KR-32570, a novel Na+/H+ exchanger-1 inhibitor, attenuates hypoxia-induced cell death through inhibition of intracellular Ca2+ overload and mitochondrial death pathway in H9c2 cells

Pharmacological profile of KR-33028, a highly selective inhibitor of Na+/H+ exchanger

“…The collapse of the Δψ m with the release of cytochrome c is followed by the activation of caspase-3, nuclear fragmentation and apoptotic cell death (Smaili et al, 2003). Consistent with this concept, our previous data have shown that hypoxia-induced reduction in the Δψ m is almost completely blocked by the inhibition of mitochondrial Ca 2+ uptake with ruthenium red (Kim et al, 2005). In the present study, treatment with KR-33028 at 1h before and during hypoxia substantially attenuated hypoxia-induced increases in both cytosolic Ca 2+ and mitochondrial Ca 2+ (Fig.…”

“…3 and 4), with a potency similar to that of cariporide (Kim et al, 2005). These results suggest that KR-33028 may prevent both necrotic and apoptotic cell death induced by hypoxia.…”

“…The percentage of cell death was calculated as the number of TUNEL-positive cells divided by the total cell count. In the caspase-3 activity assay, after the extracted proteins were incubated with the reaction buffer (100 mM HEPES, 10% sucrose, 0.1% 3-[3-cholamidopropylammonio]-1-propanesulfonate (CHAPS), pH 7.5, 10 mM DTT, and 10 μg/ml leupeptin) in a volume of 100 μl volume containing 200 μM Ac-DEVD-p-Na (Biomol, Plymouth Meeting, PA, USA), the enzyme-catalyzed release of p-nitroranilide was measured at 405 nm using a microplate reader (Molecular Devices, Palo Alto, CA, USA) as previously described (Kim et al, 2005).…”

“…After the cellular cytosolic and mitochondrial fractions were separated as previously described (Kim et al, 2005), 40 μg of protein from either the cytosolic fraction or the mitochondrial fraction were separated on 15% sodium dodecyl sulfatepolyacryamide gel electrophoresis (SDS PAGE) and transferred to a nitrocellulose membrane (Hybond-C Extra, Amersham). The membrane was blocked in 5% dry milk for 1h incubated with the primary antibody overnight, and incubated with the secondary antibody for 1h.…”

“…H9c2 cells, loaded with 5 μM fluo-3 for 20 min or 2μM rhod-2 for 30 min, were observed with a confocal microscope (Olympus, Japan) at 480nm or 560 nm, respectively. The fluorescence intensities of fluo-3 or rhod-2 were quantified using image-analysis computer software (Fluoview FV300; Olympus, Japan) as previously described (Kim et al, 2005).…”