Changes in the secretory acinar cells of the rat parotid gland during aging

The secretory acinar cells of parotid glands from rats of varying ages have been examined by electron microscopy to determine what age-related changes occur in these cells. The most prominent change noted in these cells is the progressive increase in the amount of lipofuscin granules with age. Lipofuscin granules are membrane-bound structures consisting of lipids, other subcomponents, and a matrix. In addition, these cells contain lipid droplets that are not associated with any other components and tend to accumulate at the base of the cells in older rats. Also, many acinar cells in the glands of old rats contain altered secretory granules which appear to be in the process of degeneration. The accumulation of lipid and degenerating secretory granules appears to be related to the reduced level of cellular secretory activity in the glands of older rats. It is possible that these two types of inclusions contribute to the formation of lipofuscin granules. Lipofuscin and degenerating secretory granules are associated with acid phosphatase, which is demonstrated cytochemically, indicating that these granules are lysosomal structures.     

The secretory granules of the acinar cells of the mouse submaxillary gland

Absence of membranes from the secretion granules of the acinar cells of the submaxillary gland of the mouse had led to speculation concerning mechanisms of secretion of these cells. By means of rapid perfusion fixation, smooth membranes have been identified around the secretion granules, and the mode of secretion proves to be similar to that of the other exocrine glands. The evidence suggests that potent membranolytic agents of unknown nature, capable of rapidly destroying the membranes are present in these secretory granules.     

Differential distribution of salivary agglutinin and amylase in the Golgi apparatus and secretory granules of human salivary gland acinar cells

The secretory granules of salivary glands often display complex internal substructures, yet little is known of the molecular organization of their contents or the mechanisms involved in packaging of the secretory proteins. We used post-embedding immunogold labeling with antibodies to two secretory proteins, agglutinin and α-amylase, to determine their distribution in the Golgi apparatus and secretory granules of the human submandibular gland acinar cells. With monoclonal antibodies specific for carbohydrate epitopes of the agglutinin, reactivity was found in the trans Golgi saccules, trans Golgi network, and immature and mature secretory granules. In the granules, labeling was seen in regions of low and medium electron density, but not in the dense cores. Reactivity seen on the apical and basolateral membranes of acinar and duct cells was attributed to a shared epitope on a membrane glycoprotein. Labeling with a polyclonal antibody to amylase was found in the Golgi saccules, immature and mature secretory granules, but not in the trans Golgi network. In the granules, amylase was present in the dense cores and in areas of medium density, but not in the regions of low density. These results indicate that these two proteins are distributed differently within the secretory granules, and suggest that they follow separate pathways between the Golgi apparatus and forming secretory granules. Small vesicles and tubular structures that labeled only with the antibodies to the agglutinin were observed on both faces of the Golgi apparatus and in the vicinity of the cell membrane. These structures may represent constitutive secretion vesicles involved in transport of the putative membrane glycoprotein to the cell membrane.     

Differential distribution of salivary agglutinin and amylase in the Golgi apparatus and secretory granules of human salivary gland acinar cells.

The secretory granules of salivary glands often display complex internal substructures, yet little is known of the molecular organization of their contents or the mechanisms involved in packaging of the secretory proteins. We used post-embedding immunogold labeling with antibodies to two secretory proteins, agglutinin and alpha-amylase, to determine their distribution in the Golgi apparatus and secretory granules of the human submandibular gland acinar cells. With monoclonal antibodies specific for carbohydrate epitopes of the agglutinin, reactivity was found in the trans Golgi saccules, trans Golgi network, and immature and mature secretory granules. In the granules, labeling was seen in regions of low and medium electron density, but not in the dense cores. Reactivity seen on the apical and basolateral membranes of acinar and duct cells was attributed to a shared epitope on a membrane glycoprotein. Labeling with a polyclonal antibody to amylase was found in the Golgi saccules, immature and mature secretory granules, but not in the trans Golgi network. In the granules, amylase was present in the dense cores and in areas of medium density, but not in the regions of low density. These results indicate that these two proteins are distributed differently within the secretory granules, and suggest that they follow separate pathways between the Golgi apparatus and forming secretory granules. Small vesicles and tubular structures that labeled only with the antibodies to the agglutinin were observed on both faces of the Golgi apparatus and in the vicinity of the cell membrane. These structures may represent constitutive secretion vesicles involved in transport of the putative membrane glycoprotein to the cell membrane.     

Formation of secretory granules in the Golgi apparatus of prolactin cells in the rat pituitary gland: A stereoscopic study

The mode of secretory granule formation in prolactin cells was analyzed in thin or thick sections of pituitary glands from non-lactating or lactating female as well as from male rats. In all these animals, the Golgi apparatus of prolacting cells consists of a continuous twisted ribbon-like structure that branches and anastomoses to form a hollow sphere located in the juxtanuclear area. The early signs of secretory granule formation are observed along the trans-aspect of the Golgi ribbon where progranules appear as focal distensions simultaneously occurring anywhere in the last trans thiamine pyrophosphatase (TPPase)-containing Golgi element. In the transmost Golgi saccule, such dilatations usually contain several nodular masses of electron opaque material which are separated from each other and from the saccular membrane by a less intensely stained material. While this transmost saccule becomes more fenestrated, its focal polynodular distensions seemingly yield polynodular tubular progranules which are initially closely apposed and usually parallel to the trans face of the Golgi ribbon. Subsequently, these progranules, which frequently show small membranous tubules or tubular networks attached to them, are seen some distance from the Golgi stacks and progresively transform into the more compact polymorphous granules characteristic of prolactin cells. These observations suggest that the polynodular tubular progranules arise by fragmentation of portions of the trans-Golgi elements rather than by fusion of small uninodular granules budding from the edges of a trans-Golgi saccule. Once the progranules have been liberated, the rest of the transmost Golgi element appears to break down into small residual networks, tubules, and vesicles. Thus, in prolactin cells as in other glandular cells, the whole transmost Golgi element would fragment during formation of prosecretory granules.     

Formation of secretory granules in the Golgi apparatus of prolactin cells in the rat pituitary gland: a stereoscopic study.

The mode of secretory granule formation in prolactin cells was analyzed in thin or thick sections of pituitary glands from non-lactating or lactating female as well as from male rats. In all these animals, the Golgi apparatus of prolacting cells consists of a continuous twisted ribbon-like structure that branches and anastomoses to form a hollow sphere located in the juxtanuclear area. The early signs of secretory granule formation are observed along the trans-aspect of the Golgi ribbon where progranules appear as focal distensions simultaneously occurring anywhere in the last trans thiamine pyrophosphatase (TPPase)-containing Golgi element. In the transmost Golgi saccule, such dilatations usually contain several nodular masses of electron opaque material which are separated from each other and from the saccular membrane by a less intensely stained material. While this transmost saccule becomes more fenestrated, its focal polynodular distensions seemingly yield polynodular tubular progranules which are initially closely apposed and usually parallel to the trans face of the Golgi ribbon. Subsequently, these progranules, which frequently show small membranous tubules or tubular networks attached to them, are seen some distance from the Golgi stacks and progressively transform into the more compact polymorphous granules characteristic of prolactin cells. These observations suggest that the polynodular tubular progranules arise by fragmentation of portions of the trans-Golgi elements rather than by fusion of small uninodular granules budding from the edges of a trans-Golgi saccule. Once the progranules have been liberated, the rest of the transmost Golgi element appears to break down into small residual networks, tubules, and vesicles. Thus, in prolactin cells as in other glandular cells, the whole transmost Golgi element would fragment during formation of prosecretory granules.     

Lectin histochemistry of palatine glands in the developing rat

This study examined the binding pattern of lectins, soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin-I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), and succinylated WGA (sucWGA) in the developing rat palatine glands. In adult rats, heterogeneous lectin binding patterns were revealed between the anterior and posterior portions of palatine glands, as DBA, VVA, and WGA were bound more intensely and broadly in the posterior portion. SBA, PNA, and sucWGA showed far less reactivity in the anterior than in the posterior portion. At embryonic day 18 (E18), weak labeling was observed with UEA-I and WGA at the basal membrane of terminal buds, UEA-I and PNA labeled the epithelial cord, and there was no apparent binding for SBA, DBA, VVA, and sucWGA. At E20, after acinar lumenization, all lectins were detected at the acinar cell basal membranes. After birth, all lectins detectably labeled at the mucous cell apical membranes and progressively, with maturation, extended from the apical to basal portions of the cytoplasm. Apparent serous cells were observed around postnatal day 10 (PN10) and bound UEA-I. Lectins reached peak reactivity at PN21 and the binding patterns became identical to those of adults around PN28.     

Formation of secretion granules in the Golgi apparatus of pancreatic acinar cells of the rat

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in sections of pancreatic acinar cells by using stereopairs of electron microscope photographs. Pancreatic tissue fixed in glutaraldehyde was postfixed in reduced osmium, and the sections were stained with lead citrate. Tissues were also treated to demonstrate phosphatase activity (i.e., nicotinamide adenine dinucleotide phosphatase, NADPase; thiarnine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase). The following stacked components were observed along the branching, anastomotic, continuous, ribbon like Golgi apparatus. (1) On the cis-face of the Golgi stack there was a tubular membranous network known to be osmiophilic and referred to as the cis-osmiophilic tubular network or cis-element. (2) A first, poorly fenes-trated saccule, unreactive for the phosphatases tested, was slightly distended in places and contained a fluffy granulofilamentous material. (3) The subjacent three or four saccules, reactive for NADPase and/or TPPase, showed dilated portions containing a granulofilamentous secretory material similar to that filling the rest of the saccule. They also showed nondilated portions perforated with large fenestrations, some of which were in register and formed wells containing 80-nm vesicles. The dilated portions of these saccules were present at random along the length of the saccules and were not located exclusively at their edges. (4) The remaining one or two elements of the stack, CMPase positive, showed dilated spheroidal portions or prosecretory granules containing a homogeneous secretory material and flattened fenestrated regions free of secretory material and having the appearance of networks of narrow membranous tubules. (5) Lastly on the trans-aspect of the stack there were detached prosecretory granules reactive for CMPase and surrounded by a corona of small vesicles, and smooth-surfaced spherical CMPase-negative granules having a denser content that were identified as fully formed secretion granules; there were also occasional free trans-tubular networks strongly reactive for CMPase that appeared to undergo fragmentation and numerous small vesicles free from acid-phosphatase activity. These various images were interpreted as indicating that prosecretory granules formed in relation to two or three fenestrated saccules on the trans-side of the stack. Such granules, following their detachment from the trans-face of the stack, their separation from trans-tubular networks, and condensation of their content, yielded mature secretion granules.     

Light and electron microscopic histochemistry of the peroxidase activity in the secretory granules of the developing hamster submandibular gland

On the basis of light and electron microscopic observations of the post natal development of the hamster submandibular gland, granules in the acinar cells showed considerably variations in size and shape, as well as electron density of the peroxidase-positive reaction. The present study shows that secretory granules of the hamster submandibular gland undergo changes of area and of intensity for peroxidase activity 6 months after birth.     

Changes in the distribution and fine structure of the intralobular blood vessels of the submandibular gland in the postnatally developing mouse

Previous studies have shown that the blood vessels supplying the endocrine organs and the mucosa of the intestinal canals change in terms of not only their distribution but also their structure with the development and growth of each organ. We examined changes in the distribution and structure of intralobular blood vessels, including capillaries, throughout the postnatal development of the submandibular gland, an exocrine organ. The mouse submandibular gland from days 0 (birth) to 49 was investigated chronologically and ultrastructurally. The capillaries changed from continuous to fenestrated on day 10, coincident with an increase in the number of acini to more than the number of terminal tubules. The number of sections of intralobular blood vessels per unit area gradually decreased with increasing acinar size and was lowest on day 21 when pups were weaned; the same number was maintained from then on. In contrast with the reduction in the number of intralobular blood vessels, the number of capillary pores appeared to increase gradually. Acinar size increased further till day 28. Capillary pore number also increased further, till day 35, apparently in relation to the increasing acinar size. These findings suggest that the changes in distribution and structure of the intralobular blood vessels in the submandibular gland of the postnatally developing mouse are closely related to the development of the parenchymal cells in preparation for weaning and sexual maturity.     

Differentiation of myoepithelial cells in the developing rat sublingual gland

Sublingual glands of rats were prepared for light and electron microscopy and for the histochemical demonstration of myofibrils and alkaline phosphatase (AkPase) activity. Through 17 days in utero, the epithelial cells of the glandular rudiment are relatively undifferentiated. At 18 days, the inner cells of the terminal buds begin to assemble around a lumen and accumulate secretory granules, while the outer cells flatten and form long processes. At 19 days, many of the outer cells have dilated cisternae of rough endoplasmic reticulum engorged with finely granular material. At 20 days, some of the outer cells have thin bands of microfilaments in their processes, suggesting that they are differentiating into myoepithelial cells (MEC). Though the secretory cells are almost mature at birth, only a few of the MEC have myofibrils detected with an actomyosin reaction, and AkPase activity is very weak. Progressive increases in AkPase activity and in myofibril size and number continue until the acini and intercalated ducts are fully invested with mature MEC at about 14 days after birth. Thus, the MEC and secretory cells begin to differentiate at the same time, but the MEC subsequently differentiate asynchronously with the secretory cells and with each other. Although the sublingual MEC are only partly differentiated in the newborn rat, their overall development occurs somewhat more rapidly than in the adjacent submandibular gland.     

Differentiation of myoepithelial cells in the developing rat parotid gland

Parotid glands of rats were prepared for light and electron microscopy and for the histochemical demonstration of myofibrils and alkaline phosphatase (AkPase) activity. Through 18 days in utero, the epithelial cells of the developing gland remain relatively undifferentiated. At 20 days in utero, a few cells in the outer layer of the terminal buds and adjacent segments of ducts acquire a cilium, the initial indication that they are differentiating into myoepithelial cells (MEC). Up until the time of birth, the only additional characteristics of MEC that the outer cells develop are to flatten against the underlying cells, begin to send out processes, and produce a few dilated cisternae of rough endoplasmic reticulum. Myofibrils and AkPase activity are first detected at the light microscopic level at five days after birth, around both the developing acini and intercalated ducts. Progressive increases in AkPase activity and in the size and number of myofibrils continue until the acini and intercalated ducts are invested with well-differentiated MEC at 15 days. Subsequently, as the acini undergo maturation during the weaning period (18-25 days), the MEC cease to surround the acini and assume the adult pattern of investing only the intercalated ducts. The pattern of MEC differentiation in the parotid gland differs from those in the sublingual and submandibular glands of the rat in several important respects. They begin to differentiate last, yet mature almost as early as do the MEC of the sublingual gland; they begin to differentiate prior to, rather than simultaneously with, the secretory cells; and their distribution changes as the acinar cells become mature.     

Glutamate dehydrogenase in aminoacidergic structures of the postnatally developing rat cerebellum

In the cerebellar cortex of the rat, histochemically demonstrable activity of glutamate dehydrogenase (GDH) was found to increase markedly after the second week of postnatal life. As evaluated histophotometrically, the amount of reaction product in Purkinje cell perikarya exceeded on day 40 that on day 5 by about 85%, whereas the intensity of GDH staining in granule cell bodies and in the molecular layer rose from postnatal day 5 to day 40 up to 325% and 400%, respectively. Due to the parallelism with the onset of aminoacidergic transmission processes, the results are interpreted as indicating the participation of GDH in the metabolism both of transmitter glutamate and of gamma-aminobutyric acid (GABA).     

Morphological and histochemical changes in the secretory granules of mucous cells in the early postnatal mouse parotid gland

It has previously been known that the developing parotid glands in humans and rats contain mucous cells in their terminal clusters and acini, but these cells disappear within a short period of time. Using rat parotid glands, IKEDA and AIYAMA (1997, 1999) suggested that the mucous cells might change into serous cells in the early postnatal period, but it is uncertain whether mucous cells appear only in the developing parotid gland of a few species such as humans and rats, or whether the cell transformation actually occurs. To clarify these points, the present study investigated the developing mouse parotid glands. Light microscopy showed cells with secretory granules that stained extensively with PAS and alcian blue in the terminal clusters of a 1-day-old mouse parotid gland. Mucous cell numbers in the terminal clusters and the acini reached a peak on day 5 and decreased on day 7. By day 10, the mucous cells had disappeared altogether. Thus, the presence of mucous cells in the developing mouse parotid gland was confirmed. Electron microscopy showed granules of low-electron-density and bipartite granules in the mucous cells. Bipartite granules and highly electron-dense granules sometimes co-existed in a single cell. Immuno-electron microscopy revealed a positive reaction for amylase to the low-electron-density granules and the low-electron-density portions of the bipartite granules, in addition to the highly electron-dense granules and the electrondense cores of the bipartite granules. No mucous cells with nuclei displaying characteristics of apoptosis were recognizable. Lectin histochemistry both at the light and electron microscopic levels showed that the secretory granules in the mouse parotid gland mucous cells had sugar residues similar to those of the mature serous granules. These findings demonstrate that mucous cells appear in the early postnatal mouse parotid gland, and that almost all of these cells may be converted into serous cells.     

Tissue-engineered tear secretory system: functional lacrimal gland acinar cells cultured on matrix protein-coated substrata

Dry eye is a general term that refers to a myriad of ophthalmic disorders resulting in the inadequate wetting of the corneal surface by the tear film. Dry eyes are typically treated by the application of artificial tears. However, patients with lacrimal insufficiencies such as Stevens-Johnson syndrome, chemical and thermal injuries, or ocular cicatricial pemphigoid have very limited options because of the short duration and action of lubricating agents. As a therapeutic strategy, we are working to develop a bioengineered tear secretory system for such patients. This article describes the growth and physiological properties of purified rabbit lacrimal gland acinar cells (pLGACs) on several matrix protein-coated polymers such as silicone, collagen I, copolymers of poly-D,L-lactide-co-glycolide (PLGA; 85:15 and 50:50), poly-L-lactic acid (PLLA), and Thermanox plastic cell culture coverslips. Monolayers of acinar cells were established on all of the polymeric substrata. An assay of beta-hexosaminidase activity in the supernatant medium showed significant increases in protein secretion, following stimulation with 100 microM carbachol on matrix protein-coated and uncoated polymers such as silicone, PLGA 85:15, and PLLA. Our study demonstrates that PLLA supported the morphological and physiological properties of purified rabbit lacrimal gland epithelial cells more successfully than the others.