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Comparative Study
. 2002 Sep;120(3):407-18.
doi: 10.1085/jgp.20028599.

Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances

Robert Tarran  1 , Matthew E Loewen, Anthony M Paradiso, John C Olsen, Micheal A Gray, Barry E Argent, Richard C Boucher, Sherif E Gabriel
Affiliations
Comparative Study

Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances

Robert Tarran et al. J Gen Physiol. 2002 Sep.

Abstract

Two Cl(-) conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca(2+)-activated Cl(-) conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl(-)/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca(2+) signal in response to mucosal nucleotides that may contribute to the increased Cl(-)/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca(2+) signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height.

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Figures

F<sc>igure</sc> 1.
Figure 1.
Biophysical characteristics of Ca2+-activated Cl− conductances in wild-type (WT) and CF null (CF) single ciliated cells isolated from murine nasal epithelia. (A and B, top) Current traces obtained at the start of whole cell recording (∼60 s) by holding the membrane potential at 0 mV and pulsing between ±100 mV in 20-mV steps in WT and CF cells. (A and B, bottom) Current traces obtained using the same protocol ∼2–3 min after addition of 1 μM ionomycin to the bath in WT and CF cells. (C) Mean data of ionomycin-induced CaCCs. Black bars, WT cells (n = 18); striped bars, CF cells (n = 11). (Note, no significant difference was detected between phenotypes.) Data shown as mean ± SEM.
F<sc>igure</sc> 2.
Figure 2.
Measurements of short circuit current (ISC) and ASL height in confluent WT and CF murine tracheal epithelial (MTE) monolayers. (A) Representative traces from WT (MTE7b) and CF (MTE18) monolayers after mucosal 100 μM UTP addition. (B) Mean data of UTP-induced CaCCs. Black bars, WT cells (n = 14); striped bars, CF cells (n = 13). (C) Representative traces from WT and CF monolayers after mucosal 0.1 μM thapsigargin addition. (D) Mean dose–response curves to thapsigargin for WT (circles; n = 8) and CF (squares; n = 8) monolayers taken from peak responses typified in (C). (E) Confocal images of ASL labeled with Texas red–dextran obtained before and 10 min after mucosal addition of powdered UTP (∼200 μM) suspended in PFC. (F) Percentage increases in ASL height for WT (black bars; n = 6) and CF cultures (stripped bars; n = 6). Pre-UTP heights were 3.9 and 4.1 μm for WT and CF cultures, respectively. *, significantly different (P < 0.05) between WT and CF. Data shown as mean ± SEM.
F<sc>igure</sc> 3.
Figure 3.
Ca2+-activated Cl− conductances and Ca2+ signals in WT and CF MTE cells grown as confluent monolayers. (A and B, top) Current traces obtained at the start of whole cell recording (∼120 s) by holding the membrane potential at 0 mV and pulsing from −80 to +100 mV in 20-mV steps in WT and CF cells, respectively. (A and B, bottom) Current traces obtained using the same protocol ∼2–3 min after addition of 1 μM ionomycin to the bath in WT and CF cells, respectively. (C) Mean data of ionomycin-induced CaCCs. Black bars, WT cells (n = 8); striped bars, CF cells (n = 8). (D and E) Representative traces from WT and CF MTE cells, respectively, showing changes in the intracellular Ca2+ concentration (Cai 2+) after mucosal UTP addition. (F) Mean Δ Cai 2+. Black bars, WT cells (n = 8); striped bars, CF cells (n = 8). *, significantly different (P < 0.05) from WT. Data shown as mean ± SEM.
F<sc>igure</sc> 4.
Figure 4.
Ca2+-activated Cl− conductances and Ca2+ signals in isolated WT and CF MTE cells. (A and B, top) Current traces obtained at the start of whole cell recording (∼120s) by holding the membrane potential at 0 mV and pulsing from −80 to +100 mV in 20-mV steps in WT and CF cells, respectively. (A and B, bottom) Current traces obtained using the same protocol ∼2–3 min after addition of 1 μM ionomycin to the bath in isolated WT and CF cells grown on plastic, respectively. (C) Mean data of ionomycin-induced CaCCs. Black bars, WT cells (n = 15); striped bars, CF cells (n = 15). (D and E) Representative traces from WT and CF MTE cells, respectively, grown as single cells on glass coverslips showing the change in [Ca2+]i after mucosal UTP addition. (F) Mean Δ [Ca2+]i signal. Black bars, WT cells (n = 6); striped bars, CF cells (n = 6). (Note, no significant difference was detected between phenotypes.) Data shown as mean ± SEM.
F<sc>igure</sc> 5.
Figure 5.
ISC measurements of basolaterally α toxin-permeabilized WT and CF MTE monolayers. (A) Representative traces showing stepwise reductions in mucosal Cl− to 68 mM followed by mucosal UTP addition. (B, left) Mean data taken from A showing maximal ΔISC responses after mucosal UTP addition in WT cells (Black bars; n = 8) and CF cells (striped bars; n = 8). (right) Mean data showing maximal ΔISC responses after mucosal ionomycin addition to WT cells (Black bars; n = 8) and CF cells (striped bars). *, significantly different (P < 0.05) from WTs. Data shown as mean ± SEM.
F<sc>igure</sc> 6.
Figure 6.
Stable transfection of CFTR into the CF MTE monolayers: effects on ISC and ASL height. (A) Exemplar ISC traces from CF and CF+CFTR cell lines showing 1 μM forskolin followed by 100 μM UTP added mucosally. (B, left) mean forskolin responses (CF, n = 10 and CF+CFTR, n = 8). (Right) Mean (post-forskolin) UTP responses (CF, n = 10 and CF+CFTR, n = 8). (C) Confocal images of ASL (red) obtained 24 h after PBS/Texas red–dextran addition and 10 min later after mucosal addition of powdered UTP suspended in PFC (∼200 μM). (Note, immediately after PBS Texas red–dextran addition [i.e., at t = 0] all cultures had approximately equal heights [∼4 μm]). (D) Mean ASL heights for WT (n = 4), CF (n = 6), and CF+CFTR (n = 4) cultures. Black bars, 24 h after initial volume addition; striped bars, 10 min after UTP addition. *, significantly different (P < 0.05) from pre-UTP values; #, significantly different (P < 0.05) from WTs. Data shown as mean ± SEM.
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