Immunocytochemical demonstration of nerve growth factor and histofluorescence of catecholaminergic nerves in the salivary glands of diabetic mice

Summary Nerve growth factor (NGF) was localized in the submandibular, sublingual, and parotid salivary glands of male and female diabetic mice and their normal littermates by immunoperoxidase staining usingp-phenylenediamine-pyrocatechol as a chromogen for the cytochemical demonstration of peroxidase activity. In the normal male submandibular gland, immunoreactive NGF was localized in the apical regions of granular, intercalated and collecting duct cells, while in the normal female submandibular gland, NGF was present throughout the cytoplasm of granular duct cells. The localization of NGF in the diabetic male and female submandibular glands was similar and resembled that of the normal female. NGF immunoreactivity was also observed in the striated duct cells in the sublingual and parotid glands of all four types of mice.The sympathetic innervation of the submandibular glands of normal and diabetic mice was demonstrated using glyoxylic acid-induced histofluorescence. The pattern of sympathetic innervation and the intensity of catecholamine fluorescence was consistently different in the four types of mice. In the normal male submandibular gland the fluorescence was very intense, particularly in nerves adjacent to the granular ducts. In the normal female submandibular gland, the fluorescence was weak, while in the diabetic male and female the fluorescence was moderate.The correlation between the intensity of the immunocytochemical staining for NGF and the catecholamine fluorescence adjacent to the granular ducts suggests a trophic influence of the NGF-containing granular ducts on their sympathetic innervation.     

Clcn2 encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands

Transepithelial Cl(-) transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl(-) current with the biophysical properties of ClC-2 channels dominates the Cl(-) conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl(-) current is activated by hyperpolarization and elevated intracellular Cl(-) concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl(-) current in cells from Clcn2(-/-) mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2(-/-) mice. Additionally, neither a compensatory increase in Cftr Cl(-) channel protein expression nor in Cftr-like Cl(-) currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl(-) channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium.     

Growth hormone and epidermal growth factor in salivary glands of giant and dwarf transgenic mice.

Epidermal growth factor (EGF) in rat salivary glands is regulated by testosterone, thyroxin, and growth hormone (GH). Salivary glands of 45-day-old giant and dwarf male and female transgenic mice were examined histologically and by immunohistochemistry (IHC) for EGF. Male giants showed no significant differences from wild-type (WT) parotid and submandibular glands. However, their sublingual glands expressed EGF diffusely and strongly in granular cells within the striated ducts, where they were not found in WT mice. Submandibular gland ducts of female WT were different, having individual granular cells strongly positive for EGF and distributed sporadically along the striated duct walls. Neither female GH-antagonist dwarf mice nor GH-receptor knockout mice had any granular cells expressing EGF in any gland. Obvious presence of granular duct cells in the sublingual glands of giant male mice suggests GH-upregulated granular cell EGF expression. Furthermore, absence of granular duct cells from all glands in female GH-antagonist and GH-receptor knockout transgenic mice suggests that GH is necessary for the differentiation of the granular cell phenotype in female salivary glands.     

Distinct immunohistochemical expression of osteopontin in the adult rat major salivary glands

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70–100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the compostion of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell–cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.Nicholas Obermüller and Nikolaus Gassler contributed equally to this work.     

Cytochemical correlates of structural sexual dimorphism in glandular tissues of the mouse

Summary Circulating androgens are known to effect a sexual dimorphism of the submandibular gland and kidney of the mouse. Enzyme histocytochemical differences that correlate with these structural changes have been the subject of much study, especially in the kidney. In the present study, emphasis was placed on the hypogonadic effects of diabetes mellitus on the submandibular gland and kidney of C57BL/KsJ db/db inbred mice with an autosomal recessive disease resembling maturity onset human diabetes mellitus. These glands of adult diabetic mice of both sexes were compared with those of unafflicted heterozygous littermates. The mitochondrial cytochrome oxidase and peroxisomal and cytoplasmic catalase were studied in their submandibular glands and kidneys. The parasympathetic innervation of the submandibular glands was studied by a histochemical method for acetylcholinesterase. The extensive differentiation of striated ducts of the submandibular gland into granular tubules in the postpubertal male mouse was readily evident with the cytochrome oxidase procedure. This differentiation resulted in ductal staining patterns characteristic of the sexes. Alteration of these patterns suggested that demasculinization or feminization was occuring in the male diabetic mice and that masculinization or virilization (defeminization) was occurring in the female diabetics. Similarly, in kidney, study of the parietal epithelium of Bowman's capsule revealed feminization in the male diabetics and masculinization in the female diabetics. With the catalase procedure, a dramatic sexual dimorphism was observed in the kidneys of the heterozygous unafflicted mice. Peroxisomal staining of epithelial cells of the proximal convoluted tubules was much more intense in the outer medulla of the male than of the female. In kidneys of the diabetics, the staining patterns again suggested that feminization of the male and masculinization of the female kidneys had occurred. On the other hand, neither a sexual dichotomy nor effects due to diabetes could be observed in the characteristic catalase staining observed in the luminal epithelial cells of submandibular gland distal ducts. The parasympathetic innervation of the submandibular gland, as revealed by the acetylcholinesterase method, was also markedly sexually dimorphic in the unafflicted mice. This was due to the more extensive innervation of the larger granular ducts characteristic of male than of the smaller striated ducts of the female. As a result of diabetes, the innervation and duct size decreased in the submandibular gland of the male, suggesting feminization, whereas they increased in the female suggesting masculinization. These changes were consistent with those observed in submandibular with the cytochrome oxidase procedure. Attempts were made to interrelate all of the enzyme histochemical changes observed in submandibular gland and kidney with the weights of these glands, sex, gonadal weights, diabetic status and urinary protein excretion. Generally, significant differences were recorded which suggested that the feminization of the submandibular gland and kidney in the diabetic male mice, and their masculinization in the female diabetics, were due to the hypogonadism of the disease.This investigation was supported by NIH research grants DE 02668, DE 04730, DE 00014 and RR 05333     

Immunocytochemical localization of nerve growth factor in mouse salivary glands

Summary The submandibular glands of female mice and the sublingual and parotid glands of adult male and female mice have been examined by light microscopical immunocytochemistry for nerve growth factor (NGF). In female submandibular glands, staining for NGF was observed in granular convoluted tubule and striated duct cells. Sublingual glands of the mouse contained relatively few granular cells staining for NGF compared with submandibular glands. However, such granular cells appeared to be more numerous in male sublingual glands than in female glands. The remainder of the intralobular duct cells in both male and female sublingual glands exhibited apical subluminal staining for NGF as well as light basal plasmalemmal staining. Parotid glands in both male and female mice exhibited a similar pattern of staining for NGF in striated duct cells. However, the glands did not contain granular cells nor did they exhibit any pattern of staining which reflected a sexual dimorphism. Immunodot staining of salivary gland extracts confirmed the presence of immunoreactivity for NGF in all three of the major salivary glands.     

Basic fibroblast growth factor in rat salivary glands

We studied the occurrence and localization of basic fibroblast growth factor (bFGF) in rat salivary glands using a specific monoclonal antibody. It was shown that the extract of rat salivary glands has a pronounced stimulatory activity on the growth of bovine capillary endothelial cells, which is blocked by the addition of an antibody against bFGF. The concentration of bFGF in the submandibular/sublingual gland, as determined by radioimmunoassay, was 80% that in the brain. Immunocytochemistry revealed bFGF-immunoreactivity localized primarily in the epithelial cells lining the striated ducts and excretory ducts of the parotid, sublingual and submandibular glands. In addition, intense bFGF-immunoreactivity was observed in the granular convoluted tubule of the submandibular gland, localized predominantly in the agranular pillar cells, which lay in small numbers among the majority of weakly immunostained cells containing many apical secretory granules. At the electron-microscopic level, the immunoreactive material was distributed diffusely in the cytoplasmic matrix and nuclei of all immunoreactive cells, whereas it was absent from all cytoplasmic organelles including the secretory granules. These results indicate that bFGF is localized in different cellular and subcellular compartments from those of other growth factors in the duct system of rat salivary glands.     

Immunohistochemical localization of Na+,K+-ATPase in rodent and human salivary and lacrimal glands

The enzyme Na+,K+-ATPase was localized immunohistochemically in major salivary glands of mouse, rat, and human and in exorbital lacrimal glands of the rodents. Immunoreactive Na+,K+-ATPase was abundant in the basolateral membranes of all epithelial cells lining striated and intra- and interlobular ducts of all glands. Reactivity of intercalated ducts varied among gland type and species. Cells lining granular ducts in rodent submandibular gland showed a heterogeneous staining pattern in rat but stained homogeneously in mouse. Secretory cells varied greatly in their content of immunoreactive Na+,K+-ATPase. As with all duct cells, staining was present only at the basolateral surface and was never observed at the luminal surface of reactive secretory cells. Mucous cells failed to show any reactivity in any gland examined. Serous cells showed a gradient of immunostaining intensity ranging from strongly positive in demilunes of human sublingual gland to negative in rat submandibular gland and lacrimal glands of rats and mice. The presence of basolaterally localized Na+,K+-ATPase in most serous cells but not in mucous cells suggests that the enzyme contributes to the ion and water content of copious, low-protein serous secretions. The intense immunostaining of cells in most if not all segments of the duct system supports the idea that the ducts are involved with modification of the primary saliva, and extends this concept to include all segments of the duct system.     

Androgen receptor in rat Harderian and submandibular glands

Summary Androgens regulate the development and sexual dimorphism of rodent Harderian and submandibular glands. This effect is believed to be mediated by the androgen receptor. Immunohistochemistry and immunoblotting were carried out to study the receptor in normal, castrated and dihydrotestosterone-supplemented rat Harderian and submandibular glands. Immunohistochemically, the most intense nuclear staining was observed in the acinar cells of the submandibular glands, followed by intercalated duct cells. The granular convoluted tubules showed weak immunostaining and the striated ducts were negative. In the Harderian gland, nuclear staining was seen in both type I and II secretory cells. Castration and treatment had no effect on the expression of the androgen receptor protein in either gland. A 110 K androgen receptor signal was detected by immunoblotting in the Harderian gland but not in the submandibular gland. An experiment was designed to explore the possible effect of proteinases on the receptor protein in the homogenate of submandibular gland. Our results demonstrate the cell-specific location of the receptor in Harderian and submandibular glands, and show that the expression of the receptor protein is androgen-independent.     

Accelerated tubular cell senescence in SMP30 knockout mice

An experimental model with accelerated but not drastic renal senescence seemed useful to recognize the mechanisms of how kidney function deteriorates with age. Senescence marker protein-30 (SMP30), whose expression decreased with age and was sex-independent, is mainly expressed in hepatocytes and proximal tubular cells. Therefore, we established a SMP30 deficient strain of mice with a C57BL/6 background by gene targeting to investigate whether this molecule is involved in renal tubular cell senescence. Male SMP30 knockout (SMP30Y/-) mice and male wild-type (SMPY/+) mice (n=5) aged 12 months were examined histologically. Their tubular epithelia showed the deposition of lipofuscin and the presence of senescence-associated beta-galactosidase (SA-beta-GAL). However, no tubular cells were atrophic. In electron microscopy, SMP30-KO mice showed markedly enlarged lysosomes containing an electron dense substance. These are convincing hallmarks of senescence. We recognized the early manifestation of senescence hallmarks in SMP30-KO mice at 12 months old. Thus, this model represents the first report of a mouse strain that manifests accelerated ordinal senescence in a kidney after gene manipulation.     

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.     

Morphological changes in the submandibular glands and in the X zone of the adrenal gland following ovariectomy in mice

Summary Morphological changes in submandibular glands of female mice following ovariectomy were studied morphometrically by light microscopy and ultrastructurally by electron microscopy. The X zone of the adrenal gland was examined in order to assess possible changes that might be expected to occur after ovariectomy.In submandibular glands, 1 to 4 weeks after ovariectomy, no changes were observed in percentages of the acinar, intercalated duct, and granular convoluted tubular areas occupying photomicrographs. However, an increase in the granular content of both intercalated duct and granular convoluted tubular cells was recognized. By contrast, the glandular picture 4 months after ovariectomy changed remarkably, resembling that of the male mouse both morphometrically and in terms of fine structure. In the adrenal cortex of control female mice, the X zone became thinner with aging. As compared with this, the X zone of ovariectomized mice at any time after the operation was thicker than that of controls.These observations suggest that the absence of ovarian hormones in the ovariectomized mouse may lead to prolonged functioning of X zone cells, which in turn may cause masculinization of the submandibular gland.     

Distribution of nerve growth factor in the submandibular gland of the male and female mouse

Summary Nerve growth factor (NGF) was localized in the mouse submandibular gland by means of indirect immunofluorescence applied to 0.5 mthick sections of freeze-dried, plastic-embedded tissue. The antibody to NGF (I_gG-fraction) was raised in rabbits immunized with pure 2.5 S NGF from submandibular glands of adult male mice.In the male gland anti-NGF bound selectively to the secretory granules was present in the cells of the granular ducts. Immunoreactive granules extended from the perinuclear region toward the apical pole. In the female gland immunoreactive cells and granules were considerably less abundant than in males. Immunofluorescence was confined to individual secretory cells located in the wall of the granular striated duct.In the present study no support was found for the hypothesis suggesting that immunoreactive NGF is formed within the secretory granules during their transport from the perinuclear region to the apical pole.     

Morphological changes and EGF expression in the granular convoluted tubule cells of submandibular glands of Trypanosoma cruzi infected rats

We have previously demonstrated in rats that Chagas' disease affects the salivary glands, by promoting an enlargement of the submandibular gland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study was to analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstrated that infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of the submandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistry analysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas' disease, while after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at the acute phase of Chagas' disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates with decreased EGF production by submandibular glands cells.     

Immunocytochemical evidence for the presence of somatostatin-like immunoreactivity in scattered cells of the duct system of the submandibular glands in the Monkey,Macaca irus

Summary Using the indirect immunofluorescent technique with anti-somatostatin serum, the distribution of scattered cells in the duct system of submandibular glands in the Monkey, Macaca irus has been assessed. In both males and females, these cells are located only in some portions of the duct system, e.g. striated ducts and excretory ducts. No immunoreactive cells were observed in the intercalated ducts or in secretory endpieces. The lymphatic node constantly adjacent to the submandibular gland did not contain immunoreactive cells. In the parotid glands, no immunoreactive cells to antisomatostatin immuneserum were ever observed     

Immunocytochemical evidence for the presence of somatostatin-like immunoreactivity in scattered cells of the duct system of the submandibular glands in the monkey, Macaca irus

Using the indirect immunofluorescent technique with anti-somatostatin serum, the distribution of scattered cells in the duct system of submandibular glands in the Monkey, Macaca irus has been assessed. In both males and females, these cells are located only in some portions of the duct system, e.g. striated ducts and excretory ducts. No immunoreactive cells were observed in the intercalated ducts or in secretory endpieces. The lymphatic node constantly adjacent to the submandibular gland did not contain immunoreactive cells. In the parotid glands, no immunoreactive cells to antisomatostatin immuneserum were ever observed.     

Senescence marker protein 30 (SMP30) expression in eukaryotic cells: existence of multiple species and membrane localization

Senescence marker protein (SMP30), also known as regucalcin, is a 34 kDa cytosolic marker protein of aging which plays an important role in intracellular Ca(2+) homeostasis, ascorbic acid biosynthesis, oxidative stress, and detoxification of chemical warfare nerve agents. In our goal to investigate the activity of SMP30 for the detoxification of nerve agents, we have produced a recombinant adenovirus expressing human SMP30 as a fusion protein with a hemaglutinin tag (Ad-SMP30-HA). Ad-SMP30-HA transduced the expression of SMP30-HA and two additional forms of SMP30 with molecular sizes ～28 kDa and 24 kDa in HEK-293A and C3A liver cells in a dose and time-dependent manner. Intravenous administration of Ad-SMP30-HA in mice results in the expression of all the three forms of SMP30 in the liver and diaphragm. LC-MS/MS results confirmed that the lower molecular weight 28 kDa and 24 kDa proteins are related to the 34 kDa SMP30. The 28 kDa and 24 kDa SMP30 forms were also detected in normal rat liver and mice injected with Ad-SMP30-HA suggesting that SMP30 does exist in multiple forms under physiological conditions. Time course experiments in both cell lines suggest that the 28 kDa and 24 kDa SMP30 forms are likely generated from the 34 kDa SMP30. Interestingly, the 28 kDa and 24 kDa SMP30 forms appeared initially in the cytosol and shifted to the particulate fraction. Studies using small molecule inhibitors of proteolytic pathways revealed the potential involvement of β and γ-secretases but not calpains, lysosomal proteases, proteasome and caspases. This is the first report describing the existence of multiple forms of SMP30, their preferential distribution to membranes and their generation through proteolysis possibly mediated by secretase enzymes.     

Immunohistochemical localization of S100A1 and S100A6 in postnatally developing salivary glands of rats

 S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0–5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands. Accepted: 14 July 1998     

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.     

A comparative histochemical study of peroxidase activity in the submandibular glands of five mammalian species including man

Summary A light microscopic histochemical investigation of endogenous peroxidase activity in specimens of the submandibular salivary glands of man, hamster, rabbit, dog and guinea pig was carried out. A modification of the original Graham and Karnovsky diaminobenzidine (DAB)-hydrogen peroxide method was employed at different pH's.At all pH's (6.0, 7.6, and 9.0) a positive DAB reaction was found: in serous acinar cells in four of seven human submandibular glands, in convoluted tubule cells of the hamster, in acinar tissue, in secretory granular tubule cells and in the saliva of the guinea pig. This staining pattern was not markedly affected by KCN or 2,4-dichlorophenol (DCP). Furthermore, small cytoplasmic granules in collecting ducts of the dog displayed positive, KCN- and DCP-resistant DAB staining at all pH's tested. No reaction was observed in the acinar cells of the dog and rabbit glands.Mitochondrial oxidation of DAB in the striated duct cells occurred in all of the glands examined. Optimal staining of these cells was obtained at pH 6.0, but there was also strong positive staining at pH 7.6. At pH 9.0, however, the staining of the striated duct cells was very faint. The positive reaction in the striated duct cells was completely abolished by KCN.