Active Na+ transport across Xenopus lung alveolar epithelium

29

Respiration Physiology, 81 (1990) 29-40 Elsevier RESP 01672

Active N a + transport across Xenopus lung alveolar epithelium Kwang-Jin Kim Departments of Medicine and Physiology, Cornell University Medical College, New York, NY, U.S.A. (Accepted 8 April 1990) Abstract. Bioelectricproperties and unidirectional ion fluxes of alveolar epithelium were studied by utilizing

excised Xenopus lungs mounted in the flux chamber under short-circuited conditions. Results show that the alveolar epithelium generates a potential difference (PD) of 8 mV (lumen negative) with a tissue resistance (Rt) of 1000 ohm' cm2. The short-circuit current (Isc) is inhibitable by 80~o with alveolar amiloride. Alveolar or pleural exposure of ouabain slowlydecreases Is¢ to zero. R t is slightlyincreased by either agent. Control tissues exhibit a greater unidirectional 22Na÷ flux in the alveolar to pleural (A ~ P) direction than in the opposite (P ~ A) direction, indicating a net removal of Na ÷ from the alveolar fluid. Amiloride and ouabain both decrease the A ---*P Na ÷ flux to the level of the P ~ A flux, thereby abolishing net Na ÷ absorption. In contrast, unidirectional 36C1 flUXeSare not different in either direction. Neither amiloridenor ouabain affected these 36Cl fluxes and tissue resistance appreciably,indicatingthat CI - passivelypermeates the alveolar epithelium.

Alveolar epithelium, ion fluxes, Animal, Xenopus laevis; Drugs, amiloride, ouabain; Electric properties, of alveolar epithelium; Epithelial membrane potential; Ion fluxes, in alveolar epithelium.

The alveolar air spaces must be kept relatively fluid-free for efficient gas exchange. The alveolar epithelium, which forms a c o n t i n u o u s lining of the terminal air spaces, has been found to offer the major resistance to the passage of water a n d solutes compared to other (e.g., airway a n d endothelial) barriers present in the lung (Berg et aL, 1989; K i m and Crandall, 1985; N o r m a n d e t a l . , 1971; Pietra e t a l . , 1968; Schneeberger a n d K a r n o v s k y , 1968, 1971; Taylor a n d G a a r , 1970; W a n g e n s t e e n et al., 1969). I n recent years, it has also been appreciated that the alveolar epithelium has a n u m b e r of ion transport functions which help keep the n o r m a l alveolar fluid balance. F o r example, fetal lungs in utero c o n t a i n lung liquid distinctly different from plasma, where the concentrations of C1- (higher) a n d H C O 3 (lower) are different from those expected for a simple passive distribution ( A d a m s o n et al., 1969), indicating that the fetal pul-

Correspondence to: K.-J. Kim, Will Rogers Institute Pulmonary Research Program, Departments of Medicine and Physiology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, U.S.A. 0034-5687/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

30

K.-J. KIM

monary epithelium may have mechanisms for active ion transport (Olver and Strang, 1974; Olver et al., 1981, 1986). Very recently, distal pulmonary epithelia from the adult mammalian (rat) lungs, have been shown to actively reabsorb Na + from the air spaces (Basset et aL, 1987a,b; Berg et al., 1989; Goodman et aL, 1987). In vivo lungs of sheep, rabbits, and dogs were found to remove fluid out of the distal air spaces, which is in part related to active Na + transport (Berthiaume et al., 1988; Gates and Matthay, 1987; Matthay et al., 1982). However, the information obtained from these intact mammalian lung studies in vivo and in vitro, while useful, is very difficult to interpret mechanistically, due largely to the anatomical complexity of the lung. More precise information about alveolar epithelial properties can be obtained by studying anatomically simpler preparations. Excised anuran lungs representing an intact preparation of alveolar epithelium have been used in recent years to study ion transport properties of the alveolar epithelial barrier. For example, Gatzy (1975), using bullfrog lungs as a model for the alveolar epithelium, demonstrated that the amphibian alveolar epithelium actively transports C1- into the lung lumen, a property perhaps shared by canine tracheal epithelium and fetal pulmonary epithelial barriers. In contrast, Fischer et al. (1989) have very recently reported that another amphibian (Xenopus laevis) model of isolated alveolar epithelium develops a PD of 10 mV (lumen negative), Is¢ of 12 #A. cm -2, and R t of 0.8 Kohm" cm 2. They further noted that most of the Isc (90~o) is inhibitable by apical amiloride, with an apparent K~ of 1.2/~M for amiloride, suggesting that Xenopus alveolar epithelium predominantly transports Na + out of the alveolar spaces. In this study, bioelectric properties and unidirectional fluxes of Na ÷ and C1- across the excised Xenopus lungs in the presence and absence of amiloride and ouabain, were measured to further characterize the ion transport properties of the alveolar epithelium. Xenopus alveolar epithelium was found to normally transport Na + actively out of the alveolar air spaces, with C1- following passively. Part of this study has already been reported in a preliminary form previously (Kim, 1989, 1990).

Methods

Tissue preparation. South African clawed frogs (Xenopus laevis) of either sex were obtained from Charles Sullivan, Inc. (Nashville, TN). Animals were used within a week of delivery to the laboratory. The techniques used to prepare the lung as a planar sheet mounted between two hemichambers of a flux chamber have been described in detail elsewhere (Kim and Crandall, 1985). Briefly, frogs were doubly pithed and their ventral surfaces were opened. The lung was transected at the tracheoglottis and cut open to form a planar sheet. This tissue was then placed between two reservoirs of a flux chamber (0.64-1.65 cm 2 opening) and bathed with 8 ml of identical Ringer solution on both sides. Soft silicone O-rings covered with a thin film of high vacuum grease (Dow Coming, Midland, MI), were used on each surface of the tissue to minimize the possible edge effects. The Ringer solution used has the following composition (in mM):

ACTIVE Na ÷ TRANSPORT ACROSS ALVEOLAR EPITHELIUM

31

110.0 NaC1, 2.4 KHCO3, 1.0 Ca-D-gluconate, 1.0 MgSO 4, 10.0 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] and the pH of the Ringer solution was adjusted to 7.4 with 1 N HC1/1 N N a O H after gassing with 100~o 02 for 1 h. The reservoir fluid was agitated vigorously with a magnetic stirrer.

Measurements of tissue bioelectric parameters and experimental maneuvers. The bioelectric properties of the Xenopus alveolar epithelium were determined by monitoring the transmural potential difference (PD) and the short-circuit current (Isc). Detailed description for these measurements can be found in a previous report by Kim and Crandall (1985). In brief, PD was measured via two matched calomel half-cells and two connecting 4~o agar-3 M KC1 bridges whose tips were located about 2 mm from the tissue surfaces. Current was passed across the tissue via two Ag/AgC1 electrodes and two connecting agar-KC1 bridges whose tips were positioned farther away (about 2 cm) from the tissue surfaces. Transtissue resistance (Rt) was estimated either by the relation P D . Isc - 1 or dV. dI - 1, where dV is the voltage response to a small amount of direct current, dI (5/~A). This latter relation was used when the observed Isc was too small (ca < 1/~A) to assess the R t accurately. Effects of amiloride (0.01 # M - 1 mM) and ouabain (1 mM) on bioelectric properties were determined by exposing the tissue to a Ringer solution containing either of these transport inhibitors. Solutions containing less than 1 mM amiloride were obtained by diluting the 1 mM amiloride-Ringer with a fresh Ringer solution. Measurements of unidirectional ion fluxes. Apparent permeability coefficients for sodium and chloride ions were estimated from the rate of radionuclide (/2Na or 36C1) appearance in the downstream reservoir and the source radioactivity in the upstream reservoir, following the procedures previously reported (Kim and Crandall, 1985). The tissues were continuously short-circuited using an automatic voltage clamp device (DVC 100, World Precision Instruments, New Haven, CT) during the flux experiment, except for a brief period (30 sec) at every 15 min for the measurement of PD and R t. Radionuclides were instilled into the upstream reservoir 5 min after a transport inhibitor was added to the appropriate reservoir (20 # M amiloride to the alveolar (A) side or 1 mM ouabain to the pleural (P) side). Samples (1 ml) of downstream reservoir fluid were taken every 15 min up to 120 min from the addition of radioactivity to the upstream reservoir fluid. Upstream samples (0.05 ml) were taken at 15 rain from the instillation of radioactivity and also at the end of each experiment. Immediately after each sampling from the reservoir, an equal amount of appropriate fresh Ringer solution was added back to the same reservoir to maintain a constant volume. This dilution factor was taken into account in the estimation of permeability values. Ten milliliters of premixed scintillant solution (Ecoscint, National Diagnostics, Sommerville, N J) were added to each vial containing the radioactive samples. The upstream samples were diluted with an appropriate volume of a fresh Ringer solution. All samples were assayed for radioactivity (dpm) in a liquid scintillation spectrometer (Tri-Carb 4000, Packard, Downers Grove, IL).

32

K.-J. KIM

Statisticalprocedures. All experiments were performed at room temperature. Results were expressed as means + SE. Two-way analyses of variances for three population means (control, amiloride-, and ouabain-treated tissues) with two different directions (A --* P and P ---,A) for measuring permeability coefficients, were performed following the procedures (for unequal cell sizes) described by Winer (1971). Modified Newman-Keuls tests (Wirier, 1971) were carried out for multiple comparisons between population means. Statistical significance was tested for P ~< 0.05.

Results

The control alveolar epithelium ofXenopus laevis lungs exhibits a PD of 8.2 + 2.9 mV (lumen negative) and an I~ of 9.1 + 2.1/~A. c m - 2 for 38 tissues. The estimated R t for control tissues was 1.09 _+ 0.23 K o h m . c m 2. Amiloride (>0.1 #M) exposure to the alveolar surface elicited a rapid and concomitant depression of P D and I~ with a T~/2 of about 1-1.5 rain, while slightly raising R t. Figure 1 shows the dose-response curves (for PD, I~, and Rt) obtained from alveolar exposure of amiloride, where the ordinate represents the percent normalized values of these three bioelectric parameters (measured after 10 min of amiloride exposure) against the corresponding values measured immediately before the exposure. As can be seen, the dose-response curves for PD and I~ were approximately the same, whereas a slight increase in R t was seen as the concentration of amiloride was increased. The effective half-maximal responses for PD and I~ were observed at 0.6 p M amiloride. In contrast, the pleural exposure of amiloride (up to 1 mM) did not significantly change any of the bioelectric parameters

20

_=? -20 o

._~

~

-,o

o

E o

-60

~

-ao

o "rg

-100

- - - / / ...... 0.001

0.01

0.1

Amiloride

I

10

!00

1000

(/zM)

Fig. 1. Effectsof alveolar amilorideon bioelectricproperties of Xenopus lungs. Y-axis shows the percent initial valuesof the parameters(PD, Rt, and I,c) after exposureto amiloride.The effectivedose of amiloride which reaches 50~o of maximal response is about 0.6 #M.

33

ACTIVE Na + TRANSPORT ACROSS ALVEOLAR EPITHELIUM

(fig. 2). Ouabain (1 mM) exposure to either surface of the tissue resulted in a much slower decrease in both PD and Isc (with a T~/2 of about 10-15 min) to zero with a slightly raised R t. The appearance rate of downstream radioactivity becomes linear after about 15-20 min of lag time from the instillation of radionuclides to the upstream reservoir, regardless of the experimental conditions imposed and also irrespective of the direction of flux measurements, as seen in fig. 3. Thus, slopes were estimated from linear regression of the downstream radioactivity vs time (30-120 min) and subsequently used to calculate apparent permeability (P) coefficients. Since we have used approximately the same amount of each radionuclide for individual experiment, the apparent permeability value can be taken as an index for unidirectional flux. Table 1 summarizes the PNa+ and corresponding R t values for control, amiloride (20 #M, alveolar) -treated, and ouabain (1 mM, pleural) -treated tissues under shortcircuited conditions. The control A ~ P PNa ÷ is significantly greater than the control P~A PNa÷, indicating that there is a net active Na + absorption (about 0.25 #Eq- cm - 2. h - 1) from the alveolar fluid of the control tissue. The decrements in the PN~ + observed in the A ~ P direction followed by exposures to amiloride or ouabain were significant. All the PN~+ values for the P ~ A direction, however, were not significantly different from each other. Thus, amiloride or ouabain led to an abolishment of net active Na + absorption by a significant reduction of absorptive (i.e., A ~ P) unidirectional flux to the same level of the P --, A flux, respectively. P ~ A fluxes o f N a + for amiloride- and ouabain-treated tissues were slightly increased from the control value, respectively. Both experimental conditions and direction of Na + flux measurements failed to effect a significant change in R t. Figure 4 shows the control 22Na flux data depicted as tissue conductance ( G t ) vs

--

o

o 0

,I

=o

0

oo. oE

-5

n

U

"r- g ~ g o

J

u

~

-10

.//

. . . . . . . .

I

10

100

b

1000

Arniloride (/~M) Fig. 2. Effects of pleural amiloride on Xenopus alveolar epithelium. Unlike alveolar exposure of amiloride, the pleural amiloride did not change any of the bioelectric parameters significantly from the corresponding control values. See details for fig. 1

34

K.-J. K I M 2O

E "u

15

o O 10

O

0 0 0

O

5

•

o 0

i

,

•

15

30

• * ~.5

L 8'0

7'5

90

!05

120

Time (turn) Fig. 3. Typical time courses of 22Na appearance in the downstream reservoir for the two (A ~ P, unfilled circles and P ~ A, filled circles) control tissues under short-circuited conditions. A and P denote the alveolar and pleural reservoir, respectively. As can be seen, there is a lag time of about 15-20 min after which the rate of appearance for radioactivity becomes linear. Also noted is the steeper (i.e., faster accumulation of radioactivity) slope for those data points representing A --, P transport of 22Na across the tissue.

TABLE 1 Apparent sodium permeability (PNa + ) and tissue resistance (R t) of the Xenopus alveolar epithelium. Pna ÷ has a unit of 10 - 7 cm- sec - 1 and Rt is expressed in K o h m . cm z. All entries are mean ± SE (n), where n is the number of observations. All values of R t are not different from each other at P = 0.05 by two-way analysis of variances with modified N e w m a n - K e u l s Procedures. Control A~P* P---'A

PN~÷ Rt PNa÷ Rt

10.7 1.13 4.5 1.20

+ ± + +

Amiloride 1.5 (12) '~ 0.11 (12) 0.7 (9) 0.11 (9)

6.0 1.48 6.3 1.36

+0.6 ± 0.14 + 0.7 + 0.15

Ouabain (8) (8) (8) (8)

7.2 1.24 7.5 1.14

± ± ± +

1.1 0.12 1.0 0.13

(6) (6) (6) (6)

* A and P denote the alveolar and pleural side, respectively, of the lung epithelium. ~' Significantly different from all other values of PNa (P < 0.05).

As can be seen, the data points which represent the 22Na flux measured in the A--+ P direction clearly are located to the right side of those points representing the P -~ A direction, which may indicate that the greater N a + flux in the A -+ P direction is real and not due to leakier tissues. Analyses similarly performed for amiloride- and ouabaln-treated tissues exhibit no clear-cut separation of data points representing A ~ P and P ~ A direction, indicating the abolishment of active Na + transport by these transport inhibitors. Table 2 summarizes Pc]- and R t values obtained for the tissues (control, arniloridePNa÷"

35

ACTIVE Na ÷ T R A N S P O R T A C R O S S A L V E O L A R E P I T H E L I U M 1.50 -

1.00

0

I

0 0

0 ,A

0

0

0

0

0

eo 0.50 -

0

0.00

0

5

1'0

1'5

20

PNo+ (lO-7cm.sec -1)

Fig. 4. Transtissue conductance (Gt) of the Xenopus lungs vs permeability of 22Na (PNa+) under control conditions. It can be noted that the data points for A ~ P direction are located to the fight side of those for P ~ A direction. This m a y indicate that the observed net sodium absorption in the alveolar to pleural direction across the Xenopus alveolar epithelium is not due to a small number of tissues whose conductance is extremely high.

TABLE 2 Apparent chloride permeability (Pcl-) and tissue resistance (Rt) of the Xenopus alveolar epithelium. Pc~has a unit of 10 - 7 cm" s e c - l and R t is expressed in K o h m . cm 2. All entries are mean + SE (n), where n is the number of observations. All values of R t (or P c l - ) are not different from each other at P = 0.05 by two way analysis of variances with modified N e w m a n - K e u l s Procedures. Control A-,P* P~A

Pa Rt PaRt

11.2 1.02 11.0 1.01

+2.2 + 0.05 +2.2 + 0.06

Amiloride (7) (7) (10) (10)

10.3 1.54 11.3 1.39

_+0.6 -+ 0.28 -+ 1.4 -+ 0.24

Ouabain (5) (5) (6) (6)

13.5 1.40 14.7 1.10

+1.0 + 0.10 +0.8 -+ 0.08

(4) (4) (5) (5)

* A and P denote the alveolar and pleural side, respectively, of the lung epithelium.

and ouabain-treated) under short-circuited conditions. P o - estimated for the control tissues was approximately the same in both directions, which did not change significantly by treatment with amiloride or ouabain. The two R t values (estimated for A ~ P and P ~ A directions of C1 - flux measurements) for each experimental condition were not different from each other. R t w a s not significantly affected by the experimental conditions in this series of experiments, although slight increases of R t by amiloride and ouabain were similar to those observed in the series of N a + flux experiments. Figure 5 depicts the data points for C1 - flux measurements under control conditions. As can be seen, there is no clear separation of the two groups (i.e., A ~ P vs P ~ A

36

K.-J. KIM 1.50

0

0 1.00

o

0

•

0

I

0

E o & vE 0.50

0.00

,'o

1'5

2'o

PCI- ( 1 0 - 7 c r n ' s e c - 1)

Fig. 5. Transtissue conductance (Gt) of the Xenopus lungs vs permeability of 36C1(PcI) under control conditions. As seen, the slopes (Pcl- vs Gt) obtained for the two directions are not significantlydifferent from each other.

direction) of data points. Analysis of 36C1 Flux data for amiloride- and ouabain-treated tissues also indicated that the data points are similarly bunched up together as shown in fig. 5.

Discussion The results obtained in this study demonstrate directly for the In,st time that Xenopus alveolar epithelium actively transports N a + out of alveolar air spaces, with C1following passively. The A ---,P flux of N a ÷ is significantly greater than the P --, A flux of N a ÷ in the absence of electrochemical gradients (i.e., short-circuited) across the tissue, clearly indicating that the normal alveolar epithelium is capable of removing net Na ÷ from the alveolar fluid. It appears that N a ÷ may enter the alveolar epithelial cells via apical, amiloride-sensitive channels (and perhaps in part through N a + / H ÷ exchange mechanisms), followed by subsequent extrusion via N a +, K ÷ -ATPases most likely located in the basolateral membranes of the alveolar epithelium. C1 - , on the other hand, seems to be transported passively across the alveolar epithelium, perhaps via paraceUular pathways. If P ~ A movement of N a + and C1- is assumed to occur via passive diffusional pathway alone (i.e., DNa÷/D o _ = 0.66; Robinson and Stokes, 1955), the observed ratio of PNa+ , p - A / P o - , p - A = 0.41 for control tissues may indicate the fact that N a + is somewhat less selective to the tissue than C I - . The sum of partial ionic conductances under control conditions was about 0.61 mS . c m - 2 which only represents about 70~o of the over all tis sue conductance of about 0.86 m S. cm - 2. This re suit i s in keeping with

ACTIVE Na ÷ TRANSPORT ACROSS ALVEOLAR EPITHELIUM

37

the findings that alveolar exposure of amiloride up to 1 mM led to a characteristic 80~o inhibition of the total Isc (fig. 1). Taken together, these data may indicate that there are other unknown conductive pathways for ions. One such pathway may be that for K +, since it was previously reported (Kim, 1989), that Ba 2÷ decreases Is¢ by as much as about 20Yo from the control value. Others may include active H ÷ and/or HCO3transport, which needs to be further explored. The bioelectric parameters obtained in our studies are generally in good agreement with those reported earlier by Fischer et al. (1989) for the same tissue. The half-maximal effective dose for amiloride in this study is about 0.6 #M, which is approximately half the value of K~ for alveolar amiloride reported by Fischer etal. (1989). These investigators also observed that amiloride (50#M, alveolar) and ouabain (0.1 mM, pleural) slightly raise the tissue resistance, by 8 and 13 Yo, respectively, while amiloride inhibits Isc by 60-99~o and ouabain lowers Is¢ to zero. 22Na permeability data obtained in our studies can be compared with other amphibian alveolar epithelium as well as those of mammalian alveolar epithelium. For example, Gatzy (1975) reported that bullfrog alveolar epithelium, which mostly secretes C1actively into the lumen, has PNa÷ = 5.6 ( A ~ P ) and 5.0 (P --. A), all in 10- 7 cm. sec - 1. The passive permeability for Na ÷ in the Xenopus alveolar epithelium (4.5 × 10 -7 cm" s e c - l , present study) is very similar to that estimated for bullfrog alveolar epithelium. In contrast, recent reports on passive Na ÷ permeability of isolated perfused rat lungs indicate PNa+ = 0.67 × 1 0 - T c m ' s e c -~ (Basset etal., 1987a,b), which was measured in the direction of airspace fluid to vascular perfusate by assuming alveolar surface area of 5000 cm 2. This apparent discrepancy may simply be explainable by the fact that the actual luminal surface area of the Xenopus alveolar epithelium can be as much as about 6 times the nominal surface area of the flux chamber (Czopek, 1965; Fischer et aL, 1989). Finally, the tight, Na ÷ -absorbing alveolar epithelial monolayers in primary culture conditions obtained from rat lungs, were very recently reported with a passive P N a + = 5.0 × 10 - 7 cm- sec - I (Cheek et al., 1989; Crandall et al., 1990), which is very close to that estimated for Xenopus alveolar epithelium in this study. The net active Na ÷ absorption estimated for Xenopus alveolar epithelium is also in good agreement with other reports. For example, isolated perfused rat lungs (Basset etal., 1987a,b; Berg etal., 1989; Goodman etal., 1987) have an ami[oride- and ouabain-inhibitable (i.e., active) Na ÷ flux of about 0.02 # E q . c m - a ' h - 1 which is about half the rate (0.25/6) estimated for Xenopus alveolar epithelium after correction for the actual surface area (see above). Primary cultured alveolar epithelium of rat lungs (Cheek etal., 1989; Crandall etal., 1990) has net Na ÷ absorption of about 0.19 # E q . c m - 2 . h - ~ (based on the nominal surface area of the flux chamber), which is very close to that estimated for Xenopus alveolar epithelium in this study. These comparisons indicate that the excised Xenopus alveolar epithelium is a reasonably suitable model for the studies of alveolar epithelial ion transport. The finding that C1- normally crosses the Xenopus alveolar epithelium via passive diffusion is in keeping with other studies on alveolar epithelial C1- transport. Isolated perfused rat lungs (Basset et al., 1987a,b) as well as primary cultured alveolar epithelial

38

K.-J. KIM

monolayers of rat lungs (Crandall et al., 1990) all exhibit a C1- permeation rate which is mostly passive in nature to date and also very similar to that reported in this study. Thus, it appears that Xenopus alveolar epithelium shares the typical ion transport properties found in a n u m b e r of isolated alveolar epithelial preparations in vivo and in vitro. As noted previously, bullfrog alveolar epithelium is more representative of the upper airway epithelium a n d / o r the fetal p u l m o n a r y epithelial barriers as far as ion transport characteristics are concerned. In summary, the excised Xenopus alveolar epithelium is characterized with an active resorption of N a + from the alveolar fluid, with C1- following passively. This finding may be useful to u n d e r s t a n d the n o r m a l alveolar fluid balance and the mechanisms and pathways underlying ion transport across the distal airway epithelial barrier of the adult m a m m a l i a n lungs in vivo. Xenopus lung may be a useful model for studying ion transport characteristics of the alveolar epithelium in isolation.

Acknowledgements.The author thanks for the excellenttechnical assistance offered by Ms J. Roszwadowski, Messrs D. Schoenfeldand D. Nag, who have participated in this study through Cornell Traditional Summer Student Program, and for the critical reading of the manuscript by Dr S. Desai. This study was supported in part by research grants (HL38658, HL38578, HL38621) from the National Institutes of Health, a Research Fund from the New York Lung Association, and American Heart Association Grant-in-Aid (AHA 89-749).

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Kim, K.J. and E. D. Crandall (1985). Heteropore populations of bullfrog alveolar epithelium. J. Appl. Physiol. 54: 140-146. Kim, K.J. (1989). Active Na + transport across Xenopus lung alveolar epithelium. FASEB J. 3: 562. Kim, K.J. (1990). Effects of amiloride and ouabain on ion fluxes across Xenopus alveolar epithelium. FASEB J 4: A550. Matthay, M.A., C.C. Landolt and N.C. Staub (1982). Differential liquid and protein clearance from the alveoli of anesthetized sheep. J. AppL Physiol. 53: 96-104. Normand, I. C. S., R. E. Olver, E. O. R. Reynolds and L. B. Strang (1971). Permeability of lung capillaries and alveoli to non-electrolytes in the foetal lamb. J. Physiol. 219: 303-330. Olver, R.E. and L.B. Strang (1974). Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the foetal lamb. J. Physiol. 241: 327-357. Olver, R.E., E. E. Schneeberger and D.V. Waiters (1981). Epithelial solute permeability, ion transport and tight junction morphology in the developing lung of the fetal lamb. J. Physiol. 315: 395-412. Olver, R. E., C. A. Ramsden, L.B. Strang and D.V. Waiters (1986). The role of amiloride-blockable sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb. J. PhysioL 376: 321-340. Pietra, G.G., J.P. Szidon, M.M. Leventhal and A.P. Fishman (1968). Hemoglobin as a tracer in hemodynamic pulmonary edema. Science 166: 1643-1646. Robinson, R.A. and R.H. Stokes (1955). Electrolyte Solutions. London: Butterworths Scientific Publications, pp. 293-329. Schneeberger-Keely, E.E. and M.J. Karnovsky (1968). The ultrastructural basis of alveolar-capillary membrane permeability to peroxidase used as a tracer. J. Cell Biol. 37: 781-793. Schneeberger, E.E. and M.J. Karnovsky (1971). The influence of intravascular fluid volume on the permeability of newborn and adult mouse lungs to ultrastructural protein tracers. J. Cell Biol. 49: 319-334. Taylor, A. E. and K. A. Gaar (1970). Estimation of equivalent pore radii of pulmonary capillary and alveolar membranes. Am. J. Physiol. 218: 1133-1140. Wangensteen, O. D., L. E. Wittmers Jr. and J. A. Johnson (1969). Permeability of the mammalian blood-gas barrier and its components. Am. J. Physiol. 216: 719-727. Winer, B.J. (1971). Statistical Principles in Experimental Design. Second ed., New York: McGraw-Hill, pp. 455-449.