Characterization and possible function of adenosine 5′‐triphosphate receptors 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 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--y-S and X,4-MeATP (1 -1000 jLM 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 (1-1I000 1M) 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-y-S (10 LM) 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 pM). 4-Aminopyridine (1-1O mM), however, almost abolished this current and unmasked a smaller outward current.4 The rank order of agonist potency was 2-methylthio ATP > ATP> aP-MeATP. Adenosine and UTP were inactive. Suramin (300 JLM) and reactive blue 2 (50 gLM) antagonized the effect of 2-methylthio ATP (300 tiM). 5I-V relations were determined by delivering fast voltage ramps before and during the application of 2-methylthio ATP (300 JAM). 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 (1-1I000 jM) induced the same inward current both in proliferating and nonproliferating microglia. However, the depolarizing response to 2-methylthio ATP (300 laM) 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 JAM, 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.

Activation of purinergic P2X receptors inhibits P2Y-mediated Ca2+ influx in human microglia

“…In addition, for both rodent and human microglia, activation of the P 2Y pathway has been found to enhance an outward K + current [3,4,10,14]. Thus, the net change of [Ca 2+ ] i in response to ATP is dependent on a number of modulatory factors affecting cell membrane potential and the status of intracellular Ca 2+ stores and store-operated channels.…”

“…However, recent work has documented a diversity of agents which cause transient increases in levels of cytoplasmic Ca 2+ in rodent microglia; such responses may be involved in coupling activating stimuli to particular cellular functions. Among the spectrum of stimuli found to elicit transient Ca 2+ responses in cultured rodent cells are complement [1], ATP [2][3][4], carbachol [5], platelet-activating factor (PAF [6]) and endothelin [7]. All of these stimuli (complement has not yet been tested) have also been found to cause transient Ca 2+ responses in human microglia [8][9][10][11].…”

“…The expression of purinergic receptor subtypes P 2X and P 2Y have been well documented in microglia [4,10,13,14]. In addition a P 2Z receptor, associated with a high conductance pore permeable to large hydrophylic solutes, has also been reported in microglia [15,16]; activation of this receptor may require concentrations of ATP near 1 mM [16].…”

Inhibition of store‐operated Ca2+ influx by acidic extracellular pH in cultured human microglia

“…Both compounds may play significant roles in cerebral ischemia. ATP has been deemed a putative signaling agent released by neurons after cellular damage and may serve as an activating factor for microglia (Norenberg et al, 1994). PAF has also been suggested as an activating factor for microglia (Mori et al, 1996) and is synthesized in the brain by glia, neurons, endothelial cells, and blood-borne leukocytes after ischemic injury (Yue et al, 1994).…”

Neuroprotective roles of the P2Y2 receptor

“…However, responses downstream of P2X and P2Y receptor activation can vary widely. For example, Ca 2+ influx and associated changes in membrane conductance accompanying activation of P2XRs trigger the opening of voltage-gated and Ca 2+ -dependent K + channels [192], whereas P2Y receptor activation is coupled to a variety of G protein-dependent and G protein-independent signaling pathways, as described above. Accumulation of proinflammatory cytokines and neurotoxic oligomeric Aβ peptide is associated with the progression of AD [193].…”