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...

Low-affinity A 2B adenosine receptors (A 2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A 2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A 2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-a) treatment (24-h) increased A 2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-a markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A 2B ARs desensitization. In the presence of TNF-a, A 2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A 2B AR antagonist MRS 1706 and required the presence of TNF-a. These results suggest that, in ADF cells, TNF-a selectively modulates A 2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A 2B AR in response to brain damage. Keywords: A 2B adenosine receptors, cerebral damage, cytokines, functional cross-talk, human astrocytoma cells, reactive astrogliosis. Astrocytes are the most important source of extracellular adenine-based purines in brain (Ciccarelli et al. 1999) and also express specific adenosine receptors (ARs) including the A 1 , A 2A, A 2B and A 3 subtypes (Peakman and Hill 1996;Trincavelli et al. 2002a). Recent data suggest a role for the adenosine system in the autocrine and heterocrine regulation of these cells under both physiological and pathological conditions, such as hypoxia or ischaemia, when astrocytes turn into an activated form called 'reactive astrogliosis ' (Ciccarelli et al. 1994;Brambilla and Abbracchio 2001;Brambilla et al. 2003). There is still debate as to the functional significance of this reaction. Although it is accepted that activated astrocytes contribute to isolate damaged brain areas from surrounding healthy cells and participate in neuronal recovery synthesizing neurotrophins and pleiotrophins, these cells also release several potentially toxic compounds (e.g. nitric oxide and cytokines; Neary et al. 1996). There is a suggestion that excessive and prolonged astrogliosis, such as that found in acute and chronic neurodegenerative diseases, may contribute to brain damage; hence, the characterization of the endogenous Address correspondence and reprint requests to Claudia Martini, Department Psychiatry, Neurobiology, Pharmacology and Biotechnology. Via Bonanno, 6. 56126 Pisa, Abbreviations used: ARs, adenosine receptors; ADF, hum...

This study reports the synthesis of a number of 1- and 2-alkyl derivatives of the 4-aminopyrazolo[3,4-d]pyrimidine (APP) nucleus and their evaluation as inhibitors of ADA from bovine spleen. The 2-substituted aminopyrazolopyrimidines proved to be potent inhibitors, most of them exhibiting K(i) values in the nanomolar/subnanomolar range. In this series the inhibitory activity is enhanced with the increase in length of the alkyl chain, reaching a maximum with the n-decyl substituent. Insertion of a 2'-hydroxy group in the n-decyl chain gave 3k, whose (R)-isomer displayed the highest inhibitory potency of the series (K(i) 0.053 nM), showing an activity 2 orders of magnitude higher than that of (+)-EHNA (K(i) 1.14 nM), which was taken as the reference standard. Docking simulations of aminopyrazolopyrimidines into the ADA binding site were also performed, to rationalize the structure-activity relationships of this class of inhibitors.

The Cambridge Structural Database (CSD) was searched through two 3D queries based on substructures shared by well-known antagonists at the A(1) and A(3) adenosine receptors (ARs). Among the resulting 557 hits found in the CSD, we selected five compounds to purchase, synthesize, or translate synthetically into analogues better tailored to interact with the biological targets. Binding experiments using human ARs showed that four out of five tested compounds turned out to be antagonists at the A(1)AR or A(3)AR with K(i) values between 50 and 440 nM. Lead optimizations of 2-(benzimidazol-2-yl)quinoxalines (BIQs, 3) gave the best results in terms of potency and selectivity at the A(1) and A(3) ARs. Particularly, 2-(4-ethylthiobenzimidazol-2-yl)quinoxaline (3e) exhibited K(i) values at the A(1)AR, A(2A)AR, and A(3)AR of 0.5, 3440, and 955 nM, respectively, whereas 2-(4-methylbenzimidazol-2-yl)quinoxaline (3b) displayed at the same ARs K(i) values of 8000, 833, and 26 nM, respectively.

Abstract:A 3 adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen-dependent nature of the therapeutic effects probably related to receptor desensitization and down-regulation. Here we studied the agonist-induced internalization of human A 3 adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N 6 -(4-amino-3-[ 125 I]iodobenzyl)adenosine-5Ј-Nmethyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A 3 adenosine receptor showed a profile typical of these receptors in other cell lines (K D ϭ 1.3 Ϯ 0.08 nM; B max ϭ 400 Ϯ 28 fmol/mg of proteins). The iodinated agonist, bound at 4°C to whole transfected cells, was internalized by increasing the temperature to 37°C with a rate constant of 0.04 Ϯ 0.034 min Ϫ1 . Agonist-induced internalization of A 3 adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short-term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02 Ϯ 0.0017 min Ϫ1 .