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Cytochemical study of uptake of exogenous peroxidase by the rat submandibular salivary gland
Archa md
Bioi.Vol. 22. pp.65 t0 69. Pergamon Press1917.Printed in GreatBritain
SHORT COMMUNICATIONS CYTOCHEMICAL STUDY OF UPTAKE OF EXOGENOUS PEROXIDASE BY THE RAT SUBMANDIBULAR SALIVARY GLAND S. YAMAMOTO, Y. YOSHIDA, F. KISHI
and Y.
KAKUW
Department of Physiology, Osaka Dental University, l-47, Kyobashi, Higashiku, Osaka, Japan
Summary-When horseradish peroxidase (HPO) was administered intravenously to rats, its reaction product was detected cytochemically in the basal lamella surrounding the submandibular gland cells and in the intercellular spaces between the gland cells in the first few minutes after administration. HP0 was also seen in vesicles in the cells of both the acini and duct elements. Ten min to 6 h after administration, HP0 was found only in large membrane-bound vacuoles (probably lysosomes). It is concluded that exogenous HP0 is rapidly incorporated into the cells by pinocytosis through the basal and lateral cell membranes, and then the pinocytotic vesicles are fused with lysosomes, and that exogenous HP0 is transported into the gland lumen by lysosomes rather than by penetration through tight junctions.
We killed nine adult male Wistar rats (30&4OOg) at intervals ranging from 5 min to 6 h after intravenous administration of 30 mg HP0 (type II., Sigma Chemical Co.) dissolved in 0.6 ml of 0.9 per cent NaCl. The gland immersed in 5 per cent glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) was immediately cut into small blocks, and fixed for 4 h. The blocks were then washed and kept overnight in the same buffer containing 7 per cent sucrose and frozen sections about 4Opm thick were cut. The sections were incubated for 30min at room temperature in 10ml of 0.05 M tris-HCl buffer (pH 7.6) containing 0.1-0.2 ml of 1 per cent hydrogen peroxide and 5 mg of 3,3’-diaminobenzidine (DAB) (Graham and Karnovsky, 1966). As controls, some sections were incubated in the medium of Graham and Karnovsky but without DAB or hydrogen peroxide. After incubation, the sections were postfixed in osmium tetroxide and processed for electron-microscopy. Before preparing ultrathin sections for electron-microscopy, sections of about 1 pm thick were examined by optical microscopy in order to identify the cells and evaluate the amount of the brown product of the peroxidase reaction. The submandibular glands of untreated animals, or of those after the intravenous administratien of 0.9 per cent NaCl solution, were likewise treated to detect endogenous peroxidase activities. Five min after administration of HPO, its reaction product was found in the lumen of blood capillaries, in vesicles in endothelial lining cells, in basal lamella surrounding the gland cells and outside the cell membrane at the site of basal infoldings of duct cells. HP0 reaction product was also seen in the intercellular spaces between the adjacent gland cells, but not in the spaces above the level of the tight junction (Fig. 1). Furthermore, the tracer was detected in vesicles in the cells of both the acini and duct elements. In the acinar cells, many vesicles containing HP0 were
There is a low concentration of serum proteins in mammalian saliva, suggesting the possibility that salivary glands take up macromolecules from blood and secretes them into the saliva. According to Tung and Schein (1964), who studied the transport of exogenous serum proteins into whole saliva, it is likely that in rabbits and man exogenous macromolecules are also transported from blood into their saliva through the salivary glands. On the other hand, the possibility
66
S. Yamamoto, Y. Yoshida, F. Kishi and Y. Kakudo
found near the lateral cell membrane (Fig. 2), while those in the duct cells were located near the basal membrane (Fig. 3). The pinocytotic vesicles containing HP0 found in the duct elements, particularly in the convoluted granular tubule cells, outnumbered those in the acini. In the glands examined 1&30min after administration, HP0 was still observed in the extracellular spaces. However, the pinocytotic vesicles containing HP0 which had been observed in the first few minutes after administration were no longer detected. HP0 reaction product was found in the matrix of large membrane-bound vacuoles scattered throughout the cytoplasm of the acini (Fig. 4) and of duct elements (Fig. 5). Although large membrane-bound vacuoles containing some electron-opaque materials in the matrix (Fig. 6) were also present in the gland cells from untreated control rats, they were evidently HPO-negative. Thirty min to 6 h after HPOadministration the vacuoles containing HP0 tended to move toward the lumina of the acini and of the duct elements. Acid phosphatase (AcPase) in the glands excised from the rats to which HP0 had been administered 30min earlier was detected in the large membranebound vacuoles (probably lysosomes) in the cells of both the acini and duct elements. These vacuoles had similar shapes and distribution as the vacuoles containing HPO. It thus seemed likely that the large membrane-bound vacuoles containing HP0 were lysosomes. No HP0 activity was detected when sections were incubated in the medium lacking DAB or hydrogen peroxide. Endogenous peroxidase was found in the erythrocytes in the glands taken from 0.9 per cent NaCl-injected rats and from untreated control animals, though it was detectable in the acinar cells of rat submandibular gland by Strum and Karnovsky (1970). Some speculation to explain this difference has previously been made (Yoshida, Yamamoto and Kakudo, 1974). In summary, our findings indicate that HP0 entered the intercellular spaces and was incorporated by pinocytosis into the cells of the duct elements and to a lesser extent into the acinar cells; the pinocytotic vesicles then fused with lysosomes. The fate of exogenous HP0 incorporated into the gland cells remains unclear. Presumably, a portion of HP0 was broken down by lysosomal enzymes. It is possible that the HP0 incorporated into saliva without having been disintegrated into molecules with molecular weight less than about 9000 (Yoshida, Yamamot and Kakudo, 1974) is derived from the lysosomal system rather than from a direct communication through the tight junction between the intercellular space and the glandular lumen. This interpretation is in agreement with the work of those who have studied the uptake and transport of exogenous
HP0 in mouse and rat liver (Graham, Limpert and Kellermeyer, 1969; Ma, Laird and Scott, 1974). Co& tact between lysosomes and the luminal membrank was not detected, perhaps because this is a rapid transient phenomenon. Although the uptake of exogenous HP0 of plant origin was predominantly by pinocytotic vesicles, further work is needed before conclusions can be made about the mode of incorporation of normal serum proteins by salivary gland cells.
Acknowledgement-We thank Dr. H. Ishizaki, Department of Biology, College of Science, Nagoya University for his constant support and helpful suggestions in the course of this work.
REFERENCES
Fahimi H. D. 1970. The fine structural localization of endogenous and exogenous peroxidase activity in Kunffer cells of rat liver. J. Cell Biol. 47, 247-262. Friend D. S. and Farquhar M. G. 1967. Functions of coated vesicles during protein absorption in the rat vas deferens. J. Cell Biol. 35, 357-376. Graham R. C. and Karnovsky M. J. 1966. The early stage of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. .r. Histochem. Cvtochem. 14,291-302: Graham R. C.. Limnert S. and Kellermever R. W. 1969. The uptake’ and -transport of exogenous proteins in mouse liver: ultrastructural cytochemical studies with peroxidase tracers. Lab. Invest. 20, 298-304. Ma M. H., Laird W. H. and Scott H. 1974. Cytopempsis of horseradish peroxidase in the hepatocyte. J. Histothem. Cytochem. 22, 16169. Martin K. and Burgen A. S. V. 1962. Changes in the permeability of the salivary gland caused by sympathetic stimulation and by catecholamines. J. gen. Physiol. 46, 225-243.
Strum J. and Karnovsky M. J. 1970. Ultrastructural localization of peroxidase in submaxillary acinar cells. .I. Ultrastruct. Res. 31, 323-336.
Tomasi T. B. Jr., Tan E. M., Solomon A. and Prendergast R. A. 1965. Characteristics of an immuno-system common to certain external secretions. J. exp. Med. 121, 101-124. Tourville D. R., Adler R. H., Bienenstock J. and Tomasi T. B. 1969. The human secretory immunoglobulin system: immunohistological localization of A, secretory “piece”, and lactofferin in normal human tissues. J. exp. Med. 129, 411429. Tung F. F. and Schein A. H. 1964. The appearance of administered proteins in saliva. J. dent. Res. 43,423-432. Yokovama M. and Charm J. P. 1971. An ultracvtochemical and ultrastructural study of epithelial cells in ductuli efferentes of Chinese hamster. J. Histochem. Cytochem. 19, 766774.
Yoshida Y., Yamamoto S. and Kakudo Y. 1974. Transport of intravenously administered horseradish peroxihase into the rat submaxillarv saliva. Archs oral Biol. 19. 801-806.
Peroxidase uptake by submandibular
Plate 1 overleaf.
gland
67
S. Yamamoto, Y. Yoshida, F. Kishi and Y. Kakudo
Plate 1 Figs. l-5 Electron-micrographs
of sections stained with uranyl acetate and lead citrate.
Fig. 1. Acinar cell from a rat killed 5 min after administration of HP0 showing reaction product in the intercellular space (IS). The space above the level of the tight junction (TJ) and the intercellular canaliculus (IC) is devoid of product. SG, secretory granule. x 27,432 Fig. 2. Acinar cell from a rat killed 5 min after administration of HP0 showing reaction product in the intercellular space (IS) and in the vesicles. Two vesicles seem to be in the process of endocytosis (arrows). M, mitochondria. x 41,148 Fig. 3. Convoluted granular tubule cell from a rat killed 5 min after administration of HP0 showing reaction product outside the duct, in the narrow spaces between the highly invaginated basal cell membrane (short arrows) and in the vesicles isolated in the cytoplasm (long arrows). M, mitochondria. x 80,010 Fig. 4. Acinar cell from a rat killed 20min after administration of HP0 showing reaction product in a large membrane-bound vacuoles. Arrow indicates a probable lysosome near the lumen of acinus. SG, secretory granule. x 54,864 Fig. 5. Striated duct cell from a rat killed 20 min after administration of HP0 showing reaction product in large membrane-bound vacuoles. Arrow indicates a probable lysosome in the inner cytoplasm. M, mitochondria. x 34,290 Fig. 6. Acinar cell from an untreated control rat showing a large membrane-bound vacuole. Arrow indicates a probable lysosome which contains electron-opaque material in the matrix. SG, secretory granule. x 27,432
Peroxidase uptake by submandibular
Plate 1
gland
69
Bioi.Vol. 22. pp.65 t0 69. Pergamon Press1917.Printed in GreatBritain
SHORT COMMUNICATIONS CYTOCHEMICAL STUDY OF UPTAKE OF EXOGENOUS PEROXIDASE BY THE RAT SUBMANDIBULAR SALIVARY GLAND S. YAMAMOTO, Y. YOSHIDA, F. KISHI
and Y.
KAKUW
Department of Physiology, Osaka Dental University, l-47, Kyobashi, Higashiku, Osaka, Japan
Summary-When horseradish peroxidase (HPO) was administered intravenously to rats, its reaction product was detected cytochemically in the basal lamella surrounding the submandibular gland cells and in the intercellular spaces between the gland cells in the first few minutes after administration. HP0 was also seen in vesicles in the cells of both the acini and duct elements. Ten min to 6 h after administration, HP0 was found only in large membrane-bound vacuoles (probably lysosomes). It is concluded that exogenous HP0 is rapidly incorporated into the cells by pinocytosis through the basal and lateral cell membranes, and then the pinocytotic vesicles are fused with lysosomes, and that exogenous HP0 is transported into the gland lumen by lysosomes rather than by penetration through tight junctions.
We killed nine adult male Wistar rats (30&4OOg) at intervals ranging from 5 min to 6 h after intravenous administration of 30 mg HP0 (type II., Sigma Chemical Co.) dissolved in 0.6 ml of 0.9 per cent NaCl. The gland immersed in 5 per cent glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) was immediately cut into small blocks, and fixed for 4 h. The blocks were then washed and kept overnight in the same buffer containing 7 per cent sucrose and frozen sections about 4Opm thick were cut. The sections were incubated for 30min at room temperature in 10ml of 0.05 M tris-HCl buffer (pH 7.6) containing 0.1-0.2 ml of 1 per cent hydrogen peroxide and 5 mg of 3,3’-diaminobenzidine (DAB) (Graham and Karnovsky, 1966). As controls, some sections were incubated in the medium of Graham and Karnovsky but without DAB or hydrogen peroxide. After incubation, the sections were postfixed in osmium tetroxide and processed for electron-microscopy. Before preparing ultrathin sections for electron-microscopy, sections of about 1 pm thick were examined by optical microscopy in order to identify the cells and evaluate the amount of the brown product of the peroxidase reaction. The submandibular glands of untreated animals, or of those after the intravenous administratien of 0.9 per cent NaCl solution, were likewise treated to detect endogenous peroxidase activities. Five min after administration of HPO, its reaction product was found in the lumen of blood capillaries, in vesicles in endothelial lining cells, in basal lamella surrounding the gland cells and outside the cell membrane at the site of basal infoldings of duct cells. HP0 reaction product was also seen in the intercellular spaces between the adjacent gland cells, but not in the spaces above the level of the tight junction (Fig. 1). Furthermore, the tracer was detected in vesicles in the cells of both the acini and duct elements. In the acinar cells, many vesicles containing HP0 were
There is a low concentration of serum proteins in mammalian saliva, suggesting the possibility that salivary glands take up macromolecules from blood and secretes them into the saliva. According to Tung and Schein (1964), who studied the transport of exogenous serum proteins into whole saliva, it is likely that in rabbits and man exogenous macromolecules are also transported from blood into their saliva through the salivary glands. On the other hand, the possibility
66
S. Yamamoto, Y. Yoshida, F. Kishi and Y. Kakudo
found near the lateral cell membrane (Fig. 2), while those in the duct cells were located near the basal membrane (Fig. 3). The pinocytotic vesicles containing HP0 found in the duct elements, particularly in the convoluted granular tubule cells, outnumbered those in the acini. In the glands examined 1&30min after administration, HP0 was still observed in the extracellular spaces. However, the pinocytotic vesicles containing HP0 which had been observed in the first few minutes after administration were no longer detected. HP0 reaction product was found in the matrix of large membrane-bound vacuoles scattered throughout the cytoplasm of the acini (Fig. 4) and of duct elements (Fig. 5). Although large membrane-bound vacuoles containing some electron-opaque materials in the matrix (Fig. 6) were also present in the gland cells from untreated control rats, they were evidently HPO-negative. Thirty min to 6 h after HPOadministration the vacuoles containing HP0 tended to move toward the lumina of the acini and of the duct elements. Acid phosphatase (AcPase) in the glands excised from the rats to which HP0 had been administered 30min earlier was detected in the large membranebound vacuoles (probably lysosomes) in the cells of both the acini and duct elements. These vacuoles had similar shapes and distribution as the vacuoles containing HPO. It thus seemed likely that the large membrane-bound vacuoles containing HP0 were lysosomes. No HP0 activity was detected when sections were incubated in the medium lacking DAB or hydrogen peroxide. Endogenous peroxidase was found in the erythrocytes in the glands taken from 0.9 per cent NaCl-injected rats and from untreated control animals, though it was detectable in the acinar cells of rat submandibular gland by Strum and Karnovsky (1970). Some speculation to explain this difference has previously been made (Yoshida, Yamamoto and Kakudo, 1974). In summary, our findings indicate that HP0 entered the intercellular spaces and was incorporated by pinocytosis into the cells of the duct elements and to a lesser extent into the acinar cells; the pinocytotic vesicles then fused with lysosomes. The fate of exogenous HP0 incorporated into the gland cells remains unclear. Presumably, a portion of HP0 was broken down by lysosomal enzymes. It is possible that the HP0 incorporated into saliva without having been disintegrated into molecules with molecular weight less than about 9000 (Yoshida, Yamamot and Kakudo, 1974) is derived from the lysosomal system rather than from a direct communication through the tight junction between the intercellular space and the glandular lumen. This interpretation is in agreement with the work of those who have studied the uptake and transport of exogenous
HP0 in mouse and rat liver (Graham, Limpert and Kellermeyer, 1969; Ma, Laird and Scott, 1974). Co& tact between lysosomes and the luminal membrank was not detected, perhaps because this is a rapid transient phenomenon. Although the uptake of exogenous HP0 of plant origin was predominantly by pinocytotic vesicles, further work is needed before conclusions can be made about the mode of incorporation of normal serum proteins by salivary gland cells.
Acknowledgement-We thank Dr. H. Ishizaki, Department of Biology, College of Science, Nagoya University for his constant support and helpful suggestions in the course of this work.
REFERENCES
Fahimi H. D. 1970. The fine structural localization of endogenous and exogenous peroxidase activity in Kunffer cells of rat liver. J. Cell Biol. 47, 247-262. Friend D. S. and Farquhar M. G. 1967. Functions of coated vesicles during protein absorption in the rat vas deferens. J. Cell Biol. 35, 357-376. Graham R. C. and Karnovsky M. J. 1966. The early stage of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. .r. Histochem. Cvtochem. 14,291-302: Graham R. C.. Limnert S. and Kellermever R. W. 1969. The uptake’ and -transport of exogenous proteins in mouse liver: ultrastructural cytochemical studies with peroxidase tracers. Lab. Invest. 20, 298-304. Ma M. H., Laird W. H. and Scott H. 1974. Cytopempsis of horseradish peroxidase in the hepatocyte. J. Histothem. Cytochem. 22, 16169. Martin K. and Burgen A. S. V. 1962. Changes in the permeability of the salivary gland caused by sympathetic stimulation and by catecholamines. J. gen. Physiol. 46, 225-243.
Strum J. and Karnovsky M. J. 1970. Ultrastructural localization of peroxidase in submaxillary acinar cells. .I. Ultrastruct. Res. 31, 323-336.
Tomasi T. B. Jr., Tan E. M., Solomon A. and Prendergast R. A. 1965. Characteristics of an immuno-system common to certain external secretions. J. exp. Med. 121, 101-124. Tourville D. R., Adler R. H., Bienenstock J. and Tomasi T. B. 1969. The human secretory immunoglobulin system: immunohistological localization of A, secretory “piece”, and lactofferin in normal human tissues. J. exp. Med. 129, 411429. Tung F. F. and Schein A. H. 1964. The appearance of administered proteins in saliva. J. dent. Res. 43,423-432. Yokovama M. and Charm J. P. 1971. An ultracvtochemical and ultrastructural study of epithelial cells in ductuli efferentes of Chinese hamster. J. Histochem. Cytochem. 19, 766774.
Yoshida Y., Yamamoto S. and Kakudo Y. 1974. Transport of intravenously administered horseradish peroxihase into the rat submaxillarv saliva. Archs oral Biol. 19. 801-806.
Peroxidase uptake by submandibular
Plate 1 overleaf.
gland
67
S. Yamamoto, Y. Yoshida, F. Kishi and Y. Kakudo
Plate 1 Figs. l-5 Electron-micrographs
of sections stained with uranyl acetate and lead citrate.
Fig. 1. Acinar cell from a rat killed 5 min after administration of HP0 showing reaction product in the intercellular space (IS). The space above the level of the tight junction (TJ) and the intercellular canaliculus (IC) is devoid of product. SG, secretory granule. x 27,432 Fig. 2. Acinar cell from a rat killed 5 min after administration of HP0 showing reaction product in the intercellular space (IS) and in the vesicles. Two vesicles seem to be in the process of endocytosis (arrows). M, mitochondria. x 41,148 Fig. 3. Convoluted granular tubule cell from a rat killed 5 min after administration of HP0 showing reaction product outside the duct, in the narrow spaces between the highly invaginated basal cell membrane (short arrows) and in the vesicles isolated in the cytoplasm (long arrows). M, mitochondria. x 80,010 Fig. 4. Acinar cell from a rat killed 20min after administration of HP0 showing reaction product in a large membrane-bound vacuoles. Arrow indicates a probable lysosome near the lumen of acinus. SG, secretory granule. x 54,864 Fig. 5. Striated duct cell from a rat killed 20 min after administration of HP0 showing reaction product in large membrane-bound vacuoles. Arrow indicates a probable lysosome in the inner cytoplasm. M, mitochondria. x 34,290 Fig. 6. Acinar cell from an untreated control rat showing a large membrane-bound vacuole. Arrow indicates a probable lysosome which contains electron-opaque material in the matrix. SG, secretory granule. x 27,432
Peroxidase uptake by submandibular
Plate 1
gland
69