A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland

2002 ◽  
Vol 283 (2) ◽  
pp. C438-C445 ◽  
Author(s):  
S. G. Venkatesh ◽  
S.-U. Gorr
Keyword(s):  
Parotid Gland ◽  
Secretory Granules ◽  
Direct Interaction ◽  
Preliminary Evidence ◽  
Secretory Protein ◽  
Proteoglycan Synthesis ◽  
Secretory Proteins ◽  
Synthesis Inhibitor ◽  
Rat Parotid ◽  
And Storage

Sulfated proteoglycans have been proposed to play a role in the sorting and storage of secretory proteins in exocrine secretory granules. Rat parotid acinar cells expressed a 40- to 60-kDa proteoglycan that was stored in secretory granules. Treatment of the tissue with the proteoglycan synthesis inhibitor paranitrophenyl xyloside resulted in the complete abrogation of the sulfated proteoglycan. Pulse-chase experiments in the presence of the xyloside analog showed a significant reduction in the stimulated secretion and granule storage of the newly synthesized regulated secretory proteins amylase and parotid secretory protein. Inhibition of proteoglycan sulfation by chlorate did not affect the sorting of these proteins. The effect of proteoglycan synthesis inhibition on protein sorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sorting and storage of regulated secretory proteins in the exocrine parotid gland. Preliminary evidence suggests that the mechanism involves the modulation of granule pH by the proteoglycan rather than a direct interaction with other granule components.

Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands

2004 ◽  
Vol 286 (2) ◽  
pp. C365-C371 ◽  
Author(s):  
S. G. Venkatesh ◽  
Darrin J Cowley ◽  
Sven-Ulrik Gorr
Keyword(s):  
Secretory Granules ◽  
Secretory Protein ◽  
Low Ph ◽  
Secretory Proteins ◽  
Parotid Glands ◽  
Granule Membrane ◽  
Parotid Secretory Protein ◽  
Rat Parotid ◽  
Ph Range ◽  
Induced Aggregation

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although <10% of parotid secretory protein (PSP) aggregated below pH 6.0. To test aggregation directly in isolated granules, rat parotid secretory granules were permeabilized with 0.1% saponin in the presence or absence of calcium and in the pH range of 5.0 to 8.4. In contrast to the low-pH-dependent retention of amylase in exocrine pancreatic granules, amylase was quantitatively released and most PSP was released from parotid granules under all conditions. Both proteins were completely released upon granule membrane solubilization. Thus neither amylase nor PSP show low-pH- or calcium-induced aggregation under physiological conditions in the exocrine parotid secretory granules.

scholarly journals Retention of amylase in the secretory granules of parotid gland after extensive release of Ca++ by ionophore A-23187.

1977 ◽  
Vol 74 (3) ◽  
pp. 789-793 ◽  
Author(s):  
Y Flashner ◽  
M Schramm
Keyword(s):  
Parotid Gland ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Stabilizing Agent ◽  
A 23187 ◽  
Rat Parotid Gland ◽  
Rat Parotid ◽  
Extensive Release

The effect of the ionophore A-23187 was tested on isolated secretory granules of rat parotid gland. The ionophore caused extensive release of calcium from the granules without effecting release of amylase or other secretory proteins. It is therefore concluded that the role of calcium in the granules is probably not that of a stabilizing agent.

scholarly journals SECRETORY PROTEIN SYNTHESIS IN THE STIMULATED RAT PAROTID GLAND

1973 ◽  
Vol 59 (3) ◽  
pp. 708-721 ◽  
Author(s):  
John H. Lillie ◽  
Seong S. Han
Keyword(s):  
Protein Synthesis ◽  
Parotid Gland ◽  
Latent Period ◽  
Total Protein ◽  
Feedback System ◽  
Secretory Protein ◽  
Secretory Process ◽  
Secretory Proteins ◽  
Rat Parotid Gland ◽  
Rat Parotid

Administration of the ß-adrenergic drug, isoproterenol (IPR), affects the release of 98% of stored amylase from rat parotid gland acinar cells. A period of 6 h elapses from the onset of secretion to the maximum [14C]phenylalanine (Phe) incorporation into total protein and amylase. 10 h after IPR administration the rate of [14C]Phe incorporation into total protein was no longer elevated above that of control. Incorporation into amylase, however, remained elevated above the control by 2.3 times. This latent period may reflect: (a) reduced amounts of available ATP which occurs as a result of the process of secretion as well as (b) the time required for reorganization of cellular organelles and membranes after secretion. The latent period after IPR-induced secretion appears similar to the latent period which has recently been reported to occur after physiologic release of amylase from the parotid gland during the diurnal feeding cycle of the rat. These observations support the existence of a positive feedback system operant in the parotid acinar cell linking the release of secretory proteins with their synthesis. The period of greatest protein synthesis is, however, temporally dissociated from the secretory process.

scholarly journals An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

1989 ◽  
Vol 109 (1) ◽  
pp. 17-34 ◽  
Author(s):  
P Rosa ◽  
U Weiss ◽  
R Pepperkok ◽  
W Ansorge ◽  
C Niehrs ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Intracellular Localization ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Chromogranin B ◽  
Double Labeling ◽  
Protein Protein Interaction ◽  
And Function ◽  
Secretogranin I

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

Proteoglycan sulfation and storage parallels storage of basic secretory proteins in exocrine cells

1991 ◽  
Vol 261 (5) ◽  
pp. C897-C905 ◽  
Author(s):  
E. A. Blair ◽  
A. M. Castle ◽  
J. D. Castle
Keyword(s):  
Chondroitin Sulfate ◽  
Secretory Proteins ◽  
Parotid Acinar Cells ◽  
Secretion Granules ◽  
Polypeptide Backbone ◽  
Rat Parotid ◽  
Phosphoadenosine Phosphosulfate ◽  
And Storage ◽  
Fixed Positive Charge

Increased storage of basic proline-rich secretory proteins induced in rat parotid acinar cells by isoproterenol is accompanied by increased storage of a chondroitin sulfate-containing proteoglycan. Amino acid analysis of the purified proteoglycan and the chondroitinase digestion products reveals that the polypeptide backbone is a proline-rich protein. Most sulfation occurs in Golgi elements; however, a small fraction of the proteoglycan can be labeled by incubating isolated secretion granules with [35S]phosphoadenosine phosphosulfate ([35S]PAPS), and the amount of sulfate incorporation decreases with increased granule maturity. In vitro incorporation is sensitive to inhibitors of PAPS transport and occurs in intact granules as shown by radioautography. Both the increased production of a chondroitin sulfate proteoglycan following isoproterenol treatment and its sulfation at sites of secretory condensation and storage suggest that sulfation may aid secretory packaging by reducing the known fixed positive charge that stems from the large concentration of basic secretory proteins.

scholarly journals Effects of Diabetes and Insulin on α-amylase Messenger RNA Levels in Rat Parotid Glands

1990 ◽  
Vol 69 (8) ◽  
pp. 1500-1504 ◽  
Author(s):  
S.K. Kim ◽  
L.M. Cuzzort ◽  
R.K. McKean ◽  
E.D. Allen
Keyword(s):  
Parotid Gland ◽  
Beta Cell ◽  
Messenger Rna ◽  
Secretory Protein ◽  
Diabetic Rats ◽  
Mrna Levels ◽  
Mrna Synthesis ◽  
Parotid Glands ◽  
Protein Levels ◽  
Rat Parotid

Previous studies have shown that amylase levels are reduced significantly in the pancreas and parotid gland of diabetic rats and that insulin reverses this effect and increases the secretory protein levels. In the pancreas, these changes in amylase protein levels are accompanied by parallel changes in amylase mRNA levels. In the present study, the effects of diabetes and subsequent insulin treatments on contents ( per cell) of amylase protein and its mRNA in parotid glands were compared in rats rendered diabetic with an injection of a beta-cell toxin, streptozotocin (STZ). Both amylase protein and its mRNA contents were reduced significantly in diabetic rats, compared with control rats, and this reduction was reversed following insulin injections of diabetic rats. In insulin-injected diabetic rats, amylase protein contents increased before a detectable increase in amylase mRNA levels was seen. The mRNA contents of a non-secretory protein, actin, did not change during diabetogenesis or subsequent insulin treatments. The reductions in parotid contents of amylase and its mRNA in diabetic rats and the reversal of these changes by insulin are similar to those changes that occur in the pancreas under the same conditions. However, the magnitude of these changes in parotid glands was much smaller than in the pancreas, and the effect of insulin on amylase mRNA synthesis was not as immediate as in the latter gland.

Endocytosis of Proteins by Salivary Gland Duct Cells

1987 ◽  
Vol 66 (2) ◽  
pp. 412-419 ◽  
Author(s):  
A.R. Hand ◽  
R. Coleman ◽  
M.R. Mazariegos ◽  
J. Lustmann ◽  
L.V. Lotti
Keyword(s):  
Protein A ◽  
Periodate Oxidation ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Cationized Ferritin ◽  
Cell Secretion ◽  
High Concentration ◽  
Thin Sections ◽  
Duct Cells ◽  
Rat Parotid

The ability of the intralobular duct cells of the rat parotid gland to take up protein from the lumen was examined by retrograde infusion of exogenous proteins and by immunogold localization of endogenous secretory proteins. Small amounts of native horseradish peroxidase (HRP) were taken up by intercalated and striated duct cells, and were present in small vesicles, multi vesicular bodies, and lysosomes. In contrast, HRP modified by periodate oxidation was avidly internalized by the duct cells and was present in large apical vacuoles that acquired lysosomal hydrolase activity. Native and cationized ferritin were taken up in a similar manner when infused at a high concentration (up to 10 mg/mL). At lower concentrations (0.3-1.0 mg/mL), endocytosis of cationized ferritin occurred mainly in small apical tubules and vesicles in striated duct cells. Little native ferritin was taken up at these concentrations. After stimulation of acinar cell secretion by isoproterenol, similar vacuoles were occasionally observed in both intercalated and striated duct cells. Labeling of thin sections with antibodies to amylase and to a 26,000-dalton secretory protein (protein B1), followed by protein A-gold, revealed the presence of these proteins in the vacuoles, indicating endocytosis of acinar secretory proteins by the duct cells. Although uptake of acinar proteins by duct cells occurs at a low rate in normal animals, previous work suggests that extensive endocytosis may occur in certain pathological conditions. This may be a mechanism for removing abnormal or modified proteins from saliva before it reaches the oral cavity.

Calcium Uptake in Rat Parotid Gland Secretory Granules

1991 ◽  
Vol 70 (12) ◽  
pp. 1524-1527 ◽  
Author(s):  
J.E. Porter ◽  
P. Cheung ◽  
F.J. Dowd
Keyword(s):  
Parotid Gland ◽  
Calcium Uptake ◽  
Secretory Granules ◽  
Uptake Mechanism ◽  
Atpase Inhibitors ◽  
Rat Parotid Gland ◽  
Disulphonic Acid ◽  
Rat Parotid ◽  
Hydrolysis Of ◽  
Isolated Rat

45Ca2+ uptake in isolated rat parotid secretory granules was examined in the presence of oxalate. Uptake of calcium was dependent on time, with the maximum occurring at 15 min. The uptake of calcium was dependent on adenosine-5'-triphosphate (ATP), and substitution of ATP with B,g-methylene-ATP did not stimulate calcium uptake. Enzyme marker analysis indicated that mitochondria accounted for no greater than 3.0 ±0.2% of the observed ATP-dependent calcium uptake. Calcium uptake was blocked by the ATPase inhibitors tributyltin, IC 50 = 12.2 ±0.6 nmol/L and 4-acetamido-4'-isothiocyano-2,2'-stilbene disulphonic acid (SITS), IC50 = 3.0 ±0.3 (μmol/L. These results indicate that in the parotid secretory granule there is a calcium uptake mechanism that is dependent on the hydrolysis of ATP and is suppressed by two inhibitors of granule ATPase.

scholarly journals RADIOAUTOGRAPHIC ANALYSIS OF THE SECRETORY PROCESS IN THE PAROTID ACINAR CELL OF THE RABBIT

1972 ◽  
Vol 53 (2) ◽  
pp. 290-311 ◽  
Author(s):  
J. David Castle ◽  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Intracellular Transport ◽  
Secretory Granules ◽  
Close Association ◽  
Low Frequency ◽  
Secretory Process ◽  
Secretory Proteins ◽  
Surgical Removal ◽  
Storage Granules ◽  
And Storage

Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland. Rabbit parotids were chosen on the basis of size (2–2.5 g per animal), ease of surgical removal, and amylase concentration. Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure. Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0.175 M phosphate buffer (pH 7.2) as primary fixative. Pulse labeling with leucine-3H, followed by a chase incubation, showed that the label is initially located (chase: 1–6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase: 16–36 min), condensing vacuoles (chase: 36–56 min), immature granules (chase: 56–116 min), and finally mature storage granules (chase: 116–356 min). Distinguishing features of the parotid transport apparatus are: low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules. Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged.

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