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Four major kallikreins (mK1, mK22, mK9, and mK13) were identified in the mouse submandibular gland (SMG). mK1, a true tissue kallikrein, was used as a protein marker to identify different types of SMG granular convoluted tubule (GCT) cells along with epidermal growth factor (EGF), nerve growth factor (NGF), and renin. Kallikrein mK1 was localized in a very small number (~5%) of GCT cells, which were scattered throughout the GCT, indicating that the majority of GCT cells are mK1-negative. Among mK1-positive cells, particularly strong signals were observed in a small number of narrow cells, recognized as slender granular cells (SG cells, Type IV), in the GCT. After postnatal development of the SMG, GCT cells are no longer uniform based on the bioactive substances (mK1, EGF, NGF, and renin) that they produce and secrete. GCT cells were classified into four subtypes, Types I–IV, and it became clear that these subtypes are complicatedly and reversibly converted by the endocrine hormones 5α-dihydrotestosterone (DHT) and triiodothyronine (T3). Duct segments with similar morphology or hormone dependency were recognized in the sublingual and parotid glands. The presence of duct cells with such characteristics is therefore a common feature of the three major salivary glands of rodents.

Occludin is an important tight junction (TJ) protein in pulmonary epithelial cells. In this study, we identified changes in occludin in arsenic-induced lung injury in vivo and in vitro. Upon intratracheal instillation with arsenic trioxide (As2O3) at a daily dose of 30 μg/kg for 1 week, levels of occludin mRNA and protein expression decreased significantly in mouse lung tissue. Levels of occludin mRNA and protein expression in BEAS-2B cells were reduced upon exposure to As2O3 in a concentration- and time-dependent manner. In addition, exposure to As2O3 significantly increased expression of p-p38, p-ERK1/2, p-ELK1, and MLCK in mouse lung tissue and BEAS-2B cells. Treatment with As2O3 induced oxidative stress in mouse lung tissue and BEAS-2B cells. In BEAS-2B cells, exposure to As2O3 reduced transepithelial resistance, which was partially restored with N-acetyl-cysteine (NAC) treatment. Reduced expression of occludin mRNA and protein induced by As2O3 was entirely restored with NAC and resveratrol. However, SB203580, PD98059, and ML-7 partially blocked As2O3-induced occludin reduction in BEAS-2B cells. These results indicate that As2O3 inhibits occludin expression in vivo and in vitro at least partially via the ROS/ERK/ELK1/MLCK and ROS/p38 MAPK signaling pathways.

Antimony (Sb) has been reported to lead to pulmonary damage, but the underlying mechanism remains unclear. Accumulating evidence indicates that silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+-dependent deacetylase, mediates stimuli-induced cellular apoptosis. Here, we investigated whether SIRT1 plays a role in Sb-triggered apoptosis in human bronchial epithelial cells (BEAS-2b). First, we showed that Sb initiated apoptosis. Furthermore, the expression of SIRT1 was markedly downregulated by Sb treatment, while overexpression of SIRT1 through resveratrol treatment or transfection with SIRT1-Flag plasmid attenuated the Sb-induced apoptosis. Accelerated degradation of SIRT1 protein and lower SIRT1 gene expression contributed to low expression of SIRT1. In addition, Sb activated the ERK and JNK pathways; however, inhibition of ERK rather than JNK rescued SIRT1 suppression. Subsequent analyses demonstrated that antioxidant N-acetylcysteine (NAC) attenuated SIRT1 repression, increased SIRT1 mRNA levels and decreased SIRT1 protein degradation in Sb-treated cells. In addition, NAC also inhibited JNK and ERK activation by Sb exposure. These data suggest that reactive oxygen species-dependent SIRT1 suppression mediates Sb-stimulated cell apoptosis in BEAS-2b cells via lower SIRT1 gene expression and protein stability.

Inorganic arsenic is widely present in the environment. Exposure to moderate to high concentrations of arsenic from drinking water or air can cause various cancers and multisystem dysfunction. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) stress sensor of unfolded protein response (UPR) under stress conditions and it enhances cell survival. The aim of this study is to investigate molecular mechanisms of arsenic-induced GRP78 expression in BEAS-2B cells model. We found that GRP78 protein expression was enhanced, while the level of GRP78 mRNA expression did not change under arsenic trioxide (As2O3)-induced ER stress condition in BEAS-2B cells. Cycloheximide, a protein synthesis inhibitor, completely inhibited As2O3-induced GRP78 protein expression. GRP78 mRNA expression was inhibited by actinomycin-D (Act-D). However, GRP78 protein expression was upregulated in the presence of Act-D under As2O3-induced ER stress condition. These data indicated that the upregulation of GRP78 protein under As2O3-induced UPR condition was possibly due to the increased biosynthesis of GRP78 protein. Moreover, both inositol-requiring enzyme 1α (IRE1α) RNase and kinase inhibitor KIRA6 and IRE1α kinase inhibitor APY29 completely inhibited As2O3-induced GRP78 protein expression and phosphorylation of JNK, ERK and p38 MAPK. Activation of apoptotic signaling kinase 1 (ASK1) is a downstream effector of IRE1α kinase. ASK1 inhibitor selonsertib and p38 MAPK inhibitor SB203580 partially inhibited As2O3-induced GRP78 protein expression, respectively. Our results suggested that As2O3 enhanced GRP78 protein expression in BEAS-2B cells via IRE1α kinase/ASK1/p38 MAPK signaling pathway. To our knowledge, this is the first report on illuminating the related mechanisms of increased GRP78 protein expression in As2O3-induced ER stress condition through a novel IRE1α pathway.

// Zhiyong Weng 1 , Xuefeng Liu 1 , Jiehua Hu 2 , Jingzhou Mu 3 , Jing Xie 1 , Chenjuan Yao 4 and Lihua Li 1 1 Department of Cell Biology, Jinzhou Medical University, Jinzhou, PR China 2 Naval University of Engineering, Logistics College, Information Center, Tianjin, PR China 3 Department of Physiology, Dalian Medical University, Dalian, PR China 4 Department of Molecular Oral Physiology, The University of Tokushima Graduate School, Tokushima, Japan Correspondence to: Lihua Li, email: lilihua1018@sina.com Keywords: dehydroandrographolide, obstructive cholestasis, liver adaptive response, anti-fibrosis formation Received: April 15, 2017 Accepted: August 23, 2017 Published: September 23, 2017 ABSTRACT Background: Dehydroandrographolide (DA) is the main contributor to the therapeutic properties of the medicinal plant Andrographis paniculata (AP). However, it is unknown whether DA has a hepatoprotective effect on obstructive cholestasis in mice and humans. Methods: We administered DA to mice for 5 days prior to bile duct ligation (BDL) and for the 7 days. Liver function markers, liver histology and necrosis, compensatory responses of hepatocytes, liver fibrosis and the expression of hepatic fibrogenesis markers were evaluated in BDL mice and/or human LX-2 cells. Results: Mice treated with DA demonstrated lower levels of serum alanine transarninase (ALT), milder liver damage, liver necrosis and fibrosis formation than in vehicle control with carboxymethylcellulose (CMC) mice after BDL. DA treatment also enhanced the Mrp3 expression of hepatocytes but not Mrp4 following BDL. Further, DA treatment in BDL mice significantly reduced liver mRNA and/or protein expression of Tgf-β, Col1a1, α-Sma and Mmp2. This result was also supported by hydroxyproline analysis. The molecular mechanisms of DA treatment were also assessed in human hepatic stellate cell line (LX-2 cell). DA treatment significantly inhibited Tgf-β-induced Col1a1, Mmp2 and α-Sma expression in human LX-2 cells. These data suggested that DA treatment reduced liver damage through development of a hepatic adaptive response and inhibition of the activation of HSCs, which led to a reduction in liver fibrosis formation in BDL mice. Conclusions: DA treatment protected against liver damage and fibrosis following BDL and might be an effective therapy for extrahepatic cholestasis due to bile duct obstruction.

Antimony (Sb), a naturally occurring metal present in air and drinking water, has been found in the human brain, and there is evidence of its toxic effects on neurobehavioral perturbations, suggesting that Sb is a potential nerve poison. Here, we provide the first study on the molecular mechanism underlying Sb-associated neurotoxicity. Mice exposed to antimony potassium tartrate hydrate showed significantly increased neuronal apoptosis. In vitro, Sb triggered apoptosis in PC12 cells in a dose-dependent manner. Mechanically, Sb triggered autophagy as indicated by increased expression of microtubule-associated protein 1 light chain 3-II (LC3-II) and accumulation of green fluorescent protein-tagged LC3 dots. Moreover, Sb enhanced autophagic flux and sequestosome 1 (p62) degradation. Subsequent analyses showed that Sb treatment decreased phosphorylation of protein kinase B (Akt) as well as the mammalian target of rapamycin (mTOR), while an Akt activator protected PC12 cells from autophagy. Moreover, the antioxidant N-acetylcysteine attenuated Sb-induced Akt/mTOR inhibition and decreased autophagy and apoptosis, with autophagy inhibition also playing a cytoprotective role. In vivo, mice treated with Sb showed higher expression of LC3-II and p62 in the brain, consistent with the in vitro results. In summary, Sb induced autophagic cell death through reactive oxygen species-mediated inhibition of the Akt/mTOR pathway.

β-actin, a cytoskeletal protein, is the most widely used housekeeping gene. Although housekeeping genes are expressed in all tissues, the β-actin gene is expressed in certain cell types because of differential binding of transcriptional factors to the regulatory elements of the gene. The expression and localization of β-actin protein in the submandibular glands (SMG) of mice were investigated in this study, using Western blot analysis and immunohistochemistry. In ICR and C57BL/6J mice, the levels of β-actin protein in the SMG of females are significantly higher than those in the SMG of males. β-actin protein is majorly distributed in acinar cells of SMG. There is no significant difference in the expression level of β-actin protein between females and castrated males. After castrated male ICR mice are treated with 10 mg/kg/day testosterone propionate (TP) for 3 weeks, the levels of β-actin protein in SMG decrease. The numbers of duct per unit area increase, whereas the numbers of acinus per unit area decrease after TP administration. These data suggest that β-actin protein is mainly distributed in acinar cells of SMG and results in a marked sexual dimorphism in mice.

Silica nanoparticles (SiNPs) are one of the most widely used types of nanoparticles across many industrial sectors, and are known to be present in the air year-round. In this study, we aimed to evaluate the potential adverse effects of SiNP exposure on pulmonary epithelial tight junctions, which serve as a critical barrier between the respiratory system and the circulatory system. In vivo studies confirmed that SiNPs decreased the protein expression levels of zonula occludens 1 (ZO-1), zonula occludens 2 (ZO-2), and occludin in the lungs of C57BL/6 mice. In vitro studies showed that SiNPs not only decreased the mRNA and protein expression of ZO-1 and ZO-2, but also decreased the protein expression of occludin in human bronchial epithelial (BEAS-2B) cells. In addition, SiNP exposure increased reactive oxygen species (ROS) production and activated extracellular regulated protein kinases (ERKs) and c-Jun N-terminal kinase (JNK). The inhibition of ROS and ERKs effectively protected the SiNP-induced downregulation of ZO-1 mRNA and protein expression, but had no effect on ZO-2 or occludin expression. SiNP-induced matrix metalloproteinase 9 (MMP9) protein expression appeared to be involved in occludin proteolytic degradation, in addition to SiNP-induced direct occludin protein degradation. The present study suggests that SiNPs disturb pulmonary epithelial tight junction structure and function via the ROS/ERK pathway and protein degradation.

Aquaporin 5 (AQP5) plays an important role in the salivary gland function. The mRNA and protein for AQP5 are expressed in the acini from embryonic days E13-16 and E17-18, respectively and for entire postnatal days. Ligation-reopening of main excretory duct induces changes in the AQP5 level which would give an insight for mechanism of regeneration/self-duplication of acinar cells. The AQP5 level in the submandibular gland (SMG) decreases by chorda tympani denervation (CTD) via activation autophagosome, suggesting that its level in the SMG under normal condition is maintained by parasympathetic nerve. Isoproterenol (IPR), a β-adrenergic agonist, raised the levels of membrane AQP5 protein and its mRNA in the parotid gland (PG), suggesting coupling of the AQP5 dynamic and amylase secretion-restoration cycle. In the PG, lipopolysaccharide (LPS) is shown to activate mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signalings and potentially downregulate AQP5 expression via cross coupling of activator protein-1 (AP-1) and NF-κB. In most species, Ser-156 and Thr-259 of AQP5 are experimentally phosphorylated, which is enhanced by cAMP analogues and forskolin. cAMP-dependent phosphorylation of AQP5 does not seem to be markedly involved in regulation of its intracellular trafficking but seems to play a role in its constitutive expression and lateral diffusion in the cell membrane. Additionally, Ser-156 phosphorylation may be important for cancer development.

Arsenic is associated with several adverse health outcomes, and people with diabetes may be more susceptible to arsenic. In this study, we found that arsenic levels in some tissues such as liver, kidney, and heart but not lung of type 1 diabetes mellitus (T1DM) mice were higher than in those of normal mice after a single oral dose of arsenic trioxide for 2 h. However, little is known about the molecular mechanism of the increased tissue uptake of trivalent inorganic arsenic in mice with T1DM. This study aimed to investigate the expression of the mammalian arsenic transporters aquaglyceroporins (AQPs) and glucose transporter 1 (GLUT1) in T1DM mice and compare them with those in normal mice. Results showed that the levels of AQP9 and GLUT1 mRNA and protein were higher in T1DM mouse liver than in the normal one. The levels of AQP7 mRNA and protein were higher in T1DM mouse kidney. In the heart, we observed that the levels of AQP7 and GLUT1 mRNA and protein were higher in T1DM mice, but the levels of AQP9 mRNA and protein in the lung had no significant difference between both mice. These results suggested that T1DM may increase the expression of transporters of trivalent inorganic arsenic and thus increase the arsenic uptake in specific tissues.

The kidney urea transport protein UT-B is an attractive target for the development of small-molecule inhibitors with a novel diuretic (‘urearetic’) action. Previously, two compounds in the triazolothienopyrimidine scaffold (1a and 1c) were reported as UT-B inhibitors. Compound 1c incorporates a 1,1-difluoroethyl group, which affords improved microsomal stability when compared to the corresponding ethyl-substituted compound 1a. Here, a small focused library (4a–4f) was developed around lead inhibitor 1c to investigate the requirement of an amidine-linked thiophene in the inhibitor scaffold. Two compounds (4a and 4b) with nanomolar inhibitory potency (IC50 ≈ 40 nM) were synthesized. Computational docking of lead structure 1c and 4a–4f into a homology model of the UT-B cytoplasmic surface suggested binding with the core heterocycle buried deep into the hydrophobic pore region of the protein.

We previously reported benzopyrimido-pyrrolo-oxazinedione (BPO) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in a model of polycystic kidney disease. Here, we separated the enantiomers of lead compound BPO-27, (1), which contains a single chiral center, and determined their absolute configuration, activity and metabolic stability. Following separation by chiral supercritical fluid chromatography, the R enantiomer, as determined by x-ray crystallography, inhibited CFTR chloride conductance with IC50 ~ 4 nM, while S enantiomer was inactive. In vitro metabolic stability in hepatic microsomes showed both enantiomers as stable, with

Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.

We previously reported the identification and structure-activity analysis of bithiazole-based correctors of defective cellular processing of the cystic fibrosis-causing CFTR mutant, ΔF508-CFTR. Here, we report the synthesis and uptake of a functional, fluorescently labeled bithiazole corrector. Following synthesis and functional analysis of four bithiazole-fluorophore conjugates, we found that 5, a bithazole-based BODIPY conjugate, had low micromolar potency for correction of defective δF508-CFTR cellular misprocessing, with comparable efficacy to benchmark corrector corr-4a. Intravenous administration of 5 to mice established its stability in extrahepatic tissues for tens of minutes. By fluorescence imaging of whole-body frozen slices, fluorescent corrector 5 was visualized strongly in gastrointestinal organs, with less in lung and liver. Our results provide proof-of-concept for mapping the biodistribution of a ΔF508-CFTR corrector by fluorophore labeling and fluorescence imaging of whole-body slices.

We previously reported the discovery of pyrimido-pyrrolo-quinoxalinedione (PPQ) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in an organ culture model of polycystic kidney disease (PKD) (Tradtrantip et al., J. Med. Chem. 52, 6447–6455, 2009). Here, we report related benzopyrimido-pyrrolo-oxazinedione (BPO) CFTR inhibitors. To establish structure-activity relationships and select lead compound(s) with improved potency, metabolic stability and aqueous solubility compared to the most potent prior compound 8 (PPQ-102, IC50 ~ 90 nM), we synthesized 16 PPQ analogs and 11 BPO analogs. The analogs were efficiently synthesized in 5–6 steps and 11–61 % overall yield. Modification of 8 by bromine substitution at the 5-position of the furan ring, replacement of the secondary amine with an ether bridge, and carboxylation, gave 6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid 42 (BPO-27), which fully inhibited CFTR with IC50 ~ 8 nM, and, compared to 8, had >10-fold greater metabolic stability and much greater polarity / aqueous solubility. In an embryonic kidney culture model of PKD 42 prevented cyst growth with IC50 ~ 100 nM. Benzopyrimido-pyrrolo-oxazinediones such as 42 are potential development candidates for anti-secretory therapy of PKD.

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.

Three constructs having mutated PKA-target motif at 152 SRRTS of AQP5, an exocrine type water channel, were prepared and fused to C-terminus of green fluores- cence protein cDNA to examine the effects of blocking of phosphorylation at 152 SRRTS (a consensus PKA-target motif of AQP5) on translocation or trafficking of the chimeric pro- teins expressed in the Madin-Darby canine kidney-II (MDCK-II) cells. H-89 treatment increased translocation of wild-type GFP-AQP5 to the apical membrane. All 3 mutant molecules translocated 1.5 to 2 times more than the control wild-type GFP-AQP5. Col- chicine but not cytochalasin B inhibited the translocation of wild-type GFP-AQP5. Present results suggest dephosphorylation of this consensus sequence increase GFP-AQP5 translo- cation, and that microtubules but not microfilaments are involved in this event. J. Med. Invest. 56 : 55-63, February, 2009

The submandibular gland (SMG) develops under the epithelial-mesenchymal interaction. Its process is regulated by various growth/differentiation factors, which are synthesized as inactive precursors and activated via the limited proteolysis at their multi basic amino acid site(s) such as Arg-X-Lys/Arg-Arg. Although many of these processing steps are elucidated to be catalyzed by subtilisin-like proprotein convertases (SPCs), little is known about the role of SPCs in the SMG development. Here, we focused upon the physiological role of PACE4 (SPC4), a member of SPC family, in the SMG development. In the organ culture system of rat embryonic SMG (E15), Dec-RVKR-CMK, a potent inhibitor for SPCs, inhibited the salivary branching and the expression of an exocrine gland type water channel, AQP5. However, other peptidyl-CMKs and inhibitors for trypsin-like serine proteases including leupeptin did not affect the salivary branching and AQP5 expression. Dec-RVKR-CMK also suppressed the expression of PACE4, but not furin, another member of the family. The specific antibody for the catalytic domain of PACE4 suppressed the salivary branching and AQP5 expression similarly. These inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2 whose precursor is a candidate for the physiological substrates of PACE4. Further, the transcriptional silencing of PACE4 by its specific siRNAs caused the suppression of both the salivary branching and AQP5 expression in the present organ culture system. These observations strongly support the idea that PACE4 mediates the SMG development.

The submandibular gland (SMG) develops through the epithelial–mesenchymal interaction mediated by many growth/differentiation factors including activin and BMPs, which are synthesized as inactive precursors and activated by subtilisin-like proprotein convertases (SPC) following cleavage at their R-X-K/R-R site. Here, we found that Dec-RVKR-CMK, a potent inhibitor of SPC, inhibited the branching morphogenesis of the rat embryonic SMG, and caused low expression of a water channel AQP5, in an organ culture system. Dec-RVKR-CMK also decreased the expression of PACE4, a SPC member, but not furin, another SPC member, suggesting the involvement of PACE4 in the SMG development. Heparin, which is known to translocate PACE4 in the extracellular matrix into the medium, and an antibody specific for the catalytic domain of PACE4, both reduced the branching morphogenesis and AQP5 expression in the SMG. The inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2, whose precursor is one of the candidate substrates for PACE4 in vivo. Further, the suppression of PACE4 expression by siRNAs resulted in decreased expression of AQP5 and inhibition of the branching morphogenesis in the present organ culture system. These observations suggest that PACE4 regulates the SMG development via the activation of some growth/differentiation factors.

By chorda tympani denervation (CTD, parasympathectomy), the aquaporin 5 (AQP5), but not AQP1, protein level in the rat submandibular gland (SMG) was significantly decreased, dropping to 37% of that of the contralateral gland at 4 wk. The protein levels of AQP5 and AQP1 were not significantly affected by denervation of the cervical sympathetic trunk (sympathectomy). Administration of cevimeline hydrochloride, an M3 muscarinic receptor agonist (10 mg/kg for 7 days po), but not pilocarpine (0.3 mg/kg for 7 days po), recovered the AQP5 protein level reduced by CTD and increased the AQP1 protein level above the control one. The mRNA level of AQP5 was scarcely affected by CTD and cevimeline hydrochloride administration. Administration of chloroquine (50 mg/kg for 7 days po), a denaturant of lysosomes, increased the AQP5 protein level reduced by CTD. An extract obtained from the submandibular lysosomal fraction degraded the AQP5 protein in the total membrane fraction in vitro. These results suggest the possible regulation of the AQP5 protein level in the SMG by the parasympathetic nerves/M3 muscarinic receptor agonist and imply the involvement of lysosomal enzymes, but not a transcriptional mechanism, in this regulation.

β-actin, a cytoskeletal protein, is the most widely used housekeeping gene. Although housekeeping genes are expressed in all tissues, the β-actin gene is expressed in certain cell types because of differential binding of transcriptional factors to the regulatory elements of the gene. The expression and localization of β-actin protein in the submandibular glands (SMG) of mice were investigated in this study, using Western blot analysis and immunohistochemistry. In ICR and C57BL/6J mice, the levels of β-actin protein in the SMG of females are significantly higher than those in the SMG of males. β-actin protein is majorly distributed in acinar cells of SMG. There is no significant difference in the expression level of β-actin protein between females and castrated males. After castrated male ICR mice are treated with 10 mg/kg/day testosterone propionate (TP) for 3 weeks, the levels of β-actin protein in SMG decrease. The numbers of duct per unit area increase, whereas the numbers of acinus per unit area decrease after TP administration. These data suggest that β-actin protein is mainly distributed in acinar cells of SMG and results in a marked sexual dimorphism in mice.

Generation and propagation of oscillatory activities in cortical networks are important features of the brain. However, many issues related to oscillatory phenomena are unclear. We previously reported neocortical oscillation following caffeine treatment of rat brain slices. Input to the primary visual cortex (Oc1) generates N -methyl-d-aspartate (NMDA) receptor-dependent oscillations, and we proposed that the oscillatory signals originate in the secondary visual cortex (Oc2). Because non-NMDA and NMDA receptors cooperate in synaptic transmission, non-NMDA?receptors may also play an important role in oscillatory activities. Here we investigated how non-NMDA receptor activities contribute to NMDA receptor-dependent oscillations by using optical recording methods. After induction of stable oscillations with caffeine application, blockade of NMDA receptors abolished the late stable oscillatory phase, but elicited 'hidden' non-NMDA receptor-dependent oscillation during the early depolarizing phase. An interesting finding is that the origin of the non-NMDA receptor-dependent oscillation moved from the Oc1, during the early phase, toward the origin of the NMDA receptor-dependent oscillation that is fixed in the Oc2. In addition, the frequency of the non-NMDA receptor-dependent oscillation was higher than that of the NMDA receptor-dependent oscillation. Thus, in one course of spatiotemporal oscillatory activities, the relative balance in receptor activities between non-NMDA and NMDA receptors gradually changes, and this may be due to the different kinetics of the two receptor types. These results suggest that interplay between the two receptor types in the areas of Oc1 and Oc2 may play an important role in oscillatory signal communication. Caffeine-induced oscillations are generated in the visual cortex.The main components of the oscillations consist of NMDA receptor-dependent waves.Non-NMDA receptor-dependent oscillations underlie and precede NMDA receptor-dependent oscillations.Interplay between the two receptor types may induce stable patterns of oscillatory wave propagation.

A greater than twofold diversity in the expression level of aquaporin 5 (AQP5) has been observed in the membrane fraction of the submandibular gland (SMG) in Sprague-Dawley rats (Murdiastuti K, Miki O, Yao C, Parvin MN, Kosugi-Tanaka C, Akamatsu T, Kanamori N, and Hosoi K. Pflügers Arch 445: 405–412, 2002). In the present study, breeding between brother and sister rats was repeated within high AQP5 producers and low ones to obtain inbred offspring. High- and low-producer rats from 3rd to 18th generations were used for experiments. By Western blotting, levels of AQP5 proteins in the parotid and lacrimal glands, and lungs were all low in low producers, whereas they were all high in high producers, implying genetic variations of the gene for this water channel. Despite this implication, AQP5 mRNA levels were almost the same between the two groups by Northern blotting, suggesting the irrelevance of transcriptional regulation for this diversity. AQP5 cDNAs from the SMGs of the two groups were sequenced. The nucleotide sequence of AQP5 cDNA from low producers indicated the existence of a point mutation at nt 308 (G308A), leading to a replacement of 103Gly with 103Asp in the third transmembrane domain, but no alteration was detected in the Kozak area. The existence of such a mutation was confirmed by the assessment of genomic DNA also. This mutation may have resulted in an abnormal membrane insertion or ineffective trafficking of AQP5, since the rats having this mutation showed extremely low membrane expression of AQP5 in the SMG acinar cells and decreased water secretion from their salivary glands.

The green fluorescent protein (GFP) of the jellyfish, Aeqorea victoria, was used as an autofluorescent tag to track the trafficking of aquaporin 5 (AQP5), an exocrine gland-type water channel. Two groups of chimeric proteins were constructed; one in which GFP was fused to the amino-terminus of AQP5 (GFP–AQP5) and the other, in which it was fused to the carboxyl terminus of it (AQP5–GFP). In each group, 2 chimeras were produced, a wild-type AQP5 with its normal sequence and a mutant AQP5 having a mutated amino acid at 259, i.e., GFP–AQP5–T259A and AQP5–GFP–T259A. They were used to transfect Madin–Darby canine kidney (MDCK) cells. The GFP–AQP5 chimera was localized in the intracellular vesicles, which trafficked to the plasma membrane in response to N6, 2′-O-dibutyryladenosine 3′, 5′-cyclic monophosphate (dbcAMP). Membrane trafficking was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquimolinesulfonamide (H-89) but not by palmitoyl-dl-carnitine chloride (PCC). In contrast, the AQP5–GFP chimera expressed in MDCK cells was localized constitutively on the plasma membrane. The cellular localization of the latter chimera was not affected by stimulation with dbcAMP in the presence or absence of H-89 or PCC. Replacement of Thr-259 with Ala-259 did not affect the dbcAMP-induced translocation of the chimeric protein, suggesting that phosphorylation of Thr-259 was not necessary for AQP5 trafficking under the present experimental conditions. Thus, the GFP–AQP5 chimera will be a useful tool to study AQP5 trafficking in vitro, whereas the constitutive membrane localization of the AQP5–GFP chimera suggests the importance of the carboxyl terminus of the AQP5 protein for its sorting, whether it is translocated to intracellular vesicles or to the plasma membrane.

Aquaporin-2 (AQP2) is responsible for the concentration of urine in the kidney collecting tubule under the regulation of vasopressin. The mRNA level of this water channel in polydipsic STR/N mice was extremely reduced compared with that in normal ICR mice. In male mice, reduction of the AQP2 mRNA level was not evident at 3 wk of age, at which time water intake was not increased. At 10 wk of age, however, the AQP2 mRNA level was reduced to 10% of that in control mice, whereas water intake was increased by 36%. At 44 wk, the water intake became five times that of the control ICR mice, and the AQP2 mRNA level in these polydipsic mice was only ∼5% of control. Similar changes were observed in the AQP2 protein level, suggesting that the mRNA level of AQP2 reflects the protein level of AQP2. These inverse changes in the AQP2 mRNA level and water intake were also evident in female mice. The data imply that polydipsia in STR/N mice may have affected AQP2 mRNA transcription in the kidney, resulting in reduced AQP2 expression, which would contribute to a reduction in overretention of water.

Background: The submandibular gland (SMG) is a major salivary gland, which plays an important role in maintenance the oral health. In this study, we intended to explore the role of the SMG's defense system of the animals in which experimental inflammation is induced. Methods: The levels of mRNAs for inflammation cytokines and acute phase proteins were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Results: The mRNAs for acute phase proteins were found to be increased in the SMG and extraorbital and intraorbital lacrimal gland (ELG and ILG) of rats at 24 h after subcutaneous injection of turpentine oil. The induction of mRNA for these inflammatory proteins by turpentine oil was preceded by a transient increase in the level of mRNAs for IL-1β, IL-6 and TNF-α at 6 h after subcutaneous injection of the oil. Such cytokine induction was similarly seen by lipopolysaccharide (LPS) injection, and involvement of Toll-like receptor 4 (TLR4) was strongly suggested from experiment using C3H/HeJ mice, a TLR4-deficient mutant strain. Conclusion: The up-regulation of acute phase proteins and inflammation cytokines in the SMG, ELG and ILG by experimental inflammation suggests the existence of a strict defense system via the innate immune system in the SMG and other exocrine gland.

The expression and localization of aquaporins (AQP1-AQP5), members of the water channel family, in the developing rat submandibular gland were analysed using RT-PCR, Northern blotting and immunohistochemistry to explore their relation to the development of this salivary gland. RT-PCR analysis revealed unique expression patterns of each AQP. AQP1 was expressed constitutively during prenatal development, whereas the expression of AQP5 became more intense in the course of development from embryonic day 16.5 (E16) to E20. These expression patterns concurred with the results of Northern blot analysis. AQP3 and AQP4 mRNAs in the prenatal development were not detected in Northern blots, although they were detected by RT-PCR. During postnatal development, AQP5 and AQP1 mRNAs were expressed continuously, but no message for AQP3 or AQP4 was detected. AQP2 mRNA was not detected during either prenatal or postnatal development in this tissue. Immunohistochemical studies revealed that AQP5 was first localized at the apical membrane of proacinar cells at E18, and then became clearly distributed at the apical membrane of acinar cells in accordance with the differentiation and establishment of the mature acini. In addition, some vasculature also showed immunoreactivity for AQP5. AQP1 was immunolocalized in the blood vessels, including capillaries, of the gland throughout development. These observations suggest the existence of transcriptional regulation of rat AQP5, which is one of the most probable regulators of saliva production and secretion, during the establishment of the functional submandibular salivary gland.

In the colony of Sprague-Dawley (SD) strain, we found that there were rats expressing a mutant AQP5, which has a point mutation at nt 308 (G308A), leading to a replacement of (103)Gly with (103)Asp in the 3rd transmembrane domain. The mutant molecule scarcely expressed in the acinar cells, probably because of ineffective trafficking. The mutant molecule, however, showed normal water permeability when assessed by the oocyte system.

In the membrane fraction of mouse parotid gland (PG), the protein level of aquaporin 5 (AQP5), a member of the water channel family, was increased by injection (ip) of isoproterenol (IPR), a β-adrenergic agonist, at 1 h, and stayed at high levels until 6 h; this change occurred simultaneously as amylase secretion. The AQP5 level then decreased and returned toward the original level at 12–48 h. After IPR injection, the AQP5 mRNA gradually increased and reached a maximum at 24 h. The facts suggest a rapid appearance of AQP5 at plasma membrane by IPR and subsequent degradation/metabolism by activation of proteolytic systems. Pretreatment of animals with two calpain inhibitors, N-Ac-Leu-Leu-methininal (ALLM) and calpeptin, as well as a protein synthesis inhibitor, cycloheximide (CHX), significantly suppressed the IPR-induced AQP5 degradation in the PG membrane fraction; such suppression was not observed by two proteasome inhibitors, MG132 and lactacystin, or the lysosome denaturant chloroquine, although most of these inhibitors increased AQP5 protein levels in unstimulated mice. The AQP5 protein was also degraded by μ-calpain in vitro. Furthermore, we demonstrated that μ-calpain was colocalized with AQP5 in the acinar cells by immunohistochemistry, and its activity in the PG was increased at 6 h after IPR injection. These results suggest that the calpain system was responsible for IPR-induced AQP5 degradation in the parotid gland and that such a system was coupled to the secretory-restoration cycle of amylase in the PG.

Urea transporters, which include UT-B in kidney microvessels, are potential targets for development of drugs with a novel diuretic ('urearetic') mechanism. We recently identified, by high-throughput screening, a triazolothienopyrimidine UT-B inhibitor, 1, that selectively and reversibly inhibited urea transport with IC(50) = 25.1 nM and reduced urinary concentration in mice ( Yao et al. J. Am. Soc. Nephrol. , in press ). Here, we analyzed 273 commercially available analogues of 1 to establish a structure-activity series and synthesized a targeted library of 11 analogues to identify potent, metabolically stable UT-B inhibitors. The best compound, {3-[4-(1,1-difluoroethyl)benzenesulfonyl]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-5-yl}thiophen-2-ylmethylamine, 3k, had IC(50) of 23 and 15 nM for inhibition of urea transport by mouse and human UT-B, respectively, and ∼40-fold improved in vitro metabolic stability compared to 1. In mice, 3k accumulated in kidney and urine and reduced maximum urinary concentration. Triazolothienopyrimidines may be useful for therapy of diuretic-refractory edema in heart and liver failure.

The gustatory insular cortex (IC) is connected with not only the somatosensory cortex, but also the endopiriform nucleus (EPN). We have previously revealed that low-frequency electrical stimulation to the IC can elicit membrane potential oscillations at a frequency of 8-10 Hz in the somatosensory cortex of rat brain slices under bath-application of caffeine. Using the same procedure, we investigated whether the EPN has the ability to generate oscillations, and whether such oscillations emerge age-dependently. Electrical stimulations were delivered to the IC, and field potentials were recorded from the EPN. In the case of slices made from mature rats, stable field potential oscillations at 8-10 Hz were induced in the EPN after repetitive stimulations. Optical recordings revealed that signals traveled from the IC to the EPN by way of the claustrum. Generation of oscillations was N-methyl-d-aspartate (NMDA) receptor activity-dependent, since oscillatory phases disappeared following application of NMDA receptor antagonist. In slices from immature rats, however, oscillations were not induced. IC stimulation can thus age-dependently elicit membrane potential oscillations in the EPN, and the EPN oscillations were NMDA receptor activity-dependent. These findings suggest that developmental changes in properties of the EPN might contribute to development of information integration, including gustatory information.

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.

To examine the very initial step that takes place immediately after tissue injury and is linked to tissue regeneration, we employed the submandibular gland (SMG), which was injured by ligation of its main excretory duct (MED). Ligation of the MED of the SMG in mice induced the expression of Sca-1, a protein marker of hematopoietic stem cells. In the normal gland, a low level of Sca-1 was expressed, which was localized predominantly in the excretory duct cells. At 1 day after ligation, Sca-1 expression increased prominently in almost all of cells in the duct system, but not in the acinar cells. The level of Sca-1 mRNA had begun to increase at 6 h after ligation and continuously rose thereafter until it reached a plateau, which occurred ∼12 h after ligation. STAT3 phosphorylated at its tyrosine-705 (p-STAT3) in the ligated gland increased immediately after ligation, and it was localized in the nuclei of all duct cells. The results of an EMSA revealed the specific binding of a nuclear extract to the sequence of the γ-interferon activation site (GAS) present in the Sca-1 promoter and confirmed that such binding increased after ligation. Thus the present study suggests that STAT3, having been phosphorylated following MED ligation, was transferred to the nucleus, where it bound to the GAS element in the promoter of Sca-1 gene, resulting in promotion of Sca-1 gene expression. Actual prevention of STAT3 phosphorylation reduced the ligation-induced Sca-1 elevation.

Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca2+, signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.

Background information. AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0–12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly103 > Asp103) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291, G1081–G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild-type) animals. Results. Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP-AQP5s) and expressed in polarized MDCK-II (Madin—Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP-AQP5 being translocated less efficiently. Thapsigargin and H-89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP-AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co-localized with LAMP2 (lysosome-associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co-localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation. Conclusion. Replacement of highly conserved hydrophobic Gly103 with strongly hydrophilic Asp103 in rat AQP5, though it did not affect water permeability, may possibly have resulted in less efficient membrane trafficking and increased lysosomal degradation, leading to its lower expression in the apical membrane of the acinar cells in the SMG.

S100A8 and S100A9 constitute a heterodimeric protein, calprotectin. The mRNAs of S100A8 and S100A9, being expressed at minimal levels in the submandibular and parotid glands (SMG and PG, respectively) of C3H/HeN mice, were induced strongly and transiently by lipopolysaccharide (LPS). Among the mRNAs of members of the S100 protein family examined, those of S100A8 and S100A9 were specifically induced by LPS in the salivary glands. The induction was assumed to be mediated via toll-like receptor 4 (TLR4), since their elevation was limited in C3H/HeJ mice, a TLR4-mutant strain. These proteins became expressed in the granular convoluted tubular cells and striated duct cells in the SMG, and in both acinar and duct cells in the PG (all in the cytoplasm). The salivary calprotectin level was not increased by LPS treatment, implying that elevated calprotectin was not secreted into the saliva and that they may function in microcellular environment of the salivary gland.

Chorda tympani denervation (CTD) of rats was earlier shown to result in loss of submandibular gland (SMG) weight (at only 1 wk) and in continued reduction in aquaporin 5 (AQP5) protein expression (until 4 wk), without affecting its mRNA synthesis (Li X, Azlina A, Karabasil MR, Purwanti N, Hasegawa T, Yao C, Akamatsu T, Hosoi K. Am J Physiol Gastrointest Liver Physiol 295: G112–G123, 2008). The present study indicated that despite elevation of bax, a proapoptosis protein, by CTD, the operation also increased the level of bcl-2, an antiapoptosis protein, in the SMG. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay) showed no increase in the number of apoptotic cells in the SMG. CTD, however, induced strongly and transiently (at 1–3 days) the protein expression of LC3B-II, a marker protein of autophagosomes, suggesting that the reduction in the gland weight was due to onset of autophagy by CTD. Upon CTD, Lamp2, a lysosomal marker, gradually increased in amount, reaching a peak at the 14th day. Immunohistochemical analysis revealed an increase in the number of lysosome-like structures positive for both AQP5 and Lamp2 in the acinar cells of the SMG after CTD; similar changes were observed also for AQP5 and LC3Bs. These data suggest that AQP5 in the SMG entered autophagosomes and/or lysosomes for degradation upon CTD. In vitro AQP5-degrading activity was found in the SMG extracts, and such activity was shown to be increased by CTD. Inhibitor experiments implied cathepsins B and L to be candidate enzymes for this degradation under normal and CTD conditions, respectively.

The mRNA and protein levels of aquaporin (AQP)5 in the parotid gland were found to be potentially decreased by lipopolysaccharide (LPS) in vivo in C3H/HeN mice, but only weakly in C3H/HeJ, a TLR4 mutant mouse strain. In the LPS-injected mice, pilocarpine-stimulated saliva production was reduced by more than 50%. In a tissue culture system, the LPS-induced decrease in the AQP5 mRNA level was blocked completely by pyrrolidine dithiocarbamate, MG132, tyrphostin AG126, SP600125, and partially by SB203580, which are inhibitors for IkappaB kinase, 26S proteasome, ERK1/2, JNK, and p38 MAPK, respectively. In contrast, the expression of AQP1 mRNA was down-regulated by LPS and such down-regulation was blocked only by SP600125. The transcription factors NF-kappaB (p65 subunit), p-c-Jun, and c-Fos were increased by LPS given in vivo, whereas the protein-binding activities of the parotid gland extract toward the sequences for NF-kappaB but not AP-1-responsive elements present at the promoter region of the AQP5 gene were increased by LPS injection. Co-immunoprecipitation by using antibody columns suggested the physical association of the three transcription factors. These results suggest that LPS-induced potential down-regulation of expression of AQP5 mRNA in the parotid gland is mediated via a complex(es) of these two classes of transcription factors, NF-kappaB and p-c-Jun/c-Fos.