Calcium-induced Calcium Release Mechanism Research Articles

Mathematical model for IP$_{3}$ dependent calcium oscillations and mitochondrial associate membranes in non-excitable cells

Theoretical studies on calcium oscillations within the cytosolic [Ca$^{2+}$], and mitochondria [Ca$^{2+}$]$_{mit}$ have been conducted using a mathematical model-based approach. The model incorporates the mechanism of calcium-induced calcium release (CICR) through the activation of inositol-trisphosphate receptors (IPR), with a focus on the endoplasmic reticulum (ER) as an internal calcium store. The production of 1,4,5 inositol-trisphosphate (IP$_{3}$) through the phospholipase \(C\) isoforms and its degradation via Ca$^{2+}$ are considered, with IP$_{3}$ playing a crucial role in modulating calcium release from the ER. The model includes a simple kinetic mechanism for mitochondrial calcium uptake, release and physical connections between the ER and mitochondria, known as mitochondrial associate membrane complexes (MAMs), which influence cellular calcium homeostasis. Bifurcation analysis is used to explore the different dynamic properties of the model, identifying various regimes of oscillatory behavior and how these regimes change in response to different levels of stimulation, highlighting the complex regulatory mechanisms governing intracellular calcium signaling.

Magnesium Ions Moderate Calcium-Induced Calcium Release in Cardiac Calcium Release Sites by Binding to Ryanodine Receptor Activation and Inhibition Sites.

Ryanodine receptor channels at calcium release sites of cardiac myocytes operate on the principle of calcium-induced calcium release. In vitro experiments revealed competition of Ca2+ and Mg2+ in the activation of ryanodine receptors (RyRs) as well as inhibition of RyRs by Mg2+. The impact of RyR modulation by Mg2+ on calcium release is not well understood due to the technical limitations of in situ experiments. We turned instead to an in silico model of a calcium release site (CRS), based on a homotetrameric model of RyR gating with kinetic parameters determined from in vitro measurements. We inspected changes in the activity of the CRS model in response to a random opening of one of 20 realistically distributed RyRs, arising from Ca2+/Mg2+ interactions at RyR channels. Calcium release events (CREs) were simulated at a range of Mg2+-binding parameters at near-physiological Mg2+ and ATP concentrations. Facilitation of Mg2+ binding to the RyR activation site inhibited the formation of sparks and slowed down their activation. Impeding Mg-binding to the RyR activation site enhanced spark formation and speeded up their activation. Varying Mg2+ binding to the RyR inhibition site also dramatically affected calcium release events. Facilitation of Mg2+ binding to the RyR inhibition site reduced the amplitude, relative occurrence, and the time-to-end of sparks, and vice versa. The characteristics of CREs correlated dose-dependently with the effective coupling strength between RyRs, defined as a function of RyR vicinity, single-channel calcium current, and Mg-binding parameters of the RyR channels. These findings postulate the role of Mg2+ in calcium release as a negative modulator of the coupling strength among RyRs in a CRS, translating to damping of the positive feedback of the calcium-induced calcium-release mechanism.

Pharmacogenomic Screening of Drug Candidates using Patient-Specific hiPSC-Derived Cardiomyocyte High-Throughput Calcium Imaging.

Calcium imaging is an invaluable technique to detect and characterize calcium flux in cells. The use of calcium dye provides information on the concentration and spatial distribution of calcium. Calcium imaging is a well-established technique to assess the calcium-induced calcium release mechanism in cardiomyocytes. It can also be used to characterize mutations in genes crucial for this mechanism that frequently causes arrhythmia. Here we describe a high-throughput methodology of calcium imaging that records individual calcium transients in more than 10,000 humaninduced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in less than 30min.

Metabolic syndrome inhibits store-operated Ca2+ entry and calcium-induced calcium-release mechanism in coronary artery smooth muscle

Background and purposeMetabolic syndrome causes adverse effects on the coronary circulation including altered vascular responsiveness and the progression of coronary artery disease (CAD). However the underlying mechanisms linking obesity with CAD are intricated. Augmented vasoconstriction, mainly due to impaired Ca2+ homeostasis in coronary vascular smooth muscle (VSM), is a critical factor for CAD. Increased calcium–induced calcium release (CICR) mechanism has been associated to pathophysiological conditions presenting persistent vasoconstriction while increased store operated calcium (SOC) entry appears to activate proliferation and migration in coronary vascular smooth muscle (VSM). We analyze here whether metabolic syndrome might alter SOC entry as well as CICR mechanism in coronary arteries, contributing thus to a defective Ca2+ handling and therefore accelerating the progression of CAD. Experimental approachMeasurements of intracellular Ca2+ ([Ca2+]i) and tension and of Ca2+ channels protein expression were performed in coronary arteries (CA) from lean Zucker rats (LZR) and obese Zucker rats (OZR). Key resultsSOC entry stimulated by emptying sarcoplasmic reticulum (SR) Ca2+ store with cyclopiazonic acid (CPA) was decreased and associated to decreased STIM-1 and Orai1 protein expression in OZR CA. Further, CICR mechanism was blunted in these arteries but Ca2+ entry through voltage-dependent L-type channels was preserved contributing to maintain depolarization-induced increases in [Ca2+]i and vasoconstriction in OZR CA. These results were associated to increased expression of voltage-operated L-type Ca2+ channel alpha 1C subunit (CaV1.2) but unaltered ryanodine receptor (RyR) and sarcoendoplasmic reticulum Ca2+‐ATPase (SERCA) pump protein content in OZR CA. Conclusion and implicationsThe present manuscript provides evidence of impaired Ca2+ handling mechanisms in coronary arteries in metabolic syndrome where a decrease in both SOC entry and CICR mechanism but preserved vasoconstriction are reported in coronary arteries from obese Zucker rats. Remarkably, OZR CA VSM at this state of metabolic syndrome seemed to have developed a compensation mechanism for impaired CICR by overexpressing CaV1.2 channels.

Amyloid beta 42 oligomers induce neuronal and synaptic receptor dysfunctions.

Amyloid beta 42 oligomers induce neuronal and synaptic receptor dysfunctions.

Danger signals activate a putative innate immune system during regeneration in a filamentous fungus.

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce. Here we show that activation of injury responses and hyphal regeneration in the filamentous fungus Trichoderma atroviride relies on the detection of two danger or alarm signals. As an early response to injury, we detected a transient increase in cytosolic free calcium ([Ca2+]c) that was promoted by extracellular ATP, and which is likely regulated by a mechanism of calcium-induced calcium-release. In addition, we demonstrate that the mitogen activated protein kinase Tmk1 plays a key role in hyphal regeneration. Calcium- and Tmk1-mediated signaling cascades activated major transcriptional changes early following injury, including induction of a set of regeneration associated genes related to cell signaling, stress responses, transcription regulation, ribosome biogenesis/translation, replication and DNA repair. Interestingly, we uncovered the activation of a putative fungal innate immune response, including the involvement of HET domain genes, known to participate in programmed cell death. Our work shows that fungi and animals share danger-signals, signaling cascades, and the activation of the expression of genes related to immunity after injury, which are likely the result of convergent evolution.

Nitric oxide modulates ATP-evoked currents in mouse Leydig cells.

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Ultrastructural and gene-expression changes in the calcium regulation system of rat skeletal muscles under exhausting exercise

The expression of genes responsible for the synthesis of essential proteins regulating the calcium-ion balance and ultrastructural characteristics of fast-twitch (m. extensor digitorum longus, EDL) and slow-twitch (m. soleus, SOL) skeletal muscles under prolonged exercise were studied in an experimental model of forced-swimming rats. A day after the end of the exercise, no significant changes in any of the five investigated genes were revealed in the SOL. A few triad elements (T-tubules and cisternae of sarcoplasmic reticulum) were revealed. A small number of excitation-contraction coupling (ECC) structures in the control and a slight increase in their amount after exercises were noticed. Polymorphism and mitochondrial defects within SOL muscles indicate the importance of these structures in the regulation of calcium balance. In EDL muscles, adaptation mechanisms are aimed mainly at pumping Ca2+ ions to the sarcoplasmic reticulum, where the main calcium buffer is calsequestrin. Expression of SERCA1 gene increased by an order of magnitude, and that of CASQ1 increased by three times. Electron microscopy showed a major role of triads in the maintenance of calcium homeostasis in the EDL muscles, as well as a greater destruction of these muscles compared to SOL after exhausting exercise. The high level of triads and a possible activation of the CICR (calcium-induced calcium release) mechanism in fast-twitch muscles can cause damage to them during exhausting exercise. Adaptation of SOL muscles is associated with structural rearrangements of the mitochondrial apparatus, while adaptation of the EDL muscles is caused by calcium removal from the sarcoplasm with Ca-ATPase and its retention in the sarcoplasmic reticulum by calsequestrin.

Structural Differences between Cardiac and Skeletal Ryanodine Receptors

Cardiac and skeletal muscles have different mechanisms of excitation-contraction coupling that depend on a set of tissue-specific protein isoforms. In heart, ryanodine receptor isoform 2 (RyR2), cardiac dihydropyridine receptor (DHPR) and FKBP12.6 together with other proteins couple the plasmalemmal depolarization to the release of Ca2+ from the sarcoplasmic reticulum (SR), via a calcium-induced calcium release mechanism. In skeletal muscle, RyR1, skeletal DHPR and FKBP12 together with other proteins couple these two events via an entirely different mechanism that requires a more direct RyR-DHPR coupling, and different long-range allosterism pathways.While the 3D structure of RyR1 has been solved at near-atomic resolution, the 3D structure of RyR2 lags significantly in resolution (currently ∼30 A). To start uncovering the different molecular mechanisms of these two main RyR isoforms, we have determined the 3D structure of RyR2 in complex with FKBP12.6 in the closed state at nanometer resolution. This has enabled building a pseudo-atomic model for RyR2. Comparative analysis between the RyR2 and RyR1 isoforms shows significant structural differences localized to specific domains, but not all structural differences can be explained by primary sequence divergence. These differences are interpreted in the context of the two modes of excitation-contraction coupling. The analysis of heterogeneity in the dataset allowed us to further define two conformations representing two functional states of RyR2.This work was supported by American Heart Association grant No. 14GRNT19660003 and Muscular Dystrophy Association grant No. MDA352845 (to MS).

Intracellular Calcium Mobilization in Response to Ion Channel Regulators via a Calcium-Induced Calcium Release Mechanism.

Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca2+]i. We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds.

A ROS-Assisted Calcium Wave Dependent on the AtRBOHD NADPH Oxidase and TPC1 Cation Channel Propagates the Systemic Response to Salt Stress.

Plants exhibit rapid, systemic signaling systems that allow them to coordinate physiological and developmental responses throughout the plant body, even to highly localized and quickly changing environmental stresses. The propagation of these signals is thought to include processes ranging from electrical and hydraulic networks to waves of reactive oxygen species (ROS) and cytoplasmic Ca(2+) traveling throughout the plant. For the Ca(2+) wave system, the involvement of the vacuolar ion channel TWO PORE CHANNEL1 (TPC1) has been reported. However, the precise role of this channel and the mechanism of cell-to-cell propagation of the wave have remained largely undefined. Here, we use the fire-diffuse-fire model to analyze the behavior of a Ca(2+) wave originating from Ca(2+) release involving the TPC1 channel in Arabidopsis (Arabidopsis thaliana). We conclude that a Ca(2+) diffusion-dominated calcium-induced calcium-release mechanism is insufficient to explain the observed wave transmission speeds. The addition of a ROS-triggered element, however, is able to quantitatively reproduce the observed transmission characteristics. The treatment of roots with the ROS scavenger ascorbate and the NADPH oxidase inhibitor diphenyliodonium and analysis of Ca(2+) wave propagation in the Arabidopsis respiratory burst oxidase homolog D (AtrbohD) knockout background all led to reductions in Ca(2+) wave transmission speeds consistent with this model. Furthermore, imaging of extracellular ROS production revealed a systemic spread of ROS release that is dependent on both AtRBOHD and TPC1 These results suggest that, in the root, plant systemic signaling is supported by a ROS-assisted calcium-induced calcium-release mechanism intimately involving ROS production by AtRBOHD and Ca(2+) release dependent on the vacuolar channel TPC1.

Therapeutic Applications of Calcium Metabolism Modulation in Heart Disease.

Cardiovascular disease is the leading cause of death worldwide and there is extensive research on the pathophysiology of all its clinical entities. Despite the big array of possible therapeutic modalities for cardiovascular disease, there is still a big necessity to develop novel treatments that will augment our strategies for tackling the burden of cardiovascular disease and decrease morbidity and mortality. A major player in both the physiology and pathophysiology of the cardiovascular system is calcium. Extracellular calcium is required in order to initiate cardiac muscle contraction and promote the calcium-induced calcium release mechanism from the sarcoplasmic reticulum. A lot of molecules and structures that in a direct or indirect way interact with calcium are being studied and there is a constant flow of new information that is emerging. In this review we focus on some of these calcium metabolism modulators representatives such as SERCA2a, RyR2, S100A1, phosholamban and calcineurin. We emphasize on their mechanism of action, their role in cardiovascular disease and potential therapeutic implications. We also focus on the effect the bisphosphonates might have in regression of the calcium deposition in the human arteries as well as the usage of novel biomarkers such as mircoRNAs in calcium metabolism modulation in heart disease.

Non-Ligand Mobilization of Intracellular Free Calcium by Nanosecond Pulsed Electric Field (NsPEF)

NsPEF makes membrane and intracellular structures in living cells permeable to ions and small molecules thus can be used to modulate cell's signaling pathways and homeostasis. We utilized Fura-2 fast ratiometric recordings to measure changes of intracellular free calcium concentration ([Ca2+]i) in single CHO cells stimulated by 10, 60 and 300 ns PEF. 10 and 300 ns PEF were used for comparison with 60 ns PEF in regard to their potency to make plasma membrane (PM) and/or endoplasmic reticulum (ER) permeable to Ca2+ ions. Single 60 ns PEF evoked transient [Ca2+]i rise due to Ca2+ influx via PM and Ca2+ efflux from ER with thresholds ∼9 and ∼19 kV/cm, respectively. The amplitude of Ca2+ rise increased with nsPEF intensity linearly by 8-10nM per 1 kV/cm (PM) or 5-6 nM per 1 kV/cm when permeabilization of ER was assessed in Ca2+ free solution. This rate of change increased drastically (to 174nM per 1 kV/cm) when amplitude of Ca2+ rise reached 100-150nM. Depleting Ca2+ content of ER showed that such increase was a result of calcium induced calcium release (CICR) activation. Specific blockers 2-APB and dantrolene proved that the activation of IP3 receptors was responsible for physiological mechanism of CICR triggered by nsPEF. 10, 60 and 300 ns PEF had different efficiencies in regard to PM and ER permeabilization for Ca2+ ions. The thresholds of PM and ER permeabilization were indistinguishable for 10 ns pulse while for 60 ns pulse the ER threshold was twofold higher. 300 ns pulse had further reduced potency towards ER permeabilization in comparison to either 10 or 60 ns pulses. These results show that nsPEF can be effectively used to manipulate [Ca2+]i and activate intracellular signaling pathways bypassing PM receptors.Supported by R01CA125482; R01GM088303

Large-conductance calcium-activated potassium current modulates excitability in isolated canine intracardiac neurons

We studied principal neurons from canine intracardiac (IC) ganglia to determine whether large-conductance calcium-activated potassium (BK) channels play a role in their excitability. We performed whole cell recordings in voltage- and current-clamp modes to measure ion currents and changes in membrane potential from isolated canine IC neurons. Whole cell currents from these neurons showed fast- and slow-activated outward components. Both current components decreased in the absence of calcium and following 1-2 mM tetraethylammonium (TEA) or paxilline. These results suggest that BK channels underlie these current components. Single-channel analysis showed that BK channels from IC neurons do not inactivate in a time-dependent manner, suggesting that the dynamic of the decay of the fast current component is akin to that of intracellular calcium. Immunohistochemical studies showed that BK channels and type 2 ryanodine receptors are coexpressed in IC principal neurons. We tested whether BK current activation in these neurons occurred via a calcium-induced calcium release mechanism. We found that the outward currents of these neurons were not affected by the calcium depletion of intracellular stores with 10 mM caffeine and 10 μM cyclopiazonic acid. Thus, in canine intracardiac neurons, BK currents are directly activated by calcium influx. Membrane potential changes elicited by long (400 ms) current injections showed a tonic firing response that was decreased by TEA or paxilline. These data strongly suggest that the BK current present in canine intracardiac neurons regulates action potential activity and could increase these neurons excitability.

Spaceflight regulates ryanodine receptor subtype 1 in portal vein myocytes in the opposite way of hypertension

Gravity has a structural role for living systems. Tissue development, architecture, and organization are modified when the gravity vector is changed. In particular, microgravity induces a redistribution of blood volume and thus pressure in the astronaut body, abolishing an upright blood pressure gradient, inducing orthostatic hypotension. The present study was designed to investigate whether isolated vascular smooth muscle cells are directly sensitive to altered gravitational forces and, second, whether sustained blood pressure changes act on the same molecular target. Exposure to microgravity during 8 days in the International Space Station induced the decrease of ryanodine receptor subtype 1 expression in primary cultured myocytes from rat hepatic portal vein. Identical results were found in portal vein from mice exposed to microgravity during an 8-day shuttle spaceflight. To evaluate the functional consequences of this physiological adaptation, we have compared evoked calcium signals obtained in myocytes from hindlimb unloaded rats, in which the shift of blood pressure mimics the one produced by the microgravity, with those obtained in myocytes from rats injected with antisense oligonucleotide directed against ryanodine receptor subtype 1. In both conditions, calcium signals implicating calcium-induced calcium release were significantly decreased. In contrast, in spontaneous hypertensive rat, an increase in ryanodine receptor subtype 1 expression was observed as well as the calcium-induced calcium release mechanism. Taken together, our results shown that myocytes were directly sensitive to gravity level and that they adapt their calcium signaling pathways to pressure by the regulation of the ryanodine receptor subtype 1 expression.

Mucin Secretion Induced by Titanium Dioxide Nanoparticles

Nanoparticle (NP) exposure has been closely associated with the exacerbation and pathophysiology of many respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD) and asthma. Mucus hypersecretion and accumulation in the airway are major clinical manifestations commonly found in these diseases. Among a broad spectrum of NPs, titanium dioxide (TiO2), one of the PM10 components, is widely utilized in the nanoindustry for manufacturing and processing of various commercial products. Although TiO2 NPs have been shown to induce cellular nanotoxicity and emphysema-like symptoms, whether TiO2 NPs can directly induce mucus secretion from airway cells is currently unknown. Herein, we showed that TiO2 NPs (<75 nm) can directly stimulate mucin secretion from human bronchial ChaGo-K1 epithelial cells via a Ca2+ signaling mediated pathway. The amount of mucin secreted was quantified with enzyme-linked lectin assay (ELLA). The corresponding changes in cytosolic Ca2+ concentration were monitored with Rhod-2, a fluorescent Ca2+ dye. We found that TiO2 NP-evoked mucin secretion was a function of increasing intracellular Ca2+ concentration resulting from an extracellular Ca2+ influx via membrane Ca2+ channels and cytosolic ER Ca2+ release. The calcium-induced calcium release (CICR) mechanism played a major role in further amplifying the intracellular Ca2+ signal and in sustaining a cytosolic Ca2+ increase. This study provides a potential mechanistic link between airborne NPs and the pathoetiology of pulmonary diseases involving mucus hypersecretion.

A calcium-induced calcium release mechanism supports luteinizing hormone-induced testosterone secretion in mouse Leydig cells

Leydig cells are responsible for the synthesis and secretion of testosterone, processes controlled by luteinizing hormone (LH). Binding of LH to a G protein-coupled receptor in the plasma membrane results in an increase in cAMP and in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here we show, using immunofluorescence, that Leydig cells express ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Measurements of intracellular calcium changes using the fluorescent calcium-sensitive dye fluo-3 and confocal microscopy show that both types of receptors are involved in a calcium-induced calcium release (CICR) mechanism, which amplifies the initial Ca(2+) influx through plasma membrane T-type calcium channels (Ca(V)3). The RyRs and IP(3)Rs are functional, as judged from both their activation by caffeine and IP(3) and block by ryanodine and 2-aminoethoxydiphenyl borate (2-APB), respectively. RyRs are the principal players involved in the release of Ca(2+) from the endoplasmic reticulum, as evidenced by the fact that global Ca(2+) changes evoked by LH are readily blocked by 100 muM ryanodine but not by 2-APB or xestospongin C. Finally, steroid production by Leydig cells is inhibited by ryanodine but not by 2-APB. These results not only broaden our understanding of the role played by calcium in Leydig cells but also show, for the first time, that RyRs have an important role in determining testosterone secretion by the testis.

Abstract 5282: TRPM7, a novel regulator of human nasopharyngeal carcinoma cells Ca2+ influx and migration

Abstract Ion channels are involved in various physiologic and pathologic processes, including the migration of tumor cells that is required for metastasis. To determine whether transient receptor potential melastatin 7 (TRPM7) Ca2+ channels play an important role in the migration of tumor cells, we examined the potential function of TRPM7 channels in the migration of 5-8F human nasopharyngeal carcinoma cells. The migratory potential of the cells was impaired by TRPM7 inhibitors (La3+, 2-APB), and their migration was reduced significantly after either TRPM7 was silenced or extracellular Ca2+ was removed. Conversely, addition of TRPM7 activator (BK) promoted the migration of 5-8F cells. Furthermore, the Ca2+ influx regulated by TRPM7 activated release of Ca2+ stores by a calcium-induced calcium release mechanism. This study suggests, first, that Ca2+ influx is required for the migration of human nasopharyngeal carcinoma 5-8F cells. Second, and more importantly, it identifies TRPM7 as a novel potential-regulator of the Ca2+ influx that allows migration of 5-8F cells. TRPM7, therefore, might have potential as a prognostic indicator and as a therapeutic target in cancer. Keywords: TRPM7; nasopharyngeal carcinoma cells; 5-8F; Ca2+ influx; Ca2+; migration. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5282.

TRPM7 regulates the migration of human nasopharyngeal carcinoma cell by mediating Ca 2+ influx

Ion channels are involved in various physiologic and pathologic processes, including the migration of tumor cells that is required for metastasis. To determine whether transient receptor potential melastatin 7 (TRPM7) Ca 2+ channels play an important role in the migration of tumor cells, we examined the potential function of TRPM7 channels in the migration of 5-8F and 6-10B human nasopharyngeal carcinoma cells. The migratory potential of 5-8F cells was significantly decreased by extracellular Ca 2+ chelator (EGTA), TRPM7 inhibitors (La 3+, 2-APB), or TRPM7 knockdown. Conversely, the addition of TRPM7 activator Bradykinin and overexpression of TRPM7 promoted the migration of 5-8F and 6-10B cells. Furthermore, the sustained Ca 2+ influx regulated by TRPM7 activated release of Ca 2+ stores via ryanodine receptors by a calcium-induced calcium release (CICR) mechanism. This study suggests, first, that Ca 2+ influx is required for the migration of human nasopharyngeal carcinoma 5-8F cells. Second, and more importantly, it identifies TRPM7 as a novel potential-regulator of the Ca 2+ influx that allows migration of 5-8F cells. TRPM7, therefore, might have potential as a prognostic indicator and as a therapeutic target in nasopharyngeal carcinoma.

γ-Aminobutyric Acid and Glutamate Differentially Regulate Intracellular Calcium Concentrations in Mouse Gonadotropin-Releasing Hormone Neurons

Multiple factors regulate the activity of the GnRH neurons responsible for controlling fertility. Foremost among neuronal inputs to GnRH neurons are those using the amino acids glutamate and gamma-aminobutyric acid (GABA). The present study used a GnRH-Pericam transgenic mouse line, enabling live cell imaging of intracellular calcium concentrations ([Ca(2+)](i)) to evaluate the effects of glutamate and GABA signaling on [Ca(2+)](i) in peripubertal and adult mouse GnRH neurons. Activation of GABA(A), N-methyl-d-aspartate, or alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate acid (AMPA) receptors was found to evoke an increase in [Ca(2+)](i), in subpopulations of GnRH neurons. Approximately 70% of GnRH neurons responded to GABA, regardless of postnatal age or sex. Many fewer (approximately 20%) GnRH neurons responded to N-methyl-d-aspartate, and this was not influenced by postnatal age or sex. In contrast, about 65% of adult male and female GnRH neurons responded to AMPA compared with about 14% of male and female peripubertal mice (P < 0.05). The mechanisms underlying the ability of GABA and AMPA to increase [Ca(2+)](i) in adult GnRH neurons were evaluated pharmacologically. Both GABA and AMPA were found to evoke [Ca(2+)](i) increases through a calcium-induced calcium release mechanism involving internal calcium stores and inositol-1,4,5-trisphosphate receptors. For GABA, the initial increase in [Ca(2+)](i) originated from GABA(A) receptor-mediated activation of L-type voltage-gated calcium channels, whereas for AMPA this appeared to involve direct calcium entry through the AMPA receptor. These observations show that all of the principal amino acid receptors are able to control [Ca(2+)](i) in GnRH neurons but that they do so in a postnatal age- and intracellular pathway-specific manner.