Divalent cation regulation of murine P2X7 receptors expressed by RAW264.7 macrophages

Moore, S.F & Mackenzie, A.B. Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY

Stimulation of the P2X7 receptor (P2X7R) with millimolar concentrations of ATP induces the opening of a non-selective cation channel and in many cases permeabilisation of the cell membrane to high molecular weight compounds (<900 Da) (Humphreys et al, 1998). We have studied the pharmacological properties of murine P2X7Rs and investigated the actions of extracellular divalent cations on pore formation.

Activation of P2X7Rs was evaluated by the uptake of ethidium bromide (EtBr, 25 μM, MW 314 Da) in a NaCl-containing buffer solution [147 NaCl, 2 KCl, 10 Hepes, 12 glucose, 2 CaCl2 and 1 MgCl2, pH 7.3 with NaOH]. Divalent cations were added or removed 5 mins before addition of ATP. Data were normalized to maximal fluorescence (100 %) in the presence of 0.2 % Triton-X100. In separate experiments, loss of cell viability was measured by the release of cytosolic lactate dehydrogenase (LDH). Data shown are mean ± s.e.mean from at least three experiments. Cells were plated at a concentration of 150,000 cells per well. Statistical significances were determined using either a Student’s t-test or one-way ANOVA.

ATP evoked a biphasic uptake of EtBr, Phase 1 (0 - 15 min) was found to have a pEC50 = 2.81±0.05 and a maximal rate of EtBr uptake of 0.68±0.07 % min-1 (n = 27). Phase 2 (0 - 30 min) was found to have a pEC50 = 2.64±0.03 and a maximal rate of EtBr uptake 1.00±0.08 % min-1 (n = 27). No LDH release was observed in Phase 1 (0 - 15 min) while LDH release was detected after 30 minutes stimulation with 1 - 5 mM ATP (n = 9). For the remainder of the study we characterized the properties of Phase 1 EtBr uptake. ATP (1 - 5 mM) induced EtBr uptake was inhibited by increasing concentrations of extracellular divalent cations with a potency series of Cu2+>Cd2+>Zn2+>Mg2+>Ca2+. In contrast to the action of Zn2+ at rat P2X7Rs, activated with micromolar concentrations of BzATP (Virginio et al., 1997), ≤300 μM Zn2+ did not block ATP-induced EtBr uptake (n = 9). The effect of Hg2+ on EtBr uptake was found to be distinct in comparison to the other divalents tested. The addition of Hg2+ (10 μM) induced an increase in the maximal rate of EtBr uptake to 1.09±0.09 % min-1 (158±35% increase in control rate; P<0.05, n = 9). Addition of HgCl2 (10 μM) did not alter ATP induced release of LDH (n = 9). To conclude, with the exception of Hg2+ murine P2X7Rs are inhibited by divalent cations. This inhibition has the same order of potency reported for cloned rat P2X7Rs (Virginio et al., 1997).

Virginio, C et al (1997) Neuropharmacology. 36, 1288-1294
Humphreys, B.D. et al (1998) Molecular Pharmacology. 54, 22-32

This work was funded by Heart Research UK.