Inhibition of Voltage-Gated Na+ Currents Exerted by KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea), an Inhibitor of Na+-Ca2+ Exchanging Process.

KB-R7943, an isothiourea derivative, has been recognized as an inhibitor in the reverse mode of the Na+-Ca2+ exchanging process. This compound was demonstrated to prevent intracellular Na+-dependent Ca2+ uptake in intact cells; however, it is much less effective at preventing extracellular Na+-dependent Ca2+ efflux. Therefore, whether or how this compound may produce any perturbations on other types of ionic currents, particularly on voltage-gated Na+ current (INa), needs to be further studied. In this study, the whole-cell current recordings demonstrated that upon abrupt depolarization in pituitary GH3 cells, the exposure to KB-R7943 concentration-dependently depressed the transient (INa(T)) or late component (INa(L)) of INa with an IC50 value of 11 or 0.9 µM, respectively. Likewise, the dissociation constant for the KB-R7943-mediated block of INa on the basis of a minimum reaction scheme was estimated to be 0.97 µM. The presence of benzamil or amiloride could suppress the INa(L) magnitude. The instantaneous window Na+ current (INa(W)) activated by abrupt ascending ramp voltage (Vramp) was suppressed by adding KB-R7943; however, subsequent addition of deltamethrin or tefluthrin (Tef) effectively reversed KB-R7943-inhibted INa(W). With prolonged duration of depolarizing pulses, the INa(L) amplitude became exponentially decreased; moreover, KB-R7943 diminished INa(L) magnitude. The resurgent Na+ current (INa(R)) evoked by a repolarizing Vramp was also suppressed by adding this compound; moreover, subsequent addition of ranolazine or Tef further diminished or reversed, respectively, its reduction in INa(R) magnitude. The persistent Na+ current (INa(P)) activated by sinusoidal voltage waveform became enhanced by Tef; however, subsequent application of KB-R7943 counteracted Tef-stimulated INa(P). The docking prediction reflected that there seem to be molecular interactions of this molecule with the hNaV1.2 or hNaV1.7 channels. Collectively, this study highlights evidence showing that KB-R7943 has the propensity to perturb the magnitude and gating kinetics of INa (e.g., INa(T), INa(L), INa(W), INa(R), and INa(P)) and that the NaV channels appear to be important targets for the in vivo actions of KB-R7943 or other relevant compounds.

Changes in cellular Ca2+ and Na+ regulation during the progression towards heart failure.

In adapting to disease and loss of tissue, the heart shows great phenotypic plasticity that involves changes to its structure, composition and electrophysiology. Together with parallel whole body cardiovascular adaptations, the initial decline in cardiac function resulting from the insult is compensated. However, in the long term, the heart muscle begins to fail and patients with this condition have a very poor prognosis, with many dying from disturbances of rhythm. The surviving myocytes of these hearts gain Na+ , which is positively inotropic because of alterations to Ca2+ fluxes mediated by the Na+ /Ca2+ exchange, but compromises Ca2+ -dependent energy metabolism in mitochondria. Uptake of Ca2+ into the sarcoplasmic reticulum (SR) is reduced because of diminished function of SR Ca2+ ATPases. The result of increased Ca2+ influx and reduced SR Ca2+ uptake is an increase in the diastolic cytosolic Ca2+ concentration, which promotes spontaneous SR Ca2+ release and induces delayed afterdepolarisations. Action potential duration prolongs because of increased late Na+ current and changes in expression and function of other ion channels and transporters increasing the probability of the formation of early afterdepolarisations. There is a reduction in T-tubule density and so the normal spatial arrangements required for efficient excitation-contraction coupling are compromised and lead to temporal delays in Ca2+ release from the SR. Therefore, the structural and electrophysiological responses that occur to provide compensation do so at the expense of (1) increasing the likelihood of arrhythmogenesis; (2) activating hypertrophic, apoptotic and Ca2+ signalling pathways; and (3) decreasing the efficiency of SR Ca2+ release.

The effect of iron on the expression levels of calcium related gene in cisplatin resistant epithelial ovarian cancer cells.

Aim: Anticancer drugs (chemotherapeutics) used in cancer treatment (chemotherapy) lead to drug resistance. This study was conducted to investigate the possible effect of iron on calcium homeostasis in epithelial ovarian cancer cells (MDAH-2774) and cisplatin-resistant cells of the same cell line (MDAH-2774/DDP). Methods: To develop MDAH-2774/DDP cells, MDAH-2774 (MDAH) cells were treated with cisplatin in dose increases of 5 µM between 0 µM and 70 µM. The effect of iron on the viability of MDAH and MDAH/DDP cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test at the end of 24 h incubation. Results: At increasing iron concentrations in MDAH and MDAH/DDP cells, the mRNA gene of fifteen genes [inositol 1,4,5-triphosphate receptor (IP3R)1/2/3, ryanodine receptor (RYR)1/2, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)1/2/3, Na+/Ca2+ exchange (NCX)1/2/3, and plasma membrane Ca2+ ATPase (PMCA)1/2/3/4] associated with Ca2+ differences in expression were determined by quantitative reverse transcription-polymerase chain reaction. Changes in IP3R2, RYR1, SERCA2, NCX3, PMCA1, and PMCA3 gene expressions were observed in iron treatment of MDAH/DDP cells, while changes were detected in iron treatment of MDAH cells in IP3R1/2/3, RYR1/2, SERCA1/2/3, NCX2/3, and PMCA1 expressions. Conclusions: This changes in the expression of calcium channels, pumps, and exchange proteins in the epithelial ovarian cancer cell line and in cisplatin-resistant epithelial ovarian cancer cells suggest that iron may have an important role in regulating calcium homeostasis. Due to differences in the expression of genes that play of an important role in the regulation of calcium homeostasis in the effect of iron, drug resistance can be prevented by introducing a new perspective on the use of inhibitors and activators of these genes and thus cytostatic treatment strategies.

The Effect of Estrogen on Intracellular Ca2+ and Na+ Regulation in Heart Failure.

Contradictory findings of estrogen supplementation in cardiac disease highlight the need to investigate the involvement of estrogen in the progression of heart failure in an animal model that lacks traditional comorbidities. Heart failure was induced by aortic constriction (AC) in female guinea pigs. Selected AC animals were ovariectomized (ACOV), and a group of these received 17ß-estradiol supplementation (ACOV+E). One hundred-fifty days post-AC surgery, left-ventricular myocytes were isolated, and their electrophysiology and Ca2+ and Na+ regulation were examined. Long-term absence of ovarian hormones exacerbates the decline in cardiac function during the progression to heart failure. Estrogen supplementation reverses these aggravating effects.

Mechanisms of artemether toxicity on single cardiomyocytes and protective effect of nanoencapsulation.

BACKGROUND AND PURPOSE: The artemisinin derivative, artemether, has antimalarial activity with potential neurotoxic and cardiotoxic effects. Artemether in nanocapsules (NC-ATM) is more efficient than free artemether for reducing parasitaemia and increasing survival of Plasmodium berghei-infected mice. NCs also prevent prolongation of the QT interval of the ECG. Here, we assessed cellular cardiotoxicity of artemether and how this toxicity was prevented by nanoencapsulation. EXPERIMENTAL APPROACH: Mice were treated with NC-ATM orally (120 mg·kg-1 twice daily) for 4 days. Other mice received free artemether, blank NCs, and vehicle for comparison. We measured single-cell contraction, intracellular Ca2+ transient using fluorescent Indo-1AM Ca2+ dye, and electrical activity using the patch-clamp technique in freshly isolated left ventricular myocytes. The acute effect of free artemether was also tested on cardiomyocytes of untreated animals. KEY RESULTS: Artemether prolonged action potentials (AP) upon acute exposure (at 0.1, 1, and 10 µM) of cardiomyocytes from untreated mice or after in vivo treatment. This prolongation was unrelated to blockade of K+ currents, increased Ca2+ currents or promotion of a sustained Na+ current. AP lengthening was abolished by the NCX inhibitor SEA-0400. Artemether promoted irregular Ca2+ transients during pacing and spontaneous Ca2+ events during resting periods. NC-ATM prevented all effects. Blank NCs had no effects compared with vehicle. CONCLUSION AND IMPLICATIONS: Artemether induced NCX-dependent AP lengthening (explaining QTc prolongation) and disrupted Ca2+ handling, both effects increasing pro-arrhythmogenic risks. NCs prevented these adverse effects, providing a safe alternative to the use of artemether alone, especially to treat malaria.

Membrane current evoked by mitochondrial Na+-Ca2+ exchange in mouse heart.

The electrogenicity of mitochondrial Na+-Ca2+ exchange (NCXm) had been controversial and no membrane current through it had been reported. We succeeded for the first time in recording NCXm-mediated currents using mitoplasts derived from mouse ventricle. Under conditions that K+, Cl-, and Ca2+ uniporter currents were inhibited, extra-mitochondrial Na+ induced inward currents with 1 µM Ca2+ in the pipette. The half-maximum concentration of Na+ was 35.6 mM. The inward current was diminished without Ca2+ in the pipette, and was augmented with 10 µM Ca2+. The Na+-induced inward currents were largely inhibited by CGP-37157, an NCXm blocker. However, the reverse mode of NCXm, which should be detected as an outward current, was hardly induced by extra-mitochondrial application of Ca2+ with Na+ in the pipette. It was concluded that NCXm is electrogenic. This property may be advantageous for facilitating Ca2+ extrusion from mitochondria, which has large negative membrane potential.

Mitochondrial localization of NCXs: Balancing calcium and energy homeostasis.

It is well established that mitochondria are the main source of ATP production within cells. However, mitochondria have other remarkable functions, serving as important modulators of cellular Ca2+ signaling, and it is now generally recognized that control over Ca2+ homeostasis is intrinsically interwoven with mitochondrial abilities to adjust and tune ATP production. In this review, we describe the mechanisms that mitochondria use to balance Ca2+ homeostasis maintenance and cell energy metabolism. In recent years, the knowledge on the molecular machinery mediating Ca2+ influx/efflux has been improved and, albeit still open to further investigations, several lines of evidence converge on the hypothesis that plasma membrane Na+/Ca2+ exchanger (NCX) isoforms are also expressed at the mitochondrial level, where they contribute to the Ca2+ and Na+ homeostasis maintenance. In particular, the connection between mitochondrial NCX activity and metabolic substrates utilization is further discussed here. We also briefly focus on the alterations of both mitochondrial Ca2+ handling and cellular bioenergetics in neurodegenerative diseases, such as Parkinson's and Alzheimer's disease.

NCX activity generates spontaneous Ca2+ oscillations in the astrocytic leaflet microdomain.

The synergy between synaptic Glu release and astrocytic Glu-Na+ symport is essential to the signalling function of the tripartite synapse. Here we used kinetic data of astrocytic Glu transporters (EAAT) and the Na+/Ca2+ exchanger (NCX) to simulate Glu release, Glu uptake and subsequent Na+ and Ca2+ dynamics in the astrocytic leaflet microdomain following single release event. Model simulations show that Glu-Na+ symport differently affect intracellular [Na+] in synapses with different extent of astrocytic coverage. Surprisingly, NCX activity alone has been shown to generate markedly stable, spontaneous Ca2+ oscillation in the astrocytic leaflet. These on-going oscillations appear when NCX operates either in the forward or reverse direction. We conjecture that intrinsic NCX activity may play a prominent role in the generation of astrocytic Ca2+ oscillations.

Thyroid Hormone Diminishes Ca2+ Overload Induced by Hypoxia/Reoxygenation in Cardiomyocytes by Inhibiting Late Sodium Current and Reverse-Na+/Ca2+ Exchange Current.

BACKGROUND AND PURPOSE: Intracellular calcium concentration ([Ca2+]i) overload occurs in myocardial ischemia and -reperfusion. The augmentation of the late sodium current (INaL) causes intracellular Na+ accumulation and subsequent [Ca2+]i overload via the reverse mode of the Na+/Ca2+ exchange current (reverse-INCX), which can lead to arrhythmia and cardiac dysfunction. Thus, inhibition of INaL is a potential therapeutic approach for ischemic heart disease. The aim of this study was to investigate the effects of thyroid hormone on augmented INaL, reverse-INCX, altered action potential duration (APD), and [Ca2+]i concentration in hypoxia/reoxygenation (H/R)-induced ventricular myocytes in vitro. METHODS: The transient Na+ current (INaT), INaL, reverse-INCX, and APs were recorded using a whole-cell patch-clamp technique in neonatal mouse ventricular myocytes. [Ca2+]i concentration alteration were, respectively, observed by confocal microscopy and flow cytometry. RESULTS: Triiodothyronine (T3) pretreatment decreased the INaL in a concentration-dependent manner. H/R injury aggravated the INaL, INaT, and reverse-INCX in cardiomyocytes and increased the continuous accumulation of [Ca2+]i (p < 0.05). The application of T3 prior to H/R injury significantly decreased the increased INaL, INaT, and reverse-INCX and blunted the [Ca2+]i increase. Furthermore, T3 pretreatment shortened the APD induced by H/R injury. CONCLUSION: T3 inhibited H/R-increased INaL and reverse INCX augmentation, shortened the APD, and diminished [Ca2+]i overload, indicating a potential therapeutic use of T3 as an INaL inhibitor to maintain Ca2+ homeostasis and protect cardiomyocytes against H/R injury.

Cardiac Na+/Ca2+ exchange stimulators among cardioprotective drugs.

We previously reviewed our study of the pharmacological properties of cardiac Na+/Ca2+ exchange (NCX1) inhibitors among cardioprotective drugs, such as amiodarone, bepridil, dronedarone, cibenzoline, azimilide, aprindine, and benzyl-oxyphenyl derivatives (Watanabe et al. in J Pharmacol Sci 102:7-16, 2006). Since then we have continued our studies further and found that some cardioprotective drugs are NCX1 stimulators. Cardiac Na+/Ca2+ exchange current (INCX1) was stimulated by nicorandil (a hybrid ATP-sensitive K+ channel opener), pinacidil (a non-selective ATP-sensitive K+ channel opener), flecainide (an antiarrhythmic drug), and sodium nitroprusside (SNP) (an NO donor). Sildenafil (a phosphodiesterase-5 inhibitor) further increased the pinacidil-induced augmentation of INCX1. In paper, here I review the NCX stimulants that enhance NCX function among the cardioprotective agents we examined such as nicorandil, pinacidil, SNP, sildenafil and flecainide, in addition to atrial natriuretic (ANP) and dofetilide, which were reported by other investigators.

Calcium Microdomain Formation at the Perisynaptic Cradle Due to NCX Reversal: A Computational Study.

It has recently been proposed using a multi-compartmental mathematical model that negatively fixed charged membrane-associated sites constrain the flow of cations in perisynaptic astroglial processes. This restricted movement of ions between the perisynaptic cradle (PsC), principal astroglial processes and the astrocyte soma gives rise to potassium (K+) and sodium (Na+) microdomains at the PsC. The present paper extends the above model to demonstrate that the formation of an Na+ microdomain can reverse the Na+/Ca2+ exchanger (NCX) thus providing an additional source of calcium (Ca2+) at the PsC. Results presented clearly show that reversal of the Na+/Ca2+ exchanger is instigated by a glutamate transporter coupled increase in concentration of cytoplasmic [Na+]i at the PsC, which and instigates Ca2+ influx through the NCX. As the flow of Ca2+ along the astrocyte process and away from the PsC is also constrained by Ca2+ binding proteins, then a Ca2+ microdomain forms at the PsC. The paper also serves to demonstrate that the EAAT, NKA, and NCX represent the minimal requirement necessary and sufficient for the development of a Ca2+ microdomain and that these mechanisms directly link neuronal activity and glutamate release to the formation of localized Na+ and Ca2+ microdomains signals at the PsC. This local source of Ca2+ can provide a previously underexplored form of astroglial Ca2+ signaling.

Pinacidil, a KATP channel opener, stimulates cardiac Na+/Ca2+ exchanger function through the NO/cGMP/PKG signaling pathway in guinea pig cardiac ventricular myocytes.

Pinacidil, a nonselective ATP-sensitive K+ (KATP) channel opener, has cardioprotective effects for hypertension, ischemia/reperfusion injury, and arrhythmia. This agent abolishes early afterdepolarizations, delayed afterdepolarizations (DADs), and abnormal automaticity in canine cardiac ventricular myocytes. DADs are well known to be caused by the Na+/Ca2+ exchange current (INCX). In this study, we used the whole-cell patch-clamp technique and Fura-2/AM (Ca2+-indicator) method to investigate the effect of pinacidil on INCX in isolated guinea pig cardiac ventricular myocytes. In the patch-clamp study, pinacidil enhanced INCX in a concentration-dependent manner. The half-maximal effective concentration values were 23.5 and 23.0 µM for the Ca2+ entry (outward) and Ca2+ exit (inward) components of INCX, respectively. The pinacidil-induced INCX increase was blocked by L-NAME, a nitric oxide (NO) synthase inhibitor, by ODQ, a soluble guanylate cyclase inhibitor, and by KT5823, a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor, but not by N-2-mercaptopropyonyl glycine (MPG), a reactive oxygen species (ROS) scavenger. Glibenclamide, a nonselective KATP channel inhibitor, blocked the pinacidil-induced INCX increase, while 5-HD, a selective mitochondria KATP channel inhibitor, did not. In the Fura-2/AM study pinacidil also enhanced intracellular Ca2+ concentration, which was inhibited by L-NAME, ODQ, KT5823, and glibenclamide, but not by MPG and 5-HD. Sildenafil, a phosphodiesterase 5 inhibitor, increased further the pinacidil-induced INCX increase. Sodium nitroprusside, a NO donor, also increased INCX. In conclusion, pinacidil may stimulate cardiac Na+/Ca2+ exchanger (NCX1) by opening plasma membrane KATP channels and activating the NO/cGMP/PKG signaling pathway.

TRPC6 and TRPC4 Heteromultimerization Mediates Store Depletion-Activated NCX1 Reversal in Proliferative Vascular Smooth Muscle Cells.

Store depletion has been shown to induce Ca2+ entry by Na+/Ca+ exchange (NCX) 1 reversal in proliferative vascular smooth muscle cells (VSMCs). The study objective was to investigate the role of transient receptor potential canonical (TRPC) channels in store depletion and NCX1 reversal in proliferative VSMCs. In cultured VSMCs, expressing TRPC1, TRPC4, and TRPC6, the removal of extracellular Na+ was followed by a significant increase of cytosolic Ca2+ concentration that was inhibited by KBR, a selective NCX1 inhibitor. TRPC1 knockdown significantly suppressed store-operated, channel-mediated Ca2+ entry, but TRPC4 knockdown and TRPC6 knockdown had no effect. Separate knockdown of TRPC1, TRPC4, or TRPC6 did not have a significant effect on thapsigargin-initiated Na+ increase in the peripheral regions with KBR treatment, but knockdown of both TRPC4 and TRPC6 did. Stromal interaction molecule (STIM)1 knockdown significantly reduced TRPC4 and TRPC6 binding. The results demonstrated that TRPC4-TRPC6 heteromultimerization linked Ca2+ store depletion and STIM1 accumulation with NCX reversal in proliferative VSMCs.

The Na+/Ca2+ exchanger and the Plasma Membrane Ca2+-ATPase in ß-cell function and diabetes.

The rat pancreatic ß-cell expresses 6 splice variants of the Plasma Membrane Ca2+-ATPase (PMCA) and two splice variants of the Na+/Ca2+ exchanger 1 (NCX1). In the ß-cell Na+/Ca2+ exchange displays a high capacity, contributes to both Ca2+ outflow and influx and participates to the control of insulin release. Gain of function studies show that overexpression of PMCA2 or NCX1 leads to endoplasmic reticulum (ER) Ca2+ depletion with subsequent ER stress, decrease in ß-cell proliferation and ß-cell death by apoptosis. Loss of function studies show, on the contrary, that heterozygous inactivation of NCX1 (Ncx1+/-) leads to an increase in ß-cell function and a 5 fold increase in both ß-cell mass and proliferation. The mutation also increases ß-cell resistance to hypoxia, and Ncx1+/- islets show a 2-4 times higher rate of diabetes cure than Ncx1+/+ islets when transplanted in diabetic animals. Thus, down-regulation of the Na+/Ca2+ exchanger leads to various changes in ß-cell function that are opposite to the major abnormalities seen in diabetes. In addition, the ß-cell includes the mutually exclusive exon B in the alternative splicing region of NCX1, which confers a high sensitivity of its NCX splice variants (NCX1.3 & 1.7) to the inhibitory action of compounds like KBR-7943. Heterozygous inactivation of PMCA2 leads to apparented, though not completely similar results.These provide 2 unique models for the prevention and treatment of ß-cell dysfunction in diabetes and following islet transplantation.

Peptidyl-Prolyl Isomerase 1 Regulates Ca2+ Handling by Modulating Sarco(Endo)Plasmic Reticulum Calcium ATPase and Na2+/Ca2+ Exchanger 1 Protein Levels and Function.

BACKGROUND: Aberrant Ca2+ handling is a prominent feature of heart failure. Elucidation of the molecular mechanisms responsible for aberrant Ca2+ handling is essential for the development of strategies to blunt pathological changes in calcium dynamics. The peptidyl-prolyl cis-trans isomerase peptidyl-prolyl isomerase 1 (Pin1) is a critical mediator of myocardial hypertrophy development and cardiac progenitor cell cycle. However, the influence of Pin1 on calcium cycling regulation has not been explored. On the basis of these findings, the aim of this study is to define Pin1 as a novel modulator of Ca2+ handling, with implications for improving myocardial contractility and potential for ameliorating development of heart failure. METHODS AND RESULTS: Pin1 gene deletion or pharmacological inhibition delays cytosolic Ca2+ decay in isolated cardiomyocytes. Paradoxically, reduced Pin1 activity correlates with increased sarco(endo)plasmic reticulum calcium ATPase (SERCA2a) and Na2+/Ca2+ exchanger 1 protein levels. However, SERCA2a ATPase activity and calcium reuptake were reduced in sarcoplasmic reticulum membranes isolated from Pin1-deficient hearts, suggesting that Pin1 influences SERCA2a function. SERCA2a and Na2+/Ca2+ exchanger 1 associated with Pin1, as revealed by proximity ligation assay in myocardial tissue sections, indicating that regulation of Ca2+ handling within cardiomyocytes is likely influenced through Pin1 interaction with SERCA2a and Na2+/Ca2+ exchanger 1 proteins. CONCLUSIONS: Pin1 serves as a modulator of SERCA2a and Na2+/Ca2+ exchanger 1 Ca2+ handling proteins, with loss of function resulting in impaired cardiomyocyte relaxation, setting the stage for subsequent investigations to assess Pin1 dysregulation and modulation in the progression of heart failure.

Contribution of small conductance K+ channels to sinoatrial node pacemaker activity: insights from atrial-specific Na+ /Ca2+ exchange knockout mice.

KEY POINTS: Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain. We identified all three isoforms of Ca2+ -activated small conductance K+ (SK) channels in the murine SAN. SK channel blockade slows repolarization and subsequent depolarization of SAN cells. In the atrial-specific Na+ /Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing. These findings suggest that Ca2+ -sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking. SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+ . ABSTRACT: Small conductance K+ (SK) channels have been implicated as modulators of spontaneous depolarization and electrical conduction that may be involved in cardiac arrhythmia. However, neither their presence nor their contribution to sinoatrial node (SAN) pacemaker activity has been investigated. Using quantitative PCR (q-PCR), immunostaining and patch clamp recordings of membrane current and voltage, we identified all three SK isoforms (SK1, SK2 and SK3) in mouse SAN. Inhibition of SK channels with the specific blocker apamin prolonged action potentials (APs) in isolated SAN cells. Apamin also slowed diastolic depolarization and reduced pacemaker rate in isolated SAN cells and intact tissue. We investigated whether the Ca2+ -sensitive nature of SK channels could explain arrhythmic SAN pacemaker activity in the atrial-specific Na+ /Ca2+ exchange (NCX) knockout (KO) mouse, a model of cellular Ca2+ overload. SAN cells isolated from the NCX KO exhibited higher SK current than wildtype (WT) and apamin prolonged their APs. SK blockade partially suppressed the arrhythmic burst pacing pattern of intact NCX KO SAN tissue. We conclude that SK channels have demonstrable effects on SAN pacemaking in the mouse. Their Ca2+ -dependent activation translates changes in cellular Ca2+ into a repolarizing current capable of modulating regular pacemaking. This Ca2+ dependence also promotes abnormal automaticity when these channels are hyperactivated by elevated Ca2+ . We propose SK channels as a potential target for modulating SAN rate, and for treating patients affected by SAN dysfunction, particularly in the setting of Ca2+ overload.

Identification of residues that control Li+ versus Na+ dependent Ca2+ exchange at the transport site of the mitochondrial NCLX.

BACKGROUND: The Na+/Ca2+/Li+ exchanger (NCLX) is a member of the Na+/Ca2+ exchanger family. NCLX is unique in its capacity to transport both Na+ and Li+, unlike other members, which are Na+ selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li+ or Na+ selective Ca2+ transport and map their putative location on NCLX cation transport site. METHOD: We combined molecular modeling to map transport site of NCLX with euryarchaeal H+/Ca2+ exchanger, CAX_Af, and fluorescence analysis to monitor Li+ versus Na+ dependent mitochondrial Ca2+ efflux of transport site mutants of NCLX in permeabilized cells. RESULT: Mutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca2+ exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li+/Ca2+ exchange activity but a reduced Na+/Ca2+ exchange activity. On the other hand, D471A showed dramatically reduced Li+/Ca2+ exchange, but Na+/Ca2+ exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca2+ binding residues was required to completely abolish both Na+/Ca2+ and Li+/Ca2+ exchange activities. CONCLUSIONS: We identified distinct Na+ and Li+ selective residues in the NCLX transport site. We propose that functional segregation in Li+ and Na+ sites reflects the functional properties of NCLX required for Ca2+ exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane. GENERAL SIGNIFICANCE: The results of this study provide functional insights into the unique Li+ and Na+ selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Inhibitory effect of YM-244769, a novel Na+/Ca2+ exchanger inhibitor on Na+/Ca2+ exchange current in guinea pig cardiac ventricular myocytes.

Recently, YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) has been reported as a new potent and selective Na+/Ca2+ exchange (NCX) inhibitor by using various cells transfected with NCX using the 45Ca2+ fluorescent technique. However, the electrophysiological study of YM-244769 on NCX had not been performed in the mammalian heart. We examined the effects of YM-244769 on NCX current (INCX) in single cardiac ventricular myocytes of guinea pigs by using the whole-cell voltage clamp technique. YM-244769 suppressed the bidirectional INCX in a concentration-dependent manner. The IC50 values of YM-244769 for the bidirectional outward and inward INCX were both about 0.1 µM. YM-244769 suppressed the unidirectional outward INCX (Ca2+ entry mode) with an IC50 value of 0.05 µM. The effect on the unidirectional inward INCX (Ca2+ exit mode) was less potent, with 10 µM of YM-244769 resulting in the inhibition of only about 50 %. At 5 mM intracellular Na+ concentration, YM-244769 suppressed INCX more potently than it did at 0 mM [Na+]i. Intracellular application of trypsin via the pipette solution did not change the blocking effect of YM-244769. In conclusion, YM-244769 inhibits the Ca2+ entry mode of NCX more potently than the Ca2+ exit mode, and inhibition by YM-244769 is [Na+]i-dependent and trypsin-insensitive. These characteristics are similar to those of other benzyloxyphenyl derivative NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The potency of YM-244769 as an NCX1 inhibitor is higher than those of KB-R7943 and SN-6 and is similar to that of SEA0400.

Contribution of elevated intracellular calcium to pulmonary arterial myocyte alkalinization during chronic hypoxia.

In the lung, exposure to chronic hypoxia (CH) causes pulmonary hypertension, a debilitating disease. Development of this condition arises from increased muscularity and contraction of pulmonary vessels, associated with increases in pulmonary arterial smooth muscle cell (PASMC) intracellular pH (pHi) and Ca(2+) concentration ([Ca(2+)]i). In this study, we explored the interaction between pHi and [Ca(2+)]i in PASMCs from rats exposed to normoxia or CH (3 weeks, 10% O2). PASMC pHi and [Ca(2+)]i were measured with fluorescent microscopy and the dyes BCECF and Fura-2. Both pHi and [Ca(2+)]i levels were elevated in PASMCs from hypoxic rats. Exposure to KCl increased [Ca(2+)]i and pHi to a similar extent in normoxic and hypoxic PASMCs. Conversely, removal of extracellular Ca(2+) or blockade of Ca(2+) entry with NiCl2 or SKF 96365 decreased [Ca(2+)]i and pHi only in hypoxic cells. Neither increasing pHi with NH4Cl nor decreasing pHi by removal of bicarbonate impacted PASMC [Ca(2+)]i. We also examined the roles of Na(+)/Ca(2+) exchange (NCX) and Na(+)/H(+) exchange (NHE) in mediating the elevated basal [Ca(2+)]i and Ca(2+)-dependent changes in PASMC pHi. Bepridil, dichlorobenzamil, and KB-R7943, which are NCX inhibitors, decreased resting [Ca(2+)]i and pHi only in hypoxic PASMCs and blocked the changes in pHi induced by altering [Ca(2+)]i. Exposure to ethyl isopropyl amiloride, an NHE inhibitor, decreased resting pHi and prevented changes in pHi due to changing [Ca(2+)]i. Our findings indicate that, during CH, the elevation in basal [Ca(2+)]i may contribute to the alkaline shift in pHi in PASMCs, likely via mechanisms involving reverse-mode NCX and NHE.

Nicorandil stimulates a Na⁺/Ca²âº exchanger by activating guanylate cyclase in guinea pig cardiac myocytes.

Nicorandil, a hybrid of an ATP-sensitive K(+) (KATP) channel opener and a nitrate generator, is used clinically for the treatment of angina pectoris. This agent has been reported to exert antiarrhythmic actions by abolishing both triggered activity and spontaneous automaticity in an in vitro study. It is well known that delayed afterdepolarizations (DADs) are caused by the Na(+)/Ca(2+) exchange current (I NCX). In this study, we investigated the effect of nicorandil on the cardiac Na(+)/Ca(2+) exchanger (NCX1). We used the whole-cell patch clamp technique and the Fura-2/AM (Ca(2+) indicator) method to investigate the effect of nicorandil on I NCX in isolated guinea pig ventricular myocytes and CCL39 fibroblast cells transfected with dog heart NCX1. Nicorandil enhanced I NCX in a concentration-dependent manner. The EC50 (half-maximum concentration for enhancement of the drug) values were 15.0 and 8.7 µM for the outward and inward components of I NCX, respectively. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a membrane-permeable analog of guanosine 3',5'-cyclic monophosphate (cGMP), enhanced I NCX. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor (10 µM), completely abolished the nicorandil-induced I NCX increase. Nicorandil increased I NCX in CCL39 cells expressing wild-type NCX1 but did not affect mutant NCX1 without a long intracellular loop between transmembrane segments (TMSs) 5 and 6. Nicorandil at 100 µM abolished DADs induced by electrical stimulation with ouabain. Nicorandil enhanced the function of NCX1 via guanylate cyclase and thus may accelerate Ca(2+) exit via NCX1. This may partially contribute to the cardioprotection by nicorandil in addition to shortening action potential duration (APD) by activating KATP channels.