Thyroid calorigenesis in isolated, perfused rat liver: minor role of active sodium-potassium transport.

1. The effects of ouabain on hepatic oxygen uptake, cell membrane potential, and Na-K transport were examined at 37 degrees C during non-recirculating perfusion of isolated livers from fasted normal rats and rats treated with triiodothyronine (T3). The perfusate was Krebs-Ringer bicarbonate buffer containing albumin and bovine erythrocytes. 2. Treatment with T3 increased the rate of hepatic oxygen uptake by 30% (i.e. by 0-83 (micromole/min) per gram liver). 3. After shifting to perfusate containing 2-5 mM ouabain, a 4-5 mV depolarization and maximal rates of net hepatic K release and Na uptake occurred within 2 min in both thyroid states. These changes were not accompanied by any significant change in the rates of hepatic oxygen uptake. 4. T3-treatment increased the maximal, post-ouabain net flux of K by 29% (i.e. by 0-52 (muequiv/min) per gram liver). The T3-indlced increase in the net flux of Na (19%) did not achieve statistical significance. 5. In either thyroid state, the observed passive fluxes of Na and K were calculated to be balanced by active vluxes at the expense of 5-6% of the observed rate of hepatic oxygen uptake. 6. The results indicate that hyperthyroidism may enhance the rate of hepatic Na-K transport, but the energy expenditure due to this process appears to be too small to make any important contribution to thyroid calorigenesis in perfused rat liver.     

Insulin removal by isolated perfused rat liver.

Removal of unlabeled insulin was studied in the perfused rat liver. Insulin removal followed first-order kinetics over the range of concentrations found in the portal vein of postabsorptive rats, but deviated from first-order kinetics in experiments with a wider concentration range. Clearance was more than twice as great at concentrations normally found in the portal vein in the postabsorptive state (0.40-1.1nM or about 60-100 muU/ml) than at concentrations expected after pancreatic stimulation (4.5-7.0 nM). Saturation of the liver's capacity to remove insulin, however, was not observed even at higher levels. Insulin clearance diminished when the flow rate was reduced. It was not significantly altered by prolonged starvation. Our results suggest that when the insulin concentration is high a greater percentage escapes hepatic degradation then when it is low. Hepatic insulin clearance is in part dependent on the portal flow rate. The kinetics of insulin removal by the perfused liver cannot be accounted for by the properties of insulin-degrading enzymes described by others.     

Amino acid metabolism in the isolated perfused rat liver

1. Rat livers have been perfused with homologous blood and the distribution of (15)N from labelled amino acids added to the perfusate studied.2. Each of the amino acids added, aspartate, alanine, glycine, and glutamate (unlabelled) were rapidly removed from the perfusate. Glutamate was formed from added aspartate (but not from added alanine or glycine) by transamination in the perfusate plasma; the perfusate concentration of no other amino acid was affected by addition of any of the acids investigated.3. Urea output was increased by addition of aspartate, increase in output after addition of alanine or glycine was not significant.4. After addition of labelled aspartate, alanine or glycine an increase in isotope ratio was detected in urea, ammonia, glutamine, aspartate and alanine in the perfusate and, in some experiments, in the liver. After addition of alanine an increase was also detected in lysine, tyrosine, phenylalanine, methionine, proline, tryptophan and threonine+serine (analysed together).     

Hepatotoxicity of d-Galactosamine in the isolated perfused rat liver

The hepatotoxicty of d-galactosamine was evaluated using the isolated perfused rat liver. In controls, ATP- and UDPglucose-levels decreased by about 20% during 12 hr of perfusions; UTP + UDP as well as the energy charge remained constant. d-Galactosamine reduced the contents of UDPglucose and UTP + UDP to 10 to 20% of normal within 30 min, whereas the sum of all acid-soluble uracil nucleotides increased linearly for the first 8 hr. The leakage of intracellular enzymes into the perfusion medium was small and almost linear in controls; in livers treated with d-galactosamine, however, a sharp increase occurred after 6 hr. Addition of uridine 3 hr after d-galactosamine prevented cell damage as judged by enzyme release. E. coli endotoxin, either alone or after preincubation with rat serum, did not enhance enzyme leakage. Lightmicroscopically, liver cell plates were partially dissociated and necroses of groups of liver cells were found in galactosamine-treated livers. In electron microscopy, degenerative changes of hepatocytes were seen after galactosamine perfusion.     

Ouabain-mediated sodium uptake and bile formation by isolated perfused rat liver.

Ouabain exhibits a dose-dependent choleretic effect in the isolated perfused rat liver. Its uptake from the perfusate into the liver is maintained against a concentration gradient and becomes clearly saturated at higher perfusate concentrations. A low extracellular sodium concentration inhibits the rate of ouabain transfer into liver cells, resulting in a marked decrease of the maximal transport rate. Dibucaine completely abolishes the uptake of the glycoside by the isolated liver. Determination of Na-22 tracer fluxes suggests that ouabain uptake is accompanied by a net flux of sodium into the cell, which seems to be due to a cotransport of sodium with ouabain rather than to the inhibition of the sinusoidal Na+ -K+ -ATPase. Sodium introduced into the cell in this way apparently is extruded into the bile canaliculi. The increase of isotonic bile flow, which is simultaneously observed, points to a dilution of the canalicular sodium gradient by water and electrolytes through an intercellular pathway. Our results present further evidence that bile secretion is controlled by transcellular sodium movements.     

Sodium dependence of carnitine transport in isolated perfused adult rat hearts

In heart muscle, the intracellular carnitine concentration is approximately 40 times higher than the plasma carnitine concentration, suggesting the existence of an active transport process. At physiological serum carnitine concentrations (44 microM), 80% of total myocardial carnitine uptake occurs via a carrier-mediated transport system. The mechanism of this carrier-mediated transport was studied in isolated perfused rat hearts. Carnitine transport showed an absolute dependence on the extracellular sodium concentration. The rate of carnitine transport was linearly related to the perfusate sodium concentration at every perfusate carnitine concentration examined (15-100 microM). Total removal of extracellular sodium completely abolished the carrier-mediated transport. Decreasing the perfusate potassium concentration from a control of 5.9 to 0.6 mM stimulated transport by 35%, whereas increasing the extracellular potassium concentration from 5.9 to 25 mM reduced transport by 60%. The carrier-mediated transport was inversely proportional to the extracellular potassium concentration. Acetylcholine (10(-3) M), isoproterenol (10(-7) M), or ouabain (10(-3) did not alter the rate of carnitine transport. Addition of tetrodotoxin (10(-5) stimulated carnitine transport by about 40%, while gramicidin S (5 X 10(-6) M) decreased uptake by about 18% relative to control. The data provide evidence that carnitine transport by cardiac cells occurs by a Na+-dependent cotransport mechanism that is dependent on the Na+ electrochemical gradient.     

Galactose metabolism in isolated perfused suckling-rat liver.

The metabolic conversion of 1, 2, or 4 mM galactose to glucose was studied in isolated livers of suckling rats. Whereas galactose uptake during perfusion with 1 and 2 mM galactose was linear throughout the 90-min experiment, uptake was delayed for 35 min when 4 mM galactose was perfused. Studies with radioactive galactose revealed a parallel disappearance of galactose and the appearance of [14C]glucose; about 80% of the galactose taken up was converted to glucose. Galactose perfusion appeared to reduce the basal amount of glucose derived from substrates other than galactose. The specific activities in the galactose-perfused livers of the three major galactose metabolizing enzymes, galactokinase, galactose-1-phosphate uridylyltransferase, and uridine diphosphogalactose-4-epimerase, revealed that the transferase was significantly lower, whereas that of galactokinase and epimerase were significantly higher than in livers perfused without galactose. No meaningful changes were observed in the levels of either phosphorylated or uridylated hexoses in these studies.     

Insulin and cortisol improve heat tolerance in isolated perfused rat liver

Isolated rat livers were perfused at 37 degrees, 41 degrees, 42 degrees, and 43 degrees C with and without insulin and cortisol. Two additional groups were perfused at 42 degrees C with either hormone alone. The perfusate contained red blood cells, amino acids, and albumin in Krebs-Ringer bicarbonate. Bile flow was significantly increased by hormones at 37 degrees C. Bile flow was also increased by hormones at all other temperatures. At 41 degrees C, K+ leakage was the only parameter that indicated injury. Insulin and cortisol significantly reduced K+ leakage at this temperature compared to those without hormones. At 42 degrees C, insulin and cortisol reduced K+ leakage, increased bile flow, reduced transaminase release, and improved ultrastructural integrity. The enhanced bile flow was due primarily to insulin. A reduction in K+ leakage required both insulin and cortisol. Transaminase leakage responded to either hormone alone or in combination; however, only the cortisol-treated group showed a statistically significant reduction in transaminase leakage. At 43 degrees C, indications of irreversible injury were evident and hormones had no beneficial effects. Loss of membrane homeostasis appeared to be the initial event.