High glucose and advanced glycation end products induce phospholipid hydrolysis and phospholipid enzyme inhibition in bovine retinal pericytes.

In the present study, we investigated the possible role of oxidative stress and the modulation of phospholipid turnover in two related models of pericyte injury, i.e., treatment with high glucose or advanced glycation end products (AGEs). Growing microcapillary pericytes from bovine retinas in culture were incubated, for 3 weeks, with 20-50 mM glucose or 2-20 microM AGEs, and peroxidation parameters (malondialdehyde, conjugated diene, hydroperoxide, glutathione (GSH) levels and lactate dehydrogenase (LDH) release) were evaluated. Arachidonate (AA) and choline release from membrane phospholipids was determined in pericytes prelabeled with [1-(14)C]arachidonate and [Me-(3)H]choline, respectively, and stimulated with elevated glucose or AGEs for 30 min or 2 h. [1-(14)C]arachidonate and [Me-(3)H]choline incorporation into phospholipids, for 2 h and 3 h respectively, was also studied in conditioned and serum-starved cultures. Finally, lysates of treated and control cells were assayed for cytosolic phospholipase A(2) (cPLA(2)), acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (AT), CTP:phosphocholine cytidylyltransferase (CT) and microsomal choline phosphotransferase (CPT) enzyme activities. We found that high glucose and AGEs caused neither significant production of reactive oxygen species nor cell toxicity or death, unlike other cell types. Both agents had no significant effect on the cellular ultrastructure, evaluated by light and electron microscopy, AA incorporation and release, cytosolic phospholipase A(2) (cPLA(2)) and AT activities. On the contrary, choline incorporation into phosphatidylcholine, CT and CPT activities were significantly reduced either by 50 mM glucose or 20 microM AGEs. Simultaneously, [Me-(3)H]choline release was significantly stimulated by both agents. We conclude that prolonged treatments with high glucose or AGEs are not able to induce oxidative injury in bovine retinal capillary pericytes. Nevertheless, they do induce phospholipid hydrolysis and phospholipid enzyme activity inhibition.

t-Butyl hydroperoxide and oxidized low density lipoprotein enhance phospholipid hydrolysis in lipopolysaccharide-stimulated retinal pericytes.

Free radicals induced by organic peroxides or oxidized low density lipoprotein (oxLDL) play a critical role in the development of atherosclerosis. In investigating this process, and the concomitant inflammatory response, the role of pericytes, cells supporting the endothelial ones in blood vessels, has received little attention. In this study we tested the hypothesis that tert-butyl hydroperoxide (t-BuOOH) and oxLDL, administered in sublethal doses to the culture medium of retinal pericytes, function as prooxidant signals to increase the stimulation of the peroxidation process induced by lipopolysaccharide (LPS). Confluent cell monolayers were exposed to t-BuOOH (25-400 microM), native LDL or oxLDL (3.4-340 nmol hydroperoxides/mg protein, 1-100 micro). LPS (1 microg/ml), t-BuOOH (200 microM), and oxLDL (100 microM), but not native LDL, incubated for 24 h with cells, markedly increased lipid peroxidation, cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release in a time- and dose-dependent manner. AACOCF(3), a potent cPLA2 inhibitor, and the antioxidant alpha-tocopherol strongly inhibited the prooxidant-stimulated AA release. Long-term exposure to maximal concentrations of t-BuOOH (400 microM) or oxLDL (100 microM) had a sharp cytotoxic effect on the cells, described by morphological and biochemical indices. The presence of t-BuOOH or oxLDL at the same time, synergistically increased phospholipid hydrolysis induced by LPS alone. 400 microM t-BuOOH or 100 microM oxLDL had no significant effect on the stimulation of an apoptosis process estimated by DNA laddering and light and electron microscopy. The results indicate that (i) pericytes may be the target of extensive oxidative damage; (ii) activation of cPLA2 mediates AA liberation; (iii) as long-term regulatory signals, organic peroxide and specific constituents of oxLDL increase the pericyte ability to degrade membrane phospholipids mediated by LPS which was used, in the present study, to simulate in vitro an inflammatory burst in the retinal capillaries.

Effect of CDP-choline treatment on mitochondrial and synaptosomal protein composition in different brain regions during aging.

Several age-dependent modifications of inner mitochondrial membrane and synaptosomal plasma membrane proteins from different brain regions of 4-, 12-, 18- and 24-month-old male Wistar rats, were observed. Some proteins, identified by immunoblotting assay as various subunits of mitochondrial respiratory chain complexes and calmodulin, were particularly impaired. Chronic treatment with CDP-choline at a dose of 20 mg/kg body weight per day for 28 days caused significant changes in the amounts of several of the above mentioned proteins. Most of the proteins, which decreased during aging, showed a significant increase after CDP-choline treatment compared with the corresponding control values at the same age. The effect of CDP-choline might be due to: the increased availability of cytidylic nucleotides, which in the brain are present in limited amounts compared to the other nucleotides; the increased content of total adenine nucleotides; the improvement of brain energy metabolism.

Modifications of synaptosomal plasma membrane protein composition in various brain regions during aging.

The age-dependent modifications of synaptosomal plasma membrane protein composition in three different rat brain regions (cerebral cortex, cerebellum and striatum) at various ages (4, 12 and 24 months) were studied. The proteins were separated by gel-electrophoresis and the quantity of the different polypeptides was determined densitometrically from the stained gels. In the three brain regions examined several age-related modifications in the amount of the synaptosomal plasma membrane proteins were observed. In particular a significant decrease in the content of some synaptosomal plasma membrane proteins at 24 months of age was found. The age-related modifications in the protein composition of synaptosomal plasma membrane may cause changes in many brain functions, such as neurotransmission, ionic transport and enzyme activities. Particularly interesting is the decrease of a protein with 18 kDa mol. wt. This protein has been identified as calmodulin by immunoblotting assay. The decrease in the amount of this protein may be correlated to the impairment of several Ca(2+)-requiring processes in the aging brain.

ADP-ribosylation of proteins in brain regions of rats during postnatal development.

Post-translational modifications of chromatin-bound proteins play an important role in the regulation of eukaryotic gene expression. Processes such as acetylation, methylation, phosphorylation and ADP-ribosylation may alter the interaction of these proteins with DNA and consequently affect chromatin conformation and the binding of enzymes and other molecules involved in the regulation of gene expression. In the present study the process of ADP-ribosylation of chromosomal proteins (histone and non-histone proteins) in some rat brain regions during postnatal development was investigated; also the effect of epidermal growth factor (EGF) on this process in fetal brain slices was studied. It has been found that the process of ADP-ribosylation of total histones extracted from rat cerebral cortex and cerebellum at 1, 10 and 30 days of age, increases from 1 to 10 days of age (i.e. the period of maximal cell proliferation) and decreases thereafter, while the process of ADP-ribosylation of non-histone proteins (NHPs) sharply decreases during the same developmental period. The addition of EGF to fetal brain slices causes a significant increase of ADP-ribosylation of total histones (particularly of the histone H1 fraction) and also of NHPs and microsomal proteins. This result is in agreement with the effect of EGF as a mitogen factor, previously shown in astroglial cell cultures.

Effects of Metadoxine on cellular status of glutathione and of enzymatic defence system following acute ethanol intoxication in rats.

The aim of the study was to determine whether a combination of pyridoxine and pyrrolidone carboxylate (PCA) could influence the antioxidant defence system in hepatic and extrahepatic tissues during acute ethanol administration. The results showed that ethanol treatment led to a significant depletion of reduced glutathione and increase in oxidized glutathione (GSSG) in the different organs, associated with decreased activity of glutathione reductase. Pretreatment of animals with Metadoxine (PCA + pyridoxine) one hour before ethanol administration produced a significant protection against glutathione (GSH) depletion in the different organs examined. This was consistent with an increase in glutathione reductase activity. In view of the fact that free thiol compounds such as glutathione are vital in cellular defence against oxidants and that decreases in reduced glutathione precede lethal cell injury resulting from free radical accumulation, our study supports the effectiveness of Metadoxine as a useful therapeutic agent effective in the management of all those pathological conditions where a severe imbalance of cellular redox state seems to take place as a result of the generation of free radical species.

Long-term ethanol administration enhances age-dependent modulation of redox state in central and peripheral organs of rat: protection by metadoxine.

Evidence is accumulating that intermediates of oxygen reduction may be associated with the development of alcoholic disease. In addition, free radical-induced perturbation of the oxidant/antioxidant balance in cells is widely recognized as the main causative factor of age-related disorders. In the present work, we investigated the effects of 25 months of ethanol consumption on the antioxidant defense system in different organs of rat in comparison with normal aging, in the absence and presence of treatment with metadoxine, an ion pair composed of pirrolidone carboxylate and pyridoxine. We demonstrate that aged rats underwent a significant perturbation of the antioxidant defense system, as indicated by depletion of reduced glutathione (GSH) content, and increases in oxidized GSH and free radical-induced luminescence associated with a decrease of GSH reductase and an increase of GSH transferase activities. These modifications, observed particularly in the liver and brain with respect to other organs, were enhanced by long-term alcohol exposure, and interestingly, significantly reduced after metadoxine supplementation. Our results indicate that increased GSH transferase activity and decreased GSH reductase activity, followed by thiol depletion, are important factors sustaining a pathogenic role for oxidative stress in aging and in all situations where age-correlated changes occur. Administration of metadoxine greatly reduces these metabolic abnormalities. This evidence supports the pharmacological potential of metadoxine in the management of alcoholic disturbances.

Cysteine-induced enhancement of lipid peroxidation in substantia nigra: comparative effect with exogenous administration of reduced glutathione.

It is generally accepted that reactive oxygen species have a major role in the mediation of cell damage and that free sulphydryl (SH) groups are vital in cellular defence against endogenous or exogenous oxidants. Modification of cellular oxidant/antioxidant balance has been involved in the neuropathogenesis of several diseases, e.g., stroke, Parkinson's disease, Alzheimer's disease and physiological ageing. An increasingly important area of antioxidant defence is based on sulphydryl chemistry, owing to the role of SH groups in the function of macromolecular structures such as enzymes and cellular membranes. Thiols, however, may themselves generate deleterious free radicals, and thionyl radicals, which have been demonstrated to originate in biological systems through enzymatic reactions of different peroxidases, by reacting with molecular oxygen or hydrogen peroxide are able to promote reactions of oxidatives stress. In the present study we provide experimental evidence suggesting a selective effect of cysteine in promoting reactions of oxidative stress in the brain areas of substantia nigra and septum, but not in other areas. In contrast, exogenous administration of reduced glutathione led to a significant decrease of lipoperoxidation in the brain areas of cortex and hippocampus, associated to selective changes in the endogenous pool of thiols.

Changes of nucleic acid and protein synthesis in hypertrophied guinea-pig heart during intermittent hypoxia.

The effect of intermittent normobaric hypoxia on nucleic acid and protein synthesis of guinea-pig heart was studied. De novo synthesis of purine nucleotides, of RNA and DNA, as well as RNA polymerase activities and protein synthesis, increased in the heart right ventricles of hypoxic animals compared to the controls. These results suggest an activation of cellular genetic apparatus with an increase of nucleic acid and protein syntheses during the compensatory processes following hypoxia.

Age-related chances of mitochondrial cytochrome C oxidase and F0F1-ATP synthase subunit contents in rat cerebral cortex.

The levels of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of F(0)F(1)-ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, 26 months of age were analyzed by Western blot. Age-related changes in the content of subunits, encoded either in mitochondrial or nuclear DNA, were observed.

Effect of pyrrolidone carboxylate (PCA) and pyridoxine on liver metabolism during chronic ethanol intake in rats.

Rats subjected to chronic ethanol intake for a period of 28 days showed significant elevation in blood ethanol levels, a marked decrease in hepatic reduced glutathione (GSH) content and a decrease in liver tryptophan pyrrolase (TPO) activity. A daily intraperitoneal injection of a combined solution of pyrrolidone carboxylate (PCA) and vitamin B6 (pyridoxine hydrochloride) (0.3 mmoles/kg) into ethanol-treated rats resulted in the blood ethanol levels becoming significantly reduced, while the hepatic GSH content and TPO activity were markedly elevated. Our results support the view that PCA and pyridoxine operate to restore the redox imbalance of the hepatocytes caused by chronic alcohol intake.

Effects of metadoxine on cellular formation of fatty acid ethyl esters in ethanol treated rats.

Free fatty acids (FFA) and fatty acid ethyl esters (FAEE) were extracted from different organs of rats administered ethanol, which was found to have induced FAEE formation, which reached its highest levels in the heart, followed by kidney, brain and liver; the ethanol administration resulted also in a marked increase of total FFA content, particularly in brain, kidney, heart and liver. Pretreatment of animals with Metadoxine one hour before ethanol administration inhibited significantly both FAEE and FFA accumulation in all organs examined. These effects were concomitant with the decreased levels of ethanol in blood found in alcohol-intoxicated rats pretreated with Metadoxine. Our results point to the role of fatty acid ethyl esters as possible mediators in the production of alcohol-dependent syndromes, especially in organs lacking oxidative pathways. Administration of Metadoxine, through an increment in alcohol metabolism and turnover, greatly reduces this metabolic abnormality, warranting its potential usefulness as a pharmacological tool in alcoholism management.

Changes in endogenous lipoperoxidation and antioxidant enzyme status induced by cysteine in the substantia Nigra.

It is generally accepted that reactive oxygen species have a major role in the mediation of cell damage and that free sulfhydryl groups are vital in cellular defence against endogenous or exogenous oxidants. Modification of cellular oxidant/antioxidant balance has been involved in the neuropathogenesis of several diseases, e.g., stroke, Parkinson's disease, Alzheimer's disease and physiological ageing. An increasingly important area of antioxidant defence is based on sulfhydryl chemistry, owing to the role of -SH groups in the function of macromolecular structures such as enzymes and cellular membranes. Thiols, however, may themselves generate deleterious free radicals. In the present study we provide experimental evidence suggesting a selective effect of cysteine in promoting reactions of oxidative stress in the brain areas of substantia nigra and septum, but not in other different areas which were associated with corresponding changes in the activity of antioxidant enzymes.

Long-term ethanol administration enhances urinary ultraweak luminescence and age-dependent modulation of redox in central and peripheral organs of the rat.

Numerous experimental evidence sustains a pathogenic role for oxidative stress in aging. Acute and chronic ethanol metabolism is also known to be associated with oxidative perturbation of cellular oxidant/antioxidant balance. In the present work we investigated the effects of 25 months of ethanol consumption on the antioxidant defense system in different organs of rats, in comparison with normal and aged animals. We show that aged rats underwent a significant perturbation of the antioxidant defense system, as indicated by depletion of reduced glutathione content, increases in oxidized glutathione and free radical-induced urinary luminescence associated with a decrease of glutathione reductase and increase of glutathione transferase activities. These modifications, observed particularly in the liver and brain, were enhanced by long-term alcohol exposure. Our results indicate that increased glutathione transferase activity and decreased glutathione reductase activity, followed by thiol depletion, are important factors sustaining a pathogenic role for oxidative stress in aging and in all situations where age-correlated changes occur. They also reinforce the oxidative potential of toxic compounds, such as ethanol intoxication.

Nucleic acid biosynthesis in rat embryo cells infected with X14 or H-1 parvovirus.

Nucleic acid biosynthesis was studied in rat embryo cell (REC) cultures 48 hours after infection with X14 or H-1 parvovirus. The incorporation of 14C-formate and [6-(14C]-orotic acid into purines and pyrimidines of various was lowered after infection with these parvoviruses. 14C-Formate incorporation into acid-soluble thymine was greatly inhibited in H-1 virus-infected cells whereas it was slightly inhibited in X14 virus-infected cells. These results suggest that X14 virus-infected cells can carry out the biosynthesis of thymidylic acid utilizing some endogenous pyrimidine nucleotide (e.g. deoxycytidylic acid, via deoxyuridylic acid). In the infected cells, the nucleoplasmic RNA polymerase activity was strongly inhibited. This results suggests an interference by the two viruses with hosts RNA synthesis.

Pyrimidine nucleotide synthesis in rat embryo cells infected with X14 or H-1 parvovirus.

The activity of some enzymes involved in pyrimidine nucleotide synthesis was studied in rat embryo cell (REC) cultures infected with X14 or H-1 parvovirus. dCMP aminohydrolase activity of the infected cells was 64--121% greater than that of the mock-infected cells. dTMP synthetase activity was 18% greater in X14 virus-infected cells and 34% lower in H-1 virus-infected cells. These results suggest some differences in the infected cells, as regards the biosynthesis of dTMP. Orotate phosphoribosyltransferase and orotidylate decarboxylase activities appeared nearly unmodified compared to the mock-infected cells. The addition of phosphoribosylpyrophosphate (PRPP) to the cell suspension incubated with [6-14C] orotate increased the specific radioactivity of acid-soluble uracil, 5-fold in the mock-infected cells and 15- --24-fold in the X14 or H-1 virus-infected cells (72 hr p.i.). This result suggests that the lowered pyrimidine nucleotide synthesis in infected cells depends to a large extent on the diminished PRPP pool.

Pool and synthesis of phosphoribosylpyrophosphate in rat embryo cells infected with X14 or H-1 parvovirus.

In rat embryo cell cultures infected with X14 or H-1 parvovirus the PRPP pool and the PRPP synthetase activity have been assayed. A radiometric method, prepared by Authors, based on the conversion of [6-14C) orotate to [6-14C) UMP by the mixed enzyme orotate phosphoribosyltransferase and orotidylate decarboxylase and on the separation of UMP by ascending chromatography, has been utilized. The PRPP pool and te PRPP synthetase activity appeared nearly unmodified in the cells infected with X14 or H-1 parvovirus compared to the mock-infected cells. Therefore, the lowered pyrimidine nucleotide synthesis in infected cells, shown in previous studies, may depend, rather than on the diminished PRPP pool, on the lower PRPP utilization; in fact, some inhibition by metabolites, that may be removed by added PRPP, might occur in the infected cells.