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Characterization and possible function of adenosine 5′‐triphosphate receptors 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 in culture were recorded by the whole‐cell patch‐clamp technique. These cells have two preferred resting membrane potentials, one at − 35 mV and another one at − 70 mV. 2 Most experiments were carried out in non‐proliferating cells. ATP, ATP‐γ‐S and α,β‐MeATP (1–1000 μm in all cases) evoked an inward current at a holding potential of − 70 mV, followed, in some experiments, by an outward current. At − 70 mV 2‐methylthio ATP (1–1000 μm) evoked an inward current, whereas at − 35 mV it produced an outward current only. 3 When K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mm), the main outward component of the ATP‐γ‐S (10 μm) induced response disappeared. Instead, an inward current was obtained. Replacement of K+ by Cs+ did not affect the inward current evoked by 2‐methylthio ATP (300 μm). 4‐Aminopyridine (1–10 mm), however, almost abolished this current and unmasked a smaller outward current. 4 The rank order of agonist potency was 2‐methylthio ATP >ATP>α,β‐MeATP. Adenosine and UTP were inactive. Suramin (300 μm) and reactive blue 2 (50 μm) antagonized the effect of 2‐methylthio ATP (300 μm). 5 I–V relations were determined by delivering fast voltage ramps before and during the application of 2‐methylthio ATP (300 μm). In the presence of extra‐ (1 mm) and intracellular (150 mm) Cs+, the 2‐methylthio ATP‐evoked current crossed the zero current level near 0 mV. When both Cs+ (1 mm) and 4‐aminopyridine (1 mm) were present in the bath medium, the intersection of the 2‐methylthio ATP current with the zero current level was near − 75 mV. 6 2‐Methylthio ATP (1–1000 μm) induced the same inward current both in proliferating and non‐proliferating microglia. However, the depolarizing response to 2‐methylthio ATP (300 μm) was larger and longer‐lasting in the proliferating cells. When the free Ca2+ concentration in the pipettes was increased from the standard 0.01 to 1 μm, the amplitude and duration of this depolarization was increased in non‐proliferating cells. 4‐Aminopyridine (1 mm) enhanced the duration, but not the amplitude of responses. 7 ATP and its structural analogues stimulate microglial purinoceptors of the P2Y‐type. This leads to the opening of non‐selective cationic channels and potassium channels. Depending on the resting membrane potential, depolarization or hyperpolarization prevails. Although the inward current produced by 2‐methylthio ATP is of similar amplitude in proliferating and non‐proliferating microglia, the resulting depolarization is smaller in the latter cell type because of the presence of voltage‐sensitive, outwardly rectifying potassium channels.