Biomedical subjects
Publications and source records attributed to R C Boucher.
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.
Epithelial cells from normal human nasal turbinate and cystic fibrosis (CF) nasal polyps were isolated and inoculated in denuded, heterologous tracheal grafts that were implanted in immunocompromised mice. After three weeks, 22% of grafts were repopulated by the cell inoculum and exhibited bioelectric manifestations of ion transport. Compared with normal cells, grafts repopulated with CF cells retained bioelectric responses that reflect reduced cellular permeability to chloride ions (Cl-): (1) greater inhibition of short circuit current (Isc) when exposed to amiloride, (2) reduced Cl- diffusion potential difference, and (3) failure of isoprenaline (isoproterenol) to increase Isc. These observations suggest that the Cl- permeability defect of CF respiratory epithelial cells is preserved in heterologous culture, and is intrinsic to cells rather than secondary to local or circulating mediators.
The chloride permeability of airway and sweat ductal epithelium of cystic fibrosis (CF) patients is decreased. This abnormality could represent an intrinsic characteristic of the epithelial cell or the response to a tonic extrinsic stimulus, in vivo. We cultured airway epithelial cells derived from CF and non-CF individuals under identical conditions that were free from donor-specific factors. Differences in the characteristics of cells that multiplied under these circumstances are unlikely to reflect the effects of extrinsic modulation present in the host. After 8-12 days in culture, the cells of CF and non-CF patients were similar in morphology and intracellular electrolyte content, but the CF cultures took up chloride at a reduced rate. The difference could not be attributed to a higher intracellular potential in CF cells or to the presence of a stilbene anion-sensitive chloride-chloride exchange in non-CF cells. We conclude that epithelial cells from CF patients grown in the absence of extracellular factors of the host express reduced cellular chloride permeability, a defect similar to that found in vivo and in freshly excised nasal epithelium.
The feasibility of culturing human nasal epithelial cells was tested by plating on permeable collagen matrices primary isolates of epithelial cells from normal (n = 21) and atopic (n = 5) subjects and from subjects with cystic fibrosis (CF) (n = 13). The cells were cultured in serum-free, hormone-supplemented medium for 5 days followed by maintenance in conditioned medium with 1% serum. A transepithelial electric potential difference (PD) exceeding 1.0 mV developed in most preparations (71%). The PD reached a peak by Day 10 and declined slowly thereafter. The maximal PD of CF epithelia (-32.4 +/- 5.8 mV, n = 9) on collagen matrices exceeded that of normal (-11.3 +/- 1.8 mV, n = 15) and atopic (-13.5 +/- 2.5, n = 5) epithelia. The maximal PD for both CF and normal cultures exceeded that of freshly excised preparations. This difference reflected higher transepithelial resistance of cultures as compared with intact preparations. The efficacy of amiloride (10(-4) M) to reduce the PD was greater in cultures of CF epithelia (-85 +/- 4.6%, n = 7) than it was in cultures of normal (-45.5 +/- 5.0%, n = 11) and atopic (-50.5 +/- 2.9%, n = 5) epithelia, whereas the hyperpolarization induced by replacement of Cl- in the apical bath was smaller for CF cultures (5.9 +/- 1.5 mV) than it was for normal (9.3 +/- 0.9 mV) and atopic (8.8 +/- 0.5 mV) cultures. We conclude that nasal epithelial cells will form high resistance barriers when cultured on collagen matrices.(ABSTRACT TRUNCATED AT 250 WORDS)
The transepithelial potential difference (PD) is raised across cystic fibrosis (CF) respiratory epithelia. This raised voltage reflects active sodium absorption across a relatively chloride impermeable membrane. Because relatively little is known about the regulation of the rate of sodium absorption across mammalian airways, we assessed the possible contribution of aldosterone to the PD in normal and CF respiratory epithelia. Aldosterone excretion in five CF patients was 12.2 +/- 0.9 micrograms/24 h, a mean value not different from normal control subjects (13.6 +/- 1.5 micrograms/24 h, n = 5). Despite similar aldosterone excretion rates, nasal PD was more than 2-fold greater in the CF patients (-53.6 +/- 6.4 mV) than normal subjects (-21.3 +/- 1.4 mV). The effect of an aldosterone antagonist, spironolactone, on aldosterone excretion and nasal and rectal PD was evaluated in four CF patients and five normal subjects. During spironolactone administration, aldosterone excretion increased (2- to 4-fold) and rectal PD decreased in both groups. However, nasal PD was unchanged in each group (CF = -52.1 +/- 4.3 mV pre, -53.6 +/- 1.4 mV during; normal = -21.2 +/- 3.1 mV pre, -21.6 +/- 3.2 mV during). We conclude that neither increased aldosterone secretion rates nor organ sensitivity to aldosterone can account for the abnormally raised PD that characterizes the respiratory epithelium of subjects with CF.
Recent studies have identified abnormalities in Cl- permeation across two target cystic fibrosis (CF) epithelia (sweat duct and respiratory epithelium). In the present study, anion conductances of red blood cells (RBCs) and peripheral blood lymphocytes (PBLs) from CF and normal subjects were estimated and compared. For RBCs, the valinomycin-induced rate constant for K+ loss (PK+) was taken as an index of PCl-. For PBLs, the secondary volume increase after gramicidin pretreatment and hypotonic (0.67 X isotonic) stress was used to estimate PCl-. The Cl- permeabilities of RBCs and PBLs from CF and control subjects were comparable. These findings suggest that the abnormality in PCl- reported for CF sweat ductal and respiratory epithelia is not expressed in circulating blood elements.
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.
Segments of fetal and maternal trachea, maternal bronchi from near-term sheep, and trachea and bronchi from nonpregnant adult sheep were excised and mounted as sheets in Ussing chambers. The conductance (G) for each group of tissues was similar (approximately 4 mS/cm-2); the short circuit current (Isc) ranged from 45-90 microA/cm-2. Under short-circuit or open-circuit conditions trachea and bronchi from pregnant and nonpregnant adult animals absorbed Na+, whereas fetal trachea secreted Cl-. Short-circuited maternal bronchi secreted K+, whereas maternal and fetal trachea did not. Isoproterenol induced an increase in Isc, G, and Cl- secretion of fetal trachea. Maternal trachea and bronchi were not affected. Amiloride reduced Na+ absorption and Isc of maternal trachea and bronchi, but had little effect on fetal trachea. The permeability of fetal trachea to 14C-mannitol was 17 X 10(-7) cm/s and was not affected by isoproterenol. The permeation of dextran (10 K) and horseradish peroxidase across fetal trachea and of all three probes across maternal airways did not reach steady state, but the relative rates were compatible with an equivalent pore radius greater than 4 nm. We conclude that ion transport in fetal large airways contributes to the Cl- and liquid secretion by the entire fetal pulmonary epithelium, whereas resting ion transport of large airways from adult sheep, like that of mature airways of many species, is dominated by Na+ absorption. All of these airway epithelia are characterized by large paracellular aqueous paths.
We studied factors that might be expected to influence Na+ absorption, the major active ion transport by excised rabbit trachea. Transepithelial electric potential difference (PD), short-circuit current (Isc), conductance (G), and unidirectional 22Na+ and 36Cl- flows were measured before and during exposure to a drug or after a change in bathing solution composition. Ouabain (3 X 10(-4) M) in the submucosal bath abolished Isc and Na+ absorption but increased G and unidirectional Cl- flows. Luminal amiloride (10(-3) M) abolished net Na+ absorption but reduced Isc by only 40%. Residual Isc was accounted for by induction of net Cl- secretion, which resulted from a reduction in mucosal (m)-to-submucosal (s) Cl- flux (J). Replacement of luminal Na+ by choline induced effects similar to those of amiloride. Residual Isc was not reduced by mucosal indomethacin (10(-6) M). Replacement of luminal Cl- by gluconate raised transepithelial PD fourfold, raised Isc 50%, decreased G 60%, and abolished net Na+ absorption by decreasing Jm leads to s and increasing Js leads to m. Luminal amphotericin B affected bioelectric properties and ion flows minimally, whereas monensin (10(-4) M) decreased Isc and net Na+ transport. Antidiuretic hormone (ADH, 1 U/ml) or aldosterone (10(-6) M) did not affect in Isc or PD after exposure up to 6 h. We conclude that 1) Na+ absorption across the rabbit trachea is ouabain sensitive, 2) the entry step for Na+ across the luminal membrane is amiloride sensitive, 3) Cl- secretion induced by amiloride or luminal Na+ replacement probably reflects a more favorable chemical gradient for basolateral coupled NaCl entry and/or electrical gradient for Cl- efflux across the luminal membrane, 4) the coupling of Na+ entry to the presence of Cl- in the mucosal solution is probably an electrical rather than a chemical cotransport process, and 5) the tracheal epithelium of the rabbit is not a target for aldosterone and ADH.
Bioelectric properties and 22Na+ and 36Cl- isotopic flows across rabbit trachea, an airway epithelium without submucosal glands, were measured in vitro. One hundred twenty-two excised tracheas exhibited a mean transepithelial electric potential difference (PD) of 12 mV (lumen negative), a conductance (G) of 8.5 mS X cm-2, and a short-circuit current (Isc) of 90 microA X cm-2. G remained stable for more than 3 h, but Isc and PD fell slowly (10%/h). G was inversely correlated with PD, but Isc and G were not correlated. Na+ was absorbed under both open-circuit (1.7 mueq. cm-2 X h-1) and short-circuit (2.2 mueq X cm-2 X h-1) conditions. Net Na+ transport accounted for 70% of Isc of the short-circuited trachea. Net Cl- flow in the absorptive direction approximated that of Na+ under open-circuit conditions (1.6 mueq X cm-2 X h-1). Under short-circuit conditions the small net flow of Cl- in the direction of secretion (0.4 mueq X cm-2 X h-1) was not significant. Both unidirectional Cl- fluxes were correlated with G; [14C]-mannitol permeability and Na+ flows were weakly or not correlated with G. We found no evidence of net HCO-3 or proton transport. Acetylcholine (10(-4) M), phenylephrine (10(-5) M), or isoproterenol (10(-5) M) induced no change in bioelectric properties or ion flows. We conclude that the rabbit trachea is primarily a Na+ absorbing epithelium. The absence of a correlation between mannitol permeability and G suggests that much of the Cl- conductance is transcellular. Whereas insensitivity of rabbit trachea to cholinergic and alpha-adrenergic agents is compatible with the absence of glands, the lack of response to beta-adrenergic agonists denotes a species difference (compared with canine trachea) in airway-surface epithelial cell function.
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The effects of cholinergic and adrenergic agonists and antagonists on ion transport and bioelectric properties of excised canine trachea, main-stem bronchi, and subsegmental bronchi were studied. Acetylcholine (ACh) induced in bronchi a dose-dependent reduction in net Na+ absorption, maximal (30%) at 10(-5) M, that reflected a raised Na+ flux from submucosa to mucosa. Because conductance (G) did not change we speculate that the increased flow in the "passive" direction represents the induction of bidirectional Na+ transport. ACh (10(-4) M) increased net Cl- secretion in the trachea and main-stem bronchi and reduced Na+ absorption. ACh effects in all regions were blocked by atropine. In bronchi, alpha- or beta-adrenergic agonists, 10(-5) and 10(-3) M, raised G and unidirectional Cl- fluxes without affecting short-circuit current (Isc) or inducing Cl- secretion. Small reductions in Na+ absorption were noted at 10(-5) M. In contrast, adrenergic agents increased Isc and Cl- secretion in the trachea. We concluded that neurohumoral agonists induce different patterns of effects on ion transport in central compared with more distal airways. Agonists of both classes induced Cl- secretion in the trachea and reduced but did not abolish net NaCl absorption in the bronchi.
We measured in vivo in human subjects the airway transepithelial electric potential difference (PD), a parameter that reflects passive ion permeabilities and active ion transport across respiratory epithelia. A high impedance voltmeter was used to measure PDs between a fluid-filled and perfused recording bridge located on the airway surface and a reference bridge in the subcutaneous space. Tracheal PD was a relatively high 32 +/- 5 mV (lumen negative) in subjects younger than 35 yr of age (n = 23). Tracheal PD declined after 35 yr of age at a rate of 6 mV per decade. The PDs in bronchi were lower than in the trachea in subjects of all age groups. Neither the magnitude nor the regional variation of the PD appeared influenced by the chemical agents used in general anesthesia. However, superfusion of the airway surface with a pharmacologic inhibitor of Na+ absorption, amiloride, significantly reduced PD in the trachea (-38.5 +/- 7.2%, n = 7) and bronchi (-37.6 +/- 6.3%, n = 6). Both cigarette smoking and tracheostomy appear to reduce tracheal PD when the patients were compared with age-matched control subjects. We conclude that (1) the tracheal PD in young human subjects is relatively high, (2) substantial regional variations in the magnitude of human airway PDs can be demonstrated, (3) active Na+ absorption contributes to the generation of airway PD, and (4) measurements of PD in vivo may help detect early epithelial dysfunction.
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The data reviewed demonstrate that viral and mycoplasma infections induce a spectrum of functional abnormalities in airways. Acute virus infections cause wheezing illnesses in both children and adults. Changes in peripheral airway function during infection with similar organisms are observed in other subjects, usually normal adults. The pathogenesis of these responses is unclear. Pathologic data do show that infection with these pathogens damages the airway epithelium. These changes appear to increase permeability of the respiratory epithelium to protein antigens and consequently may contribute to increased frequency of attacks in asthmatic subjects. In addition, increased mucosal permeability may enhance delivery of inhaled drugs to effector sites in airway walls to induce exaggerated bronchoconstrictor responses in clinical challenge situations. Whether changes in the epithelium during infection, inducing greater antigen entry into the interstitium, results in subsequent development of specific allergy is not known and requires further study.
Observations of freeze-fracture preparations of human nasal epithelium have provided a unique perspective of the spatial distribution of epithelial cell cilia unattainable by more conventional ultrastructural techniques. The initial stages of ciliogenesis were characterized ultrastructurally in these preparations by differentiation of the lumenal aspect of the epithelial cell membrane prior to the emergence and maturation of new cilia. Morphometric analyses of the resultant electron micrographs indicate that the development of an optimal ciliary population during differentiation of ciliated cells may be integral to the adequate functioning of respiratory mucociliary mechanisms. The frequency with which such ciliogenic structures are observed indicates that ciliogenesis is a common feature of the nasal epithelium and suggests that epithelial cell turnover in the nasal cavities is relatively rapid.
Liquid from canine airway surfaces was absorbed onto filter paper strips and analyzed. In resting conditions, tracheal surface liquid was hyperosmolal (330 mosmol/kg H2O) compared to plasma with raised Na+ (158 meq/l), Cl- (134 meq/l), K+ (28 meq/l), and HCO3- (32 meq/l) concentrations. The volume collected was increased by repetitive sampling, a response blocked by atropine, or by methacholine injection. Compared to nose breathing, tracheal surface liquid osmolality was increased by 10 min of mouth breathing (410 mosmol/kg H2O). Surface liquid from 0.5-cm diameter bronchi was nearly isosmolal (304 mosmol/kg H2O) with plasma in resting conditions, with Na and Cl concentrations lower than plasma (120 and 106 meq/l, respectively), and K+ (52 meq/l), and HCO3- (50 meq/l) concentrations higher than those of plasma or tracheal liquid. Although the K+ in tracheal fluid approaches the value for electrochemical equilibrium, K+ in fluid from the bronchi and HCO3- in both regions cannot be accounted for by passive forces. The regional difference in osmolality supports the concept that the higher osmolality of tracheal liquid reflects evaporative water loss from this site. The transepithelial osmotic gradient generated by evaporative water loss may be a driving force for hydration of the tracheal surface.
Absorption of airway liquid, as it moves from the large surface area of distal airways to more central airway surfaces, is considered to be essential for regulation of the depth of airway liquid. The short-circuited excised canine trachea, however, secretes Cl- and absorbs a lesser amount of Na+. We compared bioelectric properties and unidirectional ion flows across epithelia excised from trachea and mainstem and 4th- to 6th-generation bronchi. Compared with trachea, 4th- to 6th-generation bronchi exhibited a lower potential difference (8.9 compared with 35 mV) and higher direct-current conductance (6.9 compared with 2.1 mS . cm-2). Corresponding values for main-stem bronchus were intermediate. Under short-circuit conditions, the trachea secreted Cl- (1.75 mueq . cm-2 . h-1) and absorbed Na+ (1.15 mueq . cm-2 . h-1). K+ fluxes were symmetric. Main-stem bronchus exhibited a similar pattern of ion flow. Subsegmental bronchi absorbed 2.0 mueq Na+ . cm-2 . h-1 and secreted 0.04 mueq K+ . cm-2 . h-1, but net Cl- transport was absent. Under open circuit, Na+ absorption persisted in all regions, but net Cl- secretion disappeared in the trachea and main-stem bronchi. Na+ absorption was uniformly abolished by addition of ouabain (3 x 10(-5) M) to the submucosal bath. We conclude that NaCl absorption dominates resting ion translocation in canine bronchi and that liquid absorption probably occurs at these sites in vivo.