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 NH4C1 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 pmol/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. 4. It is concluded that (a) hepatic glutamine metabolism is under the control of liver-cell volume and/or volume-regulatory responses, thereby involving flux changes through glutaminase and glutamine synthetase, i.e. alterations of intercellular glutamine cycling; (b) volume-regulatory mechanisms may be signals for switching carbohydrate utilization to glutamine utilization as respiratory fuel and vice versa; and (c) the concentrative uptake of glutami...

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 NH4CI (0.5 mmol/l) by about 60% and a decrease of hepatic oxygen uptake by 0.43 f 0.03 pmol g-min-[from 3.09 f 0.13 pmol g-' min-' to 2.66 0.12 pmol g -l min-' (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 NH4C1 (0.2 mmol/l) was infused.2. The inhibition of urea synthesis from NH4C1 (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 NH4C1 (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.4. The data are consistent with a cell-swelling-induced inhibition of the transfer of malate and reducing equivalents into the mitochondria1 compartment, thereby inhibiting urea synthesis due to a limitation of aspartate supply for argininosuccinate synthesis. They suggest that alterations of liver cell volume and/or volume regulatory responses exert marked effects on hepatic ammonia, amin...