Abstract
SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) has recently been described as a potent and selective inhibitor of Na+-Ca2+ exchange in cardiac, neuronal, and renal preparations. The inhibitory effects of SEA0400 were investigated on the cloned cardiac Na+-Ca2+ exchanger, NCX1.1, expressed in Xenopus laevis oocytes to gain insight into its inhibitory mechanism. Na+-Ca2+ exchange currents were measured using the giant excised patch technique using conditions to evaluate both inward and outward currents. SEA0400 inhibited outward Na+-Ca2+ exchange currents with high affinity (IC50 = 78 ± 15 and 23 ± 4 nM for peak and steady-state currents, respectively). Considerably less inhibitory potency (i.e., micromolar) was observed for inward currents. The inhibitory profile was reexamined after proteolytic treatment of excised patches with α-chymotrypsin, a procedure that eliminates ionic regulatory mechanisms. After this treatment, an IC50 value of 1.2 ± 0.6 μM was estimated for outward currents, whereas inward currents became almost insensitive to SEA0400. The inhibitory effects of SEA0400 on outward exchange currents were evident at both high and low concentrations of regulatory Ca2+, although distinct features were noted. SEA0400 accelerated the inactivation rate of outward currents. Based on paired pulse experiments, SEA0400 altered the recovery of exchangers from the Na+i-dependent inactive state, particularly at higher regulatory Ca2+i concentrations. Finally, the inhibitory potency of SEA0400 was strongly dependent on the intracellular Na+ concentration. Our data confirm that SEA0400 is the most potent inhibitor of the cardiac Na+-Ca2+ exchanger described to date and provide a reasonable explanation for its apparent transport mode selectivity.
Footnotes
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This study was supported by operating grants from the Heart and Stroke Foundation of Manitoba and the Canadian Institutes of Health Research (CIHR) and by a Canada Research Chair Award (to L.V.H.). C.L. was supported by a CIHR Postdoctoral Fellowship Award and the Tailored Advanced Collaborative Training in Cardiovascular Science for Research Fellows (TACTICS) Partnership Program of the CIHR and the Heart and Stroke Foundation.
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doi:10.1124/jpet.104.070805.
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ABBREVIATIONS: NHE, Na+-H+ exchanger; [Ca2+]i, intracellular Ca2+; KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; SEA0400, 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; MES, 4-morpholineethanesulfonic acid; TEA, tetraethylammonium; MOPS, 4-morpholinepropanesulfonic acid; λ, rate of current decay; FSS, extent of current inactivation.
- Received April 29, 2004.
- Accepted July 1, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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