Role of large-conductance Ca2+-activated potassium channels in adenosine A1 receptor-mediated pharmacological preconditioning in H9c2 cells

Large-conductance Ca2+-activated potassium channels, located on the inner mitochondrial membrane, have recently been implicated in cytoprotection. Therefore, the primary aim of this study was to determine the role of large-conductance Ca2+-activated potassium channels in adenosine A(1) receptor-induced pharmacological preconditioning in the rat embryonic cardiomyoblast-derived cell line H9c2. For pharmacological preconditioning, H9c2 cells were exposed to the adenosine A(1) receptor agonist N-6-cyclopentyladenosine (100 nM) or the Ca2+-activated potassium channel opener NS1619 (10 mu M) for 30 min prior to 6 h hypoxia (0.5% O-2) in glucose-free and serum-free media. Where appropriate cells were treated (15 min) before pharmacological preconditioning with the Ca2+-activated potassium channels blockers paxilline (1 mu M) or iberiotoxin (100 nM). Cell viability following 6 h hypoxia was assessed by monitoring lactate dehydrogenase (LDH) release and caspase-3 activation. Ca2+-activated potassium channel subunit protein expression and cell survival protein kinase (ERK1/2 and PKB/Akt) activation were assessed by Western blotting. The results demonstrate that the adenosine A(1) receptor is functionally expressed in H9c2 cells and when activated protects against hypoxia-induced LDH release and caspase-3 activation. Treatment with paxilline or iberiotoxin attenuated adenosine A(1) receptor and NS1619-induced pharmacological preconditioning. Large-conductance Ca2+-activated potassium channel alpha and beta 4 protein subunits were detected in mitochondrial fractions isolated from H9c2 cells. NS1619 (10 mu M) induced no significant changes in ERK1/2 or PKB phosphorylation. These results have shown for the first time that large-conductance Ca2+-activated potassium channels are involved in adenosine A(1) receptor-induced pharmacological preconditioning in a cell model system. (C) 2009 Elsevier B.V. All rights reserved.