Guinea Pig Cardiac Ventricular Myocytes Research Articles

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 23.0μM 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.

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.

Enhanced effect of VEGF165 on L-type calcium currents in guinea-pig cardiac ventricular myocytes

The mechanisms of vascular endothelial growth factor 165 (VEGF165) on electrical properties of cardiomyocytes have not been fully elucidated. The aim of this study is to test the hypothesis that VEGF165, an angiogenesis-initiating factor, affects L-type calcium currents (ICa,L) and cell membrane potential in cardiac myocytes by acting on VEGF type-2 receptors (VEGFR2). ICa,L and action potentials (AP) were recorded by the whole-cell patch clamp method in isolated guinea-pig ventricular myocytes treated with different concentrations of VEGF165 proteins. Using a VEGFR2 inhibitor, we also tested the receptor of VEGF165 in cardiomyocytes. We found that VEGF165 increased ICa,L in a concentration-dependent manner. SU5416, a VEGFR2 inhibitor, almost completely eliminated VEGF165-induced ICa,L increase. VEGF165 had no significant influence on action potential 90 (APD90) and other properties of AP. We conclude that in guinea-pig ventricular myocytes, ICa,L can be increased by VEGF165 in a concentration-dependent manner through binding to VEGFR2 without causing any significant alteration to action potential duration. Results of this study may further expound the safety of VEGF165 when used in the intervention of heart diseases.

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 β-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 β-blockers, on Na⁺/Ca²⁺ exchange current (I_NCX) in guinea-pig cardiac ventricular cells and fibroblasts expressing dog cardiac NCX1. Carvedilol suppressed I_NCX in a concentration-dependent manner but metoprolol did not. IC₅₀ values for the Ca²⁺ influx (outward) and efflux (inward) components of I_NCX were 69.7 and 61.5 µmol/l, respectively. Carvedilol at 100 μmol/l inhibited I_NCX 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 0.1μM. YM-244769 suppressed the unidirectional outward INCX (Ca2+ entry mode) with an IC50 value of 0.05μM. The effect on the unidirectional inward INCX (Ca2+ exit mode) was less potent, with 10μM of YM-244769 resulting in the inhibition of only about 50%. At 5mM intracellular Na+ concentration, YM-244769 suppressed INCX more potently than it did at 0mM [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.

Blocking effect of carvedilol, a beta‐blocker on Na+/Ca2+ exchange current in isolated cardiac ventricular cells of guinea pig

BackgroundCarvedilol has a1, b1, and b2‐adrenergic blocking effects together with the effects such as vasodilating, anti‐ischemic, anti‐oxidant, anti‐apoptotic, anti‐proliferate and anti‐inflammatory activities. Carvedilol inhibited IKr, IKs, ICa‐L and Ito in rabbit cardiac ventricular myocytes. On the other hand, metoprolol, a nonselective b‐blocker also has blocking several channels, including IK1, ICa and Ito in hearts. Murayama et al. (2013) reported that carvedilol and its analogues inhibit delayed after depolarizations (DADs) in Langendorff‐perfused rabbit hearts. It is well known that DADs are caused by the Na+/Ca2+ exchange current (INCX).MethodsWe examined the effects of carvedilol and metoprolol, two b‐blockers, on INCX by using the whole‐cell voltage‐clamp method in isolated guinea pig ventricles and CCL39 fibroblast cells expressing dog cardiac Na+/Ca2+ exchanger (NCX1).ResultsCarvedilol suppressed INCX in a concentration‐dependent manner but metoprolol did not in isolated guinea pig cardiac ventricular myocytes. IC50 values for the Ca2+ influx (outward) and efflux (inward) components of INCX were 69.7 μM and 61.5 μM, respectively. Carvedilol at 100 μM inhibited INCX in CCL39 cells expressing wild type NCX1 similarly to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 μM abolished ouabain‐induced DADs in isolated cardiac ventricular myocytes.ConclusionsCarvedilol suppressed bi‐directional INCX in a concentration‐dependent manner with the IC50 value of approximately 65 μM. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.

Putative small conductance Ca2+‐sensitive K+ channels isoforms and splice variants in mitochondria of guinea pig cardiac ventricular myocytes

Small Ca2+‐sensitive K+ channels (SKCa) exist in many cell types including cardiac atrial and ventricular. There are 3 isoforms of SKCa, i.e. SKCa 1, 2, and 3. Activation of SKCa channels may protect against ischemia (I) and reperfusion (R) injury. Recently we found SKCa channels exist in the inner mitochondrial membrane (IMM) of guinea pig cardiac ventricular myocytes and that SKCa channel opening protects against cardiac IR injury. It is unknown which SKCa isoforms and splice variants are specific for cardiac cell IMM so we searched for the gene products that might confer their distinct structural and functional characteristics. SKCa is expressed in cardiac ventricular cells, so ventricular tissue devoid of endovascular cells were used to extract total RNA, which was then reverse transcribed to cDNA. The resulting cDNA was amplified with SKCa 1, 2 and 3 gene‐specific primers. Results showed that both SKCa 2 and 3 were amplified, which suggests both isoforms may be expressed in ventricular mitochondria. Gene and amino acid sequence alignments showed the amplified SKCa 3 to be SKCa 3.1, which lacks residues 478–499, present in SKCa 3.2. Similar to rat SKCa 3.1, guinea pig SKCa 3.1 displayed a conserved COOH‐terminal but did not have a poly‐Glu repeat region. Our results indicate that in ventricular cell IMM there may exist two SKCa isoforms and at least one SKCa 3 splice variant that can be functionally characterized. (NIH, VA)

Effects of Anandamide on Electrical Activity, Calcium Transients and Contraction in Guinea Pig Cardiac Ventricular Myocytes

Anandamide is an endocannabinoid which reduces cardiac contractility. Although the effects of endocannabinoids are generally attributed to their association with G-protein-coupled cannabinoid CB1 and CB2 receptors, the identity of the receptor(s) or target(s) responsible for the effects of anandamide in the heart are unknown.Anandamide (10 μM) reduced action potential (AP) duration and contraction in guinea pig ventricular myocytes stimulated to fire APs using an intracellular microelectrode. In addition, anandamide reduced peak amplitude of L-type Ca2+ currents over the range −30 to + 60 mV (switched voltage clamp, step depolarisations from −40 mV), with a reduction in peak current at 0 mV of 57 ± 11 % (n = 6, p < 0.01). Ca2+ transients measured in field-stimulated cells using Fluo-5F were also reduced. The reduction in AP duration in response to anandamide was partially inhibited by the CB2 receptor antagonist AM 630 (1 μM), but effects appeared non-competitive, and were smaller than predicted based on a Ki value of 31 nM for CB2 receptors. The CB1 receptor antagonist AM 281 (1 μM), the TRPV1 receptor antagonist capsazepine (1 μM), and O-1918 (an antagonist of a reported non CB1/non CB2 cannabinoid receptor; 1 μM) failed to inhibit responses to 10 μM anandamide. Furthermore, the selective CB1 receptor agonist ACEA, and the selective CB2 receptor agonist HU-308 (both 10 μM) were without significant effect on AP duration.The effects of anandamide can, at least in part, be accounted for by an inhibition of L-type Ca2+ currents. However, additional effects on other ion channels or intracellular targets cannot be ruled out. It appears that if a cannabinoid receptor mediates these effects, the characteristics of this receptor are not those expected of conventional CB1 or CB2 receptors.

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+/Ca2+ exchange current (INCX) was investigated using the patch-clamp method. Cibenzoline inhibited the bi-directional INCX in a concentration-dependent manner. The IC50 values of cibenzoline for the outward and inward components of INCX 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 INCX at pHo 6.5 was smaller than those at pHo 7.4 and pHo 8.2. We conclude that cibenzoline inhibits INCX in supra-therapeutic concentrations.

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.

Effect of YM-244769, a novel NCX inhibitor in guinea pig cardiac ventricular myocytes

Recently, it was reported that YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) is a new selective Na+/Ca2+ exchange (NCX) inhibitor (Iwamoto and Kita, 2006). We examined the effect of YM-244769 on the NCX current in isolated guinea pig ventricular myocytes with the whole-cell voltage-clamp technique. YM-244769 suppressed the bi-directional NCX current in a concentration-dependent manner. The IC50 values of YM-244769 were 0.12 μM and 0.1 μM for the bi-directional outward and inward NCX current, respectively, with a Hill coefficient of 0.7 and 0.8 for the bi-directional outward and inward NCX current, respectively.

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.

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+/Ca2+ exchanger (NCX) by hydrogen peroxide (H2O2) in isolated guinea-pig cardiac ventricular myocytes. Exposure to H2O2 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 H2O2 on NCX. In addition, both pathways were blocked by edaravone, a hydroxyl radical (•OH) scavenger; pertussis toxin, a Gαi/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 H2O2 increases the NCX current via two signal transduction pathways. The common pathway is the conversion of H2O2 to •OH, which activates Gαi/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.

Effects of SN-6, a novel NCX inhibitor on membrane currents in guinea pig cardiac ventricular myocytes

Effects of SN-6, a novel NCX inhibitor on membrane currents in guinea pig cardiac ventricular myocytes

Action Potential Duration Restitution Portraits of Mammalian Ventricular Myocytes: Role of Calcium Current

Construction of the action potential duration (APD) restitution portrait allows visualization of multiple aspects of the dynamics of periodically paced myocytes at various basic cycle lengths (BCLs). For the first time, we obtained the restitution portrait of isolated rabbit and guinea pig cardiac ventricular myocytes and analyzed the time constant, τ, of APD accommodation and the slopes of different types of restitution curves, S dyn and S 12, measured at varying BCLs. Our results indicate that both τ and the individual slopes are species and pacing dependent. In contrast, the mutual relationship between slopes S dyn and S 12 does not depend on pacing history, being a generic feature of the species. In addition, the maximum slope S 12, measured in the restitution portrait at the lowest BCL, predicts the onset of alternans. Further, we investigated the role of the L-type calcium current, I Ca-L, in the restitution portrait. We found that I Ca-L dramatically affects APD accommodation, as well as the individual slopes S dyn and S 12 measured in the restitution portrait. However, peak calcium current plays a role only at small values of BCL. In conclusion, the results demonstrate that the restitution portrait is a powerful technique to investigate restitution properties of periodically paced cardiac myocytes and the onset of alternans, in particular. Moreover, the data also show that I Ca-L plays a crucial role in multiple aspects of cardiac dynamics measured through the restitution portrait.

α 1-Acid glycoprotein reverses cocaine-induced sodium channel blockade in cardiac myocytes

α 1-Acid glycoprotein (AAG) is an acute phase protein capable of binding basic drugs. This action explains its reversal of sodium channel blockade by drugs such as amitriptyline and quinidine. We report here the reversal of cocaine-induced sodium channel blockade by AAG. The sodium channel blocking property of cocaine is a major mechanism behind cocaine-induced sudden cardiac death, since sodium channels play a key role in the initiation and regulation of the heart beat. Voltage-gated sodium current ( I Na) was recorded using whole-cell patch-clamp techniques. Guinea-pig cardiac ventricular myocytes were isolated and continuously perfused at room temperature with physiological solutions. At concentrations ranging from 5 to 320 μM cocaine showed a dose-dependent and reversible blockade of I Na with an IC 50 of 45.9 μM. The addition of equimolar amounts of AAG to cocaine produced almost complete reversal of cocaine's effects, suggesting a single binding site for cocaine on the AAG molecule. With changes of peak I Na normalized against control as 1, cocaine at 20 and 40 μM reduced I Na to 0.62 ± 0.042 ( n = 6) and 0.57 ± 0.052 ( n = 9), respectively, and the addition of an equimolar concentration of AAG reversed I Na to 0.86 ± 0.022 and 0.91 ± 0.060, respectively. In conclusion: AAG reverses cocaine-induced sodium channel blockade in a dose-dependent manner, indicating a therapeutic potential to reverse acute cocaine cardiac toxicity.

Contraction augments L-type Ca2+ currents in adherent guinea-pig cardiomyocytes.

As integrins are thought to function as mechanoreceptors, we studied whether they could mediate mechanical modulation of the L-type Ca2+ channel current (ICa) in guinea-pig cardiac ventricular myocytes (CVMs). CVMs were voltage clamped with 280 ms pulses from -45 to 0 mV at 0.5 Hz (1.8 mM [Ca2+]o, 22 degrees C). Five minutes after whole-cell access (designated as 0 min) peak ICa was determined from a current-voltage (I-V) curve. Additional recordings were made after 5, 10 and 15 min. At control, ICa was not stable, but ran down during these periods. This run-down of ICa was attenuated by soluble fibronectin (FN) and was changed to an enhancement of ICa when CVMs were attached to FN-coated coverslips. Soluble peptide containing the integrin binding sequence of FN, Arg-Gly-Asp (RGD motif), did not modulate ICa; however, ICa increased in stimulated CVMs attached to RGD peptide-coated coverslips. The effect was not specific to integrins, because attachment to poly-D-lysine-coated coverslips also augmented ICa in stimulated CVMs. Augmentation of ICa by immobilized FN required rhythmical contraction of attached CVMs, because it was attenuated without electrical stimulation and after cell dialysis with the calcium chelator BAPTA. Furthermore, contraction-induced augmentation of ICa in FN-attached CVMs was sensitive to inhibition of protein kinase C (PKC; by Ro-31-8220), inhibition of tyrosine kinase activity (herbimycin A) and cytoskeletal depolymerization (cytochalasin D or colchicine). We attribute augmentation of ICa to the activation of signalling cascades by shear forces that are generated when CVMs contract against attachment; in vivo similar signals may occur when CVMs contract against attachment of integrins to the extracellular matrix.

Effects of amiodarone on mutant Na +/Ca 2+ exchangers expressed in CCL 39 cells

Using the whole cell voltage clamp, we reported previously that amiodarone acutely inhibits Na +/Ca 2+ exchange current ( I NCX) in guinea pig cardiac ventricular myocytes. Intracellular application of trypsin via the patch pipette attenuated the blocking effect of amiodarone, suggesting that amiodarone affects the Na +/Ca 2+ exchanger (NCX) from the cytoplasmic side. Here, we attempted to detect the site of amiodarone inhibition using wild type NCX1, mutants, and NCX3 expressed in CCL39 fibroblasts. I NCX was recorded by ramp pulses. Amiodarone at 30 μM inhibited I NCX by 80% in cells expressing wild type NCX1. However, 30 μM amiodarone inhibited I NCX by about 55% in cells expressing mutant NCX1 with amino acids 217-671 (ΔXIP) or 247-671 (Δ247-671) deleted in the long intracellular loop between the transmembrane segments (TM) 5 and 6. I NCXs from NCX mutants deleted of cytoplasmic TM1-2, TM3-4 or the C-terminus were inhibited by amiodarone to a similar extent as the wild type. Amiodarone also inhibited I NCX of NCX3 by 76%. These results suggest that a long intracellular loop may be involved in the inhibition of NCX1 by amiodarone, but that other intracellular loops, XIP region or C terminus are not involved in the amiodarone inhibition of NCX1.

Effect of azimilide on Na+/Ca2+ exchange current in Guinea-pig cardiac ventricular myocytes

Effect of azimilide on Na+/Ca2+ exchange current in Guinea-pig cardiac ventricular myocytes

Inhibitory effect of fluvastatin on lysophosphatidylcholine-induced nonselective cation current in Guinea pig ventricular myocytes.

Using the whole-cell voltage-clamp method, we investigated the effect of fluvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, on lysophosphatidylcholine (LPC)-induced nonselective cation current (I(NSC)) in guinea pig cardiac ventricular myocytes. External LPC (3 to approximately 50 microM) induced I(NSC) in a dose-dependent manner with a lag. With fluvastatin (5 microM) in the external solution, LPC induced I(NSC), which was significantly smaller and with a longer lag compared with that in the absence of fluvastatin. With mevalonic acid (MVA) (100 microM) in the external solution, fluvastatin did not diminish LPC-induced I(NSC). Geranylgeranylpyrophosphate, an MVA metabolite, in the pipette solution prevented fluvastatin from diminishing LPC-induced I(NSC), suggesting that isoprenylated signaling molecules, such as the small G-protein Rho, might be involved in the LPC effect. Botulinum toxin C3, Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2 HCl (Y-27632), or pertussis toxin in the pipette solution suppressed LPC-induced I(NSC). We conclude that LPC induces I(NSC) via a Gi/Go-coupled receptor and Rho-mediated pathway. The inhibitory effect of fluvastatin on LPC-induced I(NSC) provides a new insight into the signal transduction mechanism and may have important clinical implications.