Interactions between benzylamiloride and fura-2: Studies in vitro and 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 vitro data 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). The in situ relevance of these interactions was evaluated in adult rat cardiac myocytes which exhibit Na⁺/Ca²⁺ exchange reflected by rapid [Ca²⁺](in) increase 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 noted in vitro. Together, these data show that BZA interacts with Fura-2 in vitro and in situ and suggest caution when interpreting Fura-2 fluorescence data derived in conjunction with BZA.