Effect of anoxic preconditioning on ATP-sensitive potassium channels in guinea-pig ventricular myocytes

Ischemic or hypoxic preconditioning in experimental animals and humans is described. The mechanism of preconditioning may involve several endogenous substances released from ischemic or hypoxic tissues (such as adenosine, noradrenaline and bradykinin) that stimulate protein kinase C (PKC), which then phosphorylates ATP-sensitive potassium channels (K(ATP) channels). However, the effect of hypoxic preconditioning on K(ATP) channels in guinea- pig ventricular myocytes is unclear. The uncoupler carbonyl cyanide p- (trifluoromethoxy)phenylhydrazone (FCCP) has been shown to activate K(ATP) channels in isolated cardiac cells. In the present study we tested whether anoxic preconditioning (APC) could affect the opening of K(ATP) channels activated by metabolic inhibition (MI) induced by FCCP in cell-attached and inside-out patches from guinea-pig ventricular myocytes. We measured the channel activity as NP(o)i and calculated it using the formula P(o)=I/(Ni), where P(o) is open-state probability, I is the mean patch current carried by all K(ATP) channels activated in a particular patch for a certain period of time, N is the number of functioning channels in the patch, and i is the unitary current of the K(ATP) channels. In cell-attached membrane patches, after about 5 min of initiating MI, K(ATP) channels were activated at a holding potential of +40 mV (NP(o)i=3.7±0.9 pA); APC pretreatment (3 min of anoxia followed by 7 min of reoxygenation) before MI (APC+MI group) shortened the time to activate K(ATP) channels by MI (2.3±0.5 min) and increased the activity of K(ATP) currents (NP(o)i=8.4±0.5 pA). This effect of APC was eliminated by administration of a PKC blocker, chelerythrine (5 μM), for 5 min before the APC pretreatment. In the inside-out patches, the IC50 of intracellular ATP against the K(ATP) channels in the APC+MI group was significantly increased to 642 μM compared to that in the MI group (IC50 of intracellular ATP =252 μM). Chelerythrine inhibited the effect of APC on the sensitivity of K(ATP) channels to the intracellular ATP concentration (IC50 of [ATP](i)=301 μM). Our results demonstrate that APC can increase and accelerate the opening of K(ATP) channels induced by MI, and decrease the sensitivity of K(ATP) channels to [ATP](i), which is mediated by promoting the activation of PKC induced by APC.