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1. Langendorff-perfused rat hearts were treated, either alone or in combination, with acetate, adrenaline or supraphysiological [Ca2+] to mimic increased workload. No significant increases in the PPi content of the freeze-clamped tissue were observed. 2. In contrast with liver mitochondria, isolated rat heart mitochondria incubated at a free [Mg2+] of 1.5 mM showed little or no increase in matrix PPi content or volume in the presence of 0.75 microM Ca2+; neither did 5 mM acetate cause heart mitochondrial PPi to increase. 3. At 0.1 mM Mg2+ some increase in matrix PPi content of heart mitochondria was observed, and increases in liver mitochondria were significantly greater. 4. Substantial increases in heart mitochondrial-matrix PPi content were observed at 1.5 mM Mg2+ when [Ca2+] &gt; 1 microM, especially in the presence of acetate, or at 0.75 microM Ca2+ in the presence of bongkrekic acid to inhibit the adenine nucleotide translocase. 5. Total pyrophosphatase activity was similar in heart and liver mitochondrial matrix extract and toluene-permeabilized mitochondria. 6. These data suggest that under physiological conditions Ca2+ does not regulate heart mitochondrial [PPi], because higher matrix [Mg2+] overcomes the inhibition of pyrophosphatase by Ca2+, and also significant loss of PPi from the matrix on the adenine nucleotide translocase may occur. 7. It is concluded that, in contrast with the situation in liver [Halestrap (1989) Biochim. Biophys. Acta 973, 355-382], Ca(2+)-induced PPi-mediated increases in mitochondrial volume are unlikely to play a physiological role in the heart. 8. The substantial swelling of liver and kidney mitochondria caused by addition of supraphysiological [Ca2+] (20 nmol/mg of protein) was associated with significant increases in PPi content, but was not observed in heart mitochondria. 9. This substantiates the role that we have proposed for PPi in the opening of a non-specific pore by Ca2+ overload of mitochondria [Halestrap and Davidson (1990) Biochem. J. 268, 153-160].

Pyrophosphate metabolism in the perfused heart and isolated heart mitochondria and its role in regulation of mitochondrial function by calcium