The A1/A2
adenosine agonist 5'-(N-ethylcarboxamido)
adenosine (
NECA) limits
infarction when administered at reperfusion. The present study investigated whether p70S6
kinase is involved in this anti-
infarct effect. Adult rat ventricular myocytes were isolated and incubated in
tetramethylrhodamine ethyl
ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction in TMRE fluorescence serves as an
indicator of collapse of the mitochondrial transmembrane potential. Cells were subjected to H2O2 (200 microM), which like
ischemia induces loss of mitochondrial membrane potential. Fluorescence was measured every 3 min and to facilitate quantification membrane potential was arbitrarily considered as collapsed when fluorescence reached less than 60% of the starting value. Adding
NECA (1 mM) to the cells prolonged the time to fluorescence loss (48.0+/-3.2 min in the
NECA group versus 29.5+/-2.2 min in untreated cells, P<0.001) and the mTOR/p70S6
kinase inhibitor
rapamycin (5 nM) abolished this protection (31.3+/-3.4 min). Since
cyclosporine A offered similar protection,
mitochondrial permeability transition pore formation is a likely cause of the H2O2-induced loss of potential. The direct
GSK-3beta inhibitor
SB216763 (3 microM) also prolonged the time to fluorescence loss (49.2+/-2.1 min, P<0.001 versus control), and its protection could not be blocked by
rapamycin (42.2+/-2.3 min, P<0.001 versus control).
NECA treatment (100 nM) of intact isolated rabbit hearts at reperfusion after 30 min of regional
ischemia decreased
infarct size from 33.0+/-3.8% of the risk zone in control hearts to 11.8+/-2.0% (P<0.001), and
rapamycin blocked this
NECA-induced protection (38.3+/-3.7%). A comparable protective effect was seen for
SB216763 (1 microM) with
infarct size reduction to 13.5+/-2.3% (P<0.001).
NECA treatment (200 nM) of intact rabbit hearts at reperfusion also resulted in phosphorylation of p70S6
kinase more than that seen in untreated hearts. This
NECA-induced phosphorylation was blocked by
rapamycin. These experiments reveal a critical role for p70S6
kinase in the signaling pathway of
NECA's cardioprotection at reperfusion.