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.     

Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment

The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0- 10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.     

Brefeldin A arrests the intracellular transport of viral envelope proteins in primary cultured rat hepatocytes and HepG2 cells.

We have studied the effect of brefeldin A (BFA) on the intracellular transport of the envelope proteins of vesicular stomatitis virus (VSV) and sindbis virus in primary cultured rat hepatocytes. BFA (2.5 micrograms/ml) inhibited not only the secretion of plasma proteins into the medium, but also the assembly of both G protein of VSV and E1 and E2 proteins (envelope proteins) of sindbis virus into respective virions. Concomitantly, both the acquisition of endo-beta-N-acetylglucosaminidase H resistance by the G protein and the proteolytic conversion of PE2 to E2 were found to be inhibited in the BFA-treated cells, suggesting that the intracellular transport of the envelope proteins was arrested in the endoplasmic reticulum. Such inhibitory effects of the drug were variable depending upon the culture conditions of the hepatocytes. In the 1-day-cultured cells, even in the presence of the drug, newly synthesized envelope proteins were assembled into the virions after a 3 h chase period, at the same time as secretion of plasma proteins into the medium resumes. In contrast, in 4-day-cultured hepatocytes, BFA continuously blocked the entry of the envelope proteins into the virions and the release of plasma proteins into the medium for at least 5 h. BFA also completely inhibited the exocytotic pathway in HepG2 cells. These results indicate that the duration time of the effect of BFA is different from one cell to another and may change depending upon the culture conditions of the cells.     

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     

Morphological effects of brefeldin A on the intracellular transport of secretory materials in parotid acinar cells

The morphological effects of Brefeldin A (BFA) on the parotid acinar cells of a rat were investigated at the stage of active resynthesis of secretory materials following administration of the secretogogue, isoproterenol. Incubation with BFA resulted in: a) marked dilation of the rough endoplasmic reticulum (RER), b) involution of the Golgi complex to rudimentary forms which disseminated throughout the cytoplasm, and c) agenesis of secretion granules. It appears that the primary action of BFA is inhibition of the export of secretory materials from the RER toward the Golgi complexes. Histochemical staining indicated the thiamine pyrophosphatase (TPPase) positive saccules of the Golgi stack to undergo degradation in autophagic vacuoles. In contrast, small vesicles showing the osmium reducing activity characteristic of cis elements, including osmium negative vesicles, continued to be present throughout a 4-h period of investigation, indicating the cis and, most likely, medial elements to be the components of the rudimentary Golgi complexes. On removal of the drug, a large number of transport vesicles appeared immediately from the RER and carried secretory materials to the rudimentary Golgi complex, so that the organelles were rapidly reconstructed within 30-60 min, followed by the reaccumulation of secretory granules by 90 min. It is thus indicated that the size and configuration of the Golgi complex is regulated by a dynamic equilibrium of the transport of secretory materials, and that the rudimentary Golgi complex containing cis and probably medial elements may function as the smallest units of the Golgi complex for full development as seen under normal conditions.     

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.     

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.     

Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.