calcimycin and Cystic-Fibrosis

1. An improved novel plasmid backbone, pTrial10, has been developed. We have used this vector to deliver the cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR) to cells, both in vitro and in vivo, complexed with cationic liposomes. 2. Human 293 kidney epithelial cells (HEK 293) showed expression of an immunoprecipitable 165 kDa protein corresponding to CFTR when transfected in vitro with pTrial10-CFTR2, but not when the vector pTrial10 was used. 3. HEK 293 cells transfected with pTrial10-CFTR2, but not pTrial10, demonstrated a cAMP-dependent anion conductance, measured by fluorescence microscopy using a halide-sensitive probe, SPQ. 4. The CFTR-dependent, cAMP-sensitive chloride secretory response in murine tracheal epithelium could be measured if the calcium-dependent chloride secretory process was first maximally stimulated with a mixture of the Ca(2+)-ATPase inhibitor, TBHQ, and the calcium ionophore, A23187. With these conditions wild-type and CF-null (transgenic animals in which the cystic fibrosis (CF) gene has been disrupted so that no CFTR is produced) murine tracheas could be distinguished. The difference between the current elicited by forskolin in wild-type and CF tracheas was highly significantly different (P < 0.001), giving a CFTR-dependent current of 11.2 microA cm-2. 5. Transfection of the airways with pTrial10-CFTR2, but not pTrial10, significantly (P < 0.01) increased the CFTR-dependent chloride secretory current in CF tracheas. The degree of correction was greater when intra-tracheal installation rather than nasal insufflation was used to deliver the plasmids.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Antioxidants; Avian Sarcoma Viruses; Calcimycin; Chloride Channels; Chlorides; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diuretics; Genetic Therapy; HeLa Cells; Humans; Hydroquinones; Ionophores; Mice; Mice, Inbred CFTR; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Trachea; Transfection

The mechanism(s) of chronic airway inflammation in cystic fibrosis (CF) remains poorly understood. We studied Ca2+-induced release of arachidonic acid (AA), a precursor of proinflammatory lipid mediators, in epithelial cell lines with the deltaF508 mutation in CF transmembrane conductance regulator (CFTR) gene and in those lacking this mutation or cells in which this mutation was corrected by a functional CFTR gene transfer. We found that: (i) the mutant cells manifested an abnormally high Ca2+-induced AA release as compared to controls, (ii) AA release appeared to be catalyzed by a phospholipase A2 (PLA2) but not by phospholipase C followed by diacylglycerol lipase, and (iii) either correction of the CFTR-mutation or inhibition of PLA2 activity rectified this AA release abnormality. Taken together, our results suggest that CFTR mutation is associated with an intrinsic abnormality in AA release by epithelial cells carrying the deltaF508 mutation and suggest that the mechanism of chronic airway inflammation in CF, at least in part, involves this abnormality. These results also partly explain the effectiveness of high-dose ibuprofen therapy in arresting the progression of destructive lung disease in CF. Furthermore, they raise the possibility that correction of abnormal AA release by inhibiting PLA2 activity may improve the therapeutic benefits of ibuprofen.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Calcimycin; Calcium; Cells, Cultured; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dose-Response Relationship, Drug; Epithelial Cells; Epithelium; Humans; Ibuprofen; Lipoprotein Lipase; Mutation; Phospholipases A; Phospholipases A2; Precipitin Tests; Staurosporine; Tetradecanoylphorbol Acetate

This study documents a difference between cystic fibrosis human (CF-HTG) and normal human (HTG) tracheal gland cells: the ability of histamine to induce an increase of intracellular free calcium concentration [Ca2+]i was abnormally reduced in CF-HTG cells. The magnitude of the [Ca2+]i peak rise in response to histamine is smaller in CF-HTG cells than in HTG cells, and the percentage of CF-HTG cells that increase [Ca2+]i is decreased compared with HTG cells. In contrast to histamine, the human neutrophil elastase (HNE) stimulation of both CF-HTG and HTG cells generated [Ca2+]i asynchronous oscillations and the magnitude of the peak [Ca2+]i response as well as the percentage of responding cells were similar for both groups. By videomicroscopy observations, the secretory response (exocytosis of secretion granules) of CF-HTG cells occurred with HNE, but not with histamine, thus suggesting that [Ca2+]i asynchronous oscillations may be linked to the exocytosis process in human tracheal gland cells.

Topics: Calcimycin; Calcium; Cells, Cultured; Cystic Fibrosis; Histamine; Humans; Ionophores; Leukocyte Elastase; Pancreatic Elastase; Trachea

The present microelectrode experiments on fused respiratory epithelial cells of cystic fibrosis (CF) origin and non-CF origin aim at characterizing the molecular basis of the Cl- conductances regulated by cyclic adenosine monophosphate (cAMP) or respectively Ca2+, as described in the preceding publication. Cell membrane potential (Vm) and resistance (Rm) were recorded as well as their response to substitution of 90% of bath Cl- by isethionate (delta Vm,ISE), by I- (delta Vm,I), or by other halide anions. Fused CF cells had significantly (P < 0.05) higher control Vm values (-18.0 +/- 9.4 mV, +/- SD, n = 68) than fused non-CF cells (-12.5 +/- 6.6 mV, n = 69) and responded to the Ca2+ ionophore A23187 with an increase in the Vm response to Cl- substitution, but did not respond to forskolin. This indicates that CF cells express only the Ca(2+)-stimulated Cl- conductance. Injection of the antibody M3A7 against a fusion protein containing amino acids 1195 to 1480 of the CF gene product into young, forskolin-stimulated or old non-CF cells decreased delta Vm,ISE and delta Vm,I within 15 min to values observed in CF cells. This indicates inhibition of the cAMP-stimulated Cl- conductance and supports the molecular identity of this conductance with the CF gene product. However, the slow onset of inhibition does not allow secondary effects to be excluded and a slight fall in Rm remains unexplained. Stimulation of the Ca(2+)-regulated Cl- conductance was not impaired. Injection of M3A7 into CF cells or of a control antibody in non-CF cells had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antibodies; Calcimycin; Calcium; Cell Fusion; Cells, Cultured; Chloride Channels; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Epithelium; Humans; Membrane Proteins; Nasal Polyps; Respiratory System

With the aim of further elucidating the role of the epithelial Cl- conductance and its defect in cystic fibrosis (CF) patients we studied the properties and regulation of the Cl- conductance in primary cultures of human nasal polyp epithelia. To facilitate microelectrode punctures and to gain access to the cytoplasmic compartment for injection of antibodies, we prepared giant cells using a polyethylene-glycol fusion technique. The membrane potential (Vm) and resistance (Rm) and their responses to ionic substitutions in the bath were measured under control conditions and in the presence of different secretagogues. In non-CF cells Vm averaged-12.5 mV (SD +/- 6.6 mV, n = 69) and was independent of time after fusion, while Rm dropped from 12.4 +/- 7.3 M omega (n = 51) to 3.5 +/- 5.5 M omega (n = 24) in the 2nd week after fusion. The low Vm values reflected a vanishing K+ conductance in the presence of a dominating Cl- conductance that increased with time. In young cells, a Cl- conductance prevailed which could be stimulated by application of the Ca2+ ionophore, A23187, or of carbachol. As determined in CF cells, it had an outwardly rectifying current/voltage (ilV) relationship and exhibited the selectivity sequence I- > Br- > Cl- > F- > isethionate (ISE-) both in Vm and Rm measurements. With increasing age after fusion, a Cl- conductance prevailed in non-CF cells which could be stimulated by cyclic adenosine monophosphate (cAMP) or forskolin and which was downregulated by A23187. It had a linear ilV relationship and exhibited the selectivity sequence Br- > Cl- > I- > F- > ISE- if determined from Vm measurements, but a sequence of Cl- > Br- > F- = ISE- > I- if determined from Rm measurements. This points to multiple-ion pore behaviour of the respective Cl- channel. In agreement with observations described in the following publication, the results suggest that the cAMP-regulated Cl- conductance corresponds to the CF-gene product while the molecular nature of the Ca(2+)-regulated Cl conductance is not yet known.

Topics: Anions; Calcimycin; Carbachol; Cell Fusion; Cells, Cultured; Chloride Channels; Colforsin; Cyclic AMP; Cystic Fibrosis; Electric Conductivity; Epithelium; Humans; Nasal Polyps; Respiratory System; Signal Transduction

Fluid secretion from the pulmonary epithelium may play a significant role in determining intrauterine lung development. We used suspensions of distal pulmonary epithelial cells isolated from rat fetuses to assess a shift in secretory mechanisms occurring in the lung of this species during late gestation. The impact of cAMP on distal airway epithelial cells isolated from d 18 to d 21 rat fetuses was evaluated with measurements of cell volume and 36Cl efflux rates. At d 18, 8-Br-cAMP stimulated a volume reduction measured by electronic cell sizing that was prevented by the Cl- channel blocker anthracene-9-carboxylate (A-9C) and reflected in an increased rate of A-9C sensitive 36Cl efflux. Because the cystic fibrosis transmembrane conductance regulator (CFTR) is thought to be a cAMP-regulated Cl- channel, we measured the effect of prior cell incubation with oligodeoxynucleotides antisense to the transcription site of the human CFTR gene on these events. We found that in antisense oligomer-treated cells, but not in sense oligomer-treated controls, volume and 36Cl efflux responses to 8-Br-cAMP were prevented in d 18 cells. In d 21 cells, 8-Br-cAMP did not stimulate volume reduction but the calcium ionophore A23187 did elicit cell volume reduction in cells suspended in an isotonic Ca(2+)-containing medium that was prevented by A-9C. This response to the ionophore was not found in the d 18 cells, and incubation with the antisense CFTR oligomer had no effect on the ionophore-induced responses in d 21 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Base Sequence; Calcimycin; Cell Size; Chloride Channels; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelium; Fetus; Gene Expression; Gestational Age; In Vitro Techniques; Membrane Proteins; Molecular Sequence Data; Oligonucleotides, Antisense; Pulmonary Alveoli; Rats

To investigate the effects of glucocorticoids on leukotriene (LT) generation in patients with cystic fibrosis (CF), we evaluated calcium ionophore A23187-induced LTB4 and LTC4 production by leukocytes with and without pretreatment with dexamethasone. The CF patients were in good condition and did not have acute infection. There were no significant differences in LTB4 and LTC4 production without dexamethasone pretreatment between the CF patients and controls. However, the ratios of LTB4 and LTC4 production by leukocytes preincubated with dexamethasone to those of leukocytes without dexamethasone pretreatment were significantly higher in the CF patients than in the controls (both p < 0.05). Our data suggest that the response of LTB4 and LTC4 production to dexamethasone is disturbed in patients with CF. The generation of LTs may be enhanced due to a disturbance in glucocorticoid suppression.

Topics: Adolescent; Adult; Calcimycin; Child; Child, Preschool; Cystic Fibrosis; Dexamethasone; Female; Gene Expression Regulation; Humans; Leukocytes; Leukotriene B4; Leukotriene C4; Male

X-ray micro-analysis was carried out on cultured respiratory cells from polyps removed from individuals with and without cystic fibrosis (CF). In a first set of experiments, proper experimental conditions were established. Washing the cells with 300 mmol l-1 mannitol in distilled water was found to give the best removal of the culture medium. The elemental concentrations stabilized in about 10 min after the start of the preincubation. Intracellular [Na] and [Cl] increased slightly with increasing passage number, whereas intracellular [K] decreased. Under resting conditions there were no significant differences in elemental content between CF and control cells, and there were no indications for abnormally high total [Ca] in CF cells. In normal cells, stimulation with a cAMP-analogue resulted in a decrease of cellular [Cl], whereas in CF cells an increase was measured. Exposure of both normal and CF cells to ouabain resulted in decreased [K] and increased [Na] and [Cl] level. The calcium ionophore A23187 had a similar effect on normal cells but did not affect CF cells markedly. Application of amiloride to the apical side of the cells resulted in a decrease of cellular [Na] in CF cells, whereas [Na] in control cells was not affected. The results correspond with what is known about the defective cAMP-regulated transepithelial Cl-transport in CF cells. The effect of the calcium ionophore on cellular electrolyte content is more complicated and may be the result of two separate effects: efflux of Cl- via a Ca(2+)-dependent mechanism and inhibition of the Na(+)-K(+)-ATPase by intracellular Ca2+ ions causing an influx of Na+ and Cl- ions.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amiloride; Calcimycin; Cells, Cultured; Chlorides; Cyclic AMP; Cystic Fibrosis; Electron Probe Microanalysis; Epithelium; Humans; Ouabain; Potassium; Respiratory System; Sodium

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

Topics: Calcimycin; Calcium; Carcinoma; Cations, Divalent; Colonic Neoplasms; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Half-Life; Humans; Membrane Proteins; RNA, Messenger; Terpenes; Thapsigargin; Transcription, Genetic; Tumor Cells, Cultured

Epithelial cells utilize at least two types of apical Cl- channels, the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) and the Ca2+/calmodulin-dependent Cl- channel. While phorbal ester (PMA) activates only CFTR-dependent Cl- secretion and the Ca2+ ionophore A23187 only the Ca2+/calmodulin-dependent Cl- secretion, PMA and A23187 share the ability to down-regulate expression of the CFTR gene at the transcriptional level. Since both PMA and A23187 can activate protein kinases, we hypothesized that protein kinase pathways may be involved in the regulation of CFTR gene expression. Exposure of HT-29 human colon carcinoma cells to the protein kinase C activator SC9 down-regulated CFTR mRNA levels in a dose-dependent fashion, similar to that seen with PMA. The reduction in CFTR transcript levels by SC9 and PMA was blocked by H7, an inhibitor of protein kinases. In a similar fashion, the down-regulation of CFTR transcript levels by A23187 was blocked by H7 as well as staurosporine, another protein kinase inhibitor. Interestingly, both H7 and staurosporine themselves increased CFTR mRNA levels. Quantification of CFTR gene transcription rate showed a reduction by SC9 (similar to that with PMA and A23187) that was prevented by H7 and that H7 by itself increased CFTR transcription. Together, these observations suggest that protein kinase pathways, likely including protein kinase C, are involved in the regulation of CFTR gene expression, with activation or inhibition of protein kinase activity down-regulating or up-regulating CFTR gene expression, respectively.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcimycin; Cell Line; Colonic Neoplasms; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Isoquinolines; Kinetics; Membrane Proteins; Piperazines; Protein Kinase C; RNA, Messenger; Sulfonamides; Tetradecanoylphorbol Acetate; Transcription, Genetic

Abnormal epithelial electrolyte transport has been identified in a range of cystic fibrosis (CF) organs and appears to account for the various clinical manifestations of the disease. The aim of this study was to further define the Cl- secretion defect in CF jejunum. Excised jejunum was obtained from 11 CF patients and 12 controls. Transport studies were performed on stripped epithelium in vitro under short-circuited conditions in Ussing Chambers. 3-Isobutyl-1-methylxanthine (IBMX) (300 microM) significantly increased Cl- secretion in control (-2.3 +/- 0.6 to -3.3 +/- 0.7 mueq.cm-2.h-1; P less than 0.01, paired t test; n = 5 subjects) but not in CF jejunum (-0.5 +/- 0.3 to -0.1 +/- 0.4; n = 4). However in contrast to control jejunum, net Na+ absorption in CF jejunum was higher in the IBMX (1.3 +/- 0.5 mueq.cm-2.h-1) compared with basal periods (0.6 +/- 0.3; P less than 0.05, paired t test). IBMX stimulation of tissue adenosine 3',5'-cyclic monophosphate (cAMP) was similar in both control and CF jejunum. A range of secretagogues known to induce secretion in mammalian intestine, including dibutyryl cAMP (DBcAMP), DBcGMP, Ca2+ ionophore A23187, and the protein kinase C activator 4 beta-phorbol 12,13-dibutyrate, failed to induce secretion in CF jejunum. In conclusion, CF jejunum failed to exhibit Cl- secretion and also demonstrated abnormalities of Na+ absorption. These results support the view that the defect lies at a site distal to the intracellular messengers. Moreover, these abnormalities of intestinal electrolyte transport may account for some of the gastrointestinal manifestations of the disease such as meconium ileus and distal intestinal obstruction syndrome.

Topics: 1-Methyl-3-isobutylxanthine; Adolescent; Adult; Aged; Biological Transport; Bucladesine; Calcimycin; Child; Cyclic AMP; Cystic Fibrosis; Dibutyryl Cyclic GMP; Epithelium; Humans; In Vitro Techniques; Intestinal Mucosa; Ions; Jejunum; Kinetics; Middle Aged; Phorbol 12,13-Dibutyrate; Reference Values

Continuous epithelial cell lines from individuals with cystic fibrosis (CF) and normal controls are required to understand the genetic and cellular defects in CF. We used retroviruses to transduce SV40 large T antigen into nasal epithelial cells. Transformed continuous cell lines were isolated that expressed epithelial markers, cytokeratin, and tight junctions. Northern blot analysis shows that all of the cell lines express the putative CF gene mRNA. Studies of transepithelial electrolyte transport show that CF and normal cell lines develop a transepithelial electrical resistance. Normal but not CF cell lines secreted Cl- in response to agonists that increase cellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (isoproterenol, forskolin, and a membrane-permeant analogue of cAMP) or in response to a tumor-promoting phorbol ester that activates protein kinase C. In contrast, the Ca2(+)-elevating agonist bradykinin and the Ca2+ ionophore A23187 stimulated secretion in both normal and CF cell lines. The continuous cell lines we have produced maintain their proper phenotypes and will serve as useful tools in understanding the pathophysiology of CF.

Topics: Antigens, Polyomavirus Transforming; Base Sequence; Biomarkers; Blood Proteins; Bradykinin; Calcimycin; Calgranulin A; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Chloride Channels; Chlorides; Colforsin; Cystic Fibrosis; Epithelial Cells; Epithelium; Genotype; Humans; Ion Channels; Keratins; Membrane Potentials; Membrane Proteins; Molecular Sequence Data; Nasal Mucosa; Oligonucleotide Probes; Phenotype; Polymerase Chain Reaction; Reference Values; Retroviridae; Simian virus 40; Tetradecanoylphorbol Acetate; Theophylline

In comparison to skin fibroblasts from normal subjects, those from patients with cystic fibrosis (CF): (1) bound [20-3H] phorbol 12,13-dibutyrate (PDBu) with a higher affinity Kd = 25.8 vs 12.8 nM respectively) but expressed a similar number of total phorbol ester binding sites (about 2.5 pmol PDBu bound/mg of protein); (2) exhibited a faster and higher response to 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) for the stimulation of [35S]-labelled glycoconjugate release, but were equally sensitive to the synergistic effect of A23187 on this process; and (3) secreted glycoconjugates with similar [35S]-sulfate and [14C]-leucine to [14C]-glucosamine labelling ratios. Taken together, these results provide further evidence for abnormal protein kinase C (PKC) regulation of macromolecule secretion in CF disease.

Topics: Binding Sites; Bucladesine; Calcimycin; Cells, Cultured; Colforsin; Cystic Fibrosis; Dose-Response Relationship, Drug; Fibroblasts; Glycoconjugates; Humans; Isoproterenol; Phorbol 12,13-Dibutyrate; Protein Kinase C; Skin; Tetradecanoylphorbol Acetate

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.

Topics: Adenocarcinoma; Calcimycin; Carbachol; Cell Line; Cholera Toxin; Colonic Neoplasms; Cystic Fibrosis; Histamine; Humans; Immunodiffusion; Immunoenzyme Techniques; Intestine, Small; Microscopy, Electron; Mucins; Prostaglandins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

It has widely been established that Cl- transport is defective in cystic fibrosis fibroblasts. In the present study, the effect of elevation of intracellular concentration of cyclic AMP and calcium on the efflux of Cl- from human fibroblasts has been investigated. Cyclic AMP analogs (8-bromo cAMP and dibutyryl cAMP) and a beta agonist (isoproterenol) induced only a weak stimulation (5-10%) of Cl- efflux. Conversely, elevation of cytoplasmic calcium concentration produced by addition of the Ca2+ ionophore A23187 in the efflux medium, did not affect Cl- efflux. Our data indicate that the response of Cl- efflux to elevation of cAMP and calcium is similar in normal and cystic fibrosis fibroblasts. Exposure to hypotonic medium induced a significant stimulation of Cl- efflux in fibroblasts from both normal and cystic fibrosis individuals. Substitution of Cl- in the medium by gluconate and the subsequent addition of furosemide did not inhibit the effect of hypotonicity, indicating the involvement of a conductive pathway for Cl- transport, which was insensitive to oligomicin C.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Bucladesine; Calcimycin; Calcium; Cell Line; Cells, Cultured; Chlorides; Cyclic AMP; Cystic Fibrosis; Fibroblasts; Furosemide; Gluconates; Humans; Hypotonic Solutions; Isoproterenol

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

Because the defect in Cl- secretion exhibited by cystic fibrosis (CF) epithelia reflects regulatory rather than conductive abnormalities of an apical membrane Cl- channel, we investigated the role of different regulatory pathways in the activation of Cl- secretion in freshly excised normal and CF nasal epithelia mounted in Ussing chambers. A beta agonist (isoproterenol [ISO]), a Ca2+ ionophore (A23187), and a phorbol ester (PMA) were all effective Cl- secretagogues in normal human nasal epithelia. Agonist addition studies indicated that ISO and PMA but not A23187 may share a common regulatory pathway. In contrast, only A23187 induced Cl- secretion in CF epithelia. Bradykinin raised cytosolic Ca2+ and induced Cl- secretion in both normal and CF tissues, indicating that receptor gated Ca2+ dependent Cl- secretory mechanisms were preserved in CF. The defective Cl- secretory response in CF epithelia to ISO and PMA did not reflect abnormalities in cAMP-dependent (A) and phospholipid Ca2+-dependent (C) kinase activities. We conclude that (a) a Ca2+-sensitive mechanism for regulating Cl- secretion is maintained in CF airway epithelia, and (b) a regulatory pathway shared by two distinct protein kinases is defective in CF, indicating that the CF genetic lesion is not tightly coupled to a single (e.g., cAMP dependent) regulatory mechanism.

Topics: 1-Methyl-3-isobutylxanthine; Adolescent; Adult; Amiloride; Bradykinin; Calcimycin; Calcium; Child; Chlorides; Cyclic AMP; Cystic Fibrosis; Electric Conductivity; Epithelium; Female; Humans; Isoproterenol; Male; Nasal Mucosa; Protein Kinase C; Tetradecanoylphorbol Acetate

The oxidative burst of polymorphonuclear cells and monocytes from patients with cystic fibrosis as measured by luminol-enhanced chemiluminescence was examined after in vitro activation of the cells. All patients were outpatients at the time of the assays; their median age was 25.5 years (range, 12 to 33 years) and normal controls were young healthy adults. Stimulation of polymorphonuclear cells with phorbol myristate acetate, the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, and the calcium ionophore A23187 resulted in significantly greater chemiluminescence responses from the cells of patients than from the control cells. The monocyte response of patients to opsonized zymosan was also greater than that of controls. Thus, phagocytic cells from adolescents and young adults with cystic fibrosis have a greater chemiluminescence response to a variety of stimuli. This may result in tissue damage in the lungs of these patients and thus make them more susceptible to pulmonary infections.

Topics: Adolescent; Adult; Calcimycin; Child; Cystic Fibrosis; Humans; Luminescent Measurements; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Tetradecanoylphorbol Acetate; Zymosan

Sodium ion and chloride transport was studied in vitro in small intestinal and colonic tissue from patients with cystic fibrosis (CF) and from non-CF control subjects matched as to age and sex. Normal histological appearance and substantial response to mucosal glucose (5 mM, ileum) or mucosal amiloride (10(-5) M, colon) indicated normal tissue viability in both control and CF tissues. Electroneutral NaCl absorption was demonstrated in the small intestine of control subjects and CF patients. Small intestinal and colonic tissues of control subjects responded to four secretagogues (theophylline, 5 mM; prostaglandin E2, 10(-6) M; calcium ionophore (A23187), 10(-5) M; bethanechol, 5 x 10(-5) M), with electrogenic chloride secretion. The tissues of CF patients, however, did not respond to any of the test secretagogues. These studies demonstrate that an abnormality in chloride transport is present in the small intestinal and colonic epithelia of CF patients. Unlike airway epithelia, which secrete chloride in response to Ca ionophore, the intestinal epithelia of CF patients do not respond to either cAMP- or Ca-mediated secretagogues. This abnormality in intestinal electrolyte transport may play a role in the pathogenesis of meconium impactions in CF patients.

Topics: Adult; Amiloride; Biological Transport; Calcimycin; Chlorides; Cyclic AMP; Cystic Fibrosis; Electric Conductivity; Female; Glucose; Humans; Infant; Infant, Newborn; Intestinal Absorption; Intestinal Mucosa; Intestines; Male; Membrane Potentials; Sodium; Theophylline

The secretory activity of jejunal biopsies from children with cystic fibrosis (CF) has been investigated using a modified Ussing chamber technique. Samples from six children with CF failed to respond when challenged with the intestinal secretagogues acetylcholine (10(-3) M), prostaglandin E2 (1.4 X 10(-6) M) and dibutyryl cyclic AMP (10(-3) M), while control tissues exhibited rises in short circuit current of 28.1 (7.4) (6) microA/cm2, 23.4 (4.6) (6) microA/cm2 and 10.0 (2.0) (4) microA/cm2 respectively in response to these agents. The calcium ionophore, A23187 (3.8 X 10(-6) M), increased the short circuit current in all the control tissues (mean change = 10.1 (2.7) (5) microA/cm2) and induced a small response in some of the CF tissues. Both groups of tissues generated a rise in short circuit current associated with sodium linked glucose (10 mM/l) absorption (control = 32.6 (9.3) (6) microA/cm2, CF = 36.2 (13.9) (6) microA/cm2, p greater than 0.05). These results show that the defect in chloride transport observed in other epithelia in CF also exists in the jejunum and could contribute to the intestinal effects of the disease. The technique used should permit further studies of the basic defect and may be of diagnostic value.

Topics: Acetylcholine; Adolescent; Biological Transport; Bucladesine; Calcimycin; Child; Child, Preschool; Chlorides; Cystic Fibrosis; Dinoprostone; Glucose; Humans; In Vitro Techniques; Intestinal Mucosa; Jejunum; Membrane Potentials; Prostaglandins E

Cl- efflux from normal human fibroblasts is stimulated by elevation of cAMP and by elevation of intracellular free Ca2+. In both cases the stimulated Cl- transport occurs via electrically conductive pathways. In six lines of normal human fibroblasts, dibutyryl cAMP increased total Cl- efflux by an average of 13%. In six lines of fibroblasts from patients with cystic fibrosis, dibutyryl cAMP was without effect. The electrically conductive component of Cl- transport was increased an average of 30% by dibutyryl cAMP in normal cells and was unaffected by dibutyryl cAMP in cystic fibrosis cells. Stimulation of the Ca2+-sensitive Cl- channel by addition of A23187 increased Cl- efflux by an average of 30% in normal and 30% in cystic fibrosis fibroblasts. The data indicate that there is a defect in a cAMP-activated Cl- channel in cystic fibrosis fibroblasts.

Topics: Bucladesine; Calcimycin; Calcium; Cell Line; Chlorides; Cyclic AMP; Cystic Fibrosis; Electric Conductivity; Fibroblasts; Humans; Ion Channels

In order to determine whether cystic fibrosis neutrophils are affected in their secretory functions, lysosomal enzyme release and chemiluminescence (light emission from cells) were assayed in patients' cells and compared with those in normal control cells. We observed a decreased response of cystic fibrosis neutrophils in beta-glucuronidase release and chemiluminescence after stimulation by N-formyl-methionyl-leucyl-phenylalanine. There was no significant correlation of these results with the clinical score nor with the medical treatment. On the other hand, responses to the calcium ionophore A23187 and to opsonized zymosan showed no significant difference between normal and cystic fibrosis subjects in lysosomal enzyme release. N-formyl-methionyl-leucyl-phenylalanine receptor alterations did not seem involved in the observed effect as demonstrated by Scatchard plot analysis of N-formyl-methionyl-leucyl-phenylalanine binding to these receptors. These results clearly demonstrate a difference between normal and cystic fibrosis neutrophils in release and chemiluminescence responses to N-formyl-methionyl-leucyl-phenylalanine stimulation, a difference that might be located in the plasma membrane as both responses are membrane dependent.

Topics: Adolescent; Adult; Calcimycin; Calcium; Child; Child, Preschool; Cystic Fibrosis; Exocytosis; Female; Gallic Acid; Glucuronidase; Humans; Kinetics; Luminescent Measurements; Lysosomes; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Trifluoperazine; Zymosan

Hamster trachea epithelial (HTE) cells were shown to respond to 20% cystic fibrosis serum (CFS) by secreting twice as much protein as in the presence of 20% normal human serum (NHS). Serum from obligate heterozygotes (HHS) produced an intermediate effect. A peak of Ca2+ entry into the HTE cells occurred about 30 min after exposure to 20% CFS, followed by a slow decline to basal levels. In contrast, 20% NHS did not cause an influx of Ca2+ and HHS produced an influx to about half that of CFS. Increasing concentrations (5-30%) of pooled NHS had no effect on HTE cell Ca2+ uptake or secretion, but pooled CFS and HHS caused progressive increases in Ca2+ influx and protein secretion from 10 to 25% sera. The CFS-induced Ca2+ influx and secretion were about twice those of HHS throughout the range of serum concentrations tested, suggesting the presence of a modulatory influence in HHS. When EGTA was used to chelate extracellular Ca2+ in the presence of CFS, Ca2+ influx was prevented and there was no stimulation of secretion. Ionophore A23187 allowed Ca2+ entry into HTE cells in the presence or absence of serum and a heightened level of secretory activity followed. The time course of Ca2+ influx under the influence of CFS was shown to correspond to the efflux of Na+ from the cells. Also verapamil, a Ca2+ channel blocking agent, inhibited CFS-induced Ca2+ influx by 50% at 10(-5)M and prevented secretion. Thus, it appears that CFS, but not NHS, contains an agent which stimulates Ca2+ uptake into HTE cells by means of a Ca2+ channel and/or Na+-Ca2+ exchange mechanism, and that increased intracellular Ca2+ levels then trigger secretion. The intermediate HTE cell response to HHS suggests that half of the CFS stimulatory agent is present as would be expected in a gene dose effect, lending support for a genetic basis for CF.

Topics: Adolescent; Adult; Calcimycin; Calcium; Calcium Radioisotopes; Cells, Cultured; Child; Child, Preschool; Cystic Fibrosis; Egtazic Acid; Epithelium; Humans; Infant; Sodium; Trachea; Verapamil

Ultrastructural and cytochemical observations indicate that both cystic fibrosis (CF) sera and calcium ionophore A23187 induce a swelling or an increase in the size and possibly the number of secondary lysosomes and an increase in mucus secretion in epithelium of the rabbit tracheal bioassay system. Extended incubation of the rabbit tracheal explants with either CF or control sera produces a cytotoxic effect on the tracheal epithelium, but only after the termination of the normal bioassay time period. Comparative ultrastructural study of the effect of both CF sera and calcium ionophore A23187 on the rabbit tracheal bioassay system indicates that increased membrane permeability to calcium may be important in the production of the ciliary dyskinesia response by CF serum factor(s) in the rabbit tracheal bioassay system.

Topics: Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Cilia; Cystic Fibrosis; Epithelium; Male; Rabbits; Trachea

Topics: Animals; Calcimycin; Calcium; Culture Techniques; Cystic Fibrosis; Epithelium; Heterozygote; Immunoglobulin G; Mucus; Rabbits; Trachea

An ionophore A23187-induced increase in membrane permeability to calcium ions in culture medium produced a rabbit tracheal mucociliary response indistinguishable from that caused by cystic fibrosis (CF) sera on three different occasions. Specific chelation of calcium ions with EGTA in the basal medium Eagle (BME) media with no additive or in native CF sera abolished the mucociliary disturbances in all cases. Increased membrane permeability to calcium may be important in the production of the mucociliary response by CF serum factor(s) in the tracheal assay system.

Topics: Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Cell Membrane Permeability; Cilia; Culture Techniques; Cystic Fibrosis; Egtazic Acid; Mucous Membrane; Osmolar Concentration; Rabbits; Trachea

Topics: Animals; Biological Assay; Calcimycin; Calcium; Cell Membrane Permeability; Cilia; Culture Techniques; Cystic Fibrosis; Humans; Rabbits; Trachea