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    • Although the effects of adenosine receptor

    2023-01-29

    Although the effects of adenosine receptor signaling in acute renal IRI have been attributed to the generation of high concentrations of adenosine, we have recently reported an increase in kidney levels of both AMP and adenosine, with a concomitant fall in ATP and ADP. We have also previously demonstrated that both serum AMP and adenosine concentrations were increased following the intravenous injection of pro-inflammatory collagen. This observation together with the evidence of increased A2BR expression in acute renal IRI, led to the hypothesis that AMP, in addition to adenosine, may be a ligand for the A2BR. This potential interaction is investigated here using computational modeling and calcium mobilization assays.
    Materials and methods
    Results and discussion To explore the hypothesis that AMP was a ligand for A2BR, we analyzed the docking potential of adenosine, AMP and AMPαS using computational modeling. Adenosine was chosen as the canonical ligand; AMP as the putative ligand and AMPαS as it is a non-hydrolysable analogue of AMP and was used for in vitro studies. A homology model of A2BR was created using the combined A2AR crystal structures as a template.16, 17 The quality of the model was assessed, with 98.9% of the amino acids residing in the allowed regions of the Ramachandran plot. Furthermore, Procheck states that the amino a-MSH, amide properties, including main-chain and side-chain parameters, bond lengths and bond angles were within or better than an average 2.5 Å resolution crystal structure, thus this model was deemed suitable for docking studies. All three ligands (adenosine, AMP and AMPαS) were docked convincingly into the A2BR model. AMP and AMPαS docked in an almost identical manner (Fig. 1A), suggesting that AMPαS is a good substitute for AMP. Because of this, only AMP will be discussed. Interestingly, AMP scored significantly higher than adenosine, with docking scores of 8.4 and 6.8 respectively. The adenosine rings of all ligands shared similar hydrophobic interactions with residues Leu 86, Phe 173, Glu 174, Met 182, Val 250, Met 272, Ile 276 and Asn 254. The adenosine rings also all contained hydrogen bonds between the side-chains of Asn 254 (with N1 of the adenine ring), Glu 174 (with amino group of adenine ring) and Ser 279 (with ribose oxygen) (Fig. 1B,C). Since the adenosine portion of all three ligands was orientated in the A2BR model with similar interactions, the differences in docking scores are likely to be attributed to the phosphate tail of AMP. Analysis of the top 20 docking solutions for AMP found 2 main clusters of solutions, differing only in the position of the phosphate tail. The first cluster consisted of 30% of the solutions with the phosphate tail interacting with the backbone of residues Leu 81 and Ala 82. The second cluster was significantly larger (60% of solutions) with the phosphate tail interacting with the side-chains of Thr 89, Asn 186 and His 251 (Fig. 1B,C). Based upon the higher proportion of solutions in this cluster and that these solutions more closely resembled the orientation of adenosine seen in the A2AR crystal structure, this cluster was chosen as the preferred docking orientation. To further explore the energetic contributions of AMP or adenosine binding, we then conducted molecular dynamics simulations on the docked solutions. Each representative docked solution was subjected to five independent 1 ns molecular dynamics simulations using the NAMD forcefield (27). An average energy calculation of each simulation was then conducted in VMD (28). The average total energy for the AMP docked simulation (−168,636 ± 16.07 kcal/mol) was significantly lower than the adenosine docked simulation (−150,114 ± 20.9 kcal/mol) (Fig. 2). This provides additional evidence that AMP interacts in an energetically favorable way with A2BR compared to adenosine. The A2BR is coupled to both Gs and Gq proteins, the former promoting cAMP accumulation, the latter calcium. Stimulation of CHO-hA2B cells with AMPαS had minimal effect on cAMP accumulation (Fig. 3ab) but increased intracellular calcium in a dose dependent fashion, overlapping the effect produce by adenosine (Fig. 3c) indicating equivalence potency with adenosine and preferential Gq activation. AMPαS was used in these assays given that it docked in an analogous manner to AMP and to exclude any effects that may be attributable to AMP hydrolysis. These data are at odds with those of Rittiner et al., who showed in vitro that AMP was an A1R agonist and did not activate the A2BR. An important difference between the studies is the cell type used: Rittiner et al. utilized HEK293 cells which, although commonly used for such studies, do endogenously express GPCRs and related signaling proteins. We utilized CHO cells which normally do not express adenosine receptors. Furthermore, the CHO cells used herein were engineered to stably express the human A2BR and generate a color change only with A2BR activation. Such a system eliminates the potential for rogue adenosine receptor activation.