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.

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.

Cardioprotective and functional effects of levosimendan and milrinone in mice with cecal ligation and puncture-induced sepsis.

Levosimendan and milrinone may be used in place of dobutamine to increase cardiac output in septic patients with a low cardiac output due to impaired cardiac function. The effects of the two inotropic agents on cardiac inflammation and left ventricular (LV) performance were examined in mice with cecal ligation and puncture (CLP)-induced sepsis. CLP mice displayed significant cardiac inflammation, as indicated by highly increased pro-inflammatory cytokines and neutrophil infiltration in myocardial tissues. When continuously given, levosimendan prevented but milrinone exaggerated cardiac inflammation, but they significantly reduced the elevations in plasma cardiac troponin-I and heart-type fatty acid-binding protein, clinical markers of cardiac injury. Echocardiographic assessment of cardiac function showed that the effect of levosimendan, given by an intravenous bolus injection, on LV performance was impaired in CLP mice, whereas milrinone produced inotropic responses equally in sham-operated and CLP mice. A lesser effect of levosimendan on LV performance after CLP was also found in spontaneously beating Langendorff-perfused hearts. In ventricular myocytes isolated from control and CLP mice, levosimendan, but not milrinone, caused a large increase in the L-type calcium current. This study represents that levosimendan and milrinone have cardioprotective properties but provide different advantages and drawbacks to cardiac inflammation/dysfunction in sepsis.

Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes.

Andersen-Tawil syndrome (ATS) is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs). Multi-electrode arrays (MEAs) were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca2+ imaging of cells loaded with the Ca2+ indicator Fluo-4 enabled us to examine intracellular Ca2+ handling properties, and we found a significantly higher incidence of irregular Ca2+ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca2+ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na+/Ca2+exchanger (NCX) inhibitor, KB-R7943, was also found to suppress the irregular Ca2+ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (INCX). ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of INCX.

A subset of cerebrovascular pericytes originates from mature macrophages in the very early phase of vascular development in CNS.

Pericytes are believed to originate from either mesenchymal or neural crest cells. It has recently been reported that pericytes play important roles in the central nervous system (CNS) by regulating blood-brain barrier homeostasis and blood flow at the capillary level. However, the origin of CNS microvascular pericytes and the mechanism of their recruitment remain unknown. Here, we show a new source of cerebrovascular pericytes during neurogenesis. In the CNS of embryonic day 10.5 mouse embryos, CD31+F4/80+ hematopoietic lineage cells were observed in the avascular region around the dorsal midline of the developing midbrain. These cells expressed additional macrophage markers such as CD206 and CD11b. Moreover, the CD31+F4/80+ cells phagocytosed apoptotic cells as functionally matured macrophages, adhered to the newly formed subventricular vascular plexus, and then divided into daughter cells. Eventually, these CD31+F4/80+ cells transdifferentiated into NG2/PDGFRß/desmin-expressing cerebrovascular pericytes, enwrapping and associating with vascular endothelial cells. These data indicate that a subset of cerebrovascular pericytes derive from mature macrophages in the very early phase of CNS vascular development, which in turn are recruited from sites of embryonic hematopoiesis such as the yolk sac by way of blood flow.

Suppressive Effect of Carvedilol on Na+/Ca2+ Exchange Current in Isolated Guinea-Pig Cardiac Ventricular Myocytes.

BACKGROUND AND AIMS: Carvedilol ((+/-)-1-(carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol), a ß-adrenoceptor-blocker, has multi-channel blocking and vasodilator properties. This agent dose-dependently improves left ventricular function and reduces mortality in patients with arrhythmia and chronic heart failure. However, the effect of carvedilol on the cardiac Na+/Ca2+ exchanger (NCX1) has not been investigated. METHODS AND RESULTS: We examined the effects of carvedilol and metoprolol, 2 ß-blockers, on Na+/Ca2+ exchange current (INCX) in guinea-pig cardiac ventricular cells and fibroblasts expressing dog cardiac NCX1. Carvedilol suppressed INCX in a concentration-dependent manner but metoprolol did not. IC50 values for the Ca2+ influx (outward) and efflux (inward) components of INCX were 69.7 and 61.5 µmol/l, respectively. Carvedilol at 100 µmol/l inhibited INCX in CCL39 cells expressing wild type NCX1 similar to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 µmol/l abolished ouabain-induced delayed afterdepolarizations. CONCLUSION: Carvedilol inhibited cardiac NCX in a concentration-dependent manner in isolated cardiac ventricles, but metoprolol did not. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.

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.

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.

Inhibitory effect of cibenzoline on Na+/Ca2+ exchange current in guinea-pig cardiac ventricular myocytes.

The effect of cibenzoline, a class I antiarrhythmic drug, on Na(+)/Ca(2+) exchange current (I(NCX)) was investigated using the patch-clamp method. Cibenzoline inhibited the bi-directional I(NCX) in a concentration-dependent manner. The IC(50) values of cibenzoline for the outward and inward components of I(NCX) were 77 and 84 µM, respectively, with Hill coefficients of 1. Intracellular application of trypsin via the pipette solution did not change the inhibitory effect of cibenzoline. The inhibitory effect of cibenzoline on I(NCX) at pH(o) 6.5 was smaller than those at pH(o) 7.4 and pH(o) 8.2. We conclude that cibenzoline inhibits I(NCX) in supra-therapeutic concentrations.

Inhibitory effect of azimilide on Na+/Ca2+ exchange current in guinea-pig cardiac myocytes.

We examined the effect of azimilide, a class III antiarrhythmic drug, on Na(+)/Ca(2+) exchange current (I(NCX)) in guinea-pig cardiac single ventricular cells. External application of azimilide suppressed bi-directional I(NCX) in a concentration-dependent manner. IC(50) values for outward and inward I(NCX) were 45 and 40 µM, respectively, with Hill coefficients of 1. Azimilide attenuated I(NCX) in the presence of trypsin in the patch pipette, indicating that azimilide is a trypsin-insensitive NCX inhibitor. Delayed afterdepolarization induced by electrical stimulation with ouabain disappeared in the presence of 30 µM azimilide. We conclude that azimilide inhibits NCX at supratherapeutic concentrations.

Acidic preconditioning inhibits Na+/H+ and Na+/Ca2+ exchanger interaction via PKCepsilon in guinea-pig ventricular myocytes.

An interaction between the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/H(+) exchanger (NHE) induces reperfusion injury. We investigated the effect of brief repetitive acidosis as acidic preconditioning on NCX and NHE interaction during recovery from acidosis. NCX current with the reversal potential was measured in guinea-pig ventricular myocytes using the whole-cell voltage clamp. The cells were exposed to 5 min of acidosis preceded by two episodes of brief acidosis as acidic preconditioning. Acidosis inhibited NCX current and upon recovery shifted its reversal potential in the negative direction. The shift was prevented by cariporide, but was augmented by a high concentration of phorbol 13-myristate acetate (PMA). Acidic preconditioning prevented the shift, but not in the presence of a selective PKCepsilon inhibitor. A low concentration of PMA, which activates PKCepsilon selectively, prevented the shift, but together with PKCepsilon inhibitor (epsilonV1-2) restored the shift during recovery. 5-Hydroxydecanoate inhibited the effects of acidic preconditioning and those of both low and high concentrations of PMA. The negative shift of NCX reversal potential during recovery from acidosis may be due to [Na(+)](i) accumulation by the NHE. Acidic preconditioning prevented the shift most likely by activating PKCepsilon, which in turn inhibited the NHE. The NHE-NCX interaction may be one of the important end-effectors of preconditioning.

Arsenic trioxide augments Chk2/p53-mediated apoptosis by inhibiting oncogenic Wip1 phosphatase.

The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damage-induced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.

Acute inhibitory effect of dronedarone, a noniodinated benzofuran analogue of amiodarone, on Na+/Ca2+ exchange current in guinea pig cardiac ventricular myocytes.

Using the whole-cell voltage-clamp method, we examined an acute effect of dronedarone, a noniodinated benzofuran analogue of amiodarone, on Na+/Ca2+ exchange current (INCX) in guinea pig cardiac ventricular cells. The INCX was recorded by ramp pulses with a holding potential of -60 mV using a pipette solution containing 226 nM free Ca2+ (20 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and 10 mM Ca2+) and 20 mM Na+. The external solution contained 140 mM Na+, 1 mM Ca2+, and blockers of other currents and pumps such as Cs+, nifedipine, ryanodine, and ouabain. A selective potent NCX inhibitor, KB-R7943 (100 microM), was used to completely inhibit INCX. Dronedarone inhibited INCX in a concentration-dependent manner. The IC50 values for the outward and inward INCX inhibition were about 33 and 28 microM, respectively, with the Hill coefficient of 1 for both. The inhibitory effect of dronedarone at 50 microM on INCX did not change in the presence of trypsin in the pipette solution. Therefore, dronedarone is classified as a trypsin-insensitive NCX inhibitor and distinct from amiodarone which is a trypsin sensitive. We conclude that dronedarone inhibits INCX but the potency is tenfold less than that of amiodarone. Dronedarone may modestly inhibit INCX in a therapeutic concentration range.

Na+/Ca2+ exchange inhibitors: a new class of calcium regulators.

The Na(+)/Ca(2+) exchanger (NCX) is a bidirectional transporter that normally extrudes Ca(2+) from the cell (forward mode), but also brings Ca(2+) into the cell (reverse mode) under special conditions such as intracellular Na(+) (Na(+)(i)) accumulation or membrane depolarization. There are three mammalian NCX isoforms: NCX1 is widely expressed in the heart, kidney, brain, blood vessels, and so on; whereas the expression of NCX2 and NCX3 is limited mainly to the brain and skeletal muscle. The pharmacology of NCX inhibitors has been studied extensively since the development of KB-R7943, a prototype benzyloxyphenyl NCX inhibitor, in 1996. Currently, experiments are actively progressing with more selective inhibitors: SEA0400, SN-6, and YM-244769. Intriguingly, the inhibitory potency of benzyloxyphenyl NCX inhibitors is directly coupled to the rate of Na(+)(i)-dependent inactivation. Therefore, the benzyloxyphenyl inhibitors are apparently dormant during the forward mode under normal conditions (low Na(+)(i)), but become effective during the reverse mode under pathological conditions (high Na(+)(i)). This should be an ideal profile for calcium regulators against Na(+)(i)-related diseases, such as ischemia/reperfusion injuries, salt-dependent hypertension, and digitalis arrhythmia. Existing ion channel blockers, such as amiodarone, dronedarone, bepridil, aprindine, and cibenzoline, have been found to have an NCX inhibitory action. It is possible that this property is partly responsible for their antiarrhythmic and cardioprotective effects. This article presents the characteristics of selective and non-selective NCX inhibitors and their therapeutic potential as a new calcium regulator.

Characterization of SN-6, a novel Na+/Ca2+ exchange inhibitor in guinea pig cardiac ventricular myocytes.

We examined the effect of SN-6, a new benzyloxyphenyl Na(+)/Ca(2+) exchange (NCX) inhibitor on the Na(+)/Ca(2+) exchange current (I(NCX)) and other membrane currents in isolated guinea pig ventricular myocytes using the whole-cell voltage-clamp technique. SN-6 suppressed I(NCX) in a concentration-dependent manner. The IC(50) values of SN-6 were 2.3 microM and 1.9 microM for the outward and inward components of the bi-directional I(NCX), respectively. On the other hand, SN-6 suppressed the outward uni-directional I(NCX) more potently (IC(50) value of 0.6 microM) than the inward uni-directional I(NCX). SN-6 at 10 microM inhibited the uni-directional inward I(NCX) by only 22.4+/-3.1%. SN-6 and KB-R7943 suppressed I(NCX) more potently when intracellular Na(+) concentration was higher. Thus, both drugs inhibit NCX in an intracellular Na(+) concentration-dependent manner. Intracellular application of trypsin via a pipette solution did not change the blocking effect of SN-6 on I(NCX). Therefore, SN-6 is categorized as an intracellular-trypsin-insensitive NCX inhibitor. SN-6 at 10 microM inhibited I(Na), I(Ca), I(K) and I(K1) by about 13%, 34%, 33% and 13%, respectively. SN-6 at 10 microM shortened the action potential duration at 50% repolarization (APD(50)) by about 34%, and that at 90% repolarization (APD(90)) by about 25%. These results indicate that SN-6 inhibits NCX in a similar manner to that of KB-R7943. However, SN-6 at 10 microM affected other membrane currents less potently than KB-R7943.

Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes.

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.

Electrophysiological effects of SN-6, a novel Na+/Ca2+ exchange inhibitor on membrane currents in guinea pig ventricular myocytes.

We examined the effect of SN-6 on the Na+/Ca2+ exchanger (NCX) current (I(NCX)) and other membrane currents in isolated guinea pig ventricular myocytes using the whole-cell voltage clamp technique. SN-6 suppressed the bidirectional I(NCX) in a concentration-dependent manner. The IC50 values of SN-6 were 2.3 microM and 1.9 microM for the outward and inward components of the bidirectional I(NCX), respectively. On the other hand, SN-6 suppressed the unidirectional outward I(NCX) more potently than the inward I(NCX), with an IC(50) value of 0.6 microM. SN-6 at 10 microM inhibited the unidirectional inward I(NCX) by only 22.4 +/- 3.1%. SN-6 suppressed I(NCX) more potentially when intracellular Na+ concentration became higher. SN-6 inhibited I(Na), I(Ca), I(Kr), I(Ks), and I(K1) by about 13%, 34%, 33%, 18%, and 13%, respectively. SN-6 shortened the action potential duration (APD) by about 34% and 25% at APD(50) and APD(90), respectively. These results indicate that SN-6 inhibits NCX in a similar manner to that of KB-R7943. SN-6 and KB-R7943 inhibit the unidirectional outward I(NCX) more potently than the unidirectional inward I(NCX). Both drugs inhibit NCX in an intracellular Na+ concentration-dependent manner. However, SN-6 affected other membrane currents less potently than KB-R7943.

Mechanism of Na+/Ca2+ exchanger activation by hydrogen peroxide in guinea-pig ventricular myocytes.

Using the whole-cell voltage clamp, we examined the mechanism of activation of the Na(+)/Ca(2+) exchanger (NCX) by hydrogen peroxide (H(2)O(2)) in isolated guinea-pig cardiac ventricular myocytes. Exposure to H(2)O(2) increased the NCX current. The effect was inhibited by cariporide, an inhibitor of the Na(+)/H(+) exchanger (NHE), suggesting that there are NHE-dependent and -independent pathways in the effect of H(2)O(2) on NCX. In addition, both pathways were blocked by edaravone, a hydroxyl radical (*OH) scavenger; pertussis toxin, a Galpha(i/o) protein inhibitor; and U0126, an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). On the other hand, wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited only the NHE-dependent pathway, while PP2, a Src family protein tyrosine kinase inhibitor, inhibited only the NHE-independent pathway. Taken together, our data suggest that H(2)O(2) increases the NCX current via two signal transduction pathways. The common pathway is the conversion of H(2)O(2) to *OH, which activates Galpha(i/o) protein and a mitogen-activated protein (MAP) kinase signaling pathway. Then, one pathway activates NHE with a PI3K-dependent mechanism and indirectly increases the NCX current. Another pathway involves activation of a Src family tyrosine kinase.

Protein kinase A catalytic subunit alters cardiac mitochondrial redox state and membrane potential via the formation of reactive oxygen species.

BACKGROUND: The identification of protein kinase A (PKA) anchoring proteins on mitochondria implies a direct effect of PKA on mitochondrial function. However, little is known about the relationship between PKA and mitochondrial metabolism. METHODS AND RESULTS: The effects of PKA on the mitochondrial redox state (flavin adenine dinucleotide (FAD)), mitochondrial membrane potential (DeltaPsi(m)) and reactive oxygen species (ROS) production were investigated in saponin-permeabilized rat cardiomyocytes. The PKA catalytic subunit (PKAcat; 50 unit/ml) increased FAD intensities by 56.6+/-7.9% (p<0.01), 2'7'-dichlorofluorescin diacetate (DCF) intensities by 10.5+/-3.3 fold (p<0.01) and depolarized DeltaPsi(m) to 48.1+/-9.5% of the control (p<0.01). Trolox (a ROS scavenger; 100 micromol/L) inhibited PKAcat-induced DeltaPsi(m), FAD and DCF alteration. PKAcat-induced DeltaPsi(m) depolarization was inhibited by an inhibitor of the inner membrane anion channel (IMAC), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS: 1 micromol/L) but not by an inhibitor of mitochondrial permeability transition pore (mPTP), cyclosporine A (100 nmol/L). CONCLUSIONS: PKAcat alters FAD and DeltaPsi(m) via mitochodrial ROS generation, and PKAcat-induced DeltaPsi(m) depolarization was not caused by mPTP but rather by DIDS-sensitive mechanisms, which could be caused by opening of the IMAC. The effects of PKA on mitochondrial function could be related to myocardial function under the condition of extensive beta-adrenergic stimulation.

Topics on the Na+/Ca2+ exchanger: pharmacological characterization of Na+/Ca2+ exchanger inhibitors.

Using the whole-cell voltage clamp, we examined acute effects of various agents on Na(+)/Ca(2+) exchange current (I(NCX)) in guinea-pig cardiac ventricular cells and transfected cells. Among the antiarrhythmic drugs, amiodarone, bepridil, dronedarone, cibenzoline, azimilide, and aprindine inhibited I(NCX) in a concentration-dependent manner. We also investigated the effects on NCX of 2,3-buanedione monoxim (BDM) and selective NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The presence of trypsin in the pipette solution attenuated the inhibitory effects on NCX of amiodarone, bepridil, and BDM, suggesting that these drugs inhibit NCX from the cytosolic side. In contrast, the trypsin-insensitive NCX inhibitors were aprindine, azimilide, dronedarone, cibenzoline, KB-R7943, SEA0400, and SN-6. KB-R7943, SEA0400, and SN-6 suppressed the uni-directional outward I(NCX) more potently than the uni-directional inward I(NCX). The mechanism of this mode-dependency is unknown, but is suggested to be related to intracellular Na(+) concentration.