Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice

Abstract

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.

Fig. 1.

Effect of transgenic mouse strain on the nasal potential difference (NPD) response to lubiprostone (Lubi). The standard NPD protocol was modified to replace the isoproterenol perfusion with a dose escalation of lubiprostone. Data are expressed as mean millivolts of potential difference (y-axis) in each perfusate (x-axis). Error bars mark the SD. CFTR^tm1Unc-Tg(FABPCFTR)1Jaw/J, gut corrected bitransgenic (CFKO), n = 12; C57Bl/6, n = 12; A/J, n = 8. The A/J and CFKO strains have nearly identical lubiprostone responses.

Fig. 2.

Lubiprostone-mediated chloride transport is sustained after 4 min of perfusion (x-axis units in hours:minutes elapsed) but runs down when perfusion of the compound is halted. The NPD in a CFKO mouse (1 of 2 experiments) is shown to illustrate that perfusion with lubiprostone sustained chloride secretion (∼10 min) before diminishing.

Fig. 3.

Effect of 1% DMSO vehicle control on an NPD in C57Bl/6 mouse. DMSO was added to the zero Cl⁻ (solution 3) and run for at least 3 min. DMSO-free solution 3 was then applied for 3 min, and then 1% DMSO in solution 3 was reapplied. No significant voltage difference was seen in 3 mice tested. X-axis units in hours:minutes elapsed.

Fig. 4.

Effect of washout of lubiprostone. X-axis units in hours:minutes elapsed. Lubiprostone (10 μM) (solution 5) supplied to a CFKO mouse produces the characteristic hyperpolarization (n = 2). However, substitution with zero Cl⁻ (solution 3) containing 1% DMSO (vehicle control) results in a loss of effect or washout. This can be repeated several times in the same mouse.

Fig. 5.

Lubiprostone challenge in ClC-2 knockout mouse. X-axis units in hours:minutes elapsed. After baseline NPD was recorded, amiloride superfusion led to a modest depolarization consistent with the presence of wild-type CFTR. Shortly after switching to solution 3, chloride transport was induced, consistent with the presence of functional CFTR. Addition of 20 μM lubiprostone did not activate further chloride transport, consistent with the absence of ClC-2. Confirmation of CFTR function occurred with the subsequent exposure to 100 μM isoproterenol in solution 7.

Fig. 6.

Top: photograph of RT-PCR gel examining ClC-2 mRNA (90 bp). Bottom: β-actin control (130 bp).