Morphological evidence that pentagastrin regulates secretion in the human parotid gland

Salivary secretion is principally regulated by autonomic nerves. However, recent evidence from in vivo animal experiments suggests that gastrointestinal peptide hormones can also influence saliva production. The aim of the present study was to define the secretagogue activity of the gastrin-analogue pentagastrin in human salivary glands. For this purpose, parotid tissues were exposed to pentagastrin in vitro. Morphological techniques were used to evaluate modifications to serous acinar cells associated with secretion. Using a variant of the osmium maceration method, high resolution scanning electron microscopy allowed assessment of the morphology of the cytoplasmic aspect of the plasmalemma to demonstrate secretory activity. To quantify responses to pentagastrin, we recorded morphometric data on microvilli, microbuds, and protrusions. Dose-dependent morphological changes were observed, whereas protein concentration increased in the incubate. The use of selective receptor antagonists showed pentagastrin to act principally via cholecystokinin-A receptors. The morphological responses observed following exposure to pentagastrin differed from those elicited following exposure to the pan-muscarinic agonist carbachol. This study provides the first demonstration of a direct secretory action of gastrointestinal peptides on salivary glands in humans.     

Carbonic anhydrase III — A marker of human salivary gland myoepithelial cells

Department of Pathological Anatomy and Department of Histology, S. V. Kurashov Kazan' Medical Institute. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, No. 3, pp. 320–321, March, 1991.     

Microliths in the parotid of ferret investigated by electron microscopy and microanalysis

The present investigation is an attempt to determine the occurrence, elemental composition and formation of microliths in the parotid of ferret. Parotids from four normal ferrets were examined by electron microscopy and X-ray microanalysis. Crystalline microliths were found in phagosomes of acinar cells, which occasionally contained secretory material, and in phagosomes situated between mitochondria of striated ductal cells. Crystalline microliths and microliths that consisted of granular material either without crystals or mixed with a component of crystals were found in lumina, where they were often associated with cellular debris. The crystals contained calcium and phosphorus. Phagy and stagnation related to pockets of inefficient secretory activity have been previously found to be features of the parotid of ferret. Thus, possibly persistent degradation of redundant cellular material, particularly secretory granules, in phagosomes results in accumulation of calcium and leads to calcified microliths, whereas consolidation of stagnant debris extracellularly does not involve such accumulation and leads to non-calcified or mixed microliths.     

Cytopathological features of an epithelial-myoepithelial carcinoma with predominant clear myoepithelial cells in the parotid gland

Epithelial-myoepithelial carcinoma (EMC) is a rare low-grade carcinoma occurring most frequently in the parotid gland. Most EMCs consist of two cell types that typically form double-layered ductal structures. However, occasionally EMC presents predominantly clear myoepithelial cells. A 34-year-old man visited in August 1993 and was diagnosed as having clear-cell carcinoma. The tumor was curatively resected. However, in the following 5 years, recurrence developed a total of five times. The imprint cytological feature of the recurrence at the third time showed monophasic clear cells in sheet clusters with overlapping. Most of the clear tumor cells presented an expression to alpha-smooth muscle actin (SMA). The imprint cytological feature of the recurrence at the fifth time showed increase of nuclear atypia with coarse chromatin patterns and large nucleoli. In addition to cytological findings, the cytological diagnosis of EMC with predominant clear myoepithelial cells requires a definite expression to SMA.     

Melatonin release by exocytosis in the rat parotid gland

Several beneficial effects on oral health are ascribed to melatonin. Due to its lipophilic nature, non‐protein‐bound circulating melatonin is usually thought to enter the saliva by passive diffusion through salivary acinar gland cells. Recently, however, using transmission electron microscopy (TEM), melatonin was found in acinar secretory granules of human salivary glands. To test the hypothesis that granular located melatonin is actively discharged into the saliva by exocytosis, i.e. contrary to the general belief, the β‐adrenergic receptor agonist isoprenaline, which causes the degranulation of acinar parotid serous cells, was administered to anaesthetised rats. Sixty minutes after an intravenous bolus injection of isoprenaline (5 mg kg^?1), the right parotid gland was removed; pre‐administration, the left control gland had been removed. Samples were processed to demonstrate melatonin reactivity using the immunogold staining method. Morphometric assessment was made using TEM. Gold particles labelling melatonin appeared to be preferentially associated with secretory granules, occurring in their matrix and at membrane level but, notably, it was also associated with vesicles, mitochondria and nuclei. Twenty‐six per cent of the total granular population (per 100 μm² per cell area) displayed melatonin labelling in the matrix; three‐quarters of this fraction disappeared (P < 0.01) in response to isoprenaline, and melatonin reactivity appeared in dilated lumina. Thus, evidence is provided of an alternative route for melatonin to reach the gland lumen and the oral cavity by active release through exocytosis, a process which is under the influence of parasympathetic and sympathetic nervous activity and is the final event along the so‐called regulated secretory pathway. During its stay in granules, anti‐oxidant melatonin may protect their protein/peptide constituents from damage.     

Recovery of rat submandibular salivary gland function following removal of obstruction: a sialometrical and sialochemical study

Functional recovery of the rat submandibular gland following ligation of the main excretory duct was examined. Rat submandibular glands were ligated for 1, 4 and 8 weeks using a micro-clip with a plastic tube. Micro-clips were removed and glands were allowed to recover for periods of 8, 16 and 24 weeks. Submandibular glands were stimulated with autonomimetic drugs (methacholine and isoprenaline) and salivas were collected from atrophic or de-ligated and contralateral control glands. Glands recovered almost full size (92% of control gland) following 24 weeks of de-ligation. Saliva volume secreted by ligated/de-ligated (RSM) and control (LSM) glands were similar with different doses of agonists. Protein output expressed per gram of tissue wet weight was similar from both ligated/de-ligated and control glands with all doses of agonist. Sodium and chloride levels were higher from de-ligated glands than contralateral control glands. Protein electrophoresis showed similar profiles of salivary proteins in all samples with some minor differences. Acinar cells in de-ligated glands showed a normal morphology, as indicated by light microscopy, whilst granular ductal cells were fewer and contained fewer secretory granules. Sodium potassium ATPase staining of striated ducts in de-ligated glands was similar to that of control glands. It can be concluded that rat submandibular glands can regenerate following severe atrophy and secrete normal amounts of saliva containing broadly a full profile of secretory proteins. In contrast to acinar cells, ductal cells appear not to recover full function.     

A novel role for cilia‐dependent sonic hedgehog signaling during submandibular gland development

Background: Submandibular glands (SMGs) are specialized epithelial structures which generate saliva necessary for mastication and digestion. Loss of SMGs can lead to inflammation, oral lesions, fungal infections, problems with chewing/swallowing, and tooth decay. Understanding the development of the SMG is important for developing therapeutic options for patients with impaired SMG function. Recent studies have suggested Sonic hedgehog (Shh) signaling in the epithelium plays an integral role in SMG development; however, the mechanism by which Shh influences gland development remains nebulous. Results: Using the Kif3a^f/f;Wnt1‐Cre ciliopathic mouse model to prevent Shh signal transduction by means of the loss of primary cilia in neural crest cells, we report that mesenchymal Shh activity is necessary for gland development. Furthermore, using a variety of murine transgenic lines with aberrant mesenchymal Shh signal transduction, we determine that loss of Shh activity, by means of loss of the Gli activator, rather than gain of Gli repressor, is sufficient to cause the SMG aplasia. Finally, we determine that loss of the SMG correlates with reduced Neuregulin1 (Nrg1) expression and lack of innervation of the SMG epithelium. Conclusions: Together, these data suggest a novel mechanistic role for mesenchymal Shh signaling during SMG development. Developmental Dynamics 247:818–831, 2018. © 2018 Wiley Periodicals, Inc.     

Apoptosis and proliferation during human salivary gland development

Human salivary gland (SG) branching morphogenesis is an intricate mechanism divided into stages, prebud, initial bud, pseudoglandular, canalicular, and terminal bud, to form the final lobular structure of the organ. The coordination of molecular cascades, including cell proliferation and apoptosis, are fundamental to this process. The intrinsic apoptosis pathway appears to be important in the early phases of ductal cavitation and luminisation; however, the role of the extrinsic apoptosis pathway has still to be determined. Questions remain as to whether the latter mechanism participates in the maintenance of the ductal lumen; therefore, the present study investigated the expression of proteins Prostate apoptosis response‐4 (Par‐4), Fas cell surface death receptor (Fas), Fas ligand (FasL), pleckstrin homology‐like domain family A member 1 (PHLDA1), caspase‐3, B‐cell CLL/lymphoma 2 (Bcl‐2), survivin, Ki‐67, mucin 1 (MUC1), and secreted protein acidic and cysteine‐rich (SPARC) during distinct phases of human SG development (50 specimens). This strategy aimed to draw an immunomorphological map of the proteins involved in apoptosis, cell proliferation, and tissue maturation during the SG branching morphogenesis process. Par‐4 was positive at all stages except the pre‐acinar phase. Fas and FasL were expressed in few cells. PHLDA1 was expressed in all phases but not in the terminal bud. Bcl‐2 expression was mainly negative (expressed in few cells). Survivin showed a cytoplasmic expression pattern in the early phases of development, which changed to a predominantly nuclear expression during development into more differentiated structures. Ki‐67 was expressed mainly at the pseudoglandular stage. MUC1 was positive in the pseudoglandular stage with a cytoplasmic pattern in regions of early luminal opening. Immunostaining for SPARC and caspase‐3 was negative. Our results suggest that proteins associated with the regulation of extrinsic and intrinsic apoptosis contribute to apoptosis during specific phases of the early formation of SGs in humans.     

Characterization of a folate receptor in parotid gland and a folate binding protein in saliva from humans. Epitope relatedness to human milk folate binding protein

The present study was performed to establish the antigenic identity and origin of the folate binding protein in human saliva. We identified a folate receptor in human parotid and submandibular gland which immunoreacted with antibodies against human milk folate binding protein, as evidenced by ELISA and immunostaining of ductal epithelium and secretory glandular material. The receptor concentration was 0.4-1.4 nmol 3H-folate bound/g protein. Ligand binding was of a high-affinity (K=10(10) M(-1)) type, exhibited positive cooperativity, a slow radioligand dissociation at pH 7.4, and inhibition by folate analogues. The concentration of immunoreactive folate binding protein in saliva as determined by ELISA with antibodies against human milk folate binding protein was several fold higher than that determined by radioligand binding (nil - 1 nM). This indicates that a major fraction of the immunoreactive material does not bind 3H-folate, and could represent a precursor form of the protein. In conclusion, the folate binding protein in human saliva seems to be a secretory product of the salivary glands. The protein is also epitope-related to folate binding proteins in other human mucosal secretions.     

下颌下腺主导管结扎可激活损伤组织中的干/祖细胞

背景：成体干细胞的伦理学问题较少,而且某些操作技术比较成熟,利用成体干细胞进行组织工程化涎腺的构建具有十分诱人的吸引力和极其重要的应用前景。 目的：建立下颌下腺主导管结扎的涎腺组织损伤大鼠模型,探讨涎腺组织损伤模型中成体干/祖细胞存在的可能性及可能位置。 方法：SD大鼠统一行右侧下颌下腺主导管结扎,1周后处死大鼠取出两侧腺体,通过苏木精-伊红染色、PAS糖原染色及细胞角蛋白19、Bcl-2、Ki-67等指标的免疫组织化学测定,对正常涎腺组织与建立的损伤模型组织进行比较。 结果与结论：同一只大鼠,结扎侧与对照侧体积、质量有明显的差异。对照侧下颌下腺组织呈卵圆形,色泽红润,表面光滑,有完整包膜,质地柔软;结扎后腺体萎缩,组织形态欠规整,色泽暗红,包膜充血,质地变韧,周围组织血管代偿性扩张。主导管结扎的组织损伤模型可导致PAS阳性腺细胞的消失和细胞角蛋白19阳性的小导管上皮细胞增殖,并有在未结扎的腺体中很少见到的小丛层粘连蛋白阳性细胞出现在导管周围,而抑制细胞凋亡的Bcl-2和提示增殖活跃度的Ki-67的表达均有所增强。可见下颌下腺组织中可能存在着定位于涎腺周围导管区的下颌下腺干/祖细胞;下颌下腺主导管结扎导致的组织损伤模型是一种能有效激活下颌下腺组织中干/祖细胞的方法。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Low-frequency noise effects on the rat parotid gland: A transmission electron microscopy study

Introduction: Low-frequency noise (LFN) is a ubiquitous physical stressor known to cause degenerative cellular changes and organ alterations with functional repercussions both in humans and animals. Materials and methods: After acceptance of the study protocol by a local ethics committee, 20 Wistar rats were randomly divided into two equal groups. One group was kept in silence and the other continuously exposed to LFN during 13 weeks. The rats had unlimited access to water and were fed standard rat chow. After exposure, the animals were sacrificed and the parotid glands were excised and prepared for transmission electron microscopy. Results: The acinar cells showed marked ultrastructural alterations, such as intracellular vacuolization, loss of cell polarity, increased heterochromatin, cytoplasmic inclusions, and oncocytic transformation. Conclusions: LFN induces ultrastructural changes in the rat parotid gland that correlate with previously described functional changes.     

寒冷应激对大鼠肾上腺髓质亨廷顿蛋白相关蛋白1表达的影响

目的 探讨亨廷顿蛋白相关蛋白1(HAP1)在大鼠肾上腺髓质的超微结构定位,以及寒冷应激对大鼠肾上腺髓质HAP1表达的影响. 方法 成年雄性Wistar大鼠14只,2只用于免疫电镜研究,12只用于寒冷实验研究.寒冷实验中,将动物随机分为对照组和寒冷组,每组6只,寒冷组动物放置4℃环境下,12h后用免疫组织化学和Western blotting方法 检测大鼠肾上腺髓质HAP1表达的变化. 结果 免疫电镜结果 显示,HAP1免疫反应产物分布在肾上腺髓质细胞分泌颗粒外膜及分泌颗粒间的膜性细胞器上.寒冷组大鼠肾上腺髓质HAP1的表达明显减少,和对照组比较有显著性差异( P <0.01). 结论 HAP1可能与肾上腺髓质细胞内分泌颗粒及位于分泌颗粒内的肾上腺素/去甲肾上腺素的运输和释放有关.     

Beta-adrenergic regulation of rat parotid gland exocrine protein secretion during aging

beta-Adrenergic regulation of exocrine protein secretion from the parotid gland was studied over the adult rat life span. Enzymatically dispersed cell aggregates were prepared from 3-, 6-, 12-, and 24-month-old rats and exocrine protein secretion (amylase release) measured. No age differences were seen in the time course of amylase release following (-)-isoproterenol stimulation or in the (-)-isoproterenol dose-response curve. the beta-adrenergic antagonist (+/-)-propanolol inhibited (-)-isoproterenol-stimulated protein secretion from parotid cell aggregates of young and old animals equally. Similarly dibutyryl cyclic AMP induced comparable rates of protein secretion from cells of different aged rats. Direct examination of beta-adrenergic receptor characteristics in parotid gland membranes from 3- to 24-month-old rats revealed no differences in the equilibrium dissociation constant (Kd) or maximum specific ligand binding capacity (receptor number). These results suggest that the rat parotid beta-adrenergic system remains functionally intact throughout the animals' lifetime.     

Neurokinin A in the parotid and submandibular glands of the rat: immunohistochemical localization and effect on protein and peroxidase secretion

An indirect immunofluorescence technique was used to study the distribution of neurokinin A immunoreactive (NKA-IR) nerve fibres in submandibular and parotid glands of the rat. The functional role of neurokinin A on protein and peroxidase secretion in these glands was evaluated by using in vitro methods. In the parotid gland neurokinin A immunoreactive fibres were mainly distributed around the secretory acini, but some were also in evidence around the stromal blood vessels and ducts. The number of the neurokinin A immunoreactive nerve fibres was lower in the submandibular gland than in the parotid gland. They were mainly distributed around the secretory acini and stromal blood vessels and ducts. In vitro, neurokinin A significantly stimulated the release of total amount of released proteins and peroxidase from parotid gland fragments, while in the submandibular gland only the release of peroxidase was increased. By using SDS polyacrylamide gel electrophoresis (SDS-PAGE) specific changes were found in the release of proteins after neurokinin A stimulation. The results of the present study demonstrate that neurokinin A immunoreactive nerve fibres are present in the rat parotid and submandibular glands. Their localization around the secretory elements of the glands and the effect of neurokinin A in vitro experiments indicates that neurokinin A might have a significant role in the regulation of salivary secretion.     

Expression of HER-2/neu gene and protein in salivary duct carcinomas of parotid gland as revealed by fluorescence in-situ hybridization and immunohistochemistry

AIMS: Salivary duct carcinoma is a highly malignant salivary gland tumour with aggressive clinical behaviour, characterized by histological resemblance to invasive ductal carcinoma of the breast. Amplification of HER-2/neu oncogene and over-expression of its gene product have both prognostic and therapeutic implications in breast cancer. Recent report on salivary duct carcinomas for HER-2/neu using immunohistochemistry (IHC) has shown over-expression in most cases. However, correlation between IHC and molecular genetic analysis of HER-2/neu in salivary duct carcinoma has not yet been performed. METHODS AND RESULTS: We have now evaluated 11 cases of salivary duct carcinomas for HER-2/neu status using IHC and fluorescent in-situ hybridization (FISH). To our knowledge, this is the first molecular genetic analysis of HER-2/neu in salivary duct carcinoma. CONCLUSIONS: In immunohistochemistry, over-expression of HER-2/neu protein was identified as distinct membrane staining in most carcinoma cells in all our salivary duct carcinoma cases, while only four cases revealed an amplification of HER-2/neu gene by means of FISH analysis. Both amplified and non-amplified salivary duct carcinomas with strong immunohistochemical staining for HER-2/neu protein were associated with poor clinical outcome for the patients. Apparently, HER-2/neu protein over-expression could also be controlled by mechanisms other than gene amplification. In the group of salivary gland tumours other than salivary duct carcinoma, strong over-expression was detected only in three cases of carcinoma ex pleomorphic adenoma. Thus, over-expression of HER-2/neu protein is also a useful marker of malignant transformation in pleomorphic adenomas.     

Investigation of the capillaries of the rat parotid gland during a 3-h secretory cycle

Principles governing changes in diameter of the lumen and area of the endothelium of capillaries in the parotid salivary gland of rats with an experimentally established 3-h secretory cycle, and also during circulatory ischemia of the organ for 5 min, were studied. The diameter of the lumen and area of the endothelium of the capillaries changed very little with the phase of the secretory cycle. Occlusion of the common carotid artery for 5 min likewise did not change the diameter of the lumen or the area of the endothelium of the capillaries. The results suggest that the increase in the transcapillary blood flow during secretion can be explained by the recruiting of more capillaries into the circulation.Laboratory of Electron Microscopy, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 7, pp. 104–106, July, 1975.     

Characteristics of aquaporin 1, 3, and 5 expression during early murine salivary gland development

Aquaporins (AQPs) are essential to coordinate the transit of water and ions through the cell membrane. In salivary glands (SGs), AQPs have been associated with saliva formation, facilitating water absorption through the epithelium during the formation of hypotonic saliva, which is then secreted into the oral cavity. Different members of the AQP family have been suggested to play distinct roles during embryonic development, highlighted by their specific expression patterns. Here, we have investigated the expression patterns of AQP-1, AQP-3 and AQP-5 by immunofluorescence at key stages of salivary gland development, utilising cultured mouse embryonic submandibular (SMG) and sublingual (SLG) glands. The expression of AQPs was compared to a mitotic marker, phospho-histone 3 (PH3), a myoepithelial marker, smooth muscle actin (SMA), and a vascular marker, CD31. Qualitative analysis revealed that AQP-1 and AQP-3 were primarily expressed during the earlier phases of SG morphogenesis and were associated with cells undergoing mitotic processes (PH3-positive). AQP-5, in contrast, was not associated to mitotic figures, but was predominantly expressed during late stages of SG morphogenesis. Our results highlight that AQPs are expressed from early stages of SG morphogenesis and exhibit complimentary expression patterns that may contribute to the morphogenesis of salivary glands.