Comparative analysis of ABCG2-expressing and label-retaining cells in mouse submandibular gland

The submandibular gland (SMG) is a tissue that can be regenerated in a tissue injury model and that has adult stem cells capable of self-renewal and differentiation into functional cells. We have analyzed the localization of label-retaining cells (LRCs), which are putative progenitor cells, by using the BrdU-labeling method. 5-Bromo-2′-deoxyuridine (BrdU) injection followed by a long chasing period permitted the identification of LRCs based on the slow-cycling characteristic. In order to confirm the accurate localization of LRCs, BrdU and SMG-specific markers, including aquaporin5, cytokeratin, and smooth muscle actin, were examined by double-immunofluoresence staining. We found that LRCs were distributed in the acinus, duct, myoepithelium, and connective tissue. Moreover, ABCG2 (a known stem cell marker) was used for the characterization of LRCs and the localization of cells as putative stem/progenitor cells. ABCG2-expressing cells were distributed in various regions of the SMG but did not co-localize with LRCs. We suggest that putative progenitor cells exist in various regions of the SMG and have diverse capacities to differentiate into specific cells. Yeun-Jung Kim and Hyuk-Jae Kwon contributed equally to this work. This work was supported by Korea Research Foundation Grant (KRF-2006–013-E00143).     

The immunohistolocalization of carbonic anhydrase III in the submandibular gland of rats and hamsters

Summary Carbonic anhydrase III has been localized using the avidin-biotin-glucose oxidase complex (ABC) method in the submandibular gland of the rat and hamster. This isozyme, which is predominant in skeletal muscle, was observed in intercalated duct, striated duct and excretory duct cells in the rat submandibular glands. In contrast, only some striated duct cells in hamster submandibular glands were stained.     

四川短尾唾液腺的组织学观察

利用生物显微技术观察和研究了四川短尾鼩(Anourosorex squamipes)唾液腺的组织结构。结果表明,腮腺属纯浆液腺,有闰管和分泌管,无颗粒曲管;颌下腺属混合腺,以混合性腺泡为主,有少量浆液性腺泡和黏液性腺泡,有闰管、颗粒曲管和分泌管;舌下腺属纯黏液腺,有闰管和分泌管,无颗粒曲管,但在分泌管上存在有颗粒曲管细胞。     

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.     

Effects of thymidine analogs on Syrian hamster melanoma cells: phenotypes arising from selection for analog resistance

In order to compare the biological effects of different thymidine (dT) analogs, two unusual cell lines (B-4 and HAB) previously isolated from a Syrian hamster melanoma line by selection with 5-bromodeoxyuridine (BrdU) were analyzed for their response to other analogs. B-4 cells require high concentrations of BrdU for optimal growth, and it was seen that the requirement for BrdU could be satisfied partially by 5-chlorodeoxyuridine (CldU) but not by the other dT analogs tested. HAB cells are able to grow with all the dT residues in nuclear DNA replaced by BrdU, and it was found that they could also grow with essentially all the dT residues in nuclear DNA replaced by CldU but not by other analogs. New cell lines resistant to 100 micrometer concentrations of CldU, 5-iododeoxyuridine (IdU), and 5-hydroxymethyldeoxyuridine (HMdU) were isolated from the melanoma line and tested for cross-resistance to the other dT analogs. A high level of cross-resistance was observed only with BrdU and CldU. The ability of the cell lines resistant to BrdU, CldU, and IdU to incorporate these analogs into nuclear DNA also was determined. BrdU and CldU were incorporated efficiently by all of the lines tested, but the IdU-resistant cells seemed to preferentially exclude IdU.     

Immunocytochemistry of myoepithelial cells in the salivary glands

MECs are distributed on the basal aspect of the intercalated duct and acinus of human and rat salivary glands. However, they do not occur in the acinus of rat parotid glands, and sometimes occur in the striated duct of human salivary glands. MECs, as the name implies, have structural features of both epithelial and smooth muscle cells. They contract by autonomic nervous stimulation, and are thought to assist the secretion by compressing and/or reinforcing the underlying parenchyma. MECs can be best observed by immunocytochemistry. There are three types of immunocytochemical markers of MECs in salivary glands. The first type includes smooth muscle protein markers such as -SMA, SMMHC, h-caldesmon and basic calponin, and these are expressed by MECs and the mesenchymal vasculature. The second type is expressed by MECs and the duct cells and includes keratins 14, 5 and 17, 1β1 integrin, and metallothionein. Vimentin is the third type and, in addition to MECs, is expressed by the mesenchymal cells and some duct cells. The same three types of markers are used for studying the developing gland.

Development of MECs starts after the establishment of an extensively branched system of cellular cords each of which terminates as a spherical cell mass, a terminal bud. The pluripotent stem cell generates the acinar progenitor in the terminal bud and the ductal progenitor in the cellular cord. The acinar progenitor differentiates into MECs, acinar cells and intercalated duct cells, whereas the ductal progenitor differentiates into the striated and excretory duct cells. Both in the terminal bud and in the cellular cord, the immediate precursors of all types of the epithelial cells appear to express vimentin. The first identifiable MECs are seen at the periphery of the terminal bud or the immature acinus (the direct progeny of the terminal bud) as somewhat flattened cells with a single cilium projecting toward them. They express vimentin and later -SMA and basic calponin. At the next developmental stage, MECs acquire cytoplasmic microfilaments and plasmalemmal caveolae but not as much as in the mature cell. They express SMMHC and, inconsistently, K14. This protein is consistently expressed in the mature cell. K14 is expressed by duct cells, and vimentin is expressed by both mesenchymal and epithelial cells.

After development, the acinar progenitor and the ductal progenitor appear to reside in the acinus/intercalated duct and the larger ducts, respectively, and to contribute to the tissue homeostasis. Under unusual conditions such as massive parenchymal destruction, the acinar progenitor contributes to the maintenance of the larger ducts that result in the occurrence of striated ducts with MECs. The acinar progenitor is the origin of salivary gland tumors containing MECs. MECs in salivary gland tumors are best identified by immunocytochemistry for -SMA. There are significant numbers of cells related to luminal tumor cells in the non-luminal tumor cells that have been believed to be neoplastic MECs.     

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.     

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.     

Immunolocalization of PTHrP in the submandibular glands of three rodent species.

The present study deals with immunohistochemical localization of PTHrP in bank vole, pine vole and white mouse submandibular glands. PTHrP immunoreactivity was observed in epithelial cells of all ductal segments (intercalated, striated, interlobular and main excretory ducts) of the salivary glands in all the three animal species tested. We also found PTHrP expression in myoepithelial cells surrounding the mucous alveoli of submandibular glands in those animals. The reaction was less intense than that found in the epithelial cells of excretory ducts. We occasionally observed a very slight positive reaction for PTHrP in smooth muscle cells of small blood vessels. We also found PTHrP expression in the neurons of ganglion in the submandibular gland.     

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.     

Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population

Mammary epithelium can functionally regenerate upon transplantation. This renewal capacity has been classically ascribed to the function of a multipotent mammary gland stem cell population, which has been hypothesized to be a primary target in the etiology of breast cancer. Several complementary approaches were employed in this study to identify and enrich mammary epithelial cells that retain stem cell characteristics. Using long-term BrdU labeling, a population of label retaining cells (LRCs) that lack expression of differentiation markers has been identified. LRCs isolated from mammary primary cultures were enriched for stem cell antigen-1 (Sca-1) and Hoechst dye-effluxing "side population" properties. Sca-1(pos) cells in the mammary gland were localized to the luminal epithelia by using Sca-1(+/GFP) mice, were progesterone receptor-negative, and did not bind peanut lectin. Finally, the Sca-1(pos) population is enriched for functional stem/progenitor cells, as demonstrated by its increased regenerative potential compared with Sca-1(neg) cells when transplanted into the cleared mammary fat pads of host mice.     

Apoptosis and proliferation of myoepithelial cells in atrophic rat submandibular glands.

This study was designed to determine whether apoptosis and proliferation of myoepithelial cells occur in atrophic rat submandibular glands. The excretory duct of the right submandibular gland was doubly ligated with metal clips. The atrophic right submandibular glands removed after 1-28 days of duct ligation were investigated using immunohistochemical double staining for actin as a marker for myoepithelial cells and proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells, double staining for actin immunohistochemistry, nick end-labeling (TUNEL) as a marker for apoptotic cells, and transmission electron microscopy (TEM). A few PCNA- and no TUNEL-positive myoepithelial cells were found in the control submandibular glands taken from animals with no operation. In the experimental glands, PCNA-positive myoepithelial cells were common 2 and 3 days after duct ligation and then decreased in number. TUNEL-positive myoepithelial cells appeared at 2 days and were observed most frequently at 5 days. Apoptotic myoepithelial cells were also identified by TEM. These observations suggest that both apoptosis and proliferation of myoepithelial cells occur, especially in the early phase of atrophy, in the rat submandibular gland.     

Histology and histochemistry of the parotid and the principal and accessory submandibular glands of the little brown bat

The parotid and the principal and accessory submandibular glands of the little brown bat. Myotis lucifugus (Vespertilionidae), were examined using light microscopy and staining methods for mucosubstances. The parotid gland is a compound tubuloacinar seromucous gland. Parotid gland secretory cells contain both neutral and nonsulfated acidic mucosubstances. The principal and accessory submandibular glands are compound tubuloacinar mucus-secreting glands. They contain somewhat atypical mucus-secreting demilunar cells that often appear to be interspersed between mucous tubule cells. The mucous tubule cells in both the principal and accessory submandibular glands contain sulfonmucins. Demilunar cells of the principal submandibular gland contain moderate amounts of nonsulfated acidic mucosubstances, but the corresponding cells of the accessory submandibular gland contain considerable neutral mucosubstance with very little acid mucosubstance. Intercalated ducts composed of cuboidal or low columnar epithelial cells are present in all three glands. Striated ducts in all glands are composed of columnar cells whose apices bulge into the ductal lumina. Excretory ducts are composed of simple columnar epithelium, with occasional basal cells that suggest a possible pseudostratified nature. The cells of the excretory ducts also have bulging apices. All duct types contain apical cytoplasmic secretory material that is a periodic acid-Schiff positive, neutral mucosubstance. Ductal apical secretory material is more evident in intercalated and striated ducts than in excretory ducts.     

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.     

Glycoprotein secretion in the mouse submandibular gland as revealed by radioautography after L-3H-fucose injection

Summary Glycoprotein secretion in the mouse submandibular gland was investigated by light microscope radioautography of semi-thin sections after the administration of L-³H-fucose. The incorporation of the precursor in the acini was negligible. ³H-fucose was taken up in the paranuclear region of the cells lining the intercalated, secretory, striated and excretory ducts. This labeling pattern was interpreted as addition of the precursor to glycoproteins within the Golgi apparatus. Incorporation in the intercalated duct was restricted to the cells with fine cytoplasmic granules. The glycoproteins synthesized by the intercalated and secretory ducts were transported to the saliva by the secretion granules. It is assumed that the glycoproteins synthesized in the striated and excretory ducts are plasma membrane glycoproteins which seem to renew continuously. Quantitation of the radioautographs supplied data concerning the incorporation of ³H-fucose into newly synthesized glycoproteins as well as the renewal of the labeled macromolecules in each duct.     

5-Halogenated thymidine analogues induce a senescence-like phenomenon in HeLa cells

We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.     

Apical maxi-K (KCa1.1) channels mediate K+ secretion by the mouse submandibular exocrine gland

The exocrine salivary glands of mammals secrete K+ by an unknown pathway that has been associated with HCO3(-) efflux. However, the present studies found that K+ secretion in the mouse submandibular gland did not require HCO3(-), demonstrating that neither K+/HCO3(-) cotransport nor K+/H+ exchange mechanisms were involved. Because HCO3(-) did not appear to participate in this process, we tested whether a K channel is required. Indeed, K+ secretion was inhibited >75% in mice with a null mutation in the maxi-K, Ca2+-activated K channel (KCa1.1) but was unchanged in mice lacking the intermediate-conductance IKCa1 channel (KCa3.1). Moreover, paxilline, a specific maxi-K channel blocker, dramatically reduced the K+ concentration in submandibular saliva. The K+ concentration of saliva is well known to be flow rate dependent, the K+ concentration increasing as the flow decreases. The flow rate dependence of K+ secretion was nearly eliminated in KCa1.1 null mice, suggesting an important role for KCa1.1 channels in this process as well. Importantly, a maxi-K-like current had not been previously detected in duct cells, the theoretical site of K+ secretion, but we found that KCa1.1 channels localized to the apical membranes of both striated and excretory duct cells, but not granular duct cells, using immunohistochemistry. Consistent with this latter observation, maxi-K currents were not detected in granular duct cells. Taken together, these results demonstrate that the secretion of K+ requires and is likely mediated by KCa1.1 potassium channels localized to the apical membranes of striated and excretory duct cells in the mouse submandibular exocrine gland.     

Stem cells propagate their DNA by random segregation in the flatworm Macrostomum lignano

Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts) are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized 'Label-Retaining Cells' (LRCs). Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells. We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU- double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.     

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.