Pharmacological preconditioning by diazoxide downregulates cardiac L-type Ca(2+) channels.
OBJECTIVE
Pharmacological preconditioning (PPC) with mitochondrial ATP-sensitive K(+) (mitoK(ATP) ) channel openers such as diazoxide, leads to cardioprotection against ischaemia. However, effects on Ca(2+) homeostasis during PPC, particularly changes in Ca(2+) channel activity, are poorly understood. We investigated the effects of PPC on cardiac L-type Ca(2+) channels.


METHODS
PPC was induced in isolated hearts and enzymatically dissociated cardiomyocytes from adult rats by preincubation with diazoxide. We measured reactive oxygen species (ROS) production and Ca(2+) signals associated with action potentials using fluorescent probes, and L-type currents using a whole-cell patch-clamp technique. Levels of the α(1c) subunit of L-type channels in the cellular membrane were measured by Western blot.


RESULTS
PPC was accompanied by a 50% reduction in α(1c) subunit levels, and by a reversible fall in L-type current amplitude and Ca(2+) transients. These effects were prevented by the ROS scavenger N-acetyl-L-cysteine (NAC), or by the mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). PPC significantly reduced infarct size, an effect blocked by NAC and 5-HD. Nifedipine also conferred protection against infarction when applied during the reperfusion period. Downregulation of the α(1c) subunit and Ca(2+) channel function were prevented in part by the protease inhibitor leupeptin.


CONCLUSIONS
PPC downregulated the α(1c) subunit, possibly through ROS. Downregulation involved increased degradation of the Ca(2+) channel, which in turn reduced Ca(2+) influx, which may attenuate Ca(2+) overload during reperfusion.
González
G
G
Departamento de Farmacología, Centro de Investigación y de Estudios Avanzados del I.P.N. México, México.
Zaldívar
D
D
Carrillo
Ed
E
Hernández
A
A
García
Mc
M
Sánchez
Ja
J
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D015153:Blotting, Western; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D003981:Diazoxide / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects*; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods*; D008297:Male; D009203:Myocardial Infarction / Q000517:prevention & control; D015428:Myocardial Reperfusion Injury / Q000503:physiopathology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D015221:Potassium Channels; D017382:Reactive Oxygen Species; D014665:Vasodilator Agents; C117780:mitochondrial K(ATP) channel; D003981:Diazoxide