Biomedical subjects

W B Guggino

Publications and source records attributed to W B Guggino.

At least 73 records · Page 4Linked to original sources

PubMed1996

A role for CFTR in human autosomal dominant polycystic kidney disease.

Human autosomal dominant polycystic kidney disease (ADPKD) is the most common lethal dominant hereditary disorder characterized by enormous renal enlargement and the development of multiple cysts originating from nephrons. We investigated the pathogenesis of cyst formation in ADPKD by using patch-clamp and immunocytochemical techniques. Adenosine 3',5'-cyclic monophosphate-activated Cl- currents are present in primary cultures of ADPKD cells and have characteristics such as a linear current-voltage relation, insensitivity to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, sensitivity to glibenclamide and diphenylamine carboxylic acid, and an anion selectivity sequence of Br- > Cl- > I- > glutamate, all of which are identical to cystic fibrosis transmembrane conductance regulator (CFTR). With the use of CFTR antibodies raised against the regulatory and first nucleotide-binding domains, CFTR was detected in primary cultures of ADPKD cells. Similar results were obtained in vivo in cyst-lining epithelial cells in ADPKD kidneys, where staining was seen associated with the apical membrane regions. These data indicate that the CFTR Cl- channel exists in apical membranes of ADPKD cells and may play an important role in cyst formation or enlargement.

Anions↗

PubMed1996

Apical and basolateral ATP stimulates tracheal epithelial chloride secretion via multiple purinergic receptors.

Stimulation of Cl- secretion across the airway epithelium by ATP or UTP as agonists has therapeutic implications for cystic fibrosis. Our results demonstrate that ATP stimulates Cl- secretion in rat tracheal epithelial cell monolayers in primary culture from the apical or basolateral side of the monolayer. Multiple types of ATP-sensitive Cl- conductances in intact monolayers were elucidated through inhibition by Cl- channel-blocking drugs. Multiple Cl- conductances stimulated by ATP and adenosine 3',5'-cyclic monophosphate (cAMP) (tested for comparison) were also deciphered more specifically by nystatin permeabilization of the basolateral membrane, subsequent imposition of symmetrical Cl-, I-, or Br- solutions to test halide permselectivity, inhibition by Cl- channel-blocking drugs, and construction of current-voltage plots to study time and voltage dependence of the currents. Apical ATP stimulates Cl- secretion through P2U (or P2Y2) purinergic receptors via both intracellular Ca2+ (Ca(2+)i)-dependent and Cai(2+)-independent signaling pathways by opening outwardly rectifying Cl- channels (ORCCs), cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, and Cai(2+)-dependent Cl- channels. Basolateral ATP stimulates Cl- secretion via a combination of receptor subtypes (P2T and P2U) or a novel type of receptor (P2Y3), independent of Cai2+ or cAMP signaling by opening only CFTR channels. cAMP also stimulated multiple types of Cl- conductances, consistent with simultaneous activation of CFTR and ORCCs. Together, these results suggest that ATP as an agonist stimulates Cl- secretion via multiple purinergic receptors and multiple signal transduction pathways activated in different membrane domains of tracheal epithelia.

Adenosine Triphosphate↗

PubMed1996

Both the wild type and a functional isoform of CFTR are expressed in kidney.

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

Animals↗

PubMed1996

Developmental regulation of CFTR expression during human nephrogenesis.

Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein are expressed in proximal and distal tubules of the human kidney, but CFTR expression pattern during human nephrogenesis is unknown. We have now studied CFTR expression in fetal kidneys by immunohistochemistry and Western blot analysis, using six antibodies against human CFTR. CFTR was expressed in 12-wk human fetal kidneys, mostly in the apical membrane region of the ureteric bud epithelial cells. By 15 wk, CFTR was also diffusely expressed throughout the cytoplasm of proximal tubules and loops of Henle. No glomerular staining was seen at any state. From 15 to 24 wk of gestation this staining pattern remained constant and also included immunoreactivity of the transitional epithelium. Western blot for CFTR was performed on membrane extracts of human fetal kidneys, using T84 cells as a positive control. A 165-kDa protein corresponding to the predicted size of CFTR was seen at 13 wk and throughout development. We also observed a 75-kDa protein that was distinctly regulated during development. This protein was detected with several antibodies against the first half of CFTR (including the regulatory "R" domain) but not with a COOH-terminal-specific antibody and had the predicted size of a functional splice variant of CFTR identified in the human kidney. These results show the complex regulation of CFTR during nephrogenesis and raise the question of the respective roles of the full-length and the splice variant CFTR proteins in the human kidney.

Adolescent↗

PubMed1996

Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung.

Gene therapy for cystic fibrosis (CF) would ideally be accomplished with a vector capable of long-term expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in the absence of a host inflammatory response. Recombinant adeno-associated virus (AAV)-CFTR vectors possess these characteristics in rabbits. Because the utility of AAV vectors as gene transfer agents has only been recognized recently, AAV vector-mediated transduction has never been modeled in a primate host, which is an important step before its use in humans. In order to test the safety and biological activity of AAV-CFTR, single doses of AAV-CFTR vector were administered by fiberoptic bronchoscopy to the posterior basal segment of the right lower lobe (RLL) of the lungs of 10 rhesus macaques with four matched vehicle-treated controls. Animals were followed for 10, 21, 90 or 180 days following vector instillation. Vector DNA transfer occurred in bronchial epithelial cells in the RLL of each animal that received vector as assessed by in situ DNA PCR. Vector mRNA was detectable for 180 days after administration as detected by RT-PCR and by RNase protection assay. Safety of vector administration was determined by measurements of pulmonary mechanics, arterial blood gas analysis, chest radiographs, and bronchoalveolar lavage (BAL) fluid analysis including cell count and quantification of inflammatory cytokines. Gross and microscopic pathologic examination were also performed. There was no evidence of inflammation or other toxicity, although vector DNA was found in extrapulmonary organs of some animals. These results indicate that transduction of the primate airway epithelium with the AAV-CFTR mediates long-term CFTR cDNA transfer and is relatively safe.

Animals↗

PubMed1995

Molecular cloning and characterization of AqpZ, a water channel from Escherichia coli.

The aquaporin family of molecular water channels is widely expressed throughout the plant and animal kingdoms. No bacterial aquaporins are known; however, sequence-related bacterial genes have been identified that encode glycerol facilitators (glpF). By homology cloning, a novel aquaporin-related DNA (aqpZ) was identified that contained no surface N-glycosylation consensus. The aqpZ RNA was not identified in mammalian mRNA by Northern analysis and exhibited bacterial codon usage preferences. Southern analysis failed to demonstrate aqpZ in mammalian genomic DNA, whereas a strongly reactive DNA was present in chromosomal DNA from Escherichia coli and other bacterial species and did not correspond to glpF. The aqpZ DNA isolated from E. coli contained a 693-base pair open reading frame encoding a polypeptide 28-38% identical to known aquaporins. When compared with other aquaporins, aqpZ encodes a 10-residue insert preceding exofacial loop C, truncated NH2 and COOH termini, and no cysteines at known mercury-sensitive sites. Expression of aqpZ cRNA conferred Xenopus oocytes with a 15-fold increase in osmotic water permeability, which was maximal after 5 days of expression, was not inhibited with HgCl2, exhibited a low activation energy (Ea = 3.8 kcal/mol), and failed to transport nonionic solutes such as urea and glycerol. In contrast, oocytes expressing glpF transported glycerol but exhibited limited osmotic water permeability. Phylogenetic comparison of aquaporins and homologs revealed a large separation between aqpZ and glpF, consistent with an ancient gene divergence.

Amino Acid Sequence↗

PubMed1995

A single amino acid in gamma-aminobutyric acid rho 1 receptors affects competitive and noncompetitive components of picrotoxin inhibition.

A class of bicuculline-insensitive gamma-aminobutyric acid (GABA) receptors, GABAC, has been identified in retina. Several lines of evidence indicate that GABAC receptors are formed partially or wholly of GABA rho subunits. These receptors generate a Cl- current in response to GABA but differ from GABAA receptors in a number of ways. Picrotoxin, widely accepted as a noncompetitive antagonist of GABAA receptors, displays competitive and noncompetitive antagonism of GABAC receptors in perch and bovine retina and GABA rho 1 receptors expressed in Xenopus oocytes. The aim of this study was to identify the molecular basis of the two components of picrotoxin inhibition of GABA rho 1 receptors. By using a domain-swapping and mutagenesis strategy, a difference in picrotoxin sensitivity between rho 1 and rho 2 receptors was localized to a single amino acid in the putative second transmembrane domain. Substitution of this amino acid with residues found in the analogous position in highly picrotoxin-sensitive glycine alpha and GABAA subunits increased the sensitivity of rho 1 mutants 10- to 500-fold. Importantly, the competitive component of picrotoxin inhibition of the rho 1 mutant receptors was almost eliminated. These findings demonstrate that an amino acid in the putative channel domain of GABA rho 1 receptors influences picrotoxin sensitivity and mediates agonist binding by an allosteric mechanism.

Animals↗

PubMed1995

A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine selectively activates chloride efflux from human epithelial and mouse fibroblast cell lines expressing the cystic fibrosis transmembrane regulator delta F508 mutation.

Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) stimulates 36Cl- efflux from pancreatic CFPAC-1 cells which bear the delta F508 genotype common to most cases of cystic fibrosis [Eidelman et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5562-5566]. By contrast, correction of the cystic fibrosis defect by retrovirus-mediated gene transfer renders the resulting CFPAC-PLJ-CFTR cells insensitive to CPX. We now report that CPX also activates chloride efflux from the CF tracheal epithelial cell line IB3-1 bearing a delta F508 allele, but not if the IB3-1 cells have been repaired by transfection of the wild-type CFTR gene. Similar results were obtained with recombinant NIH 3T3 cells, in which CPX activates 36Cl- efflux from cells expressing the CFTR (delta F508) gene product but not from 3T3 cells expressing the wild-type CFTR. In all three cell types expressing CFTR (delta F508), CPX was found to activate 36Cl- efflux in a dose-dependent manner over the concentration range of 1-30 nM and then gradually lose potency at higher CPX concentrations. Six CPX analogues, A1 receptor antagonists of affinity similar to that of CPX, were found to be much less effective than CPX at activating 36Cl- efflux from CFPAC-1 cells. These included 2-thio-CPX. CPT (8-cyclopentyl-1,3-dimethylxanthine),3,4-dehydro-CPX,3-F-CPX,3-1-CPX, and KW-3902 (8-noradamantyl-1,3-dipropylxanthine).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Cloning and expression of two brain-specific inwardly rectifying potassium channels.

We have cloned two inwardly rectifying K+ channels that occur selectively in neurons in the brain and are designated BIRK (brain inwardly rectifying K+) channels. BIRK1 mRNA is extremely abundant and is enriched in specific brainstem nuclei, BIRK1 displays a consensus phosphate-binding loop, and expression in Xenopus oocytes has shown that its conductance is inhibited by ATP and adenosine 5'-[gamma-thio]triphosphate. BIRK2 is far less abundant and is selectively localized in telencephalic neurons. BIRK2 has a consensus sequence for cAMP-dependent phosphorylation.

Adenosine Triphosphate↗

PubMed1995

Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR.

Animals↗

PubMed1995

CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions to regulate both Cl- and Na+ conductive pathways; however, the cellular mechanisms whereby CFTR acts as a conductance regulator are unknown. CFTR and outwardly rectifying Cl- channels (ORCCs) are distinct channels but are linked functionally via an unknown regulatory mechanism. We present results from whole-cell and single-channel patch-clamp recordings, short-circuit current recordings, and [gamma-32P]ATP release assays of normal, CF, and wild-type or mutant CFTR-transfected CF airway cultured epithelial cells wherein CFTR regulates ORCCs by triggering the transport of the potent agonist, ATP, out of the cell. Once released, ATP stimulates ORCCs through a P2U purinergic receptor-dependent signaling mechanism. Our results suggest that CFTR functions to regulate other Cl- secretory pathways in addition to itself conducting Cl-.

Adenosine Triphosphate↗

PubMed1995

Alternate translation initiation codons can create functional forms of cystic fibrosis transmembrane conductance regulator.

To evaluate the function of transmembrane domain 1 (TMD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and the methionines that function in translation initiation, a series of progressive 5' truncations in TMD1 were created to coincide with residues that might serve as translation initiation codons. Expression of the mutants in Xenopus oocytes demonstrated that internal sites in TMD1 can function as initiation codons. In addition, all of the mutants that progressively removed the first four transmembrane segments (M1-M4) of TMD1 expressed functional cAMP-regulated Cl- channels with ion selectivity identical to wild-type CFTR but with reduced open probability and single channel conductance. Further removal of transmembrane segments did not produce functional Cl- channels. These data suggest that segments M1-M4 are not essential components of the conduction pore or the selectivity filter of CFTR.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Water channel properties of major intrinsic protein of lens.

The functions of major intrinsic protein (MIP) of lens are still unresolved; however the sequence homology with channel-forming integral membrane protein (CHIP) and other Aquaporins suggests that MIP is a water channel. Immunolocalizations confirmed that Xenopus oocytes injected with bovine MIP cRNA express the protein and target it to the plasma membrane. Control oocytes or oocytes expressing MIP or CHIP exhibited small, equivalent membrane currents that could be reversibly increased by osmotic swelling. When compared with water-injected control oocytes, the coefficient of osmotic water permeability (Pf) of MIP oocytes was increased 4-5-fold with a low Arrhenius activation energy, while the Pf of CHIP oocytes increased > 30-fold. To identify structures responsible for these differences in Pf, recombinant MIP proteins were expressed. Analysis of MIP-CHIP chimeric proteins revealed that the 4-kDa cytoplasmic domain of MIP did not behave as a negative regulator. Individual residues in MIP were replaced by residues conserved among the Aquaporins, and introduction of a proline in the 5th transmembrane domain of MIP raised the Pf by 50%. Thus oocytes expressing MIP failed to exhibit ion channel activity and consistently exhibited water transport by a facilitated pathway that was qualitatively similar to the Aquaporins but of lesser magnitude. We conclude that MIP functions as an Aquaporin in lens, but the protein may also have other essential functions.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues.

The Aquaporin family of water channels plays a fundamental role in transmembrane water movements in numerous plant and animal tissues. Since the molecular pathway by which water is secreted by salivary glands is unknown, a cDNA was isolated from rat submandibular gland by homology cloning. Similar to other Aquaporins, the salivary cDNA encodes a 265-residue polypeptide with six putative transmembrane domains separated by five connecting loops (A-E); the NH2- and COOH-terminal halves of the polypeptide are sequence-related, and each contains the motif Asn-Pro-Ala. A mercurial-inhibition site is present in extracellular loop E, and cytoplasmic loop D contains a cAMP-protein kinase phosphorylation consensus. In vitro translation yielded a 27-kDa polypeptide, and expression of the cRNA in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability (Pf) which was reversibly inhibited by 1 mM HgCl2. Northern analysis demonstrated a 1.6-kilobase mRNA in submandibular, parotid, and sublingual salivary glands, lacrimal gland, eye, trachea, and lung. In situ hybridization revealed a strong hybridization over the corneal epithelium in eye and over the secretory lobules in salivary glands. These studies have identified a new mammalian member of the Aquaporin water channel family (gene symbol AQP5) which is implicated in the generation of saliva, tears, and pulmonary secretions.

Amino Acid Sequence↗

PubMed1995

Cloning of a putative human voltage-gated chloride channel (CIC-2) cDNA widely expressed in human tissues.

We have cloned a cDNA from the human epithelial cell line T84 whose predicted amino acid sequence shows 93.9% identity with rat CIC-2. Mapping by somatic cell hybrids and polymerase chain reaction localizes the gene corresponding to this cDNA to chromosome 3q26-qter. The major transcription start site assessed by RNA primer extension is 100 nt upstream of the putative translation initiation codon. Analysis of the 5' flanking sequence revealed a high GC content and lack of common transcriptional elements such as TATA and CCAAT boxes. Northern blot analysis indicated wide organ distribution including tissues affected in cystic fibrosis (CF) and expression in an airway epithelial cell line derived from a CF patient. The high degree of sequence similarity and similar tissue distribution to rat CIC-2 suggests that this cDNA encodes the human CIC-2 voltage-gated chloride channel. Since this chloride channel is present in epithelial tissues it may be amenable to manipulation to circumvent the chloride secretion defect observed in CF.

Amino Acid Sequence↗

PubMed1995

Differential expression of ORCC and CFTR induced by low temperature in CF airway epithelial cells.

When nonepithelial cell types expressing the delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) mutation are grown at reduced temperatures, the mutant protein can be properly processed. The effect of low temperatures on Cl- channel activity in airway epithelial cells that endogenously express the delta F508-CFTR mutation has not been investigated. Therefore, we examined the effect of incubation temperature on both CFTR and outwardly rectifying Cl- channel (ORCC) activity in normal, in cystic fibrosis (CF)-affected, and in wild-type CFTR-complemented CF airway epithelia with use of a combination of inside-out and whole cell patch-clamp recording, 36Cl- efflux assays, and immunocytochemistry. We report that incubation of CF-affected airway epithelial cells at 25-27 degrees C is associated with the appearance of a protein kinase A-stimulated CFTR-like Cl- conductance. In addition to the appearance of CFTR Cl- channel activity, there is, however, a decrease in the number of active ORCC when cells are grown at 25-27 degrees C, suggesting that the decrease in incubation temperature may be associated with multiple alterations in ion channel expression and/or regulation in airway epithelial cells.

Bronchi↗

PubMed1995

G protein G alpha i-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells.

Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells.

Adenosine Triphosphate↗

PubMed1995

CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells.

Previously we demonstrated that the inner medullary collecting duct cell line mIMCD-K2 secretes Cl- by an electrogenic mechanism [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995]. The goal of the present study was to characterize the Cl- channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- secretion. To this end, using the patch-clamp technique, we measured Cl- currents. In whole cell patch-clamp experiments, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) activated Cl- currents that were time and voltage independent, inhibited by diphenylamine 2-carboxylate (DPC), and had a linear current-voltage (I-V) relation. In cell-attached patches of the apical membrane, we identified 7-pS Cl- channels that were stimulated by CPT-cAMP. In inside-out patches with Cl- in the pipette and bath solutions, Cl- currents had a linear I-V relation. The halide permeability sequence was PCl = PBr > PI. The Cl- channel inhibitors DPC, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and glibenclamide blocked the 7-pS Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was ineffective. By reverse transcriptase polymerase chain reaction, we isolated a partial cDNA clone encoding the cystic fibrosis transmembrane conductance regulator in mIMCD-K2 cells. We conclude that cAMP stimulates electrogenic Cl- secretion in inner medullary collecting duct cells by activating cystic fibrosis transmembrane conductance regulator Cl- channels.

Animals↗