Glutathione status of rat thymocytes and splenocytes during the early events of their ConA proliferative responses

Glutathione plays an important role in the lymphocyte mitogenic response. We have demonstrated that 2-ME increases the ConA proliferative response of rat splenocytes and in parallel, causes an enhancement of glutathione synthesis in these cells. On the other hand, 2-ME had the same action on the glutathione level of thymocytes during the late phase of their mitogenic response, but it had no effect on the [3H]thymidine uptake of these cells. To clarify this discrepancy and the role of glutathione during the mitogenic response, we studied the glutathione status of thymus cells during the early phase of the ConA-induced proliferative response in the presence or the absence of 2-ME in parallel with that of whole spleen cells and the T cell fraction of splenocytes. During the early events of the mitogenic response, i.e., during the 24th h, we observed a normal 2 GSSG/GSH + 2 GSSG ratio in cultured cells, indicating a normal redox state, and that ConA involved an increased glutathione level in thymocytes but not in whole splenocytes and in splenic T cells. 2-ME had no effect on the glutathione level of stimulated thymocytes during the early phase of the mitogenic response. This phenomenon could be related to an absence of its effect on [3H]thymidine uptake. On the other hand, 2-ME induced an enhancement of the glutathione level and [3H]thymidine uptake in the two types of stimulated splenocytes. This study suggest that thymocytes do not have the same mechanism of glutathione synthesis induction as that which occurs in splenocytes during the ConA proliferative response. This mechanism could be related to the maturation state of the T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological oxygen tension modulates the chemically induced mitogenic response of cultured rat hepatocytes

Freshly isolated rat hepatocytes were cultured at periportal- (13% O₂) or perivenous-like (4% O₂) oxygen tension and exposed to subtoxic exposure levels of cyproterone acetate (CPA: 10–330 μM), phenobarbital (PB: 0.75-6 mM), and dimethylsulfoxide (DMSO: 0.1–3.3%) from 24–72 h after seeding. Induced alterations in ploidy, in the number of S-phase cells, the degree of binuclearity, and cellular protein content were determined by twin parameter protein/DNA flow cytometry analysis of intact cells and isolated nuclei. CPA and PB increased whereas DMSO decreased dose dependently the total number of S-phase cells. The changes differed within individual ploidy classes and were modulated by the oxygen tension. CPA increased and DMSO decreased the number of S-phase cells preferentially among the diploid hepatocytes at periportal-like oxygen tension. In contrast, PB increased binuclearity and S-phase cells mainly among the tetraploid hepatocytes at perivenous-like oxygen tension. Cellular protein content increased dose dependently after exposure to the hepatomitogens (CPA, PB) and decreased after exposure to DMSO at both oxygen tensions. Comparison with in vitro data proves that chemicals which interact with cells from the progenitor liver compartment (CPA, DMSO) exert their mitogenic activity best in cultures at periportal-like oxygen tension preferentially in diploid hepatocytes, whereas chemicals which affect cells from the functional compartment show a higher activity at perivenous-like oxygen tension. Physiological oxygen tension seems to be an effective modulator of the proliferative response of cultured rat hepatocytes similar to that expected for periportally or perivenously derived hepatocytes. © 1993 Wiley-Liss, Inc.     

Glutathione influences the proliferation as well as the extent of mitochondrial activation in rat splenocytes.

The time-dependent changes of mitochondrial membrane potential and mass have been investigated on rat splenic lymphocytes stimulated with Con A in the presence and absence of reduced glutathione (GSH). Rhodamine-123 (Rh-123) and nonyl acridine orange (NAO) were used as specific dyes to monitor the membrane potential and mass of mitochondria, respectively. The percentage of cells showing blast transformation and the level of Rh-123 or NAO uptake were analyzed by flow cytometry. Present results demonstrate that a large number of cells showed activated mitochondria already at 24 hr after Con A stimulation and the activation of these organelles was not related to blast transformation. The addition of GSH into the culture medium increased the number of cells responding to mitogenic stimulation. In parallel it augmented the percentage of lymphocytes with activated mitochondria and also prevented their depolarization.     

Limits to the early increase in free cytoplasmic calcium concentration during the mitogenic stimulation of lymphocytes.

Three aspects of the calcium hypothesis we have proposed previously [Metcalfe, Pozzan, Smith & Hesketh (1980) Biochem. Soc. Symp. 45, 1-26] for the control of mitogenic stimulation of lymphocytes are examined in studies on the mitogenic action of the Ca2+ ionophore A23187 and its effect on cap formation. (1) Pig lymphocytes that were mitogenically stimulated by continuous incubation with 3H-labelled A23187 for 48 h contained between 3 and 15 amol of ionophore per cell. Lymphocytes exposed to 3H-labelled A23187 for 2h before washing the cells and resuspending them in ionophore-free medium were only stimulated mitogenically at 48h if the residual ionophore associated with the cells after washing was in the concentration range 3-15 amol per cell. When the cells were washed repeatedly after 2h incubation with ionophore to reduce the cell-associated ionophore below the critical concentration range, no mitogenic stimulation occurred as a result of short-term exposure to any ionophore concentration. Re-addition of ionophore to within the indicated range of cell-associated concentrations restored mitogenic stimulation at 48h. We conclude that large, short-term Ca2+ fluxes into the cells induced by the ionophore cannot generate a mitogenic signal that commits the cells to enter the cell cycle. (2) Further experiments with the ionophore showed that detectable mitogenic stimulation at 48h required a minimum of 3h exposure to optimal ionophore concentrations, and that maximal stimulation required at least 20h exposure. This is consistent with the view that a prolonged increase in the free cytoplasmic calcium concentration is required to stimulate the maximum proportion of the cells into the cell cycle. (3) Mouse splenic lymphocytes treated for short periods with very high ionophore concentrations (30 microM) in the presence of various external Ca2+ concentrations showed significant inhibition of cap formation of surface immunoglobulin receptors in the range 1-10 microM-Ca2+ in normal or depolarizing medium. We conclude that mitogens at optimal concentrations for the stimulation of lymphocytes do not cause any early increase in the free cytoplasmic Ca2+ concentration above 10 microM.     

Influence of insulin status on extra-mitochondrial oxygen metabolism in the rat.

The effects of alloxan diabetes and subsequent treatment with insulin on extra-mitochondrial oxygen metabolism in terms of D-amino acid oxidase (DAAO), xanthine oxidase and catalase were examined. The DAAO activity in the liver with D-alanine and D-serine decreased by 33-62% in the diabetic group while the decrease in the kidneys was 61-74%. Insulin treatment resulted in overstimulation of DAAO activity in the liver but not in the kidneys. Tissue glycogen content was lowered in the diabetic animals but was restored by insulin treatment. Tissue glycogen content and DAAO activity showed an inverse relationship. The xanthine oxidase activity in the two tissues decreased from 40-55%; the catalase activity decreased from 34-54%. Insulin treatment was unable to restore the xanthine oxidase and catalase activities in both the tissues.     

Effects of oxygen concentration during incubation and broiler breeder age on embryonic heat production,chicken development,and 7-day performance

Older breeder flocks produce eggs with a relatively larger yolk and thereby a higher nutrient availability than young breeder flocks. To optimise nutrient utilisation and embryonic development throughout incubation and posthatch period, embryos originating from older breeder flocks may require a higher oxygen availability. The current study investigated effects of broiler breeder flock age and incubational oxygen concentration on embryonic metabolism and chicken development until 7-day posthatch. Similar sized eggs of a young (28–32 week) or old (55–59 week) Cobb 500 breeder flock were incubated at one of three oxygen concentrations (17%, 21% or 25%) from day 7 of incubation until 6 h after emergence from the eggshell. Posthatch, chickens were reared until 7 days of age. Egg composition at the start of incubation, heat production during incubation, and embryo or chicken development at embryonic day (ED)14 and ED18 of incubation, 6 h after hatch and day 7 posthatch were evaluated. An interaction was found between breeder age and oxygen concentration for yolk-free body mass (YFBM) at ED18. A higher oxygen concentration increased YFBM in the old breeder flock, whereas no difference was found between 21 and 25% oxygen in the young breeder flock. Yolk size was larger in the old compared to the young flock from ED0 until 6 h after hatch. Breeder flock age did not affect YFBM at ED14 and 6 h after hatch nor daily embryonic heat production, but there were some effects on relative organ weights. Chickens of the old compared to the young breeder flock showed a higher weight gain at day 7, but at a similar feed conversion ratio (FCR). A higher oxygen concentration during incubation stimulated embryonic development, especially between 17% and 21% of oxygen, in both flock ages_. Although this growth advantage disappeared at 7 days posthatch, a low oxygen concentration during incubation resulted in a higher FCR at 7 days posthatch. Results indicated that breeder flock age seemed to influence body development, with an advantage for the older breeder flock during the posthatch period. Oxygen concentrations during incubation affected body development during incubation and FCR in the first 7 days posthatch. Although an interaction was found between breeder flock age and oxygen concentration at ED18 of incubation, there was no strong evidence that nutrient availability at the start of incubation (represented by breeder flock ages) affected embryo and chicken development at a higher oxygen concentration.     

Secreted proteins induced by epidermal growth factor and transforming growth factor beta in EL2 rat fibroblasts. Role in the mitogenic response

Most growth active hormones and peptides are mitogenic only in the presence of other growth factors [e.g., Platelet Derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF) in "competence-progression" fibroblast model]. We have previously described that EGF alone is able to induce the signals which appear necessary for the mitogenic stimulation of EL2 rat embryo fibroblast line. Recently, we have demonstrated that Transforming Growth Factor beta (TGF beta) slightly stimulates the mitogenic response in EL2 cells. Here, we show that in EGF-treated EL2 cells the induction of at least four inducible-secreted proteins (ISPs, range from 29,000 to 68,000 Mr) is accompanied by a marked increase in DNA synthesis. In contrast, TGF beta or different concentrations of EGF induce a slow increase of the ISPs proportional to slow induction in DNA synthesis. Our results suggest that the mitogenic response in EL2 cell line may be connected with the qualitative and quantitative induction of these secreted proteins.     

Effects of hyperbaric pressure on oxygen concentration in the swim bladder of Poecilia latipinna.

Poecilia latipinna was subjected to varying hydrostatic pressures in the laboratory which demonstrated the following points. The hyperbaric chamber is a suitable apparatus for testing gas gland secretion in this teleostean fish. Oxygen concentration within the swim bladder increased with increasing hydrostatic pressure. Pressures encountered by fishes at various levels of the water column and their effects on oxygen concentration in the swim bladder may be simulated in the laboratory.     

Effects of single-dose external gamma irradiation on rat thyroid status as observed during the year

We studied the rat thyroid status depending on the dose of external radiation and the time passed after the exposure. The experiments were carried out on female albino Wistar rats. The doses absorbed amounted to 0.25; 0.5; 1.0; 2.0 and 5.0 Gy. The animals were decapitated after 3, 6, 24 hours and 7, 30, 180 and 365 days following the radiation. The blood serum was assayed for the contents of thyroxin (T4) and triiodothyronine (T3) using a radioimmunological technique. The liver tissue was assayed spectrophotometrically for the activity of thyroid-induced NADP malate dehydrogenase (NADP-MDH). No changes were found in the blood thyroid hormone contents within short periods after the radiation effect. After 6 hours the T4 levels was 1.2-1.3-fold decreased in the blood of rats receiving the radiation doses of 1.0; 2.0; and 5.0 Gy. After a day the T4 concentration was diminished by 1.21-193-fold in all the experimental animals independently of the radiation dose and that of T3--in 2.0 Gy--and 5.0 Gy--irradiated group. After 7 days following the radiation the T4 and T3 contents remained to be decreased by 1.37-1.75 fold and those of NADP-MDH--by 1.3-1.8-fold in all the animal groups. In a month, the low dose-treated animals (0.25, 0.5, 1.0 Gy) showed the level of thyroid hormones reduced to the control values, whereas the 2.0 and 5.0 Gy--treated rats demonstrated this reduction only by 6 months. The decreased concentration of blood thyroid hormones was due not to the activation of their peripheral metabolism, but, probably, to inhibition of their biosynthesis in thyroid cells under conditions of radiation-induced activation of oxidative stress.     

Effect of 2% dimethyl sulfoxide on the mitogenic properties of epidermal growth factor and hepatocyte growth factor in primary hepatocyte culture.

Two percent dimethyl sulfoxide (DMSO) reversibly inhibited DNA synthesis in primary rat hepatocyte cultures maintained with epidermal growth factor (EGF) or hepatocyte growth factor (HGF). These data suggest that, in vitro, DMSO is a non-specific inhibitor of hepatocyte proliferation, regardless of the stimulating mitogen. In addition, removal of DMSO from mitogen-free cultures resulted in an increase in DNA synthesis. Protein synthesis gradually but irreversibly declined in all cultures after DMSO removal. The relevance of these findings to regulation of hepatocyte growth is discussed.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.     

Prostaglandin E2 increases the calcium concentration in rat brown adipocytes and their consumption of oxygen

Effects of prostaglandin E₂ (PGE₁) were examined on the oxygen consumption and intracellular calcium concentration of rat brown adipose tissue (BAT). PGE₂ 0.1 nM-1 μM increased oxygen consumption of the tissue blocks of BAT, with a maximum 2–13 min after PGE2 administration. PGE₂ was most effective at 1 and 10 nM, and the oxygen consumption was elevated for over 40 min. Pretreatment of BAT with indomethacin, a prostaglandin synthesis inhibitor, did not affect the increase in oxygen consumption induced by noradrenaline. PGE₂ at 1–10 nM gradually increased the intracellular calcium concentration of freshly dispersed single brown adipocytes by 3–4 times in 30 min. PGE₂ also increased the intracellular calcium concentration of brown adipocytes in calcium-free medium. These results raise the possibility that PGE₂ and noradrenaline affect heat genesis and metabolism of BAT independently.     

Nitrogen fixation and respiration by root nodules ofAlnus rubra Bong.: Effects of temperature and oxygen concentration

Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO₂=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1     

Hepatic response to the oxidative stress induced by E. coli endotoxin: Glutathione as an index of the acute phase during the endotoxic shock

Reactive oxygen species are important mediators of cellular damage during endotoxic shock. In order to investigate the hepatic response to the oxidative stress induced by endotoxin, hepatic and plasma glutathione (total, GSH and GSSG), GSSG/GSH ratio as well as Mn-superoxide dismutase and catalase activities were determined during the acute and recovery phases of reversible endotoxic shock in the rat. A significant increase in liver and plasma total glutathione content was observed 5 h after endotoxin treatment (acute phase), followed by a diminution of these parameters below control values at 48 h (recovery phase). The significant increases of GSSG levels and GSSG/GSH ratio are indicative of oxidative stress occurring during the acute phase. Liver Mn-SOD activity showed a similar time dependency as the GSSG/GSH ratio; however, a marked decrease in the liver catalase activity was observed during the process. These results indicate the participation of liver glutathione in the response to endotoxin and the possible use of plasma glutathione levels and GSSG/GSH ratio as indicators of the acute phase during the endotoxic process. (Mol Cell Biochem 159: 115-121, 1996)