2-chloro-n(6)-(3-iodobenzyl)adenosine-5--n-methyluronamide and Astrocytoma

In previous studies, we have demonstrated that exposure of astroglial cells to A3 adenosine receptor agonists results in dual actions on cell survival, with "trophic" and antiapoptotic effects at nanomolar concentrations and induction of cell death at micromolar agonist concentrations. The protective actions of A3 agonists have been associated with a reinforcement of the actin cytoskeleton, which likely results in increased resistance of cells to cytotoxic stimuli. The molecular mechanisms at the basis of this effect and the signalling pathway(s) linking the A3 receptor to the actin cytoskeleton have never been elucidated. Based on previous literature data suggesting that the actin cytoskeleton is controlled by small GTP-binding proteins of the Rho family, in the study reported here we investigated the involvement of these proteins in the effects induced by A3 agonists on human astrocytoma ADF cells. The presence of the A3 adenosine receptor in these cells has been confirmed by immunoblotting analysis. As expected, exposure of human astrocytoma ADF cells to nanomolar concentrations of the selective A3 agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (CI-IB-MECA) resulted in formation of thick actin positive stress fibers. Preexposure of cells to the C3B toxin that inactivates Rho-proteins completely prevented the actin changes induced by CI-IB-MECA. Exposure to the A3 agonist also resulted in significant reduction of Rho-GDI, an inhibitory protein known to maintain Rho proteins in their inactive state, suggesting a potentiation of Rho-mediated effects. This effect was fully counteracted by the concomitant exposure to the selective A3 receptor antagonist MRS1191. These results suggest that the reinforcement of the actin cytoskeleton induced by A3 receptor agonists is mediated by an interference with the activation/inactivation cycle of Rho proteins, which may, therefore, represent a biological target for the identification of novel neuroprotective strategies.

Topics: Adenosine; Astrocytoma; Cytoskeleton; Enzyme Inhibitors; Guanine Nucleotide Dissociation Inhibitors; Humans; Receptor, Adenosine A3; Receptors, Purinergic P1; rho-Specific Guanine Nucleotide Dissociation Inhibitors

The pathophysiological role of the adenosine A3 receptor in the central nervous system is largely unknown. We have investigated the effects of the selective A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine, Cl-IB-MECA, in cells of the astroglial lineage (human astrocytoma ADF cells). A marked reorganization of the cytoskeleton, with appearance of stress fibers and numerous cell protrusions, was found following exposure of cells to low (nM) concentrations of Cl-IB-MECA. These "trophic" effects were accompanied by induction of the expression of Rho, a small GTP-binding protein, which was virtually absent in control cells, and by changes of the intracellular distribution of the antiapoptotic protein Bcl-XL, that, in agonist-exposed cells, became specifically associated to cell protrusions. This is the first demonstration that the intracellular organization of Bcl-XL can be modulated by the activation of a G-protein-coupled membrane receptor, such as the A3 adenosine receptor. Moreover, modulation of the astrocytic cytoskeleton by adenosine may have intriguing implications in both nervous system development and in the response of the brain to trauma and ischemia.

Topics: Actins; Adenosine; Astrocytes; Astrocytoma; bcl-X Protein; Cell Size; Cytoskeleton; GTP-Binding Proteins; Humans; Proto-Oncogene Proteins c-bcl-2; Purinergic P1 Receptor Agonists; Receptor, Adenosine A3; Receptors, Purinergic P1; Tumor Cells, Cultured