Pharmacological preconditioning by diazoxide downregulates cardiac L-type Ca(2+) channels

Br J Pharmacol. 2010 Nov;161(5):1172-85. doi: 10.1111/j.1476-5381.2010.00960.x.

Abstract

Background and purpose: 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.

Experimental approach: 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.

Key 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 and implications: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / drug effects
  • Animals
  • Blotting, Western
  • Calcium Channels, L-Type / drug effects*
  • Calcium Channels, L-Type / metabolism
  • Calcium Signaling / drug effects
  • Diazoxide / pharmacology*
  • Down-Regulation / drug effects*
  • Ischemic Preconditioning, Myocardial / methods*
  • Male
  • Myocardial Infarction / prevention & control
  • Myocardial Reperfusion Injury / physiopathology
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism
  • Patch-Clamp Techniques
  • Potassium Channels / drug effects
  • Potassium Channels / metabolism
  • Rats
  • Rats, Wistar
  • Reactive Oxygen Species / metabolism
  • Vasodilator Agents / pharmacology

Substances

  • Calcium Channels, L-Type
  • L-type calcium channel alpha(1C)
  • Potassium Channels
  • Reactive Oxygen Species
  • Vasodilator Agents
  • mitochondrial K(ATP) channel
  • Diazoxide