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Figure 1. Choline and phosphocholine inhibit BzATP-induced IL-1β release from U937 cells via nicotinic acetylcholine receptor containing α9 and α10 subunits.(a) Human monocytic U937 cells were primed with lipopolysaccharide (LPS, 1 µg/ml, 5 h) followed by stimulation with BzATP (100 µM, 30 min) in the presence or absence of phosphocholine (PC, 100 µM), choline (Cho, 100 µM) and/or different concentrations of the α-conotoxin RgIA4, a specific antagonist of α9α10 nicotinic acetylcholine receptors (nAChR). Interleukin-1β (IL-1β) released into the culture medium was measured by enzyme linked immunosorbent assay (ELISA). In control experiments, cells were left untreated (C1), primed with LPS (C2) or with LPS followed by BzATP (C3). In the presence of PC as well as Cho the IL-1β release was inhibited. The inhibitory effect of PC and Cho was dose-dependently antagonized by RgIA4 (*P ≤ 0.05, **P ≤ 0.01, significantly different from cells treated with PC or Cho alone, Mann-Whitney rank-sum test). (b) In LPS-primed U937 cells that were transfected with control siRNA (si) the BzATP-stimulated IL-1β release was inhibited by PC and Cho. In cells transfected with siRNA to Chrna9 or Chrna10, the effects of PC and Cho were blunted (*P ≤ 0.05, different from cells treated with LPS and BzATP; #P ≤ 0.05, ##P ≤ 0.01, different from respective experiments on cells treated with control siRNA; Kruskal-Wallis followed by Mann-Whitney rank-sum test). Data are presented as individual data points, bar represents median, whiskers percentiles 25 and 75.
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Figure 2. Choline and phosphocholine do not inhibit BzATP-induced IL-1β release from mononuclear leukocytes of Chrna9 and Chrna10 gene-deficient mice.(a,b) Mononuclear leukocytes were isolated from Chrna9 and Chrna10 gene-deficient mice (white circle; Chrna9 −/−; Chrna10 −/−) and corresponding wild-type mice (black circle; Chrna9 +/+; Chrna10 +/+). BzATP (100 µM) induced release of interleukin-1β (IL-1β) was investigated in the presence of phosphocholine (PC; 100 µM) or choline (Cho; 100 µM). PC and Cho suppressed BzATP-induced release of IL-1β in all WT strains investigated. In sharp contrast, no inhibition of IL-1β release was seen in Chrna9 −/− and Chrna10 −/− mice deficient in α9 or α10 subunit containing nicotinic acetylcholine receptors, suggesting that both subunits are needed (*P ≤ 0.05, **P ≤ 0.01, significantly different from cells treated with PC or Cho alone, Mann-Whitney rank-sum test). Data are presented as individual data points, bar represents median, whiskers percentiles 25 and 75.
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Figure 3. Choline inhibits BzATP-induced ion current stimulation in U937 cells.Whole-cell patch-clamp measurements were performed on human monocytic U937 cells primed with lipopolysaccharide (1 µg/ml, 5 h). Depicted are representative current curves (a,c). (a,d) In control experiments, consecutive application of the P2X7 receptor agonist BzATP (100 µM, grey bar) induced repetitive ion current stimulations (BzATP1 and 2). (b,d). After washout of the first BzATP stimulus, choline (Cho, 100 µM, dark grey bars) was applied. In presence of Cho, BzATP did not change the ion current. (c,d) Mecamylamine (Mec, 100 µM, white bar) antagonized the inhibitory effect of Cho. All BzATP-induced current changes (∆IBzATP) are shown as individual data points, bars represent median, whiskers percentiles 25 and 75. Statistical analyses were performed using the Wilcoxon signed-rank test.
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Figure 4. The inhibitory effect of phosphocholine on BzATP-mediated ion current stimulation is antagonized by α-conotoxin RgIA4.Whole-cell patch-clamp measurements were performed on human monocytic U937 cells primed with lipopolysaccharide (1 µg/ml, 5 h). (a) Application of the P2X7 receptor agonist BzATP (100 µM, grey bar) induced an ion current stimulation (BzATP1). After washout of BzATP1, phosphocholine (PC, 1 mM, dark grey bars) was applied. In presence of PC, BzATP did not change the ion current (BzATP2). (b,c,d) Mecamylamine (Mec, 100 µM, white bar, (b) as well as RgIA4 (200 nM, white bar; (c) antagonized the inhibitory effect of PC. (d) In parallel performed control experiments application of BzATP induced repetitive ion current stimulations that did not differ (current curve not shown). All ∆IBzATP values are shown as individual data points, bars represent median, whiskers percentiles 25 and 75. Statistical analyses were performed using the Wilcoxon signed-rank test.
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Figure 5. Phosphocholine does not induce ion channel functions in Xenopus laevis oocytes that heterologously express α9 or α9α10 nicotinic acetylcholine receptors.Two-electrode voltage-clamp (TEVC) measurements were performed on oocytes that heterologously expressed human α9 alone (a–c) or a combination of α9 and α10 (d–f) nicotinic acetylcholine receptor (nAChR) subunits. (a,d) Choline (Cho, 1 mM, black bars) induced repetitive stimulations of the transmembrane ion current (IM) in oocytes transfected with α9 (a) and in oocytes co-expressing α9α10 nAChR subunits (d). The second Cho-induced effect (Cho2) was smaller compared to the first one (Cho1) indicating receptor desensitization. (b,e) Initial application of phosphocholine (PC, 1 mM, white bars) had no impact on IM, whereas application of Cho thereafter induced a current stimulation. Again, oocytes expressing only α9 and those co-expressing α9α10 nAChR subunits led to similar results. (g,h) Representative current traces of water injected control oocytes (no expression of human receptors). Neither repeated application of Cho (n = 17), nor PC (n = 11) induced any changes in IM. Depicted are representative current curves (a,b,d,e,g,h). All changes of the transmembrane current (∆IM) induced by cholinergic stimulation are shown as individual data points, bars represent median, whiskers percentiles 25 and 75 (c,f). Statistical analyses were performed using the Wilcoxon signed-rank test.
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Figure 6. Phosphocholine inhibits choline-gated currents mediated by α9α10 nicotinic acetylcholine receptors heterologously expressed by Xenopus laevis oocytes.Two-electrode voltage-clamp experiments were performed on oocytes that heterologously expressed human α9α10 nicotinic acetylcholine receptors as described in Methods. (a) Representative current traces showing the inhibitory effects of phosphocholine (PC, 1 mM) on choline-gated currents (Cho, 1 mM). The current traces are each from 30 s recordings and are shown concatenated (omitting the 30 s gap between each individually recorded trace). Oocytes were continuously perfused with control solution and stimulated with 1 sec pulses of Cho once per min until steady-state baseline responses were observed (indicated by the arrow). Subsequently, the control solution was changed to one containing PC and the Cho-gated currents monitored for changes in amplitude for 20 min. Thereafter, PC was washed out and recovery from inhibition by PC monitored. (b) Analysis of inhibition and recovery from inhibition of Cho-gated currents by PC. The error bars denote the standard error of mean (SEM) from 5 oocytes.
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