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W Bieger

Publications and source records attributed to W Bieger.

35 records · Page 2Linked to original sources
PubMed1976

Studies on secretory glycoproteins in the rat exocrine pancreas. I. Fine structure of the Golgi complex and release of fucose-labeled proteins after in vivo stimulation with caerulein.

Using a double-label technique on isolated rat pancreatic lobules, the rate of synthesis and discharge of regular and fucosylated secretory proteins was studied under control conditions and after in vivo prestimulation with caerulein. Both labeled leucine and fucose were incorporated into pancreatic proteins at a linear rate, which was potentiated by in vivo stimulation. In pulse-chase experiments both regular and fucosylated secretory proteins were discharged into the medium in parallel. The in vivo pretreatment with caerulein caused an earlier discharge and increased the total amount released. Kinetic analysis of unstimulated (baseline) discharge of both classes of secretory proteins indicated in vitro sensitivity by the previous in vivo treatment with caerulein. The biochemical data were compared to the fine structure of the Golgi complex under both control and prestimulated ocnditions. The Golgi stacks were composed of four to six individual cisternae which in some cases were connected by intercisternal pores. Transporting vesicles were observed fusing along the total length of the outermost cisterna on both the cis- and trans-side and with the lateral ends of the intermediate cisternae. Under control conditions only the last trans-cisterna contained some electron opaque material; in vivo prestimulation led to distension and filling of all disternae in an individual Golgi-unit. Numerous stages of transformation of the last transcisterna into condensing vacuoles were observed, lending support to the hypothesis that during packaging of secretory products the membranes of the Golgi complex undergo a continuous turnover.

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PubMed1976

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas. V. Kinetic studies on accelerated transport following caerulein infusion in vivo.

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 x 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.

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PubMed1976

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas. IV. Stimulation by in vivo infusion of caerulein.

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.

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PubMed1975

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas. II. Inhibition of antimicrotubular agents.

The possible role of microtubules and microfilaments in the secretory process of the rat exocrine pancreas was analysed in vitro using isolated pancreatic lobules. Colchicine and vinblastine as microtubule inhibitors, hexylene glycol as a microtubule stabilizer, and cytochalasin B as a disruptive agent for microfilaments were used in increasing concentrations to test their effects on protein synthesis, intracellular transport, zymogen discharge, and cellular respiration. Colchicine only at 10(-2) M concentrations inhibits protein synthesis, while vinblastine inhibits at 10(-6) and 10(-5) M by 20% and at 10(-4) M by 55%. A similar inhibition is observed with 1.5% concentrations of hexylene glycol while cytochalasine B at 1,5 and 10 mug/ml is without effect on protein synthesis. Colchicine and vinblastine have their major effects on intracellular transport both in secretion studies and cell fractionation experiments. Colchicine in concentrations between 10(-3) to 10(-5) M inhibits discharge of newly synthesized proteins by 50%, while vinblastine shows a dose-response relationship of 40% inhibition of 10(-6) M to 90% at 10(-4) M. Discharge of amylase is uniformly reduced by 30% by both colchicine and vinblastine in the whole dose range. The pronounced effect of colchicine and vinblastine is evident in cell fractionation studies: both drugs inhibit the disappearance of protein radioactivity from microsomes and its appearance in zymogen granules; similarly the peak radioactivity in smooth microsomes (Golgi) appears delayed. No differential effect on the secretory process was observed with 1.5% concentrations of hexylene glycol or cytochalasin B at 1.5 and 10 mug/ml concentrations. A fines tructural analysis of microtubules and microfilaments in the exocrine pancreatic cell reveals their distribution in all parts of the cytoplasm and in relation to all cell organelles. Both systems (microtubules, microfilaments) seem to be connected, at least in certain areas of the cytoplasm and at the plasma membrane. The reduction of transport efficiency by microtubule inhibitors results in a deposition of secretory material in the cristernal space of the rough endoplasmatic reticulum, which leads to the formation of paracrystals. Colchicine at 10(-3) M concentrations leads to an enlargement of condensing vacuoles in the Golgi complex.

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PubMed1975

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas. III. Effect of cobalt, lanthanum and antimycin A.

The effects of cobalt and lanthanum on the secretory process of the rat exocrine pancreas was studied in vitro using isolated pancreatic lobules. Cobalt in concentrations between 10(-3) to 10(-5) M has no effect on the rate of protein synthesis, intracellular transport, or discharge of zymogen granules, if the total population of stored granules is considered. It has, however, a marked effect on the release of newly packed zymogen granules which are formed during incubation in 10(-3) M CoC1(2). Determination of specific radioactivity in amylase released under the stimulation of 5X10(-6) M carbamylcholine and of total proteins retained in the zymogen granule fraction during stimulation indicate that granules formed during incubation in CoC1(2) are excluded from discharge. Lanthanum, on the other hand, has a differential effect on protein synthesis, intracellular transport, and discharge. Incorporation of tritiated leucine into TCA-precipitable proteins is inhibited by 50% at 10(-3) M LaC1(3). Intracellular transport as studied by cell fractionation is not changed during the first 35 min post pulse but is delayed from then on. This late effect is more pronounced if pancreatic lobules are preincubated for 60 min in 10(-3) M LaC1(3). Discharge of amylase and newly synthesized proteins is inhibited dose-dependently up to 80% by 10(-3) M LaC1(3). The effects of both cobalt and lanthanum are not due to an inhibition of cellular respiration. Comparison of these results with the inhibitory action of antimycin A between 10(-4) to 10(-8) M concentrations reveals a dose-dependent diminution of the rate of protein synthesis and intracellular transport, while discharge of granules is less energy dependent. The fine structural appearance of pancreatic lobules after 3 hrs incubation in 10(-3) M CoC1(2) is not altered, while in 5X10(-3) and 10(-3) M lanthanum acinar lumina are enlarged and the apical cytoplasm contains large vacuoles. At the highest concentration of lanthanum a flocculent electron dense material is observed apposed to the external lamina of the plasma membrane. The distribution of this material on the membrane is described. Antimycin A leads to cellular changes corresponding to the irreversible inhibition of cellular respiration. It is concluded from the results that cobalt acts on the process of granule formation inside the cell, while lanthanum by its binding to the plasma membrane may alter molecules involved in secretagogue binding and transport systems into the cell.

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PubMed1975

Studies on intracellular transport in the rat exocrine pancreas. I. Inhibition by aromatic amino acids in vitro.

In vitro incubation of rat pancreatic lobules in the presence of 10 mM concentrations of 2 natural (phenylalanine, tryptophane) and 2 modified aromatic amino acids (p-fluorophenylalanine, p-chlorophenylalanine) induces paracrystal formation in the cisternal space of the rough endoplasmic reticulum and in the acinar lumen. Aggregation of secretory material in transitional elements of the rough endoplasmic reticulum suggests tubular connection to the Golgi complex. Paracrystal formation is correlated with a disturbance of the three major phases in the secretory process of the exocrine cell. Incorporation of radioactive amino acids into proteins is inhibited by 10 mM concentrations of phenylalanine and tryptophane by 20 and 50% respectively and by p-chlorophenylalanine at 1 and 10 mM concentrations by 50 and 75%. The inhibition of protein synthesis is not due to a reduced intracellular concentration of radioactive precursor amino acids. Intracellular transport of newly synthesized proteins as studied by a radioassay for zymogen discharge and by cell fractionation is similarly inhibited by phenylalanine, tryptophane and p-chlorophenylalanine at 10 mM concentrarions (20, 30, and 40% respectively). Discharge of zymogens as measured by the secretion of amylase stimulated with 5 X 10(-6) M carbamylcholine is reduced by 20% if 10 mM concentrations of phenylalanine, tryptophane or p-chlorophenylalanine are present in the medium. Paracrystals were isolated by differential centrifugation and their protein content compared with isolated zymogen granules. On sodium dodecylsulfate gel electrophoresis paracrystalline proteins show the same electrophoretic pattern as the content of zymogen granules.

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