Glutathione and glutathione conjugate efflux from cultured liver cells

Efflux of glutathione (GSH) and GSH-conjugates from cultured rat liver epithelial cell lines; the non-tumorigenic ARL-15C₁ and the -glutamyl transpeptidase containing, tumorigenic ARL-16T₂, has been assessed under basal condition and during chronic treatment with 75 and 150 M ethacrynic acid (EA). The intracellular level of GSH increased in proportion to EA concentration during chronic exposure. The rates of GSH and GSH-EA conjugate efflux increased with intracellular GSH in both ARL cell lines.Glutathione-S-transferase activity measured with EA as substrate increased over the experimental time course after treatment with 150, but not 75 M EA. When intracellular GSH content was increased by treatment with the cysteine pro-drug, 2-L-oxothiazolidine 4-carboxylic acid, the rate of GSH efflux was increased, but not the rate of GS-EA conjugate export. Inhibition of -glutamyl transpeptidase by acivicin (AT-125) increased the GSH and GS-EA conjugate efflux rate in ARL-16T₂ cells by factors of approximately 2 and 15, respectively. Acivicin treatment of ARL-16T₂ cells chronically treated with EA elevated GSH efflux rate by 10-fold and GS-EA efflux by 40-fold versus control samples. These studies show that GSH and GSH conjugate efflux are accomplished as independently regulated processes. Efflux of GSH is enhanced by increased in racellular GSH, but increase in the conjugate transport rate requires the presence of the GSH conjugate. The response of the efflux process to treatment with a chronic GSH depleting agent was identical in two cell lines in which the metabolic fate of glutathione is known to differ fundamentally.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - GS-EA the glutathione conjugate of ethacrynic acid - EA ethacrynic acid - CDNB 1-chloro 2,4-dinitrobenzene - HBS HEPES buffered saline - OTC 2-L-oxothiazolidine 4-carboxylic acid - CYSSG cysteinyl-glutathione mixed disulfide - FDNB 1-fluoro-2,4-dinitrobenzene - GCS -glutamyl cysteine synthetase - GST glutathione-S-transferase - BCA bicinchoninic acid - SDS sodium dodecyl sulfate - PCA perchloric acid     

Rat hepatocytes prepared without collagenase: Prolonged retention of differentiated characteristics in culture

Rat hepatocytes prepared by collagenase digestion or ED TA dissociation were examined in culture for comparison of culture stability and morphology, and retention of selected adult rat liver characteristics. Cells prepared by EDTA perfusion followed by Percoll cen trifugation were deemed to form confluent monolayer cultures more rapidly and monolayers remained intact for up to 21 days without signs of nonparenchymal cell growth or loss of primary hepatocyte appearance. The spectrally determined cytochrome P-450 content remained constant through eight days in culture. Collagenase prepared cells contained an identical amount of P-450 but within 72 hr lost greater than 80% of the spectrally detectable P-450. Glutathione (GSH) content was higher in the EDTA-prepared hepatocytes and remained constant with only a modest effect of transferrin and selenium (TI S) supplementation, while GSH levels in collagenaseprepared cells increased, thereafter decreased with time in culture and was dependent on TI S supplementation. Cells prepared with ED TA also displayed an increase in GSH efflux rate in response to chronic GSH depletion by ethacrynic acid. -Cystathionase (CNase) activity was retained at initial levels in ED TAprepared hepatocytes supplemented with TI S and declined only about 25% in unsupplemented cells. Collagenase-prepared cells lost 75% of CNase activity by 72 hr. The established marker of hepatocyte neoplastic transformation, -glutamyl trans-peptidase (GGT), increased rapidly in collagenase-prepared cells. The accumulation of GGT was slowed by T/S supplementation. GGT activity did not increase in EDTA-prepared hepatocytes. Evaluation of morphological and biochemical criteria suggest that hepatocytes prepared without collagenase present superior model systems for the study of biochemical events through more extended culture times.Abbreviations CNase -cystathionase - EDTA ethylenediamine tetraacetic acid - GGT -glutamyl transpeptidase - GSH reduced glutathione - GSSG oxidized glutathione - HEPES N-2-hydroxyethylpiperizine-N-2-ethane-sulfonic acid - SAH S-adenosylhomocysteine - SAM S-adenosylmethionine - T/S transferrin- and selenite-supplemented     

Regional variations in intestinal brush border membrane fluidity and function during diabetes and the role of oxidative stress and non-enzymatic glycation

The physical state (fluidity) of lipids modulates the activities of several membrane bound enzymes and transport proteins. Alteration of brush border membrane (BBM) fluidity is one of the several changes exhibited by the small intestine during diabetes. In the present study, an investigation of the diabetes induced regional changes in fluidity, oxidative damage, non-enzymatic glycation as well as the activities and the kinetic parameters of the enzymes alkaline phosphatase and -glutamyl transpeptidase was carried out on the intestinal BBM. At the end of 6 weeks of diabetes, significant increases in the extent of both oxidative damage and non-enzymatic glycation were observed along the length of the intestine along with a simultaneous decrease in membrane fluidity. A significant correlation between the decrease in BBM fluidity and increase in non-enzymatic glycation was observed in the duodenum and jejunum. Additionally regional variations in the activities and kinetic parameters of both the enzymes were observed.     

Influence of selenium on glutathione and some associated enzymes in rats with mammary tumor induced by 7,12-Dimethylbenz(a)anthracene

A recent finding in epidemiological and laboratory studies suggests that the ratio of selenium to glutathione is lower in breast cancer subjects than its control counterparts. Selenium, an antioxidant and anticarcinogen, can modify the status of glutathione and some associated enzymes by blocking peroxidation of lipids in membranes of cancer subjects. Studies were conducted using female albino rats of Wistar strain bearing mammary tumor induced by 7,12-dimethylbenz(a) anthracene to assess the biological role of selenium on some antioxidant enzymes associated with the maintenance of glutathione status. For induction of mammary tumor, 25 mg DMBA in a 1 ml emulsion of sunflower oil and physiological saline was injected subcutaneously to each rat. One group in each of control and tumor bearing rats, were fed 5 mg sodium selenite/kg diet from the day of tumor induction for 24 weeks. Increase in the reduced glutathione concentration was preceded by significant increase in the oxidized glutathione as well as in the activities of -glutamylcysteine synthetase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and glucose-6-phosphate dehydrogenase by selenium administration in rats bearing tumor. However, selenium administration to rats bearing tumor decreased the activity of -glutamyl transpeptidase. These observations clearly demonstrate the influence of dietary selenium supplementation in correcting abnormal changes in glutathione turnover and some associated enzymes in tumor induced rats.     

Identification of high molecular weight antigens structurally related to gamma-glutamyl transferase in epithelial tissues

Summary Heterologous antibodies to gamma-glutamyl transferase (GT), an ectoenzyme associated with the apical surface of many types of epithelial cells involved in secretion and transport, have been used to identify and partially characterize the spectrum of antigens in a series of epithelial tissues that exhibit a range of enzyme activities. In addition to antigens corresponding to the subunits of the active enzyme (mol wt 55K, 30K), antigens of mol wt 85–95K have been detected using an antibody raised against the enzyme purified in nonionic detergent. The latter species are shown to share antigenic determinants with and to be structurally related to the enzyme subunits; however, they do not bind significantly to antibodies raised to protease-solubilized GT. Further, they constitute the major antigens in tissues that exhibit relatively low levels of enzyme activity. These polypeptides are apparently larger than a recently characterized biosynthetic precursor of the GT subunits. Although they do not have GT activity themselves and their function is undefined, the possibility that they may represent highly glycosylated polypeptides related either to GT precursors (that persist without processing) or to the large enzyme subunit merits consideration.     

Role of Glutathione in the Response of Escherichia coli to Osmotic Stress

The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity. A mutant in -glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain. The unfavorable effect of the gsh mutation on osmoadaptation of growing E. coli cells was more pronounced at low concentrations of K⁺ in the medium. An increase in osmolarity caused an increase in the intracellular content of glutathione. Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage. The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation. Changes in the level of intracellular K⁺ were similarly biphasic: a rapid increase in the K⁺ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage. In mutant gshA cells adapted to osmotic shock, the intracellular K⁺ level was markedly higher than in the parental strain cells. The possible role of glutathione in the response of E. coli to osmotic shock is discussed.     

Influence of sample preparation on cellular glutathione recovery from adherent cells in culture

During the last decade, the unbound glutathione content of cultured adherent cells has become a very important biological marker for many pharmacological and toxicologicalin vitro studies with regard to the protective role of the tripeptide in its reduced form (GSH). However, the literature does not provide extensive information on the influence of sample preparation on cellular GSH and thiol analyses. Using the fibroblast-like V79 cell line as model, we undertook a comparative study of the efficiency of different procedures reported in the literature with respect to GSH recovery. Depending on the preanalytical step, up to 10-fold discrepancies could be observed in the recovery of intracellular GSH. Different parameters that must be controlled in order to maximize GSH recovery are discussed. The optimal strategy consisted in rapid perchloric acid deproteinization performed directly in the dish, which was extremely valuable for preparing GSH samples from adherent cells, and especially from cells expressing elevated -glutamyl transferase activity.Abbreviations EDTA ethylenediaminetetraacetic acid - GGT -glytamyl transferase (EC 2.3.2.2) - GSH reduced glutathione - HPLC high-performance liquid chromatography - PA perchloric acid - PBS Dulbecco's phosphate-buffered saline     

The Activities of Six Exo- and Endopeptidases in the Substantia Nigra,Neostriatum, and Cortex of the Rat Brain

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and -Glutamyl transpeptidase (GTP), and two endopeptidases: Postproline endopeptidase (PEP) and Trypsin-like peptidase (T-L P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) GTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a Trypsin-like peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.     

Aspartate aminotransferase and glutaminase activities in rat olfactory bulb and cochlear nucleus; Comparisons with retina and with concentrations of substrate and product amino acids

The quantitative distributions of aspartate aminotransferase and glutaminase were mapped in subregions of olfactory bulb and cochlear nucleus of rat, and were compared with similar data for retina and with the distributions of their substrate and product amino acids aspartate, glutamate, and glutamine. The distributions of both enzymes paralleled that of aspartate in the olfactory bulb and that of glutamate in the cochlear nucleus. In retina (excluding inner segments), there were similarities between aspartate aminotransferase and both glutamate and aspartate distributions. The distribution of -aminobutyrate (GABA) was similar to those of both enzymes in olfactory bulb, to aspartate aminotransferase in cochlear nucleus, and to glutaminase in retina (excluding inner segments). The results are consistent with significant involvement of aspartate aminotransferase, especially the cytosolic isoenzyme, and glutaminase in accumulation of the neurotransmitter amino acids glutamate, aspartate, and GABA, although with preferential accumulation of different amino acids in different brain regions.     

Regulation of Phospholipase C Activity by Calcium Ions and Guanine Nucleotide in the Normoxic Cat Carotid Body

The carotid bodies (CB) are a paired chemoreceptor organ located at the bifurcation of the common carotid arteries. High O₂ tension suppresses while low tension activates afferent carotid chemoreceptor activity and the chemoreflex ventilatory response in the cat. The intracellular mechanism of chemotransduction is till now unknown. Previously we have shown different activities of phospholipase C (PLC) in normoxic, hypoxic and hyperoxic cat carotid body. Now we have addressed the question whether calcium ions and G-protein could be regulators of the formation of lipid derived messenger molecules in the cat carotid body. To answer this question, the PLC acting against [³H] inositol-phosphatidylinositol (PtdIns) and [³H] inositol-phosphatidylinositol-4, 5-bisphosphate [PtdIns(4,5)P₂] in the cat CB were investigated using labelled phospholipids as a source of the substrate. CB homogenate was used as a source of the enzyme. The results indicate that PLC acting on PtdIns is Ca²⁺-dependent, in contrary to that acting on PtdIns(4,5)P₂ which remains active in the presence of 10 mM EGTA. PtdIns(4,5)P₂-PLC is stimulated by GTPS. In the presence of Ca²⁺, GTPS has a synergistic stimulatory effect. PLC acting on PtdIns is not activated by GTPS. In the presence of calcium ions dopamine and a nonhydrozylable analogue of acetylocholine, carbachol, have a small stimulatory effect of about 30 % on PLC acting on PtdIns(4,5)P₂. GTPS enhances this effect. These results allow us to suggest that there are two pathways of phosphoinositides degradation in the CB, one of them is regulated by calcium ions/PtdIns-PLC/, the other one by G-protein/PtdIns(4,5)P₂-PLC/.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Anaerobic dechlorination and degradation of hexachlorocyclohexane isomers by anaerobic and facultative anaerobic bacteria

Screening studies with strict and facultative anaerobic bacteria showed that Clostridium app. and several other representatives of Bacillaceae and Enterobacteriaceae actively degraded -hexachlorocyclohexane (-HCH) under anaerobic conditions. Representatives of Lactobacillaceae and Propronibacterium were inactive. With ³⁶Cl-labelled -HCH a nearly complete dechlorination was shown to occur in 4–6 days by Clostridium butyricum, C. pasteurianum and Citrobacter freundii, while other facultative anaerobic species were less active.Aerobically grown facultative anaerobes also dechlorinated actively -HCH during subsequent anaerobic incubation with glucose, pyruvate or formate as substrates. The -, - and -HCH isomers were also, but more slowly, dechlorinated (>>-HCH). All species active in anaerobic degradation of -HCH formed -tetrachlorocyclohexene (TCH) as the main intermediate metabolite and no -pentachlorocyclohexene (PCH) or other isomers of TCH or PCH have been found. Small amounts of tri- and tetrachlorinated benzenes have been found too. The mechanism of dechlorination is discussed.Non-Common Abbreviations Used -HCH -hexachlorocyclohexane - -TCH -2,3,4,5-tetrachlorocyclohexene - -PCH -1,2,3,4,5-pentachlorocyclohexene - GLC gas liquid chromatography     

Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 70-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity,and on cell surface protein expression

Summary The cytotoxicity of 7-hydroxycholesterol (7-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 µM 7(3-OHC over a period of 72 h whereas 30 µM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7-OHC added to the cultures at concentrations of 20 µM or 30 µM. Cellular fractionation after incubation with 20 µM or 30 µM 7-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 µM 7-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 µM 7-OHC treatment. Fluorescence anisotropy measurements indicated that 20 µM 7-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 µM 7-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 µM 7-OHC.     

Synergistic effect of BMP-2 and ascorbate on the phenotypic expression of osteoblastic MC3T3-E1 cells

Of TGF- superfamily proteins, BMP-2 enhanced alkaline phosphatase (ALP) activity in cultured osteoblastic cells, MC3T3-E1, to the same level promoted by ascorbate, whereas TGF-s (1, 2, 3) reduced ALP activity and altered cell morphology under the same conditions. Activin appeared to have no distinct effect on ALP synthesis. Ascorbate dependent increase in ALP synthesis was markedly stimulated in the presence of BMP-2. The synergistic effect of ascorbate on ALP synthesis was replaced by type I collagen coated on the culture dish. However, BMP-2 appeared not to bind to type I collagen. These findings indicate that BMP-2 acts directly on osteoblastic cells through its receptors and collagenous matrix can neither recruit BMP-2 nor modulate directly the action of BMP-2 in the pericellular area. Treatment of cells grown in ascorbate media with TGF-s decreased rapidly the cellular ALP activity indicating that TGF-s direct cells to the differentiated stage.     

Immunohistochemical and histochemical evidence for the presence of noradrenaline,serotonin and gamma-aminobutyric acid in chief cells of the mouse carotid body

The immunohistochemical study revealed tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), serotonin, glutamate decarboxylase (GAD) and -aminobutyric acid (GABA) immunoreactivities in the mouse carotid body. TH and DBH immunoreactivities were found in almost all chief cells and a few ganglion cells, and in relatively numerous varicose nerve fibers of the carotid body. The histofluorescence microscopy showed catecholamine fluorescence in almost all chief cells. However, no PNMT immunoreactivity was observed in the carotid body. Serotonin, GAD and GABA immunoreactivities were also seen in almost all chief cells of the carotid body. From combined immunohistochemistry and fluorescence histochemistry, catecholamine and serotonin or catecholamine and GABA were colocalized in almost all chief cells. Thus, these findings suggest that noradrenaline, serotonin and GABA may be synthesized and co-exist in almost all chief cells of the mouse carotid body and may play roles in chemoreceptive functions.     

Nature and regulation of pistil-expressed genes in tomato

The specialized reproductive functions of angiosperm pistils are dependent in part upon the regulated activation of numerous genes expressed predominantly in this organ system. To better understand the nature of these pistil-predominant gene products we have analyzed seven cDNA clones isolated from tomato pistils through differential hybridization screening. Six of the seven cDNAs represent sequences previously undescribed in tomato, each having a unique pistil- and/or floral-predominant expression pattern. The putative protein products encoded by six of the cDNAs have been identified by their similarity to sequences in the database of previously sequenced genes, with a seventh sequence having no significant similarity with any previously reported sequence. Three of the putative proteins appear to be targeted to the endomembrane system and include an endo--1,4-glucanase which is expressed exclusively in pistils at early stages of development, and proteins similar in sequence to -thionin and miraculin which are expressed in immature pistils and stamens, and in either sepals or petals, respectively. Two other clones, similar in sequence to each other, were expressed primarily in immature pistils and stamens and encode distinct proteins with similarity to leucine aminopeptidases. An additional clone, which encodes a protein similar in sequence to the enzyme hyoscyamine 6--hydroxylase and to other members of the family of Fe²⁺/ascorbate-dependent oxidases, was expressed at high levels in pistils, stamens and sepals, and at detectable levels in some vegetative organs. Together, these observations provide new insight into the nature and possible functional roles of genes expressed during reproductive development.     

Polyclonal activation of human lymphocytes and induction of cytotoxic lymphocytes by streptococcal preparations

Polyclonal activation of human peripheral blood lymphocytes (PBLs)in vitro by preparations ofStreptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4⁺ helper type of T cells, and CD4^–CD8^– double-negative (DN) lymphocytes, including both CD3⁺ TcR ⁺ T cells and CD2⁺CD3^– immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3⁺ and CD3^– types of CD4^–CD8^– DN lymphocytes, but not CD4⁺ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4^–CD8 T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLsin vitro to induce the proliferation/activation of CD4⁺ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.     

Immunocytochemistry of GABA and glutamic acid decarboxylase in the thoracic ganglion of the crab Eriphia spinifrons

We have used specific antisera against protein-conjugated -aminobutyric acid (GABA) and rat-brain glutamic acid decarboxylase (GAD) in immunocytochemical preparations to study the distribution of putatively GABAergic neurons in the fused thoracic ganglion of the crab Eriphia spinifrons. In the thoracic neuromeres, about 2000 neurons with somata arranged in clusters or located singly in the cell cortex exhibited both GABA-like and GAD-like immunoreactivity. In addition, more than a hundred cells showed only GABA-like immunoreactivity. Fibrous immunoreactive staining to GAD and GABA was distributed throughout the neuropil of the thoracic ganglion, and several fiber tracts contained immunoreactive processes. Sets of serially homologous neurons exhibited GABA-like and GAD-like immunoreactivity in the thoracic neuromeres. Especially prominent were one medial and four ventro-lateral clusters of somata, together with thirteen individually recognized cells in each neuromere. Six of these cells in the ventro-medial cell cortex may be the somata of inhibitory motoneurons. The leg nerves contained three immunoreactive fibers, corresponding to the previously described common inhibitory motoneuron and the two specific inhibitors. The results present further evidence for GABA being the neurotransmitter of all inhibitory leg motorneurons, and suggest its presence and role as a neurotransmitter in a considerable number of interneurons in the thoracic ganglion of the crab.