Patrizia Marraccini

GLUTATHIONE TRANSPORT SYSTEM IN HUMAN SMALL INTESTINE EPITHELIAL CELLS

The present study characterizes for the first time a GSH specific transporter in a human intestinal epithelial cell line (I407). GSH metabolism is very important for the antioxidant and detoxifying action of intestine and for the maintenance of the luminal thiol-disulfide ratio involved in regulation mechanisms of the protein activity of epithelial cells. GSH level decreases have been related to physio-pathological alterations either of intestine or other organs. GSH specific transport systems have been identified in membranes of various cell types of rat, mice and rabbit. The presence of a Na+-independent transport system of GSH is confirmed by the similar behaviour of GSH uptake time-courses when Na+ in extracellular uptake medium was replaced with choline+ or K+ as well as by kinetic saturation and by the trans-stimulation effect on GSH uptake in GSH preloaded cells. Moreover, this transporter is activated when cations are present in extracellular medium and it is affected by membrane potential changes with an increase in GSH uptake values when membrane depolarization occurs. The present results also show a remarkable affinity and specificity of this transporter for GSH; in fact, Km value is very low (90 +/- 20 microM) and only compounds strictly related to GSH structure, such as GSH S-conjugates and GSH-ethyl ester, inhibit GSH uptake in 1407 cells. Finally, a possible hormonal control and modulation by the thiol-disulfide status of GSH transporter activity is suggested.

Effect of orally administered glutathione on glutathione levels in some organs of rats: role of specific transporters

The present study reports data on absorption of orally administered glutathione (GSH) in rat jejunum and in other organs, and the possible role of specific transport systems of GSH and gamma-glutamyltranspeptidase (EC 2.3.2.1; gamma-GT) activity. GSH levels were measured simultaneously in various organs after oral GSH administration to untreated rats and rats treated with L-buthionine sulfoximine (BSO) or acivicin (AT125). BSO selectively inhibits GSH intracellular synthesis and AT125 is a specific inhibitor of gamma-GT activity. GSH levels were also measured after oral administration of an equivalent amount of the constituent amino acids of GSH to untreated and BSO-treated rats. Significant increases in GSH levels were found in jejunum, lung, heart, liver and brain after oral GSH administration to untreated rats. GSH increases were also obtained in all organs, except liver, when GSH was administered to rats previously GHS-depleted by treatment with BSO. The analysis of all results allowed us to distinguish between the increase in GSH intracellular levels due to intact GSH uptake by specific transporters, and that due to GSH degradation by gamma-GT activity and subsequent absorption of degradation products with intracellular resynthesis of GSH; both these mechanisms seemed to be involved in increasing GSH content in heart after oral GSH administration. Jejunum, lung and brain took up GSH mostly intact, by specific transport systems, while in liver GSH uptake occurred only by its breakdown by gamma-GT activity followed by intracellular resynthesis.

Age and GSH metabolism in rat cerebral cortex, as related to oxidative and energy parameters.

A comprehensive study on GSH metabolism in relation to some markers of oxidative and energy status in rat cerebral cortex as a function of age was performed. Reduced GSH, total GSH and the GSH Redox Index decreased both during growth (defined as the period between 1 and 5 months) and during aging (defined as the period between 5 and 27 months) while GSSG levels increased during the two periods, but most significantly during aging. Also GSH-associated enzymes and adenine-pyridine nucleotide levels show age characteristic changes. The obtained results suggest that decreases in oxidative and energy metabolism occur during aging. They probably contribute to decreases in the activity of the biosynthetic processes (i.e., NADP+(H) and GSH synthesis) and in the antioxidant capacity of the GSH system. However, the oxidative stress does not seem to be a typical characteristic of the aging period; as an oxidative status is present during the growth period too. Typical parameters of aging process are mainly the low levels of reduced GSH, total GSH and GSH Redox Index and the high levels of GSSG as well as the high levels of GSH peroxidase and GSH transferase and the low levels of gamma-glutamylcysteine synthetase.

Relationship between age and GSH metabolism in synaptosomes of rat cerebral cortex

A comprehensive analysis on glutathione metabolism in rat cerebral cortex synaptosomes as a function of age was performed. All different glutathione system components (GSH, GSSG, total GSH, and GSH redox index) changed significantly only during aging. GSH, total GSH, and GSH redox index decreased by about 40%, 24%, and 52%, respectively, while GSSG showed a remarkable increase of about 60%. On the contrary, some GSH-related enzyme activities showed characteristic changes both during growth and aging. GSH peroxidase and GSH-S-transferase activities significantly increased both during growth and aging, GSH reductase and gamma-glutamylcysteine synthetase activities showed lower levels only during aging, while glucose-6-phosphate dehydrogenase activity did not change throughout the life of the rat. The results obtained suggest an increase of the oxidative status due to a reduced antioxidant capacity of the GSH system in the synaptosomal compartment during aging. The main cause of these metabolic modifications is a lowering of the rates of both GSSG reduction to GSH and GSH synthesis. Moreover, an irreversible loss of GSH as GSH-S-conjugates due to a high detoxification mechanism during aging is also possible. These alterations in glutathione metabolism, found mainly during aging in rat cerebral cortex synaptosomes may contribute to clarify some aspects of cerebral diseases.

Altered metabolism of glutathione in cells transformed by oncogenes which cause resistance to ionizing radiations

We measured glutathione (GSH) metabolism in normal NIH/3T3 fibroblasts, and in cells transformed by the oncogenes sis, erbB, src, ras, dbl, and raf.erbB,src,ras and raf, but not sis and dbltransformants, showed increased level of total and reduced GSH as compared with normal NIH/3T3 fibroblasts; oxidized GSH was elevated only in src‐ and ras‐transformed cells. Increased total GSH content was associated with decreased activity of the synthetic enzyme γ‐glutamylcysteine synthetase, and oxidized GSH level with increased activity of GSH reductase. These data suggest that GSH synthesis was selectively enhanced in cells transformed by specific oncogenes, with resulting down‐regulation of its synthetic enzyme; alterations of GSH metabolism appeared to be peculiar of transformation by specific oncogenes, and not trivial epiphenomena of neoplastic transformation. Oncogenic transformants that presented elevated level of GSH were also those reported to be resistant to antineoplastic drugs and ionizing radiations, thus confirming a possible link between altered GSH metabolism and resistance to antineoplastic treatment.

GLUTATHIONE INVOLVEMENT ON THE INTESTINAL NA+-DEPENDENT D-GLUCOSE ACTIVE TRANSPORTER

Glutathione and its related enzymes are present in intestinal epithelium. Depletion or alteration of glutathione levels have been related to different physiological and pathological conditions. Glutathione also seems to be related to the regulation of some protein actvities. The present study, by in vivo experiments. shows a specific relationship between D-glucose Na+-dependent active transporter activit in rat intestine brush-border membranes and reduced glutathione/oxidized glutathione ratio levels. Changes of the kinetic parameters show that an increase of this ratio is related to an increase of the affinity of glucose for its binding sites and a higher transport capacity of the transporter. Neither alteration in the activity of other substrate transport systems nor change in the specific activity of the key enzymes related to glutathione and glucose metabolism are found. These findings suggest the possibility that D-glucose transporter activity is modulated through the change in the redox status of glutathione.

Effect of phosphotyrosine phosphatase over-expression on glutathione metabolism in normal and oncogene-transformed cells

We measured the level of reduced glutathione (GSH) and oxidized glutathione (GSSG) in normal and oncogene‐transformed NIH/3T3 fibroblasts and 32D hematopoietic cells. NIH/3T3 cells transformed by the activated oncogenes erbB, src, and raf, showed increased levels of GSH with concomitant alterations in the levels of GSH‐related enzymes. Transfection and over‐expression of a synthetic gene coding for a phosphotyrosine protein phosphatase (PTPase), which inhibited the proliferation of normal and transformed NIH/3T3 cells, was accompanied by a decrease in GSH levels in normal and erbB‐transformed fibroblasts, and by an increase in src and raf transformants. Among GSH‐related enzymes, only γ‐glutamylcysteine synthetase was altered in normal and erbB‐transformed NIH/3T3 fibroblasts following PTPase transfection. Therefore, tyrosine phosphorylation could be selectively involved in the regulation of GSH metabolism in normal and oncogene‐transformed NIH/3T3 fibroblasts, possibly by a dual‐type effect on receptor/oncoprotein‐mediated mitogenic signal transduction. However, no relationship was observed between the GSH and PTPase effect on cell growth, either after oncogene transfection or PTPase transfection. Moreover, the changes in GSH metabolism were specifically related to cell lineage. In fact GSH and related enzymes did not change in 32D hematopoietic cells transformed by the same activated erbB oncogene and in those ‐ normal or transformed over‐expressing the PTPase: in these cells also, over‐expression of the PTPase gene was not accompanied by growth inhibition.