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 benzy lamiloride (BZA), an inhibitor of Na+/Ga2+ exchange, in conjunction wi th Fura-2, a commonly used fluorescent Ca2+ indicator, might complicat e interpretation of fluorescence data obtained. In vitro data show tha t BZA decreases the Fura-2 fluorescence at all useful wavelengths in a concentration-dependent manner. The Fura-2 ratio 340/380 (used to est imate intracellular Ca2+ ([Ca2+](in))) also decreased with increasing BZA concentrations. The Stern-Volmer relation suggests that this pheno menon is due to either static or dynamic quenching. Varying temperatur es from 4 to 37 degrees C did not alter Stern-Volmer constants, consis tent instead with fluorescence resonance energy transfer (FRET), The i n situ relevance of these interactions was evaluated in adult rat card iac myocytes which exhibit Na+/Ca2+ exchange reflected by rapid [Ca2+] (in) increase following Nat removal. Pretreatment with BZA greater tha n or equal to 25 mu M decreased the magnitude of Fura-2 changes induce d by Na+ removal. Analysis of the individual Fura-2 useful wavelengths indicated that greater than or equal to 25 mu M BZA altered the Fura- 2 signal in a manner consistent with the quenching effects noted in vi tro, 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, (C) 1998 Academic Press.