Proc. Natl. Acad. Sci. USAVol. 82, pp. 6167-6171, September 1985Cell Biology

Cystic fibrosis decreases the apical membrane chloridepermeability of monolayers cultured from cells oftracheal epithelium

(Cl secretion/Na absorption/Ussing chambers/intracellular microelectrodes)

J. H. WIDDICOMBE*, M. J. WELSHt, AND W. E. FINKBEINER**Cardiovascular Research Institute, and the Departments of Physiology and Pathology, University of California, San Francisco, CA 94143; and tLaboratory ofEpithelial Transport, and the Pulmonary Division, Department of Internal Medicine, University of Iowa Hospitals, Iowa City, IA 52242

Communicated by John A. Clements, May 28, 1985

ABSTRACT The tracheal mucosa from a 12-year-old girlwas digested with collagenase 4 hr after her death from cysticfibrosis. Forty million viable cells were obtained. The cells,plated at 106 per cm2 onto four Nuclepore filters coated withhuman placental collagen, formed confluent monolayers after1 day. Their ultrastructure was similar to that of normalhuman cells. They were studied in conventional Ussing cham-bers or with intracellular microelectrodes on days 5-7 afterplating. The monolayers displayed resistance of 380 ± 50fkcm2 and short-circuit current (Isc) of 1.8 ± 0.4 ItAAcm 2.This resistance is similar to that obtained for dog or normalhuman monolayers. The 4c is less than normal human (-3IAAcm 2) or dog ('10 ,uAAcm-2) cells. The cystic fibrosis cellsresembled normal Inonolayers in that serosal ouabain andmucosal amiloride inhibited Is, while mucosal ouabain orserosal amiloride had no effect. They differed from normalhuman or dog cells in that Ic was not inhibited by bumetanideand the stimulation of Ic by isoproterenol or prostaglandin E2was greatly reduced or abolished. Addition of isoproterenoldepolarized apical membrane potential (ia) and decreasedfractional resistance (fR) in normal human and dog but had noeffect on ia or fR in cystic fibrosis cells. Reduction of mucosalchloride from 120 to 5 mM by replacement with gluconateincreased fR of dog and normal human monolayers anddepolarized l,, by 22 (dog) or 30 (human) mV. In cystic fibrosismonolayers, chloride replacement hyperpolarized *la by 2 mVand had little effect on fR. These results suggest that theprimary defect in cystic fibrosis is reduced apical membranechloride conductance.

There is evidence that the underlying genetic defect in cysticfibrosis (CF) is a reduced permeability of epithelia to chloride(Cl). Quinton (1), the first to provide such evidence, treatedsweat gland ducts with ouabain and then perfused theirlumina with 50 mM NaCl or 75 mM Na2SO4. Large lumen-negative potentials were generated in normal tissues but onlysmall potentials were found in CF ducts. From these data,Quinton calculated that the ratio of sodium to chloridepermeability (PNa/PCI) was increased to 10 times that ofnormal in CF sweat ducts. Knowles et al. (2) reported similarresults for in vivo nasal epithelium. Direct evidence that theincrease in PNa/PCI in CF nasal mucosa was due to areduction in Cl permeability came from Ussing chamberstudies (3), which showed that in CF nasal mucosa theresistance was twice normal and the unidirectional Cl fluxeswere half those of the normal tissue. These results on sweatduct and nasal mucosa provided strong evidence that epithe-lial Cl permeability is reduced in CF. However, they failed to

determine whether this defect lies in the trans- or theparacellular pathway.

It is now well established that epithelial cells will grow inculture to establish confluent monolayers with tightjunctionsseparating discrete apical and basolateral cell membranes(4-8). Furthermore, the electrical properties of cultured cellsheets resemble those of the original tissue (9-12). Cells ofdog tracheal epithelium when grown in culture also retaindifferentiated transport function (13). These monolayersresemble the original tissue in their resistance ("400 fl cm2),the presence of both active chloride secretion and activesodium absorption, and the stimulation of Cl secretion by awide variety of endogenous mediators.

In this paper we have used cell culture techniques in anattempt to localize the chloride permeability defect implicat-ed in CF. We compare ion transport by monolayers culturedfrom cells ofdog, normal human, and CF tracheal mucosa. Inparticular, the properties of the transcellular pathway havebeen investigated using intracellular microelectrodes.

METHODS

Cell cultures were obtained as described in detail elsewhere(8, 13). In brief, strips of tracheal mucosa were digested withcollagenase and the dispersed cells were plated at 2.5 or 5 x105 cells per cm2 (dog) or 106 cells per cm2 (human) ontoNuclepore filters coated with human placental collagen.Confluent monolayers were studied in conventional Ussingchambers (8, 13) or with intracellular microelectrodes.

In the Ussing chamber studies, the transepithelial potentialdifference (it) was clamped at zero and the short-circuitcurrent (Isc) was monitored continuously on a pen recorder.lit was referenced to the mucosal solution. Transepithelialresistance (Rt) was calculated from the change in current inresponse to square-wave voltage pulses of 0.2-1 mV (dura-tion, 5 sec; period, 20 sec). For the microelectrode studies,monolayers were kept at their spontaneous lit. Rt wascalculated from the change in ait produced by bipolar currentpulses (duration, 1 sec; period, 5-10 sec; amplitude, ±40 to±160 utA cm-2). The electrical potential difference across theapical membrane (ha) was measured with intracellularmicroelectrodes as described (14). The fractional resistanceof the apical membrane (fR) was determined from fR =Alla/AlIt = Ra/(Ra + Rb), where Alia and Alit refer to thechanges in mia and 4it produced by the constant current pulsesand Ra and Rb refer to apical and basolateral membraneresistance, respectively.

Abbreviations: CF, cystic fibrosis; I,, short-circuit current; P,permeability; R., Rb, and R., apical membrane, basolateral mem-brane, and transepithelial resistances; qi', 'f/, and f/t, the correspond-ing potential differences; fR, fractional resistance (= R,/(R, + Rb);PGE2, prostaglandin E2-

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Electron microscopy techniques have been described (8).All drugs were added as aliquots of 100-fold concentratedstock solutions.

RESULTS

Cell Yield and Cytology. Details of cell yield and viabilityfor dog and normal human cells have been described (8, 13).A CF trachea was obtained 4 hr after the death of a

12-year-old girl. A piece of the posterior membranous portionwas mounted in an Ussing chamber and found to have zeropotential difference and resistance. The remainder of thetrachea was used for cell isolation. Cells were collected at30-min intervals for 2 hr. In the first 30 min, 6 x 106 viablecells were obtained. These were mainly ciliated cells as werethe 7 x 106 cells obtained in the second 30-min period. In thetwo remaining 30-min periods, 41 and 5 x 106 cells wereobtained, respectively. Most of these were small (-7-,gmdiameter) and lacked cilia. We assume that they were basalcells. The cells were pooled and preplated for 1 hr to removefibroblasts (13). Following preplating, 41 x 106 cells (92%)excluded trypan blue. Four confluent monolayers wereproduced from these cells.The monolayers appeared confluent 1 day after plating.

Electron microscopy of 5-day-old cells revealed that theywere flattened and were joined by tight junctions thatseparated two distinct membranes. The membrane facing themedium contained microvilli and a pronounced glycocalyx.The membrane apposed to the filter was relatively undiffer-entiated. The morphology of the CF cells (Fig. 1) was similarto that of normal human cells. The direction of drug action(see below) led us to identify the membrane facing the

medium as the apical membrane and the membrane apposedto the filter as the basolateral membrane.

Transepithelial Electrical Properties. It took approximately1 min to mount the tissues in the Ussing chambers. Imme-diately after mounting, we obtained the following values forthe electrical parameters: Isc = 2.5 + 0.4 MA-cm-2, R = 486+ 85 fl.cm22, at = 1.3 + 0.4 mV (all values are mean ± SEM,n = 3). After 15 min, these parameters had decreased to thefollowing steady-state values: Isc = 1.8 ± 0.4 puA cm-2, Rt =380 ± 50Q cm2, at = 0.7 ± 0.2 mV. The corresponding valuesfor "normal" monolayers are 'sc = 3.1 ± 0.4 gA'cm-2 and Rt= 436 ± 45 flQcm2 (mean ± SEM of 15 filters from fourhumans). The Ic of the CF monolayers is significantly lessthan that of the normal monolayers (P < 0.05, Student's ttest). The resistances of the two groups are not statisticallydifferent.The responses of CF cells to isoproterenol (10 juM, serosal

bath) together with a pair of typical responses from normalmonolayers are shown in Fig. 2. With the CF filters, theresponses to isoproterenol were small or nonexistent, themean maximal increase in I,, induced by isoproterenol being0.22 ± 0.12 ,uA cm-2. Resistance was not affected. Incomparison, the maximal increase in Ikc in normal humantissues is 2.84 ± 0.50 gA cm-2 (mean ± SEM; five filters fromfour humans). Prostaglandin E2 (PGE2) (10 /LM, serosal andmucosal baths) was added to the tissue that failed to respondto isoproterenol (Fig. 2) and it caused a transient increase inIc of approximately 2-min duration with maximal amplitudeof 0.3 kuA cm-2. Again this is approximately 1/10th of thechange produced by PGE2 in normal tissues of 3.3 ± 1.6ttA cm-2 (mean ± SEM, three filters from three humans).The conductance of the CF monolayers of 2.63 mS-cm-2

predicts that in the absence of a transepithelial potential

A

FIG. 1. Cross sections of normal (Upper) and CF (Lower) cells. (Bar = 5 ,um.)

Proc. Natl. Acad. Sci. USA 82 (1985)

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NORMAL

5 _ CYSTIC FIBROSIS

TIME (min)

FIG. 2. Typical responses in ISc of normal and CF monolayers toisoproterenol (10 juM in the serosal bath). Drug was added at thepoint marked by the end of the horizontal scale bar.

difference, the sum of the passive ion fluxes in eitherdirection across the cells will be 2.62 ,ueqcm-2 hr-t (15, 16).The ISc of 1.8 /iA-cm-2 predicts a net active transport of ionsof 0.07 ,ueq cm-2 hr-1. The passive ion fluxes are thus some40 times greater than the active. Given this discrepancy, wefelt it unreasonable to try to measure active ion transportusing radioactive tracers; instead, we used specific transportblockers to provide indirect information about the transportprocesses present. All blockers were used at 0.1 mM.Mucosal amiloride and serosal ouabain inhibited Isc in bothCF and normal tissues (Fig. 3). Serosal amiloride or mucosalouabain were without effect on Isc. The major difference

Bm Bs NORMALI

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FIG. 3. Effects of transport inhibitors on ISC. Ouabain (0),amiloride (A), or bumetanide (B) was added to either the serosal (s)or the mucosal (m) side of the monolayers. All drugs were 0.1 mM.For the normal human cell tracing, the break is for 2 min. For the CFtracing, the breaks are for 1 min each.

between CF and normal tissues is in their responses to loopdiuretics. Furosemide, bumetanide, or MK-196 all inhibit Icin normal tissues, and bumetanide is only effective from theserosal side of the tissue. By contrast, in CF monolayers,bumetanide failed to affect ISc from either the mucosal or theserosal side of the monolayer. Typical records for the effectsof transport inhibitors are illustrated in Fig. 3.

Intracellular Microelectrode Studies. The responses in Ic ofnormal human material to mediators and transport blockersresemble the responses of dog monolayers (8, 13). Indirectevidence suggests that these responses in Is, are due toincreased chloride secretion. To obtain more direct evidencethat the cultured cells can secrete Cl, we examined the effectsof isoproterenol on the cellular electrical properties.Average values of electrical properties were obtained from

multiple impalements of electrodes in dog, normal human,and CF monolayers (Table 1). In dog and normal human cells,after isoproterenol q, increased, Oa depolarized, and fRdecreased. This pattern of response is the same as that ofintact dog tracheal epithelium (14) and suggests thatisoproterenol stimulates chloride secretion by increasingapical membrane Cl permeability. In a previous study weshowed that monolayers from dog retain the cell membraneCl secretory properties ofthe original epithelium even thoughthe transepithelial response to secretagogues is reduced (17).In contrast, values of Oa, qit, and fR were unaltered byisoproterenol in the CF monolayer.To localize the defect in chloride secretion in the CF

epithelial monolayers, we examined the responses to replac-ing Cl in the mucosal bath by gluconate. Tissues werestimulated with isoproterenol and the mucosal Cl concentra-tion was then reduced to 5 mM (Fig. 4). In dog, normalhuman, and CF monolayers, a decrease in mucosal Clconcentration produced similar increases in Aft and Rt (Table2). In dog and normal human cells, qMa depolarized and fRincreased. These are the responses expected from a Cl-permeable membrane and are the same as the changes seenin the original dog tracheal epithelium (14). In contrast, qMa ofthe CF monolayer hyperpolarized slightly and fR did notchange significantly.

DISCUSSIONMonolayers derived from tracheal mucosa of dogs or normalhumans show similar electrical properties. Monolayers de-

Table 1. Effect of isoproterenol (5 /iM, serosal side) on cellularelectrical properties

n at,mV 4a, mV fRDog

Baseline 6 0.4 ± 0.3 -38 ± 3 0.71 ± 0.06Isoproterenol 6 1.2 ± 0.4* -23 + 3* 0.24 ± 0.05*

Normal humanBaseline 16 0.1 ± 0.2 -35 ± 2 0.58 ± 0.03Isoproterenol 10 0.7 ± 0.2* -23 + 1* 0.34 ± 0.02*

CFBaseline 4 0.2 ± 0.1 -30 ± 1 0.79 ± O.5tIsoproterenol 10 0.3 ± 0.1 -32 ± 2t 0.76 ± 0.03tValues were obtained from six cell sheets cultured from dogs, four

from normal humans, and one from a patient with CF. n, Number ofelectrode impalements under each condition. For the dog the valuesbefore and after addition of drug were obtained from a singleimpalement in each cell sheet. For the normal human and the CFpatient the values represent multiple impalements obtained in eachmonolayer.*P < 0.05 compared with baseline by paired t test for the dog andunpaired t test for the humans.tP < 0.005 compared with the value under same condition in thenormal human by unpaired t test.

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FIG. 4. Effects of mucosal Cl substitution on cellular electrical properties. Each tracing is a representative recording of a monolayer froma dog (left), a normal human (middle), or a patient with CF (right). The mucosal [Cl] was decreased during the time indicated. Isoproterenol(5 AM) was present in the serosal medium throughout.

rived from a patient with CF behave in a completely differentfashion. The behavior of CF tissue can be explained by theabsence of an apical membrane chloride conductance.

In cells from dog and normal human, baseline ISc is reducedby both bumetanide and amiloride. Bumetanide acts onlyfrom the serosal side, amiloride only from the mucosal side.This suggests that both chloride secretion and sodium ab-sorption are present. In human cells, about 40% of thebaseline Ic persists in the presence of a combination ofbumetanide and amiloride. As discussed elsewhere (8), in dogtracheal epithelium amiloride blocks only -50% of active Naabsorption and bumetanide does not remove all active Clsecretion. The residual Isc in cultured cells is thereforeprobably due to a combination of amiloride-insensitive Naabsorption and bumetanide-insensitive Cl secretion.

Several types of endogenous mediators stimulate Is, acrossdog and normal human cells in culture (8, 13). This stimula-tion is probably due to increased chloride secretion. Thus,sodium absorption across intact airway epithelia is insensi-tive to mediators (8). Also, in cultured dog cells the mediator-induced increases in Ihc are greatly reduced in Cl-free(gluconate) medium (unpublished results). Finally, our intra-cellular recordings of the response to isoproterenol show thatthis drug decreases fR and depolarizes qia to -23 mV, a valueclose to the expected equilibrium potential for Cl (Table 1).Thus, in dog and normal human cells, the response toisoproterenol seems to be an increase in the apical membraneCl permeability leading to an increase in net transepithelial Clsecretion. In contrast to normal human cells, the Ic of CFcells is unaffected by bumetanide and the responses toisoproterenol and PGE2 are reduced to about 1/10th of

Table 2. Effect of mucosal chloride substitution on cellularelectrical properties

n t, mV a, mV R., fQcm2 fRDog 6

124 mM Cl 0.6 ±0.4 -27 ± 3 47 ± 7 0.22 ± 0.055 mM Cl 7.0+ 0.8* -5 + 4* 74 ± 11* 0.49± 0.07*

Normal human 11124 mM Cl 0.9 ± 0.2 -23 ± 1 284 ± 37 0.36 ± 0.025 mM Cl 7.8 ± 0.3* +7 ± 2* 357 ± 37* 0.57 ± 0.03*

CF 3124 mM Cl 0.4 ± 0.2 -28 ± 1 111 ± 7 0.69 ± 0.055 mM Cl 9.1 + 1.2* -30 + 2t 140 + 5* 0.80 + 0.03tValues were obtained from six cell sheets from dogs, three from

normal humans, and one from a patient with CF. n, Number ofelectrode impalements for each group. Values obtained with normal(124 mM) and decreased (5 mM, gluconate substitution) Cl concen-trations were obtained during single cellular impalements.*P < 0.005 compared with value in 124 mM Cl by paired t test.tp < 0.005 compared with value from the normal human with 5 mMCl by unpaired t test.

normal. Furthermore, isoproterenol does not change fR or tpa.These results suggest that in CF cells C1 secretion and itsstimulation by secretagogues are absent or greatly reduced.To localize the defect responsible for impaired chloride

secretion in CF cells we used intracellular microelectrodes tostudy the effects ofreducing mucosal Cl concentration. Whenmost of the mucosal Cl was replaced by gluconate, dog andnormal human monolayers showed the response expected forCl-selective apical membranes: ia depolarized and fR in-creased. In contrast, the same treatment in CF monolayerscaused qia to hyperpolarize slightly without much change infR. These results are consistent with a reduced Cl permeabil-ity of the apical membrane in CF.The mechanisms behind the hyperpolarization of 4fa seen in

CF cells on replacing chloride are uncertain. As Cl conduc-tance seems to be absent, amiloride-sensitive sodium chan-nels should result in the apical membrane of CF cells beinghighly Na selective. We speculate that the activity coefficientfor sodium gluconate is less than that for NaCl. In this casereplacing NaCl by an equivalent amount of sodium gluconateshould lead to a reduction in the Na activity gradient acrossa Na-selective membrane. This could account for thehyperpolarization of it and small increase in fR seen in CFcells.

Indirect evidence suggests that the ion selectivity of theparacellular pathway is unaffected in CF. In unstimulated dogtracheal epithelium, the conductance of the paracellularpathway accounts for -85% of the total tissue conductance(18). In cultured cells, Rt is much the same as in the intactepithelium but the Isc is only about 1/10th that of the originaltissue. This suggests that the conductance ofthe transcellularpathway is reduced in culture and that the paracellularconductance may be much greater than the apical membraneconductance (Ga). Thus, changes in /t in response to changesin the ion content of the mucosal solution may reflect mainlythe ion selectivity of the paracellular route. When chloridewas reduced to 5 mM by replacement with gluconate, theincreases in it were the same for dog, normal human, and CFcells (Table 2). Thus, the ion selectivity of the paracellularpathway may be little altered in CF. In sweat ducts and nasalmucosa (1, 2), the large lumen-negative potential differencesinduced by mucosal Cl replacement may reflect a relativelygreater contribution of Ga to Gt in normal tissue.

Unlike chloride secretion, there seems to be little changein sodium absorption in CF. This is illustrated in Fig. 3, wherethe decrease in IC induced by amiloride is similar in both CFand normal material. Our small sample size does not permita more detailed comparison of Na transport rates.There seem to be three possible chloride channel defects

that singly or in combination could explain the reduced apicalmembrane Cl permeability in CF. Chloride channels may bepresent in reduced numbers, they may have structural alter-ations leading to reduced ability to transport Cl, or their

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regulation may be faulty. A defect in regulation is suggestedby the results of Sato and Sato (19). They found thatmethacholine induced the same flow rates from normal andCF sweat glands and that isoproterenol caused the sameelevation of cyclic AMP in control and CF glands andstimulated sweat secretion in normal glands but did notinduce flow from CF glands. Our results with isoproterenolcould also reflect a defect in cyclic AMP-dependent regula-tory processes in CF. In addition, we found that the responseto PGE2 was reduced in CF. PGE2 resembles isoproterenol inthat it seems to stimulate Cl secretion across dog trachealepithelium by raising intracellular cyclic AMP levels (20).

In conclusion, the work described here indicates that thebasic defect in CF is a reduction in the permeability ofepithelial apical membranes to chloride. Our data support thegrowing body of evidence that epithelial cells retain differ-entiated transport function in culture and we have shownthat, although the postmortem CF tissue lacked measurableresistance and potential difference, cultured cell sheetsexhibited active ion transport and high trans-epithelial resist-ance. We suggest that cell culture will continue to be avaluable approach to the study of the Cl channel defect incystic fibrosis.

We thank Dr. J. A. Nadel for his support and interest and Drs.Brian Davis and Christopher J. Newth for helping us obtain the CFtissue. We are also grateful to Ella Highland, Timothy Ruppert, andPhil Karp for technical assistance. This study was supported in partby the National Institutes of Health Program Project Grant HL-24136and grants from the National Cystic Fibrosis Foundation, CysticFibrosis Research, Inc., and National Medical Enterprises, Inc.M.J.W. is an Established Investigator of the American HeartAssociation.

1. Quinton, P. M. (1983) Nature (London) 301, 421-422.2. Knowles, M. R., Gatzy, J. T. & Boucher, R. C. (1983) J. Clin.

Invest. 71, 1410-1417.

3. Knowles, M. R., Stutts, M. J., Spock, A., Fischer, N., Gatzy,J. T. & Boucher, R. C. (1983) Science 221, 1067-1070.

4. Mills, J. W., MacKnight, A. D. C., Dayer, J.-M. & Ausiello,D. A. (1979) Am. J. Physiol. 236, C157-C162.

5. Bisbee, C. A., Machen, T. E. & Bern, H. A. (1979) Proc.Nati. Acad. Sci. USA 76, 536-540.

6. Cereijido, M., Ehrenfeld, J., Meza, I. & Martinez-Palomo, A.(1980) J. Membr. Biol. 52, 147-159.

7. Mason, R. J., Williams, M. C., Widdicombe, J. H., Sanders,M., Misfeldt, D. S. & Berry, L. (1982) Proc. Nat!. Acad. Sci.USA 79, 6033-6037.

8. Widdicombe, J. H., Coleman, D. C., Finkbeiner, W. E. &Tuet, I. K. (1985) J. Appl. Physiol. 58, 1729-1735.

9. Handler, J. S., Steele, R. E., Sahib, M. J., Wade, J. B.,Preston, A. S., Lawson, N. L. & Johnson, J. P. (1979) Proc.Nati. Acad. Sci. USA 76, 4151-4155.

10. Johnson, J. P., Steele, R. E., Perkins, F. M., Wade, J. B.,Preston, A. S., Green, S. W. & Handler, J. S. (1981) Am. J.Physiol. 241, F129-F138.

11. Burg, M. B., Green, N., Sohraby, S., Steele, R. & Handler, J.(1982) Am. J. Physiol. 242, C229-C233.

12. Lowy, R. J., Schreiber, J. H., Dawson, D. C. & Ernst, S. A.(1984) Fed. Proc. Fed. Am. Soc. Exp. Biol. 43, 447 (abstr.).

13. Coleman, D. L., Tuet, I. K. & Widdicombe, J. H. (1984) Am.J. Physiol. 246, C355-C359.

14. Welsh, M. J., Smith, P. L. & Frizzell, R. A. (1982) J. Membr.Biol. 70, 227-238.

15. Linderholm, H. (1952) Acta Physiol. Scand. 27, Suppl. 97,1-100.

16. Schultz, S. G., Zalusky, R. & Gass, A. E. (1964) J. Gen.Physiol. 48, 375-378.

17. Welsh, M. J. (1984) Clin. Res. 32, 769A (abstr.).18. Welsh, M. J., Smith, P. L. & Frizzell, R. A. (1983) J. Membr.

Biol. 71, 209-218.19. Sato, K. & Sato, F. (1984) J. Clin. Invest. 73, 1763-1771.20. Smith, P. L., Welsh, M. J., Stoff, J. S. & Frizzell, R. A.

(1982) J. Membr. Biol. 70, 215-226.

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