The role of oxidative stress in an equine model of human asthma

Equine recurrent airway obstruction (RAO) is a naturally occurring respiratory disease in horses with many similarities to human asthma and, as a result, has been used as an animal model of this disease. Oxidative stress has been demonstrated to occur in a range of respiratory diseases in human beings including asthma. Quantitatively, horses have a greater non-enzymatic antioxidant capacity in the pulmonary epithelial lining fluid compared to human beings due to high ascorbic acid concentrations, which reflects their ability to synthesise ascorbic acid. Consequently, a greater oxidative load is likely to be required to induce oxidative stress in horses compared to human beings. Induction of acute neutrophilic airway inflammation in RAO horses by exposure to organic dust does not result in marked pulmonary oxidative stress. However, with a more prolonged inflammatory response, the antioxidant capacity is depleted and oxidative stress occurs. Despite the clear evidence of oxidative stress in RAO, there is currently limited data linking oxidative stress with a causal role in the development of the pathophysiological features of RAO, namely airway obstruction, airway hyper-responsiveness, airway inflammation and mucus accumulation. However, pathways do exist whereby oxidants could potentially augment the production of important mediators in RAO. Further work is required to ascertain the benefits of antioxidant supplementation in RAO and to determine the role of oxidative stress in the pathogenesis of the disease. Given the similarities with human asthma, results from RAO horses could enhance the understanding of the role of oxidative stress in human asthma.     

The role of oxidative stress in an equine model of human asthma

Abstract

Equine recurrent airway obstruction (RAO) is a naturally occurring respiratory disease in horses with many similarities to human asthma and, as a result, has been used as an animal model of this disease. Oxidative stress has been demonstrated to occur in a range of respiratory diseases in human beings including asthma. Quantitatively, horses have a greater non-enzymatic antioxidant capacity in the pulmonary epithelial lining fluid compared to human beings due to high ascorbic acid concentrations, which reflects their ability to synthesise ascorbic acid. Consequently, a greater oxidative load is likely to be required to induce oxidative stress in horses compared to human beings. Induction of acute neutrophilic airway inflammation in RAO horses by exposure to organic dust does not result in marked pulmonary oxidative stress. However, with a more prolonged inflammatory response, the antioxidant capacity is depleted and oxidative stress occurs. Despite the clear evidence of oxidative stress in RAO, there is currently limited data linking oxidative stress with a causal role in the development of the pathophysiological features of RAO, namely airway obstruction, airway hyper-responsiveness, airway inflammation and mucus accumulation. However, pathways do exist whereby oxidants could potentially augment the production of important mediators in RAO. Further work is required to ascertain the benefits of antioxidant supplementation in RAO and to determine the role of oxidative stress in the pathogenesis of the disease. Given the similarities with human asthma, results from RAO horses could enhance the understanding of the role of oxidative stress in human asthma.     

Both cleavage products of the mCLCA3 protein are secreted soluble proteins

Members of the chloride channels, calcium-activated (CLCA) family of proteins and in particular the murine mCLCA3 (alias gob-5) and its human ortholog hCLCA1 have been identified as clinically relevant molecules in diseases with secretory dysfunctions including asthma and cystic fibrosis. Initial studies have indicated that these proteins evoke a calcium-activated chloride conductance when transfected into human embryonic kidney cells 293 cells. However, it is not yet clear whether the CLCA proteins form chloride channels per se or function as mediators of other, yet unknown chloride channels. Here, we present a systematic biochemical analysis of the posttranslational processing and intracellular trafficking of the mCLCA3 protein. Pulse-chase experiments after metabolic protein labeling of mCLCA3-transfected COS-1 or human embryonic kidney 293 cells revealed cleavage of a primary 110-kDa mCLCA3 translation product in the endoplasmic reticulum into a 75-kDa amino-terminal and a 35-kDa carboxyl-terminal protein that were glycosylated and remained physically associated with each other. Confocal fluorescent analyses identified both cleavage products in vesicles of the secretory pathway. Neither cleavage product was associated with the cell membrane at any time. Instead, both subunits were fully secreted into the extracellular environment as a soluble complex of two glycoproteins. These results suggest that the two mCLCA3 cleavage products cannot form an anion channel on their own but may instead act as extracellular signaling molecules. Furthermore, our results point toward significant structural differences between mCLCA3 and its human ortholog, hCLCA1, which is thought to be a single, non-integral membrane protein.     

Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production,Phagocytosis and Extracellular Trap Formation

Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.     

The murine goblet cell protein mCLCA3 is a zinc-dependent metalloprotease with autoproteolytic activity

Several members of the CLCA family of proteins, originally named chloride channels, calcium-activated, have been shown to modulate chloride conductance in various cell types via an unknown mechanism. Moreover, the human (h) hCLCA1 is thought to modulate the severity of disease in asthma and cystic fibrosis (CF) patients. All CLCA proteins are post-translationally cleaved into two subunits, and recently, a conserved HEXXH zinc-binding amino acid motif has been identified, suggesting a role for CLCA proteins as metalloproteases. Here, we have characterized the cleavage and autoproteolytic activity of the murine model protein mCLCA3, which represents the murine orthologue of human hCLCA1. Using crude membrane fractions from transfected HEK293 cells, we demonstrate that mCLCA3 cleavage is zinc-dependent and exclusively inhibited by cation-chelating metalloprotease inhibitors. Cellular transport and secretion were not affected in response to a cleavage defect that was introduced by the insertion of an E157Q mutation within the HEXXH motif of mCLCA3. Interspecies conservation of these key results was further confirmed with the porcine (p) orthologue of hCLCA1 and mCLCA3, pCLCA1. Importantly, the mCLCA3E157Q mutant was cleaved after co-transfection with the wild-type mCLCA3 in HEK293 cells, suggesting that an intermolecular autoproteolytic event takes place. Edman degradation and MALDI-TOF-MS of the protein fragments identified a single cleavage site in mCLCA3 between amino acids 695 and 696. The data strongly suggest that secreted CLCA proteins have zinc-dependent autoproteolytic activity and that they may cleave additional proteins.     

The interacting binding domains of the beta(4) integrin and calcium-activated chloride channels (CLCAs) in metastasis

CLCA (chloride channel, calcium-activated) proteins are novel pulmonary vascular addresses for blood-borne, lung-metastatic cancer cells. They facilitate vascular arrest of cancer cells via adhesion to beta4 integrin and promote early, intravascular, metastatic growth. Here we identify the interacting binding domains of endothelial CLCA proteins (e.g. hCLCA2, mCLCA5, mCLCA1, and bCLCA2) and beta4 integrin. Endothelial CLCAs share a common beta4-binding motif (beta4BM) in their 90- and 35-kDa subunits of the sequence F(S/N)R(I/L/V)(S/T)S, which is located in the second extracellular domain of the 90-kDa CLCA and near the N terminus of the 35-kDa CLCA, respectively. Using enzyme-linked immunosorbent, pull-down, and adhesion assays, we showed that glutathione S-transferase fusion proteins of beta4BMs from the 90- and 35-kDa CLCA subunits bind to the beta4 integrin in a metal ion-dependent manner. Fusion proteins from fibronectin and the integrins beta1 and beta3 served as negative controls. beta4BM fusion proteins competitively blocked the beta4/CLCA adhesion and prevented lung colonization of MDA-MB-231 breast cancer cells. A disrupted beta4BM in hCLCA1, which is not expressed in endothelia, failed to interact with beta4 integrin. The corresponding CLCA-binding domain of the beta4 integrin is localized to the specific determining loop (SDL). Again enzyme-linked immunosorbent, pull-down, and adhesion assays were used to confirm the interaction with CLCA proteins using a glutathione S-transferase fusion protein representing the C-terminal two-thirds of beta4 SDL (amino acids 184-203). A chimeric beta4 integrin in which the indicated SDL sequence had been replaced with the corresponding sequence from the beta1 integrin failed to bind hCLCA2. The dominance of the CLCA ligand in beta4 activation and outside-in signaling is discussed in reference to our previous report that beta4/CLCA ligation elicits selective signaling via focal adhesion kinase to promote metastatic growth.     

The putative chloride channel hCLCA2 has a single C-terminal transmembrane segment

Calcium-activated chloride channel (CLCA) proteins were first described as a family of plasma membrane Cl(-) channels that could be activated by calcium. Genetic and electrophysiological studies have supported this view. The human CLCA2 protein is expressed as a 943-amino-acid precursor whose N-terminal signal sequence is removed followed by internal cleavage near amino acid position 680. Earlier investigations of transmembrane geometry suggested five membrane passes. However, analysis by the more recently derived simple modular architecture research tool algorithm predicts that a C-terminal 22-amino-acid hydrophobic segment comprises the only transmembrane pass. To resolve this question, we raised an antibody against hCLCA2 and investigated the synthesis, localization, maturation, and topology of the protein. Cell surface biotinylation and endoglycosidase H analysis revealed a 128-kDa precursor confined to the endoplasmic reticulum and a maturely glycosylated 141-kDa precursor at the cell surface by 48 h post-transfection. By 72 h, 109-kDa N-terminal and 35-kDa C-terminal cleavage products were detected at the cell surface but not in the endoplasmic reticulum. Surprisingly, however, the 109-kDa product was spontaneously shed into the medium or removed by acid washes, whereas the precursor and 35-kDa product were retained by the membrane. Two other CLCA family members, bCLCA2 and hCLCA1, also demonstrated preferential release of the N-terminal product. Transfer of the hCLCA2 C-terminal hydrophobic segment to a secreted form of green fluorescent protein was sufficient to target that protein to the plasma membrane. Together, these data indicate that hCLCA2 is mostly extracellular with only a single transmembrane segment followed by a short cytoplasmic tail and is itself unlikely to form a channel.     

The interleukin 4 receptor gene and its role in recurrent airway obstruction in Swiss Warmblood horses

Recurrent airway obstruction (RAO) in horses is the result of an interaction of genetic and environmental factors and shares many characteristics with human asthma. Many studies have suggested that the interleukin-4 receptor gene (IL4R) is associated with this disease, and a QTL region on chromosome 13 containing IL4R was previously detected in one of the two Swiss Warmblood families. We sequenced the entire IL4R gene in this family and detected 93 variants including five non-synonymous protein-coding variants. The allele distribution at these SNPs supported the previously detected QTL signal. Subsequently, we investigated IL4R mRNA expression in bronchoalveolar lavage fluid cells. During exacerbation, IL4R expression was increased in RAO-affected offspring in the implicated family, but not in the other family. These findings support that IL4R plays a role in some cases of RAO.     

Breath condensate hydrogen peroxide correlates with both airway cytology and epithelial lining fluid ascorbic acid concentration in the horse

The relationship between hydrogen peroxide (H2O2) concentration in expired breath condensate (EBC) and cytology of the respiratory tract obtained from tracheal wash (TW) or bronchoalveolar lavage (BAL), and epithelial lining fluid (ELF) antioxidant status is unknown. To examine this we analysed the concentration of H2O2 in breath condensate from healthy horses and horses affected by recurrent airway obstruction (RAO), a condition considered to be an animal model of human asthma. The degree of airway inflammation was determined by assessing TW inflammation as mucus, cell density and neutrophil scores, and by BAL cytology. ELF antioxidant status was determined by measurement of ascorbic acid, dehydroascorbate, reduced and oxidised glutathione, uric acid and alpha-tocopherol concentrations. RAO-affected horses with marked airway inflammation had significantly higher concentrations of breath condensate H2O2 than control horses and RAO-affected horses in the absence of inflammation (2.0 +/- 0.5 micromol/l. 0.4 +/- 0.2 micromol/l and 0.9 +/- 0.2 micromol/l H2O2, respectively; p < 0.0001). The concentration of breath condensate H2O2 was related inversely to the concentration of ascorbic acid in ELF (r = -0.80; p < 0.0001) and correlated positively with TW inflammation score (r = 0.76, p < 0.0001) and BAL neutrophil count (r = 0.80, p < 0.0001). We conclude that the concentration of H2O2 in breath condensate influences the ELF ascorbic acid concentration and provides a non-invasive diagnostic indicator of the severity of neutrophilic airway inflammation.     

Early onset airway obstruction in response to organic dust in the horse.

Equine recurrent airway obstruction (RAO) has been used as a naturally occurring model of human asthma. However, it is unknown whether there is an early-phase response in RAO. The aim of this study was to determine whether exposure to organic dust induces immediate changes in lung function in RAO-affected horses, which could be mediated by airway mast cells. Six RAO-affected horses in remission and six control horses were challenged with hay-straw dust suspension by nebulization. Total respiratory resistance at 1 Hz, measured by forced oscillation, was increased from 0.62 +/- 0.09 cmH(2)O.l(-1).s (mean +/- SE) to 1.23 +/- 0.20 cmH(2)O.l(-1).s 15 min after nebulization in control horses (P = 0.023) but did not change significantly in the RAO group. Total respiratory reactance at 1 Hz (P = 0.005) was significantly lower in the control horses (-0.77 +/- 0.07 cmH(2)O.l(-1).s) than in the RAO group (-0.49 +/- 0.04 cmH(2)O.l(-1).s) 15 min after nebulization. Bronchoalveolar lavage fluid (BALF) histamine concentration was significantly elevated 10 and 20 min postnebulization in control horses but not in RAO horses. Minimum reactance at 1 Hz in the early postnebulization period significantly correlated with both prechallenge BALF mast cell numbers (r = -0.65, P = 0.02) and peak BALF histamine concentration postnebulization (r = -0.61, P = 0.04). In conclusion, RAO horses, unlike human asthmatic patients, do not exhibit an early-phase response. However, healthy control horses do demonstrate a mild but significant early (<20 min) phase response to inhaled organic dust. This response may serve to decrease the subsequent dose of dust inhaled and as such provide a protective mechanism, which may be compromised in RAO horses.     

Spi2 gene polymorphism is not associated with recurrent airway obstruction and inflammatory airway disease in thoroughbred horses

The aim was to detect the presence of polymorphisms at exons 1, 2, 3 and 4 of the Spi2 gene, and evaluate a possible association between them and recurrent airway obstruction (RAO) or inflammatory airway disease (IAD) in thoroughbred horses, through single-strand conformational-polymorphism (SSCP) screening. Although polymorphism was not detected in exons 1, 2 and 3, three alleles and six genotypes were identified in exon 4. The frequencies of allele A (0.6388) and genotype AA (0.3888) were higher in horses affected by RAO, although no association was found between polymorphism and horses with either RAO or IAD.     

Persistent mucus accumulation: a consequence of delayed bronchial mucous cell apoptosis in RAO-affected horses?

This study examined the contribution of delayed apoptosis of bronchial mucous cells to mucus accumulation in equine recurrent airway obstruction (RAO). In pilot studies, Bcl-2, an apoptosis inhibitor, was detected in airway mucous cells of RAO-affected horses in remission and during acute disease, when most mucus was secreted. To study whether delayed apoptosis results in an increase in the number of mucous cells during disease recovery, six RAO-affected and six control horses were fed hay for 5 days to induce inflammation and then pellets for 7 days to partially resolve RAO before euthanasia. RAO-affected horses had more airway obstruction and luminal mucus than control horses under both management systems. At the time of euthanasia, RAO-affected horses had more inflammation and Bcl-2-positive bronchial mucous cells than control animals. In horses with >10 and <10 neutrophils per microliter of bronchoalveolar lavage fluid, >50% and <10% of mucous cells stained positive for Bcl-2, respectively. No differences in mucous cell number or amount of stored mucosubstance were observed between RAO-affected and control horses, but in RAO-affected animals, the amount of stored mucosubstance decreased as the number of neutrophils in bronchoalveolar lavage fluid increased. Because the number of mucous cells was similar in both groups of horses but only mucous cells of RAO-affected horses expressed Bcl-2 during recovery from acute disease, a conclusive role for Bcl-2 in prolonging bronchial mucous cell life could not be determined. Future studies are needed to compare horses that are kept in remission for prolonged periods when all mucous cells are fully developed.     

Increased hypoxia-inducible factor 1alpha expression in lung cells of horses with recurrent airway obstruction

ABSTRACT: BACKGROUND: Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition caused by exposure of susceptible horses to organic dusts in hay. The immunological processes responsible for the development and the persistence of airway inflammation are still largely unknown. Hypoxia-inducible factor (Hif) is mainly known as a major regulator of energy homeostasis and cellular adaptation to hypoxia. More recently however, Hif also emerged as an essential regulator of innate immune responses. Here, we aimed at investigating the potential involvement of Hif1-alpha in myeloid cells in horse with recurrent airway obstruction. RESULTS: In vitro, we observed that Hif is expressed in equine myeloid cells after hay dust stimulation and regulates genes such as tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A). We further showed in vivo that airway challenge with hay dust upregulated Hif1-alpha mRNA expression in myeloid cells from the bronchoalveolar lavage fluid (BALF) of healthy and RAO-affected horses, with a more pronounced effect in cells from RAO-affected horses. Finally, Hif1-alpha mRNA expression in BALF cells from challenged horses correlated positively with lung dysfunction. CONCLUSION: Taken together, our results suggest an important role for Hif1-alpha in myeloid cells during hay dust-induced inflammation in horses with RAO. We therefore propose that future research aiming at functional inactivation of Hif1 in lung myeloid cells could open new therapeutic perspectives for RAO.     

Differential Gene Expression Profiles and Selected Cytokine Protein Analysis of Mediastinal Lymph Nodes of Horses with Chronic Recurrent Airway Obstruction (RAO) Support an Interleukin-17 Immune Response

Recurrent airway obstruction (RAO) is a pulmonary inflammatory condition that afflicts certain mature horses exposed to organic dust particulates in hay. Its clinical and pathological features, manifested by reversible bronchoconstriction, excessive mucus production and airway neutrophilia, resemble the pulmonary alterations that occur in agricultural workers with occupational asthma. The immunological basis of RAO remains uncertain although its chronicity, its localization to a mucosal surface and its domination by a neutrophilic, non-septic inflammatory response, suggest involvement of Interleukin-17 (IL-17). We examined global gene expression profiles in mediastinal (pulmonary-draining) lymph nodes isolated from RAO-affected and control horses. Differential expression of > 200 genes, coupled with network analysis, supports an IL-17 response centered about NF-κB. Immunohistochemical analysis of mediastinal lymph node sections demonstrated increased IL-17 staining intensity in diseased horses. This result, along with the finding of increased IL-17 concentrations in lymph node homogenates from RAO-affected horses (P = 0.1) and a down-regulation of IL-4 gene and protein expression, provides additional evidence of the involvement of IL-17 in the chronic stages of RAO. Additional investigations are needed to ascertain the cellular source of IL-17 in this equine model of occupational asthma. Understanding the immunopathogenesis of this disorder likely will enhance the development of therapeutic interventions beneficial to human and animal pulmonary health.     

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.     

Changes in Holstein cow milk and serum proteins during intramammary infection with three different strains of Staphylococcus aureus

Background

Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions.

Results

Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P₁ cultures compared to P₀ cultures while ciliation was delayed in P₁ cultures.

Conclusions

This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.     

Phenotypic Responses of Differentiated Asthmatic Human Airway Epithelial Cultures to Rhinovirus

ObjectivesHuman airway epithelial cells are the principal target of human rhinovirus (HRV), a common cold pathogen that triggers the majority of asthma exacerbations. The objectives of this study were 1) to evaluate an in vitro air liquid interface cultured human airway epithelial cell model for HRV infection, and 2) to identify gene expression patterns associated with asthma intrinsically and/or after HRV infection using this model.MethodsAir-liquid interface (ALI) human airway epithelial cell cultures were prepared from 6 asthmatic and 6 non-asthmatic donors. The effects of rhinovirus RV-A16 on ALI cultures were compared. Genome-wide gene expression changes in ALI cultures following HRV infection at 24 hours post exposure were further analyzed using RNA-seq technology. Cellular gene expression and cytokine/chemokine secretion were further evaluated by qPCR and a Luminex-based protein assay, respectively.ConclusionsALI-cultured human airway epithelial cells challenged with HRV are a useful translational model for the study of HRV-induced responses in airway epithelial cells, given that gene expression profile using this model largely recapitulates some important patterns of gene responses in patients during clinical HRV infection. Furthermore, our data emphasize that both abnormal airway epithelial structure and inflammatory signaling are two important asthma signatures, which can be further exacerbated by HRV infection.     

pCLCA1 becomes a cAMP-dependent chloride conductance mediator in Caco-2 cells

Members of the CLCA protein family are expressed in airway and intestinal epithelium, where they may participate in secretory activity as mediators of chloride conductance. A calcium-dependent chloride conductance has been observed upon expression of CLCA proteins in non-epithelial cell lines. The pCLCA1 gene, cloned in our laboratory, codes for a product containing a unique A-kinase consensus acceptor site not found in other CLCA proteins. Calcium-dependent, but not cAMP-dependent, chloride conductance increased when pCLCA1 was expressed in NIH/3T3 fibroblasts. We transfected the Caco-2 human colon carcinoma cell line with pCLCA1 to investigate the regulation of CLCA-associated chloride conductance in this differentiated epithelial cell line. Expression of pCLCA1 in the Caco-2 cell line enhanced cAMP-responsive 36Cl efflux, short circuit current, and whole cell chloride current in these cells. This cAMP-dependent chloride conductance was localized to the apical membrane of polarized Caco-2 cells.