No.7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), a selective inhibitor of the Na+/Ca2+ exchanger (NCX1), has been newly synthesized. It dose-dependently inhibited Na+i-dependent 45Ca2+ uptake and Na+i-dependent [Ca2+]i increase in cardiomyocytes, smooth muscle cells, and NCX1-transfected fibroblasts (IC50 = 1.2-2.4 microM). Inhibition was observed without prior incubation with the agent and was completely reversed by washing cells with buffer for 1 min. Interestingly, No.7943 was much less potent in inhibiting Na+o-dependent 45Ca2+ efflux and Na+o-induced [Ca2+]i decline (IC50 = >30 microM), indicating that it selectively blocks the reverse mode of Na+/Ca2+ exchange in intact cells. In cardiac sarcolemmal preparations consisting mostly of inside-out vesicles, the agent inhibited Na+i-dependent 45Ca2+ uptake and Na+o-dependent 45Ca2+ efflux with similar, but slightly lower, potencies (IC50 = 5.4-13 microM). Inhibition was noncompetitive with respect to Ca2+ and Na+ in both cells and sarcolemmal vesicles. These results suggest that No.7943 primarily acts on external exchanger site(s) other than the transport sites in intact cells, although it is able to inhibit the exchanger from both sides of the plasma membrane. No.7943 at up to 10 microM does not affect many other ion transporters nor several cardiac action potential parameters. This agent at these concentrations also did not influence either diastolic [Ca2+]i or spontaneous beating in cardiomyocytes. Furthermore, No.7943 markedly inhibited Ca2+ overloading into cardiomyocytes under the Ca2+ paradox conditions. Thus, No.7943 is not only useful as a tool with which to study the transport mechanism and physiological role of the Na+/Ca2+ exchanger but also has therapeutic potential as a selective blocker of excessive Ca2+ influx mediated via the Na+/Ca2+ exchanger under pathological conditions.

other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.

1 We investigated the inhibitory e ect of KB-R7943 on`bi-directional' Na + /Ca 2+ exchange current (i NCX ) with the reversal potential of i NCX (E NCX ) in the middle of the ramp voltage pulse employed. 2 Bi-directional i NCX was recorded with`full' ramp pulses given every 10 s from the holding potential of 760 mV over the voltage range between 30 and 7150 mV under the ionic conditions of 140 mM [Na] 3 KB-R7943 (0.1 ± 100 mM) concentration-dependently inhibited the current, which reversed near the calculated E NCX , indicating that the blocked current was i NCX . 4 The inhibition levels were not signi®cantly di erent between outward and inward i NCX measured at 0 and 7120 mV, respectively. IC 50 of KB-R7943 was approximately 1 mM for both directions of i NCX . 5 Under the bi-directional ionic conditions, only an outward or inward i NCX was induced by positive or negative`half' ramp pulses, respectively, from the holding potential of 760 mV. KB-R7943 inhibited both direction of i NCX and the concentration-inhibition relations were superimposable to the ones obtained by`full' ramp pulses. 6 These results indicate that KB-R7943 inhibits i NCX direction-independently under bi-directional conditions. This conclusion is di erent from that of our previous results obtained from i NCX under uni-directional ionic conditions, where KB-R7943 inhibited i NCX direction-dependently. The di erence could be attributed to slow dissociation of the drug from the exchanger.

1 We investigated protective e ects of KB-R7943, a Na + /Ca 2+ exchange (NCX) inhibitor, on ouabain-induced tonotropy and arrhythmias in isolated whole atria and ouabain-induced changes in electrocardiogram (ECG) in the guinea-pig. 2 KB-R7943 (10 and 30 mM) suppressed the tonotropic e ect of ouabain, and prolonged the onset time of extra-systole induced by ouabain in isolated atria. 3 The intravenous injection of KB-R7943 (1 and 3 mg kg 71 ) signi®cantly increased the doses of ouabain required to induce ventricular premature beats (VPB), ventricular tachycardia (VT), ventricular ®brillation (VF) and cardiac arrest (CA) in anaesthetized guinea-pigs. 4 Lidocaine (Na + channel inhibitor) and R56865 (Na + and Ca 2+ overload inhibitor) also suppressed the ouabain-induced tonotropic e ect and extra-systole in isolated atria, but Hoe-694 (Na + /H + exchange inhibitor) or diltiazem (Ca 2+ channel inhibitor) did not a ect them. 5 Lidocaine also increased the doses of ouabain required to induce VPB, VT, VF and CA in anaesthetized guinea-pigs. 6 From these results, we conclude that KB-R7943 suppresses ouabain-induced arrhythmias through inhibition of the reverse-mode NCX.

1 The eect of 2,3-butanedione monoxime (BDM), a`chemical phosphatase', on Na + /Ca 2+ exchange current (I NCX ) was investigated using the whole-cell voltage-clamp technique in single guinea-pig cardiac ventricular myocytes and in CCL39 ®broblast cells expressing canine NCX1. 2 I NCX was identi®ed as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na + and 2 mM Ca 2+ in the external solution and 20 mM Na + and 433 nM free Ca 2+ in the pipette solution.3 In guinea-pig ventricular cells, BDM inhibited I NCX in a concentration-dependent manner. The IC 50 value was 2.4 mM with a Hill coecients of 1. The average time for 50% inhibition by 10 mM BDM was 124+31 s (n=5). 4 The eect of BDM was not aected by 1 mM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid-sensitive protein phosphatases. 5 Intracellular trypsin treatment via the pipette solution signi®cantly suppressed the inhibitory eect of BDM, implicating an intracellular site of action of BDM. 6 PAM (pralidoxime), another oxime compound, also inhibited I NCX in a manner similar to BDM. 7 Isoprenaline at 50 mM and phorbol 12-myristate 13-acetate (PMA) at 8 mM did not reverse the inhibition of I NCX by BDM. 8 BDM inhibited I NCX in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. 9 We conclude that BDM inhibits I NCX but the mechanism of inhibition is not by dephosphorylation of the Na + /Ca 2+ exchanger as a`chemical phosphatase'.

No abstract

In single guinea‐pig ventricular myocytes, we examined the stoichiometry of Na+‐Ca2+ exchange (NCX) by measuring the reversal potential (ENCX) of NCX current (INCX) and intracellular Ca2+ concentration ([Ca2+]i) with the whole‐cell voltage‐clamp technique and confocal microscopy, respectively. With given ionic concentrations in the external and pipette solutions, the predicted ENCX were −73 and −11 mV at 3:1 and 4:1 stoichiometries, respectively. ENCX measured were −69 ± 2 mV (n= 11), −47 ± 1 mV (n= 14) and −15 ± 1 mV (n= 15) at holding potentials (HP) of −73, −42 and −11 mV, respectively. Thus, ENCX almost coincided with HP, indicating that [Ca2+]i and/or [Na+]i changed due to INCX flow. Shifts of ENCX (ΔENCX) were measured by changing [Ca2+]o or [Na+]o. The measured values of ΔENCX were almost always smaller than those expected theoretically at a stoichiometry of either 3:1 or 4:1. Using indo‐1 fluorescence, [Ca2+]i measured under the whole‐cell voltage‐clamp supported a 3:1 but not 4:1 stoichiometry. To prevent Ca2+ accumulation, we inhibited INCX with Ni2+ and re‐examined ENCX during washing out Ni2+. With HP at predicted ENCX at a 3:1 stoichiometry, ENCX developed was close to predicted ENCX and did not change with time. However, with HP at predicted ENCX for a 4:1 stoichiometry, ENCX developed initially near a predicted ENCX for a 3:1 stoichiometry and shifted toward ENCX for a 4:1 stoichiometry with time. We conclude that the stoichiometry of cardiac NCX is 3:1.

The role of P2 receptors in synaptic transmission to the rat medial nucleus of the trapezoid body (MNTB) was studied in an in vitro brain slice preparation. Whole-cell patch recordings were made and spontaneous synaptic responses studied under voltage clamp during application of P2X receptor agonists. ATPγS (100 µM) had no effect on holding current, but facilitated spontaneous excitatory postsynaptic current (sEPSC) frequency in 41% of recordings and facilitated spontaneous inhibitory postsynaptic currents (sIPSCs) in 20% of recordings. These were blocked by the P2 receptor antagonist suramin (100 µM). α,β-meATP also facilitated sEPSC and sIPSC frequency, while L-β,γ-meATP facilitated only sIPSCs. The sEPSC facilitation by ATPγS was blocked by TTX (but did not block facilitation of sIPSCs). sEPSC facilitation was blocked by PPADS (30 µM) and the selective P2X 3 receptor antagonist A-317491 (3 µM), suggesting that modulation of sEPSCs involves P2X 3 receptor subunits. α,β-meATP-facilitated sIPSCs were also recorded in wild-type mouse MNTB neurones, but were absent in the MNTB from P2X 1 receptor-deficient mice demonstrating a functional role for P2X 1 receptors in the CNS.