Apoptotic cell death during regressive changes in salivary glands: a morphological perspective.

Background: Apoptosis was initially identified through transmission electron microscopy. Subsequent advances in morphological techniques for apoptosis detection have revealed its involvement in multiple pathological conditions in various tissues. This review summarizes previous experimental studies on apoptotic cell death during regressive changes in the salivary glands, with a focus on morphological observations.

A sustained calcium response mediated by IP3 receptor anchoring to the desmosome is essential for apoptotic cell elimination.

Efficient elimination of apoptotic cells within epithelial cell sheets is crucial for preserving epithelial barrier integrity. It is well established that immediate neighbors of an apoptotic cell actively participate in its removal by enclosing it within a wall of actomyosin, pushing it out in a purse-string manner in a process called apical extrusion. Here, we found that sustained elevation of calcium ions in neighboring epithelial cells is necessary to generate the contractility required for apoptotic cell elimination.

Local anesthetics inhibit muscarinic acetylcholine receptor-mediated calcium responses and the recruitment of β-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 β-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 Ca responses and formation of receptor-β-arrestin complexes in the HSY human parotid cell line.

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 β-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.

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.

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 Ca signaling; however, the mechanisms that increase the intracellular Ca concentration ([Ca ] ) are controversial. The current study aimed to elucidate the mechanism underlying the phenytoin-induced increase in [Ca ] in human gingival fibroblasts (HGFs).

Methods: Effects of 100 μM phenytoin on [Ca ] 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.

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).

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 (IP) 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 IP, can measure IP concentration. The FRET signal should be enhanced by attaching a FRET donor to an appropriate position.

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.

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 Ca 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). Ca imaging analysis under cell culture conditions revealed that SF2-G-GECO cells exhibited spontaneous Ca responses, which could be classified into the following three major patterns depending on the cell density: localized Ca responses at cell protrusions at a low density, a cell-wide spread of Ca responses at a medium density, and Ca responses in clusters of 3-20 cells at a high density.

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

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

Effects of full-length human amelogenin on the differentiation of dental epithelial cells and osteoblastic 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).

Bcl-2 and IP compete for the ligand-binding domain of IPRs modulating Ca signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca dynamics by directly targeting and inhibiting the IP receptor (IPR), a major intracellular Ca-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IPR activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IPR function provoked by low concentration of agonist or IP, while fails to attenuate against high concentration of agonist or IP. Surprisingly, Bcl-2 remained capable of inhibiting IPR1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.

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

New Findings: What is the central question of this study? The effects of Ca responses on salivary fluid secretion have been studied indirectly by monitoring ion channel activities and other indices. Therefore, Ca responses during salivary secretion remain poorly understood. What is the main finding and its importance? Herein, we developed a simultaneous monitoring system for Ca responses and salivary secretion in live animals using a YC-Nano50-expressing submandibular gland and a fibre-optic pressure sensor.

[Advances in methods for analyzing IP signaling and understanding of coupled Ca and IP oscillations].

Inositol 1,4,5-trisphosphate (IP) 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 Ca from intracellular stores via IP receptors. IP-mediated Ca signals often exhibit complex spatial and temporal organizations, such as Ca oscillations. Recently, new methods have become available to measure IP concentration ([IP]) using AlphaScreen technology, fluorescence polarization, and competitive ligand binding assay (CFLA).

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.

On the dynamical structure of calcium oscillations.

Oscillations in the concentration of free cytosolic Ca 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 Ca oscillations (i.

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.

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

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.

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.

SIRT1 regulates lamellipodium extension and migration of melanoma cells.

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.

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.

A fluorescence-based method for evaluating inositol 1,4,5-trisphosphate receptor ligands: determination of subtype selectivity and partial agonist effects.

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.