Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes

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Abstract
The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2'',7''-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukacs and Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung and Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for succesfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a curvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitchondria, we found the pKa of BCECF for H+ at 25.degree. c shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25.degree. C, pH 8.0, but only at [Ca2+] below 1 .mu.M. At matrix [Ca2+] > 1 .mu.M, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myoctes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.