Sodium Channel but Neither Na"-H+ nor Na-Glucose Symport Inhibitors Slow Neonatal Lung Water Clearance Hugh O'Brodovich, Vicky Hannam, and Bijan Rafii Department of Paediatrics and Hospital for Sick Children, Research Institute, University of Toronto, Toronto, Ontario, Canada

Normal clearance of alveolar liquid following birth requires active Na transport; however, the contribution of Na channels, Na-H antiports, and Na-glucose symports is unknown. We demonstrated that intraalveolar instillation of amiloride (n = 6) or the more specific Na channel blockers benzamil (n = 13) or phenamil (n = 12) before the first breath impaired lung water clearance relative to control newborns (n = 34). Benzamil and phenamil were more potent than amiloride (P < 0.05). Neither the Na-H antiport inhibitor dimethyl amiloride (n = 7) nor the Na-glucose symport inhibitor phloridzin (n = 7) impaired lung water clearance. Ion substitution experiments with fetal rat type II alveolar epithelia demonstrated that more than 95 % of their resting or terbutaline-stimulated short circuit current (L) depended upon Na bathing their apical membrane. I; was decreased by amiloride (ICso of amiloride-sensitive I; = 0.3 X 10-6 M) and benzamil (ICso of benzamil-sensitive I; = 0.3 X 10-7 M) but was unaffected by dimethyl amiloride (10-4 M). We conclude that in vivo postnatal clearance of fetal lung liquid can be impaired by Na channel blockers and is unaffected by blockers of Na-H antiports and Na-glucose symports. Na transport in fetal type II cells has high affinity for amiloride, and these cells likely contribute to normal neonatal lung liquid clearance.

Previous investigators have demonstrated that intravenously administered infusions of J3-agonists (1) or airspace instillation of membrane-permeant analogues of cyclic adenosine monophosphate (cAMP) (2) stop lung fluid secretion and induce amiloride blockable fluid absorption in the intrauterine full-term fetal lamb (3). Our laboratory has demonstrated that active transport ofNa by the pulmonary epithelium plays a physiologically important role in the clearance of fluid from the airspaces immediately after birth. When amiloride was instilled into the newborn guinea pig's airspace fluid before the first breath, the resultant impairment of epithelial Na transport caused respiratory distress, hypoxemia, and persistently elevated extravascular lung water (EVLW) contents (4). The present studies investigated several questions. First, experiments were designed to assess the mechanisms of amiloride's action as it can affect both the Na-H antiport and Na permeant ion channels: in vivo experiments

(Received in original form January 9,1991 and in revisedform March 28, 199!) Address correspondence to: Dr. Hugh O'Brodovich, Division of Respiratory Research, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Abbreviations: cyclic adenosine monophosphate, cAMP; dimethyl sulfoxide, DMSO; extravascular lung water volume, EVLW; fraction of inspired oxygen, Flo2; Hanks' balanced salt solution, HBSS; short circuit current, Ise ; n-methyl-D-glucamine, NMDG; potential difference, PO; resistance, R; systemic arterial oxygen saturation, Sao-. Am. J. Respir. Cell Mol. BioI. Vol. S. pp. 377-384, 1991

used amiloride analogues that preferentially block either the Na-H antiport (dimethyl amiloride) or the Na channel (benzamil and phenamil). Second, because the intra-alveolar amiloride only impaired approximately 50% of the fluid clearance, we questioned whether the Na-glucose symport might contribute to the clearance of the remaining fluid. Studies in adult lungs have suggested that approximately half of the instilled fluid is cleared by amiloride-inhibitable mechanisms, with the remainder being cleared by phloridzin (which blocks Naglucose symport)-inhibitable processes (5). Third, we performed additional experiments to investigate whether the alveolar epithelium might be one of the cell types participating in the clearance oflung fluid at birth. Our previous studies had indicated that the short circuit current (L) of fetal type II epithelium is only partly inhibited by amiloride and benzamil. In vitro experiments were performed on monolayers of fetal rat lung alveolar epithelium mounted in Ussing chambers to determine if their I; was totally dependent upon Na ions (as assessed by ion substitution). Dose-response studies were also performed to determine if Na transport had high or low amiloride sensitivity because a recent report suggests that adult type II cells (6), in contrast to most epithelia, have Na conductive pathways with low amiloride affinity. The above in vivo and in vitro studies demonstrated a similar qualitative response to amiloride and its analogues, yet there was a discrepancy in the dosage required to produce a biologic effect (see RESULTS). Because these compounds

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differ in their lipophilicity (hence, their lung clearance rates may be different), we measured changes in their airspace fluid concentrations during 2- to 3-h in vivo experiments in adult rabbits.

Materials and Methods Newborn Lung Water Clearance To evaluate the effect of pharmacologic agents on the clearance of lung liquid from newborn guinea pigs, we used the same approach previously described in detail (4). In brief, pregnant High Oaks Ranch F, strain (formerly Hartley) guinea pigs obtained from High Oaks Ranch (Goodwood, Ontario, Canada) were anesthetized with inhaled halothane once there was separation of their pubic rami. Although separation of the pubic rami indicates that delivery may occur anytime in the next 48 h, none of the mothers was in obvi0us labor at the time of the experiment (labor is known to alter lung water content [7] and Na/K adenosine triphosphatase activity [8]). Assisted ventilation was provided to the mother with a Harvard rodent ventilator. The neck of a fetus was then exposed through a hysterotomy to enable placement of a Teflon" tracheostomy tube through which 0.3 ml of the saline vehicle or active drug was subsequently instilled. The tube was then occluded, the fetus delivered, and the umbilical cord clamped. Assisted ventilation with a timecycled, pressure-limited ventilator (BP200; Bournes, Riverside, CA) was used for the initial 30 to 120 s after birth, and then the animals breathed room air spontaneously for the duration of the experiment. The procedure was repeated for each of the other fetuses except for the last fetus, which was immediately killed to enable subsequent measurement of the EVLW content that was present at the time of birth (subsequently referred to as birth animals). Most mothers had a total of three to four fetuses, and all were delivered and were spontaneously breathing within 15 min of the initial uterine incision. After birth, the newborn guinea pigs were anesthetized with an intraperitoneal injection of pentobarbital sodium (0.5 to 1.0 mg/lOOmg body weight), and body temperature was maintained using a servo-controlled radiant heater. The animals were then observed for chest wall retractions to evaluate the degree of clinical respiratory distress, and systemic arterial oxygen saturation (Sao.)' was measured using and Nellcor pulse oximeter (Hayward, CA) by placing the probe over the thigh of the hindleg. Sao, was recorded as the stable value averaged over 1 min at 1, 2, 3, and 4 h after birth. In five additional fetal guinea pigs, we sampled the lung liquid before the first breath to determine its glucose concentration because this would be important in our interpretation of the series of experiments in which we assessed whether the Na-glucose symport contributed to the clearance of lung liquid following birth (see below). These five animals were not otherwise used for the experiments as manipulation and removal of tracheal fluid would affect the measurement of lung water content. The fetal guinea pigs were randomly assigned to receive either 0.3 ml of an active drug solution or vehicle in different series of experiments using amiloride and its analogues (9) or phloridzin. In all experiments, we studied animals receiving the vehicle and did not rely upon historical controls. In the first series of experiments, we administered 0.3 ml of

5 1991

10-3 M amiloride (n = 6) into the animals' tracheae to confirm our previous observations that the Na transport inhibitor amiloride results in impaired clearance oflung liquid after birth (4). The second series of experiments used a similar volume and concentration of dimethyl amiloride (n = 7), an amiloride analogue that at these concentrations blocks the Na-H antiport but has no effect on the Na channel. The third series of experiments used 0.3 ml of 10-3 (n = 6) or 10-4 M (n = 7) benzamil, an amiloride analogue that at these concentrations blocks the Na channel but has no effect on the Na-H antiport. We performed another series of experiments with 0.3 ml of 10-3 (n = 7) or 10-4 M (n = 5) phenamil, an amiloride analogue with comparable specificity for the Na channel but whose inhibition of Na transport has been reported to be more difficult to reverse (10). Phenamil is the least water-soluble of all the above compounds, and it was initially dissolved in dimethyl sulfoxide (DMSO) for the 10-3 M experiments only. The final instilled fluid contained phenamil dissolved in 0.03 ml DMSO and 0.27 ml saline (control animals for these experiments received a similar DMSO-containing vehicle). Finally, a series of experiments were performed in which we administered 0.3 ml of 10-2 M phloridzin to assess the contribution of the Na-glucose symport in the clearance of neonatal lung liquid because other reports have suggested that this symport is present in the pulmonary epithelium of neonatal lambs (11) and data suggest it plays a role in the clearance of fluid instilled into the.lungs of adult rats (5). When each of these compounds is instilled into the trachea, it will be immediately diluted by the airspace fluid once ventilation is begun. Assuming the newborn animals weigh 0.1 kg and have a total airspace fluid volume of 30 ml/kg, the above concentrations of drug would be immediately decreased by approximately a factor of 10. As one would predict from the lipophilicity of amiloride and its analogues (9), and as additional experiments in adult rabbits confirmed (see below), there would be a continuing progressive and marked decrease in the drug's airspace fluid concentrations during the 4-h experiment. Four hours after birth, the newborn animal's chest was rapidly opened. After a l-ml sample of blood was obtained from the beating heart, the lungs were removed for determination of wet/dry wt ratio and EVLW/g dry blood-free lung by the technique of Pearce and associates (12). We had added 0.3 ml fluid to the lungs of fetuses that were subsequently killed at 4 h of life. To enable a valid comparison with animals killed at birth, we added 0.3 g to the wet weight of the birth fetuses (see RESULTS). Our previous study demonstrated that the degree of perivascular fluid cuffing is similar in animals receiving amiloride or the saline vehicle (4). Although the measurement of vascular cuffing is a relatively crude indicator of interstitial fluid volume, these previous findings do suggest that any differences in EVLW between groups represents differences in the amount of airspace fluid (4). To evaluate whether benzamil or phenamil might have any toxic effect on the pulmonary epithelium that could promote increases in EVLW content, we instilled 0.3 ml of 10-3 M benzamil (n = 4), 0.3 ml of 10-3 M phenamil (n = 4), or 0.3 ml of saline (n = 5) into the trachea of 9-day-old guinea pigs. After 1 to 2 min of assisted ventilation, the animals were allowed to spontaneously breathe room air for 4 h before being killed to enable measurement of EVLW contents.

O'Brodovich, Hannam, and Rafii: Na Transport Inhibitors in Fetal Lung

Airspace Fluid Concentration of Amiloride and Benzamil As discussed above, we had an estimate of the initial concentration of drug in the airspace fluid. However, the concentration of amiloride and its analogues during the subsequent 4-h period was unknown. This is important because amiloride and the analogues used in these experiments do not covalently bind to the Na channel, thus yielding a reversible inhibition of Na transport. If amiloride moved rapidly out of the airspace fluid, then epithelial Na transport may not be inhibited for the entire duration of the experiment. To address this question, we took advantage of the fluorescent nature of the amiloride molecule (9) to assess the effect of time on amiloride and benzamil concentrations in fluid instilled into the lungs of adult rabbits. Rabbits were used because preliminary experiments had demonstrated that we could not reliably sample the fluid in the lungs of the very small newborn guinea pigs. We used a protocol similar to one previously published by our laboratory (13). In brief, six adult New Zealand white rabbits were anesthetized and tracheotomized, and gas exchange was maintained with a high-frequency oscillator (Hummingbird Mera, Tokyo, Japan) operating at a rate of 15 Hz (tidal volume = 10 ml, and FIo2 = 1.0). A catheter was inserted into the ear artery to monitor systemic arterial blood pressure and sample blood to determine gas tensions and acid base status. A total of 20 rnl saline/kg body weight (n = 2) or 10-4 M amiloride (n = 2) or 10-4 M benzamil (n = 2) dissolved in saline was then instilled into the trachea followed by a single inspiratory sigh to 20 em H20 for 10 s. Ventilatory support was then continued with the oscillator at settings described above. In the two rabbits receiving saline alone, we stopped the oscillation 10 min after instillation and collected the airspace fluid that spontaneously flowed up into the tracheostomy tube. This fluid was then centrifuged at 20,000 X g for 10 min, and the supernatant stored at 4 0 C. It was subsequently analyzed to determine if the airspace fluid had spontaneous fluorescence and was also used as the solvent to generate a standard curve of amiloride and benzamil concentrations. In the other four rabbits, the oscillator was similarly briefly stopped at varying intervals ranging from 10 to 180 min after the saline-containing drug was instilled to obtain fluid to determine residual concentration of amiloride or benzamil within the airspace fluid. The concentrations were assessed with the aid of a Hitachi fluorescent spectrophotometer (model U-2000; Hitachi, Tokyo, Japan). Excitation wavelength was 360 nm, and emittance wavelength was 410 nm. Preliminary experiments determined the range over which there was a linear relation between fluorescence and the concentration of the drug. All samples were diluted so that the fluorescent measurements were obtained in this region. Bioelectric Properties of Fetal Alveolar Epithelial Cells Fetal type II alveolar epithelial cells were harvested from pregnant Wistar rats of 20 days of gestation (term = 22 days) and grown in primary culture. These methods have been described in detail by ourselves (14) and others (15, 16) and utilized a differential absorption and centrifugation technique to separate the epithelial cells from the contaminating cells.

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The only modification from our previous approach (14) was to incubate the initial cell isolate with 0.1% collagenase for 15 min at 3]0 C before differential absorption to aid in the removal of fibroblasts. These epithelial cells have been extensively characterized and have the morphologic, biochemical, intracellular filament, and lectin-binding characteristics of type II alveolar epithelial cells; epithelial cell purity is greater than 95% (14-16). In contrast to our recent work (14), we seeded the cells on Transwell'" tissue culture-treated polycarbonate filters (catalogue #3412; Costar, Cambridge, MA). A seeding density of 1.5 million cells/em- was used, and unattached cells were removed 24 h after seeding. The cells were cultured in Eagle's minimal essential medium (Dvaline substituted for L-valine) plus 10% dialyzed fetal calf serum (GIBCO, Grand Island, NY) at 37 0 C under a 5 % COi 95 % room air humidified atmosphere. Two to four days after harvest, the filters were placed into Ussing chambers (MRA, Clearwater, FL) containing 3]0 C Hanks' balanced salt solution (HBSS) and 1.8 g/liter NaHC03 , which was circulated by an airlift equilibrated with 95 % room air/5 % CO 2, KCl agar-calomel half-cells and silversilver chloride electrode-saline agar bridges were connected to a high-impedance millivoltmeter that could function as a voltage current clamp with automatic fluid resistance compensation (VCC 600; Physiologic Instruments, San Diego, CA). Transepithelial potential difference (PD) was continuously recorded, and every 100 s a bidirectional f-u.A current was passed to determine transepithelial resistance (R) using Ohm's Law. Every 10 to 15 min, the transepithelial PD was clamped to 0 mV to determine L. If baseline R was greater than 100 ohm-ern- and the PD was greater than 0.7 mV, the filters were used to study the bioelectric properties of the epithelial monolayer in the amiloride and benzamil doseresponse studies. More than 95 % of the filters met these criteria. In the ion substitution experiments, a baseline R greater than 100 ohms-ern- was the only criterion used. Our previous study using pharmacologic agents suggested that these fetal type II alveolar epithelial cells do not secrete Cl but have the characteristics of Na-absorbing cells, a process that can be upregulated by ,Bragonists (14). Amiloride decreased the I; to approximately 70 % of baseline values in these previous experiments. To confirm that these cells indeed actively transport Na and to assess whether the amiloride-insensitive component was caused by Na transport, we used ion substitution experiments in which n-methyl-D-glucamine (NMDG) (138 mM) was substituted for Na in the modified HBSS. Simultaneous experiments were performed where monolayers were bathed in either regular Na- or NMDG (sodium-free)-containing HBSS. All solutions contained Cl ions at all times. After establishing baseline PD, R, and I, values, we applied the ,B2-agonist terbutaline (10- 3 M) to the basolateral side of the monolayer and recorded the maximal change in bioelectric properties (15 to 30 min). After determining baseline values and after addition of terbutaline, we added Na-containing regular HBSS first to the basolateral side of the monolayer to determine if NMDG-induced inhibition of I; had merely resulted from impairment of an Na/K/2Cl cotransporter (this cotransporter is usually involved in Cl secretion). Na-containing regular HBSS was subsequently added to the apical side of the monolayer. In both cases, Na-containing HBSS was added in a 1:2

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ratio by volume so that the final Na concentration was 46 mM in the monolayers originally bathed by the NMDGcontaining, sodium-free HBSS. No further changes were made in the bathing media; however, amiloride (10-4 M) was then applied to the apical side followed by furosemide (10-3 M) applied to the basolateral side of the monolayer. Additional studies were performed to determine the dose response of amiloride and benzamil on 1,,, which would allow us to determine if Na transport pathways of fetal type II epithelia had high or low amiloride affinity (6, 9, 17). Separate monolayers had baseline bioelectric parameters determined and then either amiloride or benzamil in saline was added to the apical side of the monolayer at one of the log increment doses between 10-9 to 10-5 M (n = 3 filters each log dose). We then administered 10-4 M of the appropriate compound to determine that monolayer's maximal response to these inhibitors of Na transport. Amiloride, phloridzin, and furosemide were purchased from Sigma Chemical Co. (St. Louis, MO). Terbutaline was a gift from Astra Pharmaceuticals (Mississauga, Ontario), and benzamil, phenamil, and dimethyl amiloride were obtained from Dr. Cragoe of Merck, Sharp, and Dohme Research Laboratories (West Point, PA). Statistical Analyses Results are presented as mean ± SEM and were analyzed using the SAS statistical package (SAS Institute, Cary, NC). In the analysis of these experiments, we have combined the values for all of the animals that were killed before the first breath (birth group). To test for statistical significance between EVLW contents at 4 h after birth, we used a one-way ANOVA followed by Duncan's multiple range test. A similar approach was used to test for statistical significance in EVLW between 9-day-old guinea pigs receiving either benzamil, saline, or phenamil. To test for significance of any difference in the Sao, values between groups after birth, a two-way ANOVA was used. To test for an effect of the maximal concentration of amiloride and its analogues on the I; of monolayers of fetal type II epithelium, a paired Student's t test was used. A P value < 0.05 was considered significant.

Results The birth group of fetuses had a body weight of 81 ± 2.5 g, the group of fetuses that received the pharmacologic drugs had a body weight of 81 ± 1.8 g, and the group receiving the vehicles for the drugs had a body weight of 80 ± 2.1 g. The fetuses that had 0.3 ml of 10-3 M amiloride, benzamil, or phenamil placed in their airspace fluid before their first breath had similar responses. All of these groups had persistent chest wall retractions and had Sao, values that were significantly (P < 0.05) lower than the saline vehicle, dimethyl amiloride, and phloridzin groups (Figure 1). Sao, was also measured in other experiments using lower doses (see legend to Figure 1). EVLW contents per gram dry blood-free lung for each group are illustrated in Figure 2. The groups of animals that receivedthe higher doses of amiloride, benzamil, and phenamil all had delayed clearance of lung water. The amiloride analogues with high specificity for the Na channel (benzamil and

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O'Brodovich, Hannam, and Ratti: Na Transport Inhibitors in Fetal Lung

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phenamil) had a greater effect than did amiloride (P < 0.05). Neither dimethyl amiloride nor phloridzin delayed lung water clearance. The glucose concentration in lung liquid sampled before the first breath was 30 ± 5.8 mg/dl (n = 5), which was significantly lower (P < 0.05) than the simultaneously deter- . mined blood glucose concentration of 87 ± 5.7mg/dl (n =5). Together, these data suggestthat amiloride'sability to block the clearance of fetal lung liquid after birth results from its effect on the Na channel and not on the Na-H antiport or the Nglucose symport. The 9-day-old guinea pigs that received 0.3 ml of 10-3 M benzamil or phenamil intratracheally did not have prolonged respiratory distress. EVLW 4 h after instillation was slightly but statistically significantly higher (P < 0.05) in the phenamil (5.3 ± 0.14; n = 4) group relative to the benzamil (4.9 ± 0.1; n = 4) and saline (4.4 ± 0.1; n = 5) groups. We have previously reported that the instillationof a similar volumeand concentration of amiloride does not increase EVLW in 9-day-old guinea pigs (4). All rabbits survived the experiments designed to measure alveolar amiloride and benzamil concentrations. However, the protocol was ended after 120 or 180 min because the airspace fluid no longer freely flowed up the airway when the high-frequency oscillator was temporarily disconnected. The ultracentrifuged supernatant from the airspace fluid of the two animals receiving saline without amiloride did not show spontaneous fluorescence, and with the use of this fluid as the solvent for the standard curve we were able to detect amiloride or benzamil concentrations greater than 10-7 M. Figure 3 illustrates that the airspace fluid amiloride concentration fell to less than 10-5 M between 2 and 3 h after instillation. Benzarnil concentrations decreased even more rapidly, with values falling to less than 10-6 M within 1 h. Comparable baseline resistances were obtained for the primary monolayer cultures of fetal alveolar epithelium bathed in HBSS and modified HBSS with NMDG substituted for Na (450 ± ohms-em? and 420 ± 630hms·cm-2 , respectively). The monolayers that were bathed by Nacontaining HBSS demonstrated a baseline PD = 2.4 ± 0.32 mV (apically negative) and an I; = 5.2 ± 0.25 p.A/cm2 ,

Figure 4. Different monolayers of fetal alveolar epithelium grown on polycarbonate filters were placed in either regular Hanks' balanced salt solution (HBSS) (control, open bars; n = 5) containing Na or in HBSS in which n-methyl-D-glucamine (NMDG, hatched bars; n = 6) was substituted for Na. The NMDG monolayers in Na-free but Cl-containing media did not generate a significant short circuit current (L) either during baseline measurements or after addition of 10-3 M terbutaline or 46 mM Na to the basolateral side (closed arrow). After addition of 46 mM Na to the apical side of the monolayer (open arrow), there was an abrupt increase in Is< from 0.3 to 7.7 p.A/cm2 • This suggests that a minimum of 96 % of the Is< in terbutaline-treated monolayers is due to the presence of Na on the apical side of the cells. The terbutaline-stimulated L, in the control monolayers and in the other monolayers that now contained 46 mM Na and 96 mM NMDG on their basal and apical surfaces was predominantly amiloride-sensitive (10-4 M) and was unaffected by subsequent administration of the Na/KI2CI cotransport inhibitor furosemide (Lasix, 10-3 M). Values are mean ± SEM.

whereas that monolayers bathed in HBSS with NMDG replacing Na did not generate a significant baseline I; or PD. Baseline and subsequent change in I; are illustrated in Figure 4 and demonstrated that virtually all (> 95 %) of the I; in unstimulated and terbutaline-treated monolayers was dependent upon Na transport from the apical to basolateral side of the cell. We did not detect a significant diffusion potential with the addition of 46 mM Na (0.12 ± 0.02 to 0.28 ± 0.09 mY). It is unknown whether this reflects our inability to detect such a change-or if the addition of Na-induced secondary events (e.g., .electrogenic Na-calcium exchange) could now work. Cell-free filters had R < 5 ohms-ern- and an undetectable PD and L. Additional studies demonstrated that the concentration of the drug resulting in 50 % of the maximal reduction in I; (IC5o) was estimated to be 0.3 x 10-6 M for amiloride and 0.3 x 10-7 M for benzamil (Figure 5). This indicates that Na transport in fetal type II epithelium shows high amiloride affinity. Apical and basal dimethyl arniloride (10-4 M) did not affect I,e (baseline = 3.6 ± 0.45 p.A·cm-1, drug = 3.4 ± 0.48 p.A·cm-1; n = 6).

Discussion Our present study using inhibitors of several different types ofNa transport provides new information in regard to the organ and cellular mechanisms of fetal lung liquid clearance after birth. They suggest that functional Na channels are required for normal postnatal lung water clearance and when they are blocked respiratory distress and hypoxemia occur

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that drug, and it should be noted that there is a significant amount of amiloride- or benzamile-insensitive Isc . Data points are fitted by eye. See text for details. n = three monolayers each log dose.

in the newborn animal. Neither the Na-H antiport nor the Na-glucose symport appears to playa physiologically important role in fluid clearance. This contrasts with their important role in Na transport from the adult rat's alveolar space (Na-glucose symport) and the intestine (Na-H antiport). In vitro fetal type II alveolar epithelium actively transports Na from its apical to basolateral side, and this Na transport accounts for more than 95 % of the resting and I3ragoniststimulated L, Amiloride inhibits most but not all of this Na-dependent L, whereas furosemide has no effect on the L. The dose-response effects of amiloride and the more specific Na channel inhibitor benzamil suggest the presence of high and not low amiloride affinity Na channels on the apical membrane of these cells. The in vivo dose-response characteristics of Na transport inhibitors are difficult to determine. Our experiments in which we instilled large volumes of saline containing either amiloride or benzamil into the lungs of the adult rabbits suggest that these compounds rapidly leave the airspace fluid (Figure 3). The relative lipophilicity of amiloride and its analogues helps explain these results. As discussed by Kleyman and Cragoe (9), the unprotonated form of amiloride is somewhat lipid soluble but markedly less lipid soluble than its analogues benzamil and phenamil, which have an enhanced specificity for the Na channel. The percentage of compound found in an octanol or chloroform fraction in 1:1 equilibrium with water is 5% for amiloride, 6% for dimethyl amiloride, 56 % for benzamil, and 99 % for phenamil. An additional factor that could have affected the relative concentration of amiloride and benzamil is a difference in residual airspace fluid volume caused by their different potencies for Na transport inhibition. Such differences, however, would not explain the decrease in concentration of the individual drug. It would be unwise to predict what the "effective" airspace concentrations of amiloride and benzamil were in these experiments because the concentrations changed so markedly over the time course of the experiment. In the guinea pig experiments, the initial airspace concentration was estimated to be lO-4 M when 0.3 ml of lO-3 M active agent was added be-

fore the first breath. By the end of the 4-h period, the lung liquid concentration was likely several logs lower (Figure 3). These studies have important implications for those using airspace instillation of such agents to investigate alveolar epithelial ion transport (for example, see reference 5). Amiloride and its analogues would be absorbed through the alveolar-capillary membrane and distributed to the remainder of the body. The effect of these compounds on EVLW was, however, likely due to their presence within the airspace fluid. If the entire amount was immediately absorbed, the amiloride dose would be less on an mg/kg basis than that used therapeutically in humans. If a systemic effect were seen, it would likely be a diuresis, which would tend to decrease not increase lung water. Phenamil and benzamil, two different amiloride analogues with enhanced specificity for the epithelial Na channel, impaired fetal lung liquid clearance. Although we did see a more potent effect, relative to amiloride, at the higher doses we had also expected to see an effect of benzamil and phenamil at the lower doses. Our present experiments in rabbits provide one possible explanation, i.e., these reversible blockers of the Na channel rapidly leave the lung thereby decreasing their airspace concentration (see above and Figure 3). We also could not demonstrate an increased effect of phenamil over benzamil. Although Garvin and associates (lO) reported irreversible inhibition with phenamil in toad bladders, it only occurred at Na concentrations less than 120 mM and therefore their observation is likely not relevant to our present in vivo experiments. In contrast, dimethyl amiloride, which has high specificity against the Na-H antiport, did not delay lung water clearance and surprisingly accelerated lung water clearance by a small but statistically significant amount (Figure 2). There is strong evidence suggesting that there is an Na-H antiport on fetal (18) and adult (19) type II alveolar epithelial cells. The principal physiologic role of the Na-H antiport, however, is the intracellular regulation of H ion concentration. It does participate in NaCI absorption in some regions of the intestine, but it utilizes an adjacent chloride-bicarbonate exchanger. One possible reason that we did not see a response is that the Na-H antiport of adult type II alveolar epithelial cells is inactive at pH > 7.0, as demonstrated by Nord and associates (19). Phloridzin, the Na-glucose symport inhibitor, did not affect lung water clearance in the present experiments. Others have reported experiments suggesting Na-glucose symport-like activity on the pulmonary epithelium of fetal lambs (11) and that phloridzin can slow fluid reabsorption from the airspace of adult rats (5). There are potential explanations why the epithelial Na-glucose symport did not make a physiologically significant contribution to lung liquid clearance at birth. First, there are only small concentrations of glucose within the lung liquid of fetal guinea pigs (see RESULTS) and negligible amounts in fetal lambs (11). Even when Barker and co-workers (11) artificially increased the glucose concentration of the fetal lamb lung liquid, there was only a small decrease in the rate of tracheal fluid flow. Second, the Na-glucose symport may contribute little to the transepithelialtransport of Na. We have previously been unable to demonstrate any effect of phloridzin on the bio-

O'Brodovich, Hannam, and Rafii: Na Transport Inhibitors in Fetal Lung

electric properties of fetal type II alveolar epithelial cells in primary culture (14), and loris and Quinton (20) have demonstrated that less than 10% of the total!" of adult tracheal epithelium results from the Na-glucose symport. It should be stressed, however, that at least some of the apparent differences in ion transport discussed above may be a result of species differences. For example, although tracheal epithelium in the dog secretes Cl, most other species, including the adult sheep, guinea pig, and monkey, absorb Na (21). The quantitatively important site of active Na transport in the fluid absorbing lung is unknown. It likely is within the distal lung units as they have an immense surface area (1'\.12 rn' in the newborn infant and 70 to 100 m' in the adult human). Although there are no data regarding the ion transport capabilities of type I alveolar epithelium, it is known that primary cultures of adult lung Clara cells (22) and fetal (see RESULTS) and adult type II alveolar epithelium (23, 24) transport Na. The Na absorption of the type II alveolar epithelium can be increased by l3-agonists and membrane permeant analogues of cAMP and also are inhibited by amiloride and amiloride analogues that preferentially inhibit Na channels (23, 24) (see RESULTS). Recent studies (25) have also shown that the alveolar surface area covered by type II cells is approximately 5 times greater in the perinatal as opposed to the adult lung. Together, these observations suggest that type II epithelium is a good choice for the in vitro study of Na absorption in the perinatal lung. Results obtained in cultured cells should, however, only be extrapolated to the in vivo situation with caution as we do not know how isolation and culture conditions affect the cells' ion transport characteristics. Our previous studies on the bioelectric properties of monolayers of fetal alveolar type II epithelium in primary culture suggested that these cells formed an Na-absorptive rather than Cl-secreting epithelium (14). This conclusion, however, was based upon the observed formation of domelike structures when cultured on plastic and on the use of pharmacologic-blocking agents on epithelial monolayers cultured on collagen-coated nitrocellulose filters. Although this is strong evidence for Na transport, we utilized ion substitution experiments to directly demonstrate that their baseline bioelectric properties and response to a 132-adrenergic agent is essentially entirely dependent upon the presence of Na ions within the fluid bathing their apical membrane. This response is compatible with observations made in monolayers of adult type II alveolar epithelial cells in which ion substitution (23) or 22Na flux (24) experiments demonstrated the active transport of Na ions and no evidence of Cl secretion. In the present experiments, amiloride inhibited most but not all of this Na-dependent I",. This is not without precedent because amiloride blocked only 50% of the 22Na flux across tracheal epithelium (26). The dose-response curves of amiloride- and benzamilsensitive I", yielded an IC so of 0.3 X 10-6 and 0.3 X 10-7 M, respectively. This is in close agreement with the usual values for inhibition of Na transport in intact fetal lungs (3), fetal airways (27), and through Na channels of most other epithelial tissues (9). Thus, our indirect studies suggest that Na transport occurs via channels with high rather than low

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amiloride affinity. These findings contrast with recent reports demonstrating that Na conductive pathways in vesicles made from adult rat type II alveolar epithelium (6) and some other epithelium (17) have low amiloride affinity. It is unknown whether this difference between fetal and adult rat alveolar type II epithelium is a developmental difference or if it results from the differences in methodology (i.e., intact monolayers versus membrane vesicles). It is premature to assume that it is the same Na channel or that there is only one type of ion channel involved in the transcellular transport of the Na within the fetal or the adult lung. For example, much of our information regarding amiloride-sensitive ion channels involved in transepithelial active Na transport suggests that they are highly selective for Na over K and have single-channel conductance in the range of 8 pS (28). Recently, however, Light and associates described an amiloride-sensitive nonselective cation channel that appears to playa role in the active Na transport across primary cultures of the renal internal medullary collecting duct (29). Our laboratory has also used patch clamp techniques and identified a very similar amiloride-sensitive 23 pS nonselective cation channel in fetal type II epithelium (30). This study demonstrates that when drugs that block Na channels are placed in the newborn's fluid-filled airspaces, there is clinically significant respiratory distress, hypoxemia, and marked slowing of the clearance of fluid. Other investigators have previously demonstrated that the ability of l3-agonists (1), membrane permeant analogues of cAMP (2), and arginine vasopressin (31) to convert the fetal lamb lung from a fluid-secreting to a fluid-absorbing organ is dependent upon gestational age with immature fetuses not responding to these agents. Thus, it is possible that respiratory distress in some newborn infants might result from immature active Na transport mechanisms. Our studies suggest that further research is required to determine the effect of fetal lung maturation on expression and signal transduction mechanisms influencing Na conductive channels within the lung and that the fetal type II cell is a good model for studying these processes. Acknowledgments: This research was supported by Grants-in-Aid T-1509 from the Heart and Stroke Foundation of Ontario and by Program Grant PG-42 from project 5 of the Medical Research Council of Canada. Dr. O'Brodovich is a Career Scientist of the Heart and Stroke Foundation of Ontario.

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