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Ultrastructural localization of exogenous silver in the anterior pituitary gland of the rat
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
41, 58-66 (1984)
Ultrastructural Localization of Exogenous Silver in the Anterior Pituitary Gland of the Rat OLE THORLACIUS-USSING Institute
of Anatomy
B, University Received
AND J@RGEN RUNGBY of Aarhus,
November
DK-8000
Aarhus
C, Denmark
4, 1983
Silver accumulations in the anterior pituitary of argyric rats were demonstrated using a histochemical method that visualizes minute traces of the metal. Silver was localized intraand extracellularly throughout the anterior pituitary. Intracellular deposits were found within the lysosomes of somatotrophs and gonadotrophs. Extracellularly. the grains were located in basal laminae of portal veins and sinusoidal capillaries and in the membrane separating the anterior pituitary and part intermedia. The amount of deposited silver was dependent upon the dose of silver administered. Increasing the dose of silver lactate from 10 to 30 mg resulted in increased deposition, whereas a further increase to 60 mg did not alter the amount of silver deposited.
INTRODUCTION It is generally accepted that trace metals are capable of influencing the endocrine system at the level of the anterior pituitary (Henkin, 1976). The effects of silver, however, have, received only little attention, although it has been reported that the effects normally produced by chronic estradiol treatment are absent in argyric rats (Schreiber et al., 1980). In rats given silver nitrate in the drinking water, silver has been found within the connective tissue of portal vein walls in the anterior pituitary (Wislocki and Leduc, 1952), whereas in the neurohypophysis, silver is abundantly distributed intra- and extracellularly (Gatz, 1949; Wislocki and Leduc, 1952; Dempsey and Wislocki, 1955). The aim of the present paper is to describe the localization of silver in the anterior pituitary of argyric rats in light and electron microscopic preparations using a histochemical method for the demonstration of trace amounts of silver (Danscher, 1981~). MATERIAL
AND METHODS
Sixteen male Wistar rats weighing 150 g each were kept in groups of four under normal laboratory conditions. Silver lactate (5 mg dissolved in 1 ml distilled water) was injected intraperitoneally (i.p.). Three groups received a total of 10, 30, or 60 mg per rat, respectively. Injections of 1 ml per rat were given twice a week. The fourth group received two i.p. injections totaling 2 ml of 0.9% saline and served as controls. One week after the last injection, the silver-injected animals and the controls were anesthetized with Nembutal and killed by vascular perfusion at 120 mm Hg with 3% glutaraldehyde dissolved in Veronal buffer, pH 7.4. The perfusion lasted 12 min. The pituitary gland was removed, bisected, and kept in the same fixative for 1 hr. Tissue blocks were dehydrated and embedded in Epon, and semithin sections were cut according to conventional procedures. The sections were then exposed to silver amplification as described by Danscher (1981~). In summary, this method is initiated by the exposure of sections of uv light in order to reduce 58 0014-4800/84 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
SILVER
IN THE ANTERIOR
PITUITARY
GLAND
59
FIG. 1. Micrograph showing a semithin section from the anterior pituitary of a rat dosed with 30 mg silver lactate. Silver grains are seen intracellularly (big arrow) and extracellularly (small arrows). x640. FIG. 2. Semithin section showing silver in the membrane between the pars intermedia (pi) and pars anterior (pa). Rat treated with 30 mg silver lactate. x 640.
silver not bound as sulfides or selenides. Subsequently the sections were physically developed. The development was performed in a dark box, the sections being exposed to a developer containing silver ions, hydroquinone as a reducing agent, citrate buffer, and a protecting colloid based on gum Arabic. Silver traces in the sections catalyze the hydroquinone reduction of silver ions from the developer on the surface of the traces, thus enlarging these to visible size. To avoid background staining the sections are rinsed in water and sodium thiosulphate after the development. Routinely sections were developed for 60 min. After being rinsed the sections were counterstained with 1% toluidine, examined in the light microscope, stuck onto Epon blocks, and cut into ultrathin sections. These were counterstained with lead/uranyl acetate and examined in the electron microscope. RESULTS In the light microscope silver grains were evenly distributed throughout the anterior pituitary of all silver-treated rats whereas the controls were free of grains. Silver was located both intra- and extracellularly. The intracellular deposits were primarily seen in the cytoplasm of the largest secretory cells, whereas smaller secretory cells contained only few deposits (Fig. 1). Extracellularly, the silver was found in walls of the portal veins and the sinusoidal capillaries. The membrane dividing the pars intermedia and the anterior pituitary was heavily stained (Figs. 2 and 3). The number of cells stained and the number of grains per cell increased when
60
THORLACIUS-USSING
AND RUNGBY
FIG. 3. Electron micrograph showing silver deposits at the membrane dividing the pars intermedia and the anterior pituitary. Note the marginal cell (mc). This section was cut from the semithin section shown in Fig. 2. x 8800.
the dose of silver was increased from 10 to 30 mg, whereas no obvious quantitative differences were observed between doses of 30 and 60 mg. Ultrastructurally, silver grains were found in two cell types, somatotrophs (Fig. 5) and gonadotrophs (Figs. 4 and 6) of the silver treated rats but precipitates were never found in controls. Somatotrophs were identified by 350- to 400-nm secretory granules, abundant rough endoplasmic reticulum, and numerous lysosomes; gonadotrophs by their 200-nm granules, coexisting with fewer 400-nm granules, both evenly distributed throughout the cytoplasm (see Farquhar et al., 1975). In sections for both light and electron microscopy, grains of silver were found only in a portion of the two cell populations. In both somatotrophs and gonadotrophs the grains were located in bodies with a less dense and less demarcated matrix than that of the secretory granules. The bodies were variable in size, ranging from 100 to 500 nm, but most were somewhat larger than the largest secretory granules (Figs. 5 and 6). Accordingly, we have interpreted the structures as lysosomes. In very few instances a trace of silver was found in what, by their ultrastructural appearance, were identified as somatotroph secretory granules (Fig. 5). The extracellular silver was seen in basal laminae of the portal veins and sinusoidal capillaries (Fig. 7) and in the juxtaluminal part of the basal laminae surrounding the parenchymal cells (Figs. 7 and 8). Basal laminae separating the endocrine cells did not contain silver. No ultrastructural changes were observed in somatotrophs or gonadotrophs loaded with silver, nor did the total or relative number of cells of different types in the gland appear to have changed as a result of treatment with silver.
SILVER
IN THE
ANTERIOR
PITUITARY
GLAND
61
62
THORLACIUS-USSING
AND RUNGB
FIG. 5. Somatotroph from a rat dosed with 10 mg silver lactate. Silver is present in lysosomes (lower inset) and in secretory granules (upper inset). x 11,700 (insets x 33,000).
DISCUSSION The process of physical development can be catalyzed by other substances than silver. Metallic gold is a well known example, but sulfides and selenides of mercury, zinc, and other heavy metals can also catalyze the reaction (Roberts, 1935; Timm, 1958; Danscher and Schroder, 1979; Danscher, 1981a,b, 1982). It is therefore necessary to perform control experiments when using this method. Artifacts due to precipitation of lead or uranyl in the sections are easily distinguished from the silver grains since the latter have a uniform structure with characteristic edged rounded shapes and a homogeneous electron density (Danscher, 1981~; Rungby and Danscher, 1983). The darkly stained inclusions sometimes observed in lysosomes from osmium fixed tissue did not appear as a confounding factor since the procedure does not include osmification. Silver is transported by globulins and albumins in the blood (Hill and Pillsbury, 1939; Scott and Hamilton, 1950). The mechanisms responsible for the dissociation of silver from these elements and the subsequent uptake of metal into cells of certain types are as yet unexplained. The following considerations might, however, provide part of the explanation. (1) The lysosomes of somatotrophs and gonadotrophs may have an excess of available chemical groups with a high affinity for silver (e.g., sulfide groups) or they may take part in the degradation of substances with such high affinity groups, as suggested for central nervous system lysosomes by Rungby and Danscher (1983). (2) Silver could be bound in the content of secretory granules, thus the crinophagia of these substances (Farquhar, 1969, 1971) might account for the presence
SILVER
IN THE ANTERIOR
PITUITARY
GLAND
63
FIG. 6. Heavily loaded Iysosomes seen at high magnification. Gonadotroph from rat treated with 30 mg silver lactate. x 27,600.
of silver in the lysosomes (although silver was only occasionally seen in secretory granules). (3)The possibility that silver contained in basal laminae around anterior pituitary cells, vascular basal laminae, or blood-borne substances, could be taken up by phagocytosis cannot be excluded. However, phagocytic activity has been ascribed only to follicular cells of the anterior pituitary (Dingemans and Feltkamp, 1972; Farquhar et al., 1975) and these cells did not contain silver. The high affinity of basement membranes for silver has been described by Dempsey and Wislocki (1955), Walker (1972), and Scott and Norman (1980), and the present investigation confirms these results. The functional effects of the accumulation of exogenous silver in the anterior pituitary are still unknown, but the present findings suggest that silver could interfere with lysosomal functions. The lysosomes of the cells in the anterior pituitary contain protein-degrading enzymes, e.g., cathepsin A, B, B,, and D and dipeptidyl aminopeptidase I and II. These enzymes are responsible for a selective degradation of the hormones removed by crinophagia (Ellis, 1960; Ellis and Pery, 1966; Ellis and Nuenke, 1967; McDonald et al., 1971). At least one of these enzymes, dipeptidyl aminopeptidase I, can be inhibited by heavy metal compounds (mercury ions and p-chloromercuriphenyl-sulfonate). This inhibition is thought to be mediated by the binding of mercury to sulfydryl groups of the enzymes (Fruton and Mycek, 1956; McDonald et al., 1971). Both mercury and silver have a high affinity for sulfydryl groups (Petering, 1976; Danscher and Schroder, 1979); thus a possible disturbance of the crinophagia caused by silver might be mediated by this mechanism.
64
THORLACIUS-USSING
AND RUNGBY
FIG. 7. Sinosoidal capillary with silver grains in the basal lamina adjacent to a follicular cell (Fc) and an endothelial cell (EC). x 12,500. FIG. 8. Perivascular basal lamina of a portal vein with many silver deposits adjacent to a somatotroph (St). x 10,600
SILVER
IN THE ANTERIOR
PITUITARY
GLAND
65
Silver is a potent inhibitor of adenylate cyclase and cyclic AMP phosphodiesterase activity in many organs (Nathanson and Bloom, 1976). The secretory processes of endocrine cells are dependent upon respiratory energy (Jamieson and Palade, 1968, 1971). Changing the ability of the cells to produce cyclic AMP may change their secretory rate. Thus silver may interfere with the level of activity in anterior pituitary cells in at least two different ways. This might have functional significance. ACKNOWLEDGMENTS We thank Dr. G. Danscher for his inspiration and help and Dr. A. R. Lieberman for help with the manuscript. We gratefully acknowledge the technical assistance rendered by Mr. B. Krunderup, Mr. A. Meier, Ms. P. K. Melter, Mr. T. A. Nielsen, Ms. S. B. Thomsen. and Ms. K. Wiedemann.
REFERENCES DANSCHER, G. (198la). Histochemical demonstration of heavy metals. Hisrochemistry 71, l-16. DANSCHER, G. (1981b). Localization of gold in biological tissue. Hisfochemistry 71, 81-88. DANSCHER, G. (1981~). Light and electron microscopic localization of silver in biological tissue. Histochemistry 71, 177-186. DANSCHER, G. (1982). Exogenous selenium in the brain. Histochemistry 76, 281-293. DANSCHER, G., and SCHR~DER, H. D. (1979). Histochemical demonstration of mercury-induced changes in rat neurons. Histochemistry 60, l-7. DEMPSEY, E. W., AND WISLOCKI, G. B. (1955). An electron microscopic study of the blood-brain barrier of the rat, employing silver nitrate as a vital stain. J. Biophys. Biochem. Cytol. 1, 24.5-257. DINGEMANS, K. P., and FELTKAMP, C. A. (1972). Nongranulated cells in the mouse adenohypophysis. Z. Zellforsch. Mikrosk. Anat. 124, 387-405. ELLIS, S. (1960). Pituitary proteinase I: Purification and action on growth hormone and prolactin. J. Biol. Chem. 235, 1694-2001. ELLIS, S., and NUENKE, J. M. (1967). Dipeptidyl arylamidase III of the pituitary; purification and characterization. J. Biol. Chem. 242, 4623-4629. ELLIS, S., and PERY, M. (1966). Pituitary arylamidases and peptidases. J. Biol. Chem. 241, 36793687.
FARQUHAR, M. G. (1969). Lysosome function in regulating secretion: Disposal of secretory granules in cells of the anterior pituitary gland. In “Lysosomes in Biology and Pathology” (J. T. Dingle and H. B. Fell, eds.), Vol. 2, pp. 462-482. North-Holland, Amsterdam. FARQUHAR, M. G. (1971). Processing of secretory products by cells of the anterior pituitary gland. Mem. Sot. Endocrinol. 19, 79-124. FARQUHAR, M. G., SHUTELSKY, E. M., and HOPKINS, C. R. (1975) Structure and function of the anterior pituitary and dispersed pituitary cells. In vitro studies. In “The Anterior Pituitary” (A. Tixier-Vidal and M. G. Farquhar, eds.), pp. 83-135. Academic Press, New York. FRLJTON,J. S., and MYCEK, M. J. (1956). Studies on beef spleen cathepsin C. Arch. Biochem. Biophys. 65, 11-20.
GATZ, A. J. (1949). Experimental argyria in albino rats. Anat. Rec. 103, 454-455 (abstract). HENKIN, R. I. (1976). Trace metals in endocrinology. Med. C/in. North Amer. 60, 779-797. HILL, W. R., and PILLSBURY, D. M. (1939). “Argyria, the Pharmacology of Silver.” Williams & Wilkins, Baltimore. JAMIESON, J. D., and PALADE, G. E. (1968). Intracellular transport of secretory proteins in the pancreatic exocrine cells. IV. Metabolic requirements. J. Cell Biol. 39, 589-603. JAMIESON, J. D., and PALADE, G. E. (1971). Condensing vacuole conversion and zymogen granule discharge in pancreatic exocrine cells: Metabolic studies. J. Cell Biol. 48, 503-523. MCDONALD, J. K., CALLAHAN, P. X., ELLIS, S., and SMITH, R. E. (1971). Polypeptide degradation by dipeptidyl aminopeptidase I (cathepsin C) and related peptidases. In “Tissue Proteinases” (A. J. Barrett and J. T. Dingle, eds.), pp. 69-107. North-Holland, Amsterdam. NATHANSON, J. A., and BLOOM, F. E. (1976). Heavy metals and adenosine cyclic 3’,5’-monophosphate metabolism: Possible relevance to heavy metal toxicity. MO/. Pharmacol. 12, 390-398. PETERING, H. G. (1976). Pharmacology and toxicology of heavy metals: Silver. Pharmacol. Ther. Part A 1, 127-130.
66
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AND
RUNGBY
ROBERTS, W. J. (1935). A new procedure for the detection of gold in animal tissue. Proc. Roy. Acad. Sci. (Amsterdam)
38, 540-544.
RUNGBY, J., and DANSCHER, G. (1983). Localization of exogenous silver in brain and spinal cord of silver exposed rats. Acta Neuropathol. 60, 92-98. SCHREIBER,V., PFUBYL,T., and JAHODOVA, J. (1980). Modulation of oestradiol effects by silver nitrate: Inhibition of the adenohypophyseal reaction, block of the ceruloplasmin increase and block of the hypothalamic ascorbic acid depletion. Endokrinologie (Berlin) 76, 129-136. SCOTT, K. G., and HAMILTON, J. G. (1950). The metabolism of silver in the rat with radio-silver used as an indicator. Univ. Calif. Berkeley Publ. Pharmacol. 2, 241-262. SCOTT, T., and NORMAN P. M. (1980). Silver deposition in arteriolar basal laminae in the cerebral cortex of argyric rats. Acta Neuropathol. 52, 243-246. TIMM, F. (1958). Zur Histochemie der Schwermetalle. Das Sulfid-Silber-Verfahren. Dtsch. 2. Gesamte Gerichtl. Med. 46, 706-711. WALKER, F. (1972). Basement-membrane turnover in man. J. Pathol. 107, 123- 126. WISLOCKI, G. B., and LEDUC, E. H. (1952). Vital staining of the hematoencephalic barrier by silver nitrate and tryphan blue, and cytological comparisons of the neurohypophysis, pineal body, area postrema, intercolumnar tubercle and supraoptic crest. J. Camp. Neural. 96, 371-413.
AND
MOLECULAR
PATHOLOGY
41, 58-66 (1984)
Ultrastructural Localization of Exogenous Silver in the Anterior Pituitary Gland of the Rat OLE THORLACIUS-USSING Institute
of Anatomy
B, University Received
AND J@RGEN RUNGBY of Aarhus,
November
DK-8000
Aarhus
C, Denmark
4, 1983
Silver accumulations in the anterior pituitary of argyric rats were demonstrated using a histochemical method that visualizes minute traces of the metal. Silver was localized intraand extracellularly throughout the anterior pituitary. Intracellular deposits were found within the lysosomes of somatotrophs and gonadotrophs. Extracellularly. the grains were located in basal laminae of portal veins and sinusoidal capillaries and in the membrane separating the anterior pituitary and part intermedia. The amount of deposited silver was dependent upon the dose of silver administered. Increasing the dose of silver lactate from 10 to 30 mg resulted in increased deposition, whereas a further increase to 60 mg did not alter the amount of silver deposited.
INTRODUCTION It is generally accepted that trace metals are capable of influencing the endocrine system at the level of the anterior pituitary (Henkin, 1976). The effects of silver, however, have, received only little attention, although it has been reported that the effects normally produced by chronic estradiol treatment are absent in argyric rats (Schreiber et al., 1980). In rats given silver nitrate in the drinking water, silver has been found within the connective tissue of portal vein walls in the anterior pituitary (Wislocki and Leduc, 1952), whereas in the neurohypophysis, silver is abundantly distributed intra- and extracellularly (Gatz, 1949; Wislocki and Leduc, 1952; Dempsey and Wislocki, 1955). The aim of the present paper is to describe the localization of silver in the anterior pituitary of argyric rats in light and electron microscopic preparations using a histochemical method for the demonstration of trace amounts of silver (Danscher, 1981~). MATERIAL
AND METHODS
Sixteen male Wistar rats weighing 150 g each were kept in groups of four under normal laboratory conditions. Silver lactate (5 mg dissolved in 1 ml distilled water) was injected intraperitoneally (i.p.). Three groups received a total of 10, 30, or 60 mg per rat, respectively. Injections of 1 ml per rat were given twice a week. The fourth group received two i.p. injections totaling 2 ml of 0.9% saline and served as controls. One week after the last injection, the silver-injected animals and the controls were anesthetized with Nembutal and killed by vascular perfusion at 120 mm Hg with 3% glutaraldehyde dissolved in Veronal buffer, pH 7.4. The perfusion lasted 12 min. The pituitary gland was removed, bisected, and kept in the same fixative for 1 hr. Tissue blocks were dehydrated and embedded in Epon, and semithin sections were cut according to conventional procedures. The sections were then exposed to silver amplification as described by Danscher (1981~). In summary, this method is initiated by the exposure of sections of uv light in order to reduce 58 0014-4800/84 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
SILVER
IN THE ANTERIOR
PITUITARY
GLAND
59
FIG. 1. Micrograph showing a semithin section from the anterior pituitary of a rat dosed with 30 mg silver lactate. Silver grains are seen intracellularly (big arrow) and extracellularly (small arrows). x640. FIG. 2. Semithin section showing silver in the membrane between the pars intermedia (pi) and pars anterior (pa). Rat treated with 30 mg silver lactate. x 640.
silver not bound as sulfides or selenides. Subsequently the sections were physically developed. The development was performed in a dark box, the sections being exposed to a developer containing silver ions, hydroquinone as a reducing agent, citrate buffer, and a protecting colloid based on gum Arabic. Silver traces in the sections catalyze the hydroquinone reduction of silver ions from the developer on the surface of the traces, thus enlarging these to visible size. To avoid background staining the sections are rinsed in water and sodium thiosulphate after the development. Routinely sections were developed for 60 min. After being rinsed the sections were counterstained with 1% toluidine, examined in the light microscope, stuck onto Epon blocks, and cut into ultrathin sections. These were counterstained with lead/uranyl acetate and examined in the electron microscope. RESULTS In the light microscope silver grains were evenly distributed throughout the anterior pituitary of all silver-treated rats whereas the controls were free of grains. Silver was located both intra- and extracellularly. The intracellular deposits were primarily seen in the cytoplasm of the largest secretory cells, whereas smaller secretory cells contained only few deposits (Fig. 1). Extracellularly, the silver was found in walls of the portal veins and the sinusoidal capillaries. The membrane dividing the pars intermedia and the anterior pituitary was heavily stained (Figs. 2 and 3). The number of cells stained and the number of grains per cell increased when
60
THORLACIUS-USSING
AND RUNGBY
FIG. 3. Electron micrograph showing silver deposits at the membrane dividing the pars intermedia and the anterior pituitary. Note the marginal cell (mc). This section was cut from the semithin section shown in Fig. 2. x 8800.
the dose of silver was increased from 10 to 30 mg, whereas no obvious quantitative differences were observed between doses of 30 and 60 mg. Ultrastructurally, silver grains were found in two cell types, somatotrophs (Fig. 5) and gonadotrophs (Figs. 4 and 6) of the silver treated rats but precipitates were never found in controls. Somatotrophs were identified by 350- to 400-nm secretory granules, abundant rough endoplasmic reticulum, and numerous lysosomes; gonadotrophs by their 200-nm granules, coexisting with fewer 400-nm granules, both evenly distributed throughout the cytoplasm (see Farquhar et al., 1975). In sections for both light and electron microscopy, grains of silver were found only in a portion of the two cell populations. In both somatotrophs and gonadotrophs the grains were located in bodies with a less dense and less demarcated matrix than that of the secretory granules. The bodies were variable in size, ranging from 100 to 500 nm, but most were somewhat larger than the largest secretory granules (Figs. 5 and 6). Accordingly, we have interpreted the structures as lysosomes. In very few instances a trace of silver was found in what, by their ultrastructural appearance, were identified as somatotroph secretory granules (Fig. 5). The extracellular silver was seen in basal laminae of the portal veins and sinusoidal capillaries (Fig. 7) and in the juxtaluminal part of the basal laminae surrounding the parenchymal cells (Figs. 7 and 8). Basal laminae separating the endocrine cells did not contain silver. No ultrastructural changes were observed in somatotrophs or gonadotrophs loaded with silver, nor did the total or relative number of cells of different types in the gland appear to have changed as a result of treatment with silver.
SILVER
IN THE
ANTERIOR
PITUITARY
GLAND
61
62
THORLACIUS-USSING
AND RUNGB
FIG. 5. Somatotroph from a rat dosed with 10 mg silver lactate. Silver is present in lysosomes (lower inset) and in secretory granules (upper inset). x 11,700 (insets x 33,000).
DISCUSSION The process of physical development can be catalyzed by other substances than silver. Metallic gold is a well known example, but sulfides and selenides of mercury, zinc, and other heavy metals can also catalyze the reaction (Roberts, 1935; Timm, 1958; Danscher and Schroder, 1979; Danscher, 1981a,b, 1982). It is therefore necessary to perform control experiments when using this method. Artifacts due to precipitation of lead or uranyl in the sections are easily distinguished from the silver grains since the latter have a uniform structure with characteristic edged rounded shapes and a homogeneous electron density (Danscher, 1981~; Rungby and Danscher, 1983). The darkly stained inclusions sometimes observed in lysosomes from osmium fixed tissue did not appear as a confounding factor since the procedure does not include osmification. Silver is transported by globulins and albumins in the blood (Hill and Pillsbury, 1939; Scott and Hamilton, 1950). The mechanisms responsible for the dissociation of silver from these elements and the subsequent uptake of metal into cells of certain types are as yet unexplained. The following considerations might, however, provide part of the explanation. (1) The lysosomes of somatotrophs and gonadotrophs may have an excess of available chemical groups with a high affinity for silver (e.g., sulfide groups) or they may take part in the degradation of substances with such high affinity groups, as suggested for central nervous system lysosomes by Rungby and Danscher (1983). (2) Silver could be bound in the content of secretory granules, thus the crinophagia of these substances (Farquhar, 1969, 1971) might account for the presence
SILVER
IN THE ANTERIOR
PITUITARY
GLAND
63
FIG. 6. Heavily loaded Iysosomes seen at high magnification. Gonadotroph from rat treated with 30 mg silver lactate. x 27,600.
of silver in the lysosomes (although silver was only occasionally seen in secretory granules). (3)The possibility that silver contained in basal laminae around anterior pituitary cells, vascular basal laminae, or blood-borne substances, could be taken up by phagocytosis cannot be excluded. However, phagocytic activity has been ascribed only to follicular cells of the anterior pituitary (Dingemans and Feltkamp, 1972; Farquhar et al., 1975) and these cells did not contain silver. The high affinity of basement membranes for silver has been described by Dempsey and Wislocki (1955), Walker (1972), and Scott and Norman (1980), and the present investigation confirms these results. The functional effects of the accumulation of exogenous silver in the anterior pituitary are still unknown, but the present findings suggest that silver could interfere with lysosomal functions. The lysosomes of the cells in the anterior pituitary contain protein-degrading enzymes, e.g., cathepsin A, B, B,, and D and dipeptidyl aminopeptidase I and II. These enzymes are responsible for a selective degradation of the hormones removed by crinophagia (Ellis, 1960; Ellis and Pery, 1966; Ellis and Nuenke, 1967; McDonald et al., 1971). At least one of these enzymes, dipeptidyl aminopeptidase I, can be inhibited by heavy metal compounds (mercury ions and p-chloromercuriphenyl-sulfonate). This inhibition is thought to be mediated by the binding of mercury to sulfydryl groups of the enzymes (Fruton and Mycek, 1956; McDonald et al., 1971). Both mercury and silver have a high affinity for sulfydryl groups (Petering, 1976; Danscher and Schroder, 1979); thus a possible disturbance of the crinophagia caused by silver might be mediated by this mechanism.
64
THORLACIUS-USSING
AND RUNGBY
FIG. 7. Sinosoidal capillary with silver grains in the basal lamina adjacent to a follicular cell (Fc) and an endothelial cell (EC). x 12,500. FIG. 8. Perivascular basal lamina of a portal vein with many silver deposits adjacent to a somatotroph (St). x 10,600
SILVER
IN THE ANTERIOR
PITUITARY
GLAND
65
Silver is a potent inhibitor of adenylate cyclase and cyclic AMP phosphodiesterase activity in many organs (Nathanson and Bloom, 1976). The secretory processes of endocrine cells are dependent upon respiratory energy (Jamieson and Palade, 1968, 1971). Changing the ability of the cells to produce cyclic AMP may change their secretory rate. Thus silver may interfere with the level of activity in anterior pituitary cells in at least two different ways. This might have functional significance. ACKNOWLEDGMENTS We thank Dr. G. Danscher for his inspiration and help and Dr. A. R. Lieberman for help with the manuscript. We gratefully acknowledge the technical assistance rendered by Mr. B. Krunderup, Mr. A. Meier, Ms. P. K. Melter, Mr. T. A. Nielsen, Ms. S. B. Thomsen. and Ms. K. Wiedemann.
REFERENCES DANSCHER, G. (198la). Histochemical demonstration of heavy metals. Hisrochemistry 71, l-16. DANSCHER, G. (1981b). Localization of gold in biological tissue. Hisfochemistry 71, 81-88. DANSCHER, G. (1981~). Light and electron microscopic localization of silver in biological tissue. Histochemistry 71, 177-186. DANSCHER, G. (1982). Exogenous selenium in the brain. Histochemistry 76, 281-293. DANSCHER, G., and SCHR~DER, H. D. (1979). Histochemical demonstration of mercury-induced changes in rat neurons. Histochemistry 60, l-7. DEMPSEY, E. W., AND WISLOCKI, G. B. (1955). An electron microscopic study of the blood-brain barrier of the rat, employing silver nitrate as a vital stain. J. Biophys. Biochem. Cytol. 1, 24.5-257. DINGEMANS, K. P., and FELTKAMP, C. A. (1972). Nongranulated cells in the mouse adenohypophysis. Z. Zellforsch. Mikrosk. Anat. 124, 387-405. ELLIS, S. (1960). Pituitary proteinase I: Purification and action on growth hormone and prolactin. J. Biol. Chem. 235, 1694-2001. ELLIS, S., and NUENKE, J. M. (1967). Dipeptidyl arylamidase III of the pituitary; purification and characterization. J. Biol. Chem. 242, 4623-4629. ELLIS, S., and PERY, M. (1966). Pituitary arylamidases and peptidases. J. Biol. Chem. 241, 36793687.
FARQUHAR, M. G. (1969). Lysosome function in regulating secretion: Disposal of secretory granules in cells of the anterior pituitary gland. In “Lysosomes in Biology and Pathology” (J. T. Dingle and H. B. Fell, eds.), Vol. 2, pp. 462-482. North-Holland, Amsterdam. FARQUHAR, M. G. (1971). Processing of secretory products by cells of the anterior pituitary gland. Mem. Sot. Endocrinol. 19, 79-124. FARQUHAR, M. G., SHUTELSKY, E. M., and HOPKINS, C. R. (1975) Structure and function of the anterior pituitary and dispersed pituitary cells. In vitro studies. In “The Anterior Pituitary” (A. Tixier-Vidal and M. G. Farquhar, eds.), pp. 83-135. Academic Press, New York. FRLJTON,J. S., and MYCEK, M. J. (1956). Studies on beef spleen cathepsin C. Arch. Biochem. Biophys. 65, 11-20.
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