Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells

Abstract

The metabolism and intracellular compartmentalization of adenine nucleotides in a preparation of adult rat heart myocytes showing good morphology, viability, and tolerance to calcium ion has been examined by high performance liquid chromatography. These myocytes contain an average of 23 nmol adenine nucleotide per milligram protein which is about 60% of the adenine nucleotide content of intact rat heart tissue. The loss of adenine nucleotide occurs during the incubation and washing steps that increase the yield of viable cells, rather than during the collagenase perfusion. An analysis of cellular compartments shows that the adenine nucleotide of the cell consists of 17 nmol adenine nucleotide in the cytosol, 5 nmol in the mitochondria, and 1.3 nmol adenosine diphosphate bound to myofibrils per milligram cell protein. Myocytes lose both adenosine triphosphate and adenine nucleotide when incubated anaerobically in the absence of glucose, and the lost adenine nucleotide can be accounted for as increased inosine, adenosine, and inosine monophosphate. Myocytes that contain less than 0.1 nmol of cytosol adenosine triphosphate per milligram cell protein maintain an intact sarcolemma, but are unable to carry out anaerobic glycolysis. Reoxygenation of anaerobic cells results in restoration of energy charge and a net resynthesis of about 2 nmol adenine nucleotide per milligram protein. Adenosine and inosine monophosphate decrease on reaeration of anaerobic cells, whereas inosine levels increase. When iodoacetate is added to block glycolysis, the decline in adenine nucleotide and production of inosine monophosphate are accelerated and there is no resynthesis of adenine nucleotide when anaerobic cells are reoxygenated. Large accumulations of inosine monophosphate are also seen in myocytes treated with an uncoupler of oxidative phosphorylation.