ADP/ATP Antiport and ADP Phosphorylation Increase Mitochondrial Free Ca2+

Abstract

Introduction: Matrix free [Ca2+] (m[Ca2+]) is believed to be a key regulator of mitochondrial function. The effect of differential buffering of calcium by ADP, ATP and Pi on m[Ca2+] levels has not been examined. We tested how m[Ca2+] is increased by ADP/ATP transport and phosphorylation, and if increased m[Ca2+] alters the bioenergetic state. Materials and Methods: Guinea pig heart mitochondria were isolated by differential centrifugation. Respiration and m[Ca2+], using indo-1 fluorescence, and corrected for NADH autofluorescence, were measured. After energizing mitochondria with 0.5 mM pyruvic acid, 0, 10, 25 μM CaCl2 (16, 88, 130 nM [Ca2+]) was added to the suspension before adding 250 μM ADP, in the presence or absence of ADP/ATP carrier blocker carboxyatractyloside (CATR) or F1F0ATPase blocker oligomycin (OMN). Results: m[Ca2+] increased proportionately with addition of CaCl2. ADP caused an additional increase to 100±6% in m[Ca2+] after 25 μM CaCl2. This was due to lesser binding of ADP vs. ATP to Ca2+. The rise in m[Ca2+] after ADP was reversed after all ADP was converted to ATP. With OMN the increase after ADP was lower (18±6%), but remained elevated as ADP was not phosphorylated to ATP. CATR completely blocked the ADP -induced increases in m[Ca2+] because matrix ADP transport was blocked. State 2 and 4 respiration, but not state 3, increased 14% and 18% with 25 μM CaCl2. NADH decreased with ADP alone, but NADH was not altered by adding CaCl2. Discussion: These results show that ADP transport into mitochondria and ADP conversion to ATP have significant effects on m[Ca2+]. Acutely changing buffer [CaCl2] has limited effects on redox state, although m[Ca2+] is believed to stimulate several dehydrogenases. However the k0.5 (1 μM) for this effect is only reached by adding ADP after 25 μM CaCl2.