PKG activator 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (
CPT) at reperfusion protects ischemic hearts, but the mechanism is unknown. We recently proposed that in preconditioned hearts PKC lowers the threshold for
adenosine to initiate signaling from low-affinity A2b receptors during early reperfusion thus allowing endogenous
adenosine to activate survival
kinases phosphatidylinositol 3-kinase (PI3K) and ERK. We tested whether
CPT might also sensitize A2b receptors to
adenosine.
CPT (10 microM) during the first minutes of reperfusion markedly reduced
infarction in isolated rabbit hearts undergoing 30-min regional
ischemia/2-h reperfusion, and salvage was blocked by
MRS 1754, an A2b-selective antagonist. Coadministration of
wortmannin (PI3K inhibitor) or
PD-98059 (MEK1/2 and therefore ERK1/2 inhibitor) also blocked protection. In nonischemic hearts, 10-min infusion of
CPT did not change phosphorylation of Akt or ERK1/2. Neither did a subthreshold dose (2.5 nM) of the nonselective but A2b-potent receptor agonist 5'-(N-ethylcarboxamido)adenosine (
NECA). However, when 2.5 nM
NECA was combined with 10 microM
CPT, both phospho-Akt and phospho-ERK1/2 significantly increased, indicating
CPT had lowered the threshold for A2b-dependent signaling. The PKC antagonist
chelerythrine blocked this phosphorylation induced by
CPT +
NECA.
Chelerythrine also blocked the anti-
infarct effect of
CPT as did nonselective (
glibenclamide) and mitochondrial-selective (5-hydroxydecanoate) K(
ATP) channel blockers. A
free radical scavenger, N-(2-mercaptopropionyl)glycine, also blocked
CPT protection. We propose
CPT targets PKG, which activates PKC through
mitochondrial K(ATP) channel (
mitoKATP)-dependent redox signaling, a sequence mimicking that already documented in preconditioning. Activated PKC then augments sensitivity of normally low-affinity cardiac
adenosine A2b receptors so endogenous
adenosine can protect by activating Akt and ERK.