Quinidine-sensitive K+ channels in the basolateral membrane of embryonic coprodeum epithelium: regulation by aldosterone and thyroxine

Basolateral K⁺ channels and their regulation during aldosterone- and thyroxine-stimulated Na⁺ transport were studied in the lower intestinal epithelium (coprodeum) of embryonic chicken in vitro. Isolated tissues of the coprodeum were mounted in Ussing chambers and investigated under voltage-clamped conditions. Simultaneous stimulation with aldosterone (1 mol·l^-1) and thyroxine (1 mol·l^-1) raised short-circuit current after a 1- to 2-h latent period. Maximal values were reached after 6–7 h of hormonal treatment, at which time transepithelial Na⁺ absorption was more than tripled (77±11 A·cm^-2) compared to control (24±8 A·cm^-2). K⁺ currents across the basolateral membrane with the pore-forming antibiotic amphotericin B and application of a mucosal-to-serosal K⁺ gradient. This K⁺ current could be dose dependently depressed by the K⁺ channel blocker quinidine. Fluctuation analysis of the short-circuit current revealed a spontaneous and a blocker-induced Lorentzian noise component in the power density spectra. The Lorentzian corner frequencies increased linearly with the applied blocker concentration. This enabled the calculation of single K⁺ channel current and K⁺ channel density. Single K⁺ channel current was not affected by stimulation, whereas the number of quinidine-sensitive K⁺ channels in the basolateral membrane increased from 11 to 26·10⁶·cm^-2 in parallel to the hormonal stimulation transepithelial Na⁺ transport. This suggests that the basolateral membrane is a physiological target during synergistic aldosterone and thyroxine regulation of transepithelial Na⁺ transport for maintaining intracellular K⁺ homeostasis.Abbreviations f frequency - f _c Lorentzian corner frequency - g _K single K⁺ channel conductance - HEPES N-2-hydroxyethylpiperazin-N'-2-ethansulfonic acid - i _K single K⁺ channel current - I_Ampho amphotericin B induced K⁺ current - I _sc short-circuit current - I _K quinidine blockable K⁺ current - I _max maximally blocked current by quinidine - IC ₅₀ half-maximal blocker concentration - k _on, k _off on- and off-rate coefficients of reversible single channel block by quinidine - M _K number of conducting K⁺ channels - [Q] quinidine concentration - R _t transepithelial resistance - S spectral density - S _o Lorentzian plateau - TBM cells toad urinary bladder cell line Present address: University of California at Berkeley, Dept. of Molecular and Cell Biology Berkeley, CA 94720, USA     

Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.     

Acid secretion and proton conductance in human airway epithelium

Acid secretion and proton conductive pathways across primary human airway surface epithelial cultures were investigated with the pH stat method in Ussing chambers and by single cell patch clamping. Cultures showed a basal proton secretion of 0.17 +/- 0.04 micromol.h(-1).cm(-2), and mucosal pH equilibrated at 6.85 +/- 0.26. Addition of histamine or ATP to the mucosal medium increased proton secretion by 0.27 +/- 0.09 and 0.24 +/- 0.09 micromol.h(-1).cm(-2), respectively. Addition of mast cells to the mucosal medium of airway cultures similarly activated proton secretion. Stimulated proton secretion was similar in cultures bathed mucosally with either NaCl Ringer or ion-free mannitol solutions. Proton secretion was potently blocked by mucosal ZnCl(2) and was unaffected by mucosal bafilomycin A(1), Sch-28080, or ouabain. Mucosal amiloride blocked proton secretion in tissues that showed large amiloride-sensitive potentials. Proton secretion was sensitive to the application of transepithelial current and showed outward rectification. In whole cell patch-clamp recordings a strongly outward-rectifying, zinc-sensitive, depolarization-activated proton conductance was identified with an average chord conductance of 9.2 +/- 3.8 pS/pF (at 0 mV and a pH 5.3-to-pH 7.3 gradient). We suggest that inflammatory processes activate proton secretion by the airway epithelium and acidify the airway surface liquid.     

Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl? Secretion by Human Airway Epithelia

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl⁻ and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl⁻ secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca²⁺ from the endoplasmic reticulum (ER), lowering [Ca²⁺] in the ER and thereby activating the Ca²⁺-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca²⁺] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl⁻ current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca²⁺ buffer that lowers [Ca²⁺] in the ER similar to the effect of 3O-C12 also increased cAMP and I_Cl. The results suggest that 3O-C12 stimulates CFTR-dependent Cl⁻ and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca²⁺] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl⁻ and fluid secretion.     

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.     

Bridging near and remote Oceania: mtDNA and NRY variation in the Solomon Islands

Although genetic studies have contributed greatly to our understanding of the colonization of Near and Remote Oceania, important gaps still exist. One such gap is the Solomon Islands, which extend between Bougainville and Vanuatu, thereby bridging Near and Remote Oceania, and include both Austronesian-speaking and Papuan-speaking groups. Here, we describe patterns of mitochondrial DNA (mtDNA) and nonrecombining Y chromosome (NRY) variation in over 700 individuals from 18 populations in the Solomons, including 11 Austronesian-speaking groups, 3 Papuan-speaking groups, and 4 Polynesian Outliers (descended via back migration from Polynesia). We find evidence for ancient (pre-Lapita) colonization of the Solomons in old NRY paragroups as well as from M2-M353, which probably arose in the Solomons ～9,200 years ago and is the most frequent NRY haplogroup there. There are no consistent genetic differences between Austronesian-speaking and Papuan-speaking groups, suggesting extensive genetic contact between them. Santa Cruz, which is located in Remote Oceania, shows unusually low frequencies of mtDNA and NRY haplogroups of recent Asian ancestry. This is in apparent contradiction with expectations based on archaeological and linguistic evidence for an early (～3,200 years ago), direct colonization of Santa Cruz by Lapita people from the Bismarck Archipelago, via a migration that "leapfrogged" over the rest of the Solomons. Polynesian Outliers show dramatic island-specific founder events involving various NRY haplogroups. We also find that NRY, but not mtDNA, genetic distance is correlated with the geographic distance between Solomons groups and that historically attested spheres of cultural interaction are associated with the recent genetic structure of Solomons groups, as revealed by mtDNA HV1 sequence and Y-STR haplotype diversity. Our results fill an important lacuna in human genetic studies of Oceania and aid in understanding the colonization and genetic history of this region.     

Partial restoration of defective chloride conductance in DeltaF508 CF mice by trimethylamine oxide

This study was designed to test the in vivo efficacy of the chemical chaperone trimethylamine oxide (TMAO) in correcting the Cl- transport defect in a mouse model of cystic fibrosis (CF). Rectal potential difference (RPD) measurements were done in matched wild-type and DeltaF508 CF mice. Mice were treated by subcutaneous injections of TMAO. Wild-type mice demonstrated a forskolin-stimulated, Cl--dependent hyperpolarization of -6.4 +/- 0.8 mV (n = 11), which was significantly increased to -13.1 +/- 1.4 mV after treatment with TMAO. DeltaF508 CF mice showed no significant responses to forskolin. Treatment with TMAO recovered a forskolin-activated RPD in DeltaF508 CF mice (-1.1 +/- 0.2 mV; n = 17) but not in CFTR null mice. The effects of TMAO were dose dependent, resulting in a slope of -0.4 +/- 0.1 mV x g(-1) x kg(-1) in DeltaF508 CF mice. The forskolin-stimulated RPD in TMAO-treated DeltaF508 CF mice was partially blocked by glibenclamide and further stimulated by apigenin. The total response to forskolin plus apigenin was -2.5 +/- 0.45 mV (n = 6 mice), corresponding to 39% of the response evoked by forskolin only in wild-type mice.     

Ozone enhancement of lower airway allergic inflammation is prevented by gamma-tocopherol

Ozone is a commonly encountered environmental oxidant which has been linked to asthma exacerbation in epidemiological studies. Ozone induces airway inflammation and enhances response to inhaled allergen. It has been suggested that antioxidant therapy may minimize the adverse effects of ozone in asthma. We have previously shown that the antioxidant gamma-tocopherol (gammaT), an isoform of vitamin E, also has anti-inflammatory effects. We employed a Brown Norway rat model of ozone-enhanced allergic responses to test the therapeutic effects of gammaT on O(3)-induced airway inflammation. Ovalbumin (OVA)-sensitized rats were intranasally challenged with 0 or 0.5% OVA on Days 1 and 2, and exposed to 0 or 1 ppm ozone (8 h/day) on Days 4 and 5. Rats were also given 0 or 100 mg/kg gammaT on Days 2 through 5. Pulmonary tissue and bronchoalveolar lavage fluid (BALF) were collected on Day 6. OVA challenge caused increased total cells (267% increase) and eosinophils (4000%) in BALF that was unaffected by ozone exposure. Morphometric evaluation of lung tissue revealed increases in intraepithelial mucosubstances (IM) (300%) and subepithelial eosinophils (400%) in main axial airways. Ozone exposure of allergic rats enhanced IM increases in proximal axial airways (200%), induced cys-leukotrienes, MCP-1, and IL-6 production in BALF, and upregulated expression of IL-5 and IL-13 mRNA. gammaT treatment had no effect on IM increases by allergen, but blocked enhancement by ozone. gammaT attenuated both OVA- or ozone-stimulated eosinophilic infiltration, and increases of BALF cys-leukotrienes, MCP-1, and IL-6, as well as IL-5 and IL-13 mRNA. These data demonstrate broad anti-inflammatory effects of a gammaT and suggest that it may be an effective therapy of allergic airway inflammation.