The selective A2A adenosine receptors stimulation reduces delayed rectifier potassium currents (KDR) and differentiation of cultured oligodendrocyte progenitor cells

L Cellai, E Coppi, G Maraula, AM Pugliese, F Pedata. University of Florence, Department of Preclinical and Clinical Pharmacology 50139, Italy

Adenosine A2A receptor (A2AR) is a subtype of Gs-coupled receptor belonging to the P1 class of purinergic receptors. Neurons (at pre- and post-synaptic level), astrocytes, microglial cells, endothelial cells and oligodendrocytes, both in the precursor (OPCs) and mature stage, express A2ARs (Stevens B et al., (2002) Neuron 36: 855-868).

We studied the role of A2ARs on membrane currents and on differentiation of OPCs by electrophysiological recordings and by immunocytochemical labeling.

Experiments were carried out on purified primary OPC cultures isolated from postnatal day 1-2 Wistar rat cortex (Chen Y et al., (2007) Nat Protocol 2: 1044-1051). These cultures contained more than 98% of positive cells for OPC specific markers (NG2 proteoglycan and Olig2 transcription factor) and precursor cells were allowed to differentiate by appropriate conditions (i.e. by withdrawal of mitogenic factors). It has been described that the major component of membrane outward potassium (K+) currents in these cells is represented by delayed rectifier currents (KDR). Furthermore, 70% of OPCs also express the transient outward (KA) currents (Sontheimer H et al., (1989) Neuron 2: 1135-1145). We performed single-cell patch-clamp recordings from oligodendrocytes at different stages of development (0-3; 6; 12 days in culture). K+ currents were elicited by specific voltage-clamp protocols and were recorded before and after 5 min of application of the selective adenosine A2AR agonist CGS21680 (4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2 yl] amino] ethyl] benzene propanoic acid). CGS21680, applied at different concentrations (1-100 nM, n=21), decreased the amplitude of KDR currents (from 268±45 pA/pF in control conditions to 191±40 pA/pF at the agonist concentration of 100 nM, +80 mV, n=8, P<0.01 paired Student’s t-test). No significant changes were observed in the KA component (n=6). The effect on KDR was maximal after 5 min application and it was almost completely reversed after 5 min washout. Such currents were absent when intracellular and extracellular K+ ions where replaced by equimolar cesium. The CGS21680 effect on KDR currents was: i) concentration-dependent (reaching its maximum at 100 nM CGS21680); ii) blocked in the presence of the selective adenosine A2ARs antagonist SCH58261 (2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 100 nM, n=8); iii) prevented in the presence of the specific KDR blocker TEA (tetra-ethyl-ammonium, 3 mM, n=5); iv) recordable at all stages of differentiation. The application of CGS21680 (100 nM) in cultured OPCs at early stages reduced cell differentiation as evaluated by immunocytochemical labeling of specific markers of maturation (P<0.01, One-Way ANOVA followed by Newman-Keuls multiple comparison test) at 3, 6 and 9 days of culture. This effect was prevented by SCH58261 (100 nM).

Data demonstrated that cultured OPCs express functional A2ARs along their different stages of differentiation and that the activation of these receptors negatively modulates KDR currents. It is known that the block of KDR reduces oligodendrocyte differentiation (Gallo V et al., J Neurosci (1996) 16: 2659-2670). Such a mechanism may account for a reduction of OPC differentiation induced by selective A2AR stimulation.