Biomedical subjects

L E Nagy

Publications and source records attributed to L E Nagy.

31 records · Page 2Linked to original sources

PubMed1994

Regulation of hepatocyte glutathione by amino acid precursors and cAMP in protein-energy malnourished rats.

Hepatic glutathione concentration is decreased in protein-energy malnutrition. Malnourished rats are able to replenish hepatic glutathione after oral supplementation with L-2-oxothiazolidine-4-carboxylate, a cysteine pro-drug, to levels that are higher than in control rats. These results suggest that, even if a normal amount of amino acids for glutathione synthesis is provided, homeostatic control of glutathione concentration after protein-energy malnutrition is abnormal. The rate limiting enzyme for glutathione synthesis, gamma-glutamylcysteine synthetase, is subject to both short and long term hormonal control. Therefore, we used hepatocytes isolated from weanling rats fed a very low protein diet (0.5 g protein/100 g diet) or a diet adequate in protein for 2 wk to investigate whether a loss of hormonal control could contribute to abnormal regulation of hepatic glutathione. Glutathione concentration in hepatocytes isolated from protein-energy malnourished rats was 82% lower than in controls. In vitro supplementation of isolated hepatocytes with oxothiazolidine-4-carboxylate or methionine increased glutathione concentration in hepatocytes from malnourished rats to concentrations equivalent to control cells. However, when hepatocytes were incubated with cysteine, total glutathione in malnourished rats exceeded that of controls. Treatment of cells from control rats with 50 nmol/L glucagon or 1 mmol/L db-cAMP decreased glutathione concentration by 25-43%. In contrast, the glutathione concentration in hepatocytes of rats fed the low protein diet did not respond to treatment with glucagon or db-cAMP. These data indicate that glutathione synthesis is insensitive to regulation by cAMP in rats with protein-energy malnutrition.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1993

Effects of ethanol-induced changes in adenosine transport on cAMP production in rat hepatocytes.

Ethanol-induced increases in extracellular adenosine in primary cultures of rat hepatocytes are due to inhibition of adenosine uptake via the nucleoside transporter, as well as increased adenosine production resulting from ethanol metabolism. Chronic treatment of hepatocytes with ethanol during culture increases receptor-stimulated cAMP production. This increase is due, at least in part, to a decrease in Gi mediated inhibition of cAMP production and can be prevented by co-culture with the adenosine A1 receptor antagonist, cyclopentyltheophylline. These data suggest that increased extracellular adenosine, acting via adenosine A1 receptors, mediates the chronic effects of ethanol on cAMP in hepatocytes.

Adenosine↗

PubMed1992

Ethanol increases receptor-dependent cyclic AMP production in cultured hepatocytes by decreasing G(i)-mediated inhibition.

Increasing evidence suggests that ethanol-induced changes in cyclic AMP (cAMP) signal transduction play a critical role in the acute and chronic effects of ethanol. Here we have investigated the effects of ethanol on cAMP signal transduction in primary cultures of rat hepatocytes. Acute exposure to ethanol had a biphasic effect on glucagon-receptor-dependent cAMP production in intact cells: 25-50 mM-ethanol decreased cAMP, whereas treatment with 100-200 mM-ethanol increased cAMP. After chronic exposure to 50-200 mM-ethanol for 48 h in culture, glucagon-receptor-dependent cAMP levels were increased, but no change in glucagon receptor number was observed. These effects of ethanol were independent of ethanol oxidation. Chronic ethanol treatment also increased adenosine-receptor- and forskolin-stimulated cAMP production. Increased cAMP production was also observed upon stimulation of adenylate cyclase with glucagon, forskolin and F- in membranes isolated from cells cultured with 100 mM-ethanol for 48 h. However, no differences were observed in basal and MnCl2-stimulated adenylate cyclase activity. The quantity of alpha i protein was decreased by 35% after chronic ethanol treatment, but no change in the quantity of alpha s protein was detected. Decreased alpha i protein was associated with a decrease in G(i) function, as assessed by the ability of 0.1 nM-guanosine 5'-[beta gamma-imido]triphosphate and 1 microM-somatostatin to inhibit forskolin-stimulated adenylate cyclase activity. Taken together, these results suggest that chronic exposure to ethanol increases receptor-dependent cAMP production in hepatocytes by decreasing the quantity of alpha i protein at the plasma membrane and thereby decreasing the inhibitory effects of G(i) on adenylate cyclase activity.

Alcohol Dehydrogenase↗

PubMed1992

Ethanol metabolism and inhibition of nucleoside uptake lead to increased extracellular adenosine in hepatocytes.

Recent evidence suggests that adenosine mediates many of the acute and chronic effects of ethanol in both cultured cells and whole animals. These adenosine-mediated effects of ethanol result from ethanol-induced increases in extracellular adenosine. Acute exposure of primary cultures of rat hepatocytes to 12.5-200 mM ethanol increased extracellular adenosine concentrations by 20-35%. Pretreatment of hepatocytes with 100 microM 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, completely blocked ethanol-induced increases in extracellular adenosine at 12.5 and 25 mM ethanol. However, even in the presence of 4-methylpyrazole, ethanol at concentrations greater than 50 mM still increased extracellular adenosine concentrations. This increase appears to be due to ethanol inhibition of adenosine uptake via the nucleoside transporter (50% inhibitory concentration, 28 mM). After chronic treatment with 100 mM ethanol for 48 h, acute challenge with ethanol no longer inhibited adenosine uptake, i.e., the nucleoside transporter had become tolerant to ethanol. Moreover, in these chronically treated cells, ethanol-induced increases in extracellular adenosine were completely blocked by treatment with 4-methylpyrazole at all concentrations of ethanol. Taken together, these results suggest that increased extracellular adenosine in hepatocytes is dependent on both ethanol oxidation and inhibition of adenosine uptake via the nucleoside transporter.

Adenosine↗

PubMed1991

cAMP-dependent protein kinase regulates inhibition of adenosine transport by ethanol.

Ethanol inhibits adenosine uptake, thereby increasing the concentration of extracellular adenosine. Elevation of extracellular adenosine increases intracellular cAMP concentration via activation of adenosine A2 receptors. Extracellular adenosine is also required for the subsequent development of ethanol-induced heterologous desensitization. Here we report that activation of cAMP-dependent protein kinase is necessary for inhibition of adenosine uptake by ethanol and for the consequent accumulation of extracellular adenosine. Ethanol does not inhibit adenosine uptake in mutants of the S49 cell line that lack receptor-stimulated cAMP production (unc cells) or cAMP-dependent protein kinase activity (kin- cells). Forskolin, which bypasses the receptor-coupling defect in unc cells to increase cAMP levels, restores inhibition of adenosine uptake by ethanol. In contrast, in kin- cells forskolin did not restore inhibition of adenosine uptake by ethanol, despite similar increases in cAMP levels. Taken together, these results suggest that cAMP-dependent protein kinase phosphorylates a component of the nucleoside transporter, thereby regulating the sensitivity of adenosine transport to ethanol.

Adenosine↗

PubMed1990

Ethanol increases extracellular adenosine by inhibiting adenosine uptake via the nucleoside transporter.

Chronic exposure to ethanol results in heterologous desensitization of receptors coupled to adenylyl cyclase via Gs, the stimulatory guanine nucleotide regulatory protein. Ethanol-induced accumulation of extracellular adenosine is required for the development of heterologous desensitization (Nagy, L. E., Diamond, I., Collier, K., Lopez, L., Ullman, B., and Gordon, A. S., Mol. Pharmacol., in press). To understand the mechanism underlying ethanol-induced increases in extracellular adenosine, we examined the interaction of ethanol with the adenosine transport system in S49 lymphoma cells. We found that ethanol inhibited nucleoside uptake without affecting deoxyglucose or isoleucine transport. Inhibition of adenosine uptake was due to decreased influx via the nucleoside transporter. Thus, ethanol-induced increases in extracellular adenosine appear to be due to inhibition of adenosine influx. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol, i.e. ethanol no longer inhibited uptake. Consequently, ethanol no longer increased extracellular adenosine concentrations. Taken together with our previous studies, these results suggest that ethanol inhibition of adenosine influx leads to an increase in extracellular adenosine which causes an initial increase in intracellular cAMP levels and subsequent development of heterologous desensitization of cAMP signal transduction.

Adenosine↗

PubMed1989

Adenosine is required for ethanol-induced heterologous desensitization.

Recent evidence suggests that ethanol initially causes an increase in receptor-dependent cAMP levels, followed by heterologous desensitization of receptors coupled to GS after chronic exposure. Here we investigated the role of adenosine in mediating these responses. We found that ethanol caused accumulation of extracellular adenosine in NG108-15 and S49 lymphoma cells. This adenosine activated adenosine receptors to increase intracellular cAMP levels. The addition of adenosine deaminase, to degrade accumulated extracellular adenosine, or isobutyl-methylxanthine, an adenosine receptor antagonist, completely blocked ethanol-induced increases in cAMP levels in NG108-15 cells. Chronic exposure of NG108-15 and S49 wild type cells to ethanol resulted in heterologous desensitization of adenosine receptor- and prostaglandin E1 receptor-dependent cAMP signal transduction. Coincubation of NG108-15 and S49 wild type cells with adenosine deaminase and ethanol for 48 hr prevented heterologous desensitization. Moreover, mutant S49 cells, which are unable to transport adenosine, did not accumulate extracellular adenosine after incubation with ethanol and did not develop ethanol-induced heterologous desensitization. Our results suggest that adenosine is an important mediator of both the acute and chronic effects of ethanol on cAMP signal transduction.

Adenosine↗

PubMed1988

Cultured lymphocytes from alcoholic subjects have altered cAMP signal transduction.

Previous work has shown that freshly isolated lymphocytes from alcoholic subjects show significantly reduced basal and adenosine receptor-stimulated cAMP levels. This decrease could be due to ethanol-induced cellular adaptation or to a genetic difference in the regulation of cAMP signal transduction. Therefore, we cultured human lymphocytes in defined medium without ethanol for 7-8 days and then examined differences in receptor-dependent cAMP accumulation between lymphocytes from alcoholic and nonalcoholic subjects. After four to six generations in culture without ethanol, lymphocytes from alcoholic subjects have significantly higher cAMP levels than do cells from nonalcoholic subjects. Thus, a difference in cAMP signal transduction is demonstrable in cells from alcoholic subjects grown without ethanol. We also found that cultured lymphocytes from both alcoholic and nonalcoholic subjects show a decrease in receptor-stimulated cAMP levels after exposure to 200 mM ethanol for 48 hr. To determine whether alcoholic subjects have increased sensitivity to ethanol, lymphocytes were exposed to only 100 mM ethanol for 24 hr. Under these conditions, receptor-dependent cAMP levels did not change in cells from nonalcoholic subjects. However, lymphocytes from alcoholic subjects showed a 39% decrease (P less than 0.003) in adenosine receptor-stimulated cAMP levels. Taken together, the results show that (i) chronic ethanol treatment in culture reproduces the suppression of cAMP levels found in circulating lymphocytes from alcoholic subjects and (ii) despite four to six cell divisions in culture without ethanol, lymphocytes from alcoholic subjects exhibit significantly increased adenosine receptor-dependent cAMP levels and increased sensitivity to chronic exposure to ethanol. These findings suggest that the suppression of cAMP levels observed in freshly isolated lymphocytes from alcoholic subjects results from both a direct effect of chronic exposure to ethanol and a genetic difference leading to altered cAMP signal transduction.

Adenosine Deaminase↗

PubMed1988

Utilization of glutamine in the developing rat jejunum.

The activity of glutaminase was measured in the jejunum of rats during the suckling period. Activity increased significantly (P less than 0.05) from 2.37 +/- 0.31 mol glutamate/(mg jejunal protein.h) (X +/- SD) in the first week to 3.50 +/- 0.99 in the second week and 4.75 +/- 0.96 during the third week. The quantity of the glutaminase protein, measured with a dot immunobinding assay, remained constant during the first (592 +/- 174 cpm bound/g protein) and second (599 +/- 125) weeks and then increased significantly by the third week (784 +/- 270) after birth. These results indicate that the activity of glutaminase is regulated by alterations in both the quantity and activity of glutaminase protein. The oxidation of [U-14C]glutamine to 14CO2 in vitro also increased during the suckling period, with significantly (P less than 0.05) higher rates of oxidation observed by the third week after birth. The capacity of the developing rat jejunum to utilize both glutamine and -hydroxybutyrate (BHB) as fuel sources to support [methyl-3H]thymidine (3HTdR) incorporation was also determined. Addition of glutamine to jejunal homogenates in vitro resulted in a significantly (P greater than 0.05) higher rate of 3HTdR incorporation than was observed with either glucose or BHB as a fuel source during the early suckling period. In the late suckling period, the addition of BHB and glutamine together resulted in significantly (P greater than 0.05) higher rates of 3HTdR incorporation than that found with glucose as a fuel source. These data suggest that both glutamine and BHB are important fuel sources in the jejunum during the suckling period.

Animals↗

PubMed1986

Effect of diabetic ketosis on jejunal glutaminase.

The intestine is capable of shifting its major fuel source from glutamine in the fed animal to ketone bodies in the fasted animal. Glutaminase (EC 3.5.1.2), the entry enzyme of glutamine oxidation, was examined for its function as a determinant in the utilization of jejunal fuel during diabetes and fasting. Male Sprague-Dawley rats were made ketotic to varied degrees by either fasting or the induction of diabetes with graded doses of streptozotocin (SZ). Specific activity of glutaminase was decreased in the diabetic animals to 64% (p less than 0.05) of controls in the group receiving 110 mg/kg SZ and 82% of controls in the group receiving 65 mg/kg SZ and to 78% (p less than 0.05) of controls in the fasted animals. The activity of glutaminase in the small intestine was negatively correlated to the concentration of beta-hydroxybutyrate in the plasma (r = -0.97, p less than 0.025) and jejunum (r = -0.92, p less than 0.05) for the four groups of animals. Specific activity of glutaminase was decreased in all cell types isolated along the villus-crypt axis of the small intestine from diabetic and fasted rats compared with control rats. The quantity of glutaminase-protein was determined by a dot immunobinding assay using an antibody to purified glutaminase. The activity of glutaminase relative to immunoreactive glutaminase-protein was significantly decreased (p less than 0.05) to 53% of control values in the 110 mg/kg SZ group, 77% in the 65 mg/kg SZ group, and 70% in the fasted group. These data indicate that an inactivation of glutaminase-protein may play a role in the ability of the intestine to shift its fuel source from glutamine to ketone bodies during diabetes and fasting.

3-Hydroxybutyric Acid↗

PubMed1984

Postprandial energy expenditure and respiratory quotient during early and late pregnancy.

Rates of energy expenditure after a 750-kcal meal were determined by open circuit, indirect calorimetry for four women in late pregnancy (30 to 40 wk gestation), six women in early pregnancy (10 to 20 wk gestation), and six nonpregnant women. Preprandial resting metabolic rates, expressed in kcal min-1, were 22.5% higher (p less than 0.05) in the late pregnancy group compared to the early pregnancy, and 15.9% higher (p less than 0.05) in the early pregnancy compared to the nonpregnant women. No differences in preprandial energy expenditure rates were seen between groups when expressed as kcal kg-1 h-1. Rates of energy expenditure increased above preprandial levels in all groups by 15 min postprandially and remained significantly elevated for the next 175 min. The total increase in postprandial energy expenditure above preprandial levels did not differ significantly due to stage of gestation. Respiratory quotient, the ratio of VCO2:VO2, increased significantly in all groups (p less than 0.05) above preprandial levels by 15 min after the meal. Respiratory quotient values began decreasing after 95 min and returned, in all groups, to preprandial levels by 175 min. The results from this study demonstrate that the increase in the rates of energy expenditure after a mixed meal was not altered by gestation.

Adult↗

PubMed1983

Energy expenditure of pregnant women at rest or walking self-paced.

Energy expenditure during rest and self-paced walking was determined from early to late pregnancy either longitudinally or in a cross-section of women. The cross-sectional study was done with 16 women confined to a metabolic unit: six nonpregnant (NP), six early pregnant (EP 10 to 20 wk gestation), and four late pregnant (LP 30 to 40 wk gestation). In the longitudinal study, five of the six EP subjects from the cross-sectional study were studied at 5-wk intervals until parturition. Basal metabolic rate, measured by open circuit, indirect calorimetry, and expressed as kcal/min, was 13% greater (p less than 0.05) in EP compared to NP and was 28% greater (p less than 0.05) in LP compared to EP. Resting metabolism increased during gestation in the EP group from a value of 1.01 kcal/min at 15 to 25 wk to 1.15 kcal/min at 35 to 40 wk. When energy expenditure during rest is expressed as kcal/kg body weight/h, there were no significant differences due to stage of pregnancy. The time required for the women to walk 400 m at their own pace was measured. The pace of the LP women was 20% slower (p less than 0.05) than the EP women. But when the EP women were studied at 35 and 40 wk gestation their pace was only 4.5% slower than that at 15 to 25 wk. These data suggest that individual behavioral differences have a greater effect on pace than stage of gestation. A decrease in pace reduced the rate of energy expenditure per kilogram body weight for walking 400 m. But, body weight, rather than pace, was the major determinant of total energy expenditure for the walk (p less than 0.05). It is apparent from these data that body weight is the major determinant of energy expenditure during rest and self-paced weight bearing activity in pregnancy.

Adult↗

PubMedDate not supplied

Development of glutaminase along the villus-crypt axis in the jejunum of rat.

The activity of glutaminase (E.C. 3.5.1.2), the entry enzyme for oxidation of glutamine, was measured in enterocytes isolated along the villus-crypt axis from rat jejunum. Specific activity of glutaminase was 5.05 +/- 0.24 mumol glutamate/mg protein/h in villus cells (fully differentiated cells) and 4.16 +/- 0.30 in the deep crypt (undifferentiated cells). Activity of glutaminase was significantly (p less than 0.05) increased in cells isolated from the villus-crypt junction (differentiating cells) compared to the activity of the enzyme in both the villus and crypt at 6.21 +/- 0.45. A similar pattern of activity of glutaminase was observed when the cells of the villus-crypt gradient were separated by sequential horizontal sectioning with a cryostat. Oxidation of L-[U-14C]glutamine to 14CO2 was also significantly (p less than 0.01) higher in cells isolated from the villus-crypt junction compared to both villus or deep crypt cells. The quantity of glutaminase protein was determined by a dot immunobinding assay using an antibody to purified glutaminase. Immunoreactive glutaminase protein relative to total cellular protein was 6.06 +/- 0.40 cpm/microgram homogenate protein in the villus cells, 3.01 +/- 0.24 (p less than 0.05) at the villus-crypt junction, and 4.49 +/- 0.57 (p less than 0.05) in the deep crypt. Thus, the highest activity of glutaminase present in the villus-crypt junction is the result of an increase in activity of the enzyme rather than an increase in the enzyme protein.

Animals↗