Schreur J H, Figueredo V M, Miyamae M, Shames D M, Baker A J, Camacho S A
Department of Medicine, Cardiology, San Francisco General Hospital, California 94110, USA.
Biophys J. 1996 Jun;70(6):2571-80. doi: 10.1016/S0006-3495(96)79828-4.
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.
使用indo-1的乙酰氧基甲酯（AM）形式评估游离胞质[Ca2+]（[Ca2+]c）可能会受到indo-1进入非胞质区室（主要是线粒体）的影响。为了确定用indo-1 AM加载的完整心脏中非胞质荧光的比例，使用Mn2+淬灭胞质荧光。在计算[Ca2+]c之前，从总荧光中减去残余（即非胞质）荧光。非胞质荧光用于估计线粒体[Ca2+]。在以5 Hz起搏的心脏中（N = 17），在λ385和λ456处，非胞质荧光分别为总荧光的0.61±0.06和0.56±0.07。考虑到非胞质荧光后，收缩期和舒张期[Ca2+]c分别为673±72和132±9 nM，非胞质[Ca2+]为183±36 nM，当细胞外Ca2+从2 mM增加到6 mM时，增加到272±12 nM。这种非胞质[Ca2+]的增加被钌红抑制，钌红是线粒体摄取Ca2+的阻滞剂。我们得出结论，通过使用Mn2+淬灭胞质荧光，可以在加载indo-1 AM的完整心脏中测定胞质和线粒体[Ca2+]。
