Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia

Abstract

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.

FIGURE 1.

Hypotonicity-induced dye uptake and ATP release in WD-HBE cells. A, the uptake of propidium iodide (PI) was assessed in real time in response to a 33% hypotonic stress as described under “Experimental Procedures.” The images represent PI-associated nuclear fluorescence at T = 0 and 60 s after the hypotonic challenge. Scale bar = 100 μm. B, time course of hypotonic stress-promoted PI uptake (assessed as in A) and ATP release. Dye uptake is expressed as the percent of nuclei displaying red fluorescence. Similar results were obtained in at least three separate experiments performed in quadruplicate. C and D, WD-HBE cells were preincubated for 15 min with vehicle or with 10 μm carbenoxolone (CBX), 100 μm flufenamic acid (FFA), 30 μm ¹⁰Panx1 or its scrambled control (^srcPanx1), and exposed for 5 min to either isotonic (Iso) or hypotonic solution containing vehicle (Veh) or the indicated reagent. The results are mean ± S.E., n = 4. *, significant inhibition of hypotonic stress-evoked responses, p < 0.001 (analysis of variance).

FIGURE 2.

Hypotonicity induces cell swelling. Calcein-labeled WD-HBE cells were preincubated for 15 min with vehicle or 10 μm carbenoxolone (CBX). A, images of representative confocal microscopy scanning in the xz axis of cells undergoing hypotonic cell swelling. B, quantification of hypotonicity-elicited cell volume changes. The data are mean ± S.E. of three independent experiments performed in triplicate. *, significant difference from control RVD.

FIGURE 3.

Pannexin 1 mediates hypotonicity-induced ATP release in A549 cells. A549 cells were sham transfected (Control) or transfected with either pannexin 1 siRNA oligonucleotides (siRNA) or its scrambled control, as described under “Experimental Procedures.” A, Panx1 siRNA decreased pannexin 1 (but not connexin 43 (Cx43)). The data represent mean ± S.E., n = 4. B, ATP release was measured in A549 cells transfected as above and incubated for 5 min in isotonic (Iso) or hypotonic (Hypo) solutions. The data represent mean ± S.E. of three separate experiments performed in triplicate. C, representative images of PI uptake assessed under the conditions described in B. Scale bar = 100 μm. D, quantification of PI uptake (mean ± S.D., n = 4). Similar results were obtained in two independent experiments performed in quadruplicate. *, significantly different from control and scrambled, p < 0.05.

FIGURE 4.

Reduced ATP release from pannexin 1^−/− tracheas. A, Western blot analysis of cultured MTE cells (15 μg prot) from WT and pannexin 1^−/− littermates. Molecular weight standards are indicated on the right (in kDa). G-p28, a Golgi marker, was used as loading control. B, propidium iodide uptake was performed in WT and pannexin 1-deficient MTE cells incubated for 5 min under isotonic or hypotonic conditions. C, tracheas excised from WT and pannexin 1^−/− littermates were perfused (50 μl/min) with HBSS+ (isotonic) for 45 min. Tonicity was reduced to 50% (hypotonic), as indicated. The data (mean ± S.D., n = 3) is representative of two independent experiments performed with separate litters.

FIGURE 5.

Dye uptake in WD-HBE cells reflects a reversible phenomenon. Representative images (A) and quantification (B) of dye uptake in WD-HBE cells that were incubated for 5 min with isotonic (iso) or hypotonic (hypo) solutions in the presence of propidium iodide (a and b) or in its absence (c and d). Isotonicity was restored in d, and propidium iodide subsequently added to c and d for an additional 5 min. The images are representative of two independent experiments performed in quadruplicate. Scale bar = 100 μm. Nuclei staining was quantified (B) and expressed as mean ± S.E. *, significantly different from a, p < 0.01.

FIGURE 6.

Hypotonicity-induced ATP release in WD-HBE cells is associated with enhanced Rho activation and MLC phosphorylation. A, WD-HBE cells were preincubated for 45 min with 1 μm H1152 or 1 μm ML-7, and ATP release was measured after a 5-min incubation in hypotonic solution (Hypo) or isotonic control (Iso). n = 4. *, significantly different from Ctrl/Hypo, p < 0.05. B, representative Western blot for RhoA (left panel) and quantification of RhoA activation (right panel, mean ± S.E., n = 7) in response to a 5-min hypotonic challenge. C, effect of 1 μm H1152 or 1 μm ML-7 on hypotonicity-promoted MLC phosphorylation. A representative Western blot analysis and the quantification of phosphorylated MLC (MLCp) are shown in the left and right panels, respectively (mean ± S.D., n = 4). *, significantly different from Vehicle/Hypo, p < 0.05.

FIGURE 7.

Inhibition of RhoA activation reduces hypotonic stress-induced ATP release. A549 cells were transfected with empty vector or RhoA(T19N), as indicated under “Experimental Procedures.” A, hypotonicity-elicited ATP release (mean ± S.E., from three independent experiments performed in quadruplicate; *, p < 0.05). B and C, RhoA activation and phosphorylated MLC (MLCp) in response to hypotonic stress. Representative Western blot analysis and quantifications (mean ± S.E., n = 4) are shown in the left and right panels, respectively. *, significantly different from empty vector/Hypo, p < 0.05.

FIGURE 8.

Rho-dependent regulation of dye uptake. A, representative images illustrating PI uptake in WD-HBE cells preincubated with vehicle, H1152, or ML-7 (as in Fig. 5) and incubated for an additional 5 min with isotonic (Iso) or hypotonic (hypo) solutions in the presence of PI. B, quantification of PI uptake in WD-HBE cells treated as in A. Data are the mean ± S.E., n = 4. Scale bar = 200 μm. C, PI uptake was measured (as above) in A549 cells transfected with empty vector or RhoA(T19N). The results represent the mean ± S.E. of four separate experiments performed in triplicate. *, significantly different from control (p < 0.05, B) or empty vector/hypotonic (p < 0.01, C).

FIGURE 9.

Hypotonic challenge-induced Rho activation and pannexin 1-mediated ATP release is sensitive to TRPV4 inhibitors. A, WD-HBE cells were preincubated for 30 min with vehicle, 10 μm ruthenium red (RuRed), or 10 μm HC67047 followed by a 5-min hypotonic challenge, and ATP release was measured as indicated under “Experimental Procedures.” *, p < 0.05 against vehicle/hypotonic. B, cells were preincubated with 10 μm HC67047 and propidium iodide uptake was assessed as described in previous figures. Scale bar = 200 μm. The data (mean ± S.E., n = 4, right panel) are expressed as the % maximal PI uptake in response to a hypotonic challenge. *, p < 0.01. C, RhoA activation was measured in cells preincubated as above and challenged for 5 min with isotonic or hypotonic solution. Data are mean ± S.D., n = 3. *, significantly different from vehicle/hypotonic, p < 0.05.