Analysis of Ca 2+ fluxes and Ca 2+ pools in pancreatic acini

1981 ◽  
Vol 296 (1080) ◽  
pp. 105-113 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Intact Cells ◽  
Pancreatic Acini ◽  
Mitochondrial Inhibitors ◽  
Intracellular Ca ◽  
Membrane Bound ◽  
Intracellular Storage ◽  
Carbamyl Choline

45 Ca 2+ movements have been analysed in dispersed acini prepared from rat pancreas in a quasi-steady state for 45 Ca 2+ . Carbamyl choline (carbachol; Cch) caused a quick 45 Ca 2+ release that was followed by a slower 45 Ca 2+ ‘reuptake’. Subsequent addition of atropine resulted in a further transient increase in cellular 45 Ca 2+ . The data suggest the presence of a Cch-sensitive ‘trigger’ pool, which could be refilled by the antagonist, and one or more intracellular ‘storage’ pools. Intracellular Ca 2+ sequestration was studied in isolated acini pretreated with saponin to disrupt their plasma membranes. In the presence of 45 Ca 2+ (1 µM), addition of ATP at 5 mM caused a rapid increase in 45 Ca 2+ uptake exceeding the control by fivefold. Maximal ATP-promoted Ca 2+ uptake was obtained at 10 µM Ca 2+ (half-maximal at 0.32 µM Ca 2+ ). In the presence of mitochondrial inhibitors it was 0.1 µM (half-maximal at 0.014 µM). 45 Ca 2+ release could still be induced by Cch but the subsequent reuptake was missing. The latter was restored by ATP and atropine caused further 45 Ca 2+ uptake. Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca 2+ , oxalate and ATP which were absent in intact cells or cells pretreated with A23187. The data suggest the presence of a plasma membrane-bound Cch-sensitive ‘trigger’ Ca 2+ pool and ATP-dependent Ca 2+ storage systems in mitochondria and rough endoplasmic reticulum of pancreatic acini. It is assumed that Ca 2+ is taken up into these pools after secretagogue-induced Ca 2+ release.

scholarly journals Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine

1984 ◽  
Vol 219 (2) ◽  
pp. 679-685 ◽  
Author(s):  
A E Richardson ◽  
R L Dormer
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Ionophore A23187 ◽  
Uptake System ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Pancreatic Acini ◽  
Dense Fraction

Two microsomal subfractions from isolated rat pancreatic acini were produced by centrifugation through a discontinuous sucrose density gradient and characterized by biochemical markers. The denser fraction (SF2) was a highly purified preparation of rough endoplasmic reticulum; the less-dense fraction (SF1) was heterogeneous and contained Golgi, endoplasmic reticulum and plasma membranes. 45Ca2+ accumulation in the presence of ATP and its rapid release after treatment with the bivalent-cation ionophore A23187 were demonstrated in both fractions. The pH optimum for active 45Ca2+ uptake was approx. 6.8 for the rough endoplasmic reticulum (SF2) and approx. 7.5 for SF1 . Initial rate measurements were used to determine the affinity of the rough-endoplasmic-reticulum uptake system for free Ca2+. An apparent Km of 0.16 +/- 0.06 microM and Vmax. of 21.5 +/- 5.6 nmol of Ca2+/min per mg of protein were obtained. 45Ca2+ uptake by SF1 was less sensitive to Ca2+, half-maximal uptake occurring at 1-2 microM-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 microM-carbamylcholine, 45Ca2+ uptake was increased in the rough endoplasmic reticulum. The increased uptake was due to a higher Vmax. with no significant change in Km. No effect was observed on 45Ca2+ uptake by SF1 . In conclusion, two distinct non-mitochondrial, ATP-dependent calcium-uptake systems have been demonstrated in rat pancreatic acini. One of these is located in the rough endoplasmic reticulum, but the precise location of the other has not been determined. We have shown that the Ca2+-transporting activity in the rough endoplasmic reticulum may have an important role in maintaining the cytosolic free Ca2+ concentration in resting acinar cells and is involved in Ca2+ movements which occur during stimulation of enzyme secretion.

A simple biochemical approach to quantitate rough endoplasmic reticulum

1995 ◽  
Vol 268 (2) ◽  
pp. C308-C316 ◽  
Author(s):  
A. K. Rajasekaran ◽  
S. A. Langhans-Rajasekaran ◽  
R. M. Gould ◽  
E. Rodriguez-Boulan ◽  
T. Morimoto
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Line ◽  
Rough Endoplasmic Reticulum ◽  
Cell Membranes ◽  
Fold Increase ◽  
Sucrose Density Gradient ◽  
Total Cell ◽  
Cell Fractionation ◽  
Cell Line Hela ◽  
Membrane Bound

In this report we demonstrate that the changes in size of the rough endoplasmic reticulum (RER) can be determined by quantifying the membrane-bound ribosomal population separated by cell fractionation and sucrose density gradient analysis. Total cell membranes, rather than microsomes, were used as the source of membrane-bound ribosomes to eliminate potential losses during the preparation of microsomes. Bound ribosomes were assayed after quantitative release and recovery from total cell membranes using puromycin in the presence of high-salt buffer. Using this analysis, we demonstrate a 4.2-fold increase in RER in estrogen-treated male Xenopus laevis liver. Furthermore, we show that the ratio of the distribution of free to membrane-bound ribosomes in a nonsecretory cell line (HeLa) was 3.3, while this ratio in a secretory cell line (AR42J) was 1.2, indicating that cells active in secretion contain more RER. We suggest that this biochemical technique provides a simpler assay to detect changes in the size of the RER.

The Occurrence of Phenylalanine Ammonia-Lyase and Cinnamic Acid p-Hydroxylase on the Endoplasmic Reticulum of Cell Suspension Cultures of Glycine max

1978 ◽  
Vol 33 (1-2) ◽  
pp. 65-69 ◽  
Author(s):  
C. Postius ◽  
H. Kindi
Keyword(s):  
Endoplasmic Reticulum ◽  
Glycine Max ◽  
Golgi Apparatus ◽  
Cinnamic Acid ◽  
Time Course ◽  
Phenylalanine Ammonia Lyase ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Activity Increase ◽  
Membrane Bound

Abstract 1. The time course of activity of soluble and microsomal phenylalanine ammonia-lyase (PAL) was studied in dark grown cell cultures of soybean (Glycine max). A distinct activity increase of PAL in the soluble and microsomal fraction occurred prior to the stationary phase of the cell culture. Cinnamic acid p-hydroxylase and NADH : cytochrome c reductase, too, exhibited maximal activity in the log phase, 5 days after the transfer of soybean cells to fresh culture medium.2. Upon subfractionation of the once washed microsomal fraction by sedimentation velocity centrifugation on a sucrose gradient, membranes of the endoplasmic reticulum could be separated from fractions containing mainly membranes from the Golgi apparatus or plasma membranes, respectively. PAL and cinnamic acid p-hydroxylase were found in fractions of endoplasmic reticulum whereas no activity of either enzymes could be detected in fractions containing Golgi apparatus or plasma membranes.3. Repeated washing of microsomal fractions led to a residual membrane-bound PAL representing about 1% of the total PAL activity of the cells. This residual membrane-bound activity could be solubilized almost completely by Triton X-100 or digitonin at concentrations of 0.5 - 5%.

scholarly journals Redox-assisted regulation of Ca2+ homeostasis in the endoplasmic reticulum by disulfide reductase ERdj5

2016 ◽  
Vol 113 (41) ◽  
pp. E6055-E6063 ◽  
Author(s):  
Ryo Ushioda ◽  
Akitoshi Miyamoto ◽  
Michio Inoue ◽  
Satoshi Watanabe ◽  
Masaki Okumura ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Disulfide Bond ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Cellular Functions ◽  
Endoplasmic Reticulum Calcium ◽  
Disulfide Reductase ◽  
Intracellular Ca ◽  
Activity Dependent ◽  
Er Calcium

Calcium ion (Ca2+) is an important second messenger that regulates numerous cellular functions. Intracellular Ca2+ concentration ([Ca2+]i) is strictly controlled by Ca2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca2+ from the cytosol into the ER in an ATPase activity-dependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond. Notably, ERdj5 activated SERCA2b at a lower ER luminal [Ca2+] ([Ca2+]ER), whereas a higher [Ca2+]ER induced ERdj5 to form oligomers that were no longer able to interact with the pump, suggesting [Ca2+]ER-dependent regulation. Binding Ig protein, an ER-resident molecular chaperone, exerted a regulatory role in the oligomerization by binding to the J domain of ERdj5. These results identify ERdj5 as one of the master regulators of ER calcium homeostasis and thus shed light on the importance of cross talk among redox, Ca2+, and protein homeostasis in the ER.

Processing of receptor-bound somatostatin: internalization and degradation by pancreatic acini

1987 ◽  
Vol 252 (4) ◽  
pp. G535-G542 ◽  
Author(s):  
N. Viguerie ◽  
J. P. Esteve ◽  
C. Susini ◽  
N. Vaysse ◽  
A. Ribet
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Specific Radioactivity ◽  
Pancreatic Acinar Cells ◽  
Nuclear Fraction ◽  
Pancreatic Acini ◽  
Specific Binding Sites ◽  
Membrane Bound ◽  
Membrane Level

We have previously demonstrated the presence of specific binding sites for somatostatin on plasma membranes from pancreatic acinar cells. In the present study we attempted to characterize the fate of receptor-bound 125I-[Tyr11]somatostatin. Internalization of somatostatin was rapid (reaching a plateau at 20% of the cell-associated specific radioactivity) and temperature dependent. To follow the processing of bound somatostatin, acini were incubated with 125I-[Tyr11]somatostatin at 5 degrees C during 16 h then, after washing, incubated at 37 degrees C for 90 min in fresh medium. Surface-bound somatostatin decreased rapidly, whereas radioactivity increased in the cell interior and the incubation medium. Intracellular and membrane-bound radioactivity was mainly intact 125I-[Tyr11]somatostatin. Degradation occurred at the plasma membrane level and led to iodotyrosine production. After 15 min of incubation, 15% of the initially surface-bound 125I-[Tyr11]somatostatin was compartmentalized within the cell, mainly in the microsomal fraction. After 30 min, a significant increase in radioactivity appeared in the nuclear fraction. These results indicate that the major part of somatostatin cellular degradation takes place at the plasma membrane level. Within the cell, somatostatin is routed to the nucleus via particular fractions sedimenting with microsomal vesicles.

scholarly journals Early events in the cellular formation of proparathyroid hormone

1980 ◽  
Vol 85 (2) ◽  
pp. 292-298 ◽  
Author(s):  
JF Habener ◽  
R Maunus ◽  
PC Dee ◽  
JT Potts
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Bilayer ◽  
Rough Endoplasmic Reticulum ◽  
Microsomal Fraction ◽  
Polypeptide Chain ◽  
Leader Peptide ◽  
Substantial Fraction ◽  
Intact Cells ◽  
Triton X ◽  
Short Times

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.

scholarly journals Electrophoretic mobility of γ-glutamyltransferase in rat liver subcellular fractions. Evidence for structure difference from the kidney enzyme

1989 ◽  
Vol 262 (2) ◽  
pp. 535-539 ◽  
Author(s):  
B Antoine ◽  
A Visvikis ◽  
C Thioudellet ◽  
A Rahimi-Pour ◽  
N Strazielle ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Liver Enzyme ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Kidney ◽  
Immunoblot Analysis ◽  
Subcellular Fractions ◽  
Golgi Membranes

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.

Ultrastructural and cytochemical observations on the body wall of the redia of Sphaeridiotrema globulus (Rudolphi, 1819)

Parasitology ◽  
1972 ◽  
Vol 65 (3) ◽  
pp. 537-546 ◽  
Author(s):  
Trevor A. J. Reader
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Body Wall ◽  
Secretory Granules ◽  
The Body ◽  
Carbohydrate Storage ◽  
Parenchyma Cells ◽  
Membrane Bound ◽  
Cytoplasmic Processes ◽  
Uptake Of Nutrients

The ultrastructure of the body wall of the redia of Sphaeridiotrema globulus is described. The tegument, which possesses numerous microvilli, is shown to be a syncytial, cytoplasmic layer connected to underlying subtegumentary ‘cells’ by cytoplasmic processes. Between the subtegumentary ‘cells’ are located the normal parenchyma cells of the body wall, which are believed to be important in carbohydrate storage. Mitochondria, Golgi complexes, endoplasmic reticulum and beta glycogen granules are located in the tegument and subtegumentary ‘cells’. In addition, large whorled bodies and dense secretory granules appear to be formed within the subtegumentary ‘cells’ prior to their passage into the outer tegument. It is suggested that these whorled structures are contributing to the growth of the tegument. Small membrane-bound ‘vesicles’ are also seen in the tegument and some of these may be pinocytotic in nature. Following incubation, horseradish peroxidase tracer was localized within ‘vesicles’ in both the tegument, subtegumentary ‘cells’ and parenchyma cells, which indicates that the redial body wall may be important in the uptake of nutrients. Phosphatase enzymes are abundant within the tegument, particularly in association with the plasma membranes and microvilli. These enzymes, which appear to have their origin in the endoplasmic reticulum of subtegumentary cells, are believed to be associated with the uptake of nutrients through the redial tegument.

scholarly journals Co-translational excision of alpha-glucose and alpha-mannose in nascent vesicular stomatitis virus G protein.

1984 ◽  
Vol 98 (6) ◽  
pp. 2245-2249 ◽  
Author(s):  
P H Atkinson ◽  
J T Lee
Keyword(s):  
Endoplasmic Reticulum ◽  
High Resolution ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Rough Endoplasmic Reticulum ◽  
Hela Cells ◽  
Chain Elongation ◽  
Carbonyl Cyanide ◽  
Membrane Bound ◽  
Vesicular Stomatitis

Membrane bound polysomes were prepared from HeLa cells infected with vesicular stomatitis virus (VSV), after pulse labeling with [3H]mannose for various times from 15 to 90 min. Oligosaccharides on nascent chains were released from peptides by treatment with endoglycosidase H and sized by high resolution Biogel P4 chromatography. Processing on some nascent chains proceeded to the removal of all three types of alpha-linked glucose and one alpha-1,2-mannose from the Glc3Man9GlcNAc precursor showing that the enzymes responsible were not only active on nascent chains but were present in the rough endoplasmic reticulum (RER). Incubation of cells for various times in cycloheximide, where chain elongation had ceased, made no difference to the profile of oligosaccharides on the nascent chains, and trimming proceeded no further than Man8GlcNAc2Asn . Carbonyl cyanide m-chlorophenylhydrazone (CCCP), an energy inhibitor reportedly able to block the transfer of glycoproteins from the RER, increases the amount of Man8-oligosaccharides on the nascent chains and also the amount of Glc3Man9GlcNAc precursor. On completed G protein in the RER fraction from which membrane bound polysomes were prepared, processing occurred to Man6 - but not to Man5GlcNAc sized oligosaccharides in the CCCP-treated cells. By contrast, processing to Man5GlcNAc oligosaccharides was observed in unfractionated control cells.

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