Cytosolic and mitochondrial [Ca2+] in whole hearts using indo-1 acetoxymethyl ester: effects of high extracellular Ca2+.

Assessment of free cytosolic Ca2+] (Ca2+]c) using the acetoxymethyl ester (AM) form of indo-1 may be compromised by loading of indo-1 into noncytosolic compartments, primarily mitochondria. To determine the fraction of noncytosolic fluorescence in whole hearts loaded with indo-1 AM, Mn2+ was used to quench cytosolic fluorescence. Residual (i.e., noncytosolic) fluorescence was subtracted from the total fluorescence before calculating Ca2+]c. Noncytosolic fluorescence was used to estimate mitochondrial Ca2+]. In hearts paced at 5 Hz (N = 17), noncytosolic fluorescence was 0.61 +/- 0.06 and 0.56 +/- 0.07 of total fluorescence at lambda 385 and lambda 456, respectively. After taking into account noncytosolic fluorescence, systolic and diastolic Ca2+]c was 673 +/- 72 and 132 +/- 9 nM, respectively, noncytosolic Ca2+] was 183 +/- 36 nM and increased to 272 +/- 12 when extracellular Ca2+ was increased from 2 to 6 mM. This increase in noncytosolic Ca2+] was inhibited by ruthenium red, a blocker of Ca2+ uptake by mitochondria. We conclude that cytosolic and mitochondrial Ca2+] can be determined in whole hearts loaded with indo-1 AM by using Mn2+ to quench cytosolic fluorescence.