Probenecid but not pannexin-1 blockers reduce P2X7-induced IL-1β secretion from human monocytes and J774 macrophages.

<p>Magnetically isolated CD14⁺ human monocytes were primed with LPS (100 ng/ml) for 4 hours and stimulated with 3 mM ATP for 15 minutes. After stimulation supernatants were removed and tested for IL-1β. (A) representative data from one donor, (B) shows pooled data from 6 donors expressed as % of ATP control. Carbenoxolone (CBX) was used at 50 μM, probenecid (PRO) at 1 mM and 2.5 mM, ¹⁰Panx1 at 100 μM, and A-438079 at 10 μM. (C) The J774 mouse macrophage cell line was primed with LPS (1 μg/ml) and stimulated with ATP (5 mM) for 20 minutes in physiological saline. P2X7 selective antagonist AZ10606120 (AZ106) was used at 10 μM. Mean data from three independent experiments is shown. ATP-induced IL-1β released into supernatant was measured by specific ELISAs for human and mouse IL-1β respectively. Error bars represent S.E.M and * represents P<0.05.</p

Probenecid inhibits ATP-induced dye uptake in HEK-hP2X7 cells.

<p>(A) Ethidium⁺ uptake was induced in HEK-hP2X7 expressing cells by the addition of 1 mM ATP (as denoted by the arrow) in low divalent physiological solution. Control ethidium uptake to ATP is shown in black squares and ethidium uptake in the presence of 2.5 mM probenecid (pre-incubated for 10 minutes at 37°C) is shown in open circles. (B) Concentration response curves for probenecid on human P2X7 dye uptake responses induced by 0.2 mM or 1 mM ATP. Data was calculated at 300 second timepoint as baseline corrected endpoint data and normalised to the ATP control. Data was fit using a normalised response variable slope in GraphPad Prism. (C) Concentration response curves for agonist ATP in the presence of increasing concentrations of probenecid (0.5 mM, 1 mM and 5 mM) showing a rightward shift.</p

Probenecid inhibits P2X7-induced dye uptake in human CD14⁺ monocytes.

<p>Ethidium⁺ uptake was induced in CD14-APC labelled human monocytes by the addition of 1 mM ATP (denoted by the arrow) in low divalent KCl buffer in the absence and presence of 1 mM probenecid. Dye uptake was measured on a FACSCalibur flow cytometer using a heated time-resolved module. A representative uptake curve is shown from 6 donors (top panel). Data (area under the curve after 3 minutes) from all donors in shown in bottom panel scatter plot.</p

Probenecid blocks P2X7-mediated calcium influx and inward currents in HEK-hP2X7 cells.

<p>HEK-hP2X7 cells were loaded with 1 μM Fluo-4 for 30 minutes and calcium responses recorded at room temperature (26°C) using a fluorescent plate reader. ATP (1 mM) was injected and fluorescence recorded at 520 nm in the absence of inhibitors or in the presence of 1 mM probenecid (purple) or 10 μM A-438079 (blue). (B) Mean data from three independent calcium experiments. Sustained portion of the calcium response was measured and calculated as % of ATP control. Probenecid reduced response to 63±3.4% of control whereas A-438079 completely abolished the sustained calcium response. (C) Inward currents through wild-type human P2X7 receptors expressed in HEK-293 cells were recorded using whole cell patch clamp at room temperature. Membrane was clamped at −60 mV and ATP (1 mM in low divalent solution) was applied using a fast-flow delivery system. Black bars indicate ATP exposure (5 seconds). The initial ATP response was measured then the cell was exposed to probenecid for 2 minutes before re-challenge with ATP in the continued presence of probenecid.</p

Probenecid blocks lucifer yellow uptake in HEK-hP2X7 cells.

<p>Lucifer yellow (0.25 mg/ml) uptake in response to P2X7 stimulation by ATP (3 mM) was measured using a fixed time incubation at 37°C. Coverslips were briefly washed in standard extracellular solution before being imaged. (A) Phase contrast and fluorescent images for each stimulation condition; Control, ATP (3 mM) and ATP+PRO (2.5 mM) and (B) mean data from 100–300 cells (regions of interest) from multiple images.</p

Probenecid shows minimal inhibition of ATP-induced dye uptake in HEK-rat P2X7 and HEK-mouse P2X7 cells.

<p>Ethidium⁺ uptake was induced in (A) rat P2X7 and (B) mouse P2X7 stable cell lines by the addition of 1 mM ATP (denoted by the arrow) in a low divalent physiological solution. Control ethidium uptake to ATP is shown in black squares and ethidium uptake in the presence of 2.5 mM probenecid (pre-incubated for 10 minutes at 37°C) is shown in red shapes.</p