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B Stoll

Publications and source records attributed to B Stoll.

At least 55 records · Page 3Linked to original sources
PubMed1994

Effects of aniso-osmolarity and hydroperoxides on intracellular pH in isolated rat hepatocytes as assessed by (2',7')-bis(carboxyethyl)-5(6)-carboxyfluorescein and fluorescein isothiocyanate-dextran fluorescence.

Freshly isolated rat hepatocytes were plated for 4-6 h and either loaded with (2',7)-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran in order to study the effects of aniso-osmotic exposure and oxidative stress on cytosolic (pHcyt) and apparent vesicular pH (pHves) by single-cell fluorescence recordings. In the presence of normo-osmotic (305 mosmol/l) medium pHcyt was 7.23 +/- 0.03 (n = 108), whereas an apparent pH of 6.07 +/- 0.02 (n = 156) was found in the vesicular compartment accessible to endocytosed FITC-dextran. Substitution of 60 mM NaCl against 120 mM raffinose had no effect on pHcyt or apparent pHves, whereas addition of NH4Cl increased both pHcyt and apparent pHves. Hypo-osmotic cell swelling lowered pHcyt, whereas simultaneously apparent pHves increased. These effects were rapidly reversible upon re-institution of normo-osmotic media. Similarly, an increase of apparent pHves was observed when cell swelling was induced by Ba2+, glutamine or histidine. Conversely, hyperosmotic cell shrinkage due to addition of NaCl or raffinose led to a cytosolic alkalinization and a vesicular acidification. Both, H2O2 (0.2 mmol/l) and t-butyl-hydroperoxide (0.2 mmol/l) were without effect on pHcyt, but lowered apparent pHves by about 0.2 pH units. Ba2+ (1 mmol/l) diminished the acidifying effect of the hydroperoxides by about 50%. Pretreatment of the cells with colchicine, but not with lumicolchicine, largely abolished the effects of aniso-osmolarity and hydroperoxides on pHves. The data suggest that hepatocellular hydration affects the proton gradients built up across the membranes of endocytotic FITC-dextran-accessible compartments in a microtubule-dependent way. They further suggest that hydroperoxides induce vesicular acidification in a colchicine- and Ba(2+)-sensitive way. Because hydroperoxides induce Ba(2+)-sensitive cell shrinkage [Hallbrucker, Ritter, Lang, Gerok and Häussinger (1992) Eur. J. Biochem. 211, 449-458], the results are compatible with the view that hydroperoxide-induced cell shrinkage mediates vesicular acidification. It is concluded that modulation of vesicular pH by the hepatocellular hydration state may play a role in triggering some metabolic changes in response to cell swelling/shrinkage.

Ammonium Chloride↗
PubMed1994

Effects of anisotonic exposure on duck hepatitis B virus replication.

In cultured hepatocytes from in vivo duck hepatitis B virus-infected ducks the effect of medium osmolarity on viral replication was studied. A 10-day exposure to hypotonic media (277 mOsm/L due to removal of 26 mmol/L NaCl) lowered the duck hepatitis B virus DNA content of cells and of the medium by about 50%, whereas hyperosmotic exposure (421 mOsm/L by addition of 46 mmol/L NaCl) increased it about four-fold compared with normotonic standard incubation medium (329 mOsm/L). The tissue levels of viral RNA transcripts increased during the 10 days of hypertonic exposure but decreased only slightly after hypoosmotic treatment. Western-blot analysis for the production of viral pre-S/S proteins revealed a marked stimulation of viral protein synthesis in hypertonic media, whereas hypotonic exposure inhibited it. Conversely, total cellular protein synthesis as assessed from [3H]leucine incorporation into acid-precipitable material decreased during hyperosmotic exposure but increased during hypoosmotic exposure. We noted a comparable increase of duck hepatitis B virus DNA when raffinose (80 mmol/L) was added to hypotonic or normotonic media, without change in the NaCl concentrations. This suggests that the effects of anisotonicity on viral replication were not due to alterations of Na+ or Cl- activity in the incubation media, but might reflect changes of cellular volume. The effects of anisotonicity on viral replication were only seen after exposure of more than 8 hr of the cells to anisotonicity. The findings suggest that the cellular volume is an important determinant for duck hepatitis B virus replication, yet the underlying molecular mechanisms remain elusive.

Animals↗
PubMed1993

Structural reaction pattern of hepatocytes following exposure to hypotonicity.

Isolated rat hepatocytes were exposed to hypotonic media (225 mosmol/l) for 5 and 15 min and processed for a quantitative electron microscopic stereologic analysis. Within 5 min of hypotonicity, the hepatocyte volume increased by 25% and thereafter displayed a volume regulatory decrease leading to mean cellular volume, which was 16% above that of controls. Stereologic analysis of the major subcellular compartment, the cytosol, showed an identical change as the whole cell. In contrast to that, the mitochondrial compartment increased in volume by 30% within the first 5 min of exposure and returned by regulatory volume decrease back to values of the isotonic controls after 15 min of hypotonicity. In contrast, hypotonicity (220 mosmol/l)-induced stimulation of flux through mitochondrial glutaminase and the glycine cleavage enzyme complex, as assessed by 14CO2 production from [1-14C]glutamine or [1-14C]glycine in isolated perfused rat liver persisted throughout a 15-min period of hypotonic exposure. Thus hypotonicity-induced alterations of mitochondrial metabolism apparently do not parallel the time course of mitochondrial volume changes. This suggests that persistent mitochondrial swelling is not required for functional alterations, but that the latter may be triggered by the initial swelling of mitochondria. Hypotonic exposure did not alter the nuclear volume of isolated hepatocytes. Cell membrane surface nearly doubled after 5 min of hypotonic exposure, but returned within 15 min of exposure to values observed in normotonic media. This may reflect the participation of exocytosis in hepatocyte volume regulation.

Animals↗
PubMed1992

Hepatocyte swelling leads to rapid decrease of the G-/total actin ratio and increases actin mRNA levels.

Exposure of isolated rat hepatocytes to hypotonic (190 mosmol/l) incubation media lowered the cellular G-actin level without affecting the total actin content: here the G-/total actin ratio decreased by 15.5 +/- 1.4% (n = 7). Similar effects were observed following isotonic cell swelling by either addition of glutamine (10 mM) or insulin (100 nM), resulting in a decrease of the G-/total actin ratios by 13.5 +/- 2.1% (n = 5) and 14.1 +/- 1.1% (n = 11), respectively. The effects of hypotonic exposure, glutamine and insulin on the G-/total actin ratio largely occurred within 1 min and persisted for at least 2 h in presence of the respective effectors. After a 120 min exposure to hypotonic media, glutamine or insulin the actin mRNA levels were increased 2.4-, 2.0- and 3.6-fold, respectively. Hypertonic exposure lowered the G-/total actin ratio by only 4.9 +/- 2.5% (n = 4) and increased actin mRNA levels only 1.2-fold. There was a close relationship between glutamine- and hypotonicity-induced cell swelling and the decrease of G-/total actin ratios. The data suggest that cell swelling exerts rapid and marked effects on the state of actin polymerization and increases actin mRNA levels. Thus, cytoskeletal alterations in response to cell swelling may be involved in the regulation of hepatic metabolism by cell volume.

Actins↗
PubMed1992

Liver cell volume and protein synthesis.

Protein synthesis in isolated rat hepatocytes was determined from the incorporation of [3H]leucine (4 mM) into acid-precipitable material in the presence of amino acids at twice their physiological concentration. Protein synthesis increased linearly with time and incubated cell protein, and was inhibited by cycloheximide by more than 95%. In normo-osmotic incubations containing amino acids at twice the physiological concentration the rate of [3H]leucine incorporation was 5.8 +/- 0.2 nmol/h per mg of cell protein (n = 26). Hyperosmotic cell shrinkage due to addition of 60 mM-NaCl or 120 mM-raffinose inhibited [3H]leucine incorporation into acid-precipitable material by 60 and 74% respectively, whereas hypo-osmotic cell swelling was ineffective. Inhibition of protein synthesis by adding 120 mM-raffinose was largely counteracted by simultaneous lowering of the NaCl concentration by 60 mM. Glutamine (10 mM) had no effect on protein synthesis in normo-osmotic incubations (320 mosM), but stimulated protein synthesis in hyperosmotically (440 mosM) pre-shrunken cells almost to rates found in normo-osmotic (320 mosM) control incubations. Cyclic AMP and vasopressin inhibited protein synthesis by 23% and 8% respectively, whereas insulin and phenylephrine were ineffective. However, inhibition of protein synthesis by cyclic AMP was about twice as strong in the presence of vasopressin or phenylephrine. When protein synthesis was preinhibited by cyclic AMP, [3H]leucine incorporation was stimulated by glutamine (10 mM), insulin or hypo-osmotic exposure. There was a close relationship between the inhibition of protein synthesis and the extent of hepatocyte shrinkage induced by the above-mentioned effectors, suggesting a role of cell volume in the regulation of hepatic protein synthesis.

Animals↗
PubMed1992

Effect of anisotonic cell-volume modulation on glutathione-S-conjugate release, t-butylhydroperoxide metabolism and the pentose-phosphate shunt in perfused rat liver.

1. Addition of 1-chloro-2,4-dinitrobenzene to isolated perfused rat liver results in the rapid formation of its glutathione-S-conjugate [S-(2,4-dinitrophenyl)glutathione], which is released into both, bile and effluent perfusate. Anisotonic perfusion did not affect total S-conjugate formation, but release of the S-conjugate into the perfusate was increased (decreased) following hypertonic (hypotonic) exposure at the expense of excretion into bile. Stimulation of S-conjugate release into the perfusate following hypertonic exposure paralleled the time course of volume-regulatory net K+ uptake. 2. Basal steady-state release of oxidized glutathione (GSSG) into bile was 1.30 +/- 0.12 nmol.g-1.min-1 (n = 18) during normotonic (305 mOsmol/l) perfusion and was 3.8 +/- 0.3 nmol.g-1.min-1 in the presence of t-butylhydroperoxide (50 mumol/l). Hypotonic exposure (225 mOsmol/1) lowered both, basal and t-butylhydroperoxide (50 mumol/l)-stimulated GSSG release into bile by 35% and 20%, respectively, whereas hypertonic exposure (385 mOsmol/l) increased. Anisotonic exposure was without effect on t-butylhydroperoxide removal by the liver. GSSG release into bile also decreased by 33% upon liver-cell swelling due to addition of glutamine plus glycine (2 mmol/l, each). 3. Hypotonic exposure led to a persistent stimulation 14CO2 production from [1-14C]glucose by about 80%, whereas 14CO2 production from [6-14C]glucose increased by only 10%. Conversely, hypertonic exposure inhibited 14CO2 production from [1-14C]glucose by about 40%, whereas 14CO2 production from [6-14C]glucose was unaffected. The effect of anisotonicity on 14CO2 production from [1-14C]glucose was also observed in presence of t-butylhydroperoxide (50 mumol/l), which increased 14CO2 production from [1-14C]glucose by about 40%. 4. t-Butylhydroperoxide (50 mumol/l) was without significant effect on volume-regulatory K+ fluxes following exposure to hypotonic (225 mOsmol/l) or hypertonic (385 mOsmol/l) perfusate. Lactate dehydrogenase release from perfused rat liver under the influence of t-butylhydroperoxide was increased by hypertonic exposure compared to hypotonic perfusions. 5. The data suggest that hypotonic cell swelling stimulates flux through the pentose-phosphate pathway and diminishes loss of GSSG under conditions of mild oxidative stress. Hypotonically swollen cells are less prone to hydroperoxide-induced lactate dehydrogenase release than hypertonically shrunken cells. Hypertonic cell shrinkage stimulates the excretion of glutathione-S-conjugates into the sinusoidal circulation at the expense of biliary secretion.

Animals↗
PubMed1992

Anisoosmostic liver perfusion: redox shifts and modulation of alpha-ketoisocaproate and glycine metabolism.

1) In isolated perfused rat liver, 14CO2 production from [1-14C]alpha-ketoisocaproate or [1-14C]glycine as well as ketogenesis from alpha-ketoisocaproate were stimulated upon exposure to hypoosmotic perfusion media, whereas hyperosmotic exposure inhibited. The effects of anisotonicity were preserved when ketogenesis from alpha-ketoisocaproate and 14CO2 production from [1-14C]glycine were already stimulated by glucagon. On the other hand, ketogenesis from tyrosine (2 mM) or octanoate (0.1 mM) were almost unaffected by anisoosmotic exposure. 2) With all ketogenic substrates studied, hypoosmotic (hyperosmotic) cell swelling (shrinkage) decreased (increased) the beta-hydroxybutyrate/acetoacetate ratio in effluent perfusate. A shift of the mitochondrial and cytosolic NADH systems to a more oxidized (reduced) state following hypoosmotic (hyperosmotic) exposure was also found upon infusion of beta-hydroxybutyrate/acetoacetate and lactate/pyruvate as redox indicator metabolite couples. The effects of anisotonicity on the beta-hydroxybutyrate/acetoacetate ratio were reversible upon normoosmotic reexposure and persisted throughout anisoosmotic exposure despite completion of volume regulatory K+ fluxes within 10-15 min. Hepatic oxygen consumption decreased by about 10% during hyperosmotic cell shrinkage and was transiently stimulated during hypoosmotic exposure. 3) There was a close relationship between ketogenesis from alpha-ketoisocaproate (0.5 mM) and the mitochondrial redox state, as assessed by the beta-hydroxybutyrate/acetoacetate ratio in effluent, regardless of whether the pathway was modulated by anisotonicity or glucagon. 4) Isoosmotic cell swelling induced by addition of glutamine (3 mM) was without significant effect on ketogenesis from octanoate and stimulated ketogenesis and 14CO2production from [1-14C]alpha-ketoisocaproate only slightly (i.e. by less than 10%); however, in each case the hydroxybutyrate/acetoacetate ratio in effluent perfusate decreased by about 20% upon addition of glutamine. 5) Stimulation of 14CO2production from [1-14C]glycine by hypoosmotic exposure and glucagon was only slightly affected when the accompanying decrease of the beta-hydroxybutyrate/acetoacetate ratio was reversed by addition of beta-hydroxybutyrate. 6) The data are compatible with a hypotonicity (hypertonicity)-induced shift of the mitochondrial NADH system to a more oxidized (reduced) state, probably due to a alterations of respiration. Mitochondrial swelling probably also occurs under the influence of glutamine. Modulation of ketogenesis from alpha-ketoisocaproate, but not of glycine oxidation by anisoosmotic exposure and glucagon can be related to the accompanying redox shifts. The observations support the concept that cell volume may be an important parameter determining liver cell function.

Animals↗
PubMed1992

Characterization of the fetal inflammatory response to cytomegalovirus placentitis. An immunohistochemical study.

The histopathologic features of cytomegalovirus placentitis, an established cause of chronic villitis, are well documented. However, the immunologic features of the fetal inflammatory response to placental cytomegalovirus infection are largely unknown. The characterization of the fetal-derived inflammatory cell reaction may be important in our understanding of both the intrauterine as well as the antenatal immunological response of the neonate to this important viral infection. We examined formalin-fixed placentas from four cases of confirmed congenital cytomegalovirus infection using an in situ DNA probe to cytomegalovirus, and a variety of antibodies to leukocyte antigens, including anti-CD68, CD45RO, CDw75, CD74, IgG, IgM, and IgA. All four placentas showed marked hyperplasia of fetal-derived placental macrophages, termed Hofbauer cells. A lymphocytic villitis was present in all placentas, which was characterized by positive staining in all cases with T-cell antibodies. There was no evidence of positive staining of lymphocytes using B-cell antibodies in any of the cases. Two placentas showed plasmacellular villitis, which stained positively for both IgG- and IgM-secreting cells, that was present as early as the second trimester of gestation. No IgA positivity of plasma cells was observed. These data are presented in light of current theories of fetal viral immunity.

Adolescent↗
PubMed1991

Hepatocyte heterogeneity in uptake and metabolism of malate and related dicarboxylates in perfused rat liver.

1. In isolated perfused rat liver a near-maximal net malate uptake of about 120 nmol g-1 min-1 was observed at influent malate concentrations above 100 mumol l-1 and a half-maximal uptake at about 50 mumol l-1 in influent. 14CO2 production from added [U-14C]malate paralleled hepatic net malate uptake, however, 14CO2 production exceeded net malate uptake by 20-25%. This was observed in antegrade as well as in retrograde perfusions and regardless of whether NH4Cl was added to the influent perfusate. Stimulation of glutamine synthesis by NH4Cl only slightly affected net malate uptake and 14CO2 production, but resulted in a marked stimulation of [14C]glutamine release from the liver. 2. Because [U-14C]malate uptake by the liver (reflecting the influent/effluent concentration difference of labeled malate) could at least in part involve a malate/malate exchange mechanism, net malate uptake (as determined from the influent/effluent concentration difference of enzymatically assayable malate) may underestimate hepatic [U-14C]malate uptake. On the other hand, during metabolic steady states 14CO2 production from added [U-14C]malate can be considered as an upper limit estimate of [U-14C]malate uptake by the liver. Assuming that 14CO2 production equals [U-14C]malate uptake by the liver, extrapolation studies suggest that during maximal rates of NH4Cl-stimulated glutamine synthesis 80-110% of the [U-14]malate taken up by the liver was used for glutamine synthesis. This was true for retrograde and antegrade perfusions. Similar data, i.e. a 100-130% incorporation regardless of the direction of perfusion, were obtained when [U-14C]malate uptake was assumed to equal net malate uptake by the liver. 3. Substitution of Na+ in the perfusion fluid by choline abolished net malate uptake by the liver and inhibited 14CO2 production from [U-14C]malate by more than 90%. 4. 2-Oxoglutarate inhibited [14C]malate uptake and [1-14C]oxoglutarate uptake by the liver was inhibited by malate, fumarate, succinate and oxaloacetate, but not by aspartate and glutamate. Inhibition of [1-14C]oxoglutarate uptake and of 14CO2 production from added labeled 2-oxoglutarate by malate and fumarate seemed largely competitive. Malate, fumarate and succinate not only inhibited [1-14C]oxoglutarate uptake, but also stimulated the release of unlabeled 2-oxoglutarate from the liver. 5. The data are consistent with a predominant uptake of vascular malate by perivenous glutamine synthetase containing hepatocytes when glutamine synthesis is stimulated to Vmax values by NH4Cl. Malate and other citric acid cycle dicarboxylates, but not aspartate and glutamate, may compete with 2-oxoglutarate for uptake into perivenous glutamine synthesizing hepatocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Enumeration of human peripheral blood lymphocytes secreting immunoglobulins of major classes and subclasses in healthy children and adults.

The reverse enzyme-linked immunospot assay was modified to enumerate peripheral blood mononuclear cells (PBMC) secreting IgG1-4, IgA1-2, and IgM. Anti-human IgG F(ab')2 and mouse monoclonal antibodies specific to each of the isotypes were used as solid-phase capture antibodies and developing antibodies, respectively. Although attempts were also made to detect IgD- and IgE-secreting cells (SC), their numbers in the peripheral blood were too few to be reliably estimated. The assay was applied to study healthy subjects including 21 neonates within 3 days of birth, 44 1- to 48-month-old children, and 32 adults. Sixty percent of these neonates had IgM SC in small numbers (less than 20 per 10(6) PBMC), but only three neonates had IgSC of other isotypes. In contrast, by 1-2 months of age children had IgSC of all isotypes, including IgA2 and IgG4, often in higher numbers than adults. The relative frequencies of IgSC were IgA1 greater than IgG1 greater than IgM greater than IgG2 greater than IgG3 greater than IgG4 greater than IgA2 in the children and IgA1 greater than IgG1 greater than IgA2 greater than IgM greater than IgG4 greater than IgG2 greater than IgG3 in the adults. The order of the serum concentrations in the adults was IgG1 greater than IgG2 greater than IgA greater than IgM greater than IgG4 greater than IgG3. No correlation was found between the serum level and the IgSC number of individual isotypes (except for serum IgA and IgA1-SC). This new methodology should facilitate investigating the current status of immunoglobulin synthesis and the Ig repertoire in adults and children, in health and in disease.

Adult↗
PubMed1991

Functional hepatocyte heterogeneity in glutamate, aspartate and alpha-ketoglutarate uptake: a histoautoradiographical study.

[3H]glutamate, [3H]alpha-ketoglutarate or [3H]aspartate was injected in physiological concentrations into antegrade (from portal to hepatic vein) or retrograde (from hepatic to portal vein) perfused rat liver, and the tissue distribution of radioactivity was studied by histoautoradiography. Independent of the direction of perfusion, radioactivity was accumulated in a small perivenous liver parenchymal cell population, which surrounded the terminal hepatic venules as a layer of about two to five cells thick. In contrast, accumulation of radioactivity in periportal hepatocytes was low and sometimes not detectable. This distribution pattern roughly resembled that described for the immunohistochemical distribution of glutamine synthetase in liver. The present histoautoradiographic findings demonstrate a predominant uptake of vascular glutamate, aspartate and ketoglutarate into a small perivenous cell population. They confirm previous label incorporation studies in the metabolically intact liver, demonstrating an almost exclusive uptake of vascular glutamate and alpha-ketoglutarate into perivenous glutamine synthetase containing hepatocytes. In addition, evidence is presented suggesting that perivenous uptake of alpha-ketoglutarate may be one determinant for hepatic glutamine synthesis, at least under the experimental conditions used here.

Animals↗
PubMed1989

Hepatocyte heterogeneity in glutamate metabolism and bidirectional transport in perfused rat liver.

1. The metabolic fate of infused [1-14C]glutamate was studied in perfused rat liver. The 14C label taken up by the liver was recovered to 85 +/- 2% as 14CO2 and [14C]glutamine. Whereas 14CO2 production accounted for about 70% of the [1-14C]glutamate taken up under conditions of low endogenous rates of glutamine synthesis, stepwise stimulation of glutamine synthesis by NH4Cl increased 14C incorporation into glutamine at the expense of 14CO2 production. Extrapolation to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of vascular glutamate taken up by the liver for glutamine synthesis. This was observed in both, antegrade and retrograde perfusions and suggests an almost exclusive uptake of glutamate into perivenous glutamine-synthetase-containing hepatocytes. 2. Glutamate was simultaneously taken up and released from perfused rat liver. At a near-physiological influent glutamate concentration (0.1 mM), the rates of unidirectional glutamate influx and efflux were similar (about 100 and 120 nmol g-1 min-1, respectively). 3. During infusion of [1-14C]oxoglutarate (50 microM), addition of glutamate (2 mM) did not affect hepatic uptake of [1-14C]oxoglutarate. However, it increased labeled glutamate release from the liver about 10-fold (from 9 +/- 2 to 86 +/- 20 nmol g-1 min-1; n = 4), whereas 14CO2 production from labeled oxoglutarate decreased by about 40%. This suggests not only different mechanisms of oxoglutarate and glutamate transport across the plasma membrane, but also points to a glutamate/glutamate exchange. 4. Oxoglutarate was recently shown to be taken up almost exclusively by perivenous glutamine-synthetase-containing hepatocytes [Stoll, B & Häussinger, D. (1989) Eur. J. Biochem. 181, 709-716] and [1-14C]oxoglutarate (9 microM) was used to label selectively the intracellular glutamate pool in this perivenous cell population. The specific radioactivity of this intracellular (perivenous) glutamate pool was assessed by measuring the specific radioactivity of newly synthesized glutamine which is continuously released from these cells into the perfusate. Comparison of the specific radioactivities of glutamine and glutamate released from perivenous cells indicates that about 60% of total glutamate release from the liver is derived from the perivenous glutamine-synthetase-containing cell population. Following addition of unlabeled glutamate (0.1 mM), unidirectional glutamate efflux from perivenous cells increased from about 30 to 80 nmol g-1 min-1, whereas glutamate efflux from non-perivenous (presumably periportal) hepatocytes remained largely unaltered (i.e. 20-30 nmol g-1 min-1). 5. It is concluded that, in the intact liver, vascular glutamate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase.

Animals↗
PubMed1989

Functional hepatocyte heterogeneity. Vascular 2-oxoglutarate is almost exclusively taken up by perivenous, glutamine-synthetase-containing hepatocytes.

1. In isolated perfused rat liver maximal rates of 2-[1-14C]oxoglutarate uptake were about 0.4 mumol.g-1 .min-1; half-maximal rates of 2-[14C]oxoglutarate uptake were observed with influent concentrations of about 100 microM. 2-[14C]Oxoglutarate uptake by the liver was not affected by the direction of perfusion, but was decreased by about 80-90% when Na+ in the perfusion fluid was substituted by choline+, suggesting a Na+-dependence of hepatic 2-oxoglutarate uptake. In the absence of added ammonia, [14C]oxoglutarate uptake by the liver was about twice the net oxoglutarate uptake, indicating a simultaneous release of unlabeled oxoglutarate from perfused rat liver. 2. 14C-Labeled metabolites derived from [1-14C]oxoglutarate and recovered in the effluent perfusate were 14CO2 and 14C-labeled glutamate and glutamine; they accounted for 85-100% of the radiolabel taken up by the liver. 14CO2 was the major product (more than 70%) from [1-14C]oxoglutarate taken up the liver, provided glutamine synthesis was either inhibited by methionine sulfoximine or the endogenous rate of glutamine production was below 40 nmol.g-1.min-1. 3. Stimulation of glutamine synthesis by ammonia did not affect [14C]oxoglutarate uptake by the liver, but considerably increased net hepatic oxoglutarate uptake, indicating a decreased release of unlabeled oxoglutarate from the liver. Stepwise stimulation of hepatic glutamine synthesis led to a gradual decrease of 14CO2 production and radiolabel was recovered increasingly as [14C]glutamine in the effluent. At high rates of glutamine formation (i.e. about 0.6 mumol.g-1.min-1), about 60% of the [1-14C]oxoglutarate taken up by the liver was recovered in the effluent as [14C]glutamine. 14CO2 and [14C]glutamine production from added [1-14C]oxoglutarate were dependent on the rate of hepatic glutamine synthesis but not on the direction of perfusion. Extrapolation of 14C incorporation into glutamine to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of the [14C]oxoglutarate taken up by the liver for glutamine synthesis. This was again true for both the antegrade and the retrograde perfusion directions. On the other hand, addition of ammonia did not affect 14CO2 production from labeled oxoglutarate, when glutamine synthetase was inhibited by methionine sulfoximine. 4. The data suggest that vascular oxoglutarate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase, i.e. a cell population comprising only 6-7% of all hepatocytes. Thus, the findings demonstrate the existence of a, to date, uniquely zonally distributed oxoglutarate transport system which is probably Na+-dependent in the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonia↗
PubMed1988

Stimulation of thromboxane release by extracellular UTP and ATP from perfused rat liver. Role of icosanoids in mediating the nucleotide responses.

1. In isolated perfused rat liver, infusion of UTP (20 microM) led to a transient, about sevenfold stimulation of thromboxane release (determined as thromboxane B2), which did not parallel the time course of the UTP-induced stimulation of glucose release. An increased thromboxane release was also observed after infusion of ATP (20 microM). Although the maximal increase of portal pressure following ATP was much smaller than with UTP (4.2 vs 11.5 cm H2O), the peak thromboxane release was similar with both nucleotides. 2. Indomethacin (10 microM) inhibited the UTP-induced stimulation of thromboxane release and decreased the UTP-induced maximal increase of glucose output and of portal pressure by about 30%. The thromboxane A2 receptor antagonist BM 13.177 (20 microM) completely blocked the pressure and glucose response to the thromboxane A2 analogue U-46619 (200 nM) and decreased the ATP- and UTP-induced stimulation of glucose output by about 25%, whereas the maximal increase of portal pressure was inhibited by about 50% and 30%, respectively. BM 13.177 and indomethacin inhibited the initial nucleotide-induced overshoot of portal pressure increase, but had no effect on the steady-state pressure increase which is obtained about 5 min after addition of ATP or UTP. 3. The leukotriene D4/E4 receptor antagonist LY 171883 (50 microM) inhibited not only the glucose and pressure response of perfused rat liver to leukotriene D4, but also to leukotriene C4 by about 90%. This suggests that leukotriene D4 (not C4) is the active metabolite in perfused liver and the effects of leukotriene C4 are probably due to its rapid conversion to leukotriene D4. LY 171883 also inhibited the response to the thromboxane A2 analogue U-46619 by 75-80%, whereas the response of perfused liver to leukotriene C4 was not affected by the thromboxane receptor antagonist BM 13.177 (20 microM). The glucose and pressure responses of the liver to extracellular UTP were inhibited by LY 171883 and by BM 13.177 by about 30%. This suggests that the inhibitory action of LY 171883 was due to a thromboxane receptor antagonistic side-effect and that peptide leukotrienes do not play a major role in mediating the UTP response. 4. In isolated rat hepatocytes extracellular UTP (20 microM), ATP (20 microM), cyclic AMP (50 microM) and prostaglandin F2 alpha (3 microM) increased glycogen phosphorylase a activity by more than 100%.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetophenones↗