Biochimica et Biophysica Acta, 1094 (1991) 19-26 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100209T
19
BBAMCR 12980
Cation selective channel in fetal alveolar type II epithelium Beverley A. Orser 1,2, Maria Bertlik l, Ludwik Fedorko 3 and Hugh O'Brodovieh 1 i Respiratory Research Division, Research Institute, Hospital for Sick Children, Toronto (Canada), 2 Department of Anesthesia, Toronto Western Hospital, Toronto (Canada) and 3 lift. Sinai Hospital Research Institute, Toronto (Canada) (Received 28 February 1991)
Key words: Non-selective cation channel; Amiloride; Alveolar epithelium; Ion channel; Sodium channel
A cation selective channel was identified in the apical membrane of fetal rat (Wistar) alveolar type lI epithelium using the patch clamp technique. The single channel conductance was 23 ± 1.2 pS (n -- 16) with symmetrical NaC! (140 raM) solution in the bath and pipette. The channel was highly permeable to Na + and K + (Pwa/PK = 0.9) hut essentially impermeant to chloride and gluconate. Membrane potential did not influence open state probability when measured in a high Ca 2÷ (1.5 mM) bath. The channel reversibly inactivated when the bath was exchanged with a Ca2+-free ( < 10-9 M) solution. The Na + channel blocker amiloride (10-6 M) applied to the extraceilular side of the membrane reduced Popen relative to control patches; P~ont~ot= 0.57 __ 0.11 (n = 5), Pamiloride = 0.09 ~ 0.07 (n --- 4, p < 0.01), however, amiloride did not significantly influence channel conductance (g): gco~t~ot 19 _ 0.9 pS (n = S), 18 _+3.0 pS (n = 4). More than one current level was observed in 42% (16/38) of active patches; multiple current levels (ranging from 2 to 6) were of equal amplitude suggesting the presence of multiple channels or subconductance states. Channel activity was also evident in cell attached patches. Since monolayers of these cells absorb Na + via an amiloride sensitive transport mechanism we speculate that this amiloride sensitive cation selective channel is a potential apical pathway for electrogenic Na + transport in the alveolar region of the lung.
Introduction Successful transition from intrauterine to extrauterine life depends on the rapid adaptation of the fetal lung. Fluid, actively secreted into the lungs airspaces and airways [1] during uterine life, must be cleared at birth to allow the lung to function as the organ of gas exchange. Although part of this fluid is removed during labour and delivery [2,3], a significant proportion of the fluid is cleared postnatally by the pulmonary epithelium via amiloride sensitive mechanisms [4]. In vivo studies support the hypothesis that active transport of Na + by pulmonary epithelium generates an osmotic gradient which drives fetal lung fluid absorption at birth. Epinephrine stimulates fetal lung fluid absorption in utero [5], a process which can be blocked by the sodium transport inhibitor amiloride [6]. Also, our laboratory has recently showed that instillation of
Correspondence: H. O'Brodovich, Respiratory Research, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario MSG 1X8, Canada.
amiloride [4] or benzamil [7] into the tracheal fluid of fetal guinea pigs shortly before birth delays the clearance of lung water causing respiratory distress in the newborn. It is likely that the perinatal reabsorption of fetal lung liquid results, at least in part, from solute coupled active transport across the alveolar epithelium. For example, Shaw et al. showed amiloride blockable Na uptake into membrane vesicles made from fetal sheep alveolar type II epithelium [8]. Our laboratory has demonstrated that monolayers of fetal alveolar epithelium maintain a high transcellular resistance and generate a Na + dependent short-circuit current which can be inhibited by the Na + channel blockers amiloride and benzamil [7,9]. Fetal alveolar epithelium grown in organotypic culture have a significant transcellular voltage gradient ( - 12 mV, apically negative) that can be reduced by the metabolic inhibitor ouabain [10]. The above indirect evidence suggests the presence of sodium conductive ion channels in fetal alveolar epithelium. We used the patch clamp technique to identify and characterize ion channels present on the apical membrane of fetal rat (Wistar) alveolar epithelial cells which could participate in Na + transport.
20
Materials and Methods
Cell culture Primary cultures of fetal alveolar type II cells were isolated as previously described [9,11]. Briefly, cells were harvested from Wistar fetal rats (Charles River, St. Constant, Canada)delivered by Cesarean section at 20 days gestation (term 22 days), Lungs were minced and cells dissociated by enzymatic digestion with 0.125% trypsin and 0.002% DNAase in Hanks' balanced salt solution. The cell suspension was filtered through a 100 mesh Nitex Filter (Tetco Inc., Elmsford, NY, U.S.A.) and isolated cells added to minimal essential media (MEM, Gibco, Burlington, Canada) and 0.1% collagenase (Gibco) for 15 rain at 37°C. Fibroblasts were removed by a differential adherence technique. Nonadherent alveolar epithelial cells were collected, centrifuged and seeded onto collagen coated coverslips (approx. 2. l0 s cells/cm 2) in MEM plus penicillin/streptomycin 100 U / m l (Gibco). Cells were cultured in a humidified 95% air, 5% CO2 containing incubator and were studied 24 to 96 h after harvest. Collagen 0.1% used to coat the coverslips was prepared from a concentrate of rat-tail collagen in acetic acid diluted in MEM 1 : 10 buffered with NaHCO 3 to a pH 7.0. Cell viability (> 90%) and purity (> 95%) associated with this isolation procedure has been previously reported [9]. We had difficulty acquiring stable, high resistance (> 109 ,O) seals from confluent monolayers, therefore subconfluent clusters of alveolar epithelium were studied. In an effort to increase the numbers of high resistance seals, approximately one half of the cultures were 'cleaned' with coUagenase 0.2% as previously described to improve the rate of seal formation [12]. This had no effect on the number or quality of seals acquired.
latch clamp technique Patch pipettes were fabricated from boroscilicate (1.0 mm ID, 1.5 mm OD, Garner Glass, Claremont, CA, U.S.A.) or aluminoscilicate glass (0.75 mm ID, 1.2 mm OD, Sutter Instruments, Novata, CA, U.S.A.) on a two-stage vertical puller (Narashigi, Tokyo, Japan). Pipettes were Sylgard® coated (Dow Coming, Midland, Mi, U.S.A.) and firepolished yielding a final tip resistance of (5-8). 106 fJ when filled with NaC! (140 raM) solution. Experiments were performed at room temperature. Membrane current were recorded using a 50.10 9 ~'~ resistor in a current to voltage converter (Ampatch l-B, Axon Instruments, Foster City, CA, U.S.A.) Current signals were filtered at 1 kHz through a 4-pole Bessel low-pass filter, and stored on a FM tape (Sony) after digitization (A.R. Vetter Co., Rebersbeg, PA, U.S.A.). Reported holding potentials indicate the 'intracellular' membrane potential referenced to ground (Vbath- [/'pipette) . Outward current or positive
charge moving from the bath to pipette (intracellular to extracellular side of the patch) is depicted by an upward deflection in all raw current records. In most experiments, the ground electrode was a Ag-AgCl wire. When the bath was exchanged with solutions containing varying concentrations of Cl-, an agar bridge (NaCl 140 mM) was used to electrically couple the ground wire to the bath.
Solutions Amiloride was purchased from Sigma (St. Louis, MO, U.S.A.). All solutions were mixed with ultrapure water and then passed through a 0.22 p.m filter (Millipore Products, Bedford, MA, U.S.A.) prior to use. In most studies the initial bath and pipette solution conrained (raM): NaCI (140), CaCI 2 (1.5), glucose (10), Hepes (10) (pH 7.4). The contents of the bathing and pipette solutions were varied to evaluate the characteristics of the channel (see Results). The concentration of free Ca 2+ in solution was estimated using a computer program for calculating the composition of solutions containing Ca 2+ and EGTA [13]. The concentration of Ca 2+ in 'Ca2+-free solution' was measured to be < 10 -9 using the fluorescent dyes; quin2 and indo-1 [14,15].
Analyses Data were analyzed using the PClamp program version 5.0 or 5.5 (Axon Instruments, Foster City, CA, U.S.A.). Single channel amplitudes were determined using all points histograms created from at least 20 s of recorded data. Histograms were curve fitted using the least-squared method for a Gaussian distribution and current levels are reported as the amplitude difference between close and open states. The number of channels in the patch was determined by counting the number of open current levels above baseline in the raw current records when current (i) was between (n - 1/2)i and (n + 1/2)/. Single channel conductance was measured as the slope of the current to voltage (I-V) relationship and reported as mean + S.E. of the mean. Many patches contained multiple open levels of equal amplitude suggesting subconductance states or multiple channels in the patch. We made the assumption that each level above baseline represented a single channel which opened and closed independently. The probability of the channel being in the open state (Pope,) was estimated by the relative area under the all points amplitude histogram. When histograms contained multiple open amplitude peaks Pope, was estimated using the following formula: I1
1 ~Ai'i eopen =
i = n1 n
EA i=o
i
.
21 where A = area u n d e r Gaussian C u r " n = total n u m b e r o f channel,: and i -- n u m b e r o f o p e n channels. F o r example, Fig. 1B illustrates an all points histogram from a patch containing three o p e n levels above baseline (c) level: O i , 0 2 , 0 3 , r e p r e s e n t the current amplitude with o n e , two, and three c h a n n e l s open, respec-
tively. T h e probability o f channel o p e n i n g (Pope,) is calculated as: ( a r e a 0 3 + area 0 2 • 2 + a r e a O I • 1) divided by the total a r e a u n d e r the o p e n a n d closed curves times the total n u m b e r o f c h a n n e l s in the patch. This m e t h o d assumes binomial distribution and channel independence. T h e distribution o f p e a k areas in the all points histograms was similar to that predicted f r o m the binomial distribution for n c h a n n e l s with the
(my) + 60 C..~.
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-45
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z 102400 Events o3 --,p-.
1300 N/dlv Amphtude (pA)
0
125
2 46
3 67
Fig. 1. (A) Raw current recordings from an inside-out patch of fetal alveolar epithelial cell membrane. The pipette and bath solution contain mM: NaCI 140, CaCI 2 1.5, glucose 10, Hepes 10, EGTA 1. 'lntracellular' potential (mV) is referenced to the ground and is indicated to the left of the current tracings. An upward deflection represents outward current, (positive charge) moving from the membrane's cytoplasmic face into the pipette. The dashed line denotes the open state (O), the solid line the closed state (C). (B) All points amplitude histogram of a current record containing three channels (holding potential = - 7 0 mV). The dosed state (C) and three open states (O) are seen. '!.he number of events is indicated on the y-axis, amplitude on the x-axis. This histogram represents 20 s of recorded data, binned in 0.05 pA ircrements with 1300 events per division on the y-axis. The single channel current between individual levels 1, 2 and 3 was (pA) 1.25, 1.21 and 1.21, respectively.
22 2.C
open probability of the channels measured. To test for statistical significance between Popen in control and amiloride treated patches an unpaired Student's t-test was used. Results are reported as mean +_S.E. and probability (p) < 0.05 was considered statistically significant.
pA 1, =_
10 05
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."
Microscopy We wished to determine if the upward facing cell surface of FAE grown in subconfluent cultures represented the apical membrane of polarized epithelium. Light and electron micrographs were studied in order to identify the morphologic characteristics of polarized cells grown 'on collagen-coated Millipore HA filters (Millipore, Bedford, CT, U.S.A.). Cells on filters were fixed with 1% glutaraldehyde then washed in phosphate-buffered saline. Cells were stained with 1% osmium tetroxide and 3% potassium ferrocyanide and then were dehydrated in ethanol. Filters were cut into short strips and embedded in Epon/Araldite (EMS, Fort Washington, PA, U.S.A.). Light microscopy was performed on 60 /tm sections stained with toluidine blue in 1% sodium borate using a Reichert Polyvar microscope, and 100 × oil objective. Transmission electron microscopy (Phillips 400) was performed on 60 nm ultrathin sections exposed to 3% uranylacetate followed by lead citrate in ethanol. Results
Light micrographs of subconfluent cultures of fetal alveolar epithelium showed basally located nuclei in 86/130 (66%) of cells. Electron-micrographs confirmed the presence of lameUar bodies, tight junctions and microvilli, which are characteristics of polarized cells. Similar characteristics have been described in subeonfluent cultures of pancreatic acinar cells [16-18]. We assume therefore that our patches were acquired from the cell's apical membrane. Stable excised inside-out patches were difficult to acquire from both confluent and subconfluent cultures of fetal alveolar epithelium. Although we did not record the exact rate of successful high resistance (> 109 ,O) seal formation, it was estimated to be less than 10%. Patches also rarely tolerated multiple voltage steps or exchanges of the bath solution. The channel most frequently observed in inside-out patches excised from the cell's apical surface in symmetrical NaCI (140 mM) solution was a cation selective channel (Fig. IA). In 42% (16/38) of all active patches more than one (range 2-6) open level was observed suggesting the presence of more than one channel in the patch or multiple subconductive states (Fig. 1B). The single channel conductance was 23 + 1.2 pS (n = 16) with NaCI (140 mM) in the bath and pipette solution. No rectification of the current to voltage
k -80
I -60
I -40
I -20
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I
I
40
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60
..~..~.n " . O.5 mv
-2,0
Fig, 2. The current to voltage (I-V) relation of inside-out patches of FAE. The boxes (13) indicate current measured in symmetrical NaCI (140 raM) solution, slope conductance = 23+ 1.2 pS ( , = 16). Each point represents the mean single channel current; when three or more patches were recorded at the same holding potential, currents were averaged and S.E. is indicated by the vertical bars. The triangles ( • ) indicate current recorded in five patches following a 2:1 dilution of the bath with isotonic sucrose (bath mM: NaCI 46.6 sucrose 187 CaCI 2 1.5, Hepes 10, glucose 10; pipette: NaCi 140, CaCI 2 !.5, Hepes 10, glucose 10). The zero CUlTci~tpotential (ERe v) of +27.5 mV after sucrose dilution indicate a cation selective channel.
(l-V) curve was observed (Fig. 2). Anion and cation selectivity of the channel was determined by sucrose dilution and ion substitution experiments. Diluting the bath solution 2 to 1 with isotonic sucrose caused the -2.0 pA
-1.5 -1.0 ,& -0.5
I
80
I 60
I
40
I - 20
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-80
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mV
1.0 1.5 2£ Fig. 3. The K + permeability of the cation selective channel. I.V relation with high 'intracellular' K+; (raM) bath: KCI 140, CaCI 2 1.5, glucose 10, Hepes 10; pipette: NaC1 140, CaC! 2 1.5, glucose 10, Hepes 10. The channel conductance calculated from the slope of the line of linear regression (. . . . . . ) was 20+ 1.8 pS and ERev =--3-t" 0.6 mY, n = 4.
23 closed
open
-I A
B
A
B
closed
open
,o,L__ 2 0 mSec. Fig. 4, Inactivation of channel activity in low 'intraccllular' Caz+. Single channel recording of the same inside-out patch that was repetitively switched from high (1,5 raM) to low ( < 10 -9 M) Ca2+ bathing solutions, Pipette Ca2÷ concentration was not changed during the experiment. Membrane potential = - 60 mV. This is a tracing from a single patch, the break lines are time periods when bathing solution was being changed. The pipette contained (mM); NaCI 140, CaCI 2 1.5, glucose 10, Hepes 10. The bathing solutions were (raM); solution A: NaCI 140, CaCI 2 1.5, glucose 10, Hepes 10, or solution B: NaCI 140, CaCI 2 0, glucose 10, Hepes 10, EGTA 2 (free Ca 2+ < 10 -9 M). Note that replacement of the bath with a high Ca 2+ solution reversed channel inactivation.
1.0(;
3 A
4
i
4
6
0.8C
3
• 2
c
0.6C
e
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•
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ft. 0.4C
0.2C
I - 80
I - 60
I - 40
I - 20
Membrane
I 0
I 20
I 40
I 60
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potent~ol
Fig. 5. Membrane potential does not influence channel open probability (/'oven) of inside out patches in symmetrical solutions (raM): NaCI 140, CaCI 2 1.5, glucose 10, Hopes 10 (n = 6). The circle and bar represent the mean and S.E., respectively, with the number of patches recorded at each holding potential indicated above the mean value.
I-V curve to inwardly rectify and shifted the ERev to + 27.5, n = 5 (Fig. 2) indicating the channel was permeant to cations. ERev for a completely cation selective channel calculated from the Nernst equation is + 27.7 mV at a temperature of 20 ° C. The CI permeability relative to Na + permeability was negligible. In three experiments, substitution of CI- anion in the pipette with gluconate (mM: pipette, sodium gluconate 140, CaCI 2 1.5, glucose 10, Hopes 10; bath NaCI 140, CaCI 2 1.5, glucose 10, Hepes 10) shifted ERev to +5 + 1.0 mV, slope conductance = 27 + 4.0 pS. This slight shift in the reversal potential in the presence of a large chloride gradient indicates that the ion channel is essentially impermeant to anions. Cation selectivity was investigated by exchanging the bath with a high K + (140 raM) solution (Fig. 3). The single channel conductance was 20 + 1.8 pS (n ffi 4) and the reversal potential (ERev) shifted from 0 mV to - 3 . 0 + 0 . 6 inV. The estimated Na + permeability to K + permeability ratio (PNa/PK)calculated using the
24 modified Goldman-Hogkin-Katz voltage equation was 0.9. This indicates that the channel fails to discriminate between Na + and K +. In a high Ca 2+ bath the channel resided primarily in the open state (holding potential - 60 mV); Ca~ + = 1.5 mM, Pope, = 0.85:1:0.05 (n = 5), Ca~ + = 0.14 mM, Pop=, ffi 0.57 + 0.11 (n ffi 5). In three patches exchange of the bath with a Ca 2+ free solution inactivated the channel. The channel reactivated following replacement of the high Ca 2+ solution (Fig. 4). In these experiments pipette Ca 2+ concentration (1.5 mM) was not altered. The exact threshold for Ca 2+ dependent activation was not determined, Pope. was not influenced by changes in membrane potential with cytosolic Ca z+= 1.5 mM (Fig, 5). Cell attached patches were acquired from five cells with NaCl (140 raM) in the bath and pipette (Fig. 6). Single channel conductance in the cell attached configuration was 12 pS (estimated from the slope of the I-V
A closed open
o
°
•
Control
closed open Amiloride 10~6 M 2pA ! 50 mSec.
1.o[- B
curve). In four experiments) amiloride (10 -6 M) was added to the pipette solution) pipette and bath solutions were otherwise identical for control and treated patches. Extracellular amiloride significantlyreduced Pope, relative to control (Fig. 7); Pamilo~de did not influence single channel conductance (g); gamilo,ide= 18 ± 3.0 pS (n -- 4), gco..ol= 19 ± 0.9 pS (n -- 5). Intracellular amiloride (I0 -4 M ) had no effect on Po~. or single channel conductance (n = 2). A n ICso for amiloride was not determined. W e did not perform studies to
°~
o
04
0_
02 0
4, . 0
0
4
.
~
Control Amiloride n.~5 n=4
Fig. 7. (A) Single channel current from control and amiioride (10 -6 M) treated inside.out patches ( - 6 0 mV). Downward deflections indicate inward current, closed and open states are indicated. All patches with amiioride demonstrated infrequent channel openings. In control patches the bath contained (raM) bath; NaCi 140, CaCI 2 !.5, glucose 10, Hepes 10, EGTA 1; pipette NaCI 140, CaCI2 1.5, glucose 10, Hepes 10, EGTA 1). Membrane potential was -60 mV in both control and treated patches. (B) The probability of channel opening Popcn is reduced by amiloride (10 -0 M) in the extracellular solution. Po~n control = 0.57+0.11, n = 5, Pope, amiloride = 0.09+ 0.07, n = 4, * indicates p < 0.01 using Student's unpaired t-test).
20 pA 1.5
determine if amiloride could block the cation channel in Na-free solutions.
1.0 0.5
Discussion |
I
I
I
I
/
-100 -80 -60 -40 -20
6
I
J
,4,, -71o
I
40
60
I
B0 100
mV
-1.5 -2,0 Fig. 6. t-V ,elation of channels in the cell attached patch configuration. The solid squares represent the combined data from five different patches with NaCI (140 raM) in the bath and pipette solution. Applied pipette potential is indicated on the x-axis. The linear regression line has a slope of 12 pS, The applied pipette reversal potential estimated from the x-intercept = + 34 inV.
Our results demonstrate a 23 pS cation selective channel on the surface of fetal type II alveolar epithelium. The channel is almost equally permeable to Na + and K + but highly selective for cations relative to chloride anions. The channel resided primarily in the open state in a high Ca~ + bath ( > 0.1 raM) but was inactive in calcium free solution ( < 10 -9 M). Channel activity is not influenced by membrane potential in mM calcium concentrations. Extracellular amiloride in micromolar concentrations reduced Pope. but had no influence on single channel conductance. Cell attached membrane patches demonstrated channel activity at holding potentials near the resting membrane potential
25 of the cell [10] suggesting that the channel is active in the living cell. This is the first single ion channel to be characterized in alveolar epithelial cells. Its amiloride sensitivity suggests it might play a role in sodium transport across the alveolar epithelium. Stable excised inside-out patches were extremely difficult to acquire from primary cultures of these cells. The reason for this is unknown, but may relate to the properties of the cell's apical membrane. The surface of adult type II alveolar epithelium is coated with a glycocalyx (3-4). 10 -s M thick called alveolin [19]. Fetal type II cells also secrete high-molecular-weight glycoproteins which may interfere with the interaction of the glass electrode with the cell membrane [20,21]. The cation selective channel identified in fetal alveolar epithelium shares some of the properties of non selective cation channels described in other epithelial and non-epithelial cells. The channel fails to discriminate between Na + and K + but excludes anions [22-27]. Recordings frequently demonstrated multiple open levels. This may represent clustering of channels within the membrane patch, or multiple subconductance states [28]. The Popen of our cell's cation selective channel was insensitive to voltage changes, a feature that also characterizes a non selective cation channel in insulinoma, thyroid follicular cells and inner medullary cortical duct of the kidney [29-31]. The non selective cation channel from these cells contrasts with the non selective cation channel from pancreatic duct epithelium whose Pope, is voltage dependent [27). The lack of voltage effect on Popen of the alveolar epithelium's cation selective channel may result from the high concentration of C a 2 + in the bath as a previous report has shown the influence of voltage is decreased by an increase in cytosolic Ca 2+ [30]. Amiloride (10 -6 M) within the pipette and hence applied to the extracellular side of the membrane reduced the probability of channel opening. Amiloride (10 - 4 M ) applied to the intracellular membranes did not infuence channel activity. This 'sidedness' has been described in other amiloride sensitive Na + transporting epithelia [32] and suggests that the amiloride binding site is on the external side of the cell membrane. Light has described a very similar 23 pS non selective cation channel in the renal inner medullary cortical duct (IMCD) epithelium which is calcium activated, voltage insensitive and blocked by extracellular amiloride [29,33,34]. Similar amiloride blockable non selective cation channels have been identified in human sweat duct epithelium [35] and in endothelial cells from brain microvessels [36]. Similar to the alveolar epithelium [7,9] both the IMCD and human sweat duct are Na + and water reabsorbing epithelium. Channel activity was evident in alveolar epithelial cell attached patches. The applied pipette potential at which the current reversed polarity was estimated to be
+ 34 mV. Using intracellular microelectrodes we have previously shown that the resting membrane potential of fetal alveolar epithelium is approximately - 3 0 mV [I0]. The absolute reversal potential was therefore close to 0 mV, which would be predicted for a non selective channel. Gray and Argent [27] noted that the non selective cation channel from pancreatic cells was only rarely evident on cell attached patches but that there was a 9-fold increase in cell attached activity after exposure to an agonist. We did not formally study the influence of membrane excision on channel activity, however, others have observed an increase in conductance following excision and Gogelein et al. [37] suggested that intracellular factors may reduce channel conductance. We speculate that the cation selective channel present on the surface of fetal alveolar epithelium may function as an amiloride sensitive pathway for electrogenie Na + transport. Under physiological conditions opening of the NSC would primarily result in Na + influx because of the relative electrochemical gradients for Na + influx and K + efflux. Intracellular Na + could then be actively transported across the basement membrane by a N a / K pump as described by Ussing's model of Na + transport [38]. The net flux of ions from the alveolus to the interstitium would generate an osmotic gradient for absorption of alveolar fluid. Blockade of the cation selective channel by amiloride may explain amiloride's reduction of fetal alveolar epithelial short circuit current and impaired clearance of fetal lung water at birth [4,7]. Acknowledgements
The authors wish to thank B. Rafii for his assistance with the cell culture, Drs. E. Cutz and S. Eich for electronmicroscopy, Dr. A. Klip for measurements of calcium concentration, and Majorie Samuel for careful preparation of the manuscript. Dr. Orser is a Fellow of the Parker B. Francis Fellowship Program. Dr. Fedorko is a Scholar of the Medical Research Council of Canada and Dr. O'Brodovich is a Career Investigator of the Heart and Stroke Foundation of Ontario. The research was funded by a grant from the Canadian Cystic Fibrosis Foundation and Medical Research Council of Canada (PG 42). The National Sanitorium Association provided financial assistance for the purchase of the patch clamp equipment. References
10lver, R.E. and Strang, L.B. (1974)J. Physiol.241, 327-357. 2 Bland, R.D., Hansen, T.N., Haberkern, C.M., Bressack, M.A., Hazinski, T.A., Raj, J.U, and Goldberg, R.B. (1982) J. Appl. Physiol.53(4),992-1004. 3 Brown,M.J., Olver R.A., Ramsden,C.A., Strang, L.B. and Waiters, D.V. (1983)J. Physiol.344, 137-152.
26 40'Brodovich, H., Hannam, V., Seear, M. and Mullen, J.B,M. (1990) J. Appl. Physiol. 68(4), 1758-1762. 5 Brown, M.J., Olver, R.A., Ramsden, C.A., Strang, L.B. and Waiters, D.V. (1983) J. Physiol. 344, 137-152. 6 Giver, R.E., Ramsden, C.A., Strang, L.B. and Waiters, D.V. (1986) J. Physiol. 376, 321-340. 7 O'Brodovich, H., Hannam, V. and Rafii, B. (1991) Am. J. Respir. Cell Mol. Biol., in press. 8 Shaw, A.M., Steele, L.W., Butcher, P.A., Ward, M.R. and Olver, R.E. (1990) Biochim. Biophys. Acta 1028, 9-13. 90'Brodovich, H , Rafii, B. and Post, M. (1990) Am. J. Physiol. 258, L201-L206. 10 Orser, B.A., Fedorko, L., Post, M. and O'Brodovich, H. (1991) Biol. Neonate 59(2), 61-68, 11 Post, M, and Smith, B.T. (1988) Am. Rev. Respir. Dis. 137, 525-530. 12 Neher, E. (1982) in Techniques in the Life Sciences - Techniques in Cellular Physiology (Baker, P.F., ed.), pp. 1-16, Elsevier, London. 13 Fabiato, A. and Fabiato, F. (1979) J. Physiol. (Paris) 75, 463-505. 14 Klip, A., Mills, G.B., Britt, B.A. and Elliott, M.E. (1990) Am. J. Physiol. 258, C495-C503. 15 KJip, A. and Ramlal, T. (1987)J. Biol. Chem. 262(19), 9141-9146. 16 Mareyama, Y. and Petersen, O.H. (1982) Nature 299, 159-161. 17 Williams, J.A, Korc, M. and Dormer, R.L. (1978) Am. J. PhysioL 235(5), E517-E524. 18 Peikin, S.R,, Rottman, A.J., Batzri, S. and Gardner, J.D. (1978) Am. J. Physiol. 235(6), E743-E749. 19 Kuhn, f'. (1968) Am. J. Pathol. 53, 809-833. 20 Skinner, S.J.M., Post, M, Torday, J.S., Stiles, A.D. and Smith, B.T. (1987) Exp. Lung Res. 12, 253-264. 21 Skinner, S.J.M., Ashby, C.J. and Liggins, G.C. (1989) Exp. Lung Res. 15, 269-283.
22 Partridge, L.D. and Swandulla, D. (1988)Trends Neurosci. 11(2), 69-72. 23 Sakai. H., Okada, Y., Morii, M. and Takeguchi, N. (1989) Pflugers Arch. 414, 185-192. 24 Galietta, L.J.V., Mastrocola, T. and Nobile, M. (1989) FEBS Lett. 253, 43-46. 25 Weber, A. and Siemen, D. (1989) Pflugers Arch. 414, 564-570. 26 Gogelein, H. and Pfannmuller, B. (1989) Pflugers Arch. 413, 287-298, 27 Gray, M.A. and Argent, B.E. (1990) Biochim. Biophys. Acta 1029, 33-42. 28 Cook, D.L, Poronnik, P. and Young, J.A. (1990) J. Membr. Biol. 114, 37-52. 29 Light, D.B., McMann, F.V., Keller, T.M. and Stanton, B.A, (1988) Am. J. Physiol. 255, F278-F286. 30 Sturgess, N.C, Hales, C.N. and Ashford, M.L.J. (1987) Pflugers Arch. 409, 607-615. 31 Maruyama, Y., Moore, D. and Petersen, O.H. (1985) Biochim. Biophys. Acta 821, 229-232. 32 Palmer, L.G. and Frindt, G. (1986) Prec. Natl. Acad. Sci. USA 83, 2767-2770. 33 Light, D.B., Schwiebert, E.M., Karlson, K,H. and Stanton, B.A. (1989) Science 243, 383-385. 34 Light, D.B., Ausiello, D.A. and Stanton, B.A. (1989) J. Clin. Invest. 84, 352-356. 35 Joris, L., Krouse, M.E., Hagiwara, G., Bell, C.L and Wine, JJ. (1989) Pflugers Arch. 414, 369-372. 36 Vigne, P., Champigny, G., Marsault, R., Barbry, P., Frelin, C. and Lazdunski, M. (1989) J. Biol. Chem. 264, 7663-7688. 37 Gogelein, H. and Greger, R. (1986) Pflugers Arch. 407 (Supplement 2), $142-$148. 38 Ussing, H.H. and gerahn, K. (1951) Acta Physiol. Scand. 30, 110-127.
19
BBAMCR 12980
Cation selective channel in fetal alveolar type II epithelium Beverley A. Orser 1,2, Maria Bertlik l, Ludwik Fedorko 3 and Hugh O'Brodovieh 1 i Respiratory Research Division, Research Institute, Hospital for Sick Children, Toronto (Canada), 2 Department of Anesthesia, Toronto Western Hospital, Toronto (Canada) and 3 lift. Sinai Hospital Research Institute, Toronto (Canada) (Received 28 February 1991)
Key words: Non-selective cation channel; Amiloride; Alveolar epithelium; Ion channel; Sodium channel
A cation selective channel was identified in the apical membrane of fetal rat (Wistar) alveolar type lI epithelium using the patch clamp technique. The single channel conductance was 23 ± 1.2 pS (n -- 16) with symmetrical NaC! (140 raM) solution in the bath and pipette. The channel was highly permeable to Na + and K + (Pwa/PK = 0.9) hut essentially impermeant to chloride and gluconate. Membrane potential did not influence open state probability when measured in a high Ca 2÷ (1.5 mM) bath. The channel reversibly inactivated when the bath was exchanged with a Ca2+-free ( < 10-9 M) solution. The Na + channel blocker amiloride (10-6 M) applied to the extraceilular side of the membrane reduced Popen relative to control patches; P~ont~ot= 0.57 __ 0.11 (n = 5), Pamiloride = 0.09 ~ 0.07 (n --- 4, p < 0.01), however, amiloride did not significantly influence channel conductance (g): gco~t~ot 19 _ 0.9 pS (n = S), 18 _+3.0 pS (n = 4). More than one current level was observed in 42% (16/38) of active patches; multiple current levels (ranging from 2 to 6) were of equal amplitude suggesting the presence of multiple channels or subconductance states. Channel activity was also evident in cell attached patches. Since monolayers of these cells absorb Na + via an amiloride sensitive transport mechanism we speculate that this amiloride sensitive cation selective channel is a potential apical pathway for electrogenic Na + transport in the alveolar region of the lung.
Introduction Successful transition from intrauterine to extrauterine life depends on the rapid adaptation of the fetal lung. Fluid, actively secreted into the lungs airspaces and airways [1] during uterine life, must be cleared at birth to allow the lung to function as the organ of gas exchange. Although part of this fluid is removed during labour and delivery [2,3], a significant proportion of the fluid is cleared postnatally by the pulmonary epithelium via amiloride sensitive mechanisms [4]. In vivo studies support the hypothesis that active transport of Na + by pulmonary epithelium generates an osmotic gradient which drives fetal lung fluid absorption at birth. Epinephrine stimulates fetal lung fluid absorption in utero [5], a process which can be blocked by the sodium transport inhibitor amiloride [6]. Also, our laboratory has recently showed that instillation of
Correspondence: H. O'Brodovich, Respiratory Research, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario MSG 1X8, Canada.
amiloride [4] or benzamil [7] into the tracheal fluid of fetal guinea pigs shortly before birth delays the clearance of lung water causing respiratory distress in the newborn. It is likely that the perinatal reabsorption of fetal lung liquid results, at least in part, from solute coupled active transport across the alveolar epithelium. For example, Shaw et al. showed amiloride blockable Na uptake into membrane vesicles made from fetal sheep alveolar type II epithelium [8]. Our laboratory has demonstrated that monolayers of fetal alveolar epithelium maintain a high transcellular resistance and generate a Na + dependent short-circuit current which can be inhibited by the Na + channel blockers amiloride and benzamil [7,9]. Fetal alveolar epithelium grown in organotypic culture have a significant transcellular voltage gradient ( - 12 mV, apically negative) that can be reduced by the metabolic inhibitor ouabain [10]. The above indirect evidence suggests the presence of sodium conductive ion channels in fetal alveolar epithelium. We used the patch clamp technique to identify and characterize ion channels present on the apical membrane of fetal rat (Wistar) alveolar epithelial cells which could participate in Na + transport.
20
Materials and Methods
Cell culture Primary cultures of fetal alveolar type II cells were isolated as previously described [9,11]. Briefly, cells were harvested from Wistar fetal rats (Charles River, St. Constant, Canada)delivered by Cesarean section at 20 days gestation (term 22 days), Lungs were minced and cells dissociated by enzymatic digestion with 0.125% trypsin and 0.002% DNAase in Hanks' balanced salt solution. The cell suspension was filtered through a 100 mesh Nitex Filter (Tetco Inc., Elmsford, NY, U.S.A.) and isolated cells added to minimal essential media (MEM, Gibco, Burlington, Canada) and 0.1% collagenase (Gibco) for 15 rain at 37°C. Fibroblasts were removed by a differential adherence technique. Nonadherent alveolar epithelial cells were collected, centrifuged and seeded onto collagen coated coverslips (approx. 2. l0 s cells/cm 2) in MEM plus penicillin/streptomycin 100 U / m l (Gibco). Cells were cultured in a humidified 95% air, 5% CO2 containing incubator and were studied 24 to 96 h after harvest. Collagen 0.1% used to coat the coverslips was prepared from a concentrate of rat-tail collagen in acetic acid diluted in MEM 1 : 10 buffered with NaHCO 3 to a pH 7.0. Cell viability (> 90%) and purity (> 95%) associated with this isolation procedure has been previously reported [9]. We had difficulty acquiring stable, high resistance (> 109 ,O) seals from confluent monolayers, therefore subconfluent clusters of alveolar epithelium were studied. In an effort to increase the numbers of high resistance seals, approximately one half of the cultures were 'cleaned' with coUagenase 0.2% as previously described to improve the rate of seal formation [12]. This had no effect on the number or quality of seals acquired.
latch clamp technique Patch pipettes were fabricated from boroscilicate (1.0 mm ID, 1.5 mm OD, Garner Glass, Claremont, CA, U.S.A.) or aluminoscilicate glass (0.75 mm ID, 1.2 mm OD, Sutter Instruments, Novata, CA, U.S.A.) on a two-stage vertical puller (Narashigi, Tokyo, Japan). Pipettes were Sylgard® coated (Dow Coming, Midland, Mi, U.S.A.) and firepolished yielding a final tip resistance of (5-8). 106 fJ when filled with NaC! (140 raM) solution. Experiments were performed at room temperature. Membrane current were recorded using a 50.10 9 ~'~ resistor in a current to voltage converter (Ampatch l-B, Axon Instruments, Foster City, CA, U.S.A.) Current signals were filtered at 1 kHz through a 4-pole Bessel low-pass filter, and stored on a FM tape (Sony) after digitization (A.R. Vetter Co., Rebersbeg, PA, U.S.A.). Reported holding potentials indicate the 'intracellular' membrane potential referenced to ground (Vbath- [/'pipette) . Outward current or positive
charge moving from the bath to pipette (intracellular to extracellular side of the patch) is depicted by an upward deflection in all raw current records. In most experiments, the ground electrode was a Ag-AgCl wire. When the bath was exchanged with solutions containing varying concentrations of Cl-, an agar bridge (NaCl 140 mM) was used to electrically couple the ground wire to the bath.
Solutions Amiloride was purchased from Sigma (St. Louis, MO, U.S.A.). All solutions were mixed with ultrapure water and then passed through a 0.22 p.m filter (Millipore Products, Bedford, MA, U.S.A.) prior to use. In most studies the initial bath and pipette solution conrained (raM): NaCI (140), CaCI 2 (1.5), glucose (10), Hepes (10) (pH 7.4). The contents of the bathing and pipette solutions were varied to evaluate the characteristics of the channel (see Results). The concentration of free Ca 2+ in solution was estimated using a computer program for calculating the composition of solutions containing Ca 2+ and EGTA [13]. The concentration of Ca 2+ in 'Ca2+-free solution' was measured to be < 10 -9 using the fluorescent dyes; quin2 and indo-1 [14,15].
Analyses Data were analyzed using the PClamp program version 5.0 or 5.5 (Axon Instruments, Foster City, CA, U.S.A.). Single channel amplitudes were determined using all points histograms created from at least 20 s of recorded data. Histograms were curve fitted using the least-squared method for a Gaussian distribution and current levels are reported as the amplitude difference between close and open states. The number of channels in the patch was determined by counting the number of open current levels above baseline in the raw current records when current (i) was between (n - 1/2)i and (n + 1/2)/. Single channel conductance was measured as the slope of the current to voltage (I-V) relationship and reported as mean + S.E. of the mean. Many patches contained multiple open levels of equal amplitude suggesting subconductance states or multiple channels in the patch. We made the assumption that each level above baseline represented a single channel which opened and closed independently. The probability of the channel being in the open state (Pope,) was estimated by the relative area under the all points amplitude histogram. When histograms contained multiple open amplitude peaks Pope, was estimated using the following formula: I1
1 ~Ai'i eopen =
i = n1 n
EA i=o
i
.
21 where A = area u n d e r Gaussian C u r " n = total n u m b e r o f channel,: and i -- n u m b e r o f o p e n channels. F o r example, Fig. 1B illustrates an all points histogram from a patch containing three o p e n levels above baseline (c) level: O i , 0 2 , 0 3 , r e p r e s e n t the current amplitude with o n e , two, and three c h a n n e l s open, respec-
tively. T h e probability o f channel o p e n i n g (Pope,) is calculated as: ( a r e a 0 3 + area 0 2 • 2 + a r e a O I • 1) divided by the total a r e a u n d e r the o p e n a n d closed curves times the total n u m b e r o f c h a n n e l s in the patch. This m e t h o d assumes binomial distribution and channel independence. T h e distribution o f p e a k areas in the all points histograms was similar to that predicted f r o m the binomial distribution for n c h a n n e l s with the
(my) + 60 C..~.
÷ 40
-45
I~'v~l .
.
.
.
....
r
.
-
~,,,-o,r,l~,, .
.
.
.
.
.
.
.
"t .
. .
.. r m "
.
-
-
¢-~ -
.
60
rl
o _
. . . . . . . . . . . . . . . .
. . . . . . .
,pA I 5 0 mSe¢.
IB
01
E
o2
z 102400 Events o3 --,p-.
1300 N/dlv Amphtude (pA)
0
125
2 46
3 67
Fig. 1. (A) Raw current recordings from an inside-out patch of fetal alveolar epithelial cell membrane. The pipette and bath solution contain mM: NaCI 140, CaCI 2 1.5, glucose 10, Hepes 10, EGTA 1. 'lntracellular' potential (mV) is referenced to the ground and is indicated to the left of the current tracings. An upward deflection represents outward current, (positive charge) moving from the membrane's cytoplasmic face into the pipette. The dashed line denotes the open state (O), the solid line the closed state (C). (B) All points amplitude histogram of a current record containing three channels (holding potential = - 7 0 mV). The dosed state (C) and three open states (O) are seen. '!.he number of events is indicated on the y-axis, amplitude on the x-axis. This histogram represents 20 s of recorded data, binned in 0.05 pA ircrements with 1300 events per division on the y-axis. The single channel current between individual levels 1, 2 and 3 was (pA) 1.25, 1.21 and 1.21, respectively.
22 2.C
open probability of the channels measured. To test for statistical significance between Popen in control and amiloride treated patches an unpaired Student's t-test was used. Results are reported as mean +_S.E. and probability (p) < 0.05 was considered statistically significant.
pA 1, =_
10 05
...'~
T~
~
."
Microscopy We wished to determine if the upward facing cell surface of FAE grown in subconfluent cultures represented the apical membrane of polarized epithelium. Light and electron micrographs were studied in order to identify the morphologic characteristics of polarized cells grown 'on collagen-coated Millipore HA filters (Millipore, Bedford, CT, U.S.A.). Cells on filters were fixed with 1% glutaraldehyde then washed in phosphate-buffered saline. Cells were stained with 1% osmium tetroxide and 3% potassium ferrocyanide and then were dehydrated in ethanol. Filters were cut into short strips and embedded in Epon/Araldite (EMS, Fort Washington, PA, U.S.A.). Light microscopy was performed on 60 /tm sections stained with toluidine blue in 1% sodium borate using a Reichert Polyvar microscope, and 100 × oil objective. Transmission electron microscopy (Phillips 400) was performed on 60 nm ultrathin sections exposed to 3% uranylacetate followed by lead citrate in ethanol. Results
Light micrographs of subconfluent cultures of fetal alveolar epithelium showed basally located nuclei in 86/130 (66%) of cells. Electron-micrographs confirmed the presence of lameUar bodies, tight junctions and microvilli, which are characteristics of polarized cells. Similar characteristics have been described in subeonfluent cultures of pancreatic acinar cells [16-18]. We assume therefore that our patches were acquired from the cell's apical membrane. Stable excised inside-out patches were difficult to acquire from both confluent and subconfluent cultures of fetal alveolar epithelium. Although we did not record the exact rate of successful high resistance (> 109 ,O) seal formation, it was estimated to be less than 10%. Patches also rarely tolerated multiple voltage steps or exchanges of the bath solution. The channel most frequently observed in inside-out patches excised from the cell's apical surface in symmetrical NaCI (140 mM) solution was a cation selective channel (Fig. IA). In 42% (16/38) of all active patches more than one (range 2-6) open level was observed suggesting the presence of more than one channel in the patch or multiple subconductive states (Fig. 1B). The single channel conductance was 23 + 1.2 pS (n = 16) with NaCI (140 mM) in the bath and pipette solution. No rectification of the current to voltage
k -80
I -60
I -40
I -20
.."
..'
I 2O
I
I
40
I
~0
60
..~..~.n " . O.5 mv
-2,0
Fig, 2. The current to voltage (I-V) relation of inside-out patches of FAE. The boxes (13) indicate current measured in symmetrical NaCI (140 raM) solution, slope conductance = 23+ 1.2 pS ( , = 16). Each point represents the mean single channel current; when three or more patches were recorded at the same holding potential, currents were averaged and S.E. is indicated by the vertical bars. The triangles ( • ) indicate current recorded in five patches following a 2:1 dilution of the bath with isotonic sucrose (bath mM: NaCI 46.6 sucrose 187 CaCI 2 1.5, Hepes 10, glucose 10; pipette: NaCi 140, CaCI 2 !.5, Hepes 10, glucose 10). The zero CUlTci~tpotential (ERe v) of +27.5 mV after sucrose dilution indicate a cation selective channel.
(l-V) curve was observed (Fig. 2). Anion and cation selectivity of the channel was determined by sucrose dilution and ion substitution experiments. Diluting the bath solution 2 to 1 with isotonic sucrose caused the -2.0 pA
-1.5 -1.0 ,& -0.5
I
80
I 60
I
40
I - 20
I 20 /
I
- 40
I -60
I
-80
s
e"
0.5
mV
1.0 1.5 2£ Fig. 3. The K + permeability of the cation selective channel. I.V relation with high 'intracellular' K+; (raM) bath: KCI 140, CaCI 2 1.5, glucose 10, Hepes 10; pipette: NaC1 140, CaC! 2 1.5, glucose 10, Hepes 10. The channel conductance calculated from the slope of the line of linear regression (. . . . . . ) was 20+ 1.8 pS and ERev =--3-t" 0.6 mY, n = 4.
23 closed
open
-I A
B
A
B
closed
open
,o,L__ 2 0 mSec. Fig. 4, Inactivation of channel activity in low 'intraccllular' Caz+. Single channel recording of the same inside-out patch that was repetitively switched from high (1,5 raM) to low ( < 10 -9 M) Ca2+ bathing solutions, Pipette Ca2÷ concentration was not changed during the experiment. Membrane potential = - 60 mV. This is a tracing from a single patch, the break lines are time periods when bathing solution was being changed. The pipette contained (mM); NaCI 140, CaCI 2 1.5, glucose 10, Hepes 10. The bathing solutions were (raM); solution A: NaCI 140, CaCI 2 1.5, glucose 10, Hepes 10, or solution B: NaCI 140, CaCI 2 0, glucose 10, Hepes 10, EGTA 2 (free Ca 2+ < 10 -9 M). Note that replacement of the bath with a high Ca 2+ solution reversed channel inactivation.
1.0(;
3 A
4
i
4
6
0.8C
3
• 2
c
0.6C
e
'
•
I
ft. 0.4C
0.2C
I - 80
I - 60
I - 40
I - 20
Membrane
I 0
I 20
I 40
I 60
I 8(3
potent~ol
Fig. 5. Membrane potential does not influence channel open probability (/'oven) of inside out patches in symmetrical solutions (raM): NaCI 140, CaCI 2 1.5, glucose 10, Hopes 10 (n = 6). The circle and bar represent the mean and S.E., respectively, with the number of patches recorded at each holding potential indicated above the mean value.
I-V curve to inwardly rectify and shifted the ERev to + 27.5, n = 5 (Fig. 2) indicating the channel was permeant to cations. ERev for a completely cation selective channel calculated from the Nernst equation is + 27.7 mV at a temperature of 20 ° C. The CI permeability relative to Na + permeability was negligible. In three experiments, substitution of CI- anion in the pipette with gluconate (mM: pipette, sodium gluconate 140, CaCI 2 1.5, glucose 10, Hopes 10; bath NaCI 140, CaCI 2 1.5, glucose 10, Hepes 10) shifted ERev to +5 + 1.0 mV, slope conductance = 27 + 4.0 pS. This slight shift in the reversal potential in the presence of a large chloride gradient indicates that the ion channel is essentially impermeant to anions. Cation selectivity was investigated by exchanging the bath with a high K + (140 raM) solution (Fig. 3). The single channel conductance was 20 + 1.8 pS (n ffi 4) and the reversal potential (ERev) shifted from 0 mV to - 3 . 0 + 0 . 6 inV. The estimated Na + permeability to K + permeability ratio (PNa/PK)calculated using the
24 modified Goldman-Hogkin-Katz voltage equation was 0.9. This indicates that the channel fails to discriminate between Na + and K +. In a high Ca 2+ bath the channel resided primarily in the open state (holding potential - 60 mV); Ca~ + = 1.5 mM, Pope, = 0.85:1:0.05 (n = 5), Ca~ + = 0.14 mM, Pop=, ffi 0.57 + 0.11 (n ffi 5). In three patches exchange of the bath with a Ca 2+ free solution inactivated the channel. The channel reactivated following replacement of the high Ca 2+ solution (Fig. 4). In these experiments pipette Ca 2+ concentration (1.5 mM) was not altered. The exact threshold for Ca 2+ dependent activation was not determined, Pope. was not influenced by changes in membrane potential with cytosolic Ca z+= 1.5 mM (Fig, 5). Cell attached patches were acquired from five cells with NaCl (140 raM) in the bath and pipette (Fig. 6). Single channel conductance in the cell attached configuration was 12 pS (estimated from the slope of the I-V
A closed open
o
°
•
Control
closed open Amiloride 10~6 M 2pA ! 50 mSec.
1.o[- B
curve). In four experiments) amiloride (10 -6 M) was added to the pipette solution) pipette and bath solutions were otherwise identical for control and treated patches. Extracellular amiloride significantlyreduced Pope, relative to control (Fig. 7); Pamilo~de did not influence single channel conductance (g); gamilo,ide= 18 ± 3.0 pS (n -- 4), gco..ol= 19 ± 0.9 pS (n -- 5). Intracellular amiloride (I0 -4 M ) had no effect on Po~. or single channel conductance (n = 2). A n ICso for amiloride was not determined. W e did not perform studies to
°~
o
04
0_
02 0
4, . 0
0
4
.
~
Control Amiloride n.~5 n=4
Fig. 7. (A) Single channel current from control and amiioride (10 -6 M) treated inside.out patches ( - 6 0 mV). Downward deflections indicate inward current, closed and open states are indicated. All patches with amiioride demonstrated infrequent channel openings. In control patches the bath contained (raM) bath; NaCi 140, CaCI 2 !.5, glucose 10, Hepes 10, EGTA 1; pipette NaCI 140, CaCI2 1.5, glucose 10, Hepes 10, EGTA 1). Membrane potential was -60 mV in both control and treated patches. (B) The probability of channel opening Popcn is reduced by amiloride (10 -0 M) in the extracellular solution. Po~n control = 0.57+0.11, n = 5, Pope, amiloride = 0.09+ 0.07, n = 4, * indicates p < 0.01 using Student's unpaired t-test).
20 pA 1.5
determine if amiloride could block the cation channel in Na-free solutions.
1.0 0.5
Discussion |
I
I
I
I
/
-100 -80 -60 -40 -20
6
I
J
,4,, -71o
I
40
60
I
B0 100
mV
-1.5 -2,0 Fig. 6. t-V ,elation of channels in the cell attached patch configuration. The solid squares represent the combined data from five different patches with NaCI (140 raM) in the bath and pipette solution. Applied pipette potential is indicated on the x-axis. The linear regression line has a slope of 12 pS, The applied pipette reversal potential estimated from the x-intercept = + 34 inV.
Our results demonstrate a 23 pS cation selective channel on the surface of fetal type II alveolar epithelium. The channel is almost equally permeable to Na + and K + but highly selective for cations relative to chloride anions. The channel resided primarily in the open state in a high Ca~ + bath ( > 0.1 raM) but was inactive in calcium free solution ( < 10 -9 M). Channel activity is not influenced by membrane potential in mM calcium concentrations. Extracellular amiloride in micromolar concentrations reduced Pope. but had no influence on single channel conductance. Cell attached membrane patches demonstrated channel activity at holding potentials near the resting membrane potential
25 of the cell [10] suggesting that the channel is active in the living cell. This is the first single ion channel to be characterized in alveolar epithelial cells. Its amiloride sensitivity suggests it might play a role in sodium transport across the alveolar epithelium. Stable excised inside-out patches were extremely difficult to acquire from primary cultures of these cells. The reason for this is unknown, but may relate to the properties of the cell's apical membrane. The surface of adult type II alveolar epithelium is coated with a glycocalyx (3-4). 10 -s M thick called alveolin [19]. Fetal type II cells also secrete high-molecular-weight glycoproteins which may interfere with the interaction of the glass electrode with the cell membrane [20,21]. The cation selective channel identified in fetal alveolar epithelium shares some of the properties of non selective cation channels described in other epithelial and non-epithelial cells. The channel fails to discriminate between Na + and K + but excludes anions [22-27]. Recordings frequently demonstrated multiple open levels. This may represent clustering of channels within the membrane patch, or multiple subconductance states [28]. The Popen of our cell's cation selective channel was insensitive to voltage changes, a feature that also characterizes a non selective cation channel in insulinoma, thyroid follicular cells and inner medullary cortical duct of the kidney [29-31]. The non selective cation channel from these cells contrasts with the non selective cation channel from pancreatic duct epithelium whose Pope, is voltage dependent [27). The lack of voltage effect on Popen of the alveolar epithelium's cation selective channel may result from the high concentration of C a 2 + in the bath as a previous report has shown the influence of voltage is decreased by an increase in cytosolic Ca 2+ [30]. Amiloride (10 -6 M) within the pipette and hence applied to the extracellular side of the membrane reduced the probability of channel opening. Amiloride (10 - 4 M ) applied to the intracellular membranes did not infuence channel activity. This 'sidedness' has been described in other amiloride sensitive Na + transporting epithelia [32] and suggests that the amiloride binding site is on the external side of the cell membrane. Light has described a very similar 23 pS non selective cation channel in the renal inner medullary cortical duct (IMCD) epithelium which is calcium activated, voltage insensitive and blocked by extracellular amiloride [29,33,34]. Similar amiloride blockable non selective cation channels have been identified in human sweat duct epithelium [35] and in endothelial cells from brain microvessels [36]. Similar to the alveolar epithelium [7,9] both the IMCD and human sweat duct are Na + and water reabsorbing epithelium. Channel activity was evident in alveolar epithelial cell attached patches. The applied pipette potential at which the current reversed polarity was estimated to be
+ 34 mV. Using intracellular microelectrodes we have previously shown that the resting membrane potential of fetal alveolar epithelium is approximately - 3 0 mV [I0]. The absolute reversal potential was therefore close to 0 mV, which would be predicted for a non selective channel. Gray and Argent [27] noted that the non selective cation channel from pancreatic cells was only rarely evident on cell attached patches but that there was a 9-fold increase in cell attached activity after exposure to an agonist. We did not formally study the influence of membrane excision on channel activity, however, others have observed an increase in conductance following excision and Gogelein et al. [37] suggested that intracellular factors may reduce channel conductance. We speculate that the cation selective channel present on the surface of fetal alveolar epithelium may function as an amiloride sensitive pathway for electrogenie Na + transport. Under physiological conditions opening of the NSC would primarily result in Na + influx because of the relative electrochemical gradients for Na + influx and K + efflux. Intracellular Na + could then be actively transported across the basement membrane by a N a / K pump as described by Ussing's model of Na + transport [38]. The net flux of ions from the alveolus to the interstitium would generate an osmotic gradient for absorption of alveolar fluid. Blockade of the cation selective channel by amiloride may explain amiloride's reduction of fetal alveolar epithelial short circuit current and impaired clearance of fetal lung water at birth [4,7]. Acknowledgements
The authors wish to thank B. Rafii for his assistance with the cell culture, Drs. E. Cutz and S. Eich for electronmicroscopy, Dr. A. Klip for measurements of calcium concentration, and Majorie Samuel for careful preparation of the manuscript. Dr. Orser is a Fellow of the Parker B. Francis Fellowship Program. Dr. Fedorko is a Scholar of the Medical Research Council of Canada and Dr. O'Brodovich is a Career Investigator of the Heart and Stroke Foundation of Ontario. The research was funded by a grant from the Canadian Cystic Fibrosis Foundation and Medical Research Council of Canada (PG 42). The National Sanitorium Association provided financial assistance for the purchase of the patch clamp equipment. References
10lver, R.E. and Strang, L.B. (1974)J. Physiol.241, 327-357. 2 Bland, R.D., Hansen, T.N., Haberkern, C.M., Bressack, M.A., Hazinski, T.A., Raj, J.U, and Goldberg, R.B. (1982) J. Appl. Physiol.53(4),992-1004. 3 Brown,M.J., Olver R.A., Ramsden,C.A., Strang, L.B. and Waiters, D.V. (1983)J. Physiol.344, 137-152.
26 40'Brodovich, H., Hannam, V., Seear, M. and Mullen, J.B,M. (1990) J. Appl. Physiol. 68(4), 1758-1762. 5 Brown, M.J., Olver, R.A., Ramsden, C.A., Strang, L.B. and Waiters, D.V. (1983) J. Physiol. 344, 137-152. 6 Giver, R.E., Ramsden, C.A., Strang, L.B. and Waiters, D.V. (1986) J. Physiol. 376, 321-340. 7 O'Brodovich, H., Hannam, V. and Rafii, B. (1991) Am. J. Respir. Cell Mol. Biol., in press. 8 Shaw, A.M., Steele, L.W., Butcher, P.A., Ward, M.R. and Olver, R.E. (1990) Biochim. Biophys. Acta 1028, 9-13. 90'Brodovich, H , Rafii, B. and Post, M. (1990) Am. J. Physiol. 258, L201-L206. 10 Orser, B.A., Fedorko, L., Post, M. and O'Brodovich, H. (1991) Biol. Neonate 59(2), 61-68, 11 Post, M, and Smith, B.T. (1988) Am. Rev. Respir. Dis. 137, 525-530. 12 Neher, E. (1982) in Techniques in the Life Sciences - Techniques in Cellular Physiology (Baker, P.F., ed.), pp. 1-16, Elsevier, London. 13 Fabiato, A. and Fabiato, F. (1979) J. Physiol. (Paris) 75, 463-505. 14 Klip, A., Mills, G.B., Britt, B.A. and Elliott, M.E. (1990) Am. J. Physiol. 258, C495-C503. 15 KJip, A. and Ramlal, T. (1987)J. Biol. Chem. 262(19), 9141-9146. 16 Mareyama, Y. and Petersen, O.H. (1982) Nature 299, 159-161. 17 Williams, J.A, Korc, M. and Dormer, R.L. (1978) Am. J. PhysioL 235(5), E517-E524. 18 Peikin, S.R,, Rottman, A.J., Batzri, S. and Gardner, J.D. (1978) Am. J. Physiol. 235(6), E743-E749. 19 Kuhn, f'. (1968) Am. J. Pathol. 53, 809-833. 20 Skinner, S.J.M., Post, M, Torday, J.S., Stiles, A.D. and Smith, B.T. (1987) Exp. Lung Res. 12, 253-264. 21 Skinner, S.J.M., Ashby, C.J. and Liggins, G.C. (1989) Exp. Lung Res. 15, 269-283.
22 Partridge, L.D. and Swandulla, D. (1988)Trends Neurosci. 11(2), 69-72. 23 Sakai. H., Okada, Y., Morii, M. and Takeguchi, N. (1989) Pflugers Arch. 414, 185-192. 24 Galietta, L.J.V., Mastrocola, T. and Nobile, M. (1989) FEBS Lett. 253, 43-46. 25 Weber, A. and Siemen, D. (1989) Pflugers Arch. 414, 564-570. 26 Gogelein, H. and Pfannmuller, B. (1989) Pflugers Arch. 413, 287-298, 27 Gray, M.A. and Argent, B.E. (1990) Biochim. Biophys. Acta 1029, 33-42. 28 Cook, D.L, Poronnik, P. and Young, J.A. (1990) J. Membr. Biol. 114, 37-52. 29 Light, D.B., McMann, F.V., Keller, T.M. and Stanton, B.A, (1988) Am. J. Physiol. 255, F278-F286. 30 Sturgess, N.C, Hales, C.N. and Ashford, M.L.J. (1987) Pflugers Arch. 409, 607-615. 31 Maruyama, Y., Moore, D. and Petersen, O.H. (1985) Biochim. Biophys. Acta 821, 229-232. 32 Palmer, L.G. and Frindt, G. (1986) Prec. Natl. Acad. Sci. USA 83, 2767-2770. 33 Light, D.B., Schwiebert, E.M., Karlson, K,H. and Stanton, B.A. (1989) Science 243, 383-385. 34 Light, D.B., Ausiello, D.A. and Stanton, B.A. (1989) J. Clin. Invest. 84, 352-356. 35 Joris, L., Krouse, M.E., Hagiwara, G., Bell, C.L and Wine, JJ. (1989) Pflugers Arch. 414, 369-372. 36 Vigne, P., Champigny, G., Marsault, R., Barbry, P., Frelin, C. and Lazdunski, M. (1989) J. Biol. Chem. 264, 7663-7688. 37 Gogelein, H. and Greger, R. (1986) Pflugers Arch. 407 (Supplement 2), $142-$148. 38 Ussing, H.H. and gerahn, K. (1951) Acta Physiol. Scand. 30, 110-127.
