Characterization and transduction mechanisms of purinoceptors in activated rat microglia

Abstract

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. 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. 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. 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. 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). 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. 1994 British Pharmacological Society