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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.

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.

Gingival overgrowth caused by phenytoin is proposed to be associated with CaEffects of 100 μM phenytoin on [CaExposure of HGFs to 100 μM phenytoin induced a transient increase in [CaOur findings demonstrate that phenytoin induced increase in [Ca

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.

Binding of fluorescent ligand (FL) to the cyan fluorescent protein (CFP)-coupled ligand-binding domain of the inositol 1,4,5-trisphosphate (IP

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 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. ispartof: CELLULAR AND MOLECULAR LIFE SCIENCES vol:76 issue:19 pages:3843-3859 ispartof: location:Switzerland status: published

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 (Ca

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.

Inositol 1,4,5-trisphosphate (IP3) is an important intracellular messenger produced by phospholipase C via the activation of G-protein-coupled receptor- or receptor-tyrosine-kinase-mediated pathways, and is involved in numerous responses to hormones, neurotransmitters, and growth factors through the releases of Ca2+ from intracellular stores via IP3 receptors. IP3-mediated Ca2+ signals often exhibit complex spatial and temporal organizations, such as Ca2+ oscillations. Recently, new methods have become available to measure IP3 concentration ([IP3]) using AlphaScreen technology, fluorescence polarization, and competitive ligand binding assay (CFLA). These methods are useful for the high throughput screening in drug discovery. Calcium ions generate versatile intracellular signals such as Ca2+ oscillations and waves. Fluorescent sensors molecules to monitor changes in [IP3] in single living cells are crucial to study the mechanism for the spatially and temporally regulated Ca2+ signals. In particular, FRET-based IP3 sensors are useful for the quantitative monitoring intracellular [IP3], and allowed to uncovered the oscillatory IP3 dynamics in association with Ca2+ oscillations. A mathematical model of coupled Ca2+ and IP3 oscillations predicts that Ca2+ oscillations are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations. These model predictions have also been confirmed experimentally. At present, however, usefulness of FRET-based IP3 sensors are limited by their relatively small change in fluorescence. Development of novel IP3 sensors with improve dynamic range would be important for understanding the regulatory mechanism of Ca2+ signaling and for in vivo IP3 imaging.

We present a novel method, termed competitive fluorescent ligand assay for inositol 1,4,5-trisphosphate (CFLA-IP

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.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca

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.

What is the central question of this study? The effects of CaWe monitored changes in [Ca

New Findings What is the central question of this study? Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the Ca2+-mobilizing effect of pilocarpine in salivary gland cells is extremely small. Therefore, we examined the effect of pilocarpine on Ca2+ responses in submandibular gland cells and on secretion in vitro and in vivo. What is the main finding and its importance? Pilocarpine induces small Ca2+ responses and reduces the effects of other mAChR agonists on Ca2+ responses via its partial agonistic effects. These effects of pilocarpine on Ca2+ responses in the submandibular gland were further established in vivo with a novel Ca2+ imaging system and a genetically encoded Ca2+ indicator. Pilocarpine stimulates salivary secretion via muscarinic ACh receptors (mAChRs), although the effect of pilocarpine on Ca2+ responses in dispersed salivary gland cells is extremely small. Here, we demonstrate the effect of pilocarpine on Ca2+ responses and salivary secretion in the rat submandibular gland (SMG). In fura-2-loaded SMG cells, the maximal effect of pilocarpine on [Ca2+]i elevation was 16% of that of carbachol, and pilocarpine attenuated carbachol- and bethanechol (Bet)-induced [Ca2+]i increases, indicating that pilocarpine acts as a partial agonist for mAChR-mediated Ca2+ responses. The partial agonistic effect of pilocarpine on Ca2+ dynamics in the SMG was also confirmed in live animals using the genetically encoded Ca2+ indicator, YC-Nano50. Administration of pilocarpine (3 mg kg−1, i.p.) elicited a small increase in [Ca2+]i in the SMG. Quantitative analyses demonstrated that resting [Ca2+]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 Ca2+ 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 Ca2+ 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 [Ca2+]i. Furthermore, pilocarpine exhibits an antagonistic effect on mAChR-induced Ca2+ responses and salivary secretion.

Background Salivary glands are comprised predominantly of two epithelial cell types, acinar and ductal cells. Activation of muscarinic acetylcholine receptors on acinar cells induces inositol 1,4,5-trisphosphate-mediated Ca 2+ release from intracellular Ca 2+ stores, followed by Ca 2+ influx across the plasma membrane. The increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) activates ion channels and transporters involved in fluid secretion; thus [Ca 2+ ] i acts as the primary fluid secretion signal in acinar cells. Highlight The development of Ca 2+ imaging techniques has enabled extensive study of spatiotemporal Ca 2+ dynamics in isolated acinar cells; however, the relationship between Ca 2+ responses and fluid secretion in vivo is not yet fully understood. In vivo Ca 2+ imaging in salivary gland cells is necessary for determining Ca 2+ responses during salivary secretion. Recently, we developed an intravital imaging system for monitoring Ca 2+ responses in rat submandibular gland (SMG) acinar cells in live animals using retrograde ductal injection of adenoviral vectors expressing the genetically encoded Ca 2+ indicator, YC-Nano50. Expression of YC-Nano50 was observed throughout the SMG, and we confirmed that YC-Nano50 was functional for monitoring Ca 2+ responses in SMG cells. Administration of acetylcholine significantly increased [Ca 2+ ] i in the SMG, and this increase gradually decreased to resting levels. Conclusion We successfully visualized agonist-induced Ca 2+ dynamics in SMG acinar cells in live animals. This novel experimental system represents a powerful tool for understanding the relationships between Ca 2+ responses and salivary secretion in vivo .

Background Developments in fluorescent molecular imaging techniques have enabled characterization of the localization and dynamics of particular molecules at the cellular and subcellular levels. Highlight Advances in imaging devices (fluorescence microscopy) and fluorescent indicators have contributed to the development of fluorescence imaging techniques. In particular, cloning of green fluorescent protein from the bioluminescent jellyfish Aequorea victoria and the subsequent development of other fluorescent proteins have revolutionized imaging technologies and accelerated their application in molecular and cell biological studies. There are two types of genetically encoded fluorescent indicators, namely, fluorescence resonance energy transfer-based ratiometric indicators and single fluorescent protein (FP)-based intensiometric indicators. The principal advantages of FP-based indicators are their superior sensitivities and specificities for molecular and physiological events, which are achieved by the incorporation of naturally evolved protein sensor domains. Consequently, numerous fluorescent indicators that enable monitoring of intracellular signaling, enzyme activities, apoptosis, cell cycle progression, and other cellular events have been developed. FP-based indicators can be expressed in specific cells in a temporally controlled manner; hence, they are favorable for long-term in vitro and in vivo experiments. Conclusion The advantages of FP-based indicators extend their application to understanding the functions of particular molecules and cellular events during physiological responses in live animals.

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.

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., Author summary We construct a mathematical model of Ca2+ and IP3 oscillations in HSY cells, a salivary ductal cell line from human parotid. The model reproduces the experimental data that exhibit coupled oscillations of [Ca2+] and [IP3] with the peak of each Ca2+ spike being followed by the peak of an IP3 spike. Recently, it was conjectured that IP3 oscillations in HSY cells are not necessary for Ca2+ oscillations. We corroborate this statement with our model and show that Ca2+ oscillations can occur without oscillating [IP3]. Further to this, based on our model simulation, we hypothesise that IP3 oscillations in HSY cells may affect the frequency of Ca2+ oscillations. Indeed, experimental data verify that oscillating [IP3] lengthens the period of Ca2+ oscillations.

Melanoma is highly metastatic, but the mechanism of melanoma cell migration is still unclear. We found that melanoma cells expressed the nicotinamide adenine dinucleotide–dependent protein deacetylase SIRT1 in the cytoplasm. Cell membrane extension and migration of melanoma cells were inhibited by SIRT1 inhibitors or SIRT1 knockdown, whereas SIRT1 activators enhanced elongation of protrusion and cellular motility. In B16F1 cells, growth factor stimulation induced lamellipodium extension, a characteristic feature at the leading edge of migrating cells, and SIRT1 was found in the lamellipodium. SIRT1 inhibitor nicotinamide (NAM) or SIRT1 small interfering RNAs suppressed the lamellipodium extension by serum or platelet-derived growth factor (PDGF). The lamellipodium formation by dominant-active Rac1 was also inhibited by NAM, a SIRT1 inhibitor. NAM inhibited the accumulation of phosphorylated Akt at the submembrane by serum or PDGF. Using fluorescence resonance energy transfer, we found that NAM impaired PDGF-dependent increase in the phosphatidylinositol-3,4,5-trisphosphate level at the leading edge. NAM inhibited the abdominal metastasis of transplanted B16F1 melanoma cells in C57BL6/J mice and improved survival. Finally, SIRT1-knockdown B16F1 cells showed significantly reduced metastasis in transplanted mice compared with that in control B16F1 cells. These results indicate that SIRT1 inhibition is a strategy to suppress metastasis of melanoma cells.

Background and objective Amelogenins are major components of extracellular matrix proteins in developing teeth, and regulate the growth of enamel crystals. They also function as signaling molecules in cell differentiation. This study aimed to determine the biological effects of amelogenins on the differentiation of HAT-7 dental epithelial cells and MC3T3-E1 pre-osteoblastic cells using full-length recombinant human amelogenin (rh-AMEL). Design rh-AMEL was expressed in a mammalian cell line (Expi293F™) and was purified by DDK agarose beads. Effects of rh-AMEL on differentiation were evaluated by Mineralization and Alkaline phosphatase (ALP) activity using Alizarin Red S staining and colorimetric substrate p-nitrophenol, respectively. Results Western blotting and silver staining confirmed the successful purification of rh-AMEL. Mineralization and ALP activity in HAT-7 cells were significantly higher after treatment with 4 μg/mL rh-AMEL, but not after treatment with Emdogain ® (EMD). In MC3T3-E1 cells, on the other hand, rh-AMEL showed biphasic effects on differentiation. Treatment with low concentrations of rh-AMEL (0.001–0.1 μg/mL) and EMD (0.01–1 μg/mL) increased mineralization and ALP activity in MC3T3-E1 cells, whereas treatment with high concentrations of rh-AMEL (4 μg/mL) and EMD (100 μg/mL) had the opposite effect. Conclusion High concentrations of rh-AMEL and EMD decreased the differentiation of MC3T3-E1 cells. By contrast, a high concentration of rh-AMEL, but not that of EMD, promoted the differentiation of HAT-7 cells. This study demonstrates that the effects of rh-AMEL on cell differentiation differ between HAT-7 and MC3T3-E1 cells, and suggests that different regions on AMEL may induce the differentiation of these cell types.

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 (IP 3 ) 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.

Inositol 1,4,5-trisphosphate (IP₃) receptors consist of three subtypes: IP₃R1, IP₃R2, and IP₃R3. Although numerous IP₃ receptor ligands have been synthesized, none of the subtype-selective ligands are known. We have developed a simple fluorescence method to examine the subtype selectivity of IP₃ receptor ligands using FRET-based IP₃ biosensors LIBRAvI, LIBRAvII, and LIBRAvIII. The addition of IP₃ or adenophostin A (ADA) to permeabilized biosensor-expressing cells increased the fluorescence ratios of these biosensors in a concentration-dependent manner, and the potency of ADA relative to that of IP₃ in terms of the changes in the fluorescence ratios of LIBRAvI, LIBRAvII, and LIBRAvIII was 43-, 22-, and 28-fold, respectively. This fluorescence-based method further showed that several ADA analogs had significant differences with respect to subtype selectivity and potency. These results highlight the important role played by the O-glycosidic structure of ADA in the selectivity of the ligands for IP₃R1, as evidenced by the modified selectivity following replacement of the 5'-hydroxyl with a phenyl or phenethyl group. We also found that one ADA analog 5'-deoxy-5'-phenyladenophostin A possessed a partial agonistic effect on IP₃R1. Together, the novel fluorescent methods described herein are useful for the evaluation of properties of IP₃R ligands, including potency, efficacy, and subtype selectivity.

Store-operated Ca 2+ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca 2+ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca 2+ release from intracellular Ca 2+ stores and Ca 2+ entry from the extracellular space. Ca 2+ release was not affected by treatment with ST, but Ca 2+ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca 2+ sequestration into Ca 2+ stores. The Ca 2+ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca 2+ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca 2+ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La 3+ and 2-APB. The large increase in Ca 2+ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca 2+ stores. These results show that SOCE remains activated even after the refilling of Ca 2+ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.

High resolution imaging of intracellular calcium (Ca 2+ ) concentrations ([Ca 2+ ];) has revealed that Ca 2+ signals show diverse responses, such as Ca 2+ oscillations and Ca 2+ waves ; these signals are exploited to control diverse cellular processes. Ca 2+ responses in non-excitable cells are primarily regulated by inositol 1,4,5-trisphosphate (IP 3 ). To elucidate the mechanisms of these diverse Ca 2+ responses, we have developed a series of fluorescent IP 3 biosensors, the LIBRAv series, using cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) in combination with fluorescence resonance energy transfer (FRET) technologies. These fluorescent IP 3 biosensors are specific to IP 3 and permit the monitoring of IP 3 dynamics in single living cells. Studies conducted with IP 3 biosensors during agonist-induced Ca 2+ oscillations revealed cell type-specific differences in IP 3 dynamics, non-fluctuating rises in cytosolic IP 3 concentrations ([IP 3 ]) and repetitive IP 3 spikes. These results provide important experimental information for resolving long-standing questions regarding the mechanisms responsible for Ca 2+ oscillations. IP 3 biosensors also demonstrated an intracellular gradient of IP 3 and its wave-like expansion during mechanical stimulation-induced Ca 2+ waves. As such, continued improvements in IP 3 biosensors and the development of additional novel fluorescent biosensors are highly likely to provide a better understanding of the regulatory mechanisms of various forms of Ca 2+ signals and clarification of their physiologic roles.

Ca2+ responses in salivary acinar cells are mostly homogeneous and synchronous, whereas the timing and magnitude of Ca2+ responses in salivary ductal cells are variable. Imaging analysis using multiphoton microscopy revealed the heterogeneity of salivary ductal cell sensitivity to Ca2+-mobilizing agonists. Stimulation of isolated rat parotid ducts with a low concentration (0.1 μM) of epinephrine (Epi) elevated the intracellular Ca2+ levels ( [Ca2+] i) in ∼30% of the ductal cells. Similarly, threshold concentrations (0.5 or 1 μM) of phenylephrine (PhL), carbachol (CCh), or ATP induced responses in∼20% of the ductal cells. Sequential stimulation with threshold concentrations of PhL, CCh, and ATP suggested that low concentrations of PhL, CCh, and ATP activate different subpopulations of parotid ductal cells. In addition, certain rat parotid ductal cells exhibit spontaneous oscillations in [Ca2+] i. Electron microscopic analysis indicated that spontaneously oscillating ducts contained numerous granules on the luminal side, which is characteristic of granular ducts. These Ca2+ oscillations were completely blocked by the purinergic receptor inhibitors, PPADS and suramin. Differential interference contrast images and a plasma membrane fluorescence probe, synaptogreen C4, revealed that the spontaneous elevations in [Ca2+] i were closely correlated with spontaneous swelling of ductal cells. The present findings suggest that purinergic receptors mediate spontaneous Ca2+ oscillations in parotid ductal cells and regulate electrolyte reabsorption from the primary saliva under resting conditions.

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.

Using multiphoton microscopy, we established that rat parotid ductal cells exhibit spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i). These oscillatory Ca2+ responses were observed during continuous perfusion with a physiological salt solution at 37°C in the absence of calcium mobilizing agonist stimulation. The timing and patterns of these spontaneous Ca2+ oscillations varied among individual ductal cells, and the average number of Ca2+ responses in a single responding ductal cell was 2.1 in a 10-min recording period. High-speed scanning (0.6 s/image) revealed that most spontaneous elevations in [Ca2+]i were initiated at the luminal side of ductal cells and spread toward the basal side within 2 s. Electron microscopic analysis after Ca2+ imaging indicated that spontaneously oscillating ducts contained numerous granules at the luminal side, which is characteristic of granular ducts. These Ca2+ oscillations were completely blocked by the purinergic receptor inhibitors 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) and suramin but were not blocked by the muscarinic antagonist atropine or the α-adrenergic antagonist phentolamine. Simultaneous observation of fura-2 fluorescence and differential interference contrast (DIC) images showed that spontaneous elevations of [Ca2+]i were well correlated with changes in shape of ductal cells. Using a plasma membrane fluorescence probe, SynaptoGreen C4, we found that the changes in DIC images reflected spontaneous cell swelling of ductal cells. Our findings present the possibility that purinergic receptors mediate spontaneous Ca2+ oscillations in parotid ductal cells and regulate electrolyte reabsorption from the primary saliva in the resting state.

The depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) entry (CCE), which is a Ca(2+)-selective and La(3+)-sensitive entry pathway. Here, we report a novel mechanism of La(3+)-resistant Ca(2+) entry that is synergistically regulated by B-cell-receptor (BCR) stimulation and Ca(2+) store depletion. In DT40 cells, stimulation of BCRs with anti-IgM antibodies induced Ca(2+) release and subsequent Ca(2+) entry in the presence of 0.3 microM La(3+), a condition in which CCE is completely blocked. This phenomenon was not observed in inositol 1,4,5-trisphosphate receptor-deficient DT40 (IP3R-KO) cells. However, in response to thapsigargin pretreatment, BCR stimulation induced La(3+)-resistant Ca(2+) entry into both wild-type and IP3R-KO cells. These results indicate that BCR stimulation alone does not activate Ca(2+) entry, whereas BCR stimulation and depleted Ca(2+) stores (either due to IP3R-mediated Ca(2+) release or Ca(2+) uptake inhibition) work in concert to activate La(3+)-resistant Ca(2+) entry. This Ca(2+) entry was inhibited by genistein. In addition, BCR-mediated Ca(2+) entry was completely abolished in Stim1-deficient DT40 cells and was restored by overexpression of YFP-Stim1, but was unaffected by double knockdown of Orai1 and Orai2. These results demonstrate a unique non-CCE pathway, in which Ca(2+) entry depends on Stim1- and BCR-mediated activation of tyrosine kinases.