Concentration–Response Relationships for Adenosine Agonists during Preconditioning of Rabbit Cardiomyocytes☆

Abstract Although adenosine receptors have been implicated in the induction of preconditioning in a variety of experimental models, there is controversy concerning the specific adenosine receptor subtypes mediating this effect. Concentration–protection relationships for adenosine and adenosine agonists in rabbit cardiomyocytes were used to characterize the role of adenosine receptor subtypes in preconditioning. Isolated cells were ischemically preconditioned or pre-incubated for 10 min with increasing concentrations of adenosine, CCPA (2-chloro-N 6 -cyclopentyladenosine), APNEA (N 6 -2-(4-aminophenyl)ethyladenosine), or BNECA (N 6 -benzyl-5′-N-ethylcarboxamidoadenosine) in the presence or absence of 1 or 10 μ m of the selective A 1 -adenosine antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine). Following a 30-min post-incubation period, cells were pelleted, layered with oil and ischemically incubated for 180 min. Injury was assessed by osmotic swelling and trypan blue exclusion of sequential samples, and determination of the areas beneath the mortality curves. Adenosine produced a broad concentration–protection curve which was displaced to the right by DPCPX. The curve for A 1 -selective agonist CCPA was biphasic, with an initial response below 1 n m and a second above 1 μ m . DPCPX abolished the early response leaving a steep monophasic curve between 0.1 and 10 μ m CCPA. The APNEA curve appeared monophasic, the major slope occurring between 1–100 nμ m ; DPCPX (1 μ m ) shifted the concentration–response curve ≈ 30-fold and decreased the slope. Adenosine receptor agonist BNECA produced preconditioning characterized by a shallow monophasic concentration–protection curve with a maximal effect of 49% and an EC 50 of ≈5 n m ; DPCPX shifted the BNECA concentration–protection relationship ≈40-fold with only a modest increase in slope. Analysis of the data suggests that induction of preconditioning results from interaction of agonists with the A 1 receptor and a second adenosine receptor having properties consistent with the A 3 receptor. Adenosine, CCPA, APNEA, BNECA and DPCPX each appear to be selective for the A 1 adenosine receptor subtype in isolated rabbit cardiomyocytes.