DUAL ACTIVATION OF ADENOSINE A(1) AND A(3) RECEPTORS MEDIATES PRECONDITIONING OF ISOLATED CARDIAC MYOCYTES

Ischemic preconditioning reduces post-ischemic myocardial injury by ac tivating myocellular adenosine A(1) receptors. Adenosine A(3) receptor s have also been implicated but there is no evidence for A(3) receptor s in cardiac myocytes. The aim of this study was to develop a model of preconditioning in isolated cardiac myocytes to evaluate the role of the adenosine A(1) and A(3) receptors in preconditioning-induced prote ction from ischemic injury. Reverse transcription polymerase chain rea ction (PCR) was also employed to establish the presence of adenosine A (3) receptors in these cells. In the preconditioning studies, ischemic injury was simulated by exposing isolated rabbit myocytes (placed in the cell chamber and paced at 1 Hz) to buffer containing (in mM) 2'-de oxyglucose (20), NaCN (1), Na+-lactate (20), KCl (10) at pH 6.6 (37 de grees C). Changes of diastolic and systolic cell length were monitored with an optical-video edge imaging system, and hypercontracture was a ssessed as an index of irreversible cell injury. Preconditioning (2 mi n brief ischemia and 15 min reperfusion) significantly reduced cell in jury resulting from a subsequent prolonged ischemia (10 min) and reper fusion (15 min), as indicated by a reduction in the incidence of cell hypercontracture from 67+/-6% to 29+/-5% (P <0.001). Preconditioning-i nduced cardioprotection was only partially blocked by a maximally effe ctive concentration (100 nM) of the adenosine A(1) receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (cell hypercontracture=43/-3%, P <0.05 vs. control) but completely blocked by either the combin ation of DPCPX (100 nM) with the adenosine A(1)/A(3) receptor antagoni st CPXS+8-(4-carboxyethylphenyl)-1,3-dipropylxanthine (BWA1433; 1 mu M ) or the non-selective adenosine receptor antagonist, 8-(p-sulfophenyl )theophylline (8-SPT; 100 mu M) (cell hypercontracture=64+/-4%, 59+/-5 %, respectively; P=NS vs. control). In non-hypercontractured myocytes, preconditioning also substantially enhanced the recovery of the contr actile amplitude and, similarly, this effect was only partially blocke d by DPCPX but completely blocked by either the combination of DPCPX w ith BWA1433, or 8-SPT. These studies suggest that preconditioning prot ects isolated cardiac myocytes from ischemic injury independent of oth er cell types. and that maximal preconditioning-induced cardioprotecti on requires activation of both adenosine A(1) and A(3) receptors. Reve rse transcription-PCR using primers for the rabbit receptor provide ev idence for the presence of adenosine A(3) receptors in these cells.