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Morphological study of the extrusion of secretory materials by the parotid glands of mouse and rat
J. ULXRASTRVCTUl~ZRESEARCr~6, 449-465 (1962)
449
Morphologicol Study of the Extrusion of Secretory Moterials by the Porotid Glonds of Mouse ond Rot 1'~ HAROLD F. PARKS3
Department of Anatomy, University of Rochester School of Medicine and Dentistry, Rochester 20, New York Received August 21, 1961 Ultrastructural changes that were interpreted as manifestations of the extrusion of secretory materials are described in acinous and striated-duct cells of parotid glands of mouse and rat; the changes appeared to be identical in both species. In acinous cells: secretion granules discharged their contents by a process in which the membrane of the secretion granule became a part of the plasma membrane of the cell; small masses of cytoplasm were separated from the remaining cytoplasm by the coalescence of numerous tiny membranous vesicles, and extruded into the lumen; vacuoles, which formed by the swelling of various pre-existent membranous structures, extruded their contents in the same manner as secretion granules. In the striated ducts the apical ends of epithelial cells, which were usually characterized by a microvillous surface, lost their microvilli and swelled, sending large bleb-like projections of cytoplasm into the lumen. The blebs were apparently separated from their cells of origin, becoming a part of the secretion of the gland.
T h e p r o b l e m of the m a n n e r in which secretion antecedents are e x t r u d e d f r o m gland u l a r cells is one of the oldest in cytology; a t t e m p t s of light microscopists to observe the discharge of secretion granules f r o m the living p a n c r e a s date b a c k some 85 years (4, 5, 7, 8, 10, 16). Because of the l i m i t a t i o n s of r e s o l u t i o n of light m i c r o s c o p y , h o w ever, it r e m a i n e d for electron m i c r o s c o p y (11) to elucidate the u l t r a s t r u c t u r a l basis of z y m o g e n granule extrusion. This p a p e r describes a n u m b e r of processes t h a t a p p e a r to represent secretory p h e n o m e n a , confirming P a l a d e ' s (11) d e s c r i p t i o n of the m o d e of extrusion of z y m o g e n granules a n d r e p o r t i n g a similar m e c h a n i s m in salivary a n d l a c r i m a l g l a n d u l a r cells. T h e m e c h a n i s m s of discharge of vacuoles a n d of small q u a n t a of c y t o p l a s m f r o m 1 Supported by Grant D 689 from the Public Health Service. 2 Partially based on an abstract (15) of a paper read at a meeting of the International Association for Dental Research, 1960. Present address: Department of Zoology, Cornell University, Ithaca, New York.
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p a r o t i d acinous cells are described for the first time. Structural modifications of striated duct cells are described, b u t their physiological significance in relation to electrolyte secretion remains obscure.
MATERIALS A N D METHODS The observations recorded here were taken from a study of the parotid glands of approximately 60 white mice and 10 rats. Five of the rats were given pilocarpine hydrochloride (10 mg/kg) subcutaneously at intervals of 15, 30, 60, or 240 minutes before death, while the others received no treatment. The mice were used in the following ways: 13 animals were given a subcutaneous injection of 0.2-0.3 mg pilocarpine hydrochloride 10 minutes to 4 hours before parotid tissue was taken for examination. Of 18 animals that were starved for 24 hours, three were fed 5-30 minutes before death and three were given pilocarpine 5-10 minutes before death. Four animals were starved 4 days and then fed 2.5 to 4 hours before tissue was taken for examination. Two animals were given adrenaline (0.15 ml, subcutaneous) 8-10 minutes before death. The remaining animals were not treated in any way. Parotid tissue was removed from animals anesthetized with urethane or nembutal, usually after the gland was injected by hypodermic syringe with ice-cold 1% OsO~ fixative containing 0.22 M sucrose and buffered to p H 7.2-7.4 (3). Injection of the fixative had the effect of partially "dissecting" the lobules apart so that they could subsequently be separated from the main glandular mass with a razor blade without mechanical trauma. Tissues were imbedded in butyl methacrylate catalyzed with 1% benzoyl peroxide and polymerized in a 50°C oven over a period of 36 hours: a few specimens were imbedded in Araldite, but to no discernible advantage. Sections were cut with a glass knife, mounted on carbon grids, stained with lead hydroxide solution for 20 minutes (21), sandwiched with Formvar (20), and examined with a Siemens Elmiskop la operated at 60 kV.
OBSERVATIONS ACINOUS CELLS
Extrusion of secretion granules. The secretion granules i n p a r o t i d acinous cells of m o u s e a n d rat show certain i n t e r n a l differences in structure a n d consistency (13, 17, 19), b u t are similar in the respect that all are enclosed by a m e m b r a n e that resembles the p l a s m a m e m b r a n e of the cell. The appearance of electron micrographs of acinous cells that were stimulated to secretory activity indicated that the process of granule extrusion takes place as FIGS, 1 and 2. "Secretion figures" in parotid acinous cells of mice (starved 24 hours). Each picture shows, in addition to numerous "intact" secretion granules (G), a secretion granule (G') whose membrane has become continuous with the plasma membrane of the acinous cell around a constricted opening through which the granule's contents are discharged into the lumen (L). In Fig. 1, two transversely sectioned microvilli pass across the opening of the secretion granule, but do not obscure the basic relation of granule membrane to cell membrane, x 22,000.
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follows: (1) the m e m b r a n e s u r r o u n d i n g a secretion granule fuses with the cell m e m b r a n e lining the l u m e n of the acinus; (2) a b r e a k t h r o u g h occurs at the site of fusion, leaving the cell m e m b r a n e c o n t i n u o u s with the m e m b r a n e of the granule a r o u n d a c o n s t r i c t e d o p e n i n g t h r o u g h which (3) the contents of the granule flow into the l u m e n (Figs. 1 a n d 2). This m a n n e r of extrusion, which was first described in the guinea pig p a n c r e a s (ll), has also been seen b y the writer in acini of the s u b m a x i l l a r y a n d e x o r b i t a l glands of the rat a n d in the p a n c r e a s of the mouse. P i l o c a r p i n e injection p r o d u c e d i m m e d i a t e a n d extensive extrusion of secretion granules in the p a r o t i d glands of mice a n d rats, as was m a n i f e s t e d b y the a p p e a r a n c e of large n u m b e r s of "secretion figures" (i.e., profiles showing secretion granules whose i n t e r n a l contents h a d achieved access to the acinous lumen) in the g l a n d u l a r tissue; a similar effect was p r o d u c e d in mice b y feeding after a 24-hour fast. F a s t i n g a p p e a r e d in m a n y cases to p r o d u c e a sort of " h u n g e r secretion"; a fairly large n u m ber of secretion figures were seen in a n i m a l s t h a t were starved for 24 hours. P a r o t i d glands f r o m the mice t h a t received a d r e n a l i n e 8 or 10 m i n u t e s before d e a t h also showed a c o n s i d e r a b l e n u m b e r of secretion figures (e.g., 30 were seen in one section). Secretion figures were very rarely seen in the glands of a n i m a l s t h a t h a d received no experimental treatment. The a p p e a r a n c e of secretion figures in the m o u s e p a r o t i d i n d i c a t e d that, in stimulated glands, the granules t h a t were a b o u t to be discharged i m b i b e d w a t e r f r o m the cytoplasm, o r t h a t once a granule gained access to the lumen, its contents were quickly dissolved or dispersed a n d w a s h e d o u t into the lumen. The m a j o r i t y of secretion granules in fixed m o u s e p a r o t i d tissue were i n h o m o g e n e o u s , their contents consisting of a dense cortical layer a n d a less-dense central p a r t t h a t o c c a s i o n a l l y
Note: Figs. 3-10 are a selected series of micrographs showing what appear to be progressive stages in the extrusion of quanta of cytoplasm from the parotid acinous cell of the mouse. In the interest of completeness of description, the experimental treatment is given in each legend, but no causal relationship between the stage of extrusion depicted and the nature or duration of experimental treatment is implied. Fro. 3. Quanta of specialized cytoplasm (8 minutes after injection of adrenaline). The border of each quantum is delineated by an arrangement of tiny membranous vesicles. The quanta appear to be spherical, and are approximately 1 # in diameter. Four quanta (Q) are seen in the field; the smallest profile, near the lumen (L), is presumably sectioned tangentially, x 25,000. FIG. 4. Quantum of cytoplasm (10 minutes after injection of adrenaline.) A number of the small vesicles surrounding the quantum have apparently coalesced to form a slit-like membrane-enclosed space (arrow). x 35,000. FIG. 5. Quantum of cytoplasm (8 minutes after adrenaline). The slit-like space (arrow) appears to be expanding in the direction of the lumen (L) of an intercellular secretory canaliculus. The small mass of apparently free cytoplasm in the lumen may represent a quantum of cytoplasm that was extruded at a different level of the canal. × 18,000. FIG. 6. Quantum of cytoplasm (24 hours' starvation). Here the slit-like space has apparently become continuous with the lumen (L), and a part of the quantum has thus become directly exposed to the lumen, x 22,000.
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J . Ultraetructure Research
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contained small isolated masses of dense material. W h e n such granules were seen in the process of being extruded, their contents were usually h o m o g e n e o u s and of a low density (Figs. 1 and 2), but occasionally some of the dense cortical material was discernible. In some instances, granules that were situated near the lumen, but were apparently not in contact with it, showed a similar homogeneous, low-density content. In an unstimulated gland, however, an acinus was seen in which b o t h the lumen and the secretion granules that were in process of extruding their contents were filled with uniformly dense material. This last-named instance was interpreted as a secretory p h e n o m e n o n in which the acinus was very slowly discharging organic material but was not secreting water.
Extrusion of quanta of cytoplasm Some relatively infrequently encountered cytoplasmic formations that appeared to represent secretion antecedents are illustrated in Fig. 3. Such formations consisted of a presumably spherical mass of cytoplasm that was demarcated f r o m the surr o u n d i n g cytoplasm by a sheet-like arrangement of small m e m b r a n o u s vesicles (appearing linear in section) so disposed as to enclose a sphere (and thus appearing in section as a circular arrangement). By collecting a large n u m b e r of micrographs showing these formations, it was possible to arrange a series showing what appears to be a sequence of stages in an extrusion process (Figs. 4-10). The extrusion process apparently occurs in the following manner. A n u m b e r of the vesicles coalesce to f o r m a membrane-enclosed slit-like space that partially separates the secretion antecedent f r o m the ambient cytoplasm (Figs. 4 and 5). The outer layer of the m e m b r a n e surrounding the slit-like space fuses with the plasma m e m b r a n e at the lumen of the acinus; and a b r e a k t h r o u g h of the fused m e m b r a n e occurs (it will be noted that this process of fusion-and-breakthrough is identical to that by which a secretion granule releases its contents). Since the m e m b r a n e lining the slit-like space thus becomes a part of the plasma m e m b r a n e of the cell, a portion of the surface of the special q u a n t u m of cytoplasm becomes exposed to the lumen (Figs. 6 and 7). The mereFIG. 7. Quantum of cytoplasm (8 minutes after adrenaline). The relationship between quantum and lumen (L) is the same as that in Fig. 6, but here a larger part of the quantum has become exposed to the lumen. × 30,000. FIG. 8. Quantum of cytoplasm (one hour after pilocarpine). Approximately half of the quantum is projecting into the lumen; the other half remains imbedded within, and attached to, the cytoplasm. Some virus-like bodies (V), and an apparently free mass of cytoplasm immediately beneath them, are seen in the lumen. The mass of cytoplasm may be a previously-extruded "quantum". x 26,000. F:G. 9. Quantum of cytoplasm (3.5 hours after feeding, following a 92-hour fast). The quantum (Q) is almost completely contained within the lumen, but remains broadly attached at its base. An apparently free mass of cytoplasm of similar texture is seen in the upper part of the picture, x 22,000. FIG. 10. Quantum of cytoplasm (3.5 hours after feeding, following a 92-hour fast). The quantum appears to be almost free in the lumen; its attachment to the cell has become constricted. × 22,0130.
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branous vesicles associated with the deeper parts of the secretion antecedent continue to coalesce with one another and to fuse with the plasma membrane lining the lumen, with the effect that the secretion antecedent is gradually separated from the cell, moved into the lumen (Figs. 8, 9 and 10), and ultimately freed from the cell (Figs. 5, 8 and 9 show what appear to be free masses of cytoplasm in the acinous lumen). There is reason to suspect that the process of extrusion is a slow one: virus-like bodies that have been described as forming on the luminal microvilli of acinous cells (13) were also seen forming on the free edge of a partially extruded secretion antecedent of this kind. The cytoplasm constituting these secretion antecedents was of a very light density and was free of membranous contents, but occasionally contained a number of granules resembling RNP particles. It was remarked above that these quanta of cytoplasm were demarcated from the surrounding cytoplasm by a group of vesicles; an additional demarcating structure was sometimes visible in the form of a layer of denser cytoplasm immediately outside the layer of vesicles (Figs. 3, 5, 6 and 7). It is possibly significant that secretion antecedents of this kind were usually seen in close proximity to a glandular lumen. The complete 3-dimensional relationship of these quanta to the ambient cytoplasm was not fully established by serial-section study, and, although in the majority of cases they appeared to be completely surrounded by cytoplasm, it is possible that they were actually in contact with the acinous lumen at a level not shown by the section. It is therefore acknowledged that these specialized quanta of cytoplasm may originate in relation to the luminal surface of a cell, being merely "countersunk" instead of submerged in the depths of the cytoplasm, in which case profiles like those in Fig. 3 would appear only in sections passing approximately parallel to the glandular lumen. Cytoplasmic formations of the kind described above were seen in the parotid glands of animals that were starved 24 hours, starved-and-fed, given pilocarpine or adrenaline, or not treated in any way; they therefore appear not to be peculiar to any experimental treatment unless it be urethane or nembutal anesthesia. Similar formations have also been seen very rarely by the writer in mouse pancreas: one of the pancreases was obtained from a 5-day-old animal that was killed by decapitation. without anesthesia. An idea of the frequency of occurrence of these structures is conveyed by the following account. A section of parotid gland from a mouse that had received an injection of adrenaline seemed to have more than the usual number of such inclusions: the whole section was scanned, and 35 inclusions were counted. In a section from a similarly treated animal, five such structures were found in one acinus, but a total of only 14 were found while scanning the remainder of the section during one hour at a magnification of 10,000.
SECRETORY ACTIVITY OF PAROTID GLAND
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Vacuoles The term "vacuole" is used here to signify a membranous vesicle whose content consists mainly of water and is therefore of a lighter density than ground cytoplasm (in distinction to a "granule", whose content is denser than ground cytoplasm). The formation of vacuoles has not been studied thoroughly, and will therefore not be dealt with in detail. Vacuoles were not seen in the parotid glands of animals that had received no experimenta[ treatment or that had been starved 24 hours. They were present, however, in animals that were given pilocarpine (Figs. 13 and 14) or were fed after a 24hour fast (Figs. 11 and 12). The vacuoles in the glands of starved-and-fed animals rarely exceeded 2.5 # in diameter, while those of pilocarpine-treated glands sometimes reached a diameter of 5 #. As to the mode of vacuole formation, the basic phenomenon could be described as a swelling of some pre-existent membranous vesicle, presumably by imbibition of water. Smaller vacuolar formations sometimes exhibited structural peculiarities indicating the nature of the structure from which they had arisen. (1) Certain of the smaller vacuoles in acinous cells of the mouse had a homogeneous content that was intermediate in density between the dense part and the "light" part of an intact secretion granule; these were interpreted as forming from secretion granules by imbibition of water and dissolution of dense content. (2)Other smallvacuolar formations were of an irregular shape suggesting that they had formed by the swelling of a previously flat membranous vesicle such as a Golgi cisterna or similar structure (Fig. 11, lower left). (3) Some vacuoles had a protuberance of low-density cytoplasm bulging into them (Fig. 11, top center; also of Fig. 14) suggesting that they might have formed by a disproportionate enlargement or swelling of the slit-like space sometimes seen in relation to the quantum-of-cytoplasm type of secretion antecedent (Figs. 4 and 5). Although the majority of profiles of large vacuoles gave no indication as to their provenance, it seems probable that the smaller vacuoles were younger or immature forms of the large ones. A common, but not constant, feature of vacuoles was the presence of a mantle of relatively low-density cytoplasm surrounding them (Figs. 12 and 13). In a few cases of pilocarpine-induced vacuole formation, the cytoplasm related to a part of the surface of a vacuole had apparently swollen and become lighter in density (presumably by increased water content): cisternae of the endoplasmic reticulum located within this special region of cytoplasm had also become swollen and the membranes forming the swollen parts of the cisternae had become degranulated (Fig. 14). Beyond acknowledging the obvious point that such a change in cytoplasmic structure is most probably abnormal and deleterious, little can be said about its significance.
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STRIATED DUCTS Striated d u c t cells are c h a r a c t e r i z e d m o r p h o l o g i c a l l y b y the presence of n u m e r o u s r a d i a l l y a r r a n g e d m i t o c h o n d r i a a n d infoldings of the p l a s m a m e m b r a n e in the b a s a l regions of the cells (13, 17, 19). Their apical c y t o p l a s m c o n t a i n s granules a n d vesicles whose a p p e a r a n c e a n d l o c a t i o n suggest t h a t t h e y are secretion antecedents, a n d the l u m i n a l surface is usually t h r o w n into microvillous extensions (Fig. 15). A s t r u c t u r a l m o d i f i c a t i o n of striated duct cells, which was i n t e r p r e t e d as p r o b a b l y being a m a n i f e s t a t i o n of secretory activity because it was m o s t p r o m i n e n t l y d e v e l o p e d in glands t h a t h a d been s t i m u l a t e d b y p i l o c a r p i n e or b y feeding after a 24-hour fast, o c c u r r e d at the apical ends of the cells. This m o d i f i c a t i o n consisted in a swelling of the c y t o p l a s m j u s t apical to the " s e c r e t i o n " granules a n d the a c c o m p a n y i n g loss of microvilli, which were p r e s u m a b l y flattened out b y the swelling process. The cytop l a s m i c " b l e b " thus f o r m e d often c o n t a i n e d some tiny particles r e s e m b l i n g R N P granules (Fig. 16) and, occasionally, a small n u m b e r of " s e c r e t i o n " granules o r vesicles (Fig. 17): it was of a b o u t the same density as the g r o u n d c y t o p l a s m of the cells, b u t a p p e a r e d lighter because it was relatively free of p r o m i n e n t c y t o p l a s m i c inclusions. I t was a p p a r e n t l y the fate of these blebs to be s e p a r a t e d f r o m their cells of origin a n d to disintegrate in the lumen, j u d g i n g b y the o c c a s i o n a l l y seen presence of m e m b r a n o u s debris in the lumen. A l l duct cells were n o t u n i f o r m l y affected b y secretory stimulus. Fig. 17 shows a section t h r o u g h the l u m e n of a striated d u c t of a p i l o c a r p i n e - t r e a t e d mouse. M a n y
Fla. 11. Vacuoles (V) and secretion granules in mouse parotid acinus (30 minutes after feeding, following a 24-hour fast). The shape of the vacuole at the lower left suggests that it may have arisen by swelling of a previously flat membranous precursor. The vacuole at the top of the picture contains a projection of low-density cytoplasm, suggesting that it may have arisen by a disproportionate swelling of the slit-like space that is sometimes seen in relation to the quantum-of-cytoplasm secretion antecedents (cf. Figs. 4 and 5). One vacuole has become continuous with the lumen (L) of an intercellular secretory canaliculus. × 14,500. FIG. 12. Vacuoles (V) and secretion granules in mouse parotid acinus (5 minutes after feeding, following a 24-hour fast). In the lower left a secretion granule whose membrane has become continuous with that lining the lumen (L) of a secretory canaliculus is seen. x 14,500. FI6. 13. Vacuole (V) in parotid acinous cell of mouse (15 minutes after pilocarpine). This vacuole is surrounded by a mantle of cytoplasm that is devoid of all the membranous and granular components that are usually evenly distributed in the cytoplasm of acinous cells, x 21,000. FIG. 14. Vacuole (V) and secretion granule (G) in parotid acinous cell of mouse (15 minutes after pilocarpine). This vacuole has no mantle of modified cytoplasm like that in Fig. 13, but has a mass of low-density cytoplasm related to it at one side. Two of the membranous vesicular structures in the low-density cytoplasm (arrows) are seen to be the ends of endoplasmic-reticulum cisternae that have lost their RNP granules and become swollen. × 21,000. FIG. 15. Apical portion of striated duct cells of mouse parotid gland (no experimental treatment). The micrograpb illustrates the usual microvillous luminal surface of these cells. Many of the microvilli have been cut in oblique or transverse section, and thus appear free in the lumen. The large granular body (M) is a mitochondrion whose internal membranes lie parallel to the plane of the section, x 32,500.
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of the cells have large swollen ends which, in the aggregate, almost occlude the lumen, but other cells have apparently maintained their microvillous surfaces. In addition to being a prominent feature of the strongly stimulated gland, bleb formation was occasionally seen, usually in a milder f o r m (Fig. 16), in animals that had been starved for 24 hours or had received no experimental treatment. The adrenaline-treated animals contained only a few blebs; these were very small, affecting only a part, rather than the whole, of a cell's apical surface.
DISCUSSION The term "secretion", which has been variously defined in the literature (6, 12), is used in the context of this paper to refer both to the extrusion of materials synthesized by the glandular cells and to the active transport of materials f r o m blood to saliva. In this investigation no attempt has been made to study the effects of any particular secretion stimulus (pilocarpine, adrenaline, or feeding-after-starvation) per se. The goal in this preliminary study has been merely to record the various manifestations of secretory activity.
Granules and vacuoles The process of extrusion of secretion granule material was seen in this study to be fundamentally the same as that described for the pancreas by Palade (11). Indications were seen, however, of a possible difference between the parotid and pancreatic acinus in one feature of their secretory process. The pancreatic z y m o g e n granule has dense h o m o g e n e o u s contents. In micrographs showing "secretion figures" in pilocarpine-treated mouse pancreas (14), as well as in Palade's (11) illustration of a secretion figure in guinea pig pancreas, the material in the lumen of the acinus was as dense as that in an intact z y m o g e n granule. A similar equality of density, as manifested by equal intensity of staining, of luminal content and intact z y m o g e n granule FIG. 16. Apical end of a striated duct cell from mouse parotid gland (starved 24 hours). The cytoplasm immediately apical to the vesicular and granular "secretion antecedents" (arrows) has become swollen, and microvilli have disappeared from the luminal surface. Since fasting appeared to constitute an adequate stimulus to secretion in acinous cells, it seems not unlikely that this structural modification is a manifestation of secretory activity (also cf. Fig. 17). Two fat droplets (F) with their centers dissolved out appear in the section. × 28,000. FIG. 17. Section through lumen of striated duct (pilocarpine given in two half-doses; 60 minutes and 30 minutes before death). The lumen (L) is almost occluded with large bleb-like extensions of cytoplasm (B) from the duct cells. There is no marked tendency for the "secretion antecedents" to enter the blebs (note the cell associated with bleb B); however, a few of the granules and vesicles do so (arrows) and suffer the fate of the blebs, which appears to he detachment from cell of origin and disintegration in the duct. Some of the duct cells are seen to have intact, microvillous apical ends. × 8000.
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has been noted in light-microscopic preparations of pancreas from the pilocarpinetreated mouse (14). These considerations indicate that not a great deal of water passes through the pancreatic acinous cell during the process of extrusion. On the other hand, the great majority of secretion figures in sections of parotid gland have shown a low density of material in the lumen and in secretion granules discharging their content--considerably less than the denser content of an intact secretion granule. This consideration argues that an appreciable amount of water passes through the parotid acinous cell while secretion granules are being discharged. An obvious question relating to the mode of granule extrusion described above is, given the fact that the area of surface membrane of the cell is increased every time a granule is discharged, how does the cell maintain a reasonably constant surface area? Palade (11) offers the reasonable hypothesis that membrane is taken into the cell to be re-used, and mentions small membranous vesicles near the surface membrane as possibly representing invaginated surface membrane. While no better answer has presented itself in this study, neither were obvious indications seen of membrane invagination from the apical ends of acinous cells. Although indications of invagination have been seen at the basal and lateral surfaces of acinous cells (13), it cannot be decided at present whether apical surface membrane can migrate to the sides of acinous cells. If the "desmosomes" that are seen in sections passing through the apical parts of acinous cells actually represent continuous "terminal bars" that pass completely around the perimeter of a cell near its apex, then a migration of membrane from apical surface to lateral surface would seem improbable.
Vacuole formation has been described at the light-microscopic level by Covell (4), Hirsch (6), and Ries (16) in the pilocarpine-stimulated pancreatic cell, and by Duthie (who incorrectly believed himself to be dealing with the sublingual gland) in the pilocarpine-treated parotid gland of mouse and rat (5). As for the mode of vacuole formation, (1) Covell (4) has vacuoles forming by liquefactian of zymogen granules; (2) Ries (16) and Hirsch (cited by Ries, but without reference to a specific publication) agree in stating that vacuoles are produced by coalescence of zymogen granules; and (3) Hirsch (6) further distinguishes between two kinds of vacuole--those formed by the solution of zymogen granules, and "water vacuoles" formed by imbibition of water by the cell. At the electron-microscopic level, some evidence may be said to exist in support of each of these claims: viz., this study has shown that some of the smaller vacuoles were indeed swollen secretion granules and that some vacuoles appear to arise from membranous formations other than secretion granule membranes; also, Palade (11) has illustrated the coalescence of pancreatic zymogen granules during the act of extrusion. Electron microscopic evidence disagrees, however, with
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the claims of the earlier workers who saw granules and vacuoles being discharged bodily, i.e., in an intact condition, from the acinous cell: both forms of secretory entity appear to be discharged by means of a fusion-and-breakthrough of membranes (cf. Figs. 1 and 2 for granule, Fig. 11 for vacuole). A possible explanation for this discrepancy is provided by Ries's (16) observation that in "stiirmische Extrusion" caused by pilocarpine whole shreds of cytoplasm are lost from the apex of the cell; he refers to the secretion illustrated by Covell (4) as "allm~ihliche Sekretion", but the secretory phenomena observed by Covell were probably more "sttirmisch" than Ries imagined. A definitive study of vacuole formation has not been made at the time of this writing, but is envisioned as a future undertaking. Perhaps the most important point regarding the relation of vacuole formation to cell function that has been brought out in this study is that vacuole formation is apparently a normal phenomenon, if one may so judge from the fact that vacuoles do appear in the glands of animals fed after a 24-hour fast (Figs. 11 and 12) as well as in pilocarpine-treated animals. Thus this observation agrees with Ries's (16) statement that pilocarpine evokes normal cell activities in an extreme form (at least at the morphologicallevel). Babkin (1) points out, however, that pilocarpine-stimulated saliva has different concentrations of constituents from chorda saliva in dog and cat submaxillary glands, and warns that pilocarpine cannot be regarded as a true substitute for parasympathetic stimulation. In pilocarpine-stimulated parotid glands of the mouse, Rutberg (17) found large vacuoles that were not enclosed by a membrane, but appeared as mere spaces in the cytoplasm. This difference, and other differences between his observations and the author's on pilocarpine-treated glands, may be due to the fact that Rutberg used a pilocarpine dosage %10 times stronger than that used in this study: it seems likely that extreme effects of pilocarpine stimulation can be deleterious (cf. Fig. 14).
Quanta of cytoplasm The process by which an acinous cell separates from itself a quantum of cytoplasm and discharges it into the lumen is, to the best of the writer's knowledge, described for the first time in this paper. Whether such fragments of cytoplasm constitute a useful contribution to the saliva or represent a casting-off process by which the cell rids itself of unwanted material cannot be decided at present. No direct evidence concerning the source of the tiny membranous vesicles surrounding the secretion antecedents was seen in the parotid acinous cells; however, in an inclusion of this type in a pancreatic acinous cell, one of the vesicles, which had a smooth surface itself, appeared to be in the act of pinching off from a granular cisterna of endoplasmic reticulum.
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Striated ducts A morphological modification of striated duct cells of mouse and rat parotid glands that was interpreted as of probable secretory significance was the enlargement of the apical ends of the cells to form relatively large cytoplasmic blebs, a process that was accompanied by the loss of the microvilli ordinarily present on the luminal surfaces of these cells. Blebs were conspicuous in mice and rats that were given pilocarpine and in mice that were fed after a 24-hour fast, but were also occasionally seen in mice that had been starved 24 hours or that had not received experimental treatment. It is difficult at present to assess the morphological validity of striated-duct bleb formation as a secretory phenomenon. The observation of blebs is not peculiar to this investigation; they have been seen in the rat parotid at the electron-microscopic level (19) and in the human submaxillary gland at the light-microscopic level (22). Rutberg (17), however, failed to find apical blebs in striated duct ceils of mouse parotid following pilocarpine stimulation; and in the present study it was noted that not all of the striated duct cells formed blebs. In a problem of this kind the electron microscopist is hampered by the smallness of the sample of tissue he can examine in a reasonable amount of time. The over-all occurrence of the large striated-duct blebs in relation to the various secretory stimuli can probably be more effectively investigated by light-microscopic methods, in which larger samples of tissue can be examined, and by which a wider variety of fixatives can be used to investigate the possibility that blebs are a fixation artifact. It would be equally difficult to evaluate the physiological significance of bleb formation in striated ducts. There is as yet no complete agreement on the exact functional nature of the striated duct: e.g., Rutberg (17) argues the hypothesis that striated ducts act mainly by absorbing sodium from a primary saliva produced by acini and intercalated ducts; Burgen (2), who finds abundant secretion in glands of young dogs in which there seems not to be enough acinous tissue to account for the flow, thus suspects a secretory function for striated ducts; and Langley and Brown (9) find evidence for both secretion and reabsorption of sodium in different parts of the duct system in the canine parotid. This divergence of ideas concerning function makes it impossible to relate bleb formation to any particular kind of cellular activity. Furthermore, it is not possible to know whether bleb formation, which apparently does not affect all cells (or at least does not affect all ceils simultaneously), is a manifestation of an active physiological process or represents merely a passive response to the medium in contact with the apical surface of the ceil. Data is now available (18) showing that pilocarpine-induced parotid secretion in the rat varies from hypotonic at low rates of secretion to hypertonic at high rates; however, it is not known whether
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the blebs described above were formed while the duct contents were hypertonic or while they were hypotonic. Blebs apparently become detached from their parent cells and become a part of the saliva. In doing this they sometimes carry a few "secretion" granules or vesicles with them. No evidence of extrusion of secretion granules by striated duct cells in the manner described for acinous cells has been seen. Rutberg (17) suggests that the apical granules may be manifestations of an absorptive, rather than a secretory, process. REFERENCES 1. BABKIN,B. P., Secretory Mechanism of the Digestive Glands, 2nd ed. Paul B. Hoeber, Inc., New York, 1950. 2. BURGEN, A. S. V. (personal communication). 3. CAULHELD,J., J. Biophys. Biochem. Cytol. 3, 827 (1957). 4. COVELL,W. P., Anat. Record 40, 213 (1928). 5. DUTmE, E. S., Proc. Roy. Soc. London B 114, 20 (1933). 6. HIRSCH, G. C., Biol. Revs. Cambridge Phil. Soc. 6, 88 (1931). 7. - - - - Z. ZellJbrsch. u. rnikroskop. Anat. 15, 36 (1932). 8. K/3I~NE, W. and LEA, A., Verhandl. Naturhistorischmedizinischen Ver. Heidelberg N.F. 1, 445 (1876). 9. LANGLEY,L. L. and BROWN, R. S., Am. J. Physiol. 199, 59 (1960). 10. MATHEWS, A., J. Morphol., suppl. 15, 171 (1899). 11. PALADE,G. E., Subcellular Particles, p. 64. American Physiological Society, Washington, D.C., 1959. 12. PALAY, S. L., Frontiers in Cytology, p. 305, Yale University Press, New Haven, 1958. 13. PARKS, H. F., Am. J. Anat. 108, 303 (1961). 14. - (unpublished observations). 15. PARKS, H. F. and JOHANSEN,E., J. Dent. Research 39, 714 (1960). 16. RI~S, E., Z. Zellforsch. u. mikroskop. Anat. 22, 523 (1935). 17. RUT~ERG, U., Acta Odontol., Scand. 19, suppl. 30 (1961). 18. SCHbrEVER,C. A. and SCHNEYER, L. H., Am. J. Physiol. 199, 55 (1960). 19. SCOTT, B. L. and PEASE, D. C., Am. J. Anat. 104, 115 (1959). 20. WATSON, M. L., J. Biophys. Biochem. Cytol. 3, 1017 (1957). 21. - ibid. 4, 727 (1958). 22. ZIMMERMANN,K. W., Arch. mikroskop. Anat. u. Entwicklungsmech. 52, 552 (1898).
449
Morphologicol Study of the Extrusion of Secretory Moterials by the Porotid Glonds of Mouse ond Rot 1'~ HAROLD F. PARKS3
Department of Anatomy, University of Rochester School of Medicine and Dentistry, Rochester 20, New York Received August 21, 1961 Ultrastructural changes that were interpreted as manifestations of the extrusion of secretory materials are described in acinous and striated-duct cells of parotid glands of mouse and rat; the changes appeared to be identical in both species. In acinous cells: secretion granules discharged their contents by a process in which the membrane of the secretion granule became a part of the plasma membrane of the cell; small masses of cytoplasm were separated from the remaining cytoplasm by the coalescence of numerous tiny membranous vesicles, and extruded into the lumen; vacuoles, which formed by the swelling of various pre-existent membranous structures, extruded their contents in the same manner as secretion granules. In the striated ducts the apical ends of epithelial cells, which were usually characterized by a microvillous surface, lost their microvilli and swelled, sending large bleb-like projections of cytoplasm into the lumen. The blebs were apparently separated from their cells of origin, becoming a part of the secretion of the gland.
T h e p r o b l e m of the m a n n e r in which secretion antecedents are e x t r u d e d f r o m gland u l a r cells is one of the oldest in cytology; a t t e m p t s of light microscopists to observe the discharge of secretion granules f r o m the living p a n c r e a s date b a c k some 85 years (4, 5, 7, 8, 10, 16). Because of the l i m i t a t i o n s of r e s o l u t i o n of light m i c r o s c o p y , h o w ever, it r e m a i n e d for electron m i c r o s c o p y (11) to elucidate the u l t r a s t r u c t u r a l basis of z y m o g e n granule extrusion. This p a p e r describes a n u m b e r of processes t h a t a p p e a r to represent secretory p h e n o m e n a , confirming P a l a d e ' s (11) d e s c r i p t i o n of the m o d e of extrusion of z y m o g e n granules a n d r e p o r t i n g a similar m e c h a n i s m in salivary a n d l a c r i m a l g l a n d u l a r cells. T h e m e c h a n i s m s of discharge of vacuoles a n d of small q u a n t a of c y t o p l a s m f r o m 1 Supported by Grant D 689 from the Public Health Service. 2 Partially based on an abstract (15) of a paper read at a meeting of the International Association for Dental Research, 1960. Present address: Department of Zoology, Cornell University, Ithaca, New York.
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p a r o t i d acinous cells are described for the first time. Structural modifications of striated duct cells are described, b u t their physiological significance in relation to electrolyte secretion remains obscure.
MATERIALS A N D METHODS The observations recorded here were taken from a study of the parotid glands of approximately 60 white mice and 10 rats. Five of the rats were given pilocarpine hydrochloride (10 mg/kg) subcutaneously at intervals of 15, 30, 60, or 240 minutes before death, while the others received no treatment. The mice were used in the following ways: 13 animals were given a subcutaneous injection of 0.2-0.3 mg pilocarpine hydrochloride 10 minutes to 4 hours before parotid tissue was taken for examination. Of 18 animals that were starved for 24 hours, three were fed 5-30 minutes before death and three were given pilocarpine 5-10 minutes before death. Four animals were starved 4 days and then fed 2.5 to 4 hours before tissue was taken for examination. Two animals were given adrenaline (0.15 ml, subcutaneous) 8-10 minutes before death. The remaining animals were not treated in any way. Parotid tissue was removed from animals anesthetized with urethane or nembutal, usually after the gland was injected by hypodermic syringe with ice-cold 1% OsO~ fixative containing 0.22 M sucrose and buffered to p H 7.2-7.4 (3). Injection of the fixative had the effect of partially "dissecting" the lobules apart so that they could subsequently be separated from the main glandular mass with a razor blade without mechanical trauma. Tissues were imbedded in butyl methacrylate catalyzed with 1% benzoyl peroxide and polymerized in a 50°C oven over a period of 36 hours: a few specimens were imbedded in Araldite, but to no discernible advantage. Sections were cut with a glass knife, mounted on carbon grids, stained with lead hydroxide solution for 20 minutes (21), sandwiched with Formvar (20), and examined with a Siemens Elmiskop la operated at 60 kV.
OBSERVATIONS ACINOUS CELLS
Extrusion of secretion granules. The secretion granules i n p a r o t i d acinous cells of m o u s e a n d rat show certain i n t e r n a l differences in structure a n d consistency (13, 17, 19), b u t are similar in the respect that all are enclosed by a m e m b r a n e that resembles the p l a s m a m e m b r a n e of the cell. The appearance of electron micrographs of acinous cells that were stimulated to secretory activity indicated that the process of granule extrusion takes place as FIGS, 1 and 2. "Secretion figures" in parotid acinous cells of mice (starved 24 hours). Each picture shows, in addition to numerous "intact" secretion granules (G), a secretion granule (G') whose membrane has become continuous with the plasma membrane of the acinous cell around a constricted opening through which the granule's contents are discharged into the lumen (L). In Fig. 1, two transversely sectioned microvilli pass across the opening of the secretion granule, but do not obscure the basic relation of granule membrane to cell membrane, x 22,000.
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follows: (1) the m e m b r a n e s u r r o u n d i n g a secretion granule fuses with the cell m e m b r a n e lining the l u m e n of the acinus; (2) a b r e a k t h r o u g h occurs at the site of fusion, leaving the cell m e m b r a n e c o n t i n u o u s with the m e m b r a n e of the granule a r o u n d a c o n s t r i c t e d o p e n i n g t h r o u g h which (3) the contents of the granule flow into the l u m e n (Figs. 1 a n d 2). This m a n n e r of extrusion, which was first described in the guinea pig p a n c r e a s (ll), has also been seen b y the writer in acini of the s u b m a x i l l a r y a n d e x o r b i t a l glands of the rat a n d in the p a n c r e a s of the mouse. P i l o c a r p i n e injection p r o d u c e d i m m e d i a t e a n d extensive extrusion of secretion granules in the p a r o t i d glands of mice a n d rats, as was m a n i f e s t e d b y the a p p e a r a n c e of large n u m b e r s of "secretion figures" (i.e., profiles showing secretion granules whose i n t e r n a l contents h a d achieved access to the acinous lumen) in the g l a n d u l a r tissue; a similar effect was p r o d u c e d in mice b y feeding after a 24-hour fast. F a s t i n g a p p e a r e d in m a n y cases to p r o d u c e a sort of " h u n g e r secretion"; a fairly large n u m ber of secretion figures were seen in a n i m a l s t h a t were starved for 24 hours. P a r o t i d glands f r o m the mice t h a t received a d r e n a l i n e 8 or 10 m i n u t e s before d e a t h also showed a c o n s i d e r a b l e n u m b e r of secretion figures (e.g., 30 were seen in one section). Secretion figures were very rarely seen in the glands of a n i m a l s t h a t h a d received no experimental treatment. The a p p e a r a n c e of secretion figures in the m o u s e p a r o t i d i n d i c a t e d that, in stimulated glands, the granules t h a t were a b o u t to be discharged i m b i b e d w a t e r f r o m the cytoplasm, o r t h a t once a granule gained access to the lumen, its contents were quickly dissolved or dispersed a n d w a s h e d o u t into the lumen. The m a j o r i t y of secretion granules in fixed m o u s e p a r o t i d tissue were i n h o m o g e n e o u s , their contents consisting of a dense cortical layer a n d a less-dense central p a r t t h a t o c c a s i o n a l l y
Note: Figs. 3-10 are a selected series of micrographs showing what appear to be progressive stages in the extrusion of quanta of cytoplasm from the parotid acinous cell of the mouse. In the interest of completeness of description, the experimental treatment is given in each legend, but no causal relationship between the stage of extrusion depicted and the nature or duration of experimental treatment is implied. Fro. 3. Quanta of specialized cytoplasm (8 minutes after injection of adrenaline). The border of each quantum is delineated by an arrangement of tiny membranous vesicles. The quanta appear to be spherical, and are approximately 1 # in diameter. Four quanta (Q) are seen in the field; the smallest profile, near the lumen (L), is presumably sectioned tangentially, x 25,000. FIG. 4. Quantum of cytoplasm (10 minutes after injection of adrenaline.) A number of the small vesicles surrounding the quantum have apparently coalesced to form a slit-like membrane-enclosed space (arrow). x 35,000. FIG. 5. Quantum of cytoplasm (8 minutes after adrenaline). The slit-like space (arrow) appears to be expanding in the direction of the lumen (L) of an intercellular secretory canaliculus. The small mass of apparently free cytoplasm in the lumen may represent a quantum of cytoplasm that was extruded at a different level of the canal. × 18,000. FIG. 6. Quantum of cytoplasm (24 hours' starvation). Here the slit-like space has apparently become continuous with the lumen (L), and a part of the quantum has thus become directly exposed to the lumen, x 22,000.
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contained small isolated masses of dense material. W h e n such granules were seen in the process of being extruded, their contents were usually h o m o g e n e o u s and of a low density (Figs. 1 and 2), but occasionally some of the dense cortical material was discernible. In some instances, granules that were situated near the lumen, but were apparently not in contact with it, showed a similar homogeneous, low-density content. In an unstimulated gland, however, an acinus was seen in which b o t h the lumen and the secretion granules that were in process of extruding their contents were filled with uniformly dense material. This last-named instance was interpreted as a secretory p h e n o m e n o n in which the acinus was very slowly discharging organic material but was not secreting water.
Extrusion of quanta of cytoplasm Some relatively infrequently encountered cytoplasmic formations that appeared to represent secretion antecedents are illustrated in Fig. 3. Such formations consisted of a presumably spherical mass of cytoplasm that was demarcated f r o m the surr o u n d i n g cytoplasm by a sheet-like arrangement of small m e m b r a n o u s vesicles (appearing linear in section) so disposed as to enclose a sphere (and thus appearing in section as a circular arrangement). By collecting a large n u m b e r of micrographs showing these formations, it was possible to arrange a series showing what appears to be a sequence of stages in an extrusion process (Figs. 4-10). The extrusion process apparently occurs in the following manner. A n u m b e r of the vesicles coalesce to f o r m a membrane-enclosed slit-like space that partially separates the secretion antecedent f r o m the ambient cytoplasm (Figs. 4 and 5). The outer layer of the m e m b r a n e surrounding the slit-like space fuses with the plasma m e m b r a n e at the lumen of the acinus; and a b r e a k t h r o u g h of the fused m e m b r a n e occurs (it will be noted that this process of fusion-and-breakthrough is identical to that by which a secretion granule releases its contents). Since the m e m b r a n e lining the slit-like space thus becomes a part of the plasma m e m b r a n e of the cell, a portion of the surface of the special q u a n t u m of cytoplasm becomes exposed to the lumen (Figs. 6 and 7). The mereFIG. 7. Quantum of cytoplasm (8 minutes after adrenaline). The relationship between quantum and lumen (L) is the same as that in Fig. 6, but here a larger part of the quantum has become exposed to the lumen. × 30,000. FIG. 8. Quantum of cytoplasm (one hour after pilocarpine). Approximately half of the quantum is projecting into the lumen; the other half remains imbedded within, and attached to, the cytoplasm. Some virus-like bodies (V), and an apparently free mass of cytoplasm immediately beneath them, are seen in the lumen. The mass of cytoplasm may be a previously-extruded "quantum". x 26,000. F:G. 9. Quantum of cytoplasm (3.5 hours after feeding, following a 92-hour fast). The quantum (Q) is almost completely contained within the lumen, but remains broadly attached at its base. An apparently free mass of cytoplasm of similar texture is seen in the upper part of the picture, x 22,000. FIG. 10. Quantum of cytoplasm (3.5 hours after feeding, following a 92-hour fast). The quantum appears to be almost free in the lumen; its attachment to the cell has become constricted. × 22,0130.
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branous vesicles associated with the deeper parts of the secretion antecedent continue to coalesce with one another and to fuse with the plasma membrane lining the lumen, with the effect that the secretion antecedent is gradually separated from the cell, moved into the lumen (Figs. 8, 9 and 10), and ultimately freed from the cell (Figs. 5, 8 and 9 show what appear to be free masses of cytoplasm in the acinous lumen). There is reason to suspect that the process of extrusion is a slow one: virus-like bodies that have been described as forming on the luminal microvilli of acinous cells (13) were also seen forming on the free edge of a partially extruded secretion antecedent of this kind. The cytoplasm constituting these secretion antecedents was of a very light density and was free of membranous contents, but occasionally contained a number of granules resembling RNP particles. It was remarked above that these quanta of cytoplasm were demarcated from the surrounding cytoplasm by a group of vesicles; an additional demarcating structure was sometimes visible in the form of a layer of denser cytoplasm immediately outside the layer of vesicles (Figs. 3, 5, 6 and 7). It is possibly significant that secretion antecedents of this kind were usually seen in close proximity to a glandular lumen. The complete 3-dimensional relationship of these quanta to the ambient cytoplasm was not fully established by serial-section study, and, although in the majority of cases they appeared to be completely surrounded by cytoplasm, it is possible that they were actually in contact with the acinous lumen at a level not shown by the section. It is therefore acknowledged that these specialized quanta of cytoplasm may originate in relation to the luminal surface of a cell, being merely "countersunk" instead of submerged in the depths of the cytoplasm, in which case profiles like those in Fig. 3 would appear only in sections passing approximately parallel to the glandular lumen. Cytoplasmic formations of the kind described above were seen in the parotid glands of animals that were starved 24 hours, starved-and-fed, given pilocarpine or adrenaline, or not treated in any way; they therefore appear not to be peculiar to any experimental treatment unless it be urethane or nembutal anesthesia. Similar formations have also been seen very rarely by the writer in mouse pancreas: one of the pancreases was obtained from a 5-day-old animal that was killed by decapitation. without anesthesia. An idea of the frequency of occurrence of these structures is conveyed by the following account. A section of parotid gland from a mouse that had received an injection of adrenaline seemed to have more than the usual number of such inclusions: the whole section was scanned, and 35 inclusions were counted. In a section from a similarly treated animal, five such structures were found in one acinus, but a total of only 14 were found while scanning the remainder of the section during one hour at a magnification of 10,000.
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Vacuoles The term "vacuole" is used here to signify a membranous vesicle whose content consists mainly of water and is therefore of a lighter density than ground cytoplasm (in distinction to a "granule", whose content is denser than ground cytoplasm). The formation of vacuoles has not been studied thoroughly, and will therefore not be dealt with in detail. Vacuoles were not seen in the parotid glands of animals that had received no experimenta[ treatment or that had been starved 24 hours. They were present, however, in animals that were given pilocarpine (Figs. 13 and 14) or were fed after a 24hour fast (Figs. 11 and 12). The vacuoles in the glands of starved-and-fed animals rarely exceeded 2.5 # in diameter, while those of pilocarpine-treated glands sometimes reached a diameter of 5 #. As to the mode of vacuole formation, the basic phenomenon could be described as a swelling of some pre-existent membranous vesicle, presumably by imbibition of water. Smaller vacuolar formations sometimes exhibited structural peculiarities indicating the nature of the structure from which they had arisen. (1) Certain of the smaller vacuoles in acinous cells of the mouse had a homogeneous content that was intermediate in density between the dense part and the "light" part of an intact secretion granule; these were interpreted as forming from secretion granules by imbibition of water and dissolution of dense content. (2)Other smallvacuolar formations were of an irregular shape suggesting that they had formed by the swelling of a previously flat membranous vesicle such as a Golgi cisterna or similar structure (Fig. 11, lower left). (3) Some vacuoles had a protuberance of low-density cytoplasm bulging into them (Fig. 11, top center; also of Fig. 14) suggesting that they might have formed by a disproportionate enlargement or swelling of the slit-like space sometimes seen in relation to the quantum-of-cytoplasm type of secretion antecedent (Figs. 4 and 5). Although the majority of profiles of large vacuoles gave no indication as to their provenance, it seems probable that the smaller vacuoles were younger or immature forms of the large ones. A common, but not constant, feature of vacuoles was the presence of a mantle of relatively low-density cytoplasm surrounding them (Figs. 12 and 13). In a few cases of pilocarpine-induced vacuole formation, the cytoplasm related to a part of the surface of a vacuole had apparently swollen and become lighter in density (presumably by increased water content): cisternae of the endoplasmic reticulum located within this special region of cytoplasm had also become swollen and the membranes forming the swollen parts of the cisternae had become degranulated (Fig. 14). Beyond acknowledging the obvious point that such a change in cytoplasmic structure is most probably abnormal and deleterious, little can be said about its significance.
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STRIATED DUCTS Striated d u c t cells are c h a r a c t e r i z e d m o r p h o l o g i c a l l y b y the presence of n u m e r o u s r a d i a l l y a r r a n g e d m i t o c h o n d r i a a n d infoldings of the p l a s m a m e m b r a n e in the b a s a l regions of the cells (13, 17, 19). Their apical c y t o p l a s m c o n t a i n s granules a n d vesicles whose a p p e a r a n c e a n d l o c a t i o n suggest t h a t t h e y are secretion antecedents, a n d the l u m i n a l surface is usually t h r o w n into microvillous extensions (Fig. 15). A s t r u c t u r a l m o d i f i c a t i o n of striated duct cells, which was i n t e r p r e t e d as p r o b a b l y being a m a n i f e s t a t i o n of secretory activity because it was m o s t p r o m i n e n t l y d e v e l o p e d in glands t h a t h a d been s t i m u l a t e d b y p i l o c a r p i n e or b y feeding after a 24-hour fast, o c c u r r e d at the apical ends of the cells. This m o d i f i c a t i o n consisted in a swelling of the c y t o p l a s m j u s t apical to the " s e c r e t i o n " granules a n d the a c c o m p a n y i n g loss of microvilli, which were p r e s u m a b l y flattened out b y the swelling process. The cytop l a s m i c " b l e b " thus f o r m e d often c o n t a i n e d some tiny particles r e s e m b l i n g R N P granules (Fig. 16) and, occasionally, a small n u m b e r of " s e c r e t i o n " granules o r vesicles (Fig. 17): it was of a b o u t the same density as the g r o u n d c y t o p l a s m of the cells, b u t a p p e a r e d lighter because it was relatively free of p r o m i n e n t c y t o p l a s m i c inclusions. I t was a p p a r e n t l y the fate of these blebs to be s e p a r a t e d f r o m their cells of origin a n d to disintegrate in the lumen, j u d g i n g b y the o c c a s i o n a l l y seen presence of m e m b r a n o u s debris in the lumen. A l l duct cells were n o t u n i f o r m l y affected b y secretory stimulus. Fig. 17 shows a section t h r o u g h the l u m e n of a striated d u c t of a p i l o c a r p i n e - t r e a t e d mouse. M a n y
Fla. 11. Vacuoles (V) and secretion granules in mouse parotid acinus (30 minutes after feeding, following a 24-hour fast). The shape of the vacuole at the lower left suggests that it may have arisen by swelling of a previously flat membranous precursor. The vacuole at the top of the picture contains a projection of low-density cytoplasm, suggesting that it may have arisen by a disproportionate swelling of the slit-like space that is sometimes seen in relation to the quantum-of-cytoplasm secretion antecedents (cf. Figs. 4 and 5). One vacuole has become continuous with the lumen (L) of an intercellular secretory canaliculus. × 14,500. FIG. 12. Vacuoles (V) and secretion granules in mouse parotid acinus (5 minutes after feeding, following a 24-hour fast). In the lower left a secretion granule whose membrane has become continuous with that lining the lumen (L) of a secretory canaliculus is seen. x 14,500. FI6. 13. Vacuole (V) in parotid acinous cell of mouse (15 minutes after pilocarpine). This vacuole is surrounded by a mantle of cytoplasm that is devoid of all the membranous and granular components that are usually evenly distributed in the cytoplasm of acinous cells, x 21,000. FIG. 14. Vacuole (V) and secretion granule (G) in parotid acinous cell of mouse (15 minutes after pilocarpine). This vacuole has no mantle of modified cytoplasm like that in Fig. 13, but has a mass of low-density cytoplasm related to it at one side. Two of the membranous vesicular structures in the low-density cytoplasm (arrows) are seen to be the ends of endoplasmic-reticulum cisternae that have lost their RNP granules and become swollen. × 21,000. FIG. 15. Apical portion of striated duct cells of mouse parotid gland (no experimental treatment). The micrograpb illustrates the usual microvillous luminal surface of these cells. Many of the microvilli have been cut in oblique or transverse section, and thus appear free in the lumen. The large granular body (M) is a mitochondrion whose internal membranes lie parallel to the plane of the section, x 32,500.
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of the cells have large swollen ends which, in the aggregate, almost occlude the lumen, but other cells have apparently maintained their microvillous surfaces. In addition to being a prominent feature of the strongly stimulated gland, bleb formation was occasionally seen, usually in a milder f o r m (Fig. 16), in animals that had been starved for 24 hours or had received no experimental treatment. The adrenaline-treated animals contained only a few blebs; these were very small, affecting only a part, rather than the whole, of a cell's apical surface.
DISCUSSION The term "secretion", which has been variously defined in the literature (6, 12), is used in the context of this paper to refer both to the extrusion of materials synthesized by the glandular cells and to the active transport of materials f r o m blood to saliva. In this investigation no attempt has been made to study the effects of any particular secretion stimulus (pilocarpine, adrenaline, or feeding-after-starvation) per se. The goal in this preliminary study has been merely to record the various manifestations of secretory activity.
Granules and vacuoles The process of extrusion of secretion granule material was seen in this study to be fundamentally the same as that described for the pancreas by Palade (11). Indications were seen, however, of a possible difference between the parotid and pancreatic acinus in one feature of their secretory process. The pancreatic z y m o g e n granule has dense h o m o g e n e o u s contents. In micrographs showing "secretion figures" in pilocarpine-treated mouse pancreas (14), as well as in Palade's (11) illustration of a secretion figure in guinea pig pancreas, the material in the lumen of the acinus was as dense as that in an intact z y m o g e n granule. A similar equality of density, as manifested by equal intensity of staining, of luminal content and intact z y m o g e n granule FIG. 16. Apical end of a striated duct cell from mouse parotid gland (starved 24 hours). The cytoplasm immediately apical to the vesicular and granular "secretion antecedents" (arrows) has become swollen, and microvilli have disappeared from the luminal surface. Since fasting appeared to constitute an adequate stimulus to secretion in acinous cells, it seems not unlikely that this structural modification is a manifestation of secretory activity (also cf. Fig. 17). Two fat droplets (F) with their centers dissolved out appear in the section. × 28,000. FIG. 17. Section through lumen of striated duct (pilocarpine given in two half-doses; 60 minutes and 30 minutes before death). The lumen (L) is almost occluded with large bleb-like extensions of cytoplasm (B) from the duct cells. There is no marked tendency for the "secretion antecedents" to enter the blebs (note the cell associated with bleb B); however, a few of the granules and vesicles do so (arrows) and suffer the fate of the blebs, which appears to he detachment from cell of origin and disintegration in the duct. Some of the duct cells are seen to have intact, microvillous apical ends. × 8000.
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has been noted in light-microscopic preparations of pancreas from the pilocarpinetreated mouse (14). These considerations indicate that not a great deal of water passes through the pancreatic acinous cell during the process of extrusion. On the other hand, the great majority of secretion figures in sections of parotid gland have shown a low density of material in the lumen and in secretion granules discharging their content--considerably less than the denser content of an intact secretion granule. This consideration argues that an appreciable amount of water passes through the parotid acinous cell while secretion granules are being discharged. An obvious question relating to the mode of granule extrusion described above is, given the fact that the area of surface membrane of the cell is increased every time a granule is discharged, how does the cell maintain a reasonably constant surface area? Palade (11) offers the reasonable hypothesis that membrane is taken into the cell to be re-used, and mentions small membranous vesicles near the surface membrane as possibly representing invaginated surface membrane. While no better answer has presented itself in this study, neither were obvious indications seen of membrane invagination from the apical ends of acinous cells. Although indications of invagination have been seen at the basal and lateral surfaces of acinous cells (13), it cannot be decided at present whether apical surface membrane can migrate to the sides of acinous cells. If the "desmosomes" that are seen in sections passing through the apical parts of acinous cells actually represent continuous "terminal bars" that pass completely around the perimeter of a cell near its apex, then a migration of membrane from apical surface to lateral surface would seem improbable.
Vacuole formation has been described at the light-microscopic level by Covell (4), Hirsch (6), and Ries (16) in the pilocarpine-stimulated pancreatic cell, and by Duthie (who incorrectly believed himself to be dealing with the sublingual gland) in the pilocarpine-treated parotid gland of mouse and rat (5). As for the mode of vacuole formation, (1) Covell (4) has vacuoles forming by liquefactian of zymogen granules; (2) Ries (16) and Hirsch (cited by Ries, but without reference to a specific publication) agree in stating that vacuoles are produced by coalescence of zymogen granules; and (3) Hirsch (6) further distinguishes between two kinds of vacuole--those formed by the solution of zymogen granules, and "water vacuoles" formed by imbibition of water by the cell. At the electron-microscopic level, some evidence may be said to exist in support of each of these claims: viz., this study has shown that some of the smaller vacuoles were indeed swollen secretion granules and that some vacuoles appear to arise from membranous formations other than secretion granule membranes; also, Palade (11) has illustrated the coalescence of pancreatic zymogen granules during the act of extrusion. Electron microscopic evidence disagrees, however, with
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the claims of the earlier workers who saw granules and vacuoles being discharged bodily, i.e., in an intact condition, from the acinous cell: both forms of secretory entity appear to be discharged by means of a fusion-and-breakthrough of membranes (cf. Figs. 1 and 2 for granule, Fig. 11 for vacuole). A possible explanation for this discrepancy is provided by Ries's (16) observation that in "stiirmische Extrusion" caused by pilocarpine whole shreds of cytoplasm are lost from the apex of the cell; he refers to the secretion illustrated by Covell (4) as "allm~ihliche Sekretion", but the secretory phenomena observed by Covell were probably more "sttirmisch" than Ries imagined. A definitive study of vacuole formation has not been made at the time of this writing, but is envisioned as a future undertaking. Perhaps the most important point regarding the relation of vacuole formation to cell function that has been brought out in this study is that vacuole formation is apparently a normal phenomenon, if one may so judge from the fact that vacuoles do appear in the glands of animals fed after a 24-hour fast (Figs. 11 and 12) as well as in pilocarpine-treated animals. Thus this observation agrees with Ries's (16) statement that pilocarpine evokes normal cell activities in an extreme form (at least at the morphologicallevel). Babkin (1) points out, however, that pilocarpine-stimulated saliva has different concentrations of constituents from chorda saliva in dog and cat submaxillary glands, and warns that pilocarpine cannot be regarded as a true substitute for parasympathetic stimulation. In pilocarpine-stimulated parotid glands of the mouse, Rutberg (17) found large vacuoles that were not enclosed by a membrane, but appeared as mere spaces in the cytoplasm. This difference, and other differences between his observations and the author's on pilocarpine-treated glands, may be due to the fact that Rutberg used a pilocarpine dosage %10 times stronger than that used in this study: it seems likely that extreme effects of pilocarpine stimulation can be deleterious (cf. Fig. 14).
Quanta of cytoplasm The process by which an acinous cell separates from itself a quantum of cytoplasm and discharges it into the lumen is, to the best of the writer's knowledge, described for the first time in this paper. Whether such fragments of cytoplasm constitute a useful contribution to the saliva or represent a casting-off process by which the cell rids itself of unwanted material cannot be decided at present. No direct evidence concerning the source of the tiny membranous vesicles surrounding the secretion antecedents was seen in the parotid acinous cells; however, in an inclusion of this type in a pancreatic acinous cell, one of the vesicles, which had a smooth surface itself, appeared to be in the act of pinching off from a granular cisterna of endoplasmic reticulum.
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Striated ducts A morphological modification of striated duct cells of mouse and rat parotid glands that was interpreted as of probable secretory significance was the enlargement of the apical ends of the cells to form relatively large cytoplasmic blebs, a process that was accompanied by the loss of the microvilli ordinarily present on the luminal surfaces of these cells. Blebs were conspicuous in mice and rats that were given pilocarpine and in mice that were fed after a 24-hour fast, but were also occasionally seen in mice that had been starved 24 hours or that had not received experimental treatment. It is difficult at present to assess the morphological validity of striated-duct bleb formation as a secretory phenomenon. The observation of blebs is not peculiar to this investigation; they have been seen in the rat parotid at the electron-microscopic level (19) and in the human submaxillary gland at the light-microscopic level (22). Rutberg (17), however, failed to find apical blebs in striated duct ceils of mouse parotid following pilocarpine stimulation; and in the present study it was noted that not all of the striated duct cells formed blebs. In a problem of this kind the electron microscopist is hampered by the smallness of the sample of tissue he can examine in a reasonable amount of time. The over-all occurrence of the large striated-duct blebs in relation to the various secretory stimuli can probably be more effectively investigated by light-microscopic methods, in which larger samples of tissue can be examined, and by which a wider variety of fixatives can be used to investigate the possibility that blebs are a fixation artifact. It would be equally difficult to evaluate the physiological significance of bleb formation in striated ducts. There is as yet no complete agreement on the exact functional nature of the striated duct: e.g., Rutberg (17) argues the hypothesis that striated ducts act mainly by absorbing sodium from a primary saliva produced by acini and intercalated ducts; Burgen (2), who finds abundant secretion in glands of young dogs in which there seems not to be enough acinous tissue to account for the flow, thus suspects a secretory function for striated ducts; and Langley and Brown (9) find evidence for both secretion and reabsorption of sodium in different parts of the duct system in the canine parotid. This divergence of ideas concerning function makes it impossible to relate bleb formation to any particular kind of cellular activity. Furthermore, it is not possible to know whether bleb formation, which apparently does not affect all cells (or at least does not affect all ceils simultaneously), is a manifestation of an active physiological process or represents merely a passive response to the medium in contact with the apical surface of the ceil. Data is now available (18) showing that pilocarpine-induced parotid secretion in the rat varies from hypotonic at low rates of secretion to hypertonic at high rates; however, it is not known whether
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the blebs described above were formed while the duct contents were hypertonic or while they were hypotonic. Blebs apparently become detached from their parent cells and become a part of the saliva. In doing this they sometimes carry a few "secretion" granules or vesicles with them. No evidence of extrusion of secretion granules by striated duct cells in the manner described for acinous cells has been seen. Rutberg (17) suggests that the apical granules may be manifestations of an absorptive, rather than a secretory, process. REFERENCES 1. BABKIN,B. P., Secretory Mechanism of the Digestive Glands, 2nd ed. Paul B. Hoeber, Inc., New York, 1950. 2. BURGEN, A. S. V. (personal communication). 3. CAULHELD,J., J. Biophys. Biochem. Cytol. 3, 827 (1957). 4. COVELL,W. P., Anat. Record 40, 213 (1928). 5. DUTmE, E. S., Proc. Roy. Soc. London B 114, 20 (1933). 6. HIRSCH, G. C., Biol. Revs. Cambridge Phil. Soc. 6, 88 (1931). 7. - - - - Z. ZellJbrsch. u. rnikroskop. Anat. 15, 36 (1932). 8. K/3I~NE, W. and LEA, A., Verhandl. Naturhistorischmedizinischen Ver. Heidelberg N.F. 1, 445 (1876). 9. LANGLEY,L. L. and BROWN, R. S., Am. J. Physiol. 199, 59 (1960). 10. MATHEWS, A., J. Morphol., suppl. 15, 171 (1899). 11. PALADE,G. E., Subcellular Particles, p. 64. American Physiological Society, Washington, D.C., 1959. 12. PALAY, S. L., Frontiers in Cytology, p. 305, Yale University Press, New Haven, 1958. 13. PARKS, H. F., Am. J. Anat. 108, 303 (1961). 14. - (unpublished observations). 15. PARKS, H. F. and JOHANSEN,E., J. Dent. Research 39, 714 (1960). 16. RI~S, E., Z. Zellforsch. u. mikroskop. Anat. 22, 523 (1935). 17. RUT~ERG, U., Acta Odontol., Scand. 19, suppl. 30 (1961). 18. SCHbrEVER,C. A. and SCHNEYER, L. H., Am. J. Physiol. 199, 55 (1960). 19. SCOTT, B. L. and PEASE, D. C., Am. J. Anat. 104, 115 (1959). 20. WATSON, M. L., J. Biophys. Biochem. Cytol. 3, 1017 (1957). 21. - ibid. 4, 727 (1958). 22. ZIMMERMANN,K. W., Arch. mikroskop. Anat. u. Entwicklungsmech. 52, 552 (1898).