In the present study, we sought to model the dysregulated Na+ absorptive phenotype of CF airway epithelia in primary cultures of normal bronchial epithelial cells with acute and chronic mucosal Nys treatment.
To characterize our model, we first studied the bioelectric response of HBEs to acute Nys challenge. We found that acute Nys challenge in primary HBEs produced a dose-dependent, rapid increase in Isc
followed by a gradual relaxation of the response (). Diverse responses to acute Nys challenge in Ussing chambers have been described in the literature. Although a pattern similar to the one we observed has been described in toad bladder A6 cells and rat colonic epithelium (13
), other model systems (e.g., Calu-3 cells) responded to acute Nys exposure with a slower rise in Isc
(10–20 min) and a plateau phase that was sustained for over 30 minutes (26
). Since Nys binding to the plasma membrane is strictly dependent on sterol content (28
), these differences might be attributed to differences in the apical membrane sterol composition in the different cell types studied.
Detailed characterization using ion substitution and specific inhibitors of ion transport revealed that the acute bioelectric response of HBE to Nys challenge reflected multiple ion transport responses, with Na+ absorption being the dominant one. The initial, steep increase in Isc was dependent on the presence of Na+ in the luminal bath () and on the activity of the basolateral Na+/K+ATPase (). The failure of amiloride pretreatment to inhibit the Nys-induced Isc peak () suggested that Nys challenge elicits ENaC-independent Na+ absorption.
was not affected by bumetanide () or HCO3−
substitution (). However, there was a significant blunting of this response by bilateral removal of Cl−
(). These results suggest that either Cl−
secretion was a component of the Isc
response or that Cl−
substitution had an adverse effect on the electrochemical gradient for Na+
entry across the apical membrane. The former possibility seems unlikely for three reasons. First, previous patch clamp studies showed that Nys forms pores that have a small, finite Cl−
permeability only when used at high concentrations (150–400 μM) (9
). In dog bronchial epithelial cells, mucosal Nys increased the Cl−
permeability across the apical membrane only at concentrations higher than 90 μM (29
). Lower concentrations of Nys (50 μM), comparable to the one used in our studies, have been shown to selectively affect Na+
transport in immortalized Calu-3 human airway epithelial cells (26
). Second, the Nys-induced Isc
was insensitive to bumetanide (). Third, the possibility that Cl−
secretion was a component of the Isc
response of HBE to acute Nys challenge is inconsistent with our data showing that Nys treatment enhanced the volume absorptive properties of HBE both acutely and chronically ( and ).
We could also detect evidence of K+ secretion concurrent with Na+ absorption upon acute Nys treatment (). However, a greater net rate of Na+ absorption dominated, as evidenced by the polarity of the Isc response and the liquid absorption measurements. In agreement with this notion, acute Nys administration produced cell swelling (), consistent with cellular accumulation of Na+ ions and H2O, whereas an increase in K+ secretion is predicted to produce cell shrinkage. Cell swelling was not observed in thin film conditions (), suggesting that Na+ influx through Nys pores, followed by osmotically driven H2O influx, requested large luminal volumes to produce cell swelling. Also note that the Nys-induced swelling was restricted to the luminal columnar cell layer, whereas the basal cell layer appeared to be unaffected, suggesting that Nys pores were confined to the apical membrane of the “mucosal” cell layer ().
In agreement with a previous study (30
), we showed that acute Nys-dependent Na+
hyperabsorption was coupled to a rapid increase in the rate of mucosal fluid absorption (). More importantly, the data shown in suggest that Nys can reproduce both the Na+
transport and the ASL volume regulation defects associated with CF. Specifically, cultures exposed to Nys exhibited more rapid initial rate of volume absorption, consistent with an accelerated rate of Na+
absorption. Moreover, Nys-treated cultures were unable to inhibit volume absorption as the ASL volume approached the critical 7 μm threshold required for efficient ciliary beating and mucus transport. This latter defect (i.e., failure to inhibit ENaC at low ASL volume) may be the most critical defect in the Na+
transport path in CF (31
) and appears to be mimicked by Nys. In CF the failure to regulate ENaC at low ASL volume reflects the absence of functional CFTR (1
). With Nys, the absence of Na+
/volume absorption regulation likely reflects the inability of endogenous regulators of ENaC to inhibit Nys pores (30
). Regardless of mechanistic differences, Nys appears to model both features of dysregulated Na+
transport exhibited by CF airway epithelia.
To study the effect of chronic apical membrane permeabilization, we treated HBE cultures with 40 μM Nys for 4 days. In the experimental design for the chronic Nys treatment, we considered two conditions to control for the volume of liquid and mass of NaCl applied to the Nys-treated cultures, that is, non–volume-treated (naïve, 0 μl) and volume-treated (KBR, 120 μl) cultures compared with Nys/volume-treated (Nys+KBR, 120 μl) cultures. We showed that chronic Nys treatment significantly increased VT and Ieq over time, while affecting RT only at early time points () compared with either control, suggesting that the functional integrity of the epithelia was preserved.
transport driven by the basolateral Na+
ATPase is an energy-consuming process. Although we found no signs of overt cytotoxicity induced by the chronic Nys treatment (), we observed increased levels of lactate in the serosal media of KBR- and Nys-treated cultures, suggesting that both conditions imposed an increased energy demand on HBE cultures (). We speculate that Nys-treated cultures faced the highest metabolic energy expenditure as a consequence of the increased Na+
transport due the increased Na+
permeability in the apical membrane after insertion of Nys pores. In the case of the KBR-treated cultures, the increase in lactate concentration in comparison to naïve cultures could reflect the hypoxic conditions created by the continuous presence of liquid on the mucosal surface of these cultures. Alternatively, the higher metabolic energy demand could reflect an increase in Na+
transport due to the persistent high activation state of ENaC elicited in airway epithelia by the dilution of ENaC inhibitory factors normally present in the ASL (30
A recent study has shown that chronic mucosal treatment of normal nasal epithelial cells with 50 μM amphotericin B (AmphoB) significantly reduced VT
, and amiloride-sensitive Isc
). AmphoB treatment did not affect cellular metabolism or integrity, but decreased expression of αENaC, α1
ATPase, and NKCC1. These findings were interpreted to result from a feedback mechanism aimed to limit cellular Na+
overload due to membrane permeabilization with AmphoB. In contrast, our data indicate that Nys produced a sustained increase in Isc
() coupled to increased volume absorption () without significant effects on the expression of major components of transcellular Na+
transport system (). In the same experimental conditions used in our study, acute AmphoB challenge caused a rapid increase in Isc
similar to the one elicited by acute Nys (see
Figure E1A in the online supplement). Although both Nys and AmphoB belong to the polyene antibiotic family and, at least acutely, have similar effects on the bioelectric properties of airway epithelial cells, a comparison between our results and those of Jornot and coworkers (23
) suggests that the different outcomes might be dependent on the different interactions of AmphoB versus Nys with the plasma membrane. For example, our data showed that Nys binding to the plasma membrane was rapidly reversible, and even after chronic exposure the bioelectric properties of Nys-treated cultures after significant Nys dilution, were comparable to those of naïve HBEs. In contrast, AmphoB interactions with the plasma membrane are likely stronger, since the time required to recover normal bioelectric properties depends on the duration of the AmphoB-exposure (23
), regardless of the removal of the drug from the mucosal solution. Due to the rapid reversibility of the induced hyperabsorptive phenotype, Nys could be the reagent of choice when secondary modification of the bioelectric properties of the cultures or long recovery time after treatment are unwanted.
Importantly, during the chronic Nys treatment protocol, we observed a striking difference in ASL volume homeostasis between KBR- and Nys-treated cultures that persisted over 4 days (). The persistence of this hyperabsorptive response over 4 days suggests that long-term Nys treatment produced a sustained increase in HBE Na+
transport. Consistent with short-term ASL volume experiments in which the mucus layer was removed from the cell surface (), we found that after 4 days of chronic Nys treatment, the PCL was depleted and the cilia were not fully extended (, left panels
). In contrast, naïve and KBR-treated cultures exhibited a normal ASL phenotype, with the cilia outstretched in an approximately 7-μm high periciliary layer. Further, the cilia and the apical surface of Nys-treated cultures were covered with a thick layer of condensed, strongly AB-PAS–positive material (, right panels
). This phenotype strongly resembles the appearance of mucus-plugged CF airways (33
). It is worth stressing that our ability to still detect the mucous material on the cell surface of Nys-treated cultures after the repeated washings required for 4% PFA fixation indicates that the interaction between the condensed layer and the epithelial surface was strong.
Assessment of the percentage of solids of mucus plaques collected after long-term Nys treatment quantitatively showed that the material lining the surface of Nys-treated cultures was dehydrated compared with that harvested from naïve or KBR-treated cultures (). In humans, mucus from bronchiectatic individuals ranges from 4 to 8% solids, whereas CF mucus ranges from 11 to 20% solids (34
). The “% solids” values measured for the material lining the Nys-treated cultures were higher than those noted in CF HBE cultures after 3 days in culture (24
) or in CF lungs in vivo
. The higher value could reflect the presence of dead cells in the plugs, as evident in the histologic sections where cell debris entrapped in the mucus layer can be easily seen (, and Figure E1B in the online supplement). Overall, chronic Nys treatment did not appear to promote goblet cell hyperplasia in HBEs. Nevertheless, at this point we can not rule out the possibility that chronic Nys treatment also promoted mucin hypersecretion, perhaps through activation of voltage-sensitive Ca2+
), compounding the effect of dehydration in the accumulation of concentrated mucus at the mucosal surface.
In conclusion, we have shown that Nys treatment of normal primary airway epithelial cells recapitulates two key features characterizing CF airway epithelia: (1
) abnormal Na+
transport, as reflected by both accelerated rate of Na+
absorption and, most critically, failure to regulate Na+
absorption at low ASL volume, producing ASL volume depletion on airway surfaces; and (2
) accumulation of dehydrated, “thickened” mucous material that adheres to the cell surface. The Nys model presents some advantages in comparison to cultured CF HBEs. Besides being inexpensive and readily available, Nys treatment is not influenced by culture conditions and it is applicable to a wide variety of cultured cells, for example, tracheal and bronchial primary, passage 1 and passage 2 cells, primary nasal cells, and mouse tracheal cells. However, the Nys model also suffers from limitations. First, the lack of selectivity on cation transport by Nys pores, (i.e., the parallel increase in K+
as well as Na+
permeability) does not strictly model the CF defect. Second, Nys treatment may exert a subtle pro-inflammatory action on airway epithelial cells (36
) of different nature as the inflammation observed in CF airways. Third, although we provided evidence of lack of cytotoxicity, the details about how HBE cells respond to long-term intracellular Na+
load are still unknown. Despite these potential limitations, this simple in vitro
model that features luminal thickened mucus may be useful for characterizing the interactions of bacteria, inflammatory cells, and soluble factors in a CF-like luminal milieu. Potentially, this model may also be used to study the adhesive interaction between mucus layers and airway epithelial surface, providing data on the mechanisms involved in mucus adherence and, hence, clues as to novel therapies for CF lung disease.