Granular Convoluted Tubule Research Articles

Expression, localization and developmental regulation of insulin-like growth factor I mRNA in rat submandibular gland

The mRNA for insulin-like growth factor I (IGF-I) in the submandibular gland of mature and developing rats was examined by Northern blotting and in situ hybridization with an oligonucleotide probe. In the mature adult rat, IGF-I mRNA was expressed at a higher level in the submandibular gland than in the liver, and was localized primarily in the granular convoluted tubule (GCT) cells. A 4.7-kb mRNA on Northern blots, which was expressed only slightly in the liver, proved to be the predominant size species of IGF-I transcripts in the GCT cells, and its level increased progressively with the postnatal development of GCTs in the gland. In addition, a 1.8-kb mRNA for IGF-I was also expressed at a much lower level throughout the acinar and duct systems, irrespective of age. These results have shed a light on the status of IGF-I as one of the many biologically active polypeptides that are produced in the rodent submandibular gland.

Changes of Calmodulin, S-100 Protein .ALPHA./.BETA. and Keratin in Duct-Ligated Salivary Glands of Rats. An Immunohistochemical Study.

Immunohistochemical study of expression of calmodulin, S-100 α, β protein and cytokeratin K8.12 was carried out on normal and duct-ligated submandibular (SMG) and sublingual glands (SLG) of rats. The normal rat SMG showed calmodulin and S-100 α immunoreactivity in the pillar and transition cells of the granular convoluted tubule (GCT) and striated duct cells. S-100 β staining was positive in acinar cells of SMG as fine granular deposition. A trace immunoreaction product of keratin K8.12 was observed in GCT segments and striated ducts. Three days following duct ligation, immunostaining for calmodulin and S-100 β disappeared in both SMG and SLG, however, S-100 α staining persisted in pillar, transition and striated duct cells. The altered GCT cells of SMG and duct-like structures of SLG in the duct-ligated glands also showed S-100 α staining. In addition, K8.12 immunoreactivity was noted in the duct-like structures, particularly in those cells located luminally. The day 5 duct-ligated specimen of SMG retained positive S-100 α protein in the newly formed duct-like structures and the day 7, 10 and 14 specimens showed squamous metaplasia with positive K8.12 staining at the luminal side of the ducts. Myoepithelial cells in duct-ligated SLG stained positively for K8.12. It is concluded that loss of calmodulin from the ductal cells of SMG and SLG, and S-100 β from acini of SMG marks the earliest and predominant pathological alteration followed by increased proliferation of duct-like structures with cells containing cytokeratin K8.12 as pathological alteration of duct ligation of the salivary glands.

Secretory and synthetic changes in the secretory proteinases of mouse submandibular glands after methoxamine stimulation, assessed histochemically by means of D-Val-Leu-Arg-methoxy-naphthylamide.

The secretion of proteinases and their resynthesis in the granular convoluted tubule (GCT) cells of mouse submandibular glands were studied after in vivo methoxamine (Methox) treatment (15mg/kg, i. p.), using a histochemical reaction with D-Val-Leu-Arg-4-methoxy-2-naphthylamide. The amidase reaction product was confined to the secretory granules in GCT cells, in unstimulated and stimulated glands. Methox caused extensive secretion of the granules. An increased staining reaction by the proteinases seemed to occur in granules prior to secretion, and fusions had often taken place. Reaccumulation of proteinases in the newly forming granules was found to start between 6 to 12hr after injection and the process was still incomplete at 48hr, but the granules showed less staining than at the earlier times. The variations observed in staining intensity may relate to the extent of binding with other proteins, and an unbinding of proteinases appears to be a possibility prior to the secretion of granules.

Secretion of glandular kallikrein and renin from the basolateral pole of mouse submandibular duct cells: an immunocytochemical study.

Glandular kallikrein from salivary glands in rats has been measured in the circulation and has been shown to have local vasoactive effects. In mice, renin and epidermal growth factor from the submandibular gland (SM) also reach the circulation, as both have been measured in plasma. The route by which these peptides enter the blood from their site of synthesis in ducts of the SM is unclear. We have investigated by immunocytochemistry the secretory pathways for kallikrein and renin from salivary duct cells in mice. The renal/pancreatic kallikrein-secreting cells of the striated and excretory ducts of the SM were distinguished from the granular convoluted tubule (GCT) cells which secrete other glandular kallikreins on the basis of data obtained in previous studies, in which we used gene-specific oligonucleotide probes to identify the expressing cell types. Renal/pancreatic kallikrein was apparently secreted constitutively from the basolateral surface of striated duct cells and in secretory vesicles from excretory duct cells, whereas apical secretion occurred via the regulated pathway in both cell types. Glandular kallikreins and renin synthesized in GCT cells were secreted from the basolateral surface by dissolution of granules at the cell membrane. There were fenestrated capillaries underlying the duct tree which would enable the secreted products to reach the circulation.

Immunoreaction of keratin, actin, S‐100 protein and rat‐EGF in ductligated rat salivary glands

Duct-ligated submandibular and sublingual glands of rats were evaluated immunohistochemically for changes in keratin (MoAb 1164), actin, S-100 protein and rat-EGF (rEGF). Normal salivary glands were reactive for keratin, S-100 protein and rEGF in the granular convoluted tubule (GCT) and duct cells, and for actin in the myoepithelium. Submandibular glands showed a marked reduction of S-100 protein and rEGF staining following duct ligation, and no increased staining of proliferating epithelial cells of the late stage in duct ligated glands. Sublingual glands revealed no marked changes for actin staining in myoepithelial cells, irrespective of atrophic changes occurring in acinar and duct cells after duct ligation. Immunohistochemical patterns differed for each type of gland; changes associated with the obstructive lesion were more prominent in the submandibular gland.

Neuron-specific enolase reduction in irradiated salivary glands of the rat--an immunohistochemical study.

Neuron-specific enolase (NSE) in the salivary glands of normal and irradiated rats was studied by immunohistochemical methods. The normal salivary gland of Sprague-Dawley rats (465 g) showed positive staining for NSE in striated ducts and granular convoluted tubule (GCT) cells. A single radiation, TDF (time, dose, fractionation factor) 100 and 200 (18.82 Gy and 27.97 Gy, respectively) was done, and 4 groups (1, 2, 3 and 4 weeks after radiation) of 5 rats each were used. Irradiated salivary glands indicated a remarkable reduction of NSE staining in GCT cells and a reduction but to a lesser degree in the striated duct of the submandibular gland. Immunohistochemical deposition of NSE was not changed in the sublingual glands of irradiated rats. The reduction of NSE immunodeposition was irradiation dose dependent.

Immunohistochemical expression of chromogranin in postnatal developmental salivary glands of mice and rats.

The immunoreactivity of chromogranin was studied in postnatal salivary glands of the mice and rats. Chromogranin was observed in proacinar cells in both sexes during the postnatal developmental stage of the submandibular glands; the strongest reactivity was seen in 4th to 7th day specimens of mouse, and in 7th to 21st day specimens of rat. Acinar cells of mouse parotid glands showed positive staining in 1 to 42 days. No chromogranin staining was seen in granular convoluted tubule (GCT) cells of the submandibular glands in mice and rats. In sublingual glands of rat, reactivity of chromogranin was found in undifferentiated serous acinar cells, whereas in mice, it was absent in these cells. No sex difference of chromogranin staining was observed in submandibular glands in mice and rats. Immunoblotting of chromogranin using polyclonal anti-bovine SP-1/chromogranin antiserum showed in submandibular glands and adrenal glands of the rat consisted of chromogranin A, B and C. The results of this study suggest that the existence of chromogranin in proacinar cells of salivary glands is probably associated with the developmental phenomenon during the postnatal development.

Expression of transforming growth factor beta 1 in the submandibular gland of the rat.

We employed immunocytochemical and in situ hybridization techniques to study the expression of transforming growth factor beta 1 (TGF-beta 1) in rat submandibular gland. Immunoreactivity for TGF-beta 1 was observed in the cells of granular convoluted tubules (GCTs), striated ducts, and excretory ducts, whereas it was absent in the intercalated ducts and secretory acini in both male and female rats. Immunoelectron microscopy revealed the ultrastructural localization of TGF-beta 1 in the secretory granules of GCT cells. On the other hand, signals for rat TGF-beta 1 mRNA were abundant in the GCT and striated duct cells but were lacking in the excretory duct cells. These results provided evidence for the production of TGF-beta 1 in the GCTs and striated ducts of rat submandibular gland.

Ultrastructure of the submandibular gland in the African multimammate rodent, Praomys natalensis

Praomys natalensis, an African rodent that is phenotypically and cytogenetically intermediate to rats and mice, possesses a submandibular gland that is histologically similar to that in both of these near relatives, but is ultrastructurally unique. Acinar cells, which are seromucous in nature, contain secretory granules that often contain a perfect "bull's eye" inclusion (or some variant of this configuration) suspended in a dense matrix. The Golgi apparatus in these cells has an unusual structure, with the Golgi saccules often being doubled over, so that the outermost saccule also is the innermost. This peculiar architecture apparently arises fairly late in the secretory process, i.e., a Golgi apparatus of conventional structure gives rise to a nascent granule (condensing vacuole), then its saccules secondarily fold over. Intercalated ducts are preceded by a ring of specialized cells that have a number of serous-type granules, the duct cells themselves being devoid of such granules. Granular convoluted tubules (GCT) contain large dense granules that appear to be spontaneously involved in chain exocytosis. These GCT granules probably are the repositories of nerve growth factor, which is particularly abundant in Praomys. Striated ducts for the most part are typical in appearance, but they and, to a lesser extent, GCTs contain prominent, membrane-bound crystalloids with a periodicity of about 15 nm.

Effects of Castration and Sex Steroids on Sexually Dimorphic Development of the Mouse Submandibular Gland

The aims of this study were to characterize sexual dimorphism in the submandibular glands of young adult mice and to determine how sex differences arise during postnatal development. In the mouse submandibular glands, prominent sexual dimorphism was observed at 30 days of age, when the male gland was superior in both the relative occupied area (ROA) and the mitotic rate of the granular convoluted tubules (GCT) to those of the female. By neonatal castration, this sexual dimorphism was abolished, and the intraglandular structures of castrated males were similar to those of normal females. In castrated mice of both sexes, daily treatment with testosterone and 5 alpha-dihydrotestosterone for 10 days from 20 days induced only the ROA of the GCT to increase to the normal male levels but not those of the other three regions of the glands, the acini, intercalated ducts and excretory striated ducts. Testosterone responsiveness of the glands, considering both the glandular weight gain and the mitotic rate of the GCT, was significantly higher in castrated males than in castrated females. On the other hand, 17 beta-estradiol had no effect on the glands of castrated mice. Therefore, the present study suggests that the testicular hormones are responsible for the masculine development of GCT of the glands, but not the ovarian hormones, and that there is a sex difference in the responsiveness of the glands to testosterone, which is more effective in males than in females.

Localization of Chromogranins, Non-neuron-specific Enolase, and Different Forms of Somatostatins in the Submandibular Salivary Glands of Mice

The localizations of chromogranins A, B, and C, neuron-specific enolase (NSE, gamma gamma-type) and non-NSE (alpha alpha-type), and different forms of somatostatins were immunocytochemically identified. The localizations were compared with those of epidermal growth factor (EGF) and nerve growth factor (NGF) in the submandibular salivary glands (SMG) of male mice at five to six weeks of age, with use of a variety of antibodies and the peroxidase-antiperoxidase (PAP) and avidin-biotin complex (ABC) detection methods. In the SMG of male mice, the major chromogranin present was chromogranin A, whereas chromogranins B and C were not detected at these ages by either method. Chromogranin A-like immunoreactivity was located in the granular convoluted tubule (GCT) cells of the SMG, whereas non-NSE immunoreactivity was observed throughout the duct system and in some acinar-associated cells. NSE was not detected in any part of the SMG. The distribution of chromogranin A and somatostatins in the GCT cells was similar to that of EGF and NGF. Our results strongly suggest that chromogranin A and somatostatins, but not chromogranin B or C, may be useful as a means of differentiation of the cells in the duct system of the SMG responsible for the production of biologically-active factors.

Morphological study of granular convoluted tubules in the submandibular gland of the mouse during the growth of a sarcoma-180 subcutaneous tumor.

Histological and cytological changes in the submandibular glands of adult male mice arising during the growth of sarcoma-180 subcutaneous tumors were studied. The submandibular glands of the mice were examined by morphometric analysis at 1, 3, 6, 10, 20, 30 and 64 days after inoculation of the tumor cells. There was a slow increase in the relative cross-sectional area of the granular convoluted tubule (GCT) in the section of the submandibular gland of the animals as the tumors grew. The increased proportional area of the GCT was significantly different from that of the control's from day 30. However, the mean weight of the glands was not increased. The proportional area of the granular cluster in the cells of the GCT of tumor cells in inoculated animals decreased about 5% on the first day and then quickly increased by 16% on the third day in comparison with those of the controls, eventually reaching a maximum of 74% (control, 54%) by day 30. In addition, the average number of granules per GCT cell decreased in the first three days, then increased to normal levels from day 6, going above the normal level from day 20 of the tumor growth. These changes in the glands of tumor-bearing animals disappeared within 20 days after removal of the tumor. These results indicate that the growth of the sarcoma-180 subcutaneous tumor caused morphological changes in the GCT and GCT cells, suggesting an alternation in the requirements of the secretions contained in the granules, such as the epidermal growth factor, during the growth of the tumor.

Changes with senescence in the fine structure of the granular convoluted tubule of the submandibular gland of the mouse.

Cells of the granular convoluted tubules (GCTs) of the submandibular gland of senescent male mice show structural changes indicative of functional decline. In order to define the nature of these age-related changes more clearly, the fine structure of GCT cells of 12- and 28-month-old males was compared. In old mice, there was cell-to-cell variation in the extent of these changes, with some cells of senescent males appearing no different from those of young adults. In affected cells the most striking alterations were seen in secretion granules and lysosomal elements. Secretion granules varied greatly in size, with some GCT cells having only very fine apical granules. Secondary lysosomes and large lipofuscin granules were frequent in the basal cytoplasm. Very large dense bodies (3-5 micron) occurred in many cells. These possibly represent intracellular pools of released secretory materials, as they were occasionally seen in continuity with the luminal contents. Structures whose appearance was intermediate between the very large dense bodies and lipofuscin granules were common, suggesting crinophagic activity. There was an apparent decrease in numbers of polysomes and in the extent of the Golgi apparatus. These fine structural changes are consistent with impairments with advanced age in synthesis and posttranslational processing of secretory products by affected GCT cells. In addition to cell-to-cell variation in any one male, there was also interanimal variation in the degree and extent of these senescent changes.

Localization of epidermal growth factor and nerve growth factor mRNAs in mouse submandibular gland by a simplified in situ hybridization with riboprobes

We simplified procedures of in situ hybridization with riboprobes on serial sections of paraffin-embedded tissues. We found that when tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut serial sections with 5 microns thick, excellent results were obtained without proteolytic, heat, or HCl treatment. With our simplified method, both EGF and NGF mRNAs were detected intensively and specifically in granular convoluted tubule (GCT) cells of a male mouse submandibular gland, but not detected in either the acini or ducts other than the GCT. Accumulation of the silver grains was localized in the basal side of the GCT cells. No significant hybridization was observed in those cells with the sense probe and in the parotid and sublingual gland even with the anti-sense probe. Since our results were not only very consistent with other data, but also much more excellent than other results obtained by in situ hybridization on the similar tissues, our simplified method should be very useful to study gene expression on tissue sections.

Depletion of salivary gland epidermal growth factor by sialodacryoadenitis virus infection in the Wistar rat.

Male and female Wistar rats 2 to 15 months of age were inoculated intranasally with sialoda-cryoadenitis (SDA) virus and killed at 8 to 21 days post-inoculation (PI). Submandibular glands were evaluated by light and electron microscopy, and levels of salivary gland epidermal growth factor (EGF) were quantitated by cytochemistry and competitive radioreceptor assay. Apical granules in the epithelial cells of the granular convoluted tubules (GCT) were selectively depleted during the acute and convalescent stages of the disease. In addition, levels of immunoreactive EGF were reduced in affected submandibular glands, especially at 8 to 14 days PI with SDA virus, but some evidence of EGF depletion was seen at up to 3 weeks PI. A corresponding transient depletion of EGF receptor reactive salivary EGF was seen between 1 and 3 weeks after experimental SDA infection. These studies suggest that a clinical (or subclinical) infection with SDA virus could have significant effects on experimental studies on EGF-dependent functions, including reproductive physiology and carcinogenesis.

Electron microscopic immunostaining of nerve growth factor: Secretagogue stimulated submandibular glands

Immunocytochemical localization of nerve growth factor (NGF) was assessed on thin sections of plastic-embedded male mouse submandibular glands by electron microscopy. Both control and secretagogue-stimulated glands were examined. NGF was localized in granules of both granular convoluted tubule (GCT) cells and transition cells. The latter were intermediate in morphology between GCT cells and striated duct cells. Both large and small granules were immunostained in GCT cells; however, considerable variability in immunostaining intensity was observed in both sizes of granules but especially in the small granules of transition cells. Rough endoplasmic reticulum (RER) in both cell types exhibited NGF immunoreactivity. No Golgi-associated immunostaining was observed. Following alpha-adrenergic stimulation with phenylephrine, NGF-containing granules were sharply reduced because of extensive degranulation. Pools of immunostained secretory material suggested intracellular fusion of NGF-containing granules. Immunostaining was also observed on membrane fragments found within large vacuoles in GCT cells. Evidence of NGF secretion after beta-adrenergic or cholinergic stimulation was less dramatic. In isoproterenol-stimulated GCT cells there was evidence of fusion of small, apical, NGF-stained granules. These cells also possessed heavily immunostained apical membrane blebs. Pilocarpine-stimulated cells exhibited pleomorphic immunostained apical granules but less apical membrane immunostaining. Abundant basal lysosomes appearing in GCT cells after pilocarpine stimulation did not stain for NGF.

Autoradiographic localization of dihydrotestosterone binding in the major salivary glands and other androgen-responsive organs of the mouse.

Mouse submandibular glands show an androgen-dependent sexual dimorphism, reflected in higher concentrations in males than in females of bioactive peptides, such as epidermal growth factor (EGF), nerve growth factor, and renin in the cells of the granular convoluted tubules (GCT). Biochemical studies have demonstrated androgen receptors in submandibular gland and other androgen-responsive organs in mouse. We have determined the cellular localization of these receptors using steroid autoradiography. Fifteen adult gonadectomized male mice were injected intravenously with 0.13 microgram or 0.26 microgram [3H]-dihydrotestosterone (SA 135 Ci/mM); some animals were pre-treated with cyclocytidine to stimulate secretion by GCT cells. Animals were killed 15 min, 1, 2, or 3 hr after isotope injection. Steroid autoradiographs were prepared, and some were stained immunocytochemically for EGF. Of the different cell types of submandibular gland, the acinar cells most frequently and intensely concentrated [3H]-DHT; GCT cells also concentrated the hormone, as did a small number of striated duct cells. In the other major salivary glands, the only cells that concentrated the androgen were interlobular striated duct cells in sublingual gland. In prostate, anterior pituitary, and brain a large number of cells concentrated androgen, as has been previously reported. Androgen binding by the GCT cells was a predictable finding, since androgen-induced alterations in composition and form of these cells are well documented. The intense androgen concentration by the acinar cells was an unexpected finding and suggests a hitherto unknown androgen regulation of these cells. An incidental finding was intense concentration of [3H]-DHT in the nuclei of the endothelial cells of the post-capillary venules of the cervical lymph nodes.

Inductive Effects of Triiodothyronine or Dihydrotestosterone on EGF in the Submandibular Glands of Young, Middle-Aged, and Old C57BL/6NNia Female Mice

Effects of advancing age on responsiveness to triiodothyronine (T3) or dihydrotestosterone (DHT) by granular convoluted tubule (GCT) cells in submandibular glands were studied in C57BL/6NNia female mice of 4, 12, and 24 months of age. In untreated mice, absolute GCT volume and the frequency of cells strongly immunostained for EGF increased with age. Concentrations of EGF rose fourfold. Both T3 and DHT had inductive effects at each age: GCT cells were enlarged, contained more granules, and stained very strongly for EGF; EGF concentrations and the volume of GCTs also increased. However, the pattern of induction differed with age for the two hormones. After T3 treatment EGF concentration declined with age with respect to the degree of induction over same age control levels. By contrast, after DHT treatment the level of induction of EGF over control levels was the same at all three ages. The pattern of aging of GCT cells and their responsiveness to T3 or DHT in female mice differ from that previously reported for males.

Cellular basis for the differential response of mouse kallikrein genes to hormonal induction.

The expression of many mouse kallikrein genes in the salivary gland is sexually dimorphic and inducible in females by administration of testosterone or thyroxine. Induction is slow (3-7 days) and is accompanied by the non-uniform differentiation of the cell type expressing these genes from striated duct (SD) cells (female) to granular convoluted tubule (GCT) cells (male). One kallikrein gene, mGK-6, is expressed at an apparently constant total level in male and female and is not induced by either hormone. In situ hybridization histochemistry shows that all kallikrein genes analyzed exhibit uniform cellular distribution of expression in the SD cells of the female. The hormonally mediated differentiation of some, but not all, of these cells has different effects on kallikrein gene expression--mGK-6 is repressed while other kallikreins are induced--leading to non-uniform distribution of expression.

Zinc-containing 7S-NGF complex. Evidence from zinc histochemistry for localization in salivary secretory granules.

7S-NGF is a pro-protein containing a neurotrophic subunit, beta-NGF, which has been localized by immunocytochemistry to the granules of granular convoluted tubule (GCT) cells in certain murine salivary glands [Watson et al., Anat Rec (1985) 213:365]. The 7S-NGF pro-protein contains zinc and is stabilized by zinc ions [Pattison and Dunn, Biochemistry (1976) 15:3696]. In the present work, dithizone, toluene sulfonamide quinoline (TSQ), and neo-Timm's methods for zinc were used to determine whether zinc histochemistry could be used to visualize the zinc associated with the 7S-NGF complex and, if so, whether zinc histochemistry might corroborate the reported localization of the 7S-NGF complex in GCT secretory granules. The results indicate that intensity of zinc staining varies with the reported variations in NGF levels in different salivary glands, and that the zinc is selectively concentrated in the GCT secretory granules. We suggest that zinc histochemistry may be a useful marker for the presence of the zinc-stabilized 7S-NGF pro-protein.