A₃Adenosine Receptors in Human Astrocytoma Cells: Agonist-Mediated Desensitization, Internalization, and Down-Regulation

Abstract

A 3 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 A 3 adenosine receptors in human astrocytoma cells after exposure to the agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5Ј-carbamoyladenosine (Cl-IBMECA). Cl-IBMECA induced a concentrationdependent inhibition of adenylyl cyclase activity with an EC 50 value of 2.9 Ϯ 0.1 nM. The effect was suggested to be mediated by A 3 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 A 3 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: A 3 adenosine receptor internalized with rapid kinetics, within 30 min, after cell exposure to 100 nM Cl-IB-MECA. The localization of A 3 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 A 3 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 A 3 adenosine receptors that reached 21.9 Ϯ 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 A 3 receptors, in astrocytoma cells, are regulated after short-and long-term agonist exposure.Actions of adenosine are mediated by four G protein-coupled membrane receptors (GPCRs): A 1 , A 2A , A 2B , and A 3 receptors (Fredholm et al., 1994). Although expressed at very low levels in mammalian brain, the A 3 adenosine receptor (AR) subtype has been implicated in behavioral depression and modulation of ischemic cerebral damage (for review, see von Lubitz, 1999). Through the development of selective A 3 AR agonists (e.g., Cl-IBMECA) and antagonists (e.g., MRS 1191 and MRS 1220) (Kim et al., 1994(Kim et al., , 1996Jacobson et al., 1997), the putative pathophysiological roles of this receptor have became clear. It has been demonstrated that A 3 AR agonists profoundly affect cell survival, by promoting cell protection or cell death, depending upon the cell type and/or agonist concentration (for reviews, see Jacobson, 1998;Jacobson et al., 1999). In human astrocytoma cells (ADF cells), exposure to nanomolar Cl-IBMECA concentrations increased resistance to apoptosis, by a mechanism...

Role of adenosine A3 receptors on CA1 hippocampal neurotransmission during oxygen–glucose deprivation episodes of different duration

“…Studies using CHO cells stably expressing the A 3 receptors have demonstrated that, following a few minutes of exposure to the agonist, there is rapid phosphorylation of the A 3 receptor, a reduction in number of A 3 high affinity agonist binding sites [16], and receptor internalization [19]. In human astrocytoma cells, brief exposure (1-60 min) to nanomolar concentrations of Cl-IB-MECA results in a rapid uncoupling of A 3 receptors and receptor internalization while a long-lasting (1-24 h) Cl-IB-MECA treatment leads to receptor down-regulation which recovers after several hours [20]. On this basis, in our experiments, 9 to 15 min of treatment with Cl-IB-MECA likely produces uncoupling and internalization of the A 3 receptor.…”

“…Because it has been demonstrated that A 3 adenosine receptors undergo a rapid desensitization that is more evident and persistent when the agonist is applied for long periods [20], we chose to examine a different protocol, shortening the time of Cl-IB-MECA application. As shown in Fig.…”

“…This effect may be attributable to desensitization of A 3 receptors. In fact, both human and rat A 3 receptors are desensitized within a few minutes after agonist exposure [16][17][18][19][20][21].…”

Peripheral Benzodiazepine Receptor: Characterization in Human T-Lymphoma Jurkat Cells

“…Jurkat cells were processed for electron microscopy immunocytochemistry as described previously (Trincavelli et al, 2002) using a rabbit polyclonal anti-PBR antibody (Trevigen) diluted 1:10 in PBS containing 0.1% gelatin and 0.5% bovine serum albumin. Negative controls were performed in the absence of anti-PBR antibody.…”

Translocation of arrestin induced by human A3 adenosine receptor ligands in an engineered cell line: Comparison with G protein-dependent pathways

“…It is important to know how these agonists and antagonists behave in A 3 AR-mediated pathways that are independent of G proteins, such as arrestin. The A 3 AR has been reported to be a rapidly desensitizing receptor [10,11], and receptor regulation appears to be important in the clinical actions of A 3 AR agonists [12]. GRKs have been suggested to be involved in the desensitization and internalization process.…”