Rat Sublingual Glands Research Articles

Degenerative changes induced by paradoxical sleep deprivation in rat sublingual glands.

The aim of this study was to evaluate whether sleep deprivation can induce degenerative changes in rat sublingual glands. For this purpose, a total of 24 males were distributed into three groups: control (n = 8), in which the animals were not subjected to any procedure; sleep deprivation (n = 8) in which the animals were submitted to sleep deprivation for 96h; recovery (n = 8), in which the animals were subjected to paradoxical sleep deprivation for 96 consecutive hours followed by 96h without intervention. Morphological changes in sublingual glands as well as the immunoexpressions of some proteins, such as Ki-67, p16, cleaved caspase-3 and BCL-2 were investigated in this setting. The results showed that paradoxical sleep deprivation induced tissue degeneration as a result of the presence of pyknosis, vacuoles and areas of salivary retention, in the experimental groups. Expression of cleaved caspase 3 and BCL-2 were increased in both sleep deprivation and recovery groups. The analysis of Ki-67 showed an increase in expression only in the recovery group, associated with a decrease in p16 levels. Sleep deprivation can induce a degenerative process in the parenchyma of sublingual gland by means of dysregulation of apoptosis associated with proliferative activity.

Immunohistochemical Expression of Transforming Growth Factor-Alpha between Rat Submandibular and Sublingual Salivary Glands during Post-Natal Development

Immunohistochemical Expression of Transforming Growth Factor-Alpha between Rat Submandibular and Sublingual Salivary Glands during Post-Natal Development

Regulation of Transforming Growth Factor-Alpha Expression by Thyroid Hormone during the Post-Natal Development of Rat Submandibular and Sublingual Salivary Glands

Regulation of Transforming Growth Factor-Alpha Expression by Thyroid Hormone during the Post-Natal Development of Rat Submandibular and Sublingual Salivary Glands

Are Demilunes in Mixed Salivary Glands Real or Fixation Artifacts? A Critique

Demilunes have historically been considered to be integral components of the secretory tree of mixed salivary glands. Based on preservation of rat sublingual glands by rapid freezing and freeze-substitution fixation, Yamashina et al. have advanced the notion that serous demilunes are artifactual products of chemical fixation, that swollen mucous cells cause a relocation of serous cells to the endpiece periphery. This concept has been uncritically accepted by several authors of histology textbooks. This note presents a compendium of observations and unanswered questions that render this proposition untenable.

Effects of glucocorticoid on postnatal development of rat sublingual glands

Hormones have been reported to be involved in salivary gland's growth and development, but few studies have investigated the effects of glucocorticoids on the morphology of the sublingual glands around the weaning period. The objective of this study was to ascertain the effects of glucocorticoid administration on rat sublingual glands around the weaning period. Male Wistar rats were administered triamcinolone, a glucocorticoid, once every other day from 8 days after birth (experimental group). A control group was given vehicle only. The sublingual glands were then extracted at 15, 20, 25, and 30 days after birth. Samples thus obtained were subjected to Alcian blue and periodic acid-Schiff staining, lectin staining, and immunohistochemical staining to assess cellular proliferative potential. And acinar cell circumferences were measured. We found that glucocorticoid had no effect on the production of acid or neutral mucopolysaccharides by acinar cells around the weaning period. Glucocorticoid administration resulted in hypertrophy of acinar cells between 15 and 30 days after birth. Early appearance of changes in α-mannose, α-glucosamine, and N-acetylglucosamine in secretory granules suggested that glucocorticoid may have acted to promote cell differentiation. The glucocorticoid had no effect on the proliferative potential of sublingual gland acinar cells around the weaning period.

CD38/ADP-ribosyl cyclase in the rat sublingual gland: Subcellular localization under resting and saliva-secreting conditions

CD38 is a 42–45kDa transmembrane glycoprotein that exhibits ADP-ribosyl cyclase enzyme activity. In the rat, we have previously reported strong ADP-ribosyl cyclase activity in the sublingual salivary gland (Masuda W. and Noguchi T. Biochem. Biophys. Res. Commun. (2000) 270, 469–472). Here, we have examined the specific localization of CD38/ADP-ribosyl cyclase activity in this gland and whether that localization changes upon saliva-secretary stimulation. Under resting conditions, CD38/ADP-ribosyl cyclase activity in the post-nuclear fraction of SLG homogenates was separated into two major peaks by sucrose density gradient centrifugation. The first peak included the plasma membrane proteins Na+/K+ ATPase and aquaporin 5, while the second peak included mucous secretory protein mucin and vesicle-associated membrane protein 2. When rats were subjected to the muscarinic agonist pilocarpine, the CD38/ADP-ribosyl cyclase activity disappeared from the second peak, as did mucin and vesicle-associated membrane protein 2. Pre-treatment of rats with the muscarinic antagonist atropine before pilocarpine administration, or adrenergic stimulation with isoproterenol, the sucrose density gradient separation profiles were same as that seen under resting condition. Using an immunofluorescent strategy, we observed the preferential localization of CD38 in the basolateral plasma membrane and intracellular granule-like membrane in sublingual acinar cells under resting conditions.

Morphological and Histochemical Changes in the Parenchyma of the Rat Parotid and Sublingual Glands with Growth and Aging

Morphological and Histochemical Changes in the Parenchyma of the Rat Parotid and Sublingual Glands with Growth and Aging

Cyclooxygenase-2 S-nitrosylation in salivary gland acinar cell inflammatory responses to <i>Porphyromonas gingivalis</i>: modulatory effect of ghrelin

Disturbances in nitric oxide synthase (NOS) system and the excessive prostaglandin (PGE2) generation are well-recognized features of oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis. Employing rat sublingual gland acinar cells, we show that P. gingivalis LPS-induced up-regulation in PGE2 generation and the enhancement in inducible (i) iNOS activity was associated with COX-2 activation through S-nitrosylation, and accompanied by the suppression in cSrc activity and the impairment in constitutive (c) cNOS phosphorylation. Further, we demonstrate that the countering effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in the increased cNOS activation through phosphorylation, repression in iNOS induction, and the reduction in PGE2 generation associated with the loss of COX-2 protein S-nitrosylation. Moreover, the effect of ghrelin on cNOS phosphorylation and the LPS-induced COX-2 S-nitrosylation was susceptible to the blockage by cSrc inhibition. Our findings suggest that P. gingivalis-induced up-regulation in iNOS leads to COX-2 S-nitrosylation and up-regulation in PGE2 generation, and that the countering effect of ghrelin is mediated through Src-dependent cNOS activation that is obligatory for the maintenance of iNOS gene suppression.

Ghrelin Protects against the Detrimental Consequences ofPorphyromonas gingivalis-Induced Akt Inactivation through S-Nitrosylation on Salivary Mucin Synthesis

Disturbances in nitric oxide synthase isozyme system and the impairment in salivary mucin synthesis are well-recognized features associated with oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis. In this study, using rat sublingual gland acinar cells, we report that P. gingivalis LPS-induced impairment in mucin synthesis and associated suppression in Akt kinase activity were accompanied by a decrease in constitutive nitric oxide synthase (cNOS) activity and an induction in inducible nitric oxide synthase (iNOS) expression. The LPS effect on Akt inactivation was manifested in the kinase S-nitrosylation and a decrease in its phosphorylation at Ser473. Further, we demonstrate that a peptide hormone, ghrelin, countered the LPS-induced impairment in mucin synthesis. This effect of ghrelin was reflected in the suppression of iNOS and the increase in Akt activation, associated with the loss in S-nitrosylation and the increase in phosphorylation, as well as cNOS activation through phosphorylation. Our findings suggest that induction in iNOS expression by P. gingivalis-LPS leads to Akt kinase inactivation through S-nitrosylation that detrimentally impacts cNOS activation through phosphorylation as well as mucin synthesis. We also show that the countering effect of ghrelin on P. gingivalis-induced impairment in mucin synthesis is associated with Akt activation through phosphorylation.

Ghrelin-induced cSrc activation through constitutive nitric oxide synthase-dependent S-nitrosylation in modulation of salivary gland acinar cell inflammatory responses to <i>Porphyromonas gingivalis</i>

A peptide hormone, ghrelin, recognized for its role in the regulation of nitric oxide production has emerged as an important modulator of oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis. As cSrc kinase plays a major role in controlling the activity of nitric oxide synthase (NOS) system, in this study we investigated the influence of P. gingivalis LPS on the processes of Src activation in rat sublingual gland acinar cells. The LPS-induced enhancement in the activity of inducible (i) iNOS and the impairment in constitutive (c) cNOS were reflected in the suppression in cSrc activity and the extent of its phosphorylation at Tyr416. Further, we show that the countering effect of ghrelin on the LPS-induced changes in cSrc activity and the extent of its phosphorylation was accompanied by a marked reduction in iNOS and the increase in cNOS activation through phosphorylation at Ser1179. Moreover, the effect of ghrelin on cSrc activation was associated with the kinase S-nitrosylation that was susceptible to the blockage by cNOS inhibition. Our findings suggest that P. gingivalis-induced up-regulation in iNOS leads to disturbances in cNOS phosphorylation that exerts the detrimental effect on the processes of cSrc activation through cNOS mediated S-nitrosylation. We also show that the effect of ghrelin on P. gingivalis-induced inflammatory changes are manifested in the enhancement in cSrc activation through S-nitrosylation and the increase in its phosphorylation at Tyr416.

Postmortem acinar autolysis in rat sublingual gland: a morphometric study

ObjectiveTo analyze and to quantify morphological acinar postmortem changes in rat sublingual glands (SLG).Material and MethodsFifty rats were divided into two groups of 25 animals each. Group I was used for morphological and morphometric evaluations and group II for the determination of gland density and processed gland volume. Acinar autolytic changes were studied at 0 (control group), 3, 6, 12 and 24 h postmortem periods. The morphometric analysis of the volume density (Vv) and total volume (VT) of intact (ia) and autolyzed (aa) acini was performed under light microscopy using a Zeiss II integration grid with 100 symmetrically distributed points.ResultsMorphologically, temporal progressive nuclear alterations and gradual loss of the structural architecture of acinar cells were found. Regarding quantitative results, both the Vvaa and the Vvia showed statistically significant differences among all postmortem periods (p<0.05). Vvaa increased from 0.42% at 0 h to 75.84% at 24 h postmortem and Vvia decreased from 71.16% to 0% over the same period. For VTaa and VTia, no statistically significant differences occurred between 12-24 h and 0-3 h (p>0.05), respectively. Vtaa increased from 0.18 mm3 at 0 h to 38.17 mm3 at 12 h, while Vtia showed a decrease from 33.47 mm3 to 0 mm3 between 3-24 h postmortem. Data concerning VTaa were adjusted by twovariable linear regression, obtaining the equation: y=-3.54 + 3.38x (r2=0.90). The VTaa growth rate calculated by this equation was 3.38 mm3/h between 0-12 h.ConclusionAcinar autolysis on rat SLG demonstrated the most significant signs during the first 6 h postmortem and was widely spread through the gland at 12 h.

Suppression by Ghrelin ofPorphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells

Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS) activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS). We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

Growth rate of the cell populations of the rat sublingual gland during the early postnatal period

The development of the rat sublingual gland during the first month of postnatal life was analyzed by morphometry. The absolute number of cells in the various morphological glandular compartments - mixed acini with serous demilunes, intercalated ducts, striated ducts and stroma - was determined by the Aherne II morphometric method for particle counting. The data of fresh glandular volume and the number of cells were analyzed by linear, exponential and parabolic regression, with the best fits being obtained with the linear equation. Based on these equations, the growth rate of glandular volume and of each cell population was calculated. Glandular volume increased about 12-fold during the period from 2 to 30 days of postnatal life, corresponding to a rate of 1.34 mm3/day. This volumetric growth of the gland was mainly due to significant 16-, 10-, 4-, 7- and 8-fold increases in the absolute number of mucous acinar, serous demilune, intercalated duct, striated duct and stromal cells, corresponding to a growth rate of 301 x 103, 228 x 103, 19 x 103, 54 x 103 and 247 x 103 cells/day, respectively. Based on the present results, we conclude that the mucous and serous cell populations of the mixed acini grew at a rate close to that of stromal cells, but considerably higher than that obtained for the intercalated duct and striated duct populations, with the lowest growth rate being observed for the ntercalated duct cell population.

Ghrelin Protection against Cytotoxic Effect of Ethanol on Rat Salivary Mucin Synthesis involves Cytosolic Phospholipase A2 Activation through S-Nitrosylation

Recent advances in identifying the salivary constituents of significance to the maintenance of soft oral tissue integrity have brought to focus the importance of a 28-amino acid peptide hormone, ghrelin. Here, we report on the role of ghrelin in countering the disturbances in salivary mucin synthesis caused by ethanol cytotoxicity in rat sublingual gland acinar cells. We show that the countering effect of ghrelin on mucin synthesis was associated with the increase in NO and PGE2 production, and the enhancement in cytosolic phospholipase A2 (cPLA2) activity. The ghrelin-induced up-regulation in mucin synthesis, like that of cPLA2 activity, was subject to suppression by Src inhibitor, PP2, ERK inhibitor, PD98059, as well as Akt inhibitor, SH-5 and ascorbate. Moreover, the loss in countering effect of ghrelin on the ethanol cytotoxicity and mucin synthesis was attained with cNOS inhibitor, L-NAME as well as COX-1 inhibitor, SC-560. Furthermore, while the effect of L-NAME was also reflected in the inhibition of the acinar cell capacity for NO and PGE2 generation, and cPLA2 S-nitrosylation, the COX-1 inhibitor caused the inhibition in PGE2 only. Our findings demonstrate that ghrelin protection of the acinar cells against ethanol cytotoxicity and the impairment in salivary mucin synthesis involves Src kinase activation of the Akt/cNOS pathway that leads to up-regulation in cNOS activity. We also show that cNOS-derived NO induction of the cPLA2 activation through S-nitrosylation, for the increase in PGE2 generation, is an essential element of the protective mechanism of ghrelin action.

Mechanism of Cytosolic Phospholipase Activation in Ghrelin Protection of Salivary Gland Acinar Cells against Ethanol Cytotoxicity

Ghrelin, a peptide hormone, newly identified in oral mucosal tissues, has emerged recently as an important mediator of the processes of mucosal defense. Here, we report on the mechanism of ghrelin protection against ethanol cytotoxicity in rat sublingual salivary gland cells. The protective effect of ghrelin was associated with the increase in NO and PGE2, and upregulation in cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release. The loss in countering effect of ghrelin occurred with cNOS inhibitor, L-NAME, as well as indomethacin and COX-1 inhibitor, SC-560, while COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced cell capacity for NO production, cPLA2 activation and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA2 phosphorylation and S-nitrosylation. Inhibition in ghrelin-induced S-nitrosylation was attained with L-NAME, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA2 protein phosphorylation as well as S-nitrosylation. Thus, ghrelin protection of salivary gland cells against ethanol involves cNOS-derived NO induction of cPLA2 activation through S-nitrosylation for the increase in AA release at the site of COX-1 action for PGE2 synthesis.

Role of ghrelin in modulation of s-nitrosylation-Dependent akt inactivation induced in salivary gland acinar cells by porphyromonas gingivalis

Ghrelin, a peptide hormone, newly identified in oral mucosal tissue, has emerged re-cently as a principal modulator of the in-flammatory responses to bacterial infection through the regulation of nitric oxide syn-thase system. In this study, using rat sub-lingual salivary gland acinar cells, we report that lipopolysaccharide (LPS) of periodon-topathic bacterium, P. gingivalis- induced enhancement in the activity of inducible ni-tric oxide synthase (iNOS) was associated with the suppression in Akt kinase activity and the impairment in constitutive (c) cNOS phosphorylation. Further, we show that the detrimental effect of the LPS on Akt activa-tion, manifested in the kinase protein S-nitrosylation and a decrease in its phos-phorylation at Ser473, was susceptible to suppression by iNOS inhibitor, 1400W. Moreover, we demonstrate that a peptide hormone, ghrelin, countered the LPS- induced changes in Akt activity and NOS system. This effect of ghrelin was reflected in the decreased in Akt S-nitrosylation and the increase in its phosphorylation at Ser473, as well as cNOS activation through phos-phorylation. Our findings suggest that P. gingivalis-induced up-regulation in iNOS leads to Akt kinase inactivation through S-nitrosylation that impacts cNOS activation through phosphorylation. We also show that the countering effect of ghrelin on P. gingivalis-induced disturbances in Akt ac-tivation are manifested in a decrease in the kinase S-nitrosylation and the increase in its phosphorylation.

Glycomic mapping of O- and N-linked glycans from major rat sublingual mucin

Carbohydrate moieties of salivary mucins play various roles in life processes, especially as a microbial trapping agent. While structural details of the salivary O-glycans from several mammalian sources are well studied, very little information is currently available for the corresponding N-glycans. The existence of N-glycans alongside O-glycans on mucin isolated from rat sublingual gland has previously been implicated by total glycosyl compositional analysis but the respective structural data are both lacking. The advent of facile glycomic mapping and sequencing methods by mass spectrometry (MS) has enabled a structural reinvestigation into many previously unsolved issues. For the first time, high energy collision induced dissociation (CID) MALDI-MS/MS as implemented on a TOF/TOF instrument was applied to permethyl derivatives of mucin type O-glycans and N-glycans, from which the linkage specific fragmentation pattern could be established. The predominant O-glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types whereas the N-glycans were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain. The masking effect of terminal sialylation on the tight binding of rat sublingual mucin to Galbeta1-->4GlcNAc specific lectins and three oligomannose specific lectins were clearly demonstrated in this study.

Leptin modulates the detrimental effect of Porphyromonas gingivalis lipopolysaccharide-induced cytosolic phospholipase A2 activation on salivary mucin synthesis via ERK-signal transduction

Activation of cytosolic phospholipase A2 (cPLA2) by bacterial LPS is considered a key step in the generation of proinflammatory lipid messengers, including platelet-activating factor (PAF), recognized as the most proximal mediator of inflammatory events triggered by bacterial infection. In this study, we report on the role of leptin in modulation of the detrimental consequences of cPLA2 activation in salivary gland acinar cells by the LPS of a periodontopathic bacterium, P. gingivalis. Employing mucous cells of rat sublingual gland, we show that the LPS-induced cPLA2 activation is associated with up-regulation in PAF generation and the impairment in mucin synthesis, and was subject to suppression by leptin. A potentiation in the countering capacity of leptin on the LPS-induced arachidonic acid release and PAF generation was attained in the presence of ERK inhibitor, PD98059, while the PI3K inhibitor, wortmannin had no effect. However, the prevention by leptin of the LPS detrimental effect on mucin synthesis was subject to suppression by the inhibitors of both PI3K and ERK. Moreover, amplification in the effect of leptin on the LPS-induced decrease in mucin synthesis was attained with cPLA2 inhibitor, MAFP as well as PAF receptor antagonist, BN52020, while the reversal of the leptin effect occurred in the presence of exogenous PAF. These findings demonstrate the involvement of leptin in countering the pathological consequences of cPLA2 activation by P. gingivalis LPS on salivary mucin synthesis through the involvement in MAPK/ERK and PI3K signaling events.

Morphological Changes in the Rat Sublingual Gland Parenchyma with Aging

Background: The characteristics of mucous cells in the aging rat sublingual gland were investigated in this study. Particular attention was paid to accumulated amyloid protein and changes of the properties of the secretory granules at the histochemical and ultrastructural level. Objective: This study was designed to examine age-related morphological changes in the sublingual gland of male Wistar rats from 12 to 27 months. Methods: For light microscopy, the sublingual glands were fixed with 10% neutral-buffered formalin, embedded in paraffin, and processed for Alcian blue, Congo red, and TUNEL staining. For transmission electron microscopy, some of the samples were fixed with Karnovsky solution, postfixed with 2% osmium tetroxide, and embedded in epoxy resin for pronase treatment. Results: The sublingual gland showed slight shrinkage after 21 months. After 24 months, Congo red staining showed positive reaction to the intralobular connective tissue surrounding the terminal portions and to the interlobular connective tissue around the blood vessels and the excretory ducts. At 27 months, some of the granules in the serous demilunes had difficulty in digesting with pronase treatment. The appearance rate of TUNEL-positive cells was low in both mucous and serous portions during the observation period, though the positive cell number was higher in the serous than in the mucous portion. Conclusions: These findings indicate that the rat sublingual gland accumulates amyloid protein in the parenchyma and changes the properties of secretory granules of the acinar cells in the serous demilune with aging, though apoptosis of the parenchymal cells and the decrease of the gland weight are slight.

Roles of Protease-Activated Receptor-2 (PAR-2), a G Protein-Coupled Receptor, in Modulation of Exocrine Gland Functions

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor, is activated by proteolytic unmasking of the N-terminal extracellular tethered ligand that presumably binds to the extracellular loop 2 of the receptor itself. PAR-2 is widely distributed in the mammalian body and plays various roles in biological events in the cardiovascular, respiratory, alimentary, and central neurons systems. PAR-2-activating peptides administered systemically to mice and rats trigger prompt salivation in vivo. In an in vitro study, PAR-2 agonists including the endogenous PAR-2 activator trypsin induce secretion of amylase and mucin from isolated rat parotid glands and sublingual glands, respectively. PAR-2-activating peptides administered systemically also modulate pancreatic exocrine secretion in vivo as well as in vitro. In the gastric mucosa, PAR-2 stimulation enhances secretion of mucus and pepsinogen and suppresses acid secretion. Tear secretion can also be caused by PAR-2-related peptides in PAR-2-dependent and -independent manners. PAR-2 thus plays a general or key role in the regulation of exocrine secretion. This review focuses on the physiologic and/or pathophysiologic roles of PAR-2 in glandular exocrine secretion. The possibility of PAR-2 as a target for drug development is also discussed.