Differential coupling of the P2Y1 receptor to Galpha14 and Galphaq/11 proteins during the development of the rat salivary gland

In rat submandibular gland (SMG), the P2Y1 receptor (P2Y1R) mediates increases in the intracellular calcium concentration, [Ca2+]i that diminish as the animal ages from 1 to 4-6 weeks. However, P2Y1R mRNA levels do not change with age, suggesting that the age-dependent decrease in the [Ca2+]i response to P2Y1R agonists may be due to alterations in the activity of a component of the P2Y1R signalling pathway. OBJECTIVES: To assess whether the decrease in P2Y1R-mediated intracellular calcium signalling in SMG cells as rats age is due to a decrease in P2Y1R coupling to G proteins or to a decrease in the expression of a cognate G protein. DESIGN: SMG cells were isolated from Sprague-Dawley rats. P2Y1R function was assessed by measuring 2-MeSADP-induced increases in [Ca2+]i and ERK1/2 activation. P2Y(1)R-mediated activation of G proteins was determined by the [35S]GTPgammaS binding assay. Gq protein expression was determined by RT-PCR, Northern, and Western analysis. RESULTS: In SMG cells from 1-week-old rats, two bands (52 and 42kDa) were detected using anti-Galpha14 antibody, whereas in SMG cells from 4- to 6-week-old rats only the 42 kDa band was detected. Furthermore, 2-MeSADP-induced GTPgamma35S binding to Galpha14 and Galphaq/11 decreases in SMG cells from 4- to 6-week-old rats as compared to 1-week-old rats. CONCLUSIONS: These findings suggest that the age-dependent decrease in P2Y1R-mediated intracellular calcium signalling in rat SMG cells is due to a loss of 52 kDa Galpha14 and indicate the differential coupling of the P2Y1R to Galpha14 and Galphaq/11 as the gland develops.     

Unfolded protein response-related gene regulation in inflamed periodontal tissues with and without Russell bodies

ObjectiveTo examine the expression of unfolded protein response (UPR) genes, a set of genes that are activated to assist in protein trafficking and cellular homeostasis when endoplasmic reticulum (ER) stress occurs, in inflamed and uninflamed periodontal tissues, with or without Russell bodies (RB). RB are a histologically apparent extension of the ER that represents an accumulation of abnormal proteins that cannot be secreted or degraded and may serve as a marker of ER stress.DesignPeriodontal tissue specimens were collected and categorised histologically based on the presence of inflammation and the quantity of RB. The differential regulation of 84 UPR-related genes was examined by qRT²-PCR.ResultsUPR genes related to the inositol-requiring ER-to-nucleus signal kinase (IRE)-1 pathway, molecular chaperones and ER quality control were up-regulated in RB⁺ tissues compared with RB⁻ tissues, irrespective of inflammation. Inflamed periodontal tissues showed a marked down-regulation of heat shock protein (HSP)-70 family members.ConclusionThe presence of RB in inflamed periodontal tissues correlated with the expression of a unique set of ER stress-related genes and therefore may serve as a marker of UPR response in periodontal inflammation. Inflamed periodontal tissues showed a marked down-regulation of UPR genes, in particular HSP70. This may be contributory to disease progression in periodontal disease.     

Expression and localization of calpain 3 in the submandibular gland of mice

ObjectivesCalpains comprise a family of intracellular Ca²⁺-dependent cysteine proteases and are considered to play roles in various physiological phenomena with limited proteolytic activities against specific substrates. We herein revealed the expression and localization of calpain 3, the muscle-type calpain, in the submandibular gland (SMG) of mice.DesignThe expression of the mRNA for conventional, ubiquitous calpains 1 and 2 and skeletal muscle-specific calpain 3 was examined in the major salivary glands of mice using RT-PCR, and the expression and localization of calpain 3 protein was examined in the SMG of mice using immunohistochemistry and Western blotting.ResultsThe large catalytic subunits of calpains 1 and 2 and the small regulatory subunit common to calpains 1 and 2 were weakly expressed in the parotid gland, sublingual gland, and SMG at similar levels in males and females. In contrast, the single large catalytic subunit of calpain 3 was expressed predominantly in the SMG at markedly higher levels in males than in females and in a manner dependent on androgens. Immunoreactivity for calpain 3 was mainly localized in cells of the granular convoluted tubules (GCT) that developed preferentially in the male SMG. In GCT cells, calpain 3 immunoreactivity was localized predominantly in the cytosolic region and was absent in the secretory granules.ConclusionsThese results revealed that the GCT is the primary site of production of calpain 3 in the mouse SMG.     

Induction of Sca-1 in the duct cells of the mouse submandibular gland by obstruction of the main excretory duct

The effect of ligation of the main excretory duct (MED) of the mouse submandibular gland (SMG) on the expression of Sca-1, a stem cell antigen, was examined by Western blotting and immunohistochemistry. By Western blotting, the expression of Sca-1 with a molecular weight of 18 kDa was identified in the normal gland. At 1 day post-ligation, the expression level of Sca-1 was strongly increased in the experimental gland and weakly in the contralateral gland, and such expression in both glands decreased at 6 days. By immunohistochemistry, Sca-1 was detected weakly in the apical membrane of excretory duct (ED) cells of the SMG under the normal condition. By duct ligation, Sca-1 became expressed strongly in most cells of the two major duct systems, i.e., the striated duct (SD) and granular convoluted tubules (GCT), but was not detected in the acinar (Ac) cells. By fluorescence-activated cell sorter (FACS) analysis, the number of side population (SP) cells in this gland was found to be increased by ligation. These results imply that Sca-1-positive cells may have a role in the duct cell proliferation in the regeneration step elicited by MED ligation-induced injury.     

Expression and localization of receptor protein tyrosine phosphatase β and its ligand pleiotrophin in the submandibular gland of mice

ObjectivesThe family of receptor protein tyrosine phosphatase β (RPTPβ) is composed of 4 splice variants and thought to play roles in the neural migration and outgrowth. Several ligands including the growth factor pleiotrophin (PTN) bind to RPTPβ and inhibit its phosphatase activity, thereby activating cellular signalling pathways. We examined the expression and localization of RPTPβ and its ligands in the submandibular gland (SMG) of mice, which is known for a prominent sexual dimorphism in the duct system.DesignThe homogenates and tissue sections of male and female mouse SMG were analysed with RT-PCR, Western blotting, and immunohistochemistry.ResultsThe short receptor type of RPTPβ (RPTPβ-S) was dominantly expressed in the SMG, and the male gland had significantly higher levels of RPTPβ-S expression than the female gland. In the male, RPTPβ-S was localized predominantly in intercalated duct (ID) cells, but was not found in granular convoluted tubule (GCT) cells or acinar cells. In the female, weaker reactivity was demonstrated in both ID and striated duct (SD) cells. Of the known ligands for RPTPβ, PTN was expressed in the SMG, without sexual difference in levels. In the male, PTN was localized in ID cells as well as in cells located in the distal ends of GCT that are in close vicinity to the ID, whereas in the female PTN was colocalized with RPTPβ-S throughout ID and SD cells.ConclusionsThese results indicated that the distribution of RPTPβ-S and its ligand PTN has a close relation to the sexual dimorphism in the duct system of mouse SMG.     

小鼠颌下腺器官体外培养研究

目的建立小鼠颌下腺器官半固态体外培养系统，观察培养效果，初步研究小鼠颌下腺器官体外培养和体内生长差异。方法利用DMEM-F12和明胶建立半固态体外培养系统，选用出生后1天的C57小鼠颌下腺在该培养系统中进行培养，分别于培养后1、2．3天取出颌下腺，进行形态学观察，通过HE染色和AB-PAS染色观察组织学以及进行细胞培养，与体内生长的同期小鼠颌下腺进行比较。结果体外培养的小鼠颌下腺体积逐渐增大，组织进一步成熟，培养后的细胞能够正常生长，形态正常。与体内生长的同期小鼠颌下腺进行比较，其体积增大稍慢，在组织成熟，细胞形态上无明显差异。结论首次应用该半固态培养系统进行出生后小鼠颌下腺器官培养，与体内生长的同期小鼠颌下腺相比，形态学、组织学和细胞形态上无明显差异，但体积增长速度稍慢，可用于模拟体内环境进行相关研究。     

The effect of salivary gland extracts on the histology of lymphoid organs and salivary glands in mice

Ligation of the excretory duct and artery of the submandibular gland (SMG) of male Swiss mice led to atrophy of the gland within 7 days. The effects of a crude extract of the SMG and sublingual gland (SLG) on the histology of spleen, thymus, lymph nodes, SMG and SLG were studied. Four groups of mice were used. Three groups were injected intraperitoneally for 7 consecutive days with SLG extract (Group I), SMG extract (Group II), saline (Group III) and Group IV was not treated. On day 7, the animals were killed and the spleen, thymus, salivary glands with the regional lymph nodes were weighed and fixed. Histological studies and histometric measurements were made of the thymus, the spleen and of the acinar and tubular areas of the SMG. The volumes of the SMG and SLG were also estimated. In the SMG and SLG, infiltration of mononuclear cells occurred in Groups I and II. At the same time there was a reduction of the tubular part of the SMG and a decrease in the volume of the SMG and SLG, compared with Groups III and IV. An immune response was detected in the spleen and regional lymph nodes and the number of lymphoblasts had increased in the cortex of the thymus. Injections of SMG and SLG extracts induced both an antibody and a cell-mediated immune response.     

Localization and expression pattern of amelotin,odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva

The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.     

The diagnostic utility of biopsies from the submandibular and labial salivary glands in IgG4-related dacryoadenitis and sialoadenitis,so-called Mikulicz's disease

IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) is characterized by serum IgG4 elevation and the infiltration of IgG4-positive plasma cells in glandular tissues. For definitive diagnosis of IgG4-DS, biopsies of local lesions are recommended to exclude Sjögren's syndrome (SS), malignant tumours, and similar disorders. In this study, we examined the diagnostic utility of submandibular gland (SMG) and labial salivary gland (LSG) biopsies in IgG4-DS. Fourteen patients presenting with swelling of the SMG (eight females and six males) underwent both SMG and LSG biopsies. The sensitivity, specificity, and accuracy of SMG biopsies were all 100.0%. In contrast, those of LSG biopsies were 69.2%, 100.0%, and 71.4%, respectively. Thirty-three out of 61 LSG biopsies (54.1%) from all 14 patients were positive for the diagnostic criteria of IgG4-DS (IgG4-positive/IgG-positive plasma cells >0.4). None of the patients experienced complications such as facial nerve palsy, sialocele, or hyposalivation. The IgG4/IgG ratio showed no significant correlation between the LSG and SMG. The final diagnosis was IgG4-DS in 13 patients and marginal zone B-cell lymphoma (MZL) in one. These results suggest that incisional biopsy of the SMG is useful and appropriate for the definitive diagnosis of IgG4-DS, while diagnosis by LSG biopsy alone requires more caution.     

A biochemical analysis of parotid and submandibular salivary gland function with age after simultaneous stimulation with pilocarpine and isoproterenol in female NIA Fischer 344 rats.

This analysis of physiological, biochemical and molecular changes related to aging was made in 3-, 12- and 24-month-old rats. The salivary gland weight/body weight ratio and the structural membrane proteins did not change with age for either gland, but a significant age-related decline in DNA synthesis for both glands was detected, unrelated to the hormonal responsiveness at the level of the plasma membrane. There was a marked increase in the concentration of soluble proteins in adolescent parotid gland and, for the two older age groups, in submandibular gland. The saliva flow rate was different when expressed as volume per time, as volume per time and g glandular wet weight, and/or kg body weight. The concentration of secreted proteins was not affected by age in either gland. The total amount of proteins secreted over 30 min revealed no age-related perturbation for the parotid gland, but showed a significant age-related increase in submandibular saliva. Sodium dodecyl sulphate-polyacrylamide gel analysis revealed changes in the protein bands between 39 and 50 kDa in the Coomassie blue-stained gels from 12-month-old animals. Amylase showed an initial increase (12 months), followed by a marked decline in its activity in parotid saliva. The glandular supernatant had low residual cellular amylase activity after stimulation. Therefore, secretory impairment with age after pilocarpine-isoproterenol stimulation was excluded. Analysis of total RNA showed a pronounced decrease of amylase mRNA in the parotid gland between 12 and 24 months of age. No amylase mRNA was expressed in any of the submandibular samples. For epidermal growth factor, total saliva showed a decrease with age. It seemed that the submandibular gland followed the same picture with age as the parotid gland, with a specific decline in the biosynthesis of single secretory proteins.     

Sodium-phosphate cotransporter in human salivary glands: molecular evidence for the involvement of NPT2b in acinar phosphate secretion and ductal phosphate reabsorption

OBJECTIVE: In order to elucidate the cellular and molecular mechanisms of phosphate secretion by human salivary glands, the expression and intracellular distribution of sodium-phosphate cotransporters was investigated. DESIGN: Total RNA was extracted from 33 parotid gland (PG) and 35 submandibular gland (SMG) samples and RT-PCR was performed using gene specific primers for all known sodium-phosphate cotransporters. An antibody was raised against an NPT2b epitope and the cellular and intracellular distribution was investigated by immunohistochemistry. RESULTS: No mRNA for the type I cotransporter NPT1 was found. Out of the type II phosphate cotransporters only message for NPT2b but not for NPT2a or NPT2c could be detected in about the same number of samples (76% in PG versus 69% in SMG). Type III cotransporter mRNA was also found in both glands, PIT1 gave positive results for 93% of PG samples compared to 69% of SMG samples. For PIT2 also, a higher expression was found in PG than in SMG, although the difference was smaller (79% versus 51%). Immunostaining for NPT2b was found both in the acini and in the ducts, with a stronger reaction in the latter. In acinar cells, NPT2b was restricted to the basal-lateral plasma membrane, in duct cells, a broad band of reactivity was located in the apical part of the cell. CONCLUSIONS: These findings suggest a secondary active secretion of phosphate into the primary saliva. Ductal cells appear to be able to reabsorb phosphate, thereby modifying the phosphate concentration in the final saliva.     

Compensatory hyperplasia of the rat submandibular gland following unilateral extirpation

This study investigated the morphological changes in rat submandibular glands undergoing compensatory hyperplasia. Fifteen adult male rats underwent left submandibulectomy, after which they were killed in groups of five (at days 3, 7, and 14), and their right submandibular glands (SMG) were excised. Fifteen control rats were killed in groups of five (at days 0, 7, and 14), and their right SMG were removed. Sections of 3 microns were cut, and the parenchymal and stromal cells were counted in 50 microscopic fields and sorted according to their morphological features and "class". Class is equivalent to the number of nuclei in an acinar or tubular cross-section. No change in glandular weight was noted post-surgery. Total cell count/field rose to 138.5 +/- 7.1% of control values on day 3 after gland extirpation, remaining almost constant thereafter until the end of the experiment. Acinar cell count and class showed a 154.1% peak on day 3, followed by a 30% drop in cell count by day 7 and an equivalent decline in class by day 14. Tubular cell count increased gradually to 146.5% by day 14, without a change in class. In the first week, the increase in tubular cells was mainly due to intercalated duct (ID) cells, while in the second week, there was a sharp rise in granular duct (GD) cells. This diverging cellular behavior indicates that the GD cell stems from the ID cell. The cellular changes in the hyperplastic SMG indicate death of newly generated acinar cells and expansion of the glandular progenitor compartment, as expressed in elongation of the ID.     

Effect of castration and androgen supplementation on sialic acid levels in rat submandibular gland

The effects of castration and testosterone propionate (TP) on rat submandibular gland (SMG) size and sialic acid levels were compared. Castration resulted in a slight decrease in SMG size and a significant reduction in both the whole gland content and concentration of sialic acid. These changes were reversed completely by TP.     

Effect of inflammation upon human gingival cyclic AMP metabolism

The effect of the increasing degree of human gingival inflammation on adenylate cyclase (basal, fluoride stimulated) and low Km and high Km cAMP phosphodiesterase activities were evaluated in separate studies. Human gingival biopsies were classified by the Löe Bleeding Index as (1) mildly, (2) moderately, and (3) markedly inflamed. Basal and F stimulated adenylate cyclase (cAMP synthesis) activities were found to be unaltered by the increasing degree of inflammation when the data were expressed on either a mg wet wt, or mg protein basis. A significant loss of F stimulated adenylate cyclase (mg protein) activity was observed in the moderately inflamed group when the data were compared with either the mildly or markedly inflamed groups of tissue. The low Km, and high Km cAMP phosphodiesterase activities (cAMP degradation) were found to he unaffected by gingival inflammation. This suggests that neither cAMP synthesis, nor degradation are stimulated in human gingiva by inflammation.     

Protein free diet feeding: effects on sympathetic activity and salivary evoked secretion in the submandibular gland of the rat

Protein restriction impairs the salivary flow rate and composition in human and rats. The aim of the present work was to establish the effect of low protein (casein 5%) and protein free (casein 0%) isocaloric diets on sympathetic activity and salivary evoked secretion in the submandibular gland (SMG) of the rat. After 21 days, rats fed casein 0% presented: (a) a significant shift to the left of the dose-response curves (DRC) to the autonomic agonists-norepinephrine (NE), methoxamine, isoproterenol (ISO) and methacholine; (b) increased food consumption (p<0.001); (c) decreased body (p<0.001) and SMG (p<0.001) weights maintaining SMG/body (w/w) relation; (d) enhanced submandibular alpha1-adrenoceptor number without changes in the apparent dissociation constant (Kd); (e) increased submandibular NE content (p<0.05) and phosphoinositoside hydrolysis (p<0.001); (f) decreased submandibular tyrosine hydroxylase activity (TH) (p<0.01). Casein 5% feeding increased food consumption (p<0.01) and reduced body weight (p<0.05). This protein restriction increased metacholine-evoked salivation, but it altered neither submandibular sympathetic activity nor sympathetic-induced salivary secretion as compared to the Control group (C) fed a similar diet containing 25.5% protein. Present results suggest that in the adult rat, a protein free diet during 21 days lowers SMG sympathetic and cholinergic activity leading to supersensitivity as revealed by up-regulation of alpha1-adrenergic receptor number and increased autonomic-evoked salivation.     

人β-防御素-2在涎腺肿瘤及涎腺炎症中的表达及其意义

目的 研究涎腺良性肿瘤组织、恶性肿瘤组织和涎腺炎症中人β-防御素-2（HBD-2）mRNA和蛋白的表达特征。方法 对不同涎腺组织,采用聚合酶链反应（PCR）、实时聚合酶链反应（Real-Time PCR）和免疫组化检测HBD-2的表达,并分析HBD-2 mRNA和蛋白在涎腺良性肿瘤组织、恶性肿瘤组织、炎症组织和正常涎腺组织中的表达差异。结果 与涎腺正常组织比较,良性肿瘤组HBD-2 mRNA表达量为其6.468倍,显著高于涎腺正常组织组（P<0.05）;恶性肿瘤组为其0.334倍,显著低于涎腺正常组织组（P<0.05）;涎腺炎症组为其10.563倍,显著高于涎腺正常组织组（P<0.05）。HBD-2不仅在这些组织的细胞质中有表达,而且在恶性组织中的细胞核也有表达。结论 HBD-2在涎腺良性肿瘤组织及涎腺炎症组织中高表达,在涎腺恶性肿瘤组织中低表达,其蛋白发生核转移。