Normal Airway Epithelial Cells Research Articles

Expression of gelatinase A, tissue inhibitor of metalloproteinases-2, matrilysin, and trypsin(ogen) in lung neoplasms: An immunohistochemical study

Lung cancer is a heterogeneous tumor in terms of clinical and biological behavior, and its aggressiveness depends on its invasive and metastatic properties. Matrix metalloproteinases and serine proteinases are believed to play a crucial role in invasion and metastasis of malignant tumor cells. In the present study, the authors evaluated inununohistochemically the expression of gelatinase A; tissue inhibitor of metalloproteinases—2 (TIMP-2), an inhibitor of gelatinase A; matrilysin; and trypsin(ogen) in 67 lung tumors from a variety of histological types including 17 squamous cell carcinomas, 16 adenocarcinomas, 15 small cell carcinomas, and 12 carcinoids. Interestingly, normal bronchial, bronchiolar, and alveolar epithelial cells expressed gelatinase A, TIMP-2, matrilysin, and trypsin(ogen) at varying frequencies and intensities. Bronchial smooth muscle cells and cartilage cells expressed gelatinase A alone, whereas endothelial cells, fibroblasts, and macrophages expressed gelatinase A and TIMP-2. Gelatinase A was expressed at high levels in most lung tumors examined (47% to 80%). TIMP-2 was also expressed at high levels except in the small cell carcinomas, which showed TIMP-2 expression at a lower frequency (60%) compared with other types of lung tumors (80% to 100%). Although matrilysin was expressed by tumor cells of all the histological types at various frequencies (13% to 63%), its expression was most common in adenocarcinomas. Expression of trypsin(ogen) was observed almost exclusively in adenocarcinomas (56%); other types of lung tumors expressed trypsin(ogen) far less frequently (0% to 12%). The present results, taken together with those of previous studies, suggest that gelatinase A is associated with malignant behavior of all the types of lung tumors, whereas its activity may be controlled by the endogenous inhibitor TIMP-2. The aggressive clinical behavior of small cell carcinoma may be attributable, at least in part, to a loss of the inhibitory effect of TIMP-2, as a significant proportion of these tumors showed negative or low levels of TIMP-2 expression. Matrilysin and trypsin(ogen) expressions are unlikely to be correlated with the aggressiveness of lung tumors. The expression of trypsin(ogen) may rather reflect the differentiation of adenocarcinoma cells toward normal airway epithelial cells.

C-type natriuretic peptide increases chloride permeability in normal and cystic fibrosis airway cells.

C-type natriuretic peptide (CNP), a hormone which stimulates particulate guanylate cyclase activity, was studied for its ability to stimulate chloride permeability through the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. Two cell lines, Calu-3 and CF-T43, were used as models of normal and cystic fibrosis (CF) airway epithelial cells, respectively. Calu-3 cells, derived from a lung carcinoma, express relatively high levels of wild-type CFTR. CF-T43 is a transformed line derived from a nasal polyp and expresses the mutant CFTR, deltaF508. Calu-3 cells exposed to the nucleotide guanosine-3',5'-monophosphate (cGMP) analogue 8-Br-cGMP exhibit increased 36Cl- efflux, demonstrating that cGMP can mediate changes in chloride permeability. CNP induces a bumetanide-sensitive short circuit current across Calu-3 monolayers. Whole-cell currents stimulated by CNP display linear current-voltage relationships and have inhibitor pharmacology and ion selectivity consistent with CFTR channel activity. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, and CNP both increase cGMP levels and short circuit current in Calu-3 cells. In contrast, exposure of CF-T43 cells to CNP resulted in an increased 36Cl- efflux rate only when combined with the adenylate cyclase agonist isoproterenol and the response was sensitive to kinase inhibitors. CF-T43 cells exposed to isoproterenol and SNP showed no increase in chloride efflux. Together, these data indicate that CNP can activate wild-type and mutant CFTR through a cAMP-dependent protein kinase pathway and that the sensitivity of Calu-3 cells for this stimulation is greater than that of the CF-T43 cells.

Migration of guinea pig airway epithelial cells in response to bombesin analogues.

Bombesin-like peptides within neuroepithelial cells elicit proliferation of normal and malignant airway epithelial cells. It is not clear that these peptides also elicit epithelial cell migration, a necessary component of airway repair after injury. We studied the effects of the bombesin analogues, gastrin releasing peptide (GRP) and neuromedin B (NMB), on guinea pig tracheal epithelial cell (GPTEC) migration. Primary GPTEC were allowed to migrate through 8-microm-pore gelatin-coated filters for 6 h in a chemotaxis chamber, after which the number of migrated cells per 10 high power fields (10 hpf) were counted. Both neuropeptides elicited migration of GPTEC: 24.8 +/- 4.5 cells for 10(-11) M NMB (P < 0.001 versus control, n = 4) and 16.8 +/- 1.2 cells for 10(-12) M GRP (P < 0.001 versus control, n = 8). Migration was attenuated substantially by a bombesin receptor antagonist. To investigate further the relationship of migration through a filter to the repair of a damaged epithelium, we studied the repair of epithelial cells by video microscopy. A 0.3- to 0.5-microm2 wound was created in a confluent monolayer of GPTEC, and wound closure was followed over 24 h. There was no significant acceleration in the rate of repair of GRP- or NMB-stimulated monolayers compared to control. These data demonstrate that GRP and NMB elicit migration of airway epithelial cells but may not play a significant role in the early repair of the airway epithelium in culture.

Airway epithelial cells produce stem cell factor

Airway epithelial cells modulate the inflammatory response in asthmatic, allergic and fibrotic lung diseases through the secretion of cytokines that regulate the movement and activation of inflammatory cells. Mast cells play an important role in the pathogenesis of these lung diseases. In this study we report that normal airway epithelial cells express stem cell factor which is a critical mediator of mast cell growth and differentiation and that transforming growth factor-β inhibits secretion of stem cell factor by airway epithelial cells.

N-Acetyl-L-cysteine and its derivatives activate a Cl- conductance in epithelial cells.

N-Acetyl-L-cysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders including cystic fibrosis (CF). The beneficial effects of NAC are empirical and the exact mechanism of action in the airways remains obscure. In the present study we examined the effects on whole-cell (wc) conductance (Gm) and voltage (Vm) of NAC and the congeners S-carboxymethyl-L-cysteine (CMC) and S-carbamyl-L-cysteine (CAC) and L-cysteine in normal and CF airway epithelial cells. L-Cysteine (1 mmol/l) had no detectable effect. The increase in Gm (delta Gm) by the other compounds was concentration dependent and was (all substances at 1 mmol/l) 3.8 +/- 1.4 nS (NAC; n = 11), 4.2 +/- 1.0 nS (CMC; n = 16) and 3.8 +/- 1.6 nS (CAC; n = 18), respectively. The changes in Gm were paralleled by an increased depolarization (delta Vm) when extracellular Cl- concentration was reduced to 34 mmol/l: under control conditions = -4.1 +/- 2.1 versus 10.2 +/- 2.1 mV in the presence of NAC, CMC, CAC (n = 36). In the presence of NAC, CMC and CAC, the reduction in Cl- concentration was paralleled by a reduction of Gm by 2.1 +/- 0.4 nS (n = 35), indicating that all substances acted by increasing the Cl- conductance. Analysis of intracellular pH did not reveal any changes by any of the compounds (1 mmol/l). A Cl- conductance was also activated in HT29 colonic carcinoma and CF tracheal epithelial (CFDE) cells but not in CFPAC-1 cells, which do not express detectable levels of delta F508-CFTR, suggesting that the presence of CFTR may be a prerequisite for the induction of Cl- currents. Next we examined the ion currents in Xenopus oocytes microinjected with CFTR-cRNA. Water-injected oocytes did not respond to activation by forskolin and 3-isobutyl-1-methylxanthine (IBMX) (delta Gm = 0.08 +/- 0.04 microS; n = 10) and no current was activated when these oocytes were exposed to NAC or CMC. In contrast, in CFTR-cRNA-injected oocytes Gm was enhanced when intracellular adenosine 3',5'-cyclic monophosphate (cAMP) was increased by forskolin and IBMX (Gm = 4.5 +/- 1.3 microS; n = 8). Gm was significantly increased by 0.74 +/- 0.2 microS (n = 11) and 0.46 +/- 0.1 microS (n = 10) when oocytes were exposed to NAC and CMC, respectively (both 1 mmol/l). In conclusion, NAC and its congeners activate Cl- conductances in normal and CF airway epithelial cells and hence induce electrolyte secretion which may be beneficial in CF patients. CFTR appears to be required for this response in an as yet unknown fashion.

Evidence for impaired retinoic acid receptor-thyroid hormone receptor AF-2 cofactor activity in human lung cancer.

Retinoic acid (RA) is required for normal airway epithelial cell growth and differentiation both in vivo and in vitro. One of the earliest events following the exposure of bronchial epithelial cells to RA is the strong induction of RA receptor beta (RAR beta) mRNA. Previous work established that many lung cancer cell lines and primary tumors display abnormal RAR beta mRNA expression, most often absence or weak expression of the RAR beta 2 isoform, even after RA treatment. Restoration of RAR beta 2 into RAR beta-negative lung cancer cell lines has been reported to inhibit tumorigenicity. Since RAR beta 2 inactivation may contribute to lung cancer, we have investigated the molecular mechanism of defective RAR beta 2 expression. Nuclear run-on assays and transient transfections with RAR beta 2 promoter constructs indicate the presence of trans-acting transcriptional defects in most lung cancer cell lines, which map to the RA response element (RARE). These defects cannot be complemented by RAR-retinoid X receptor cotransfection and can be separated into two types: (i) one affecting transcription from direct repeat RAREs, but not palindromic RAREs, and (ii) another affecting transcription from both types of RARE. Studies using chimeras between RAR alpha, TR alpha, and other transcription factors suggest the existence of novel RAR-thyroid hormone receptor AF-2-specific cofactors, which are necessary for high levels of transcription. Furthermore, these factors may be frequently inactivated in human lung cancer.

Identification and function of A1 adenosine receptors in normal and cystic fibrosis human airway epithelial cells

A role for adenosine in the regulation of ion transport in pulmonary epithelial cells has recently been proposed. Although evidence exists documenting the presence and function of adenosine A2 receptors in airway epithelia, the presence of adenosine A1 receptors remains controversial. The present study used reverse transcriptase-polymerase chain reaction (PCR) and whole cell patch-clamp analysis to investigate A1 receptor presence and function in normal and cystic fibrosis (CF) human airway epithelial cells. Oligonucleotide primers complementary to the human brain A1 receptor sequence generated a PCR product of the predicted size (311 bp) in normal tracheal (9HTEo-) and CF submucosal (2CFSMEo-) airway cell lines and in primary cultures of CF nasal polyp epithelial cells. An oligonucleotide probe internal to the PCR primers hybridized with the 311-bp cDNAs by Southern blot analysis. cDNA sequencing demonstrated that the normal and CF airway cell PCR products are 100% identical to the corresponding sequence of the human brain adenosine A1 receptor. Northern blot analysis of 9HTEo-and 2CFSMEo- poly(A)+ RNA revealed the presence of two bands of approximately 3.0 and approximately 5.5 kb corresponding to the A1 receptor. Whole cell patch-clamp analyses demonstrated that 8-cyclopentyl-1,3-dipropylxanthine, a specific A1 receptor antagonist, increases adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance in 9HTEo-airway cells and allows cAMP to increase Cl- conductance in 2CFSMEo- CF airway cells and CF nasal polyp epithelial cells in primary culture. These results provide evidence for the presence and function of A1 receptors in normal and CF airway epithelial cells and provide support for a role of adenosine A1 receptors in modulating airway epithelial cell Cl- transport.

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

Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.

Heterotrimeric G proteins, vesicle trafficking, and CFTR Cl- channels

Previously (E.M. Schwiebert, N. Kizer, D. C. Gruenert, and B. A. Stanton, Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992), we showed that heterotrimeric G proteins regulate adenosine 3',5'-cyclic monophosphate (cAMP)-activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in human airway epithelial cells. The goal of the present study was to test the hypothesis that heterotrimeric G proteins regulate vesicle trafficking and exocytosis and that these events are critical for cAMP activation of CFTR-mediated Cl- secretion. We report that cAMP stimulates exocytosis and CFTR Cl- conductance (GCl) in normal but not in CF cells. Stimulation of the heterotrimeric G protein G alpha i-2 inhibited cAMP-activated CFTR GCl and exocytosis in normal cells. In contrast, inhibition of G alpha i-2 stimulated exocytosis and allowed cAMP to stimulate CFTR GCl in cells isolated from patients with cystic fibrosis (CF). Brefeldin A and nocodazol prevented cAMP-induced exocytosis and also blocked cAMP stimulation of CFTR GCl in normal airway epithelial cells. Our studies suggest that the heterotrimeric G protein G alpha i-2 regulates CFTR GCl in human airway epithelial cells by modulating vesicle trafficking and the delivery of CFTR Cl- channels from an intracellular vesicular pool to the plasma membrane. Inhibition of G alpha i-2 may be a useful therapeutic approach to target mutant delta F508 CFTR Cl- channels from an intracellular vesicular pool to the plasma membrane and thereby correct defective Cl- secretion in CF airway epithelial cells.

ATP and A1 adenosine receptor agonists mobilize intracellular calcium and activate K+ and Cl- currents in normal and cystic fibrosis airway epithelial cells.

The effect of purinergic compounds on [Ca2+]i and membrane currents of cell lines derived from the airway epithelium of normal and cystic fibrosis individuals has been investigated. 2-Chloroadenosine (2-CADO), as well as other agonists of the A1 adenosine receptors, causes a transient elevation of cytosolic [Ca2+] that is antagonized by the A1 adenosine receptor antagonist 8-cyclopentyl-1,3 dipropylxanthine (DPCPX). ATP is also effective, but at a lower extent. The [Ca2+]i increase induced by 2-CADO and ATP is abolished by preincubation with phorbol 12-myristate 13-acetate and the Ca(2+)-ATPase inhibitor thapsigargin. This latter result suggests that purinergic agonists mobilize Ca2+ from inositol 1,4,5-trisphosphate-sensitive stores. Pertussis toxin completely inhibits the effect of 2-CADO, whereas only it partially affects that of ATP, suggesting the involvement of different types of G proteins. Perforated patch clamp experiments carried out in both current clamp and voltage clamp modes show that 2-CADO and ATP activate K(+)- and Cl(-)-selective membrane currents, with a mechanism inhibited by preincubation with DPCPX and thapsigargin. These data indicate that activation of adenosine A1 receptor, in a similar way to ATP receptor, causes [Ca2+]i increase and ion channels activation through a transduction mechanism that is not impaired in cystic fibrosis airway epithelial cells.

Protein phosphorylation responses in normal and cystic fibrosis airway epithelial cell lines.

Monolayers of SV-40 immortalized human airway epithelial cell lines were stimulated with bradykinin and isoproterenol to study protein phosphorylation responses that accompany ion transport regulation in normal (BEAS) and cystic fibrosis (CF/T43) cells. Phosphorylation responses were analyzed by two-dimensional gel electrophoresis of postmicrosomal supernatant fractions of 32Pi-labeled cells. Isoproterenol increased the labeling of three phosphoproteins of M(r) 17,000, 18,000, and 37,000 that were equivalent to proteins known to undergo cAMP-dependent phosphorylation in T84 cell monolayers. Distinct proteins showed increased phosphorylation with bradykinin, including acidic proteins of M(r) 15,000 and 29,000. These resembled proteins exhibiting Ca(2+)-dependent phosphorylation T84 cells. The CF/T43 and BEAS cell protein phosphorylation responses were indistinguishable. These findings support the concept that the regulation and function of protein kinase A is normal in cystic fibrosis airway epithelia and that the abnormal cAMP-mediated regulation of chloride permeability in these cells is due to altered regulatory or effector proteins.

Relationship of Secretory Granule Content and Proliferative Intensity in the Secretory Compartment of the Hamster Bronchial Epithelium

We have previously shown that normal hamster airway epithelial secretory cells have a lower proliferative intensity than basal cells, but because of their high frequency are a major contributor to cell renewal (Am. J. Respir. Cell Mol. Biol. 1990; 2:51-58). In the present experiment, the relation between proliferative intensity and secretory granule content in bronchial epithelial cells is studied. [3H]thymidine (2 microCi/g wt) was given intraperitoneally, 1 h before killing, to 5 hamsters treated 21 days earlier with intratracheal saline and to six hamsters in which secretory cell metaplasia had been induced by intratracheal treatment with 300 micrograms of human neutrophil elastase given 21 days earlier. Light microscopic autoradiograms were prepared from 2-microns-thick Epon sections of left intrapulmonary hilar bronchi. Cells were categorized as basal, ciliated, secretory, or indeterminate. Secretory cells were classified as either: S1, with 0 to 4 granules; S2, with > or = 5 granules with intervening cytoplasm; or S3, with abundant granules and no apparent supranuclear cytoplasm. Proliferative intensity was defined by the categorical labeling index (LIc) at 1 h after [3H]thymidine injection. LIc was determined by the number of labeled cells in a category as the percent of labeled and unlabeled cells of that category. LIc of each of the cell categories were similar in the elastase and saline groups. LIc was highest for basal cells, reflecting their proliferative intensity, and lowest for ciliated cells. In the saline group, LIc of S1 (0.25%) was significantly higher compared with S2 (0.13%); S3 cells were rare (0.2%) and none were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)

GTP-binding proteins inhibit cAMP activation of chloride channels in cystic fibrosis airway epithelial cells.

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl- secretion by airway epithelial cells. In CF, cAMP does not activate Cl- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[beta-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl- currents and restore cAMP-activated Cl- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[gamma-thio]triphosphate and AlF4- reduce Cl- currents and inhibit cAMP from activating Cl- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[beta-thio]diphosphate and in normal cells, cAMP activates a Cl- conductance that has properties similar to CF transmembrane-conductance regulator Cl- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl- secretion in CF.

Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium.

Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.

Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells.

Using whole cell patch-clamp and perforated patch recording techniques on human cystic fibrosis (CF) and non-CF airway epithelial cells, we sought to determine whether a single Cl- conductance (GCl) could be modulated via different regulatory pathways or whether multiple conductances could be identified. Cl- current in both CF and non-CF cells was activated by cellular swelling as well as by an elevation in intracellular calcium ([Ca2+]i). While the adenosine 3',5'-cyclic monophosphate (cAMP)-activated GCl was absent in CF cells, its activation in non-CF cells was only observed in the perforated patch configuration at lower temperatures (24 degrees C) or infrequently in the whole cell configuration at elevated temperatures (33 degrees C). Currents activated by all three regulatory pathways were sensitive to the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Further increases in current activation could be produced by cellular swelling after maximal Ca2+ or cAMP-induced current activation. Intracellular application of a peptide inhibitor of Ca(2+)-calmodulin-dependent protein kinase selectively blocked the Ca(2+)-dependent current activation while leaving the swelling-induced current increase intact. These results are consistent with the presence of multiple anion conductances in both CF and non-CF airway cells. The heterogeneity of the responses to the three regulatory stimuli, however, prevented the correlation of a specific anion conductance with a separate modulatory pathway based on characteristic voltage-dependent kinetics and conductance.

Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.

The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia.

Alpha 1-adrenergic signaling in human airway epithelial cells involves inositol lipid and phosphate metabolism.

A role for phospholipase C (PLC) hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) as a mechanism of alpha 1-adrenergic signal transduction in human airway epithelial cells (AEC) was investigated in isolated normal tracheal and cystic fibrosis (CF) nasal epithelial cells grown in in vitro culture and prelabeled with 3 muCi myo-[3H]inositol/ml for 72 h. Breakdown of polyphosphoinositides was measured using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and PIP2. Inositol phosphates were separated by ion-exchange column chromatography. In normal AEC, the addition of the endogenous catecholamine l-epinephrine produced a rapid, transient accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2) and breakdown of PIP and PIP2. IP3 increased 1.7-fold and IP2 1.6-fold after 20 and 40 s, respectively. A maximal decrease of 35% PIP2 and 30% PIP is observed after 20 and 40 s, respectively. The effects of l-epinephrine were not blocked by the beta-adrenergic antagonist dl-propranolol but were mimicked by the alpha 1-adrenergic agonist methoxamine. Prazosin, an alpha 1-adrenergic antagonist, and pertussis toxin (PTX) blocked the effects of l-epinephrine and methoxamine. Addition of l-epinephrine and methoxamine to CF nasal epithelial cells also induced prazosin-sensitive polyphosphoinositide breakdown and inositol phosphate accumulation. A 2.2-fold accumulation of IP3 was observed after 10 s and 2.0-fold increase in IP2 after 20 s. Maximal decreases of 32% PIP2 and 23% PIP levels were observed after 20-s incubation with l-epinephrine. PTX reduced the effects of l-epinephrine and significantly blocked the effects of methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characterization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells.

Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.

Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.

Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase

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.