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.

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.

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.

Glutamine metabolism stimulates intestinal cell MAPKs by a cAMP-inhibitable, Raf-independent mechanism.

Background & Aims: Infectious diarrhea caused by viruses plus enterotoxigenic bacteria is often more severe than diarrhea induced by either pathogen alone. We postulated that the increased cell adenosine 3',5'-cyclic monophosphate (cAMP) concentration observed during infection by enterotoxigenic organisms retards the intestinal repair process by blocking activation of mitogen-activated protein kinases (MAPKs) in proliferating intestinal cells.

Methods: We evaluated the effects of glutamine on MAPK activity, thymidine incorporation, and cell number in glutamine-starved and -sufficient rat intestinal crypt cells (IEC-6).

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%).

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.

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.

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.

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.

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.

A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism.

Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the absorption of sodium through the highly selective, amiloride-sensitive epithelial sodium channel (ENaC) made of three homologous subunits (alpha, beta, and gamma). In human, autosomal recessive mutations of alpha, beta, or gammaENaC subunits cause pseudohypoaldosteronism type 1 (PHA-1), a renal salt-wasting syndrome characterized by severe hypovolemia, high plasma aldosterone, hyponatremia, life-threatening hyperkaliemia, and metabolic acidosis. In the mouse, inactivation of alphaENaC results in failure to clear fetal lung liquid at birth and in early neonatal death, preventing the observation of a PHA-1 renal phenotype.

Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice.

The amiloride-sensitive epithelial sodium channel, ENaC, is a heteromultimeric protein made up of three homologous subunits (alpha, beta and gamma) (1,2). In vitro, assembly and expression of functional active sodium channels in the Xenopus oocyte is strictly dependent on alpha-ENaC--the beta and gamma subunits by themselves are unable to induce an amiloride-sensitive sodium current in this heterologous expression system (2). In vivo, ENaC constitutes the limiting step for sodium absorption in epithelial cells that line the distal renal tubule, distal colon and the duct of several exocrine glands.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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-.