Characterization and transduction mechanisms of purinoceptors in activated rat microglia.

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 microM), 2-methylthio ATP (3-3000 nM), ATP (0.1-1000 microM), and ATP-gamma-S (1-10 microM), but not alpha,beta-methylene ATP (alpha,beta-MeATP; 100 microM) 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 microM) evoked a fast inward current at a holding potential of -70 mV in nonproliferating microglia, while adenosine (100-1000 microM) 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 microM), but not by 8-(p-sulphophenyl)-theophylline (100 microM), while the adenosine- (1 microM) 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-beta-S(200 microM) 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 microM) induced inward current was not changed by intracellular GDP-beta-S (200 microM). The outward current response to adenosine (1 microM) 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.