Regulation of Ca signals in a parotid cell line Par-C5

The Ca²⁺ signaling system in an established immortalized rat parotid acinar cell line, Par-C5, was examined using the Ca²⁺-sensitive fluorescent indicator fura-2 and by measuring inositol 1,4,5-trisphosphate (IP₃) formation. Agonist-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i) by mobilization of intracellular stores and influx across the cell membrane was stimulated by acetylcholine (ACh) and ATP, whereas noradrenaline-(NA)-induced a small [Ca²⁺]_i increase mediated primarily by release from intracellular Ca²⁺ stores. [Ca²⁺]_i increase by ACh and ATP was meditated through the phosphoinositide signal pathway since both agonists significantly increased 1,4,5-IP₃ formation and Ca²⁺ mobilization was abolished by the phospholipase C inhibitor U73122. In Ca²⁺-free medium, ACh or ATP discharged the IP₃-sensitive Ca²⁺ store and essentially abolished subsequent [Ca²⁺]_i response to thapsigargin (TG). Exposure to ionomycin and monensin after TG induced a further mobilization of Ca²⁺, suggesting IP₃-insensitive stores are present. Furthermore, depletion of IP₃-sensitive Ca²⁺ stores by TG, ACh and ATP enhanced plasmalemmal Ca²⁺-entry pathways. Exposure to tumor necrosis factor-α (TNF-α), a cytokine associated with lymphocyte invasion of salivary epithelial cells in autoimmune disorders, significantly reduced ACh-stimulated Ca²⁺ mobilization. TNF-α inhibitory effect on Ca²⁺ mobilization was not directly due to an interaction on muscarinic receptors since ACh-induced 1,4,5-IP₃ formation was not altered. These results in the Par-C5 cell line indicate 1) [Ca²⁺]_i is regulated by muscarinic and P2Y-nucleotide receptors and partly by α₁-adrenergic receptors; 2) IP₃-sensitive and -insensitive Ca²⁺ stores exist; 3) Ca²⁺ influx activated by ACh, ATP or TG is mediated by the store-operated Ca²⁺ entry pathway; and 4) muscarinic agonist-stimulated Ca²⁺ mobilization is altered by the cytokine TNF-α.     

The effect of magnolol on Ca2+ homeostasis and its related physiology in human oral cancer cells

ObjectiveMagnolol, a polyphenol compound from herbal medicines, was shown to alter physiology in various cell models. However, the effect of magnolol on Ca²⁺ homeostasis and its related physiology in oral cancer cells is unclear. This study examined whether magnolol altered Ca²⁺ signaling and cell viability in OC2 human oral cancer cells.MethodsCytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in suspended cells were measured by using the fluorescent Ca²⁺-sensitive dye fura-2. Cell viability was examined by 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay.ResultsMagnolol at concentrations of 20–100 μM induced [Ca²⁺]_i rises. Ca²⁺ removal reduced the signal by approximately 50%. Magnolol (100 μM) induced Mn²⁺ influx suggesting of Ca²⁺ entry. Magnolol-induced Ca²⁺ entry was partially suppressed by protein kinase C (PKC) regulators, and inhibitors of store-operated Ca²⁺ channels. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished magnolol-evoked [Ca²⁺]_i rises. Conversely, treatment with magnolol abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 partially inhibited magnolol-induced [Ca²⁺]_i rises. Magnolol at 20–100 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM).ConclusionsTogether, in OC2 cells, magnolol induced [Ca²⁺]_i rises by evoking partially PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Magnolol also caused Ca²⁺-independent cell death. Therefore, magnolol-induced cytotoxicity may not be involved in activation mechanisms associated with intracellular Ca²⁺ mobilization in oral cancer cells.     

Histamine H1 receptor-stimulated Ca2+ signaling pathway in human periodontal ligament cells

We studied histamine-induced Ca²⁺ mobilization in human periodontal ligament (HPDL) cells. Histamine induced a transient rise in intracellular Ca²⁺ ([Ca²⁺]_i) and maintained a sustained phase in the presence of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, the transient peak was slightly reduced and the sustained phase was decreased to the basal level. The initial rise in [Ca²⁺]_i was attributed to two components: intracellular Ca²⁺ release and Ca²⁺ influx, whereas the sustained phase was due to Ca²⁺ influx. After depletion of intracellular Ca²⁺ stores with thapsigargin, a known Ca²⁺-ATPase inhibitor, histamine-induced increase in [Ca²⁺]_i was significantly reduced, suggesting histamine induces Ca²⁺ release from inositol 1,4,5-trisphosphate [Ins(l,4,5)P₃]- and thapsigargin-sensitive Ca²⁺ stores. Histamine-induced peak in [Ca²⁺]_i was increased dose-dependently in the presence and absence of extracellular Ca²⁺. The histamine-mediated response in [Ca²⁺]_i was specifically attenuated by chlorpheniramine (H₁ antagonist) but not by cimetidine (H₂ antagonist), clearly indicating that activation of H₁ receptor mediates histamine-induced Ca²⁺ mobilization. We next examined the effect of histamine on inositol phosphates formation. Histamine stimulated the formation of inositol phosphates which changed time-dependently. In particular, the formation of Ins(1,4,5)P₃ was increased significantly for 10 s. The histamine-induced Ca²⁺ mobilization caused an increase of prostaglandin E₂ (PGE₂) release which was reduced in excluding extracellular Ca²⁺. These results indicate that activation of histamine H₁ receptor induces the accumulation of Ins(l,4,5)P₃ and the following transient increase in [Ca²⁺]_i, and elicits the release of PGE₂ which may be coupled with Ca²⁺ influx.     

Are salivary glands cell lines in culture a good model for purinergic receptors in salivary glands?

A major obstacle in studying the physiological and biochemical processes of salivary secretion is the lack of a good ductal cell line model. HSY, an immortalised cell line originating from human parotid gland intercalated ducts, provides a possible model for purinergic, mechanisms in ductal cells. Unlike the biphasic dose response to ATP of isolated submandibular ductal cells, the rise in [Ca2+]i in MY cells shows single Michaelis-Menten kinetics with an apparent K_a of 0.8 μM. Pre-incubation with thapsigargin inhibited the ATP induced [Ca²⁺]_i rise. Both ATP (10 μM) and carbachol (100 μM) increased IP₃ production. Intercalated duct cells may differentiate into acinar or ductal cells in response to appropriate stimuli from extracellular matrix. We therefore attempted to induce a duct-like phenotype in the striated duct-derived HSY cells by growing them on microcarrier beads coated with type I collagen. In Ca-containing medium cells grown on all substrates showed similar responses to ATP. In contrast, in Ca-free medium, [Ca2+]i rose only slightly in cells grown on beads relative to those on glass. This probably resulted from reduced IP₃ Production. Carbachol also induced a much smaller increase in [Ca2+]i and less IP₃ production in cells grown on Cytodex-3. The HSY response to purinergic stimuli by an increase in [Ca²⁺]_i and IP₃ means that they can be used to study the metabotropic purinergic pathway. The impairment in the HSY responses grown on Cytodex-3 can be used to probe phosposinositol signal transduction in salivary cells.     

The impact of nitric oxide on calcium homeostasis in PE/CA-PJ15 cells

ObjectiveNitric oxide (NO) production and Ca²⁺ homeostasis are key determinants for the control of many cell functions. NO is known to be a mediator of Ca²⁺ homeostasis in a highly complex and cell-specific manner and although Ca²⁺ homeostasis has been explored in human oral cancer cells, the exact mechanisms are not completely understood. In this study we investigated the impact of exogenous NO on [Ca²⁺]_c homeostasis in PE/CA-PJ15 cells.DesignCells were treated with S-nitrosocysteine as NO-donor and the determinations of cytosolic Ca²⁺ concentrations were performed using FURA-2 AM. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin were used to challenge mitochondrial functionality, whereas thapsigargin (TG) and La³⁺ were employed to perturb intracellular calcium levels.ResultsNO derived from S-nitrosocysteine (CySNO) induced a dose-dependent reduction of cytosolic calcium [Ca²⁺]_c whereas oxy-haemoglobin (oxyHb) completely counteracted this effect. Subsequently, we assessed possible relationships between NO and cellular structures responsible for Ca²⁺ homeostasis. We found that uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and oligomycin strongly reduced the effect of NO on [Ca²⁺]_c. Moreover, we found that during this mitochondrial energetic deficit, the effect of NO on [Ca²⁺]_c was also reduced in the presence of La³⁺ or thapsigargin.ConclusionsNO induces a concentration-dependent [Ca²⁺]_c reduction in PE/CA-PJ15 human oral cancer cells and potentiates mitochondrial Ca²⁺ buffering in the presence of TG or La³⁺.Further, we show that exogenous NO deregulates Ca²⁺ homeostasis in PE/CA-PJ15 cells with fully energized mitochondria.     

Diversity and Spatio-Temporal Properties of Calcium Responses in Salivary Ducts

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.     

Spontaneous Ca2+ Oscillations in Rat Parotid Ducts Are Associated with Cell Swelling

Imaging analysis using multiphoton microscopy revealed that rat parotid ductal cells exhibit spontaneous Ca²⁺ oscillations in the absence of calcium mobilizing agonist stimulation. This spontaneous Ca²⁺ release was first observed during the monitoring of Ca²⁺ transients during continuous perfusion at 37°C, and the protocol for cell preparation was modified to allow consistent observation of spontaneous oscillations. Spontaneous Ca²⁺ oscillations were completely blocked by application of the purinergic receptor inhibitors PPADS and suramin. Simultaneous observation of fura-2 fluorescence and differential interference contrast (DIC) images showed that spontaneous elevations in intracellular Ca²⁺ concentration ( [Ca²⁺] _i) were well correlated with changes in the shape of ductal cells. Using a plasma membrane fluorescence probe we found that the changes in DIC images reflected spontaneous cell swelling of ductal cells. The present findings suggest the possibility that purinergic receptors mediate spontaneous Ca²⁺ oscillations in parotid ductal cells and regulate electrolyte reabsorption from the primary saliva under resting conditions. Cell swelling concomitant with a spontaneous increase in [Ca²⁺] _i was an unexpected result because an agonist-induced increase in [Ca²⁺] _i has been shown to induce cell shrinkage in ductal cells. When spontaneous Ca²⁺ release was compared to the carbachol-induced Ca²⁺ response, there were significant differences in the speed of Ca²⁺ elevation and duration of the Ca²⁺ response. Our data suggest that the different patterns of Ca²⁺ responses in parotid ducts might activate different ion channels and/or ion transporters and cause opposite cell shape changes.     

The Development of FRET-Based IP3 Biosensors and Their Use for Monitoring IP3 Dynamics during Ca2+ Oscillations and Ca2+ Waves in Non-Excitable Cells

High resolution imaging of intracellular calcium (Ca²⁺) concentrations ([Ca²⁺];) has revealed that Ca²⁺ signals show diverse responses, such as Ca²⁺ oscillations and Ca²⁺ waves ; these signals are exploited to control diverse cellular processes. Ca²⁺ responses in non-excitable cells are primarily regulated by inositol 1,4,5-trisphosphate (IP₃). To elucidate the mechanisms of these diverse Ca²⁺ responses, we have developed a series of fluorescent IP₃ 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 IP₃ biosensors are specific to IP₃ and permit the monitoring of IP₃ dynamics in single living cells. Studies conducted with IP₃ biosensors during agonist-induced Ca²⁺ oscillations revealed cell type-specific differences in IP₃ dynamics, non-fluctuating rises in cytosolic IP₃ concentrations ([IP₃]) and repetitive IP₃ spikes. These results provide important experimental information for resolving long-standing questions regarding the mechanisms responsible for Ca²⁺ oscillations. IP₃ biosensors also demonstrated an intracellular gradient of IP₃ and its wave-like expansion during mechanical stimulation-induced Ca²⁺ waves. As such, continued improvements in IP₃ 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 Ca²⁺ signals and clarification of their physiologic roles.     

Human salivary gland cells express bradykinin receptors that modulate the expression of proinflammatory cytokines

Bradykinin is an important peptide modulator that affects the function of neurons and immune cells. However, there is no evidence of the bradykinin receptors and their functions in human salivary glands. Here we have identified and characterized bradykinin receptors on human submandibular gland cells. Both bradykinin B1 and B2 receptors are expressed on human submandibular gland cells, A253 cells, and HSG cells. Bradykinin increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a concentration‐dependent manner. Interestingly, a specific agonist of the B1 receptor did not have any effect on [Ca²⁺]_i in HSG cells, whereas specific agonists of the B2 receptor had a Ca²⁺ mobilizing effect. Furthermore, application of the B1 receptor antagonist, R715, did not alter the bradykinin‐mediated increase in cytosolic Ca²⁺, whereas the B2 receptor antagonist, HOE140, showed a strong inhibitory effect, which implies that bradykinin B2 receptors are functional in modulating the concentration of cytosolic Ca²⁺. Bradykinin did not affect a carbachol‐induced rise of [Ca²⁺]_i and did not modulate translocation of aquaporin‐5. However, bradykinin did promote the expression of proinflammatory cytokines, including tumor necrosis factor‐α (TNF‐α), implying the role of bradykinin in salivary gland inflammation. These data suggest that bradykinin receptors are involved in Ca²⁺ signaling in human submandibular gland cells and serve a unique role, which is separate from that of other salivary gland G protein–coupled receptors.     

Preconditioning with Porphyromonas gingivalis lipopolysaccharide may confer cardioprotection and improve recovery of the electrically induced intracellular calcium transient during ischemia and reperfusion

Fan MHM, Wong KL, Wu S, Leung WK, Yam WC, Wong TM. Preconditioning with Porphyromonas gingivalis lipopolysaccharide may confer cardioprotection and improve recovery of the electrically induced intracellular calcium transient during ischemia and reperfusion. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01206.x. © 2009 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis lipopolysaccharide (LPS) is a ligand for cell surface toll‐like receptors (TLR), TLR2 and TLR4 while stimulation of either leads to cardioprotection. We hypothesized that: (1) pretreatment with P. gingivalis LPS at appropriate concentrations would induce cardioprotection against injury induced by ischemia and reperfusion; and (2) P. gingivalis LPS pretreatment at cardioprotective concentrations may reduce Ca²⁺ overload, which is a precipitating cause of injury, and improve recovery of contractile function. Material and Methods: Male Sprague–Dawley rats were randomly selected to receive intraperitoneal saline or hot phenol–water‐extracted P. gingivalis LPS at 0.2, 0.5, 1.0, 2.0 or 4.0 mg/kg 24 h before the experiment. The hearts were isolated and subjected to regional ischemia by coronary artery ligation followed by reperfusion. In isolated rat ventricular myocytes, the cytosolic Ca²⁺ level and the electrically induced intracellular calcium (E[Ca²⁺]_i) transient, which reflects contractile function, were determined after pretreatment with a cardioprotective dose of P. gingivalis LPS. Results: Pretreatment with 0.5 mg/kg P. gingivalis LPS significantly reduced, while pretreatment with 1.0–4.0 mg/kg significantly increased infarct size. The Ca²⁺ overload induced by ischemia–reperfusion was attenuated in myocytes from rats pretreated with 0.5 mg/kg P. gingivalis LPS. Pretreated myocytes also showed an increased amplitude of the E[Ca²⁺]_i transient, no prolongation of the time to reach the peak E[Ca²⁺]_i transient and shorter 50% decay time during reperfusion. Conclusion: At a dosage of 0.5 mg/kg, P. gingivalis LPS confers cardioprotection against ischemia–reperfusion‐induced injury and improved intracellular E[Ca²⁺]_i transient recovery, hence improving myocyte contractile recovery.     

The effect of angiotensin II on the proliferation of human gingival fibroblasts

Angiotensin not only raises blood pressure and modifies body fluids and electrolytes but also induces differentiation and proliferation of fibroblasts in the circulatory system in order to repair damage. The purpose of the present study was to observe the influence of the addition of angiotensin II (AngII) or nifedipine (NIF) alone or both sequentially on proliferative activity, the intracellular Ca²⁺ concentration ([Ca²⁺]i), and the inositol-1,4,5-triphosphate (IP3) level in cultivated human gingival fibroblasts. Addition of 10⁻⁸−10⁻⁴ M NIF or 10⁻⁵−10⁻⁴ M AngII alone increased the proliferation of cultivated gingival fibroblasts, and the interaction of NIF and AngII suppressed proliferation. Addition of AngII alone increased [Ca²⁺]i, with a peak 60 s afterward and a return to a level slightly higher than the pretreatment level at 120 s. Addition of both AngII and NIF did not increase [Ca²⁺]i as much as the addition of AngII alone. When Ca²⁺ was absent from the extracellular environment, the AngII-induced increase in [Ca²⁺]i was suppressed. AngII increased the concentration of IP₃, with a peak at 120 s after its addition. From these results we concluded that AngII increased the proliferation of gingival fibroblasts by causing an influx of Ca²⁺, which increased [Ca²⁺]i.     

Functional and molecular characterization of transmembrane intracellular pH regulators in human dental pulp stem cells

ObjectiveHomeostasis of intracellular pH (pH_i) plays vital roles in many cell functions, such as proliferation, apoptosis, differentiation and metastasis. Thus far, Na⁺-H⁺ exchanger (NHE), Na⁺-HCO₃⁻ co-transporter (NBC), Cl⁻/HCO₃⁻ exchanger (AE) and Cl⁻/OH⁻ exchanger (CHE) have been identified to co-regulate pH_i homeostasis. However, functional and biological pH_i-regulators in human dental pulp stem cells (hDPSCs) have yet to be identified.DesignMicrospectrofluorimetry technique with pH-sensitive fluorescent dye, BCECF, was used to detect pH_i changes. NH₄Cl and Na⁺-acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pH_i-regulators were detected by Western blot technique.ResultsThe resting pH_i was no significant difference between that in HEPES-buffered (nominal HCO₃⁻-free) solution or CO₂/HCO₃-buffered system (7.42 and 7.46, respectively). The pH_i recovery following the induced-intracellular acidosis was blocked completely by removing [Na⁺]_o, while only slowed (−63%) by adding HOE694 (a NHE1 specific inhibitor) in HEPES-buffered solution. The pH_i recovery was inhibited entirely by removing [Na⁺]_o, while adding HOE 694 pulse DIDS (an anion-transporter inhibitor) only slowed (−55%) the acid extrusion. Both in HEPES-buffered and CO₂/HCO₃-buffered system solution, the pH_i recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl⁻]_o. Western blot analysis showed the isoforms of pH_i regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs.ConclusionsWe demonstrate for the first time that resting pH_i is significantly higher than 7.2 and meditates functionally by two Na⁺-dependent acid extruders (NHE and NBC), two Cl⁻-dependent acid loaders (CHE and AE) and one Na⁺-independent acid extruder(s) in hDPSCs. These findings provide novel insight for basic and clinical treatment of dentistry.     

The biphasic effect of extracellular glucose concentration on carbachol‐induced fluid secretion from mouse submandibular glands

Cholinergic agonists evoke elevations of the cytoplasmic free‐calcium concentration ([Ca²⁺]_i) to stimulate fluid secretion in salivary glands. Salivary flow rates are significantly reduced in diabetic patients. However, it remains elusive how salivary secretion is impaired in diabetes. Here, we used an ex vivo submandibular gland perfusion technique to characterize the dependency of salivary flow rates on extracellular glucose concentration and activities of glucose transporters expressed in the glands. The cholinergic agonist carbachol (CCh) induced sustained fluid secretion, the rates of which were modulated by the extracellular glucose concentration in a biphasic manner. Both lowering the extracellular glucose concentration to less than 2.5 mM and elevating it to higher than 5 mM resulted in decreased CCh‐induced fluid secretion. The CCh‐induced salivary flow was suppressed by phlorizin, an inhibitor of the sodium–glucose cotransporter 1 (SGLT1) located basolaterally in submandibular acinar cells, which is altered at the protein expression level in diabetic animal models. Our data suggest that SGLT1‐mediated glucose uptake in acinar cells is required to maintain the fluid secretion by sustaining Cl^? secretion in real‐time. High extracellular glucose levels may suppress the CCh‐induced secretion of salivary fluid by altering the activities of ion channels and transporters downstream of [Ca²⁺]_i signals.     

Autoantibodies in Sjögren's syndrome patients acutely inhibit muscarinic receptor function

Oral Diseases (2012) 18 , 132–139 Objectives: Autoantibodies from the sera of Sjögren’s syndrome patients (SS IgG) have been suggested to inhibit muscarinic receptor function. However, the acute nature of such an inhibitory effect remains controversial. In this study, we investigated the acute effects of SS IgG on muscarinic receptor function in human submandibular gland (HSG) cells. Methods: The effects of autoantibodies on muscarinic receptor function were studied using microspectrofluorimetry, whole‐cell patch clamp, immunofluorescence confocal microscopy, and a co‐immunoprecipitation assay. Results: Carbachol (CCh) was found to consistently increase intracellular calcium concentration ([Ca²⁺]_i) and activate K⁺ current in HSG cells. However, pretreatment of the cells with SS IgG for 5 or 30 min significantly attenuated these responses, with a substantially more prominent effect after 30 min of treatment. Like CCh, adenosine 5′‐triphosphate (ATP) also increased [Ca²⁺]_i and activated K⁺ currents in HSG cells, although pretreatment with SS IgG did not affect the cellular response to ATP. CCh was found to reorganize α‐fodrin in HSG cells in a Ca²⁺‐dependent manner. However, pretreatment with SS IgG prevented the cytoskeletal reorganization of α‐fodrin induced by CCh. Conclusions: SS IgG acutely and reversibly inhibited muscarinic receptor function, thereby inhibiting the Ca²⁺ mobilization necessary for the activation of K⁺ currents and α‐fodrin reorganization in HSG cells.     

Hyperglucose Contributes to Periodontitis: Involvement of the NLRP3 Pathway by Engaging the Innate Immunity of Oral Gingival Epithelium

Background: The NLRP3 inflammasome is essentially a family of intracellular innate immune sensors that can respond to bacterial challenge and initiate early host immunity responses. However, the involvement and possible molecular mechanism of the NLRP3 pathway in the context of chronic periodontitis (CP) and diabetes mellitus have yet to be fully elucidated. Methods: Gingival tissues were collected from patients with CP and/or type 2 diabetes mellitus (T2DM), and the expression of NLRP3 and interleukin (IL)‐1β was analyzed by immunohistochemistry. To explore the possible molecular mechanism, human gingival epithelial cells (HGECs) were established in vitro and challenged with lipopolysaccharide (LPS) and/or high glucose. High extracellular K⁺ was applied as an inhibitor of NLRP3. The NLRP3 pathway was analyzed by immunocytochemistry and quantitative polymerase chain reaction. Results: Compared with control individuals, NLRP3 and IL‐1β were significantly upregulated in oral gingival epithelium of patients with CP and/or T2DM (P <0.05). The expression of NLRP3 was significantly upregulated in HGECs when stimulated in vitro by LPS or high glucose (P = 0.00). The simultaneous stimulation of LPS and high glucose contributed to significant upregulation of NLRP3 expression versus LPS or high glucose alone (P = 0.00). Although expression of caspase 1 and IL‐1β protein were increased in HGECs when stimulated by LPS, they were partially inhibited after the NLRP3 was successfully blocked. Conclusion: For patients with T2DM and CP, hyperglycemic status may exacerbate the inflammation state of gingival tissue by activating the NLRP3 pathway, and this abnormal host inflammatory response may contribute to further tissue breakdown.     

Purines,a new class of agonists in salivary glands?

The response of rat submandibular glands to extracellular purines was tested. In crude cellular suspensions, ATP increased the [Ca²⁺]_i mostly by promoting uptake of extracellular calcium. ATP caused the pH_i to drop, a response blocked by chloride channel inhibitors. ATP also inhibited the basal and isoproterenol-stimulated activity of the Na⁺-K⁺-2Cl-cotransporter. These effects were reproduced by benzoyl-ATP, an agonist of ionotropic purinoceptors. In pure ductal suspensions, ATP activated a metabotropic P2Y₁ purinergic receptor coupled to phospholipase C and opened a non-specific cation channel coupled to a P2X₇ receptor. Activation of these receptors stimulated a Ca²⁺-dependent and a Ca²⁺-independent phospholipase A₂, the latter resulting in kallikrein secretion.We conclude that purinergic agonists can modulate the activity of both acinar and ductal phases of secretion. Activation of metabotropic receptors coupled to phospholipase C could lead to responses resembling those to muscarinic or adrenergic agonists. Activation of ionotropic receptors could stimulate new intracellular responses also involved in secretory function.     

Ca2+ Channels in Odontoblasts

Odontoblasts, well-polarized columnar cells at the periphery of the dental pulp, originate from neural crest cells. They are primarily involved in dentin formation (dentinogenesis) as sites of the synthesis and secretion of collagenous and non-collagenous matrix proteins, and also participate in the directional transport of Ca²⁺ from the circulation to the dentin/mineralizing front. Dentinogenesis is activated by : 1) pulpal patho-physiological events, such as intra-pulpal inflammatory responses, physiological or developmental processes, and 2) events at the dentin surface, such as variousimechanical/heat/coldjstimuli applied to the tooth surface. Therefore, the aim of this communication is to give an overview of the Ca²⁺-signaling system, which may have a major role in dentin formation in both physiological and pathological settings. Special attention will be given to discussion of the following : 1) sequential Ca²⁺ signaling pathways from internal inositol 1,4,5-trisphosphate (IP₃) production via the activation of phospholipase C-coupled receptors to the release of Ca²⁺ from IP₃-sensitive Ca²⁺ stores, 2) subsequent Ca²⁺ influx via store-operated Ca²⁺ channels, and 3) the expression of transient receptor potential channels coupled with Na⁺-Ca²⁺ exchangers in odontoblasts.     

Influence of calcium, phosphate and complexing substances on inorganic pyrophosphatase in mineralizing hamster molars.

The influence of calcium and phosphorus on PP_i-ase activity in mineralizing hamster molars was studied in the presence and absence of several complexing substances. These complexing substances inhibited the PP_i-ase activity. Only Zn²⁺ completely reversed this inhibition. The Ca²⁺ stimulation of the PP_i-ase was substrate-dependent with an optimal activity at a Ca:PP_i ratio of 1. At higher concentrations, Ca²⁺ was strongly inhibitory, possibly by forming neutral salts with PP_i. The influence of complexing substances on the enzyme was modified by Ca²⁺, which changes the mol Ca²⁺:PP_i ratio or competes slightly with Zn²⁺ for the complexing agent. There was a small inhibition of the PP_i-ase activity by P_i which was not influenced by the substrate used (PP_i^4?, Mg-PP_i^2?or Ca-PP_i^2?. P_i can reverse the inhibiting effects of complexing substances to a small extent, except for stronger inhibitions obtained for 0.1 mM EHDP and 2mM Cl₂ MDP. These small in-vitro effects of Ca²⁺ and P_i on the PP_i-ase activity could not explain the great changes in PP_i-ase activity in mineralizing molars during normal development and at P_i depletion. This supports a stimulating role for the PP_i-ase in mineralization. It was concluded that, during mineralization, different Ca²⁺ and P_i concentrations can modify the inhibitory effects of the complexing substances on the PP_i-ase only to a very small extent.     

Etching effect of acidic fluorides on human tooth enamel in vitro

ObjectiveThis in vitro study aimed to examine the etching effect of acidic fluoride solutions on enamel.Materials and methods24 human teeth divided into 48 enamel-specimens were partly isolated with impression material. Specimens were exposed for 10 min to 20 ml of the following solutions: 1.6% TiF₄, 3.9% SnF₂, 0.2% HF and 1.8% citric acid (CA). The isolation was removed and 24 specimens analysed by profilometry (Δheight: exposed/isolated enamel surfaces, surface roughness parameters). For the remaining 24 specimens [Ca²⁺] in the test solutions was analysed by atomic absorption spectroscopy.ResultsMedian Δheights (μm) after exposure were: TiF₄ 0.07, SnF₂ −0.03, HF −0.14 and CA −5.92. TiF₄-exposed surfaces showed both deposits and etched areas and exhibited statistically significant different surface roughness parameters compared to the HF- and SnF₂-exposed surfaces. Median [Ca²⁺] values (ppm): TiF₄ 1.88, SnF₂ 0.11, HF 0.10 and CA 2.17.ConclusionAt the [F] tested in this study it can be concluded that SnF₂- and HF solutions had negligible erosive effects on enamel. TiF₄ solution resulted in an incomplete surface deposition associated with calcium dissolution suggesting that TiF₄ applied as solution may not be advisable.