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. 2011 Nov 4;585(21):3430-5.
doi: 10.1016/j.febslet.2011.09.033. Epub 2011 Oct 1.

Two non-vesicular ATP release pathways in the mouse erythrocyte membrane

Feng Qiu  1 , Junjie Wang, David C Spray, Eliana Scemes, Gerhard Dahl
Affiliations

Two non-vesicular ATP release pathways in the mouse erythrocyte membrane

Feng Qiu et al. FEBS Lett. .

Abstract

Erythrocytes are exceptionally suited for analysis of non-exocytotic release mechanisms of ATP, because these cells under physiological conditions lack vesicles. Previous studies have indicated, that Pannexin1 (Panx1) provides a key ATP permeation pathway in many cell types, including human and frog erythrocytes. Here we show that erythrocytes of Panx1(-/-) mice lend further support to this conclusion. However, ATP release, although attenuated, was still observed in Panx1(-/-) mouse erythrocytes. In contrast to Panx1(+/+) cells, this release was not correlated with uptake of extracellularly applied dyes, was insensitive to Panx1 channel blockers, and was inhibited by dipyridamole and stimulated by iloprost. Thus, in erythrocytes, two independent pathways mediate the release of ATP. We also show that glyburide is a strong inhibitor of Panx1 channels.

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Figures

Figure 1
Figure 1
a) Example of a Western blot of erythrocyte membranes of Panx1+/+ and Panx1-/- mice. The double band characteristic for Panx1 at ~50 kD was present in Panx1+/+ erythrocytes but not in Panx1-/- cells. b) ATP release from Panx1+/+ (white bars) and Panx1-/- (grey bars) erythrocytes. ATP release, as determined with a luciferase assay, is stimulated by hypotonic K+ solution (K+) more profoundly in Panx1+/+ erythrocytes than in Panx1-/- erythrocytes. The Panx1 channel inhibitor probenecid (prob, 1 mM) attenuated ATP release in Panx1+/+ cells but not significantly (p>0.05) in Panx1-/- cells.
Figure 2
Figure 2
a) Iloprost (25 μM) stimulated ATP release in Panx1+/+ (white bars) and Panx1-/- (grey bars) erythrocytes to similar levels. The iloprost-induced release remained unaffected by probenecid (1 mM). b) Dipyridamole inhibited the iloprost-induced ATP release by Panx1+/+ mouse erythrocytes. c) ATP release by Panx1+/+ mouse erythrocytes induced by hypotonic K+ solution (KGlu) was inhibited by dipyridamole or by probenecid (1 mM). The combination of the drugs was not more effective than either drug alone. Note, that different hematocrits were used for part (a) (20%) as compared to parts (b and c) (2%). Significance levels (Anova with Newman-Keul’s post test): * < 0.05, **<0.01.
Figure 3
Figure 3
a) Panx1 channel currents in oocytes expressing mouse Panx1 exogenously were inhibited by probenecid (1 mM) but remained unaffected by dipyridamole (10 μM). The inhibition by probenecid was less pronounced when dipyridamole was also present. Currents were induced by voltage pulses from -60 to + 20 mV. The traces were selected from a continuous record from the same oocyte, The interval between the traces was 30 minutes (for full recovery from the probenecid exposure). b) Quantitative analysis of current inhibition by probenecid (1 mM) in the absence and presence of dipyridamole. Means ± SE are shown, * p<0.05.
Figure 4
Figure 4
Hypotonic K+ solution (K+) stimulated the uptake of extracellularly applied carboxyfluorescein in Panx1+/+ (wt) erythroctes but not in Panx1-/- (KO) erythrocytes. Iloprost (25 μM) stimulation of Panx1-/- erythrocytes did not result in uptake of the dye.
Figure 5
Figure 5
a) Membrane currents in oocytes expressing mouse Panx1 induced by voltage pulses from -50 to + 50 mV. Glyburide (glibenclamide) attenuated the currents in a dose-dependent fashion. n=3-4. b) Connexin 46 currents in oocytes were not affected by glyburide. c) Dose-response relationship of Panx1 current inhibition by glyburide. d) ATP release by Panx1 expressing ooctes was stimulated by 150 mM potassium gluconate solution (KGlu) and inhibited by glyburide (100 μM).
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