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1Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom; 2Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois; and 3Department of Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania
Submitted 11 August 2005 ; accepted in final form 5 October 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S, UTP, ionomycin, and PMA induced robust increases in total cellular capacitance that were demonstrated to be dominated by a specific increase in apical membrane surface area. The UTP-induced increase in capacitance occurred in parallel with goblet cell emptying and the secretion of mucin and was associated with decreases in apical and basolateral membrane resistances. The magnitude and kinetics of the capacitance increases were dependent on the agonist and the sidedness of the stimulation. The peak increase in capacitance induced by UTP was 
30 mucin granule fusions per goblet cell. Secretagogue-induced decreases in apical membrane resistance were independent of exocytosis, although each of the secretagogues induced profound reductions in basolateral membrane resistance. Transepithelial impedance analysis offers the potential to study morphological and conductance changes in cultured human bronchial epithelial cells. degranulation; exocytosis; goblet cell; impedance analysis; mucus
Mucin secretion is commonly measured by quantification of the release of mucin granule contents after degranulation of surface epithelial goblet cells or glandular mucous cells. The biochemical detection of secreted mucins has been the most widely reported technique (17, 22), although the unique biophysical and biochemical properties of this class of glycoproteins, combined with a general lack of well-characterized tools, do not lend themselves to routine quantitation. These methods can provide information relating to secretion of specific mucus components, although they reveal little in terms of the kinetics of degranulation. The magnitude and kinetics of secretion can also be dependent on the choice of mucin detection ligand (12). Morphometric analysis (15, 19, 20) can provide information relating to the degree of emptying of mucin-containing cells, although only at the time of fixation, again yielding little information regarding the kinetics of secretion. At the single-cell level, patch-clamp studies utilizing capacitance (surface area) measurements with the colonic goblet cell line HT-29-Cl.16E (5, 26) and Calu-3 airway epithelial cells (7) have illustrated some dynamics of exocytosis, endocytosis, and associated ion secretory phenomena. Similarly, single goblet cell imaging using video microscopy has been described (1, 8, 23) and has enabled an assessment of the magnitude and kinetics of degranulation, with the advantage of studying cells while they reside in the native epithelium. However, a more routine technique that would permit the real-time measurement of degranulation in a functional, well-differentiated epithelium would be desirable to enable further characterization of secretory mechanisms.
Transepithelial impedance analysis (TIA) has been used to study the static, bioelectric properties of a variety of epithelia (see Ref. 22 for review). More recently, the technique has been utilized to characterize the dynamic behavior of membrane surface area during degranulation in HT-29-Cl.16E cell monolayers (2, 3). The aims of the present study were, therefore, 1) to assess the viability of using TIA to measure mucin secretagogue-induced capacitance changes as a surrogate of goblet cell degranulation in the human airway epithelium and 2) to characterize conductance changes occurring in concert with exocytosis. To this end, we have used a recently described impedance analysis technique (33, 37) to measure the real-time kinetics of exocytosis in air-liquid interface cultures of human bronchial epithelial (HBE) cells. We demonstrate that mucin secretagogues induce robust increases in total cellular capacitance (CT) that can be attributed to specific increases in the apical membrane capacitance (CA). Furthermore, the magnitude and kinetics of the capacitance increases were consistent with the published reports of single goblet cell degranulations using video microscopy. The increases in capacitance were dependent on the secretagogue as well as the sidedness of the stimulation. In concert with exocytosis, each of the secretagogues induced a decrease in the basolateral membrane resistance that may represent a mechanistic component of degranulation and/or a mechanism to drive anion secretion to maintain adequate mucin hydration during secretion.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HBE cells (Clonetics) were cultured as described by Gray and colleagues (14). At passage 2, HBE cells were seeded into plastic T-162 flasks (Costar) and grown in bronchial epithelial cell growth medium (BioWhittaker) supplemented with bovine pituitary extract (52 µg/ml), hydrocortisone (0.5 µg/ml), human recombinant epidermal growth factor (0.5 ng/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), retinoic acid (0.1 µg/ml), triiodothyronine (6.5 µg/ml), gentamicin (50 µg/ml), and amphotericin B (50 ng/ml). The medium was changed every 48 h until the cells were 90% confluent. The cells were then passaged and seeded onto culture inserts in differentiation medium containing 50% DMEM in bronchial epithelial cell growth medium with the supplements described above, but without amphotericin B or triiodothyronine and at a final retinoic acid concentration of 50 nM (all-trans-retinoic acid; Sigma). Cells were seeded at a density of 75 x 103 cells/cm2 onto 1.1-cm2 Snapwell (Costar) or 0.33-cm2 Transwell (Costar) inserts. HBE cells were maintained submerged for the first 7 days in culture; then they were exposed to an air-liquid interface for the remainder of the culture period. Medium was refreshed three times each week: on Monday, Wednesday, and Friday. At least once each week, the apical surface of the cells was rinsed with PBS (37°C) to remove accumulated mucus and debris. At all stages of culture, cells were maintained at 37°C in 5% CO2 in an air incubator. Under these conditions, HBE cells formed a well-differentiated mucociliary phenotype with the classical ion transport phenotype associated with this tissue (10). Three HBE cell donors were used for these studies.
Transepithelial Impedance Analysis
Measurement of CT. TIA of HBE cells was performed as recently described in frog skin (25), Xenopus A6 cells (37), T84 colonic epithelial cells (33), and HBE cells (19). HBE cells on culture inserts were mounted in vertical diffusion chambers (Costar) and bathed with continuously gassed Ringer solution (5% CO2 in O2, pH 7.4) maintained at 37°C containing (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose (all from Sigma). The solution osmolarity was 280–300 mosmol/kgH2O. Electrodes [agar bridged (5% in 3 M KCl) Ag-AgCl] were positioned such that the voltage-measuring electrodes were as close to the insert as possible, and after placement the inserts were voltage clamped to 0 mV. The hardware for impedance analysis was based on two digital signal-processing (DSP) boards (model 310B, Dalanco Spry, Rochester, NY) equipped with two high-speed (300-kHz) analog-to-digital converters (14 bit) and two digital-to-analog converters (12 bit) connected to a personal computer. The interface between the DSP boards and the high-speed voltage clamp consisted of antialiasing filters, programmable gain amplifiers, and digital control circuits controlled by the DSP boards. One DSP board recorded transepithelial conductance (GT) and short-circuit current (Isc). For measurement of GT, a 1-Hz sine wave (5 mV) was applied to the cells and the change in Isc was recorded. The second DSP board measured CT at five selectable frequencies (2, 4.1, 8.2, 11.0, and 16.5 kHz) via the high-pass filter. Data in this study illustrate CT records at 8.2 kHz.
Estimation of apical and basolateral capacitances and resistances.
Under experimental conditions identical to those described above for the measurement of CT, impedance spectra were obtained using the second DSP board while Isc, GT, and CT signals were interrupted (33). The technique is based on the principle that an epithelium can be modeled as an equivalent electrical circuit (Fig. 1). This "lumped model" depicts two parallel resistor-capacitor circuits connected in series with a parallel shunt (paracellular) resistance (RP) and a series (i.e., solution between the electrodes and the epithelium) resistance (Rs). This represents the simplest, morphologically correct model that can be used to study the behavior of epithelia and has been used to investigate the membrane properties of a variety of epithelia (2, 3, 25, 31, 33, 37). Impedance spectra were acquired to obtain estimates of apical (RA and CA) and basolateral (RB and CB) resistances and capacitances during the secretagogue-stimulated changes in membrane properties. Impedance spectra were analyzed using a custom MatLab program that enabled the impedance spectra to be fitted to a one- or two-membrane equivalent circuit model, with the results presented as Nyquist plots. This approach required an estimate of RP that was assumed to remain constant throughout the experiment. Support for the assumption that Rp remained constant was obtained from unidirectional flux studies of the extracellular marker mannitol. For these studies, Snapwell inserts were mounted in an Ussing chamber, and the monolayers were continuously short circuited after fluid resistance compensation with use of automatic voltage clamps as described above. Mannitol (5 mM) was added to the bath solution for the flux studies. At 15 min after the filters were mounted, forskolin (0.6 µM) and [3H]mannitol (5 µCi) were added to the apical bath. When Isc had stabilized, two 0.1-ml samples were taken from the mucosal bath. After an additional 2 min, two 0.4-ml samples were taken from the serosal bath, and 0.8 ml of fresh, unlabeled solution was returned to the serosal bath to maintain a constant volume. This time was considered time 0, and duplicate 0.4-ml samples were taken at 15-min intervals for the next 60 min. UTP (30 µM) was added to the mucosal bath at 30 min, and the mucosal-to-serosal flux of mannitol before and after the addition of UTP was compared. Isotope activities were determined in a liquid scintillation counter. All samples were weighed, and these volumes were used to correct the chamber volume and to calculate the mucosal-to-serosal flux of mannitol with use of standard equations (6). The mucosal-to-serosal flux of mannitol before and after the addition of UTP was unchanged: 0.011 ± 0.0013 and 0.010 ± 0.0011 µmol·cm–2·h–1, respectively. Under the same conditions, GT vs. Isc plots were linear and provided a y-intercept estimate of Rp of 328 ± 38.2 
·cm2 (n = 4). Rp was estimated in this way for each filter, and this value used to calculate the membrane parameters from the impedance results.
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For all studies, group sizes were between four and seven inserts. Data are expressed as absolute means ± SE or mean ± SE changes in Isc, transepithelial resistance (RT), RA, RB, CT, CA, and CB from baseline. A summary measures approach was used to analyze kinetic data. At the times of the peak secretagogue-induced changes in membrane properties, differences from the vehicle-treated cells were assessed using Student's t-test with Bonferroni's correction, and significance was assumed when P < 0.05.
Compound Additions
Compounds were added to the HBE cells when Isc, Gt, and CT had stabilized. All compounds were prepared as 1,000x stock concentrations. The method of compound addition to the Ussing chamber was critical to the measurement of capacitance changes. Ringer solution (500 µl) was removed from the appropriate side (apical or basolateral) of the HBE cells and immediately mixed with 5 µl of the secretagogue stock solution or vehicle in a 1-ml plastic tube. All the solution was then gently reintroduced into the Ussing chamber to be further mixed by the gas-lift circulation. Excessive mixing or stirring of the bathing fluid often resulted in sudden and significant changes in CT. ATPS, UTP, and PMA were freshly prepared each day in Ringer solution. Ionomycin and forskolin were prepared in DMSO and stored at 4°C and room temperature, respectively. All compounds were purchased from Sigma.
Assessment of Mucin Secretion
Biochemical detection of secreted mucin by enzyme-linked lectin assay. Initial studies indicated that the concentrations of any secreted mucins in the 5-ml volume of the Ussing chamber were below the limit of detection of the assay. Consequently, paired studies were performed using HBE cells from the same batch and donor for capacitance measurements, mucin secretion, and histological analysis. In the tissue culture plate format, the apical surface of the cells was rinsed with PBS (37°C). The cells were then incubated with vehicle or 30 µM UTP (500 µl) for 30 min at 37°C, and the apical solution was collected and stored at –80°C. The mucin concentration in the apical solution was quantified as previously described (18). The mucin concentration was determined by comparison with a human mucin standard and expressed as equivalent units of mucin per milliliter. Student's t-test was used to test for a significant difference between the mucin concentrations in the vehicle and UTP-stimulated samples, with significance assumed when P < 0.05.
Quantification of mucin secretion by histological evaluation of goblet cell density.
HBE cells were mounted in Ussing chambers and prepared for capacitance measurements (see above). When the Isc had stabilized, vehicle or 30 µM UTP was added to the apical chamber. After 30 min, the HBE cells were removed from the Ussing chamber and fixed in 10% neutral buffered formalin and embedded in wax. The inserts were then cut into 4-µm-thick sections and stained with 1% Alcian blue (in 3% aqueous acetic acid, pH 2.5) for detection of total mucins. Finally, all sections were counterstained with Cole's hematoxylin. The goblet cell density was quantified by counting the total number of Alcian blue-stained cells (Axioplan 2 microscope, Zeiss; x20 magnification) across the entire length of two to three sections of each insert (20–30 mm total epithelial length). The number of Alcian blue-stained cells was then expressed as percentage of the total number of epithelial cells lining the apical surface. All samples were blinded before the cells were counted to remove the potential for operator bias. A paired Student's t-test was used to test for a significant difference between vehicle- and UTP-stimulated goblet cell emptying, with significance assumed when P < 0.05.
Routine Determination of Goblet Cell Density
Goblet cell density of HBE cells was assessed on a weekly basis to confirm the development of a well-differentiated epithelium. The preparation and scoring procedure were identical to those described above, except the cells were not placed in the Ussing chamber.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the course of these studies, HBE cells developed a basal Isc, RT, and CT of 18.1 ± 1.0 µA/cm2, 547 ± 32 ·cm2, and 2.718 ± 0.084 µF/cm2, respectively (n = 76). The HBE cells were routinely assessed for goblet cell density and revealed a mean goblet cell density of 30.7 ± 2.6% (n = 42).
Mucin Secretagogues Stimulate Increases in CT
Initial studies were designed to assess the effects of mucin secretagogues on simultaneous changes in Isc, RT, and CT. The individual secretagogue studies were not performed in parallel, nor were they designed to assess quantitative differences between secretagogues. Variations between donors and intradonor variation on a week-to-week basis were apparent in terms of capacitance and conductance changes.
ATPS.
ATPS (500 µM, n = 4), when added to the apical side of HBE cells, induced a mean peak increase in Isc of 18.1 ± 2.5 µA/cm2 that was associated with a peak decrease in RT of 162.8 ± 10.5 ·cm2 (Fig. 2, A and B). In parallel with these changes was a rapid increase in CT of 0.207 ± 0.012 µF/cm2 that peaked within 3 min of compound addition (Fig. 2, C and D). The increase in CT was transient and decreased toward the starting baseline 5–10 min after agonist addition.
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·cm2), and an increase in CT (0.365 ± 0.033 µF/cm2; Fig. 2, E–H). As with ATPS, the UTP-stimulated increase in CT was transient.
Ionomycin.
Addition of 4 µM ionomycin (n = 4) to the apical membrane induced a peak increase in CT of 0.199 ± 0.017 µF/cm2 (Fig. 3, C and D). The increase in CT was initially rapid over the first 4 min after ionomycin addition and was followed by a more gradual increase over 6–8 min before peaking. The increase was again transient, although the recovery of CT toward the starting baseline was slower than that observed with the trinucleotides, taking 20 min after the peak to recover to the original baseline level. In contrast to the effects of ATPS and UTP, ionomycin-induced a reduction in Isc of 7.9 ± 1.6 µA/cm2 below the starting current and a transient increase in RT of 40.3 ± 8.7 ·cm2 (Fig. 3, A and B).1
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15 min after PMA addition. The effect of PMA on Isc was similar to that observed with ionomycin, inducing a decrease of 7.2 ± 1.5 µA/cm2 below the basal level (Fig. 3E). In each of these studies, addition of the vehicle (Ringer solution or 0.1% DMSO) was without effect on Isc, RT, or CT (data not shown).
UTP-Induced Mucin Secretion and Goblet Cell Emptying
These studies were performed in parallel with those illustrated in Fig. 2, E–H. Addition of 30 µM UTP to the apical membrane of HBE cells induced a 295 ± 11% (P < 0.0001, n = 6) increase in mucin secretion compared with vehicle over a 30-min period (Fig. 4A). The goblet cell densities of batch- and time-matched HBE cell cultures were assessed after 30 min of incubation during which the cells were voltage clamped in Ussing chambers with or without 30 µM UTP in the apical solution. The number of goblet cells staining positive for mucins was reduced from 12.1 ± 1.7% in controls2 to 4.3 ± 0.4% in the presence of UTP (P < 0.002, n = 6; Fig. 4B). A reduction in goblet cell staining suggests emptying of cell mucins (Fig. 4C).
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The observations that ATPS, UTP, ionomycin, and PMA induced increases in CT within the same time frame that UTP was able to induce mucin secretion suggested that the increase in CT could be due to an increase in apical membrane surface area (CA) after goblet cell degranulation. To test this hypothesis, the bioelectric properties of the apical and basolateral membranes required independent characterization during agonist-induced stimulation. To this end, impedance spectra of HBE cells were acquired to establish whether CA, CB, RA, and RB could be estimated.
Initial studies indicated that, under basal conditions, the time constants of apical and basolateral membranes were such that the impedance data could not be reproducibly fitted to the two-membrane model (see MATERIALS AND METHODS; see sample Nyquist plot from an untreated HBE insert in Fig. 5D). To enable calculation of estimates of both membranes, it was necessary to alter the time constants of the two membranes. The addition of 0.6 µM forskolin (apical and basolateral) altered the time constants and revealed two membranes in the Nyquist plot (Fig. 5E), enabling estimation of the bioelectric properties of apical and basolateral membranes with the use of equations that describe the two-membrane model (32, 36). Mean data for the bioelectric properties of forskolin-treated HBE cells are shown in Table 1 (n = 36), and, as would be predicted for an epithelium, CB was 10 times CA. This also indicated that changes in CT would be dominated by CA, because CT–1 = CA–1 + CB–1. Forskolin induced a small and variable peak increase in CT of 0.060 ± 0.006 µF/cm2 that reached a plateau within 5 min of addition. Further evidence in support of the inference that CT is dominated by CA was obtained by nystatin permeabilization of individual membranes in the presence of amiloride and forskolin. These experiments demonstrated that permeabilization of the basolateral membrane resulted in only a 15% change in CT, whereas permeabilization of the apical membrane resulted in a 170% change in CT, consistent with CT being dominated by CA under these conditions.
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S (Fig. 5F, corresponding with the peak increase in CT). After the addition of ATPS, the two membranes remained identifiable from the Nyquist plot, and estimates of the capacitance and conductances indicated an increase in CA (0.216 µF/cm2) and decreases in RA (98 ·cm2) and RB (112 ·cm2). There was no significant difference between the increases in CT and CA, further supporting the hypothesis that the observed increase in CT is dominated by a change in CA. In light of this and previous studies indicating that pretreatment of HBE cells with forskolin did not affect the subsequent UTP-induced increase in CT (data not shown), the effects of each of the mucin secretagogues were examined under conditions of forskolin pretreatment to enable measurement of capacitance and conductance changes in apical and basolateral membranes.
UTP.
Apical.
When added to the apical side of HBE cells, 30 µM UTP induced a rapid and significant (P < 0.0001 compared with vehicle control) increase in CA of 0.242 ± 0.026 µF/cm2 (Fig. 6A) that reached a peak within 3 min of agonist addition. As previously observed with CT measurements, the increase in CA was transient and decreased to below the prestimulated baseline within 15–20 min of agonist addition. In parallel with the increase in CA, there were transient reductions in RA and RB of 51.8 ± 10.3 ·cm2 (Fig. 6B; P < 0.001) and 443 ± 160 ·cm2 (Fig. 6D; P < 0.0001), respectively, that also peaked within 3 min of UTP addition. CB increased slowly after compound addition to a peak of 3.8 ± 0.6 µF/cm2 (Fig. 6C; P < 0.005) by 20 min.
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20 min after the addition of UTP (P < 0.0001). There was no significant effect of basolateral UTP on RA (P > 0.05). RB was transiently reduced by 1,018 ± 210 ·cm2 within 60 s of UTP addition (P < 0.0001) and recovered to the starting baseline within 10 min. The observed decline in CB over the course of the study was variable although significantly different from the vehicle control (P < 0.01).
Ionomycin.
Addition of 4 µM ionomycin to the apical surface of HBE cells induced a mean peak increase in CA of 0.236 ± 0.008 µF/cm2 within 3–4 min (Fig. 7A; P < 0.0001). In contrast to apical UTP, CA remained elevated for the duration of the study. Associated with the increase in CA was a decrease in RB of 983 ± 36 ·cm2 (Fig. 7D; P < 0.0001). Addition of ionomycin induced an initial increase in RA of 60 ·cm2 that decreased to a minimum of 50 ·cm2 below the starting baseline resistance value, although these changes did not reach statistical significance. Ionomycin had no robust effect on CB.
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10–12 min and was followed by a recovery toward baseline. Associated with this increase was a significant reduction in RB of 539 ± 163 ·cm2 (Fig. 7H; P < 0.0005). There was a trend toward a reduction in RA induced by PMA, although this did not reach statistical significance. CB was unaffected. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Airway goblet cells have been estimated to contain 40–280 1-µm-diameter mucin granules per cell (8, 23), indicating that if a substantial secretion of mucins could be induced from our HBE cell cultures (20–30% apical goblet cells), degranulation might be quantifiable as an increase in capacitance (surface area). Initial experiments indicated that the addition of four mucin secretagogues (ATPS, UTP, ionomycin, and PMA) to HBE cells each induced robust increases in CT (Figs. 2 and 3). In parallel HBE cell cultures, UTP also induced mucin secretion and goblet cell emptying quantified using a biochemical assay and histological assessment, respectively (Fig. 4). Impedance spectra revealed that CB >> CA (Table 1), indicating that a change in CT was likely to be dominated by changes in surface area at the apical membrane. To confirm that the CT increase was dominated by an increase in CA, impedance spectra were acquired over the course of these secretory events. These experiments confirmed that mucin secretagogues induced robust increases in CA (Figs. 5– 7) that were similar to the magnitude of CT changes. CA could be estimated only in the presence of 0.6 µM forskolin. Thus the presence of forskolin may influence the subsequent secretory response to ATPS, UTP, ionomycin, and PMA. This concentration of forskolin induced a small increase in CT before the subsequent addition of any of the mucin secretagogues (Fig. 5C), suggesting that forskolin was able to induce an increase in membrane surface area. It has been suggested that elevation of intracellular cAMP concentration may result in the insertion of new CFTR into the plasma membrane (which could increase CT) in airway epithelial cells (4, 31). However, 1 µM forskolin has also been demonstrated to induce mucin secretion from HBE cell cultures (unpublished observations). Irrespective of the mechanism of the forskolin-induced rise in cellular capacitance, the magnitude of the subsequent stimulated increases in CT by ATPS, UTP, ionomycin, and PMA was unaffected, and the increases in CA mirrored the increases in CT (Fig. 5G).
A central question, therefore, is whether the magnitude and kinetics of the observed increase in CA could be accounted for by goblet cell degranulation. With UTP as the example and the assumption that HBE cells are cylinders with a mean diameter of 12 µm (measured in our cultures), the total cell density would be 1.1 x 106 cells/cm2, of which 0.12–0.3 x 106 cells/cm2 (12–30%) would be goblet cells.3 Human goblet cell granules have been reported to be 1-µm spheres (23), and if it is assumed that 1 cm2 of plasma membrane = 1 µF, i.e., a dielectric constant identical to that of the plasma membrane (31). A granule would equate to a capacitance of 31 fF per granule. An increase in capacitance of 0.2–0.36 µF/cm2 would therefore require 20–90 (at peak change in capacitance) degranulation events per goblet cell. This value is in agreement with the numbers of degranulation events in canine and human airway goblet cells observed using video microscopy (1, 8, 23). The kinetics of the induced changes in CT and CA also mirror those reported in the literature for goblet cell degranulation. The rapid increases in capacitance observed with ionomycin and the apical addition of nucleotide triphosphates were also described as a rapid initial rate of degranulation events in video microscopy studies (1, 8, 23). In canine goblet cells, the kinetics of the degranulation induced by serosal ATP were also generally slower than those induced by apical ATP (8), consistent with our observations from basolateral stimulation with UTP (Fig. 6E). A relatively slow time to the peak in CT and CA observed with PMA (Figs. 3H and 7E) was also reported by Abdullah and colleagues (1). The recovery of CT and CA indicates recovery (endocytosis) or shedding of the membrane (36) after degranulation.
Two important additional factors require consideration when interpreting the data. 1) HBE cells form a heterogeneous population of cells, and it is likely that goblet cells are not the only population of cells that respond to secretagogues with changes in membrane surface area. However, the magnitude, kinetics, and pharmacology of the stimulated increase in capacitance suggest that goblet cell exocytosis is a major contributor. Furthermore, range analysis performed by allowing RP to be as low as RT and as high as 20% greater than the estimated value demonstrated that there was very little change in the estimated value of CA. Therefore, even if the nongoblet cell population is considered part of RP, the estimates of CA appear not to change.4 2) A capacitance change will always reflect a balance between degranulation (exocytosis) and membrane retrieval (endocytosis). Exocytosis and endocytosis most likely occur simultaneously (34), indicating that the calculated changes in CT and CA may actually underestimate exocytosis. Finally, the different kinetic profiles of capacitance increases observed with use of these secretagogues also offer potential insights into the intracellular mechanisms responsible for degranulation. The functional sidedness of purinergic receptor agonists on the human airway epithelium have been studied in terms of changes in intracellular Ca2+ concentration ([Ca2+]i), PKC signaling, and associated ion transport events (29, 30). The present published paradigm suggests that stimulation of the serosal or mucosal membranes with UTP/ATP induces a localized, ipsilateral elevation of [Ca2+]i and activation of a Ca2+-independent PKC pathway (30). In contrast to the elevation of [Ca2+]i, activation of the PKC pathway by serosal UTP/ATP has been demonstrated to induce effects at the apical membrane. This observation of a PKC-mediated communication between the apical and basolateral membranes would support the concept that the increase in capacitance induced by basolateral UTP is potentially through a PKC-dependent, rather than Ca2+-dependent, pathway. That PMA, an activator of PKC-dependent pathways, induced a capacitance increase at the apical membrane with a kinetic profile similar to that of basolateral UTP would further support this notion.
In addition to secretagogue-induced increases in CA, we also observed changes in CB. For example, apical stimulation with nucleotide triphosphates induced an increase in CB. The stimulation of ion secretion has been demonstrated to cause a reduction in cell cytoplasmic volume (13, 35), and such a decrease in cellular volume can open the lateral interspace (37). Opening of the lateral interspace would be expected to increase the apparent surface area of the basolateral membrane (37), potentially accounting for the observed increase in CB. Goblet cell degranulation in the gut has also been demonstrated to significantly reduce cell volume (16). On the basis of the estimated numbers of mucin granules in airway goblet cells, exocytosis of 30 granules, as observed in the present study, could account for up to 75% of the cellular mucin granule pool, which would presumably result in a profound decrease in cell volume and an apparent increase in CB. The effects of ionomycin and PMA on CB were variable. In addition to stimulating exocytosis, the direct membrane-permeabilizing activity of ionomycin could also potentially influence cell volume regulation. In some studies (data not shown), ionomycin induced a decrease in CB suggestive of cell swelling. The lack of a significant effect of PMA on CB may reflect the lack of an effect on anion secretion or the relatively small increase in CA, suggesting a reduced capacity to induce exocytosis.
An additional facet of the impedance analysis technique is the ability to quantify changes in membrane conductances and the consequent ion transport responses in temporal proximity to measurements of morphological changes. Several studies have suggested a link between mucin secretion and anion secretion (15, 26). Evidence from HT-29-Cl.16E cells suggests that Cl– conductances in the mucin granule membrane can be inserted into the cell membrane during degranulation and serve to "flush" the condensed granules of their contents (26). However, Bertrand et al. (5) recently demonstrated in this same HT-29 cell line that the Cl– channel blocker niflumic acid attenuated exocytosis but was without effect on the secretory Cl– current, suggesting that the two events may not be linked. Similarly, in Calu-3 cells, an elevation of [Ca2+]i resulted in exocytosis but a reduction in membrane conductance and current (7). In the presence of forskolin (i.e., conditions under which CFTR is activated), the apical application of ATPS, UTP, or PMA induced a decrease in RA. These observations are consistent with the activation of an apical Ca2+-activated Cl– conductance and/or the further activation of CFTR through a PKC-dependent mechanism. However, these changes may underestimate the potential for the secretagogues to decrease RA, inasmuch as CFTR would already be considered to be activated in the presence of forskolin. This may also explain the lack of effect of basolateral UTP on RA, because Paradiso and colleagues (30) observed that basolateral stimulation of HBE cell cultures with nucleotide triphosphates induced the PKC-dependent opening of apical CFTR. That basolateral UTP and apical ionomycin induced increases in CA but had no effect on RA further suggests that any putative granule-associated conductance does not play a major role in the determination of RA during exocytosis. The most pronounced conductance change observed during exocytosis was a profound increase in the basolateral membrane conductance, which was apparent irrespective of secretagogue examined or the sidedness of the stimulation. The opening of basolateral K+ conductances is required to establish the driving force for Cl– secretion from the apical membrane and is rate limiting for anion secretion in HBE cells (9). Physiologically, it would therefore be desirable that a mucin secretagogue also induce fluid secretion to ensure adequate hydration of the airway surface liquid. With the present knowledge, this would appear to be the most likely explanation for involvement of the basolateral conductance(s), although a role in the mechanics of degranulation cannot be ruled out.
These studies have therefore demonstrated that mucin secretagogues induce profound changes in the apical and basolateral membrane properties of human airway epithelial cells. Morphological changes associated with exocytosis and cell shrinkage are accompanied by the opening of conductances of the epithelial cells, potentially to ensure the appropriate hydration of secreted mucins. This TIA technique therefore offers the opportunity to advance our understanding of the physiology of stimulus-evoked secretion in an intact, near primary, airway epithelial cell system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Studies were performed in the absence of amiloride and under these conditions. The decrease in Isc is likely due to a decrease in electrogenic sodium transport (8). 
2 The lower density of goblet cells staining positive for mucins (12%) than that of cells before incubation in Ussing chambers (30%) probably reflects loss of mucin due to endogenous release of nucleotides, which would stimulate mucin depletion before addition of exogenous secretagogues.
3 In Fig. 4, cells of various morphologies appear to stain positive with Alcian blue. The most likely explanation is that the Alcian blue-labeled cells represent goblet cells that have been imaged in different coronal planes, particularly when one takes into account the appearance of the nuclei at the base of the cells. Alternatively, these may be goblet cells of varying morphology, and we cannot rule out that they are surface cells expressing high levels of mucin. Even if these cells are not a pure goblet cell population, they appear morphologically to secrete mucin at the apical surface. Therefore, although such a change in definition may alter the semantics, we believe these considerations do not change the conclusions.
4 Mean peak CA measurements after UTP stimulation for Rp = 372, 409, and 491 ·cm2 were 3.49 ± 0.066, 3.45 ± 0.062, and 3.41 ± 0.057 µF/cm2, respectively (n = 4, P = 0.37).
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, Secretagogue; 
, vehicle control.