Bile acid transport and metabolism in rat liver slices

To further characterise precision-cut liver slices from 34- to 40-day-old male rats as an in vitro model for bile acid (BA) metabolism and transport, the effect of the primary BAs cholic (CA, 5 microM) and chenodeoxycholic acid (CDCA, 0.15 and 0.75 microM) as well as of the therapeutically used tauroursodeoxycholic acid (T-UDCA, 5 microM) on BA profiles was investigated. After 4 h incubation in 5 ml Krebs-Henseleit buffer (KHB) 26 individual BAs were determined in slices (50 mg liver/5 ml KHB) and medium by HPLC with postcolumn derivatisation and fluorescence detection. In control incubations, mean total BA concentrations were 5.09 nmol/50 mg liver (101.80 nmol/g liver) in slices and 25.71 nmol/5 ml KHB, among them 72% taurine-(T-), 22% glycine-(G-) conjugated and 6% free BAs in tissue and medium. The main BAs were beta-muricholic (beta-MCA and conjugates) and cholic acids (CA and conjugates) in tissue and medium. The following results were obtained after addition of CDCA, CA, and T-UDCA, respectively, to the KHB. The toxic CDCA was quantitatively converted mainly to T-UDCA and taurohyodeoxycholic (T-HDCA) acid. CA was conjugated in equal shares to T- and G-CA, whereas T-UDCA was enriched in slices and hydroxylated half to T-beta-MCA, which is the main BA in rats. In conclusion, rat liver slices are highly effective not only in uptake, conjugation and excretion of BAs but also in conversion of strong detergent into less toxic BAs.     

pH-temperature dependence of organic acid transport in rat kidney slices.

Uptake of p-aminohippurate (PAH) by rat kidney slices was optimal at a pH of around 7.3 when incubation temperature was 37 degrees C. At 25 degrees C, however, optimal pH was increased to about 7.5. When the uptake was evaluated as a function of the OH/H ratio of the medium, it was found that optimal uptake is at a constant OH/H. Data showing the same characteristics of OH/H dependence were also observed for phenol red. Passive uptake of PAH was pH independent. Efflux rates for PAH also showed the pH optimal which was identical to that of uptake. At both 25 and 37 degrees C the Km appeared to be independent of pH while the maximum rate of active influx. An optimal OH/H ratio determines maximal velocity and this ratio is fixed and independent of temperature.     

Stimulated amino acid imbalance and histidine transport in rat brain slices.

Histidine concentration in the brain decreases rapidly when rats are fed a low protein diet in which an amino acid imbalance is created by addition of an amino acid mixture devoid of histidine. Competition for histidine transport into the brain was suggested as an explanation for this effect. Therefore, animo acid mixtures simulating composition of plasma from rats fed basal or histidine-imbalanced diets were added to media to evaluate their effects on uptake of histidine by brain slices during a 60-min incubation period. At the concentrations actually found in plasma, the unbalanced mixture decreased histidine uptake significantly more than did the basal mixture. Two distinct inhibition patterns were observed with different groups of amino acids: a linear decrease in histidine uptake with a mixture of the small neutral, hydroxyl, basic, and acidic amino acids, and a hyperbolic decrease with a mixture of large neutral amino acids, and a hyperbolic decrease with a mixture of large neutral amino acids. Inhibition of histidine transport by the complete mixtures reflected these two effects. Plasma patterns and concentrations of competitive amino acids as well as the concentration of histidine appear to be factors involved in decreasing histidine transport into the brain.     

Canalicular bile salt-independent bile formation: concepts and clues from electrolyte transport in rat liver

Studies on canalicular electrolyte transport are reviewed with reference to the concept that hepatocellular inorganic ion secretion may provide an osmotic drive for canalicular water flow. Cellular transport of electrolytes and of some nonelectrolytes appears directly or indirectly (cotransport or potential-sensitive transport) related to the activity of Na+-K+-ATPase of the sinusoidal cell membrane, but the role of the enzyme in regulating bile flow remains undetermined. Bile secretion of the isolated rat liver continues in the absence of either Na+, K+, Cl-, or HCO-3 when these ions are replaced in the perfusion medium by other permanent ions. Transepithelial salt concentration gradients, established experimentally, cause transient changes of bile flow and dissipate very quickly. Isotopic ion equilibration between sinusoids and bile proceeds faster than between sinusoids and liver cells. Both observations indicate extensive electrolyte diffusion through a paracellular shunt pathway. This pathway appears preferentially permeable to cations, and it restricts permeation of molecules of the size of sucrose (no apparent diffusion or effects of solvent drag) or bile acids (no backleak). In promoting canalicular osmotic water flow, transepithelial concentration gradients of NaCl are less effective than those of sucrose, revealing a reflection coefficient of NaCl of 0.3. By perfusion with hypertonic medium containing sucrose, bile flow is reduced. Bile production against this opposing osmotic gradient is accomplished by an increase in biliary organic anion concentration. Inorganic ion concentrations essentially conform to a Gibbs-Donnan distribution across the canalicular epithelium, established by the presence of impermeant anions in bile. Hence, the luminal electrical potential is expected to be negative with respect to the sinusoids. It is concluded that biliary secretion of endogenous organic anions is the major osmotic driving force for canalicular bile salt-independent bile flow and that transport of inorganic ions into bile results mainly from diffusion and solvent drag.     

Effects of bile acids on biliary epithelial cell proliferation and portal fibroblast activation using rat liver slices

During cholestasis, bile acids accumulate in the liver, and induce cellular alterations. Cholestasis is a major cause of liver fibrosis. We have used precision-cut liver slices (PCLS) in culture to investigate the effects of bile acids on hepatic cells. Rat PCLS were placed on an insert in a vial containing culture medium, and gently agitated on a roller platform. PCLS were treated with 100 microM taurolithocholate (TLC), taurodeoxycholate (TDC) or taurocholate (TC) for 24 or 48 h. PCLS viability was measured, and immunohistochemistry was performed with antibodies against active caspase 3, platelet-derived growth factor (PDGF) receptor-beta and ED-A fibronectin. TDC and TLC, two hydrophobic bile acids, induced hepatocyte necrosis and apoptosis, whereas TC, an hydrophilic bile acid, improved slice viability as compared with controls. Both TDC and TC induced biliary epithelial cell proliferation, together with portal fibroblast proliferation and activation, as shown by PDGF receptor-beta and ED-A fibronectin expression. TLC induced biliary epithelial cell apoptosis. Our results indicate that individual bile acids induce cell type-specific effects in a complex liver microenvironment. The fact that PCLS support biliary epithelial cell and portal fibroblast proliferation will make this model very useful for the study of the mechanisms involved in portal fibrosis.     

Influence of bile acids on stimulated lipid peroxidation and hydrogen peroxide production in rat liver microsomes.

Bile acids were found to be effective antioxidants in bile and intestine. The influence of different bile acids on the NADPH-Fe(++)-stimulated lipid peroxidation (LPO) and cytochrome P-450 dependent hydrogen peroxide production (H2O2) in rat liver microsomes was investigated in vitro. LPO was determined as production of thiobarbituric acid reactants (TBAR). Different tri-, di- and monohydroxylated bile acids and cholesterol were given to the incubation mixture in concentrations ranging from 10(-5) to 10(-3) M. Sodium salts of cholic, tauroglycocholic and deoxycholic acids as well as cheno-deoxycholic, ursodeoxycholic, lithocholic acids and cholesterol did not alter the microsomal production of TBAR. H2O2 formation was significantly decreased by sodium deoxycholate whereas cholesterol increased H2O2 production up to 4 times. These results show that bile acids were not able to protect microsomal membrane lipids against peroxidative damage. Cholesterol mediated H2O2 formation as a source of hydroxyl radicals had no toxic effect concerning LPO, TBAR were not enhanced significantly.     

Influence of liver injury on the catecholamine metabolism in rat brain

The present study investigated the brain catecholamine metabolism of rats with liver injury induced either by malnutrition or with CCl4. In the malnutrition group, the plasma tyrosine concentration was low, while it showed a tendency to be high in the cerebral cortex. Dopamine concentrations were low in both the cerebral cortex and diencephalon. Norepinephrine concentrations were low in the cerebral cortex, striatum and diencephalon. Tyrosine hydroxylase activity was elevated while monoamine oxidase activity was decreased in the striatum. In the CCl4 group, tyrosine concentrations in the plasma and cerebral cortex did not change. The dopamine concentration in the cerebral cortex increased five days after, and the norepinephrine concentration in the diencephalon increased 24 h after the last administration of CCl4. These data suggest that catecholaminergic neurons in the brain may be substantially affected by liver injury. It may be considered that malnutrition disturbs brain development, particularly in young rats.     

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).     

Relationship between biliary calcium and bile acid secretion in the regenerating liver of the rat.

Biliary calcium secretion during liver regeneration following two-thirds hepatectomy was studied in Wistar rats. Calcium output per 100 g body weight was significantly reduced at 24 h post-hepatectomy but not at other times. Values per gram of liver were significantly increased at 48 h and 96 h. A significant relationship was found between bile acid output and calcium output into bile both in controls and hepatectomized animals. The number of nmol of calcium secreted per nmol of bile acids was increased at 24 h post-hepatectomy parallel to an increased choleretic capacity of bile acids and a decrease in the fraction of bile acid independent flow. Our data confirm that bile acid secretion is an important determinant in the secretion of calcium into bile after partial hepatectomy in the rat.     

Age-related changes of long-chain fatty acid metabolism in rat liver

Palmitate oxidation activity and the activities of several enzymes involved in long-chain fatty acid metabolism were examined in the liver of young adult (2-month-old) and senescent (32-month-old) female rats. Palmitate oxidation activity in rat liver mitochondria showed age-related decrease, as judged by the rates of both 14CO2 production and formation of radioactive acid-soluble products from [1-14C]palmitate. In addition, long-chain acyl-coenzyme A synthetase activity was found to be decreased in liver mitochondria and increased in liver microsomes in senescent rats. These results suggest that, in the rat liver, preferential channeling of long-chain fatty acids through the triacylglycerol synthetic pathway may increase with age, and as a result, energy production by their oxidation may decrease.