Intracellular transport of albumin through the secretory apparatus of rat liver parenchymal cells. An immunocytochemical study

The fine structural localization of albumin in rat liver parenchymal cells was determined by an improved immunocytochemical method and serial sectioning. Albumin in the secretory apparatus of the parenchymal cells was present in segments of the rough endoplasmic reticulum, interrupted with negative segments, in transport vesicles, Golgi saccules, finely anastomosed tubules and vesicles on the trans side of the Golgi complex, and in secretion granules. Horizontally sectioned Golgi saccules contained lipoprotein particles on one side and albumin on the other side. After transport, the vesicles that contained albumin fused with the so-called rigid lamellae on the trans-side of the Golgi complex. Ultrathin serial sections revealed no true structural continuity between the endoplasmic reticulum and the cis-aspect of the Golgi complex. We concluded that secretory proteins are transported from the endoplasmic reticulum to the Golgi complex by transport vesicles that bud from the endoplasmic reticulum and fuse with the Golgi saccules. These vesicles fuse regularly with the Golgi saccules on the cis-side and occasionally with tubular elements on the trans-aspect that may belong to the so-called GERL.     

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas

Summary Prolonged secretory stimulation of the exocrine pancreas in the rat by in vivo infusion of caerulein leads to a rapid degranulation of the organ associated with a progressive reduction in the size of the zymogen granules. During the first six to twelve hours of stimulation Golgi complexes are enlarged and several structural forms of multivesicular bodies are found indicating a lysosomal degradation of membrane material in the Golgi area. Maximum secretory activity is obtained after a 24 hour infusion, Golgi complexes appear fragmented, the secretory granules measure only 1/3 to 1/4 their normal size. Thereafter, in spite of a continuous stimulation, the exocrine cells regranulate progressively up to 72 hours of infusion. This regranulation is associated with massive enlargement of the Golgi complexes.The phasic adaptation of the exocrine pancreas to prolonged stimulation, concluded from the structural studies, was confirmed by biochemical analysis of protein synthesis, intracellular transport and enzyme discharge. Pancreatic protein synthesis as measured by the incorporation of tritiated leucine remained unchanged during the first six hours of stimulation, then increased reaching a maximum of 230% of the control levels after 24 hours of infusion. After 48 and 72 hours the rate of protein synthesis decreased again to normal values. Most pronounced changes were observed in the kinetics of intracellular transport of newly synthesized proteins. Using pulse-chase incubation of prestimulated pancreatic lobules, the rate of transition of secretory proteins through the cell increased consistently with prolonged infusion periods reaching maximal acceleration after 24 hours. Newly synthesized proteins were transported and segregated up to ten times faster than in controls. After a maximum at 24 hours transport returned to normal rates after 72 hours of infusion. Enzyme secretion, measured for amylase, followed a similar pattern of stimulation.The results suggest a phasic adaptation of the exocrine pancreatic cell to prolonged stimulation. They demonstrate for the first time the possibility of an acceleration of intracellular transport by means of secretagogues.Dedicated to Professor W. Bargmann on the occasion of his 70th birthday.Supported by a grant from Deutsche Forschungsgemeinschaft (Ke 113/8). A preliminary communication was presented at the 9th annual meeting of the European Society for Clinical Investigation, Rotterdam (April 24–26, 1975). The expert technical assistance of Miss Helga Hollerbach and Miss Hiltraud Hosser is gratefully acknowledged.     

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas

Summary The previous finding that intracellular transport of secretory proteins in the rat exocrine pancreas is accelerated by in vivo stimulation with a pancreatic secretagogue has been further analyzed. Using a radioassay for discharge of newly synthesized proteins, the rate of release was compared in control and prestimulated lobules. In control preparations discharge occurred with an initial lag period of 30 minutes and a maximum after two hours of incubation. After in vivo infusion of 5 × 10^-8 g/hr. caerulein for 24 h in vitro discharge started after 10 minutes of in vitro incubation and attained a maximal rate after one hour. Using the same radioassay and several inhibitors of intracellular transport and granule discharge, it could be demonstrated that both processes were reduced to the same extent in controls and in lobules with accelerated transport. To obtain direct evidence for the degree of acceleration of the different transport steps between rough endoplasmic reticulum, Golgi complex and zymogen granules, the respective subcellular fractions of these organelles prepared and characterized ultrastructurally and biochemically. The rate of disappearance of newly formed proteins from rough microsomes and the appearance in smooth microsomes and zymogen granules were significantly increased after in vivo stimulation. The data substantiate an acceleration of the regular transport steps by the secretagogue. There was no indication that a high level of secretory activity leads to a rerouting of secretory proteins or to an omission of one of the regular steps in intracellular transport.Supported by a grant from Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg (Ke 113/10) The expert technical assistance of Miss Hiltraud Hosser and Miss Helga Hollerbach is gratefully acknowledged     

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.     

Disparate effects of monensin and colchicine on intracellular processing of secretory proteins in cultured rat hepatocytes

We have studied the biosynthesis and intracellular processing of three major secretory proteins, albumin, alpha 1-protease inhibitor and alpha 2u-globulin, in cultured rat hepatocytes. The effect of secretion-blocking agents, monensin, a monovalent ionophore, and the microtubule-affecting agents colchicine and taxol was determined. In the control cells, alpha 1-protease inhibitor, a glycoprotein, was first synthesized as an endoglycosidase-H-sensitive form with Mr 51 000, and then processed to two endoglycosidase-H-resistant forms having Mr 51 000 and 56 000, the latter of which was secreted into the medium. Initially synthesized proalbumin was converted with chase to serum-type albumin, while no pro-type precursor was identified for alpha 2u-globulin. In the cells treated with colchicine or taxol, in which secretion was greatly inhibited, the fully processed alpha 1-protease inhibitor and albumin accumulated and were finally secreted into the medium. In the monensin-treated cells, however, most of the newly synthesized alpha 1-protease inhibitor and albumin were not processed to the final mature forms, resulting in accumulation of two 51 000-Mr forms and proalbumin, respectively. Moreover in treated cells, proalbumin and the endoglycosidase-H-resistant alpha 1-protease inhibitor were finally secreted into the medium. Such an effect was not caused by NH4Cl which also inhibited the secretion and is known to exert the similar effect as monensin on the receptor-mediated endocytosis pathway. Based on these results, the use of monensin may prove valuable for more detailed analysis of intracellular processing of various proteins.     

Comparative studies of intracellular transport of secretory proteins

The physiology of protein intracellular transport and secretion by cell types thought to be free from short-term control has been compared with that of the pancreatic acinar cell, using pulse-chase protocols to follow biosynthetically-labeled secretory products. Data previously obtained (Tartakoff, A.M., and P. Vassalli. J. Exp. Med. 146:1332-1345) has shown that plasma-cell immunoglobulin (Ig) secretion is inhibited by respiratory inhibitors, by partial Na/K equilibration effected by the carboxylic ionophore monensin, and by calcium withdrawal effected by the carboxylic ionophore A 23187 in the presence of ethylene glycol bis (beta-aminoethylether)-N,N,N'',N''-tetraacetic acid (EGTA) and absence of calcium. We report here that both inhibition of respiration and treatment with monensin slow secretion by fibroblasts, and also macrophages and slow intracellular transport (though not discharge per se) by the exocrine pancreatic cells. Attempted calcium withdrawal is inhibitory for fibroblasts but not for macrophages. The elimination of extracellular calcium or addition of 50 mM KCl has no major effect on secretory rate of either fibroblasts or macrophages. Electron microscopic examination of all cell types shows that monensin causes a rapid and impressive dilation of Golgi elements. Combined cell fractionation and autoradiographic studies of the pancreas show that the effect of monensin is exerted at the point of the exit of secretory protein from the Golgi apparatus. Other steps in intracellular transport proceed at normal rates. These observations suggest a common effect of the cytoplasmic Na/K balance at the Golgi level and lead to a model of intracellular transport in which secretory product obligatorily passes through Golgi elements (cisternae?) that are sensitive to monensin. Thus, intracellular transport follows a similar course in both regulated and nonregulated secretory cells up to the level of distal Golgi elements.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Summary Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

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.     

Receptor-mediated endocytosis of ovalbumin by two carbohydrate-specific receptors in rat liver cells. The intracellular transport of ovalbumin to lysosomes is faster in liver endothelial cells than in parenchymal cells.

1. The uptake of ovalbumin (OVA) in rat liver parenchymal cells (PC) and non-parenchymal cells was studied in vivo and in vitro in order to compare the cellular expression of glycoprotein receptors and the kinetics of intracellular transport of ligand endocytosed by these receptors. 2. Ovalbumin was labelled with 125I or with 125I-tyramine-cellobiose (125I-TC). By using 125I-TC-OVA the labelled degradation products were trapped in the cells. 3. 125I-TC-OVA was rapidly cleared from blood mainly by receptor-mediated uptake in the liver. At 30 min after injection, 50% of the ligand was recovered in the liver. The endothelial cells (EC) and the PC were the predominant cell types responsible for uptake. 4. The uptake in PC was strongly inhibited by asialo-orosomucoid (AOM), but not by mannan, indicating that the uptake in these cells was mediated by the galactose receptor and not by the mannose receptor. This finding is compatible with the observation that a proportion of the OVA contains terminal galactose residues in the carbohydrate moiety. 5. In vitro uptake of OVA in cultured EC was saturable and inhibited by mannan, mannose, fructose, N-acetylglucosamine, EDTA or monensin, but not by galactose or AOM. The uptake of OVA in these cells was therefore mediated by the mannose receptor. 6. To label the organelles involved in endocytosis in PC and EC, 125I-TC-OVA was injected intravenously together with an excess of either AOM or mannan. In this way the labelled ligand could be directed selectively to EC or PC respectively. Subcellular fractionation of total liver in sucrose and Nycodenz gradients revealed that in EC the intracellular transport of OVA is so fast that endocytosed ligand accumulates and thus increases the density of the lysosomes. Conversely, in PC transfer of ligand is slower, with the result that accumulation of undegraded ligand in the lysosomes does not occur. These findings are interpreted to mean that in EC the rate-limiting step of handling of endocytosed ligand is intralysosomal degradation, whereas in PC the rate-limiting step is transport of ligand to the lysosomes. 7. Altogether, these findings suggest that endocytosis of OVA by the liver EC and PC is mediated by mannose and galactose receptors respectively, and that the kinetics of intracellular transport of OVA differ in the two cell types.     

Immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells

The bovine exocrine pancreatic cell produces a variety of enzymes and proenzymes for export. Biochemical studies by Greene L.J., C.H. Hirs, and G.E. Palade (J. Biol. Chem. 1963. 238:2054) have shown that the mass proportions of several of these proteins in resting pancreatic juice and zymogen granule fractions are identical. In this study we have used immunocytochemical techniques at the electron microscope level to determine whether regional differences exist in the bovine gland with regard to production of individual secretory proteins and whether specialization of product handling occurs at the subcellular level. The technique used is a modification of one previously reported (McLean, J.D., and S.J. Singer. 1970. Proc. Natl. Acad. Sci U.S.A. 69:1771) in which immunocytochemical reagents are applied to thin sections of bovine serum albumin-imbedded tissue and zymogen granule fractions. A double antibody technique was used in which the first step consisted of rabbit F(ab')2 antibovine secretory protein and the detection step consisted of sheep (F(ab')2 antirabbit F(ab')2 conjugated to ferritin. The results showed that all exocrine cells in the gland, and all zymogen granules and Golgi cisternae in each cell, were qualitatively alike with regard to their content of secretory proteins examined (trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggest that these secretory proteins are transported through the cisternae of the Golgi complex where they are intermixed before copackaging in zymogen granules; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins, and is independent of extent of glycosylation, e.g., trypsinogen, a nonglycoprotein vs. DNase, a glycoprotein.     

Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes

We examined the effect of brefeldin A, an antiviral antibiotic, on protein synthesis, intracellular processing, and secretion in primary culture of rat hepatocytes. The secretion was strongly blocked by the drug at 1 microgram/ml and higher concentrations, while the protein synthesis was maintained fairly well. Pulse-chase experiments with [35S]methionine demonstrated that brefeldin A completely blocked the proteolytic conversion of proalbumin to serum albumin up to 60 min of chase, although its conversion was observed as early as 20 min in the control cells. The drug also inhibited the terminal glycosylation of oligosaccharide chains of alpha 1-protease inhibitor and haptoglobin. These two modifications have been shown to occur at the trans region of the Golgi complex. The drug, however, had no effect on the proteolytic processing of the haptoglobin proform which takes place within the endoplasmic reticulum. Such an effect by brefeldin A is very similar with that induced by the carboxylic ionophore monensin. However, in contrast to evidence that monensin causes a delayed secretion of the unprocessed forms of these proteins, brefeldin A allowed the completely processed forms to be secreted after a prolonged accumulation of the unprocessed forms. Morphological observations demonstrated that the endoplasmic reticulum was markedly dilated by treatment with the drug at 10 micrograms/ml which continuously blocked the secretion. On the other hand, brefeldin A caused no inhibitory effect on the endocytic pathway as judged by cellular uptake and degradation of 125I-asialofetuin. These results indicate that brefeldin A is a unique agent which primarily impedes protein transport from the endoplasmic reticulum to the Golgi complex by a mechanism different from those considered for other secretion-blocking agents so far reported.     

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.     

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.     

Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-³H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.     

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.     

The role of cytosolic proteins in the intracellular transport of haem in rat liver. A dual-label approach.

After consecutive injections of delta-amino[3H]- and -[14C]-laevulinic acid, the incorporation of the two labels into haem associated with different subfractions of the liver was determined. Marked differences in the 14C/3H ratios were observed between haem associated loosely and tightly with microsomes and mitochondria and haem associated with three subfractions of the cytosol obtained by gel filtration. The effect of changing the amounts of delta-aminolaevulinic acid injected and of changing the interval between injections and killing of the animal on the ratios of labels in the haem of each subfraction was studied. The results are discussed in terms of the flow of haem from the mitochondria to other parts of the cell via putative cytosolic carrier proteins.