Ion transport across the murine intestine in the absence and presence of CFTR.

CF mice, i.e., mice without functional CFTR (cystic fibrosis transmembrane conductance regulator) exhibit a very low basal Isc in all regions of the intestinal tract.

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units.

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.

A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis.

Background: Cystic fibrosis is a monogenic disease that deranges multiple systems of ion transport in the airways, culminating in chronic infection and destruction of the lung. The introduction of a normal copy of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the airway epithelium through gene transfer is an attractive approach to correcting the underlying defects in patients with cystic fibrosis. We tested the feasibility of gene therapy using adenoviral vectors in the nasal epithelium of such patients.

CFTR as a cAMP-dependent regulator of sodium channels.

Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results.

Relative expression of the human epithelial Na+ channel subunits in normal and cystic fibrosis airways.

The availability of the newly cloned subunits (alpha, beta, gamma) of the epithelial Na+ channel (ENaC) permits molecular studies of the pathogenesis of the abnormal Na+ transport rates of cystic fibrosis (CF) airway epithelia. Northern analyses of airway epithelia showed that both normal and CF airway epithelia express ENaC subunit mRNAs in a ratio of alpha > beta > gamma. In situ hybridization studies revealed expression of all three ENaC subunits in the superficial epithelium and the alpha- and beta-subunits in the gland ductular and acinar epithelium of both normal and CF airways.

Effect of adenoviral vector infection on cell proliferation in cultured primary human airway epithelial cells.

Although recombinant adenoviruses are used as vectors for delivering therapeutic genes to the airways of cystic fibrosis (CF) patients, the effects of these vectors on the kinetics of airway epithelial cell growth have not been investigated. We tested whether E1, E3-deleted Ad vectors (Ad5-CMV-lacZ) affect the kinetics of cell proliferation of human airway epithelial cells in primary culture. There was a dose-dependent relationship between the vector multiplicity of infection (moi) and the efficiency of Ad-mediated lacZ gene transfer.

Ion transport across the jejunum in normal and cystic fibrosis mice.

Cystic fibrosis (CF) mice created by targeted disruption of the murine cystic fibrosis transmembrane conductance regulator gene lack adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and exhibit marked intestinal complications secondary to inadequate fluid secretion. The basal short-circuit current (Isc) in the normal murine jejuna [43.2 +/- 5.

Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.

Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control.

Pharmacologic modulation of salt and water in the airway epithelium in cystic fibrosis.

Cystic fibrosis (CF) is a recessive genetic disease with thickened airway secretions that result from abnormal airway epithelial ion transport, including defective cyclic AMP-mediated Cl- (liquid) secretion and excessive Na+ (liquid) absorption. These abnormalities reflect mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which normally functions as a cyclic AMP-regulated Cl- channel. Aerosolized pharmacologic agents are being tested as novel treatment for these genetic ion transport defects.

Activation of CFTR Cl- conductance in polarized T84 cells by luminal extracellular ATP.

Luminal extracellular ATP evoked a bumetanide-sensitive short-circuit current in cultured T84 cell epithelia (90.2 +/- 18.2 microA/cm2 at 100 microM ATP, apparent 50% effective concentration, 11.

Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP.

Cultured normal and cystic fibrosis (CF) airway epithelia were exposed to 5'-(N-ethylcarboxamido)-adenosine (NECA), ATP, or ionomycin. NECA activated a sustained, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-insensitive Cl- secretory response in normal but not CF, consistent with stimulation of the CF transmembrane conductance regulator (CFTR). In normal and CF, ionomycin or ATP induced Cl- secretion with an initial peak that was inhibited > 50% by DIDS, but in normals there was a prolonged current that was not inhibited by DIDS.

Inefficient gene transfer by adenovirus vector to cystic fibrosis airway epithelia of mice and humans.

The success of adenoviral vectors for gene therapy of lung disease in cystic fibrosis (CF) depends on efficient transfer of the complementary DNA encoding the correct version of the cystic fibrosis transmembrane regulator (CFTR) to the affected columnar epithelial cells lining the airways of the lung. Pre-clinical studies in vitro suggest that low doses of adenovirus vectors carrying this CFTR cDNA can correct defective Cl- transport in cultured human CF airway epithelia. Here we use mice carrying the disrupted CF gene to test the efficacy of this transfer system in vivo.

Modulation of the ionic milieu of the airway in health and disease.

Airway surface liquid (ASL) is an integral part of lung defense mechanisms. Ion transport by airway epithelia regulates the volume and composition of this fluid. A better understanding of the mechanisms of ion transport will enable the development of new therapies for airway diseases associated with defects in these mechanisms.