Abstract

The A₃ adenosine receptor (A₃AR) contributes to several cardiovascular effects of adenosine, including antihypertensive and cardioprotective effects. Although several studies have detailed the mechanisms underlying agonist-mediated desensitization of the rat A₃AR, the regulation of the human A₃AR, which displays only a 70% amino acid identity with the rat homologue, has not been addressed. Using a Chinese hamster ovary cell line stably expressing a recombinant human A₃AR, we demonstrated that prolonged treatment with the AR agonist 5′-N-ethylcarboxamidoadenosine induces uncoupling of the A₃AR from G proteins and functional desensitization. In addition to A₃AR desensitization, a 1.5–2.5-fold increase was noted in the adenylyl cyclase (AC) activity achieved in the presence of GTP with or without forskolin. This sensitization of AC activity was not a consequence of the down-regulation of G_iproteins induced by NECA treatment and was not associated with sustained or transient increases in the expression of G_s. Time course experiments revealed that the onset of sensitization was half-maximal between 2 and 3 hr but was not due to the synthesis of new proteins because cycloheximide treatment failed to inhibit sensitization. The inability of the sensitization process to alter the AC activity obtained in the presence of manganese chloride suggests that prolonged A₃AR activation increases the coupling efficiency between G_s and AC catalytic units. This phenomenon has implications for long term cellular adaptation to agonist because in agonist-treated cells, the extent to which a suboptimal concentration of forskolin could increase phosphorylation of the cAMP-responsive element binding protein was elevated compared with vehicle-treated controls.