The loading of fura 2 into mitochondria in intact rat heart and its use to estimate matrix Ca2+.

Ca2+ is a widely used hormonal second-messenger in the cytosol of mammalian cells [ 11. The processes stimulated by increased cytosolic Ca2+ are often energy-requiring, e.g. contraction, secretion, yet rarely are decreases in cellular ATP/ADP ratios observed under such circumstances 121. There is now good evidence that a compensatory increase in oxidative metabolism and hence ATP production is responsible for maintaining energy homeostasis under such conditions which involves a relay of the Ca2+ signal into the mitochondrial matrix [3]. Ca2+ is thus a key regulator of intramitochondrial oxidative metabolism in mammalian cells through its activation of the pyruvate (PDH), NAD+-isocitrate, and 2-oxoglutarate dehydrogenases [ 31 which are major sites of NADH production for the respiratory chain. Ca2+ activates PDH by increasing the amounts of active, non-phosphorylated enzyme (PDH,) through its stimulation of PDH phosphate phosphatase, whereas the other two enzymes are activated non-covalently through marked decreases in their K, values for isocitrate and oxoglutarate respectively. In extracts or after purification, the Ca2+ activatory ranges for the enzymes is approximately 0.1 -SpM, although NAD+-isocitrate dehydrogenase may respond to a higher range 141. The fluorescent CaWndicator fura 2 can be loaded into isolated rat heart mitochondria and can thus be used to measure matrix Ca2+ directly, and hence to correlate this with the activities of these Ca2+dependent enzymes in intact, viable mitochondria [S,6]. There are also reports that some fura 2 localises to mitochondria in intact cells from heart and other mammalian tissues (e.g. [7,8]). We have previously shown that the positive inotropic activation of the perfused rat heart (via raised cytosolic Ca2+) is associated with increases in the amounts of PDH, [9]. Moreover, these activations of PDH were found to persist through to isolated and then subsequently incubated (at 30.C in KCI-based buffer containing EGTA) mitochondria [lo]. Parallel activations of 2-oxoglutarate dehydrogenase were also observed in mitochondria prepared from stimulated hearts, and others showed that these activations were accompanied by increases in total mitochondrial Ca content within the range associated with enzyme activation (0-5 nmol/mg protein) [ 111. Furthermore these persistent activations could be diminished by adding Na+ ions to the incubations to promote Caz+-egress from the mitochondria [ 101. The present studies were therefore undertaken to see whether some fura 2 could be loaded into the mitochondrial mamx in intact perfused hearts, and whether this could be used to report on any persistent changes in matrix Ca2+ concentration (due to prior inotropic intervention) in subsequently isolated mitochondria. Male rat (200-2508) hearts were perfused at 370C with a gassed (02/C02 ; 19/1) Krebs-bicarbonate buffer containing l0mM glucose 191. After an initial Smin flow-through period, the perfusions were switched to a ( pumped) S0ml re-circulating system and the acetoxymethyl ester of fura 2 (fura 2/AM) added to a concentration of 10pM. Perfusions were then continued at 12ml/min for 58min. and then for a further 2min with no change (control), or for 2min at 24ml/min to produce a positive inotropic response ('high work'). A small portion of each heart (0.1-0.2g) was then quickly freezeclamped for susequent tissue PDH analysis (as in [9]), and the remainder rapidly homogenised, under conditions and in buffer designed to minimise any artefactual Ca2+ movements across the mitochondrial inner membrane (see [lo]). After mitochondrial isolation [lo], matrix entrapped fura 2 was evident (results not shown) and this was then used to measure matrix Ca2+ and was calibrated as described previously [6].