Existence and role of substrate cycling between AMP and adenosine in isolated rabbit cardiomyocytes under control conditions and in ATP depletion.

BACKGROUND Adenosine, a physiological coronary vasodilator, has been proposed to regulate coronary circulation according to myocardial oxygen demand. In the present study, we investigated the mechanisms of adenosine formation and utilization in isolated rabbit cardiomyocytes and, in particular, the existence and the role of substrate cycling between AMP and adenosine in the regulation of its concentration. METHODS AND RESULTS Rabbit cardiomyocytes were isolated by collagenase perfusion and incubated in HEPES-buffered Krebs-Henseleit solution at 37 degrees C, pH 7.4, in control conditions and in ATP depletion achieved by inhibiting glycolysis with 5 mmol/L iodoacetate. Under control conditions, adenosine accumulated at a rate of 4 pmol.min-1.10(-6) cells. The 13-fold elevation of adenosine accumulation induced by iodotubercidin (ITu), an inhibitor of adenosine kinase, proves that adenosine is normally recycled into AMP. This recycling involves 95% of the adenosine formed. In ATP depletion, adenosine accumulated at the rate of 335 pmol.min-1.10(-6) cells and was no longer rephosphorylated after 20 minutes, as shown by the absence of effect of ITu after this time interval. Moreover, adenosine was deaminated, as indicated by the twofold increase of its accumulation induced by deoxycoformycin (dCF), an inhibitor of adenosine deaminase. Both in control conditions and in ATP depletion, adenosine-dialdehyde, an inhibitor of S-adenosylhomocysteine (SAH) hydrolase, had no significant effect on adenosine formation, indicating that the transmethylation pathway is not an important source of adenosine in rabbit cardiomyocytes. CONCLUSIONS The results indicate that recycling of adenosine into AMP is essential for the maintenance of low, nonvasodilatory concentrations of the nucleoside under control conditions and that interruption of recycling plays an important role in elevating adenosine during ATP depletion.