J. T. Gatzy

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.

Increased Bioelectric Potential Difference across Respiratory Epithelia in Cystic Fibrosis

To investigate respiratory epithelial function in cystic fibrosis, we measured the transepithelial electrical potential difference across the upper and lower respiratory mucosa in patients with cystic fibrosis and control subjects. The nasal potential difference in the 24 patients with cystic fibrosis exceeded by more than 3 standard deviations the mean voltage in healthy controls, subjects with other diseases, and subjects heterozygous for cystic fibrosis. Potential differences in lower airways were measured in four patients and were significantly greater than in controls (P less than 0.05). Superfusion of the luminal surface with amiloride, an inhibitor of active sodium absorption, induced greater reductions in both nasal and airway potential difference in patients than in controls. We conclude that the increased respiratory-epithelial potential differences appear to be a specific abnormality in homozygotes for cystic fibrosis. The greater reduction in potential difference in response to amiloride suggests that absorption of excess salt and perhaps liquid from respiratory epithelial surfaces contributes to the pathogenesis of lung disease in cystic fibrosis.

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.

Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures.

1. Employing a primary cell culture system and intracellular microelectrodes, we quantitated and compared the Na+ and Cl‐ pathways in apical membranes of normal and cystic fibrosis (CF) human airway epithelia. 2. Like the transepithelial difference (PD) in situ, the PD of CF epithelia in culture (‐27 +/‐ 4 mV, mean +/‐ S.E.M.; n = 28) exceeded the PD of normal epithelia (‐10 +/‐ 1 mV; n = 22). The raised PD principally reflected an increase in the rate of active transport (equivalent short circuit, Ieq) for CF epithelia (61 +/‐ 9 microA cm‐2) as compared with normal epithelia (23 +/‐ 3 microA cm‐2). No significant differences in transepithelial resistance were detected. 3. As indicated by ion replacement studies (gluconate for Cl‐), the apical membrane of normal cells exhibits an apical membrane Cl‐ conductance (GCl) that can be activated by isoprenaline. CF cells do not exhibit an apical membrane GCl, nor can a GCl be activated by isoprenaline. 4. CF cells exhibited a larger amiloride‐sensitive Ieq and amiloride‐sensitive apical membrane conductance (GNa) than normal cells. Further, the amiloride‐sensitive Ieq was increased by isoprenaline in CF but not normal airway epithelia. 5. Equivalent circuit analysis yielded evidence for a more positive electromotive force (EMF) across the apical membrane and a more negative EMF across the basolateral membrane of CF cells as compared with normal cells. Baseline resistances of the apical (Ra) and basolateral (Rb) membranes did not differ for normal and CF cells. 6. Estimates of the resistance of the paracellular path to ion flow (Rs) by equivalent circuit analysis or ion substitution detected no differences in Rs between CF and normal cells. 7. We conclude that abnormalities in both cellular Cl‐ permeability (reduced) and Na+ permeability (increased) are characteristic of the cultured CF respiratory epithelial cell. These data suggest that a defect in the regulation of apical membrane permeabilities is a central feature of this disease.

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.

Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H+,K+-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} secretory pathway that was defective in CF: (i) ASL [\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}] was higher in NL than CF ASL; (ii) activating CFTR with forskolin/3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii) NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHASL is abnormally regulated under basal conditions because of absent CFTR-dependent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.

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.

Electrical potential difference and ion transport across nasal epithelium of term neonates: Correlation with mode of delivery, transient tachypnea of the newborn, and respiratory rate

We studied the change in ion transport function by measuring the basal transepithelial potential difference (PD) across the ciliated epithelium of the nose in 85 term neonates during the first 72 hours of life. Differences in PD associated with the mode of delivery or the presence of respiratory disease and differences in the PD response to the superfusion of amiloride (10(-5) mol/L) were assessed. We also studied term neonates with transient tachypnea of the newborn (TTN) and acute respiratory insufficiency. Basal PDs during the first 24 hours of life were higher in neonates delivered by cesarean section without prior labor (-29.7 +/- 2.5 mV) and in those with TTN (-38.5 +/- 6.0 mV) than in neonates born during normal spontaneous vaginal delivery (-23.0 +/- 2.9 mV) or cesarean section with prior labor (-23.7 +/- 0.7 mV) or in those with respiratory insufficiency (-22.4 +/- 2.3 mV). The percentage inhibition of PD by amiloride superfusion (less than 24 hours) was significantly lower in infants with TTN (30.9 +/- 4.9%) and after cesarean section without prior labor (31.8 +/- 2.2%) than in other groups (37.6 +/- 1.6%). By 48 hours, nasal PDs after cesarean section without prior labor and in neonates with TTN had declined; and by 72 hours, values were similar to those in other groups; respiratory rate paralleled the decline in PD. The respiratory rate of neonates with respiratory insufficiency remained high and paralleled the persistence of respiratory distress. Amiloride sensitivity was similar for all groups by 72 hours. These findings indicate (1) that PDs vary with the mode of delivery and support a role for labor in the normal transition of respiratory epithelial ion transport and (2) that TTN is associated with abnormal epithelial ion transport.

Oxygen consumption and ouabain binding sites in cystic fibrosis nasal epithelium

ABSTRACT. Ion transport by the epithelium lining the airways of patients with cystic fibrosis (CF) is characterized by a raised transepithelial PD and an increased amiloride sensitivity (1). These properties could arise from normal sodium transport across an epithelium with decreased cell chloride permeability and limited chloride secretion. Alternatively, a higher than normal rate of sodium absorption could contribute to these abnormalities. We investigated the latter possibility by measuring oxygen consumption and specific ouabain binding of CF and atopic polyp epithelia and normal turbinate epithelium. Tissue from CF patients consumed oxygen at a rate that was two to three times that of non-CF tissues and had 60% more ouabain binding sites than non-CF epithelium. These results are not consistent with an isolated defect in chloride permeability but support recent findings that the sodium conductance of the apical cell membrane and net sodium absorption by CF nasal epithelium are greater than those of non-CF nasal epithelium.

Increased nasal potential difference and amiloride sensitivity in neonates with cystic fibrosis

Patients with cystic fibrosis (CF) have an increased nasal transepithelial potential difference (PD) which reflects increased sodium absorption across epithelium relatively impermeable to chloride. To evaluate nasal epithelial function in neonates with CF, the PD was recorded and the voltage response to superfusion of 10 −5 M amiloride, an inhibitor of sodium transport, measured between a Ringer perfused bridge on the nasal mucosa and a reference electrode in the subcutaneous space. We studied three neonates with CF with meconium ileus and compared the results with those in 24 term healthy neonates, including one obligate heterozygote for CF, and 27 control neonates with disease. All three CF neonates had raised sweat chloride values (mean 100 mEq/L) at 2 months. The CF neonates had higher PDs (−64.0±8.4 mV) than those in normal (−24.4±2.0 mV) or control (−25.8±2.0 mV) neonates. Superfusion with amiloride induced a 72% reduction in PD in the CF neonates as compared with healthy (37.5±1.0%) and diseased (36.0±1.3%) neonates. The PD and amiloride response in CF neonates are similar to those in CF infants (2–24 months), older CF children (>6 years), and CF adults (−64.9±9.3 mV; 77.7±1.8%, n=51). These results suggest that (1) nasal epithelial dysfunction is present in patients with CF shortly after birth, and (2) the nasal PD may be a diagnostic adjunct to the sweat test in the early diagnosis of CF.