CHARACTERIZATION AND TRANSDUCTION MECHANISMS OF PURINOCEPTORS IN ACTIVATED RAT MICROGLIA

1 Purinoceptor agonist-induced currents in untreated (proliferating) a nd lipopolysaccharide- (LPS; 100 ng m(-1)) treated (nan-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp tech nique. 2 In non-proliferating microglia, adenosine (0.01-100 mu M), 2- methylthio ATP (3-3000 nM), ATP (0.1-1000 mu M), and ATP-gamma-S (1-10 mu M), but not alpha,beta-methylene ATP (alpha,beta-MeATP; 100 mu M) produced a slow outward current at a holding potential of 0 mV. When K + was replaced in the pipette solution by an equimolar concentration o f Cs+ (150 mM), the 2-methylthio ATP- (300 nM) induced outward current disappeared. The effect of 2-methylthio ATP (300 nM) did not depend o n the presence of extracellular Mg2+ (1 mM). The outward current respo nse to 2-methylthio ATP (300 nM) was larger in proliferating than in n on-proliferating microglia. 3 ATP (1-1000 mu M) evoked a fast inward c urrent at a holding potential of - 70 mV in nonproliferating microglia , while adenosine (100-1000 mu M) was inactive. When the effects of AT P were compared at 0 and - 70 mV, it became evident that ATP is much m ore potent in evoking the outward current. 4 The 2-methylthio ATP- (30 0 nM) induced outward current was blocked by suramin (300 mu M), but n ot by 8-(p-sulphophenyl)-theophylline (100 mu M), while the adenosine- (1 mu M) 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 mu M) 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 PM) induced inward current was not changed by intracellular GDP-beta-S (200 mu M). The o utward current response to adenosine (1 mu M) was also abolished after pretreatment with pertussis toxin (50 ng ml(-1)). 6 Rat microglia pos sess both ATP-sensitive P-2Y and adenosine-sensitive Pt-purinoceptors. The ATP-evoked inward current is mediated by P-2Y-purinoceptors, whil e the ATP- and adenosine-evoked outward currents are mediated by P-2Y- and P-1-purinoceptors, respectively. The transduction mechanisms of t he outward, but not the inward current activation involve a pertussis toxin-sensitive G protein.