scholarly journals LOCALIZATION OF PEROXIDASE ACTIVITY IN THE DEVELOPING SUBMANDIBULAR GLAND OF NORMAL AND ISOPROTERENOL-TREATED RATS

1972 ◽  
Vol 20 (11) ◽  
pp. 855-872 ◽  
Author(s):  
SHOHEI YAMASHINA ◽  
TIBOR BARKA
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Submandibular Gland ◽  
Peroxidase Activity ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Chronic Administration ◽  
Secretory Material ◽  
Old Rats ◽  
Tubule Cells

The localization of peroxidase activity was studied in the developing submandibular gland of normal and isoproterenol-treated rats using a diaminobenzidine-H2O2 method. During postnatal development peroxidase activity was localized in the proacinar and acinar cells. The proacinar cells were characterized by the presence of polymorphic secretory granules that gave a strong peroxidase reaction, particularly in the glands of 1-day-old rats. In addition to the secretory granules, enzyme activity was demonstrated in the rough endoplasmic reticulum and nuclear envelope. The terminal tubule cells and duct cells were devoid of peroxidase activity. The secretory granules in the fully developed acinar cells revealed little or no enzyme activity. Isoproterenol stimulated the secretion of the peroxidase-positive granules from the proacinar cells. The stimulation was followed by a reaccumulation of peroxidase-positive secretory material. During this process enzyme activity was demonstrable in the Golgi complex. Isoproterenol had no effect on the terminal tubule cells. A less effective depletion of the secretory granules from the proacinar cells was seen after pilocarpine administration. Chronic administration of isoproterenol to 5-day-old rats led to an acceleration of the differentiation of acinar cells and to a hypertrophy of the gland, without significant change in the localization of peroxidase activity.

scholarly journals DEVELOPMENT OF ENDOGENOUS PEROXIDASE IN FETAL RAT SUBMANDIBULAR GLAND

1973 ◽  
Vol 21 (1) ◽  
pp. 42-50 ◽  
Author(s):  
SHOHEI YAMASHINA ◽  
TIBOR BARKA
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Submandibular Gland ◽  
Rough Endoplasmic Reticulum ◽  
Peroxidase Activity ◽  
Secretory Granules ◽  
Prenatal Development ◽  
Reaction Products ◽  
Cell Type ◽  
Endogenous Peroxidase

The prenatal development of endogenous peroxidase activity in the submandibular gland of rat was investigated by means of the diaminobenzidine-H2O2 histochemical method. The submandibular gland of a 16-day-old fetus was composed of cords of uniform, undifferentiated cells which contained no secretory granules and revealed no peroxidase activity. Peroxidase activity first appeared at the 17th day of gestation in the cisternae of the rough endoplasmic reticulum and nuclear envelope in a few cells. At the 18th day of gestation cells which exhibited reaction products in the rough endoplasmic reticulum and nuclear envelope also contained secretory granules with a strong peroxidase activity. During the last days of gestation the number of peroxidase positive cells, which contained numerous secretory granules, increased. The peroxidase-containing cells are the immediate precursors of the proacinar cells of early postnatal stages. During the same time period, when the peroxidase-containing cells differentiated, a second cell type also differentiated in the cellular cords. The development of this cell type was marked by the appearance of secretory granules stainable with toluidine blue. Through the prenatal development, this cell type revealed no peroxidase activity and was identified with the terminal tubule cell of the newborn. The morphologic and cytochemical findings indicate that terminal tubule cells and proacinar cells are committed cells; the former differentiate toward 2nd order intercalated duct cells and the latter transform to mature acinar cells.

scholarly journals Monoclonal antibodies against rat saliva and salivary gland antigens.

1985 ◽  
Vol 33 (3) ◽  
pp. 209-218 ◽  
Author(s):  
T Barka ◽  
E W Gresik ◽  
H van der Noen
Keyword(s):  
Submandibular Gland ◽  
Solid Phase ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Adult Rats ◽  
Saliva Secretion ◽  
Old Rats ◽  
Hybridoma Cell Lines ◽  
Ascites Tumors ◽  
Submandibular Saliva

Hybridomas were produced by the fusion of NS1 myeloma cells with spleen cells of a BALB/c mouse immunized with rat submandibular saliva. Growth of hybridomas was evident in 60/96 wells, and colonies secreting antibodies against saliva components were identified in 20 wells by using a solid phase enzyme-linked immunoassay. Cloning of cells from 12 wells yielded originally 43 hybridoma cell lines secreting anti-saliva antibodies. After recloning, one hybridoma (4Cl3) was selected for further studies. The hybridoma (4Cl3) cells were grown as ascites tumors, and the antibodies were purified from the ascitic fluid by diethylaminoethyl Affi-gel Blue chromatography. The purified antibody (MA4), immunoglobulin G1, immunoprecipitated a 39K dalton protein from submandibular saliva, and also reacted with a protein of the same electrophoretic mobility on immunoblots. From extracts of submandibular gland slices, incubated with [3H]leucine, the antibody again immunoprecipitated a 39K protein, indicating that this protein is synthesized in the gland. MA4 was used for immunocytochemical stainings of submandibular glands of rats of different ages. In general, immunostaining was seen only in acinar cells. Thus, there was no staining in the glands of 1-day-old rats that lack differentiated acinar cells. In the glands of 1- to 4-week-old rats the number of immunoreactive cells and the extent of immunostaining paralleled the differentiation of the acinar cells. In the glands of adult rats a uniform staining of the secretory granules of the acinar cells was observed. The immunoreactive 39K protein seemed to be restricted to the acinar cells in the submandibular gland; there was no immunostaining in the parotid, sublingual, or lingual salivary glands, or in the pancreas, colon, and duodenum. Stimulation of saliva secretion by isoproterenol resulted in a virtual depletion of the antigen from the acinar cells. These results indicate the feasibility of producing mouse hybridomas that secrete antibodies against rat saliva components. The monoclonal antibody at hand will be useful in analyzing the differentiation of the acinar cells, and the factors that influence this differentiation process.

scholarly journals THE LOCALIZATION OF ENDOGENOUS PEROXIDASE IN THE LACRIMAL GLAND OF THE RAT DURING POSTNATAL DEVELOPMENT

1972 ◽  
Vol 53 (3) ◽  
pp. 662-680 ◽  
Author(s):  
V. Herzog ◽  
F. Miller
Keyword(s):  
Endoplasmic Reticulum ◽  
Postnatal Development ◽  
Rough Endoplasmic Reticulum ◽  
Lacrimal Gland ◽  
Peroxidase Activity ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Secretory Cells ◽  
Lacrimal Fluid ◽  
Endogenous Peroxidase

The distribution of endogenous peroxidase activity in the lacrimal gland of the rat during postnatal development was investigated by electron microscope cytochemistry Peroxidase activity is first found 6 hr after birth in only a few acinar cells At this stage, reaction product fills only localized segments of the scant rough endoplasmic reticulum and of the perinuclear cisternae. Peroxidase activity thus develops asynchronously in a given cell as well as in the secretory cell population as a whole 2 days after birth, all cisternae of the rough endoplasmic reticulum of a peroxidase-positive cell contain reaction product, but the majority of the acinar cells is still negative During the next days, the number of peroxidase-positive cells and the amount of the rough endoplasmic reticulum increase rapidly. By 15 days postparturition, all secretory cells are peroxidase-positive. Reaction product is then found in all cisternae of the rough endoplasmic reticulum including the perinuclear cisternae, in smooth surface vesicles located mainly between the rough endoplasmic reticulum and the Golgi stacks, in condensing vacuoles, and in all secretory granules The Golgi cisternae rarely contain reaction product In total homogenates and in fractions of glandular tissue of adult rats, peroxidatic and catalatic activities are demonstrable. The microsomal fractions and the postmicrosomal supernatants were used to separate peroxidase from catalase by precipitation with ammonium sulfate, and the following parameters were determined: substrate (H2O2-) optimum (∼ 2.0 x 10-4M), pH-optimum (pH 6 5), temperature-optimum (42°C), and the absorption maximum (415 nm before and 425 nm after addition of H2O2) The same parameters were obtained from lacrimal fluid peroxidase. Both peroxidase from lacrimal gland and that from lacrimal fluid are almost completely inhibited by 10-3M aminotriazole and are possibly identical enzymes. Peroxidase is secreted into lacrimal fluid, which does not contain catalase.

scholarly journals INDUCTION OF ENDOMETRIAL PEROXIDASE SYNTHESIS AND SECRETION BY ESTROGEN AND ESTROGEN ANTAGONIST

1974 ◽  
Vol 62 (2) ◽  
pp. 449-459 ◽  
Author(s):  
Andrew Churg ◽  
Winston A. Anderson
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Peroxidase Activity ◽  
Secretory Granules ◽  
Combined Treatment ◽  
Initial Injection ◽  
Immature Rats ◽  
Glandular Cells ◽  
Endogenous Peroxidase ◽  
Estrogen Antagonist

Synthesis of peroxidase was induced in the uterine epithelium of immature rats by multiple doses over a 24–96-h period of either 17 ß-estradiol, the estrogen-antagonist Parke-Davis CI-628, or a combination of estradiol plus antagonist. Endogenous peroxidase activity first appeared in the cisternae of the rough endoplasmic reticulum of surface epithelial and glandular cells within 24–48 after the initial injection. Uterine peroxidase activity was also visible in the cisternae of the Golgi apparatus, in Golgi-derived secretory granules, and within the uterine and glandular lumen. Some cells of the epithelium produced little or no peroxidase, even after 96 h. Whereas the antagonist appeared to induce synthesis and secretion of peroxidase, neither the antagonist alone nor the combined treatment (estradiol plus antagonist) reproduced the estradiol-mediated growth in organ size and increased lumen diameter.

scholarly journals Relationship between the golgi apparatus, gerl, and secretory granules in acinar cells of the rat exorbital lacrimal gland

1977 ◽  
Vol 74 (2) ◽  
pp. 399-413 ◽  
Author(s):  
AR Hand ◽  
C Oliver
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Golgi Apparatus ◽  
Lacrimal Gland ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Secretory Proteins ◽  
Thiamine Pyrophosphatase ◽  
Variable Distance ◽  
Secretory Enzyme

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats.

1981 ◽  
Vol 29 (10) ◽  
pp. 1189-1195 ◽  
Author(s):  
T Tanaka ◽  
E W Gresik ◽  
T Barka
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Acinar Cells ◽  
Gold Colloid ◽  
Parotid Glands ◽  
Adult Rats ◽  
Old Rats ◽  
Rat Parotid ◽  
Unlabeled Antibody ◽  
Number Of Cells

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.

scholarly journals Retrovirus-mediated Gene Transfer into Rat Salivary Gland Cells In Vitro and In Vivo

1997 ◽  
Vol 45 (11) ◽  
pp. 1533-1545 ◽  
Author(s):  
Tibor Barka ◽  
Hendrika M. van der Noen
Keyword(s):  
Enzyme Activity ◽  
Salivary Gland ◽  
Gene Transfer ◽  
Cell Line ◽  
Submandibular Gland ◽  
Acinar Cells ◽  
Retroviral Vector ◽  
Placental Alkaline Phosphatase

A retroviral vector DAP that encodes the human placental alkaline phosphatase (PLAP) and the neomycin-resistant gene was used to transduce the salivary gland-derived cell line A5 in vitro and acinar cells in rat submandibular gland in vivo. Expression of the transduced PLAP gene was established by histochemical staining for heat-resistant AP and by determination of enzyme activity. From the in vitro experiments, we concluded that the salivary gland-derived cell line A5 can be infected by the retroviral vector DAP. In the transduced cells the viral long terminal repeat (LTR) promoter was effective, and the cells expressed heat-stable PLAP which was localized mostly in the plasma membrane and could be released by treatment with bromelain or phosphatidyinositol-specific phospholipase C. A5-DAP cells secreted PLAP into the medium. Clones of A5-DAP cells expressed various levels of the enzyme. The level of enzyme activity in different clones was unrelated to growth rate. Retrograde ductal injection of the viral vector into the duct of the submandibular gland of rats resulted in integration and long-term expression of PLAP gene in acinar cells. Expression of PLAP was seen up to 25 days, the limit of the observation period. To facilitate integration of the viral DNA, cell division of acinar cells was induced by administration of the β-adrenergic agonist isoproterenol before administration of the virus. PLAP was secreted into submandibular saliva. The data support the notion that salivary glands are suitable targets for gene transfer in vivo by a retroviral vector. (J Histochem Cytochem 45:1533–1545, 1997)

Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells

1994 ◽  
Vol 107 (3) ◽  
pp. 539-549 ◽  
Author(s):  
C.S. Velez-Granell ◽  
A.E. Arias ◽  
J.A. Torres-Ruiz ◽  
M. Bendayan
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Tissue ◽  
Secretory Proteins ◽  
Trans Golgi Network ◽  
Hsp70 Protein ◽  
Golgi Network

Three chaperones, the chaperonins cpn10 and cpn60, and the hsp70 protein, were revealed by immunochemistry and cytochemistry in pancreatic rat acinar cells. Western immunoblotting analysis of rat pancreas homogenates has shown that antibodies against cpn10, cpn60 and hsp70 protein recognize single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpn10 and cpn60 were also detected in pancreatic juice. Immunofluorescence studies on rat pancreatic tissue revealed a strong positive signal in the apical region of the acinar cells for cpn10 and cpn60, while an immunoreaction was detected at the juxtanuclear Golgi region with the anti-hsp70 antibody. Immunocytochemical gold labeling confirmed the presence of these three chaperones in distinct cell compartments of pancreatic acinar cells. Chaperonin 10 and cpn60 were located in the endoplasmic reticulum, Golgi apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 followed the increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway. On the contrary, the labeling for hsp70 was mainly concentrated in the endoplasmic reticulum and the Golgi apparatus. In the latter, the hsp70 was found to be primary located in the trans-most cisternae and to colocalize with acid phosphatase in the trans-Golgi network. The three chaperones were also present in mitochondria. In view of the role played by the chaperones in the proper folding, sorting and aggregation of proteins, we postulate that hsp70 assists the adequate sorting and packaging of proteins from the ER to the trans-Golgi network while cpn10 and cpn60 play key roles in the proper packaging and aggregation of secretory proteins as well as, most probably, in the prevention of early enzyme activation in secretory granules.

scholarly journals Immunoelectron microscopy of carbonic anhydrase isozyme VI in rat submandibular gland: comparison with isozymes I and II.

1992 ◽  
Vol 40 (6) ◽  
pp. 807-817 ◽  
Author(s):  
Y Ogawa ◽  
C K Chang ◽  
H Kuwahara ◽  
S S Hong ◽  
S Toyosawa ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Submandibular Gland ◽  
Secretory Granules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Acinar Cells ◽  
Enzyme Digestion ◽  
Immunoperoxidase Technique ◽  
Carbonic Anhydrase Isozyme ◽  
Indirect Immunoperoxidase ◽  
Rat Submandibular Gland

Carbonic anhydrase (CA) was purified from the saliva of pilocarpine-treated rats by inhibitor-affinity chromatography, and its localization in the rat submandibular gland was studied by the indirect immunoperoxidase technique using a monoclonal antibody (MAb) raised against the enzyme. SDS-polyacrylamide gel electrophoresis of the CA VI gave three bands of 33, 39, and 42 KD. Enzyme digestion experiment showed that the 42 KD molecule was degraded into the 39 KD molecule and the 39 KD molecule into the 33 KD molecule. The cleavage of the 42 KD molecule was independent and that of the 39 KD molecule was dependent on endo-beta-N-acetylglucosaminidase F. The 42 KD molecule was detected in the CA purified from the pilocarpine-treated but not the untreated salivary gland. The MAb recognized all the three components of the enzyme. Immunostaining for CA VI was seen in the cytosol and secretory granules of serous acinar cells and in the duct luminal contents. Staining specific for erythrocyte CA (CA I and CA II) was observed in the cytosol of the epithelial cells of granular, striated, and excretory ducts. Among these duct cells, the agranular varieties in the granular and excretory ducts were essentially devoid of the immunoreactivity.

scholarly journals Immunoelectron microscopy of carbonic anhydrase isozyme VI in human submandibular gland: comparison with isozymes I and II.

1993 ◽  
Vol 41 (3) ◽  
pp. 343-351 ◽  
Author(s):  
Y Ogawa ◽  
S S Hong ◽  
S Toyosawa ◽  
H Kuwahara ◽  
M Shimazaki ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Carbonic Anhydrase ◽  
Submandibular Gland ◽  
Polyclonal Antibodies ◽  
Secretory Granules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Acinar Cells ◽  
Protein Band ◽  
Immunoperoxidase Technique ◽  
Dot Blotting

Carbonic anhydrase VI (CA VI) was purified from human saliva by inhibitor-affinity chromatography, and its distribution was studied in human submandibular gland by the indirect immunoperoxidase technique with a rabbit polyclonal antibody raised against the isozyme. Polyclonal antibodies to human CA I and CA II purified from erythrocytes were also raised and used for immunostaining. SDS-polyacrylamide gel electrophoresis of the purified isozymes revealed a single protein band (CA VI, 42 KD; CA I and CA II, 30 KD). Antibody raised against CA VI did not crossreact with CA I or CA II either by Western or by dot-blotting. However, antibodies against CA I and CA II showed slight crossreaction with each other's antigen by dot-blotting. In a Western blot of purified submandibular gland CA, antibody to CA VI stained the 42 and 30 KD bands, and antibodies to CA I and CA II stained the 30 KD band. The 42 KD but not the 30 KD molecule was cleaved by endo-beta-N-acetylglucosaminidase F, indicating that the former contains N-linked oligosaccharides. Immunostaining for CA VI was seen in the secretory granules and cytosol of serous acinar cells and in the duct luminal contents. Staining specific for CA II was observed in the cytosol of serous acinar and duct epithelial cells. Antibody to CA I reacted only with the walls of small blood vessels. These results suggest that (a) serous acinar cells secrete 42 KD CA VI which functions in the oral cavity and that (b) serous acinar and duct epithelial cells possess cytosolic CA (30 KD CA VI and CA II) which functions in situ.

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