Forward Mode Of NCX Research Articles

Astrocytes Reduce Store-Operated Ca2+ Entry in Microglia under the Conditions of an Inflammatory Stimulus and Muscarinic Receptor Blockade.

Inflammation and loss of cholinergic transmission are involved in neurodegenerative diseases, but possible interactions between them within neurons, astrocytes, and microglia have not yet been investigated. We aimed to compare store-operated Ca2+ entry (SOCE) in neurons, astrocytes, and microglia following cholinergic dysfunction in combination with (or without) an inflammatory stimulus and to investigate the effects of linalyl acetate (LA) on this process. We used the SH-SY5Y, U373, and BV2 cell lines related to neurons, astrocytes, and microglia, respectively. Scopolamine or lipopolysaccharide (LPS) was used to antagonize the muscarinic receptors or induce inflammatory responses, respectively. The concentration of intracellular Ca2+ was measured using Fura-2 AM. Treatment with scopolamine and LPS significantly increased SOCE in the neuron-like cells and microglia but not in the scopolamine-pretreated astrocytes. LA significantly reduced SOCE in the scopolamine-pretreated neuron-like cells and microglia exposed to LPS, which was partially inhibited by the Na+-K+ ATPase inhibitor ouabain and the Na+/Ca2+ exchanger (NCX) inhibitor Ni2+. Notably, SOCE was significantly reduced in the LPS plus scopolamine-pretreated cells mixed with astrocytes and microglia, with a two-fold increase in the applied number of astrocytes. LA may be useful in protecting neurons and microglia by reducing elevated SOCE that is induced by inflammatory responses and inhibiting the muscarinic receptors via Na+-K+ ATPase and the forward mode of NCX. Astrocytes may protect microglia by reducing increased SOCE under the conditions of inflammation and a muscarinic receptor blockade.

SAR296968, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Ca2+ Handling and Contractile Function in Human Atrial Cardiomyocytes.

Background: In reverse-mode, cardiac sodium-calcium exchanger (NCX) can increase the cytoplasmic Ca2+ concentration in response to high intracellular Na+ levels, which may contribute to diastolic contractile dysfunction. Furthermore, increased spontaneous Ca2+ release from intracellular stores can activate forward mode NCX. The resulting transient inward current causes delayed afterdepolarization (DAD)-dependent arrhythmias. Moreover, recently, NCX has been associated with impaired relaxation and reduced cardiac function in heart failure with preserved ejection fraction (HFpEF). Since NCX is upregulated in human chronic atrial fibrillation (AF) as well as heart failure (HF), specific inhibition may have therapeutic potential. Objective: We tested the antiarrhythmic, lusitropic and inotropic effects of a novel selective NCX-inhibitor (SAR296968) in human atrial myocardium. Methods and Results: Right atrial appendage biopsies of 46 patients undergoing elective cardiac surgery in a predominant HFpEF cohort (n = 24/46) were investigated. In isolated human atrial cardiomyocytes, SAR296968 reduced the frequency of spontaneous SR Ca2+ release events and increased caffeine transient amplitude. In accordance, in isolated atrial trabeculae, SAR296968 enhanced the developed tension after a 30 s pause of electrical stimulation consistent with reduced diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Moreover, compared to vehicle, SAR296968 decreased steady-state diastolic tension (at 1 Hz) without impairing developed systolic tension. Importantly, SAR296968 did not affect the safety parameters, such as resting membrane potential or action potential duration as measured by patch clamp. Conclusion: The novel selective NCX-inhibitor SAR296968 inhibits atrial pro-arrhythmic activity and improves diastolic and contractile function in human atrial myocardium, which may have therapeutic implications, especially for treatment of HFpEF.

Sodium-Calcium Exchanger Reverse Mode Sustains Dichotomous Ion Fluxes Required for Procoagulant COAT Platelet Formation.

Procoagulant collagen-and-thrombin (COAT)-activated platelets represent a subpopulation of activated platelets, which retain a coat of prohemostatic proteins and express phosphatidylserine on their surface. Dichotomous intracellular signaling generating procoagulant platelet activity instead of traditional aggregating endpoints is still not fully elucidated. It has been demonstrated that secondary messengers such as calcium and sodium play a critical role in platelet activation. Therefore, we developed a flow cytometric analysis to investigate intracellular ion fluxes simultaneously during generation of aggregating and procoagulant platelets. Human platelets were activated by convulxin-plus-thrombin. Cytosolic calcium, sodium, and potassium ion fluxes were visualized by specific ion probes and analyzed by flow cytometry. We observed high and prolonged intracellular calcium concentration, transient sodium increase, and fast potassium efflux in COAT platelets, whereas aggregating non-COAT platelets rapidly decreased their calcium content, maintaining higher cytosolic sodium, and experiencing lower and slower potassium depletion. Considering these antithetical patterns, we investigated the role of the sodium-calcium exchanger (NCX) during convulxin-plus-thrombin activation. NCX inhibitors, CBDMB and ORM-10103, dose-dependently reduced the global calcium mobilization induced by convulxin-plus-thrombin activation and dose-dependently prevented formation of procoagulant COAT platelets. Our data demonstrate that both NCX modes are used after convulxin-plus-thrombin-induced platelet activation. Non-COAT platelets use forward-mode NCX, thus pumping calcium out and moving sodium in, while COAT platelets rely on reverse NCX function, which pumps additional calcium into the cytosol, by extruding sodium. In conclusion, we described for the first time the critical and dichotomous role of NCX function during convulxin-plus-thrombin-induced platelet activation.

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.

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.

Heparin Oligosaccharides Have Antiarrhythmic Effect by Accelerating the Sodium-Calcium Exchanger.

Background: Blockage of the Na+/Ca2+ exchanger (NCX) is used to determine the role of NCX in arrhythmogenesis. Trisulfated heparin disaccharide (TD) and Low Molecular Weight Heparins (LMWHs) can directly interact with the NCX and accelerate its activity.Objective: In this work, we investigated the antiarrhythmic effect of heparin oligosaccharides related to the NCX activity.Methods: The effects of heparin oligosaccharides were tested on the NCX current (patch clamping) and intracellular calcium transient in rat cardiomyocytes. The effects of heparin oligosaccharides were further investigated in arrhythmia induced in isolated rat atria and rats in vivo.Results: The intracellular Ca2+ concentration decreases upon treatment with either enoxaparin or ardeparin. These drugs abolished arrhythmia induction in isolated atria. The NCX antagonist KB-R7943 abolished the enoxaparin or ardeparin antiarrhythmic effects in isolated atria. In the in vivo measurements, injection of TD 15 min both before coronary occlusion or immediately after reperfusion, significantly prevented the occurrence of reperfusion-induced arrhythmias (ventricular arrhythmia and total AV block) and reduced the lethality rate. The patch clamping experiments showed that, mechanistically, TD increases the forward mode NCX current.Conclusion: Together, the data shows that heparin oligosaccharides may constitute a new class of antiarrhythmic drug that acts by accelerating the forward mode NCX under calcium overload.

Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca(2+) dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca(2+) mediated by the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca(2+) influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca(2+) dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.

Functional contributions of the plasma membrane calcium ATPase and the sodium–calcium exchanger at mouse parallel fibre to Purkinje neuron synapses

We investigated how two calcium clearance mechanisms, the sodium-calcium exchanger-NCX, and the plasma membrane calcium ATPase-PMCA2, function at the facilitating cerebellar parallel fibre to Purkinje neuron (PF-PN) synapse. Forward mode NCX helped recover PF presynaptic calcium elevations when the PFs received a double stimulation and the calcium load was sufficiently high. A smaller presynaptic calcium load evoked by a single PF stimulation failed to recruit NCX in wild-type mice but did so when PMCA2 was absent in PFs from PMCA2 knockout mice. Simulated calcium dynamics using a simple single-compartment model reported qualitatively similar effects. Functionally, reduced NCX activity in the absence of PMCA also prolonged the recovery of facilitation at the PF-PN synapse, beyond that seen by reduced NCX activity alone. We conclude that PMCA and NCX work in parallel to accurately shape residual presynaptic calcium recovery dynamics and fine-tune facilitation at this important cerebellar synapse.

Can the Sodium-Calcium Exchanger Initiate or Suppress Calcium Sparks in Cardiac Myocytes?

Positive feedback of Calcium (Ca)-induced Ca release is the mechanism of Ca spark formation in cardiac myocytes. To initiate this process, a certain amount of Ca in the cleft space is necessary. When the membrane potential becomes higher during excitation-contraction coupling, Ca can enter through both Ca current (ICaL) and sodium-calcium exchanger (NCX) and may activate ryanodine receptors to initiate a Ca spark. On the other hand, at the resting membrane potential (Vm ∼–80 mV), NCX removes Ca from the cell (forward mode). If Ca released from the sarcoplasmic reticulum is quickly removed via forward mode NCX before Ca-induced Ca release starts, the Ca release becomes nonspark Ca leak. This would also be influenced by the cleft/noncleft distribution of NCX, which is unknown. Using a physiologically detailed mathematical model of subcellular Ca cycling, we analyze how NCX strength and distribution alter Ca spark formation. During excitation-contraction coupling, most Ca sparks are induced by ICaL with very few due to NCX current. At the resting membrane potential if most NCX is localized to the cleft, spontaneous Ca sparks are significantly reduced.

Can the Sodium-Calcium Exchanger Initiate or Supress Calcium Sparks in Cardiac Myocytes?

Calcium (Ca) sparks in cardiac myocyte are formed by positive feedback (Ca induced Ca release). At the resting membrane potential (Vm ∼-80 mV), the sodium-calcium exchanger (NCX) removes Ca from the cell (forward mode). If Ca released from the sarcoplasmic reticulum (SR) is quickly removed via forward mode NCX before the positive feedback process starts, the Ca release becomes non-spark Ca leak. However, at higher Vm, Ca enters through the NCX (reverse mode) and Ca entry through reverse mode NCX may activate ryanodine receptors (RyRs) and initiate Ca sparks. These are also influenced by the cleft/non-cleft distribution of NCX, which is still unknown. In this study, using physiologically detailed mathematical model of the subcellular Ca cycling, we investigate how the magnitude of NCX current and the distribution of NCXs alter Ca spark formation. We find that at the resting membrane potential if all NCXs are localized to the cleft, Ca sparks are significantly reduced (at [Ca]SR=700 μM, there is a 40% reduction vs. the case where all NCXs locate outside of the cleft). During excitation-contraction coupling, most Ca sparks are induced by L-type Ca current (ICaL) and only a small fraction of Ca sparks are due to NCX current at Vm = 0mV. If Vm is higher (+40 mV), since ICaL becomes smaller and NCX becomes larger, more sparks are induced by NCX. But the absolute number of Ca sparks is limited. These results also strongly depend on the distribution of NCXs. If many NCXs locate close to RyRs, NCX currents initiate Ca sparks much more efficiently. This underscores the criticality of NCX localization regarding functional impact on SR Ca release.

Cytosolic calcium elevation induced by orexin/hypocretin in granule cell domain cells of the rat cochlear nucleus in vitro

Using rat brain slice preparations, we examined the effect of orexin on cytosolic Ca 2+ concentrations ([Ca 2+] i) in the granule cell domain (GCD) cells of the cochlear nucleus that carry non-auditory information to the dorsal cochlear nucleus. Application of orexin concentration-dependently increased [Ca 2+] i, and in two thirds of GCD cells these increases persisted in the presence of tetrodotoxin. There was no significant difference between the dose–response curve for orexin-A and that for orexin-B. Extracellular Ca 2+ removal abolished the [Ca 2+] i elevation induced by orexin-B, whereas depletion of intracellular Ca 2+ stores had no effect. The orexin-B-induced elevation of [Ca 2+] i was not blocked by inhibitors of reverse-mode Na +/Ca 2+ exchanger (NCX) and nonselective cation channel, whereas it was blocked by lowering the extracellular Na + or by applying inhibitors of forward-mode NCX and voltage-gated R- and T-type Ca 2+ channels. The ORX-B-induced increase in [Ca 2+] i was also blocked by inhibitors of adenylcyclase (AC) and protein kinase A (PKA), but not by inhibitors of phosphatidylcholine-specific and phosphatidylinositol-specific phospholipase C. In electrophysiological experiments using whole-cell patch clamp recordings, half of GCD cells were depolarized by orexin-B, and the depolarization was abolished by a forward-mode NCX inhibitor. These results suggest that orexin increases [Ca 2+] i postsynaptically via orexin 2 receptors, and the increase in [Ca 2+] i is induced via the AC–PKA–forward-mode NCX-membrane depolarization-mediated activation of voltage-gated R- and T-type Ca 2+ channels. The results further support the hypothesis that the orexin system participates in integrating neural systems that are involved in arousal, sensory processing, energy homeostasis and autonomic function.

M-Calpain-mediated cleavage of Na+/Ca2+ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle

Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na(+)/Ca(2+) exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 microM) in presence of CaCl(2) (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca(2+) chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca(2+) overload, which could arise due to inhibition of Ca(2+) efflux by the forward-mode NCX and that could lead to sustained Ca(2+) overload in the smooth muscle leading to pulmonary hypertension.

Sodium Ion Transporters as New Therapeutic Targets in Heart Failure

Sodium ion transporters in sarcolemma are involved in numerous vital cell functions, such as excitability, excitation-contraction coupling, energy metabolism, pH and volume regulation, development and growth. In a number of cardiac pathologies, the intracellular sodium concentration ([Na+]i) is elevated. Since [Na+]i and intracellular Ca2+ concentration ([Ca2+]i are coupled through the Na+/Ca(2+)-exchanger, these cardiac pathologies display disturbed calcium handling. For instance, [Na+]i is increased in heart failure (HF) leading to Na+/Ca(2+)-exchanger mediated increase in [Ca2+]i, reduced contractility and increased propensity to arrhythmias. Several studies support the contention that an increase in [Na+]i and [Ca2+]i transduces a signal the nucleus, that triggers development of cardiac remodelling and hypertrophy. Pharmacological intervention, which favourably interferes with [Na+]i and [Ca2+]i homeostasis, might prevent hypertrophy, cardiac remodelling, arrhythmias and HF. The most important sodium transport mechanisms that may underlie increased [Na+]i are: Na+/H(+)-exchanger (NHE-1), Na+-HCO(3)(-) co-transporter (NBC), Na(+)-K(+)-Cl(-) co-transporter (NKCC), Na(+)-channel, Na+/K(+)-ATPase and Na+/Ca(2+)-exchanger (NCX). Preclinical studies showed that pharmacological interventions, targeted against sarcolemmal sodium ion transporters, proved effective in ameliorating heart failure. In this respect: 1) NHE-1 inhibition reduces cardiac remodelling, hypertrophy and HF, although, in the patients following coronary artery bypass graft surgery, it was associated with an increase of stroke. 2) The activity of NBC is up-regulated, during the development of hypertrophy and may be a therapeutic strategy to prevent the development of hypertrophy and HF. 3) NKCC is increased in post-infarction HF, and the inhibition of NKCC attenuated post-infarction remodelling. 4) Inactivation of sodium channels is impaired in HF, which may result, in increased Na+ influx and prolongation of the action potential. 5) Blockade of NCX may be useful as a part of a combined therapeutic approach. Inhibition of reversed mode, or activation of forward mode NCX reduce Ca2+ overload. 6) Inhibition of Na+/K(+)-ATPase (digoxin), is used to increase contractility, however, it enhances progression of HF. Oppositely, new drugs which increase activity of Na+/K(+)-ATPase may prevent the development of cardiac remodelling hypertrophy and HF.

Hypoxic pulmonary vasoconstriction in intact rat intrapulmonary arteries is not initiated by inhibition of Na+-Ca2+ exchange

It has been proposed that a hypoxia-induced inhibition of the Na(+)-Ca(2+) exchanger (NCX) contributes to hypoxic pulmonary vasoconstriction (HPV). By recording isometric tension development in rat intrapulmonary arteries (IPA), we examined the effect on HPV of maneuvers that reduce the ability of NCX to regulate intracellular Ca(2+) concentration ([Ca(2+)](i)). In some experiments, fura pentakis(acetoxymethyl) ester-3 (fura PE-3) was also used to monitor [Ca(2+)](i). HPV was elicited in IPA that were pretreated with 10 microM diltiazem and slightly preconstricted with PGF(2alpha), which enhances the hypoxic response. Substitution of Na(+) with Li(+) increased HPV and the associated rise in [Ca(2+)](i). Pretreatment with ouabain (100 microM) to diminish the Na(+) gradient or with the reverse-mode NCX inhibitor KB-R7943 (3 or 10 microM) had no significant effect on HPV. Combined treatment with ouabain and low-[Na(+)] (24 mM) solution enhanced HPV strongly. The role of NCX in Ca(2+) extrusion was examined by assessing the decrease in [Ca(2+)](i) in Ca(2+)-free physiological saline solution either containing or lacking Na(+) following a high K(+)-induced loading of cellular [Ca(2+)]. Although the large initial rapid fall in [Ca(2+)] was Na(+) independent, final recovery of [Ca(2+)] to its basal level was delayed in the absence of Na(+). Therefore, HPV persisted or was increased under conditions in which forward-mode NCX was already attenuated or prevented, demonstrating that inhibition of NCX by hypoxia is unlikely to initiate HPV. Instead, NCX appears to act to inhibit HPV as would be expected if it is functioning to extrude Ca(2+).

Endothelin-1 decreases [Ca 2+] i via Na +/Ca 2+ exchanger in CHO cells stably expressing endothelin ET A receptor

Endothelin ET A receptor couples to G q/11 protein that transduces a variety of receptor signals to modulate diverse cellular responses including Ca 2+ mobilization. Stimulation of endothelin ET A receptor with endothelin-1 is generally believed to induce an increase in intracellular Ca 2+ concentration ([Ca 2+] i ) via G q/11 protein. Here we provide the first convincing evidence that endothelin-1 elicited G q/11 protein-dependent and -independent ‘decrease’ in [Ca 2+] i via Na +/Ca 2+ exchanger (NCX) in Chinese hamster ovary (CHO) cells stably expressing human endothelin ET A receptor. In the cells treated with 1 μM thapsigargin, an inhibitor of endoplasmic Ca 2+ pump, that induces an increase in [Ca 2+] i via capacitative Ca 2+ entry, endothelin-1 induced a decrease in [Ca 2+] i which was partially inhibited by YM-254890, a specific inhibitor of G q/11, indicating that G q/11-dependent and independent pathways are involved in the decrease. The endothelin-1-induced decrease in [Ca 2+] i was markedly suppressed by 3′,4′-dichlorobenzamil hydrochloride, a potent NCX inhibitor, and also by a replacement of extracellular Na + with Li +, which was not transported by NCX, indicating a major role of NCX operating in the forward mode in the endothelin-1-induced decrease in [Ca 2+] i . Molecular approach with RT-PCR demonstrated the expression of mRNA for NCX1, NCX2 and NCX3. These results suggest that stimulation of endothelin ET A receptor with endothelin-1 activates the forward mode NCX through G q/11-dependent and -independent mechanisms: the NCX exports Ca 2+ out of the cell depending on Na + gradient across the cell membrane, resulting in the decrease in [Ca 2+] i .

Propofol Modulates Na+–Ca2+Exchange Activity via Activation of Protein Kinase C in Diabetic Cardiomyocytes

The authors' objective was to identify the role of the Na+-Ca2+ exchanger (NCX) in mediating the contractile dysfunction observed in diabetic cardiomyocytes before and after exposure to propofol. Freshly isolated ventricular myocytes were obtained from normal and diabetic rat hearts. Intracellular concentration of Ca2+ and cell shortening were simultaneously measured in electrically stimulated, ventricular myocytes using fura-2 and video-edge detection, respectively. Postrest potentiation (PRP) and sarcoplasmic reticulum Ca2+ load were used to assess propofol-induced changes in the activity of the NCX. Propofol (10 microM) increased PRP in diabetic cardiomyocytes but had no effect on PRP in normal cardiomyocytes. Removal of sodium enhanced and KB-R7943 (reverse mode NCX inhibitor) blocked PRP in both normal and diabetic cardiomyocytes. In the absence of sodium, propofol enhanced PRP in diabetic cardiomyocytes but had no additional effect in normal cardiomyocytes. KB-R7943 completely blocked propofol-induced potentiation of peak intracellular concentration of Ca2+ and shortening in both cell types. Propofol increased sarcoplasmic reticulum Ca2+ load and prolonged removal of cytosolic Ca2+ in diabetic cardiomyocytes, but not in normal cardiomyocytes. Removal of sodium enhanced propofol-induced increases in sarcoplasmic reticulum Ca2+ load and further prolonged removal of cytosolic Ca2+, whereas KB-R7943 completely blocked propofol-induced increase in sarcoplasmic reticulum Ca2+ load. Protein kinase C inhibition with bisindolylmaleimide I prevented the propofol-induced increase in PRP and prolongation in Ca2+ removal. These data suggest that propofol enhances PRP via activation of reverse mode NCX, but attenuates Ca2+ removal from the cytosol via inhibition of forward mode NCX in diabetic cardiomyocytes. The actions of propofol are mediated via a protein kinase C-dependent pathway.

Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes

Store-operated Ca2+ entry (SOCE), which is Ca2+ entry triggered by the depletion of intracellular Ca2+ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca2+ loading (load(SR)) that was not altered by inhibition of L-type Ca2+ channels, reverse mode Na+/Ca2+ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca2+] to 0 mM or adding either 0.5 mM Zn2+ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 microM) inhibited load(SR) at rest. Interestingly, inhibition of forward mode NCX with 30 microM KB-R7943 stimulated SOCE significantly and resulted in enhanced load(SR). In addition, manipulation of the extracellular and intracellular Na+ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca2+ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca2+ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca2+ overload and arrhythmogenesis.

Quantitative Fluorescence Measurement of Cardiac Na+/Ca2+ Exchanger Inhibition by Kinetic Analysis in Stably Transfected HEK293 Cells

We developed a method to quantitatively evaluate the potency of Na+/Ca2+ exchanger (NCX) inhibitors with fluorescence microscopy in NCX1-transfected HEK 293 cells. The reverse mode and forward mode NCX activities were measured as the ascending slope of the early phase increase in cytoplasmic Ca2+ concentration after change to low Na+ extracellular solution and the descending rate (inverse of the exponential time constant) on return to normal solution, respectively. Both modes of NCX were inhibited by SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) and KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), and the concentration-inhibition relationships for both inhibitors were in good agreement with those previously reported in voltage clamped cardiomyocytes.

Effects of Na +-Ca 2+ exchanger activity on the α-amino-3-hydroxy-5-methyl-4-isoxazolone-propionate-induced Ca 2+ influx in cerebellar Purkinje neurons

Variations in intracellular calcium activity ([Ca 2+] i) play crucial roles in information processing in Purkinje neurons such as synaptic plasticity. Although Na +-Ca 2+ exchanger (NCX) has been shown to participate in the regulation of homeostasis and secretion in neuronal cells, the physiological role of NCX in Purkinje neurons, such as a role in cerebellar synaptic plasticity, is not well understood. NCX in acutely dissociated rat Purkinje neurons was identified by double staining with anti-calbindin D-28k antibody and anti-NCX antibody. The physiological activity of NCX was examined by measuring transient intracellular Ca 2+ changes resulting from the Ca 2+ influx via reverse mode of NCX (with 0 mM Na +/2.5 mM Ca 2+ solutions) and the efflux via the forward mode of NCX (with 140 mM Na +/0 mM Ca 2+ solutions). This transient increase in Ca 2+ concentration was not elicited in the cells pretreated with NCX antisense oligodeoxynucleotides. And the Ca 2+ influx resulting from the reverse mode of NCX was significantly reduced by 2-[2-[4-(4-nitrobenyloxy) phenyl] ethyl] isothiourea methanesulfonate, while the Ca 2+ efflux via forward mode was inhibited by bepridil. The physiological role of NCX in synaptic function was studied by measuring Ca 2+ transients induced by α-amino-3-hydroxy-5-methyl-4-isoxazolone-propionate (AMPA) receptor activation. This AMPA-evoked response was decreased with the inhibition of NCX forward mode and also, to less degree, with the inhibition of reverse mode. In antisense oligodeoxynucleotides pretreated cells, the AMPA-evoked response was also reduced, as was the case in NCX-inhibitor treated cells. The inhibition of NCX activity had depressant effects on Ca 2+ transients induced by AMPA receptor activation. These results suggest that NCX plays a physiological role in modulating the activity of cerebellar Purkinje neurons, such as synaptic plasticity, via interaction with AMPA receptors in Purkinje neurons.

Synthesis and structure–activity relationships of phenoxypyridine derivatives as novel inhibitors of the sodium–calcium exchanger

The sodium–calcium exchanger (NCX) is known as the transporter that controls the concentration of Ca 2+ in cardiac myocytes. In the setting of heart failure and myocardial ischemia-reperfusion, NCX underlies an arrhythmogenic transient inward current responsible for delayed after––depolarizations and nonreentrant initiation of ventricular tachycardia. NCX is an attractive target for treatment in heart failure and myocardial ischemia-reperfusion. We have designed and synthesized a series of phenoxypyridine derivatives, based on compound 3. These derivatives have been evaluated for their inhibitory activity against both the reverse and forward mode of NCX in CCL39 cells. We have discovered several novel potent NCX inhibitors ( 39q, 48k), which have a high selectivity for reverse NCX inhibitory activity.