Aquaporins (AQPs) are membrane water channels that are involved in a diverse set of functions in mammalian physiology including epithelial fluid transport, brain water balance, cell migration, cell proliferation, neuroexcitation, fat metabolism, epidermal hydration, and others. Phenotype analysis of knockout mice has demonstrated an important role for AQPs in transepithelial fluid transport in kidney tubules, salivary and airway submucosal glands, choroid plexus and ciliary epithelium. The physiological functions of these epithelia, such as absorption of glomerular filtrate by proximal tubule and secretion of saliva by salivary gland, involve rapid transcellular water transport across epithelial cell barriers. Studies in knockout mice have also provided evidence that AQPs are not physiologically important in some epithelia where they are expressed, including lacrimal gland, sweat gland, gallbladder, alveoli and airways. Rates of transepithelial fluid transport per unit membrane surface area in these epithelia are substantially lower than transepithelial fluid transport rates in proximal tubule and salivary gland. Pharmacological inhibition of AQP water permeability in epithelia, with consequent reduced fluid transport, offers potential therapy for human diseases involving water imbalance such as congestive heart failure, hypertension and glaucoma. J. Med. Invest. 56 Suppl.: 179-184, December, 2009

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Recent evidences suggest that the apoptotic pathway plays a central role in tolerazing T cells to tissue-specific self antigen, and may drive the autoimmune phenomenon in the salivary glands. We found that retinoblastoma-associated protein RbAp48 overexpression induces p53-mediated apoptosis in the salivary glands caused by estrogen deficiency. We demonstrated that transgenic (Tg) expression of RbAp48 resulted in the development of autoimmune exocrinopathy resembling Sjögren’s syndrome (SS). CD4⁺T cell-mediated autoimmune lesions in the salivary glands were aggravated with age, in association with autoantibody productions. We obtained evidences that salivary epithelial cells can produce interferon-γ (IFN-γ) besides interleukin (IL)-18, which activates interferon regulatory factor-1 (IRF-1), and class II transactivator (CIITA). Indeed, the autoimmune lesions into Rag2^-/- mice were induced by the adoptive transfer of lymph node cells from RbAp48-Tg mice. These results indicate a novel immunocompetent role of epithelial cells that can produce IFN-γ, resulting in loss of local tolerance prior to developing gender-based autoimmunity. The studies reviewed the molecular mechanisms on the development of salivary gland autoimmunity, and gender-related differences in SS. J. Med. Invest. 56 Suppl.: 185-191, December, 2009

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Fluid secretion by exocrine glands requires the coordinated activity of multiple water and ion transporter and channel proteins. The molecular cloning of many of the transporter molecules involved in fluid secretion has yielded a better understanding of the fluid secretion process. Mouse salivary glands are easily accessible model systems for the study of exocrine gland secretion at the cellular and organ level. Indeed, the characterization of mice with null mutations in many of the water and ion transporter and channel genes has demonstrated the physiological roles of individual proteins. This overview will focus on recent developments in determining the molecular identification of the proteins that are involved in the fluid secretion process. J. Med. Invest. 56 Suppl.: 192-196, December, 2009

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The oral gustatory perception during a meal has very important physiological roles such as inducing appetite, smoothing mastication and swallowing, promoting digestion and each nutrient availability. One hundred years ago, L-glutamate was discovered as a new taste substance in Japan. Since then, Japanese taste physiologists have lead the research to establish L-glutamate as the prototype molecule for the fifth basic taste (umami taste), in addition to saltiness, sweetness, bitterness and sourness. Meanwhile, various lines of evidence demonstrated that taste perception is linked to taste stimuli-oral/pharyngeal reflexes. In this review, we focus on the efficacy of L-glutamate for human salivation and discuss the possible application of umami taste simulation to the nutritional management for the elderly due to amelioration of their quality of life (QOL). J. Med. Invest. 56 Suppl.: 197-204, December, 2009

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Type III cells in taste buds form chemical synapses with intragemmal afferent nerve fibers and are characterized by the presence of membrane-bound vesicles in the cytoplasm. Although the vesicles differ in shape and size among species, they are primarily categorized into small clear (40 nm in diameter) and large dense-cored (90-200 nm) types. As such vesicles tend to be closely juxtaposed to the synaptic membrane of the cells, it is reasonable to consider that the vesicles include transmitter(s) towards the gustatory nerve. In the guinea-pig taste bud, stimulation with various taste substances (sucrose, sodium chloride, quinine hydrochloride, or monosodium L-glutamate) causes ultrastructural alterations of the type III cells. At the synapse, the presynaptic plasma membrane often displays invaginations of 90 nm in a mean diameter towards the cytoplasm, which indicates the dense-cored vesicles opening into the synaptic cleft by means of exocytosis. The vesicles are also exocytosed at the non-synaptic region into the intercellular space. These findings strongly suggest that the transmitters presumably contained in the vesicles are released to conduct the excitement of the type III cells to the nerves and also to exert their paracrine effects upon the surroundings, such as the Ebner’s salivary gland, acting as local hormones. J. Med. Invest. 56 Suppl.: 205-208, December, 2009

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Chemical substances of foods drive the cognitive recognition of taste with the subsequent regulation of digestion in the gastrointestinal (GI) tract. Tastants like glutamate can bind to taste membrane receptors on the tip of specialized taste cells eliciting umami taste. In chemical-sensing cells diffused through the GI tract, glutamate induces functional changes. Most of the taste-like receptor-expressing cells from the stomach and intestine are neuroendocrine cells. The signaling molecules produced by these neuroendocrine cells either activate afferent nerve endings or release peptide hormones that can regulate neighboring cells in a paracrine fashion or travel through blood to their target receptor. Once afferent sensory fibers transfer the chemical information of the GI content to the central nervous system (CNS) facilitating the gut-brain signaling, the CNS regulates the GI through efferent cholinergic and noradrenergic fibers. Thus, this is a two-way extrinsic communication process. Glutamate within the lumen of the stomach stimulates afferent fibers and increases acid and pepsinogen release; whereas on the duodenum, glutamate increases the production of mucous to protect the mucosa against the incoming gastric acid. The effects of glutamate are believed to be mediated by G protein-coupled receptors expressed at the lumen of GI cells. The specific cell-type and molecular function of each of these receptors are not completely known. Here we will examine some of the glutamate receptors and their already understood role on GI function regulation. J. Med. Invest. 56 Suppl.: 209-217, December, 2009

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Dietary L-glutamate (Glu), an amino acid abundant in many foodstuffs in a free form, is able to modulate physiological functions in the stomach, including secretion and motility. Recently, specific receptors for Glu were identified in the apical membrane of chief cells in the lower region of fundic glands and in the somatostatin-secreting D-cell fraction of the gastric mucosa. This Glu-sensing system in the stomach is linked to activation of the vagal afferents. Among 20 kinds of amino acid, luminal Glu alone activated the vagal afferents in the stomach through a paracrine cascade led by nitric oxide and followed by serotonin (5-HT). In dogs with Pavlov pouches, found that supplementation of an amino acid-rich diet lacking Glu with monosodium Glu (MSG) enhanced the secretion of acid, pepsinogen, and fluid. However, MSG did not affect these secretions induced by a carbohydrate-rich diet and it had no effect on basal secretion when MSG was applied alone without the diet. Enhancement of gastric secretion by MSG was abolished by blockage of the gastric afferents using intra-gastric applied lidocaine. This effect of MSG was due in part to stimulation of 5-HT₃ receptors in the gastric mucosa. J. Med. Invest. 56 Suppl.: 218-223, December, 2009

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Proteome analysis is a popular method to discover biomarkers for the prevention and diagnosis of diseases. Since saliva is a non-invasively available body fluid, gathering of saliva causes minimal harm to patients. Therefore, detection of proteins for the prevention and diagnosis from the saliva sample may be the preferred method, especially for children and elderly people. However, the abundance of salivary proteins and contaminant proteins from food and mouth bacteria obscure identification of proteins present in the saliva at low concentrations. To address this problem, we developed a shotgun proteomic method using two-dimensional nano-flow LC tandem mass spectrometry. We report here that our method is able to detect proteins quantitatively even in small sample volumes of saliva. J. Med. Invest. 56 Suppl.: 224-227, December, 2009

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Branching morphogenesis (BrM) is a basic developmental process for the formation of the lung, kidney, and all exocrine glands, including the salivary glands. This process proceeds as follows. An epithelial downgrowth invaginates into underlying mesenchyme, and forms a cleft at its distal end, which is the site of dichotomous branching and elongation; this process of clefting and elongation is repeated many times at the distal ends of the invading epithelium until the desired final extent of branching is reached. The distal ends of the epithelium differentiate into the secretory endpieces, and the elongated segments become the ducts. This presentation is a brief historical review of studies on BrM during the development of the submandibular gland (SMG). J. Med. Invest. 56 Suppl.: 228-233, December, 2009

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Many organs such as the salivary glands, kidney and lung form multiple epithelial clefts during the process of branching morphogenesis in embryonic development. The salivary gland provides an excellent model for clarifying the mechanism of this phenomenon. Extracellular matrix molecules regulate the dynamics of this complex process of organ formation. Nevertheless, the mechanism of cleft formation is poorly understood. Here we describe that a combination of laser microdissection with T7-SAGE has been established as a method for gene discovery of candidate molecules that may be essential for early organ morphogenesis. We show a current approach that promises to improve our understanding of the early morphological changes. We are attempting to find novel regulators of branching morphogenesis. Fibronectin, laminin γ2, and TIMP-3 are differentially expressed in developing clefts or buds of submandibular glands. Fibronectin-mediated crosstalks between integrin cell-to-matrix and cadherin cell-to-cell adhesion systems promote the formation of clefts. We have started studies to identify and characterize new molecules that may be important for cleft formation and fibronectin expression. We will introduce recent findings and future plans to identify new functional regulators. Progress in understanding the mechanisms of branching morphogenesis will provide novel approaches to future tissue engineering or regeneration of damaged organs. J. Med. Invest. 56 Suppl.: 234-238, December, 2009

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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. J. Med. Invest. 56 Suppl.: 241-246, December, 2009

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The submandibular gland (SMG) of the fetal mouse is a well-studied model for the epithelial-mesenchymal interactions required for branching morphogenesis (BrM), which involves cleft formation and stalk elongation. In a previous report, we showed that α6 integrin subunit is involved in BrM of this gland rudiment, since the neutralizing antibody against α6 integrin subunit, GoH3, strongly inhibited branching of cultured intact E13 SMG. In this study, we investigated whether GoH3 inhibits cleft formation and/or stalk elongation during BrM of cultured mesenchyme-free epithelial rudiments of fetal SMG and also analyzed by Western blotting the levels of phosphorylation of ERK1/2, which is a signaling molecule known to regulate BrM of the fetal mouse SMG. J. Med. Invest. 56 Suppl.: 247-249, December, 2009

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Fetal murine submandibular salivary gland (SMG) is known as a model to study organogenesis including branching morphogenesis, which is a basic developmental process for formation of a wide variety of arborized organs. Branching morphogenesis is under the control of a complex network of regulatory proteins, such as the ErbB family of tyrosine kinase receptors, activated by members of the epidermal growth factor (EGF) family of ligands. Recent reports identify critical roles for micro RNAs (miRNAs) on many developmental processes through regulation of gene expression. We hypothesize that miRNAs regulating branching morphogenesis are expressed in fetal murine SMG and that expression of the miRNAs associated with branching morphogenesis is modulated in part by EGF. Using cloning methods, we obtained the expression profiles on miRNAs derived from fetal murine SMG under three different conditions: (1) native E13 SMGs (freshly isolated), (2) E13 SMGs cultured for 24 hours with no added EGF (controls), or (3) cultured with EGF. There were 44 known miRNAs and four novel miRNAs candidates in native SMG at E13. Comparing the three profiles revealed that several miRNAs were expressed specifically at each condition. These results suggested that these miRNAs were associated with regulating organogenesis, possibly including branching morphogenesis. J. Med. Invest. 56 Suppl.: 250-252, December, 2009

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Ligation of the main excretory duct (MED) of the mouse submandibular gland (SMG) induced the expression of Sca-1, a stem cell marker. Sca-1 expression increased prominently in almost all of cells in the duct system, except the acinar cells. Sca-1 induction was accompanied with phosphorylated-STAT3 (Y705) elevation, which was localized in the nuclei of all duct cells. Electrophoretic mobility shift assay (EMSA) confirmed the specific binding of STAT3 to the GAS sequence, a biding site of gamma interferon activating site. Present study suggested one of the initial steps of the tissue regeneration after injury includes STAT3 pathway. J. Med. Invest. 56 Suppl.: 253-254, December, 2009

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Parotid glands secrete about 25% of all saliva produced in the salivary glands. In the presence of a stimulus, the amount of saliva secreted from the parotid gland increases to 50% (1). In human adults, the parotid duct, approximately 6-8 cm long, traverses the masseter muscle and penetrates through the buccinator muscle into the oral cavity. Although various studies have been conducted on the parotid gland, there are only few suggesting the functional roles of the parotid duct, especially of the area penetrating the buccinator muscle. In the present study, we observed parotid ducts of human fetuses to morphologically analyze the function of the buccinator muscles in the flux of parotid saliva. Thirty fetal specimens ranging from five to ten months of age were dissected for anatomical and histological examinations. The area of the parotid duct penetrating the buccinator muscle was fully formed in six-month-old fetuses. Furthermore, this study confirms the existence of thin buccinator muscle fibers underneath the epithelium of the parotid duct’s distal portion. Results suggest that the buccinator muscle may play a major role in preventing the reflux of salivary secretions by assisting the contraction of the parotid duct. J. Med. Invest. 56 Suppl.: 255-257, December, 2009

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Hyposecretion of saliva and consequent dry mouth lead to severe caries and periodontal disease. Therapeutic radiation for head and neck cancer and sialadenitis result in atrophy and fibrosis of salivary glands, but the mechanism is not clear. As a model for dysfunction of salivary glands, we examined the change of gene expression patterns in primary cultured parotid acinar cells. The expression levels of acinar markers such as amylase and aquaporin-5 rapidly decreased during culture. At the same time, ductal markers began to be expressed although their expression was transient. In the late phase of culture, markers of epithelial-mesenchymal transition began to be expressed and increased. Inhibitor for Src or p38 MAP kinase suppressed these changes. These results suggest that parotid acinar cells transiently change to duct-like cells during epithelial-mesenchymal transition and that these changes are induced by signal transduction via Src-p38 MAP kinase pathway. There is a possibility that parotid acinar cells retain a plasticity of differentiation. J. Med. Invest. 56 Suppl.: 258-259, December, 2009

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Saliva contains a wide variety of secretory proteins, including α-amylase, lysozyme, peroxidase, immunoglobulins, and mucins. Hyposecretion of saliva and consequent dry mouth will lead to severe dental caries, periodontal disease, and mucosal infections, resulting in degrade of quality of life. Polyposia development is one of sign usually seen in dry mouth patients. However, little is reported in dry mouth-model animal regarding the entire process of polyposia development. We investigated the behavior of polyposia in E2F1-deficient non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice, as a dry mouth-model. E2F1-deficient NOD/SCID mice secreted small amount of saliva under the stimulation with a cholinergic agonist, pilocarpine, compared with control mice. The frequency of water intake by E2F-1-deficient NOD/SCID mice was more than that by control mice. These results suggest that E2F-1-deficient NOD/SCID mice show a behavior similar to polyposia and are very useful experimental model of dry mouth patients. J. Med. Invest. 56 Suppl.: 260-261, December, 2009

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Extracellular nucleotides are local, short-lived signaling molecules that inhibit renal tubular transport via both luminal and basolateral P2 receptors (1, 2). Apparently, the renal epithelium itself is able to release nucleotides (3, 4). The mechanism and circumstances under which epithelia nucleotide release is stimulated remains elusive (5, 6). Here, we investigate the phenomenon of nucleotide secretion in intact perfused mouse medullary thick ascending limb (mTAL) and cortical collecting duct (CCD). The nucleotide secretion was monitored by a biosensor cell placed to register nucleotides in the tubular out-flow. [Ca²⁺]_i was measured simultaneously in the biosensor cells and the renal tubule with fluo-4. We were able to identify spontaneous tubular nucleotide secretion in resting perfused mTAL. This was seen as lively [Ca²⁺]_i oscillations in the nucleotide biosensor cells when the tubular outflow fluid engulfed the sensing cells. In mouse mTAL 10 nM AVP and dDAVP induced robust [Ca²⁺]_i oscillations, whereas AVP in the CCD induced large, slow and transient [Ca²⁺]_i elevations. Importantly, we identify that AVP/dDAVP triggers tubular secretion of nucleotides in mTAL. After addition of AVP/dDAVP the biosensor cells registered bursts of nucleotides originating from the tubular perfusate. The approximated tubular nucleotide concentration reached peak values of ∼0.2-0.3 μM. A very similar response was observed after AVP stimulation of CCDs. Thus, AVP stimulated tubular secretion of nucleotides in a burst like pattern with peak tubular nucleotide concentrations in the low micromolar range. Luminal nucleotides are prone to activate luminal P2 receptors (1) which in turn are well described to inhibit AVP-augmented aquaporin-2-dependent water absorption (7) or ENaC-mediated Na⁺ transport (8). Therefore, we speculate that local nucleotide signaling is an intrinsic feed-back element of hormonal control of renal tubular transport (9). J. Med. Invest. 56 Suppl.: 262-263, December, 2009

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The superior salivatory nucleus (SSN) is the primary parasympathetic center controlling submandibular salivatory secretion. Our previous electrophysiological study revealed that many SSN neurons receive GABAergic and glycinergic synaptic inputs. In the present study, we examined the distribution of GABAergic and glycinergic nerve terminals, GABA_A receptors in the SSN, and the origin of GABAergic nerve terminals innervating the SSN. Glutamic acid decarboxylase (GAD) and glycine transporter 2 (GLYT2) were used as markers of GABAergic and glycinergic nerve terminals, respectively. GAD- and GLYT2-positive nerve terminals and GABA_A receptors were examined immunohistochemically in SSN neurons labeled by the retrograde axonal transport of FastBlue (FB) injected into the chorda-lingual nerve. The SSN neurons abundantly contained GAD-positive nerve terminals and GABA_A receptors, suggesting that SSN neurons undergo strong GABAergic inhibition. The origin of GABAergic terminals was examined in neurons labeled by the retrograde transport of FluoroGold (FG) injected into the SSN. GAD was used as a marker of GABAergic neurons. Numerous FG-labeled neurons were found in the forebrain and brainstem. However, in FG-labeled neurons, GAD-positive neurons were occasionally observed in the reticular formation of the brainstem. These findings suggest that SSN neurons mainly receive GABAergic projections from the reticular formation. J. Med. Invest. 56 Suppl.: 264-266, December, 2009

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Cevimeline, a therapeutic drug for xerostomia, is an agonist of muscarinic acetylcholine receptors (mAChRs), and directly stimulates the peripheral mAChRs of the salivary glands. Since cevimeline is distributed in the brain after its oral administration, it is possible that it affects the central nervous system. However, it is unknown how cevimeline affects the superior salivatory (SS) neurons, which control submandibular salivation. In the present study, we examined the effects of cevimeline on the SS neurons using the whole-cell patch-clamp technique in brain slices. In Wistar rats (6-10 days), the SS neurons were retrogradely labeled by Texas Red applied to the chorda-lingual nerve. Two days after injection, whole-cell recordings were obtained from the labeled cells, and miniature excitatory postsynaptic currents (mEPSCs) were examined. Cevimeline induced the inward currents dose-dependently and increased the frequency of mEPSCs. Therefore, it is suggested that cevimeline enhances the excitability via post- and presynaptic muscarinic receptors in the rat SS neurons. In conclusion, cevimeline may enhance the excitability of the SS neurons. J. Med. Invest. 56 Suppl.: 267-269, December, 2009

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The primary parasympathetic center of the submandibular and sublingual salivary glands is the superior salivatory (SS) nucleus, and its neurons receive excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) synaptic transmissions in rats. In the present study, we focused on the postnatal development of inhibitory transmission to SS neurons. Gramicidin-perforated whole-cell patch-clamp recordings were performed in rat brainstem slices on postnatal day 2 (P2)-P14. Developmental changes in the intracellular Cl^- concentration ([Cl^-]_in) were examined based on the reversal potentials of total inhibitory postsynaptic currents (GABAergic plus glycinergic), which were evoked by electrical stimulation near the recording neuron. The [Cl^-]_in in the P8-P14 group was significantly lower than in the P2-P7 group. The effect of GABA application at the resting potentials changed from depolarization to hyperpolarization around P8, suggesting that SS neurons acquired mature inhibitory systems around P8. The period at which GABA responses change from excitatory to inhibitory in SS neurons was discussed compared with those of the forebrain, brainstem, and spinal neurons. J. Med. Invest. 56 Suppl.: 270-272, December, 2009

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Following chorda tympani denervation (CTD, parasympathetomy), the protein levels of aquaporin5 (AQP5) as well as AQP1 and Na⁺K⁺ATPase α-subunit in the rat submandibular gland (SMG) were found to be decreased significantly. However, the level of another membrane protein, dipeptidyl peptidase IV was not affected by CTD, suggesting a selective reduction of AQP5, AQP1, and Na⁺K⁺ATPase α -subunit proteins by CTD. However, the AQP5 mRNA level was scarcely affected by CTD, which suggested that transcription process of AQP5 was unaffected by this operation. AQP5 protein was shown to be degraded in vitro by the extract of the SMG obtained from normal rat; inhibitor experiments in vitro suggested cathepsin B was a responsible enzyme. Co-localization of AQP5 and LAMP-2, a lysosomal marker, implicated AQP5 is degraded in lysosomes. A significant increase in the protein levels of LC3-II, an autophagy marker, at day 1 after CTD, and co-localization of the LC3 protein and AQP5, suggested that CTD activated autophagy of SMG, leading to AQP5 degradation. J. Med. Invest. 56 Suppl.: 273-276, December, 2009

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The act of brushing teeth induces salivary secretion (1). However, there is no evidence to show which particular regions of the teeth and oral cavity in brushing induces the most effective salivary secretion. We investigated the effects of tooth brushing in different oral regions on parotid salivary secretion in humans. Saliva was collected using a modified Lashley cup. Brushing of the gingival margin was performed according to the Bass method, which may stimulate periodontal mechanoreceptors. The occulsal surface of the molars, gingiva, tongue, and palatal rugae were also brushed with a toothbrush. Compared to the flow rate of saliva from the unstimulated parotid gland, the salivary flow rates were enhanced when every oral region was brushed. The flow rate produced by brushing the palatal gingival margin of the ipsilateral maxillary molars was greater than that produced by brushing the occlusal surface of ipsilateral maxillary molars and palatal gingiva beside the molars. The flow was also significantly greater than that produced by brushing the palatal gingival margins of the contralateral maxillary molars, the maxillary/mandibular incisors, the dorsum of tongue and the palatal rugae. No significant difference was observed between brushing the gingival margins of the ipsilateral maxillary versus mandibular molars. These data indicate that brushing the gingival margin of molars is the most effective region to stimulate parotid saliva secretion. This likely activates periodontal mechanoreceptors, and this technique may be a simple way to improve salivary secretion in oral rehabilitation. The degree of salivary secretion caused by brushing the teeth is specific to particular oral regions. Brushing of the gingival margins is the most effective location to induce parotid salivary secretion. J. Med. Invest. 56 Suppl.: 277, December, 2009

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Neurokinin A (NKA) evokes salivary secretion. Despite such reports, the direct effect of NKA on salivary secreteion in submandibular gland has not been clarified. Here we studied characterization of salivary fluid secretion induced by NKA in the perfused submandibular grand (SMG) of the rat. NKA (3-100 nM) stimulated salivary fluid secretion in a dose-dependent manner. The profile of secretion induced by NKA consisted of two phases, transient and sustained phases. When the gland was perfused with Lucifer yellow (LY)-containing perfusate buffer and stimulated by NKA, concentration of LY in saliva was increased. In the absence of Ca²⁺ in the perfusate, NKA induced only a transient salivary fluid and a transient LY secretion. When the gland was treated with BAPTA, NKA failed to induce both salivary fluid secretion and LY secretion. These results suggest that NKA induces salivary secretion via both transcellular and paracellular pathways, which depends on intracellular Ca²⁺ mobilization. J. Med. Invest. 56 Suppl.: 278-280, December, 2009

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Xerostomia is the symptom of dry mouth often seen in patients who receive head and neck radiation therapy or in patients who have Sjögren’s syndrome. The primary treatment to relieve xerostomia symptom is oral administration of pilocarpine, a parasympathomimetic agent with muscarinic action. Increase in salivary secretion induced by systemic administration of pilocarpine is considered to be mediated by actions on muscarinic cholinergic receptors in the central nervous system and salivary glands. In this study, we investigated the direct effect of pilocarpine on salivary fluid secretion in the isolated, perfused rat submandibular gland. Pilocarpine provoked salivary fluid secretion in a dose-dependent manner. The Na⁺-channel blocker tetrodotoxin had almost no effect on the pilocarpine-induced salivary fluid secretion, indicating that pilocarpine directly stimulates submandibular gland. Pilocarpine induced an increase in intracellular Ca²⁺ concentration in dispersed submandibular gland cells at 37°C , but not 25°C. The salivary fluid secretion induced by pilocarpine was consisted of a rapid and transient phase and a subsequent sustained phase, which profile was different from that evoked by carbachol, another typical muscarinic agonist. Pilocarpine also induced Lucifer yellow secretion via paracellular route. J. Med. Invest. 56 Suppl.: 281-283, December, 2009

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In parotid acinar cells, activation of β-adrenergic receptors provokes exocytotic amylase release via the accumulation of intracellular cAMP. Cellular redox status plays a pivotal role in the regulation of various cellular functions. Cellular redox imbalance caused by the oxidation of cellular antioxidants, as a result of oxidative stress, induces significant biological damages. In this study, we examined effect of diamide, a thiol-oxidizing reagent, on amylase release in rat parotid acinar cells. In the presence of diamide, isoproterenol (IPR)-induced cAMP formation and amylase release were partially reduced. Diamide had no effect on amylase release induced by forskolin and mastoparan, an adenylate cyclase activator and heterotrimeric GTP binding protein activator, respectively. In the cells pretreated with diamide, the binding affinity of [³H]dihydroalprenolol to β-receptors was reduced. These results suggest that oxidative stress results in reduction of binding affinity of ligand on β-receptor and consequently reduces protein secretory function in rat parotid acinar cells. J. Med. Invest. 56 Suppl.: 284-286, December, 2009

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Calprotectin is a major cytosolic calcium-binding protein of leukocytes which belongs to the S100 protein family. S100A8 and S100A9, major types of calprotectin are heterodimeric complexes being composed of light- and heavy-chain subunits. The calprotectin levels in the plasma, feces, synovial fluid, gingival crevicular fluid, dental calculus and saliva change when the host animal suffers from several inflammatory diseases. Members of Toll-like receptor (TLR) family are pattern-recognition receptors for lipopolysaccharide (LPS) and other pathogens. Here we examined if the biological role of TLR receptor is reflected to the calprotectin expression in the salivary gland. Time course study by using real-time RT-PCR detected higher levels of S100A8 and S100A9 mRNA at 1.5-3 h after injection of LPS in both the submandibular gland (SMG) and parotid gland (PG) of C3H/HeN mice but not in the same tissues of C3H/HeJ, a TLR-4 mutant strain, indicating that this induction is mediated via the TLR-4. These results indicate that, an inflammatory marker, calprotectin, is expressed in the mouse salivary gland and that LPS stimulated its synthesis. Calprotectin (S100A8/A9) showed minimum expression in all cellular segments in the SMG except excretory duct cells, which showed strong signal at the cytoplasm. LPS induced their expressions in the granular convoluted tubular cells and striated duct cells. In the PG, these proteins were expressed very weakly in both duct and acinar cells with a little stronger staining for the former cells. LPS injection induced calprotectin (S100A8/A9) in both duct and acinar cells especially in the former cells. J. Med. Invest. 56 Suppl.: 287-289, December, 2009

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Most striated duct (SD) cells in the adult mouse parotid gland (PAG) have a few small secretory granules. These granules, however, are usually too small and sparse to be detected using light microscopy. Our serial studies have suggested that these PAG SD cells belong to a group of hormone-responsive granular duct cells, similar to the granular convoluted tubule (GCT) cells found in the submandibular gland. These studies also indicate that and some PAG SD cells may be capable of developing a granular cell phenotype under supraphysiological conditions of androgenic and thyroid hormones, leading to more abundant, and more kinds of GCT-specific secretory polypeptides. Here, the cytology of hormone-modulated SD cells, the immunocytochemistry of their secretory products, and their secretory responses to some autonomic agents are reviewed. Finally, the close similarity of the duct systems of the three major salivary glands in mice is critically emphasized. J. Med. Invest. 56 Suppl.: 290-295, December, 2009

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Leptin is a hormone, which plays a central role in inhibiting food intake and body weight gain. Leptin is secreted from exocrine as well as endocrine cells. Circulating leptin activates JAK-STAT tyrosine kinases through Ob-Rb leptin receptor in the hypothalamus and brain stem. In recent years, “leptin resistance” has been considered to be one of the main causes of obesity. However, the detailed mechanisms of leptin resistance are not well understood. Recently, we hypothesized possibility that endoplasmic reticulum (ER) stress is involved in leptin resistance. In the present manuscript, we would like to mention possible mechanisms of ER stress-induced leptin resistance and possible implication in obesity. In addition, pathophysiological role of leptin’s action in regulating endocrine as well as exocrine functions at the state of ER stress are discussed. J. Med. Invest. 56 Suppl.: 296-298, December, 2009

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Distal colonic K⁺ excretion is determined by the balance of K⁺ absorption and K⁺ secretion of the enterocytes. K⁺ secretion occurs via active basolateral K⁺ uptake mostly via the NKCC1 co-transporter followed by K⁺ exit via a luminal K⁺ channel. The specific focus here is directed towards the luminal secretory K⁺ channel (1). Several recent observations highlight the pivotal role of the large conductance, Ca²⁺-activated K_Ca1.1 (BK, KCNMA) channel as the only functionally relevant luminal K⁺ efflux pathway in mouse distal colon (2, 3). This conclusion was based on defining results from BK knock-out mice. The following key observations were made: 1. BK channels mediate the resting distal colonic K⁺ secretion (2, 4), 2. They are acutely stimulated by activation of luminal nucleotide receptor and elevations of intracellular Ca²⁺ (2, 4, 5), 3. Colonic BK channels are up-regulated by increases of plasma aldosterone (3), 4. In addition, the cAMP-stimulated distal colonic K⁺ secretion is apparently mediated via BK channels, 5. Finally, aldosterone was found to up-regulate specifically the ZERO (e.g. cAMP activated) C-terminal splice variant of the BK channel. In summary, we suggest that the sole exit pathway for transcellular ^K+ secretion in mammalian distal colon is the BK channel, which is the target for short term intracellular Ca²⁺ and cAMP activation and long term aldosterone regulation. J. Med. Invest. 56 Suppl.: 301, December, 2009

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Polarized protein deposition at the apical and basolateral membranes of epithelial cells is critical for the asymmetrical transport of ions and fluids across the epithelia. PDZ-based modular adaptor proteins are expressed in the junctional areas in epithelial cells and are generally part of a molecular scaffold that determines the localization and activity of ion channels, receptors, and other signaling molecules to their correct spatial arrangement for proper response to diverse stimuli. Hence, understanding the regulatory mechanisms of channels and receptors via PDZ-based adaptors will provide valuable insights into the physiology of epithelial cells as well as pathophysiology of many human diseases including cystic fibrosis. J. Med. Invest. 56 Suppl.: 302-305, December, 2009

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Pancreatic duct cells express Na⁺-dependent glucose transporter, SGLT1 and Na⁺-independent glucose transporters, GLUT1, GLUT2, and GLUT8. We examined transepithelial glucose transport by pancreatic duct. Interlobular ducts were isolated from rat pancreas. During overnight culture both ends of the duct segments sealed spontaneously. The lumen of the sealed duct was micropunctured and the luminal fluid was replaced by HEPES-buffered solution containing 10.0 mM or 44.4 mM glucose. The bath was perfused with HEPES-buffered solution at 37°C containing 10.0 or 44.4 mM glucose. Transepithelial differences in osmolality were balanced with mannitol. Glucose transport across ductal epithelium was measured by monitoring changes in luminal volume. When the lumen was filled with 44.4 mM glucose, with either 10.0 or 44.4 mM glucose in the bath, the luminal volume decreased to 65∼70% of the initial volume in 15 min. Luminally-injected phlorizin, an inhibitor of SGLT1, abolished the decrease in luminal volume. With 10.0 mM glucose in the lumen and 44.4 mM glucose in the bath, the luminal volume did not change significantly. Luminal application of phlorizin under identical condition led to an increase in luminal volume. The data suggest that both active and passive transport mechanisms of glucose are present in pancreatic ductal epithelium. J. Med. Invest. 56 Suppl.: 308-311, December, 2009

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AQP11 is a member of a new aquaporin subfamily which includes many aquaporin homologs with low amino acid identities, around 20% of previously identified AQPs. Although these AQPs have unusual NPA sequences, these AQPs have a completely conserved and functionally indispensable cysteine residue downstream of the second NPA box, suggesting that they belong to a specific AQP subfamily, which we propose to name the group III AQPs. On the other hand, the NPA boxes are highly conserved in previous AQP subfamilies: the group I AQPs, original water-selective aquaporin family and the group II AQPs, aquaglyceroporin family. Currently the roles of the group III AQPs are only known with AQP11 as the disruption of intracellularly located AQP11 in mice produced huge vacuoles in the proximal tubule leading to fatal polycystic kidneys at one month old. This review focused on the classification of AQPs based on primary structures to obtain insights into the function and the role of AQPs. With the accumulation of new AQP-like sequences through genome projects, this classification will be useful to predict their functions as each group may have specific characteristics in its function, distribution and regulation. J. Med. Invest. 56 Suppl.: 312-317, December, 2009

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Chronic pancreatitis with all kinds of etiologies is characterized by pancreatic exocrine dysfunction especially impaired fluid secretion from pancreatic ducts. However, the molecular mechanism of this impaired fluid secretion in chronic pancreatitis is largely unknown. Aquaporin water channels are intrinsic membrane proteins expressed most of the cell types which have high osmotic water permeability. Among them aquaporin 1 (AQP1) is a predominant water channel expressed in the plasma membranes of human pancreatic ducts. Exocrine function test revealed that fluid secretion was severely impaired in AIP. immunohistochemical analysis revealed that AQP1 is localized mainly in the apical and lateral membranes of small pancreatic ducts in control subjects. AQP1 expression was significantly increased in plasma membranes of pancreatic ducts in AIP. Upregulation of AQP1 expression seen in pancreatic ducts of patient with AIP may be caused by the reduced fluid secretion from the pancreas as compensation. Further study would be required to elucidate the precise molecular mechanism for the role of AQP1 in pancreatic fluid secretion from the pancreas in diseases characterized by the impaired ductal fluid secretion such as cystic fibrosis. J. Med. Invest. 56 Suppl.: 318-321, December, 2009

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A part of the salivary components are shifted from the blood via a trans- and/or paracellular route. The isolated, arterially perfused, salivary glands (submandibular and parotid glands) were used to assess paracellular transport functionally and morphologically. In the present study, the hydrostatic pressure of the perfusion was changed and the fluid secretion and paracellular transport of fluorescent dye in the isolated perfused submandibular gland were examined. The present findings lead to the conclusion that part of the paracellular transport could be driven by hydrostatic pressure, and that fluid movement drags the solutes. J. Med. Invest. 56 Suppl.: 322-324, December, 2009

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Pancreatic duct cells secrete a HCO₃^--rich (∼140 mM) fluid. Using a computer model of the pancreatic duct, Sohma, et al. have demonstrated that the activity of a Cl^-/HCO₃^- exchanger with a 1: 1 stoichiometry at the apical membrane would have to be suppressed in order to achieve such a HCO₃^--rich secretion. Recently the apical exchanger in pancreatic ducts has been identified as SLC26A6 and this probably mediates most of Cl^--dependent HCO₃^- secretion across the apical membrane. SLC26A6 is reported to mediate electrogenic Cl^-/2HCO₃^- exchange when expressed in Xenopus oocytes. To assess the implications of this 1: 2 stoichiometry for HCO₃^- secretion, we have reconstructed the Sohma model using MATLAB/Simulink. To do this we have formulated an expression for the turnover rate of Cl^-/2HCO₃^- exchange using network thermodynamics and we have estimated the constants from published experimental data. Preliminary data suggest that the 1: 2 stoichiometry of SLC26A6 would favor HCO₃^- secretion at higher concentrations. J. Med. Invest. 56 Suppl.: 325-328, December, 2009

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Pancreatic duct epithelium secretes HCO₃^--rich fluid, which is dependent on cystic fibrosis transmembrane conductance regulator (CFTR). HCO₃^- transport across the apical membrane is thought to be mediated by both SLC26A6 Cl^--HCO₃^- exchange and CFTR HCO₃^- conductance. In this study we examined the relative contribution and interaction of SLC26A6 and CFTR in apical HCO₃^- transport. Interlobular pancreatic ducts were isolated from slc26a6 null mice. Intracellular pH (pH_i) was measured by BCECF microfluorometry. Duct cells were stimulated with forskolin and alkalinized by acetate pre-pulse in the presence of HCO₃^--CO₂. Apical HCO₃^- secretion was estimated from the recovery rate of pH_i from alkaline load. When the lumen was perfused with high-Cl^- solution, the rate of apical HCO₃^- secretion was increased by luminal application of CFTRinh-172 in ducts from wild-type mice but it was decreased in ducts from slc26a6 -/- mice. This suggests that slc26a6 and CFTR compensate/compete with each other for apical HCO₃^- secretion with high Cl^- in the lumen. With high HCO₃^- in the lumen, luminal CFTRinh-172 reduced the rate of apical HCO₃^- secretion in both wild-type and slc26a6 -/- ducts. This suggests that HCO₃^- conductance of CFTR mediates a significant portion of apical HCO₃^- secretion with high HCO₃^- in the lumen. J. Med. Invest. 56 Suppl.: 332-335, December, 2009

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The secretory epithelia of the pancreatic duct and airway share the ability to generate HCO₃^--rich fluids. They both express CFTR (cystic fibrosis transmembrane conductance regulator) at the apical membrane and both are adversely affected by cystic fibrosis. CFTR is predominantly a Cl^- channel, and it is widely believed that HCO₃^- secretion in the pancreatic duct is mediated mainly by a Cl^-/HCO₃^- exchanger at the apical membrane. Studies on airway epithelia, however, have suggested that CFTR, despite its low permeability to HCO₃^-, may nonetheless be directly responsible for HCO₃^- secretion across the apical membrane. This article reviews recent work that has re-examined both of these hypotheses. J. Med. Invest. 56 Suppl.: 336-342, December, 2009

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Aquaglyceroporins (AQGPs) are members of aquaporin (AQP) family and belong to a subgroup of this water channel family; they are transmembrane proteins that transport water as well as glycerol and other solutes of small molecules. Recent studies have also identified that AQGPs are important transporters of trivalent metalloid in some mammalian cells. However, the uptake routes of arsenite in mammals are still less defined. In this study, to understand the routes of arsenite intake in mammals, mice were treated with Hg(II), glycerol, and As(III) and uptake of As(III) into the gastrointestinal tissues was measured. The level of inorganic arsenic (iAs) in gastrointestinal tissues after As(III) stimulation was much higher than Hg(II) +As(III) or glycerol+As(III) group. RT-PCR results showed that AQGPs were extensively expressed in gastrointestinal tissues of mice. We also treated Caco-2 cells with Hg(II) and As(III); the level of iAs in a group treated with Hg(II)+As(III) decreased compared with As(III)-treated group. Our results suggested that AQGPs could be important transporters in arsenite uptake into gastrointestinal tissues of mice, but more data are need to prove if AQGPs is the only pathway involved in As transport in mammals or just one of them. J. Med. Invest. 56 Suppl.: 343-346, December, 2009

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Salivary gland acinar cells secrete large amounts of water and electrolytes, where aquaporins (AQPs) are thought to be involved in the secretion. In the present study, we investigated expression/localization of AQP6, and the anion transporting properties of AQP6 in mouse parotid acinar cells. RT-PCR, western blotting and immunohistochemical analyses revealed expression of AQP6 in acinar cells, localized in apical membrane. Voltage ramp from -100 mV to +100 mV at a holding potential of -60 mV elicited outwardly-rectifying currents, in the presence of extracellular Cl^- channel blockers and intracellular solution with 150 mM Cs⁺. These outward currents were increased when extracellular Cl^- was replaced by Br^-, NO₃^-, I^-, or SCN^-, accompanying a negative shift of reversal potentials. The outward current was enhanced by extracellular Hg²⁺. These results were consistent with the biophysical properties of transfected AQP6 oocytes or HEK cells, which indicate that the AQP6 channel is functionally expressed in parotid acinar cells, and suggest that AQP6 contributes to secretion of anions in parotid acinar cells. J. Med. Invest. 56 Suppl.: 347-349, December, 2009

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Saliva samples are useful for noninvasive diagnosis of oral and systemic diseases. The water channel protein aquaporin-5 (AQP5) is released into human saliva. Salivary AQP5 levels show a diurnal variation with the secretion of high levels during the waking hours. An age-related decrease in salivary AQP5 levels parallels a decrease in the volume of saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induces the release of AQP5. Changes in salivary AQP5 levels after cevimeline administration occur simultaneously with changes in saliva flow rate. AQP5 and lipid rafts are released separately from human salivary glands upon M₃ mAChR stimulation. In patients with diabetes mellitus or Sjögren’s syndrome, a decrease in salivary secretion occurs concomitantly with low salivary AQP5 levels. Salivary AQP5 levels correlate with salivary secretion in both healthy and disease states, suggesting that changes in salivary AQP5 levels can be used as an indicator of salivary flow rate and the effect of M₃ mAChR agonists on human salivary glands. J. Med. Invest. 56 Suppl.: 350-353, December, 2009

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The ductal system of the exocrine pancreas produces HCO₃^--rich fluid in response to secretin and other stimuli. HCO₃^- efflux across the luminal membrane is mediated by a Cl^--HCO₃^- exchanger operating in parallel with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel. Basolateral K⁺ channels provide an exit pathway for K⁺ and play a vital role in maintaining the membrane potential, which is a crucial component of the driving force for anion secretion. Measurements of membrane potential with intracellular microelectrodes suggested that Ba²⁺-sensitive K⁺ conductance accounts for more than 60% of the total basolateral ionic conductance in resting ducts (1). To identify the Ba²⁺-sensitive K⁺ channels, we isolated ducts from normal rat pancreas by collagenase digestion. We first demonstrated that the ducts did not express a vascular endothelial marker PECAM-1 (platelet/endothelial cell adhesion molecule-1), but expressed cytokeratin 20, a marker of duct cells (2), using immunofluorescent staining. In addition, monoclonal anti-CFTR antibody was detected near the luminal membrane of these cells. In cell-attached single-channel recordings, we observed three types of K⁺ channels on basolateral membrane in unstimulated duct cells. The 40 pS K⁺ channels are likely to mediate whole-cell inwardly rectifying K⁺ (Kir) currents, which were blocked by extracellular Ba²⁺ in a voltage-dependent manner. The properties of 90 pS and 170 pS K⁺ channels are similar to those of Ca²⁺-activated K⁺ channels. We then identified Kir2.0 and SK4/IK1 (intermediate conductance Ca²⁺-activated K⁺ channel) subunits as molecular candidates of the K⁺ channels using RT-PCR analysis. The present results suggest that these subunits may mediate native K⁺ currents in resting duct cells. Further functional studies with specific blockers are required to evaluate which of these K⁺ channels contribute to the resting membrane potential and might be involved in HCO₃^- secretion. J. Med. Invest. 56 Suppl.: 354, December, 2009

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Inositol 1,4,5-trisphosphate (IP₃) is an intracellular messenger that elicits a wide range of spatial and temporal Ca²⁺ signals, and this signaling versatility is exploited to regulate diverse cellular responses. In the present study, we have developed a series of IP₃ biosensors that exhibit strong pH stability and varying affinities for IP₃, as well as a method for the quantitative measurement of cytosolic concentrations of IP₃ ([IP₃]_i) in single living cells. We applied this method to elucidate IP₃ dynamics during agonist-induced Ca²⁺ oscillations, and demonstrated cell type-dependent differences in IP₃ dynamics; a non-fluctuating rise in [IP₃]_i and repetitive IP₃ spikes during Ca²⁺ oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP₃ spikes in HSY-EA1 cells varied from 10 to 100 nM, and the [IP₃]_i spike peak was preceded by a Ca²⁺ spike peak. These results suggest that repetitive IP₃ spikes in HSY-EA1 cells are passive reflections of Ca²⁺ oscillations, and are unlikely to be essential for driving Ca²⁺ oscillations. The novel method described herein as well as the quantitative information obtained by using this method should provide a valuable and sound basis for future studies on the spatial and temporal regulations of IP₃ and Ca²⁺. J. Med. Invest. 56 Suppl.: 357-361, December, 2009

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