOA), methoxy acetate, ethoxy acetate and l-malate. Alcohols and other organic acids were not oxidized by the enzyme. The apparent K_m values for DGA, GA and GOA were about 26.7, 0.5 and 4.4 mM, respectively. The reaction products from DGA were supposed to be GOA and GA by the enzymatic assays. The reaction mechanism of GA oxidase in oxidation of DGA was supposed to be as follows: HOOCH₂COCH₂COOH+H₂O+acceptor→HOOCCHO+HOOCCH₂OH+ reduced acceptor.     

Docosahexaenoic acid (22:6, n-3) is metabolized to lipoxygenase reaction products in the retina

Docosahexaenoic acid (22:6, n-3), a major component of retinal phospholipids, is a substrate for active lipoxygenation in intact canine retinas incubated in vitro with [U-14C]docosahexaenoic acid. The major lipoxygenase reaction product was identified by high performance liquid chromatography and gas chromatography-mass spectrometry as 11-hydroxy-4,7,9-(trans)13,16,19 docosahexaenoic acid. Other mono- and di-hydroxy derivatives also were detected. The synthesis of these compounds was inhibited by the antioxidant and lipoxygenase inhibitor, nordihydroguaiaretic acid, but was not inhibited by indomethacin or esculetin.     

Arachidonic acid metabolism of the murine eosinophil. II. Involvement of the lipoxygenase pathway in the response to the lymphokine eosinophil stimulation promoter

Eosinophil stimulation promoter (ESP) is a murine lymphokine that enhances the migration of eosinophils. Exogenous arachidonic acid between 0.5 and 2 micrograms/ml potentiated the activity of ESP on murine eosinophil migration, whereas such concentrations did not affect migration in the absence of ESP. Among the lipoxygenase products identified from an enriched population of murine eosinophils, leukotriene B4 (optimal activity at 100 ng/ml) and 12-HETE (optimal activity at 2 micrograms/ml) stimulated migration of these cells. Another lipoxygenase product from these cells 15-HETE inhibited ESP-induced migration; between 5 and 10 micrograms/ml 15-HETE decreased by one-half both stimulated migration and 12-HETE biosynthesis. Structurally diverse drugs at concentrations that inhibited HETE biosynthesis inhibited ESP-induced migration. The concentrations that decreased migration activity by one-half were 5 microM NDGA, 10 microM ETYA, and 150 microM BW755C. Aspirin and indomethacin at concentrations reported to inhibit prostaglandin biosynthesis did not substantially inhibit ESP activity, but concentrations of indomethacin above 20 microM caused concentration-dependent inhibition of migration. The selective lipoxygenases inhibitor 134,7,10,13-eicosatetraynoic acid was more potent than ETYA in inhibition of ESP-induced migration, and the selective cyclooxygenase inhibitor 6,9,12-octadecatriynoic acid did not effect inhibition. These results are consistent with the hypothesis that stimulation of eosinophils by the lymphokine ESP involves the generation of lipoxygenase products from arachidonic acid, which positively and negatively regulate the migratory activities of these cells.     

Lipoxygenase activity in rat kidney glomeruli, glomerular epithelial cells, and cortical tubules

We examined the possibility that renal glomerular and cortical tubular tissue has lipoxygenase activity in addition to the well established cyclo-oxygenase pathway of arachidonic acid metabolism. Homogenized rat kidney glomeruli, in the presence of meclofenamate (33 microM) and divalent cation ionophore A23187 (3 microM), metabolized octatritiated arachidonic acid to 12-hydroxyeicosatetraenoic acid and lesser amounts of 80 and/or 9-hydroxyeicosatetraenoic acid. These products were identified by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectroscopy. In order to rule out the synthesis of hydroxylated fatty acids by platelets and leukocytes entrapped in the glomeruli, we studied lipoxygenase products in glomerular epithelial cells after 9 days in cell culture. Homogenized glomerular epithelial cells converted octatritiated arachidonic acid to 12-hydroxyeicosatetraenoic acid solely. The lipoxygenase activity in cortical tubules was substantially less than in glomeruli and only 12-hydroxyeicosatetraenoic acid was synthesized. The production of hydroxyeicosatetraenoic acid by lipoxygenase inhibitors, nordihydroguaiaretic acid, 5,-homogenized glomeruli, glomerular epithelial cells, and cortical tubules was inhibited by three 8,11,14-eicosatetraynoic acid, and 1-phenyl-3-pyrazolidone. These data demonstrate that there is lipoxygenase activity in rat kidney glomeruli, glomerular epithelial cells and to a lesser extent cortical tubules, and may imply a role of the lipoxygenase products in the regulation of normal glomerular function and inflammatory disease of the kidney.     

Enhancement of endogenous production of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid in aortic smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF) has a chemotactic effect on smooth muscle cells, which is inhibited by lipoxygenase inhibitor caffeic acid. In order to study the role of endogenous lipoxygenase products of arachidonic acid on the chemotactic action of PDGF, effects of PDGF on the lipoxygenase pathway in smooth muscle cells were examined. Lipoxygenase products were analyzed by high-performance liquid chromatography. 15-, 5- and 12-lipoxygenase activities, in order of magnitude, were found in smooth muscle cell homogenate. However, when the lipoxygenase products were analyzed using intact cells prelabelled with [14C]arachidonic acid, only 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) was found to be produced endogenously. In addition, 12-HETE was not released into the medium. Treatment of the cells with PDGF increased the endogenous production of 12-HETE. The amounts of intracellular 12-HETE in PDGF-treated cells were 126, 132 and 146% at 1, 3, and 10 hr's after the initiation of PDGF treatment, respectively, when control value at each time point was considered as 100%. Caffeic acid (10(-4) M) completely inhibited the PDGF effect on 12-HETE production. However, PDGF treatment did not significantly alter the 12-lipoxygenase activity. These results suggest that the stimulatory effect of PDGF on 12-HETE production was not mediated by the activation of 12-lipoxygenase activity. Since 12-HETE itself is a potent chemoattractant for smooth muscle cells, the present dat strongly suggest that 12-HETE could be an important intracellular mediator of the chemotactic action of PDGF on aortic smooth muscle cells.     

Larvicidal activity of (-)-dihydroguaiaretic acid derivatives against Culex pipiens

The larvicidal activity against Culex pipiens of all stereoisomers of dihydroguaiaretic acid (DGA) and secoisolariciresinol was measured, and these DGAs were found to be potent. Sixteen (-)-DGA derivatives were then newly synthesized to analyze their structure-activity relationship. Two derivatives monohydroxylated at the 3- or 4-position of the 7-phenyl group of DGA induced acute paralytic activity in the mosquitoes. Derivatives with several hydroxyl groups had lower activity than the natural compound, suggesting that hydrophobicity was probably an important factor for their insecticidal activity.     

Effect of tert-butyl hydroperoxide on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in rabbit platelets

The effect of tert-butyl hydroperoxide (t-BOOH) on the formation of thromboxane (TX) B2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid (AA) in washed rabbit platelets was examined. t-BOOH enhanced TXB2 and HHT formation at concentrations of 8 microM and below, and at 50 microM it inhibited the formation, suggesting that platelet cyclooxygenase activity can be enhanced or inhibited by t-BOOH depending on the concentration. t-BOOH inhibited 12-HETE production in a dose-dependent manner. When the platelets were incubated with 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) instead of AA, t-BOOH failed to inhibit the conversion of 12-HPETE to 12-HETE, indicating that the inhibition of 12-HETE formation by t-BOOH occurs at the lipoxygenase step. Studies utilizing indomethacin (a selective cyclooxygenase inhibitor) and desferrioxamine (an iron-chelating agent) revealed that the inhibitory effect of t-BOOH on the lipoxygenase is not mediated through the activation of the cyclooxygenase and that this effect of t-BOOH is due to the hydroperoxy moiety. These results suggest that hydroperoxides play an important role in the control of platelet cyclooxygenase and lipoxygenase activities.     

Inhibition of platelet arachidonic acid 12-lipoxygenase by acetylenic acid compounds

Eighteen acetylenic fatty acids were tested as inhibitors of human platelet arachidonic acid 12-lipoxygenase. 4,7,10,13-Eicosatetraynoic (4,7,10,13-ETYA) acid emerged as the most potent compound. Additional experiments have shown that 4,7,10,13-ETYA selectively blocked the 12-lipoxygenase in washed human platelets with lesser activity against the cyclooxygenase. The ID50 value for lipoxygenase was 7.8 microM in comparison with an ID50 of 100 microM for the cyclooxygenase. The commonly used inhibitor 5,8,11,14-eicosatetraynoic acid inhibited both enzymes with equal potency. It appears that 4,7,10,13-ETYA may be a valuable lead for selective modulation of the 12-lipoxygenase pathway in platelet or other target tissues.     

Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by D-2-hydroxyglutaric acid in vitro.

L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria.     

Use of Tetraethylthiuram Disulfide to Discriminate between Alternative Respiration and Lipoxygenase

Mitochondria from axes of Glycine max (L.) Merr. cv. Chippewa 64 seedlings purified on discontinuous Percoll gradients exhibited classical cyanide-resistant respiration. These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 0.8 millimolar linoleic acid. This activity is inhibited by most known inhibitors of alternative respiration (i.e. hydroxamates and propyl gallate). Tetraethylthiuram disulfide (disulfiram) at 50 micromolar inhibited cyanide-resistant succinate oxidation by 90 per cent, whereas concentrations as high as 100 micromolar had no effect on lipoxygenase activity. Use of tetraethylthiuram disulfide allows discrimination between alternative respiration and lipoxygenase activity in mitochondria.     

The possible role of 9(S)-hydroperoxyoctadecatrienoic acid as a suicide substrate of soybean lipoxygenase

While incubation of soybean lipoxygenase with alpha-linolenic acid resulted in the gradual decrease of lipoxygenase activity, the incubation with linoleic acid had no change. The inactivation of soybean lipoxygenase during incubation with alpha-linolenic acid was markedly observed at pH 6.5, but not at pH 9.0. Among the lipoxygenation products of alpha-linolenic acid, only 9(S)-hydroperoxyoctadecatrienoic acid caused the inactivation of lipoxygenase. 9(S)-Hydroxyoctadecatrienoic acid, 13(S)-hydroperoxyoctadecatrienoic acid or 9,16-dihydroperoxy conjugated trienoic acid was without effect. Accordingly, it is suggested that the epoxide intermediate, one conversion product of 9(S)-hydroperoxyoctadecatrienoic acid, might be involved in the direct inactivation of lipoxygenase.     

Features of lipoxygenase from cells of Mortierella species mushrooms

The specific activity of lipoxygenase from several strains of the zygomycete Mortierella varied from 1.02 to 2.02 microMol diene per min per mg protein). The enzyme equally used linoleic or arachidonic acid as a substrate. An increase in lipoxygenase activity was observed after adding corn oil to the culture medium. Tests with inhibitors having different chemical structures revealed that the lipoxygenase activity from Mortierella cells was inhibited only by esculetin, ethanol and nordihydroguaiaretic acid (NDGA). NDGA inhibited the enzyme in vitro (IC50 = 142 microM), but its addition in the exponential phase of growth activated the enzyme.     

13-Hydroxyoctadeca-9,11-dienoic acid (13-HODE) inhibits thromboxane A2 synthesis, and stimulates 12-HETE production in human platelets

The effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-HODE), a major lipoxygenase product of endothelial cell linoleic acid metabolism on thrombin-induced platelet thromboxane B2 (TxB2), and 12-hydroxyeico-satetraenoic acid (12-HETE) production was evaluated. 13-HODE inhibited thrombin-induced TxB2 production in human platelets in a concentration-dependent manner. At concentrations of 10 and 30 microM, 13-HODE inhibited TxB2 production by 28 +/- 8% (1SE, n = 5; P less than 0.05) and 48 +/- 6% (P less than 0.01) respectively. 13-HODE (30 microM) also inhibited the production of platelet hydroxyheptadecatrienoic acid (38 +/- 5%, P less than 0.01). A concomitant stimulation of 12-HETE production by 13-HODE was observed (25 +/- 5% and 49 +/- 22% over control values at 10 and 30 microM respectively, P less than 0.01). Our results demonstrate a differential effect of 13-HODE on thrombin stimulated platelet cyclooxygenase and lipoxygenase metabolites.     

Effect of inhibitors of the lipoxygenase pathway on mouse myoblast fusion

In this study we examined the effects of inhibitors of the lipoxygenase and cyclooxygenase pathways on mouse myoblast fusion. The fusion of cloned mouse myoblasts was markedly inhibited, in a dose-dependent manner, when cells were cultured in medium supplemented with either phenidone (1-phenyl-3-pyrazolidione) or BW755c (3-amino-1-(3-tri-fluoromethylphenyl)-2-pyrazoline), drugs which have been reported to inhibit lipoxygenase and cyclo-oxygenase activities. Fusion was also inhibited when these cells were cultured in medium supplemented with esculetin (6,7-dihydroxycoumarin) which has been reported to inhibit lipoxygenase activity. Removal of the above inhibitors resulted in a return to control levels of fusion. Fusion was not demonstrably inhibited with aspirin (acetylsalicylic acid) and only inhibited to a minor extent with indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid); both of these drugs are inhibitors of cyclo-exygenase activity.