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Adenosine is not a direct GHSR agonist--artificial cross-talk between GHSR and adenosine receptor pathways
N-O Hermansson1, D G A Morgan, T Drmota
1Department of Lead Generation, AstraZeneca R&D Mölndal, Mölndal, Sweden.
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 Galpha(s)/adenylyl cyclase and Galpha(q/11)/phospholipase C pathways. Importantly, the GHSR inverse agonist [D-Arg(1) D-Phe(5) D-Trp(7,9) Leu(11)]-Substance P (SP-analogue) 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.
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