AMP579 is revealed to be a potent A2b-adenosine receptor agonist in human 293 cells and rabbit hearts

Abstract

The mixed A1/A2a-adenosine agonist AMP579 given at reperfusion is protective in animal models of myocardial infarction. Receptor-blocking studies have indicated that the protection came from an adenosine receptor (AR), but neither A1- nor A2a-selective agonists could duplicate its protection. We recently found that A2b-selective agonists given at reperfusion are protective, and, therefore, tested whether AMP579 might also be an A2b agonist. We used human embryonic kidney cells overexpressing human A2b receptors as an assay system. In these cells, A2b receptor occupancy causes phosphorylation of ERK. AMP579 induced ERK phosphorylation with an EC50 of 250 nM and this phosphorylation could be blocked by MRS1754 or PSB1115, two highly selective blockers of human A2b receptors. We attempted to confirm our A2b hypothesis in a rabbit heart model of ischemia-reperfusion. AMP579 (500 nM) for 1 h starting at reperfusion reduced infarct size in isolated rabbit hearts exposed to 30 min of regional ischemia and 2 h of reperfusion (12.9 +/- 2.2% infarction of risk zone vs. 32.0 +/- 1.9% in untreated hearts). PSB1115 (500 nM) given for the first 15 min of reperfusion blocked AMP579's protection (32.2 +/- 3.1% infarction) which is consistent with an A2b mechanism. We conclude that AMP579 is a non-selective, but potent A2b-AR agonist, and that its protection against infarction is through that receptor.

Fig. 1

Experimental protocols

Fig. 2

Transfected HEK cells overexpressing A_2b receptors treated with A_2b antibodies coupled to a fluorochrome show intense fluorescence of the plasma membrane (a), while no fluorescence is seen in negative control cells (b) where IgG was substituted for the primary anti-A_2b antibody. c Representative western blot of A_2b-transfected HEK and unaltered HEK cells showing detection of adenosine A_2bAR only in the transfected cells

Fig. 3

a Agonists of A₁ (CCPA), A_2a (CGS), A_2b (Bay 60), and A₃ (MECA) adenosine receptors induced ERK1/2 phosphorylation, but the A_2b agonist BAY 60-6583 was the most potent. b Phosphorylation of ERK1/2 by either CCPA or CGS 21680 was not affected by the A_2b-selective antagonist MRS 1754. c PSB 1115 and MRS 1754 could both dramatically attenuate phosphorylation of ERK1/2 induced by Bay 60. d AMP579-induced phosphorylation of ERK 1/2 could also be attenuated by both A_2b-selective antagonists MRS 1754 and PSB 1115. In b–d total ERK 1/2 was unchanged by any intervention. Con control

Fig. 4

a Dose-dependent increase in ERK1/2 phosphorylation with increasing concentrations of AMP579 in an individual experiment. Total ERK 1/2 is unchanged as AMP concentrations are increased. b Summary of ERK 1/2 phosphorylation data following increasing doses of AMP579. The plot of blot density against concentration indicates the EC₅₀ for ERK1/2 phosphorylation by AMP579 is about 250 nM (n = 4)

Fig. 5

Myocardial infarct size expressed as a percentage of risk zone in isolated rabbit hearts treated with AMP579 alone or in addition to the selective A_2b receptor blocker PSB1115. Open and gray circles represent individual experiments while black circles depict group mean ± SEM. Gray circles depict previously obtained data with AMP579 [28] or NECA [31] and are presented for comparison. Infusion of AMP579’s at reperfusion was protective. PSB115 blocked both AMP579s and 5′-(N-ethylcarboxamido) adenosine’s (NECA) protection. PSB1115 alone had no effect on infarct size. Thus, A_2b receptors are involved in the AMP579 signaling that leads to protection. *P < 0.05 versus control

Fig. 6

Structure of AMP579 and 5′-(N-ethylcarboxamido) adenosine (NECA). The dotted lines on the NECA structure show where the molecules differ