Airway surface liquid recovered by lavage with perfluorocarbon liquid in cats.

BACKGROUND: Airway surface liquid (ASL) is difficult to sample. Lavage with an immiscible perfluorocarbon (PFC) liquid to recover ASL was evaluated in cats. MATERIALS AND METHODS: Six wild-type cats underwent bronchoscopic lavage with a PFC (perfluorohexane), with the bronchoscope wedged in the feline equivalent of the right lower lobe. Two cats (control animals) were lavaged with a saline vehicle only. Four procedures were performed on each animal at 2-3-week intervals. Ionic composition of ASL was determined by flame photometry. RESULTS: Cats lavaged with PFC showed significantly more acute respiratory distress than those lavaged with saline (respiratory rate following procedure: PFC, 47 +/- 5 min-1 vs. saline, 27 +/- 2 min-1, P < 0.05; O2 saturation: PFC 80 +/- 1% vs. saline, 91 +/- 1%, P < 0.01). The PFC group also had clinical evidence of chronic respiratory compromise (mean respiratory rate before next anaesthetic; PFC, 37 +/- 2 min-1 vs. saline, 20 +/- 3 min-1, P < 0.01). The PFC-lavaged lungs demonstrated persistent radiographic changes and histological evidence of small airways obstruction with distal alveolar damage. Six PFC lavages yielded ASL samples (> 100 microL) which were sufficient for analysis. Mean (+/- SEM) ionic concentrations in these samples were Na+ 157.4 +/- 14.5 mmol L-1, Cl- 150.5 +/- 16.8 mmol L-1 and K+ 10.1 +/- 1.7 mmol L-1. CONCLUSIONS: Perfluorocarbon lavage can be used to collect unmodified ASL from the distal lung. However, repeated lavage with perfluorohexane was associated with significant pathological changes in this study.

The relative roles of passive surface forces and active ion transport in the modulation of airway surface liquid volume and composition.

Two hypotheses have been proposed recently that offer different views on the role of airway surface liquid (ASL) in lung defense. The "compositional" hypothesis predicts that ASL [NaCl] is kept low (<50 mM) by passive forces to permit antimicrobial factors to act as a chemical defense. The "volume" hypothesis predicts that ASL volume (height) is regulated isotonically by active ion transport to maintain efficient mechanical mucus clearance as the primary form of lung defense. To compare these hypotheses, we searched for roles for: (1) passive forces (surface tension, ciliary tip capillarity, Donnan, and nonionic osmolytes) in the regulation of ASL composition; and (2) active ion transport in ASL volume regulation. In primary human tracheobronchial cultures, we found no evidence that a low [NaCl] ASL could be produced by passive forces, or that nonionic osmolytes contributed substantially to ASL osmolality. Instead, we found that active ion transport regulated ASL volume (height), and that feedback existed between the ASL and airway epithelia to govern the rate of ion transport and volume absorption. The mucus layer acted as a "reservoir" to buffer periciliary liquid layer height (7 microm) at a level optimal for mucus transport by donating or accepting liquid to or from the periciliary liquid layer, respectively. These data favor the active ion transport/volume model hypothesis to describe ASL physiology.

Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel.

The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimer of three homologous subunits (alpha-, beta-, and gamma-subunits). To study the role of the beta-subunit in vivo, we analyzed mice in which the betaENaC gene locus was disrupted. These mice showed low levels of betaENaC mRNA expression in kidney (approximately 1%), lung (approximately 1%), and colon (approximately 4%). In homozygous mutant betaENaC mice, no betaENaC protein could be detected with immunofluorescent staining. At birth, there was a small delay in lung-liquid clearance that paralleled diminished amiloride-sensitive Na+ absorption in tracheal explants. With normal salt intake, these mice showed a normal growth rate. However, in vivo, adult betaENaC m/m mice exhibited a significantly reduced ENaC activity in colon and elevated plasma aldosterone levels, suggesting hypovolemia and pseudohypoaldosteronism type 1. This phenotype was clinically silent, as betaENaC m/m mice showed no weight loss, normal plasma Na+ and K+ concentrations, normal blood pressure, and a compensated metabolic acidosis. On low-salt diets, betaENaC-mutant mice developed clinical symptoms of an acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and decreased blood pressure), indicating that betaENaC is required for Na+ conservation during salt deprivation.

Acetylcholine-induced liquid secretion by bronchial epithelium: role of Cl- and HCO3- transport.

Inhibitors of Cl- and HCO-3 secretion reduce acetylcholine-induced liquid, but not mucin, secretion by bronchial submucosal glands [S. K. Inglis, M. R. Corboz, A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L372-L377, 1997]. The present study quantified contributions of Cl- and HCO-3 transport to volume and composition of acetylcholine-induced liquid secretion by airway epithelium. When distal bronchi were excised from 33 pigs and treated with 10 microM acetylcholine, the airways secreted 13.4 +/- 0.7 microliter. cm-2. h-1. Bumetanide (10 microM) pretreatment reduced acetylcholine-induced liquid and Cl- secretion rates by approximately 70%, but HCO-3 secretion fell by only 40%. Dimethylamiloride (DMA; 100 microM) pretreatment reduced Cl- secretion rates by approximately 15%, but HCO-3 secretion fell 47%. DMA alone had little effect on liquid secretion. When airways were pretreated with both bumetanide and DMA, acetylcholine-induced liquid secretion was nearly abolished. We conclude that about three-fourths of acetylcholine-induced liquid secretion in distal bronchi is dependent on Cl- secretion. Most of the remaining response is driven by HCO-3 secretion. We speculate that the principal source of this liquid is submucosal glands. Crossover inhibition of bumetanide on HCO-3 secretion and DMA on Cl- secretion implies modulation of anion secretion secondary to changes in cell electrolyte composition.

Role of gammaENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism.

Genetic evidence supports a critical role for the epithelial sodium channel (ENaC) in both clearance of fetal lung liquid at birth and total body electrolyte homeostasis. Evidence from heterologous expression systems suggests that expression of the alphaENaC subunit is essential for channel function, whereas residual channel function can be measured in the absence of beta or gamma subunits. We generated mice without gammaENaC (gammaENaC -/-) to test the role of this subunit in neonatal lung liquid clearance and total body electrolyte balance. Relative to controls, gammaENaC (-/-) pups showed low urinary [K+] and high urinary [Na+] and died between 24 and 36 h, probably from hyperkalemia (gammaENaC -/- 18.3 mEq/l, control littermates 9.7 mEq/l). Newborn gammaENaC (-/-) mice cleared lung liquid more slowly than control littermates, but lung water at 12 h (wet/dry = 5.5) was nearly normal (wet/dry = 5.3). This study suggests that gammaENaC facilitates neonatal lung liquid clearance and is critical for renal Na+ and K+ transport, and that low level Na+ transport may be sufficient for perinatal lung liquid absorption but insufficient to maintain electrolyte balance by the distal nephron. The gammaENaC (-/-) newborn exhibits a phenotype that resembles the clinical manifestations of human neonatal PHA1.

Effects of adenosine, ATP, and UTP on chloride secretion by epithelia explanted from fetal rat lung.

Catecholamines trigger the switch from liquid secretion to absorption by perinatal lung, but regulation of Cl- and liquid secretion by pulmonary epithelia early in lung development (low [catecholamine]) is unknown. We looked for evidence for P1 and P2 receptors that mediate Cl- secretion in 14-d distal lungs and 14- and 18-d tracheas explanted from fetal rats (term = 22 d). We measured amiloride-insensitive transepithelial voltage changes induced by ATP, UTP, or adenosine. Explants were hyperpolarized by all three agonists and by terbutaline, a beta-adrenergic agonist and Cl- secretagogue. Whereas adenosine, ATP, or UTP injected into 14-d explant lumena, or adenosine added to the tracheal bath, induced hyperpolarization with EC50 of 2-15 microM EC50, values for all three agonists in the distal lung bath or ATP or UTP in the tracheal bath were five times greater. By 18 d, EC50 values for agonists in the bath were comparable to those for lumenal agonists (3-12 microM). In contrast, microinjection of terbutaline into all explant lumena (final concentration = 3 x 10(-5) M) induced minimal hyperpolarization, whereas the same concentration in the bath raised bioelectric potential difference maximally. We conclude that 1) beta-adrenergic receptors are present on the basolateral membranes of cells of the pulmonary epithelium early in lung development, and 2) adenosine, ATP, and UTP receptors are present in apical membranes throughout lung epithelial development, but basolateral receptors for these agonists in distal lung or ATP/UTP in trachea function later in gestation. The putative distribution of P1 and P2 receptors suggests a role for agonists released from pulmonary epithelial cells in the regulation of liquid secretion early in lung development.

Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease.

The pathogenesis of cystic fibrosis (CF) airways infection is unknown. Two hypotheses, "hypotonic [low salt]/defensin" and "isotonic volume transport/mucus clearance," attempt to link defects in cystic fibrosis transmembrane conductance regulator-mediated ion transport to CF airways disease. We tested these hypotheses with planar and cylindrical culture models and found no evidence that the liquids lining airway surfaces were hypotonic or that salt concentrations differed between CF and normal cultures. In contrast, CF airway epithelia exhibited abnormally high rates of airway surface liquid absorption, which depleted the periciliary liquid layer and abolished mucus transport. The failure to clear thickened mucus from airway surfaces likely initiates CF airways infection. These data indicate that therapy for CF lung disease should not be directed at modulation of ionic composition, but rather at restoring volume (salt and water) on airway surfaces.

Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects.

To test whether a major contribution of airways epithelial ion transport to lung defense reflects the regulation of airway surface liquid (ASL) ionic composition, we measured ASL composition using the filter paper technique. On nasal surfaces, the Cl- concentration (approximately 125 meq/liter) was similar to plasma, but the Na+ concentration (approximately 110 meq/liter) was below plasma, and K+ concentration (approximately 30 meq/liter) above plasma. The resting ASL osmolarity [2(Na+ + K+); 277 meq/liter] approximated isotonicity. There were no detectable differences between cystic fibrosis (CF) and normal subjects. In the lower airways, the Na+ concentrations were 80-85 meq/liter, K+ levels approximately 15 meq/liter, and Cl- concentrations 75-80 meq/liter. Measurements of Na+ activity with Na(+)-selective electrodes and osmolality with freezing point depression yielded values consistent with the monovalent cation measurements. Like the nasal surfaces, no differences in cations were detected between CF, normal, or chronic bronchitis subjects. The tracheobronchial ASL hypotonicity was hypothesized to reflect collection-induced gland secretion, a speculation consistent with observations in which induction of nasal gland secretion produced hypotonic secretions. We conclude that there are no significant differences in ASL ion concentrations between CF, normal, and chronic bronchitis subjects and, because ASL ion concentrations exceed values consistent with defensin activity, the failure of CF lung defense may reflect predominantly factors other than salt-dependent defensins.

Cl- secretion by trachea of CFTR (+/-) and (-/-) fetal mouse.

The absence of pathologic changes in newborn cystic fibrosis (CF) lung suggests that the fetal CF lung is inflated with a normal volume of liquid and that Cl- is secreted through paths other than the cystic fibrosis transmembrane conductance regulator (CFTR)-associated Cl- channel. We studied liquid content of distal lung and transepithelial electrical potential difference (PD) of cultured cystic tracheal explants from 16 to 19 day gestation fetal mice of CFTR (+/-)(heterozygous) females that were mated with CFTR (-/-) "knockout" males. Distal lung water content was not affected by fetal genotype. Basal PDs were not different (CFTR (+/-), 8.6 mV, and CFTR (-/-), 9.1 mV), and PDs of both groups were inhibited by intraluminal injection of amiloride (10(-4) M) (-25%) and after addition of bumetanide (10(-4) M) to the bath (-40%). Terbutaline (3 x 10(-5) M) induced a similar increase in PD (about 65%) in both groups. Intraluminal injection of ionomycin (2 x 10(-5) and 5 x 10(-6) M) raised PD in both groups (CFTR (+/-) by 32 and 27% and CFTR (-/-) by 41 and 11%). All of the increase in PD induced by terbutaline and ionomycin was inhibited by bumetanide. The PD response to terbutaline was not attenuated by pretreatment with ionomycin or the Ca2+ chelator BAPTA (10(-4) M). Ionomycin or ATP, but not terbutaline, increased intracellular Ca2+ concentration of isolated cultured tracheal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of gas composition on liquid secretion by explants of distal lung of fetal rat in submersion culture.

Throughout gestation, fetal lung is filled with liquid secreted by the pulmonary epithelium. Factors responsible for secretion and for induction of liquid absorption by postnatal lung are poorly understood. We studied effects of "fetal" (3% O2-8% CO2) and "postnatal" (21% O2-5% CO2) gas tensions and of low hormone concentrations [media with 10% charcoal-stripped fetal bovine serum (stFBS) or unstripped FBS] on water content and morphology of distal lung explanted from 14-, 20-, and 22-day fetal (term, 22 days) and 2-day neonatal rats and incubated in submersion culture. Water-to-dry weight ratios of freshly excised fetal whole lung at 20 (6.2) or 22 days (7.0) were greater than that of 2-day postnatal lung (4.7). Culture conditions did not affect water-to-dry weight ratio of cyst-forming 14-day explants (10.8) or acystic 2-day postnatal explants (2.4). Fetal gases and stFBS supported cyst formation in [and high water/dry weight (9.2, 12.6) of] 20- and 22-day explants. Cysts also formed in 20-day explants exposed to postnatal gases and stFBS (water/dry weight = 6.5). Other conditions resulted in minimal cyst formation by 20- and 22-day explants and in water/dry weight similar to that of freshly excised and drained distal lung from 22-day fetuses (2.1). Cysts were lined with cuboidal and thin epithelial cells. No cells were ciliated. We conclude that 1) secretion dominates liquid flow across epithelia of fetal rat lung until birth, 2) alveolar epithelium contributes to this secretion, and 3) liquid secretion by fetal distal lung late in gestation is regulated by gas composition.

Functional consequences of heterologous expression of the cystic fibrosis transmembrane conductance regulator in fibroblasts.

We studied the consequences of cystic fibrosis transmembrane conductance regulator (CFTR) expression in NIH-3T3 fibroblasts as a model for the effects of virally transduced CFTR expression in non-epithelial cells. Fibroblasts were infected with a retrovirus vector that contained the human CFTR and neor cDNAs. We selected and expanded G418-resistant clones that encompassed a range of CFTR expression. CFTR-mediated Cl-conductance function was measured as whole cell current, and CFTR protein was quantitated by immunoblot analysis. Overall, there was a good relationship between CFTR protein levels and CFTR-mediated Cl- conductance. Some clones had consistently high basal levels of CFTR-mediated Cl- conductance. This variation in function was partially explained by CFTR protein levels and was not due to clonal variation in cAMP metabolism. High levels of CFTR expression were associated with depolarization of fibroblast membrane potential. The CFTR-expressing clones with the largest basally active CFTR Cl- conductances and the most depolarized membrane potentials also exhibited slower growth rates. These results suggest that potential side effects of gene replacement therapy for cystic fibrosis include functional consequences of CFTR expression in non-epithelial cells.

Effects of hormones on potential difference and liquid balance across explants from proximal and distal fetal rat lung.

1. Fetal rat tracheas and lung buds form liquid-filled cysts in submersion culture. The volume that accumulates in cysts is driven by active Cl- secretion. 2. We examined the effects, on these explants, of hormones that induce liquid absorption by fetal sheep lung in vivo. Explants were impaled with microelectrodes to measure potential difference (PD). Liquid was estimated from explant weight. 3. Water/dry weight ratio of lung buds and tracheas after 8 days in culture averaged 12 and 22. Exposure of triiodothyronine (T3) and hydrocortisone (HC) followed by a physiological dose of adrenaline on day 7 for 24 h or by a maximal dose of terbutaline on day 8 for 4 h induced a 35% decrease in water/dry weight ratio of distal buds but not tracheas. No hormone, or combination of two hormones, affected the ratio for tracheas or lung buds. 4. Basal PDs of tracheas (18.9 mV) and lung buds (3.7 mV) were increased about 50% by terbutaline. The terbutaline response was inhibited by bumetanide, but not by amiloride injected into the cysts. 5. T3 and HC pretreatment reduced basal PD by one-third. Subsequent exposure to terbutaline raised the PD of hormone-pretreated lung buds by more than 150%, a response that was blocked by amiloride, but was antagonized minimally by bumetanide. Responses of hormone-pretreated tracheas were not different from those of untreated tracheas. 6. We conclude that: (a) absorption of liquid from lung buds is driven by an amiloride-sensitive process (active Na+ transport?) and (b) only distal lung contributes to the adrenaline-sensitive reabsorptive process required for perinatal adaptation to air breathing.

L-glutamine with D-glucose stimulates oxidative metabolism and NaCl absorption in piglet jejunum.

To explore the relationship between intestinal fluid absorption and oxidative metabolism, we measured the effects of amino acids and glucose on piglet jejunal ion transport and oxygen consumption (QO2) in vitro. Jejunal QO2 was stimulated by L-glutamine and D-glucose but not by the nonmetabolizable organic solutes methyl beta-D-glucoside or L-phenylalanine. QO2 was maximally enhanced by the combination of D-glucose and L-glutamine (5 mM). Even though 5 mM L-glutamine was previously found to be insufficient to stimulate NaCl absorption, 5 mM L-glutamine enhanced jejunal NaCl flux when combined with equimolar mucosal D-glucose. Either D-glucose or methyl beta-D-glucoside caused an increase in short-circuit current (Isc), an increase in Na+ absorption in excess of Isc, and a decrease in Cl- secretion, when L-glutamine was substituted for D-glucose (10 mM) on the serosal side. This relationship suggests that mucosal sugars, if combined with L-glutamine, enhance neutral NaCl absorption as well as electrogenic Na+ flow. (Aminooxy)acetate, an inhibitor of alanine aminotransferase, abolished the stimulation of QO2 and the NaCl-absorptive response to L-glutamine. We conclude that the oxidative metabolism fueled by L-glutamine is linked to a NaCl-absorptive mechanism in the intestine. We propose that the CO2 produced by glutamine metabolism yields carbonic acid, which dissociates to H+ and HCO3-, which may stimulate parallel antiports in the apical membrane.

Bioelectric properties of cultured epithelial monolayers from distal lung of 18-day fetal rat.

In vivo studies of fetal sheep suggest that the liquid present in the lumen of the lung throughout fetal life is derived from Cl- secretion by the pulmonary epithelium. Monolayer preparations of enriched epithelial cells from distal fetal rat (18-day gestation) lung, grown in serum-free media, were histologically similar to acinar (prealveolar) structures of fresh tissue. In Ussing chambers, basal transepithelial potential difference (PD), calculated equivalent short-circuit current (Ieq), and transepithelial resistance (R) were 4.4 +/- 0.3 mV (matrix positive), 35.6 +/- 1.6 microA/cm2, and 120.0 +/- 4.0 omega cm2, respectively. Ouabain (10(-3) M) eliminated 57% of basal Ieq within 30 min, amiloride (10(-4) M) induced a 13% fall in Ieq, and phlorizin (10(-4) M) had no effect on bioelectric properties. Diphenylamine-2-carboxylate (DPC, 3 x 10(-3) M) inhibited Ieq by 50%. Bumetanide had no effect on baseline bioelectric parameters. The hyperpolarization that accompanied apical or bilateral replacement of Cl- and was enhanced by terbutaline suggested an apical Cl- permselectivity. Effects of Na+ replacement on amiloride-pretreated monolayers were consistent with Na(+)-dependent Cl- secretion or amiloride-insensitive pathways. Under these growth conditions, this preparation exhibits bioelectric characteristics that are compatible with Cl- secretion and Na+ absorption. The mechanism of Cl- secretion may be similar to that of airways but is uniquely bumetanide insensitive.

Volume flow across the alveolar epithelium of adult rat lung.

We separated the solute and water flow across the alveolar epithelium from flow across airway epithelia of the adult rat. Small volumes (0.5-1.0 ml) of Krebs-Ringer bicarbonate (KRB) were trapped in the distal air space of the isolated vascular-perfused left lung lobes while the airways were blocked by immiscible O2-carrying fluorocarbon. Lobe weight was lost or gained in response to colloid gradients and was raised by metabolic inhibitors but did not change with only fluorocarbon in the air space or in response to modifiers of epithelial ion transport. When serum was added to the KRB-colloid perfusion, weight loss occurred in the absence of a colloid gradient (3.4 ml/min) and was Na+ dependent (inhibited by luminal Na(+)-free KRB). The change in the concentration of blue dextran in liquid sampled by micropuncture from subpleural alveoli was smaller than expected from lobe weight under basal conditions or with a colloid gradient, even though the volume marker accurately detected edema formation (weight gain) induced by metabolic inhibitors. We conclude that 1) weight changes represent volume absorption from the air spaces, 2) serum stimulates a Na+ absorptive process, and 3) by exclusion, small airways and/or other subpopulations of alveoli are the site of this absorption.

Alveolar transepithelial potential difference and ion transport in adult rat lung.

The complex morphology of the mammalian lung complicates characterization of solute transport across the intact alveolar epithelium. We impaled the subpleural alveolar epithelium with microelectrodes and measured the transepithelial potential difference (PD) of the liquid-filled vascular-perfused left lobe of the rat lung. When the air space was filled entirely with Krebs-Ringer-bicarbonate, the PD was 4.7 mV (lumen negative). The PD was not affected significantly by agents that modify either Na+ or Cl- transport, but replacement of luminal Cl- with gluconate resulted in a fourfold hyperpolarization, a response also noted for large airways. When the airways were blocked by an immiscible nonconducting fluorocarbon, basal PD was not different from unblocked lobes (4.0 mV) but was inhibited 73% by luminal amiloride. Cl(-)-free Krebs-Ringer-bicarbonate blocked in the alveoli with fluorocarbon did not induce hyperpolarization. This result suggests that 1) Cl- permselectivity of the alveolar epithelium is less than that of large airway epithelium and 2) airway PD dominates the voltage across the liquid-filled lung, even when measurements are made from alveoli. When airways are blocked by fluorocarbon, the PD across the alveolar epithelium is largely dependent on Na+ flow through a path with amiloride-sensitive channels.

Effects of Na saccharin feeding and urine on barrier properties of excised rat urinary bladder.

When male rats of certain strains are fed a diet with 3% or more Na saccharin, their urinary bladders develop epithelial hyperplasia and a greater incidence of tumors. Since the daily dose of saccharin is high, a link between tumor formation and the disruption of urothelial physiologic and biochemical processes has been sought. We fed male and female Sprague-Dawley rats a saccharin-free or 7.5% Na saccharin diet for 1 month. Excised bladders were mounted in flux chambers and exposed to Krebs-Ringer bicarbonate solution (KRB) or urine. Bioelectric properties and 22Na, 36Cl, and [14C]mannitol or [3H]mannitol unidirectional fluxes were measured by conventional techniques. No differences were noted between bladders from male and female animals or between Na saccharin-fed animals and animals fed the saccharin-free diet. When both surfaces of the epithelium were exposed to KRB, transepithelial dc conductance fell over 4 hr to 50% of the initial value. Conductance averaged 1.4 mS/cm2. Transepithelial potential difference (PD) was usually lumen negative and averaged 0.7 mV. Unidirectional permeability coefficients for 36Cl, 22Na, and radiomannitol were symmetric, proportional to conductance, and followed a rank order compatible with unrestricted passive diffusion. Exposure of the bladder lumen to urine from animals fed saccharin-free or Na saccharin diet hyperpolarized the transepithelial PD by more than 5 mV and raised conductance nearly threefold. Permeability coefficients remained symmetric and compatible with passive diffusion. Exposure of the lumen to solutions with the K+, Na+, and Cl concentrations and osmolality of urine simulated the conductance and PD effects of urine. We conclude that Na saccharin feeding or urine with saccharin does not uniquely affect the permeability of the excised preparation. Small hydrophilic solutes appear to cross the bladder epithelium through paracellular channels which increase in aggregate area during exposure of the lumen to urine. The hyperpolarization induced by lumenal urine is the consequence of the transepithelial K+ gradient.

Volume and ion transport by fetal rat alveolar and tracheal epithelia in submersion culture.

The complex architecture of the mammalian lung has hindered measurements of permeability of and transport by the intact alveolar epithelium. We compared properties of fetal rat alveolar buds and tracheas in submersion culture by microelectrode and micropuncture techniques. Both alveolar buds and tracheas form cysts that accumulate liquid for up to 3 wk in culture. The transepithelial electric potential difference (PD) of alveolar buds and tracheas (lumen negative, 1.2-3.5 and 8-21 mV, respectively) was abolished by metabolic inhibitors or ouabain in the bath. Despite the difference in PDs, both epithelial regions, after 1 or 2 wk in culture, exhibited the following. 1) Cl- activities and concentrations in luminal liquid were 13 and 34% higher than the bathing medium; Na+ and K+ concentrations were not different. 2) Bumetanide inhibited PD by 70%, whereas terbutaline increased PD by 45%. 3) Amiloride injected into the cyst lumen induced a 20% decrease in the PD of 1-wk explants but did not affect 2-wk preparations. Replacement of bath Na+ by choline decreased the PD of tracheas by 85% but did not change alveolar PD in the presence or absence of bumetanide. The pattern of cyst liquid composition and PD responses to drugs suggests that fetal rat alveolar and tracheal epithelia secrete Cl-, which drives volume flow.

Paths of ion transport across canine fetal tracheal epithelium.

Fluid secretion by the fetal sheep lung is thought to be driven by secretion of Cl- by the pulmonary epithelium. We previously demonstrated Cl- secretion by tracheal epithelium excised from fetal dogs and sheep. In this study we characterized the ion transport pathways across fetal canine tracheal epithelium. The transport of Na+ and Cl- across trachea excised from fetal dogs was evaluated from transepithelial electrical properties and isotope fluxes. Under basal conditions the tissues were characterized by a lumen-negative potential difference (PD) of 11 mV and conductance of 5.2 mS/cm2. The short-circuit current (Isc) was 43 microA/cm2 (1.6 mueq.cm-2.h-1). Basal Na+ flows were symmetrical, but net Na+ absorption (1.1 mueq.cm-2.h-1) could be induced by exposure of the luminal surface to amphotericin B (10(-6) M). Bilateral replacement of Na+ reduced Isc by 85%. Replacement of submucosal Na+ or exposure to submucosal furosemide (10(-4) M) reduced net Cl- secretion by 60-70%. Luminal exposure to indomethacin (10(-6) M) induced a 50% decrease in Isc, whereas isoproterenol (10(-6) M) increased Isc by 120%. The properties of the Cl- secretory pathway across fetal dog trachea are consistent with the model proposed for Cl- secretion across adult dog trachea and other Cl- -secreting tissues (e.g., bullfrog cornea and shark rectal gland). The absence of basal Na+ absorption by fetal dog trachea probably reflects limited apical membrane Na+ permeability.

Bioelectric properties and ion transport across excised canine fetal and neonatal airways.

Knowledge of liquid secretion by fetal lung stems from studies of sheep. We extended these studies to dogs and examined the persistence of the fetal pattern of airway epithelial permeability and ion transport in the neonatal animal. Plasma and lung liquid from fetal dogs were analyzed for Na+, K+, Cl-, and HCO3-. Only the Cl- concentration of fetal lung liquid (129 meq/l) was significantly different from that of fetal plasma (111 meq/l). Segments of trachea from fetal and neonatal (less than 1, 7-10, and 21-46 days after birth) dogs were excised and mounted in flux chambers. The transepithelial potential difference (PD) of all tissues was oriented lumen negative (9.8-14.8 mV). Under short-circuit conditions, unidirectional Na+ flows were symmetrical. Cl- was secreted, and the secretion was equivalent to short-circuit current (Isc). Cl- secretion persisted under open-circuit conditions. Lobar bronchi from 21- to 46-day neonates absorbed Na+ (1.9 mueq.cm-2.h-1), but unidirectional flows of Cl- were symmetrical. Amiloride (10(-4) M) reduced Isc of neonatal bronchi by 47% but did not affect fetal bronchi. Isoproterenol increased Isc of both fetal (33%) and neonatal (40%) bronchi. These responses suggest that fetal bronchi do not absorb Na+ but can be stimulated to secrete Cl-. We conclude that Cl- secretion by epithelium of large airways may contribute to fetal lung liquid production, but it is unlikely that the tracheal epithelium is involved in fluid absorption at birth. Whereas fetal bronchi appear to secrete Cl-, neonatal bronchi absorb Na+.(ABSTRACT TRUNCATED AT 250 WORDS)