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 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 acid metabolism in mammals. VIII. Biliary secretion of cholylarginine by the isolated perfused rat liver.

In studies of cholic acid metabolism using the isolated perfused rat liver system, an unknown conjugate of cholic acid was observed. This conjugate comprised 15-27% of the biliary bile acids in these experiments, was less polar than cholylglycine on thin-layer chromatography using butanol, acetic acid, and water, and had an apparent molecular weight greater than that of cholyltaurine on gas-liquid chromatography. Amino acid analysis of the hydrolyzed conjugate demonstrated the presence of arginine. Perfusion studies with radioactive arginine, and mass spectrometric analysis proved that the conjugate was cholylarginine. Secretion of this conjugate does not represent a deficiency of available glycine and taurine.     

Bile secretion is centrally regulated by C-type natriuretic peptide

1. Current evidence supports that C-type natriuretic peptide (CNP) is the brain natriuretic peptide. Natriuretic peptide receptors and mRNA CNP have been reported in the liver and in discrete areas and nucleus of the central nervous system involved in the regulation of gastrointestinal physiology. In the present work, we sought to establish the role of CNP in the central regulation of bile secretion in the rat and to delineate the possible pathways and mechanisms involved.2. To examine the role of CNP on bile secretion, the peptide was applied in the brain lateral ventricle (1, 10, and 100 ng/L) and bile samples were collected every 15 min for 60 min. The role of the autonomic nervous system in CNP response was assessed by atropine or combined phentolamine and propranolol administration.3. Centrally applied CNP diminished basal as well as bile salt-evoked bile flow in a dose-dependent manner. CNP reduced bile acid output as well as sodium and potassium excretion, supporting CNP effect on bile acid-dependent flow. CNP also decreased chloride excretion and increased bile pH. The excretion of total glutathione was not affected by centrally applied CNP suggesting that this peptide does not alter bile acid-independent flow. Neither parasympathetic nor sympathetic blockade abolished CNP inhibitory response on bile secretion. Mean arterial pressure and portal venous pressure were not modified by CNP.4. Present findings show that centrally applied CNP modulates bile secretion in a dose-dependent fashion. CNP alkalinized bile and reduced bile acid-dependent flow without affecting bile acid-independent flow. The inhibitory response of CNP on bile secretion was not mediated by the autonomic nervous system. Present findings give further support to the role of CNP as the brain natriuretic peptide.     

Bile protein secretion in the rat stimulated by taurocholate: effect of chloroquine

The biliary protein excretion during sodium taurocholate induced choleresis was studied in normal rats and in rats treated with the lysosomotropic agent, chloroquine. The analysis of the protein component in bile was made on SDS-polyacrilamide gel, and the individual polypeptides were quantitated by densitometry. The excretion of bile polypeptides was compared with that of lysosomal acid phosphatase. The biliary excretion of polypeptides of molecular mass lower than and equal to 54 kDa was markedly stimulated by taurocholate-induced choleresis. Chloroquine treatment of rats diminished the biliary excretion of such polypeptides and also inhibited their excretion induced by taurocholate. The behaviour of these polypeptides was well correlated to that of the lysosomal marker. The biliary excretion of polypeptide bands of a higher molecular mass (up to 140 kDa) did not show major changes during taurocholate-induced choleresis in any of the groups. The results indicate that biliary excretion of proteins in the rat may be either stimulated by taurocholate or may be independent of the bile salt. The former requires the functional integrity of chloroquine-sensitive hepatocyte compartments, which may involve the lysosomes.     

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.     

Bile acid supplementation improves established liver steatosis in obese mice independently of glucagon-like peptide-1 secretion

Bile acids or its derivatives may influence non-alcoholic fatty liver disease development through multiple mechanisms. Intestinal L-cells secrete glucagon-like peptide-1 (GLP-1) and can be activated by bile acids (BA) influencing insulin resistance and hepatic steatosis development and progression. The aim of the present study was to assess the effects of cholic acid (CA) or ursodeoxycholic acid (UDCA) administration on portal and systemic levels of GLP-1 in genetically obese mice with established hepatic steatosis. Eight-week-old ob/ob mice were fed CA or UDCA during 4 weeks. Systemic and portal GLP-1 levels were measured as well as glucose tolerance test, serum and biliary parameters, hepatic triglyceride content, liver histology, and hepatic gene expression of relevant genes related to bile secretion. Eight-week-old ob/ob mice exhibited marked obesity, hyperinsulinemia, and fasting hyperglycemia. Administration of both CA and UDCA was associated to decreased hepatic triglyceride content and complete reversion of histological steatosis. BA-fed animals did not exhibit significant differences in glucose tolerance. In addition, neither CA nor UDCA administration significantly influenced portal or systemic GLP-1 levels. CA and UDCA strongly ameliorated established fatty liver in ob/ob mice independently of the GLP-1 incretin pathway. Thus, the anti-steatotic action of these bile acids is likely related to direct hepatic effects.     

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.     

Inhibition of very-low-density lipoprotein secretion by chloroquine, verapamil and monensin takes place in the Golgi complex

The effects of chloroquine, verapamil and monensin on secretion of very-low-density lipoproteins (VLDLs) were studied in cultured rat hepatocytes. Maximum inhibition of VLDL-triacylglycerol secretion by 50–90% of control was reached at 200 μM chloroquine, 200 μM verapamil and 5 μM monensin, whereas no effect on cellular triacylglycerol synthesis was observed. The inhibition could be seen within 15 min and was reversible after washout of the drugs. Chloroquine and verapamil inhibited both cellular protein synthesis and protein secretion, whereas monesin reduced protein secretion without any effect on protein synthesis. Control experiments with cycloheximide revealed that intact protein synthesis was not necessary for secretion of VLDL-triacylglycerol during 2 h. Electron micrographs of cells treated with chloroquine, verapamil or monensin showed swollen Golgi cisternae containing VLDL-like particles. By morphometry, a more than 2-fold increase in volume fractions and size indices of Golgi complexes and secondary lysosomes was observed, except that monensin had no significant effect on these parameters of secondary lysosomes. These results suggest that the inhibition of VLDL secretion by chloroquine, verapamil and monensin which takes place in the Golgi complex might be due to disruption of trans-membrane proton gradients. An increase in pH of acidic Golgi vesicles may cause swelling and disturb sorting and membrane flow through this organelle.     

Bile salt sulfotransferase in guinea pig liver

Bile salt sulfotransferase from guinea pig liver is purified by the procedures of ammonium sulfate fractionation, Sephadex G-100 column chromatography, agarose-hexane-adenosine 3′,5′-diphosphate affinity chromatography and polyacrylamide gel electrophoresis. The purified enzyme exhibits a pH optimum of 6.8, an isoelectric point of 5.6 and a molecular weight of 7600 estimated by gel filtration technique. The apparent K_m values of the enzyme are 7.7·10^?5 M for taurolithocholate and 1.4·10^?6 M for 3′-phosphoadenosine 5′-phosphosulfate. It requires Mg²⁺ and free sulfohydryl group(s) for activity. The enzyme reacts with hydroxy groups of bile salts at both 3α and 3β positions. No activity is found in the kidney of guinea pig. The purified enzyme does not react with estrone, estradiol, testosterone, dehydroepiandrosterone, cholesterol, phenol, tyramine, and serotonin. The results indicate that bile salt sulfotransferase is distinct from other hepatic sulfotransferases.     

Analysis of the inhibitory effect of verapamil on adrenal medullary secretion

Verapamil blocked catecholamine (CA) secretion evoked by acetylcholine (ACh), Ba²⁺ or Ca²⁺ in isolated perfused bovine adrenals. This inhibitory effect was irreversible and not modified by increasing the Ca²⁺ concentration of the perfusion fluid. Tetracaine also inhibited CA secretion, although no additive effect was found when both verapamil and tetracaine were present simultaneously in the perfusion medium. It is concluded that verapamil and tetracaine inhibit CA secretion presumably at the same site, but verapamil effect cannot be reverted by excess of calcium ions.     

Separation of rabbit ileum mucus secretion from electrolyte and water secretion by cholera enterotoxin, verapamil and A23187

Net water, Na+, Cl- and HCO3- fluxes were measured in in vivo rabbit ileal loops, while mucus secretion was assessed by measuring the glycoprotein or total sialic acid secreted into the lumen, or by measuring the luminal fluid viscosity. Inoculating loops with cholera enterotoxin (CT) produced a sustained secretion of electrolytes and water, but a more transient secretion of mucus. A dose of verapamil was found which, when included in the luminal fluid, inhibited or delayed the CT-induced mucus secretion while not affecting the ongoing electrolyte and water secretion. Exposure of the ileal mucosa to the ionophore, A23187, in the presence of 2mM Ca++ resulted in a brief secretion of mucus, with no change in basal water absorption. Verapamil inhibited this A23187-induced mucus secretion. The ionophore was not effective in the absence of luminal Ca++. Thus rabbit ileum mucus secretion can be separated from electrolyte and water secretion by agents that affect Ca++ movement.     

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.     

A morphologic analysis of changes in the lungs and kidneys caused by acetyl glyceryl ether phosphorylcholine and limited by verapamil

Histological analysis of lung and kidney after multiple PAF i.v. injection into rabbits was performed. Under these conditions the effect of previously injected of verapamyl, calcium channel blocker, was studied. It was shown that RAF causes inflammatory reaction with cell infiltration, microcirculation disorders and thrombosis in lung and kidney, which may classify as interstitial nephritis, pneumonitis and vasculitis. Verapamyl inhibits vasospasm, abolishes fibrinogen but does not affect cell infiltration and oedema development. Thus, Ca channel-dependent mechanism of vasospasm and thrombogenesis induced by PAF in vivo is confirmed. Perhaps, another possibilities for realising PAF action in vivo may occur.