Effect of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells. Immunocytochemical observations

We studied the effects of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells using the direct immunoenzyme technique. Livers were perfusion-fixed 0.5, 1, and 2 h after injection of colchicine. Vibratome sections of the fixed liver were stained using peroxidase-conjugated Fab' of anti-albumin or anti-fibrinogen. By light microscopy, reaction deposits showing albumin and fibrinogen were observed in the cytoplasmic granules of hepatocytes. Such stained granules decreased 30 min after injection, but later increased gradually and crowded in the cytoplasm. The Golgi complex stained for the proteins decreased after 30 min but increased in the juxtanuclear region after 60 min. The analysis of serial sections showed that colchicine severely disturbed the spatial relationship between the Golgi apparatus and the bile canaliculus. We obtained similar results by electron microscopy; a positive reaction for albumin and fibrinogen was observed in a small number of the cytoplasmic granules after 30 min. After 1 h of treatment, most of the Golgi complexes were fragmented and lost their stacked cisternae. However, they reappeared accompanied with vacuolated cisternae and secretory granules, which were partially stained for albumin and fibrinogen. After 2 h, the secretory granules positive for both proteins accumulated further. Some of them lined a long the plasma membrane, and others made a cluster in the cytoplasm. The profiles showing exocytosis were very rarely seen. These results showed that in the first 30 min, colchicine primarily disturbs partially the Golgi assembly but does not affect the post Golgi secretory pathway much. Later, the drug affects both the post Golgi pathway and the Golgi assembly, and it causes a marked accumulation of secretory granules.     

Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.     

Intracellular transport and degradation of I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Intracellular transport of endocytosed proteins in rat liver endothelial cells.

1. Receptor-mediated endocytosis of mannose-terminated glycoproteins in rat liver endothelial cells has been followed by means of subcellular fractionation and by immunocytochemical labelling of ultrathin cryosections after intravenous injection of ovalbumin. For subcellular-fractionation studies the ligand was labelled with 125-tyramine-cellobiose adduct, which leads to labelled degradation products being trapped intracellularly in the organelle where the degradation takes place. 2. Isopycnic centrifugation in sucrose gradients of a whole liver homogenate showed that the ligand is sequentially associated with three organelles with increasing buoyant densities. The ligand was, 1 min after injection, recovered in a light, slowly sedimenting vesicle and subsequently (6 min) in larger endosomes. After 24 min the ligand was recovered in dense organelles, where also acid-soluble degradation products accumulated. 3. Immunocytochemical labelling of ultrathin cryosections showed that the ligand appeared rapidly after internalization in coated vesicles and subsequently in two larger types of endosomes. In the 'early' endosomes (1 min after injection) the labelling was seen closely associated with the membrane of the vesicle; after 6 min the ligand was evenly distributed in the lumen. At 24 min after injection the ligand was found in the lysosomes. 4. A bimodal distribution of endothelial cell lysosomes with different buoyant densities was revealed by centrifugation in iso-osmotic Nycodenz gradients, suggesting that two types of lysosomes are involved in the degradation of mannose-terminated glycoproteins in liver endothelial cells. Two populations of lysosomes were also revealed by sucrose-density-gradient centrifugation after injection of large amounts of yeast invertase. 5. In conclusion, ovalbumin is transferred rapidly through three endosomal compartments before delivering to the lysosomes. The degradation seems to take place in two populations of lysosomes.     

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study.

The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 microns in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absorption of elastase through the jejunal mucosa of the rat. An immunocytochemical study

In order to reveal the absorption process of elastase from the intestine, hog pancreatic elastase was injected into the ligated jejunum lumen of the rat, and the tissues were cytochemically observed at various times after injection. The peroxidase anti-peroxidase (PAP) method using anti-hog-elastase rabbit antibody was used for light microscopy, and the anti-elastase Fab'-peroxidase conjugate was used for electron microscopy. The tissues stained by the PAP method exhibited a dense deposition of reaction products on the luminal surface of epithelial cells and a moderate deposition in the blood and lymph capillaries of the intestinal villi. Immunoelectron microscopy revealed that the reaction product was deposited on the surface of the microvilli and in their pocketing; some was found in the pinocytotic vesicles in the terminal-web area and on the inner surface of the enlarged smooth endoplasmic reticulum. Round droplets which gave a positive reaction were found in the widened intercellular cleft and the thick basement membrane lining the blood capillaries and lymphatics. The jejunum retained its normal ultrastructure. The results indicate that the elastase molecules, which were introduced into the rat jejunum lumen, were absorbed without being decomposed through healthy intestinal epithelial cells by pinocytosis and translocated into blood and lymph capillaries.     

Intracellular transport of formaldehyde-treated serum albumin in liver endothelial cells after uptake via scavenger receptors.

Endocytosis of formaldehyde-treated serum albumin (FSA) mediated by the scavenger receptor was studied in rat liver endothelial cells. Suspended cells had about 8000 receptors/cell, whereas cultured cells had about 19,000 receptors/cell. Kd was 10(-8) M in both systems. Cell-surface scavenger receptors were found exclusively in coated pits by electron microscopy, by using ligand labelled with colloidal gold. Cell-surface-bound FSA could be released by decreasing the pH to 6.0; it was therefore possible to assess the rate of internalization of surface-bound ligand. This rate was very high: t1/2 for internalization of ligand prebound at 4 degrees C was 24 s. The endocytic rate constant at 37 degrees C, Ke, measured as described by Wiley & Cunningham [(1982) J. Biol. Chem. 257, 4222-4229], was 2.44 min-1, corresponding to t1/2 = 12 s. Uptake of FSA at 37 degrees C after destruction of one cell-surface pool of receptors by Pronase was decreased to 60%. This finding is compatible with a relatively large intracellular pool of receptors. The intracellular handling of 125I-tyramine-cellobiose-labelled FSA (125I-TC-FSA) was studied by subcellular fractionation in sucrose gradients, Nycodenz gradients or by differential centrifugation. The density distributions of degraded and undegraded 125I-TC-FSA after fractionation of isolated non-parenchymal cells and whole liver were similar, when studied in Nycodenz and sucrose gradients, suggesting that the subcellular distribution of the ligand was not influenced by the huge excess of non-endothelial material in a whole liver homogenate. Fractionation in sucrose gradients showed that the ligand was sequentially associated with organelles banding at 1.14, 1.17 and 1.21 g/ml. At 9-12 min after intravenous injection the ligand was in a degradative compartment, as indicated by the accumulation of acid-soluble radioactivity at 1.21 g/ml. A rapid transfer of ligand to the lysosomes was also indicated by the finding that a substantial proportion of the ligand could be degraded by incubating mitochondrial fractions prepared 12 min after intravenous injection of the ligand. The results indicate that FSA is very rapidly internalized and transferred through an endosomal compartment to the lysosomes. The endosomes are gradually converted into lysosomes between 9 and 12 min after injection of FSA. The rate-limiting step in the intracellular handling of 125I-TC-FSA is the degradation in the lysosomes.     

Prohormone transport through the secretory pathway of neuroendocrine cells.

En route through the secretory pathway of neuroendocrine cells, prohormones pass a series of membrane-bounded compartments. During this transport, the prohormones are sorted to secretory granules and proteolytically cleaved to bioactive peptides. Recently, progress has been made in a number of aspects concerning secretory protein transport and sorting, particularly with respect to transport events in the early regions of the secretory pathway. In this review we will deal with some of these aspects, including: i) selective exit from the endoplasmic reticulum via COPII-coated vesicles and the potential role of p24 putative cargo receptors in this process, ii) cisternal maturation as an alternative model for protein transport through the Golgi complex, and iii) the mechanisms that may be involved in the sorting of regulated secretory proteins to secretory granules. Although much remains to be learned, interesting new insights into the functioning of the secretory pathway have been obtained.     

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study

Summary The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 m in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled. From these findings, it is suggested that the granules of rat Clara cells consist of two types of granules of distinct origin; one appears to derive from condensing vacuoles of Golgi origin, whereas the other may be formed by membranefusions with small cytoplasmic vesicles of unknown source.     

Zinc uptake by isolated rat liver parenchymal cells.

Primary cultures of rat liver parenchymal cells maintained as a monolayer in serum-free culture medium were used to investigate the characteristics of zinc accumulation in vitro. Liver parenchymal cells accumulated zinc by a temperature-dependent, saturable process that was inhibited by cyanide, azide, oligomycin, N-ethylmaleimide and iodoacetamide. Cadmium reversibly inhibited zinc accumulation in both serum-free and serum-containing media. Gel filtration chromatographic studies showed that recently accumulated intracellular zinc was present as a low molecular weight complex smaller than metallothionein, the zinc storage protein, but larger than individual amino acids. The quantity of zinc accumulated was affected by preincubation of the cells with various hor?ONES. Dexamethasone, prednisone and prednisolone each increased zinc uptake by 40--50% when either insulin or glucagon was also present. Hydrocortisone, cortisone and sex steroids did not influence zinc accumulation. Removal of the polypeptide hormones from the medium abolished the stimulatory effect of the synthetic glucocorticoid steroid hormones on zinc accumulation.     

A study of intracellular transport of secretory glycoproteins in rat liver

To study the transport of secretory glycoproteins in the endoplasmic reticulum of rat liver, the distribution of nascent glycoproteins in the membrane and luminal fraction of rough and smooth microsomes has been examined after a short-time incorporation of radioactive glucosamine in vivo. 50--60% of the radioactivity was associated with the membranes of rough and smooth microsomes, whereas about 10% of the serum albumin was found in the same fractions. The relative amount of radioactivity in the membranes was the same whether the luminal content of the microsomal vesicles was released by sonication, French press, Triton X-100, Brij 35 or sodium deoxycholate. The distribution of labeled glycoproteins between the membrane and luminal fraction of rough and smooth microsomes did not change during the time interval of 15--120 min after administration of the isotope. The similarity of the labeling patterns obtained after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the same set of glycoproteins were located in the lumen and the membrane of rough and smooth microsomes. A specific precipitation of nascent glycoproteins from both the membrane and luminal fractions of rough and smooth microsomes were obtained with rabbit antiserum against rat serum. The nascent glycoproteins associated with the membranes were not released by high ionic strength or treatment with mercaptoethanol. A slow exchange between [14C]glucosamine-labeled glycoproteins in the lumen and membrane fraction was, however, found.     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

The mechanism of pantothenate transport by rat liver parenchymal cells in primary culture

The mechanism of pantothenate transport across the plasma membrane was investigated with initial velocity studies of [14C]pantothenate uptake and efflux in rat liver parenchymal cells maintained in primary culture. At 116 mM sodium, double-reciprocal plots of the initial velocity of uptake versus [pantothenate] were linear from 0.3 to 36.5 microM pantothenate and gave an apparent Km,pant of 11 +/- 2 microM. The rate of pantothenate uptake at 0 [sodium] was about 14% of the rate at 116 mM sodium, and the reciprocal of the apparent Km,pant was a linear function of [sodium]. Vmax obtained by extrapolation to infinite [pantothenate] was independent of [sodium]. Ouabain, gramicidin D, cyanide, azide, and 2,4-dinitrophenol inhibited uptake, but preloading cells with pantothenate did not. Pantothenate derivatives or carboxylic acids were only weak inhibitors of uptake. Efflux was measured in cells preloaded with [14C]pantothenate. The apparent Km for efflux was 85 +/- 29 microM, and the rate of efflux was unaffected by addition of pantothenate, sodium, ouabain, gramicidin D, or 2,4-dinitrophenol to the external medium. These features are consistent with a mechanism for pantothenate transport in which sodium and pantothenate are cotransported in a 1:1 ratio on a carrier highly specific for pantothenate; sodium decreases the apparent Km for pantothenate, and a sodium-carrier complex forms only on the intracellular side of the membrane.     

Transendothelial transport of serum albumin: a quantitative immunocytochemical study

The steady-state distribution of endogenous albumin in mouse diaphragm was determined by quantitative postembedding protein A-gold immunocytochemistry using a specific anti-mouse albumin antibody. Labeling density was recorded over vascular lumen, endothelium, junctions, and subendothelial space. At equilibrium, the volume density of interstitial albumin was 18% of that in circulation. Despite this large difference in albumin concentration between capillary lumen and interstitium, plasmalemmal vesicles labeling was uniformly distributed across the endothelial profile. 68% of the junctions displayed labeling for albumin, which was however low and confined to the luminal and abluminal sides. The scarce labeling of the endothelial cell surface did not confirm the fiber matrix theory. The kinetics of albumin transcytosis was evaluated by injecting radioiodinated and DNP-tagged BSA. At 3, 10, 30, and 60 min, and 3, 5, and 24 h circulation time, blood radioactivity was measured and diaphragms were fixed and embedded. Anti-DNP antibodies were used to map the tracer in aforementioned compartments. A linear relationship between blood radioactivity and vascular labeling density was found, with a detection sensitivity approaching 1 gold particle per DNP-BSA molecule. Tracer presence over endothelial vesicles reached rapidly (10 min) a saturation value; initially localized near the luminal front, it evolved towards a uniform distribution across endothelium during the first hour. An hour was also needed to reach the saturation limit within the subendothelial space. Labeling of the junctions increased slowly, out of phase with the inferred transendothelial albumin fluxes. This suggests that they play little, if any, role in albumin transcytosis, which rather seems to proceed through the vesicular way.     

Cytoplasmic proteases of rat liver parenchymal cells

Soluble extracts of isolated rat liver parenchymal cells contained three proteases with alkaline pH optima. One protease was a high molecular weight (Mr = 500,000) enzyme which was stimulated by ATP. The other two proteases were totally dependent on calcium for activity and displayed different calcium concentration requirements. One was half-maximally activated by 150 μM Ca²⁺ while the other required only 10 μM Ca²⁺ for half-maximal activation.     

Amino acid transport in plasma membrane vesicles from isolated rat liver parenchymal cells

Plasma membrane vesicles were prepared from isolated rat liver parenchymal cells. The transport of several amino acids was studied and found to be identical to that in membrane vesicles from whole liver tissue.     

Induction of amino acid transport in primary cultures of adult rat liver parenchymal cells by insulin.

Amino acid transport was studied in primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion technique and maintained as a monolayer in a serum-free culture medium. Amino acid transport was assayed by measuring the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid. Rat liver parenchymal cells transported alpha-aminoisobutyric acid by an energy-dependent Na+-requiring system which displayed Michaelis-Menten kinetics. Addition of insulin to cultured rat liver parenchymal cells resulted in an increased influx of alpha-aminoisobutyric acid which was reflected in a higher initial rate of alpha-aminoisobutyric acid transport as well as an increased accumulation of alpha-aminoisobutyric acid at later time points. Cycloheximide effectively blocked the increase while results with actinomycin D were equivocal. Insulin at concentrations as low as 50 pM was effective in stimulating alpha-aminoisobutyric acid transport while the maximal response was observed at 80 nM.