Abstract
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.
Highlights
The Naϩ/Ca2ϩ exchanger is a major regulator of [Ca2ϩ]i1 in excitable as well as in many nonexcitable cells [1, 2]
The inhibitory potency of No.7943 was identical whether 45Ca2ϩ uptake was measured for 15 s in cardiac NCX1-transfected CCL39 cells after preincubation with this agent for 5 min (Fig. 2A) or without such preincubation (IC50 ϭ 2.1 Ϯ 0.4 M (n ϭ 3))
The same agent produced only a weak inhibitory effect on the Naϩo-dependent Ca2ϩ efflux (45Ca2ϩ efflux and [Ca2ϩ]i decrease) even at a high concentration (Figs. 4 and 5). These effects of No.7943 are most likely to reflect a direct action on the Naϩ/Ca2ϩ exchanger, because of the following. (i) In cardiomyocytes and smooth muscle cells, both Naϩi-dependent increase and Naϩodependent decrease in [Ca2ϩ]i are due to the activity of the Naϩ/Ca2ϩ exchanger [1]
Summary
The Naϩ/Ca2ϩ exchanger is a major regulator of [Ca2ϩ]i1 in excitable as well as in many nonexcitable cells [1, 2]. Blanks were obtained by measuring 45Ca2ϩ loss in the efflux medium containing no NaCl. Assays of Other Ion Transporters—L-type Ca2ϩ channel activity was assayed by measuring DHP-sensitive 45Ca2ϩ uptake into cultured smooth muscle cells as described previously [25].
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