Characterization and transduction mechanisms of purinoceptors in activated rat microglia

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Abstract
1 Purinoceptor agonist-induced currents in untreated (proliferating) and lipopolysaccharide- (LPS; 100 ng ml−1) treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. 2 In non-proliferating microglia, adenosine (0.01–100 μm), 2-methylthio ATP (3–3000 nm), ATP (0.1–1000 μm), and ATP-γ-S (1–10 μm), but not α,β-methylene ATP (α,β-MeATP; 100 μm) produced a slow outward current at a holding potential of 0 mV. When K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mm), the 2-methylthio ATP- (300 nm) induced outward current disappeared. The effect of 2-methylthio ATP (300 nm) did not depend on the presence of extracellular Mg2+ (1 mm). The outward current response to 2-methylthio ATP (300 nm) was larger in proliferating than in non-proliferating microglia. 3 ATP (1–1000 μm) evoked a fast inward current at a holding potential of − 70 mV in non-proliferating microglia, while adenosine (100–1000 μm) was inactive. When the effects of ATP were compared at 0 and − 70 mV, it became evident that ATP is much more potent in evoking the outward current. 4 The 2-methylthio ATP- (300 nm) induced outward current was blocked by suramin (300 μm), but not by 8-(p-sulphophenyl)-theophylline (100 μm), while the adenosine- (1 μm) induced outward current had the reverse sensitivity to these antagonists. 5 The 2-methylthio ATP- (300 nm) induced outward current was inhibited by inclusion of GDP-β-S (200 μm) into the pipette solution or by preincubation of microglial cells with pertussis toxin (50 ng ml−1) for 12 h. The 2-methylthio ATP- (300 μm) induced inward current was not changed by intracellular GDP-β-S (200 μm). The outward current response to adenosine (1 μm) was also abolished after pretreatment with pertussis toxin (50 ng ml−1). 6 Rat microglia possess both ATP-sensitive P2Y- and adenosine-sensitive P1-purinoceptors. The ATP-evoked inward current is mediated by P2Y-purinoceptors, while the ATP- and adenosine-evoked outward currents are mediated by P2Y- and P1-purinoceptors, respectively. The transduction mechanisms of the outward, but not the inward current activation involve a pertussis toxin-sensitive G protein.