Cells of mouse submandibular salivary gland in culture in vitro

A cell culture that preserves its phenotype up to the 20th passage was obtained from mouse submandibular salivary glands. An analysis of the heterogeneous culture indicates the existence of several morphological types of cells, including small, densely packed cells of cuboidal or polygonal shapes and large, rounded cells. Epithelial cells of the submandibular gland cultured for several weeks were able to form tubular structures. Our studied cell culture of glandulocytes (cells of glandular epithelium) was represented by K19- and NGF-positive cells. It is important to note that, using both immunocytochemical staining and PCR, the expression of genes that encode the proinsulin and insulin proteins is revealed in the studied cell population.     

Study of cell culture of mouse submandibular salivary gland in vitro

Cell culture keeping its phenotype till the 20th passage was obtained from mouse submandibular salivary glands. The analysis of the heterogeneous culture showed that there were some morphological types of cells: densely packed small cells that had cuboidal or polygonal form and also large round cells. Epithelial cells of submandibular gland cultured during several weeks were able to form tubular strucures. It was shown that glandulocyte culture was presented by K19-positive and NGF-positive cells. It is important to note that expression of genes coding proinsulin and insulin was detected by immunocytochemical staining and by PCR as well.     

Human submandibular gland (HSG) cell line as a model for studying salivary gland Ca2+ signalling mechanisms

The human submandibular gland cell line (HSG) has been used as a model for studying the molecular mechanisms of salivary cells. The aim of this study was to investigate some aspects of salivary Ca2+ signalling. We focused on the presence and function of specific molecular markers of salivary cells to see whether this cell line retained normal salivary characteristics, despite the neoplastic changes. We detected the M3 acetylcholine receptor and intracellular salivary amylase mRNA with RT-PCR. Carbachol treatment caused a rapid, transient elevation of [Ca2+]i, showing that the cholinergic receptors are functional in HSG cells. Protein kinase C activation by phorbol-esther PMA, prior to carbachol treatment, inhibited the normal Ca2+ signalling pathway in HSG cells. Using selective antagonists, we also identified the dominant muscarinic receptor subtype M3 on HSG cells. We also observed that functional extracellular purinergic receptors were present on HSG cells and coupled to intracellular Ca2+ signalling. Our results suggested that the coupling mechanisms of these receptors remained relatively intact despite the neoplastic transformation. This enables us to use this cell line to model the role of muscarinic and purinergic control of salivary gland function, cell proliferation and differentiation.     

High-resolution scanning electron microscopic study of the mouse submandibular salivary gland.

The submandibular gland of the mouse was studied by high-resolution scanning electron microscopy, using the osmium-dimethylsulfoxide-osmium method. The three-dimensional structures of the intracellular membranous organelles of acinar cells were clearly revealed. The luminal surface of cisterns of the granular endoplasmic reticulum and Golgi apparatus exhibited particles of 8-15 nm in diameter. The secretory canaliculi presented short microvilli which were irregularly arranged. The striated duct cells were characterized by rich mitochondria arranged vertically in the basal portion. The lamellar mitochondrial cristae were noted in three-dimensional images. The luminal surface extended short microvilli, while that of the excretory duct cell presented complicated microplicae. The capillary endotheliocytes showed a few short microvilli, and their fenestrated areas were bordered by cytoplasmic crests. Fenestrae were 50-80 nm in diameter and showed a plug in their center. The basement membranes of the acini and capillaries showed a spongy structure with various strands and meshes. Collagenous fibrils crisscrossed on their surface.     

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.     

Potassium release from submandibular salivary gland in vitro

Rat submandibular gland slices, incubated in continuously-gassed Krebs-Ringer bicarbonate buffer, were shown to release K⁺ in response to α-adrenergic and muscarinic cholinergic stimulation. The system employed the specific α-, β-adrenergic and cholinergic receptor-blocking agents phentolamine, propranolol and atropine, respectively, in combination with the agonists l-epinephrine and carbamylcholine both of which required the presence of Ca²⁺ for their effect. The introduction of Ca²⁺ into the cell via the ionophore A23187, with all neurotransmitter receptors blocked, resulted in K⁺ release. Ouabain also allowed extensive K⁺ release which was in addition to, and hence independent of, that elicited by epinephrine and carbamylcholine. Ethacrynic acid, a potent inhibitor of salivary secretion in vivo, had no influence on K⁺ movement. K⁺ was released by both physalaemin and an eledoisin-related peptide independently of normal neurotransmitter receptors. The activity of the eledoisin-related peptide did not require the presence of extracellular Ca²⁺. The implication of cyclic GMP at some stage of K⁺ release was suggested by experiments with a phosphodiesterase inhibitor.The results support an hypothesis where the initial stimulus at either α-adrenergic or muscarinic cholinergic receptors causes an immediate permeability change such that Ca²⁺ enters the cells resulting in K⁺ release. The loss of K⁺ is quickly countered by the ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$) ATPase which would be activated by the lowered intracellular K⁺ levels.     

Benign neurilemmoma (schwannoma) masquerading as a pleomorphic adenoma of the submandibular salivary gland

A case of solitary benign neurilemmoma (schwannoma) arising in the submandibular region is presented. The tumor was mistaken clinically for an enlarged submandibular salivary gland. Fine needle aspiration cytology made an erroneous diagnosis of a pleomorphic adenoma, predominantly stromal in composition. Histology of the resection specimen resulted in the correct diagnosis of a benign schwannoma. Review of the needle aspirate demonstrated cytologic features that should enable both the correct diagnosis of this neoplasm and its distinction from pleomorphic adenoma, which it mimicked in this location.     

Estrogen-androgen antagonism in the regulation of epidermal growth factor in mouse submandibular salivary gland and kidneys

To eludicate hormonal regulation of epidermal growth factor (EGF) concentration we studied the effects in adult female mice of ovariectomy and postovariectomy treatments with testosterone plus estradiol on the EGF concentrations in submandibular salivary gland (SMG), plasma, kidneys and urine. In the tissues, we also studied the location of EGF immunohistochemically and measured EGF mRNA. After ovariectomy, SMG EGF first decreased to one third of preovariectomy level. After postovariectomy day 10 it started to increase and reached by day 80 3.5-fold the preovariectomy level. Simultaneously, EGF mRNA increased. Testosterone treatment further strongly augmented the levels of both EGF mRNA and EGF. A small dose of estradiol counteracted slightly the mRNA effect of testosterone. After ovariectomy plasma EGF first increased 1.3-fold by day 10, then returned to the initial levels, and rose again 1.6-fold by day 80. Testosterone treatment induced a further 1.5-fold increase. Estradiol did not counteract this effect. Kidney EGF decreased 15% by postovariectomy day 20. This was preceded by a decrease in EGF mRNA from day 10 onwards. The EGF concentration recovered during the 80 days, but the EGF mRNA level stayed low. Testosterone treatment further reduced the levels of both EGF mRNA and EGF. This effect was counteracted by estradiol. Urine EGF increased after ovariectomy to a peak (1.7-fold) by day 40. It then returned to the preovariectomy levels by day 80. Testosterone treatment increased urinary EGF 1.9-fold; concomitant estradiol had no effect.     

Constructing an organ: the Drosophila salivary gland as a model for tube formation

Tubes are required in metazoans to transport the liquids and gases that sustain life. The conservation of molecules and mechanisms involved in tube formation suggests that what we learn by studying simple systems will apply to related processes in higher animals. Studies over the past 10 years have revealed the molecules that specify cell fate in Drosophila salivary gland and the cellular events that mediate tube morphogenesis. Here, we discuss how anterior-posterior and dorsal-ventral patterning information specifies both the position of salivary-gland primordia and how many cells they contain. We examine the transformation of a polarized epithelial sheet into an elongated, unbranched tube, and the intrinsic and extrinsic factors that influence the final position of the salivary gland.     

Enzymatic sulfation of mucus glycoprotein in rat submandibular salivary gland

1. The enzymic activity which catalyzes transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to mucus glycoprotein was found associated with Golgi-rich membrane fraction of rat submandibular salivary gland. 2. Optimum enzyme activity was obtained with 0.5% Triton X-100, 4 mM MgCl2 and 25 mM NaF at a pH of 6.8 using desulfated submandibular salivary mucus glycoprotein. The apparent Km of the enzyme for mucus glycoprotein was 11.1 mg/ml. 3. Alkaline borohydride reductive cleavage of the synthesized 35S-labeled glycoprotein led to the liberation of the label into reduced oligosaccharides. A 75.4% of the label was found incorporated in four oligosaccharides. These were identified in order of abundance as sulfated penta-, tri-, hepta- and nonsaccharides. 4. Based on the results of chemical and enzymatic analyses of the intact and desulfated compounds the pentasaccharide was characterized as SO3H----GlcNAc beta----Gal beta----GlcNAc(NeuAc alpha----)GalNAc-ol and the trisaccharide as SO3H----GlcNAc beta----Gal beta----GalNAc-ol.     

FGF8 dose-dependent regulation of embryonic submandibular salivary gland morphogenesis

FGF8 has been shown to play important morphoregulatory roles during embryonic development. The observation that craniofacial, cardiovascular, pharyngeal, and neural phenotypes vary with Fgf8 gene dosage suggests that FGF8 signaling induces differences in downstream responses in a dose-dependent manner. In this study, we investigated if FGF8 plays a dose-dependent regulatory role during embryonic submandibular salivary gland (SMG) morphogenesis. We evaluated SMG phenotypes of Fgf8 hypomorphic mice, which have decreased Fgf8 gene function throughout embryogenesis. We also evaluated SMG phenotypes of Fgf8 conditional mutants in which Fgf8 function has been completely ablated in its expression domain in the first pharyngeal arch ectoderm from the time of arch formation. Fgf8 hypomorphs have hypoplastic SMGs, whereas conditional mutant SMGs exhibit ontogenic arrest followed by involution and are absent by E18.5. SMG aplasia in Fgf8 ectoderm conditional mutants indicates that FGF8 signaling is essential for the morphogenesis and survival of Pseudoglandular Stage and older SMGs. Equally important, the presence of an initial SMG bud in Fgf8 conditional mutants indicates that initial bud formation is FGF8 independent. Mice heterozygous for either the Fgf8 null allele (Fgf8(+/N)) or the hypomorphic allele (Fgf8(+/H)) have SMGs that are indistinguishable from wild-type (Fgf8(+/+)) mice which suggest that there is not only an FGF8 dose-dependent phenotypic response, but a nonlinear, threshold-like, epistatic response as well. We also found that enhanced FGF8 signaling induced, and abrogated FGF8 signaling decreased, SMG branching morphogenesis in vitro. Furthermore, since FGF10 and Shh expression is modulated by Fgf8 levels, we postulated that exogenous FGF10, Shh, or FGF10 + Shh peptide supplementation in vitro would largely "rescue" the abnormal SMG phenotype associated with decreased FGF8 signaling. This is as expected, though there is no synergistic effect with FGF10 + Shh peptide supplementation. These in vitro experiments model the principle that mutations have different effects in the context of different epigenotypes.     

Developmental expression of survivin during embryonic submandibular salivary gland development

Background  

The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival.     

Structural features of the submandibular salivary gland of gnotobiotic rats

The submandibular salivary gland was histochemically and electron microscopically studied in gnotobiotic and conventional Wistar rats at the age of 15 days--10 months. By the first month of age, the submandibular salivary glands in both groups of animals complete differentiation of their acini, and the gland, according to the type of its secretion, becomes seromucous with predominance of albuminous component in it. Succinate dehydrogenase activity and nonspecific esterase predominate in epithelium of the excretory ducts. At the second month of life some signs of moreasing ductal part of the gland appear in the gnotobiotic rats. This part greatly increases by the 4th--10th month of life, mainly at the expense of twisted granular sections. This increase in number and volume of the twisted granular ducts results in compressing acini and in ultrastructural disorders of granulocytes, with destruction of some mitochondria, increasing number of lysosomes, decreasing size and alterations in character of secretory granules, in structural disorders of the laminar complex. All these manifest depressing secretory function of granulocytes in the gland of gnotobiotic animals.     

Development of secretory units of mouse submandibular gland

Summary The fine structure of the secretory units of the mouse submandibular gland was studied according to the developmental sequence. The embryonic submandibular gland consists of terminal tubules and ducts. Myoepithelium is associated only with the terminal tubules, and the cells of the primary intercalated ducts show characteristics of the young striated duct cells. The major changes shortly after birth consist of: 1) opening of the secretory lumina, 2) increasing rough ER and its altered configuration, 3) dilatation of Golgi cisternae and 4) changes in the granular structure. These findings suggest that the salivary secretion first occurs after birth, and acinar differentiation or transformation of the secretory cells of the terminal tubules is induced and profoundly affected by the commencement of the secretory activity. In the intercalated ducts this process is somehow inhibited, and the granular cells found in the adult can be considered as the remnants of the secretory cells of the terminal tubules.     

ATP-induced calcium signalling in acinar cells of the submandibular salivary gland

To study changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and the total amount of calcium in cells, we used, respectively, the fluorescent dye fura 2/AM and the metallochrome dye arsenazo III. The total amount of calcium in acinar cells after their incubation in calcium-free ATP-containing extracellular solution decreased. The action of ATP induced a dose-dependent increase in the [Ca²⁺]_i; the EC₅₀ was, on average, 130 ± ± 36 μM. Calcium transients induced by ATP demonstrated no desensitization. Against the background of a blocker of ionotropic P2X receptors, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, we observed a decrease in the ATP-induced calcium transients by 72%. In addition, these transients were reduced by 65% in the calcium-free milieu, while after thapsigargin-induced exhaustion of the endoplasmic reticulum store they disappeared. This is indicative of the involvement of metabotropic P2Y receptors in the formation of the above calcium transients. Therefore, P2X and P2Y receptors participate in ATP-induced calcium signalling in acinar cells of the submandibular salivary gland; activation of these channels results in a rise in the [Ca²⁺]_i. The P2X receptors to a higher extent contribute to the formation of calcium signals; the P2Y-determined increase in the [Ca²⁺]_i is smaller (equal to about 35%). Therefore, the functionally active ligand-operated ionotropic P2Y receptors and metabotropic G protein-related P2Y receptors do exist in acinar cells of the submandibular salivary gland and play an important role in the control of functioning of this gland. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 395–402, September–December, 2005.