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Introduction: Dynamic physiological changes in brain extracellular calcium ([Ca2+]o) occur when high levels of neuronal activity lead to substantial Ca2+ entry via ion channels reducing local [Ca2+]o. Perturbations of the extracellular microenvironment that increase [Ca2+]o are commonly used to study how [Ca2+] regulates neuronal activity. At excitatory synapses, the Ca2+-sensing receptor (CaSR) and other G-protein coupled receptors link [Ca2+]o and spontaneous glutamate release. Phospholipase C (PLC) is activated by G-proteins and is hypothesized to mediate this process. Methods: Patch-clamping cultured neocortical neurons, we tested how spontaneous glutamate release was affected by [Ca2+]o and inhibition of PLC activity. We used hypertonic sucrose (HS) to evaluate the readily releasable pool (RRP) and test if it was affected by inhibition of PLC activity. Results: Spontaneous glutamate release substantially increased with [Ca2+]o, and inhibition of PLC activity, with U73122, abolished this effect. PLC-β1 is an abundant isoform in the neocortex, however, [Ca2+]o-dependent spontaneous release was unchanged in PLC-β1 null mutants (PLC-β1-/-). U73122 completely suppressed this response in PLC-β1-/- neurons, indicating that this residual [Ca2+]o-sensitivity may be mediated by other PLC isoforms. The RRP size was substantially reduced after incubation in U73122, but not U73343. Phorbol esters increased RRP size after PLC inhibition. Discussion: Together these data point to a strong role for PLC in mediating changes in spontaneous release elicited by [Ca2+]o and other extracellular cues, possibly by modifying the size of the RRP. [ABSTRACT FROM AUTHOR]

IntroductionDynamic physiological changes in brain extracellular calcium ([Ca2+]o) occur when high levels of neuronal activity lead to substantial Ca2+ entry via ion channels reducing local [Ca2+]o. Perturbations of the extracellular microenvironment that increase [Ca2+]o are commonly used to study how [Ca2+] regulates neuronal activity. At excitatory synapses, the Ca2+-sensing receptor (CaSR) and other G-protein coupled receptors link [Ca2+]o and spontaneous glutamate release. Phospholipase C (PLC) is activated by G-proteins and is hypothesized to mediate this process.MethodsPatch-clamping cultured neocortical neurons, we tested how spontaneous glutamate release was affected by [Ca2+]o and inhibition of PLC activity. We used hypertonic sucrose (HS) to evaluate the readily releasable pool (RRP) and test if it was affected by inhibition of PLC activity.ResultsSpontaneous glutamate release substantially increased with [Ca2+]o, and inhibition of PLC activity, with U73122, abolished this effect. PLC-β1 is an abundant isoform in the neocortex, however, [Ca2+]o-dependent spontaneous release was unchanged in PLC-β1 null mutants (PLC-β1–/–). U73122 completely suppressed this response in PLC-β1–/– neurons, indicating that this residual [Ca2+]o–sensitivity may be mediated by other PLC isoforms. The RRP size was substantially reduced after incubation in U73122, but not U73343. Phorbol esters increased RRP size after PLC inhibition.DiscussionTogether these data point to a strong role for PLC in mediating changes in spontaneous release elicited by [Ca2+]o and other extracellular cues, possibly by modifying the size of the RRP.

Background/Aims: In kidney, extracellular [Ca2+] can modulate intracellular [Ca2+] to control key cellular processes. Hence, extracellular [Ca2+] is normally maintained within narrow range. We tested effect of extracellular ATP on viability of human proximal (HK-2) cells at high calcium. Modulation of intracellular calcium was assessed by imaging cytosolic [Ca2+], and expression of calcium-binding proteins (CaBPs). We present an artificial intelligence enabled deep learning model for prediction of injury and protection against extracellular [Ca2+] in HK-2 cells. Methods: HK-2 cells were cultured in calcium-free DMEM supplemented with CaCl2. Morphological changes were detected using light microscopy. Cell viability was determined using MTT Assay. Intracellular [Ca2+] was detected using fluorescence microscopy. For easy detection of HK-2 cells injury, we performed light microscopy image classification based on Convolutional Neural Network. Expression of CaBPs, p21, and Mcl-1 was measured using realtime PCR. Results: We show decreased viability of HK-2 cells cultured in elevated calcium levels, which was prevented by adenosine triphosphate (ATP). Exposure of cells to elevated extracellular [Ca2+] correlated with increasing fluorescence of intracellular calcium indicator, which was attenuated in presence of ATP. Since features cannot be detected easily by human eyes, we propose a customized deep learning-based CNN model for classification of HK-2 cells injury by extracellular calcium with high accuracy of 98%. Our data demonstrated significant increase in mRNA levels of calmodulin, S100A8, S100A14 and CaBP28k, with elevated extracellular [Ca2+]. Expression of these genes was enhanced with ATP. Conclusion: The results suggest that ATP protects human proximal (HK-2) cells against elevated extracellular calcium levels. We present a CNN model as user friendly tool to study calcium dependent injury in (HK-2) cells. Finally, we show that ATP-mediated protection is correlated with enhanced expression of calcium-binding proteins. [ABSTRACT FROM AUTHOR]

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

In this study, we aimed to investigate relaxant effect of flavanol (-)-epicatechin on the isolated human saphenous vein (HSV), as a part of its cardioprotective action, and to define the mechanisms underlying this vasorelaxation. (-)-Epicatechin induced a concentration-dependent relaxation of HSV pre-contracted by phenylephrine. Among K+ channel blockers, 4-aminopyridine, margatoxin, and iberiotoxin significantly inhibited relaxation of HSV, while glibenclamide considerably reduced effects of the high concentrations of (-)-epicatechin. Additionally, (-)-epicatechin relaxed contraction induced by 80 mM K+ , whereas in the presence of nifedipine produced partial relaxation of HSV rings pre-contracted by phenylephrine. In Ca2+ -free solution, (-)-epicatechin relaxed contraction induced by phenylephrine, but had no effect on contraction induced by caffeine. A sarcoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin, significantly reduced relaxation of HSV produced by (-)-epicatechin. These results demonstrate that (-)-epicatechin produces endothelium-independent relaxation of isolated HSV rings. Vasorelaxation to (-)-epicatechin probably involves activation of 4-aminopyridine- and margatoxin-sensitive KV channels, BKCa channels, and at least partly, KATP channels. In addition, not only the inhibition of extracellular Ca2+ influx, but regulation of the intracellular Ca2+ release, via inositol-trisphosphate receptors and reuptake into sarcoplasmic reticulum, via stimulation of Ca2+ -ATPase, as well, most likely participate in (-)-epicatechin-induced relaxation of HSV. [ABSTRACT FROM AUTHOR]

Whereas severe Cd stress (150 µM Cd) causes root growth arrest as a consequence of marked superoxide generation leading to extensive cell death in the root tips, mild Cd stress (15 µM Cd) evokes morphogenic responses, such as reduced root elongation and radial root expansion, resulting in shorter and thicker roots. Similar to the low Cd concentration-caused mild stress, treatment of roots with either Ba to remove exchangeable or EDTA to remove both exchangeable and tightly bound cations, including Ca and Mg, from the apoplast, induced root growth inhibition and swelling. However, pre-treatment of roots with Ba had a synergistic effect on the development of these mild Cd stress-induced morphogenic responses, but without the development of any other symptoms in the root tips. In turn, EDTA pre-treatment markedly increased the toxicity of Cd in barley root tips via enhanced Cd uptake-mediated superoxide generation, which evoked extensive cell death in the transition zone of root tips identically to the high Cd concentration-induced severe stress. While the mild stress-induced responses were alleviated by the inhibition of auxin signalling pathway, the severe stress-induced symptoms were prevented by Ca, but not Mg, supplementation or by the inhibition of Cd uptake into the root symplasm. Therefore, the appropriate concentration of Ca in the apoplast is crucial to prevent the rapid accumulation of Cd in the symplasm, which above a certain threshold level leads to the huge superoxide generation and cell death. [ABSTRACT FROM AUTHOR]

Calcium homeostasis modulator 1 (CALHM1), formerly known as FAM26C, was recently identified as a physiologically important plasma membrane ion channel. CALHM1 and its Caenorhabditis elegans homolog, CLHM-1, are regulated by membrane voltage and extracellular Ca concentration ([Ca]). In the presence of physiological [Ca] (∼1.5 mM), CALHM1 and CLHM-1 are closed at resting membrane potentials but can be opened by strong depolarizations. Reducing [Ca] increases channel open probability, enabling channel activation at negative membrane potentials. Together, voltage and Ca allosterically regulate CALHM channel gating. Through convergent evolution, CALHM has structural features that are reminiscent of connexins and pannexins/innexins/LRRC8 (volume-regulated anion channel (VRAC)) gene families, including four transmembrane helices with cytoplasmic amino and carboxyl termini. A CALHM1 channel is a hexamer of CALHM1 monomers with a functional pore diameter of ∼14 Å. CALHM channels discriminate poorly among cations and anions, with signaling molecules including Ca and ATP able to permeate through its pore. CALHM1 is expressed in the brain where it plays an important role in cortical neuron excitability induced by low [Ca] and in type II taste bud cells in the tongue that sense sweet, bitter, and umami tastes where it functions as an essential ATP release channel to mediate nonsynaptic neurotransmitter release. CLHM-1 is expressed in C. elegans sensory neurons and body wall muscles, and its genetic deletion causes locomotion defects. Thus, CALHM is a voltage- and Ca-gated ion channel, permeable to large cations and anions, that plays important roles in physiology. [ABSTRACT FROM AUTHOR]

As an essential indicator of allergic reactions, mast cell (MC) activation involves FcεRI-mediated signaling and the release of allergic mediators. In FcεRI signaling, Ca2+ is located at the intersection of multiple cellular signaling pathways. However, the effect of extracellular Ca2+ (exCa2+) on MCs during anaphylaxis remains unclear, along with its exact mechanisms. Therefore, we sought to determine whether and how elevated exCa2+ amplifies allergic reactions. In vitro experiments used immunoglobulin E (IgE)/antigen (Ag)-induced activation of rat and mouse MCs in vitro. The levels of MC degranulation mediators were used to evaluate the effect of exCa2+. In vivo experiments used MC-mediated passive systemic anaphylaxis (PCA) Balb/c mice. After stimulation, anaphylaxis indexes such as rectal temperature and allergic symptom score were detected. In vitro experiments revealed that exCa2+ is a stimulus signal for the aggravation of allergic reactions in MCs. When antagonists or siRNA inhibited GPRC6, MCs released fewer inflammatory mediators. Moreover, in vivo experiments confirmed in vitro results. Allergic symptoms were alleviated by antagonists NPS2143 in PCA mice, demonstrating that exCa2+ aggravates allergic reactions through GPRC6A. Our study provides an essential theoretical basis for targeting Ca2+ and GPRC6A as therapeutic options for allergies. [Display omitted] [ABSTRACT FROM AUTHOR]

Calcium signaling plays an essential role in chondrocyte mechanotransduction. Guilak and colleagues have revealed the roles of TRPV4 and Piezo channels in chondrocyte calcium signaling and metabolism. This study compared the calcium responses of primary chondrocytes and ATDC5 cells induced by two different stimuli: osmotic stress and intense mechanical stimulus. Roles of three essential calcium signaling pathways, including extracellular calcium source, intracellular ER calcium store and mechanical-sensitive ion channels, were also investigated and compared between cells. Primary chondrocytes showed more vigorous calcium peaks under osmotic stress than under mechanical stimuli, while an opposite trend was found for ATDC5 cells. Extracellular calcium source, intracellular ER store, and PLC/IP 3 pathway each played significant roles in the calcium responses of ATDC5 cells under both osmotic and mechanical stimuli. However, high level shear stress can directly cause ER release in primary cells without the presence of extracellular Ca2+ or involvement of PLC-IP 3 pathway. TRPV4 channel is essential for the responses of ATDC5 cells, but not for primary chondrocytes. In contrast, inhibition of mechano-sensitive channels had no significant effects on the ATDC5 cells. Therefore, primary chondrocytes and ATDC5 cells rely on distinct calcium sources and ion channels to initiate intracellular calcium signaling. Together, these results contribute to our understanding of stimulation-induced calcium signaling in primary chondrocytes and ATDC5 cells, and the different roles of three essential pathways between the two cell types. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction: Experimental myotonia induced in rat muscle by ClC-1 chloride channel-inhibited has been shown to be related inversely to extracellular concentrations of Mg2+ and Ca2+ ([Mg2+]o and [Ca2+]o) within physiological ranges. Because this implicates a role for [Mg2+]o and [Ca2+]o in the variability of symptoms among myotonia congenita patients, we searched for similar effects of [Mg2+]o and [Ca2+]o on myotonia in human muscle. Methods: Bundles of muscle fibers were isolated from abdominal rectus in patients undergoing abdominal surgery. Myotonia was induced by ClC-1 inhibition using 9-anthracene carboxylic acid (9-AC) and was assessed from integrals of force induced by 5-H z stimulation for 2 seconds. Results: Myotonia disappeared gradually when [Mg2+]o or [Ca2+]o were elevated throughout their physiological ranges. These effects of [Mg2+]o and [Ca2+]o were additive and interchangeable. Conclusions: These findings suggest that variations in symptoms in myotonia congenita patients may arise from physiological variations in serum Mg2+ and Ca2+. Muscle Nerve 51: 65-71, 2015 [ABSTRACT FROM AUTHOR]

Abstract: Bone is the main store of calcium and progenitor cells in the body. During the resorption process, the local calcium concentration reaches 8–40mM, and the surrounding cells are exposed to these fluctuations in calcium. This stimulus is a signal that is detected through the calcium sensing receptor (CaSR), which modulates chemotactic and proliferative G protein-dependent signaling pathways. The objective of the present work is to evaluate the roles of extracellular calcium ([Ca2+]o) and the CaSR in osteoinduction. Rat bone marrow mesenchymal stromal cells (rBMSCs) were stimulated with 10mM of Ca2+. Several experiments were conducted to demonstrate the effect of [Ca2+]o on chemotaxis, proliferation and differentiation on the osteoblastic lineage. It was found that [Ca2+]o induces rBMSCs to migrate and proliferate in a concentration-dependent manner. Real-time polymerase chain reaction and immunofluorescence also revealed that 10mM Ca2+ stimulates overexpression of osteogenic markers in rBMSCs, including alkaline phosphatase (ALP), bone sialoprotein, collagen Ia1 and osteocalcin. Functional assays determining ALP activity and mineralization tests both corroborate the increased expression of these markers in rBMSCs stimulated with Ca2+. Moreover, CaSR blockage inhibited the cellular response to stimulation with high concentrations of [Ca2+]o, revealing that the CaSR is a key modulator of these cellular responses. [Copyright &y& Elsevier]

Abstract: Prolonged exposure of oligodendrocyte progenitor cultures to non-toxic concentrations of glutamate receptor agonists for 24 h decreased cellular proliferation mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Since prolonged agonist stimulation can regulate the expression of various families of receptors, we examined this possibility. Pretreatment of progenitor cultures with 100 μM kainic acid (KA) for 1–24 h caused a time-dependent decrease in AMPA receptor activity, determined by agonist-induced 45Ca2+ uptake. The maximum effect (70–80% decrease), observed in the 24 h-pretreated cells, was accompanied by a significant reduction in AMPA receptor subunits, as determined by Western blotting. GluR2/3 and GluR4 subunits were the most affected. Receptor down-regulation and 45Ca2+ uptake were only partially reversible upon KA removal. Furthermore, 24 h co-treatment of cultures with CNQX blocked the KA-induced decreases in calcium uptake. To address whether calpain, a calcium-activated protease, was implicated in the regulation of the AMPA receptor subunits, cultures were treated with the specific inhibitor PD150606 alone or in combination with KA for 24 h. Calpain inhibition significantly increased GluR1 in both conditions and partly reversed downregulation of GluR4 by KA. Collectively, these results indicate that calpain is not involved in the agonist-induced down-regulation of AMPA receptors subunits 2/3 in oligodendrocyte progenitors, while it downregulates GluR1 and GluR4. [Copyright &y& Elsevier]

The article reports a study which investigated the effects of local anesthetic bupivacaine (BV) on primary rat tendon cells regarding potential toxicity and potential underlying mechanisms. The results revealed that BV has severely detrimental effect on tendon cells in vitro. Administration of BV in vivo has impairing effects on cells and elicits considerable but temporary functional damage.

Abstract: Loss-of-function mutations in the ClC-1 Cl− channel trigger skeletal muscle hyperexcitability in myotonia congenita. For reasons that remain unclear, the severity of the myotonic symptoms can vary markedly even among patients with identical ClC-1 mutations, and may become exacerbated during pregnancy and with diuretic treatment. Since both these conditions are associated with hypomagnesemia and hypocalcemia, we explored whether extracellular Mg2+ and Ca2+ ([Mg2+]o and [Ca2+]o) can affect myotonia. Experimental myotonia was induced in isolated rat muscles by ClC-1 inhibition and effects of [Mg2+]o or [Ca2+]o on myotonic contractions were determined. Both cations dampened myotonia within their physiological concentration ranges. Thus, myotonic contractile activity was 6-fold larger at 0.3 than at 1.2mM [Mg2+]o and 82-fold larger at 0.3 than at 1.27mM [Ca2+]o. In intracellular recordings of action potentials, the threshold for action potential excitation was raised by 4–6mV when [Mg2+]o was elevated from 0.6 to 3mM, compatible with an increase in the depolarization of the membrane potential necessary to activate the Na+ channels. Supporting this notion, mathematical simulations showed that myotonia went from appearing with normal Cl− channel function to disappearing in the absence of Cl− channel function when Na+ channel activation was depolarized by 6mV. In conclusion, variation in serum Mg2+ and Ca2+ may contribute to phenotypic variation in myotonia congenita patients. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: The dry extract of Hedera helix leaves, due to its secretolytic and antispasmodic effects, is commonly used to produce pharmaceuticals applied in case of cough and other respiratory symptoms. The results of some in vitro studies as well as the clinical signs of poisoning caused by Hedera helix suggest however strong contractile effect on smooth muscle. In order to clarify the impact of α-hederin (the main active agent of ivy extract) on smooth muscle, the origin of activated calcium involved in α-hederin-induced contraction of gastric smooth muscle preparations was studied. Materials and methods: The study was carried out on rat isolated stomach corpus and fundus strips, under isotonic conditions. The effect of α-hederin (100μM) on smooth muscle preparations was measured before and after the treatment with verapamil during the incubation in modified Krebs–Henseleit solution (M K–HS). Besides, the effect of saponin was measured during the incubation of preparation in Ca2+-free modified Krebs–Henseleit solution or Ca2+-free EGTA-containing modified Krebs–Henseleit solution. Results: The obtained results revealed that the application of verapamil significantly inhibited the reaction evoked by α-hederin. The incubation of stomach strips in calcium-free modified Krebs–Henseleit solution did not change the force of the observed contraction in comparison to the reaction of the preparations incubated in regular incubation solution (M K–HS). In contrary, the replacement of M K–HS by calcium-free chelator-containing solution inhibited totally the reaction to α-hederin. Conclusions: The results indicated that α-hederin-induced contraction results from the influx of calcium which is located in intercellular spaces or bound to the outside of the cell membrane. The Ca2+ influx occurs predominantly through voltage-dependent calcium channels of L-type. [Copyright &y& Elsevier]

To learn whether epileptic seizures in Rasmussen encephalitis ( RE) may be promoted by insufficient γ-aminobutyric acid ( GABA) release. 3 H- GABA was released from neocortical synaptosomes through transporter reversal following intrasynaptosomal Na+ accumulation by veratridine that prevents inactivation of Na+ channels. Tissues of three RE patients were compared with those of nine non- RE. In RE, the release was markedly reduced. In non- RE, the extracellular Ca2+ concentration ([ Ca2+]e) was inversely related to the amount of release. In RE, the percental decline of additional release upon [ABSTRACT FROM AUTHOR]

Abstract: Endothelins (ETs) are widely expressed in the olfactory bulb (OB) and other brain areas where they function as neuropeptides. In a previous study we reported that in the OB ET-1 and ET-3 participate in the long-term regulation of tyrosine hydroxylase (TH), the key enzyme in catecholamine biosynthesis. ETs stimulate TH activity by increasing total and phosphorylated enzyme levels as well as its mRNA. ET-1 response is mediated by a super high affinity ETA receptor coupled to adenylyl cyclase/protein kinase A and Ca2+/calmodulin-dependent protein kinase II (CaMK-II) activation whereas that of ET-3 through an atypical receptor coupled not only to these signaling pathways but also to phospholipase C (PLC)/protein kinase C pathway. Given the participation of PLC and CaMKII in the regulation of TH by ETs in the OB we sought to establish the contribution of calcium to ETs response. Present findings show that calcium released from ryanodine-sensitive channels and extracellular calcium were necessary to stimulate TH by ETs through CaMK-II. On the other hand, intracellular calcium released by the endoplasmic reticulum partially mediated ETs-evoked increase in TH mRNA but calcium influx and CaMK-II inhibition abolished the response. However calcium mechanisms were not involved in ETs-evoked increase in TH protein content. Present findings support that different sources of calcium contribute to the long-term modulation of TH activity and expression mediated by ETs in the rat OB. [Copyright &y& Elsevier]