cAMP-mediated Cl Research Articles

CFTR channels in immortalized human airway cells.

The cystic fibrosis (CF) gene codes for CF transmembrane regulator (CFTR), a small-conductance linear Cl- channel, but numerous studies have identified a larger conductance, rectifying Cl- channel as the adenosine 3',5'-cyclic monophosphate (cAMP)-regulated channel that is defective in airway cells. We examined Cl- conductance in a bronchial epithelial cell line that expresses CFTR, 16HBE14o-, (CFTR+) and in an airway cell line that does not, 9HTEo-/S, (CFTR-). Ionomycin or hypotonic Ringer increased iodide efflux from both cell lines; however, forskolin increased iodide efflux or whole cell Cl- currents only in CFTR+ cells. Forskolin-stimulated whole cell currents were linear, voltage independent, and blocked by iodide. Cell-attached and outside-out patches from confluent CFTR+ but not CFTR- cells revealed 6-pS channels having linear current-voltage relations, permselectivity Cl > I (partial block by external iodide), and little or no inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate. The number of active channels per patch increased from 0.6 to 3.0 after forskolin. Channels closed after excision with tau = 4 s, but activity could be prolonged with ATP or protein kinase A plus ATP. Channels were modeled with one open and four closed states and show apparent cooperativity in gating. Rectifying Cl- channels previously implicated in CF were not seen in cell-attached recordings from either cell line but were abundant in excised patches from both cell lines. Thus CFTR channels are the pathway for cAMP-mediated Cl- conductance in these human airway cells, Ca2+ and swelling-induced channels do not require CFTR, and CFTR-cells display a CF phenotype.

The outwardly rectifying Cl- channel is not involved in cAMP-mediated Cl- secretion in HT-29 cells: evidence for a very-low-conductance Cl- channel.

The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl- channel (ORDIC) during cAMP-mediated Cl- secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl- channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4'-dinitro-2,2'-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ODIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl- secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC50 = 0.5 +/- 0.3 mumol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl- secretion unless high concentrations were used (IC50 = 240 +/- 10 mumol/l). In order to exclude effects of NPPB other than on the apical Cl- channel, transepithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl- gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl- current showed a very-low-frequency Lorentzian noise component (fc = 1.4 +/- 0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC.(ABSTRACT TRUNCATED AT 250 WORDS)

CFTR!

Cystic fibrosis (CF) is a fatal genetic disease primarily affecting Caucasians, although cases have been reported from other ethnic groups. CF has a complex etiology, but it is chiefly a disease of electrolyte transport and is characterized by defects in fluid secretion by several epithelia, including the sweat duct, exocrine pancreas, and the pulmonary airways. The link between CF and a defect in cAMP-mediated Cl- transport in secretory epithelia was established in the early 1980s. Since then, numerous electrophysiological studies have focused on the characterization and regulation of individual Cl- channels underlying the macroscopic Cl- currents of secretory epithelia in the airways, sweat ducts, and gut. In this review the results of these studies in the light of current knowledge of the function of the CF gene product, the CF transmembrane conductance regulator (CFTR) protein, will be analyzed. The CFTR protein is a member of a family of ATP-binding proteins that act as unidirectional solute pumps. These proteins are membrane spanning, are found in both prokaryotic and eukaryotic cells, and have two ATP-binding domains. The family includes the p-glycoproteins that are involved with the expression of multidrug resistance in certain tumor cells. The majority of CF chromosomes (70%) have a single codon deletion that translates to a missing phenylalanine residue at position 508 (delta F508) of the protein. Unique for this family of proteins, the CFTR protein possesses an additional highly charged domain (the R domain) containing several consensus polypeptide sequences for kinase phosphorylation. Although CFTR bears structural resemblance to this family of ATP-dependent pumps, overexpression of the protein in a variety of different cell types is associated with the appearence of a cAMP-sensitive Cl- channel. We critically examine current information concerning the structure-function relationships of the CFTR protein obtained from both electrophysiological and biochemical approaches. We also summarize recent evidence suggesting that the CFTR protein may act as a pump and a channel, a hypothesis in keeping with the multifaceted nature of the disease.

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium

Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl − permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11–14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.

Developing fetal alveolar epithelial cells secrete fluid in primary culture

The developing pulmonary epithelium secretes a Cl(-)-rich fluid during fetal life by a process involving active transport. To determine if alveolar cells contribute to this fluid production, we studied developing fetal rat alveolar epithelial cells (18-day gestation) in primary culture. Fetal alveolar epithelial cells aggregated to form cystic, alveolar-like structures with a fluid-filled lumen. On light and transmission electron microscopy, the cells were polarized with microvilli facing the lumen. Dexamethasone and triiodothyronine stimulated lamellar body production in many of the epithelial cells of the cyst wall. The transepithelial voltage in the cyst was -2.4 +/- 0.4 mV (lumen negative), suggesting the presence of active electrolyte transport. Bumetanide, an inhibitor of Cl- secretion in other systems, decreased the size and number of cysts. A membrane-permeant analogue of adenosine 3',5'-cyclic monophosphate (cAMP) and 3-isobutyl-1-methylxanthine increased the size of cysts, an effect that was blocked by coincubation with bumetanide. The increased size of the cysts did not result from stimulation of cell growth; in fact, [3H]thymidine incorporation was inhibited to 40% control values by cAMP, suggesting that growth was inhibited rather than stimulated. These results suggest that fetal alveolar epithelial cells secrete fluid via a cAMP-mediated Cl(-)-secretory process. Secretion of fluid by the developing alveolar epithelium may play an important role in lung development.

Sodium-dependent chloride secretion across rabbit descending colon.

Electrogenic, cAMP-mediated Cl secretion across rabbit descending colon in vitro is independent of the rate or presence of active Na absorption. Yet, several observations indicate that this process is Na dependent: a) Cl secretion requires the presence of Na in the serosal solution alone, b) the kinetics of Cl transport as a function of external Na concentration are virtually identical to the Cl concentration dependence, and c) exchange of cell Cl with isotopic Cl added to the serosal solution is inhibited by Na-free media and by addition of furosemide to the serosal solution; the diuretic also inhibits Cl secretion. These findings suggest that Cl entry into the secretory cells across the basolateral membrane is mediated by NaCl cotransport. Addition of ouabain to, or removal of K from, the serosal solution inhibits Cl secretion so that Na entering the secretory cell across the basolateral membrane may be returned to the serosal solution by the Na-K pump. Finally, increasing the K concentration of the serosal solution inhibits Cl secretion under short-circuit conditions. This appears to result from K-induced depolarization of the electrical potential difference across the apical membrane so that diffusional Cl exit from cell to mucosal solution is reduced.