Inhibition of apolipoprotein B secretion by taurocholate is controlled by the N-terminal end of the protein in rat hepatoma McArdle-RH7777 cells

Bile salts (BS) inhibit the secretion of apolipoprotein B (apoB) and triacylglycerol (TG) in primary rat, mouse and human hepatocytes and in mice in vivo. We investigated whether lipidation of apoB into a lipoprotein particle is required for this inhibitory action of BS. The sodium/taurocholate co-transporting polypeptide (Ntcp) was co-expressed in McArdle-RH7777 (McA-RH7777) cells stably expressing the full-length human apoB100 (h-apoB100, secreted as TG-rich lipoprotein particles) or carboxyl-truncated human apoB18 (h-apoB18, secreted in lipid-free form). The doubly transfected cell lines (h-apoB/r-Ntcp) effectively accumulated taurocholic acid (TC). TC incubation decreased the secretion of endogenous rat apoB100 (-50%) and h-apoB18 (-35%), but did not affect secretion of rat apoA-I. Pulse-chase experiments (35S-methionine) indicated that the impaired secretion of radiolabeled h-apoB18 and h-apoB100 was associated with accelerated intracellular degradation. The calpain protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) partially inhibited intracellular apoB degradation but did not affect the amount of either h-apoB18 or h-apoB100 secreted into the medium, indicating that inhibition of apoB secretion by TC is not due to calpain-dependent proteasomal degradation. We conclude that TC does not inhibit apoB secretion by interference with its lipidation, but rather involves a mechanism dependent on the N-terminal end of apoB.     

Bile secretion in hemoglobin-free perfused rat liver

Hemoglobin-free perfused rat liver was demonstrated to be a suitable experimental model in studying bile secretion. Bile flow slowly decreased to more than 3 h of perfusion. Despite differences in metabolic states, the bile flow was the same in the recirculating as in the nonrecirculating mode of perfusion. Sulfobromophthalein stimulated bile flow at high rates of infusion. In bile, the ratio conjugated to unconjugated sulfobromophthalein also increased with sulfobromophthalein infusion rate. The access of [14C]insulin, [14C] sucrose, and inorganic [32P] phosphate from perfusate into bile was restricted. Bile flow, secretion of taurocholate and sulfobromophthalein, and bile pressure are compared with values from anesthetized animals and from isolated livers perfused with medium containing erythrocytes.     

Bile acids secretion and synthesis by isolated rat hepatocytes.

Bile acids secretion and their distribution were studied in isolated rat hepatocytes. Bile acids secretion was linearly related with time for first three hours of incubation and the net secretion rate was 23.2 ± 2.74 nmoles per g cells (wet weight) per minute. Isolated hepatocytes synthesized relatively more chenodeoxycholic acid than cholic acid compared to whole animal. These results suggest that isolated hepatocytes synthesize and secrete bile acids and thus provide experimental system to study the effect of drugs on bile acids secretion and synthesis at cellular level.     

Inhibition by chloroquine of the secretion of very low density lipoproteins by cultured rat hepatocytes

Cultured rat hepatocytes were incubated in medium containing 1.0 mM oleic acid. The incorporation of [3H]glycerol into cell-associated and medium triacylglycerols was measured after 2 h incubation. More than 95% of the secreted [3H]triacylglycerols were recovered in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Chloroquine and other lysosomotropic amines promoted a marked decrease in [3H]triacylglycerol secretion from the hepatocytes while the synthesis was unaffected. At 50-200 microM final concentration, chloroquine inhibited secretion of triacylglycerols by 70-90% of the control. Similar results were obtained when the mass of secreted triacylglycerols was measured. Chloroquine caused decreased secretion of [3H]triacylglycerols after 15-30 min incubation and the inhibitory effect was completely reversible within 1-2 h after washout of chloroquine. The reduced triacylglycerol secretion was not due to increased reuptake of secreted lipoproteins or decreased protein synthesis caused by chloroquine. Electron microscopy of chloroquine-treated cells showed that the inhibition of VLDL secretion occurs at or prior to the level of the Golgi apparatus. These results suggest that chloroquine interferes with crucial steps in the secretory process and/or that lysosomal function could be essential for secretion of VLDL.     

Bile acid secretion during rat liver carcinogenesis

Retro-differentiation of liver parenchyma during neoplastic processes is characterized by the expression of tumor antigens, such as alpha-fetoprotein and the placental isoenzyme of glutathione-S-transferase (GST-P). To investigate whether this may also affect a typical liver function such as bile acid secretion was the aim of this work. Rat hepatocarcinogenesis was induced by diethylnitrosamine (i.p., 200 mg/Kg body weight at day 0) and promoted by two-thirds partial hepatectomy (at day 21) plus 2-acetamidofluorene administration (50 mg/Kg body weight, subcutaneously, twice a week from day 14 to day 35). In order to carry out planimetric measurements of neoplastic tissue after immunohistochemical staining, a novel monoclonal antibody (MAb 14.1.3) against GST-P with no cross-reactivity against the major liver isoform of GST (GST-H) was raised. Analysis of total biliary bile acid output using the 3alpha-hydroxysteroid dehydrogenase method indicated that a significant reduction (-26%) occurred during the formation of GST-P-positive foci (12 wk). This was restored to normal values during adenoma formation (16-20 wk), but decreased again during carcinoma transformation (32 wk). These changes were not parallel to that observed in bile flow, which was progressively but slightly decreased throughout the whole period under study. HPLC analysis of bile samples collected for 1 h at different time points during hepatocarcinogenesis revealed that in contrast to what happens during cholestatic disease, a continuous and progressive increase in the cholic acid-to-chenodeoxycholic acid ratio (from 4.4+/-0.5 in control animals to 15.1+/-1.9 in rats with hepatocellular carcinoma) occurs. A significant and transient increase at 16 wk (+120%) in the proportion of bile acids amidated with glycine as compared to those conjugated with taurine was also observed. These results indicate that the mechanisms accounting for the secretion of major bile acids are modified differently at various steps of rat liver tumor development.     

Bile secretion in the liver caused by verapamil

Verapamil was studied for its effects on secretory function of liver in rats. In the animals with low initial level of bile secretion, infusion of verapamil resulted in increase of the bile flow conjugated with taurine bile salts and ester of cholesterol, and in reduction of the non-conjugated bile salts secretion, as well as bile salts conjugated with glycine. In the animals with high initial level of the liver secretory function, verapamil decreased the bile flow, the secretion of unconjugated bile salts, and bile salts conjugated with taurine and glycine, phospholipids, cholesterol and its ester. The changes of bile flow and biliary secretion of bile acids and lipids in two groups of animals suggest that verapamil could be influenced in regulation of bile secretion depending on its initial level. Possible mechanisms of the bile secretion regulation by verapamil, are discussed.     

Bile acids inhibit secretion of very low density lipoprotein by rat hepatocytes

The influence of taurocholate on very low density lipoprotein (VLDL) triacylglycerol synthesis and secretion was studied by isolated rat liver-parenchymal cells. The incorporation of [3H]glycerol into cell-associated and VLDL triacylglycerols were measured after incubation in medium containing 0.75 mM oleate. Taurocholate caused a maked decrease in VLDL [3H]triacylglycerol secretion from the hepatocytes: 50-150 microM taurocholate inhibited secretion of VLDL [3H]triacylglycerols by 70-90%. Similar results were obtained when the mass of secreted VLDL triacylglycerols was measured. Taurocholate caused a decreased secretion of VLDL [3H]triacylglycerols after 15-30 min incubation. A higher amount of cellular triacylglycerols was found in taurocholate-supplemented cells. Furthermore taurocholate did not change the intracellular lipolysis of triacylglycerols. These results suggest that bile acids interfere more probably with the assembly and/or secretion of VLDL-particles and not with earlier stages of VLDL formation, e.g. triacylglycerol synthesis.     

Bile salt related secretion of acid phosphatase in rat bile

The biliary excretion of bile salts, lysosomal acid phosphatase, and total proteins were studied in rats under different experimental conditions: during bile salt loss through a bile fistula and after loading with exogenous sodium taurocholate. The experimental models were suitable to demonstrate that variations in the excretion of bile salts were associated with those of acid phosphatase output. During bile salt depletion, acid phosphatase output showed a decrease parallel to that of bile salts. Following a single i.v. injection of sodium taurocholate and during its i.v. infusion, a rapid increase of acid phosphatase excretion in bile was seen. The patterns of enzyme outputs observed after administration of sodium taurocholate suggested a bulk discharge in bile of lysosomal contents. The profiles of protein output were similar to those of acid phosphatase suggesting an association between the secretory mechanism of these bile constituents. In contrast to sodium taurocholate, 4-methylumbelliferone, which also increases canalicular bile flow, did not produce changes in the excretory patterns of the bile components studied. Therefore, the results suggested a bile salt related secretion of acid phosphatase in the rat, which may involve protein secretion in bile.     

Bile acid sulfonate and 7-alkylated bile acid analogs: effect on intestinal absorption of taurocholate and cholesterol 7alpha-hydroxylase activity in cultured rat hepatocytes

The effects of sulfonate analogs of cholic (C), chenodeoxycholic (CDC), and ursodeoxycholic acid (UDC) and three 7-alkylated CDCs--7-methyl-, 7-ethyl-, and 7-propyl-CDCs--on taurocholate absorption from rat terminal ileum in situ and on cholesterol 7alpha-hydroxylase activity in primary culture of the rat liver were investigated. The sulfonate analogs of two dihydroxy bile acids CDC and UDC, but not C, significantly decreased the absorption of taurocholate. Taurine conjugates of 7-alkylated CDC slightly decreased the taurocholate absorption, and tauro-7-propyl-CDC significantly suppressed the absorption. Although the sulfonate analogs of C and CDC reduced cholesterol 7alpha-hydroxylase activity by 40% and 60% compared to control, UDC-sulfonate analog did not affect enzymatic activity. These results were consistent with those of the lead compounds, C, CDC, and UDC. The introduction of methyl group at C-7 position of CDC attenuated the reduction in cholesterol 7alpha-hydroxylase activity by CDC. However, elongation of the alkyl group resulted in an inhibitory effect. The present study revealed the following: 1) bile acid sulfonates act on cholesterol and bile acid metabolism in a similar manner as taurine conjugated bile acids; and 2) the biologic properties of CDC could be altered by the introduction of alkyl group at C-7 position.     

Bile secretion in the fistulated pig: effect of the method used for bile reinfusion

The aim of this work was to investigate the effects on bile secretion of flow rate and site of reinfusion of the collected bile to the animal. Thirty-two pigs weighing 50 +/- 3 kg at the beginning of the experiment were fitted with a reentrant fistula in the lower common bile duct and in the upper duodenum. Bile collected from the bile duct was reinfused in four different ways (four groups of 8 animals each): into the duodenum or the lower common bile duct at a constant flow rate using a peristaltic pump, or into the duodenum or the lower common bile duct at a rate mimicking the flow rate of the secretion using an automatic apparatus. Reinfusing the bile into the lower common bile duct at a rate mimicking the secretion rate provided a daily bile acid production about 21% higher than the level recorded with the other three methods. This was mainly due to a higher bile acid concentration since the bile flow was only slightly affected by the treatment.     

Growth hormone secretion of the neonatal rat pituitaries is stimulated by gamma-aminobutyric acid in vitro

Growth hormone secretion from pituitaries of neonatal rats was stimulated by gamma-aminobutyric acid (GABA) and the GABA agonist muscimol $\text{in vitro}$. This response to GABA was absent after the 9th postnatal day. The stimulation of growth hormone secretion by GABA was antagonized by bicuculline-methiodide and by picrotoxin. Diazepam stimulated while baclophen had no effect on growth hormone secretion. This stimulatory GABA effect might be related to a certain developmental stage of the pituitary GABA receptors or to the lack of hypothalamic regulatory influence(s) in the newborn.     

Bile acid synthesis and secretion by rabbit hepatocytes in primary monolayer culture: comparison with rat hepatocytes

Rabbit hepatocytes isolated after liver perfusion with collagenase were maintained in primary monolayer culture for periods up to 96 h. Bile acid synthesis and secretion was measured by capillary gas-liquid chromatography and by a rapid enzymatic-bioluminescence assay. As expected from the bile acid profile of rabbit gallbladder bile, cholic acid was the only bile acid synthesized in detectable amounts and was produced at a linear rate of 170 pmol/h per mg cell protein from 24 to 96 h in culture. Ketoconazole (20 microM) inhibited cholic acid synthesis and secretion by 78%, whereas the bile acids chenodeoxycholic acid (100 microM), deoxycholic acid (100 microM) or lithocholic acid (2 microM) had no effect. When rat hepatocytes were cultured under identical conditions, the rate of bile acid synthesis was found to be only 12 pmol/h per mg cell protein, a value in agreement with previous work. The large difference in rates of bile acid synthesis between rabbit and rat hepatocytes may be due to rapid loss of cytochrome P-450 from rat hepatocytes when placed in monolayer culture. Although reportedly active in cholesterol 7 alpha-hydroxylation, form 4 cytochrome P-450 levels in rabbit hepatocytes did not correlate with rates of bile acid synthesis.     

Inhibition of protein phosphorylation by chloroquine

The rapid phase of fructose-1,6-bisphosphatase (FBPase) inactivation following glucose addition to starved yeast cells [reported previously] is inhibited on addition of 10 mM chloroquine (CQ) at about pH 8. This inhibition of inactivation was shown to be due to the prevention of phosphorylation of the enzyme. CQ was also found to inhibit general protein phosphorylation in the yeast cells. Glycolysis, as observed by changes in intracellular glucose-6-phosphate and extracellular glucose and ethanol concentrations, was shown to be significantly inhibited in cells treated with CQ. Similarly, a decrease in ATP concentrations was observed. However, during the early stages of phosphorylation of FBPase, levels of ATP were similar in cells containing CQ as in those without CQ. Thus, decrease in ATP levels is not thought to be significantly responsible for the inhibition of protein phosphorylation. However, the phosphorylating activity of cyclic AMP-dependent protein kinases is inhibited in vitro by relatively low concentrations of CQ. Thus, prevention of protein phosphorylation by CQ is believed to be due to inhibition of protein kinases in yeast cells.Abbreviations FBPase fructose-1,6-bisphosphatase - CQ chloroquine - SDS sodium dodecyl sulfate - G6P glucose-6-phosphate - TCA trichloroacetic acid