The interaction between cell volume and taurocholate excretion into bile was studied in isolated perfused rat liver. Cell swelling due to hypo-osmotic exposure, addition of amino acids or insulin stimulated taurocholate excretion into bile and bile flow, whereas hyperosmotic cell shrinkage inhibited these. These effects were explained by changes in Vmax of taurocholate excretion into bile: Vmax. increased from about 300 to 700 nmol/min per g after cell swelling by 12-15% caused by either hypo-osmotic exposure or addition of amino acids under normo-osmotic conditions. Steady-state taurocholate excretion into bile was not affected when the influent K+ concentration was increased from 6 to 46 mM or decreased to 1 mM with iso-osmoticity being maintained by corresponding changes in the influent Na+ concentration. Replacement of 40 mM-NaCl by 80 mM-sucrose decreased taurocholate excretion into bile by about 70%; subsequent hypo-osmotic exposure by omission of sucrose increased taurocholate excretion to 160%. Only minor, statistically insignificant, effects of aniso-osmotic cell volume changes on the appearance of bolus-injected horseradish peroxidase in bile were observed. Taurocholate (400 microM) exhibited a cholestatic effect during hyperosmotic cell shrinkage, but not during hypo-osmotic cell swelling. Both taurocholate and tauroursodeoxycholate increased liver cell volume. Tauroursodeoxycholate stimulated taurocholate (100 microM) excretion into bile. This stimulatory effect was strongly dependent on the extent of tauroursodeoxycholate-induced cell swelling. During continuous infusion of taurocholate (100 microM) further addition of tauroursodeoxycholate at concentrations of 20, 50 and 100 microM increased cell volume by 10, 8 and 2% respectively, in parallel with a stimulation of taurocholate excretion into bile by 29, 27 and 9% respectively. There was a close relationship between the extent of cell volume changes and taurocholate excretion into bile, regardless of whether cell volume was modified by tauroursodeoxycholate, amino acids or aniso-osmotic exposure. The data suggest that: (i) liver cell volume is one important factor determining bile flow and biliary taurocholate excretion; (ii) swelling-induced stimulation of taurocholate excretion into bile is probably not explained by alterations of the membrane potential; (iii) bile acids modulate liver cell volume; (iv) taurocholate-induced cholestasis may depend on cell volume; (v) stimulation of taurocholate excretion into bile by tauroursodeoxycholate can largely be explained by tauroursodeoxycholate-induced cell swelling.

An increase of the hepatocellular hydratation state, induced by hypotonic exposure, amino acids or tauroursodeoxycholate, was shown to increase within minutes the Vmax of transcellular taurocholate transport and excretion into bile [Häussinger, Hallbrucker, Saha, Lang and Gerok (1992) Biochem. J. 288, 681-689]. This stimulatory effect of cell swelling on taurocholate excretion into bile is abolished in the presence of colchicine (5 microM). On the other hand, colchicine did not affect the stimulatory action of hypotonic cell swelling on 14CO2 production from [1-14C]glycine or [1-14C]glucose. Likewise, volume regulatory K+ fluxes following anisotonic exposure were not influenced in the presence of colchicine. Lumicolchicine (5 microM), a stereoisomer of colchicine without an inhibitory effect on microtubules, did not abolish the stimulation of taurocholate excretion into bile following hypo-osmotic exposure. Hypertonic cell shrinkage decreased taurocholate excretion into bile by about 35%; this effect was fully reversible upon normotonic re-exposure. With colchicine pretreatment, however, the hypertonicity-induced inhibition of taurocholate excretion was blunted and was no longer reversible upon normotonic re-exposure. The results suggest that stimulation of taurocholate excretion into bile in response to cell swelling involves a colchicine-sensitive, probably microtubule-dependent, mechanism, but not the stimulation of other cell-volume-sensitive pathways such as glycine oxidation or the pentose-phosphate shunt. It is hypothesized that the swelling-induced stimulation of taurocholate excretion into bile is due to a microtubule-dependent insertion of bile acid transporter molecules into the canalicular membrane.

Exposure of isolated single-pass-perfused rat liver to hypo-osmotic media resulted in liver cell swelling and an inhibition of release of branched-chain amino acids. Similarly, cell swelling inhibited [3H]leucine release from perfused livers from rats in which liver proteins were prelabelled in vivo by intraperitoneal injection of L-[4,5-3H]leucine 16-20 h before the experiment. The effects of cell swelling on [3H]leucine release were fully reversible. [3H]Leucine release was also inhibited when cell swelling was induced by addition of glutamine (0.5-2 mM). There was a close relationship between the inhibition of [3H]leucine release and the degree of liver cell swelling, regardless of whether cell swelling was induced by hypo-osmotic perfusion or addition of glutamine. The data suggest that the known anti-proteolytic effect of glutamine is in large part due to glutamine-induced hepatocyte swelling.

1. Proteolysis in isolated perfused rat liver was monitored as [3H]leucine release into effluent perfusate after in vivo labeling by intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. Exposure of the livers to hypotonic perfusion media (175 -295 mOsmol . 1-I) increased liver mass due to cell swelling and inhibited [3H]leucine release. The extent of inhibition of [3H]leucine release was linearly related to the liver-mass increase, regardless of whether livers from fed or 24-h-starved rats were studied.2. Infusion of glycine (0.5-3 mmol . 1-I) or glutamine (0.5-3 mmol . 1-') during normotonic perfusions (305 mOsmol . 1-') led to a concentration-dependent increase of liver mass and inhibition of [3H]leucine release. The inhibition of [3H]leucine release was again strongly dependent upon the increase of liver mass, regardless of whether cell swelling was induced by glutamine or glycine in normotonic perfusions, by exposure of the liver to hypotonic media or whether amino-acid-induced cell swelling was modified by the nutritional state. The effects of glutamine and glycine on [3H]leucine release were additive to the same extent as that found when the liver-mass increase was observed.3. Alanine, serine and proline inhibited [3H]leucine release in parallel to the extent of amino-acid-induced liver-mass increase; however, the inhibition of [3H]leucine release was about twice that found when comparable degrees of cell swelling were induced either by hypotonic exposure or by addition of glutamine or glycine. The relationship between alanine-induced liver-mass increase and the inhibition of [3H]leucine release was also maintained in presence of aminooxyacetate (0.2 mmol . 1-I).4. Infusion of an amino acid mixture, roughly mimicking the concentrations found in portal venous blood, to livers from 24-h-starved or fed rats inhibited [3H]leucine release by 56.0 & 2.4% (n = 6) or 31.1 f 2.3% (n = 3), respectively, and increased liver mass by 5.0 & 0.1 YO (n = 6) or 2.2 f 0.3% (n = 3), respectively. Regardless of the nutritional state, there was a close relationship between the amino-acid-mixture-induced (and also phenylalanineinduced) increase of liver mass and the degree of inhibition of [3H]leucine release; however, the inhibition of [3H]leucine release was about fourfold higher than that found when comparable degrees of cell swelling were induced by hypotonic exposure. The amino-acid-mixture-induced inhibition of [3H]leucine release was reversed by hypertonic (385 mOsmol . 1-I) cell shrinkage and stimulated by glutamine/glycine-induced swelling to an cxtcnt predicted by the relationship between liver mass and anisotonic cell swelling.5. The data are consistent with the following: (a) a role of liver-cell volume in the control of hepatic proteolysis; (b) inhibition of proteolysis by glutamine and glycine can largely be ascribed to cell-volume changes, whereas the effects of alanine, serine, proline and especially of phenylalanine or of an amino acid mixture on proteolysis can only be explain...

Hepatic proteolysis is inhibited by insulin, araino acids and hypoosmotic cell swdlinll and is stimulated by ljluCal~on. Thc~ ©ffeetor~ simultaneously modulate cell volume in the intact liver, as shown by measurements 0f the intraeellular water space. A close relationship exists between tile effect on prot¢01ysis and the accompanyin$ cell volume chanlle, regardless t~f whether hepatic proteolysis was modified by Insulin, lllUCallon, cyclic AMP, iltlatamine, 81ycine, barium of hypo0~motic ¢~xposure. It i~ sullsested that cell volume changes exerted by hormones and amino acids play a crucial role in the rel~ulation of hepatic prot¢olysis,