Interactions between Benzylamiloride and Fura-2: Studiesin Vitroand in Cardiac Myocytes

Amiloride derivatives are commonly used inhibitors of Na+/H+- and Na+/Ca2+-exchange. Because they are fluorescent molecules the use of benzylamiloride (BZA), an inhibitor of Na+/Ca2+exchange, in conjunction with Fura-2, a commonly used fluorescent Ca2+indicator, might complicate interpretation of fluorescence data obtained.In vitrodata show that BZA decreases the Fura-2 fluorescence at all useful wavelengths in a concentration-dependent manner. The Fura-2 ratio 340/380 (used to estimate intracellular Ca2+([Ca2+]in)) also decreased with increasing BZA concentrations. The Stern–Volmer relation suggests that this phenomenon is due to either static or dynamic quenching. Varying temperatures from 4 to 37°C did not alter Stern–Volmer constants, consistent instead with fluorescence resonance energy transfer (FRET). Thein siturelevance of these interactions was evaluated in adult rat cardiac myocytes which exhibit Na+/Ca2+exchange reflected by rapid [Ca2+]inincrease following Na+removal. Pretreatment with BZA ≥ 25 μM decreased the magnitude of Fura-2 changes induced by Na+removal. Analysis of the individual Fura-2 useful wavelengths indicated that ≥ 25 μM BZA altered the Fura-2 signal in a manner consistent with the quenching effects notedin vitro.Together, these data show that BZA interacts with Fura-2in vitroandin situand suggest caution when interpreting Fura-2 fluorescence data derived in conjunction with BZA.