Adenosine receptor specificity in preconditioning of isolated rabbit cardiomyocytes: evidence of A3 receptor involvement

OBJECTIVE: The aim was to further characterise an experimental model of preconditioning of isolated rabbit cardiomyocytes and to determine the role of adenosine receptor subtypes in initiation of the protective response. METHODS: Isolated myocytes were subjected to 5 min preincubation in the presence or absence of glucose and various agonists and antagonists of adenosine receptors. Ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury during ischaemia was determined by sequential sampling of the pelleted cells and assessment of trypan blue permeability following 85 mOsm swelling. RESULTS: Myocytes were preconditioned with a 30-50% reduction of injury by a 5 min glucose-free preincubation. Substitution of 5 mM pyruvate for glucose during preincubation did not prevent the protective response. Protection was maintained over a 60-180 min postincubation period. Protection was blocked by 100 microM of the non-specific adenosine A1/A2 antagonist SPT, both when added only during preincubation or only into the ischaemic pellet. Calphostin C, a specific protein kinase C inhibitor at 200 nM, added to the ischaemic pellet blocked protection. Preincubation with R-PIA, the adenosine A1 agonist, did not precondition at an A1 selective dose of 1 microM, but did at 100 microM. The selective A2 agonist CGS 12680 (1 microM) did not precondition. The selective A1/A3 adenosine agonist, APNEA, preconditioned at 1 microM and 200 nM dose levels. Preconditioning induced either by 200 nM APNEA or by glucose-free preincubation was not blocked by 200 nM or 10 microM of the A1 antagonist DPCPX, which has extremely low affinity for A3 receptors, but was blocked by 1 microM of the A1/A3 adenosine antagonist BW 1433U83. CONCLUSIONS: Preconditioning can be induced in isolated myocytes by a 5 min preincubation/30 min postincubation protocol, and a similar protection induced by adenosine agonists with A3, but not A1 selectivity. Preconditioning is blocked by non-selective or selective A1/A3 adenosine antagonists and a specific protein kinase C inhibitor, but not by A1 antagonists with little affinity for A3 receptors. The results suggest that preconditioning in isolated rabbit myocytes requires participation of adenosine receptors with agonist/antagonist binding characteristics of the A3 subtype, and is likely to be mediated by activation of protein kinase C.