The critical time for opening mitochondrial (mito) K(
ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional
ischemia. Ischemic PC or 5-minute exposure to 10 micromol/L
diazoxide, a mito K(
ATP) channel opener, reduced
infarction to 3+/-1% and 8+/-1%, respectively. The mito K(
ATP) channel closer
5-hydroxydecanoate (200 micromol/L), bracketing either 5-minute PC
ischemia or
diazoxide infusion, blocked protection (24+/-3 and 28+/-6%
infarction, respectively). However,
5-hydroxydecanoate starting 5 minutes before long
ischemia did not affect protection.
Glibenclamide (5 micromol/L), another K(
ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(
ATP) channel opening triggers protection but is not the final step. Five minutes of
diazoxide followed by a 30-minute washout still reduced
infarct size (8+/-3%), implying memory as seen with other PC triggers. The protection by
diazoxide was not blocked by 5 micromol/L
chelerythrine, a
protein kinase C antagonist, given either to bracket
diazoxide infusion or just before the index
ischemia. Bracketing preischemic exposure to
diazoxide with 50 micromol/L
genistein, a
tyrosine kinase antagonist, did not affect
infarction, but
genistein blocked the protection by
diazoxide when administered shortly before the index
ischemia. Thus, although it is not
protein kinase C-dependent, the protection by
diazoxide involves
tyrosine kinase. Bracketing
diazoxide perfusion with N:-(2-mercaptopropionyl)
glycine (300 micromol/L) or Mn(III)tetrakis(4-benzoic acid)
porphyrin chloride (7 micromol/L), each of which is a
free radical scavenger, blocked protection, indicating that
diazoxide triggers protection through
free radicals. Therefore, mito K(
ATP) channels are not the end effectors of protection, but rather their opening before
ischemia generates
free radicals that trigger entrance into a preconditioned state and activation of
kinases.