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The localisation of the vacuolar proton pump (V-H+-ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+-ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+-ATPase and CAII in the secretory coil clear cells suggests that (the) formation of HCO− 3 and H+ by carbonic anhydrase II and the transport of H+ by V-H+-ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption. [ABSTRACT FROM AUTHOR]

Purpose: Cystic fibrosis (CF) is a multisystem genetic disease caused by dysfunction of the epithelial anionic channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Decreased mucociliary clearance because of thickened mucus is part of the pulmonary disease pathophysiology. It is controversial if the thickened airway surface liquid (ASL) is caused by the deficient chloride secretion and excessive sodium (through ENaC) and water hyperabsorption from the periciliar fluid or by the lack of bicarbonate secretion with relative acidification of the ASL. Correlations between the magnitude of in vivo chloride conductance with phenotypic characteristics and CF genotype can help to elucidate these mechanisms and direct to new treatments., Methods: Nasal potential difference was measured in 28 CF patients (age from 0.3 to 28 year) and correlated with pulmonary function, pancreatic phenotype, pulmonary colonization and genotype severity., Results: The CFTR-chloride conductance was better in older patients (r = 0.40; P = 0.03), in patients with better pulmonary function (r = 0.48; P = 0.01), and was associated with genotype severity. Higher chloride diffusion in the presence of a favorable chemical gradient was associated with Pseudomonas aeruginosa negativity (P < 0.05). More negative NPDmax was associated with pancreatic insufficiency (P < 0.01) as well with genotype severity, but not with the pulmonary function., Conclusions: The anion permeability through CFTR, mainly chloride, but bicarbonate as well, is the most critical factor in CF airway pathophysiology. Treatments primarily directed to correct CFTR function and/or airway acidity are clearly a priority.

Background: Among the many consequences of loss of CFTR protein function, a significant reduction of the secretion of bicarbonate (HCO 3 - ) in cystic fibrosis (CF) is a major pathogenic feature. Loss of HCO 3 - leads to abnormally low pH and impaired mucus clearance in airways and other exocrine organs, which suggests that NaHCO 3 inhalation may be a low-cost, easily accessible therapy for CF., Objective: To evaluate the safety, tolerability, and effects of inhaled aerosols of NaHCO 3 solutions (4.2% and 8.4%)., Methods: An experimental, prospective, open-label, pilot, clinical study was conducted with 12 CF volunteer participants over 18 years of age with bronchiectasis and pulmonary functions classified as mildly to severely depressed. Sputum rheology, pH, and microbiology were examined as well as spirometry, exercise performance, quality-of-life assessments, dyspnea, blood count, and venous blood gas levels., Results: Sputum pH increased immediately after inhalation of NaHCO 3 at each clinical visit and was inversely correlated with rheology when all parameters were evaluated: [G' (elasticity of the mucus) = - 0.241; G″ (viscosity of the mucus) = - 0.287; G* (viscoelasticity of the mucus) = - 0.275]. G* and G' were slightly correlated with peak flow, forced expiratory volume in 1 s (FEV 1 ), and quality of life; G″ was correlated with quality of life; sputum pH was correlated with oxygen consumption (VO 2 ) and vitality score in quality of life. No changes were observed in blood count, venous blood gas, respiratory rate, heart rate, peripheral oxygen saturation of hemoglobin (SpO 2 ), body temperature, or incidence of dyspnea. No adverse events associated with the study were observed., Conclusion: Nebulized NaHCO 3 inhalation appears to be a safe and well tolerated potential therapeutic agent in the management of CF. Nebulized NaHCO 3 inhalation temporarily elevates airway liquid pH and reduces sputum viscosity and viscoelasticity.

Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a "capillary"-Ussing chamber (area ≈1 mm 2 ) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ( HCO 3 - ), which can be enhanced pharmacologically by cAMP as well as Ca 2+ -mediated agonists. Concurrent secretion and absorption of surface liquid along with HCO 3 - secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.

We investigated the effects of bicarbonate on the growth of several different bacteria as well as its effects on biofilm formation and intracellular cAMP concentration in Pseudomonas aeruginosa . Biofilm formation was examined in 96-well plates, with or without bicarbonate. The cAMP production of bacteria was measured by a commercial assay kit. We found that NaHCO 3 (100 mmol l -1 ) significantly inhibited, whereas NaCl (100 mmol l -1 ) did not influence the growth of planktonic bacteria. MIC and MBC measurements indicated that the effect of HCO 3 - is bacteriostatic rather than bactericidal. Moreover, NaHCO 3 prevented biofilm formation as a function of concentration. Bicarbonate and alkalinization of external pH induced a significant increase in intracellular cAMP levels. In conclusion, HCO 3 - impedes the planktonic growth of different bacteria and impedes biofilm formation by P. aeruginosa that is associated with increased intracellular cAMP production. These findings suggest that aerosol inhalation therapy with HCO 3 - solutions may help improve respiratory hygiene in patients with cystic fibrosis and possibly other chronically infected lung diseases.

The small airways of the lungs are under constant assault from the pathogens and debris in the air that they must conduct to alveoli. Although hygiene is of paramount importance for respiratory health, the underlying principles of airway clearance have not been well integrated or established. Newly emerging concepts of simultaneous absorption and secretion of airway surface liquid (ASL) and the role of [Formula: see text] in the maturation of mucins have advanced from experimental evidence as well as observations from the congenital disease cystic fibrosis (CF) to present a novel model that integrates microanatomy with organ physiology to meet the constant challenge of cleaning small airways., (Copyright © 2017 the American Physiological Society.)

The secretion and management of readily transportable airway surface liquid (ASL) along the respiratory tract is crucial for the clearance of debris and pathogens from the lungs. In proximal large airways, submucosal glands (SMGs) can produce ASL. However, in distal small airways, SMGs are absent, although the lumens of these airways are, uniquely, highly plicated. Little is known about the production and maintenance of ASL in small airways, but using electrophysiology, we recently found that native porcine small airways simultaneously secrete and absorb. How these airways can concurrently transport ASL in opposite directions is puzzling. Using high expression of the Na-K-2Cl cotransport (NKCC) 1 protein (SLC12a2) as a phenotypic marker for fluid secretory cells, immunofluorescence microscopy of porcine small airways revealed two morphologically separated sets of luminal epithelial cells. NKCC1 was abundantly expressed by most cells in the contraluminal regions of the pleats but highly expressed very infrequently by cells in the luminal folds of the epithelial plications. In larger proximal airways, the acini of SMGs expressed NKCC1 prominently, but cells expressing NKCC1 in the surface epithelium were sparse. Our findings indicate that, in the small airway, cells in the pleats of the epithelium secrete ASL, whereas, in the larger proximal airways, SMGs mainly secrete ASL. We propose a mechanism in which the locations of secretory cells in the base of pleats and of absorptive cells in luminal folds physically help maintain a constant volume of ASL in small airways.

Since the discovery of Cl(-) impermeability in cystic fibrosis (CF) and the cloning of the responsible channel, CF pathology has been widely attributed to a defect in epithelial Cl(-) transport. However, loss of bicarbonate (HCO3(-)) transport also plays a major, possibly more critical role in CF pathogenesis. Even though HCO3(-) transport is severely affected in the native pancreas, liver, and intestines in CF, we know very little about HCO3(-) secretion in small airways, the principle site of morbidity in CF. We used a novel, mini-Ussing chamber system to investigate the properties of HCO3(-) transport in native porcine small airways (∼ 1 mm φ). We assayed HCO3(-) transport across small airway epithelia as reflected by the transepithelial voltage, conductance, and equivalent short-circuit current with bilateral 25-mM HCO3(-) plus 125-mM NaGlu Ringer's solution in the presence of luminal amiloride (10 μM). Under these conditions, because no major transportable anions other than HCO3(-) were present, we took the equivalent short-circuit current to be a direct measure of active HCO3(-) secretion. Applying selective agonists and inhibitors, we show constitutive HCO3(-) secretion in small airways, which can be stimulated significantly by β-adrenergic- (cAMP) and purinergic (Ca(2+)) -mediated agonists, independently. These results indicate that two separate components for HCO3(-) secretion, likely via CFTR- and calcium-activated chloride channel-dependent processes, are physiologically regulated for likely roles in mucus clearance and antimicrobial innate defenses of small airways.

Evidence from the pathology in cystic fibrosis (CF) and recent results in vitro indicate that HCO3- is required for gel-forming mucins to form the mucus that protects epithelial surfaces. Mucus formation and release is a complex process that begins with an initial intracellular phase of synthesis, packaging and apical granule exocytosis that is followed by an extracellular phase of mucin swelling, transport and discharge into a lumen. Exactly where HCO3- becomes crucial in these processes is unknown, but we observed that in the presence of HCO3-, stimulating dissected segments of native mouse intestine with 5-hydroxytryptamine (5-HT) and prostaglandin E2 (PGE2) induced goblet cell exocytosis followed by normal mucin discharge in wild-type (WT) intestines. CF intestines that inherently lack cystic fibrosis transmembrane conductance regulator (CFTR)-dependent HCO3- secretion also demonstrated apparently normal goblet cell exocytosis, but in contrast, this was not followed by similar mucin discharge. Moreover, we found that even in the presence of HCO3-, when WT intestines were stimulated only with a Ca2+-mediated agonist (carbachol), exocytosis was followed by poor discharge as with CF intestines. However, when the Ca2+-mediated agonist was combined with a cAMP-mediated agonist (isoproterenol (isoprenaline) or vasoactive intestinal peptide) in the presence of HCO3- both normal exocytosis and normal discharge was observed. These results indicate that normal mucus formation requires concurrent activation of a Ca2+-mediated exocytosis of mucin granules and an independent cAMP-mediated, CFTR-dependent, HCO3- secretion that appears to mainly enhance the extracellular phases of mucus excretion.

This study was designed to assess the relative importance of Cl(-) and HCO(3)(-) secretion to mucociliary transport rate (MCT) in ex vivo porcine tracheas. MCT was measured in one group of tissues that was exposed to adventitial HCO(3)(-)-free solution while a parallel group was exposed to adventitial HCO(3)(-)-replete solution. After measurement of baseline MCT rates, acetylcholine (ACh) was added to stimulate submucosal gland mucous liquid secretion, and MCT rates were again measured. Before ACh addition, the mean MCT was higher in the HCO(3)(-)-free group (4.2 ± 0.9 mm/min) than in the HCO(3)(-)-replete group (2.3 ± 0.3 mm/min), but this difference was not statistically significant. ACh addition significantly increased MCT in both groups, but ACh-stimulated MCT was significantly lower in the HCO(3)(-)-free group (11.0 ± 1.5 mm/min) than in the HCO(3)(-)-replete group (17.0 ± 2.0 mm/min). A second series of experiments examined the effect on MCT of blocking Cl(-) secretion with 100 μM bumetanide. Before adding ACh, MCT in the bumetanide-treated group (1.0 ± 0.2 mm/min) was significantly lower than in the control group (3.8 ± 1.1 mm/min). ACh addition significantly increased MCT in both groups, but there was no significant difference between the bumetanide-treated group (21.4 ± 1.7 mm/min) and control group (19.5 ± 3.4 mm/min). These results indicate that ACh-stimulated MCT has greater dependence on HCO(3)(-) secretion, whereas the basal MCT rate has greater dependence on Cl(-) secretion.

Native small airways must remain wet enough to be pliable and support ciliary clearance, but dry enough to remain patent for gas flow. The airway epithelial lining must both absorb and secrete ions to maintain a critical level of fluid on its surface. Despite frequent involvement in lung diseases, the minuscule size has limited studies of peripheral airways. To meet this challenge, we used a capillary to construct an Ussing chamber (area <1 mm(2)) to measure electrolyte transport across small native airways (∼1 mm ø) from pig lung. Transepithelial potentials (V(t)) were recorded in open circuit conditions while applying constant current pulses across the luminal surface of dissected airways to calculate transepithelial electrical conductance (G(t)) and equivalent short circuit current (I(eq)(sc)) in the presence and absence of selected Na(+) and Cl(-) transport inhibitors (amiloride, GlyH-101, Niflumic acid) and agonists (Forskolin + IBMX, UTP). Considered together the responses suggest an organ composed of both secreting and absorbing epithelia that constitutively and concurrently transport fluids into and out of the airway, i.e. in opposite directions. Since the epithelial lining of small airways is arranged in long, accordion-like rows of pleats and folds that run axially down the lumen, we surmise that cells within the pleats are mainly secretory while the cells of the folds are principally absorptive. This structural arrangement could provide local fluid transport from within the pleats toward the luminal folds that may autonomously regulate the local surface fluid volume for homeostasis while permitting acute responses to maintain clearance.

The invitation to present the 2010 Hans Ussing lecture for the Epithelial Transport Group of the American Physiological Society offered me a unique, special, and very surprising opportunity to join in saluting a man whom I met only once, but whose work was the basis, not only for my career, but also for finding the molecular defect in the inherited disease cystic fibrosis (CF). In this context, I will venture to make the tribute with a new explanation of why a mutation in a single gene that codes for an anion channel can cause devastation of multiple epithelial systems with pathogenic mucus. In so doing, I hope to raise awareness of a new role for that peculiar anion around which so much physiology revolves, HCO(3)(-). I begin by introducing CF pathology as I question the name of the disease as well as the prevalent view of the basis of its pathology by considering: 1) mucus, 2) salt, and 3) HCO(3)(-). I then present recent data showing that HCO(3)(-) is required for normal mucus discharge, and I will close with conjecture as to how HCO(3)(-) may support mucus discharge and why the failure to transport this electrolyte is pathogenic in CF.

The impact of small anions on the physical properties of gel-forming mucin has been almost overlooked relative to that of cations. Recently, based on the coincident abnormalities in HCO(3)(-) secretion and abnormal mucus formed in the hereditary disease cystic fibrosis (CF), HCO(3)(-) was hypothesized to be critical in the formation of normal mucus by virtue of its ability to sequester Ca(2+) from condensed mucins being discharged from cells. However, direct evidence of the impact of HCO(3)(-) on mucus properties is lacking. Herein, we demonstrate for the first time that mucin diffusivity (∼1/viscosity) increases as a function of [HCO(3)(-)]. Direct measurements of exocytosed mucin-swelling kinetics from airway cells showed that mucin diffusivity increases by ∼300% with 20 mM extracellular HCO(3)(-) concentration. Supporting data indicate that HCO(3)(-) reduces free Ca(2+) concentration and decreases the amount of Ca(2+) that remains associated with mucins. The results demonstrate that HCO(3)(-) enhances mucin swelling and hydration by reducing Ca(2+) cross-linking in mucins, thereby decreasing its viscosity and likely increasing its transportability. In addition, HCO(3)(-) can function as a Ca(2+) chelator like EGTA to disperse mucin aggregates. This study indicates that poor HCO(3)(-) availability in CF may explain why secreted mucus remains aggregated and more viscous in affected organs. These insights bear on not only the fundamental pathogenesis in CF, but also on the process of gel mucus formation and release in general.

Cervical mucus thinning and release during the female reproductive cycle is thought to rely mainly on fluid secretion. However, we now find that mucus released from the murine reproductive tract critically depends upon concurrent bicarbonate (HCO(3)(-)) secretion. Prostaglandin E(2) (PGE(2))- and carbachol-stimulated mucus release was severely inhibited in the absence of serosal HCO(3)(-), HCO(3)(-) transport, or functional cystic fibrosis transmembrane conductance regulator (CFTR). In contrast to mucus release, PGE(2)- and carbachol-stimulated fluid secretion was not dependent on bicarbonate or on CFTR, but was completely blocked by niflumic acid. We found stimulated mucus release was severely impaired in the cystic fibrosis F508 reproductive tract, even though stimulated fluid secretion was preserved. Thus, CFTR mutations and/or poor bicarbonate secretion may be associated with reduced female fertility associated with abnormal mucus and specifically, may account for the increased viscosity and lack of cyclical changes in cervical mucus long noted in women with cystic fibrosis.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels are constitutively activated in sweat ducts. Since phosphorylation-dependent and -independent mechanisms can activate CFTR, we sought to determine the actual mechanism responsible for constitutive activation of these channels in vivo. We show that the constitutively activated CFTR Cl(-) conductance (gCFTR) in the apical membrane is completely deactivated following alpha-toxin permeabilization of the basolateral membrane. We investigated whether such inhibition of gCFTR following permeabilization is due to the loss of cytoplasmic glutamate or due to dephosphorylation of CFTR by an endogenous phosphatase in the absence of kinase activity (due to the loss of kinase agonist cAMP, cGMP or GTP through alpha-toxin pores). In order to distinguish between these two possibilities, we examined the effect of inhibiting the endogenous phosphatase activity with okadaic acid (10(-8) M) on the permeabilization-induced deactivation of gCFTR. We show that okadaic acid (1) inhibits an endogenous phosphatase responsible for dephosphorylating cAMP but not cGMP or G protein-activated CFTR and (2) prevents deactivation of CFTR following permeabilization of the basolateral membrane. These results indicate that distinctly different phosphatases may be responsible for dephosphorylating different kinase-specific sites on CFTR. We conclude that the phosphorylation by PKA alone appears to be primarily responsible for constitutive activation of gCFTR in vivo.

The mechanisms underlying mucus-associated pathologies in cystic fibrosis (CF) remain obscure. However, recent studies indicate that CF transmembrane conductance regulator (CFTR) is required for bicarbonate (HCO3-) transport and that HCO3- is critical for normal mucus formation. We therefore investigated the role of HCO3- in mucus secretion using mouse small intestine segments ex vivo. Basal rates of mucus release in the presence or absence of HCO3- were similar. However, in the absence of HCO3-, mucus release stimulated by either PGE2 or 5-hydroxytryptamine (5-HT) was approximately half that stimulated by these molecules in the presence of HCO3-. Inhibition of HCO3- and fluid transport markedly reduced stimulated mucus release. However, neither absence of HCO3- nor inhibition of HCO3- transport affected fluid secretion rates, indicating that the effect of HCO3- removal on mucus release was not due to decreased fluid secretion. In a mouse model of CF (mice homozygous for the most common human CFTR mutation), intestinal mucus release was minimal when stimulated with either PGE2 or 5-HT in the presence or absence of HCO3-. These data suggest that normal mucus release requires concurrent HCO3- secretion and that the characteristically aggregated mucus observed in mucin-secreting organs in individuals with CF may be a consequence of defective HCO3- transport.

The activities of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel and the amiloride-sensitive epithelial Na(+) channel (ENaC) are acutely coordinated in the sweat duct. However, the mechanisms responsible for cross-talk between these ion channels are unknown. Previous studies indicated that luminal pH of sweat ducts varies over 3 pH units and that the cytoplasmic pH affects both CFTR and ENaC. Therefore, using basolaterally alpha-toxin-permeabilized apical membrane preparations of sweat ducts as an experimental system, we tested the hypothesis that the cytosolic pH may mediate the cross-talk between CFTR and ENaC. We showed that while luminal pH had no effect, cytosolic pH acutely affected ENaC activity. That is, acidic pH inhibited, while basic pH activated, ENaC. pH regulation of ENaC appears to be independent of CFTR or endogenous kinase activities because basic pH independently stimulated ENaC (1) in normal ducts even when CFTR was deactivated, (2) in CF ducts that lack CFTR in the plasma membranes and (3) after blocking endogenous kinase activity with staurosporine. Considering the evidence of Na(+)/H(+) exchange (NHE) activity as shown by the expression of mRNA and function of NHE in the basolateral membrane of the sweat duct, we postulate that changes in cytosolic Na(+) ([Na(+)]( i )) may alter cytosolic pH (pH( i )) as salt loads into the cell during electrolyte absorption. These changes may play a role in coordinating the activities of ENaC and CFTR during transepithelial salt transport.

For more than 20 years, the abnormally thick mucus (mucoviscidosis) in cystic fibrosis has been widely shown to be linked to a genetic defect in the cystic fibrosis transmembrane conductance regulator Cl(-) channel. The defect is widely thought to cause mucus to become dehydrated as a result of basic defects in Cl(-) dependent fluid transport. However, this widely held explanation is inconsistent with the known physiological properties and functions of organs affected by cystic fibrosis. During the process of releasing highly condensed mucins from intracellular granules, Ca(2+) and H(+) cations must be removed to enable the mucins to expand by as much as 1000 times, forming extracellular mucus-gel networks. Over the past few years, that HCO(3)(-) transport is also defective in patients with cystic fibrosis has become apparent. I propose that HCO(3)(-) is crucial to normal mucin expansion because it forms complexes with these cations. Thus, because HCO(3)(-) secretion is defective in cystic fibrosis, mucins in organs affected by cystic fibrosis tend to remain aggregated, poorly solubilised, and less transportable. If the hypothesis is valid, pathogenesis in cystic fibrosis could be due as much to defective transport of HCO(3)(-) as to defective Cl(-) transport.

With the advent of numerous candidate drugs for therapy in cystic fibrosis (CF), there is an urgent need for easily interpretable assays for testing their therapeutic value. Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) abolished beta-adrenergic but not cholinergic sweating in CF. Therefore, the beta-adrenergic response of the sweat gland may serve both as an in vivo diagnostic tool for CF and as a quantitative assay for testing the efficacy of new drugs designed to restore CFTR function in CF. Hence, with the objective of defining optimal conditions for stimulating beta-adrenergic sweating, we have investigated the components and pharmacology of sweat secretion using cell cultures and intact sweat glands. We studied the electrical responses and ionic mechanisms involved in beta-adrenergic and cholinergic sweating. We also tested the efficacy of different beta-adrenergic agonists. Our results indicated that in normal subjects the cholinergic secretory response is mediated by activation of Ca(2+)-dependent Cl(-) conductance as well as K(+) conductances. In contrast, the beta-adrenergic secretory response is mediated exclusively by activation of a cAMP-dependent CFTR Cl(-) conductance without a concurrent activation of a K(+) conductance. Thus, the electrochemical driving forces generated by beta-adrenergic agonists are significantly smaller compared with those generated by cholinergic agonists, which in turn reflects in smaller beta-adrenergic secretory responses compared with cholinergic secretory responses. Furthermore, the beta-adrenergic agonists, isoproprenaline and salbutamol, induced sweat secretion only when applied in combination with an adenylyl cyclase activator (forskolin) or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, aminophylline or theophylline). We surmise that to obtain consistent beta-adrenergic sweat responses, levels of intracellular cAMP above that achievable with a beta-adrenergic agonist alone are essential. beta-Adrenergic secretion can be stimulated in vivo by concurrent iontophoresis of these drugs in normal, but not in CF, subjects.

Cystic fibrosis (CF) of the pancreas is the most widely accepted name of the most common fatal inherited single gene defect disease among Caucasians. Its incidence among other races is thought to be significantly less, but mutations in the gene have been reported in most, if not all, major populations. This review is intended to give general concepts of the molecular as well as physiological basis of the pathology that develops in the disease. First, an overview of the organ pathology and genetics is presented, followed by the molecular structure of the gene product (cystic fibrosis transmembrane conductance regulator, CFTR), its properties, functions, and controls as currently understood. Second, since mutations appear to be expressed primarily as a defect in electrolyte transport, effects and mechanisms of pathology are presented for two characteristically affected organs where the etiology is best described: the sweat gland, which excretes far too much NaCl ("salt") and the pancreas, which excretes far too little HCO3(- )("soda"). Unfortunately, morbidity and mortality in CF develop principally from refractory airway infections, the basis of which remains controversial. Consequently, we conclude by considering possible mechanisms by which defects in anion transport might predispose the CF lung to chronic infections.

Lessons from the sweat gland on cystic fibrosis (CF) began long before modern medicine became a science. In European folklore, the curse that "a child that taste salty when kissed will soon die" (Alonso y de los Ruyzes de Fonteca J. Diez Previlegios para Mugeres Prenadas. Henares, Spain, 1606) has been taken by many as a direct reference to cystic fibrosis [Busch R. Acta Univ Carol Med (Praha) 36: 13-15, 1990]. The high salt concentration in sweat from patients with CF is now accepted as almost pathognomonic with this fatal genetic disease, but the earliest descriptions of cystic fibrosis as a disease entity did not mention sweat or sweat glands (Andersen DH. Am J Dis Child 56: 344-399, 1938; Andersen DH, Hodges RG. Am J Dis Child 72: 62-80, 1946). Nonetheless, defective sweating soon became an inseparable, and major, component of the constellation of symptoms that diagnose "cystic fibrosis" (Davis PB. Am J Respir Crit Care Med 173: 475-482, 2006). The sweat gland has played a foremost role in diagnosing, defining pathophysiology, debunking misconceptions, and increasing our understanding of the effects of the disease on organs, tissues, cells, and molecules. The sweat gland has taught us much.

Absorptive epithelial cells must admit large quantities of salt (NaCl) during the transport process. How these cells avoid swelling to protect functional integrity in the face of massive salt influx is a fundamental, unresolved problem. A special preparation of the human sweat duct provides critical insights into this crucial issue. We now show that negative feedback control of apical salt influx by regulating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity is key to this protection. As part of this control process, we report a new physiological role of K(+) in intracellular signaling and provide the first direct evidence of acute in vivo regulation of CFTR dephosphorylation activity. We show that cytosolic K(+) concentration ([K(+)](c)) declines as a function of increasing cellular NaCl content at the onset of absorptive activity. Declining [K(+)](c) cause parallel deactivation of CFTR by dephosphorylation, thereby limiting apical influx of Cl(-) (and its co-ion Na(+)) until [K(+)](c) is stabilized. We surmise that [K(+)](c) stabilizes when Na(+) influx decreases to a level equal to its efflux through the basolateral Na(+)-K(+) pump thereby preventing disruptive changes in cell volume.

Gene therapy vectors are mostly studied in cultured cells, rodents, and sometimes in non-human primates, but it is useful to test them in human tissue prior to clinical trials. In this study, we investigated the possibility of using human sweat glands as a model for testing cystic fibrosis (CF) gene therapy vectors. Human sweat glands are relatively easy to obtain from skin biopsy, and can be tested for CFTR function. Using patients' sweat glands could provide a safe model to study the efficacy of CF gene therapy. As the first step to explore using sweat glands as a model for CF gene therapy, we examined various ex vivo gene delivery methods for a helper-dependent adenovirus (HD-Ad) vector. Gene delivery to sweat glands in skin organ culture was studied by topical application, intradermal injection or submerged culture. We found that transduction efficiency can be enhanced by pretreating isolated sweat glands with dispase, which suggests that the basement membrane is a critical barrier to gene delivery by adenoviral vectors. Using this approach, we showed that Cftr could be efficiently delivered to and expressed by the epithelial cells of sweat glands with our helper-dependent adenoviral vector containing cytokeratin 18 regulatory elements. Based on this study we propose that sweat glands might be used as an alternative model to study CF gene therapy in humans.

We previously showed that activation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl- conductance (gCFTR) supports parallel activation of amiloride-sensitive epithelial Na+ channel (ENaC) in the native human sweat duct. However, it is not clear whether phosphorylated CFTR, phosphorylated ENaC, or only Cl(-) -channel function is required for activation. We used basilaterally alpha-toxin-permeabilized human sweat ducts to test the hypothesis that ENaC activation depends only on Cl(-) -channel function and not on phosphorylation of either CFTR or ENaC. CFTR is classically activated by PKA plus millimolar ATP, but cytosolic glutamate activation of gCFTR is independent of ATP and phosphorylation. We show here that both phosphorylation-dependent (PKA) and phosphorylation-independent (glutamate) activation of CFTR Cl- channel function support gENaC activation. We tested whether cytosolic application of 5 mM ATP alone, phosphorylation by cAMP, cGMP, G-protein dependent kinases (all in the presence of 100 microM ATP), or glutamate could support ENaC activation in the absence of gCFTR. We found that none of these agonists activated gENaC by themselves when Cl- current (I(Cl-)) through CFTR was blocked by: 1) Cl- removal, 2) DIDS inhibition, 3) lowering the ATP concentration to 100 microM (instead of 5 mM required to support CFTR channel function), or 4) mutant CFTR (homozygous DeltaF508 CF ducts). However, Cl- gradients in the direction of absorption supported, while Cl- gradients in the direction of secretion prevented ENaC activation. We conclude that the interaction between CFTR and ENaC is dependent on activated I(Cl-) through CFTR in the direction of absorption (Cl- gradient from lumen to cell). But such activation of ENaC is independent of phosphorylation and ATP. However, reversing I(Cl-) through CFTR in the direction of secretion (Cl- gradient from cell to lumen) prevents ENaC activation even in the presence of I(Cl-) through CFTR.

Computerized assays on cultured cells ex vivo have been used to screen thousands of compounds for their effectiveness in correcting the basic physiological defect in cystic fibrosis (CF). While a number of these compounds appear promising, their effectiveness will almost certainly need to be demonstrated in animals before therapeutic tests in humans will be possible. We show herein that the function of salivary secretion in the mouse model for CF could be used as a simple, easy and rapid in vivo assay for drug effects. We demonstrate that salivary secretory capacity stimulated with a beta-adrenergic agonist closely reflects the genotype of origin. Specifically, the mean maximal secretory rate of saliva in normal wild type (+/+) mice was about 1.5 times higher than that of the mean rate in heterozygote (+/-) mice and more than 50 times greater than in CF (-/-) mice. Total saliva secreted per stimulated period obeyed a similar phenotype-genotype segregation. The data indicate that salivary secretory rates in CF mice could be used to assay potential drugs for their effectiveness in correcting the secretory defect in cystic fibrosis.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel function is required for activating amiloride-sensitive epithelial Na(+) channels (ENaC) in salt-absorbing human sweat duct. It is unclear whether ENaC channel function is also required for CFTR activation. The dysfunctional ENaC mutations in type-1 pseudohypoaldosteronism (PHA-1) provided a good opportunity to study this phenomenon of ion channel interaction between CFTR and ENaC. The PHA-1 ducts completely lacked spontaneous ENaC conductance (gENaC). In contrast, the normal ducts showed large spontaneous gENaC (46 +/- 10 ms, mean +/- SE: ). After permeabilization of the basolateral membrane with alpha-toxin, cAMP + ATP activation of CFTR Cl(-) conductance (gCFTR) or alkalinization of cytosolic pH (6.8 to 8.5) stimulated gENaC of normal but not PHA-1 ducts. In contrast, both spontaneous gCFTR in intact ducts and (cAMP + ATP)-activated gCFTR of permeabilized ducts appeared to be similar in normal and PHA-1 subjects. Lack of gENaC completely blocked salt absorption and caused dramatic reversal of skin potentials associated with pilocarpine-induced sweat secretion from significantly negative in normal subjects (-13 +/- 7.0 mV) to significantly positive (+22 +/- 11.0 mV) in PHA-1 patients. We conclude that virtual lack of ENaC in PHA-1 ducts had little effect on CFTR activity and that the positive skin potentials could potentially serve as a diagnostic tool to identify type-1 pseudohypoaldosteronism.

Background: The pathological hallmarks of chronic obstructive pulmonary disease (COPD) are inflammation of the small airways (bronchiolitis) and destruction of lung parenchyma (emphysema). These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter., Methods: We introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole., Results: In bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential (TEP; lumen to bath) of the bronchiole was small (mean +/- sem: -3 +/- 1 mV; n = 25), but when gluconate replaced luminal Cl-, the bionic Cl- diffusion potentials (-58 +/- 3 mV; n = 25) were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl- permeability was at least 5 times greater than Na+ permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 microM)+IBMX (100 microM), ATP (100 microM), or adenosine (100 microM), but not by ionomycin. The TEP was partially inhibited by NPPB (100 microM), GlyH-101* (5-50 microM), and CFTRInh-172* (5 microM). RT-PCR gave identifying products for CFTR, alpha-, beta-, and gamma-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways., Conclusion: These results indicate that the small airway of the pig is characterized by a constitutively active Cl- conductance that is most likely due to CFTR.

Effective and specific inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel in epithelia has long been needed to better understand the role of anion movements in fluid and electrolyte transport. Until now, available inhibitors have required high concentrations, usually in the millimolar or high micromolar range, to effect even an incomplete block of channel conductance. These inhibitors, including 5-nitro-2(3-phenylpropyl-amino)benzoate (NPPB), bumetamide, glibenclamide and DIDS, are also relatively non-specific. Recently a new anion channel inhibitor, a thiazolidinone derivative, termed CFTRInh-172 has been synthesized and introduced with apparently improved inhibitory properties as shown by effects on anion conductance expressed in cell lines and on secretion in vivo. Here, we assay the effect of this inhibitor on a purely salt absorbing native epithelial tissue, the freshly isolated microperfused human sweat duct, known for its inherently high expression of CFTR. We found that the inhibitor at a maximum dose limited by its aqueous solubility of 5 microm partially blocked CFTR when applied to either surface of the membrane; however, it may be somewhat more effective from the cytosolic side (approximately 70% inhibition). It may also partially inhibit Na+ conductance. The inhibition was relatively slow, with a half time for maximum effect of about 3 min, and showed very slow reversibility. Results also suggest that CFTR Cl- conductance (GCl) was blocked in both apical and basal membranes. The inhibitor appears to exert some effect on Na+ transport as well., (Copyright 2004 The Physiological Society)

Cystic fibrosis is caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel. Phosphorylation and ATP hydrolysis are generally believed to be indispensable for activating CFTR. Here we report phosphorylation- and ATP-independent activation of CFTR by cytoplasmic glutamate that exclusively elicits Cl-, but not HCO3-, conductance in the human sweat duct. We also report that the anion selectivity of glutamate-activated CFTR is not intrinsically fixed, but can undergo a dynamic shift to conduct HCO3- by a process involving ATP hydrolysis. Duct cells from patients with DeltaF508 mutant CFTR showed no glutamate/ATP activated Cl- or HCO3- conductance. In contrast, duct cells from heterozygous patients with R117H/DeltaF508 mutant CFTR also lost most of the Cl- conductance, yet retained significant HCO3- conductance. Hence, not only does glutamate control neuronal ion channels, as is well known, but it can also regulate anion conductance and selectivity of CFTR in native epithelial cells. The loss of this uniquely regulated HCO3- conductance is most probably responsible for the more severe forms of cystic fibrosis pathology.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a significant role in transepithelial salt absorption as well as secretion by a number of epithelial tissues including sweat glands, airways and intestine. Early studies suggested that in absorption significant cross talk occurs between CFTR Cl(-) channels and epithelial Na(+) channels (ENaC). Studies based primarily on cultured cells of the airways and on ex vivo expression systems suggested that activating CFTR inhibits ENaC channels so that activation of CFTR and deactivation of ENaC seem reciprocal. Lack of CFTR Cl(-) conductance (g(CFTR)) in the plasma membranes was seen to enhance ENaC conductance (g(ENaC)) and Na(+) absorption from the airway surface liquid causing airway pathology in cystic fibrosis (CF). To determine if these events hold true for a purely absorptive epithelium, we investigated the role of CFTR in regulating g(ENaC) in native human sweat gland ducts. After permeabilizing the basilateral membrane of the duct with alpha-toxin, the relative activities of ENaC and CFTR in the apical membrane were characterized by correlating the effect of activating CFTR with ENaC function. We found that in contrast to reciprocal activities, activating g(CFTR) by either cAMP, cGMP or the G-proteins plus 5 mM ATP was accompanied by a concomitant activation, not inhibition, of g(ENaC). The activation of g(ENaC) appeared to be critically dependent on CFTR Cl(-) channel function because removal of Cl(-) from the medium, blockage of CFTR with inhibitor DIDS or the absence of CFTR in the DeltaF508 CF ducts prevented activation of g(ENaC) by cAMP, GMP or G-proteins. Most significantly, g(ENaC) was dramatically reduced, not increased, in CF as compared to non-CF sweat ducts. These results showed that lack of CFTR in the plasma membranes is not characteristically coupled to elevated ENaC activity or to increased Na(+) absorption in CF epithelial cells. Not only are CFTR and ENaC activated together in duct salt absorption, but ENaC activation depends on functioning CFTR. NaCl is poorly absorbed in the CF duct because CFTR activity appears to impose a loss of ENaC activity as well.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A (PKA) and ATP regulated Cl- channel. Studies using mostly ex vivo systems suggested diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and glybenclamide inhibit CFTR Cl- conductance (CFTR GCl). However, the properties of inhibition in a native epithelial membrane have not been well defined. The objective of this study was to determine and compare the inhibitory properties of the aforementioned inhibitors as well as the structurally related anion-exchange blockers (stilbenes) including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) in the microperfused intact and basilaterally permeabilized native sweat duct epithelium. All of these inhibitors blocked CFTR in a dose-dependent manner from the cytoplasmic side of the basilaterally permeabilized ducts, but none of these inhibitors blocked CFTR GCl from the luminal surface. We excluded inhibitor interference with a protein kinase phosphorylation activation process by "irreversibly" thiophosphorylating CFTR prior to inhibitor application. We then activated CFTR GCl by adding 5 mM ATP. At a concentration of 10(-4) M, NPPB, DPC, glybenclamide, and DIDS were equipotent and blocked approximately 50% of irreversibly phosphorylated and ATP-activated CFTR GCl (DIDS = 49 +/- 10% > NPPB = 46 +/- 10% > DPC = 38 +/- 7% > glybenclamide = 34 +/- 5%; values are mean +/- SE expressed as % inhibition from the control). The degree of inhibition may be limited by inhibitor solubility limits, since DIDS, which is soluble to 1 mM concentration, inhibited 85% of CFTR GCl at this concentration. All the inhibitors studied primarily blocked CFTR from the cytoplasmic side and all inhibition appeared to be independent of metabolic and phosphorylation processes.

While cystic fibrosis transmembrane conductance regulator (CFTR) is well known to function as a Cl(-) channel, some mutations in the channel protein causing cystic fibrosis (CF) disrupt another vital physiological function, HCO(3)(-) transport. Pathological implications of derailed HCO(3)(-) transport are clearly demonstrated by the pancreatic destruction that accompany certain mutations in CF. Despite the crucial role of HCO(3)(-) in buffering pH, little is known about the relationship between cause of CF pathology and the molecular defects arising from specific mutations. Using electrophysiological techniques on basolaterally permeabilized preparations of microperfused native sweat ducts, we investigated whether: a) CFTR can act as a HCO(3)(-) conductive channel, b) different conditions for stimulating CFTR can alter its selectivity to HCO(3)(-) and, c) pancreatic insufficiency correlate with HCO(3)(-) conductance in different CFTR mutations. We show that under some conditions stimulating CFTR can conduct HCO(3)(-). HCO(3)(-) conductance in the apical plasma membranes of sweat duct appears to be mediated by CFTR and not by any other Cl(-) channel because HCO(3)(-) conductance is abolished when CFTR is: a) deactivated by removing cAMP and ATP, b) blocked by 1 mM DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the cytoplasmic bath and, c) absent in the plasma membranes of DeltaF508 CF ducts. Further, the HCO(3)(-)/Cl(-) selectivity of CFTR appears to be dependent on the conditions of stimulating CFTR. That is, CFTR activated by cAMP + ATP appears to conduct both HCO(3)(-) and Cl(-) (with an estimated selectivity ratio of 0.2 to 0.5). However, we found that in the apparent complete absence of cAMP and ATP, cytoplasmic glutamate activates CFTR Cl(-) conductance without any HCO(3)(-) conductance. Glutamate activated CFTR can be induced to conduct HCO(3)(-) by the addition of ATP without cAMP. The non-hydrolysable AMP-PNP (5'-adenylyl imidodiphosphate) cannot substitute for ATP in activating HCO(3)(-) conductance. We also found that a heterozygous R117H/DeltaF508 CFTR sweat duct retained significant HCO(3)(-) conductance while a homozygous DeltaF508 CFTR duct showed virtually no HCO(3)(-) conductance. While we suspect that the conditions described here are not optimal for selectively activating CFTR Cl(-) and HCO(3)(-) conductances, we surmise that CFTR may be subject to dramatic alterations in its conductance, at least to these two anions under distinctly different physiological conditions which require distinctly different physiological functions. That is physiologically, CFTR may exhibit Cl(-) conductance with and/or without HCO(3)(-) conductance. We also surmise that the severity of the pathogenesis in CF is closely related to the phenotypic ability of a mutant CFTR to express a HCO(3)(-) conductance.

It is generally believed that cAMP-dependent phosphorylation is the principle mechanism for activating cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. However, we showed that activating G proteins in the sweat duct stimulated CFTR Cl(-) conductance (G(Cl)) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G(Cl) is independent of protein kinase A. We activated G proteins and monitored CFTR G(Cl) in basolaterally permeabilized sweat duct. Activating G proteins with guanosine 5'-O-(3-thiotriphosphate) (10-100 microM) stimulated CFTR G(Cl) in the presence of 5 mM ATP alone without cAMP. G protein activation of CFTR G(Cl) required Mg(2+) and ATP hydrolysis (5'-adenylylimidodiphosphate could not substitute for ATP). G protein activation of CFTR G(Cl) was 1) sensitive to inhibition by the kinase inhibitor staurosporine (1 microM), indicating that the activation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM) and SQ-22536 (100 microM); and 3) independent of Ca(2+), suggesting that Ca(2+)-dependent protein kinase C and Ca(2+)/calmodulin-dependent kinase(s) are not involved in the activation process. Activating AC with 10(-6) M forskolin plus 10(-6) M IBMX (in the presence of 5 mM ATP) did not activate CFTR, indicating that cAMP cannot accumulate sufficiently to activate CFTR in permeabilized cells. We concluded that heterotrimeric G proteins activate CFTR G(Cl) endogenously via a cAMP-independent pathway in this native absorptive epithelium.

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl- conductance (CFTR G(Cl)) in the native sweat duct (SD). We permeabilized the basolateral membrane with alpha-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G(Cl) activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-gamma-S (100 microm) also activates CFTR G(Cl) in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-gamma-S increased CFTR G(Cl) by 44 +/- 20 mS/cm(2) (mean +/- se; n = 7). GDP (10 mm) inhibited G-protein activation of CFTR G(Cl) even in the presence of GTP-gamma-S. The heterotrimeric G-protein activator (AlF(4-) in the cytoplasmic bath activated CFTR G(Cl) (increased by 51.5 +/- 9.4 mS/cm(2) in the presence of 5 mm ATP without cAMP, n = 6), the magnitude of which was similar to that induced by GTP-gamma-S. Employing immunocytochemical-labeling techniques, we localized Galphas, Galphai, Galphaq, and Gbeta at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G(Cl) in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G(Cl) activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G(Cl) activity.

It is increasingly being recognized that cells coordinate the activity of separate ion channels that allow electrolytes into the cell. However, a perplexing problem in channel regulation has arisen in the fatal genetic disease cystic fibrosis, which results from the loss of a specific Cl- channel (the CFTR channel) in epithelial cell membranes. Although this defect clearly inhibits the absorption of Na+ in sweat glands, it is widely accepted that Na+ absorption is abnormally elevated in defective airways in cystic fibrosis. The only frequently cited explanation for this hypertransport is that the activity of an epithelial Na+ channel (ENaC) is inversely related to the activity of the CFTR Cl- channel. However, we report here that, in freshly isolated normal sweat ducts, ENaC activity is dependent on, and increases with, CFTR activity. Surprisingly, we also find that the primary defect in Cl- permeability in cystic fibrosis is accompanied secondarily by a Na+ conductance in this tissue that cannot be activated. Thus, reduced salt absorption in cystic fibrosis is due not only to poor Cl- conductance but also to poor Na+ conductance.

Bumetanide is well known for its ability to inhibit the nonconductive Na+-K+-2Cl- cotransporter. We were surprised in preliminary studies to find that bumetanide in the contraluminal bath also inhibited NaCl absorption in the human sweat duct, which is apparently poor in cotransporter activity. Inhibition was accompanied by a marked decrease in the transepithelial electrical conductance. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is richly expressed in the sweat duct, we asked whether bumetanide acts by blocking this anion channel. We found that bumetanide 1) significantly increased whole cell input impedance, 2) hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membrane potential, 4) increased the ratio of apical-to-basolateral membrane resistance, and 5) decreased transepithelial Cl- conductance (GCl). These results indicate that bumetanide inhibits CFTR GCl in both cell membranes of this epithelium. We excluded bumetanide interference with the protein kinase A phosphorylation activation process by "irreversibly" phosphorylating CFTR [by using adenosine 5'-O-(3-thiotriphosphate) in the presence of a phosphatase inhibition cocktail] before bumetanide application. We then activated CFTR GCl by adding 5 mM ATP. Bumetanide in the cytoplasmic bath (10(-3) M) inhibited approximately 71% of this ATP-activated CFTR GCl, indicating possible direct inhibition of CFTR GCl. We conclude that bumetanide inhibits CFTR GCl in apical and basolateral membranes independent of phosphorylation. The results also suggest that >10(-5) M bumetanide cannot be used to specifically block the Na+-K+-2Cl- cotransporter.

Physiological Basis of Cystic Fibrosis: A Historical Perspective. Physiol. Rev. 79, Suppl.: S3-S22, 1999. - Cystic fibrosis made a relatively late entry into medical physiology, although references to conditions probably reflecting the disease can be traced back well into the Middle Ages. This review begins with the origins of recognition of the symptoms of this genetic disease and proceeds to briefly review the early period of basic research into its cause. It then presents the two apparently distinct faces of cystic fibrosis: 1) as that of a mucus abnormality and 2) as that of defects in electrolyte transport. It considers principal findings of the organ and cell pathophysiology as well as some of the apparent conflicts and enigmas still current in understanding the disease process. It is written from the perspective of the author, whose career spans back to much of the initial endeavors to explain this fatal mutation.

Our objective in this study was to determine the effect of changes in luminal and cytoplasmic pH on cystic fibrosis transmembrane regulator (CFTR) Cl- conductance (GCl). We monitored CFTR GCl in the apical membranes of sweat ducts as reflected by Cl- diffusion potentials (VCl) and transepithelial conductance (GCl). We found that luminal pH (5.0-8.5) had little effect on the cAMP/ATP-activated CFTR GCl, showing that CFTR GCl is maintained over a broad range of extracellular pH in which it functions physiologically. However, we found that phosphorylation activation of CFTR GCl is sensitive to intracellular pH. That is, in the presence of cAMP and ATP [adenosine 5'-O-(3-thiotriphosphate)], CFTR could be phosphorylated at physiological pH (6.8) but not at low pH (approximately 5.5). On the other hand, basic pH prevented endogenous phosphatase(s) from dephosphorylating CFTR. After phosphorylation of CFTR with cAMP and ATP, CFTR GCl is normally deactivated within 1 min after cAMP is removed, even in the presence of 5 mM ATP. This deactivation was due to an increase in endogenous phosphatase activity relative to kinase activity, since it was reversed by the reapplication of ATP and cAMP. However, increasing cytoplasmic pH significantly delayed the deactivation of CFTR GCl in a dose-dependent manner, indicating inhibition of dephosphorylation. We conclude that CFTR GCl may be regulated via shifts in cytoplasmic pH that mediate reciprocal control of endogenous kinase and phosphatase activities. Luminal pH probably has little direct effect on these mechanisms. This regulation of CFTR may be important in shifting electrolyte transport in the duct from conductive to nonconductive modes.