Jr. Berlin et M. Konishi, CA2-AFFINITY CA2+ INDICATORS( TRANSIENTS IN CARDIAC MYOCYTES MEASUREDWITH HIGH AND LOW), Biophysical journal, 65(4), 1993, pp. 1632-1647
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.