1 Department of Clinical Genetics and 3 Department of Otolaryngology, Head and Neck Surgery, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen; and 2 Department of Medical Physiology and 4 Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark
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ABSTRACT |
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Airway
epithelium explants from cystic fibrosis (CF) patients and non-CF
subjects formed monolayered spheres, with the apical ciliated cell
membrane facing the bath and the basolateral cell membrane pointing
toward a fluid-filled lumen. With the use of two microelectrodes,
transepithelial potential difference and changes in potential
difference in response to passage of current pulses were recorded, and
epithelial resistance and the equivalent short-circuit current were
calculated. Non-CF control potential difference and short-circuit
current values were significantly lower than the CF values, and
amiloride inhibited both values. Fluid transport rates were calculated
from repeated measurements of spheroid diameters. The results showed
that 1) non-CF and CF spheroids
absorbed fluid at identical rates (4.4 µl · cm2 · h
1),
2) amiloride inhibited fluid
absorption to a lower residual level in non-CF than in CF spheroids,
3)
Cl
-channel inhibitors
increased fluid absorption in amiloride-treated non-CF spheroids to a
level equal to that of amiloride-treated CF spheroids,
4) hydrochlorothiazide reduced
the amiloride-insensitive fluid absorption in both non-CF and CF
spheroids, and 5) osmotic water
permeabilities were equal in non-CF and CF spheroids (~27 × 10
7
cm · s
1 · atm
1).
amiloride; hydrochlorothiazide; osmotic permeability; current-voltage relationship; cystic fibrosis
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INTRODUCTION |
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THE DEPTH OF THE PERICILIARY FLUID LAYER (5-10
µm) in the upper airways (bronchi, trachea, and nasal cavity) is of
paramount importance for ciliary activity and, thereby, for mucociliary clearance in the airways. The mechanisms by which both the thickness and composition of this fluid layer are regulated are still largely unknown. Fluid may be added from distal airways, submucosal glands, and
surface epithelium secretion. On the other hand, fluid may be removed
and the composition may be changed by mucociliary clearance, evaporation, and, importantly, surface epithelium absorption (3, 4).
Changes in more than one of these mechanisms seem to be involved in the
pathogenesis of cystic fibrosis (CF), characterized by a defective gene
product leading to missing cAMP-dependent Cl-channel function and
increased activity of amiloride-sensitive Na+ channels (4). During the last
few years, two views have evolved for the regulation of volume and
composition of the airway surface fluid layer (ASL), leading to
different predictions regarding the physiology of CF and non-CF airway
epithelia. One hypothesis predicts that the ASL in CF epithelium is
more hypertonic than that in non-CF epithelium, which may inhibit the
function of a bacterial defense mechanism in CF (11, 29).
The second hypothesis predicts that the volume of ASL in CF epithelium
is decreased compared with the non-CF epithelium, resulting in a
decrease in mucociliary clearance (16). These hypotheses are derived
from the diverging results obtained by different techniques for the measurement of volume and composition of the pericellular fluid and
transepithelial solute transport. The development of an additional experimental model for each of the components, especially fluid transport might contribute to the knowledge of these controversial topics.
We (18) recently described the structural and basic ion transport characteristics of spheroid-shaped explants of human airway epithelium from CF patients and normal (non-CF) subjects. Suspension-cultured sheets of protease-released epithelium derived from resected nasal polyps formed free-floating, matrix-independent, monolayered epithelial spheres, with the apical ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. Measurements of the transepithelial potential difference (PD) with microelectrodes demonstrated that the spheroid explant preparation discloses ion transport characteristics similar to those previously demonstrated in CF and non-CF airway epithelia (3, 4).
In the present study, the capacity of the dominating
Na+ absorption was evaluated in
non-CF (NCFSs) and CF (CFSs) spheroids from measurement of the
equivalent short-circuit current
(Isc) before
and after application of amiloride. Furthermore, the ability of these
airway epithelial preparations to absorb fluid was determined from
repeated measurement of spheroid diameters, and the relationship between ion and water transport was studied with inhibitors of Na+ absorption and
Cl secretion. Finally, the
ability of the airway explant preparations to maintain osmotic
gradients was evaluated from the measurement of hydraulic permeability.
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MATERIALS AND METHODS |
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Cellular material. Nasal polyps were
resected from 15 non-CF subjects (8 women and 7 men) and 7 CF patients
(5 men and 2 women; all F508 homozygous). The procedures for
epithelial isolation and culturing were recently described (18). In
brief, the polyps were placed in a 10-ml test tube with 5 ml of
Dulbecco's modified Eagle's medium (room temperature; pH
7.30-7.40) containing 0.1% protease type XIV (Sigma, St. Louis,
MO), 105 U/l of penicillin, 100 mg/l of streptomycin, and 50 mg/l of gentamicin. After ~1 h of gentle
shaking, fetal bovine serum (10% vol/vol; GIBCO BRL, Life
Technologies, Grand Island, NY) was added to neutralize the protease.
The solid parts of the polyps were isolated and discarded, and the
remaining epithelial suspension was washed twice (5 min each at 110 g) in Ham's F-12 culture medium
containing 1% Ultroser G serum substitute (IBF Biotechnics, Savage,
MD) and antibiotics as above. The pellet was resuspended in 5 ml of
culture medium in a 50-ml tissue culture flask and incubated at
37°C with 5% CO2. During the
first 3-4 h, the flask was gently moved every 30 min to reduce
attachment of the cell sheets to the bottom of the flask. The medium
was changed daily for the first two days and thereafter twice a week by
gentle centrifugation (20 g for 2 min)
or simple sedimentation for 10 min in a test tube. From the third day,
gentamicin was omitted from the medium. Hydrocortisone (5 × 10
6 M) was added 1-4
days before experiments.
Electrophysiology. The electrical
parameters were measured in epithelial spheroids transferred to 800 µl of a HEPES-buffered Ringer solution (containing in mM: 140 Na+, 5 K+, 1.2 Ca2+, 1.2 Mg2+, 131.2 Cl, 1.6 HPO2
4, 0.4 H2PO
4, 10 glucose, and 10 HEPES; titrated to pH 7.35) at 37°C in a thermostated chamber and
placed on the stage of an inverted microscope [Nikon Diaphot 300 equipped with Hoffman modulation contrast (HMC) optics]. Spheroids
were kept in position by a Ringer-filled holding pipette (Swemed
Laboratory, Billdal, Sweden) with an internal tip diameter of ~25
µm applying gentle suction with a microinjector (IM-6, Narishige).
Spheroids were impaled by microelectrodes pulled from filamented
borosilicate glass tubes (1 mm OD; Clark Electromedical, Reading, UK)
on a horizontal puller (P-97, Sutter Instruments, Novato, CA) and
backfilled with 1 M KCl (tip resistance 50-100 M
). Impalements
were performed perpendicular to the external apical surface of the
spheroids with a 3-dimensional piezoelectric micromanipulator
(PCS-3200/Pz-301, Burleigh), and a 3+1-dimensional hydraulic
micromanipulator (MHW-3/MHW-4, Narishige). The microelectrode for PD
measurement was connected to a high-impedance electrometer (Duo-773,
World Precision Instruments, Sarasota, FL), and the bath was grounded
via a Ringer-agar bridge and a Ag-AgCl electrode. Thus recorded
transepithelial PDs express spheroid lumen (serosal side) with respect
to bath (mucosal side). Current was applied to the
spheroids via an additional microelectrode placed in the lumen of the
spheroids. Microelectrodes for current passage had the same
characteristics as those used for PD measurements. Because of the
relatively high resistance of these electrodes, a dual microiontophoresis current generator (model 260, World Precision Instruments) was used together with a second Ringer-agar bridge. The
amount of current applied by this equipment was controlled by a serial
attached ammeter. In each preparation, a range of brief (1-s) current
pulses was applied to obtain current-voltage relationships
(I-V
plots) for accurate estimates of the equivalent Isc and
transepithelial resistance
(Rt). The
experimental station was placed on a vibration-free table equipped with
a Faraday cage (TMC-63-500, Technical Manufacturing, Peabody,
MA). All fine adjustments and final steps in the
impalement procedure were performed with remote controls outside the
station guided by the electrical response and a monitor (Sony, Japan)
attached via a camera (CCV-931, VideV Euroline, Dortmund, Germany) to
the microscope. All experiments were videotaped (AG-7355-E, Panasonic,
Osaka, Japan) for later inspection.
Spheroid volume changes and fluid
absorption. Fluid absorption was estimated from
measurements of the outer diameter of individual spheroids at intervals
of 5-20 min. The spheroids were placed in 10-µl droplets of the
original culture medium on the bottom of a plastic dish and then
covered with paraffin oil (Uvasol, Merck) that had been
"saturated" in contact with the culture medium for several days
in the incubator. In this way, evaporation and gas exchange from the
droplet to the atmosphere during diameter measurements was minimized.
Two pictures of each spheroid, focusing on the spheroid perimeter and
on the bottom surface of the spheroid, and a picture of the calibration
microscale were grabbed and stored in a computer via the video camera
with a ×10 objective (Nikon MCI 10, HMC, 0.25 long working
distance) and a software-controlled frame grabber
(Flashpoint 3.0, Integral Technologies, Indianapolis, IN). From the
calibration microscale picture and the diameter at the spheroid
perimeter (measured manually on a 20-inch video screen), spheroid
volume and surface area were calculated. The fluid transport rate
(JV; in
µl · cm2 · h
1)
was then calculated from the volume change
(
V/
t) in a period of
measurement (normally 20 min) related to the surface area
(A) that was calculated from the
average diameter during that period: JV =
V/(
t · A).
From images focusing on the spheroid surface, it was possible in some spheroids to select areas with clearly demarcated cells where surface cell densities were estimated. Simultaneously calculated JV values per area (described above) then allowed for estimation of JV values per cell.
When JV values in spheroids were studied under the influence of amiloride, diphenylamine-2-carboxylate (2-DPC), 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), or hydrochlorothiazide, the drug was added from stock solutions to prewarmed medium. Then a number of spheroids were added, and individual spheroids were transferred in a droplet to the plastic dish for measurement of volume changes as described above.
For estimates of spheroid epithelial water permeability, fluid absorption was measured (as described above) in response to an inwardly directed 75 mosmol/l gradient. Thus 15 µl of oil-covered culture medium (~300 mosmol/l) containing a spheroid were diluted with 5 µl of water with the use of a micropipette driven by a set of hydraulic and motorized micromanipulators (MM-188 and MO-188, Narishige) connected to a microinjector (IM-6).
Solutions and chemicals. Cell culture media were obtained from ICN Biochemicals (Costa Mesa, CA). 2-DPC and NPPB were gifts from Dr. R. Greger (Albert-Ludwigs-Universitet, Freiburg, Germany). All other chemicals were purchased from Sigma (St. Louis, MO). Stock solutions of amiloride were prepared in Ringer solution, whereas stock solutions of 2-DPC, NPPB, and hydrochlorothiazide were prepared in DMSO at 1,000 times the final concentration.
Statistics. All quantitative data are means ± SE from individual observations. Multiple groups were compared with ANOVA. If the ANOVA was significant, the individual values were compared with Student's t-test or the Mann-Whitney rank sum test. A P value < 0.05 was considered significant.
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RESULTS |
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In agreement with our previous observations (18), the present study demonstrated that protease-released sheets of nasal airway epithelium form fluid-filled spheroids with diameters of 50-800 µm when incubated in a defined serum-free medium. All spheroids showed a fully differentiated monolayered epithelium, with apical cell membranes containing microvilli and cilia facing the outside bath and basolateral membranes pointing toward the central, fluid-filled lumen (18). An intense ciliary activity, confirming the state of high differentiation, was consistently observed.
Spheroid electrophysiology.
Application of a range of brief (~1-s) transepithelial current pulses
within a short time period (2 min), with simultaneous measurements of
spheroid areas (from video recordings of diameters) and changes in
transepithelial PD (
PD), allowed us to calculate transepithelial
current densities (I; in
µA/cm2) and to obtain the
relationship between I and PD
(I-V
curves). The I values used resulted in
PDs of up to ±50 mV. In this range, linear
I-V
curves were obtained in both CFSs and NCFSs, showing that the spheroid
epithelium behaves as an ohmic resistor. Examples of the
I-V
relationships obtained from a single NCFS before and after apical
application of amiloride
(10
4 M) are shown in Fig.
1. From the
I-V
curves, Rt values
were calculated as
Rt =
PD/
I. Furthermore, the
equivalent Isc
value was calculated as
Isc = PD/Rt. The
electrical parameters obtained in this way from 52 NCFSs (7 donors) and
46 CFSs (6 donors) are presented in Table
1. In comparison with the NCFSs, the CFSs
exhibited significantly higher values of basolateral side-positive PD
(155%; P < 0.001) and
Isc (170%;
P < 0.001), whereas the slightly
higher Rt of CFSs
(115%) was not significantly different from the
Rt of NCFSs. The
addition of amiloride (10
4
M) to the mucosal bath of 16 NCFSs (5 donors) significantly decreased (by ~90%) both the
Isc (from 37.4 ± 3.8 to 3.3 ± 0.6 µA/cm2) and the PD (from 15.4 ± 1.4 to 1.7 ± 0.3 mV) but increased the Rt (by 40%; from
432 ± 47 to 602 ± 66
· cm2). In
13 CFSs (3 donors), amiloride abolished the PD and
Isc but increased
the Rt (by 33%;
from 454 ± 71 to 602 ± 98
· cm2).
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Spheroid volume changes and fluid
absorption. We (18) previously demonstrated that the
average size of spheroids appeared to be relatively constant when
measured daily for several weeks. This average constancy, however,
covered the fact that the size of individual spheroids fluctuated,
probably due to inward fluid transport, interrupted by periods of
shrinkage due to stretch-induced leakage (18). Initial observations in
the present study demonstrated that this also applied when the size of
the spheroids was measured within a shorter period of time. Thus in 66 NCFSs (4 donors) incubated in smaller groups in culture medium,
measurements of spheroid size with time intervals of 1-1.5 h over
a period of 5 h demonstrated that the average diameter only changed
minimally and insignificantly (from 242 ± 8 to 251 ± 9 µm).
However, in a few cases, it was possible to identify large volume
changes in individual spheroids equivalent to fluid absorption of up to
6 µl · cm2 · h
1.
We therefore started measurements of diameters of individual spheroids
at short time intervals (5-20 min) over periods of 3-4 h. It
became obvious that the volume of spheroids generally fluctuated, with
a long period of increasing volume followed by a brief period of
shrinkage. However, in several spheroids, the volume at maximum level
as well as at minimum level could be stable for >1 h. An example of
regular fluctuations is shown in Fig. 2,
where the diameter of a single NCFS was measured every 10 min. The
diameter increased in two periods (representing fluid absorption)
interrupted by a brief reduction in diameter (representing leakage and
collapse of the spheroid). In the two fluid absorption periods, an
identical increase in diameter of 0.98 µm/min was observed,
corresponding to a fluid uptake of 3 µl · cm
2 · h
1.
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Changes in individual spheroid diameters were measured in a series of
34 NCFSs (4 donors) at 20-min intervals over a period of 4 h during
incubation in culture medium. Of these 408 measurements, 56 (14%)
demonstrated a decrease in diameter of >10 µm and 195 (48%)
demonstrated changes in diameters of less than ±10 µm. In the
remaining 157 (38%) 20-min periods, an increase in diameter of >10
µm was observed. Calculation of the increase in volume from the
increase in diameter and the average diameter in each of these 157 20-min periods resulted in a
JV value of 3.35 ± 0.12 µl · cm2 · h
1.
When fluid absorption was calculated from the maximal value of volume
increase in each of the 34 NCFSs, a value of 4.42 ± 0.29 µl · cm
2 · h
1
was obtained. In a similar series of measurements of diameters in 65 CFSs (5 donors), an increase in diameter of >10 µm was observed in
a similar fraction of observations as in NCFSs
(n = 284 measurements; 40%). The
average JV in
these periods was 3.25 ± 0.07 µl · cm
2 · h
1,
whereas the JV
calculated from the maximal value of volume increase in each of the 65 CFSs was 4.35 ± 0.17 µl · cm
2 · h
1.
Thus JV values
were of equal magnitude in NCFSs and CFSs.
In a number of the spheroids incubated under control conditions in
culture medium, the
JV per cell was
estimated. In these experiments, video-stored images with the focus on
the perimeter of a spheroid allowed measurement of total surface area
and fluid absorption per square centimeter from recorded diameters and
increments in diameter as described above. Simultaneously stored images
from spheroids, with the focus on the surface closest to the objective, allowed counting of the cells in a smaller, nearly plane area and thus
the number of cells per square centimeter (Fig.
3). The cell densities obtained in this way
were ~5 × 105
cells/cm2. The calculated average
JV per cell in
periods where spheroid diameters increased >10 µm/20 min was
6.4 ± 0.2 pl/h in NCFSs (n = 19 measurements; 3 donors). A
similar single-cell absorption rate of 6.6 ± 0.3 pl/h was obtained
in CFSs (n = 18 measurements; 3 donors), again emphasizing that fluid absorption in NCFSs and CFSs were
of equal magnitude.
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The volume change of a spheroid during an increase in diameter from 410 to 460 µm over a period of 30 min is shown in Fig. 4. This illustrates the problem of
calculating absolute fluid absorption per square centimeter because the
epithelial cells are stretched as the result of spheroid fluid
absorption. Thus fluid absorption in this spheroid calculated in
relation to surface area at the initial diameter (410 µm) was 5.49 µl · cm2 · h
1,
whereas the calculated
JV in relation to
surface area at the average diameter (435 µm) was 4.88 µl · cm
2 · h
1.
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To investigate a coupling between water flux and
Na+ absorption, spheroids were
incubated in the presence of amiloride (100 µM) in the bathing
medium. This resulted in
JV values
considerably lower than those under control conditions. Consequently,
estimates of fluid absorption were based on longer periods of diameter
measurements (40-200 min) than the 20-min periods used for
measurements of maximal fluid absorption under control conditions. The
amiloride-insensitive fluid absorption was 0.51 ± 0.07 µl · cm2 · h
1
in NCFSs (n = 23 measurements; 5 donors). This value is significantly lower than the fluid absorption of
1.27 ± 0.08 µl · cm
2 · h
1
observed in amiloride-treated CFSs (P < 0.001; n = 44 measurements; 5 donors). This difference in fluid absorption may be related to a
defective Cl
secretion in
CFSs due to a missing CF transmembrane conductance regulator (CFTR)
function (1).
We (18) previously demonstrated that apical cell membrane
Cl-channel blockade by
2-DPC eliminated the amiloride-insensitive PD in NCFSs. We therefore
tested the effects on NCFS fluid transport of the
Cl
-channel inhibitors 2-DPC
(250 µM) and NPPB (100 µM) in combination with amiloride. With
these inhibitors, we obtained values of
JV (2-DPC plus
amiloride: 1.11 ± 0.12 µl · cm
2 · h
1,
n = 8 measurements, 2 donors; NPBB
plus amiloride: 1.11 ± 0.12 µl · cm
2 · h
1,
n = 7 measurements, 2 donors)
significantly higher than those obtained with amiloride alone
(P < 0.001; Fig.
5). This suggests that
amiloride-insensitive water absorption in NCFSs is counterbalanced by
an amiloride-induced Cl
and
water secretion. Interestingly, the
JV values in
NCFSs treated with amiloride and
Cl
-channel blockers were
not significantly different from that in CFSs treated with amiloride
only (Fig. 5).
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In CFSs, addition of amiloride abolished the
Isc (and the PD;
Table 1). The presence of a significant amiloride-insensitive residual
fluid absorption in CFSs (as well as in NCFSs treated with amiloride or
amiloride plus Cl-channel
inhibitors; see above) therefore suggested that an electroneutral absorptive process was responsible for this fluid absorption. The
nature of such a transport mechanism is unknown, but a plausible candidate could be an apical membrane
Na+/Cl
cotransporter (19). We therefore tested the effects of the specific
Na+/Cl
cotransport inhibitor hydrochlorothiazide (22). Figure 5 shows that in
29 CFSs exposed to mucosal amiloride (100 µM) and hydrochlorothiazide (100 µM), fluid absorption (0.85 ± 0.07 µl · cm
2 · h
1)
was significantly lower than in CFSs treated only with amiloride (1.27 ± 0.08 µl · cm
2 · h
1;
P < 0.001). Treatment of NCFSs in
the same way with amiloride plus hydrochlorothiazide also resulted in
significantly lower fluid absorption (0.15 ± 0.04 µl · cm
2 · h
1;
n = 17 measurements) compared with
that obtained with amiloride only (0.51 ± 0.07 µl · cm
2 · h
1;
P < 0.001). Thus in both CFSs and
NCFSs, an electroneutral, hydrochlorothiazide-sensitive absorptive
mechanism is responsible for a fluid absorption of ~0.4
µl · cm
2 · h
1,
which is equal to ~10% of the control
JV value.
In a series of experiments, we estimated the osmotic water permeability
of the spheroid airway epithelium. Single spheroids were placed in a
15-µl droplet of incubation medium (osmolarity 300 mosmol/l)
covered with paraffin oil in the thermostated chamber on the microscope
stage. The diameter of the spheroid was videorecorded before and after
the addition of 5 µl of water to the droplet [resulting in a
lowering droplet (apical) osmolarity of
75 mosmol/l]. In
response to this imposed transepithelial osmotic gradient
(corresponding to an equivalent
P of 1.91 atm), spheroid volume
increased much faster than during spontaneous fluid absorption. From
the fast increase in volume during the first 5 min (in those spheroids that remained intact during the increased fluid absorption), we calculated JV
values of 21.14 ± 1.53 µl · cm
2 · h
1
in NCFSs (n = 22 measurements; 3 donors) and 21.82 ± 1.96 µl · cm
2 · h
1
in CFSs (n = 10 measurements; 2 donors). On the basis of these equal
JV values, we
calculated a corresponding hydraulic conductivity (LP = JV/
P) of ~27 × 10
7
cm · s
1 · atm
1
and a filtration (osmotic) permeability of ~45 × 10
4
cm/s (Pf = LPRT/VW,
where R is the gas constant, T the absolute temperature
in °K, and VW the partial molar volume of
water) in both NCFSs and CFSs.
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DISCUSSION |
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We (18) recently introduced a spheroid preparation for the study of ion
transport in CF and non-CF airway epithelia and demonstrated that the
basolateral side (inside)-positive transepithelial PD of this
preparation during the influence of various agonists and ion channel
blockers mimics that of the native tissue and conventionally cultured
airway cells. The values obtained in the present study are in agreement
with those previously obtained (18). The results from passage of
current pulses show that the epithelia of both NCFSs and CFSs behave as
ohmic resistors, i.e., that the instantaneous
I-V
curves were linear in the range of at least ±150
µA/cm2 (Fig. 1). This is in
agreement with previous reports (6, 24). Calculation of
Rt values from
the slope of the
I-V
curve demonstrated that the
Rt of the
spheroid airway epithelium is relatively high (non-CF control
Rt = 400-450
· cm2; Table
1). In freshly isolated preparations of nasal airway epithelium,
considerably lower
Rt values
(50-100
· cm2)
have been observed (6, 15, 27), whereas high
Rt values (200-1,000
· cm2) are
generally observed in cell culture preparations of nasal and other
airway epithelia (14, 24, 26, 27). As previously emphasized, the
present spheroid-type preparation of airway epithelium is not a primary
culture but an explant of fully differentiated epithelium. However,
like primary cultures, the spheroids were incubated in culture medium
during preparation and storage. It seems that incubation in culture
medium may result in a decrease in the conductance of the paracellular
pathway; i.e., the epithelium is transformed from a low-resistance
epithelium in the direction of a high-resistance epithelium (28). In
the present spheroid preparation, the conductance of the cellular
pathway is not insignificant compared with the conductance of the
paracellular (shunt) pathway. This interpretation is supported by the
observations 1) that closure of
apical Na+ channels by amiloride
consistently increased
Rt in NCFSs (by ~40%) and CFSs (by ~33%) and
2) that the
Rt in CFSs
(lacking the cellular conductance from CFTR
Cl
channels) was higher
than that in NCFSs (the difference was not significant due to the
scatter in Rt
values). Similarly, slightly higher or equal
Rt values in CF
airway epithelia compared with those in non-CF epithelia have been
previously reported (6, 14, 16, 24).
The Isc in upper
airway epithelia is primarily a measure of amiloride-sensitive
Na+ absorption (3, 5, 6). In
accordance with this, we observed that apical amiloride decreased
Isc (measured as
equivalent Isc = PD/Rt) by 91%
in NCFSs and 100% in CFSs. Also, the observation that
Isc in untreated
preparations was significantly higher in CFSs than in NCFSs (Table 1)
is in accordance with a reported increase in epithelial capacity for
Na+ absorption in CF airway
epithelia caused by upregulation of apical membrane
Na+-channel permeability
(4-6). The residual, amiloride-insensitive Isc (and PD) in
NCFSs may at least partly be accounted for by Cl secretion through apical
(CFTR) Cl
channels
triggered by amiloride-induced apical membrane hyperpolarization (3,
26) as demonstrated by its sensitivity to the
Cl
-channel inhibitor 2-DPC
in NCFSs (18). However, it should be emphasized that in the present
preparation of NCFSs, such a
Cl
secretion is rather
small because amiloride inhibited
Isc (and PD) by
~90%. This could be due to a small driving force for
Cl
movement across the
apical membrane (26) or to a low activation of
Cl
channels. In CFSs, the
lack of a residual
Isc after
amiloride may obviously be related to the lack of CFTR
Cl
channels.
We (18) previously observed that the size of individual spheroids fluctuated around an average value that appeared to be almost constant for several weeks. It was suggested that these volume fluctuations reflected periods of inward fluid transport interrupted by episodes of shrinkage caused by pressure- and/or stretch-induced openings of tight junctions. We furthermore suggested that the fluid absorption was driven by active inward transport of Na+. The present study confirms these suggestions. When maximal rates of volume increase were related to the simultaneous average spheroid surface, JV values of equal magnitude were obtained in NCFSs and CFSs. Similar magnitudes of JV values by non-CF airway epithelial preparations have been reported in some studies (2, 14, 23, 29, 30), whereas other studies (12, 16, 20, 21) have demonstrated lower values. Most frequently, increased levels of JV values are observed in CF airway epithelial preparations (14, 16, 30). Although decreased levels have also been reported in CF preparations (29), our finding of equal JV values in NCFSs and CFSs is therefore at variance with some previous studies. The values of JV in the present study were calculated in relation to surface area at a time when the spheroids had already been stretched to some degree. Thus absolute absorption rates may be underestimated considerably. This is also suggested from the observed surface cell density of ~5 × 105 cells/cm2 during maximal spheroid fluid absorption compared with reported cell densities of up to ~45 × 105 cells/cm2 in unstretched airway epithelium (17). These considerations may suggest that fluid absorption in the present explant preparation may be considerably higher than in conventional primary cultures if it is related to unstretched epithelial surface. However, they do not explain the similarity in fluid absorption in CFSs and NCFSs. Thus equal JV values in the two types of spheroids were found not only when related to surface area but also when fluid absorption per cell was calculated. Stretching of the spheroid surface area may, in a similar way and to a similar degree, lead to an underestimation of the above-mentioned Isc values. For a comparison of ion and water transport capacities (see below), corrections for surface stretching are therefore not necessary.
The mechanism of coupling between net salt and water transport in
airway epithelia is still unknown. Thus it is not known whether a
coupling is at the intraepithelial or the transepithelial level.
Studies on fluid transport across varying types of airway epithelial
preparations demonstrate no general agreement that water follows net
solute movements in the absence of transepithelial solute concentration
gradients (16, 20, 29). A study (23), however, has
demonstrated parallel changes in net solute and water movements that
are compatible with an intraepithelial, isosmotic coupling. However, in
the present spheroid preparation, water uptake probably has to follow
net salt uptake in isosmotic proportions. The inside (basolateral)
volume of spheroids is small. Consequently, if water does not follow
net salt absorption at the intraepithelial level, salt absorption will
tend to increase the inside osmolarity, and provided the epithelial
osmotic water permeability is sufficiently high, water will follow
passively. Indications of high water permeability have been presented
in intact airway preparations (8) and in primary cultures of human
airway epithelium (7, 9, 12, 16). The osmotic water permeabilities
observed in the present study demonstrate identical values in CF and
non-CF epithelia (Lp = ~27 × 107
cm · s
1 · atm
1).
These values are as high as those measured in the highly
water-permeable epithelia in the mammalian small intestine,
gallbladder, and plexus chorioideus (13). They even have to be regarded
as minimum values due to the likely presence of unstirred layers and to
the fact that the osmotic water flows were related to a slightly
stretched surface area. Furthermore, a comparison of calculated net ion transport rates with
JV values
indicates that absorbed fluid may be close to isosmotic with the
bathing medium. With the assumptions that the equivalent
Isc of NCFSs
represents Na+ absorption, that
anions follow passively, and that water follows NaCl isosmotically, a
fluid absorption of ~7
µl · cm
2 · h
1
was calculated, a value close to observed
JV values.
The ion transport mechanisms reflecting the
JV values under
open-circuit conditions may be explained as follows: in untreated NCFSs, transcellular, active Na+
absorption (of a size reflected by the
Isc) is
followed by passive anion absorption via the paracellular pathway
and/or a cellular pathway (basolateral and cAMP-stimulated apical
membrane Cl channels; 21, 23, 25). Water follows the resulting net NaCl absorption. In CFSs, NaCl
and water absorption are expected to be higher than in NCFSs due to
upregulation of apical membrane
Na+-channel conductance and
transepithelial Na+ transport
capacity (reflected by an increase in
Isc) (4). However, the present observed
JV values are not
higher in CFSs than in NCFS, which indicate that the actual
Na+ absorption during open-circuit
conditions is not increased. The reason for this may be that under
open-circuit conditions, the apical membrane
Na+ uptake is slowed down due to a
smaller driving force [the PD across the apical membrane is
considerably smaller in CF airway cells compared with a cellular PD of
approximately
25 mV across the apical membrane in non-CF cells
(5, 6, 24)]. Furthermore, the lack of apical membrane CFTR
Cl
channels excludes the
possibility of coupled, passive transport of the counterion
Cl
via the cellular pathway.
The effects of amiloride in the bathing medium also disclose
relationships between ion transport and water absorption in the spheroid preparations. Amiloride treatment of NCFSs resulted in a
considerably lower fluid absorption (0.46 µl · cm2 · h
1)
than in nontreated NCFSs (4.4 µl · cm
2 · h
1).
Amiloride reduces fluid absorption in CFSs but to a significantly smaller degree (from 4.5 to 1.27 µl · cm
2 · h
1).
Other studies have also shown that amiloride reduces fluid absorption
in non-CF (2, 14, 20, 23, 29) and CF (14, 29) epithelia. In
amiloride-treated CFSs, Na+
absorption via apical Na+ channels
is abolished and cellular
Cl
secretion (via CFTR
Cl
channels) is not
possible. This situation is characterized by abolition of
transepithelial PD and
Isc. The fact
that spheroid fluid uptake did not vanish in response to amiloride in
either CFSs or NCFSs suggests that a nonelectrogenic NaCl absorption could be responsible for the amiloride-insensitive fluid absorption. In
rabbit nasal airway epithelium, a small, nonelectrogenic, and hydrochlorothiazide-sensitive Na+
absorption was previously demonstrated (19). Apical application of
hydrochlorothiazide (100 µM) reduced fluid absorption to the same
extent (0.3-0.4
µl · cm
2 · h
1)
in amiloride-treated preparations of both CFSs and NCFSs, suggesting the presence of an electroneutral
Na+/Cl
cotransporter (22) in the apical cell membrane.
The difference in fluid absorption (~ 0.8 µl · cm2 · h
1)
between amiloride-treated CFSs and NCFSs may suggest the presence in NCFSs of a NaCl secretion superimposed on the residual
amiloride-resistant NaCl absorption. Thus in NCFSs, the inhibition of
apical Na+ channels (and
electrogenic Na+ absorption) by
amiloride is expected to hyperpolarize the apical cell membrane and
thereby induce a small secondary active cellular Cl
secretion via apical
membrane (CFTR) Cl
channels
(and a basolateral membrane
Na+/K+/2Cl
cotransporter). In this situation, a passive, paracellular
Na+ movement may follow the
cellular Cl
secretion, and
a component of fluid movement is expected to occur in the secretory
direction. This interpretation is supported by the observation that
apical applications of
Cl
-channel inhibitors
(2-DPC or NPPB) to amiloride-treated NCFSs resulted in a significantly
higher JV (1.11 µl · cm
2 · h
1
with either 2-DPC or NPPB) than in the absence of
Cl
-channel inhibitors (0.46 µl · cm
2 · h
1).
Interestingly, these values are not different from the
JV values in
amiloride-treated CFSs (1.27 µl · cm
2 · h
1)
lacking the possibility of
Cl
secretion through CFTR
Cl
channels.
The present investigation as well as other recent studies on primary
cultures of airway cells (23, 29) suggests that a part of the
Cl absorption in non-CF
airway epithelium under open-circuit conditions may take a cellular
route via apical and basolateral membrane Cl
channels. This seems to
be at variance with previous suggestions from studies (3, 5, 19) in
freshly isolated natural airway epithelia where the pathway for passive
Cl
absorption was
interpreted to be paracellular. This discrepancy may be related to the
above-mentioned fact that natural epithelia are low-resistance
epithelia, whereas primary cultured airway cell layers (and the present
spheroid explant) are high-resistance epithelia. The difference in
resistance probably reflects a difference in the resistance of the
paracellular tight junction pathway (10). In low-resistance epithelia,
this pathway serves as an intraepithelial shunt that hyperpolarizes the
apical cell membrane (as under short-circuit conditions or by blocking
apical Na+ channels with
amiloride), thereby favoring apical
Cl
exit and
Cl
secretion instead of
Cl
uptake. Under
high-resistance conditions (with less shunting), the apical
Cl
gradient may be
favorable for absorption, and, simultaneously, the relative
contribution to Cl
absorption from paracellular passage may be small due to a lower Cl
permeability.
It should be emphasized that even in the absence of knowledge about coupling mechanisms between salt and water flows, we may expect that absorption in the in vivo upper airways has to be isosmotic because 1) the volume of the apical periciliary liquid layer is small, 2) the epithelial osmotic water permeability is high, and 3) the time of contact between surface liquid and epithelium is long due to a relatively slow mucociliary clearance. It is therefore unlikely that in vivo airway epithelia can maintain transepithelial osmotic gradients. Our results with spheroids being exposed to a sudden reduction of tonicity (by 75 mosM) of the apical solution support this conclusion. Thus osmotically driven fluid absorption was approximately five times larger than the spontaneous JV.
In conclusion, the present study on spheroid preparations of airway
surface epithelium demonstrates that
1) unstimulated
JV values under
open-circuit conditions are equal in CFSs and NCFSs; 2) water absorption is closely
related to net NaCl absorption as determined by the concerted function
of amiloride-sensitive Na+
absorption, Cl secretion,
and an electroneutral NaCl cotransport mechanism; and
3) that the high water permeability
of spheroids makes it unlikely that the human airway epithelium can
maintain osmotic gradients.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. N. Rasmussen, E. Hviid-Larsen, and R. Sinding for help during this study.
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FOOTNOTES |
---|
This work was supported by grants from the Danish Research Council (SSVF), the Novo-Nordisk Foundation, the Lundbeck Foundation, and the Velux Foundation.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. S. Pedersen, Dept. of Clinical Genetics (4061), Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark (E-mailpsp{at}dadlnet.dk).
Received 26 May 1999; accepted in final form 4 August 1999.
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