Transcriptional and Cell Signaling Cross-Talk between Adenosine and Opioid Receptors in Cardiac Cells

Abstract

A1 adenosine receptors (A1ARs) and {delta}-opioid receptors ({delta}-ORs) are involved in cardiovascular control, and mediate tissue protection during periods of stress. These G-protein coupled receptors (GPCRs) may employ similar signalling paths to elicit cardioprotection, and our prior studies support interactions between the two classes of GPCR in mediating protection. We further investigated this interaction in cardiac cells, assessing 'cross-regulation' of receptor mRNA expression in addition to signalling 'cross-talk' of kinase effectors downstream of the receptors. Murine HL-1 cardiomyocytes were stimulated with either an A1AR agonist, (100 nM CCPA) or {delta}-OR agonist (1 µM BW 373U86) for varying periods (3, 6, 12, or 24 hrs). Quantitative real-time PCR was used to determine time-dependent changes in mRNA expression for A1ARs and {delta}-ORs. Both agonists triggered a transient repression of A1AR transcription, which recovered to baseline by 24 hrs. Transcription of {delta}-ORs also responded to both A1AR and {delta}-OR agonism, albeit in a differing pattern involving initial repression (by ≥50%) in the first 3– 6 hrs followed by recovery to slightly above baseline levels at 24 hrs. To assess 'cross-talk', at the level of cell signalling, HL-1 cells were again incubated with CCPA or BW 373U86, either alone or in the presence of a selective A1AR or {delta}-OR antagonist (1 µM DPCPX or 1 µM BNTX, respectively). Both CCPA and BW 373U86 triggered Erk1/2 phosphorylation which, predictably, was blocked by their respective antagonists. Interestingly, however, the A1AR response was abrogated by {delta}-OR antagonism and, conversely, the {delta}-OR response was eliminated by A1AR antagonism. Erk1/2 phosphorylation with either A1AR or {delta}-OR agonsim was abrogated by an EGFR inhibitor (0.5 µM AG1478). These data highlight essential cross-talk between the two receptors systems, involving both cross-regulation of receptor mRNA expression, and an essential interaction in regulation of downstream kinase signalling. The nature of these interactions requires further investigation, but may in part involve EGFR signalling. These findings have important implications regarding cardiac actions of both receptors, particularly in terms of cardioprotection during ischemia-reperfusion.