A Novel Isothiourea Derivative Selectively Inhibits the Reverse Mode of Na+/Ca2+ Exchange in Cells Expressing NCX1

No.7943 (2-2-[4-(4-nitrobenzyloxy)phenylethyl]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 µM). 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 µM), 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 µM). 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 µM 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. No.7943 (2-2-[4-(4-nitrobenzyloxy)phenylethyl]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 µM). 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 µM), 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 µM). 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 µM 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.