BRIEF REPORT / RAPPORT BREF

Effect of K channel blockers on basal and @-agonist stimulated ion transport by fetal distal lung epithelium

HUGH O'BRODOVICH~ AND BIJAW RAFII Resplrcrtorq! Research DDrsion rpf the Ho.~pit~pl Sick Children S Researc-h Ii~stitute arad Deportinent of Pebiutrics,

U/~ivcr.sity of Toronto, Toronto, Ont., Canado M5@ 1x8

Received October 8. 1992

~ 'BRODOVHCH, H., and WAFIH, B. 1993. Effect of K channel blockers on basal and 6-agonist stimulated ion transport by fetal distal lung epithelium. Can. S . Physiol. Pharmacol. '71: 54 -57.

To determine whether basolateral K channels play an important role in the basal and 0-agonist stimulated ion transport by fetal distal lung epithelium we harvested these cells from fetal rats (20 days gestation, term = 22 days) and studied them in Ussing chambers. Short-circuit current (I,,) fell with basal 3 mM BaCl, (3.0 + 0.2 ( f SEM) to 2.0 rt: 0.2 yA . cm-?, r.r = 18, g < 0.01) without affecting ramnolayer resistance (R = 693 f 57 9t - cm2). Basal quinine sulfate ( I mM) also decreased I,, (3.7 + 0.15 to 3.0 + 6). 18 p A . crns2; n = 4, p < 0.81). None of apical BaCl, (3 mM), apical quinine (1 mM), nor bilaterally applied tetraethylammonium (10 mM), lidocaine (1 mM). or 4-arninopyridine (2 rnM) decreased I,,. Cell monolayers treated with basal BaCl, (3 mM) demonstrated an impaired ability to increase their LC in response to the P,-agonist terbutaline (1 mM). Basal 3 mM BaCl, also decreased I,, in arniloride (0.1 mM) and hrosernide (1 mM) treated monolayers, indicating that barium also affected the previously described amiloride-insensitive Na transport by these cells (rz - 8, p < 0.01). Together these experiments suggest that normal basolateral K channel function is required for normal and 0,-stimulated Na transport in fetal distal lung epithelium.

Key ~vords: type I1 alveolar epithelium, potassium channels, P-agonist, sodium transport, Na channels.

O ' B ~ o ~ c s v r c ~ , H., et RAFII, B. 1993. Effect of K channel blockers an basal and P-agonist stimulated ion transport by fetal distal lung epithelium. Can. 9. Physiol. Pharmacol. 71 : 54-57.

Pour dtterminer si les canaux K basolat6raux jouent un r81e important dans le transport ionique, tant stimuli par les 0-agonistes que basal, dans 1'CpithClium des poumons distaux foetaux, nous avons pr6leve ces cellules chez des rats foetaux (20 jours de gestation, terme = 22 jours) et les avons examides dans des chambres de Ussing. Ee courant de court circuit (I,,) a chutC avec 3 mM de BaCl, (3,0 + 0,2 i?i 2,0 f 0,2 P A . cm-', n = 18? p < 0,Ol). sans affecter la rksistance (R) des rnonscouches (693 & 57 5& a crn2. Le sulfate de quinine basal (1 mM) a aussi diminuC l'Ic, (3,7 -t 0,15 S 3,0 + 0,10 ,uA ern-,, rz = 4, g < 6),01). Ni le BaCl, apical (3 mM). ni la quinine apicale (1 mM) ni la 4-aminopyridine (2 mM), la lidocai'ne (1 mM) ou le tktra6thylamrnonium (10 mM) appliquks de manikre bilatkrale n'ont dirninu6 I'B,,.. Les mono- couches cellulaires traitCes au BaCl, basal (3 mM) ant dCmontrC une capacit6 alttrCe d'augmenter leur I,, en rCponse au 0,-agoniste terbutaline (1 rnM). Le BaCl, basal (3 mM) a aussi diminuC l'Ic, des monocouches traitkes au furoskrnide (1 mM) et 2 lqami80ride (0,l mM), indiquant que le baryurn a aussi affect6 le transport celltilaire de Na insensible B I'aimiloride (n = $, p < 0,01), qui a 6tC anterieurement. Ces experiences suggkrent qu'un transport de Na tant stirnu16 par les 0,-agonistes que normal dCpend d'un fonctionnement normal des canaux K basolatiraux dans 1'6pithClium pulmonaire distal foetal.

Mots elks : CpithClium alvColaire de type BI, canaux potassium, P-agonistes, transport de sodium. canaux Na. [Traduit par la rCdaction]

Intrsdepction reasonable to assume that these cells participate in the Na

The lung is entirely filled with fetal lung liquid at birth and absovtive process at the time of birth.

this must be efficiently cleared for there to be a normal transi- AS discussed in detail in recent reviews (Dawson f?f

tion to postnatal air-breathing like Our laboratory has previ- 1990; McCann and Welsh 1990a), basolateral K conductance

O U S ~ Y demonstrated that active Na transport by lung epithelium may play a role in the regulation of NaCl secretion or absorp- is required for normal lung water clearance afier bib tion. The present study determined if the ion-transporting (07Brodovich et al. 1990a, 1991) and other laboratories have capabilities of FI3J.X were altered by agents known to block demonstrated that Na transport is increased by release of K d~annels and vd~ether such agents would affect the ability pmagonists during labour ( B ~ ~ ~ ~ et 1983). Since we have of these cells to increase ion transport in response to 8-agonists.

pre;iously demonstrated that primary monolayer cultures of rat fetal distal lung epithelium (FDLE) transport Na via Na channels in their apical membrane (O'Brodovich et ab. 1990tla, 199 1 ; Orser er a!. 199 1) and that this transport can be upregu- lated by 0,-agonists (B'Brodovich et a!. B998b, 1991) or cyclic AMP analogues (O'Brodovich et a!. 19921, it is

'Correspondence may be sent to the author at the following address: Director, Respiratory Research Division, Hospital for Sick Children, 555 University Avenue, Toronto, Ont., Canada M5G 1x8.

Methods Primary culture of FDLE

FDLE were isolated and grown in primary culture using techniques previously described (O'Brodovich et a/. 1990b, 199 1, 1992). In brief, lungs were obtained from 20 days gestation Wistar fetal rats (term = 22 days), diced into small pieces, and incubated with 0-0'2% DNase (Wrthington Biochemical Corp.. Freehold, N.J.) and 0.125 % trypsin (Cibco, Burlington, Ont.). After neutralization of proteolytic activity with fetal bovine semm (FBS) we incubated the cells with 0.1 % collagenase (Wortkington Biochemical Corp., Hree-

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BRIEF REPORT 1 RAPPORT BREF

hold, N.J.). After addition of FBS and centrifugation the cells were passed through a HC3-100 Nitex mesh (Tetko Inc., EErnsford, N.Y.), and the fibroblasts were removed by two differential adkerance steps. The final cell preparation has > 95 % epithelial cells as assessed by cytokeratin immunofluorescent staining. Because the fetal lung is at the transition between the cannalicular and saccular stage of lung development, not all harvested cells are mature type I1 epithelium; therefore we refer to these cells as fetal distal lung epithelium (FDLE) .

The FDLE cell suspension was seeded onto Transwellm tissue cul- ture treated polycarbonate filters (Costar, Cambridge, Mass.) at an estimated seeding density of 1 .Ba million cells . cm-'. Cells were cul- tured in Eagle's minimal essential media with 10% FFBS (Gibco, Grand Island, N.Y.) at 3T°C under a 5% 6 B 2 - balance air humid- ified atmosphere. Penicillin (100 UlmL) and streptomycin (100 pgl mL) were present in the culture media. One day after seeding the excess unattached cells were removed and the medium was replaced.

Measurement sf biselectric prc>perties Two to 4 days after harvest the primary monolayer cultures of FDLE

were studied in Ussing chambers where each fluid reservoir had 15 mL of 3T°C Hanks balanced salt solution (MBSS) containing 1.8 g NaHCB,/L, which was circulated by an airlift using a 5% C 0 2 - balance room air gas mixture. KC1 agar - calomel half-cells and sil- ver - silver chloride electrode saline agar bridges were connected to a high impedance millivoltmeter which functioned as a voltage- current clamp with automatic fluid resistance compensation (VCC 600, Physiologic Instruments. San Biego, Calif.). In all experiments the rnonolayers were short circuited so that transepithelial potential difference (PD) = 0 nmV and short-circuit current (Is,) was continu- ously recorded. Every 10 s the monolayer was clamped to + 1 mV for 0.5 s so that transepithelial resistance fR) could be calculated using Ohm's law. The monolayers were returned to open-circuit con- dition for 5 s every 10 min so that PD could be directly measured. Reported I,, values refer to the movement of positive charge from the apical to basolateral side of the membrane and the PD is refer- enced to the apical side (basolateral side was positive relative to the apical side).

Our laboratory has previously demonstrated using ion substitution experiments and pharmacologic agents, that rnonolayers of FDLE cultured in this manner are Wa-absorbing epithelium under basal and P,-stimulated conditions and do not secrete chloride (O'Brdovich ct ak. 1990b, 1991). To determine the importance of basolateral K channels on this Na transport initial experiments used several differ- ent potential K channel blockers under baseline conditions. After establishing baseline bioelectric properties BaCJ, (3 mM), tetraethyl- ammonium sulfate (TEA, 10 mM), quinine sulfate (1 mM), lidocaine (1 mM), or 4-aminopyridine (2 mM) was applied to the scplutions bathing the apical and (or) basoratera1 surhces of different cell mono- layers. Wdter-soluble drugs were dissolved in HBSS, whereas qui- nine was dissolved in 1:200 BNISB (we have previously shown that DMSO at this concentration does not affect the bioelectric properties of FBEE (09Brdovich er cal. 19906)). Next, to demonstrate that normal basolateral K channel function is required for 6,-agonist induced increases in I,, we added BaCl, (3 mM) or vehicle to the basolateral bath of two different groups of FDLE monolayers after establishing baseline bioelectric parameters. Terbutaline (1 mM) was then added to both sides of the monolayers; although we continuously recorded Is,, we report the value 60 min after addition of the terbuta- line as the post-terbutaline value. Finally, since we have previously shown that a minor part of Wa-dependent I,, of FDEE (O'Brodovich et cal. 1991) is not inhibited by amiloride and others have previously shown that there is both an amiloride-sensitive and -insensitive transport in bovine trachea (Langridge-Smith 1986), we wished to determine if basolateral BaC1, can also decrease I,, in amiloride (0.1 nlM, apical) and hrosemide (1 mM. basolateral) treated mono- layers (hrosemide was used to decrease the likelihood that any changes in I,, would be due to chloride transport in inonolayers exposed to amiloride and BaC1,). The above studies demonstrated that BaC1, decreased Is, and, since precipitation of barium can occur

+ +

T VEHICLE

AGENT AGENT +TERB

FIG. 1. The HBSS vehicle (open bars) or 3 mM BaCE, (hatchcd bars) was placed in the basolatcral solution s f FDEE monolayers mounted in Ussing chambers. The vehicle did not affect I,, values, and subsequent terbutaline (TERB) treatment increased I,,, as has been previously described (O'Brodovich st ul. 1990b, 199 1). BaCl, decreased 1, from its respective baseline value and blunted the response to terbutalinc such that @,-stimulated values were not sig- nificantly different from original bascline (pre-BaC%, values). Results are means + SEM. *p < 0.01 from respective baseline values; Pp < 0.0% between vehicle- and BaC12-treated monolayers. Student's t-test with Bonferonni correction for repeated measures was used to determine statistical significance.

in bicarbonate-ci~ntaining solutions, we did additional experiments using bathing fluids where we substituted 10 mM Hepes for the bicar- bonate and equilibrated the fluid with room air.

Results are expressed as means + SEM. To determine the statisti- cal significance of changes in bioelectric parameters from baseline conditions in the same monolayer a paired Student's t-test was used. When nnultiple time points were assessed, the significance was deter- mined by using a one-way analysis of variance (.lawsv~i) followed by the LSD test or by a Bonferonni correction of the Student's t-test (Blossum statpack, National Institute of Health. Bethesda, Md.). Probability (g) less than 0.05 was considered to be significant.

Basolateral application of 3 mM BaCI2 decreased baseline Is, in each FDLE monolayer (baseline I,, = 3.0 f 0.24 pA

BaC12 Is, = 2.0 k 0.22 pA = n = 18, p < 0.81). In these experiments BaC12 similarly decreased PD (baseline PD = 2.8 -b 0.22 naV; BaCl7 PB = 1.4 + 0.18 mV; p < 0.01) but did not affect R (baseline R = 693 a 5'7 Q cm2: BaClz R = '708 f 61 C2 . cm"). Smaller but qualitatively similar results were seen with basslaterally applied I mM qui- nine (baseline Is, = 3.7 + 0.15 pA - cms2 VS. quinine Is, = 3.0 + 0.11 pA n = 4, p < 0.05). To verify the reversability s f BaClz and quinine efkets additional experi- ments were performed. BaC12 ( 3 mM) decreased Is, to 69 + 3.1% of baseline values, but 15 min after lowering the BaC12 concentration to 0.4 rnM the Is, returned to 88 + 1.5% s f baseline values (n = 4). Similarly, B mM quinine decreased Is, to 47% of baseline values, but within 20 min of diluting quinine to 8.1 mM, Is, returned to '77 % of baseline values (n = I). TEA applied apically and basolaterally did not affect I,, (baseline Is, = 3.9 + 0.53 pPB TEA I,, = 3-7 + 0 . 6 3 pA . ern-?.; n = 4, p = ns). Finally, neither 1 mM lids- caine (la = 6) nor 2 mM 4-aminopyridine ( n = 6) affected baseline Is, (data not shown).

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CAW. J. PHYSIOL. PHARMACOL. VOL. 71, 1993

BASELINE AMlLORlDE + FUROSEMIBE APICAL BASAL BARIUM BARIUM

FIG. 2. Measurement s f Is, in monolayers of FDLE mounted in Ussing chambers demonstrates that basolateral (basal) bud not apical application of 3 mM BaCl, decreases I,, in amiloride (lov4 M) and furosemide (1 0-%) treated cells. One-way analysis of variance fol- lowed by LSD test was used to assess statistical significance. *p < 0.05 from baseline values; Tp < 0.645 from amilsride and furosemide alone values.

In experiments that evaluated the effect of BaC12 on &-agon- ist induced increases in Is,, basolateral BaCl? (3 mM) did, but the HBSS vehicle did not, decrease I\, (Fig. 1). The subse- quent addition of terbutaline t s the bathing media increased Is, in both vehicle- and BaC12-treated monolayers; however, the amount of the increase was significantly blunted in the BaC12-treated relative to vehicle-treated monolayers ( p < 0.01, Fig. 1).

Additional experiments were performed where the FDLE were first treated with amiloride (0.1 mM, apical bath) and hrosemide (1 mM, basal bath). Hn these monolayers ( p a = 8) the baseline bioelectric parameters were Is, = 3.9 f 0.18 pA . cms2, PD = 1.3 f 0.12 mV, and R = 354 f 37 $& - cm2. As illustrated in Fig. 2, 3 mM BaClz applied to the basolateral, but not apical side of the amiloride- and hrosemide-pretreated rnsnolayers decreased I,,. These experiments were repeated in five additional monolayers where Hepes was substituted for the bicarbonate in the bathing media. BaC12 decreased Is, from 1.9 f 0.46 to 1.1 f 0.48 pA . cm-' ( p < 8.05) in the amiloride- and furosemide-treated monolayers.

The results from our experiments show that agents known to block K channels decrease the Is, of primary monolayer cul- tures of the Na-transporting cells that are present in the distal lung regions of the late-gestation fetal rat. In addition, K chan- nel blockers blunt the ability of FDLE monolayers to respond to the P2-agonist terbutaline. Together these data suggest that K channels on the basolateral membrane of these cells must hnc- tion noranally for them to optimally transport Na.

As recently reviewed (Dawson eb a!. 1990; McCann and Welsh 1990a), basolateral K conductance plays an important role when epithelial cells are either Cl-secreting or Na-absorbing cells. During both C1 secretion and Na absorption the epithelial cells' basolateral Na-I9 ATPase maintains a low intracellular Na concentration by extruding Na ions from the cell in exchange for K entry into the cell. The K is then recy- cled back out across the basolatcral menlbrane via highly

selective K channels. It seems logical therefore that such basolateral K channels might play an important role when the distal lung epithelium absorbs fluid via active Na transport dependent mechanisms during stress (Olver et al. 1986) and labour (Brown et al. 1983) and at birth (O'Brodovich et a / . 1990~1, 1991).

The ability of basolateral barium to decrease 15, in FDLE is consistent with the presence of K-permeant channels on the basolateral membrane sf the cells. Other investigators have used different techniques to study K channels in epithelial cells fron~ various levels of the respiratory system. The whole-cell patch-clamp technique has been used to study cultures of adult type HI pneumonscytes. DeCoursey et a/. (1988) and Peers ef a / . (1990) demonstrated the presence of two different types of K currents, which were characterized by different electro- physiologic characteristics and by their sensitivity to TEA. Most cells studied by DeCoursey et (el . (1988) demonstrated highly selective K currents with very low sensitivity to TEA (ICS0 - 15 mM): very few cells demonstrated high serasitiv- ity to TEA (IC,, - 108 p M ) . This would agree with our present study, where 10 mM TEA had no effect on the bioelec- tric properties of a FDLE monolayer. Although Peers st al. (1990) similarly demonstrated that the K channels in adult type IT cells were sensitive to TEA and quinine, they found, in con- trast to our present data. that 4-aminopyridine also decreased K currents. Whether this difference reflects adult versus fetal alveolar epithelial cells, culture conditions, or the experimen- tal approach is unknown. Kunzelmann et uk. (1989a' 198962) also used the patch-clamp technique to characterize K chan- nels, in nasal polyp epithelium. As in the whole-cell studies of adult type I1 pneumonocytes, the K channels were highly selective for K. The channels were also blockable by barium, TEA, quinidine, and lidocaine. but since these investigators (Kunzelmann et a / . 1989a, 19896) used the excised inside-out patch-clamp configuration. these blockers were applied to the cytoplasmic aspect rather than the outside aspect of the cell membrane. Studies of primary cultures of dog tracheal epithel- ium (McCann et a / . 1990; McCann and Welsh 199062), have shown the presence of two different isoproterenol-induced K channels, one sf which is charybdotoxin sensitive.

Present and previous work (09Brodovich st a/ . 1990.b9 199 1, 1992) have demonstrated that amiloride inhibits most but not all of the I,, of FDLE. This amiloride-insensitive Is, is, how- ever, Na transport, since our previous work using ion substitu- tion experiments indicates that the Is, of FBLE is dependent upon Na ions being present on the apical side of the membrane and that there is no detectable 15, even when both Na and Cl ions are present on the basolateral surfaces of these cells (O'Brodovich et a!. 1991). These previous results (O'Brodo- vich et a&. 1991) agree with work that indicated that a portion of the 22Na transport seen in bovine trachea is similarly amilo- ride insensitive (Langridge-Smith 1986). In the present experi- ments basolateral but not apical BaC1' decreased the amount of the amiloride-insensitive I,,. Because the apical entry route is likely different for the amiloride-sensitive and -insensitive components of the I,,, the data support the idea that the effect of the barium is on K channels, which then indirectly affects Na transport (i.e., rather than affecting two different Na entry mechanisms).

Our present demonstration of the importance sf basolateral K channels and the ability of FDLE to increase their ion trans- port are relevant to previous observations made in premature lamb fetuses (Brown et a&. 1983; Barker et a!. 1988). These

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BRIEF REPORT !' RAPPORT BREF 5 7

previous studies demonstrated that full-term mature, but not immature, lamb fetuses can convert from lung liquid secretion to amiloride-sensitive fluid absorption in response to 6-agon- ists or mernbrane-permanent cyclic AMP analogues (Brown 9% a!. 1983; Barker et a/ . 1988). Although immaturity in the expression of the lung epithelial Na channel or its regulatory pathways most likely contributes to this phenomenon, an associated i~mrnaturity of the basolateral K channel may also contribute to the inability of the immature fetal lung to initiate Na transport to induce fluid absorption.

Acknowledgements The work was supported by a grant-in-aid from the Medical

Research Council of Canada. Dr. 05Brodovich is a Career Investigator of the Heart and Stroke Foundation of Ontario.

Barker, P. RI., Brown. M. J.. Ramsden, C. A., Strang, L. B., and Walters, B. V. 1988. The effect of thyrsidectomy in the fetal sheep on lung liquid reabsorption induced by adrenaline or cyclic AMP. J. Physiol. (London), 407: 373 - 383.

Brown, M. J . , Olver, R. A., Ramsden, C. A., Strang, L. B., and Walters, D. V. 1983. Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb. J . Physiol. (London), 344: 137- 152.

Dawson, D. C . , and Richards, N. &by. 1996. Basolateral K conduc- tance: role in regulation of NaCl absorption and secretion. Am. 9. Physiol. 259: C181 -C195.

DeCoursey, T. E., Jacobs, E. R., and Silver, M. R. 1988. Potassium currents in rat type II alveolar epithelial cells. J. Physioi. (Lon- don), 395: 487 -505.

Kunzelmann, K., Pavenstidt, H., Beck, C., ~ n a l , 6, Ernmrich, P., Arndt, H. J., and Greger, 8. 1989a. Characterization of potassium channels in respiratory cells. I. General properties. Pfluegers Arch. 484: 291 -296.

Kunzelmann, K., Pavensaade, H., and Greger , R. 19896. Charactcri- zation of potassium channels in respiratory cells. 11. Inhibitors and regulation. Pfluegers Arch. 414: 297 - 303.

Langridge-Smith, d. E. 1984. Interaction between sodium and chlo- ride transpow in bovine tracheal epithelium. J. Physiol. (London), 376: 299-319.

McCann, J . D., and Welsh, M. J . 1990~. Regulation of C1 and K channels in airway epithelium. Annu. Rev. Physisl. 12: 1 15 - 135.

McCann, J. D., and Welsh, M. J. i9Wb. Basolateral K' channels in airway epithelia. 11. Role in C1- secretion an-ad evidence for two typcs of K' channel. Am. J. Physiol. 258: L343-L348.

McCann, J. B., Matsuda, J . , Garcia. M., Kaczorc~wski, G., and &byelsh. M. J. 1990. Basolateral K ' channels in alrway epithelia. I. Regu- lation by CaL ' and block by charyhdotoxin. Am. S . Physiol. 258: E334 - E342.

O'Brodovich, H., Hannam, %I., Seear, M.. and Mullen, 3. B. M. 1 9 9 0 ~ . Amiloride impairs lung water clearance in newborn guinea pigs. J . Appl. Physiol. 68(4): 1758 - 1762.

O'Brodovich, H., Rafii, B., and Post, M. 19906. Bioelectric proper- ties of fetal alveolar epithelial monolayers. Am. J. Physiol. 258: L201 -L206.

07Brdovich, H., Hannam. %I., and Rafii, B. 1991. Sodium channel but neither Na'-H' nor Na-glucose syrnport inhibitors slow neonatal lung water clearance. Am. J. Respir. Cell Mol. Biol. 5: 377 - 384.

QqBrodovich9 H., Rafii, B., and Perlon, P. 1992. Arginine vasogres- sin and atrial mtriuretic peptide do not alter ion transport by cul- tured fetal distal lung epithelium. Pediatr. Res. 38: 3 18 -322.

Olver, R. E., Rarnsdea, C . A . , Strang, L. B . , and Miters , D. V. 1986. The role of amiloride-blockage sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb. J. Physiol. (London), 376: 32 1 -340.

Orser, B. A., Bertlik, M., Fedorko, L . , and O'Brodovich, H. 1991. Non-selective cation channel in fetal alveolar type II epithelium. Biochim. Bi~ghys. Acta, 1094(1): 19 -26.

Peers, C . , Kemp, P. J . , Boyd. C. A. R., and Nye, P. C. G . 1990. Whole-cell K + currents in type II pneumocytes freshly isolated from rat lung: pharmacological evidence for two subpopulations of cells. Biochim. Biaphys. Asta, 1052: 1 13 - 1 18.

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