Tri-phasic modulation of ACh- and NA-maintained calcium plateau by high potassium in isolated mouse submandibular granular convoluted tubular cells

Objectives

The fact that submandibular glands secrete a large amount of potassium ion upon nerve stimulation has long been recognized, but a physiological role for such high potassium in the saliva has never been systematically investigated. In the present work, high potassium effect has been investigated in the freshly isolated mouse submandibular granular convoluted tubules.

Design

Isolated intact mouse submandibular granular convoluted tubules were loaded with Fura-2, and cytosolic calcium concentration in individual tubular cells was measured by microscopic fluorospectrometry.

Results

It was found that high potassium had no effect on basal cytosolic calcium concentration, but had a tri-phasic modulation of the calcium plateau maintained by continued stimulation by acetylcholine (ACh) or noradrenaline (NA): a minor initial transient depression, followed by steady increase, completed by a robust calcium rebound spike upon removal of high potassium. The phase of steady increase was blocked without major effect on the plateau by KB-R 7943, a sodium/calcium exchange (NCX) inhibitor.

Conclusion

These data together suggest that high potassium in saliva bathing the mouse submandibular granular convoluted tubular cells has a potent feedback effect on ACh and NA stimulation, and sodium/calcium exchange is likely to play a major role in this process. Such positive feedback actions of high potassium may suggest a role for enhancing ACh- or NA-stimulated protein factor secretion from the granulated convoluted tubular cells.     

Thyroid hormone regulation of granular intercalated duct cells in the submandibular glands of female mice

Intercalated ducts (IDs) in the submandibular glands (SMGs) of mice exhibit a sexual dimorphism, in which a few cells in the IDs of females, but not of males, possess secretory granules. The effects of a hypophysectomy (Hypox) followed by the administration of triiodo-l-thyronine (T₃) on such granular intercalated duct (GID) cells in the female gland were histologically examined. Semithin sections stained with Heidenhain's iron hematoxylin revealed that Hypox resulted in the complete disappearance of the GID cells. In Hypox females, electron-microscopy examination of the ID cells whose localization corresponded to that of the GID cells in normal females showed that these cells had a pale, centric nucleus, poorly developed rough-surfaced endoplasmic reticulum (RER) and Golgi apparatus, and no secretory granules. When T₃ (1mg/kg body weight) was given to Hypox female mice every other day for 2 weeks, the GID cells reappeared in most of the ID segments. Electron microscopy revealed that these cells had abundant secretory granules in their apical cytoplasm, a nucleus located near the base of the cell, and layered cisterna of RER and segments of Golgi apparatus in the perinuclear cytoplasm. The localization and distribution of the GID cells in the T₃-treated Hypox females were almost the same as those in normal females. Taken together, these results suggest that thyroid hormones upregulate the GID phenotype, and that thyroid hormones are essential for the exocrine activities of GID cells.     

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.     

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。     

Repeated androgen and thyroid hormone injection modulates the morphology of hormone-responsive duct cells in the mouse parotid gland

When the parotid glands of normal male and female ICR mice (12 weeks of age) were examined under a light microscope, no granular cells were seen in the duct system. However, transmission electron microscopy revealed that, in both sexes, many striated duct cells contained a few electron-dense secretory granules in their subluminal cytoplasm and had formed so-called granular striated tubules (GSTs) in some of the striated duct segments. These secretory granules were not large enough to be visible with a light microscope. Fully fledged granular cells, containing large secretory granules visible with a light microscope, could be induced in the GST segments of the glands of males by injection with 5α-dihydrotestosterone (DHT), triiodothyronine (T₃), and dexamethasone (Dex), given alone or in combination every other day for 2 weeks. Dex alone showed no effect on the GSTs in this study. Both DHT and T₃, either individually or with Dex, were moderately effective, inducing a few scattered fully fledged granular cells. A stronger effect was detected after concomitant injection of DHT and T₃, with or without Dex, with more abundant fully developed granular cells appearing in the GST segments. Electron microscopy revealed that these granular cells had abundant large secretory granules in their apical two-thirds, a basal nucleus, and modest basal infoldings. By contrast, the effect of the same hormones was very weak in the glands of females, and even the concomitant injection of DHT and T₃, with or without Dex, rarely induced fully fledged granular cells. These results indicate a close similarity between the ductal systems of the major salivary glands of the mouse, in terms of some of the striated duct segments containing secretory granules, being under the same multihormonal regulation, and being sexually dimorphic.     

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.     

Immunocytochemical study of granular duct cells with a hormonally enhanced granular cell phenotype in the mouse parotid gland

In the parotid glands (PGs) of intact male mice (12 weeks of age, ICR strain), immunofluorescence labels for a true tissue kallikrein, mK1, and for nerve growth factor (NGF) were recognized through the subluminal edges of the striated duct (SD) segments and interlobular duct segments. Because of their small size, secretory granules were not detectable by light microscopy in any of the duct cells. Full-fledged granular cells, containing large secretory granules that were visible by light microscopy, were induced in the SD segments of male mice after the injection of 5α-dehydrotestosterone (DHT) and triiodothyronine (T₃), given either alone or in combination every other day for 2 weeks. A stronger effect was detected in the mice that were concomitantly injected with DHT and T₃, and more abundant, fully developed granular cells appeared in the SD segments of these mice. These full-fledged granular cells were immunoreactive for mK1, NGF, and epidermal growth factor, but not for renin. The present results indicate that some of the SD cells with small granules in the mouse PG can develop a granular cell phenotype, producing more kinds of growth factors, as a result of the actions of androgen and thyroid hormone.     

Histological changes in the mouse submandibular gland subjected to parasympathetic nerve block or ischemia: Comparison between chorda tympani resection and trophic vessel transection

The purpose of this study was to determine the effect of disorders of parasympathetic innervation or trophic vessels on the parenchymal tissue of the submandibular gland. ICR mice underwent resection of the chorda tympani nerve or transection of the trophic vessels. After treatment, the submandibular glands were removed at intervals and processed for examination using light and electron microscopy.

Immunohistochemical localization of cannabinoid receptor 1 (CB1) in the submandibular gland of mice under normal conditions and when stimulated by isoproterenol or carbachol

ObjectiveWe wished to investigate the subcellular localization of CB1, a receptor for the endocannabinoids in mouse submandibular glands (SMGs) under normal conditions and when stimulated by adrenergic or cholinergic agonists.Materials and methodsSMGs of both male and female adult mice were utilized for immunoblotting and immuno-light and –electron microscopic analyses. Isoproterenol and carbachol were used as adrenergic and cholinergic stimulants, respectively. SMGs were examined at 15, 30, 60 and 120 min after intraperitoneal injection of these agents.ResultsSelective localization of intense immunoreactivity for CB1 in the granular convoluted ductal cells was confirmed by immunoblotting and the antigen absorption test. In SMGs of control male mice, CB1-immunoreactivity was evident on the basolateral plasma membranes, including the basal infoldings, but was absent on the apical membranes in the ductal cells. Localization and intensity of CB1-immunoreactivity were essentially the same in SMGs of female mice. The immunoreactivity was transiently localized in the apical plasmalemma of some acinar and granular ductal cells of male SMGs shortly after stimulation by isoproterenol, but not by carbachol.ConclusionThe present finding suggests that CB1 functions primarily in the basolateral membranes of the granular convoluted ductal cells of SMGs under normal conditions, and that the CB1 can function additionally in the apical membrane of acinar and granular ductal cells for modulation of the saliva secretory condition via adrenoceptors.     

Parasympathectomy induces morphological changes and alters gene-expression profiles in the rat submandibular gland

Objective

The chorda-lingual (CL) nerve carries parasympathetic fibers to the hilum of the sublingual and submandibular glands (SMGs) and evokes the secretion of saliva. The effect of cutting the CL nerve on the biological processes in SMGs was investigated by examining the gene-expression profiles in the SMGs after a surgical parasympathectomy.

Methods

At day 3 after the CL nerve cut, the changes in the SMGs at both the experimental cut and contralateral control sides were analysed by microarray and light microscopy. The expression levels of 6 selected genes were confirmed by real-time PCR, Western blot and immunofluorescence staining.

Results

The wet weight of the parasympathectomised SMGs decreased significantly compared to that of the contralateral side (p < 0.05). Histological analyses after the parasympathectomy showed a widened interacinar space as well as some atropic changes to the acini of the SMGs in the cut side. Microarray analysis revealed that twofold differential expression in mRNA expression in the parasympathectomized SMGs were detected in 88 genes (0.004%): 41 genes were overexpressed, 11 were underexpressed and 36 were unknown. Changes of the expression of 6 selected genes detected by Western blot and/or real-time PCR were consistent with the microarray data.

Conclusion

The important genes involved in biological processes for salivation were identified through a large-scale gene expression analysis.     

小型猪颌下腺自体再植的实验研究

目的 在小型猪体内建立颌下腺自体再植模型，并观察再植后颌下腺的组织学变化。方法 小型猪8只(16侧颌下腺)分为三组：第一组，4侧颌下腺移植到自体腹股沟区；第二组，6侧颌下腺原位再植到颌下区，导管外置于皮肤；第三组，6侧颌下腺原位再植到颌下区，导管开口于口腔前庭。术后以导管口分泌情况及移植腺体病理检查确定手术效果。结果 第一组术后腺体全部发生血栓，腺体未能成活。第二组腺体成活有分泌3例，导管阻塞3例。第三组术后腺体成活有分泌5例，导管阻塞1例。病理检查结果显示成活腺体术后一周内，部分浆液细胞和粘液细胞可见明显的凋亡和变性。结论 小型猪领下腺不适合移植到腹股沟区。采用自体原位再植可成功建立小型猪颌下腺再植模型，其中导管开口于皮肤手术操作相对简单，适于短期观察。导管开口于口腔前庭的远期效果优于导管外置，适于长期观察。移植后腺细胞凋亡及变性可能是自体颌下腺移植术后发生休眠期时腺体分泌减少、变粘稠的病理学基础。     

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.     

Immunocytochemical localization of mK1, a true tissue kallikrein,in the mouse parotid gland: sexual dimorphism and effects of castration and hypophysectomy

In the normal parotid glands of mice at 12 weeks of age, mK1, a true tissue kallikrein, was detected at the apical rim of the striated ducts (SDs). Sexual dimorphism in the immunostaining intensity in parotid glands was seen, i.e., immunostaining was more intense in males than in females. Under electron microscopy, secretory granules, being small in size, and condensed at the subluminal cytoplasm, were labeled with immunogold particles showing the presence of mK1. These secretory granules were rather abundant and large in males. Castration in males reduced the immunoreactivity of mK1 in the SD cells because of a decrease in the number and size of secretory granules as revealed by electron microscopy. Hypophysectomy in male mice resulted in considerable loss of immunoreactivity for mK1, which was characterized under electron microscopy by complete disappearance or significant reduction of secretory granules in many SD cells. These results suggest that mK1 expression in the SD cells of murine parotid glands is regulated by pituitary-dependent hormones, and sexual dimorphism of mK1 expression is regulated by androgens.     

Increases in nerve growth factor immunoreactivity in the submandibular gland, but not in the parotid gland, of the rat following sympathetic denervation

The effect of sympathetic denervation on nerve growth factor (NGF) immunoreactivity in the submandibular and parotid glands of adult female rats using a two-site, enzyme-linked immunosorbent assay was investigated. Unilateral sympathetic denervation through the avulsion of the superior cervical ganglion resulted in elevated levels of total amounts (decreased from 40-20%) and concentrations (decreased from 45-32%) of NGF in the submandibular glands, but not in the parotid glands, on the operated side 7-28 days post-operatively. Sympathetic decentralisation had no effect. The present findings support the hypothesis that increased levels of NGF play an important part in the increased activity of the acetylcholine-synthesising enzyme, choline acetyltransferase, in the rat submandibular gland following sympathetic denervation.     

大鼠下颌下腺细胞接种到脱细胞气管基质及PGA膜上的细胞相容性比较

目的:通过体外下颌下腺细胞/材料复合体的共培养,探讨SD大鼠下颌下腺细胞接种到脱细胞气管基质及PGA膜上的组织相容性。方法:将5mm×5mm×2mm大小的PGA膜(Polyglycolide)、脱细胞处理的衍生生物气管支架材料内壁表面分别接种SD大鼠的下颌下腺腺细胞,共培养后行扫描电镜观察,检测其细胞上清液中淀粉酶含量、单位面积材料上生长的细胞数、材料上细胞的增殖能力(MTT法)等。采用SPSS11.0统计软件包对数据进行相关分析。结果:脱细胞气管基质与PGA膜对大鼠下颌下腺细胞均有较好的细胞相容性。细胞在生物材料上生长的密度从大到小依次为SD大鼠气管、家兔气管、PGA膜。气管材料与PGA膜上细胞数有显著差异(P<0.05);家兔、SD大鼠气管基质组在第7天与对照组有显著差异(P<0.05),而PGA组与对照组无显著差异(P>0.05)。扫描电镜下见天然衍生生物材料上的下颌下腺细胞增殖活跃,见分泌颗粒。家兔、SD大鼠气管支架材料上培养4d后的唾液腺细胞上清液淀粉酶活性显著高于对照组(P<0.05),而PGA膜的上清液酶活性与对照组无显著差异(P>0.05)。结论:自制的脱细胞气管基质与下颌下腺细胞有较好的细胞相容性。     

Transglutaminase 2 expression in the salivary myoepithelial cells of mouse embryo

Earlier a strong transient expression of transglutaminase 2 (TGase 2) localized at the anchoring sites of muscle bundles in human embryo was observed. In this study, we report a similar transient expression of the TGase 2 in the salivary myoepithelial cells of mouse embryo by immunohistochemistry, RNA in situ hybridisation, and RT-PCR. From 35 submandibular glands of mouse embryos and postnatal mice, a consistent expression of TGase 2 in the myoepithelial cells via a stage-specific manner was identified by mono-clonal antibody to TGase 2 immunostaining. A similar expression pattern of TGase 2 in the myoepithelial cells was also observed by RNA in situ hybridisation analysis. The expression of TGase 2 in the salivary epithelium and mesenchyme during the prenatal 14.5-15.5 days was found minimally diffusely spread and became intensely focalised in the myoepithelial cells of salivary acini and ducts during the prenatal 16.5-18.5 days but thereafter gradually decreased until postnatal 7 days and remained weak in postnatal 3 weeks. Such transient rise and fall expressions of TGase 2 were also found with the sequential amount of RT-PCR products during the same period. The alpha-smooth muscle actin (alpha-SMA) as a positive control in the myoepithelial cells of mouse submandibular glands was consistently expressed during the prenatal and postnatal period. These results of transient expression of TGase 2 in the myoepithelial cells coincided with the formation of the dendritic basket structure in the periphery of acini and ducts, suggest a possible catalytic role of transglutaminase in a newly formed cellular matrixes during the cytodifferentiating stage of mouse prenatal and neonatal submandibular glands.     

小鼠下颌下腺发育关键基因的筛选及功能预测

目的:通过筛选小鼠下颌下腺发育起关键作用的基因,并预测其基因功能,为唾液腺组织工程奠定实验基础.方法:选取小鼠胚胎第18.5天、19.5天、出生后当天、出生后3天等4个不同时期的下颌下腺,进行基因芯片检测,BeadStatio'500Svstem扫描仪(illumina,Inc.)对芯片扫描,然后采用BeadStudio Gene Expression Module图像分析软件(illumina,Inc.)对芯片灰度扫描图进行分析,构建小鼠下颌下腺的全基因表达谱.应用生物信息学STC法分析基因表达趋势,建立发育时间与基因表达相关的调控网络图,从网络中筛选关键基因,预测其基因功能,并对关键基因Skp2进行实时荧光定量PCR验证.结果:应用STC生物学方法,得到差异基因的8种显著性表达趋势,其中有4种与表型相关.构建网络图筛选关键基因,得到Ddxl、Cenpl、Fanei、Skp2、Rbm4、Dak.Skp2基因检测数据与实时荧光定量PCR验证结果一致.其中Skp2与胚胎期细胞的分化、增殖密切相关.结论:6个关键基因在小鼠下颌下腺发育过程中起重要作用.     

大鼠下颌下腺细胞与柞蚕丝素蛋白膜体外复合培养的实验研究

目的:通过大鼠下颌下腺细胞(rat submandibular gland cells,RSMGs)与柞蚕丝素蛋白(antheraea pernyi silkfibroin,ApSF)膜的体外共培养,探讨RSMGs在支架上的形态特征、黏附、增殖及分泌功能。方法:将柞蚕丝素蛋白膜、桑蚕丝素蛋白(bombyx mori silk fibroin,BmSF)膜分别接种SD大鼠的RSMGs进行共培养,并以24孔培养板单独培养的RSMGs作为阴性对照组。免疫细胞化学抗角蛋白抗体(cytokratin 8,CK8)、淀粉酶抗体(amylase)染色鉴定细胞来源;扫描电镜、荧光显微镜观察细胞支架复合生长情况;检测细胞在支架上的黏附率;MTT比色法检测材料上细胞的增殖能力;Amano法测定上清液中淀粉酶含量,检测细胞支架共培养后下颌下腺细胞的功能。采用SPSS13.0软件包对数据进行统计学分析。结果:免疫细胞化学染色观察共培养后的细胞,上皮细胞特异性抗体CK8染色阳性,腺泡上皮细胞特异性抗体Amylase染色阳性。扫描电镜可见,共培养后ApSF膜上细胞增殖活跃,表面呈现出微绒毛等超微结构,并向支架材料伸出伪足。荧光显微镜下可见,随着共培养时间的延长,支架上锚定的种子细胞数量增多,形态规则。2组支架接种细胞1 h后,RSMGs开始附着于支架上且黏附率差异不显著(P>0.05);4～12 h 2组黏附率逐渐增大,ApSF组较BmSF组更显著(P<0.05);24 h时,95%细胞黏附于2种材料上,2组差异不显著(P>0.05)。MTT提示,RSMGs与ApSF复合培养3、5 d后增殖迅速,第7天时达到峰值;ApSF组的细胞在第3、5和7天增殖速度与BmSF组差异显著(P<0.05),2组细胞增殖均与阴性对照组差异显著(P<0.05)。Amano法提示,随着接种后培养时间的延长,2组淀粉酶含量均有不同程度的增加;ApSF组在第3、5、7天时唾液淀粉酶浓度与BmSF组相比有显著差异(P<0.05),2组酶浓度均与阴性对照组差异显著(P<0.05)。结论:RSMGs与ApSF复合培养后,可保持细胞表型、增殖分化能力及分泌功能。ApSF对于RSMGs具有较好的细胞相容性。