Horst Fischer

Mechanisms of Acid and Base Secretion by the Airway Epithelium

One of the main functions of the airway epithelium is to inactivate and remove infectious particles from inhaled air and thereby prevent infection of the distal lung. This function is achieved by mucociliary and cough clearance and by antimicrobial factors present in the airway surface liquid (ASL). There are indications that airway defenses are affected by the pH of the ASL and historically, acidification of the airway surfaces has been suggested as a measure of airway disease. However, even in health, the ASL is slightly acidic, and this acidity might be part of normal airway defense. Only recently research has focused on the mechanisms responsible for acid and base secretion into the ASL. Advances resulted from research into the airway disease associated with cystic fibrosis (CF) after it was found that the CFTR Cl− channel conducts HCO3− and, therefore, may contribute to ASL pH. However, the acidity of the ASL indicated parallel mechanisms for H+ secretion. Recent investigations identified several H+ transporters in the apical membrane of the airway epithelium. These include H+ channels and ATP-driven H+ pumps, including a non-gastric isoform of the H+-K+ ATPase and a vacuolar-type H+ ATPase. Current knowledge of acid and base transporters and their potential roles in airway mucosal pH regulation is reviewed here.

Anion selectivity of apical membrane conductance of Calu 3 human airway epithelium

Abstract Anion selectivity of the cystic fibrosis conductance transmembrane conductance regulator (CFTR) and other channels and parallel pathways expressed endogenously in apical membranes of polarized Calu-3 epithelial monolayers was studied under control conditions and during cAMP stimulation. Basolateral membranes were eliminated using alpha-toxin. The cAMP-stimulated, gradient-driven currents had the sequence Br≥Cl≥NO3>SCN> I≥F>formate>HCO3>acetate>propionate=butyrate=ATP= PPi=PO4=SO4=0. Selectivity of parallel cAMP-independent pathway(s) was Br>Cl=SCN=NO3>I>formate=F >HCO3>acetate>propionate. SCN, I, F or formate blocked cAMP-stimulated, but not control, Cl currents. Anions >0.53 nm in diameter were impermeant, suggesting that the apical CFTR channel has a limiting diameter of 0.53 nm. The selectivity, blocking patterns and pore size of the cAMP-stimulated conductance pathway were very similar to those in previous reports in which CFTR was heterologously expressed in non-epithelial cells. Thus, CFTR appears to be the major apical anion conductance pathway in Calu-3 cells, and its conduction properties are independent of the expression system. CFTR in Calu-3 cells also conducts physiologically relevant anions, but not ATP, PO4 or SO4. A pathway parallel (probably a tight junction) showed a different selectivity than CFTR.

Flavonoids stimulate Cl conductance of human airway epithelium in vitro and in vivo

The ability of the flavonoids genistein, apigenin, kaempferol, and quercetin to activate cystic fibrosis transmembrane conductance regulator-mediated Cl currents in human airway epithelium was investigated. We used the patch-clamp technique on single Calu-3 cells, transepithelial measurements in Calu-3 monolayers, and in vivo measurements of nasal potential difference. All flavonoids stimulated Cl currents in transepithelial experiments dose dependently. Half-maximal stimulatory concentrations were kaempferol (5.5 +/- 1.7 microM) </= apigenin (11.2 +/- 2.1 microM) </= genistein (13.6 +/- 3.5 microM) </= quercetin (22.1 +/- 4.5 microM). Stimulation of monolayers with forskolin significantly increased their sensitivity to flavonoids: kaempferol (2.5 +/- 0.7 microM) </= apigenin (3.4 +/- 0.9 microM) </= quercetin (4.1 +/- 0.7 microM) </= genistein (6.9 +/- 2.2 microM). Forskolin pretreatment significantly reduced the Hill coefficient (nH) for all flavonoids. Control monolayers showed nH = 2.00 +/- 0.21 (all flavonoids combined), and forskolin-stimulated monolayers showed nH = 1.07 +/- 0.07, which was not different among the flavonoids. These data imply that the activation kinetics and the binding site(s) for flavonoids were significantly altered by forskolin stimulation. In whole cell patch-clamp experiments, maximal flavonoid-stimulated currents (percentage of forskolin-stimulated currents) were apigenin (429 +/- 86%) >/= kaempferol (318 +/- 45%) >/= genistein (258 +/- 20%) = quercetin (256 +/- 26%). Stimulation of the currents was caused by an increase in channel open probability. No other Cl conductances contributed significantly to the flavonoid-activated Cl currents in Calu-3 cells. In vivo, flavonoids significantly stimulated nasal potential difference by, on average, 27.8% of isoproterenol responses.The ability of the flavonoids genistein, apigenin, kaempferol, and quercetin to activate cystic fibrosis transmembrane conductance regulator-mediated Cl currents in human airway epithelium was investigated. We used the patch-clamp technique on single Calu-3 cells, transepithelial measurements in Calu-3 monolayers, and in vivo measurements of nasal potential difference. All flavonoids stimulated Cl currents in transepithelial experiments dose dependently. Half-maximal stimulatory concentrations were kaempferol (5.5 ± 1.7 μM) ≤ apigenin (11.2 ± 2.1 μM) ≤ genistein (13.6 ± 3.5 μM) ≤ quercetin (22.1 ± 4.5 μM). Stimulation of monolayers with forskolin significantly increased their sensitivity to flavonoids: kaempferol (2.5 ± 0.7 μM) ≤ apigenin (3.4 ± 0.9 μM) ≤ quercetin (4.1 ± 0.7 μM) ≤ genistein (6.9 ± 2.2 μM). Forskolin pretreatment significantly reduced the Hill coefficient ( n H) for all flavonoids. Control monolayers showed n H = 2.00 ± 0.21 (all flavonoids combined), and forskolin-stimulated monolayers showed n H = 1.07 ± 0.07, which was not different among the flavonoids. These data imply that the activation kinetics and the binding site(s) for flavonoids were significantly altered by forskolin stimulation. In whole cell patch-clamp experiments, maximal flavonoid-stimulated currents (percentage of forskolin-stimulated currents) were apigenin (429 ± 86%) ≥ kaempferol (318 ± 45%) ≥ genistein (258 ± 20%) = quercetin (256 ± 26%). Stimulation of the currents was caused by an increase in channel open probability. No other Cl conductances contributed significantly to the flavonoid-activated Cl currents in Calu-3 cells. In vivo, flavonoids significantly stimulated nasal potential difference by, on average, 27.8% of isoproterenol responses.

Function of the HVCN1 proton channel in airway epithelia and a naturally occurring mutation, M91T.

Airways secrete considerable amounts of acid. In this study, we investigated the identity and the pH-dependent function of the apical H+ channel in the airway epithelium. In pH stat recordings of confluent JME airway epithelia in Ussing chambers, Zn-sensitive acid secretion was activated at a mucosal threshold pH of ∼7, above which it increased pH-dependently at a rate of 339 ± 34 nmol × h−1 × cm−2 per pH unit. Similarly, H+ currents measured in JME cells in patch clamp recordings were readily blocked by Zn and activated by an alkaline outside pH. Small interfering RNA–mediated knockdown of HVCN1 mRNA expression in JME cells resulted in a loss of H+ currents in patch clamp recordings. Cloning of the open reading frame of HVCN1 from primary human airway epithelia resulted in a wild-type clone and a clone characterized by two sequential base exchanges (452T>C and 453G>A) resulting in a novel missense mutation, M91T HVCN1. Out of 95 human genomic DNA samples that were tested, we found one HVCN1 allele that was heterozygous for the M91T mutation. The activation of acid secretion in epithelia that natively expressed M91T HVCN1 required ∼0.5 pH units more alkaline mucosal pH values compared with wild-type epithelia. Similarly, activation of H+ currents across recombinantly expressed M91T HVCN1 required significantly larger pH gradients compared with wild-type HVCN1. This study provides both functional and molecular indications that the HVCN1 H+ channel mediates pH-regulated acid secretion by the airway epithelium. These data indicate that apical HVCN1 represents a mechanism to acidify an alkaline airway surface liquid.

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells.

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.

REGULATION OF CFTR BY PROTEIN PHOSPHATASE 2B AND PROTEIN KINASE C

Abstract The activity of the CFTR Cl– channel is dependent on its phosphorylation status set by kinases and phosphatases. We report here that protein phosphatase 2B (PP2B) and protein kinase C (PKC) are potential regulators of the cystic fibrosis conductance regulator (CFTR). Treating CFTR-expressing 3T3 cells with either of the two specific PP2B blockers cyclosporin A (CsA, 1 μM) or deltamethrin (DM, 30 nM) caused rapid activation of CFTR in cell-attached patches. As determined by noise analysis of multi channel patches, DM- or CsA-activated CFTR displayed gating kinetics comparable to those of forskolin-activated CFTR. After activation of CFTR by blocking PP2B, CFTR still inactivated. CFTR-mediated currents were, on average, 6.1 times larger when cells were stimulated by forskolin during PP2B block compared to stimulation by forskolin alone. This suggests that, in CFTR-expressing 3T3 cells, a phosphorylation site of CFTR is regulated by cellular PKA, PP2B and another phosphatase. However, in the epithelial cell lines Calu-3 and HT-29/B6, CsA and DM had no effect on CFTR activity in both cell-attached patch-clamp and transepithelial experiments. In contrast, when exogenous PP2B was added to patches excised from 3T3 or Calu-3 cells, PKA-activated CFTR currents were quickly inactivated. This indicates that free exogenous PP2B can inactivate CFTR in patches from both cell types. We propose that in order to regulate CFTR in an intact cell, PP2B may require a selective subcellular localization to become active. When excised patches were PKC-phosphorylated, the gating kinetics of CFTR were significantly different from those of PKA-phosphorylated CFTR. Addition of PP2B also inactivated PKC-activated CFTR showing the indiscriminate dephosphorylation of different phosphorylation sites by PP2B.

Cl Transport in Complemented CF Bronchial Epithelial Cells Correlates with CFTR mRNA Expression Levels

Little is known about the relationship between CF transmembrane conductance regulator (CFTR) gene expression and the corresponding transport of Cl. The phenotypic characteristics of polarized ΔF508 homozygote CF bronchial epithelial (CFBE41o-) cells were evaluated following transfection with episomal expression vector containing either full-length (6.2kb) wild type (wt) and (4.7kb) ΔF508CFTR cDNA. Forskolin-stimulated Cl secretion in two clones expressing the full-length wild type CFTR was assessed; clone c7-6.2wt gave 13.4±2.5 µA/cm2 and clone c10-6.2wt showed 41.3±25.3 µA/cm2. Another clone (c4-4.7ΔF) complemented with the ΔF508 CFTR cDNA showed high and stable expression of vector-derived ΔF508 CFTR mRNA and a small cAMP-stimulated Cl current (4.7±0.7 µA/cm2) indicating ΔF508CFTR trafficking to the plasma membrane at physiological temperatures. Vector-driven CFTR mRNA levels were 5-fold (c7-6.2wt), 14-fold (c10-6.2wt), and 27-fold (c7-4.7ΔF) higher than observed in normal bronchial epithelial cells (16HBE14o-) endogenously expressing wtCFTR. Assessment of CFTR mRNA levels and CFTR function showed that cAMP-stimulated CFTR Cl currents were 33%, 167% and 24%, respectively, of those in 16HBE14o- cells. The data suggest that transgene expression needs to be significantly higher than endogenously expressed CFTR to restore functional wtCFTR Cl transport to levels sufficient to reverse CF pathology.

The actin filament disrupter cytochalasin D activates the recombinant cystic fibrosis transmembrane conductance regulator Cl- channel in mouse 3T3 fibroblasts.

1. Cytochalasin D (CD; 5 microM) readily stimulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl‐ channel activity in cell‐attached and whole‐cell patch recordings from 3T3 fibroblasts expressing recombinant CFTR but not in mock‐transfected cells. CD‐stimulated currents were indistinguishable from those evoked by forskolin stimulation. Kinetic analysis of CFTR gating showed identical channel behaviour independent of the agonist used. 2. To elucidate the mechanism of action of CD we tested its effects on cAMP, protein kinase A, and the CFTR itself during CD stimulation. In contrast to forskolin treatment, CD did not increase cellular cAMP content. 3. A direct interaction of CD with the CFTR was ruled out because CD showed no effect on CFTR in excised inside‐out patches. 4. CD effects were fully blocked when the cellular protein kinase A was inhibited by treatment of cells with RpcAMPS in cell‐attached patches or when protein kinase inhibitor peptide was dialysed into cells in whole‐cell experiments. 5. Addition of G‐actin to excised patches had no effects on CFTR. 6. We conclude that the stimulatory effect of CD is cAMP independent, but needs a functional protein kinase A in order to activate the CFTR. We propose that cytochalasin D activates CFTR by releasing a cellular inhibitor, e.g. a phosphatase, that is held in place by F‐actin.

Regulated gene expression in cultured type II cells of adult human lung

Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at approximately 95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days on collagen-coated dishes with or without DCI for the final 3 days. In freshly isolated cells, highly expressed genes included SFTPA/B/C, SCGB1A, IL8, CXCL2, and SFN in addition to ubiquitously expressed genes. Transcript abundance was correlated between fetal and adult cells (r = 0.88), with a subset of 187 genes primarily related to inflammation and immunity that were expressed >10-fold higher in adult cells. During control culture, expression increased for 8.1% of expressed genes and decreased for approximately 4% including 118 immune response and 10 surfactant-related genes. DCI treatment promoted lamellar body production and increased expression of approximately 3% of probed genes by > or =1.5-fold; 40% of these were also induced in fetal cells. Highly induced genes (> or =10-fold) included PGC, ZBTB16, DUOX1, PLUNC, CIT, and CRTAC1. Twenty-five induced genes, including six genes related to surfactant (SFTPA/B/C, PGC, CEBPD, and ADFP), also had decreased expression during control culture and thus are candidates for hormonal regulation in vivo. Our results further define the adult human type II cell molecular phenotype and demonstrate that a subset of genes remains hormone responsive in cultured adult cells.

Proton secretion in freshly excised sinonasal mucosa from asthma and sinusitis patients.

Background Proton (H+) secretion and the HVCN1 H+ channel are part of the innate host defense mechanism of the airways. The objective of this study was to determine H+ secretion in asthmatic and nonasthmatic patients with chronic rhinosinusitis (CRS) in freshly excised human sinonasal tissue. Methods Nasal or sinus mucosa from subjects with three different conditions (normal, CRS, and CRS with asthma) was harvested during sinus surgery. The equilibrium pH and the rate of H+ secretion were measured in an Ussing chamber using the pH-stat titration technique. Results Nasal epithelia isolated from subjects with CRS and asthma had a mucosal equilibrium pH = 6.95 (n = 5), which was significantly lower than in normal subjects (7.35 ± 0.21; n = 5) or from subjects with CRS without asthma (7.33 ± 0.15 In = 5). Nasal epithelia from CRS with asthma (n = 5) secreted H+ at a rate of 135 ± 46 nmol·min–1·cm–2. This rate was significantly higher compared with normal (73 ± 39 nmol·min–1·cm–2; n = 8) or CRS without asthma (51 ± 28 nmol·min–1·cm–2; n = 7). Mucosal addition of the HVCN1 blocker ZnCl2 blocked H+ secretion by 70% in normal, 53% in CRS without asthma, and by 51% in CRS with asthma. In contrast, measures in sinus tissues were unaffected by the disease condition. Conclusion Freshly excised human nasal and sinus epithelia secrete acid. Nasal (but not sinus) tissues from asthmatic CRS patients showed lower mucosal pH values and higher rates of H+ secretion than CRS and normal subjects. The increased acid secretion might contribute to epithelial injury in CRS patients with asthma.