Activation of the Cardiac Sarcolemmal ATP-sensitive Potassium Channel by A1 and A3 Receptor Agonists: Confirmation from knockout mice studies

Abstract

Activation of the A1 adenosine receptor (AR) provides cardioprotection against ischemia/reperfusion injury most likely by facilitating opening of the cardiac sarcolemmal KATP (sarcKATP) channel. Recently, A3AR agonists have also been reported to protect the myocardium against ischemia/reperfusion injury. Though the functional coupling between the A1AR and sarcKATP is well documented, the coupling between the A3AR and the sarcKATP channel is unknown due to a lack of direct evidence. In the present study, we characterized the ability of the respective AR agonists to elicit opening of the sarcKATP channel. To activate A1 or A3AR, CPA (1μM) or CP-532,903 (1μM), respectively, were used. Whole-cell sarcKATP channel current, IKATP, was recorded from ventricular myocytes enzymatically isolated from hearts obtained from wild-type (WT) and A1 and A3AR gene knock-out (A1KO and A3KO, respectively) mice. In all studies, potential input from A2A and A2BARs was blocked by the extracellular application of ZM 241385 (100nM) and PBS 663 (100nM). In WT myocytes, CPA and CP-532,903 elicited IKATP with current densities of 2.6±0.7pA/pF (mean ± SEM, n=6) and 2.4±0.7pA/pF (n=7), respectively. To confirm the effects of the respective agonists, experiments were repeated in A1KO and A3KO myocytes. In the A1KO myocytes, CP-532,903, but not CPA, elicited IKATP with a current density of 2.2±0.4pA/pF (n=6). This confirmed that the activation of IKATP by CP-532,903 was via the A3AR. On the other hand, in the A3KO myocytes, CPA, but not CP-532,903, elicited IKATP with a current density of 2.3±0.7pA/pF (n=4). These results provide strong evidence of the functional coupling between A3AR and the sarcKATP channel. They further confirm the specificities of the A1AR and A3AR agonists to activate the sarcKATP channel via the A1 and A3AR, respectively.