Enhancement of receptor-mediated calcium responses by phenytoin through the suppression of calcium excretion in human gingival fibroblasts.

BACKGROUND AND OBJECTIVES: Gingival overgrowth caused by phenytoin is proposed to be associated with Ca2+ signaling; however, the mechanisms that increase the intracellular Ca2+ concentration ([Ca2+ ]i ) are controversial. The current study aimed to elucidate the mechanism underlying the phenytoin-induced increase in [Ca2+ ]i in human gingival fibroblasts (HGFs). METHODS: Effects of 100 µM phenytoin on [Ca2+ ]i in HGFs were examined at the single-cell level using fluorescence images of fura-2 captured by an imaging system consisting of an EM-CCD camera coupled to an inverted fluorescence microscope at room temperature. RESULTS: Exposure of HGFs to 100 µM phenytoin induced a transient increase in [Ca2+ ]i in the absence of extracellular Ca2+ , indicating that the phenytoin-induced increase in [Ca2+ ]i does not require an influx of extracellular Ca2+ . In addition, phenytoin increased [Ca2+ ]i in HGFs depleted of intracellular Ca2+ stores by thapsigargin, indicating that neither Ca2+ release from stores nor inhibition of Ca2+ uptake is involved. Furthermore, the phenytoin-induced [Ca2+ ]i elevation was reduced to 18.8% in the absence of extracellular Na+ , and [Ca2+ ]i elevation upon removal of extracellular Na+ was reduced to 25.9% in the presence of phenytoin. These results imply that phenytoin increases [Ca2+ ]i of HGFs by suppressing the Na+ /Ca2+ exchanger. Suppression of intracellular Ca2+ excretion is thought to enhance the Ca2+ responses induced by various stimuli. Analysis at the single-cell level showed that stimulation with 1 µM ATP or 3 µM histamine increased [Ca2+ ]i in 20-50% of cells, and [Ca2+ ]i increased in many unresponsive cells in the presence of phenytoin. CONCLUSION: Our findings demonstrate that phenytoin induced increase in [Ca2+ ]i by the inhibition of Ca2+ efflux in HGFs. It was also found that phenytoin strongly enhanced small Ca2+ responses induced by stimulation with a low concentration of ATP or histamine by inhibiting Ca2+ efflux. These findings suggest a possibility that phenytoin causes drug-induced gingival overgrowth by interacting with inflammatory bioactive substances in the gingiva.

Association Between N-Desmethylclozapine and Clozapine-Induced Sialorrhea: Involvement of Increased Nocturnal Salivary Secretion via Muscarinic Receptors by N-Desmethylclozapine.

Clozapine-induced sialorrhea (CIS) is a common side effect of clozapine. There is no established standard treatment of CIS since the underlying mechanism remains unknown. This study aimed to elucidate the mechanisms involved in CIS. In our clinical study, a prospective observational study evaluated the association between serum and saliva concentrations of clozapine or its metabolites and Drooling Severity and Frequency Scale (DSFS) score. In our in vivo study, we first developed a new CIS animal model; subsequently, we measured salivary secretion and concentrations of clozapine or its metabolites in the animal model. In our in vitro study, we measured the calcium ion (Ca2+) response to evaluate the effect of clozapine or its metabolites on human salivary gland cell line (HSY cells) and then examined whether their effect was inhibited by atropine. In our clinical study, serum and saliva N-desmethylclozapine concentrations were significantly correlated with nocturnal DSFS score. In our in vivo study, daily single oral administration of 100 mg/kg clozapine for 7 days significantly increased salivary secretion in rats. Furthermore, N-desmethylclozapine concentrations in serum and submandibular glands of the rats were higher than clozapine concentrations. In our in vitro study, N-desmethylclozapine only elicited an increase in the intracellular Ca2+ in HSY cells. N-desmethylclozapine-induced Ca2+ responses were inhibited by atropine. These results suggest that N-desmethylclozapine is implicated in CIS by increasing nocturnal salivation via the muscarinic receptors. Moreover, our developed animal model that reflects CIS in clinical condition plays a key role as a bridge between basic and clinical research. SIGNIFICANCE STATEMENT: Clozapine-induced sialorrhea (CIS) is a severe and frequent adverse reaction, but the mechanism underlying CIS is less well understood. This paper reports that N-desmethylclozapine, a metabolite of clozapine, is implicated in CIS by increasing nocturnal salivation via the muscarinic receptors and that oral administration of clozapine at 100 mg/kg once daily for 7 days to rat is the optimum method for establishing the new animal model reflecting the clinical scenario of CIS.

Bcl-2 and IP3 compete for the ligand-binding domain of IP3Rs modulating Ca2+ signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca2+-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP3R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP3R function provoked by low concentration of agonist or IP3, while fails to attenuate against high concentration of agonist or IP3. Surprisingly, Bcl-2 remained capable of inhibiting IP3R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP3R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP3 and adenophostin A, ligands of the IP3R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP3R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2's inhibitory properties on IP3Rs that is tunable to the level of IP3 signaling in cells.

On the dynamical structure of calcium oscillations.

Oscillations in the concentration of free cytosolic Ca2+ are an important and ubiquitous control mechanism in many cell types. It is thus correspondingly important to understand the mechanisms that underlie the control of these oscillations and how their period is determined. We show that Class I Ca2+ oscillations (i.e., oscillations that can occur at a constant concentration of inositol trisphosphate) have a common dynamical structure, irrespective of the oscillation period. This commonality allows the construction of a simple canonical model that incorporates this underlying dynamical behavior. Predictions from the model are tested, and confirmed, in three different cell types, with oscillation periods ranging over an order of magnitude. The model also predicts that Ca2+ oscillation period can be controlled by modulation of the rate of activation by Ca2+ of the inositol trisphosphate receptor. Preliminary experimental evidence consistent with this hypothesis is presented. Our canonical model has a structure similar to, but not identical to, the classic FitzHugh-Nagumo model. The characterization of variables by speed of evolution, as either fast or slow variables, changes over the course of a typical oscillation, leading to a model without globally defined fast and slow variables.

Simultaneous monitoring of Ca2+ responses and salivary secretion in live animals reveals a threshold intracellular Ca2+ concentration for salivation.

NEW FINDINGS: What is the central question of this study? The effects of Ca2+ responses on salivary fluid secretion have been studied indirectly by monitoring ion channel activities and other indices. Therefore, Ca2+ responses during salivary secretion remain poorly understood. What is the main finding and its importance? Herein, we developed a simultaneous monitoring system for Ca2+ responses and salivary secretion in live animals using a YC-Nano50-expressing submandibular gland and a fibre-optic pressure sensor. This new approach revealed a clear time lag between the onset of Ca2+ responses and salivary secretion. We also estimated the [Ca2+ ]i and provided direct evidence for the regulation of salivary secretion by small increases in [Ca2+ ]i in submandibular gland acinar cells. ABSTRACT: We monitored changes in [Ca2+ ]i during salivary secretion in the rat submandibular gland in live animals using a combination of intravital Ca2+ imaging with the ultrasensitive Ca2+ indicator YC-Nano50 and a fibre-optic pressure sensor. Intravenous infusion of ACh (10-720 nmol min-1 ) increased [Ca2+ ]i and salivary flow rate in a dose-dependent manner. Repetitive stimulation with ACh induced equivalent Ca2+ responses and salivary secretion in the same individual animals. The accurate ACh stimulation experiments revealed a clear time lag between the onset of the increase in [Ca2+ ]i and salivary secretion. The time lag with the lowest dose of ACh (30 nmol min-1 ) was 106 s, which shortened to 19 s with the dose used for maximal salivary secretion (360 nmol min-1 ). This time lag might reflect the time required for [Ca2+ ]i to reach the level required to activate molecules for fluid secretion. The resting [Ca2+ ]i in submandibular gland was 37 nm, and [Ca2+ ]i at the onset of salivary secretion was 45-57 nm, irrespective of ACh dose. These results indicate that low [Ca2+ ]i is sufficient to trigger fluid secretion in the rat submandibular gland in vivo.

A mathematical model of calcium dynamics in HSY cells.

Saliva is an essential part of activities such as speaking, masticating and swallowing. Enzymes in salivary fluid protect teeth and gums from infectious diseases, and also initiate the digestion process. Intracellular calcium (Ca2+) plays a critical role in saliva secretion and regulation. Experimental measurements of Ca2+ and inositol trisphosphate (IP3) concentrations in HSY cells, a human salivary duct cell line, show that when the cells are stimulated with adenosine triphosphate (ATP) or carbachol (CCh), they exhibit coupled oscillations with Ca2+ spike peaks preceding IP3 spike peaks. Based on these data, we construct a mathematical model of coupled Ca2+ and IP3 oscillations in HSY cells and perform model simulations of three different experimental settings to forecast Ca2+ responses. The model predicts that when Ca2+ influx from the extracellular space is removed, oscillations gradually slow down until they stop. The model simulation of applying a pulse of IP3 predicts that photolysis of caged IP3 causes a transient increase in the frequency of the Ca2+ oscillations. Lastly, when Ca2+-dependent activation of PLC is inhibited, we see an increase in the oscillation frequency and a decrease in the amplitude. These model predictions are confirmed by experimental data. We conclude that, although concentrations of Ca2+ and IP3 oscillate, Ca2+ oscillations in HSY cells are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations.

Highly Sensitive Measurement of Inositol 1,4,5-Trisphosphate by Using a New Fluorescent Ligand and Ligand Binding Domain Combination.

Based on the results of our previous adenophostin A structure-activity relationship studies, two fluorescent inositol 1,4,5-trisphosphate (IP3 ) receptor ligands, fluorescent adenophostin A (FADA) and fluorescent low-affinity ligand (FLL), were designed. Binding of the fluorescent ligands to the fluorescent IP3 sensor in protein-expressing cells caused FRET. This principle was extended to a cell-free assay system by using the fluorescent IP3 sensor bound to agarose beads. The effect of FLL on the FRET signal was reduced by subsequent addition of IP3 . The IC50 values of IP3 on the FRET signals were 139.7 and 352.1 nm for 30 and 100 nm FLL, respectively. This method allowed quantitative measurement of IP3 concentrations below 10 nm and was applied to measure cytosolic IP3 concentrations in COS-7 cells and to examine the potency of synthesized adenophostin A analogues.

Partial agonistic effects of pilocarpine on Ca(2+) responses and salivary secretion in the submandibular glands of live animals.

NEW FINDINGS: What is the central question of this study? Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the Ca(2+) -mobilizing effect of pilocarpine in salivary gland cells is extremely small. Therefore, we examined the effect of pilocarpine on Ca(2+) responses in submandibular gland cells and on secretion in vitro and in vivo. What is the main finding and its importance? Pilocarpine induces small Ca(2+) responses and reduces the effects of other mAChR agonists on Ca(2+) responses via its partial agonistic effects. These effects of pilocarpine on Ca(2+) responses in the submandibular gland were further established in vivo with a novel Ca(2+) imaging system and a genetically encoded Ca(2+) indicator. Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the effect of pilocarpine on Ca(2+) responses in dispersed salivary gland cells is extremely small. Here, we demonstrate the effect of pilocarpine on Ca(2+) responses and salivary secretion in the rat submandibular gland (SMG). In fura-2-loaded SMG cells, the maximal effect of pilocarpine on [Ca(2+) ]i elevation was 16% of that of carbachol, and pilocarpine attenuated carbachol- and bethanechol (Bet)-induced [Ca(2+) ]i increases, indicating that pilocarpine acts as a partial agonist for mAChR-mediated Ca(2+) responses. The partial agonistic effect of pilocarpine on Ca(2+) dynamics in the SMG was also confirmed in live animals using the genetically encoded Ca(2+) indicator, YC-Nano50. Administration of pilocarpine (3 mg kg(-1) , i.p.) elicited a small increase in [Ca(2+) ]i in the SMG. Quantitative analyses demonstrated that resting [Ca(2+) ]i was ∼37 nm, which was increased by pilocarpine (3 mg kg(-1) ) and Bet (10 mg kg(-1) ) to 44 and 69 nm, respectively. The inhibitory effects of pilocarpine on Bet-induced Ca(2+) responses were also elucidated in vivo. We further examined real-time changes in pilocarpine-induced SMG salivary secretion and showed that pilocarpine induced an extremely weak secretory response and reduced Bet-induced secretion. Unlike Ca(2+) responses, pilocarpine failed to reduce the effect of Bet on SMG blood flow. Our results demonstrate that pilocarpine acts as a partial agonist of mAChRs to induce weak salivary secretion that is correlated with small increases in [Ca(2+) ]i . Furthermore, pilocarpine exhibits an antagonistic effect on mAChR-induced Ca(2+) responses and salivary secretion.

Key components of store-operated Ca2+ entry in non-excitable cells.

Store-operated Ca(2+) entry (SOCE) is a ubiquitous Ca(2+) entry pathway in non-excitable cells. It is activated by the depletion of Ca(2+) from intracellular Ca(2+) stores, notably the endoplasmic reticulum (ER). In the past 9 years, it has been established that two key proteins, stromal interacting molecule 1 (STIM1) and Orai1, play critical roles in SOCE. STIM1 is a single-pass transmembrane protein located predominantly in the ER that serves as a Ca(2+) sensor within the ER, while Orai1 is a tetraspanning plasma membrane (PM) protein that functions as the pore-forming subunit of store-operated Ca(2+) channels. A decrease in the ER Ca(2+) concentration induces translocation of STIM1 into puncta close to the PM. STIM1 oligomers directly interact with Orai1 channels and activates them. This review summarizes the molecular basis of the interaction between STIM1 and Orai1 in SOCE. Further, we describe current findings on additional regulatory proteins, such as Ca(2+) release-activated Ca(2+) regulator 2A and septin, novel roles of STIM1, and modulation of SOCE by protein phosphorylation.

Muscarinic acetylcholine receptor-mediated phosphorylation of extracellular signal-regulated kinase in HSY salivary ductal cells involves distinct signaling pathways.

OBJECTIVES: Typical agonists of G protein-coupled receptors (GPCRs), including muscarinic acetylcholine receptors (mAChRs), activate both G-protein and ß-arrestin signaling systems, and are termed balanced agonists. In contrast, biased agonists selectively activate a single pathway, thereby offering therapeutic potential for the specific activation of that pathway. The mAChR agonists carbachol and pilocarpine are known to induce phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) via G-protein-dependent and -independent pathways, respectively. We investigated the involvement of ß-arrestin and its downstream mechanisms in the ERK1/2 phosphorylation induced by carbachol and pilocarpine in the human salivary ductal cell line, HSY cells. METHODS: HSY cells were stimulated with pilocarpine or carbachol, with or without various inhibitors. The cell lysates were analyzed by western blotting using the antibodies p44/p42MAPK and phosphor-p44/p42MAPK. RESULTS: Western blot analysis revealed that carbachol elicited greater stimulation of ERK1/2 phosphorylation compared to pilocarpine. ERK1/2 phosphorylation was inhibited by atropine and gefitinib, suggesting that mAChR activation induces transactivation of epidermal growth factor receptors (EGFR). Moreover, inhibition of carbachol-mediated ERK1/2 phosphorylation was achieved by GF-109203X (a PKC inhibitor), a ßARK1/GRK2 inhibitor, barbadin (a ß-arrestin inhibitor), pitstop 2 (a clathrin inhibitor), and dynole 34-2 (a dynamin inhibitor). In contrast, pilocarpine-mediated ERK1/2 phosphorylation was only inhibited by barbadin (a ß-arrestin inhibitor) and PP2 (a Src inhibitor). CONCLUSION: Carbachol activates both G-protein and ß-arrestin pathways, whereas pilocarpine exclusively activates the ß-arrestin pathway. Additionally, downstream of ß-arrestin, carbachol activates clathrin-dependent internalization, while pilocarpine activates Src.

Local anesthetics inhibit muscarinic acetylcholine receptor-mediated calcium responses and the recruitment of ß-arrestin in HSY human parotid cells.

OBJECTIVES: Local anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling via GPCRs involves both G-protein- and ß-arrestin-mediated pathways. To determine the effects of local anesthetics on muscarinic acetylcholine receptors (mAChR), a family of GPCRs, we analyzed the effects of local anesthetics on mAChR-mediated Ca2+ responses and formation of receptor-ß-arrestin complexes in the HSY human parotid cell line. METHODS: Ca2+ responses were monitored by fura-2 spectrofluorimetry. Ligand-induced interactions between mAChR and ß-arrestin were examined using a ß-arrestin GPCR assay kit. RESULTS: Lidocaine reduced mAChR-mediated Ca2+ responses but did not change the intracellular Ca2+ concentration in non-stimulated cells. The membrane-impermeant lidocaine analog QX314 and procaine inhibited mAChR-mediated Ca2+ responses, with EC50 values of 48.0 and 20.4 µM, respectively, for 50 µM carbachol-stimulated Ca2+ responses. In the absence of extracellular Ca2+, the pretreatment of cells with QX314 reduced carbachol-induced Ca2+ release, indicating that QX314 reduced Ca2+ release from intracellular stores. Lidocaine and QX314 did not affect store-operated Ca2+ entry as they did not alter the thapsigargin-induced Ca2+ response. QX314 and procaine reduced the carbachol-mediated recruitment of ß-arrestin, and administration of procaine suppressed pilocarpine-induced salivary secretion in mice. CONCLUSION: Local anesthetics, including QX314, act on mAChR to reduce carbachol-induced Ca2+ release from intracellular stores and the recruitment of ß-arrestin. These findings support the notion that local anesthetics and their derivatives are starting points for the development of functional allosteric modulators of mAChR.

Increase in muscarinic stimulation-induced Ca(2+) response by adenovirus-mediated Stim1-mKO1 gene transfer to rat submandibular acinar cells in vivo.

Adenoviruses have been used for gene transfer to salivary gland cells in vivo. Their use to study the function of salivary acinar cells was limited by a severe inflammatory response and by the destruction of fluid-secreting acinar cells. In the present study, low doses of adenovirus were administered to express Stim1-mKO1 by retrograde ductal injection to submandibular glands. The approach succeeded in increasing muscarinic stimulation-induced Ca(2+) responses in acinar cells without inflammation or decreased salivary secretions. This increased Ca(2+) response was notable upon weak muscarinic stimulation and was attributed to increased Ca(2+) release from internal stores and increased Ca(2+) entry. The basal Ca(2+) level was higher in Stim1-mKO1-expressing cells than in mKO1-expressing and non-expressing cells. Exposure of permeabilized submandibular acinar cells, where Ca(2+) concentration was fixed at 50 nM, to inositol 1,4,5-trisphosphate (IP3) produced similar effects on the release of Ca(2+) from stores in Stim1-mKO1-expressing and non-expressing cells. The low toxicity and relative specificity to acinar cells of the mild gene transfer method described herein are particularly useful for studying the molecular functions of salivary acinar cells in vivo, and may be applied to increase salivary secretions in experimental animals and human in future.

Angiogenesis Assays for the Analysis of CCN Proteins.

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the actin of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) and CCN2-neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM). In addition, we introduce an autofluorescence imaging of blood vessels in the subcutaneous tumor xenograft mouse model. These assays can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

Role of aquaporin 5 and glandular blood flow in the acetylcholine-induced secretion of saliva in rats.

To clarify the role of the aquaporin 5 (AQP5) in salivary secretion, we evaluated acetylcholine (ACh)-induced secretion in Sprague-Dawley (SD) rats, rats expressing a low level of AQP5 protein (AQP5/low SD) which developed from SD rats, and Wistar/ST rats. The salivary secretion in AQP5/low SD rats in response to infusions of low-dose ACh (60-120 nmol/min) was 27-42% of that in SD rats. By contrast, Wistar/ST rats exhibited comparable secretion to that of SD rats in response to low-doses ACh, despite their low-level expression of AQP5. Experiments using spectrofluorometry and RT-PCR demonstrated no differences in the ACh-induced Ca2+ responses or the mRNA expression of muscarinic receptor, Cl- channel, or cotransporter between these strains. These findings imply that factors other than the function of salivary acinar cells regulates the secretion in response to weak stimuli. Monitoring of the hemodynamics in the submandibular gland revealed that low-doses ACh induced different patterns of the fluctuations in the blood flow in these strains. The blood flow decreased below the resting level in AQP5/low SD rats, but remained mostly above the resting level in Wistar/ST rats. The present study reveals that the contribution of AQP5-dependent transport of water is altered by stimulus intensity and blood flow.

Visualization of Ins(1,4,5)P3 dynamics in living cells: two distinct pathways for Ins(1,4,5)P3 generation following mechanical stimulation of HSY-EA1 cells.

In the present study, the contribution of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] generation on the mechanical-stimulation-induced Ca(2+) response was investigated in HSY-EA1 cells. Mechanical stimulation induced a local increase in the cytosolic concentration of Ins(1,4,5)P(3) ([IP(3)](i)), as indicated by the Ins(1,4,5)P(3) biosensor LIBRAvIII. The area of this increase expanded like an intracellular Ins(1,4,5)P(3) wave as [IP(3)](i) increased in the stimulated region. A small transient [IP(3)](i) increase was subsequently seen in neighboring cells. The phospholipase C inhibitor U-73122 abolished these Ins(1,4,5)P(3) responses and resultant Ca(2+) releases. The purinergic receptor blocker suramin completely blocked increases in [IP(3)](1) and the Ca(2+) release in neighboring cells, but failed to attenuate the responses in mechanically stimulated cells. These results indicate that generation of Ins(1,4,5)P(3) in response to mechanical stimulation is primarily independent of extracellular ATP. The speed of the mechanical-stimulation-induced [IP(3)](i) increase was much more rapid than that induced by a supramaximal concentration of ATP (1 mM). The contribution of the Ins(1,4,5)P(3)-induced Ca(2+) release was larger than that of Ca(2+) entry in the Ca(2+) response to mechanical stimulation in HSY-EA1 cells.

Responses of salivary glands to intake of soft diet.

BACKGROUND: Modernization has made individuals prefer processed and cooked foods (soft food), but this eating habit may have negative effects on the oral cavity. However, laboratory animals fed with soft diet are commonly used in an attempt to clarify this issue, and various oral tissues, including the salivary glands have been examined. In this review, we summarize the findings of previous studies concerning the responses of salivary glands to daily intake of soft diet. HIGHLIGHT: The weight of the parotid glands decreased in rodents fed with soft diet (liquid or powder). In atrophic parotid glands, acinar cell shrinkage is histologically observed and the DNA content is reduced, showing that the atrophy is caused by a decrease in the size and number of acinar cells. Immunohistochemical examinations showed that the decrease in the acinar cell number was induced by suppression of acinar cell proliferation and acceleration of apoptosis. The atrophic parotid glands recovered following a change from soft to pellet diet. Other salivary glands, such as the submandibular, sublingual, and palatine glands, responded only slightly to the soft diet feeding. CONCLUSION: Accumulated research data showed that a soft diet negatively affects the parotid glands much more than other salivary glands and that atrophic parotid glands are able to recover by switching to a hard diet. Therefore, it should be emphasized that good eating habits are important for not only digestion but also the health of oral tissues, including the salivary glands.

Insertion of circularly permuted cyan fluorescent protein into the ligand-binding domain of inositol 1,4,5-trisphosphate receptor for enhanced FRET upon binding of fluorescent ligand.

Binding of fluorescent ligand (FL) to the cyan fluorescent protein (CFP)-coupled ligand-binding domain of the inositol 1,4,5-trisphosphate (IP3) receptor (CFP-LBP) produces fluorescence (Förster) resonance energy transfer (FRET). A competitive fluorescent ligand assay (CFLA), using the FRET signal from competition between FLs and IP3, can measure IP3 concentration. The FRET signal should be enhanced by attaching a FRET donor to an appropriate position. Herein, we inserted five different circularly permuted CFPs in the loop between the second and third α-helices to generate membrane-targeted fluorescent ligand-binding proteins (LBPs). Two such proteins, LBP-cpC157 and LBP-cpC173, localized at the plasma membrane, displayed FRET upon binding the high-affinity ligand fluorescent adenophostin A (F-ADA), and exhibited a decreased fluorescence emission ratio (480 nm / 535 nm) by 1.6- to 1.8-fold that of CFP-LBP. In addition, binding of a fluorescent low-affinity ligand (F-LL) also reduced the fluorescence ratio in a concentration-dependent manner, with EC50 values for LBP-cpC157 and LBP-cpC173 of 34.7 nM and 27.6 nM, respectively. These values are comparable to that with CFP-LBP (29.2 nM), indicating that insertion of cpC157 and cpC173 did not disrupt LBP structure and function. The effect of 100 nM F-LL on the decrease in fluorescence ratio was reversed upon addition of IP3, indicating binding competition between F-LL and IP3. We also constructed cytoplasmic fluorescent proteins cyLBP-cpC157 and cyLBP-cpC173, and bound them to DYK beads for imaging analyses. Application of F-ADA decreased the fluorescence ratio of the beads from the periphery to the center over 3 - 5 min. Application of F-LL also decreased the fluorescence ratio of cyLBP-cpC157 and cyLBP-cpC173 by 20-25%, and subsequent addition of IP3 recovered the fluorescence ratio in a concentration-dependent manner. The EC50 value and Hill coefficient obtained by curve fitting against the IP3-dependent recovery of fluorescence ratio can be used to estimate the IP3 concentration.

Spontaneous calcium responses of SF2 rat dental epithelial cells stably expressing the calcium sensor G-GECO.

Genetically-encoded calcium indicators such as G-GECO are useful for studying Ca2+ responses during long-term processes. In this study, we employed a lentiviral vector and established a rat dental epithelial cell line that stably expressed G-GECO (SF2-G-GECO). Ca2+ imaging analysis under cell culture conditions revealed that SF2-G-GECO cells exhibited spontaneous Ca2+ responses, which could be classified into the following three major patterns depending on the cell density: localized Ca2+ responses at cell protrusions at a low density, a cell-wide spread of Ca2+ responses at a medium density, and Ca2+ responses in clusters of 3-20 cells at a high density. The P2Y receptor inhibitor suramin (10 µM), the ATP-degrading enzyme apyrase (5 units/mL), and the fibroblast growth factor (FGF) receptor inhibitor FIIN-2 (1 µM) decreased the frequency of spontaneous Ca2+ responses. These results indicate that ATP and FGF are involved in the spontaneous Ca2+ responses. SF2 cells differentiate into ameloblasts via interactions with mesenchymal cells. Therefore, SF2-G-GECO cells are expected to be a useful tool for studying the functions of Ca2+ responses in regulating gene expression during tooth development.

Competitive Fluorescent Ligand Assay for Inositol 1,4,5-Trisphosphate.

We present a novel method, termed competitive fluorescent ligand assay for inositol 1,4,5-trisphosphate (CFLA-IP3), to measure inositol 1,4,5-trisphosphate (IP3). This method is based on fluorescence resonance energy transfer (FRET) between two fluorescent molecules, a fluorescent IP3-binding protein and its fluorescent ligand. Binding of these fluorescent molecules generates a FRET signal, and the IP3-dependent decrease in the FRET signal due to displacement of the fluorescent ligand is detected by fluorescence microscopy.

Role of VAMP8/endobrevin in constitutive exocytotic pathway in HeLa cells.

To evaluate the role of VAMP8/endobrevin in constitutive exocytosis, we have examined the exocytotic pathways of VAMP8 and human growth hormone, both GFP-tagged, by total internal reflection fluorescence microscopy (TIRF-M). Human GH-GFP and VAMP8-GFP were similarly expressed in small round vesicles and elongated tubular vesicles in HeLa cells, and were mostly exocytosed at the peripheral area of the cells. VAMP8-GFP gave 2 types of exocytotic images: a burst type and a non-burst type. The burst type showed a sharp transient increase in the peak fluorescence intensity and a much slower decrease in the average intensity in the active windows, where exocytosis took place, as observed in the "full-fusion" type of exocytosis. The non-burst type showed a relatively long-lasting fusion to the plasma membrane with little transfer of VAMP8-GFP to the plasma membrane, as observed in the so-called "kiss-and-run" type of exocytosis. Endogenous VAMP8 and hGH-GFP were colocalized on the same vesicles at least in part. However, the constitutive exocytosis of hGH-GFP and CLuc, a secreted luciferase from Cypridina noctiluca, was normal, even when siRNAs for VAMP8 and VAMP3 robustly decreased their proteins. These results suggest that VAMP8 is not essential for constitutive exocytosis, although it can be involved in the exocytosis.