Effects of spaceflight on the mouse submandibular gland.

OBJECTIVE: This study was conducted to determine if the morphology and biochemistry of the mouse submandibular gland is affected by microgravity and the spaceflight environment. DESIGN: Tissues from female mice flown on the US space shuttle missions Space Transportation System (STS)-131 and STS-135 for 15 and 13 d, respectively, and from male mice flown on the 30 d Russian Bion-M1 biosatellite, were examined using transmission electron microscopy and light and electron microscopic immunohistochemistry. RESULTS: In contrast to the parotid gland, morphologic changes were not apparent in the submandibular gland. No significant changes in protein expression, as assessed by quantitative immunogold labeling, occurred in female mice flown for 13-15 d. In male mice, however, increased labeling for salivary androgen binding protein alpha (in acinar cell secretory granules), and epidermal growth factor and nerve growth factor (in granular convoluted duct cell granules) was seen after 30 d in space. CONCLUSION: These results indicate that spaceflight alters secretory protein expression in the submandibular gland and suggest that the sex of the animals and the length of the flight may affect the response. These findings also show that individual salivary glands respond differently to spaceflight. Saliva contains proteins secreted from salivary glands and is easily collected, therefore is a useful biofluid for general medical analyses and in particular for monitoring the physiology and health of astronauts.

Response of the mouse sublingual gland to spaceflight.

The ultrastructure and immunohistochemistry of secretory proteins of sublingual glands were studied in mice flown on the US space shuttles Discovery [Space Transportation System (STS)-131] and Atlantis (STS-135). No differences in mucous acinar or serous demilune cell structure were observed between sublingual glands of ground (control) and flight mice. In contrast, previous studies showed autophagy and apoptosis of parotid serous acinar cells in flight mice. The expression of parotid secretory protein (PSP) in sublingual demilune cells of STS-131 flight mice was significantly increased compared with ground (control) mice but decreased in STS-135 flight mice. Similarly, expression of mucin (MUC-19) in acinar cells and expression of the type II regulatory subunit of protein kinase A (PKA-RII) in demilune cells were increased in STS-131 flight mice and decreased in STS-135 flight mice, but not significantly. Demilune cell and parotid protein (DCPP) was slightly decreased in mice from both flights, and nuclear PKA-RII was slightly increased. These results indicate that the response of salivary glands to spaceflight conditions varies among the different glands, cell types, and secretory proteins. Additionally, the spaceflight environment, including the effects of microgravity, modifies protein expression. Determining changes in salivary proteins may lead to development of non-invasive methods to assess the physiological status of astronauts.

Responses to spaceflight of mouse mandibular bone and teeth.

OBJECTIVE: To determine if spaceflight and microgravity affect non-weight bearing bones and development and mineralization of teeth, reasoning that combining an organ and a cellular level approach can lead to greater insights about these effects. DESIGN: Mandibles and incisors of mice flown on the US STS-135 space shuttle mission and the Russian Bion-M1 satellite were studied using micro-computed tomography and immunohistochemistry. Ground controls were mice housed in standard vivarium cages and flight habitats. RESULTS: Incisor length was greater in the 13-day STS-135 flight mice than in either control group. Initial incisor mineralization occurred more posteriorly, and incisor, enamel and dentin volumes and enamel and dentin thicknesses were greater in the 30-day Bion-M1 flight and habitat control mice than in vivarium control mice. Mandibular bone volume (BV) was increased in STS-135 flight and habitat groups and decreased in Bion-M1 flight and habitat groups compared to vivarium controls. No significant histological alterations occurred, but changes were seen in the bone and tooth proteins dentin sialoprotein, amelogenin and the type II regulatory subunit of protein kinase A. The percentage of sclerostin positive osteocytes was greatest in flight mice, and greater in STS-135 flight and habitat control mice than in the corresponding Bion-M1 groups. TRAP staining, representing osteoclastic bone remodeling, differed between the two flights and corresponded with changes in BV. Interpretation of the findings was limited by a small number of flight mice, different sex and ages of the mice in the two missions, and different habitats and diets. CONCLUSIONS: Microgravity has measurable effects on mandibular bone physiology and incisor development and mineralization. The results also showed that the habitat had an effect either in flight or ground control samples, as demonstrated by the changes in BV and apparent slowing of incisor eruption. Therefore, developing appropriate habitats is critical for future spaceflight missions.

Localization of cystic fibrosis transmembrane conductance regulator signaling complexes in human salivary gland striated duct cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity.

Microgravity alters the expression of salivary proteins.

Spaceflight provides a unique opportunity to study how physiologic responses are influenced by the external environment. Microgravity has been shown to alter the function of a number of tissues and organ systems. Very little, however, is known about how microgravity affects the oral cavity. The rodent model is useful for study in that their salivary gland morphology and physiology is similar to that of humans. Useful also is the fact that saliva, a product of the salivary glands with a major role in maintaining oral health, can be easily collected in humans whereas the glands can be studied in experimental animals. Our working hypothesis is that expression of secretory proteins in saliva will respond to microgravity and will be indicative of the nature of physiologic reactions to travel in space. This study was designed to determine which components of the salivary proteome are altered in mice flown on the US space shuttle missions and to determine if a subset with predictive value can be identified using microscopy and biochemistry methods. The results showed that the expression of secretory proteins associated with beta-adrenergic hormone regulated responses and mediated via the cyclic AMP pathway was significantly altered, whereas that of a number of unrelated proteins was not. The findings are potentially applicable to designing a biochemical test system whereby specific salivary proteins can be biomarkers for stress associated with travel in space and eventually for monitoring responses to conditions on earth.

Amylase and cyclic amp receptor protein expression in human diabetic parotid glands.

BACKGROUND: Salivary dysfunction and oral disorders have been described in both type 1 and type 2 diabetes mellitus. However, the cellular and molecular consequences of diabetes on oral tissues remain to be ascertained. The purpose of this investigation was to study, by means of electron microscopy, the morphologic and molecular changes that occur in salivary glands during diabetes. METHODS: Biopsy samples of parotid glands were excised from non-diabetic and diabetic (type 1 and type 2) consenting patients and processed by standard methods for routine morphology and electron microscopic immunogold labeling. Specific antibodies were used to determine and quantify the expression of secretory proteins (alphaamylase and the regulatory subunit of type II protein kinase A). RESULTS: Morphologic changes in the diabetic samples included increased numbers of secretory granules, and alterations in internal granule structure. Quantitative analysis of immunogold labeling showed that labeling densities were variable among the parotid gland samples. In type 1 diabetes amylase expression was greater than in non-diabetic glands, whereas in type 2 diabetes it was not significantly changed. Expression of type II regulatory subunits was slightly, although not significantly, increased in acinar secretory granules of type 1 diabetic samples and was unchanged in type 2 diabetic samples. CONCLUSIONS: Our data show that diabetes elicits specific changes in secretory protein expression in human salivary glands, thus contributing to the altered oral environment and oral disease associated with diabetes.

Secretory proteins in the saliva of children.

The protein composition of oral fluid is modulated by environmental factors and physiological states, i.e. chemical, mechanical and pharmacologic stimuli, pathologic conditions, and psychological stress. Secretory protein concentrations in samples of whole saliva (WS) from children were measured and the results were subjected to statistical analysis. Protein expression was determined using electrophoresis and Western blotting. Protein profiles of children were significantly different from those of adults (n = 50, P < 0.05). All samples of saliva from children contained a group of high-molecular-weight (>90 kDa) proteins, whereas fewer than 5% of samples from adults had comparable bands. The ratio of the regulatory subunits (RII) of type II protein kinase A (an enzyme that regulates secretion) to total protein was stable in children's saliva, but variable in saliva from adults. Alpha amylase (alpha-amylase), an enzyme that digests carbohydrates, was less degraded in WS of children than in that of adults. Gingival crevicular fluid of both children and adults did not contain alpha-amylase or RII. No significant gender-based differences were found, but Caucasian children had higher salivary protein levels than children with an African background. Saliva collection is rapid, painless, non-invasive, economical, and yields findings that are reproducible. Objective, biochemical monitoring of secretory proteins in oral fluid of children may reveal responses to stressful stimuli.

Protein expression in salivary glands of rats with streptozotocin diabetes.

Diabetes mellitus (DM) is a widespread disease with high morbidity and health care costs. An experimental animal model was employed, using morphological and biochemical methods, to investigate the effects of DM on the expression and compartmentation of salivary gland proteins. The distribution of proline-rich proteins (PRP), submandibular mucin (Muc10) and the regulatory (RI and RII) subunits of cyclic AMP-dependent protein kinase type I and type II was determined in the parotid and submandibular (SMG) glands of rats treated with streptozotocin. Quantitative immunocytochemistry of secretory granules in diabetic glands revealed decreases of 30% for PRP in both the parotid and SMG, and a 40% decrease in Muc10 in the SMG. Immunogold labelling showed that RII decreased in nuclei and the cytoplasm in diabetic acinar cells while labelling of secretory granules was similar in control and diabetic parotid. Electrophoresis and Western blotting of tissue extracts of two secretory proteins showed that the response to DM and insulin treatment was gland specific: PRP showed little change in the SMG, but decreased in the parotid in DM and was partially restored after insulin treatment. Photoaffinity labelling showed only RI present in the SMG and mainly RII in the parotid. The results of this and previous studies demonstrating highly specific changes in salivary protein expression indicate that the oral environment is significantly altered by DM, and that oral tissues and their function can be compromised. These findings may provide a basis for future studies to develop tests using saliva for diabetic status or progression in humans.

Immunocytochemical analysis of cyclic AMP receptor proteins in the developing rat parotid gland.

UNLABELLED: Previous studies showed that regulatory subunits of type II cyclic AMP-dependent protein kinase (RII) are present in adult rat parotid acinar cells, and are secreted into saliva. If the synthesis and intracellular distribution of RII exhibit developmental specificity, then RII can be an indicator of secretory and regulatory activity of salivary glands. OBJECTIVE: To determine the expression and distribution of RII in the rat parotid at specific ages representing defined developmental stages. METHODS: Parotid glands of fetal, neonatal and adult rats were prepared for morphologic and immunocytochemical study. The cellular distribution of RII was studied using light microscopic immunogold silver staining with anti-RII, and its intracellular distribution using electron microscopic immunogold labeling. RESULTS: In utero, parotid RII levels were low; 5-18 days after birth, labeling of secretory granules and cytoplasm rose to a peak, followed by a rapid decrease in both compartments at 25 days. At 60 days, granule labeling increased to levels near those at 18 days, whereas cytoplasmic labeling remained low. Nuclear labeling was highest during the first 3 weeks after birth, and then declined. CONCLUSIONS: The higher nuclear and cytoplasmic labeling during the neonatal period may reflect RII involvement in acinar cell differentiation. The accumulation of RII in secretory granules is similar to the pattern of the major salivary proteins, amylase and PSP. The redistribution of RII in these compartments during development may reflect changing gene expression patterns, and may be useful for identification of genetic or metabolic abnormalities.

Expression of secretory proteins in oral fluid after orthodontic tooth movement.

Orthodontic treatment alters the expression of secretory proteins at the local level in bone and the oral cavity, but its systemic effects are not well understood. Total secretory proteins and a cyclic adenosine monophosphate (AMP)-dependent protein kinase subunit (RII) were measured in saliva and gingival crevicular fluid (GCF) after the placement of orthodontic separators to determine if mechanical force applied to teeth affects protein secretion. Whole saliva and GCF were collected before and 1 day after treatment. Electrophoresis and Western blotting were carried out to establish the banding patterns of total proteins and to measure the isotype and amount of RII that serves as an apparent stress indicator. Digitized image files were used for densitometric analyses of the relative concentrations of RII and total protein. Individual protein values showed no statistically significant changes in saliva or GCF. Western blots, however, showed a dramatic difference in RII after the placement of separators: the 50-to-55 kilodaltons (kd) band virtually disappeared and was replaced by a fragment in the 20-kd range. These results suggest that although the expression of total proteins is not altered by mechanical force applied to teeth, a systemic response via the cyclic AMP signaling pathway might have been activated.