Ultrastructural changes during cholestasis induced by chlorpromazine in the isolated perfused rat liver.

Addition of cholestatic doses of chlorpromazine-HC1 to the perfusate of isolated rat livers produces widespread changes in hepatocyte membrane structure. These findings include a marked increase in intrasinusoidal cytoplasmic bullae, appearance of intracellular vacuoles within hepatocytes at both sinusoidal and biliary poles, dilation of bile canaliculi and evagination of canalicular diverticuli, and the formation of myeloid bodies within hepatocytes. These findings obtained in the bile acid depleted perfused liver may result from physiochemical interactions between chlorpromazine or its metabolites and lipid-protein components of cell membranes, consistent with chlorpromazine's properties as a cationic detergent. They occur independently of the vasoconstrictive effects of chlorpromazine and suggest that chlorpromazine may produce cholestasis by altering hepatocyte membrane function.     

Effects of bile salt infusion on chlorpromazine-induced cholestasis in the isolated perfused rat liver.

The present study has demonstrated that tauroursodeoxycholate (TUDC), but not taurocholate, can reverse chlorpromazine (CPZ)-induced cholestasis in the isolated perfused rat liver. At an infusion rate of 1.5 mumol/min, TUDC led to restoration of bile flow in the perfused rat liver made cholestatic by the addition of 250 microM CPZ. This reversal was accompanied by an increased excretion of CPZ and its metabolites. A higher infusion rate of 5.0 mumols TUDC/min, however, led to only a transient increase in bile flow and to no increase in CPZ excretion. In contrast to the effects of TUDC, infusion of taurocholate led to an exacerbation of CPZ-induced cholestasis. The differences in the efficacy of the two bile salts may be due to their relative detergent (hydrophobic) properties.     

Cell surface changes and enzyme release during hypoxia and reoxygenation in the isolated, perfused rat liver

We examined the effects of hypoxia and reoxygenation in isolated, perfused rat livers. Hypoxia induced by a low rate of perfusion led to near anoxia confined to centrilobular regions of the liver lobule. Periportal regions remained normoxic. Within 15 min, anoxic centrilobular hepatocytes developed surface blebs that projected into sinusoids through endothelial fenestrations. Periportal hepatocytes were unaffected. Both scanning and transmission electron microscopy suggested that blebs developed by transformation of preexisting microvilli. Upon reoxygenation by restoration of a high rate of perfusion, blebs disappeared. Other changes included marked shrinkage of hepatocytes, enlargement of sinusoids, and dilation of sinusoidal fenestrations. There was also an abrupt increase in the release of lactate dehydrogenase and protein after reoxygenation, and cytoplasmic fragments corresponding in size and shape to blebs were recovered by filtration of the effluent perfusate. We also studied phalloidin and cytochalasin D, agents that disrupt the cytoskeleton. Both substances at micromolar concentrations caused rapid and profound alterations of cell surface topography. We conclude that hepatic tissue is quite vulnerable to hypoxic injury. The morphological expression of hypoxic injury seems mediated by changes in the cortical cytoskeleton. Reoxygenation causes disappearance of blebs and paradoxically causes disruption of cellular volume control and release of blebs as cytoplasmic fragments. Such cytoplasmic shedding provides a mechanism for selective release of hepatic enzymes by injured liver tissue.     

Xylitol metabolism in the isolated perfused rat liver

1. Loading the isolated perfused liver from well-fed rats with xylitol (20mm) caused a depletion of adenine nucleotides and P_i and an accumulation of α-glycerophosphate. The ATP content fell to 66% of the control value after 10min and to 32% after 80min. The ADP and AMP contents also fell. After 80min 63% of the total adenine nucleotides and 59% of the P_i had been lost. 2. The α-glycerophosphate content rose from 0.13 to 4.74μmol/g at 10min and reached 8.02μmol/g at 40min. 3. Xylitol was rapidly metabolized, the main products being glucose, lactate and pyruvate. 4. The [lactate]/[pyruvate] ratio in the presence of xylitol rose to 30–40. 5. On perfusion of livers from starved animals the main product of xylitol metabolism was glucose and the mean ratio xylitol removed/glucose formed was 1.29 (corrected for endogenous glucose and lactate production). This is close to the predicted value of 1.2. 6. Evidence is presented indicating that the loss of adenine nucleotides caused by xylitol is not due to the increased ATP consumption but to the accumulation of α-glycerophosphate and depletion of P_i. 7. The loss of adenine nucleotides accounts for the hyperuricaemia which can occur after xylitol infusion in man. 8. The relevance of the findings to the clinical use of xylitol as an energy source is discussed.     

Serum antitrypsin synthesis by the isolated perfused rat liver.

The isolated perfused rat liver synthesizes antitrypsin activity in a linear fashion as long as 24 hr. The rate of synthesis may be ten times the rate necessary for physiologic levels in vivo. These data are compatible with the liver as the principal site of synthesis of serum antitryptic activity in the rat. The antitrypsin activity of the rat perfusate resembles that of normal human plasma in liability to heat (56 degrees) and to acid (below pH 6) and in apparent molecular size (elution from Sephadex G-200).     

Hepatocyte heterogeneity in glutamate uptake by isolated perfused rat liver

Glutamate is simultaneously taken up and released by perfused rat liver, as shown by 14CO2 production from [1-14C]glutamate in the presence of a net glutamate release by the liver, turning to a net glutamate uptake at portal glutamate concentrations above 0.3 mM. 14CO2 production from portal [1-14C]glutamate is decreased by about 60% in the presence of ammonium ions. This effect is not observed during inhibition of glutamine synthetase by methionine sulfoximine. 14CO2 production from [1-14C]glutamate is not influenced by glutamine. Also, when glutamate accumulates intracellularly during the metabolism of glutamine (added at high concentrations, 5 mM), 14CO2 production from [1-14C]glutamate is not affected. If labeled glutamate is generated intracellularly from added [U-14C]proline, stimulation of glutamine synthesis by ammonium ions did not affect 14CO2 production from [U-14C]proline. After induction of a perivenous liver cell necrosis by CCL4, i.e. conditions associated with an almost complete loss of perivenous glutamine synthesis but no effect on periportal urea synthesis, 14CO2 production from [1-14C]glutamate is decreased by about 70%. The results are explained by hepatocyte heterogeneity in glutamate metabolism and indicate a predominant uptake of glutamate (that reaches the liver by the vena portae) by the small perivenous population of glutamine-synthesizing hepatocytes, whereas glutamate production from glutamine or proline is predominantly periportal. In view of the size of the glutamine synthetase-containing hepatocyte pool [Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567-570], glutamate transport capacity of these hepatocytes would be about 20-fold higher as compared to other hepatocytes.     

Futile cycles in isolated perfused rat liver and in isolated rat liver parenchymal cells.

Isolated livers from fed rats were perfused with a medium containing glucose labeled uniformly with ¹⁴C and specifically with ³H. There was considerable formation of glucose from endogenous sources but simultaneously uptake of about half of the ¹⁴C in glucose. After 2 hours the ${}^3\text{H}{}^{14}\text{C}$ ratios in perfusate glucose decreased by 55–60% with (2-³H, U-¹⁴C), 40–50% with (5-³H, U-¹⁴C), 25–30% with (3-³H or 4-³H, U-¹⁴C) and by 10–15% with (6-³H, U-¹⁴C) glucose. Qualitatively comparable patterns were obtained with rat hepatocytes. These results demonstrate recycling of carbon between glucose and pyruvate. Superimposed upon this there is an extensive futile cycle between glucose and glucose 6-P. There is also futile cycling between fructose 6-P and fructose 1,6 P₂ and to a small extent between phosphoenol pyruvate and pyruvate.     

Naproxen-induced oxidative stress in the isolated perfused rat liver

We previously showed that naproxen induced the oxidative stress in the liver microsomes and the isolated hepatocytes of rats. In this study, the in situ effect of naproxen on the rat liver tissue was investigated, using the isolated perfused liver from the view-point of the naproxen-induced hepatotoxicity. The leakage of glutamic-oxaloacetic transaminase (GOT) from the perfused liver and appearance of thiobarbituric acid reactive substances (TBARS) in the perfusate increased with the progress of perfusion after a lag time of about 1h. The naproxen-perfusion of the liver decreased the biliary excretion of glutathione (GSH) and oxidized glutathione, glutathione disulfide (GSSG) prior to TBARS production and GOT leakage. GSSG content in the naproxen-perfused liver was significantly higher than in the control. TBARS appeared in the perfusate of the naproxen-perfused liver for 30 min, but not in the control. The biliary excretion clearance (CL(bile)) of indocyanine green (ICG), a reagent for testing the liver function, in the liver perfused with naproxen decreased to a half of that in the liver perfused without naproxen. Thus, the naproxen-induced oxidative stress in the liver was shown to affect the physiological function of liver through the impairment of biliary excretion, which is recognized as a detoxification system.     

Uptake of asialo-glycophorin by the perfused rat liver and isolated hepatocytes

Perfused rat livers took up asialo-glycophorin, a glycoprotein derived from human erythrocyte membraneds, with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the clearance of 7 min. As a comparison, asialo-orosomucoid was taken up by this system with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 3.5 min. Both proteins were digested and their ¹²⁵I labels were released to the perfusate as free ¹²⁵I^?. EGTA completely inhibited uptake of these glycoproteins, but not uptake of denatured bovine serum albumin. Addition of Ca²⁺ reversed the inhibition nearly completely. Isolated hepatocytes had an uptake rate of approximately 3 ng/min per 10⁶ cells for the asialo forms of glycophorin, orosomucoid and fetuin. Cellular uptake of each of these asialoglycoproteins could be inhibited by one of the other proteins. Asialo-fetuin caused a 95% inhibition of the uptake rate of asialo-orosomucoid by the perfused liver. This fetal calf glycoprotein had a similar inhibitory effect on asialo-glycophorin, but only after an initial 40% of the asialo-glycophorin had been taken up by the liver at an almost normal rate during the first 30 min of perfusion. The possiblity of an alternative hepatic removal system for asialo-glycophorin is suggested.     

Synthesis of factor II antigen by isolated perfused rat liver

Rat Factor II (prothrombin), isolated and purified by chromatography on Blue Dextran-agarose, was used to raise an antiserum in rabbits. On the basis of single radial immunodiffusion measurements. Factor II synthesis by isolated perfused rat liver amounted to 0.54 mg/300 cm² body surface area of the liver donor in 10 h. Corresponding measurements of Factor II coagulant activity revealed cumulative synthesis of 802 Iowa units. Coumadin added to the liver perfusate blocked production of Factor II coagulant activity, but did not change synthesis of the immunologically measured protein. In perfusions in which either heparin or citrate was used as anticoagulant, synthesis of albumin was not affected by the choice of anticoagulant but bile production and synthesis of Factor II were significantly less in citrate perfusions.