Search

Mast cells in the peritoneal membrane (PM) may degranulate to release preformed inflammatory mediators including histamine which is capable of diffusing into the surrounding interstitium, modulating cells in their vicinity including, human peritoneal mesothelial cells (hPMC). The present study aimed to investigate the quorum intracellular calcium ([Ca 2+ i ]) response to histamine compared to the membrane soluble ionophore, A23187, in adherent cultured hPMC. To examine [Ca 2+ i ] handling, Fura - 2 loaded cells were exposed to histamine and A23187. Agonist induced transient [Ca 2+ i ] event(s) (TCE) were defined and compared including, resting calcium, peak height, recovery and transient kinetics. Changes in cell shape were examined with immunocytochemistry of the cortical actin (CA) and microtubule (MT) cytoskeleton. To investigate whether histamine induced changes in cell shape were mediated by [Ca 2+ i ], mobilization of [Ca 2+ i ] was prevented with 20 μmol/l of the calcium chelator 1,2-bis-(2-aminophenoxy) ethane- N,N,N',N' -tetraacetic acid (BAPTA-AM). Histamine produced a dose dependent increase of [Ca 2+ i ], maximal at 1.0 mmol/l which recovered to the pre-challenge resting value. Transient multiplicity with repeated challenge was evident below a histamine threshold of 10 −2  mmol/l. Morphometric analysis of MTs and CA showed significant cell elongation plus histamine and A23187. The histamine induced cell elongation was eliminated with [Ca 2+ i ] clamping. This data indicated that increased [Ca 2+ i ] was essential for cell elongation and the formation of stress fibres and therefore has a pivotal role in the regulation of the PM barrier. [ABSTRACT FROM AUTHOR]

Several members of the transient receptor channel (TRP) family can mediate a calcium-dependent cytotoxicity. In sensory neurons, vanilloids like capsaicin induce neurotoxicity by activating TRPV1. The closely related ion channel TRPA1 is also activated by irritants, but it is unclear if and how TRPA1 mediates cell death. In the present study we explored cytotoxicity and intracellular calcium signalling resulting from activation of TRPV1 and TRPA1, either heterologously expressed in HEK 293 cells or in native mouse dorsal root ganglion (DRG) neurons. While activation of TRPV1 by the vanilloids capsaicin, resiniferatoxin and anandamide results in calcium-dependent cell death, activation by protons and the oxidant chloramine-T failed to reduce cell viability. The TRPA1-agonists acrolein, carvacrol and capsazepine all induced cytotoxicity, but this effect is independent of TRPA1. Activation of both TRPA1 and TRPV1 triggers a strong influx of external calcium, but also a strong calcium-release from intracellular stores most likely including the endoplasmic reticulum (ER). Activation of TRPV1, but not TRPA1 also results in a strong increase of mitochondrial calcium both in HEK 293 cells and mouse DRG neurons. Our data demonstrate that activation of TRPV1, but not TRPA1 mediates a calcium-dependent cell death. While both receptors mediate a release of calcium from intracellular stores, only activation of TRPV1 seems to mediate a robust and probably lethal increase in mitochondrial calcium. [ABSTRACT FROM AUTHOR]

During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and α-smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca 2+ ] i ) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y 4 agonist, MRS4062, caused a fast desensitizing [Ca 2+ ] i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca 2+ in the extracellular fluid. The biphasic [Ca 2+ ] i response to UTP was attenuated respectively by P2Y 4 blockers, like reactive blue-2 and suramin, and by the P2Y 11 antagonist, NF340. UTP and the P2Y 2 receptor agonist MRS2768 increased, whereas the selective P2Y 11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y 11 receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y 2 , P2Y 4 and P2Y 11 receptors. Data indicate that besides P2Y 4 and P2Y 2 receptors which are responsible for UTP-induced [Ca 2+ ] i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y 11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling. [ABSTRACT FROM AUTHOR]

As an intracellular Ca 2+ release channel at the endoplasmic reticulum membrane, the ubiquitous inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) plays a crucial role in the generation, propagation and regulation of intracellular Ca 2+ signals that regulate numerous physiological and pathophysiological processes. This review provides a concise account of the fundamental single-channel properties of the InsP 3 R channel: its conductance properties and its regulation by InsP 3 and Ca 2+ , its physiological ligands, studied using nuclear patch clamp electrophysiology. [ABSTRACT FROM AUTHOR]

Prion diseases are neurodegenerative disorders where infectious prion proteins (PrP) accumulate in brain leading to aggregation of amyloid fibrils and neuronal cell death. The amino acid sequence 106–126 from prion proteins, PrP(106–126), is highly amyloidogenic and implicated in prion-induced pathologies. As PrP is known to be expressed in blood following leakage from brain tissue, we sought to investigate its biological effects on human platelets, which have been widely employed as ‘peripheral’ model for neurons. Our findings suggested that, PrP(106–126) (20 μM) induced dramatic 30-fold rise in intracellular calcium (from 105 ± 30 to 3425 ± 525 nM) in platelets, which was attributable to influx from extracellular fluid with comparatively less contribution from intracellular stores. Calcium mobilization was associated with 8–10-fold stimulation in the activity of thiol protease calpain that led to partial cleavage of cytoskeleton-associated protein talin and extensive shedding of microparticles from platelets, thus transforming platelets to ‘activated’ phenotype. Both proteolysis of talin and microparticle release were precluded by calpeptin, a specific inhibitor of calpain. As microparticles are endowed with phosphatidylserine-enriched surface and hence are pro-coagulant in nature, exposure to prion favored a thrombogenic state in the organism. [ABSTRACT FROM AUTHOR]

Cannabidiol (CBD), a major nonpsychotropic cannabinoid found in Cannabis plant, has been shown to influence cardiovascular functions under various physiological and pathological conditions. In the present study, the effects of CBD on contractility and electrophysiological properties of rat ventricular myocytes were investigated. Video edge detection was used to measure myocyte shortening. Intracellular Ca 2+ was measured in cells loaded with the Ca 2+ sensitive fluorescent indicator fura-2 AM. Whole-cell patch clamp was used to measure action potential and Ca 2+ currents. Radioligand binding was employed to study pharmacological characteristics of CBD binding. CBD (1 μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca 2+ transients. However, the amplitudes of caffeine-evoked Ca 2+ transients and the rate of recovery of electrically evoked Ca 2+ transients following caffeine application were not altered. CBD (1 μM) significantly decreased the duration of APs. Further studies on L-type Ca 2+ channels indicated that CBD inhibits these channels with IC 50 of 0.1 μM in a voltage-independent manner. Radioligand studies indicated that the specific binding of [ 3 H]Isradipine, was not altered significantly by CBD. The results suggest that CBD depresses myocyte contractility by suppressing L-type Ca 2+ channels at a site different than dihydropyridine binding site and inhibits excitation–contraction coupling in cardiomyocytes. [ABSTRACT FROM AUTHOR]

A synthetic compound, termed pirfenidone (PFD), is considered promising for the treatment of cardiac disease. It leads to beneficial effects in animal models of diabetes mellitus (DM); as well as in heart attack, atrial fibrillation, muscular dystrophy, and diabetic cardiomyopathy (DC). The latter is a result of alterations linked to metabolic syndrome as they promote cardiac hypertrophy, fibrosis and contractile dysfunction. Although reduced level of fibrosis and stiffness represent an essential step in the mechanism of PFD action, a wide range of functional effects might also contribute to the therapeutic benefits. For example, PFD stimulates L-type voltage-gated Ca 2+ channels (LTCCs), which are pivotal for a process known as excitation–contraction coupling (ECC). Recent evidence suggests that these two types of actions – namely structural and functional – aid in treating both cardiac disease and DM. This view is supported by the fact that in DC, for example, systolic dysfunction arises from both cardiac stiffness linked to fibrosis and down-regulation of ECC. Thus, not surprisingly, clinical trials have been conducted with PFD in the settings of DM, for treating not only cardiac but also renal disease. This review presents all these concepts, along with the possible mechanisms and pathophysiological consequences. [ABSTRACT FROM AUTHOR]

Abstract: A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca2+ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically evoked Ca2+ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca2+-uptake and Ca2+-ATPase activity in a biphasic manner. [3H]-ryanodine binding and passive Ca2+ release from SR vesicles were not altered by 10μM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1μM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2μg/ml for 4h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3μM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3μM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors. [Copyright &y& Elsevier]

Abstract: L-type calcium channels are modulated by a host of mechanisms that include voltage, calcium ions (Ca2+ dependent inactivation and facilitation), cytosolic proteins (CAM, CAMKII, PKA, PKC, etc.), and oxygen radicals. Here we describe yet another Ca2+ channel regulatory mechanism that is induced by pressure-flow (PF) forces of ∼25dyn/cm2 producing 35–60% inhibition of channel current. Only brief periods (300ms) of such PF pulses were required to suppress reversibly the current. Recombinant Ca2+ channels (α1c77/β2a/α2δ and α1c77/β1/α2δ), expressed in HEK293 cells, were similarly suppressed by PF pulses. To examine whether Ca2+ released by PF pulses triggered from different sub-cellular compartments (SR, ER, mitochondria) underlies the inhibitory effect of PF on the channel current, pharmacological agents and ionic substitutions were employed to probe this possibility. No significant difference in effectiveness of PF pulses to suppress ICa or IBa (used to inhibit CICR) was found between control cells and those exposed to U73122 and 2-APB (PLC and IP3R pathway modulators), thapsigargin and BAPTA (SERCA2a modulator), dinitrophenol, FCCP and Ru360 (mitochondrial inhibitors), l-NAME (NOS inhibitor signaling), cAMP and Pertussis toxin (Gi protein modulator). We concluded that the rapid and reversible modulation of the Ca2+ channel by PF pulses is independent of intracellular release of Ca2+ and Ca2+ dependent inactivation of the channel and may represent direct mechanical regulatory effect on the channel protein in addition to previously reported Ca2+-release or entry dependent mechanism. [Copyright &y& Elsevier]

Abstract: Glucocorticoids are steroid hormones which act through the glucocorticoid receptor. They regulate a wide variety of biological processes. Two glucocorticoids, the naturally occurring corticosterone and chemically produced dexamethasone, have been used to investigate the effect of glucocorticoids on Ca2+-signalling in cortical co-cultures of neurons and astrocytes. Dexamethasone and to a lesser degree corticosterone both induced a decrease in cytosolic Ca2+ concentration in neurons and astrocytes. The effect of both compounds can be blocked by inhibition of the plasmamembrane ATPase, calmodulin and by application of a glucocorticoid receptor antagonist, while inhibition of NMDA receptors or the endoplasmic reticulum calcium pump had no effect. Glucocorticoid treatment further protects against detrimental calcium signalling and cell death by modulating the delayed calcium deregulation in response to glutamate toxicity. At the concentrations used dexamethasone and corticosterone did not show cell toxicity of their own. Thus, these results indicate that dexamethasone and corticosterone might be used for protection of the cells from calcium overload. [Copyright &y& Elsevier]

Abstract: The contractile vacuole complex (CVC) of freshwater protists serves the extrusion of water and ions, including Ca2+. No vesicle trafficking based on SNAREs has been detected so far in any CVC. SNAREs (soluble NSF [N-ethylmaleimide sensitive factor] attachment protein receptors) are required for membrane-to-membrane interaction, i.e. docking and fusion also in Paramecium. We have identified three v-/R- and three t/Q-SNAREs selectively in the CVC. Posttranscriptional silencing of Syb2, Syb6 or Syx2 slows down the pumping cycle; silencing of the latter two also causes vacuole swelling. Increase in extracellular Ca2+ after Syb2, Syb6 or Syx2 silencing causes further swelling of the contractile vacuole and deceleration of its pulsation. Silencing of Syx14 or Syx15 entails lethality in the Ca2+ stress test. Thus, the effects of silencing strictly depend on the type of the silenced SNARE and on the concentration of Ca2+ in the medium. This shows the importance of organelle-resident SNARE functions (which may encompass the vesicular delivery of other organelle-resident proteins) for Ca2+ tolerance. A similar principle may be applicable also to the CVC in widely different unicellular organisms. In addition, in Paramecium, silencing particularly of Syx6 causes aberrant positioning of the CVC during de novo biogenesis before cytokinesis. [Copyright &y& Elsevier]

Abstract: Arsenic is highly toxic to living cells, especially skin, and skin cancer is induced by drinking water containing arsenic. The molecular mechanisms of arsenic-induced cancer, however, are not well understood. To examine the initial processes in the development of arsenic-induced cancer, we analyzed calcium signaling at an early stage of arsenic treatment of human primary cells and compared the effects with those observed with arsenic treatment in carcinoma-derived cells. We found that arsenic inhibited inositol trisphosphate receptor (IP3R) function in the endoplasmic reticulum by inducing phosphorylation, which led to decreased intracellular calcium levels. Blockade of IP3R phosphorylation by the serine/threonine protein kinase Akt inhibitor wortmannin rescued calcium signaling. In contrast, arsenic treatment of cells derived from a carcinoma (human squamous carcinoma; HSC-1) for 1h had no obvious effect. Taken together, these results suggest that arsenic-induced reduction in calcium signaling is one of the initial mechanisms underlying the malignant transformation in the development of skin cancer. [Copyright &y& Elsevier]

Abstract: The fluo family of indicators is frequently used in studying Ca2+ physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms—the acetoxymethyl (AM) ester and the K+ salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca2+ transients and fractional shortening kinetics. Loading the K+ salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns—suggesting differential loading into organelles. Ca2+ dissociation constants (K d,Ca), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K d,Ca (decrease in Ca2+ affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca2+ affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments. [Copyright &y& Elsevier]

Abstract: The spatiotemporal dynamics of elementary Ca2+ release events, such as “blips” and “puffs” shapes the hierarchal Ca2+ signaling in many cell types. Despite being the building blocks of Ca2+ patterning, the mechanism responsible for the observed properties of puffs, especially their termination is incompletely understood. In this paper, we employ a data-driven approach to gain insights into the complex dynamics of blips and puffs. We use a model of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) derived directly from single channel patch clamp data taken at 10μM concentration of IP3 to simulate calcium puffs. We first reproduce recent observations regarding puffs and blips and then investigate the mechanism of puff termination. Our model suggests that during a puff, IP3R s proceed around a loop through kinetic states from “rest” to “open” to “inhibited” and back to “rest”. A puff terminates because of self-inhibition. Based on our simulations, we rule out the endoplasmic reticulum (ER) Ca2+ depletion as a possible cause for puff termination. The data-driven approach also enables us to estimate the current through a single IP3R and the peak Ca2+ concentration near the channel pore. [Copyright &y& Elsevier]

Abstract: Every cell or neuronal type utilizes its own specific organization of its Ca2+ homeostasis depending on its specific function and its physiological needs. The magnocellular neurones, with their somata situated in the supraoptic and paraventricular nuclei of the hypothalamus and their nerve terminals populating the posterior hypophysis (neural lobe) are a typical and classical example of a neuroendocrine system, and an important experimental model for attempting to understand the characteristics of the neuronal organization of Ca2+ homeostasis. The magnocellular neurones synthesize, in a cell specific manner, two neurohormones: arginine-vasopressin (AVP) and oxytocin (OT), which can be released, in a strict Ca2+-dependent manner, both at the axonal terminals, in the neural lobe, and at the somatodendritic level. The two types of neurones show also distinct type of bioelectrical activity, associated with specific secretory patterns. In these neurones, the Ca2+ homeostatic pathways such as the Na+/Ca2+ exchanger (NCX), the endoplasmic reticulum (ER) Ca2+ pump, the plasmalemmal Ca2+ pump (PMCA) and the mitochondria are acting in a complementary fashion in clearing Ca2+ loads that follow neuronal stimulation. The somatodendritic AVP and OT release closely correlates with intracellular Ca2+ dynamics. More importantly, the ER Ca2+ stores play a major role in Ca2+ homeostatic mechanism in identified OT neurones. The balance between the Ca2+ homeostatic systems that are in the supraoptic neurones differ from those active in the terminals, in which mainly Ca2+ extrusion through the Ca2+ pump in the plasma membrane and uptake by mitochondria are active. In both AVP and OT nerve terminals, no functional ER Ca2+ stores can be evidenced experimentally. We conclude that the physiological significance of the complexity of Ca2+ homeostatic mechanisms in the somatodendritic region of supraoptic neurones and their terminals can be multifaceted, attributable, in major part, to their specialized electrical activity and Ca2+-dependent neurohormone release. [Copyright &y& Elsevier]

Abstract: Calcium ions and cyclic adenosine monophosphate (cAMP) are virtually ubiquitous intracellular signaling molecules in mammalian cells. This paper will focus on the cross-talk between Ca2+ and cAMP mobilizing signaling pathways and summarize the underlying molecular mechanisms. Subsequently, workings of adenohypophyseal corticotrope cells will be reviewed to highlight the physiological relevance of a Ca2+ cAMP interactions in neuroendocrinology. [Copyright &y& Elsevier]

Abstract: The secretion of adrenocorticotrophin (ACTH) from corticotropes is a key component in the endocrine response to stress. The resting potential of corticotropes is set by the basal activities of TWIK-related K+ (TREK)-1 channel. Corticotrophin-releasing hormone (CRH), the major ACTH secretagogue, closes the background TREK-1 channels via the cAMP-dependent pathway, resulting in depolarization and a sustained rise in cytosolic [Ca2+] ([Ca2+]i). By contrast, arginine vasopressin and norepinephrine evoke Ca2+ release from the inositol trisphosphate (IP3)-sensitive store, resulting in the activation of small conductance Ca2+-activated K+ channels and hyperpolarization. Following [Ca2+]i rise, cytosolic Ca2+ is taken into the mitochondria via the uniporter. Mitochondrial inhibition slows the decay of the Ca2+ signal and enhances the depolarization-triggered exocytotic response. Both voltage-gated Ca2+ channel activation and intracellular Ca2+ release generate spatial Ca2+ gradients near the exocytic sites such that the local [Ca2+] is ∼3-fold higher than the average [Ca2+]i. The stimulation of mitochondrial metabolism during the agonist-induced Ca2+ signal and the robust endocytosis following stimulated exocytosis enable corticotropes to maintain sustained secretion during the diurnal ACTH surge. Arachidonic acid (AA) which is generated during CRH stimulation activates TREK-1 channels and causes hyperpolarization. Thus, corticotropes may regulate ACTH release via an autocrine feedback mechanism. [Copyright &y& Elsevier]

Abstract: Endocrine pituitary cells express numerous voltage-gated Na+, Ca2+, K+, and Cl− channels and several ligand-gated channels, and they fire action potentials spontaneously. Depending on the cell type, this electrical activity can generate localized or global Ca2+ signals, the latter reaching the threshold for stimulus–secretion coupling. These cells also express numerous G-protein-coupled receptors, which can stimulate or silence electrical activity and Ca2+ influx through voltage-gated Ca2+ channels and hormone release. Receptors positively coupled to the adenylyl cyclase signaling pathway stimulate electrical activity with cAMP, which activates hyperpolarization-activated cyclic nucleotide-regulated channels directly, or by cAMP-dependent kinase-mediated phosphorylation of K+, Na+, Ca2+, and/or non-selective cation-conducting channels. Receptors that are negatively coupled to adenylyl cyclase signaling pathways inhibit spontaneous electrical activity and accompanied Ca2+ transients predominantly through the activation of inwardly rectifying K+ channels and the inhibition of voltage-gated Ca2+ channels. The Ca2+-mobilizing receptors activate inositol trisphosphate-gated Ca2+ channels in the endoplasmic reticulum, leading to Ca2+ release in an oscillatory or non-oscillatory manner, depending on the cell type. This Ca2+ release causes a cell type-specific modulation of electrical activity and intracellular Ca2+ handling. [Copyright &y& Elsevier]

Abstract: This special issue on Ca2+ signalling in neuroendocrine cells is an opportunity to assess, through a range of first-class review articles, the complex world of endocrine signalling, a complexity that is probably best captured by calling it “diversity in unity”. The unity comes from the fact that all the endocrine cells are excitable cells, able to generate action potentials and are using Ca2+ as an essential informational molecule, coupling cell stimulation with the activation of secretion, through the exocytotic process. The ‘diversity’ element, illustrated by almost all the reviews, stems from the modalities employed to achieve the increase in cytosolic Ca2+ signal, the balance between the participation of Ca2+ entry through the plasma membrane voltage-operated Ca2+ channels and the release of Ca2+ from intracellular Ca2+ stores, and the cross-talk between the Ca2+ and cyclic AMP signalling pathways. [Copyright &y& Elsevier]

Abstract: In rat pancreatic β cells, arachidonic acid (AA) triggered intracellular Ca2+ release. This effect could be mimicked by eicosatetraynoic acid, indicating that AA metabolism is not required. The AA-mediated Ca2+ signal was not affected by inhibition of ryanodine receptors or emptying of ryanodine-sensitive store but was reduced by ∼70% following the disruption of acidic stores (treatment with bafilomycin A1 or glycyl-phenylalanyl-β-naphthylamide (GPN)). The action of AA did not involve TRPM2 channels or NAADP receptors because intracellular dialysis of adenosine diphosphoribose (ADPR; an activator of TRPM2 channels) or NAADP did not affect the AA response. In contrast, stimulation of IP3 receptors via intracellular dialysis of adenophostin A, or exogenous application of ATP largely abolished the AA-mediated Ca2+ signal. Intracellular dialysis of heparin abolished the ATP-mediated Ca2+ signal but not the AA response, suggesting that the action of AA did not involve the IP3-binding site. Treatment with the SERCA pump inhibitor, thapsigargin, reduced the amplitude of the AA-mediated Ca2+ signal by ∼70%. Overall, our finding suggests that AA mobilizes Ca2+ from the endoplasmic reticulum as well as an acidic store and both stores could be depleted by IP3 receptor agonist. The possibility of secretory granules as targets of AA is discussed. [Copyright &y& Elsevier]

Abstract: Alzheimer''s disease (AD) is a progressive neurodegenerative disorder that leads to debilitating cognitive deficits. Recent evidence demonstrates that glutamate receptors are dysregulated by amyloid beta peptide (Aβ) oligomers, resulting in disruption of glutamatergic synaptic transmission which parallels early cognitive deficits. Although it is well accepted that neuronal death in AD is related to disturbed intracellular Ca2+ (Ca2+ i) homeostasis, little is known about the contribution of NMDARs containing GluN2A or GluN2B subunits on Aβ-induced Ca2+ i rise and neuronal dysfunction. Thus, the main goal of this work was to evaluate the role of NMDAR subunits in dysregulation of Ca2+ i homeostasis induced by Aβ 1–42 preparation containing both oligomers (in higher percentage) and monomers in rat cerebral cortical neurons. The involvement of NMDARs was evaluated by pharmacological inhibition with MK-801 or the selective GluN2A and GLUN2B subunit antagonists NVP-AAM077 and ifenprodil, respectively. We show that Aβ, like NMDA, increase Ca2+ i levels mainly through activation of NMDARs containing GluN2B subunits. Conversely, GluN2A-NMDARs antagonism potentiates Ca2+ i rise induced by a high concentration of Aβ (1μM), suggesting that GluN2A and GluN2B subunits have opposite roles in regulating Ca2+ i homeostasis. Moreover, Aβ modulate NMDA-induced responses and vice versa. Indeed, pre-exposure to Aβ (1μM) decrease NMDA-evoked Ca2+ I rise and pre-exposure to NMDA decrease Aβ response. Interestingly, simultaneous addition of Aβ and NMDA potentiate Ca2+ I levels, this effect being regulated by GluN2A and GluN2B subunits in opposite manners. This study contributes to the understanding of the molecular basis of early AD pathogenesis, by exploring the role of GluN2A and GluN2B subunits in the mechanism of Aβ toxicity in AD. [Copyright &y& Elsevier]

Abstract: Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H+-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H+-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H+-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca2+-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H+-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H+-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP3. Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H+-ATPase activation. [Copyright &y& Elsevier]

Abstract: In various cell types, depletion of intracellular Ca2+-stores results in store-operated Ca2+-entry (SOCE) across the cellular membrane. However, the effects of SOCE on neuronal membrane excitability and mitochondrial functions in central neurons are not well defined. We investigated such cellular downstream effects in pyramidal neurons of rat organotypic hippocampal slice cultures by applying electrophysiological and fluorescence imaging techniques. We report that SOCE is associated with (i) elevations of Ca2+-concentration in individual neuronal mitochondria ([Ca2+]m). In addition, SOCE can result in (ii) hyperpolarizing neuronal membrane currents, (iii) increase in extracellular K+-concentration ([K+]o), (iv) mitochondrial membrane depolarization, and (v) changes in intracellular redox state (NAD(P)H and FAD fluorescence), the latter reflecting responses of energy metabolism. These additional downstream effects of SOCE required concomitant muscarinic receptor activation by carbachol or acetylcholine, and were suppressed by agonist washout or application of antagonist, atropine. We conclude that muscarinic receptor activation determines the downstream effects of SOCE on neuronal membrane excitability and energy metabolism. This mechanism might have significant impact on information processing and neurometabolic coupling in central neurons. [Copyright &y& Elsevier]

Abstract: Voltage-gated calcium (CaV) channels are transmembrane proteins that form Ca2+-selective pores gated by depolarization and are essential regulators of the intracellular Ca2+ concentration. By providing a pathway for rapid Ca2+ influx, CaV channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. CaV channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α1 subunit, which typically contains associated β and α2δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α2δ-1 is post-translationally cleaved into an extracellular α2 polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α2δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α2) and Cys1047 (in the extracellular domain of δ) prevented the association of the α2 and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α2δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (CaV2.2α1/β3) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α2δ-1 function. [Copyright &y& Elsevier]

Abstract: Normal rat kidney (NRK) fibroblasts exhibit growth-dependent changes in electrophysiological properties and intracellular calcium dynamics. The transition from a quiescent state to a density-arrested state results in altered calcium entry characteristics. This coincides with modulation of the expression of the genes encoding the calcium channels Trpc1, Trpc6 and Orai1, and of the intracellular calcium sensor Stim1. In the present study we have used gene selective short hairpin (sh) RNAs against these various genes to investigate their role in (a) capacitative store-operated calcium entry (SOCE); (b) non-capacitative OAG-induced receptor-operated calcium entry (ROCE); and (c) prostaglandin F2α (PGF2α)-induced Ca2+-oscillations in NRK fibroblasts. Intracellular calcium measurements revealed that knockdown of the genes encoding Trpc1, Orai1 and Stim1 each caused a significant reduction of SOCE in NRK cells, whereas knockdown of the gene encoding Trpc6 reduced only the OAG-induced ROCE. Furthermore, our data show that knockdown of the genes encoding Trpc1, Orai1 and Stim1, but not Trpc6, substantially reduced the frequency (up to 60%) of PGF2α-induced Ca2+ oscillations in NRK cells. These results indicate that in NRK cells distinct calcium channels control the processes of SOCE, ROCE and PGF2α-induced Ca2+ oscillations. [Copyright &y& Elsevier]

Abstract: Calcimycin (A23187) is an ionophore widely used in studies related to calcium dynamics in cells, but its fluorometric potential to reveal intracellular physiology has not been explored. Exploiting the microenvironment-induced changes in its fluorescence, we show that a brief exposure of cells to non-toxic concentrations (≤3μM) of the ionophore results in the characteristic organization of the ionophore forming brightly fluorescent cytoplasmic bodies termed “I-Bodies”, which are closely related to stress linked disturbances/changes in calcium homeostasis. “I-Bodies” appear to be Ca2+ rich intracellular sites formed during stress-induced release of intracellular Ca2+, causing dysfunction and aggregation of mitochondria, providing scaffold for high density packing of A23187. Formation of “I-Bodies” in cells exposed to ionizing radiation and certain anticancer drugs suggest their potential in revealing alterations in calcium signaling and mitochondrial function during (related to) macromolecular damage-induced cell death. The absence of “I-Bodies” in non-malignant cells and their varying numbers in malignant cells with 5 fold increase in fluorescence imply that they can be potential biomarkers of cancer. Thus, “I-Bodies” are novel indicators of endogenous and induced stress linked to disturbances in calcium homeostasis in cells, with a potential to serve as biomarker of cancer. [Copyright &y& Elsevier]

Abstract: Albumin, which is the most abundant component of urine proteins, exerts injurious effects on renal cells in chronic kidney diseases. However, the toxicity of albumin to podocytes is not well elucidated. Here, we show that a high concentration of albumin triggers intracellular calcium ([Ca2+]i) increase through mechanisms involving the intracellular calcium store release and extracellular calcium influx in conditionally immortalized podocytes. The canonical transient receptor potential-6 (TRPC6) channel, which is associated with a subset of familial forms of focal segmental glomerulosclerosis (FSGS) and several acquired proteinuric kidney diseases, was shown to be one of the important Ca2+ permeable ion channels in podocytes. Therefore we explored the role of TRPC6 on albumin-induced functional and structural changes in podocytes. It was found that albumin-induced increase in [Ca2+]i was blocked by TRPC6 siRNA or SKF-96365, a blocker of TRP cation channels. Long-term albumin exposure caused an up-regulation of TRPC6 expression in podocytes, which was inhibited by TRPC6 siRNA. Additionally, the inhibition of TRPC6 prevented the F-actin cytoskeleton disruption that is induced by albumin overload. Moreover, albumin overload induced expression of the endoplasmic reticulum (ER) stress protein GRP78, led to caspase-12 activation and ultimately podocyte apoptosis, all of which were abolished by the knockdown of TRPC6 using TRPC6 siRNA. These results support the view that albumin overload may induce ER stress and the subsequent apoptosis in podocytes via TRPC6-mediated Ca2+ entry. [Copyright &y& Elsevier]

Abstract: The appropriate regulation of intracellular calcium is a requirement for proper cell function and survival. This review focuses on the effects of proinflammatory cytokines on calcium regulation in the insulin-producing pancreatic beta-cell and how normal stimulus-secretion coupling, organelle function, and overall beta-cell viability are impacted. Proinflammatory cytokines are increasingly thought to contribute to beta-cell dysfunction not only in type 1 diabetes (T1D), but also in the progression of type 2 diabetes (T2D). Cytokine-induced disruptions in calcium handling result in reduced insulin release in response to glucose stimulation. Cytokines can alter intracellular calcium levels by depleting calcium from the endoplasmic reticulum (ER) and by increasing calcium influx from the extracellular space. Depleting ER calcium leads to protein misfolding and activation of the ER stress response. Disrupting intracellular calcium may also affect organelles, including the mitochondria and the nucleus. As a chronic condition, cytokine-induced calcium disruptions may lead to beta-cell death in T1D and T2D, although possible protective effects are also discussed. Calcium is thus central to both normal and pathological cell processes. Because the tight regulation of intracellular calcium is crucial to homeostasis, measuring the dynamics of calcium may serve as a good indicator of overall beta-cell function. [Copyright &y& Elsevier]

Abstract: Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouse lenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on the intracellular Ca2+ ([Ca2+]i) level. We have found that these cultures express specific cellular markers for lens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thus recapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-B receptors (GABAAR and GABABR) with the specific agonists muscimol and baclofen, respectively induces [Ca2+]i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and were dependent on extracellular Ca2+. Bicuculline did not change the GABA-evoked Ca2+ responses in Ca2-containing buffers, but suppressed them significantly in Ca2+-free buffers suggesting the two receptors couple to convergent Ca2+ mobilization mechanisms with different extracellular Ca2+ sensitivity. Prolonged activation of GABABR induced wave propagation of the Ca2+ signal and persistent oscillations. The number of cells reacting to GABA or GABA+bicuculline in P30 mouse LEC cultures expressing predominantly the synaptic type GABAAR did not differ significantly from the number of reacting cells in P0 mouse LEC cultures. The GABA-induced Ca2+ transients in P30 (but not P0) mouse LEC could be entirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca2+ signaling may be involved in a variety of Ca2+-dependent cellular processes during lens growth and epithelial cell differentiation. [Copyright &y& Elsevier]

Abstract: Physiological platelet activation and thrombus formation are essential to stop bleeding in case of vascular injury, whereas inadequate triggering of the same process in diseased vessels can lead to fatal thromboembolism and tissue ischemia of vital organs. A central step in platelet activation is agonist-induced elevation of the intracellular Ca2+ concentration. This happens on the one hand through the release of Ca2+ from intracellular stores and on the other hand through Ca2+ influx from the extracellular space. In platelets, the major Ca2+ influx pathway is the so-called store operated Ca2+ entry (SOCE), induced by store depletion. Studies in the last five years discovered the molecular background of platelet SOCE. Stromal interaction molecule 1 (STIM1) and Orai1, two so far unknown molecules, got in the focus of research. STIM1 was found to be the Ca2+ sensor in the endoplasmic reticulum (ER) membrane, whereas Orai1 was identified as the major store operated Ca2+ (SOC) channel in the plasma membrane. These two molecules and their role in platelet function and thrombus formation are the topic of the present review with a special focus on apoptosis and apoptosis-like processes in platelet physiology. [Copyright &y& Elsevier]

Abstract: Bax inhibitor-1 (BI-1) was initially identified for its ability to inhibit BAX-induced apoptosis in yeast cells and is the founding member of a family of highly hydrophobic proteins localized in diverse cellular membranes. It is evolutionarily conserved and orthologues from plants can substitute for mammalian BI-1 in regard to its anti-apoptotic function suggesting a high degree of functional conservation. BI-1 interacts with BCL-2 and BCL-XL and, similar to these two anti-apoptotic proteins, the effect of BI-1 on cell death involves changes in the amount of Ca2+ releasable from intracellular stores. However, BI-1 is also a negative regulator of the endoplasmic reticulum stress sensor IRE1 α, it interacts with G-actin and increases actin polymerization, enhances cancer metastasis by altering glucose metabolism and activating the sodium-hydrogen exchanger, and reduces the production of reactive oxygen species through direct interaction with NADPH-P450 reductase. In this contribution, we summarize what is known about the expression, intracellular localization and structure of BI-1 and specifically illuminate its effects on the intracellular Ca2+ homeostasis and how this might relate to its other functions. We also present a thorough phylogenetic analysis of BI-1 proteins from major phyla together with paralogues from all BI-1 family members. [Copyright &y& Elsevier]

Abstract: Autophagy is a cellular process responsible for delivery of proteins or organelles to lysosomes. It participates not only in maintaining cellular homeostasis, but also in promoting survival during cellular stress situations. It is now well established that intracellular Ca2+ is one of the regulators of autophagy. However, this control of autophagy by intracellular Ca2+ signaling is the subject of two opposite views. On the one hand, the available evidence indicates that intracellular Ca2+ signals, and mainly inositol 1,4,5-trisphosphate receptors (IP3Rs), suppress autophagy. On the other hand, elevated cytosolic Ca2+ concentrations ([Ca2+]cyt) were also shown to promote the autophagic process. Here, we will provide a critical overview of the literature and discuss both hypotheses. Moreover, we will suggest a model explaining how changes in intracellular Ca2+ signaling can lead to opposite outcomes, depending on the cellular state. [Copyright &y& Elsevier]

Abstract: Research during the past several decades has provided convincing evidence for a crucial role of the Ca2+ ion in cell signaling. Hence, intracellular Ca2+ transients have been implicated in most aspects of cell physiology, including gene transcription, cell cycle regulation and cell proliferation. Further, the Ca2+ ion has been found to also play an important role in cell death regulation. Thus, necrotic cell death was early associated with intracellular Ca2+ overload, and multiple functions in the apoptotic process have subsequently been found to be governed by Ca2+ signaling. More recently, other modes of cell death, notably anoikis and autophagic cell death, have been demonstrated to also be modulated by Ca2+ transients. Characteristics, interrelationship and mechanisms involved in Ca2+ regulation of these cell death modalities are discussed in this review. [Copyright &y& Elsevier]

Abstract: Ischemia/reperfusion (I/R) damage in the heart occurs mainly during the first minutes of reperfusion. Urocortin (Ucn) is a member of the corticotrophin-releasing factor that has been identified as a potent endogenous cardioprotector peptide when used in pre- and postconditioning protocols. However, the underlying mechanisms are not completely elucidated. Here, we focused on intracellular calcium ([Ca2+]i) handling by Ucn when applied in early reperfusion. We used Langendorff-perfused rat hearts to determine hemodynamic parameters, and confocal microscopy to study global [Ca2+]i transients evoked by electrical stimulation in isolated cardiomyocytes loaded with fluorescence Ca2+ dye fluo-3AM. We found that the acute application of Ucn at the onset of reperfusion, in isolated hearts submitted to ischemia, fully recovered the hearts contractility and relaxation. In isolated cardiac myocytes, following ischemia we observed that the diastolic [Ca2+]i was increased, the systolic [Ca2+]i transients amplitude were depressed and sarcoplasmic reticulum (SR) Ca2+ load was reduced. These effects were correlated to a decrease in the Na+/Ca2+ exchanger (NCX) activity. Importantly, Ucn applied at reperfusion produced a complete recovery in diastolic [Ca2+]i and global [Ca2+]i transient amplitude, which were due to NCX activity improvement. In conclusion, we demonstrated that [Ca2+]i handling play an essential role in postconditioning action of Ucn. [Copyright &y& Elsevier]

Abstract: A change in intracellular free calcium is a common signaling mechanism that modulates a wide array of physiological processes in most cells. Responses to increased intracellular Ca2+ are often mediated by the ubiquitous protein calmodulin (CaM) that upon binding Ca2+ can interact with and alter the functionality of numerous proteins including a family of protein kinases referred to as CaM-kinases (CaMKs). Of particular interest are multifunctional CaMKs, such as CaMKI, CaMKII, CaMKIV and CaMKK, that can phosphorylate multiple downstream targets. This review will outline several protocols we have used to identify which members and/or isoforms of this CaMK family mediate specific cellular responses with a focus on studies in neurons. Many previous studies have relied on a single approach such as pharmacological inhibitors or transfected dominant-negative kinase constructs. Since each of these protocols has its limitations, that will be discussed, we emphasize the necessity to use multiple, independent approaches in mapping out cellular signaling pathways. [Copyright &y& Elsevier]

Abstract: Synaptosomal expression of NCX1, NCX2, and NCX3, the three variants of the Na+–Ca2+ exchanger (NCX), was investigated in Alzheimer''s disease parietal cortex. Flow cytometry and immunoblotting techniques were used to analyze synaptosomes prepared from cryopreserved brain of cognitively normal aged controls and late stage Alzheimer''s disease patients. Major findings that emerged from this study are: (1) NCX1 was the most abundant NCX isoform in nerve terminals of cognitively normal patients; (2) NCX2 and NCX3 protein levels were modulated in parietal cortex of late stage Alzheimer''s disease: NCX2 positive terminals were increased in the Alzheimer''s disease cohort while counts of NCX3 positive terminals were reduced; (3) NCX1, NCX2 and NCX3 isoforms co-localized with amyloid-beta in synaptic terminals and all three variants are up-regulated in nerve terminals containing amyloid-beta. Taken together, these data indicate that NCX isoforms are selectively regulated in pathological terminals, suggesting different roles of each NCX isoform in Alzheimer''s disease terminals. [Copyright &y& Elsevier]

Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the molecular mechanisms whereby these mutations induce motor neuron death remain controversial. Here, we show that stable overexpression of mutant human SOD1 (G37R) – but not wild-type SOD1 (wt-SOD1) – in mouse neuroblastoma cells (N2a) results in morphological abnormalities of mitochondria accompanied by several dysfunctions. Activity of the oxidative phosphorylation complex I was significantly reduced in G37R cells and correlated with lower mitochondrial membrane potential and reduced levels of cytosolic ATP. Using targeted chimeric aequorin we further analyzed the consequences of mitochondrial dysfunction on cellular Ca2+ handling. Mitochondrial Ca2+ uptake, elicited by IP3-induced Ca2+ release from endoplasmic reticulum (ER) was significantly reduced in G37R cells, while uptake induced by a brief Ca2+ pulse was not affected in permeabilized cells. The decreased mitochondrial Ca2+ uptake resulted in increased cytosolic Ca2+ transients, whereas ER Ca2+ load and resting cytosolic Ca2+ levels were not affected. Together, these findings suggest that the mechanism linking mutant G37R SOD1 and ALS involves mitochondrial respiratory chain deficiency resulting in ATP loss and impairment of mitochondrial and cytosolic Ca2+ homeostasis. [Copyright &y& Elsevier]

Abstract: Ubiquitin-proteasome system has emerged a central player in regulation of diverse cellular processes. However, relevance of proteasome activity in platelets, which are terminally differentiated enucleate cells, is not clear. In this report we show that activation of platelets with physiological agonists was associated with 7–10 -fold rise in proteasomal activity. Elevation of cytosolic calcium with A23187 or thapsigargin resulted in significant increase in enzymatic activity, while treatment with intracellular calcium chelator or inhibitor of inositol trisphosphate receptor attenuated proteasomal enzymes in collagen-stimulated platelets. Specific inhibitors of protein kinase C as well as calpain, too, downregulated proteasome function. To conclude, proteasomal enzymatic activity in platelets is regulated by cytosolic calcium through Ca2+-dependent downstream effectors like calpain and protein kinase C. [Copyright &y& Elsevier]

Abstract: It is well established that astrocytes release gliotransmitters and moderate neuronal activity in the central nervous system via intracellular Ca2+ dynamics. Astrocytic Ca2+ oscillations are one type of spontaneous Ca2+ mobilization that occurs in astrocytes. However, the modulation of spontaneous astrocytic Ca2+ oscillations, especially in pathophysiological conditions, is not yet fully understood. Here, we demonstrate that activation of adenosine receptors induces a long-lasting increase in the frequency of astrocytic Ca2+ oscillations in rat hippocampal slice cultures. The long-term facilitation of the frequency of Ca2+ oscillations was mediated by endogenous adenosine generated via breakdown of extracellular ATP by ecto-ATPase. We also demonstrate that local tissue injury with ultraviolet irradiation can cause this long-term facilitation of Ca2+ oscillations via endogenous adenosine. Our data suggest that endogenous adenosine is one of the modulators of spontaneous astrocytic Ca2+ oscillations in the rat hippocampus, and may play a significant role in altered Ca2+ dynamics in astrocytes observed during pathophysiological conditions. [Copyright &y& Elsevier]

Abstract: Amyloid beta (Aβ) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which Aβ oligomers cause neurotoxicity remain unknown. We recently reported that Aβ oligomers cause intracellular Ca2+ overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether Aβ oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, Aβ oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to Aβ oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca2+ induced by Aβ oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that Aβ oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent Aβ damage to neurons in Alzheimeŕs disease. [Copyright &y& Elsevier]

Abstract: Inositol 1,4,5-trisphosphate (IP3) receptor is a central unit in intracellular Ca2+ signaling. Regulation of the IP3 receptor by calcium is well characterized. High open probability values are reported for a single IP3 receptor in nuclear patch clamp experiments. These experimental observations are in contrast with the lower open probability values of the lipid bilayer experiments. Most theoretical models do not account for high open probabilities of the receptor. But more recently, new models of the IP3 receptor have been put forward which are constrained by single-channel nuclear patch clamp recordings, which generate the larger open probability with the aid of an additional agonist-independent conformational transformation (AICT)—‘active’ state. The main aim of this work is to constrain the AICT models with a wealth of experimental data characterizing calcium release from IP3 receptor clusters. Our results suggest that consistency of cluster release between theory and experiments constrains the kinetics of the agonist-independent conformational transition rates (AICT) to values which lead to small open probabilities for the IP3 receptor inconsistent with nuclear patch clamp experimental data. [Copyright &y& Elsevier]

Abstract: Cross-talk between subcellular organelles is essential for cellular Ca2+ homeostasis. We have studied the effects of knocking down STIM1, the Ca2+ sensor of the endoplasmic reticulum (ER), on several homeostatic Ca2+-handling mechanisms, including plasma membrane Ca2+ entry and transport by ER, mitochondria and nucleus. We have used targeted aequorins to selectively measure calcium fluxes in different organelles. Actions of STIM1 were extremely selective, restricted to store operated Ca2+ channels (SOC) and Ca2+ uptake by the ER. No interactions with uptake or release of Ca2+ by mitochondria or nucleus were detected. Ca2+ exit from the ER, including passive leak, release via inositol 1,4,5-trisphosphate and ryanodine receptors, was unaffected. STIM1 knock-down inhibited ER Ca2+ uptake in intact but not in permeabilized cells, suggesting a privileged calcium entry-calcium refilling (CECR) coupling between plasma membrane SOC and ER calcium pump in the intact cell. As a result a large part of the entering Ca2+ is taken up into the ER without reaching the bulk cytosol. The tightness of CECR, as measured by the slope of the stimulus-signal strength function, was comparable to classic excitation–response coupling mechanisms, such as excitation–contraction, excitation–secretion or excitation–transcription coupling. [Copyright &y& Elsevier]

Abstract: LH increases the intracellular Ca2+ concentration ([Ca2+]i) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca2+ channels. Here we show that LH modulates both T-type Ca2+ currents and [Ca2+]i transients through the effects of PKA and PKC. LH increases the peak calcium current (at −20mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca2+]i changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca2+]i the subsequent addition of LH still triggered the typical transients in [Ca2+]i. Chelerythrine also does not avoid the Ca2+ transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca2+]i rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH. [Copyright &y& Elsevier]

Abstract: We show that lobster olfactory receptor neurons (ORNs), much like their vertebrate counterparts, generate a transient elevation of intracellular calcium (Cai) in response to odorant activation that can be used to monitor ensemble ORN activity. This is done in antennal slice preparation in situ maintaining the polarity of the cells and the normal micro-environment of the olfactory cilia. The Cai signal is ligand-specific and increases in a dose-dependent manner in response to odorant stimulation. Saturating stimulation elicits a robust increase of up to 1μM free Cai within 1–2s of stimulation. The odor-induced Cai response closely follows the discharge pattern of extracellular spikes elicited by odorant application, with the maximal rise in Cai matching the peak of the spike generation. The Cai signal can be used to track neuronal activity in a functional subpopulation of rhythmically active ORNs and discriminate it from that of neighboring tonically active ORNs. Being able to record from many ORNs simultaneously over an extended period of time not only allows more accurate estimates of neuronal population activity but also dramatically improves the ability to identify potential new functional subpopulations of ORNs, especially those with more subtle differences in responsiveness, ligand specificity, and/or transduction mechanisms. [Copyright &y& Elsevier]

Abstract: Disruptions of mitochondrial dynamics have been implicated in the pathogenesis of neurodegenerative diseases. The regulation mechanisms of mitochondrial dynamics have not been fully elucidated; however, calcium has been suggested to play a role. In the present study, we examined the role of intracellular calcium in regulating mitochondrial morphology and motility in cortical astrocytes employing different concentrations of a calcium ionophore. High levels of calcium caused a dramatic reduction in mitochondrial length, the result of two distinct phenomena: mitochondrial remodelling (or “rounding”) and fission. Quantitative analysis revealed that mitochondrial remodelling/rounding was the predominant process. In addition, mitochondrial motility was reduced, as reported previously in neurons. By contrast, prolonged, more modest levels of intracellular calcium resulted in a reduction in mitochondrial length without significant effects upon mitochondrial motility. This calcium-induced reduction in mitochondrial length was not affected by the presence of calcineurin inhibitors; however, when mitochondrial fission events were specifically examined, calcineurin inhibitors had a significant inhibitory effect. This suggests that changes in mitochondrial length were primarily due to mitochondrial remodelling as opposed to fission. In the present study, we have therefore dissected the effects of calcium on mitochondrial motility, remodelling and fission. Our results suggest independent mechanisms for regulating these processes. [Copyright &y& Elsevier]

Abstract: Cyclic AMP regulates the late step of Ca2+-dependent exocytosis in many secretory cells through two major mechanisms: a protein kinase A-dependent and a cAMP-GEF/Epac-dependent pathway. We designed a protocol to characterize the role of these two cAMP-dependent pathways on the Ca2+ sensitivity and kinetics of regulated exocytosis in mouse pancreatic beta cells, using a whole-cell patch-clamp based capacitance measurements. A train of depolarizing pulses or slow photo-release of caged Ca2+ were stimuli for the exocytotic activity. In controls, due to exocytosis after slow photo-release, the C m change had typically two phases. We observed that the Ca2+-dependency of the rate of the first C m change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9±0.2μM. The intracellular depletion of cAMP did not change amp1, while rate1 and amp2 were strongly reduced. This manipulation pushed the Ca2+-dependency of the exocytotic burst to significantly lower [Ca2+]i. To address the question of which of the cAMP-dependent mechanisms regulates the observed shifts in Ca2+ dependency we included regulators of PKA and Epac2 activity in the pipette solution. PKA activation with 100μM 6-Phe-cAMP or inhibition with 500μM Rp-cAMPs in beta cells significantly shifted the EC50 in the opposite directions. Specific activation of Epac2 did not change Ca2+ sensitivity. Our findings suggest that cAMP modulates Ca2+-dependent exocytosis in mouse beta cells mainly through a PKA-dependent mechanism by sensitizing the insulin releasing machinery to [Ca2+]i; Epac2 may contribute to enhance the rates of secretory vesicle fusion. [Copyright &y& Elsevier]

Abstract: Elevations in reactive oxygen species are implicated in many disease states and cause systolic and diastolic myocardial dysfunction. To understand the underlying cellular dysfunction, we characterised the effects of H2O2 on [Ca2+]i handling and contractility in the rat ventricular myocyte. This was achieved using patch clamping, [Ca2+]i measurement using Fluo-3, video edge detection and confocal microscopy. All experiments were performed at 37°C. 200μM H2O2 resulted in a 44% decrease in the [Ca2+]i transient amplitude, a 30% increase in diastolic [Ca2+]i and an 18% decrease in the rate of systolic Ca2+ removal. This was associated with a 61% reduction in systolic shortening, a contracture of 3μm and a 42% increase in relaxation time respectively. The decrease in the [Ca2+]i transient amplitude could be explained by a 27% decrease in SR Ca2+ content. This, in turn results from a 22% decrease of SERCA activity. The decreased SR Ca2+ content also provides a mechanism for a reduction in [Ca2+]i spark frequency with no evidence for a Ca2+ independent modification of ryanodine receptor open probability. We conclude that decreased SERCA activity is the major factor responsible for the changes of the systolic [Ca2+]i transient. [ABSTRACT FROM AUTHOR]

Abstract: The spatial organisation of the Ca2+ signal in microdomains enables the regulation of various processes in specific regions of the cell and is essential for the versatility of cell responses to various stimuli. Ca2+ signals can be independently regulated in the cytoplasm and in the nucleoplasm. Increases in the concentration of Ca2+ in the nucleus can have specific effects different from those due to increases of Ca2+ in the cytoplasm. We investigated the influence of cell polarity on the subcellular distribution of molecules responsible for intracellular Ca2+ homeostasis (Ca2+ release channels, Ca2+ pumps and Ca2+ binding proteins) and its influence on the intracellular Ca2+ signal in MDCK cells with respect to its cytoplasmic or nucleoplasmic localisation. The intracellular Ca2+ store was largely reorganised during cell polarisation, with a differential redistribution of IP3R, Ca2+-binding proteins and SERCA between the nuclear envelope and the periphery of the cell. This was accompanied by morphological changes in cell shape, which condense the cytoplasm around the nucleus, and in the shape of the nucleus, resulting in invaginations of the nuclear envelope. This facilitates Ca2+ exchanges between the cytoplasm and the nucleoplasm, and preserves the ability to generate nucleoplasmic Ca2+ transients in agonist-stimulated polarised MDCK cells. [ABSTRACT FROM AUTHOR]

Abstract: The pulmonary vein is surrounded by an external sleeve of cardiomyocytes that are widely recognised to play an important role in atrial fibrillation. While intracellular Ca2+ is thought to influence the electrical activity of cardiomyocytes, there have been relatively few studies examining Ca2+ signalling in these cells. Therefore, using fluo-4 and fluorescence imaging microscopy, we have investigated Ca2+ signalling in an intact section of the rat pulmonary vein. Under resting conditions cardiomyocytes displayed spontaneous Ca2+ transients, which were variable in amplitude and had a frequency of 1.6±0.03Hz. The Ca2+ transients were asynchronous amongst neighbouring cardiomyocytes and tended to propagate throughout the cell as a wave. Removing extracellular Ca2+ produced a slight reduction in the amplitude and frequency of the spontaneous Ca2+ transients; however, ryanodine (20μM) had a much greater effect on the amplitude and reduced the frequency by 94±2%. Blocking IP3 receptors with 2-aminoethoxydiphenyl borate (20μM) also reduced the amplitude and frequency (by 73±11%) of these events, indicating the importance of Ca2+ release from the SR. Electrical field stimulation of the pulmonary vein produced Ca2+ transients in cardiomyocytes that were significantly reduced by either voltage-gated Ca2+ channel blockers or ryanodine. [ABSTRACT FROM AUTHOR]

Abstract: In obesity and the early stages of type 2 diabetes (T2D), proinflammatory cytokines are mildly elevated in the systemic circulation. This low-grade systemic inflammation exposes pancreatic islets to these circulating cytokines at much lower levels than seen within the islet during insulitis. These low-dose effects have not been well described. We examined mouse islets treated overnight with a low-dose cytokine combination commonly associated with inflammation (TNF-alpha, IL-1 beta, and IFN-gamma). We then examined islet function primarily using intracellular calcium ([Ca2+] i ), a key component of insulin secretion and cytokine signaling. Cytokine-treated islets demonstrated several features that suggested dysfunction including excess [Ca2+] i in low physiological glucose (3mM), reduced responses to glucose stimulation, and disrupted [Ca2+] i oscillations. Interestingly, islets taken from young db/db mice showed similar disruptions in [Ca2+] i dynamics as cytokine-treated islets. Additional studies of control islets showed that the cytokine-induced elevation in basal [Ca2+] i was due to both greater calcium influx through L-type-calcium-channels and reduced endoplasmic reticulum (ER) calcium storage. Many of these cytokine-induced disruptions could be reproduced by SERCA blockade. Our data suggest that chronic low-grade inflammation produces circulating cytokine levels that are sufficient to induce beta-cell dysfunction and may play a contributing role in beta-cell failure in early T2D. [ABSTRACT FROM AUTHOR]