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


CONCLUSIONS
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.
Hermansson
N-O
NO
Department of Lead Generation, AstraZeneca R&D Mölndal, Mölndal, Sweden.
Morgan
D G A
DG
Drmota
T
T
Larsson
N
N
D000241:Adenosine / Q000502:physiology*; D058908:Adenosine A2 Receptor Agonists; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000502:physiology; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D020239:Receptor Cross-Talk / Q000502:physiology*; D043704:Receptor, Adenosine A2B / Q000502:physiology*; D054440:Receptors, Ghrelin / Q000819:agonists* / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
D058908:Adenosine A2 Receptor Agonists; D053496:Inositol 1,4,5-Trisphosphate Receptors; D043704:Receptor, Adenosine A2B; D054440:Receptors, Ghrelin; D005576:Colforsin; D019830:Adenosine-5'-(N-ethylcarboxamide); D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D000241:Adenosine; D002118:Calcium