Control of hepatic nitrogen metabolism and glutathione release by cell volume regulatory mechanisms

1. Urea synthesis was studied in isolated perfused rat liver during cell volume regulatory ion fluxes following exposure of the liver to anisotonic perfusion media. Lowering of the osmolarity in influent perfusate from 305 mOsm/l to 225 mOsm/l (by decreasing influent [NaCl] by 40 mmol/l) led to an inhibition of urea synthesis from NH4Cl (0.5 mmol/l) by about 60% and a decrease of hepatic oxygen uptake by 0.43 +/- 0.03 mumol g-1 min-1 [from 3.09 +/- 0.13 mumol g-1 min-1 to 2.66 +/- 0.12 mumol g-1 min-1 (n = 9)]. The effects on urea synthesis and oxygen uptake were observed throughout hypotonic exposure (225 mOsm/l). They persisted although volume regulatory K+ efflux from the liver was complete within 8 min and were fully reversible upon reexposure to normotonic perfusion media (305 mOsm/l). A 42% inhibition of urea synthesis from NH4Cl (0.5 mmol/l) during hypotonicity was also observed when the perfusion medium was supplemented with glucose (5 mmol/l). Urea synthesis was inhibited by only 10-20% in livers from fed rats, and was even stimulated in those from starved rats when an amino acid mixture (twice the physiological concentration) plus NH4Cl (0.2 mmol/l) was infused. 2. The inhibition of urea synthesis from NH4Cl (0.5 mmol/l) during hypotonicity was accompanied by a threefold increase of citrulline tissue levels, a 50-70% decrease of the tissue contents of glutamate, aspartate, citrate and malate, whereas 2-oxoglutarate, ATP and ornithine tissue levels, and the [3H]inulin extracellular space remained almost unaltered. Further, hypotonic exposure stimulated hepatic glutathione (GSH) release with a time course roughly paralleling volume regulatory K+ efflux. NH4Cl stimulated lactate release from the liver during hypotonic but not during normotonic perfusion. In the absence of NH4Cl, hypotonicity did not significantly affect the lactate/pyruvate ratio in effluent perfusate. With NH4Cl (0.5 mmol/l) present, the lactate/pyruvate ratio increased from 4.3 to 8.2 in hypotonicity, whereas simultaneously the 3-hydroxybutyrate/acetoacetate ratio slightly, but significantly decreased. 3. Addition of lactate (2.1 mmol/l) and pyruvate (0.3 mmol/l) to influent perfusate did not affect urea synthesis in normotonic perfusions, but completely prevented the inhibition of urea synthesis from NH4Cl (0.5 mmol/l) induced by hypotonicity. Restoration of urea production in hypotonic perfusions by addition of lactate and pyruvate was largely abolished in the presence of 2-cyanocinnamate (0.5 mmol/l). Addition of 3-hydroxybutyrate (0.5 mmol/l), but not of acetoacetate (0.5 mmol/l) largely reversed the hypotonicity-induced inhibition of urea synthesis from NH4Cl.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of vasectomy on the blood-testis barrier of the hamster.

The effects of vasectomy on the blood-testis and blood-epididymal barriers to 3H2O, [3H]inulin, and [14C]urea were examined by study of the radioactivity appearing in micropuncture samples of fluids from the seminiferous tubules and cauda epididymidis. By 4 months after vasectomy, there were changes in the blood-seminiferous tubule barrier to [3H]water and [14C]urea (increased entry) and in the blood-epididymal barrier to [3H]water and [3H]inulin (increased entry) and to [14C]urea (decreased entry). These subtle changes could have an impact on spermatogenesis and/or sperm maturation after vasectomy.     

Interactions between glutamine metabolism and cell-volume regulation in perfused rat liver

1. In the presence of near-physiological glutamine concentrations, exposure of perfused rat liver to hypotonic perfusion media switched glutamine balance across the liver from net release to net uptake. This was due to both stimulation of flux through glutaminase and inhibition of flux through glutamine synthetase. Conversely, during exposure to hypertonic media, net glutamine release from the liver increased due to inhibition of glutaminase flux and slight stimulation of flux through glutamine synthetase. The effect of perfusate osmolarity on glutaminase flux was observed at an NH4Cl concentration (0.5 mM) sufficient for near-maximal ammonia stimulation of glutaminase. This indicates the involvement of different mechanisms of glutaminase flux control by extracellular osmolarity changes and ammonia. The effects of anisotonicity on flux through glutamine-metabolizing enzymes were fully reversible. Glutamine (0.6 mM) stimulated urea synthesis from NH4Cl (0.5 mM) during hypotonic and normotonic conditions. 2. Exposure to hypotonic and hypertonic media led, after initial liver-cell swelling and shrinkage, respectively to volume-regulatory K+ fluxes which largely restored the initial liver-cell volume despite the continuing osmotic challenge. Even after completion of cell-volume regulatory K+ fluxes, the effects of perfusate osmolarity on hepatic glutamine metabolism persisted. This indicates that in anisotonicity the liver cell is left in an altered metabolic state, even after completion of volume-regulatory responses. 3. During perfusion with isotonic media, addition of glutamine (3 mM) led to an increase of liver mass by about 4% within 2 min, which was accompanied by a net K+ uptake by the liver. Thereafter, the new steady state of increased liver mass was maintained throughout glutamine infusion. When the liver mass had reached this new steady state, a net release of K+ from the liver of about 3 mumol/g liver was observed during the following 10 min. Withdrawal of glutamine was followed by a slow reuptake of K+ and the liver mass returned to its initial value. Following exposure to glutamine (3 mM), the intracellular glutamine concentration (as calculated from glutamine tissue levels, taking into account the extracellular space determined with the [3H]inulin technique) rose from about 1 mM to 30-35 mM within about 12 min, indicating a 10-12-fold concentrative uptake of glutamine into the liver cells and an osmotic challenge for the hepatocyte. When intracellular glutamine had reached its steady-state concentration, net K+ efflux from the liver was also terminated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Flow rates in the isolated perfused rat liver by pulse labeling with radioactive substrates and mathematical analysis of the wash-out kinetics

The multiple indicator dilution technique (Goresky and Bach) is critically evaluated and its application to the isolated, hemoglobin-free perfused rat liver is described. From the results of pulse labelling experiments using indicator substances for the total aqueous space of the liver ([3H]water and [14C]urea) an for the extracellular space ([14C]sucrose, [3H]-inulin and [3H]dextrane), it is concluded that the mathematical model of the liver in situ according to Goresky and Bach is sufficient to also describe the hemodynamics of the isolated liver perfused with a saline solution. The data indicate that the multiple indicator dilution technique in combination with the available mathematical basis is applicable to the study of transport across the liver cell membrane. The method, however, is restricted to compounds which are metabolized very slowly, such as D-lactate. A possible extension of this method to metabolic processes is discussed in view of pulse labelling experiments with [3H]-and [1-14C]L-lactate. Since the involvement of metabolism in the available mathematical model is not differentiated and the release of products is not taken into consideration, the method in its present state is not applicable to studies of metabolism. Moreover, even the parameters of transport derived by this technique are of limited value, when the transported compounds are rapidly metabolized by reversible reactions. Despite these uncertainties, the present data indicate that the transport of L-lactate is ten times faster than that of D-lactate.     

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.     

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 thereatter 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)-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. © 1993 Wiley-Liss, Inc.     

Hepatic albumin and urea synthesis: The mathematical modelling of the dynamics of [14C]carbonate-derived guanidine-labelled arginine in the isolated perfused rat liver.

A mathematical model was constructed to define the dynamics of incorporation of radioactivity into urea carbon and the guanidine carbon of arginine in plasma albumin after the rapid intraportal-venous administration of Na214CO3 in the isolated perfused rat liver. 2. The model was formulated in terms of compartmental analysis and additional experiments were designed to provide further information on subsystem dynamics and to discriminate between alternative model structures. 3. Evidence for the rapid-time-constant of labelling of intracellular arginine was provided by precursor-product analysis of precursor [14C]carboante and product [14C]urea in the perfusate. 4. Compartmental analysis of the dynamics of newly synthesized urea was based on the fate of exogenous [13C]urea, endogenous [14C]urea and the accumulation of [12C]urea in perfusate water, confirming the early completion of urea carbon labelling, the absence of continuing synthesis of labelled urea, and the presence of a small intrahepatic urea-delay pool. 5. Analysis of the perfusate dynamics of endogenously synthesized and exogenously administered [6-14C]arginine indicated that although the capacity for extrahepatic formation of [14C]-urea exists, little or no arginine formed within the intrahepatic urea cycle was transported out of the liver. However, the presence of a rapidly turning-over intrahepatic arginine pool was confirmed. 6. On the basis of these subsystem analyses it was possible to offer feasible estimations for the parameters of the mathematical model. However, it was not possible to stimulate the form and magnitude of the dynamics of newly synthesized labelled urea and albumin which were simultaneously observed after administration of [14C]carbonate on the basis of a preliminary model which postulated that both products were derived from a single hepatic pool of [16-14C]arginine. On the other hand these observed dynamics could be satisfied to a two-compartment arginine model, which also provided an explanation for discrepancies observed between albumin synthesis measured radioisotopically and immunologically. This was based on a relative overestimation of [14C]urea specific radioactivity resulting from the rapid dynamics of [14C]carbonate and the [14C]urea subsystem relative to the labelled albumin subsystem. The effects of arginine compartmentalization could be minimized in the model by minor slowing of the rate of [14C]carbonate turnover or by constant infusion of [14C]carbonate, both of which permitted valid determination of albumin-synthesis rates.     

Albumin synthesis by the perfused rat liver. A comparison of methods with special reference to the effect of dietary protein deprivation

1. Isolated perfused rat livers were used to study synthesis of albumin after donors had been fed on normal or protein-free diets. 2. Methods of determining the liver's ability to produce albumin included incorporation of [¹⁴C]carbonate, [³H]lysine and [¹⁴C]arginine, as well as a direct method based on a heterologous perfusing system of rat erythrocytes and rabbit plasma. 3. Livers from protein-deprived rats were found to form extremely little urea and not to incorporate ¹⁴CO₂ into [¹⁴C]urea, but they were capable of producing [¹⁴C]urea from [¹⁴C]arginine and of incorporating the latter and [³H]lysine into albumin. 4. By immunological means these lives were found to synthesize less albumin than normal, but their ability was only slightly impaired when related to body weight or liver weight. 5. These findings are consistent with a block in urea-cycle enzymes with relative integrity of arginase activity and of amino acid activation.     

Restricted permeability of rat liver for glutamate and succinate

1. When rat liver slices were incubated aerobically with [U-¹⁴C]glutamate the concentration of ¹⁴C within the slices remained lower (about 50%) than in the medium. The maximal concentration of ¹⁴C in the liver was reached within minutes. In rat kidney-cortex slices by contrast, ¹⁴C reached concentrations more than six times those of the medium. 2. In both liver and kidney ¹⁴C appeared in the respiratory CO₂, indicating penetration of glutamate carbon into the mitochondria. In kidney slices the rate of glutamate oxidation per unit weight was about five times that in liver slices. 3. Taking into account the conversion of glutamate into glucose that occurs in the kidney but not in the liver, the flux rates of glutamate through the kidney were calculated to be about 15 times those through the liver when the external glutamate concentration was 5mm. 4. Anaerobically the glutamate concentrations in medium and tissue rapidly became equal in both liver and kidney. Thus the maintenance of concentration gradients depended on the expenditure of energy. 5. [U-¹⁴C]Succinate behaved similarly to glutamate. [U-¹⁴C]Serine was taken up more rapidly by the kidney than by the liver slices, but the concentrations reached in the liver did not remain below those of the medium. [¹⁴C]Urea was distributed evenly between medium and tissue water. 6. Incubation of liver slices with [³H]inulin indicated an extracellular space of liver slices of 26%. 7. When glutamate was generated within liver slices or the perfused liver on addition of oxaloacetate, pyruvate and a source of nitrogen, the concentration of glutamate in the tissue after 1hr. was 70–97 times that in the medium. Thus the exit of glutamate from the liver cell, like its entry, is restricted. This is borne out by measurements of the specific activity of extra- and intra-cellular glutamate on addition of [U-¹⁴C]glutamate medium. 8. Liver homogenates removed added glutamate and dicarboxylic acids 20–30 times as fast as did the perfused liver. 9. It is concluded that a major permeability barrier restricts the entry and exit through the outer liver cell membrane.     

Sugar transport in rat liver lysosomes. Direct demonstration by using labelled sugars.

Purified rat liver lysosomes ('tritosomes') were prepared from rats injected with Triton WR-1339. 2. The water space of tritosomes, measured by using [3H]water and [14C]sucrose, was 2.15 +/- 0.72 microliter/mg of protein (mean +/- S.E.M., n = 12). 3. Tritosomes, when compared with a crude preparation of normal lysosomes by an indirect method of study, showed sugar specificity but decreased stereospecificity of sugar uptake. 4. At 125 mM the relative rates of net uptake of D-[14C]ribose, D-[14C]- or D-[3H]glucose and 2-deoxy-D-[3H]glucose were the same as that inferred from the indirect study. 5. The entry of D-[3H]glucose into tritosomes showed concentration-dependence suggestive of saturation, with a Km of 48 +/- 18 mM (4). 6. D- and L-glucose, D-ribose, 2-deoxy-D-glucose and D-mannose competed with D-[14C]glucose or D-[14C]ribose for uptake. 7. Cytochalasin B inhibited D-[3H]glucose uptake. 8. Uptake of 1 mM-L-[14C]glucose was slower than for 1 mM-D-[14C]glucose. 9. It is concluded that a facilitated-diffusion transport system is present in purified rat liver lysosomes.     

The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.     

Leakiness of brush-border vesicles

From the water content of pelleted brush-border vesicles and from a comparison of the aqueous volume within the pellet that is available to [3H]inulin (58%), inulin [14C]carboxylic acid (34%, both approx. 5000 daltons), [3H]raffinose (97%, 540 daltons) and [3H]glucose (94%, 180 daltons) it is concluded that only 1 in 4 to 6 of the brush-border vesicles is sealed. The implication of this finding for labelling and transport studies and for vesicle formation is discussed.     

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 & H?ussinger, 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.     

The metabolic significance of pentose cycle measurements in perfused liver

The controversial dissension concerning the nature of the pentose cycle in liver is investigated. The metabolism of [2-14C]Glc and [1-14C]Rib in chronically perfused normal and regenerating rabbit liver and acutely perfused rat liver are used to test the mechanistic predictions and contribution of the F-type pentose cycle. 14C was traced in Glc, Glc 6-P, Fru 6-P, glycogen and Rib 5-P. None of the data complied with the critical theoretical limits set for the C-1/C-3 ratio (the identity badge of the F-type pentose cycle or pathway) for all values of F-type PC from 0-100%. Thus apparent F-type PC measurements using the Katz & Wood method gave a wide scatter of calculated values. The 14C distributions in Rib 5-P do not conform with the predictions of the F-type PC but are in agreement with the many previous results of similar experiments reported by Hiatt and co-workers. In perfused rat liver the C-1/C-3 constants in Glc 6-P and glycogen also failed to conform with F-PC theory following the metabolism of [2-14C]Glc. The metabolism of [5-14C]Glc and distribution of 14C in Glc 6-P and glycogen showed that L-type PC was 18%, in close agreement with a previous published value of 22% for rat hepatocytes. Metabolism of [6-14C]Glc and [4-14C]Glc (as [4,5,6-14C]Glc) showed that Pyruvate Recycling was active in perfused rat liver. None of the data from these comprehensive investigations can confirm the results of the recent study reported by the Landau laboratory on the pentose pathway metabolism of Glc and Rib in perfused rat liver.     

Vectorial production of adenosine by 5'-nucleotidase in the perfused rat heart

Rat hearts were perfused simultaneously with [8-3H] AMP and [8-14C]adenosine. [8-3H] AMP was hydrolzyed by 5'-nucleotidase to produce intra- and extracellular [8-3H] adenosine. Comparison of the specific activities of [3H]- and [14C]adenosine in the heart cells with the specific activities of [3H]- and [14C]adenosine in the effluent perfusate showed that much more [3H]adenosine accumulated in the tissue than would be expected if extracellular adenosine were the immediate precursor of intracellular adenosine. Conversely, perfusion of rat hearts with [8-14C]AMP and [8-3H]adenosine led to a much greater accumulation of intracellular [14C]adenosine than would be expected from an uptake of adenosine from the perfusate. These results are interpreted to be due to hydrolysis of extracellular AMP by 5'-nucleotidase, located in the plasma membrane, and release of the resulting adenosine inside the cell. Measurements of the specific activities of 3H and 14C in ATP, ADP, AMP, and inosine support this interpretation.     

Transport of carnitine by the epididymis of the cynomolgus macaque (Macaca fascicularis)

Unravelled tubules from the monkey caput and cauda epididymidis were perfused through the lumen in vitro during immersion in an organ bath kept at scrotal temperature and containing [3H]carnitine and [14C]inulin. The specific transport of carnitine from the bath to the lumen was constant for 4 h and reached a steady-state value of about 90 pmol/30 min per cm perfused length in the cauda and about 30 pmol/30 min/cm in the caput. These regional variations in carnitine transport differ from those found in the rat epididymis but may be relevant to human epididymal physiology.     

Regulation of the glycine cleavage system in the isolated perfused rat liver

The catabolism of glycine in the isolated perfused rat liver was investigated by measuring the production of 14CO2 from [1-14C]- and [2-14C]glycine. Production of 14CO2 from [1-14C]glycine was maximal as the perfusate glycine concentration approached 10 mM and exhibited a maximal activity of 125 nmol of 14CO2 X g-1 X min-1 and an apparent Km of approximately 2 mM. Production of 14CO2 from [2-14C]glycine was much lower, approaching a maximal activity of approximately 40 nmol of 14CO2 X g-1 X min-1 at a perfusate glycine concentration of 10 mM, with an apparent Km of approximately 2.5 mM. Washout kinetic experiments with [1-14C]glycine exhibited a single half-time of 14CO2 disappearance, indicating one metabolic pool from which the observed 14CO2 production is derived. These results indicate that the glycine cleavage system is the predominant catabolic fate of glycine in the perfused rat liver and that production of 14CO2 from [1-14C]glycine is an effective monitor of metabolic flux through this system. Metabolic flux through the glycine cleavage system in the perfused rat liver was inhibited by processes which lead to reduction of the mitochondrial NAD(H) redox couple. Infusion of beta-hydroxybutyrate or octanoate inhibited 14CO2 production from [1-14C]glycine by 33 and 50%, respectively. Alternatively, infusion of acetoacetate stimulated glycine decarboxylation slightly and completely reversed the inhibition of 14CO2 production by octanoate. Metabolic conditions which are known to cause a large consumption of mitochondrial NADPH (e.g. ureogenesis from ammonia) stimulated glycine decarboxylation by the perfused rat liver. Infusion of pyruvate and ammonium chloride stimulated production of 14CO2 from [1-14C]glycine more than 2-fold. Lactate plus ammonium chloride was equally as effective in stimulating glycine decarboxylation by the perfused rat liver, while alanine plus ammonium chloride was ineffective in stimulating 14CO2 production.     

Intracellular digestion of saturated and unsaturated phospholipid liposomes by mucosal cells. Possible mechanism of transport of liposomally entrapped macromolecules across the isolated vascularly perfused rabbit ileum

The mechanism of intestinal absorption of liposomally entrapped [14C]inulin and 125I-labelled poly(vinylpyrrolidone) was studied using the isolated rabbit intestinal loop with intact perfused vasculature, a system more closely resembling an in vivo system than the everted sac technique. [14C]Inulin or 125I-poly(vinylpyrrolidone) was entrapped in liposomes prepared from unsaturated egg phosphatidylcholine and soya phosphatidylcholine, and saturated distearoylphosphatidylcholine (18:0), dipalmitoylphosphatidylcholine (16:0) and dimyrostoylphosphatidylcholine (14:0). Free and liposomally entrapped macromolecules were introduced in the ileum and the transport of liposomes and entrapped macromolecules into the venous effluent was monitored by measuring the presence of the aqueous marker 125I-poly(vinylpyrrolidone) or [14C]inulin, and lipid marker [3H]cholesterol. The results show that intact liposomes are not transported across intestine into the venous effluent, but they are taken up by mucosal cells and digested intracellularly, releasing the entrapped markers 125I-poly(vinylpyrrolidone) and [14C]inulin. These markers are then transported into the venous effluent as free molecules. The absorption of liposomally entrapped [14C]inulin into the venous effluent is biphasic, first slow for 30 min (i.e., a lag period of 30 min), followed by a rapid linear increase. The duration of the lag period and the rate of absorption of the entrapped [14C]inulin are dependent on the degree of saturation and the transition temperature of the phospholipids used to prepare liposomes. The possible explanation of the lag period based on the evidence presented here is that it is the time required for the liposomes to be taken up by mucosal cells and digested intracellularly. Intracellular digestion of liposomes prepared from saturated phospholipids is more rapid than from those prepared from unsaturated phospholipids, and the greater the fatty acid chain length of the saturated phospholipids the more rapid the intracellular degradation of liposomes.     

Effects of anisotonicity on pentose-phosphate pathway,oxidized glutathione release and t-butylhydroperoxide-induced oxidative stress in the perfused liver of air-breathing catfish,<Emphasis Type="Italic">Clarias batrachus</Emphasis>

Both hypotonic exposure (185 mOsmol/l) and infusion of glutamine plus glycine (2 mmol/l each) along with the isotonic medium caused a significant increase of¹⁴CO₂ production from [1-¹⁴C]glucose by 110 and 70%, respectively, from the basal level of 18.4 ± 1.2 nmol/g liver/min from the perfused liver ofClarias batrachus. Conversely, hypertonic exposure (345 mOsmol/l) caused significant decrease of¹⁴CO₂ production from [1-¹⁴C]glucose by 34%.¹⁴CO₂ production from [6-¹⁴C]glucose was largely unaffected by anisotonicity. The steady-state release of oxidized glutathione (GSSG) into bile was 1.18 ±0.09 nmol/g liver/min, which was reduced significantly by 36% and 34%, respectively, during hypotonic exposure and amino acid-induced cell swelling, and increased by 34% during hypertonic exposure. The effects of anisotonicity on¹⁴CO₂ production from [1-¹⁴C]glucose and biliary GSSG release were also observed in the presence of t-butylhydroperoxide (50 (Amol/1). The oxidative stress-induced cell injury, caused due to infusion of t-butylhydroperoxide, was measured as the amount of lactate dehydrogenase (LDH) leakage into the effluent from the perfused liver; this was found to be affected by anisotonicity. Hypotonic exposure caused significant decrease of LDH release and hypertonic exposure caused significant increase of LDH release from the perfused liver. The data suggest that hypotonically-induced as well as amino acid-induced cell swelling stimulates flux through the pentose-phosphate pathway and decreases loss of GSSG under condition of mild oxidative stress; hypotonically swollen cells are less prone to hydroperoxide-induced LDH release than hypertonically shrunken cells, thus suggesting that cell swelling may exert beneficial effects during early stages of oxidative cell injury probably due to swelling-induced alterations in hepatic metabolism.     

Urea synthesis and CO2/HCO3- compartmentation in isolated perfused rat liver

With physiological portal HCO3- and CO2 concentrations of 25mM and 1.2mM in the perfusate, respectively, acetazolamide inhibited urea synthesis from NH4Cl in isolated perfused rat liver by 50-60%, whereas urea synthesis from glutamine was inhibited by only 10-15%. A decreased sensitivity of urea synthesis from glutamine to acetazolamide inhibition was also observed when the extracellular HCO3- and CO2 concentrations were varied from 0-50mM and 0-2.4mM, respectively. Stimulation of intramitochondrial CO2 formation at pyruvate dehydrogenase with high pyruvate concentrations (7mM) was without effect on the acetazolamide sensitivity of urea synthesis from NH4Cl. Urea synthesis was studied under conditions of a limiting HCO3- supply for carbamoyl-phosphate synthesis. In the absence of externally added HCO3- or CO2, when 14CO2 was provided intracellularly by [U-14C]glutamine or [1-14C]-glutamine oxidation, acetazolamide had almost no effect on label incorporation into urea, whereas label incorporation from an added tracer H14CO3- dose was inhibited by about 70%. 14CO2 production from [U-14C]glutamine was about twice as high as from [1-14C]glutamine, indicating that about 50% of the CO2 produced from glutamine is formed at 2-oxoglutarate dehydrogenase. The fractional incorporation of 14CO2 into urea was about 13% with [1-14C]-as well as with [U-14C]glutamine. Addition of small concentrations of HCO3- (1.2mM) to the perfusate increased urea synthesis from glutamine by about 70%. This stimulation of urea synthesis was fully abolished by acetazolamide. The carbonate-dehydratase inhibitor prevented the incorporation of added HCO3- into urea, whereas incorporation of CO2 derived from glutamine degradation was unaffected. Without HCO3- and CO2 in the perfusion medium, when 14CO2 was provided by [1-14C]-pyruvate oxidation, acetazolamide inhibited urea synthesis from NH4Cl as well as 14C incorporation into urea by about 50%. Therefore carbonate-dehydratase activity is required for the utilization of extracellular CO2 or pyruvate-dehydrogenase-derived CO2 for urea synthesis, but not for CO2 derived from glutamine oxidation. This is further evidence for a special role of glutamine as substrate for urea synthesis.