This experiments investigated the signaling cascade responsible for anti-infarct effect by an A₂ adenosine receptor (AR) agonist 5'-N-Ethylcarboxaminidoadenosine (NECA).

Langendorff perfused isolated rat hearts were subjected to 30 minutes of regional ischemia and 120 minutes of reperfusion. Drugs were perfused for a period of 5 minutes before and 60 minutes after reperfusion. For comparison of cardioprotection among groups, area at necrosis (AN) and area at risk (AAR) were measured by triphenyltetrazolium chloride staining.

NECA significantly attenuated AN/AAR (14.1 ± 1.9%, P < 0.001) compared with control hearts (30.7 ± 2.8%). Anti-infarct effect by NECA was attenuated by an A_2AAR antagonist 8-(3-chlorostyryl)caffeine (23.7 ± 3.4%, P < 0.05) and an A_2BAR antagonist MRS1706 (29.9 ± 3.3%, P < 0.001). Cardioprotection by NECA was blocked by a guanylyl cyclase inhibitor (23.1 ± 2.9%, P < 0.05) and a protein kinase G (PKG) inhibitor KT5823 (30.3 ± 3.2%, P < 0.001). Glycogen synthase kinase-3β (GSK-3β) inhibitor SB216763 attenuated the AN/AAR in both NECA with MRS (17.8 ± 2.7%, P < 0.01 vs. control) and NECA with KT5823 treated hearts (8.2 ± 1.8%, P < 0.001 vs. control). The mitochondrial permeability transition pore (mPTP) opener atractyloside also aborted NECA's anti-infarct effect (24.7 ± 2.4% P < 0.05).

The signaling pathway by NECA administered at reperfusion involves the activation of both A_2AAR and A_2BAR and cGMP/PKG pathway, which in turn depends on inactivation of GSK-3β and inhibition of mPTP opening.