Presence of BPIFB1 in saliva from non-obese diabetic mice.

We previously showed that mRNA expression of BPIFB1 (Bpifb1), an antibacterial protein in the palate, lung, and nasal epithelium clone protein family, was increased in parotid acinar cells in non-obese diabetic (NOD, NOD/ShiJcl) mice, which is an animal model for Sjögren's syndrome. However, we did not previously assess the protein levels. In this report, we confirmed the expression of BPIFB1 protein in the parotid glands of NOD mice. Immunoblotting of subcellular fractions revealed that BPIBB1 was localised in secretory granules in parotid glands from NOD mice, and was almost not in parotid glands from the control mice. BPIFB1 had N-linked glycan that reacted with Aleuria aurantia lectin, which caused two types of spots with a slightly different pI and molecular weight. The expression of BPIFB1 protein was also demonstrated by immunohistochemistry. BPIFB1 was detected in the saliva from NOD mice but not in the saliva from the control mice, indicating individual constitution. BPIFB1 in saliva may be applied to other research as a diagnostic marker.

The Role of Genetic Factors in the Outbreak Mechanism of Dental Caries.

OBJECTIVE: The aim of the present study was to investigate the relationships between cariogenic bacterial infection and single nucleotide polymorphisms (SNPs) in candidate genes associated with dental caries, and to explore the factors related to caries in children. STUDY DESIGN: Children aged 3 to 11 years were selected. Detection of cariogenic bacteria (Streptococcus mutans, Streptococcus oralis, Streptococcus sobrinus and Lactobacillus) from the plaque of each patient, and SNP analyses of five candidate genes (MBL2, TAS2R38, GLUT2, MMP13 and CA6) were performed using DNA isolated from buccal mucosal cells. The dental caries experience in primary and permanent teeth was determined using the decayed, missing and filled teeth (DMFT) index, and the effects of the observed factors on the DMFT value were analyzed by multiple regression analysis. RESULTS: The results of the multiple regression analysis showed that the DMFT value significantly increased in the presence of S. mutans or S. sobrinus (p < 0.001), while the dmft/DMFT value decreased in the presence of nucleobase C in MBL2 (p < 0.05). CONCLUSION: These results suggest that the MBL2 gene is related to the pathogenesis of dental caries.

Characterization of cysteine string protein in rat parotid acinar cells.

Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid acinar cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid acinar cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized acinar cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP11-112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP11-82, containing the J-domain only, nor GST-CSP183-112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in acinar cell exocytosis.

MADD/DENN/Rab3GEP functions as a guanine nucleotide exchange factor for Rab27 during granule exocytosis of rat parotid acinar cells.

We previously reported that the small GTPase Rab27 and its effectors regulate isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Although activation of Rab27 by a specific guanine nucleotide exchange factor (GEF) is thought to be required for amylase release, its activation mechanism is poorly understood, because GEF for Rab27 has not been reported in parotid acinar cells. In the present study, we investigated the possible involvement of MADD/DENN/Rab3GEP, which was recently described as a Rab27-GEF in melanocytes, in amylase release from rat parotid acinar cells. Reverse transcription-PCR analyses indicated that mRNA of DENND family members, including MADD, was expressed in parotid acinar cells. MADD protein was also expressed in the cytosolic fraction of parotid acinar cells. Incubation of an antibody against the C-terminal 150 amino acids of MADD (anti-MADD-C antibody) with streptolysin O-permeabilized parotid acinar cells caused not only inhibition of IPR-induced amylase release but also reduction in the amount of GTP-Rab27. Our findings indicated that MADD functions as a GEF for Rab27 in parotid acinar cells and that its GEF activity for Rab27, i.e., GDP/GTP cycling, is required for IPR-induced amylase release.

Antigen-presenting cells in parotid glands contain cystatin D originating from acinar cells.

Cystatin D encoded by Cst5 is a salivary classified type II cystatin. We investigated the dynamism of cystatin D by examining the distribution of cystatin D protein and mRNA in rats, to identify novel functions. The simultaneous expression of Cst5 and cystatin D was observed in parotid glands, however in situ hybridization showed that only acinar cells produced cystatin D. Synthesized cystatin D was localized in small vesicles and secreted from the apical side to the saliva, and from the basolateral side to the extracellular region, a second secretory pathway for cystatin D. We also identified antigen-presenting cells in the parotid glands that contained cystatin D without the expression of Cst5, indicating the uptake of cystatin D from the extracellular region. Cystatin D was detected in blood serum and renal tubular cells with megalin, indicating the circulation of cystatin D through the body and uptake by renal tubular cells. Thus, the novel dynamism of cystatin D was shown and a function for cystatin D in the regulation of antigen-presenting cell activity was proposed.

EPI64 protein functions as a physiological GTPase-activating protein for Rab27 protein and regulates amylase release in rat parotid acinar cells.

Rab27, a small GTPase, is generally recognized as an important regulator of secretion that interacts with Rab27-specific effectors to regulate events in a wide variety of cells, including endocrine and exocrine cells. However, the mechanisms governing the spatio-temporal regulation of GTPase activity of Rab27 are not firmly established, and no GTPase-activating protein (GAP) specific for Rab27 has been identified in secretory cells. We previously showed that expression of EPI64, a Tre-2/Bub2/Cdc16 (TBC)-domain-containing protein, in melanocytes inactivates endogenous Rab27A on melanosomes (Itoh, T., and Fukuda, M. (2006) J. Biol. Chem. 281, 31823-31831), but the EPI64 role in secretory cells has never been investigated. In this study, we investigated the effect of EPI64 on Rab27 in isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Subcellular fractionation and immunohistochemical analyses indicated that EPI64 was enriched on the apical plasma membrane of parotid acinar cells. We found that an antibody against the TBC/Rab-GAP domain of EPI64 inhibited the reduction in levels of the endogenous GTP-Rab27 in streptolysin-O-permeabilized parotid acinar cells and suppressed amylase release in a dose-dependent manner. We also found that the levels of EPI64 mRNA and EPI64 protein increased after IPR stimulation, and that treatment with actinomycin D or antisense-EPI64 oligonucleotides suppressed the increase of EPI64 mRNA/EPI64 protein and the amount of amylase released. Our findings indicated that EPI64 acted as a physiological Rab27-GAP that enhanced GTPase activity of Rab27 in response to IPR stimulation, and that this activity is required for IPR-induced amylase release.

Exocyst subunits are involved in isoproterenol-induced amylase release from rat parotid acinar cells.

Exocytosis of secretory granules in parotid acinar cells requires multiple events: tethering, docking, priming, and fusion with a luminal plasma membrane. The exocyst complex, which is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that are conserved in yeast and mammalian cells, is thought to participate in the exocytotic pathway. However, to date, no exocyst subunit has been identified in salivary glands. In the present study, we investigated the expression and function of exocyst subunits in rat parotid acinar cells. The expression of mRNA for all eight exocyst subunits was detected in parotid acinar cells by RT-PCR, and Sec6 and Sec8 proteins were localized on the luminal plasma membrane. Sec6 interacted with Sec8 after 5 min of stimulation with isoproterenol. In addition, antibodies to-Sec6 and Sec8 inhibited isoproterenol-induced amylase release from streptolysin O-permeabilized parotid acinar cells. These results suggest that an exocyst complex of eight subunits is required for amylase release from parotid acinar cells.

Evidence for amylase release by cyclin-dependent kinase 5 in the rat parotid.

Cyclin-dependent kinase 5 (Cdk5) plays no apparent role in cell cycle regulation, and Cdk5 is not activated by cyclins but only p35 or p39. Although the enzymatic activity of Cdk5 is highest in the central nervous system, recent reports indicate that it also has important functions in non-neuronal cells. In the present study, we investigated whether Cdk5 and its activators are expressed in rat parotid acinar cells, whether a ß-adrenergic agonist enhances the expression of Cdk5, and whether Cdk5 mediates amylase release. We found that Cdk5 and its activator, cyclin I, were expressed in rat parotid acinar cells, and that the expression of Cdk5 was enhanced by treatment of the cells with isoproterenol. Amylase release stimulated by isoproterenol was depressed by the addition of olomoucine, a Cdk5 inhibitor, or by the introduction of an anti-Cdk5 antibody. Cdk5 activity was enhanced by treatment with isoproterenol and this enhanced activity was attenuated by the addition of olomoucine. Olomoucine also attenuated both phosphorylation of Munc18c and translocation of Munc18c from the plasma membrane induced by isoproterenol. These results indicated that ß-stimulation of rat parotid acinar cells enhanced the expression of Cdk5, and that this Cdk5 activation may mediate amylase release through phosphorylation of Munc18c.

Measurement of Oral Moisture on Oral Dryness Patients.

Many elderly patients have oral dryness; thus, it is necessary to evaluate the oral moisture in a clinical setting. The aim of this study was to clarify the importance of controlling the measuring pressure of the oral moisture-checking device. The influence of the measuring pressure of the oral moisture-checking device was examined using agar under 10 measuring pressure conditions (Kruskal-Wallis test). Fifty-five oral dryness patients were examined the lingual moisture using the device with and without a tongue depressor. The tongue depressor was placed underneath the tongue to support it during the measurement. The mean value and the coefficient of variation of five measurements was evaluated (paired t-test or Wilcoxon signed-ranks test). The agar moisture values changed according to the measuring pressure (p < 0.05). The lingual moisture value with the tongue depressor was higher than that without the tongue depressor (p < 0.05). The coefficient of variation with the tongue depressor was smaller than that without the tongue depressor (p < 0.01). The results of this study indicated that the measuring pressure of oral moisture-checking device influenced the measurement value, and it is necessary to support the tongue for the measurement of lingual mucosal moisture in a uniform manner.

Analysis of salivary factors related to the oral health status in children.

Early detection of oral disease is important to reduce its severity and increase the likelihood of successful treatment. This study aimed to perform a quantitative assessment of the saliva components as a first stage of the research to screen oral homeostasis. Here, saliva secretions collected from children were evaluated, and their constituents were analyzed to investigate the potential correlations between the buffering capacity and a range of salivary factors. Subjects aged 3-16 years in the primary, mixed, or permanent dentition stage, were selected for this study. The following salivary factors were analyzed: flow rate, total protein, total sugar quantifications, and constituent analyses using RT-PCR and western blotting. The associations between each factor and the buffering capacity were then analyzed using multiple regression analysis. Flow rate, BPIFA2 RNA level, histatin 1 and BPIFB1 protein levels as well as female sex were positively associated with buffering capacity. In contrast, total sugar concentration and MUC7 RNA levels showed a negative relationship with the buffering capacity. Some of these constituents may indicate oral homeostasis and are therefore potential biomarkers of oral health status. These results suggest that the analyses of the correlations between oral homeostasis and salivary factors are an effective strategy for identifying the susceptibility to oral diseases.

Transferrin secretory pathways in rat parotid acinar cells.

Transferrin is the major iron transporter in blood plasma, and is also found, at lower concentrations, in saliva. We studied the synthesis and secretion of transferrin in rat parotid acinar cells in order to elucidate its secretory pathways. Two sources were identified for transferrin in parotid acinar cells: synthesis by the cells (endogenous), and absorption from blood plasma (exogenous). Transferrin from both sources is secreted from the apical side of parotid acinar cells. Endogenous transferrin is transported to secretory granules. It is secreted from mature secretory granules upon stimulation with a beta-adrenergic reagent and from smaller vesicles in the absence of stimulation. Exogenous transferrin is internalized from the basolateral side of parotid acinar cells, transported to the apical side by transcytosis, and secreted from the apical side. Secretory processes for exogenous transferrin include transport systems involving microfilaments and microtubules.

Chemical and immunological characterization of the two alpha-N-acetylgalactosaminidases from squid liver.

Based on the inherent alpha-galactosidase activity, squid liver contains two different alpha-N-acetylgalactosaminidases (alpha-GalNAcases): alpha-N-acetylgalactosaminidase I (alpha-GalNAcase I), which typically exhibits the alpha-galactosidase activity and alpha-N-acetylgalactosaminidase II (alpha-GalNAcase II), which is devoid of such activity. The molecular properties of the alpha-GalNAcases that may account for their enzymological differences are as yet unknown. In this study, we have characterized and compared the chemical and immunological properties of alpha-GalNAcase I and alpha-GalNAcase II. Analysis of the N-terminal sequence of the first twenty amino acids revealed the striking homology between alpha-GalNAcase I and alpha-GalNAcase II. Digestion of alpha-GalNAcase I and alpha-GalNAcase II generated the peptide maps that display similarities in peptide pattern, indicating their close relationship in structure. Polyclonal antibodies were generated in rabbits against the purified alpha-GalNAcase I and alpha-GalNAcase II for comparison of the immunological properties. Both Western blot and surface plasmon resonance (SPR) studies showed that the anti-alpha-GalNAcase II antibody reacted with both alpha-GalNAcase I and alpha-GalNAcase II, whereas the anti-alpha-GalNAcase I antibody reacted only with alpha-GalNAcase I, indicating the presence of common as well as unique antigenic determinants on alpha-GalNAcase I and alpha-GalNAcase II. Taken together, these results suggest that alpha-GalNAcase I and alpha-GalNAcase II are closely related with regard to structure and that their nonhomologous domains are possibly responsible for the differences in enzymatic properties.

Redistribution of small GTP-binding protein, Rab27B, in rat parotid acinar cells after stimulation with isoproterenol.

Small GTP-binding protein, Rab27, has been implicated in the regulation of different types of membrane trafficking, including melanosome transport in melanocytes and regulated secretion events in a wide variety of secretory cells. We have previously shown that Rab27 is involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar cells. Although Rab27 is predominantly localized on secretory granules under resting conditions, changes to its intracellular localization after beta-stimulation have never been elucidated. The present study investigated IPR-induced redistribution of Rab27B in the parotid acinar cells, revealing translocation from secretory granules to the subapical region after 5 min of IPR treatment and then diffusion into the cytosol after 30 min of IPR treatment. Dissociation of Rab27B from the apical plasma membrane is probably mediated through the Rab GDP dissociation inhibitor (GDI) in the cytosol extracting GDP-bound Rab protein from membranes, as a dramatic increase in the amount of the Rab27B-GDI complex in the cytosol was observed 30 min after stimulation with IPR. These results indicate that, in parotid acinar cells, Rab27B is translocated, in a time-dependent manner, from secretory granules into the apical plasma membrane as a result of exposure to IPR, and then into the cytosol through binding with the GDI.

Unstimulated amylase secretion is proteoglycan-dependent in rat parotid acinar cells.

It is well-known that amylase is secreted in response to extracellular stimulation from the acinar cells. However, amylase is also secreted without stimulation. We distinguished vesicular amylase as a newly synthesized amylase from the accumulated amylase in secretory granules by short time pulse and chased with (35)S-amino acid. The newly synthesized amylase was secreted without stimulation from secretory vesicles in rat parotid acinar cells. The secretion process did not include microtubules, but was related to microfilaments. p-Nitrophenyl beta-xyloside, an inhibitor of proteoglycan synthesis, inhibited the newly synthesized amylase secretion. This indicated that the newly synthesized amylase was secreted from secretory vesicles, not via the constitutive-like secretory route, which includes the immature secretory granules, and that proteoglycan synthesis was required for secretory vesicle formation.

Evidence for amylase release by cGMP via cAMP-dependent protein kinase in rat parotid acinar cells.

Amylase release from the rat parotid gland is primarily mediated by a cAMP-dependent protein kinase (PKA). We previously reported that cGMP/cGMP-dependent protein kinase (PKG) signaling evokes amylase release. In the present study, we investigated whether cGMP-mediated amylase release might be due to cGMP/PKA signaling, as well as cGMP/PKG pathway. Activation of PKA by cGMP was required 100-1000-fold greater concentration than activation by cAMP in a parotid cytosol fraction. Synergistic activation of PKA by the combination of physiological cAMP and low concentration of cGMP was observed. Amylase release from intact acinar cells was synergistically stimulated by the combination of diBu-cAMP and 8-pCPT-cGMP. cGMP dose-dependently stimulated amylase release from saponin-permeabilized parotid acinar cells. Phosphorylation by cGMP produced phosphorylated proteins of the same size as those produced by cAMP. Phosphorylation by cGMP was inhibited by the addition of PKA inhibitor, H-89. These results suggest that cGMP activates both PKG and PKA. Thus, it appears that both cGMP/PKG and cGMP/PKA pathways mediate amylase release from rat parotid acinar cells.

Relation of Rab26 to the amylase release from rat parotid acinar cells.

Amylase secretion from rat parotid acinar cells is induced by the accumulation of cAMP in response to beta-adrenergic agonists as well as by the elevation of intracellular Ca2+ in response to muscarinic cholinergic stimulation. Several proteins including the low molecular weight GTP-binding protein Rab may participate in these exocytic processes. In the current studies, we investigated the role of Rab26 in the process of amylase secretion. Secretory granules were separated by centrifugation on a Percoll-sucrose density gradient into mature and immature granule fractions. Rab26 and two other type III Rab proteins, Rab3D and Rab27, were present in the mature granule membrane fraction. Also, Rab26 was absent in immature granule membrane fractions. Isoproterenol-induced amylase release from streptolysin-O-permeabilised acinar cells was inhibited by an anti-Rab26 antibody, but this antibody had no effect on the Ca2+-induced release of amylase. Finally, in the early stage of beta-adrenergic stimulation, Rab26 was condensed in the secretory granule membrane. These results indicate that Rab26 is involved in the recruitment of mature granules to the plasma membrane upon beta-adrenergic stimulation.

Intracellular localisation of SNARE proteins in rat parotid acinar cells: SNARE complexes on the apical plasma membrane.

Intracellular localisation of soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) is an important factor in clarifying whether SNAREs regulate exocytosis in salivary glands. We investigated intracellular localisation of syntaxins 2, 3 and 4 and SNAP-23, which are thought to be target membrane (t)-SNAREs, in rat parotid gland by Western blotting and immunocytochemistry. Syntaxins 2 and 3 were localised in the apical plasma membrane (APM), and syntaxin 4 was localised in the plasma membrane. SNAP-23 was localised in the APM and intracellular membrane (ICM). In a yeast two-hybrid assay, syntaxins 2, 3 and 4 interacted with SNAP-23 and VAMP-3. Using immunoprecipitation methods, syntaxins 3 and 4 were seen to interact with VAMP-8 and SNAP-23 at the APM, respectively. SNAP-23 interacted with syntaxin 3, syntaxin 4, VAMP-2, VAMP-3 and VAMP-8. Many SNARE complexes were detected under non-stimulated/basic conditions in the parotid APM. Some of these complexes may have a role in exocytosis from parotid acinar cells.

Presence of cytoskeleton proteins in parotid glands and their roles during secretion.

Amylase secretion is induced by the accumulation of cAMP in response to beta-adrenergic stimulation and by the augmentation of intracellular Ca2+ in response to muscarinic-cholinergic stimulation in rat parotid glands. The roles of cytoskeleton and motor proteins in the secretory process are not yet known. We examined the effects of cytoskeleton-modulating reagents on the amylase release induced by isoproterenol (IPR) and carbamylcholine (Cch) in rat parotid acinar cells. The amylase release induced by Cch was decreased by the microtubule-disrupting reagent colchicine (Colch) and the myosin ATPase inhibitor 2,3-butanediene monoxime (BDM), but the release induced by IPR was not. The actin filament-stabilizing reagent jasplakinolide (Jasp) and actin filament-disrupting reagent cytochalasin D (CytoD) decreased the amylase release induced by both the beta-adrenergic and the muscarinic-cholinergic stimulants. Pretreatment with CytoD affected the shape of the acinar cells, which showed an intermediate state between the fusion of the secretory granules with the apical membrane and the retrieval of the membranes only after stimulation with IPR. Myosin and Dynein/dynactin complex were detected in the secretory granule membrane fraction. We concluded from this study that the cytoskeleton played different roles in the beta-adrenergic and the muscarinic-cholinergic secretory processes.

Soluble guanylyl cyclase is localised in the acinar cells and participates in amylase secretion in rat parotid gland.

It is well known that the muscarinic cholinergic agonists, carbachol and methacholine, enhance nitric oxide synthase (NOS) activity, and also stimulate salivary secretion. In the present study, we investigated whether salivary secretion by muscarinic cholinergic stimulation is mediated through the NO/cGMP signaling pathway in rat salivary glands. Since NO activates soluble guanylyl cyclase (sGC) and cGMP may function as a mediator, the localisation of sGC was investigated in the salivary glands. sGC was localized in both the acinar and duct cells of the rat parotid and sublingual glands, and localized only in the acinar cells of the submandibular glands. S-Nitroso-glutathione (NO generator; GSNO) and YC-1 (NO-independent sGC activator) stimulated sGC in the cytosol to synthesise cGMP. The combination of GSNO and YC-1 stimulated sGC synergistically. Carbachol, GSNO and YC-1 enhanced amylase release from the rat parotid glands. Amylase release stimulated by carbachol and GSNO was inhibited by addition of the sGC inhibitor, ODQ, and cGMP-dependent protein kinase inhibitor, KT-5823. These results indicate that amylase release may be mediated through the NO/cGMP signaling pathway.

Atrophy of myoepithelial cells in parotid glands of diabetic mice; detection using skeletal muscle actin, a novel marker.

In mouse parotid glands, we found expression of skeletal muscle actin (actin-α1) protein and mRNA. We isolated myoepithelial cells from the mouse parotid glands and investigated their actin-α1 expression because smooth muscle actin (actin-α2) has been used as a marker for myoepithelial cells. We used actin-α1 expression to identify pathological changes in diabetic non-obese diabetic (NOD; NOD/ShiJcl) mice-a mouse model for Sjögren's syndrome-and found myoepithelial cells to be decreased or atrophied in the diabetic state.