NCX-mediated Ca2 Research Articles

Structural dynamics of Na+ and Ca2+ interactions with full-size mammalian NCX

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

Spontaneous Ca2+ Fluctuations Arise in Thin Astrocytic Processes With Real 3D Geometry.

Fluctuations of cytosolic Ca2+ concentration in astrocytes are regarded as a critical non-neuronal signal to regulate neuronal functions. Although such fluctuations can be evoked by neuronal activity, rhythmic astrocytic Ca2+ oscillations may also spontaneously arise. Experimental studies hint that these spontaneous astrocytic Ca2+ oscillations may lie behind different kinds of emerging neuronal synchronized activities, like epileptogenic bursts or slow-wave rhythms. Despite the potential importance of spontaneous Ca2+ oscillations in astrocytes, the mechanism by which they develop is poorly understood. Using simple 3D synapse models and kinetic data of astrocytic Glu transporters (EAATs) and the Na+/Ca2+ exchanger (NCX), we have previously shown that NCX activity alone can generate markedly stable, spontaneous Ca2+ oscillation in the astrocytic leaflet microdomain. Here, we extend that model by incorporating experimentally determined real 3D geometries of 208 excitatory synapses reconstructed from publicly available ultra-resolution electron microscopy datasets. Our simulations predict that the surface/volume ratio (SVR) of peri-synaptic astrocytic processes prominently dictates whether NCX-mediated spontaneous Ca2+ oscillations emerge. We also show that increased levels of intracellular astrocytic Na+ concentration facilitate the appearance of Ca2+ fluctuations. These results further support the principal role of the dynamical reshaping of astrocyte processes in the generation of intrinsic Ca2+ oscillations and their spreading over larger astrocytic compartments.

Mitochondrial Calcium Uniporter Overexpression Exacerbates Arrhythmogenic Calcium Mishandling in Diseased Rat Ventricular Myocytes

Systolic dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with diminished Ca2+ transient amplitude and impaired mitochondrial function due to reduced mitochondrial [Ca2+]. Pharmacological or genetic facilitation of mito-Ca2+ uptake was shown to restore Ca2+ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that acute pharmacological enhancement of mito-Ca2+ uptake can exacerbate spontaneous Ca2+ release in myocytes from diseased animals, increasing propensity to stress-induced arrhythmias. To test whether chronic recovery of mito-[Ca2+] restores systolic Ca2+ release without adverse effects in diastole, we overexpressed mitochondrial Ca2+ uniporter (MCU) in ventricular myocytes (VMs) from rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca2+] using genetic probe mtRCamp1h revealed that mito-[Ca2+] in TAB VMs paced at 1 Hz under β-adrenergic stimulation is significantly lower than in Shams. Adenoviral MCU overexpression in TAB VMs restored mito-[Ca2+]. Confocal imaging of VMs loaded with fluorescent indicator Rhod-2 revealed that MCU overexpression in TAB VMs reversed the decrease in Ca2+ transient amplitude. Importantly, the decay of caffeine-induced Ca2+ transient in MCU-overexpressing TAB VMs was prolonged, indicative of decreased NCX activity. Moreover, MCU overexpression in TAB VMs was accompanied by a further increase in incidence of proarrhythmic spontaneous Ca2+ waves, suggesting exacerbation of RyR2 dysfunction. Experiments using genetic probe OMM-HyPer showed a 10-fold increase in mito-ROS emission in TAB VMs overexpressing MCU. Importantly, application of mito-ROS scavenger mitoTEMPO (20 µM, 10 min) restored Ca2+ transient amplitude, reduced spontaneous Ca2+ waves and restored mito-[Ca2+] in TAB VMs, rendering them normal. These findings suggest that restoration of mito-[Ca2+] in VMs from diseased hearts by MCU overexpression improves systolic Ca2+ release possibly via reduced NCX-mediated Ca2+ efflux, but exacerbates increased mito-ROS and RyR2 activity, increasing arrhythmic potential.

Human platelets use a cytosolic Ca2+ nanodomain to activate Ca2+-dependent shape change independently of platelet aggregation

Human platelets use a rise in cytosolic Ca2+ concentration to activate all stages of thrombus formation, however, how they are able to decode cytosolic Ca2+ signals to trigger each of these independently is unknown. Other cells create local Ca2+ signals to activate Ca2+-sensitive effectors specifically localised to these subcellular regions. However, no previous study has demonstrated that agonist-stimulated human platelets can generate a local cytosolic Ca2+ signal. Platelets possess a structure called the membrane complex (MC) where the main intracellular calcium store, the dense tubular system (DTS), is coupled tightly to an invaginated portion of the plasma membrane called the open canalicular system (OCS). Here we hypothesised that human platelets use a Ca2+ nanodomain created within the MC to control the earliest phases of platelet activation. Dimethyl-BAPTA-loaded human platelets were stimulated with thrombin in the absence of extracellular Ca2+ to isolate a cytosolic Ca2+ nanodomain created by Ca2+ release from the DTS. In the absence of any detectable rise in global cytosolic Ca2+ concentration, thrombin stimulation triggered Na+/Ca2+ exchanger (NCX)-dependent Ca2+ removal into the extracellular space, as well as Ca2+-dependent shape change in the absence of platelet aggregation. The NCX-mediated Ca2+ removal was dependent on the normal localisation of the DTS, and immunofluorescent staining of NCX3 demonstrated its localisation to the OCS, consistent with this Ca2+ nanodomain being formed within the MC. These results demonstrated that human platelets possess a functional Ca2+ nanodomain contained within the MC that can control shape change independently of platelet aggregation.

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na+, Ca2+) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca2+ at the expense of the Na+ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca2+ extrusion and thus to the recovery from intracellular Ca2+ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na+ to 1 Ca2+, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na+ concentration or minor depolarisations switch it into the “reverse mode” (Ca2+ import/Na+ export). Notably, transient Na+ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na+-permeable ion channels in response to neuronal activity. Activity-related Na+ transients result in NCX reversal, which mediates Ca2+ influx from the extracellular space, thereby generating astrocyte Ca2+ signalling independent from InsP3-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca2+ overload, while dampening Na+ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na+-driven, reverse NCX-mediated astrocyte Ca2+ signals are involved neurone-glia interaction. Na+ transients, translated by the NCX into Ca2+ elevations, thereby emerge as a new signalling pathway in astrocytes.

Long-term effects of Na+ /Ca2+ exchanger inhibition with ORM-11035 improves cardiac function and remodelling without lowering blood pressure in a model of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is increasingly common but there is currently no established pharmacological therapy. We hypothesized that ORM-11035, a novel specific Na+ /Ca2+ exchanger (NCX) inhibitor, improves cardiac function and remodelling independent of effects on arterial blood pressure in a model of cardiorenal HFpEF. Rats were subjected to subtotal nephrectomy (NXT) or sham operation. Eight weeks after intervention, treatment for 16 weeks with ORM-11035 (1mg/kg body weight) or vehicle was initiated. At 24 weeks, blood pressure measurements, echocardiography and pressure-volume loops were performed. Contractile function, Ca2+ transients and NCX-mediated Ca2+ extrusion were measured in isolated ventricular cardiomyocytes. NXT rats (untreated) showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV end-diastolic pressure (LVEDP) elevation, increased brain natriuretic peptide (BNP) levels, preserved ejection fraction and pulmonary congestion. In cardiomyocytes from untreated NXT rats, early relaxation was prolonged and NCX-mediated Ca2+ extrusion was decreased. Chronic treatment with ORM-11035 significantly reduced LV hypertrophy and cardiac remodelling without lowering systolic blood pressure. LVEDP [14 ± 3 vs. 9± 2 mmHg; NXT (n= 12) vs. NXT + ORM (n= 12); P= 0.0002] and BNP levels [71 ± 12 vs. 49 ± 11 pg/mL; NXT (n= 12) vs. NXT + ORM (n= 12); P< 0.0001] were reduced after ORM treatment. LV cardiomyocytes from ORM-treated rats showed improved active relaxation and diastolic cytosolic Ca2+ decay as well as restored NCX-mediated Ca2+ removal, indicating NCX modulation with ORM-11035 as a promising target in the treatment of HFpEF. Chronic inhibition of NCX with ORM-11035 significantly attenuated cardiac remodelling and diastolic dysfunction without lowering systemic blood pressure in this model of HFpEF. Therefore, long-term treatment with selective NCX inhibitors such as ORM-11035 should be evaluated further in the treatment of heart failure.

NCX-Mediated Subcellular Ca2+ Dynamics Underlying Early Afterdepolarizations in LQT2 Cardiomyocytes

Long QT syndrome type 2 (LQT2) is a congenital disease characterized by loss of function mutations in hERG potassium channels (IKr). LQT2 is associated with fatal ventricular arrhythmias promoted by triggered activity in the form of early afterdepolarizations (EADs). We previously demonstrated that intracellular Ca2+ handling is remodeled in LQT2 myocytes. Remodeling leads to aberrant late RyR-mediated Ca2+ releases that drive forward-mode Na+-Ca2+ exchanger (NCX) current and slow repolarization to promote reopening of L-type calcium channels and EADs. Forward-mode NCX was found to be enhanced despite the fact that these late releases do not significantly alter the whole-cell cytosolic calcium concentration during a vulnerable period of phase 2 of the action potential corresponding to the onset of EADs. Here, we use a multiscale ventricular myocyte model to explain this finding. We show that because the local NCX current is a saturating nonlinear function of the local submembrane calcium concentration, a larger number of smaller-amplitude discrete Ca2+ release events can produce a large increase in whole-cell forward-mode NCX current without increasing significantly the whole-cell cytosolic calcium concentration. Furthermore, we develop novel insights, to our knowledge, into how alterations of stochastic RyR activity at the single-channel level cause late aberrant Ca2+ release events. Experimental measurements in transgenic LTQ2 rabbits confirm the critical arrhythmogenic role of NCX and identify this current as a potential target for antiarrhythmic therapies in LQT2.

α2 isoform of Na+,K+-ATPase via Na+,Ca2+ exchanger modulates myelin basic protein synthesis in oligodendrocyte lineage cells in vitro

Oligodendrocytes in the CNS myelinate neuronal axons, facilitating rapid propagation of action potentials. Myelin basic protein (MBP) is an essential component of myelin and its absence results in severe hypomyelination. In oligodendrocyte lineage cell (OLC) monocultures MBP synthesis starts at DIV4. Ouabain (10 nM), a Na+,K+-ATPase (NKA) blocker, stimulates MBP synthesis. As OLCs express the α2 isoform of NKA (α2-NKA) that has a high affinity for ouabain, we hypothesized that α2-NKA mediates this effect. Knockdown of α2-NKA with small interfering (si)RNA (α2-siRNA) significantly potentiated MBP synthesis at DIV4 and 5. This effect was completely blocked by KB-R7943 (1 μM), a Na+,Ca2+ exchanger (NCX) antagonist. α2-NKA ablation increased the frequency of NCX-mediated spontaneous Ca2+ transients ([Ca2+]t) at DIV4, whereas in control OLC cultures comparable frequency of [Ca2+]t was observed at DIV5. At DIV6 almost no [Ca2+]t were observed either in control or in α2-siRNA-treated cultures. Immunocytochemical analyses showed that α2-NKA co-localizes with MBP in proximal processes of immature OLCs but is only weakly present in MBP-enriched membrane sheets. Knockdown of α2-NKA in cortical slice cultures did not change MBP levels but reduced co-localization of neurofilament- and MBP-positive compartments. We conclude that α2-NKA activity in OLCs affects NCX-mediated [Ca2+]t and the onset of MBP synthesis. We suggest therefore that neuronal activity, presumably in form of local extracellular [K+] changes, might locally influence NCX-mediated [Ca2+]t in OLC processes thus triggering local MBP synthesis in the vicinity of an active axon.

Sodium-Calcium Exchanger in Pig Coronary Artery.

This review focuses on the sodium-calcium exchangers (NCX) in the left anterior descending coronary artery smooth muscle. Bathing tissues in Na+-substituted solutions caused them to contract. In cultured smooth muscle cells, it increased the cytosolic Ca2+ concentration and extracellular entry of 45Ca2+. All three activities were attributed to NCX since they were inhibited by NCX inhibitors. The tissues also expressed the sarco/endoplasmic reticulum (SER) Ca2+ pump SERCA2b whose activity was much greater than that of NCX. Inhibiting SERCA2b with thapsigargin decreased the NCX-mediated 45Ca2+ accumulation by the cells. The decrease was not observed in cells loaded with the Ca2+-chelator BAPTA. The results are consistent with a limited diffusional space model with a proximity between NCX and SERCA2b. NCX molecules appear to be colocalized with the subsarcolemmal SERCA2b based on studies on membrane flotation experiments and microscopic fluorescence imaging of antibody-labeled cells. Thapsigargin inhibition of SERCA2b moved NCX even closer to SER. This provides a model for the NCX-mediated Ca2+ refilling of SER in the arterial smooth muscle. The model for the NCX-mediated refilling of the depleted SER proposed for smooth muscle did not apply to endothelium in which NCX levels were greater and SERCA levels were lower than in smooth muscle. The effect of thapsigargin on the NCX-mediated Ca2+ accumulation which was observed in smooth muscle was absent in the endothelium. We propose that the coupling between NCX and smooth muscle may be tissue dependent.

Cardiac expression of the CREM repressor isoform CREM-IbΔC-X in mice leads to arrhythmogenic alterations in ventricular cardiomyocytes

Chronic β-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to β-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the β-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca2+ releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca2+ waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca2+ transport and NCX1 protein level in TG. An increase in INCX and decrease in Ito and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-016-0532-y) contains supplementary material, which is available to authorized users.

Doxorubicin Stimulates the Na+/Ca2+ Exchanger in Ventricular Cardiomyocytes

Doxorubicin (DOX) is a widely used antineoplastic agent that exerts cardiotoxic effects. This study analyzed the effects of a clinically relevant DOX concentration (30 μM) on Ca2+ transport in rat ventricular myocytes. DOX decreased the sarcoplasmic reticulum (SR) Ca2+ content ([Ca2+]SR) (15%, p<0.001), however without significant change in Ca2+ transient amplitude and kinetics, or in the fraction of [Ca2+]SR released at a twitch. DOX abbreviated [Ca2+]i decline of caffeine-evoked transients (p<0.001). Estimates of the integrated Ca2+ fluxes attributable to individual transporters during twitch [Ca2+]i decline indicated a small depression of SR Ca2+ uptake (12%, p<0.05), but great enhancement of Ca2+ efflux via the Na+/Ca2+ exchanger (NCX) (90%, p<0.05), so that the contribution of the latter to cytosolic Ca2+ removal more than doubled. To promote NCX-mediated Ca2+ influx in the absence of electrical stimulation, extracellular [Na+] was lowered from 140 to 105 mM. In these conditions, DOX exposure increased both the rate constant of [Ca2+]i rise (12.8±1.4 vs. 4.8±0.8 nM.s-1) and the rate of spontaneous Ca2+ transients (0.18±0.04 vs. 0.05±0.01 Hz, p<0.05). After SR function inhibition with thapsigargin, spontaneous Ca2+ transients were absent, but NCX-mediated increase in [Ca2+]i remained greater in the presence of DOX (190±35 vs. 74±18 nM.min-1, p<0.01), which was abolished by inhibition of reverse NCX with 10 μM KB-R7943 (−29±6 nM.min-1, p<0.01). The results indicate that DOX altered mainly diastolic Ca2+ handling, causing mild inhibition of SR Ca2+ uptake, but marked increase of Ca2+ transport via NCX. The latter effect does not seem to be due to thermodynamic factors, but probably to NCX stimulation, as both Ca2+ influx and efflux were enhanced.

N‐arachidonoyl glycine suppresses Na+/Ca2+ exchanger‐mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

Background and PurposeN-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly's action on unstimulated and agonist-stimulated endothelial cells.Experimental ApproachThe effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.Key ResultsIn EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na+/Ca2+ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs+-based Na+-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na+ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca2+]i elevation evoked by Na+ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca2+-activated K+ (BKCa) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BKCa channel activity.Conclusion and ImplicationsOur data showed that NCX is a Ca2+ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca2+ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BKCa channels.

Role of Intracellular Ca2+and Na+/Ca2+Exchanger in the Pathogenesis of Contrast-Induced Acute Kidney Injury

The precise mechanisms underlying contrast-induced acute kidney injury (CI-AKI) are not well understood. Intracellular Ca2+ overload is considered to be a key factor in CI-AKI. Voltage-dependent Ca2+ channel (VDC) and Na+/Ca2+ exchanger (NCX) system are the main pathways of intracellular Ca2+ overload in pathological conditions. Here, we review the potential underlying mechanisms involved in CI-AKI and discuss the role of NCX-mediated intracellular Ca2+ overload in the contrast media-induced renal tubular cell injury and renal hemodynamic disorder.

The Mechanism of Injury-Induced Intracellular Calcium Concentration Oscillations in the Endothelium of Excised Rat Aorta

Endothelial injury is the primary event that leads to a variety of severe vascular disorders. Mechanical injury elicits a Ca²⁺ response in the endothelium of excised rat aorta, which comprises an initial Ca²⁺ release from inositol-1,4,5-trisphosphate (InsP₃)-sensitive stores followed by a long-lasting decay phase due to Ca²⁺ entry through uncoupled connexons. The Ca²⁺ signal may also adopt an oscillatory pattern, the molecular underpinnings of which are unclear. In the light of the role played by Ca²⁺ spiking in tissue regeneration, this study aimed to unveil the mechanisms underlying injury-induced Ca²⁺ oscillations. The latter reversibly ceased upon removal of extracellular Ca²⁺ or addition of the gap junction blockers heptanol, 18 α,β-glycyrrhetinic acid, La³⁺ and Ni²⁺, but were insensitive to BTP-2 and SKF 96365. The spiking response was abolished by inhibiting the Ca²⁺ entry mode of the Na⁺/Ca²⁺ exchanger (NCX). The InsP₃-producing agonist ATP resumed Ca²⁺ oscillations in silent cells, while the phospholipase C inhibitor U73122 suppressed them. Injury-induced Ca²⁺ transients were prevented by the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) blockers thapsigargin and cyclopiazonic acid, while they were unaffected by suramin and genistein. These data show for the first time that the coordinated interplay between NCX-mediated Ca²⁺ entry and InsP₃-dependent Ca²⁺ release contributes to injury-induced intracellular Ca²⁺ concentration oscillations.

SEA0400, a specific Na+/Ca2+ exchange inhibitor, prevents dopaminergic neurotoxicity in an MPTP mouse model of Parkinson’s disease

We have recently shown that the Na+/Ca2+ exchanger (NCX) is involved in nitric oxide (NO)-induced cytotoxicity in cultured astrocytes and neurons. However, there is no in vivo evidence suggesting the role of NCX in neurodegenerative disorders associated with NO. NO is implicated in the pathogenesis of neurodegenerative disorders such as Parkinson’s disease. This study examined the effect of SEA0400, the specific NCX inhibitor, on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity, a model of Parkinson’s disease, in C57BL/6J mice. MPTP treatment (10 mg/kg, four times at 2-h intervals) decreased dopamine levels in the midbrain and impaired motor coordination, and these effects were counteracted by S-methylthiocitrulline, a selective neuronal NO synthase inhibitor. SEA0400 protected against the dopaminergic neurotoxicity (determined by dopamine levels in the midbrain and striatum, tyrosine hydroxylase immunoreactivity in the substantia nigra and striatum, striatal dopamine release, and motor deficits) in MPTP-treated mice. SEA0400 had no radical-scavenging activity. SEA0400 did not affect MPTP metabolism and MPTP-induced NO production and microglial activation, while it attenuated MPTP-induced increases in extracellular signal-regulated kinase (ERK) phosphorylation and lipid peroxidation product, thiobarbituric acid reactive substance. These findings suggest that SEA0400 protects against MPTP-induced neurotoxicity probably by blocking ERK phosphorylation and lipid peroxidation which are downstream of NCX-mediated Ca2+ influx.

Sodium-Calcium Exchange in Intracellular Calcium Handling of Human Airway Smooth Muscle

Enhanced airway contractility following inflammation by cytokines such as tumor necrosis factor alpha (TNFα) or interleukin-13 (IL-13) involves increased intracellular Ca2+ ([Ca2+]i) levels in airway smooth muscle (ASM). In ASM, plasma membrane Ca2+ fluxes form a key component of [Ca2+]i regulation. There is now growing evidence that the bidirectional plasma membrane Na+/Ca2+ exchanger (NCX) contributes to ASM [Ca2+]i regulation. In the present study, we examined NCX expression and function in human ASM cells under normal conditions, and following exposure to TNFα or IL-13. Western blot analysis showed significant expression of the NCX1 isoform, with increased NCX1 levels by both cytokines, effects blunted by inhibitors of nuclear factor NF-κB or mitogen-activated protein kinase. Cytokine-mediated increase in NCX1 involved enhanced transcription followed by protein synthesis. NCX2 and NCX3 remained undetectable even in cytokine-stimulated ASM. In fura-2 loaded human ASM cells, NCX-mediated inward Ca2+ exchange as well as outward exchange (measured as rates of change in [Ca2+]i) was elicited by altering extracellular Na+ and Ca2+ levels. Contribution of NCX was verified by measuring [Na+]i using the fluorescent Na+ indicator SBFI. NCX-mediated inward exchange was verified by demonstrating prevention of rising [Ca2+]i or falling [Na+]i in the presence of the NCX inhibitor KBR7943. Inward exchange-mode NCX was increased by both TNFα and IL-13 to a greater extent than outward exchange. NCX siRNA transfection substantially blunted outward exchange and inward exchange modes. Finally, inhibition of NCX expression or function blunted peak [Ca2+]i and rate of fall of [Ca2+]i following histamine stimulation. These data suggest that NCX-mediated Ca2+ fluxes normally exist in human ASM (potentially contributing to rapid Ca2+ fluxes), and contribute to enhanced [Ca2+]i regulation in airway inflammation.

The mechanism of injury-induced [Ca2+]i oscillations in the endothelium of excised rat aorta

Endothelial injury is the primary event that leads to a variety of severe vascular disorders. The signal transduction pathway which drives the subsequent healing process is far from being fully elucidated. Mechanical injury elicits a Ca2+ response in the endothelium of intact rat aorta, which comprises an initial Ca2+ release from inositol-1,4,5-trisphosphate-sensitive (IP3Rs) stores followed by a long-lasting decay phase due to Ca2+ entry through uncoupled connexons. In a minor fraction of cells, the Ca2+ signal adopts an oscillatory pattern whose molecular underpinnings are yet to be elucidated. In the light of the role played by repetitive Ca2+ spikes in regulating tissue regeneration, the present study aims at elucidating the mechanisms underlying injury-induced Ca2+ oscillations. The repetitive Ca2+ signal reversibly ceased upon removal of extracellular Ca2+ or addition of the inorganic cations, La3+ and Ni2+. Moreover, the spiking response was abolished by the gap-junction blockers, heptanol and 18 beta-glycyrrhetinic acid and by interfering with the Ca2+ entry-mode mode of the Na+/Ca2+ exchanger (NCX). The InsP3-producing agonist, ATP, resumed Ca2+ oscillations in silent cells, while the phospholipase C inhibitor, U73122, inhibited the oscillatory signal. The latter was also prevented by the SERCA inhibitors, thapsigargin and cyclopiazonic acid acid. These data show that injury-induced [Ca2+]i oscillations require the coordinated interplay between NCX-mediated Ca2+ entry and InsP3-dependent Ca2+ release. Besides directly gating Ca2+ inflow, uncoupled connexons might let Na+ into the cells and stimulate Ca2+ entry through NCX by increasing submembranal Na+ levels.

Adverse Bioenergetic Consequences of Na + -Ca 2+ Exchanger–Mediated Ca 2+ Influx in Cardiac Myocytes

In heart failure, the Na+-Ca²+ exchanger (NCX) is upregulated and mediates Ca²+ influx (instead of efflux) during the cardiac action potential. Although this partly compensates for impaired sarcoplasmic reticulum Ca²+ release and supports inotropy, the energetic consequences have never been considered. Because NCX-mediated Ca²+ influx is rather slow and mitochondrial Ca²+ uptake (which stimulates NADH production by the Krebs cycle) is thought to be facilitated by high Ca²+ gradients in a "mitochondrial Ca²+ microdomain," we speculated that NCX-mediated Ca²+ influx negatively affects the bioenergetic feedback response. Methods and Results- With the use of a patch-clamp-based approach in guinea-pig myocytes, cytosolic and mitochondrial Ca²+ ([Ca²+](c) and [Ca²+](m), respectively) was determined within the same cell after varying Ca²+ influx via L-type Ca²+ channels (I(Ca,L)) or the NCX. The efficiency of mitochondrial Ca²+ uptake, indexed by the slope of plotting [Ca²+](m) against [Ca²+](c) during each Ca²+ transient, was maximal during I(Ca,L)-triggered sarcoplasmic reticulum Ca²+ release. Depletion of sarcoplasmic reticulum Ca²+ load and increased contribution of the NCX to cytosolic Ca²+ influx independently reduced the efficiency of mitochondrial Ca²+ uptake. The upstroke velocity of cytosolic Ca²+ transients closely correlated with the efficiency of mitochondrial Ca²+ uptake. Despite comparable [Ca²+](c), sarcoplasmic reticulum Ca²+ release, but not NCX-mediated Ca²+ influx, led to stimulation of Ca²+-sensitive dehydrogenases of the Krebs cycle. Conclusions- Increased contribution of the NCX to cytosolic Ca²+ transients, which occurs in cardiac myocytes from failing hearts, impairs mitochondrial Ca²+ uptake and the bioenergetic feedback response. This mechanism could contribute to energy starvation of failing hearts.

The Na +/Ca 2+ exchanger-mediated Ca 2+ influx triggers nitric oxide-induced cytotoxicity in cultured astrocytes

Nitric oxide (NO) is involved in many pathological conditions including neurodegenerative disorders. We have previously found that sodium nitroprusside (SNP), an NO donor, stimulates mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulating kinase (ERK), c-jun N-terminal protein kinase (JNK) and p38 MAPK, leading to caspase-independent apoptosis in cultured astrocytes. In view of the previous observation that NO stimulates the activity of the Na +/Ca 2+ exchanger (NCX), this study examines the involvement of NCX in cytotoxicity. The specific NCX inhibitor SEA0400 blocked SNP-induced phosphorylation of ERK, JNK and p38 MAPK, and decrease in cell viability. SNP-induced phosphorylation of ERK, JNK and p38 MAPK was blocked by removal of external Ca 2+, and SNP treatment caused an increase in 45Ca 2+ influx. This increase in 45Ca 2+ influx was blocked by SEA0400, but not the Ca 2+ channel blocker nifedipine. In addition, SNP-induced 45Ca 2+ influx and cytotoxicity were reduced in NCX1-deficient cells which were transfected with NCX1 siRNA. Inhibitors of intracellular Ca 2+-dependent proteins such as calpain and calmodulin blocked SNP-induced ERK phosphorylation and decrease in cell viability. Furthermore, the guanylate cyclase inhibitor LY83583 and the cGMP-dependent protein kinase inhibitor KT5823 blocked SNP-induced cytotoxicity. These findings suggest that NCX-mediated Ca 2+ influx triggers SNP-induced apoptosis in astrocytes, which may be mediated by a cGMP-dependent pathway.

Sodium accumulation promotes diastolic dysfunction in end-stage heart failure followingSerca2knockout

Alterations in trans-sarcolemmal and sarcoplasmic reticulum (SR) Ca(2+) fluxes may contribute to impaired cardiomyocyte contraction and relaxation in heart failure. We investigated the mechanisms underlying heart failure progression in mice with conditional, cardiomyocyte-specific excision of the SR Ca(2+)-ATPase (SERCA) gene. At 4 weeks following gene deletion (4-week KO) cardiac function remained near normal values. However, end-stage heart failure developed by 7 weeks (7-week KO) as systolic and diastolic performance declined. Contractions in isolated myocytes were reduced between 4- and 7-week KO, and relaxation was slowed. Ca(2+) transients were similarly altered. Reduction in Ca(2+) transient magnitude resulted from complete loss of SR Ca(2+) release between 4- and 7-week KO, due to loss of a small remaining pool of SERCA2. Declining SR Ca(2+) release was partly offset by increased L-type Ca(2+) current, which was facilitated by AP prolongation in 7-week KO. Ca(2+) entry via reverse-mode Na(+)-Ca(2+) exchange (NCX) was also enhanced. Up-regulation of NCX and plasma membrane Ca(2+)-ATPase increased Ca(2+) extrusion rates in 4-week KO. Diastolic dysfunction in 7-week KO resulted from further SERCA2 loss, but also impaired NCX-mediated Ca(2+) extrusion following Na(+) accumulation. Reduced Na(+)-K(+)-ATPase activity contributed to the Na(+) gain. Normalizing [Na(+)] by dialysis increased the Ca(2+) decline rate in 7-week KO beyond 4-week values. Thus, while SERCA2 loss promotes both systolic and diastolic dysfunction, Na(+) accumulation additionally impairs relaxation in this model. Our observations indicate that if cytosolic Na(+) gain is prevented, up-regulated Ca(2+) extrusion mechanisms can maintain near-normal diastolic function in the absence of SERCA2.