Phenotype and cell proliferation activity of duct-like structures in human sublingual glands: a histological and immunohistochemical study.

UNLABELLED: There are several age-related microscopic changes in the salivary glands, including the increase in the number of duct-like structures (DLS). However, the true origin and the phenotype of the DLS are not known. OBJECTIVE: To evaluate the phenotype and the cell proliferation index of the DLS of human sublingual glands. MATERIAL AND METHODS: Sixty sublingual glands obtained from human cadavers were divided into two groups - 0-30 and 61-90 years old. The phenotype was estimated by immunostaining for cytokeratin 19 (CK 19) and the S-100 protein as well as by the presence of mucin and glycogen. The cell proliferation index was determined by the Ki-67 antibody. The histochemical techniques used periodic acid-Schiff (PAS) and Alcian Blue. In each captured microscopic field, the DLS were counted to establish a percentage for the staining profile. The statistical analysis was accomplished using Student's t-test, the Mann-Whitney test and Pearson's correlation coefficient (p<0.05). RESULTS: Comparing both groups, only CK 19 showed a statistically significant difference (p=0.033), with the strongest expression in the elderly group. There was no significant difference between PAS and Alcian Blue (p=0.270). In both groups, the immunostaining for CK 19 was stronger than that for S-100 (p=0.004;p<0.001), but there was no correlation between the two immunomarkers (ρ=-0.163; p=0.315). There was no immunostaining for Ki-67. CONCLUSIONS: DLS demonstrate a ductal phenotypic profile and do not present cell proliferation activity. DLS may represent a regressive process arising from acini or represent the result of metaplasia.

Phenotype and cell proliferation activity of duct-like structures in human sublingual glands: a histological and immunohistochemical study

There are several age-related microscopic changes in the salivary glands, including the increase in the number of duct-like structures (DLS). However, the true origin and the phenotype of the DLS are not known. Objective To evaluate the phenotype and the cell proliferation index of the DLS of human sublingual glands. Material and Methods Sixty sublingual glands obtained from human cadavers were divided into two groups - 0-30 and 61-90 years old. The phenotype was estimated by immunostaining for cytokeratin 19 (CK 19) and the S-100 protein as well as by the presence of mucin and glycogen. The cell proliferation index was determined by the Ki-67 antibody. The histochemical techniques used periodic acid-Schiff (PAS) and Alcian Blue. In each captured microscopic field, the DLS were counted to establish a percentage for the staining profile. The statistical analysis was accomplished using Student's t-test, the Mann-Whitney test and Pearson's correlation coefficient (p<0.05). Results Comparing both groups, only CK 19 showed a statistically significant difference (p=0.033), with the strongest expression in the elderly group. There was no significant difference between PAS and Alcian Blue (p=0.270). In both groups, the immunostaining for CK 19 was stronger than that for S-100 (p=0.004;p<0.001), but there was no correlation between the two immunomarkers (ρ=-0.163; p=0.315). There was no immunostaining for Ki-67. Conclusions DLS demonstrate a ductal phenotypic profile and do not present cell proliferation activity. DLS may represent a regressive process arising from acini or represent the result of metaplasia. .

Constitutive nitric oxide synthase-mediated caspase-3 S-nitrosylation in ghrelin protection against Porphyromonas gingivalis-induced salivary gland acinar cell apoptosis.

Recent advances in identifying the salivary constituents capable of influencing the oral mucosal inflammatory responses have brought to focus the importance of a peptide hormone, ghrelin. Here, we report on the involvement of ghrelin in controlling the apoptotic processes induced in sublingual salivary gland acinar cells by the lipopolysaccharide (LPS) of a periodontopathic bacterium, Porphyromonas gingivalis. We show that the countering effect of ghrelin on the LPS-induced acinar cell apoptosis was associated with the increase in constitutive nitric oxide synthase (cNOS) activity, and the reduction in caspase-3 and inducible nitric oxide synthase (iNOS). The loss in countering effect of ghrelin on the LPS-induced changes in apoptosis and caspase-3 activity was attained with Src kinase inhibitor, PP2, as well as Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME, but not the iNOS inhibitor, 1400W. The effect of ghrelin on the LPS-induced changes in cNOS activity, moreover, was reflected in the increased cNOS phosphorylation that was sensitive to PP2 as well as SH-5. Furthermore, the ghrelin-induced up-regulation in cNOS activity was associated with the increase in caspase-3 S-nitrosylation that was susceptible to the blockage by SH-5 and L-NAME. The findings point to the involvement of ghrelin in Src/Akt kinase-mediated cNOS activation and the apoptogenic signal inhibition through the NO-induced caspase-3 S-nitrosylation.

Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation.

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.

Platelet-activating factor mediates Porphyromonas gingivalis lipopolysaccharide interference with salivary mucin synthesis via phosphatidylinositol 3-kinase-dependent constitutive nitric-oxide synthase activation.

Platelet -activating factor (PAF), a phospholipid-derived messenger molecule, is now recognized as the most proximal mediator of cellular events triggered by bacterial lipopolysaccharide (LPS) stimulation. In this study, we assessed the role of PAF in the disturbances in salivary mucin synthesis evoked by LPS of periodontopathic bacterium, P. gingivalis. Using primary culture of mucous acinar cells of sublingual salivary gland, we show that a specific PAF antagonist, BN52020, prevents in a dose-dependent fashion (up to 83.7%) the LPS-induced reduction in mucin synthesis, and the effect is reflected in a marked decrease in the LPS-induced apoptosis (74.8%), NO generation (82.6%), and the expression of TNF-alpha (76.1%). The impedance by BN52020 of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), which also obviated the inhibitory effect of BN52020 on the LPS-induced upregulation in apoptosis, TNF-alpha, and NO. A potentiation in the impedance by BN52020 of the LPS detrimental effect on mucin synthesis was however attained with NOS-2 inhibitor, 1400W, while cNOS inhibitor, L-NNA caused a reduction in the impedance effect of BN52020. However, while 1400W and BN52020 countered the potentiating effect of wortmannin on the LPS-induced decrease in mucin synthesis, a further exacerbation of the effect of wortmannin occurred in the presence of L-NNA. The findings implicate PAF as a pivotal factor affecting the extent of pathological consequences of P. gingivalis infection on salivary glands capacity for mucin production, and suggest that its release in response to the LPS serves as a negative regulator of PI3K controlling the pathway of cNOS activation.

Morphometric characterization of sexual differences in the rat sublingual gland

A ocorrência de diferenças morfológicas entre sexos na glândula sublingual de ratos adultos foi verificada pela morfometria. As massas glandular absoluta e relativa das fêmeas foi, respectivamente, 21% menor e 31% maior que as dos machos. As frações de volume glandular ocupadas pelos ácinos mistos, ductos intercalares e ductos estriados não mostraram diferenças significantes entre sexos, no entanto, os seus volumes absolutos foram, respectivamente, 29%, 42% e 58% maiores nos machos. Apesar dessas diferenças nos volumes compartimentais, os seus conteúdos em número de células não apresentaram diferenças significantes entre sexos, exceto o compartimento dos ductos excretores, que mostrou maior número nos machos. Quanto ao volume celular, as células acinosas mucosas, as dos ductos estriados e as dos ductos excretores mostraram volumes, respectivamente, 13%, 33% e 47% maiores nos machos, e o das células das semiluas serosas foi 38% maior nas fêmeas. A relação superfície-volume dos ácinos e dos ductos estriados foi, respectivamente, 16% e 35% maior nas fêmeas. Baseados nos resultados obtidos, concluímos que as glândulas sublinguais das fêmeas exibem ácinos menores e ductos mais curtos e menos calibrosos e células acinosas mucosas menores e serosas maiores do que nos machos, indicando a ocorrência de dimorfismo sexual, e sugerindo que possa haver também diferenças na qualidade do produto secretado.

Involvement of hepatocyte growth factor in branching morphogenesis of murine salivary gland.

We investigated the involvement of hepatocyte growth factor (HGF) in salivary gland (SG) branching morphogenesis. The mouse submandibular gland (SMG) starts to develop at embryonic day 11.5-12 (E11.5-E12), and branching morphogenesis occurs in the area between the mandibular bone and tongue between E14 and E16.5. Real-time reverse transcriptase-polymerase chain reaction showed that the expression of the c-met/HGF receptor gene in SMG increased and peaked between E14 and E16.5, concomitant with epithelial branching, and high levels of HGF mRNA were detected in the surrounding mesenchyme at E14-E15.5. Although strong expression of the HGF and c-met transcripts was observed in the tongue muscles, this expression was limited at E13.5-E14.5. Serum-free organ cultures were established, in which SG rudiments that contained SMG and sublingual gland (SLG) primordia (explant 1) and SMG/SLG rudiments with peripheral tissue that included part of the tongue muscle (explant 2) were isolated from E13.5 or E14 embryos. Mesenchyme-free SMG epithelium was obtained by the removal of mesenchymal tissue from explant 1. In the explant 1 and 2 organ cultures, SMG/SLG rudiments showed growth and branching morphogenesis, while mesenchyme-free epithelium failed to grow. When E13.5 or E14 mesenchyme-free epithelium and a recombinant human HGF (rh-HGF) -soaked bead were placed on Matrigel, the epithelium migrated toward the bead and formed branches, while the E13 epithelium failed to branch. The exogenous application of rh-HGF and anti-HGF antibody to the SMG/SLG rudiment cultures resulted in stimulation and inhibition, respectively, of branching morphogenesis. However, the response of E13.5 SMG to rh-HGF was very weak, while the branching of E14 SMG was enhanced strongly by rh-HGF. The branching morphogenesis of SMG was also inhibited by the addition of either antisense HGF or c-met oligodeoxynucleotides to the cultures. The development of SMG in explant 2, which was significantly better than in explant 1, was comparable to that seen in vivo. Moreover, the expression of both HGF and c-Met in the SMG of explant 2 was higher than in the SMG of explant 1. These findings provide the first demonstration that the branching morphogenesis of SMG is regulated by interactions with the surrounding mesenchyme-derived HGF and c-met expression in SMG, which occur concomitant with epithelial branching. The present data also suggest that the HGF that is released transiently from tongue muscles may contribute to the rapid development of SMG at the branching stage.

Proliferation and distribution of myoepithelial cells during atrophy of the rat sublingual gland.

BACKGROUND: The present study was aimed to determine the proliferation and distribution of myoepithelial cells during atrophy of rat sublingual glands. METHODS: The excretory duct of the right sublingual gland of rats was doubly ligated with metal clips to induce atrophy in the gland. The atrophic sublingual glands were taken from 1 to 28 days after duct ligation and examined with single immunohistochemistry for actin as a marker of myoepithelial cells and with immunohistochemical double staining for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells. RESULTS: In unligated sublingual glands, myoepithelial cells embraced acini and intercalated ducts, but not striated and interlobular excretory ducts. In the early stages of atrophy, myoepithelial cells surrounded small ducts but not large ones. However, in the later stages of atrophy, myoepithelial cells were also observed at the periphery of the large ducts. The immunohistochemical double staining showed that there were PCNA-positive myoepithelial cells in the normal as well as in the atrophic sublingual glands. However, the PCNA labeling indices of myoepithelial cells were low in the unligated and atrophic sublingual glands, and there were no statistically significant differences in these labeling indices. CONCLUSION: The observations suggest that the distribution of myoepithelial cells change during atrophy of rat sublingual glands and that myoepithelial cells have low proliferative activity in both the normal and atrophic condition of rat sublingual glands.

Tumor-associated glycoprotein 72 (TAG-72) expression in salivary gland neoplasia: an immunohistochemical study using the monoclonal antibody (MAb) CC49.

OBJECTIVES: The purpose of this study was to investigate immunohistochemically the expression of tumor-associated glycoprotein 72 (TAG-72) using the monoclonal antibody (MAb) CC49 in salivary gland neoplasia and normal salivary glands in an attempt to determine the potential usefulness of MAb CC49 in diagnostic and therapeutic applications. MATERIALS AND METHODS: Eighty-six specimens (21 benign tumors, 41 malignant, and 24 normal salivary glands), fixed in 10% formalin and embedded in paraffin, were retrieved from the files of the Department of Oral Medicine and Oral Pathology at the Dental School of Aristotle University, Thessaloniki, Greece, and were retrospectively studied with hematoxylin and eosin and with the streptavidin-biotin-complex method using the MAb CC49. RESULTS: Strong immunoreactivity for TAG-72 was observed in salivary duct carcinoma, adenocarcinoma, papillary cystadenocarcinoma, low-grade mucoepidermoid carcinoma, normal submandibular, sublingual, and minor salivary glands. Weak or no immunoreactivity was found in adenoid cystic carcinoma, basal cell adenocarcinoma, polymorphous low-grade adenocarcinoma, and normal parotid gland. CONCLUSIONS: Our results suggest the potential use of MAb CC49 in the differential diagnosis of some salivary gland neoplasms in which their histopathologic features overlap, and in the radiation immunolocalization and immunotherapy of malignant tumors that are localized in the parotid gland.

Salivary gland nucleotide receptors: evidence for functional expression of both P2X and P2Y subtypes.

A growing body of information now supports the suggestion that P2 receptors for extracellular nucleotides (primarily ATP) have a role in regulating salivary gland function. There is solid pharmacological and molecular evidence for the presence of P2X ligand-gated ion channel nucleotide receptors (P2X4 and P2X7/P2Z). More recently, our group and others have obtained evidence that multiple P2Y G protein-coupled nucleotide receptors (P2Y1 and P2Y2) are also expressed. Our studies have focused on defining the conditions under which P2Y receptors are expressed, the functional consequences of their activation, and the importance of co-expression of P2X and P2Y receptors. Functional and molecular approaches have been used to identify the P2 subtypes in salivary glands and in salivary cell lines. Assays include measurement of changes in [Ca2+]i, changes in transcellular short circuit current in monolayers, and RT-PCR to assess changes in receptor mRNA levels. The main observations are: (1) P2Y1 receptor activity is present in the submandibular gland (SMG) of immature rats but decreases over the first four weeks following birth, although mRNA levels remain relatively constant; (2) P2Y2 receptors are present in the cell lines and are up-regulated during short-term culture of normal parotid, sublingual, and SMG cells and following ligation of the main excretory duct of SMG; and (3) the P2X subtypes, P2X4 and P2X7, and the P2Y subtypes, P2Y1 and P2Y2, are co-expressed in salivary glands and salivary cell lines, and exhibit distinct basolateral versus apical localization in polarized cell monolayers as well as discrete patterns of intracellular signaling.

In vitro studies of the regulation of mucous gland secretion.

Our laboratory is concerned with elucidation of mechanisms regulating the exocrine secretion of mucin glycoproteins by mucous cells of salivary mucous glands. As a model system for our studies we use short-term cultures of acinar structures isolated from rat sublingual glands. Recent results are discussed from an on-going study of cross-talk between the two primary signaling pathways regulating exocrine secretion from isolated sublingual acini: the muscarinic cholinergic and vasoactive intestinal peptide (VIP) pathways. The combination of muscarinic agonist (carbachol) and VIP elicits a secretion equivalent to about 150% of the additive sum of the secretory responses to each agonist alone. This synergistic secretory response is only observed at submaximal concentrations of carbachol. VIP thus serves to decrease the EC50 for carbachol nearly three-fold. Results are thus far consistent with the hypothesis that a sustained rise in the intracellular concentration of calcium ions induced by VIP accounts for synergistic secretion and the heightened sensitivity to carbachol.

Confocal and electron microscopy to characterize sialoglycoconjugates in mouse sublingual gland acinar cells.

Double lectin labeling for confocal microscopy and lectin-protein A-gold binding for electron microscopy were applied to the mouse sublingual gland in order to study surface and cytoplasmic sialoglycoconjugates. For this purpose, serially cut sections were submitted to sialidase followed by incubation with lectins recognizing usually acceptor sugars for terminal sialic acids. At the electron microscope level, the residues subtended to sialic acid were individually identified on adjacent sections by an indirect technique of labeling, whereas with confocal microscopy the above sugars were simultaneously visualized on the same section by a double staining method using fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated lectins. Acinar cells were found to contain the terminal sequence sialic acid-beta-galactose in abundance while the sequence sialic acid-alpha-N-acetylgalactosamine appeared to be present in modest amounts. Both sialoglycoconjugates were homogeneously codistributed inside acinar cells. The combination with a saponification method also allowed the occurrence of C4 acetylated sialic acids linked to beta-galactose to be discovered, at the electron microscope level, on acinar cell secretory products.

Immunohistochemical localization of neurotrophins and neurotrophin receptors in human and mouse salivary glands.

This study was undertaken to analyze the occurrence of low- (p75) and high-affinity (TrkA, TrkB and TrkC) neurotrophin receptor proteins in human and mouse salivary glands using immunohistochemistry. Furthermore, the presence of neurotrophins was also investigated. The study was carried out on 14 human (4 parotid, 6 submandibular and 4 sublingual glands) and 5 mouse salivary glands, using polyclonal antibodies against Trk proteins. The intensity of immunostaining was calculated automatically and evaluated in arbitrary units of grey levels. In human tissues no immunoreactivity (IR) for the assessed antigens was observed in the serous or mucous acinar cells, although TrkA IR was found in the acini of the submandibular gland. The cells of the intercalated ducts showed p75 IR (sublingual) and TrkA IR (parotid gland). The striated and excretory ducts displayed p75 IR, TrkA IR and TrkC IR in all glands, but TrkB IR was never detected. No neurotrophins were detected. In the mouse glands the ductal cells display IR for p75 (submandibular) and Trks A and C (parotid and submandibular) but not the sublingual gland. Acinar cells of the submandibular gland also show p75 IR. The only neurotrophin found in the mouse salivary glands was NGF (submandibular gland). These results suggest that neurotrophins may be involved in controlling the physiology of epithelial salivary cells.

Extracellular Mg2+ regulates the intracellular Na+ concentration in rat sublingual acini.

The intracellular free Na+ concentration ([Na+]i) increases during muscarinic stimulation in salivary acinar cells. The present study examined in rat sublingual acini the role of extracellular Mg2+ in the regulation of the stimulated [Na+]i increase using the fluorescent sodium indicator benzofuran isophthalate (SBFI). The muscarinic induced rise in [Na+]i was approximately 4-fold greater in the absence of extracellular Mg2+. When Na+ efflux was blocked by the Na+,K+-ATPase inhibitor ouabain, the stimulated [Na+]i increase was comparable to that seen in an Mg2+-free medium. Moreover, ouabain did not add further to the stimulated [Na+]i increase in an Mg2+-free medium suggesting that removal of extracellular Mg2+ may inhibit the Na+ pump. In agreement with this assumption, ouabain-sensitive Na+ efflux and rubidium uptake were reduced by extracellular Mg2+ depletion. Our results suggest that extracellular Mg2+ may regulate [Na+]i in sublingual salivary acinar cells by modulating Na+ pump activity.

Estudo morfométrico e bioquímico do crescimento das glândulas sublinguais do rato durante a vida pós-natal / Morphometrical and biochemical study of the rat sublingual glands growth during postnatal period

As glândulas sulinguais do rato albino crescem marcadamente nos primeiros 40 dias de vida pós-natal. Na presente pesquisa estudamos a participaçäo da atividade proliferativa e do aumento do volume celular neste crescimento. A atividade proliferativa foi medida pelo aumento do DNA total avaliado bioquimicamente e o aumento do volume celular foi determinado por métodos morfométricos. A análise dos resultados mostrou que: a) o DNA aumentou 717 por cento (P < 0,01) no período de 2 a 40 dias de vida pós-natal; b) o volume das células acinosas mucosas exibiu flutuaçöes estatisticamente näo significativas até o 15§ dia, sofrendo, a partir daí, um aumento de 101 por cento (P < 0,05) até o 40§ dia; c) o volume das células das semiluas serosas mostrou decréscimo de 27 por cento (P < 0,05) no período de 10 a 15 dias, seguido de aumento de 71 por cento (P < 0,05) no período de 15 a 30 dias. Esses resultados indicam que o aumento significativo de massa de glândulas sublinguais do rato durante o período inicial de vida pós-natal, ocorreu principalmente por atividade proliferativa, e também, com menor participaçäo, por aumento de volume celular, notadamente das células acinosas mucosas.

Tuft cells in the main excretory duct epithelia of the three major rat salivary glands.

We report the tuft cells in the main excretory duct epithelia of rat salivary glands. These cells exhibit similar fundamental characteristics in the salivary glands and in other organs. However, numerous membrane-bound electron-dense granules are present among the microvilli of the tuft cells in the submandibular gland, but not in other organs. The apical cytoplasm contains numerous vesicles with a filamentous substance that reacts positively for glycoconjugates. The vesicles frequently are close to the apical plasma membrane and seem to open into the lumen. Nerve endings with synaptic vesicles are seen close to the basal portion of the tuft cells. The ratio of the tuft cells to principal cells is highest in the submandibular gland and lowest in the sublingual gland. The functions of the tuft cells in the salivary glands are suggested to be secretion, absorption, and reception.

Mononuclear cells in salivary glands of normal and isoproterenol-treated rats.

The purpose of this study was to analyse the phenotypical distribution of resident cells of the mononuclear phagocyte system in rat salivary glands, and to determine whether isoproterenol induces alterations in macrophage and lymphocyte surface-marker expression. Frozen sections of gland tissues were prepared from five normal rats, and from six rats treated with 20 mg/kg isoproterenol/day for 10 days. A panel of six monoclonal antibodies was used to identify membrane markers associated primarily with monocytes (ED1), mature tissue macrophages (ED2), lymphoid macrophages (ED3), MHC class II (Ia) antigens (OX6), CD5-positive T lymphocytes (OX19), and rat B lymphocytes (OX33). Double-labelling techniques were used to detect the coexpression of ED1/ED2 and OX6/ED2 mononuclear cell markers in the major salivary glands. ED2-positive macrophages were predominant in all three major glands, ranging from 96 cells/0.87 mm2 field in the parotid gland to 165 cells/0.87 mm2 in the submandibular. OX19-positive T lymphocytes were rarely observed in submandibular and parotid glands but represented a distinguishing feature of the sublingual. Moderate numbers of ED3-positive macrophages also were detected in sublingual tissues. In the submandibular and parotid glands, isoproterenol resulted in a decrease in ED2-positive cells, but ED2-positive macrophages increased in sublingual glands with isoproterenol. Isoproterenol resulted in a decrease in MHC class II antigen expression on submandibular and sublingual mononuclear cells but an induction of Ia antigen in the parotid gland. Double labelling revealed that isoproterenol induced coexpression of ED1/ED2 markers on mononuclear cells in the submandibular glands, but ED1/ED2-positive cells were absent from other glands. However, coexpression of MHC class II markers on ED2-positive cells in the sublingual and parotid glands of normal rats was frequently observed, with isoproterenol decreasing coexpression in the sublingual gland and increasing it in the parotid. B lymphocytes were not detected in any of the glands examined. These findings indicate that important differences exist in normal resident mononuclear cell subsets among the major salivary glands of the rat. The differential effects of isoproterenol on inflammatory cells may reflect important differences in local salivary gland immunoregulation. Although salivary gland inflammation induced by isoproterenol does not appear to result from immune mechanisms, the rich population of T lymphocytes and ED3-positive macrophages, and presence of MHC class II antigens, suggest that the sublingual gland may function as an immune organ and have a role in mucosal immunity.

Regulation by extracellular Na+ of cytosolic Mg2+ concentration in Mg(2+)-loaded rat sublingual acini.

The regulation of cytosolic free Mg2+ concentration ([Mg2+]i) in Mg(2+)-loaded rat sublingual mucous acini was examined using the Mg(2+)-sensitive fluorescent indicator mag-fura-2. Loading sublingual acini with 5 mM Mg2+ elevated the [Mg2+]i from 0.35 +/- 0.01 mM to 0.66 +/- 0.01 mM. Removal of extracellular Mg2+ resulted in a significantly faster [Mg2+]i decrease in Mg(2+)-loaded acini than in unloaded acini. Membrane depolarization with high extracellular [K+] and inhibition of P-type ATPases by vanadate did not alter the [Mg2+]i decrease, indicating that the Mg2+ efflux mechanism is not electrogenic. Na(+)-free medium inhibited 80% of the [Mg2+]i decrease suggesting that a Na(+)-dependent Mg2+ efflux pathway mediates the [Mg2+]i decrease. Accordingly, the Na(+)-dependent antiport inhibitor quinidine reduced > 80% of the [Mg2+]i decrease, suggesting that the Na(+)-dependent Mg2+ efflux is mediated by the Na+/Mg2+ antiport system. Mg2+ efflux was also partly driven by K+. The [Mg2+]i decreased was significantly inhibited by carbachol, a muscarinic agonist, but not by cAMP. These results indicate that in sublingual acinar cells a Na(+)-dependent pathway mediates Mg2+ efflux and that muscarinic stimulation may regulate Mg2+ extrusion.

Estudo estereológico dos ácinos de glândulas sublinguais de ratos jovens e adultos / Stereological study of the acini of the sublingual gland of young and adult rats

Em glândulas sublinguais, provenientes de ratas com 60 e com 140 dias de idade, avaliamos as dimensöes dos ácinos e dos seus constituintes celulares. Nos animais jovens os ácinos exibiram um volume total de 36,89 ñ 1,278mmü e uma superfície total de 23,24 ñ 0,965 cm², e estavam constituídos por 263,96 ñ 16,499 X 10(5) células mucosas com volume celular médio de 942,25 ñ 69,568µmü e 155,91 ñ 5,510 X 10(5) células serosas com volume celular médio de 633,33 ñ 26,017µmü. Por outro lado, as dimensöes morfométricas obtidas nos animais de 140 dias foram : volume e superfície totais de 51,61 ñ 3,131mmü e 34,84 ñ 1,838cm² , número absoluto de células mucosas e serosas de, respectivamente, 318,98 ñ 27,960 X 10(5) e 220,29 ñ 9,554X 10(5) e volume celular médio de 1002,83 ñ 89,062µmü e 661,29 ñ37,342µmü para as células mucosas e serosas. Esses resultados mostraram que o volume total dos ácinos cresceu, no período estudado, exclusivamente por atividade proliferativa, tanto de células mucosas como de células serosas

cDNA cloning, sequencing and in situ localization of a transcript specific to both sublingual demilune cells and parotid intercalated duct cells in mouse salivary glands.

A cDNA clone derived from mouse sublingual gland was isolated from lambda-phage cDNA library. Northern blot hybridization indicated that the transcript from which it was derived was approx. 700 nucleotides in length. This mRNA encoded a protein of about 20 kDa, as determined by hybrid selection and cell-free translation. Conceptual translation of the cDNA clones showed that p20 is 170 amino acids in length. The putative protein is hydrophobic in nature, is neither a mucin-like protein nor does its amino acid sequence or composition resemble the other known mouse proteins. However, the amino acid sequence of p20 suggests that it may be from a gene or gene family homologous to rat common salivary protein 1. The p20 mRNA also appears to share a non-random degree of sequence homology with the cysteine-rich domains of bovine and porcine submandibular mucins. The p20 mRNA is abundant in the mouse sublingual gland, and its expression is approx. nine times greater than in the parotid gland. In situ hybridizations localized the p20 mRNA exclusively in the demilune cells of the sublingual gland and in the intercalated duct cells of the parotid gland. It is detectable in the neonatal and adult submandibular gland at very low levels, but is absent from liver, heart, brain, thymus, spleen, lens and lacrimal glands.