Abnormal ion permeation through cystic fibrosis respiratory epithelium.

The epithelium of nasal tissue excised from subjects with cystic fibrosis exhibited higher voltage and lower conductance than tissue from control subjects. Basal sodium ion absorption by cystic fibrosis and normal nasal epithelia equaled the short-circuit current and was amiloride-sensitive. Amiloride induced chloride ion secretion in normal but not cystic fibrosis tissue and consequently was more effective in inhibiting the short-circuit current in cystic fibrosis epithelia. Chloride ion-free solution induced a smaller hyperpolarization of cystic fibrosis tissue. The increased voltage and amiloride efficacy in cystic fibrosis reflect absorption of sodium ions across an epithelium that is relatively impermeable to chloride ions.

Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation.

The transepithelial potential difference (PD) of cystic fibrosis (CF) airway epithelium is abnormally raised and the Cl- permeability is low. We studied the contribution of active Na+ absorption to the PD and attempted to increase the Cl- permeability of CF epithelia. Nasal epithelia from CF and control subjects were mounted in Ussing chambers and were short-circuited. The basal rate of Na+ absorption was raised in CF polyps compared with control tissues. Whereas beta agonists induced Cl- secretion in normal and atopic epithelia, beta agonists further increased the rate of Na+ absorption in CF epithelia without inducing Cl- secretion. This unusual effect is not due to an abnormal CF beta receptor because similar effects were induced by forskolin, and because cAMP production was similar in normal and CF epithelia. We conclude that CF airway epithelia absorb Na+ at an accelerated rate. The abnormal response to beta agonists may reflect a primary abnormality in a cAMP-modulated path, or a normal cAMP-modulated process in a Cl- impermeable epithelial cell.

Abnormal apical cell membrane in cystic fibrosis respiratory epithelium. An in vitro electrophysiologic analysis.

The transepithelial chloride permeability of airway and sweat ductal epithelium has been reported to be decreased in patients with cystic fibrosis (CF). In the present study, we investigated whether the airway epithelial defect was in the cell path by characterizing the relative ion permeabilities of the apical membrane of respiratory epithelial cells from CF and normal subjects. Membrane electric potential difference (PD) and the responses to luminal Cl- replacement, isoproterenol, and amiloride were measured with intracellular microelectrodes. The PD across the apical barrier was smaller for CF (-11 mV) than normal (-29 mV) epithelia whereas the PD across the basolateral barrier was similar, (-26 and -34 mV respectively). In contrast to normal nasal epithelium, the apical membrane in CF epithelia was not Cl- permselective and was not responsive to isoproterenol. Amiloride, a selective Na+ channel blocker, induced a larger apical membrane hyperpolarization and a greater increase in transepithelial resistance in CF epithelia. Both reduced apical cell membrane Cl- conductance and increased Na+ conductance appear to contribute to the abnormal function of respiratory epithelia of CF patients.

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.

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.

Isolated epithelial cells of the toad bladder. Their preparation, oxygen consumption, and electrolyte content.

Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. Q(OO2), sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O(2) at a rate of 4 microl hr(-1) dry wt(-1). Q(OO2) was increased 50% by ADH (100 U/liter) or by cyclic 3',5'-AMP (10 mM/liter). Na(+)-free Ringer's depressed the Q(OO2) by 40%. The Q(OO2) of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na(+)-free Ringer's but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na(+) lack or ADH. The intracellular Na(+) and K(+) concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H(2)O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na(+) concentrations. Cells unresponsive to ADH and Na(+) lack had high sucrose spaces and low transcellular membrane gradients of Na(+), K(+), and Cl(-). The results suggest that trypsin and EDTA disaggregation damage the active Na(+) transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally.

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.

Chemical modulation of alveolar epithelial permeability.

The volume and composition of fluid on the surface of the alveoli can affect alveolar ventilation, gas diffusion, and macrophage function. The passive permeability and active processes of the alveolar epithelial lining play a role in regulating surface fluid and are a potential site of damage by airborne chemicals. Like other epithelial barriers, the alveolar lining is permeable to lipophilic substances but restricts the transmural flow of small ions and hydrophilic nonelectrolytes (equivalent pore radius ca. 0.5-1.5 nm). The mammalian fetal lung and alveolar sacs of the adult bullfrog secrete Cl- and K+ into the airspace. Secretion by the fetal lung ceases at birth. Many environmental agents increase the permeability of the capillary endothelium and/or respiratory epithelium and induce pulmonary edema. Studies with bullfrog alveolar sacs have demonstrated that selective effects may or may not be followed by general derangement of the epithelial barrier. Exposure of the luminal surface to HgCl2 (10(-6) to 10(-4) M) induces a selective increase in Cl- secretion that is followed by a fall in transport and a general increase in ion permeation. CdCl2 (10(-5) to 10(-3) M) depresses ciliomotion on cells on the trabecula of the alveolus but does not affect Cl- secretion or transepithelial conductance. HNO3, like other mineral acids, increases conductance and the radii or pores in the barrier, whereas NaNO3 selectively inhibits Cl- secretion. Amphotericin B(10(7) to 10(-5) MJ) induces K+ secretion into the lumen of both bullfrog and rat lung. We conclude that environmental agents induce changes in epithelial function that may compromise the lung's ability to regulate respiratory fluid without destroying the characteristic permeability of the epithelial lining.

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.

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.

Effects of raised osmolarity on canine tracheal epithelial ion transport function.

Evaporation of water from upper airway surfaces increases surface liquid osmolarity. We studied the effects of raised osmolarity of the solution bathing the luminal surface of excised canine tracheal epithelium. Osmolarity was increased by adding NaCl or mannitol. NaCl addition induced a concentration-dependent fall in short-circuit current and a rise in transepithelial conductance (-33% and +14% per 100 mosM, respectively). Unidirectional isotopic fluxes of 22Na, 36Cl, and [14C]mannitol were measured in short-circuited tissues in the base-line state and after addition of NaCl or mannitol to an isotonic mucosal solution. NaCl addition (75 mM) caused a 50% increase in conductance (G) and a parallel increase in [14C]mannitol permeability (Pmann), indicating an increase in paracellular permeability. Net Cl- secretion was reduced 50%, and net Na+ absorption was unchanged despite an increased chemical gradient for absorption, indicating an inhibition of active ion transport. Mannitol addition (150 mM) abolished net Na+ absorption but did not increase G or Pmann or change net Cl- secretion. These results suggest that responses to increased tracheal surface liquid osmolarity during spontaneous breathing may occur in both the cellular (inhibition of active Na+ and Cl- transport) and paracellular (increased [14C]mannitol permeability) compartments of the mucosa.

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)

Effects of metabolic inhibition on ion transport by dog bronchial epithelium.

Mammalian bronchial epithelium absorbs Na+ under basal conditions, but Cl- secretion can be induced. We studied the effects of several modes of metabolic inhibition on the bioelectric properties and solute permeability of dog bronchial epithelium mounted in Ussing chambers. Net Na+ absorption and short-circuit current were inhibited by approximately 75% by hypoxia or by 10(-3) M NaCN. The reduced net Na+ absorption was characterized by a decrease in absorptive flux and an increase in backflux. The latter change was proportional to an increase in permeability to [14C]mannitol, implying that solute flow through a paracellular shunt was increased. In contrast, the reduction of conductance expected from exposure to amiloride (0.94 +/- 0.15 ms/cm2 or 12%) was abolished by NaCN pretreatment. Metabolic inhibition also decreased epithelial conductance and unidirectional Cl- fluxes by approximately 25%. NaCN rapidly and reversibly inhibited the hyperpolarization of potential difference (PD) induced by low luminal bath [Cl-]. This effect was mimicked by the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid. Because the transepithelial Cl- diffusion PD reflects, in part, the depolarization of the Cl- -conductive apical cell membrane, metabolic inhibition appears to affect this path. We conclude that metabolic inhibition not only decreased net ion transport by dog bronchial epithelium but also inhibited cellular Na+- and Cl- -conductive pathways and increased paracellular permeability.

Abnormal respiratory epithelial ion transport in cystic fibrosis.

The respiratory epithelium of cystic fibrosis patients exhibits excessive sodium (and volume) absorption and an absence of chloride (and volume) secretion in response to usual stimuli. These abnormalities likely contribute to the pathophysiology of cystic fibrosis lung disease, and provide a rationale for a novel therapeutic approach to this problem.

Ultrastructural and electrophysiological maturation of the chick tegmentum vasculosum.

We have examined the ultrastructural and electrophysiological events associated with the embryonic development of the tegmentum vasculosum, the ion-transporting epithelium in the chick cochlear duct. The cytodifferentiation of the light and dark cells in the epithelium from embryonic day 6 through post-hatching day 7 was studied with transmission electron microscopy. The predominant ultrastructural change in the developing tegmentum vasculosum was the elaboration of the complex basolateral infoldings on the dark cells from embryonic day 11 through post-hatching day 7. The relationship between the development of the endocochlear transepithelial electrical potential difference (PD) and the cytodifferentiation of the tegmentum vasculosum was examined with in vitro studies. Microelectrode impalements of the scala media showed that the positive endocochlear PD was first detectable on embryonic day 20 but did not reach a mature value of +16 mV until post-hatching day 7. Maturation of the endocochlear PD paralleled the time during which the dark cells in the tegmentum vasculosum displayed the most extensive increase in basolateral infoldings. This correlation suggests that the development of the endocochlear PD may result from an increase in the number of Na+-K+ pump sites located on the basolateral infoldings of the dark cells.

Ion transport across the excised bullfrog lung.

Fluxes of ions and water across the short-circuited, excised bullfrog lung were determined by radioisotope techniques. The unidirectional flows of Na+, K+, Ca++, TcO4 minus, HCO3 minus, gluconate, rho-aminohippurate, dinitrophenolate, SO4 equal to, and water were symmetrical. Both HCO3 minus fluxes were reduced by acetazolamide. In contrast, Cl minus, Br minus I minus, and SCN minus movement from serosa to mucosa exceeded the flux in the opposite direction. Net Cl minus transport followed the kinetics of a saturable process and was inhibited by dinitrophenol and hypoxia. These results indicate an active secretion of halide anions and SCN minus into the lumen. Attempts to demonstrate Br minus anatagonism of Cl minus transport were equivocal. Cl minus transport accounted for 50 percent minus of the early short-circuit current but after 90 min the two measurements were equal. Incubation of the lung in bicarbonate-free Ringer revealed unequal decreases in the H+ concentration of the bathing solutions. Net "base" addition to the serosal solution was reduced by prior removal of the blood from the pulmonary vasculature. Therefore, "base" release could not be localized to the epithelia. The Na+, K+, Ca++ and Cl minus composition of the lung tissue was unchanged over 3 h. Since tissue and, hence cell Cl minus is lower than the concentration in the bathing solution the Cl pump is probably located in the luminal border of the alveolar epithelial cell.

Paths of hydrophilic solute flow across excised bullfrog lung.

The rate of permeation of radiolabeled glycerol, Cl-, urea, Na+, choline, mannitol, sucrose, raffinose, cyancobalamin, and inulin across the alveolar epithelium of the excised short-circuited bullfrog lung was measured. In addition, the flows of cyancobalamin, angiotensin I, and human calcitonin were estimated by radioimmunoassay. In general, the flow of most solutes was compatible with passive permeation through aqueous channels but estimates of equivalent pore radius were hampered by contamination of tritiated and C-labeled solutes by small radioactive breakdown products. The more rapid permeation of fragments spuriously raised the apparent permeability coefficient of the larger parent compound and, usually, the estimate of the pore radius, Other limitations of the evaluation of equivalent pore radius from relative rates of probe molecule flow across excised epithelia are discussed. The most reliable data can be accounted for by a single population of pores of 1.1 nm radius, a value that lies within the range suggested for the adult mammalian lung.

Ion transport across amphibian lung.

The simple architecture of the amphibian lung makes it possible to study the movement of substances across a barrier with permeability and bioelectric properties that are dominated by the alveolar epithelium. When mounted as a planar sheet between identical Ringer solutions the excised lung of the bullfrog exhibited a transmural electrical potential difference of nearly 20 mV (pleural surface positive) and a resistance of about 700 omega cm2. Unidirectional fluxes of 36Cl, Br-, I-, and SCN- across the short-circuited lung were asymmetrical. The net 36Cl- flow from pleura to lumen matched the short-circuit current after 1.5 h of voltage clamping, followed the kinetics of a saturable process, and was reduced by inhibitors of oxidative metabolism. These results suggest that halide and certain pseudohalide anions are secreted by the frog alveolar epithelium. Fluxes of Na+, K+, Ca+, HCO3-, TcO4-, SO42-, p-aminohippurate, gluconate, dinitrophenolate and water were compatible with passive diffusion of the probe molecules across the barrier. Measurements of lung oxygen consumption, ion fluxes and bioelectric properties have helped to pinpoint possible sites and modes of action of airborne agents, such as heavy metals, sulphates and nitrates, that may damage the mammalian pulmonary barrier.