Subcellular and molecular mechanisms of bile secretion.

One of the liver's principal functions is the formation of bile, which is requisite for digestion of fat and elimination of detoxified drugs and metabolites. Bile is a complex fluid made up of water, electrolytes, bile acids, pigments, proteins, lipids, and a multitude of chemical breakdown products. In this review, we have summarized the source of various biliary components, the route by which they end up in bile, including the underlying subcellular and molecular mechanisms, and their contribution to bile formation. One of the reasons why bile formation is so complex is that there are many mechanisms with overlapping substrate specificities, i.e., many biochemically unrelated biliary constituents share common transport mechanisms. Additionally, biliary constituents may reach bile by more than one pathway. Some biliary components are critical for bile formation; others are of minor significance for bile formation but play a major physiological role. The major driving force for bile formation is the uptake and transcellular transport of bile salts by hepatocytes. The energy for bile formation comes from the sodium gradient created by the basolateral Na+/K(+)-ATPase, to which bile salt transport is coupled. The secretory pathway for bile salts involves uptake at the basolateral surface of the hepatocyte, vectorial transcellular movement, and transport across the canalicular membrane into the canalicular lumen. Hydrophilic bile salts are taken up via a sodium-dependent, saturable, carrier-mediated process coupled to the Na+/K(+)-ATPase. This uptake mechanism is also shared by other substrates, such as electroneutral lipids, cyclic oligopeptides, and a wide variety of drugs. Hydrophobic bile acids are taken up by a sodium-independent facilitated carrier-mediated mechanism in common with other organic ions, including sulfated bile acids, sulfobromophthalein, bilirubin, glutathione, and glucuronides, or by nonsaturable passive diffusion. Two major carrier proteins have been identified on the hepatocyte basolateral membrane: a 48-kDa protein that appears to be involved with Na(+)-dependent bile salt uptake, and a 54-kDa protein, thought to be associated with Na(+)-independent bile salt uptake. The intracellular transport of bile salts may involve cytosolic carrier proteins, of which several have been identified. Some evidence suggests a vesicular transport mechanism for bile salts. Since bile acids clearly do not enter the cell by endocytosis, formation of transport vesicles must be a more distal event in the transcellular translocation process. Some bile salts appear to be transported within the same unilamellar vesicles that are involved in the secretion of cholesterol and phospholipid.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of epidermal growth factor receptor in hepatocyte nuclei.

Experiments undertaken to investigate the binding of epidermal growth factor by hepatocyte nuclei showed that: (a) isolated nuclei from both normal and regenerating rat liver are capable of binding 125I-epidermal growth factor, (b) the nuclear epidermal growth factor-binding protein is similar in molecular weight to the plasma membrane epidermal growth factor receptor, (c) monoclonal antibodies produced against the plasma membrane epidermal growth factor receptor recognize the nuclear epidermal growth factor receptor and (d) the nuclear receptor has an affinity for epidermal growth factor comparable to that of the plasma membrane receptor, but fewer (approximately 10%) nuclear receptors are available per protein unit compared with the plasma membrane.

Hepatocellular processing of endocytosed proteins.

In conclusion, proteins of hepatobiliary transport utilize receptor-mediated endocytosis and intracellular vesicles and rely on functionally dynamic microtubules for their transport by hepatocytes. The many diverse transport pathways in hepatocytes reflect the many functions served by the uptake of various proteins from the blood. The mechanisms of sorting of ligands and their receptors in endosomes and the factors that regulate the intracellular transport pathways are not yet known. Future investigations in this area promise to yield many exciting discoveries about the hepatocellular processing of proteins.

Effect of oxidative iodination of epidermal growth factor on its binding and secretion by hepatocytes.

Experiments were undertaken to determine whether the method of iodination of epidermal growth factor (EGF) affects its binding to rat liver plasma membranes and its uptake, processing, and secretion into bile by intact rat hepatocytes. EGF was iodinated using one of three oxidative reagents: chloramine T (CT), lactoperoxidase (LP), or monochloride (MC). Quantitative receptor binding studies on plasma membranes isolated from male rat livers with either CT-, LP-or MC-125I-EGF indicated no significant difference in the apparent binding constants of the three preparations. To determine whether these three preparations were capable of forming a covalent-like complex with the EGF receptor, they were individually incubated with isolated plasma membranes and subjected to polyacrylamide gel electrophoresis under reducing conditions, followed by autoradiography. Each preparation formed a major radioactive protein band of approximately 180 kD, identified as the EGF receptor by immunoprecipitation with monoclonal anti-EGF receptor antibodies. Furthermore, even unlabeled EGF incubated with plasma membranes formed this same 180 kD band, as revealed on Western blots using anti-EGF antibody. The biliary secretion of CT-, LP-, and MC-125I-EGF was compared by injecting each one into rat portal veins and measuring the total and immunoprecipitable radioactivity in bile. The amount of immunologically intact CT-125I-EGF in bile was significantly greater than the others, whereas MC-125I-EGF transport was significantly reduced. We conclude that the method of iodination does not affect the covalent-like binding properties of EGF. Furthermore, since unlabeled EGF displayed these same binding properties, oxidative iodination procedures per se do not account for the covalent-like association between EGF and its receptor. However, the method of iodination used did affect the intracellular transport and processing of EGF by hepatocytes. The structural modification responsible for this alteration in transport properties has yet to be determined.

Biological effects of epidermal growth factor, with emphasis on the gastrointestinal tract and liver: an update.

Epidermal growth factor (EGF) is a 6,000 Da polypeptide hormone produced by glands of the gastrointestinal tract, namely the salivary and Brunner's glands. It is found in a wide variety of external secretions as well as in blood and amniotic fluid. In fetal and neonatal life, EGF appears to play an important role in the development of the oral cavity, lungs, gastrointestinal tract and eyelids. Its presence in cells of the central nervous system suggests that it also plays a role in modulating the development of this system. In adult animals, the function of EGF is much less well understood. In rodents, it apparently modulates acid secretion from parietal cells in the stomach, and it undoubtedly plays an important role in wound healing, either through its localization within skin or by the licking of wounds with EGF-containing saliva. Considerable evidence now suggests that it may be one of the key factors in initiating liver regeneration after partial hepatectomy or chemical injury. The liver appears to be the principal organ which regulates the circulating level of EGF. In fact, EGF is cleared so efficiently by the liver that only the peripheral cells of the lobule (zone 1) sequester EGF, and little remains in the circulation for cells in the more distal zones (zones 2 and 3). In the liver, EGF normally binds to a plasma membrane receptor and is internalized within the liver cell, where the vast majority of EGF and its receptor are destroyed in lysosomes. A small but consistent quantity of EGF enters the bile intact. In the regenerating liver, however, the lysosomal pathway appears to be shut down, and the EGF is diverted to hepatocyte nuclei prior to the initiation of DNA synthesis. Nuclear EGF is found free as well as bound to a high-molecular-weight protein which has many characteristics identical to the plasma membrane EGF receptor. The plasma membrane receptor is a large transmembrane glycoprotein of 170,000 Da containing four domains: an extracellular EGF-binding portion, a hydrophobic membrane-spanning segment, a proximal cytoplasmic domain which binds ATP and protein substrates containing tyrosine for phosphorylation and a terminal cytoplasmic portion with 3 tyrosines which undergo autophosphorylation after EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Isoflurane as an anesthetic for experimental animal surgery.

Isoflurane is an inhalational anesthetic agent associated with no known hepatic toxicity. Despite this fact, isoflurane has not been widely utilized as an anesthetic agent in studies of liver structure and function in experimental animals. For this reason, livers from rats treated with pentobarbital or diethylether were compared to those from rats treated with isoflurane to determine differences in biochemical and morphologic parameters. Liver from pentobarbital-treated rats showed a significant decline in glutathione-S-transferase activity compared to liver from isoflurane/O2 or ether-treated rats. Liver microsomes from isoflurane/O2-treated rats retained more cytochrome-C(P450)-reductase activity than did those from pentobarbital-treated, ether-treated, or decapitated rats. Despite these biochemical alterations, morphometric analysis of liver from isoflurane/O2 and pentobarbital-treated rats showed no quantitative or qualitative differences in liver structure or organelle volume densities. Neither were differences detected in uptake and distribution of 125I-epidermal growth factor when analyzed by electron microscopic autoradiography. These data show that isoflurane with supplemental O2 has no effects on hepatic structure and fewer effects on hepatic function than other anesthetics and may be a better experimental anesthetic than any currently in use.

Translocation of epidermal growth factor to the hepatocyte nucleus during rat liver regeneration.

To determine the fate of exogenous epidermal growth factor (EGF) in regenerating liver, 125I-labeled EGF was injected into rat portal veins at various times after 70% hepatectomy. Epidermal growth factor was taken up by the liver remnant at all time points studied (0, 4, 8, 16, and 36 h), but at 8 h after hepatectomy a large quantity was retained by the liver and EGF degradation products appearing in the bile decreased markedly. Electron microscopic autoradiography of the regenerating livers 1 h after injection of 125I-EGF demonstrated that 27% of the grains were associated with hepatocyte nuclei compared to 0.5% in shamoperated controls. There was also a concomitant decrease in grains associated with the lysosomal compartment. Nuclei isolated from regenerating livers exposed to 125I-EGF also demonstrated a three-fold increase in radioactivity compared to nuclei from control livers. Nearly 70% of nuclear radioactivity was precipitable with a specific antibody to EGF, and a small fraction appeared to be part of a high molecular weight complex. These data support the hypothesis that during the pre-S phase of liver regeneration, EGF is translocated to the nucleus rather than to lysosomes, and may participate in the initiation of deoxyribonucleic acid synthesis or alteration of gene expression.

The effect of concentration on hepatic transport of exogenous epidermal growth factor.

Epidermal growth factor (EGF), taken up by rat liver hepatocytes, is primarily transported to the lysosomes and degraded. However, a small but significant percentage of endocytosed EGF is transported by a nonlysosomal pathway and is secreted intact into bile. There is no information as to the mechanisms that regulate the selection of transport pathway and thereby determine the different metabolic fates for EGF. The experiments reported here were undertaken to determine whether the amount of exogenous EGF administered to the liver (the transport load) might affect the selection of the transport pathway. If "excess" EGF, exceeding some as yet undetermined threshold, is preferentially transported by the lysosomal pathway, then the proportion of degraded EGF secreted into bile should increase as a function of the amount of EGF administered. 125I-EGF (3 to 175 ng) was injected into rat portal veins, and bile samples were collected via cannula. The radioactivity secreted into bile was measured, and the bile samples were immunoprecipitated with anti-EGF antiserum. The proportion of intact vs. degraded EGF in bile was determined by the percentage of immunoprecipitable radioactivity. Regardless of the amount of EGF injected, the pattern of its secretion was unaltered. The percentage of immunoprecipitable EGF in bile was the same for all doses. Therefore, the amount of EGF that was degraded did not change as a function of EGF concentration, implying that the lysosomal pathway was not preferentially utilized as the transport load increased. In conclusion, transport load does not appear to be a regulatory mechanism in the selection of transport pathway utilized by EGF.

Transport of epidermal growth factor by rat liver: evidence for a nonlysosomal pathway.

Epidermal growth factor (EGF), circulating in the blood, is taken up by rat liver hepatocytes by means of specific and saturable receptor-mediated endocytosis. These experiments were undertaken to determine (a) the transport pathway(s) of EGF taken up by rat liver and (b) the effects of lysosomal inhibition on its transport. 125I-EGF was injected into rat portal veins, and bile samples were collected and analyzed for both total and immunoprecipitable radioactivity. In addition, the livers were examined by electron microscopic autoradiography. Some animals received injections of chloroquine before surgery, to disrupt lysosomal function. The results indicate that most of the EGF taken up by the hepatocytes is transported to lysosomes and degraded. However, a small but significant percentage of endocytosed EGF is transported by a pathway independent of the lysosomal system, resulting in secretion of intact EGF: (a) Both degraded and immunoprecipitable EGF are secreted into bile. (b) Immunoprecipitable radioactivity peaks at 20 min after EGF injection, whereas degradation-associated radioactivity does not peak until 40 min postinjection. (c) EGF isolated from bile is specifically taken up by isolated hepatocytes in monolayer culture, indicating that it is still recognizable by the EGF receptor. (d) When the lysosomal system is inhibited with chloroquine, secretion of degraded EGF is significantly inhibited, whereas the amount of intact EGF secreted into bile is unchanged. The utilization by liver of a dual transport process for EGF represents an unusual system of intracellular ligand processing, whose physiological significance has yet to be determined.

Vesicular transport of horseradish peroxidase during chronic bile duct obstruction in the rat.

The vesicular transport system for biliary secretion of plasma-derived proteins was investigated in rats with chronic bile duct obstruction. Horseradish peroxidase, previously demonstrated to be a suitable tracer for vesicular transport, was employed in these studies. Both the time course of horseradish peroxidase secretion into bile and the morphological events in its uptake, transport, and biliary secretion were found to proceed in a manner essentially identical to that of sham-operated control animals. In addition, fragmentation of hepatocytes leading to sloughing into bile of large pieces of cytoplasm bearing horseradish peroxidase-containing endocytic transport vesicles frequently was observed in the cholestatic animals. These data suggest that the vesicular transport system for the secretion into bile of plasma-derived proteins remains intact and functional during chronic bile duct obstruction and that another mechanism, possibly fragmentation and solubilization of hepatocyte membranes followed by regurgitation of proteins released from endocytic vesicles, may be responsible for the elevation of biliary proteins within plasma seen during cholestasis.

The perfused human liver wedge biopsy: a new in vitro model for morphological and functional studies.

We developed a simple and inexpensive method of perfusing small specimens of human liver in vitro that maintains short-term tissue viability as judged by protein transport function and morphological features. The technique allows investigation of liver function at the cellular level in normal specimens and those with hepatobiliary disease. Electron microscopy of specimens perfused with this system demonstrates the presence of an incomplete basement membrane within the space of Disse and shows that human microbodies contain crystalline-like cores morphologically similar to those found in rat liver.

Recycling of mast cells following degranulation in vitro: an ultrastructural study.

Mature mast cells, isolated from the rat peritoneal cavity, were placed into suspension culture, either as resting or after degranulation by exposure to compound 48/80, and were maintained for up to 63 hr. No mitotic cells were observed, and cell number was conserved. The culture conditions did not cause spontaneous degranulation and cell survival was better than 80%. However, with time in culture, an increasing percentage of cells acquired a vesiculated appearance, characterized by a Golgi area with distended cisternae, the accumulation of lysosomal or autophagic-like vesicles, and enlarged, irregular or fused secretory granules. In the degranulated group, about one-fourth of the cells recovered the morphological appearance of resting cells by 63 hr, indicating that they are capable of 'recycling'. A cell type with a unique morphology, characterized by a large central vacuole containing secretory product, an eccentric nucleus, and mature secretory granules at the cell periphery appeared in the stimulated group after 22 hr of culture. In may be a possible intermediate stage in the mast cell regranulation process, based on its occurrence exclusively in the stimulated group, the correlation between its distribution and the recovery of mast cells to the resting state, and the morphological resemblance of its granule contents to stages in granule maturation in differentiating embryonic mast cells.

Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture.

Epithelial cells dissociated from mammary glands of midpregnant mice and cultured with lactogenic hormones on plastic or collagen gel substrates have been shown to vary in their extent of differentiation, as identified by the presence of secretory organelles and accumulation and secretion of casein. Morphological and biochemical differentiation was obtained on floating collagen gels. At least four unique factors provided by the floating collagen gel substrates are not found on plastic substrates: access of nutrients to basolateral cell surfaces, close proximity of cells to the medium surface and gas phase, interaction of epithelial cells with stromal elements, and substrate flexibility permitting cell shape change. In this study, we have attempted to assess the relative contributions of these factors in the ultrastructural differentiation of mammary cells in culture. None of these factors alone is responsible for the differentiation achieved when all are present. The novel aspect of this research is the identification of the cells' apparent requirements for basolateral access to nutrients and for freedom to assume a preferred shape in order to achieve differentiation.

Plasma membrane folds on the mast cell surface and their relationship to secretory activity.

Changes in the surface morphology of secreting mast cells have been followed by scanning electron microscopy. Mast cells isolated from the rat peritoneal cavity have folds of plasma membrane that form snake-like ridges on their surfaces. Fold length varies considerably from cell to cell, whereas fold width and depth appear to remain relatively constant. To assess the possible relationship between secretory activity and surface folding, a seimquantitative method was used for measuring fold length in control and secreting populations. A positive correlation is found between secretion of histamine and the extent of membrane folds on the mast cell surface. The source of the membrane required for fold formation is probably secretory granule membrane incorporated into the plasma membranene as a result of exocytosis. Furthermore, a distinct cell type devoid of surface folds, designated as a raspberry-type cell, is found to occur as an integral part of a normal population of mast cells. This cell type is resistant to stimulation by polymyxin.

A freeze-fracture study of early membrane events during mast cell secretion.

The early membrane events taking place during mast cell secretion were followed in transmission and freeze-fracture electron microscopy. In order to slow down exocytosis and capture intermediate stages of membrane fusion, special conditions of incubation and stimulation were used. These were as follows: (a) the use of incubation media with altered ionic composition, and (b) stimulation with a low dosage of polymyxin B sulfate (4 microgram/ml) at low temperature (18 degrees C) for very short incubation times (30-60 s), with or without the presence of formaldehyde (0.8%). Under these conditions, unetchable circular impressions are found on the E face of the plasma membrane, 80-100 nm in diameter, with particles associated with their perimeters. In granule-to-granule fusion, the zone involved is demarcated by one or two rows of particles on the E face. In addition, raised circular areas of varying diameters (43-87 nm) surrounded by similar particles, also found on the E face, may represent potential sites before completion of fusion. Neither the circular impressions on the plasma membrane nor the sites on the granule membrane are permanent, but their appearance coincides with initiation of membrane fusion.