Overexpression of eCLCA1 in Small Airways of Horses with Recurrent Airway Obstruction
Department of Pathology, School of Veterinary Medicine Hannover, Hannover, Germany (FA,IL,LM,ADG), and Departments of Ophthalmology & Visual Sciences and Pharmacology, University of Nebraska Medical Center, Durham Research Center, Omaha, Nebraska (WBT)
Correspondence to: Achim D. Gruber, Department of Veterinary Pathology, Free University Berlin, Robert-von-Ostertag-Str. 15, 14163 Berlin, Germany. E-mail: gruber.achim{at}vetmed.fu-berlin.de
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Summary |
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Key Words: asthma chronic obstructive pulmonary disease calcium-activated chloride channels goblet cells mucus overproduction
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Introduction |
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Recurrent airway obstruction (RAO; COPD, heaves) in horses is the only common spontaneous disease in animals with high clinical, functional, and pathological similarities to human asthma and COPD (Snapper 1986; Bice et al. 2000
). The key mechanism is thought to be based on allergen-induced hypersensitivity with Th2 cytokinemediated chronic airway pathology including goblet cell metaplasia in small bronchioles with massive mucus overproduction (Leguillette 2003
; Davis and Rush 2002
). Importantly, equine RAO can be induced experimentally by environmental challenge under standardized conditions (Gerber et al. 2004
). Thus equine RAO is regarded as a valuable model for human asthma and COPD (Snapper 1986
; Bice et al. 2000
).
In this study, we have identified, cloned, and characterized the first equine member of the CLCA gene family, here designated as eCLCA1. Specific antibodies directed against synthetic peptides were used to biochemically characterize the protein in vitro and in vivo and to systematically immunolocalize the protein in equine tissues. The eCLCA1 mRNA and protein are strongly upregulated in bronchioles of horses with RAO, extending the use of equine RAO as a model for human asthma and COPD.
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Materials and Methods |
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Cloning of the eCLCA1 cDNA
Total RNA was extracted from the rectal mucosa of eight unrelated warm-blooded horses using the Trizol method (Invitrogen; Carlsbad, CA) and poly-A+ mRNA was purified using the Nucleotrap mRNA purification system (Macherey-Nagel; Duren, Germany). One µg of each poly (A)+ mRNA was reverse transcribed using Omniscript reverse transcriptase (Qiagen; Hilden, Germany) for 30 min at 37C in a 40-µl reaction volume using random hexamer priming (Random Primers; Promega, Madison, WI) in the presence of 100 units of RNase inhibitor (RNase OUT; Invitrogen). Sixteen different PCR primers were designed and synthesized from cDNA regions conserved between the human hCLCA1 and the murine mCLCA3 cDNA sequences (GenBank accession numbers NM001285 and NM017474, respectively). A pilot study was conducted to select a set of 2 primers from these 16 primers that yielded the best amplification results using the equine cDNA pool as template. The sequences of the primers ultimately used to generate a fragment of the homologous equine CLCA cDNA were 5'-CACATAAAGGACATGGTGAC-3' (upstream) and 5'-GTGACTCCTCCCAGAGCCC-3' (downstream), generating a 1894 base-pairs product from both the murine and equine CLCA homologs. Taq polymerase (Promega) was used for PCR (0.5 units per 50 µl reaction), and PCR conditions were 35 cycles at 95C for 2 min, 55.5C for 40 sec, and 72C for 2 min with a time increment of 3 sec per cycle and a final extension at 72C for 10 min. The bulk amplification product was sequenced (SeqLab; Göttingen, Germany), and three internal PCR primer sequences chosen to perform 5'- and 3'- rapid amplification of cDNA ends (RACE; GeneRacer Kit, Invitrogen) to obtain the entire cDNA sequence. The PCR primer sequences used for RACE were 5'-CATCGTAGATGAGCCCATTCGTGG-3' (downstream nested primer for 5'-RACE), 5'-CTTCCCTTGTGGTCCATATTCATCCAACC-3' (downstream primer for 5'-RACE), and 5'-GATTCACCAAGGAGGCTTACCAATTCTCAGG-3' (upstream primer for 3'-RACE). The open reading frame (ORF) comprising 2742 base pairs was selectively amplified using Pwo proof reading activity DNA polymerase (PeqLab Biotechnology; Erlangen, Germany) and NotI-linked primers flanking the ORF (upstream 5'-TAGCGGCCGCGATGGGGTCATTTAAGAGTTCTGT-3'; downstream 5'-TAGCGGCCGCCTCAGCCCAAGGCTACTGAC-3' with NotI sites underlined). PCR conditions were 94.5C for 40 sec, 70C for 40 sec, and 72C for 2 min for 35 cycles with a final extension at 72C for 10 min. The bulk amplification product was sequenced, digested with NotI and cloned into the NotI site of the expression vector pcDNA3.1 (Invitrogen). Three individual clones were sequenced to exclude PCR-induced sequence errors. Single nucleotide polymorphisms were identified in 14 unrelated horses by sequencing three separate bulk RT-PCR products derived from each horse.
Sequence Analyses
Nucleic acid and protein sequence analyses were performed using the DNAStar software package version 5.0 (Lasergene; Madison, WI).
Northern Blot Hybridization
Poly (A)+ RNA from murine colon, equine colon, equine normal lung, and equine lung with recurrent airway obstruction was extracted as described previously, electrophoresed (1 µg/lane) on a denaturing formaldehyde gel, blotted onto nitrocellulose, and hybridized with a (
-32P)dCTP nick-labeled (RTS RadPrime; Life Technologies, Gaithersburg, MD) cDNA probe corresponding to the ORF of mCLCA3 or a probe corresponding to the ORF of the newly cloned equine homolog. The same samples were also hybridized with a probe corresponding to the ORF of the housekeeping gene EF-1a (Schmidbauer et al. 2004
) to control for RNA degradation and loading amounts. Two stringent washes were performed with 2x saline sodium citrate, 0.1% SDS at 55C for 20 min, followed by two washes with 0.1x saline sodium citrate, 0.1% SDS at 55C for 20 min. Autoradiographs were exposed to film using an intensifying screen at 70C.
Generation of Antibodies
Regions of predicted high immunogenicity were selected from the eCLCA1 polypeptide using computer-aided antigenicity analysis. Two oligopeptides were synthesized (eCa, corresponding to amino acids 8195: VPENWKTKPEYERPK, and eCb, corresponding to amino acids 278292: DSEDFKKTTPMTAQP), conjugated to keyhole limpet hemocyanin and used for standard immunization of two rabbits each. Preimmune sera were collected before immunization and used as controls in the immunodetection experiments. The four antisera were designated -eCa1,
-eCa2,
-eCb1, and
-eCb2. The immune sera were affinity immunopurified using the respective peptides coupled to an EAH-Sepharose column.
Native Tissue Sample Preparation and Immunoblotting
Fresh tissue samples from equine ileum and colon mucosa were lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 0.5% Triton-X-100, and 0.5% sodium desoxycholate in the presence of protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 1 µg/ml pepstatin, 5 µg/ml leupeptin, 5 µg/ml antipain, and 1 µg/ml aprotinin). SDS-PAGE (10%) was performed in the presence of 2 mM dithiothreitol following standard protocols. After electroblotting onto nitrocellulose membranes and blocking of the membranes with TBS containing 0.1% Tween 20 and 5% nonfat milk, membranes were probed at 4C overnight with the immunopurified antibodies or with the preimmune sera diluted in blocking buffer (dilutions ranging from 1:500 to 1:4,000). Membranes were then incubated with horseradish peroxidaseconjugated swine anti-rabbit immunoglobulins (0.4 µg/ml; Dako, Hamburg, Germany) and developed using enhanced chemiluminescence (Amersham).
In Vitro Translation
The eCLCA1 ORF cloned into pcDNA3.1 was transcribed and translated with the TNT T7 Coupled Reticulocyte Lysate System (Promega). Reactions of 25 µl were carried out at 30C for 90 min without or with (2 µl) canine pancreatic microsomal membranes (Promega). Samples were analyzed by 10% SDS-PAGE and immunoblotting as described in the following section.
Electrophysiology
The eCLCA1/v1 or eCLCA1/v2 cDNAs were cotransfected with pEGFP-N1 into HEK293 cells plated on cover slips using GenePorter transfection reagent (Gene Therapy Systems, San Diego, CA). CLCA-expressing cells were identified by EGFP fluorescence 1224 hr after transfection. Whole cell recordings were obtained using patch electrodes pulled from borosilicate pipettes (1.2 mm outer diameter, 0.95 mm inner diameter, with internal filament) using a Narishige PP-830 vertical puller. The recording pipettes had tips of 1.5 µm outer diameter (R = 812 M
) and were filled with a solution containing (in mM): 98 KCH3SO4, 44 KCl, 3 NaCl, 5 HEPES, 3 MgCl2, 1 CaCl2, 3 EGTA, 2 glucose, 1 Mg-ATP, 1 GTP, and 1 reduced glutathione (pH 7.8). Free [Ca2+] in this solution was estimated to be 57 nM using MaxChelator. Cells were voltage clamped at 50 mV using an Axopatch 200B amplifier (Axon Instruments; Foster City, CA). Test pulses were applied and currents acquired using PClamp 8.2 with a Digidata 1322 interface (Axon Instruments). During recording, cells were perfused at room temperature using a single-pass, gravity-feed perfusion system (1 ml/min) with an oxygenated medium containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4). Ionomycin (10 µM) and niflumic acid (100 µM) were diluted 1:10,000 into this solution from stock solutions prepared in DMSO. Experiments were conducted at room temperature. All chemicals were obtained from Sigma Chemicals (St Louis, MO) except KCH3SO4, which was obtained from Pfaltz and Bauer (Waterbury, CT).
Immunohistochemistry
Fresh tissue samples from three adult healthy horses and three adult horses with RAO were fixed in 4% neutral-buffered formaldehyde and routinely embedded in paraffin. The following organs and tissues were processed: lung (four different locations: cranial right lobe and cranial, middle, and caudal region of right main lobe), nasal cavity, trachea, liver, spleen, kidneys, renal pelvis, urinary bladder, heart, adrenal glands, thyroid glands, ovaries, oviducts, uterus, cervix, vagina, mammary glands, testes, epididymides, pancreas, parotid salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, ascending colon, descending colon, rectum, lymph nodes, brain (cortex, cerebellum, stem, medulla), eyes, skin, adipose tissue, skeletal muscle, bone, and aorta. Paraffin-embedded tissues were cut at 3 µm and mounted on SuperfrostPlus adhesive glass slides (Menzel-Gläser; Braunschweig, Germany). In addition to the immunohistochemical analyses, consecutive tissue sections were routinely stained with hematoxylin and eosin for histological examination and with periodic acidSchiff (PAS) reaction to stain the mucins. The avidin-biotin-peroxidase complex (ABC) method was applied for immunohistochemical staining. After dewaxing the mounted tissue sections in xylene and rehydration in isopropanol and graded ethanol, the following antigen retrieval methods were tested: (a) 15 min microwave heating (700 W) in 10 mM citric acid pH 6.0 or (b) 20 min treatment with 0.05% pronase E (Merck) in PBS at 37C. Because of superior results of the pronase E-pretreatment, method (b) was used for the systematic tissue analyses. Endogenous peroxidase activity was inhibited by incubating the slides with 85% ethanol containing 0.5% H2O2, followed by washes in PBS containing 0.05% Tween 20 (PBS/Tween 20) and blocking in PBS/Tween 20 containing 20% heat-inactivated normal goat serum. After repeated washes, the sections were incubated with the purified antibodies or the respective preimmune sera in PBS/Tween 20 containing 1% BSA (dilutions ranging from 1:500 to 1:10,000) in a humid chamber at 4C overnight. Sections were washed in PBS/Tween 20 and incubated at room temperature for 30 min with biotinylated goat anti-rabbit immunoglobulins (5 µg/ml; Vector Laboratories) diluted in PBS/Tween 20, followed by repeated washes in PBS/Tween. Color was developed for 30 min using freshly prepared ABC solution (Vectastain Elite ABC Kit; Vector Laboratories) diluted in PBS, followed by repeated washes in PBS and rinsing in tap water. Diaminobenzidine was used as substrate for color development. The slides were counterstained with hematoxylin, dehydrated through graded ethanol, cleared in xylene, and cover slipped.
Quantitative Real-time RT-PCR
Total RNA was extracted (Trizol method; Invitrogen) from equine tissues, digested with RQ1 RNase free DNase (Promega), purified using a silica gelbased membrane (RNeasy Mini; Qiagen), and reverse transcribed as described previously. All primers and probes used for RT quantitative PCR were designed using the Beacon Designer 2 software (Premier Biosoft International; Palo Alto, CA) and purchased from PE Biosystems (Weiterstadt, Germany). Primer sequences were 5'-AAACACTCATTCAACAAATAAAGGA-3' (upstream) and 5'-TTGGTCTCTCATACTCAGGTTT-3' (downstream). The corresponding Taqman probe sequence was 5'-FAM-CCCAGGCATCTCCATATCTCTTTGAAGC-TAMRA-3'. To correct for variations of RNA amounts and cDNA synthesis efficacy in subsequent quantitative PCR assays, primers and a probe for the detection of a fragment of the equine housekeeping gene elongation factor-1a (EF-1a; Schmidbauer et al. 2004) were generated (upstream 5'-CAAAAACGACCCACCAATGG-3', downstream 5'-GGCCTGGATGGTTCAGGATA-3'; probe 5'-FAM-AGCAGCTGGCTTCACTGCTCAGGTG-TAMRA-3') and used in parallel in all experiments. Calculated melting temperatures of all primers and probes ranged between 55.0C and 61.2C (eCLCA1) and between 60.0C and 62.4C (EF-1a), respectively. Amplicon sizes were 145 bp (eCLCA1) and 98 bp (EF-1a). Real-time RT quantitative PCR and data analysis were performed using the Mx4000 Multiplex Quantitative PCR System (Stratagene; La Jolla, CA). The reactions were carried out in MicroAmp Optical 96-well plates covered with MicroAmp Optical caps (Stratagene). In addition to the cDNA samples, 10-fold serial dilutions of cloned eCLCA1 cDNA samples (open reading frame cloned into pcDNA3.1) ranging from 108 to 102 copies per sample were used as templates to generate standard curves for estimation of copy numbers on each plate. Standard curves for EF-1a cDNA copy numbers were generated as described earlier (Schmidbauer et al. 2004
). The plates contained triplicates of each cDNA sample, duplicates of serially diluted control samples for the standard curves, and a duplicate no-template control. During initial optimization runs, the exact composition of the PCR reaction mix (Brilliant QPCR Core Reagent Kit; Stratagene) and the PCR time and temperature conditions were determined using the serially diluted cloned cDNA templates and random tissue cDNA templates. Optimized 25-µl reactions contained template corresponding to 12.5 ng of total RNA, 1x core PCR buffer, 200 µM of each dNTP, 5.6 mM MgCl2, 300 nM of the respective primers, 200 nM of the respective probe, 80 nM Rox as reference dye, and 0.625 U AmpliTaq Gold polymerase (PE Biosystems). Optimized thermal cycling conditions were 10 min at 95C, followed by 42 cycles of 15 sec at 95C and 1 min at 55C. Results were quantified using the analysis software of the Mx4000 Multiplex Quantitative PCR System applying the adaptive baseline and moving average algorithm enhancements. For each sample, the cycle threshold value (Ct value) was calculated based on the normalized baseline corrected fluorescence (
Rn).
Quantitation of Target Gene Expression
For each sample analyzed, the mean Ct value based on the results of all experiments was given together with the corresponding standard deviation (SD). cDNA copy numbers (means and SD) were calculated on the basis of the results of the standard curve of the same run (correlation coefficients were always higher than 0.95). The eCLCA1 cDNA copy numbers (means and SD) were then normalized using the calculated EF-1a cDNA copy number of the same sample. This number was obtained by applying the respective Ct value for EF-1a in the standard EF-1a dilution curve (EF-1a copy number = 10(Ct EF-1a 37.578)/3.2454); correlation coefficient = 0.99). The curve was generated by plotting the average EF-1a Ct values from three different experiments, each run with duplicate samples and separate serial dilutions of EF-1a DNA.
Statistical Analyses
For each cDNA sample analyzed, arithmetic mean, SD, and coefficient of variation of the results of three different experiments were calculated.
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Results |
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Single Nucleotide Polymorphisms
Sequencing cDNA clones derived from 14 different, unrelated horses revealed three SNPs at nucleotide positions 1518 (G/A), 1533 (C/G), and 1837 (G/C), respectively. Seven horses were heterozygous at all three positions (Figure 3B)
, and seven horses were homozygous at all three positions (three and four horses for each genotype, respectively, Figures 3A and 3C), suggesting the existence of two distinct eCLCA1 alleles, namely 1518G/1533C/1837G (designated eCLCA1/v1) and 1518A/1533G/1837C (designated eCLCA1/v2). The SNPs 1518 (G/A) and 1533 (C/G) result in amino acid changes (485 H/R and 490 V/L, respectively) whereas the SNP 1837 (G/C) is silent (587 T), with no alteration of the amino acid sequence. Both amino acid changes result in only minor size differences of the amino acid residues, but no alteration of charge properties with histidine and arginine both possessing basic side chains and valine and leucine both possessing nonpolar side chains.
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Discussion |
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As the first member among the CLCA family of genes, three SNPs were identified in the coding region of the eCLCA1 mRNA. Only two of these (485 His/Arg and 490 Val/Leu) result in amino acid changes. However, both changes result only in minor size differences of the amino acid residues, but no alteration of charge properties with histidine and arginine both possessing basic side chains and valine and leucine, both possessing nonpolar side chains. No differences were noted in the electrophysiological properties of the two variants after heterologous expression in this study. Nevertheless, whether the allelic variations are associated with altered protein structure or function in vivo cannot be predicted based on these experiments and will have to be addressed in future studies. Interestingly, the two nucleotide combinations, 1518G/1533C/1837G and 1518A/1533G/1837C, were invariably found in all 14 horses, suggesting the existence of only two distinct genomic alleles. This observation could be explained by the close chromosomal proximity of the SNPs compared with the hCLCA1 genomic structure (Gruber et al. 1998), making crossover events unlikely. The equal allelic distribution in 14 unrelated horses suggests that these SNPs could be useful as highly informative markers for genetic studies. We have recently shown that certain allelic variations in the human CLCA gene locus are significantly associated with the severity of the intestinal disease phenotype in cystic fibrosis patients (Ritzka et al. 2004
), raising the question of whether a genetic analysis for different eCLCA SNPs or alleles may be of diagnostic or prognostic value for RAO in horses. Clearly, this issue has to be addressed in a larger population study in horses that may even be of comparative significance for asthma and COPD in humans.
The tissue and cellular expression patterns of the eCLCA1 protein are similar but not identical with the mCLCA3 protein in the mouse. Identical immunohistochemical staining patterns derived from antibodies against two distinct eCLCA1 synthetic peptides confirmed specificity of the results. Importantly, expression in respiratory goblet cells and glands and in intestinal goblet cells appears to be identical with the expression pattern of mCLCA3. However, detection of eCLCA1 in tubular sweat glands in the equine skin is at variance with observations in the mouse, in which mCLCA3 was not detected in the skin (Leverkoehne and Gruber 2002). This difference can clearly be explained by species-specific physiology and anatomy: tubular sweat glands in all parts of the skin and the ability to sweat via the entire skin surface are limited to humans and horses but not present in any other domestic or laboratory animal species (Talukdar et al. 1972
; Banks 1993
). Thus expression of eCLCA1 in the equine sweat glands may be of comparative importance for human skin. The eCLCA1 protein was also found in epithelial cells of the renal papilla, which does not express mCLCA3 in the mouse (Leverkoehne and Gruber 2002
). Likewise, this discrepancy can be explained by anatomic species differences because the renal papilla contains mucous glands only in equids but not in mice or humans (Dellmann and Eurell 1998
). Expression of eCLCA1 was without exception associated with PAS-positive, mucin-producing cells, strongly suggesting an essential role in mucin synthesis, condensation, or secretion. We have previously shown by immune electron microscopy that the mCLCA3 protein is located in the mucin granule membrane of goblet cells, where it may be involved in the acidification of the granule content, a critical process in the packing and secretion of mucins (Leverkoehne and Gruber 2002
). The data obtained here for eCLCA1 would be consistent with a similar function in horses.
A detailed comparison with the cellular distribution pattern of the human hCLCA1 is impossible at this point because its expression pattern has not yet been systematically studied. However, based on the mRNA data available for hCLCA1, the cellular expression patterns for eCLCA1 and hCLCA1 seem to be identical in the respiratory and intestinal tracts (Gruber et al. 1998; Hoshino et al. 2002
), particularly in the scenario of chronic inflammatory airway obstruction. No polymorphisms have been reported for the hCLCA1 protein so far. When the observed allelic amino acid polymorphisms of eCLCA1 are compared with the corresponding regions of the hCLCA1 protein, the first polymorphism of two basic side chains (485 His/Arg) correlates with a basic amino acid (485 Arg) in the hCLCA1 sequence (Gruber et al. 1998
), suggesting strong conservation of charge. However, the second polymorphism in eCLCA1 of two nonpolar residues (490 Val/Leu) aligns with a glutamic acid residue in hCLCA1 (490 Glu) and in bCLCA1, mCLCA1, and mCLCA3 (Gruber et al. 1998
), indicating some variance between the species. The functional significance of this variation remains to be determined in future studies.
Strong upregulation of the eCLCA1 mRNA and protein was observed by Northern blot hybridization, Western blotting, immunohistochemistry, and quantitative RT-PCR in the lungs of three horses with RAO, similar to the upregulation of hCLCA1 in human asthma patients and mCLCA3 in murine asthma models (Zhou et al. 2001; Hoshino et al. 2002
; Toda et al. 2002
). The immunohistochemical data clearly demonstrated eCLCA1 overexpression to be located in metaplastic goblet cells of small airways, primarily in bronchioli, which are responsible for mucus overexpression and airway plugging in equine RAO, human and murine asthma, and human COPD (Leguillette 2003
; Rogers 2003
). Thus horses with either spontaneous or experimentally induced RAO may serve as models for human COPD, including investigations on the pathophysiologic role of eCLCA1 and hCLCA1 and their suitability as therapeutic targets, both in horses and people.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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Banks W (1993) Applied Veterinary Histology. 3rd ed. St Louis, Mosby Year Book
Bice DE, Seagrave J, Green FH (2000) Animal models of asthma: potential usefulness for studying health effects of inhaled particles. Inhal Toxicol 12:829862[CrossRef][Medline]
Davis E, Rush BR (2002) Equine recurrent airway obstruction: pathogenesis, diagnosis and patient management. Vet Clin North Am Equine Pract 18:453467[CrossRef][Medline]
Dellmann HD, Eurell J (1998) Textbook of Veterinary Histology. 5th ed. Philadelphia, Lippincott Williams & Wilkins
Fuller CM, Ji HL, Tousson A, Elble RC, Pauli BU, Benos DJ (2001) Ca(2+)-activated Cl() channels: a newly emerging anion transport family. Pflugers Arch 443(suppl 1):S107110[CrossRef][Medline]
Gerber V, Lindberg A, Berney C, Robinson NE (2004) Airway mucus in recurrent airway obstructionshort-term response to environmental challenge. J Vet Intern Med 18:9297[CrossRef][Medline]
Gruber AD, Elble RC, Ji HL, Schreur KD, Fuller CM, Pauli BU (1998) Genomic cloning, molecular characterization and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated Cl channel proteins. Genomics 54:200214[CrossRef][Medline]
Gruber AD, Schreur KD, Ji HL, Fuller CM, Pauli BU (1999) Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea and mammary gland. Am J Physiol 276:C12611270[Medline]
Gruber AG, Elble RC, Pauli BU (2002) Discovery and cloning of the CLCA gene family. Curr Top Membranes 53:367387
Hoshino M, Morita S, Iwashita H, Sagiya Y, Nagi T, Nakanishi A, Ashida Y, et al. (2002) Increased expression of the human Ca2+-activated Cl channel 1 (CaCC1) gene in the asthmatic airway. Am J Respir Crit Care Med 165:11321136
Kamada F, Suzuki Y, Shao C, Tamari M, Hasegawa K, Hirota T, Shimizu M, et al. (2004) Association of the hCLCA1 gene with childhood and adult asthma. Genes Immun 5:540547[CrossRef][Medline]
Leguillette R (2003) Recurrent airway obstructionheaves. Vet Clin North Am Equine Pract 19:6386[CrossRef][Medline]
Leverkoehne I, Gruber AD (2002) The murine mCLCA3 (alias gob-5) protein is located in the mucin granule membranes of intestinal, respiratory and uterine goblet cells. J Histochem Cytochem 50:829838
Nakanishi A, Morita S, Iwashita H, Sagiya Y, Ashida Y, Shirafuji H, Fujisawa Y, et al. (2001) Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma. Proc Natl Acad Sci USA 98:51755180
Ritzka M, Weinel C, Stanke F, Tümmler B (2003) Sequence comparison of the whole murine and human CLCA locus reveals conserved synteny between both species. Genome Lett 2:149154[CrossRef]
Ritzka M, Stanke F, Gruber AD, Pusch L, Woefle S, Veeze HJ, Halley DJ, et al. (2004) The CLCA gene locus as a modulator of the gastrointestinal basic defect in cystic fibrosis. Human Genet 115:483491[CrossRef][Medline]
Snapper JR (1986) Large animal models of asthma. Am Rev Respir Dis 133:351352[Medline]
Rogers DF (2003) The airway goblet cell. Int J Biochem Cell Biol 35:16[CrossRef][Medline]
Schmidbauer SM, Venner M, von Samson-Himmelstjerna G, Drommer W, Gruber AD (2004) Compensated overexpression of procollagens 1(I) and
1(III) following perilla mint ketone-induced acute pulmonary damage in horses. J Comp Pathol 131:186198[CrossRef][Medline]
Talukdar AH, Calhoun ML, Stinson AW (1972) Microscopic anatomy of the skin of the horse. Am J Vet Res 33:23652390[Medline]
Toda M, Tulic MK, Levitt RC, Hamid Q (2002) A calcium-activated chloride channel (HCLCA1) is strongly related to IL-9 expression and mucus production in bronchial epithelium of patients with asthma. J Allergy Clin Immunol 109:246250[CrossRef][Medline]
Zhou Y, Dong Q, Louahed J, Dragwa C, Savio D, Huang M, Weiss C, et al. (2001) Characterization of a calcium-activated chloride channel as a shared target of Th2 cytokine pathways and its potential involvement in asthma. Am J Respir Cell Mol Biol 25:486491
Zhou Y, Shapiro M, Dong Q, Louahed J, Weiss C, Wan S, Chen Q, et al. (2002) A calcium-activated chloride channel blocker inhibits goblet cell metaplasia and mucus overproduction. Novartis Found Symp 248:150165; discussion 165170, 277282[Medline]
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