Cells Of Mouse Submandibular Gland Research Articles

Effects of polyphenols in non-centrifugal cane sugar on saliva secretion: in vitro and in vivo experiments and a randomized controlled trial.

This study examined the bioactivities and mechanisms of the non-centrifugal cane sugar polyphenols saponarin, schaftoside, and isoschaftoside in the salivary gland and their effects on salivation. In acute isolated C57BL/6N mouse submandibular gland cells, these polyphenols led to a higher increase in intracellular calcium after stimulation with the muscarinic agonist carbachol. Stimulation of these cells with polyphenols enhanced ATP production, aquaporin-5 translocation to the plasma membrane and eliminated intracellular reactive oxygen species generated by H2O2. In addition, phosphorylation of endothelial nitric oxide synthase and increased nitric oxide production in vascular endothelial cells were observed. In vivo administration of these polyphenols to C57BL/6N male mice resulted in significantly increased blood flow (saponarin, p = 0.040; isoschaftoside, p = 0.010) and salivation (saponarin, p = 0.031). A randomized controlled trial showed that intake of non-centrifugal cane sugar significantly increased saliva secretion compared with placebo (p = 0.003). These data suggest that non-centrifugal cane sugar polyphenols affect several pathways that support salivation and increase saliva secretion by enhancing vasodilation. Hence, non-centrifugal cane sugar polyphenols can be expected to maintain saliva secretion and improve reduced saliva flow.

Laminin-111 Peptides Conjugated to Fibrin Hydrogels Promote Formation of Lumen Containing Parotid Gland Cell Clusters.

Previous studies showed that mouse submandibular gland cells form three-dimensional structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is complicated due to its lack of purity. By contrast, the use of synthetic peptides derived from Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111 peptides have been identified for salivary cells: the A99 peptide corresponding to the α1 chain from Laminin-111 and the YIGSR peptide corresponding to the β1 chain from Laminin-111, which are important for cell adhesion and migration. We created three-dimensional salivary cell clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides. Results indicate that the YIGSR peptide improved morphology and lumen formation in rat parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a combination of both peptides not only allowed the formation of functional three-dimensional salivary cell clusters but also increased attachment and number of cell clusters. In summary, we demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and differentiation of salivary gland cell clusters with mature lumens.

The Regulatory Role of Rolipram on Inflammatory Mediators and Cholinergic/Adrenergic Stimulation-Induced Signals in Isolated Primary Mouse Submandibular Gland Cells.

Exposure to bacterial lipopolysaccharides (LPS) induces inflammatory signals in salivary glands. We investigated the regulatory role of phosphodiesterase 4 (PDE4) inhibitor rolipram on inflammatory mediators and cholinergic/adrenergic stimulation-induced intracellular Ca2+ signaling in salivary acinar and ductal cells. Submandibular gland (SMG) expressed PDE4A through 4D mRNA and PDE4 was localized in the luminal membrane of SMG. LPS induced Ca2+ signaling and ROS production in SMG. Treatment with rolipram blocked LPS-induced Ca2+ increase and ROS production. The application of histamine evoked Ca2+ signals and ROS production, which were attenuated by rolipram in SMG cells. Moreover, LPS-induced NLRP3 inflammasome and cleaved caspase-1 were inhibited by rolipram. The inhibitory role of rolipram in ROS-induced Ca2+ signaling was mainly observed in acinar cells and not in ductal cells. Rolipram also protected SMG acinar but not ductal cells from LPS-induced cell membrane damage. In the case of cholinergic/adrenergic stimulation, carbachol/isoproterenol-induced Ca2+ signals were upregulated by the treatment of rolipram in SMG. In the case of cAMP-dependent ductal bicarbonate secretion by rolipram, no effect was observed on the modulation of ductal chloride/bicarbonate exchange activity. Rolipram could suppress the inflammatory signals and could be a potential therapeutic strategy against LPS-induced inflammation to protect the salivary gland cells.

Sexual dimorphism and hormonal regulation of granular convoluted tubule (GCT) cells of mouse submandibular gland

Sexual dimorphism and hormonal regulation of granular convoluted tubule (GCT) cells of mouse submandibular gland

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-immunofluorescence 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.

Dynamic Assembly of TRPC1-STIM1-Orai1 Ternary Complex Is Involved in Store-operated Calcium Influx: EVIDENCE FOR SIMILARITIES IN STORE-OPERATED AND CALCIUM RELEASE-ACTIVATED CALCIUM CHANNEL COMPONENTS

Store-operated calcium entry (SOCE) is a ubiquitous mechanism that is mediated by distinct SOC channels, ranging from the highly selective calcium release-activated Ca2+ (CRAC) channel in rat basophilic leukemia and other hematopoietic cells to relatively Ca2+-selective or non-selective SOC channels in other cells. Although the exact composition of these channels is not yet established, TRPC1 contributes to SOC channels and regulation of physiological function of a variety of cell types. Recently, Orai1 and STIM1 have been suggested to be sufficient for generating CRAC channels. Here we show that Orai1 and STIM1 are also required for TRPC1-SOC channels. Knockdown of TRPC1, Orai1, or STIM1 attenuated, whereas overexpression of TRPC1, but not Orai1 or STIM1, induced an increase in SOC entry and I(SOC) in human salivary gland cells. All three proteins were co-localized in the plasma membrane region of cells, and thapsigargin increased co-immunoprecipitation of TRPC1 with STIM1, and Orai1 in human salivary gland cells as well as dispersed mouse submandibular gland cells. In aggregate, the data presented here reveal that all three proteins are essential for generation of I(SOC) in these cells and that dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in activation of SOC channel in response to internal Ca2+ store depletion. Thus, these data suggest a common molecular basis for SOC and CRAC channels.

Drug Forecast—Solifenacin: An Investigational Anticholinergic for Overactive Bladder

To review a new anticholinergic for overactive bladder undergoing review by the U.S. Food and Drug Administration, solifenacin. A MEDLINE/PUBMED search was conducted to identify pertinent studies in the English language. In addition, proceedings of meetings of the Incontinence Society, European Association of Urology, American Urological Association, and American College of Obstetrics and Gynecology were reviewed for relevant abstracts. Additional references were obtained from the bibliographies of these sources. Data over the time period of 1986 through October 2003 were reviewed. All studies evaluating any aspect of solifenacin in animals and humans. Preclinical studies demonstrated that solifenacin was an antagonist at muscarinic cholinergic M1, M2, and M3 receptors. On the basis of comparative preclinical studies evaluating the effects of solifenacin and oxybutynin on guinea pig detrusor muscle cells, mouse submandibular gland cells, and the intact rat, solifenacin was felt to be a "uroselective" antimuscarinic. Solifenacin is predominantly eliminated via metabolism, with urinary excretion of parent compound being less than 10%. Solifenacin, dosed once daily, is significantly superior to placebo in reducing the number of micturitions, urge episodes, and urge incontinence episodes per day and increasing the volume voided per micturition. In two active-controlled trials, solifenacin was at least equivalent to tolterodine in efficacy and tolerability. The most problematic adverse effects of solifenacin are the anticholinergic effects of dry mouth, blurred vision, and constipation. Although promising in preclinical studies, the uroselectivity of the anticholinergic activity of solifenacin has not been validated in clinical trials. No comparative efficacy/tolerability data with oxybutynin are available. On the basis of available data, solifenacin does not appear to be a substantial advance upon existing anticholinergics in the management of overactive bladder.

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. 5 alpha-Dihydrotestosterone (DHT); 3,5,3'-triiodo- l-thyronine (T(3)); and dexamethasone (Dex) each enhanced the GCT phenotype of hypophysectomized males to some degree. Combined hormonal replacement with DHT + T(3) in hypophysectomized males restored a nearly normal male GCT phenotype with a full complement of secretory granules and rare basal infoldings, whereas T(3) 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 T(3) 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.

Kininogenase secretion from dispersed cells of mouse submandibular gland

Kininogenase activity in both male and female submandibular gland (SMG) began to increase at 4 weeks of age and reached a peak at 6 weeks of age, and then showed a stable level of activity until 10 weeks of age. The release of kininogenase was stimulated via activation of a-adrenoceptors alone in male SMG and via both α-adrenoceptors and muscarinic cholinergic receptors in female SMG. This indicates that there is a marked difference between male and female SMG in response to methacholine, e.g., the less granular convoluted tubule (GCT) there is in the SMG, the higher is the percentage release of kininogenase evoked by methacholine. It is suggested from these data that the site of localization of kininogenase in the male and female mouse submandibular gland is different; probably predominantly in the GCT in the male and in the striated duct in the female.

Nerve growth factor synthesis by mouse submandibular gland cells in culture

Mouse submandibular gland (SMG) cells in culture rarely retian functional properties of SMG cells in vivo. We demonstrate that both primary SMG cells and the mouse SMG cell line SCA-9 secrete biologically active nerve growth factor (NGF). However, primary cells secrete 40-fold more NGF than SCA-9 cells, demonstrating that SCA-9 cells cannot substitute for primary SMG cells for the study of SMG NGF in vitro.

Coexistence of renin and cathepsin B in secretory granules of granular duct cells in male mouse submandibular gland.

Cathepsin B, a representative lysosomal cysteine proteinase, has been demonstrated to coexist with renin in secretary granules of rat pituitary LH/FSH cells and renal juxtaglomerular cells. We investigated immunocytochemically the localization of cathepsins B, H, and L in the submandibular gland of male mice, in which active renin is also produced. By light microscopy, granular immunodeposits for cathepsin B were detected in epithelial cells of the gland, particularly in granular duct cells and interstitial cells. Immunoreactivity for cathepsins H and L was mainly found in interstitial cells, although that for cathepsin H was weakly seen in acinar cells. By electron microscopy, immunogold particles indicating cathepsin B intensely labeled small granules near the Golgi complex of granular duct cells and weakly labeled large secretory granules, whereas those showing renin labeled both granules. Double immunostaining co-localized immunogold particles showing renin and cathepsin B in small perinuclear granules near the Golgi complex. Some immunopositive granules seemed to be closely associated with the Golgi elements. These results indicate that the co-localization of renin and cathepsin B is also seen in secretory granules of granular duct cells in the mouse submandibular gland, as seen in rat juxtaglomerular and LH/FSH cells. This suggests that cathepsin B is one of the possible candidates for the renin-processing enzyme.

Proliferative response of mouse submandibular gland to androgen but not to thyroid hormone.

Hormone-induced differentiation and proliferation of cells were investigated in the submandibular gland of castrated female mice, by determining the esteroprotease activity and 3H-thymidine labelling index, respectively. Injections of 5 alpha-dihydrotestosterone (4 micrograms/g body weight/day) or L-thyroxine (0.5 microgram/g body weight/day) induced a significant increase in the activity of esteroprotease, which has been shown to be localized in the convoluted tubule cells of the submandibular gland. Injections of the above-mentioned dose of 5 alpha-dihydrotestosterone for 3 days induced a 43-fold increase in the labelling index of the convoluted tubule cells, but injections of the above-mentioned dose of L-thyroxine for any duration did not induce a significant increase in the labelling index. The present result suggests that hormones which induce differentiation of cells in mouse submandibular gland do not necessarily induce cell proliferation.

Methacholine-stimulated release of benzoyl-arginine-ethylester esterase from mouse submandibular gland cells

The possible existence of cholinoceptor for inducing benzoyl-arginine-ethylester (BAEe) esterase release in the mouse submandibular gland was investigated using dispersed-cell preparations from normal male and female, castrated male and testosterone-treated female mice. Methacholine (a cholinoceptive agonist) induced esterase release only in normal female and castrated male mice, both of which had low esterase activity in the submandibular glands, whereas norepinephrine (an alpha-adrenoceptor agonist) induced esterase release in all of the mice. Methacholine-induced esterase release was completely inhibited by atropine, a cholinoceptive antagonist, but not by phenoxybenzamine, an alpha-adrenoceptor antagonist. The effects of metacholine and norepinephrine on esterase release were additive. The results suggest the coexistence of cholinoceptor and alpha-adrenoceptors for esterase release in the mouse submandibular gland. Calcium was essential for esterase release induced by either methacholine or norepinephrine.

Release of benzoyl-arginine-ethylester esterase from dispersed cells of mouse submandibular gland

Viable dispersed cells were prepared from male mouse submandibular gland by a tissue-dissociation procedure using collagenase and trypsin, and mild shearing force. These cells were incubated in a fortified Tyrode medium under a variety of experimental conditions and the secretion of benzoyl-arginine-ethylester esterase in response to various secretagogues was investigated. The cells released the esterase time-dependently upon stimulation with norepinephrine (1.5 × 10 −6 M) up to 30min at 30 °C and up to 15 min at 37 °C. The extent of esterase release was dependent on the concentration of agonist and was not influenced by varying the number of cells. The release of esterase was caused only by α-adrenergic agonists such as norepinephrine, phenylephrine and methoxamine, and the release by norepinephrine was completely abolished by α-adrenergic blockers such as phenoxybenzamine, ergotamine and dibenamine. Calcium was essential for the esterase release mediated by norepinephrine. The secretory pattern of esterase was similar to that of kallikrein (kininogenase).

Secretion of epidermal growth factor, nerve growth factor, and renin-like enzymes by dispersed male mouse submandibular gland cells in vitro.

The submandibular gland of the mouse contains a variety of hormone-like proteins and enzymes, such as epidermal growth factor (EGF), nerve growth factor (NGF), and submandibular gland renin-like enzymes (SGR). Dispersed cells from the submandibular gland of male mice were prepared by treatment with collagenase to study the mechanisms by which these specific proteins are secreted in vitro. All three proteins were secreted concomitantly in vitro by the dispersed submandibular gland cells. Norepinephrine, epinephrine, and phenylephrine, all α-adrenergic agonists, stimulated release of these three proteins in a dose-dependent manner, whereas isoproterenol, a β-adrenergic agonist, and carbamylcholine, a cholinergic agonist, did not. The effect of α-adrenergic agonists was inhibited either by removal of calcium ions from the incubation medium or by the addition of verapamil, a selective blocker of extracellular calcium influx. The divalent cation ionophore, A-23187, stimulated release of all three proteins in a...

Effect of autonomic agents on the amount of androgen-dependent granules in convoluted tubular cells of the mouse submandibular gland.

The convoluted tubular cells of the male mouse submandibular gland contain many serous-like granules in their apical cytoplasm. The autonomic regulation of the secretory process of the contents of these granules was studied by the following two methods: (1) immunochemical method using an antiserum specific to the granular components; and (2) histometric observations using light and electron microscopes. The results obtained by these two methods were well in agreement. When male mice were administered either phenylephrine or norepinephrine, the amount of granules in the glands significantly decreased. These two adrenergic stimulators were very effective, whereas synephrine was less effective. When mice were injected with a beta-adrenergic agent(isoproterenol) or a parasympathomimetic agent (pilocarpine), the amount of granules in the glands did not change. The alpha-adrenergic blockers phenoxybenzamine and phentolamine almost completely neutralized the effect of alpha-adrenergic agents on the glands, whereas another alpha-blocker (ergotamine) was less effective. These facts suggest that the secretion of the granular components is mediated by way of alpha-adrenergic receptor sites in the glands.

Cytophotometric analysis of DNA in enzymatically-dissociated submandibular gland cells of the mouse.

ABSTRACT – DNA ia isolated secretory cells of mouse submandibular gland was analyzed by absorption microspectrophotometry and microfluoromotry. Cells were obtained by dissociation of the gland using 0.1 % collagenase and 0.2 % hyaluronidase in calcium‐ and magnesium‐free Hanks Balanced Salt Solution. Both cytophotometric techniques showed approximately 40 % of the cells in glands of 20–25 g prepubertal male mice to be involved in DNA synthesis.