Role of large-conductance Ca(2+) -activated potassium channels in adenosine A(1) receptor-mediated pharmacological preconditioning in H9c2 cells.
Large-conductance Ca(2+)-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 Ca(2+)-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 Ca(2+)-activated potassium channel opener NS1619 (10 microM) 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 Ca(2+)-activated potassium channels blockers paxilline (1 microM) or iberiotoxin (100 nM). Cell viability following 6 h hypoxia was assessed by monitoring lactate dehydrogenase (LDH) release and caspase-3 activation. Ca(2+)-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 Ca(2+)-activated potassium channel alpha and beta4 protein subunits were detected in mitochondrial fractions isolated from H9c2 cells. NS1619 (10 microM) induced no significant changes in ERK1/2 or PKB phosphorylation. These results have shown for the first time that large-conductance Ca(2+)-activated potassium channels are involved in adenosine A(1) receptor-induced pharmacological preconditioning in a cell model system.
Fretwell
Laurice
L
School of Science and Technology, Nottingham Trent University, Nottingham, UK.
Dickenson
John M
JM
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D058907:Adenosine A1 Receptor Agonists; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D015687:Cell Hypoxia; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D015640:Ion Channel Gating / Q000187:drug effects; D019157:Ischemic Preconditioning, Myocardial; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000819:agonists / Q000378:metabolism*; D032386:Myoblasts, Cardiac / Q000187:drug effects / Q000378:metabolism; D009271:Naltrexone / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000819:agonists / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D043682:Receptor, Adenosine A1 / Q000378:metabolism*; D013997:Time Factors
D058907:Adenosine A1 Receptor Agonists; D051036:Large-Conductance Calcium-Activated Potassium Channels; D021122:Protein Subunits; D043682:Receptor, Adenosine A1; C051843:binaltorphimine; C048599:N(6)-cyclopentyladenosine; D009271:Naltrexone; D007770:L-Lactate Dehydrogenase; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D053148:Caspase 3; D000241:Adenosine