Vol. 62, Issue 6, 1373-1384, December 2002

A₃ Adenosine Receptors in Human Astrocytoma Cells: Agonist-Mediated Desensitization, Internalization, and Down-Regulation

M. L. Trincavelli, D. Tuscano, M. Marroni, A. Falleni, V. Gremigni, S. Ceruti, M. P. Abbracchio, K. A. Jacobson, F. Cattabeni, and C. Martini

Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universitá Pisa, Pisa, Italy (M.L.T., D.T., M.M., C.M.); Dipartimento di Morfologia Umana e Biologia Applicata, Universitá Pisa, Pisa, Italy (A.F., V.G.); Dipartimento di Scienze Farmacologiche, Universitá Milano, Milan, Italy (S.C., M.P.A., F.C.); and Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (K.A.J.)

A₃ adenosine receptor activation has been previously demonstrated to result in both neuroprotective and neurodegenerative effects, depending upon specific pathophysiological conditions. This dual effect may depend on receptor regulation mechanisms that are able to change receptor availability and/or function. In the present study, we investigated desensitization, internalization, and down-regulation of native A₃ adenosine receptors in human astrocytoma cells after exposure to the agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (Cl-IBMECA). Cl-IBMECA induced a concentration-dependent inhibition of adenylyl cyclase activity with an EC₅₀ value of 2.9 ± 0.1 nM. The effect was suggested to be mediated by A₃ adenosine receptor subtype by the use of selective adenosine receptor antagonists. Cell treatment with pertussis toxin abolished Cl-IBMECA-mediated inhibition of adenylyl cyclase activity, evidencing an A₃ receptor coupling to inhibitory G protein. Short-term exposure to the agonist Cl-IBMECA (100 nM) caused rapid receptor desensitization, within 15 min. Agonist-induced desensitization was accompanied by receptor internalization: A₃ adenosine receptor internalized with rapid kinetics, within 30 min, after cell exposure to 100 nM Cl-IBMECA. The localization of A₃ adenosine receptors on the plasma membrane and in intracellular compartments was directly revealed by immunogold electron microscopy. After desensitization, the removal of agonist led to the restoration of A₃ adenosine receptor functioning through receptor recycling to the cell surface within 120 min. Prolonged agonist exposure (1-24 h) resulted in a marked down-regulation of A₃ adenosine receptors that reached 21.9 ± 2.88% of control value after 24 h. After down-regulation, the recovery of receptor functioning was slow (24 h) and associated with the restoration of receptor levels close to control values. In conclusion, our results demonstrated that A₃ receptors, in astrocytoma cells, are regulated after short- and long-term agonist exposure.