CA2-AFFINITY CA2+ INDICATORS( TRANSIENTS IN CARDIAC MYOCYTES MEASUREDWITH HIGH AND LOW)

Intracellular calcium ion ([Ca2+]i) transients were measured in voltag e-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate r apid changes in [Ca2+]i. Patch electrode solutions contained the K+ sa lt of fura-2 (50 muM) or furaptra (300 muM). With identical experiment al conditions, peak amplitude of stimulated [Ca2+]i transients in fura ptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-load ed cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical propertie s were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference betwee n the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded m yocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these condition s; however, Ca2+ buffering is not the only factor that explains the di fferent amplitudes of the [Ca2+]i transients measured with these indic ators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2 binding of fura-2 was at least 65s-1, much faster than previously rep orted in skeletal muscle fibers. These binding kinetics do not explain the differences in the size of the [Ca2+]i transients reported by fur a-2 and furaptra. Parameters for fura-2 calibration, R(min), R(max), a nd beta, were obtained in salt solutions (in vitro) and in myocytes ex posed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions ( in situ). Calibration of fura-2 fluorescence signals with these in sit u parameters yielded [Ca2+]i transients whose peak amplitude was 50-10 0% larger than those calculated with in vitro parameters. Thus, in vit ro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the diffe rence in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in v itro calibration parameters.