Characterization and transduction mechanisms of purinoceptors in activated rat microglia

Abstract

1 Purinoceptor agonist-induced currents in untreated (proliferating) and lipopolysaccharide-(LPS; 100 ng ml-') treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. 2 In non-proliferating microglia, adenosine (0.01-100 PM), 2-methylthio ATP (3-3000 nM), ATP (0.1-1000 jlM), and ATP-T-S (1-10 gLM), but not a,p-methylene ATP (a,P-MeATP; 100 ftM) 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 (1-1I000 1~M) evoked a fast inward current at a holding potential of -70 mV in nonproliferating microglia, while adenosine (100-1000 giM) 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 f1M), but not by 8-(p-sulphophenyl)-theophylline (100 J4M), while the adenosine-(1 tiM) 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-P-S (200 ELM) into the pipette solution or by preincubation of microglial cells with pertussis toxin (50 ng ml-') for 12 h. The 2-methylthio ATP-(300 gAM) induced inward current was not changed by intracellular GDP-P-S (200 giM). The outward current response to adenosine (1 ,UM) was also abolished after pretreatment with pertussis toxin (50 ng ml-'). 6 Rat microglia possess both ATP-sensitive P2y-and adenosine-sensitive P,-purinoceptors. The ATPevoked inward current is mediated by P2y-purinoceptors, while the ATP-and adenosine-evoked outward currents are mediated by P2y-and P,-purinoceptors, respectively. The transduction mechanisms of the outward, but not the inward current activation involve a pertussis toxin-sensitive G protein.

Physiology of Microglia

“…This current had a reversal potential at Ϫ70 mV and was produced by the activation of K ϩ channels. The effects of adenosine were antagonized by an incubation with PTX and inhibited by the broad P1 receptors antagonist 8-(p-sulfophenyl)theophylline (497). A proliferative response of cultured rat microglia required the simultaneous expression of A 1 and A 2 receptors (309).…”

Properties of K + and Cl − channels and their involvement in proliferation of rat microglial cells

“…Most studies have been on cultured rat microglia (Banati et al, 1991;Kettenmann et al, 1990Kettenmann et al, , 1991Kettenmann et al, , 1993Langosch et al, 1994;Norenberg et al, 1992Norenberg et al, , 1993Norenberg et al, , 1994aSchmidtmayer et al, 1994, Visentin et al, 1995Walz et al, 1993), with some work on mouse (Bocchini et al, 1992;Brockhaus et al, 1993;Fischer et al, 1995;Walz et al, 1993), human (Norenberg et al, 1994b), and bovine microglia (McLarnon et al, 1995). External ATP, via P2-purinergic receptors, activates a ligand-gated, non-selective cation channel and causes a rise in cytoplasmic Ca2+ Langosch et al, 1994;Norenburg et al, 1994c;. Less frequently reported have been Ca2+-dependent K+ channels (&a) Norenberget al, 1994c;McLarnon et al, 19951, voltageactivated Na' (Korotzer and Cotman, 1992;Norenberg et al, 199413;Schmidtmayer et al, 1994) and Ca2+ currents (Colton et al, 19941, and anion currents (Ballyk et al, 1994;McLarnon et al, 1995;Schlichter et al, 1995;Visentin et al, 1995).…”

Evidence for P2-purinoceptor-mediated uptake of Ca2+ across a fish (Oreochromis mossambicus) intestinal brush border membrane

“…values reported for different preparations [7,14,15]. Suramin is generally employed at concentrations equal to or higher than the agonist concentration [2,16,17]. We added suramin at 0.1 and 1.0 mM to the assay medium.…”