Expression and localization of calpain 3 in the submandibular gland of mice

ObjectivesCalpains comprise a family of intracellular Ca²⁺-dependent cysteine proteases and are considered to play roles in various physiological phenomena with limited proteolytic activities against specific substrates. We herein revealed the expression and localization of calpain 3, the muscle-type calpain, in the submandibular gland (SMG) of mice.DesignThe expression of the mRNA for conventional, ubiquitous calpains 1 and 2 and skeletal muscle-specific calpain 3 was examined in the major salivary glands of mice using RT-PCR, and the expression and localization of calpain 3 protein was examined in the SMG of mice using immunohistochemistry and Western blotting.ResultsThe large catalytic subunits of calpains 1 and 2 and the small regulatory subunit common to calpains 1 and 2 were weakly expressed in the parotid gland, sublingual gland, and SMG at similar levels in males and females. In contrast, the single large catalytic subunit of calpain 3 was expressed predominantly in the SMG at markedly higher levels in males than in females and in a manner dependent on androgens. Immunoreactivity for calpain 3 was mainly localized in cells of the granular convoluted tubules (GCT) that developed preferentially in the male SMG. In GCT cells, calpain 3 immunoreactivity was localized predominantly in the cytosolic region and was absent in the secretory granules.ConclusionsThese results revealed that the GCT is the primary site of production of calpain 3 in the mouse SMG.     

Expression and localization of receptor protein tyrosine phosphatase β and its ligand pleiotrophin in the submandibular gland of mice

ObjectivesThe family of receptor protein tyrosine phosphatase β (RPTPβ) is composed of 4 splice variants and thought to play roles in the neural migration and outgrowth. Several ligands including the growth factor pleiotrophin (PTN) bind to RPTPβ and inhibit its phosphatase activity, thereby activating cellular signalling pathways. We examined the expression and localization of RPTPβ and its ligands in the submandibular gland (SMG) of mice, which is known for a prominent sexual dimorphism in the duct system.DesignThe homogenates and tissue sections of male and female mouse SMG were analysed with RT-PCR, Western blotting, and immunohistochemistry.ResultsThe short receptor type of RPTPβ (RPTPβ-S) was dominantly expressed in the SMG, and the male gland had significantly higher levels of RPTPβ-S expression than the female gland. In the male, RPTPβ-S was localized predominantly in intercalated duct (ID) cells, but was not found in granular convoluted tubule (GCT) cells or acinar cells. In the female, weaker reactivity was demonstrated in both ID and striated duct (SD) cells. Of the known ligands for RPTPβ, PTN was expressed in the SMG, without sexual difference in levels. In the male, PTN was localized in ID cells as well as in cells located in the distal ends of GCT that are in close vicinity to the ID, whereas in the female PTN was colocalized with RPTPβ-S throughout ID and SD cells.ConclusionsThese results indicated that the distribution of RPTPβ-S and its ligand PTN has a close relation to the sexual dimorphism in the duct system of mouse SMG.     

Morphologic changes in the granular convoluted tubule cells of the mouse submandibular gland following hypophysectomy and hormonal replacement

 Morphological changes in the granular convoluted tubule (GCT) cells of the male mouse submandibular gland (SMG) were examined following hypophysectomy and hormonal replacement. Semithin sections stained with Heidenhain's iron hematoxylin showed that hypophysectomy severely regressed the GCT phenotype. Although only a few dispersed cells containing secretory granules were observed in the GCT segments under a light microscope, electron microscopy revealed that many regressed cells continued to constitutively elaborate apical secretory granules (although they were very small) and contained a euchromatic nucleus at the center of the cell, poor rough endoplasmic reticulum (RER) and Golgi apparatus in the perinuclear region, and well-developed basal infoldings. These findings suggest that hypophysectomy resulted in atrophy of GCT cells, but that they retained evidence of being secretory cells. 5α-Dihydrotestosterone (DHT); 3,5,3′-triiodo-l-thyronine (T₃); and dexamethasone (Dex) each enhanced the GCT phenotype of hypophysectomized males to some degree. Combined hormonal replacement with DHT + T₃ in hypophysectomized males restored a nearly normal male GCT phenotype with a full complement of secretory granules and rare basal infoldings, whereas T₃ alone induced a normal female-like GCT phenotype, with considerably abundant secretory granules and the usual short basal infoldings, in hypophysectomized male glands. Furthermore, Dex was found to synergistically enlarge secretory granules when administered with T₃ and/or DHT, although it was only weakly effective in enhancing the GCT phenotype when used alone. Taken together, the above findings confirmed that the GCT phenotype of the mouse SMG is regulated by the synergistic action of pituitary-dependent hormones. Received: October 1, 2001 / Accepted: May 13, 2002     

Postnatal expression of sialin in the mouse submandibular gland

Objective

Sialin has been identified as a sialic acid and aspartate/glutamate transporter. Both cytoplasmic localization and the plasma membrane labelling pattern suggested that sialin may possess multiple transport functions in different cell types. In mouse embryos, sialin expression was primarily detected in the central nervous system. However, sialin shows widespread and high-level expression in adult tissues. Despite its ubiquitous expression and important functions, the postnatal expression profile and subcellular localization of sialin in the salivary gland remains elusive. The aim of the present study was to investigate the expression and subcellular distribution of sialin during postnatal development in the mouse submandibular gland (SMG).

Design

Six SMGs from both female and male C57BL/6 mice were collected at P10, P30 and P90, and the material from each littermate of either sex was pooled to extract total RNA and tissue protein. The remaining tissues were immediately fixed in 10% neutral buffered formalin for histological analysis. The mRNA and protein expression levels of sialin were examined by quantitative real-time RT-PCR and Western blot analysis. The subcellular distribution of sialin was analysed by immunohistochemistry and immunofluorescence.

Results

The postnatal expression level of sialin in the mouse SMG was comparable with that in brain at each time point tested. The temporal expression of sialin in the SMG gradually increased during postnatal maturation. Immunohistochemical and immunofluorescence analysis demonstrated that sialin was predominantly expressed on the basal cytoplasmic membrane of acini and ducts, as well as in some myoepithelial cells in the SMG.

Conclusions

The high-level expression and subcellular distribution pattern of sialin in the SMG suggest that sialin may play an important role in the transport and secretion of saliva.     

小鼠颌下腺器官体外培养研究

目的建立小鼠颌下腺器官半固态体外培养系统，观察培养效果，初步研究小鼠颌下腺器官体外培养和体内生长差异。方法利用DMEM-F12和明胶建立半固态体外培养系统，选用出生后1天的C57小鼠颌下腺在该培养系统中进行培养，分别于培养后1、2．3天取出颌下腺，进行形态学观察，通过HE染色和AB-PAS染色观察组织学以及进行细胞培养，与体内生长的同期小鼠颌下腺进行比较。结果体外培养的小鼠颌下腺体积逐渐增大，组织进一步成熟，培养后的细胞能够正常生长，形态正常。与体内生长的同期小鼠颌下腺进行比较，其体积增大稍慢，在组织成熟，细胞形态上无明显差异。结论首次应用该半固态培养系统进行出生后小鼠颌下腺器官培养，与体内生长的同期小鼠颌下腺相比，形态学、组织学和细胞形态上无明显差异，但体积增长速度稍慢，可用于模拟体内环境进行相关研究。     

Impaired induction of cystatin S gene expression by isoproterenol in the submandibular gland of hypophysectomized rats

Cystatin S, an inhibitor of cysteine proteases, is produced and secreted by acinar cells of the rat submandibular gland. Expression of the cystatin S gene is known to be induced at high levels by the beta-adrenergic agonist isoproterenol. In the present study, we revealed that in the submandibular gland of hypophysectomized adult male rats, the levels of induced cystatin S mRNA 24 h after a single administration of isoproterenol are strikingly lower than those in the gland of normal rats. Administration of one of the pituitary-dependent hormones testosterone, estradiol, dexamethasone and thyroxine, together with isoproterenol resulted in marked enhancement of the isoproterenol-induced cystatin S mRNA expression in hypophysectomized rats, whereas administration of any of these hormones alone had no significant effect. These results suggested the existence of cross-talk between the signaling pathways of steroid hormones and isoproterenol in inducing cystatin S gene expression in the rat submandibular gland.     

Ultrastructural study of hormonally responsive striated duct cells in the mouse sublingual gland

In semithin sections stained with Heidenhain's iron hematoxylin, a few scattered granular cells were observed in the striated ducts (SDs) of sublingual glands (SLGs) of the mouse; they were seen normally only in the glands of adult males. However, it was shown by electron microscopy that many SD cells, other than these granular cells, had apical secretory granules, thus forming a granular striated tubule (named the GST in this study) in a portion of SD segments in both sexes. Sublingual GST cells had very small dense secretory granules near the apical surface, with the nucleus in the apical one-third to one-half of the cell; small Golgi apparatus; sparse rough endoplasmic reticulum (RER); and well-developed basal infoldings. However, some granular cells in male GSTs had abundant large dense secretory granules in the apical two-thirds of the cell, a basal nucleus, and modest basal infoldings. Such granular SD cells disappeared after castration in males. Granular SD cells could be induced in the GSTs of females by the injection of 5α-dihydrotestosterone (DHT), triiodothyronine (T₃), and/or dexamethasone (Dex); given simultaneously, these hormones acted synergistically in this induction. These results indicate a close similarity between the duct systems of the SLG and those of the submandibulan gland (SMG) of the mouse: granular SD cells of the GST in the SLG resemble GCT cells in the SMG in expressing some of the same biologically active polypeptides, in being sexually dimorphic, and in being under the same multihormonal regulation. Received: October 19, 2000 / Accepted: April 24, 2001     

AQP6和AQP5在人下颌下腺上的免疫组化定位

目的检测水通道蛋白亚型AQP6和AQP5在人下颌下腺的分布情况。方法对源自10例行颈淋巴清扫术患者的正常颌下腺组织,通过免疫组织化学染色方法,标记确定人下颌下腺组织中AQP6和AQP5的分布情况。结果 AQP6在部分颌下腺腺泡细胞质中表达。AQP5在浆液性腺泡和粘液性腺泡顶膜以及分泌小管均有表达,而闰管和纹状管未见表达。结论 AQP6在部分颌下腺腺泡细胞中表达,与AQP5相协调,参与唾液中水和阴离子的分泌调节。     

颌下腺鳞状细胞癌动物模型的建立

研究涎鳞状细胞癌的发生和发展规律,建立涎 鳞癌动物模型。方法：采用化学致癌剂－二甲基苯丙蒽植入颌下腺体内的方法,对５０只ＳＤ大白鼠颌下腺进行诱癌实验,观察肿瘤诱发情况。结果：共诱发肿瘤２１例,均为鳞状细胞癌。结论：用含有二甲基苯丙蒽丙蒽的明胶海绵植入大鼠颌下腺体内能成功地建立颌下腺鳞癌的动物模型。     

α1-肾上腺素受体在颌下腺的表达及苯肾上腺素对唾液分泌调节的动物实验

目的研究α-肾上腺素受体亚型在家兔颌下腺的表达和分布，及其激动剂——苯肾上腺素促家兔颌下腺唾液分泌的相关机制。方法应用RT—PCR和Western blot检测家兔正常颌下腺α1-肾上腺素受体亚型mRNA及蛋白质的表达；免疫组化法检测颌下腺α1-肾上腺素受体亚型的分布及水通道蛋白5的表达；经颌下腺导管插管给予（1×10^-8）-（1×10^-6）moL／L的苯肾上腺素，观察家兔心率和血压的变化及促颌下腺唾液分泌的量效关系。结果家兔颌下腺有α1A-α1B和α1D-肾上腺素受体3种亚型的mRNA及蛋白质的表达，并广泛分布于导管和腺泡细胞的胞膜及胞质中。给予1×10^-7moL／L苯肾上腺素7d，促进颌下腺唾液分泌增加，而对心率、血压无明显影响；水通道蛋白5在腺泡及导管细胞的顶膜和侧膜的表达增加。结论家兔颌下腺存在α1A-、α1B-和α1D-肾上腺素受体3种亚型的mRNA和蛋白质的表达。经颌下腺导管给予低剂量苯肾上腺素促进唾液分泌是安全有效的；水通道蛋白5的表达增加可能与苯肾上腺素促唾液分泌的机制有关，该研究为临床治疗领下腺功能低下提供了初步依据。     

Induction of Sca-1 in the duct cells of the mouse submandibular gland by obstruction of the main excretory duct

The effect of ligation of the main excretory duct (MED) of the mouse submandibular gland (SMG) on the expression of Sca-1, a stem cell antigen, was examined by Western blotting and immunohistochemistry. By Western blotting, the expression of Sca-1 with a molecular weight of 18 kDa was identified in the normal gland. At 1 day post-ligation, the expression level of Sca-1 was strongly increased in the experimental gland and weakly in the contralateral gland, and such expression in both glands decreased at 6 days. By immunohistochemistry, Sca-1 was detected weakly in the apical membrane of excretory duct (ED) cells of the SMG under the normal condition. By duct ligation, Sca-1 became expressed strongly in most cells of the two major duct systems, i.e., the striated duct (SD) and granular convoluted tubules (GCT), but was not detected in the acinar (Ac) cells. By fluorescence-activated cell sorter (FACS) analysis, the number of side population (SP) cells in this gland was found to be increased by ligation. These results imply that Sca-1-positive cells may have a role in the duct cell proliferation in the regeneration step elicited by MED ligation-induced injury.     

Quantitative histochemistry of sex differences of periodic acid-schiff reaction in mouse submandibular gland

Sex differences and the effects of androgen on periodic acid-Schiff (PAS) positive substance in the submandibular gland of ICR strain mice were investigated with microspectrophotometry. The relative amount of PAS-positive groups per unit volume and per cell in the secretory tubule portion accorded with tubule-cell size which was larger in untreated male mice than in female or castrated male mice; PAS-positive groups increased following administration of testosterone propionate to female and castrated male mice.The relative amount of PAS-positive groups per unit volume in the acinar portion was larger in male mice and in the testosterone propionate-administered mice than in female or castrated male mice, though this difference was less pronounced than in the secretory tubules. However, no obvious difference was observed when the relative amount of PAS-positive groups per acinar cell was compared among these groups.The cause of the difference between the relative amount of PAS-positive groups per unit volume and per cell in the acinar portion may be attributed to the changes of cell size. The acinar cell size was smaller in untreated male mice and in the testosterone propionate administered mice than in female or in castrated male mice. It seems that androgen has little effect on the PAS-positive substance in the acinar cell, in contrast to its action on this substance in the secretory tubule cell.     

SD大鼠颌下腺肿瘤动物模型的建立

目的　用不同方法构建颌下腺肿瘤动物模型。方法　将80只大鼠按干预方式不同均分为A、B、C、D 4组,A组采用2 %二甲基苯丙蒽分析纯丙酮溶液注射入颌下腺,每只0 .0 5ml;B组采用4 %二甲基苯丙蒽分析纯丙酮溶液注射入颌下腺,每只0 .0 5ml;C组采用4 %二甲基苯丙蒽分析纯丙酮溶液注射入颌下腺,每只0 .0 5ml,连续3次,每次间隔两周;D组将实验动物的胸腺摘除,其余实验条件等同C组。各实验组在开始实验1周后给予含0 .0 5 %苯巴比妥的饲料喂养3个月,且均采用自身左右对照。结果　各实验侧先后都有肿瘤出现,诱瘤率分别约为2 2 %、30 %、4 8%、6 1% ,病理分析大部分是鳞状细胞癌,另有部分纤维肉瘤。结论　用D组方法构建的动物模型有易建立、成瘤率高的特点,可以较好地观察肿瘤的发生、发展过程。     

Epiregulin is critical for the acinar cell regeneration of the submandibular gland in a mouse duct ligation model

Acinar cell regeneration from tubular structures has been reported to occur in duct‐deligated salivary glands. However, the detailed process of acinar cell regeneration has not been clarified. We have developed a mouse duct ligation model to clarify the mechanisms underlying acinar cell regeneration, and we analyzed the epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF) ligands using the model. We studied these ligands expressions in the course of acinar cell regeneration using immunohistochemistry and RT‐PCR methods. In the duct‐ligated portion of the submandibular gland (SMG) that underwent atrophy, newly formed acinar cells were observed arising from the tubular structures after the release of the duct obstruction. The constitutive expression of EGFR was observed by immunohistochemistry in both the duct‐ligated and duct‐deligated animals as well as in normal controls. The EGFR phosphorylation detected on the tubular structures after duct ligation paralleled the acinar cell regeneration. RT‐PCR showed an increase in the epiregulin and heparin‐binding EGF levels from day 0 to day 3 after the release of the duct obstruction. The EGF level was increased only after day 7. In vitro, cultured cells isolated from ligated SMGs proliferated and produced EGF ligands following the addition of epiregulin to the culture medium. These findings suggest that the tubular structures localized in an atrophic gland are the source of acinar cell regeneration of the salivary gland. The induction of EGF ligands, in particular epiregulin, may play an important role in acinar cell regeneration in this model.     

The effect of ageing on parenchymal cell populations in adult female mouse submandibular gland

The submandibular gland shows an array of responses that accompany ageing, which are usually modest. However, the submandibular acinar-cell mucin shows a substantial decline in total amount per gland. In the submandibular gland, there is also a loss of secretory parenchyma. A number of factors that could influence either parenchymal loss or a change in its cellular composition were examined in three ages of adult female mice. The goal was to see if there are ageing-related cellular changes that might have an effect on mucin production or secretion. The factors examined included DNA, protein, rates of cell division and apoptosis, cell volume and cellular composition of the parenchymal population. The parenchymal cell composition showed significant differences during ageing, with a substantial decrease in the percentage of acinar cells and increases in the percentage of both types of ductal cell components. This decline in the proportion of acinar cells in the parenchyma also reflected an overall reduction in the total number of acinar cells in the gland. Thus, the change in proportions of cells may potentially be a direct cause of the ageing-related decline in the submandibular acinar-cell mucin. The alteration in cellular composition was not attributable to changes in the cell-division indices; however, there was an increased rate of apoptosis for acinar cells that was significantly different between 3 and 28 months. The apoptotic rate doubled for acinar cells but showed no significant change in ductal cells. This selective change in the rate of apoptosis with ageing suggests that it is one of the main reasons for the decline in the proportion of acinar cells in the submandibular gland.     

Endothelin expression in salivary gland

The present paper reviews the roles of endothelin (ET) in the rat submandibular gland (SMG). ET and its mRNA are expressed in the granular convoluted tubule (GCT) segment by immunostaining and in situ hybridization, respectively. ET is synthesized in granular cells of the GCT segment, stored in secretory granules, and secreted into the oral cavity. It is well known that granular cells in the GCT segment of the SMG in mice and rats express many kinds of growth factor such as epidermal growth factor and nerve growth factor. These growth factors are discharged into saliva and thought to regulate oral-esophageal and gastrointestinal mucosa. ET acts as a potent vasoconstrictor with mitogenic property and is excreted from Weibel-Palade bodies in vascular endothelial cells. ET in the salivary origin may regulate its own functions as noted in the endothelial origin. This review deals with comparative discussion of ET and other growth factors, which originate from GCT segments in rodents.     

Unilateral maxillary molar extraction influences AQP5 expression and distribution in the rat submandibular salivary gland

ObjectiveMastication has been regarded as a crucial factor for maintaining the morphology and function of secretion in salivary glands. Although it is known that occlusion affects mastication, the detailed process for how occlusal changes affect the secretory function of salivary glands is still unknown. Aquaporin 5 (AQP5) is a membrane protein that forms water channels, and plays an important role in water transport. In this study, we investigated the structural changes and alterations in the expression and distribution of AQP5 in the rat submandibular salivary gland (SMG) under occlusal hypofunction after unilateral molar extraction.MethodsSeven-week-old male Wistar rats (n = 36) were used in the study. In the experimental group, all of the right maxillary molars were extracted. Rats with no molar extraction were used as the control group. The rats were euthanized at 7, 14 or 28 days after the procedure, and the right SMGs were isolated and subjected to histological analyses. The expression and distribution of AQP5 were detected by immunohistochemistry.ResultsMorphological analyses revealed hypertrophic changes in the acinar cells in the experimental group. Immunohistochemical staining of AQP5 was detected in the apical membrane (APM) and intercellular secretory canaliculi of acinar cells in both groups. On the other hand, the AQP5 expression in the APM and intercellular secretory canaliculi of acinar cells was less prominent in the experimental group than in the control group.ConclusionsThe present findings suggest that unilateral molar extraction has significant influences on the function of water transport in the rat SMG.