Liquid movement across the surface epithelium of large airways

Abstract

The cystic fibrosis transmembrane conductance regulator CFTR gene is found on chromosome 7 [Kerem, B., Rommens, J.M., Buchanan, J.A., Markiewicz, D., Cox, T.K., Chakravarti, A., Buchwald, M., Tsui, L.C., 1989. Identification of the cystic fibrosis gene: genetic analysis. Science 245, 1073-1080; Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.L., et al., 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066-1073] and encodes for a 1480 amino acid protein which is present in the plasma membrane of epithelial cells [Anderson, M.P., Sheppard, D.N., Berger, H.A., Welsh, M.J., 1992. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am. J. Physiol. 263, L1-L14]. This protein appears to have many functions, but a unifying theme is that it acts as a protein kinase C- and cyclic AMP-regulated Cl(-) channel [Winpenny, J.P., McAlroy, H.L., Gray, M.A., Argent, B.E., 1995. Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells. Am. J. Physiol. 268, C823-C828; Jia, Y., Mathews, C.J., Hanrahan, J.W., 1997. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J. Biol. Chem. 272, 4978-4984]. In the superficial epithelium of the conducting airways, CFTR is involved in Cl(-) secretion [Boucher, R.C., 2003. Regulation of airway surface liquid volume by human airway epithelia. Pflugers Arch. 445, 495-498] and also acts as a regulator of the epithelial Na(+) channel (ENaC) and hence Na(+) absorption [Boucher, R.C., Stutts, M.J., Knowles, M.R., Cantley, L., Gatzy, J.T., 1986. Na(+) transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. J. Clin. Invest. 78, 1245-1252; Stutts, M.J., Canessa, C.M., Olsen, J.C., Hamrick, M., Cohn, J.A., Rossier, B.C., Boucher, R.C., 1995. CFTR as a cAMP-dependent regulator of sodium channels. Science 269, 847-850]. In this chapter, we will discuss the regulation of these two ion channels, and how they can influence liquid movement across the superficial airway epithelium.

Figure 1. Regulation of ASL height requires a balancing of Na⁺ absorption and Cl^- secretion

A, XZ confocal images of ASL labeled with Texas red-dextran. B, mean ASL height taken with time under control conditions, i.e. following the addition of 20 μl PBS labeled with Texas Red dextran. C, ASL height measured after 48 h under control conditions, and following addition of 100 μM serosal bumetanide or 10 μM mucosal nystatin to inhibit Cl^- secretion or activate Na⁺ absorption respectively. Data shown as mean ± standard error. * p ≤ 0.05 different to control. † p ≤ 0.05 different between normal & CF.

Figure 2. Interaction between protease and adenosine pathways for ASL regulation

A, Change in transepithelial electric potential difference (V_t) across normal (open bars) and CF cultures (closed bars) after 30 min apical trypsin exposure (1.5 U/ml) at 0 or 48 h after PBS (20 μl) addition (all n = 10 and 6 for normal, respectively; and 11 and 8 for CF, respectively). B, Change in V_t after 30 min apical aprotinin exposure (2 U/ml) to normal (open bars) or CF (closed bars) cultures at t = 0 or 48 h after PBS addition (n = 5 and 5 for normals; 4 and 4 for CFs, respectively). N.B., all significant changes in V_t were abolished by amiloride pretreatment (3×10^-4 M; all n = 4). C & D, XZ confocal images of normal & CF cultures respectively acutely prewashed with PBS containing Texas red-dextran and either trypsin (1.5 U/ml) or aprotinin (2 U/ml) 0, 10 and 60 min post-adenosine addition (300 μM). E& F, mean data taken from C & D. Open bars, normal cultures. Closed bars, CF cultures. All data points are n = 6. * Different (p < 0.05) from t=0. † Different (p < 0.05) from equivalent time point in the presence of trypsin. ‡ Different (p < 0.05) between normal and CF cultures. Scale bars are 7 μm.

Figure 3. Cartoon depicting changes occurring during Normal ASL volume regulation under static conditions and how this process may be probed pharmacologically

A, in normal airways under high volume conditions, any soluble regulatory molecules such as adenosine (ADO) or secreted protease inhibitors are diluted to such an extent that CFTR is inactive and anion secretion does not occur and ENaC is near to fully active, leading to Na⁺-led isotonic ASL absorption with Cl^- following through the paracellular pathway. B, as ASL volume diminishes, adenosine accumulate in the ASL sufficiently to activate CFTR (likely by stimulation of A2B adenosine receptors) and protease inhibitors (red circles) are secreted to mostly inactivate ENaC via inhibition of channel-activating proteases (CAPS). This leads to a steady state ASL height which approximates the height of outstretched cilia (7 μm) which is maintained by continued anion secretion through CFTR that is offset by a moderate amount of Na⁺ absorption through ENaC. C, to confirm that steady state ASL height is maintained by active ion transport, bumetanide can be applied serosally to block Cl^- entry into the epithelia which is predicted to result in ASL collapse to CF levels (i.e. 3-4 μm). N.B., ASL HCO₃^- (< 10 mM) is insufficient to maintain a suitable ASL height in the absence of Cl^-secretion. D, the physiological inhibition of ENaC with time is also required for maintenance of a steady-state ASL height and addition of a cationophore (nystatin) allows unregulated Na⁺-led ASL absorption to occur which is also predicted to cause ASL collapse.

Figure 4. Cartoon showing how abnormal Cl^- secretion and Na⁺ absorption in CF airways under static conditions lead to ASL volume depletion

A, in CF airways under high volume conditions, as in normal airways, any soluble regulatory molecules such as adenosine or secreted protease inhibitors are diluted and ineffective. Due to the lack of CFTR and its inhibitory effects on ENaC, ENaC is hyperactive leading to a more rapid baseline Na⁺ led volume absorption than in NL airways under high volume conditions with Cl^- following through the paracellular pathway. B, as ASL volume is reduced, regulatory molecules such as ADO and CAP-inhibitors accumulate in the ASL. However, in the absence of CFTR activation of A2B-R is predicted to raise cAMP and stimulate rather than inactivate ENaC. Further, for reasons that are currently unknown, the CAP system is also dysfunctional and ENaC remains proteolytically cleaved despite a reduction in ASL volume. Thus, unlike NL airways, CF airways are unable to make the switch from an absorbing to a secreting epithelia and ASL volume depletion results. C, D, unlike NL airways, since ASL volume is already depleted, bumetanide and nystatin addition are without affect. Note, since ASL volume does not fall below ∼3 μm in airway epithelia, transcellular Na⁺ absorption will likely be offset by a backflux of Na⁺ through the paracellular pathway.

Figure 5. Phasic motion-induced changes in PCL volume in normal vs. CF cultures

A, XZ confocal images of PCL immediately (0) and 48 h after mucosal PBS addition to normal and CF epithelia cultured under phasic motion. B, Mean PCL heights after 48 h of phasic motion culture for normal (open bars, n = 7) and CF (closed bars, n = 8). C,. Mean PCL height after 3 h of phasic motion in the presence of a CFTR antagonist (CFTR_inh172; 10 μM) or CFTR_inh172 and a CaCC antagonist (DIDS; 100 μM). Normals (open bars; n = 6) and CFs (closed bars; n = 6). Data shown as mean ± S.E.M. *Data significantly different between normal and CF cultures. † Data significantly different from control. ‡Significantly different between static and phasic motion or significantly different from CFTR_inh172.

Figure 6. Schema describing ASL height regulation by phasic shear stress

A, Normal airway epithelia under static conditions coordinately regulate the rates of Na⁺ absorption and Cl^- secretion to set ASL volume at 7 μm. B, Normal airway epithelia under phasic motion respond to increased nucleotide/nucleoside release into the ASL by shifting the balance further towards Cl^- secretion via CFTR and Ca²⁺ activated Cl^- channel (CaCC) resulting in a greater ASL height. C, in CF epithelia, the higher basal rate of Na⁺ absorption, the failure to inhibit Na⁺ transport rates, and the failure to initiate Cl^- secretion under static conditions lead to PCL depletion (note “flattened” cilia). D, CF cultures under phasic motion conditions release sufficient ATP into the ASL to inhibit Na⁺ absorption and initiate CaCC mediated Cl^- secretion to restore ASL to a physiologically adequate height.

Figure 7. Normal and CF airway epithelia respond differently to hypertonic saline and to amiloride

∼ 1000 mM NaCl is delivered to the mucosal surface of well-differentiated normal and CF bronchial cultures and ASL height monitored by XZ confocal microscopy. Normal cultures with NaCl (■), CF cultures with NaCl (●), NaCl and 10 μM Inh₁₇₂ (▲), Na gluconate (◆).

Figure 8. Cartoon predicting a differential response to hypertonic saline addition in normal vs. CF airway epithelia

A, 1000 mM NaCl is deposited on airway surfaces following inhalation of nebulized ∼6% hypertonic saline. B, in NL airways, the increase in the NaCl concentration following HS deposition increases the electrochemical driving force for Na⁺ to move transcellularly through ENaC (orange arrows) as well as increasing the electrochemical driving force for Na⁺ to move through the paracellular pathway (not shown). Importantly, the unphysiologically large increase in the ASL Cl^- concentration now generates a sufficiently large electrochemical driving force for transcellular Cl^- absorption via CFTR, with the some Cl^- also moving through the paracellular path (∼33%; not shown). The Na⁺ and Cl^- that enters the cell in response to the HS-induced NaCl concentration gradient on the airway surface exits the cell through the basolateral Na⁺-K⁺-ATPase, a basolateral Cl^- channel and the Na⁺-K⁺-2Cl^- cotransporter (not shown). Concomitantly, H₂O moves in the opposite direction from the serosa into the ASL both through aquaporins and paracellularly (green arrows) to increase ASL height, although due to the rapid, passive transcellular absorption of NaCl, the NaCl-dependent increase in ASL height is less than would be predicted if all 1000 mM NaCl remained on airway surfaces. C, following this period of rapid equilibration, ASL height is moderately raised as compared to A. D, Due to dilution of sensor molecules in the ASL, isotonic Na⁺ led absorption returns ASL height to a depth of 7 μm. E, even though an increased electrochemical gradient for Cl^- entry is also generated by HS addition in CF airways, Cl^- cannot enter the cell due to the absence of the CFTR Cl^- channel in the apical membrane. Since Cl^- is not absorbed to preserve electroneutrality, this also retards Na⁺ absorption (orange arrows). Thus, NaCl absorption must occur in CF airways via the paracellular pathway (not shown) which has only ∼33% of the capacity of the transcellular pathway. This results in a smaller dissipation of available osmoles and a larger steady state ASL height (F). G, Isotonic Na⁺ hyperabsorption results in isonoic removal of this ASL with time.