Adenosine is not a direct GHSR agonist ? artificial cross-talk between GHSR and adenosine receptor pathways

Aim: To assess if adenosine is a direct growth hormone secretagogue receptor (GHSR) agonist by investigating the mechanism behind adenosine induced calcium release in human embryonic kidney 293s (HEK) cells expressing GHSR. Methods: Calcium mobilization, cyclic adenosine monophosphate (cAMP) and IP 3 experiments were performed using HEK cells stably expressing GHSR and/or adenosine A 2B receptor (A 2B R). Results: Adenosine has been widely reported as a GHSR agonist. In our hands, adenosine and forskolin stimulated calcium release from IP 3 controlled stores in HEK-GHSR cells but not in non-transfected HEK cells. This release was not accompanied by increased IP 3 levels. The calcium release was both cholera toxin and U73122 sensitive, indicating the involvement of both Gα s /adenylyl cyclase and Gα q/11 /phospholipase C pathways. Importantly, the GHSR inverse agonist [D-Arg 1 D-Phe 5 D-Trp 7,9 Leu 11 ]-Substance P (SPanalogue) blocked the adenosine stimulated calcium release, demonstrating that GHSR is involved. Assessment of the GHSR-dependent calcium release using adenosine receptor agonists and antagonists resulted in a rank order of potencies resembling the profile of A 2B R. A 2B R over-expression in HEK-GHSR cells enhanced potency and efficacy of the adenosine induced calcium release without increasing IP 3 production. Moreover, A 2B R over-expression in HEK cells potentiated NECA-induced cAMP production. However, GHSR expression had no effect on intracellular cAMP production. Conclusion: In HEK-GHSR cells adenosine activates endogenously expressed A 2B R resulting in calcium mobilization. We hypothesize that the responsible mechanism is cAMP-dependent sensitization of IP 3 receptors for the high basal level of IP 3 caused by GHSR constitutive activity. Altogether, our results demonstrate that adenosine is not a direct GHSR agonist.