Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

We investigated the pharmacological properties and interaction domains of N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide (YM-244769), a novel potent Na /Ca exchange (NCX) inhibitor, using various NCX-transfectants and neuronal and renal cell lines. YM-244769 preferentially inhibited intracellular Na -dependent Ca uptake via NCX3 (IC50 18 nM); the inhibition was 3.8to 5.3-fold greater than for the uptake via NCX1 or NCX2, but it did not significantly affect extracellular Na -dependent Ca efflux via NCX isoforms. We searched for interaction domains with YM-244769 by NCX1/NCX3-chimeric analysis and determined that the -2 region in NCX1 is mostly responsible for the differential drug response between NCX1 and NCX3. Further cysteine scanning mutagenesis in the -2 region identified that the mutation at Gly833 markedly reduced sensitivity to YM-244769. Mutant exchangers that display either undetectable or accelerated Na -dependent inactivation, had markedly reduced sensitivity or hypersensitivity to YM-244769, respectively. YM-244769, like 2-[2-[4-(4-nitrobenzyloxyl)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), protected against hypoxia/reoxygenation-induced cell damage in neuronal SH-SY5Y cells, which express NCX1 and NCX3, more efficiently than that in renal LLC-PK1 cells, which exclusively express NCX1, whereas 2-[4(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester (SN-6) suppressed renal cell damage to a greater degree than neuronal cell damage. These protective potencies consistently correlated well with their inhibitory efficacies for the Ca uptake via NCX isoforms existing in the corresponding cell lines. Antisense knockdown of NCX1 and NCX3 in SH-SY5Y cells confirmed that NCX3 contributes to the neuronal cell damage more than NCX1. Thus, YM-244769 is not only experimentally useful as a NCX inhibitor that preferentially inhibits NCX3, but also has therapeutic potential as a new neuroprotective drug. The Na /Ca exchanger (NCX) can transport Ca either out of cells (i.e., forward mode) or into cells (i.e., reverse mode) in exchange for three Na . This exchanger is driven by membrane potential as well as ion gradients through plasma membrane (Blaustein and Lederer, 1999; Philipson and Nicoll, 2000; Shigekawa and Iwamoto, 2001; Annunziato et al., 2004). In cardiac muscle, NCX primarily pumps intracellular Ca (Cai 2 ) to outside the cell during repolarization and diastole, which balances Ca entry via L-type Ca channels during cardiac excitation (Blaustein and Lederer, 1999). Under pathological conditions such as ischemia/reperfusion injury in the heart, brain, and kidney, the exchanger is believed to cause Ca overload as a result of elevated intracellular Na concentration ([Na ]i), leading to cell damage (Blaustein and Lederer, 1999; Annunziato et al., 2004; Iwamoto, 2005; Lee et al., 2005). Recent evidence suggests that Ca entry through vascular exchangers is involved in the development of salt-dependent hypertension (Iwamoto et