A2A adenosine-receptor-mediated facilitation of noradrenaline release in rat tail artery involves protein kinase C activation and βγ subunits formed after α2-adrenoceptor activation

This work aimed to investigate the molecular mechanisms involved in the interaction of α2-adrenoceptors and adenosine A2A-receptor-mediated facilitation of noradrenaline release in rat tail artery, namely the type of G-protein involved in this effect and the step or steps where the signalling cascades triggered by α2-adrenoceptors and A2A-receptors interact.The selective adenosine A2A-receptor agonist 2-p-(2-carboxy ethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow evoked by trains of 100 pulses at 5 Hz. This effect was abolished by the selective adenosine A2A-receptor antagonist 5-amino-7-(2-phenyl ethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine (SCH 58261; 20 nM) and by yohimbine (1 μM). CGS 21680-mediated effects were also abolished by drugs that disrupted Gi/o-protein coupling with receptors, PTX (2 μg/ml) or NEM (40 μM), by the anti-Gsα peptide (2 μg/ml) anti-Gβγ peptide (10 μg/ml) indicating coupling of A2A-receptors to Gsα and suggesting a crucial role for Gβγ subunits in the A2A-receptor-mediated enhancement of tritium overflow. Furthermore, phorbol 12-myristate 13-acetate (PMA; 1 μM) or forskolin (1 μM), direct activators of protein kinase C and of adenylyl cyclase, respectively, also enhanced tritium overflow. In addition, PMA-mediated effects were not observed in the presence of either yohimbine or PTX.Results indicate that facilitatory adenosine A2A-receptors couple to Gsα subunits which is essential, but not sufficient, for the release facilitation to occur, requiring the involvement of Gi/o-protein coupling (it disappears after disruption of Gi/o-protein coupling, PTX or NEM) and/or Gβγ subunits (anti-Gβγ). We propose a mechanism for the interaction in study suggesting group 2 AC isoforms as a plausible candidate for the interaction site, as these isoforms can integrate inputs from Gsα subunits (released after adenosine A2A-receptor activation; prime-activation), Gβγ subunits (released after activation of Gi/o-protein coupled receptors) which can directly synergistically stimulate the prime-activated AC or indirectly via Gβγ activation of the PLC-PKC pathway.