Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation.

The transepithelial potential difference (PD) of cystic fibrosis (CF) airway epithelium is abnormally raised and the Cl- permeability is low. We studied the contribution of active Na+ absorption to the PD and attempted to increase the Cl- permeability of CF epithelia. Nasal epithelia from CF and control subjects were mounted in Ussing chambers and were short-circuited. The basal rate of Na+ absorption was raised in CF polyps compared with control tissues. Whereas beta agonists induced Cl- secretion in normal and atopic epithelia, beta agonists further increased the rate of Na+ absorption in CF epithelia without inducing Cl- secretion. This unusual effect is not due to an abnormal CF beta receptor because similar effects were induced by forskolin, and because cAMP production was similar in normal and CF epithelia. We conclude that CF airway epithelia absorb Na+ at an accelerated rate. The abnormal response to beta agonists may reflect a primary abnormality in a cAMP-modulated path, or a normal cAMP-modulated process in a Cl- impermeable epithelial cell

Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.

The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia

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. CFTR-mediated Cl-conductance function was measured as whole cell current, and CFTR protein was quantitated by immunoblot analysis. Overall, there was a good relationship between CFTR protein levels and CFTR-mediated Cl- conductance. Some clones had consistently high basal levels of CFTR-mediated Cl- conductance. This variation in function was partially explained by CFTR protein levels and was not due to clonal variation in cAMP metabolism. High levels of CFTR expression were associated with depolarization of fibroblast membrane potential. The CFTR-expressing clones with the largest basally active CFTR Cl- conductances and the most depolarized membrane potentials also exhibited slower growth rates. These results suggest that potential side effects of gene replacement therapy for cystic fibrosis include functional consequences of CFTR expression in non-epithelial cells

A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (coenzyme Q) in \u3ci\u3eArabidopsis\u3c/i\u3e

Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and liposoluble antioxidant. In plants, it is not known how the C-6 hydroxylation of demethoxyubiquinone, the penultimate step in ubiquinone biosynthesis, is catalyzed. The combination of cross-species gene network modeling along with mining of embryo-defective mutant databases of Arabidopsis thaliana identified the embryo lethal locus EMB2421 (At1g24340) as a top candidate for the missing plant demethoxyubiquinone hydroxylase. In marked contrast with prototypical eukaryotic demethoxyubiquinone hydroxylases, the catalytic mechanism of which depends on a carboxylatebridged di-iron domain, At1g24340 is homologous to FADdependent oxidoreductases that instead use NAD(P)H as an electron donor. Complementation assays in Saccharomyces cerevisiae and Escherichia coli demonstrated that At1g24340 encodes a functional demethoxyubiquinone hydroxylase and that the enzyme displays strict specificity for the C-6 position of the benzoquinone ring. Laser-scanning confocal microscopy also showed that GFP-tagged At1g24340 is targeted to mitochondria. Silencing of At1g24340 resulted in 40 to 74% decrease in ubiquinone content and de novo ubiquinone biosynthesis. Consistent with the role of At1g24340 as a benzenoid ring modification enzyme, this metabolic blockage could not be bypassed by supplementation with 4-hydroxybenzoate, the immediate precursor of ubiquinone’s ring. Unlike in yeast, in Arabidopsis overexpression of demethoxyubiquinone hydroxylase did not boost ubiquinone content. Phylogenetic reconstructions indicated that plant demethoxyubiquinone hydroxylase is most closely related to prokaryotic monooxygenases that act on halogenated aromatics and likely descends from an event of ho

Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel whose absence leads to dehydration and acidification of CF airways. A contributing factor to CF lung disease is dysregulation of the epithelial Na+ channel (ENaC), which exacerbates mucus dehydration. Here, we show that ENaC hyperactivity in CF airways is direct consequence of acidic airway surface liquid (ASL) and that ASL hydration is restored by raising ASL pH. Additionally, we show that short palate lung and nasal epithelial clone 1, the most abundant gene in airway epithelia, is the extracellular pH-sensitive factor that inhibits ENaC in normal but not CF airways. We suggest that future CF therapy be directed toward raising the pH of CF airways

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

BACKGROUND: The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na(+)-K(+)-ATPase. METHODS: Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na(+)-K(+)-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection. RESULTS: The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α(1)Na(+)-K(+)-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains. CONCLUSIONS: These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs

Hypersomnolence and Sleep-related Complaints in Metropolitan, Urban, and Rural Georgia

Persistent daytime hypersomnolence is associated with significant morbidity and mortality, but its prevalence in the population has been poorly documented. This study sought to characterize the prevalence of persistent daytime hypersomnolence, difficulties initiating and maintaining sleep, unrefreshing sleep, snoring, and the presence of physician-diagnosed sleep disorders in metropolitan, urban, and rural US Georgia populations. Between September 2004 and July 2005, a total of 6,530 randomly selected well and unwell adults, identified by screening interviews of 10,837 households (contacted by random digit dialing), completed a detailed phone interview. Sixteen percent reported persistent problems staying awake during the day; 26% reported persistent problems falling asleep at night; 31% experienced problems sleeping through the night; 34% were bothered by unrefreshing sleep; and 33% reported that they snored. In spite of the high occurrence of reported persistent sleep problems, only 10% of the survey participants reported having been diagnosed with a sleep disorder. These study findings highlight the need for increased public and clinician awareness with respect to proactively indentifying signs and symptoms of sleep disorders, a better understanding of their adverse impact upon morbidity and mortality, and their negative impact upon socioeconomic and academic potential

Ileal mucosal bile acid absorption is increased in Cftr knockout mice

BACKGROUND: Excessive loss of bile acids in stool has been reported in patients with cystic fibrosis. Some data suggest that a defect in mucosal bile acid transport may be the mechanism of bile acid malabsorption in these individuals. However, the molecular basis of this defect is unknown. This study examines the expression of the ileal bile acid transporter protein (IBAT) and rates of diffusional (sodium independent) and active (sodium dependent) uptake of the radiolabeled bile acid taurocholate in mice with targeted disruption of the cftr gene. METHODS: Wild-type, heterozygous cftr (+/-) and homozygous cftr (-/-) mice were studied. Five one-cm segments of terminal ileum were excised, everted and mounted onto thin stainless steel rods and incubated in buffer containing tracer (3)H-taurocholate. Simultaneously, adjacent segments of terminal ileum were taken and processed for immunohistochemistry and Western blots using an antibody against the IBAT protein. RESULTS: In all ileal segments, taurocholate uptake rates were fourfold higher in cftr (-/-) and two-fold higher in cftr (+/-) mice compared to wild-type mice. Passive uptake was not significantly higher in cftr (-/-) mice than in controls. IBAT protein was comparably increased. Immuno-staining revealed that the greatest increases occurred in the crypts of cftr (-/-) animals. CONCLUSIONS: In the ileum, IBAT protein densities and taurocholate uptake rates are elevated in cftr (-/-) mice > cftr (+/-) > wild-type mice. These findings indicate that bile acid malabsorption in cystic fibrosis is not caused by a decrease in IBAT activity at the brush border. Alternative mechanisms are proposed, such as impaired bile acid uptake caused by the thick mucus barrier in the distal small bowel, coupled with a direct negative regulatory role for cftr in IBAT function