sq-23377 and Cystic-Fibrosis

Ca(2+)-dependent Cl(-) secretion (CaCC) in airways and other tissues is due to activation of the Cl(-) channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca(2+) -activated Cl(-) channels are regulated by membrane lipid inositol phosphates, and that 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester (INO-4995) augments CaCC. Here we examined whether TMEM16A is the target for INO-4995 and if the channel is regulated by inositol phosphates.. The effects of INO-4995 on CaCC were examined in overexpressing HEK293, colonic and primary airway epithelial cells as well as Xenopus oocytes. We used patch clamping, double electrode voltage clamp and Ussing chamber techniques.. We found that INO-4995 directly activates a TMEM16A whole cell conductance of 6.1 ± 0.9 nS pF(-1) in overexpressing cells. The tetrakisphosphates Ins(3,4,5,6)P(4) or Ins(1,3,4,5)P(4) and enzymes controlling levels of InsP(4) or PIP(2) and PIP(3) had no effects on the magnitude or kinetics of TMEM16A currents. In contrast in Xenopus oocytes, human airways and colonic cells, which all express TMEM16A endogenously, Cl(-) currents were not acutely activated by INO-4995. However incubation with INO-4995 augmented 1.6- to 4-fold TMEM16A-dependent Cl(-) currents activated by ionomycin or ATP, while intracellular Ca(2+) signals were not affected. The potentiating effect of INO-4995 on transient ATP-activated TMEM16A-currents in cystic fibrosis (CF) airways was twice of that observed in non-CF airways.. These data indicate that TMEM16A is the target for INO-4995, although the mode of action appears different for overexpressed and endogenous channels. INO-4995 may be useful for the treatment of CF lung disease.

Topics: Animals; Anoctamin-1; Bronchi; Cells, Cultured; Chloride Channels; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; HEK293 Cells; Humans; Inositol Phosphates; Ionomycin; Neoplasm Proteins; Oocytes; Patch-Clamp Techniques; Xenopus

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gamma-glutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail.. The present study was aimed to evaluate the origin and the biochemical characteristics of the GGT detectable in CF sputum. We found GGT activity both in neutrophils and in the fluid, the latter significantly correlating with myeloperoxidase expression. In neutrophils, GGT was associated with intracellular granules. In the fluid, gel-filtration chromatography showed the presence of two distinct GGT fractions, the first corresponding to the human plasma b-GGT fraction, the other to the free enzyme. The same fractions were also observed in the supernatant of ionomycin and fMLP-activated neutrophils. Western blot analysis confirmed the presence of a single band of GGT immunoreactive peptide in the CF sputum samples and in isolated neutrophils.. In conclusion, our data indicate that neutrophils are able to transport and release GGT, thus increasing GGT activity in CF sputum. The prompt release of GGT may have consequences on all GGT substrates, including major inflammatory mediators such as S-nitrosoglutathione and leukotrienes, and could participate in early modulation of inflammatory response.

Topics: Adolescent; Adult; Calcium Ionophores; Cells, Cultured; Cystic Fibrosis; Female; gamma-Glutamyltransferase; Humans; Ionomycin; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Peroxidase; Sputum; Young Adult

Gammadelta T cells have an important immunoregulatory and effector function through cytokine release. They are involved in the responses to Gram-negative bacterium and in protection of lung epithelium integrity. On the other hand, they have been implicated in airway inflammation.. The aim of the present work was to study intracytoplasmic IL-2, IL-4, IFN-gamma and TNF-alpha production by gammadelta and alphabeta T lymphocytes from cystic fibrosis patients and healthy donors in response to Pseudomonas aeruginosa (PA). Flow cytometric detection was performed after peripheral blood mononuclear cells (PBMC) culture with a cytosolic extract from PA and restimulation with phorbol ester plus ionomycine. Proliferative responses, activation markers and receptor usage of gammadelta T cells were also evaluated.. The highest production of cytokine was of TNF-alpha and IFN-gamma, gammadelta being better producers than alphabeta. No differences were found between patients and controls. The Vgamma9delta2 subset of gammadelta T cells was preferentially expanded. CD25 and CD45RO expression by the alphabeta T subset and PBMC proliferative response to PA were defective in cystic fibrosis lymphocytes.. Our results support the hypothesis that gammadelta T lymphocytes play an important role in the immune response to PA and in the chronic inflammatory lung reaction in cystic fibrosis patients. They do not confirm the involvement of a supressed Th1 cytokine response in the pathogenesis of this disease.

Topics: Adolescent; Adult; Case-Control Studies; Cell Proliferation; Cells, Cultured; Child; Cystic Fibrosis; Cytokines; Female; Flow Cytometry; Humans; Interferon-gamma; Intracellular Membranes; Ionomycin; Kinetics; Leukocyte Common Antigens; Male; Monocytes; Phenotype; Pseudomonas Infections; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; Receptors, Interleukin-2; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

In view of the occurrence of hepatobiliary disorders in cystic fibrosis (CF) this study addresses the role of the cystic fibrosis transmembrane conductance regulator (CFTR) and of Ca(2+)-activated Cl(-) channels in promoting HCO3- secretion in bile ductular cells. Human cholangiocytes were isolated from control livers and from 1 patient with CF (DeltaF508/G542X mutations). Single channel and whole cell currents were analyzed by patch clamp techniques, and HCO3- secretion was determined by fluorometric analysis of the rate of recovery of intracellular pH following alkaline loading. In control cholangiocytes, both cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) catalytic subunit, activated CFTR Cl(-) channels that exhibited a nonrectifying conductance of 8 pS and appeared in clusters. Activation of Cl(-) current by cAMP was associated with an increase in the rate of HCO3- secretion. The basal rate of HCO3- secretion was lower in CF than in control cholangiocytes. In both control and CF cholangiocytes, raising intracellular Ca(2+) concentrations with ionomycin led to a parallel activation of Cl(-) current and HCO3- secretion. Consistent with reports that premature stop codon mutations (class I; e.g., G542X) can be read over by treatment with aminoglycoside antibiotics, exposure of CF cholangiocytes to gentamicin restored activation by cAMP of Cl(-) current and HCO3- secretion. The observation that activation of Ca(2+)-dependent Cl(-) channels can substitute for cystic fibrosis transmembrane conductance regulator (CFTR) in supporting HCO3- secretion and the efficacy of gentamicin in restoring CFTR function and HCO3- secretion in class I mutations are of potential clinical interest.

Topics: Anti-Bacterial Agents; Bicarbonates; Bile Ducts, Intrahepatic; Calcium; Cells, Cultured; Chloride Channels; Codon; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Gentamicins; Humans; Hydrogen-Ion Concentration; Ionomycin; Mutation; Patch-Clamp Techniques

The study investigated the role of protein kinase C (PKC) in the modulation of agonist-induced Ca2+-dependent anion secretion by pancreatic duct cells. The short-circuit current (ISC) technique was used to examine the effect of PKC activation and inhibition on subsequent ATP, angiotensin II and ionomycin-activated anion secretion by normal (CAPAN-1) and cystic fibrosis (CFPAC-1) pancreatic duct cells. The ISC responses induced by the Ca2+-mobilizing agents, which had been previously shown to be attributed to anion secretion, were enhanced in both CAPAN-1 and CFPAC-1 cells by PKC inhibitors, staurosporine, calphostin C or chelerythrine. On the contrary, a PKC activator, phorbol 12-myristate 13-acetate (PMA), was found to suppress the agonist-induced ISC in CFPAC-1 cells and the ionomycin-induced ISC in CAPAN-1 cells. An inactive form of PMA, 4alphad-phorbol 12, 13-didecanote (4alphaD), was found to exert insignificant effect on the agonist-induced ISC, indicating a specific effect of PMA. Our data suggest a role of PKC in modulating agonist-induced Ca2+-dependent anion secretion by pancreatic duct cells. Therapeutic strategy to augment Ca2+-activated anion secretion by cystic fibrosis pancreatic duct cells may be achieved by inhibition or down-regulation of PKC.

Topics: Adenosine Triphosphate; Angiotensin II; Anions; Calcium; Cell Line; Cystic Fibrosis; Electric Conductivity; Enzyme Activation; Humans; Ionomycin; Pancreatic Ducts; Protein Kinase C

Gallbladders from cystic fibrosis (CF) mice (Cftrtm1Cam and Cftrtm2Cam) were examined with the short-circuit current technique. The tissues failed to show any electrogenic anion transport in response to forskolin (cAMP stimulus) but responded to the Ca2+ ionophore ionomycin. Administration of the plasmid pTrial10-CFTR2 complexed with cationic liposomes (3beta-[N-(dimethylaminoethane)-carbamoyl]cholesterol and L-alpha-phosphatidylethanolamine dioleolyl) to the airways restored the phenotype of CF gallbladders to that of the wild type, but did not do so when given orally. Formation of human CFTR mRNA in gallbladders of transfected CF null mice was demonstrated. Using the reporter genes pCMV-luc and pCMV-LacZ, we showed that 1) the intratracheal route was more effective than the oral,intravenous, intramuscular, subcutaneous, or intraperitoneal routes in expressing luciferase activity in the gallbladder and 2) beta-galactosidase staining after pCMV-LacZ was confined to the columnar epithelium lining the gallbladder without any discernible activity in it smooth muscle. The discovery of an unusual route for gene transfer to the biliary system may give useful insight into counteracting the consequences of biliary fibrosis in human CF patients.

Topics: Animals; beta-Galactosidase; Bicarbonates; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Gallbladder; Gene Expression; Gene Transfer Techniques; Humans; Ionomycin; Liposomes; Luciferases; Mice; Mice, Inbred CFTR; Polymerase Chain Reaction; RNA, Messenger; Trachea

CFTR is a chloride channel that is required for fluid secretion and salt absorption in many exocrine epithelia. Mutations in CFTR cause cystic fibrosis. CFTR expression influences some ion channels, but the range of channels influenced, the mechanism of the interaction and the significance for cystic fibrosis are not known. Possible interactions between CFTR and other ion channels were studied in C127 mouse mammary epithelial cell lines stably transfected with CFTR, delta F508-CFTR, or vector. Cell lines were compared quantitatively using an 125I efflux assay and qualitatively using whole-cell patch-clamp recording. As expected, 125I efflux was significantly increased by forskolin only in the CFTR line, and forskolin-stimulated whole-cell currents were time- and voltage independent. All three lines responded to hypotonic challenge with large 125I efflux responses of equivalent magnitude, and whole-cell currents were outwardly rectified and inactivated at positive voltages. Unexpectedly, basal 125I efflux was significantly smaller in the delta F508-CFTR cell line than in either the CFTR or control cell lines (P < 0.0001), and the magnitude of the efflux response to ionomycin was largest in the vector cell line and smallest in the cell line expressing delta F508-CFTR (P < 0.01). Whole-cell responses to ionomycin had a linear instantaneous I-V relation and activated at depolarizing voltages. Forskolin responses showed simple summation with responses to ionomycin or hypotonic challenge. Thus, we found no evidence for interactions between CFTR and the channels responsible for swelling-mediated responses. Differences were found in basal and ionomycin-stimulated efflux, but these may arise from variations in the clonally selected cell lines that are unrelated to CFTR expression.

Topics: Animals; Biological Transport, Active; Bovine papillomavirus 1; Calcium; Cell Line; Cell Size; Chloride Channels; Colforsin; Cystic Fibrosis; Dose-Response Relationship, Drug; Drug Interactions; Electric Conductivity; Female; Hypotonic Solutions; In Vitro Techniques; Ionomycin; Mice

Cystic fibrosis (CF) reflects the loss of adenosine 3',5'-cyclic monophosphate (cAMP)-regulated Cl- secretion consequent to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In humans, but not mice, with CF, the disease is associated with male infertility. The present study investigated the relative magnitudes of the cAMP pathways and an alternative Ca(2+)-regulated Cl- secretory pathway in primary cultures of the epididymides and the seminal vesicles of normal and CF mice. The basal equivalent short-circuit currents (Ieq) of cultures derived from the epididymides and the seminal vesicles from the CF mice were lower (6.0 +/- 0.6 and 4.0 +/- 1.0 muA/cm2, respectively) than those from normal mice (11.1 +/- 1.0 and 6.6 +/- 0.6 muA/cm2, respectively). Forskolin induced significant Ieq responses in both the epididymis (8.0 +/- 0.7 muA/cm2) and seminal vesicles (4.0 +/- 0.5 muA/cm2) from normal mice, whereas forskolin-induced changes in Ieq in CF mouse epididymis and seminal vesicles were absent, consistent with defective cAMP-CFTR-mediated Cl- secretion in CF mice. Ieq responses to agonists (ionomycin, ATP) that raise intracellular Ca2+ (Ca2+i) were larger than forskolin responses in normal animals (6.6 +/- 0.9 and 13.4 +/- 1.8 muA/cm2, respectively) and were preserved in CF (6.5 +/- 0.9 and 17.1 +/- 1.0 muA/cm2, respectively). We speculate that the fertility of male CF mice is maintained by persistent expression of the predominant alternative Ca(2+)-mediated Cl- transport system in the epididymides and seminal vesicles.

Topics: Adenosine Triphosphate; Animals; Calcium; Chlorides; Colforsin; Cyclic AMP; Cystic Fibrosis; Electric Conductivity; Epididymis; Ionomycin; Male; Mice; Mice, Inbred C57BL; Reference Values; Seminal Vesicles

Defective adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- transport in cystic fibrosis (CF) reflects defects in the cystic fibrosis transmembrane conductance regulator (CFTR). A moderate level of CFTR mRNA expression has been found in rodent and human oviductal epithelium, but unlike other CFTR-expressing tissues, the oviduct in CF patients is apparently normal. The present study was carried out to investigate the relative magnitude of the cAMP- and intracellular Ca2+ (Cai2+)-regulated Cl- secretion in primary cultures of the oviduct from normal and CF mice generated by targeted disruption of the murine CF gene. Normal oviductal epithelium exhibited a basal equivalent short-circuit current (Ieq) of 20.3 +/- 1.7 muA/cm2. CF oviduct exhibited a lower basal Ieq of 4.5 +/- 1.9 muA/cm2. In normal mice, forskolin (10(-5) M, apical) elicited a slowly developing sustained rise in Ieq, whereas ionomycin (5 x 10(-6) M, apical) and ATP (10(-4) M, apical) induced larger increases in Ieq consisting of a prompt, transient response followed by a slowly decreasing component. The Ieq response to forskolin was totally abolished in CF mouse oviducts, but the magnitudes of the peak Ieq responses to ionomycin and ATP were not different from normal. The time courses of the ionomycin- and ATP-evoked responses, however, were significantly more transient in CF than in normal oviducts. These results demonstrate that CF mouse oviduct exhibits defective cAMP- but not Cai(2+)-mediated Cl- secretion. The relatively high level of functional expression of the alternative Cai(2+)-activated Cl- secretory pathway in the mouse oviduct may contribute to the absence of major pathology in the CF oviduct.

Topics: Adenosine Triphosphate; Animals; Blotting, Western; Calcium; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Fallopian Tubes; Female; Indomethacin; Ionomycin; Kinetics; Membrane Proteins; Mice

The elementary K+ conductance activated by the cAMP or the Ca2+ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl(-)-free conditions, an inwardly rectifying whole-cell K+ current was evoked by either forskolin 10 (mumol/l) or acetylcholine 1 (mumol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8 +/- 2.5 pS (n = 26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K+ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mumol/l free Ca2+ at the inner side of the membrane. This channel was blocked by Ba2+ (5 mumol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca2+ with and EC50 of 0.25 mumol/l and strongly reduced by intracellular MgATP with an IC50 of 600 mumol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K+ channel was also recorded, with similar characteristics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K+ channel regulated by intracellular Ca2+ and phosphorylation supports the main K+ current activated by secretory agonists in normal cystic fibrosis cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Adenosine Triphosphate; Barium; Biotransformation; Calcium; Cell Line; Charybdotoxin; Colforsin; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelium; Humans; Ionomycin; Membrane Proteins; Patch-Clamp Techniques; Phosphorylation; Potassium Channels; Scorpion Venoms

Calcium-activated anion secretion is elevated in the pancreatic ductal epithelium of transgenic cf/cf mice which lack the cystic fibrosis transmembrane conductance regulator (CFTR). To elucidate whether this effect is due to increased activity of calcium-activated chloride channels, we have studied the relationship between CFTR and calcium-activated chloride currents in pancreatic duct cells isolated from Cambridge cf/cf mice. CFTR chloride currents activated by cAMP were detected in 59% (29/49) of wild-type cells and in 50% (20/40) of heterozygous cells. However, we could not detect any CFTR currents in the homozygous cf/cf cells (0/25). The maximum CFTR current density measured at a membrane potential of 60 mV was 23.5 +/- 2.8 pA/pF (n = 29) in wild-type cells, and about half that value, i.e. 12.4 +/- 1.6 pA/pF (n = 20) in heterozygotes (P = 0.004). Calcium-activated chloride currents were detected in 73% (24/33) of wild-type, 75% (21/28) of heterozygous and in 58% (7/12) of homozygous cf/cf cells. There was no significant difference between the steady-state calcium-activated current densities in the three genotypic groups; the current measured at 60 mV being 527 +/- 162 pA/pF (n = 24) from wild-type, 316 +/- 35 pA/pF (n = 21) from heterozygote and 419 +/- 83 pA/pF (n = 7) from homozygous cells. Our data suggest that lack of CFTR does not enhance the calcium-activated chloride conductance in murine pancreatic duct cells.

Topics: Animals; Calcium; Cells, Cultured; Chloride Channels; Chlorides; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Female; In Vitro Techniques; Ion Transport; Ionomycin; Male; Membrane Potentials; Membrane Proteins; Mice; Mice, Transgenic; Mutagenesis; Pancreatic Ducts; Patch-Clamp Techniques

Phase one clinical trials for gene therapy of cystic fibrosis are in progress using either liposomes or adenoviral vectors for CFTR gene transfer to epithelial cells in the airways. In addition to electrophysiological measurements, expression of vector CFTR is usually assessed by RT-PCR. We have developed a CFTR-expression vector, pCFAS, that simplifies the distinction of transgene-derived CFTR mRNA from endogenous mRNA. Two point mutations were introduced into CFTR cDNA which eliminated a SphI restriction site and created a new, unique AgeI restriction site. Neither mutation altered the predicted amino acid sequence of the protein. Restriction digestion of RT-PCR products from cells transfected with pCFAS allowed the differentiation of transgene and endogenous CFTR transcripts. To verify function of the mutated CFTR, the plasmid was transferred into freshly obtained nasal epithelial cells from CF patients ex vivo using cationic liposomes. Fluorescence microscopy using the halide-sensitive fluorophore SPQ demonstrated restoration of cAMP-mediated Cl- secretion. This plasmid will be useful for CFTR gene transfer studies in vitro and in vivo.

Topics: Base Sequence; Cell Line; Chlorides; Colforsin; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA, Complementary; Gene Transfer Techniques; Genetic Vectors; Humans; Ionomycin; Molecular Sequence Data; Mutagenesis; Nasal Mucosa; Transcription, Genetic

Functional assessment of the efficacy of CFTR gene transfer protocols in humans has previously involved measurement of in vivo potential difference. We have studied whether freshly obtained airway epithelial cells may provide suitable tissue for studies of in vivo gene transfer using fluorescent digital imaging microscopy. Nasal epithelial cells from non-cystic fibrosis subjects (n = 6) and from cystic fibrosis (CF) patients (delta F508: delta F508, n = 5) were obtained by brushing and loaded with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Addition of the cAMP-agonists forskolin (20 microM) and 3-isobutyl-1-methylxanthine (IBMX, 100 microM) produced an increased efflux of iodide from the cells which was significantly (P < 0.05) greater in non-CF than in CF cells. Efflux following addition of the calcium ionophore, ionomycin (100 microM) was similar in both non-CF and CF cells. Liposome-mediated transfection of CF nasal epithelial cells in vitro with CFTR-cDNA restored the cAMP-stimulated efflux to non-CF values. Bronchial epithelial cells from non-CF subjects showed responses to forskolin and ionomycin that were not different to those in non-CF nasal epithelia. These data demonstrate that the assay provides a useful method for assessing correction of abnormal ion transport in non-cultured CF epithelium and is likely to provide a further assay for assessment of in vivo gene transfer efficiency in protocols of gene therapy for CF.

Topics: Adult; Aged; Bronchi; Cells, Cultured; Chlorides; Colforsin; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelium; Fluorescent Dyes; Genetic Therapy; Humans; Ionomycin; Kinetics; Middle Aged; Nasal Mucosa; Quinolinium Compounds; Reference Values; Transfection; Turbinates

We have studied the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride currents in pancreatic duct cells isolated from a transgenic cf/cf mouse created by targeted insertional mutagenesis. Adenosine 3',5'-cyclic monophosphate (cAMP)-activated CFTR chloride currents were detected in 78% (29/37) of wild-type cells, in 81% (35/43) of heterozygote cells, and in 61% (29/47) of homozygous cf/cf duct cells (P > 0.05, cf/cf vs. wild-type and heterozygote). The CFTR current density measured at membrane potentials of +/- 60 mV averaged 22-26 pA/pF in wild-type and heterozygote groups but only 13 pA/pF in cells derived from cf/cf animals (P < 0.05, cf/cf vs. wild-type and cf/cf vs. heterozygotes). In contrast, duct cells from animals of all three genotypic groups exhibited calcium-activated chloride currents that were of similar magnitude and up to 11-fold larger than the CFTR currents. We speculate that these transgenic insertional null mice do not develop the pancreatic pathology that occurs in cystic fibrosis patients because their duct cells contain 1) some wild-type CFTR generated by exon skipping and aberrant splicing and 2) a separate anion secretory pathway mediated by calcium-activated chloride channels.

Topics: Animals; Calcium; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; Female; Heterozygote; Homozygote; Ionomycin; Male; Membrane Proteins; Mice; Mice, Transgenic; Pancreatic Ducts

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. The ATP and ionomycin responses in CF were of greater magnitude, and the prolonged phase was inhibited by DIDS. Although we expected ATP to regulate Cl- conductance through intracellular Ca2+ activity, ATP further stimulated Cl- secretion in tissues pretreated to maximally elevate intracellular Ca2+ activity. ATP also activated whole cell Cl- currents in cells dialyzed with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thus ATP and ionomycin regulate a Cl- conductance that is distinct from CFTR, but the regulation by ATP is not tightly coupled to intracellular Ca2+ activity. Alternatively, ATP regulates separate Ca(2+)-sensitive and Ca(2+)-insensitive Cl- conductances. Furthermore, extracellular ATP activates DIDS-resistant Cl- secretion in normal but not CF cultured epithelia, consistent with activation of CFTR by extracellular ATP.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Calcium; Cells, Cultured; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Egtazic Acid; Extracellular Space; Humans; Intracellular Membranes; Ionomycin; Membrane Proteins; Reference Values; Respiratory System

The cystic fibrosis (CF) mouse trachea has become a model for gene transfer. To characterize ion transport properties of tracheal epithelium from normal and CF mice, tracheas were excised, mounted in Ussing chambers, and basal properties and responses to pharmacological agents and/or ion substitution protocols measured. No difference in basal short-circuit (Isc) was observed between normal (29.1 +/- 3.8 muA/cm2, n = 21) and CF (34.7 +/- 4.5 muA/cm2, n = 16) tracheas. The relative contribution of Na+ transport to basal Isc was small (30-40%). Ionomycin stimulated large increases in Isc in both normal and CF murine tracheas [change in Isc (delta Isc) with ionomycin: 30.5 +/- 8.8 muA/cm2, n = 11, normal; 27.3 +/- 6.7 muA/cm2, n = 6, CF]. Unexpectedly, forskolin increased Isc in both CF and normal amiloride-pretreated tracheas (delta Isc: 10.5 +/- 2.1 muA/cm2, n = 21, normal; 13 +/- 2.3 muA/cm2, n = 16, CF). Forskolin was observed to increase intracellular Ca2+ in both normal and CF tracheal cells, suggesting this as a mechanism to induce Cl- secretion. These similarities in ion transport, in part reflecting the dominance of Ca(2+)-regulated Cl- conductance, suggest that the murine trachea is not an ideal target for assessment of CF correction by gene transfer.

Topics: Animals; Biological Transport; Calcium; Chlorides; Colforsin; Cystic Fibrosis; Electrophysiology; Epithelium; Female; Ionomycin; Male; Mice; Mice, Mutant Strains; Sodium; Trachea

This study examines the conductive properties of the plasma membrane of cells isolated from the intrahepatic portion of bile ducts. Membrane Cl- conductance was measured in single cells using whole-cell patch clamp recording techniques and in cells in short-term culture using 36Cl and 125I efflux. Separate Ca(2+)- and cAMP-dependent Cl- currents were identified. Ca(2+)-dependent Cl- currents showed outward rectification of the current-voltage relation, time-dependent activation at depolarizing potentials, and reversal near the equilibrium potential for Cl-. Ionomycin (2 microM) increased this current from 357 +/- 72 pA to 1,192 +/- 414 pA (at +80 mV) in 5:7 cells, and stimulated efflux of 125I > 36Cl in 15:15 studies. Ionomycin-stimulated efflux was inhibited by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) (150 microM). A separate cAMP-activated Cl- current showed linear current-voltage relations and no time dependence. Forskolin (10 microM) or cpt-cAMP (500 microM) increased this current from 189 +/- 50 pA to 784 +/- 196 pA (at +80 mV) in 11:16 cells, and stimulated efflux of 36Cl > 125I in 16:16 studies. cAMP-stimulated efflux was unaffected by DIDS. Because the cAMP-stimulated Cl- conductance resembles that associated with cystic fibrosis transmembrane conductance regulator (CFTR), a putative Cl- channel protein, the presence of CFTR in rat liver was examined by immunoblot analyses. CFTR was detected as a 150-165-kD protein in specimens with increased numbers of duct cells. Immunoperoxidase staining confirmed localization of CFTR to bile duct cells but not hepatocytes. These findings suggest that Ca(2+)- and cAMP-regulated Cl- channels may participate in control of fluid and electrolyte secretion by intrahepatic bile duct epithelial cells, and that the cAMP-regulated conductance is associated with endogenous expression of CFTR. Abnormal ductular secretion may contribute to the pathogenesis of cholestatic liver disease in cystic fibrosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bile Ducts; Calcium; Cells, Cultured; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelium; Iodides; Ionomycin; Kinetics; Liver; Male; Membrane Potentials; Membrane Proteins; Rats; Rats, Sprague-Dawley; Thionucleotides

The development of transformed human airway epithelial cell lines has been important in advancing the understanding of the biochemical and genetic mechanisms underlying the cystic fibrosis (CF) defect. Since the most common mutation associated with CF is a phenylalanine deletion at position 508 (delta F508) in the CF transmembrane conductance regulator (CFTR) gene, a transformed airway epithelial cell line homozygous for this mutation will be important for determining the biologic significance of this mutation in the airways. We report the genotypic and phenotypic characterization of a delta F508 homozygote cell line derived from luminal epithelium in the trachea. The cells were transformed with a plasmid containing an origin of replication defective SV40 genome and have progressed through crisis. Immunocytochemical characterization of the cells shows that they express keratin, indicating epithelial cell origin, and that a calcium-dependent cell adhesion molecule, cellCAM 120/80, is present at plasma membrane junctions between cells. Electrophysiologically, the cells show no cAMP-dependent Cl transport. However, after treatment with the calcium ionophore, ionomycin, cells secrete Cl, albeit at a lower level than that observed in normal cells. Genetically, the cells express CFTR mRNA as determined by polymerase chain reaction amplification and CFTR protein as determined by Western hybridization analysis. Karyotypic analysis shows that 70% of the cells contain two copies of chromosome 7.

Topics: Adult; Base Sequence; Biological Transport; Blotting, Southern; Cell Line, Transformed; Cell Transformation, Viral; Chlorides; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; Epithelium; Homozygote; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Ionomycin; Kinetics; Male; Membrane Proteins; Molecular Sequence Data; Mutation; Trachea

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.

Topics: Anions; Bronchi; Cell Line, Transformed; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Humans; Hypotonic Solutions; Iodides; Ion Channels; Ionomycin; Membrane Proteins

The gene responsible for cystic fibrosis (CF) has recently been cloned and sequenced. When transfected into CF epithelial cells, normal transcripts of this gene correct the underlying defect in CF, i.e. cAMP-dependent Cl- secretion is restored. Thus, the protein encoded by this gene, designated "cystic fibrosis transmembrane conductance regulator" (CFTR), somehow participates in the Cl- secretory response. In this paper we have correlated CFTR gene expression with cAMP and Ca(2+)-dependent Cl- secretion in unpolarized (parental) and polarized (Cl.19A) clones of the human colonic adenocarcinoma cell line HT-29. These cell lines were found to express equally high levels of CFTR mRNA at 4 days post-passage. In addition, protein expression (determined by immunoprecipitation) was also identical. The cAMP-generating agonist forskolin had little effect on 125I efflux from the unpolarized cells. In contrast, this agonist increased 125I efflux 3-fold in polarized cells. The lack of response in the unpolarized cells was not due to the inability of forskolin to raise cAMP levels. Neurotensin, a Ca(2+)-mobilizing agonist, stimulated 125I efflux from both cell lines. In the polarized cells, the magnitude of this response was attenuated at 8 days post-seeding. At this time, the undifferentiated line attained some cAMP responsiveness. This latter effect was paralleled by the appearance of monolayers within areas of the multicell layer. Cell-attached patch-clamp recording from apical membrane patches of polarized cells revealed the presence of a forskolin-stimulated 8-pS Cl- channel; no channel activity was observed in forskolin-stimulated unpolarized cells. Ca(2+)-activated Cl- channels were found in both cell lines. In agreement with the 125I efflux data, the single-channel activation response to [Ca2+]i was smaller in the polarized cell line. From these studies, we can conclude that CFTR expression, measured both at the mRNA and protein level, does not correlate with the colonocyte's ability to secrete chloride ions in response to a cAMP-generating agonist. Cyclic AMP-dependent Cl- secretion requires cellular polarization; specifically, the delineation of an apical membrane. Differences in the cellular location of CFTR during differentiation are likely to explain our results. In contrast, Ca(2+)-stimulated Cl- secretion occurred independently of cellular polarization but was reduced when the cells formed tight junctions.

Topics: Adenocarcinoma; Calcium; Cell Differentiation; Cell Line; Chloride Channels; Chlorides; Colforsin; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelium; Humans; Ion Channels; Ionomycin; Kinetics; Membrane Potentials; Membrane Proteins; Microscopy, Electron; Neurotensin; Poly A; RNA, Messenger

Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway.

Topics: Base Sequence; Calcium; Cell Line; Chloride Channels; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Ion Channels; Ionomycin; Membrane Proteins; Models, Biological; Molecular Sequence Data; Oligonucleotides, Antisense; Phosphorylation; RNA, Messenger; Thionucleotides

We describe a simple and rapid technique for assaying both constitutive and regulated plasma membrane Cl- conductances. The method uses right-angle light scattering to measure the rate of swelling of cells in suspension, in which the anion conductance is rate limiting for swelling, due to introduction of high plasma membrane cation conductance using gramicidin. The technique was verified using Chinese hamster ovary cells and mouse L cells, both stably transfected with the cystic fibrosis transmembrane conductance regulator (CFTR), to confer a specific cAMP-activated Cl- conductance not normally present in these cell types. In agreement with results obtained using other methods for assaying Cl- permeability in these cells, forskolin stimulated a significant increase in plasma membrane Cl- conductance in CFTR-expressing cells, as indicated by an increase in light scattering. That the enhanced light scattering by the cells was the result of cell swelling due to NaCl influx was shown by ion substitution experiments, in which no forskolin-induced increase in light scatter occurred in N-methyl-D-glucamine Cl- or Na+ gluconate medium. Enhanced light scattering was also observed in both CFTR-expressing and control cells stimulated with the Ca2+ ionophore, ionomycin. Extracellular anion substitution, to exploit the inwardly directed halide gradient utilized in this protocol, enabled determination of the anion selectivities of both the cAMP- and Ca(2+)-activated Cl- channels. Thus this technique provides a simple optical method for rapidly assaying not only constitutive and regulated Cl- conductance pathways but also their anion selectivities.

Topics: Animals; Calcium; Cell Line, Transformed; Cell Membrane; Chlorides; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; Electric Conductivity; Gramicidin; Ionomycin; Light; Membrane Proteins; Reference Values; Scattering, Radiation

The regulation of chloride conductance was investigated in the T84 human colon carcinoma cell line by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium. The permeable cAMP analog 8-Br-cAMP (100 microM) and the calcium ionophore ionomycin (1 microM) activate a chloride conductance. A prolonged (4 h) preincubation of cells with phorbol 12-myristate 13-acetate (100 nM) or with the diacylglycerol analog 1-oleoyl-2-acetyl-glycerol (100 microM): (i) down-modulates to almost zero the protein kinase C activity in the membranes; (ii) inhibits the activation of the chloride conductance mediated by 8-Br-cAMP but not by calcium; (iii) reduces the mRNA without changing the expression of the protein product of the cystic fibrosis gene. The data suggest that PKC is essential for the activation of the cAMP-dependent chloride conductance in T84 cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Biological Transport; Chloride Channels; Chlorides; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diglycerides; Down-Regulation; Electric Conductivity; Fluorescence; Fluorescent Dyes; Humans; Ionomycin; Membrane Proteins; Protein Kinase C; Protein Kinases; Quinolinium Compounds; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A method that maximises the yield of viable enterocytes has been developed for the isolation of enterocytes from human jejunal biopsy specimens. These enterocytes have been used to study the values of intracellular free calcium and the rises in adenosine 3'5'-cyclic monophosphate (cAMP) induced by secretagogues in normal and cystic fibrosis cells. Basal intracellular free calcium of cystic fibrosis enterocytes, measured fluorimetrically with fura-2, was within the range of the basal intracellular free calcium of non-cystic fibrosis enterocytes (cystic fibrosis 263 nmol/l; non-cystic fibrosis 287 nmol/l). Changes in intracellular free calcium were observed after exposure to ionomycin: a 100 nmol/l solution induced a 2.5 fold increase in intracellular free calcium in the cystic fibrosis enterocytes and a 2.2 fold increase in the intracellular free calcium concentration of the non-cystic fibrosis enterocytes. Basal cAMP values were not significantly different between cystic fibrosis and non-cystic fibrosis enterocytes (cystic fibrosis 575 fmol/100,000 cells; non-cystic fibrosis 716 fmol/100,000 cells, p greater than 0.05) and the enterocyte cAMP value increased in response to stimulation with prostaglandin E2 (7 mumol/l) (cystic fibrosis 2.2 fold increase over basal, p less than 0.05; non-cystic fibrosis 1.9 fold stimulation over basal, p less than 0.05) and vasoactive intestinal polypeptide (100 nmol/l) (cystic fibrosis 7.1 fold increase over basal, p less than 0.05; non-cystic fibrosis 5.8 fold increase over basal, p less than 0.05). There was no significant difference in the magnitude of the response between cystic fibrosis and non-cystic fibrosis enterocytes (p greater than 0.05). These results indicate that the cystic fibrosis defect in the small intestine, as in other affected epithelia, seems to be distal to the production of second messengers. The small intestine is therefore an appropriate model in which to study the biochemical defect in cystic fibrosis.

Topics: Animals; Calcium; Cell Survival; Child; Child, Preschool; Cyclic AMP; Cystic Fibrosis; Dinoprostone; Humans; Infant; Intestinal Mucosa; Ionomycin; Jejunum; Male; Rats; Vasoactive Intestinal Peptide

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Chloride Channels; Cystic Fibrosis; Epithelium; Humans; Ion Channels; Ionomycin; Membrane Potentials; Membrane Proteins; Protein Kinases; Reference Values; Respiratory Physiological Phenomena; Respiratory System

The genetic disease cystic fibrosis (CF) causes decreased Cl- transport in several epithelia. cAMP-dependent regulation of apical membrane Cl- channels is defective in CF airway epithelia; as a result, CF epithelia fail to secrete Cl-. In contrast, Ca(2+)-stimulated Cl- secretion is intact in CF airway epithelia and thus has the potential to bypass the CF Cl- secretory defect. For a Cl- channel to govern Cl- secretion, it must be located in the apical membrane. To specifically investigate apical membrane Cl- channels, we studied cells grown on permeable filter supports and measured Cl- currents across the apical membrane. We found that Ca2+ and cAMP activate different Cl- channels in the apical membrane. (i) Ca(2+)-activated Cl- channels were present in the apical membrane of airway but not in intestinal epithelia. (ii) cAMP- but not Ca(2+)-activated Cl- channels were defective in CF airway epithelia. (iii) Ca(2+)- but not cAMP-activated Cl- channels were blocked by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate. (iv) Ca(2+)- and cAMP-activated apical channels had different anion permeabilities. (v) An increase in both second messengers produced an additive increase in Cl- current. These results also explain the puzzling observation that Ca(2+)-stimulated Cl- secretion is defective in CF intestine: the Ca(2+)-activated Cl- channels that could circumvent the Cl- secretory defect in CF airway are missing from the apical membrane of intestinal epithelia.

Topics: Calcium; Cell Line; Cell Membrane; Cells, Cultured; Chloride Channels; Cyclic AMP; Cystic Fibrosis; Epithelium; Humans; Intestines; Ion Channels; Ionomycin; Kinetics; Membrane Potentials; Membrane Proteins; Models, Biological; Reference Values; Respiratory Physiological Phenomena; Respiratory System

Intestinal epithelial cells were isolated from a fetus with cystic fibrosis (CF) and transfected with a plasmid vector recombined with the ori- mutant of SV40. A population of proliferative cells was then subcloned and designated as CFI-3. These cells had a doubling time of 24 h and were maintained in culture for up to 25 passages. At passage 8, CFI-3 cells did not produce any tumors in nude mice. Northern blot and immunofluorescence studies indicated that the extended lifespan of CFI-3 cells results in genomic insertion of SV40 LT. Intestinal CFI-3 cells are epithelial, according to the expression of the human cytokeratin 18 gene and poorly differentiated by phase-contrast and electron microscopy. Functional membrane receptors activated by vasoactive intestinal peptide (VIP), its natural analogue pituitary adenylate cyclase activating peptide (PACAP-38), and isoproterenol were observed in CFI-3 cells. Restriction fragment length polymorphism analysis of the PstI KM19 site revealed that the cftr locus was identical in the chorionic villi and in CFI-3 cells. The manifestation of CF in this family was not related to the common mutation delta F508, since this fetus was heterozygous for the substitutions S549N and N1303K. Chloride transport, assessed by the 125I efflux, was induced in CFI-3 cells by the calcium inophore ionomycin, but not by the adenylate cyclase activator forskolin, and was inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid. These results were confirmed in patch clamp studies in which the cpt cAMP analogue failed to stimulate membrane currents, while the calcium ionophore ionomycin stimulated inward currents. We conclude that intestinal CFI-3 cells retain the CF phenotype relating to defective regulation of Cl- channels, and therefore constitute a suitable model, 1) for elucidating the function of CFTR protein, 2) developing new therapeutic agents, and 3) correcting the CF defect by gene replacement therapy in vitro.

Topics: Antigens, Polyomavirus Transforming; Base Sequence; Cell Line; Cell Transformation, Viral; Chloride Channels; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression; Humans; In Vitro Techniques; Intestinal Mucosa; Ionomycin; Isoproterenol; Membrane Proteins; Molecular Sequence Data; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Potassium Channels; Transfection; Vasoactive Intestinal Peptide

Cystic fibrosis (CF) is a genetic disease characterized by abnormal regulation of epithelial cell chloride channels. Nonepithelial cells, including lymphocytes and fibroblasts, may exhibit a similar defect. Two independent techniques were used to assess the macroscopic chloride permeability (PCl) of freshly isolated B lymphocytes and of B and T lymphocyte cell lines. Values for PCl increased specifically during the G1 phase of the cell cycle and could be further enhanced by increasing intracellular adenosine 3',5'-monophosphate (cAMP) or calcium. In lymphocytes from CF patients, regulation of PCl during the cell cycle and by second messengers was absent. Characterization of the cell cycle-dependent expression of the chloride permeability defect in lymphocytes from CF patients increases the utility of these cells in the analysis of the functional consequences of mutations in the CF gene.

Topics: B-Lymphocytes; Calcium; Cell Cycle; Cell Line; Cell Membrane Permeability; Chlorides; Cyclic AMP; Cystic Fibrosis; Electric Conductivity; Humans; Interphase; Ionomycin; Lymphocytes; Microscopy, Fluorescence; Second Messenger Systems; T-Lymphocytes; Thionucleotides

Cystic fibrosis (CF) airway epithelia express a defect in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of apical membrane Cl- channels. Recent patch-clamp studies have raised the possibility that Ca2+ -dependent mechanisms for the activation of Cl- secretion may be preserved in CF airway epithelia. To determine 1) whether intact normal (N1) and CF airway epithelia exhibit a Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanisms initiate Cl- secretion via activation of an apical membrane Cl- conductance (GCl-), nasal epithelia from N1 and CF subjects were cultured on collagen membranes, and responses to isoproterenol or Ca2- ionophores [A23187 10(-6) M; ionomycin (10(-5)M)] were measured with transepithelial and intracellular techniques. Isoproterenol induced activation of an apical membrane GCl- in N1 cultures but was ineffective in CF. In contrast, in both N1 and CF amiloride-pretreated cultures, A23187 induced an increase in the equivalent short-circuit current that was associated with an activation of an apical membrane Gc1- and was bumetanide inhibitable. A23187 addition during superfusion of the lumen with a low Cl- (3 mM) solution reduced intracellular Cl- activity of CF cells. A Ca2+ ionophore of different selectivity properties, ionomycin, was also an effective Cl- secretagogue in both N1 and CF cultures. We conclude that 1) the A23187 induced Cl- secretion via activation of an apical GCl- in N1 human nasal epithelium, and 2) in contrast to an isoproterenol-dependent path, a Ca2+ -dependent path for GCl- activation is preserved in CF epithelia.

Topics: Adolescent; Adult; Amiloride; Calcimycin; Calcium; Cells, Cultured; Chloride Channels; Chlorides; Cystic Fibrosis; Electric Conductivity; Ethers; Female; Humans; Ion Channels; Ionomycin; Isoproterenol; Male; Membrane Potentials; Membrane Proteins; Nasal Polyps; Reference Values; Turbinates

An airway epithelial cell line (CF/T43) was developed by infecting cultured airway epithelial cells from patients with cystic fibrosis (CF) with the pZIPneoSV(X)1/SV40T retrovirus and selecting for G418 resistance and ion transport properties. The distinctive chloride secretory phenotypes of the CF cell line CF/T43 and a normal cell line (NL/T4) were not perturbed by SV40T-induced cell transformation. Epithelial cell lines generated from CF cells with the SV40T gene can be used to test candidate CF genes and to evaluate the molecular mechanisms responsible for the CF phenotype.

Topics: Amiloride; Antigens, Polyomavirus Transforming; Calcimycin; Cell Line; Cell Membrane; Chloride Channels; Chlorides; Colforsin; Cystic Fibrosis; Electric Conductivity; Epithelium; Ethers; Freeze Fracturing; Humans; Intercellular Junctions; Ion Channels; Ionomycin; Membrane Proteins; Microscopy, Electron; Nasal Polyps; Simian virus 40; Transformation, Genetic

To determine whether epithelial ion transport abnormalities in cystic fibrosis (CF) might reflect abnormal regulation of intracellular Ca2+ levels, cytosolic free calcium (Cai2+) was measured using fura-2 or quin2 in suspensions of normal or CF nasal epithelial cells derived from primary cell culture. The basal Cai2+ level measured with fura-2 in CF nasal epithelia was 155 +/- 9 nM (n = 5), a value not significantly different from normal nasal epithelia (143 +/- 16 nM, n = 5). Total cell calcium was measured by atomic absorption spectroscopy and no differences were observed between CF (6.3 +/- 0.5 nmol/mg protein; n = 3) and normal (6.2 +/- 1.2 nmol/mg protein; n = 3) nasal epithelial cells. Placing Na+ loaded cells in a low (10 mM) extracellular Na+ solution resulted in a rapid increase in Cai2+ consistent with Ca2+ uptake via a plasmalemmal Na+-Ca2+ exchanger. The level of Cai2+ achieved by this low Na+ maneuver was not significantly different in CF cells compared to normal cells. Neither isoproterenol (10(-5) M) nor forskolin (10(-6) M) had any effect on Cai2+ in normal or CF nasal epithelial cells. Thus, it appears that differences in cell Cai2+, as measured by fluorescent chelators in suspensions of cultured cells, do not account for the abnormalities in basal or isoproterenol stimulated ion transport in CF tissues.

Topics: Adolescent; Adult; Aged; Aminoquinolines; Benzofurans; Calcium; Cells, Cultured; Child; Cyclic AMP; Cystic Fibrosis; Cytosol; Epithelium; Ethers; Female; Fluorescent Dyes; Fura-2; Humans; Ionomycin; Isoproterenol; Male; Middle Aged; Nasal Mucosa; Ouabain; Sodium; Spectrophotometry, Atomic