Physiological levels of lipoxin A4 inhibit ENaC and restore airway surface liquid height in cystic fibrosis bronchial epithelium

Abstract

In cystic fibrosis (CF), the airway surface liquid (ASL) is depleted. We previously demonstrated that lipoxin A4 (LXA4) can modulate ASL height (ASLh) through actions on Cl(-) transport. Here, we report novel effects of lipoxin on the epithelial Na(+) channel ENaC in this response. ASL dynamics and ion transport were studied using live-cell confocal microscopy and short-circuit current measurements in CF (CuFi-1) and non-CF (NuLi-1) cell cultures. Low physiological concentrations of LXA4 in the picomolar range produced an increase in ASLh which was dependent on inhibition of an amiloride-sensitive Na(+) current and stimulation of a bumetanide-sensitive Cl(-) current. These ion transport and ASLh responses to LXA4 were blocked by Boc-2 an inhibitor of the specific LXA4 receptor ALX/FPR2. LXA4 affected the subcellular localization of its receptor and enhanced the localization of ALX/FPR2 at the apical membrane of CF cells. Our results provide evidence for a novel effect of low physiological concentrations of LXA4 to inhibit airway epithelial Na(+) absorption that results in an ASL height increase in CF airway epithelia.

Keywords: Cystic fibrosis; ENaC; lipoxin A4.

Figure 1.

LXA₄ pretreatment increases ASL height in NuLi‐1 and CuFi‐1 monolayers. (A) ASL height was measured over a period of 30 h to identify the time at which the ASL had stabilized. (B) LXA₄ dose response (15 min treatment) on the ASL height increase in CuFi‐1 epithelium. (C) Typical confocal Z‐sections of Nuli‐1 and CuFi‐1 (D) cell monolayers obtained by confocal microscopy in control conditions and after 15‐ and 60‐min treatment by LXA₄ (1 nmol/L). The ASL was stained with Dextran‐Texas Red and cell monolayer (stained green with Calcein‐AM). The effect of LXA₄ (1 nmol/L) on the ASL height depth in NuLi‐1 nontreated epithelium and after 15, 30, 45, 60, min, 24 and 48‐h treatments (E). (F) The effect of LXA₄ (1 nmol/L) on the ASL height measured 24 h after ASL labeling in CuFi‐1 in nontreated epithelium and after 15, 30, 45, 60 min, 24, and 48‐h treatments (***P < 0.001).

Figure 2.

Boc‐2 attenuates the LXA₄‐induced increase in ASL. Effect of Boc‐2 pretreatment to attenuate the LXA₄ (1 nmol/L, 15‐min treatment) induced ASL height increase in NuLi‐1 (A) and CuFi‐1 (B) epithelial monolayers (***P < 0.001).

Figure 3.

LXA₄ induced apical membrane ALX/FPR2 localization in CuFi‐1 monolayers. LXA₄ (1 nmol/L, 15‐min treatment) induced an apical increase of ALX/FPR2 (green) expression (B). Primary rabbit anti‐ALX/FPR2 antibody and secondary Alexa‐Fluor 488 anti‐rabbit were used to label the ALX/FPR2 receptor. Localization of the receptor at the apical surface is shown in the merged fluorochrome images in yellow (A). Rhodamine‐phalloidin was used to stain f‐actin and DAPI used to stain the nuclei. (C) FACS analysis of surface versus cytosolic localization of the ALX/FPR2 receptor in response to LXA₄ (1 nmol/L, 15‐min treatment) in Nuli‐1 and CuFi‐1 cells (n = 4, *P < 0.05).

Figure 4.

Effect of LXA₄ on amiloride‐sensitive ion transport and ASL height regulation. Response of the amiloride‐sensitive current to LXA₄ (1 nmol/L) pretreatment in NuLi‐1 (A) & CuFi‐1 (B) monolayers. Effect of apical amiloride (1 μmol/L, 15 min treatment) application on the LXA₄ mediated increase in ASL height in NuLi‐1 (C) CuFi‐1 (D) monolayers (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5.

Effect of LXA₄ on bumetanide‐sensitive ion transport and ASL height. (A) The response of bumetanide‐sensitive current to LXA₄ (1 nmol/L) pretreatment in NuLi‐1 (A) & CuFi‐1 (B) monolayers. Effect of apical bumetanide (1 μmol/L, 15‐min treatment) application on the LXA₄ mediated ASL height increase in NuLi‐1 (C) and CuFi‐1 (D) monolayers.