Elizabeth Cowley - Academia.edu (original) (raw)
Papers by Elizabeth Cowley
The Prostaglandin E2 type 4 receptor participates in the response to acute oxidant stress in airw... more The Prostaglandin E2 type 4 receptor participates in the response to acute oxidant stress in airway epithelial cells- REVISED
Exposure to sodium butyrate leads to functional downregulation of calcium-activated potassium cha... more Exposure to sodium butyrate leads to functional downregulation of calcium-activated potassium channels in human airway epithelial cells
The American Journal of Pathology, 2014
Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortalit... more Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we examined whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection in a mouse model. We found that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice displayed greatly increased epithelial permeability, bacterial dissemination, and neutrophil accumulation compared with wild-type animals after P. aeruginosa infection; these defects were corrected on reconstitution with mast cells. An in vitro Transwell co-culture model further demonstrated that a secreted mast cell factor decreased epithelial cell apoptosis and tumor necrosis factor production after P. aeruginosa infection. Together, our data demonstrate a previously unrecognized role for mast cells in the maintenance of epithelial integrity during P. aeruginosa infection, through a mechanism that likely involves prevention of epithelial apoptosis and tumor necrosis factor production. Our understanding of mechanisms of the host response to P. aeruginosa will open new avenues for the development of successful preventative and treatment strategies.
Biochimica et biophysica acta. Biomembranes, 2017
The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator ... more The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are determined predominantly by interactions between permeant anions and the narrow region of the channel pore. This narrow region has therefore been described as functioning as the "selectivity filter" of the channel. Multiple pore-lining transmembrane segments (TMs) have previously been shown to contribute to the selectivity filter region. However, little is known about the three-dimensional organization of this region, or how multiple TMs combine to determine its functional properties. In the present study we have used patch clamp recording to identify changes in channel function associated with the formation of disulfide cross-links between cysteine residues introduced into different TMs within the selectivity filter. Cysteine introduced at position L102 in TM1 was able to form disulfide bonds with F337C and T338C in TM6, two positions that are known...
Cellular and molecular life sciences : CMLS, Aug 1, 2018
Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibr... more Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilize...
The Journal of biological chemistry, Jan 13, 2018
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl channel that apparently ha... more The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl channel that apparently has evolved from an ancestral active transporter. Key to the CFTR's switch from pump to channel function may have been the appearance of one or more "lateral portals." Such portals connect the cytoplasm to the transmembrane channel pore, allowing a continuous pathway for the electrodiffusional movement of Cl ions. However, these portals remain the least well-characterized part of the Cl transport pathway; even the number of functional portals is uncertain, and if multiple portals do exist, their relative functional contributions are unknown. Here, we used patch-clamp recording to identify the contributions of positively charged amino acid side chains located in CFTR's cytoplasmic transmembrane extensions to portal function. Mutagenesis-mediated neutralization of several charged side chains reduced single-channel Cl conductance. However, these same mutations differentially...
The Faseb Journal, Apr 1, 2009
Pflugers Archiv European Journal of Physiology, Mar 1, 2006
Potassium channels are required for the absorption and secretion of fluids and electrolytes in ep... more Potassium channels are required for the absorption and secretion of fluids and electrolytes in epithelia. Calu-3 cells possess a secretory phenotype, and are a model human airway submucosal gland serous cell. Short-circuit current (I sc) recordings from Calu-3 cells indicated that basal anion secretion was reduced by apical application of the K + channel inhibitors bupivicaine, lidocaine, clofilium, and quinidine. Application of riluzole resulted in a large increase in I sc , inhibited by apical application of either bupivicane or the cystic fibrosis transmembrane conductance regulator (CFTR) Cl À channel blocker DPC. These results suggested that one or more members of the two-pore-domain K + (K 2P) channel family could influence anion secretion. Using RT-PCR, we found that Calu-3 cells express mRNA transcripts for TASK-2 (KCNK5), TWIK-1 (KCNK1), TWIK-2 (KCNK6) and TREK-1 (KCNK2). TASK-2, TWIK-2 and TREK-1 protein were detected by Western blotting, while immunolocalization of polarized cells confirmed protein expression of TREK-1 and TWIK-2 at the plasma cell membrane. TASK-2 protein staining was localized to intracellular vesicles, located beneath the apical membrane. While the pro-secretory role of basolateral K + channels is well established, we suggest that apically located K 2P channels, not previously described in airway epithelial cells, also play an important role in controlling the rate of transepithelial anion secretion.
J Soc Gynecol Investigation, 1995
Amer J Respir Cell Molec Biol, 2007
Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the... more Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the peroxidation of membrane polyunsaturated fatty acids. Previous work has demonstrated that the isoprostane 8-iso-prostaglandin E(2) (PGE(2)) stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial anion secretion across the human airway epithelial cell line, Calu-3. Since isoprostanes predominantly achieve their effects via binding to prostanoid receptors, we hypothesized that this 8-iso-PGE(2) stimulation of CFTR activity was the result of the isoprostane binding to a prostanoid receptor. Using RT-PCR, immunoblotting, and immunofluorescence, we here demonstrate that Calu-3 cells express the EP(1-4) and FP receptors, and localize these proteins in polarized cell monolayers. Using iodide efflux as a marker for CFTR-mediated Cl(-) efflux, we investigate whether prostanoid receptor agonists elicit a functional response from Calu-3 cells. Application of the agonists PGE(2), misoprostol (EP(2), EP(3), and EP(4)) and PGE(1)-OH (EP(3) and EP(4)) stimulate iodide efflux; however, iloprost, butaprost, sulprostone, and fluoprostenol (agonists of the EP(1), EP(2), EP(3), and FP receptors, respectively) have no effect. The iodide efflux seen with 8-iso-PGE(2) is abolished by the EP(4) receptor antagonist AH23848, the CFTR inhibitor 172, and inhibition of PKA and the PI3K pathway. In conclusion, we demonstrate that although Calu-3 cells possess numerous prostanoid receptors, only the EP(4) subtype appears capable of eliciting a functional iodide efflux response, which is mediated via the EP(4) receptor. We propose that 8-iso-PGE(2), acting via EP(4) receptor, could play an important role in the CFTR-mediated response to oxidant stress, and which would be compromised in the CF airways.
Brit J Pharmacol, 2010
Accumulating data point to K þ channels as relevant players in controlling cell cycle progression... more Accumulating data point to K þ channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K þ channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IK Ca1) in human PCa as well as their involvement in cell proliferation. We showed that IK Ca1 mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IK Ca1 potassium channel suppressed PCa cell proliferation. The activation of IK Ca1 hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IK Ca1 channel is likely to promote carcinogenesis in human prostate tissue.
Clinical and investigative medicine. Médecine clinique et experimentale
Molecular pharmacology, 2003
Isoprostanes are liberated when reactive oxygen species (ROS) mediate the peroxidation of arachid... more Isoprostanes are liberated when reactive oxygen species (ROS) mediate the peroxidation of arachidonic acid or other polyunsaturated fatty acids. Because exposure to ROS is associated with tissue damage in the lung, we examined whether exposure to isoprostanes elicited a response in airway epithelial cells, potentially implicating isoprostane production in the epithelial response to oxidant stress. Application of the isoprostane 8-iso-prostaglandin E2 (8-iso-PGE2) produced an increase in transepithelial anion secretion across monolayers of the human airway epithelial cell line Calu-3, measured as an increase in short circuit current (Isc). This increase in Isc was greater when 8-iso-PGE2 was applied to the basolateral rather than the apical face of the Calu-3 monolayers and was almost entirely abolished by the addition of diphenylamine-2-carboxylate, implicating the cystic fibrosis transmembrane conductance regulator Cl- channel in the response. Experiments with electrically isolated...
Journal of applied physiology (Bethesda, Md. : 1985), 1998
The constriction of pulmonary airways is limited by the tethering effect exerted by parenchymal a... more The constriction of pulmonary airways is limited by the tethering effect exerted by parenchymal attachments. To characterize this tethering effect at the scale of intraparenchymal airways, we studied the pattern of parenchymal distortion due to bronchoconstriction in a rat lung explant system. First, we measured the elastic modulus under tension for 2% (wt/vol) agarose alone (37.6 +/- 1.5 kPa) and for agarose-filled lung (5.7 +/- 1.3 kPa). The latter is similar to the elastic modulus of air-filled lung at total lung capacity (4.5-6 kPa) (S. J. Lai-Fook, T. A. Wilson, R. E. Hyatt, and J. R. Rodarte. J. Appl. Physiol. 40: 508-513, 1976), suggesting that explants can be used as a model of lung tissue distortion. Subsequently, confocal microscopic images of fluorescently labeled 0.5-mm-thick explants prepared from agarose-filled rat lungs inflated to total lung capacity (48 ml/kg) were acquired. Images were taken before and after airway constriction was induced by direct application of ...
Therapeutic delivery, 2013
The majority of drugs cross epithelial cells by either passive diffusion or via carrier-mediated ... more The majority of drugs cross epithelial cells by either passive diffusion or via carrier-mediated drug transporters. The aim of this study was to investigate the transport characteristics, protein expression and localization of organic cation transporters in human nasal epithelium. The expression, localization and transport characteristics of the transporters were investigated using permeation, PCR and immunohistochemistry. The uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide followed Michaelis-Menten kinetics. Its intracellular accumulation of the compound was inhibited by organic cation transporters (OCTs) and carnitine/organic cation transporter (OCTNs) inhibitors. Detected OCT1-3, OCTN1 and OCTN2 gene transcripts correlated with immunohistological staining for OCT1-3, OCTN1 and OCTN2 antibodies. Except for OCTN1, the antibodies were generally localized on the apical side of the epithelial cells. Based on the immunohistochemical and uptake/transport studies, we conc...
The Journal of Physiology, 2002
Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels... more Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels. Using monolayers of the Calu-3 cell line, a human submucosal serous cell model mounted in an Ussing chamber apparatus, we investigated the nature of the K + channels involved in basal, cAMPand Ca 2+-stimulated anion secretion, as reflected by the transepithelial short circuit current (I sc). The non-specific K + channel inhibitor Ba 2+ inhibited the basal I sc by either 77 or 16 % when applied directly to the basolateral or apical membranes, respectively, indicating that a basolateral K + conductance is required for maintenance of basal anion secretion. Using the K + channel blockers clofilium and clotrimazole, we found basal I sc to be sensitive to clofilium, with a small clotrimazolesensitive component. By stimulating the cAMP and Ca 2+ pathways, we determined that cAMPstimulated anion secretion was almost entirely abolished by clofilium, but insensitive to clotrimazole. In contrast, the Ca 2+-stimulated response was sensitive to both clofilium and clotrimazole. Thus, pharmacologically distinct basolateral K + channels are differentially involved in the control of anion secretion under different conditions. Isolation of the basolateral K + conductance in permeabilized monolayers revealed a small basal and forskolin-stimulated I sc. Finally, using the reverse transcriptase-polymerase chain reaction, we found that Calu-3 cells express the K + channel genes KCNN4 and KCNQ1 and the subunits KCNE2 and KCNE3. We conclude that while KCNN4 contributes to Ca 2+-activated anion secretion by Calu-3 cells, basal and cAMP-activated secretion are more critically dependent on other K + channel types, possibly involving one or more class of KCNQ1-containing channel complexes.
The Journal of Physiology, 2002
Exposure to reactive oxygen species (ROS) is associated with tissue damage in the lung and may be... more Exposure to reactive oxygen species (ROS) is associated with tissue damage in the lung and may be a common element in the pathogenesis of all inflammatory lung diseases. Exposure to the ROS hydrogen peroxide (H 2 O 2) evoked a rapid increase in transepithelial anion secretion across monolayers of the human submucosal gland serous cell line Calu-3. This increase was almost entirely abolished by the addition of diphenylamine-2-carboxylate (DPC), implicating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl _ channel in the response. The response was also reduced by inhibitors of basolateral K + channels. Studies of electrically isolated apical and basolateral membranes revealed that H 2 O 2 stimulated both apical Cl _ and basolateral K + conductances (G Cl and G K). Apical G Cl was sensitive to DPC, but unaffected by 4,4‚-diisothiocyanatostilbene-2,2‚-disulfonic acid (DIDS), suggesting that CFTR is the major anion conduction pathway mediating the response to H 2 O 2. Additionally, H 2 O 2 had no effect on G Cl in the presence of the adenylate cyclase inhibitor SQ22536 or following maximal stimulation of G Cl with forskolin, implicating the cAMP-dependent protein kinase pathway in the apical response to H 2 O 2. Basolateral G K was reduced by the K + channel inhibitors clotrimazole and clofilium, indicating roles for KCNN4 and KCNQ1 in the H 2 O 2-stimulated response. We propose that ROS-stimulated anion secretion from serous cells plays an important role in keeping the airways clear from damaging radicals that could potentially initiate tissue destruction. Our finding that this response is CFTR dependent suggests that an important host defence mechanism would be dysfunctional in the cystic fibrosis (CF) lung. Loss of this compensatory protective mechanism could expose the CF lung to ROS for extended periods, which could be important in the pathogenesis of CF lung disease.
The Prostaglandin E2 type 4 receptor participates in the response to acute oxidant stress in airw... more The Prostaglandin E2 type 4 receptor participates in the response to acute oxidant stress in airway epithelial cells- REVISED
Exposure to sodium butyrate leads to functional downregulation of calcium-activated potassium cha... more Exposure to sodium butyrate leads to functional downregulation of calcium-activated potassium channels in human airway epithelial cells
The American Journal of Pathology, 2014
Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortalit... more Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we examined whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection in a mouse model. We found that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice displayed greatly increased epithelial permeability, bacterial dissemination, and neutrophil accumulation compared with wild-type animals after P. aeruginosa infection; these defects were corrected on reconstitution with mast cells. An in vitro Transwell co-culture model further demonstrated that a secreted mast cell factor decreased epithelial cell apoptosis and tumor necrosis factor production after P. aeruginosa infection. Together, our data demonstrate a previously unrecognized role for mast cells in the maintenance of epithelial integrity during P. aeruginosa infection, through a mechanism that likely involves prevention of epithelial apoptosis and tumor necrosis factor production. Our understanding of mechanisms of the host response to P. aeruginosa will open new avenues for the development of successful preventative and treatment strategies.
Biochimica et biophysica acta. Biomembranes, 2017
The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator ... more The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are determined predominantly by interactions between permeant anions and the narrow region of the channel pore. This narrow region has therefore been described as functioning as the "selectivity filter" of the channel. Multiple pore-lining transmembrane segments (TMs) have previously been shown to contribute to the selectivity filter region. However, little is known about the three-dimensional organization of this region, or how multiple TMs combine to determine its functional properties. In the present study we have used patch clamp recording to identify changes in channel function associated with the formation of disulfide cross-links between cysteine residues introduced into different TMs within the selectivity filter. Cysteine introduced at position L102 in TM1 was able to form disulfide bonds with F337C and T338C in TM6, two positions that are known...
Cellular and molecular life sciences : CMLS, Aug 1, 2018
Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibr... more Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilize...
The Journal of biological chemistry, Jan 13, 2018
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl channel that apparently ha... more The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl channel that apparently has evolved from an ancestral active transporter. Key to the CFTR's switch from pump to channel function may have been the appearance of one or more "lateral portals." Such portals connect the cytoplasm to the transmembrane channel pore, allowing a continuous pathway for the electrodiffusional movement of Cl ions. However, these portals remain the least well-characterized part of the Cl transport pathway; even the number of functional portals is uncertain, and if multiple portals do exist, their relative functional contributions are unknown. Here, we used patch-clamp recording to identify the contributions of positively charged amino acid side chains located in CFTR's cytoplasmic transmembrane extensions to portal function. Mutagenesis-mediated neutralization of several charged side chains reduced single-channel Cl conductance. However, these same mutations differentially...
The Faseb Journal, Apr 1, 2009
Pflugers Archiv European Journal of Physiology, Mar 1, 2006
Potassium channels are required for the absorption and secretion of fluids and electrolytes in ep... more Potassium channels are required for the absorption and secretion of fluids and electrolytes in epithelia. Calu-3 cells possess a secretory phenotype, and are a model human airway submucosal gland serous cell. Short-circuit current (I sc) recordings from Calu-3 cells indicated that basal anion secretion was reduced by apical application of the K + channel inhibitors bupivicaine, lidocaine, clofilium, and quinidine. Application of riluzole resulted in a large increase in I sc , inhibited by apical application of either bupivicane or the cystic fibrosis transmembrane conductance regulator (CFTR) Cl À channel blocker DPC. These results suggested that one or more members of the two-pore-domain K + (K 2P) channel family could influence anion secretion. Using RT-PCR, we found that Calu-3 cells express mRNA transcripts for TASK-2 (KCNK5), TWIK-1 (KCNK1), TWIK-2 (KCNK6) and TREK-1 (KCNK2). TASK-2, TWIK-2 and TREK-1 protein were detected by Western blotting, while immunolocalization of polarized cells confirmed protein expression of TREK-1 and TWIK-2 at the plasma cell membrane. TASK-2 protein staining was localized to intracellular vesicles, located beneath the apical membrane. While the pro-secretory role of basolateral K + channels is well established, we suggest that apically located K 2P channels, not previously described in airway epithelial cells, also play an important role in controlling the rate of transepithelial anion secretion.
J Soc Gynecol Investigation, 1995
Amer J Respir Cell Molec Biol, 2007
Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the... more Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the peroxidation of membrane polyunsaturated fatty acids. Previous work has demonstrated that the isoprostane 8-iso-prostaglandin E(2) (PGE(2)) stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial anion secretion across the human airway epithelial cell line, Calu-3. Since isoprostanes predominantly achieve their effects via binding to prostanoid receptors, we hypothesized that this 8-iso-PGE(2) stimulation of CFTR activity was the result of the isoprostane binding to a prostanoid receptor. Using RT-PCR, immunoblotting, and immunofluorescence, we here demonstrate that Calu-3 cells express the EP(1-4) and FP receptors, and localize these proteins in polarized cell monolayers. Using iodide efflux as a marker for CFTR-mediated Cl(-) efflux, we investigate whether prostanoid receptor agonists elicit a functional response from Calu-3 cells. Application of the agonists PGE(2), misoprostol (EP(2), EP(3), and EP(4)) and PGE(1)-OH (EP(3) and EP(4)) stimulate iodide efflux; however, iloprost, butaprost, sulprostone, and fluoprostenol (agonists of the EP(1), EP(2), EP(3), and FP receptors, respectively) have no effect. The iodide efflux seen with 8-iso-PGE(2) is abolished by the EP(4) receptor antagonist AH23848, the CFTR inhibitor 172, and inhibition of PKA and the PI3K pathway. In conclusion, we demonstrate that although Calu-3 cells possess numerous prostanoid receptors, only the EP(4) subtype appears capable of eliciting a functional iodide efflux response, which is mediated via the EP(4) receptor. We propose that 8-iso-PGE(2), acting via EP(4) receptor, could play an important role in the CFTR-mediated response to oxidant stress, and which would be compromised in the CF airways.
Brit J Pharmacol, 2010
Accumulating data point to K þ channels as relevant players in controlling cell cycle progression... more Accumulating data point to K þ channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K þ channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IK Ca1) in human PCa as well as their involvement in cell proliferation. We showed that IK Ca1 mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IK Ca1 potassium channel suppressed PCa cell proliferation. The activation of IK Ca1 hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IK Ca1 channel is likely to promote carcinogenesis in human prostate tissue.
Clinical and investigative medicine. Médecine clinique et experimentale
Molecular pharmacology, 2003
Isoprostanes are liberated when reactive oxygen species (ROS) mediate the peroxidation of arachid... more Isoprostanes are liberated when reactive oxygen species (ROS) mediate the peroxidation of arachidonic acid or other polyunsaturated fatty acids. Because exposure to ROS is associated with tissue damage in the lung, we examined whether exposure to isoprostanes elicited a response in airway epithelial cells, potentially implicating isoprostane production in the epithelial response to oxidant stress. Application of the isoprostane 8-iso-prostaglandin E2 (8-iso-PGE2) produced an increase in transepithelial anion secretion across monolayers of the human airway epithelial cell line Calu-3, measured as an increase in short circuit current (Isc). This increase in Isc was greater when 8-iso-PGE2 was applied to the basolateral rather than the apical face of the Calu-3 monolayers and was almost entirely abolished by the addition of diphenylamine-2-carboxylate, implicating the cystic fibrosis transmembrane conductance regulator Cl- channel in the response. Experiments with electrically isolated...
Journal of applied physiology (Bethesda, Md. : 1985), 1998
The constriction of pulmonary airways is limited by the tethering effect exerted by parenchymal a... more The constriction of pulmonary airways is limited by the tethering effect exerted by parenchymal attachments. To characterize this tethering effect at the scale of intraparenchymal airways, we studied the pattern of parenchymal distortion due to bronchoconstriction in a rat lung explant system. First, we measured the elastic modulus under tension for 2% (wt/vol) agarose alone (37.6 +/- 1.5 kPa) and for agarose-filled lung (5.7 +/- 1.3 kPa). The latter is similar to the elastic modulus of air-filled lung at total lung capacity (4.5-6 kPa) (S. J. Lai-Fook, T. A. Wilson, R. E. Hyatt, and J. R. Rodarte. J. Appl. Physiol. 40: 508-513, 1976), suggesting that explants can be used as a model of lung tissue distortion. Subsequently, confocal microscopic images of fluorescently labeled 0.5-mm-thick explants prepared from agarose-filled rat lungs inflated to total lung capacity (48 ml/kg) were acquired. Images were taken before and after airway constriction was induced by direct application of ...
Therapeutic delivery, 2013
The majority of drugs cross epithelial cells by either passive diffusion or via carrier-mediated ... more The majority of drugs cross epithelial cells by either passive diffusion or via carrier-mediated drug transporters. The aim of this study was to investigate the transport characteristics, protein expression and localization of organic cation transporters in human nasal epithelium. The expression, localization and transport characteristics of the transporters were investigated using permeation, PCR and immunohistochemistry. The uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide followed Michaelis-Menten kinetics. Its intracellular accumulation of the compound was inhibited by organic cation transporters (OCTs) and carnitine/organic cation transporter (OCTNs) inhibitors. Detected OCT1-3, OCTN1 and OCTN2 gene transcripts correlated with immunohistological staining for OCT1-3, OCTN1 and OCTN2 antibodies. Except for OCTN1, the antibodies were generally localized on the apical side of the epithelial cells. Based on the immunohistochemical and uptake/transport studies, we conc...
The Journal of Physiology, 2002
Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels... more Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels. Using monolayers of the Calu-3 cell line, a human submucosal serous cell model mounted in an Ussing chamber apparatus, we investigated the nature of the K + channels involved in basal, cAMPand Ca 2+-stimulated anion secretion, as reflected by the transepithelial short circuit current (I sc). The non-specific K + channel inhibitor Ba 2+ inhibited the basal I sc by either 77 or 16 % when applied directly to the basolateral or apical membranes, respectively, indicating that a basolateral K + conductance is required for maintenance of basal anion secretion. Using the K + channel blockers clofilium and clotrimazole, we found basal I sc to be sensitive to clofilium, with a small clotrimazolesensitive component. By stimulating the cAMP and Ca 2+ pathways, we determined that cAMPstimulated anion secretion was almost entirely abolished by clofilium, but insensitive to clotrimazole. In contrast, the Ca 2+-stimulated response was sensitive to both clofilium and clotrimazole. Thus, pharmacologically distinct basolateral K + channels are differentially involved in the control of anion secretion under different conditions. Isolation of the basolateral K + conductance in permeabilized monolayers revealed a small basal and forskolin-stimulated I sc. Finally, using the reverse transcriptase-polymerase chain reaction, we found that Calu-3 cells express the K + channel genes KCNN4 and KCNQ1 and the subunits KCNE2 and KCNE3. We conclude that while KCNN4 contributes to Ca 2+-activated anion secretion by Calu-3 cells, basal and cAMP-activated secretion are more critically dependent on other K + channel types, possibly involving one or more class of KCNQ1-containing channel complexes.
The Journal of Physiology, 2002
Exposure to reactive oxygen species (ROS) is associated with tissue damage in the lung and may be... more Exposure to reactive oxygen species (ROS) is associated with tissue damage in the lung and may be a common element in the pathogenesis of all inflammatory lung diseases. Exposure to the ROS hydrogen peroxide (H 2 O 2) evoked a rapid increase in transepithelial anion secretion across monolayers of the human submucosal gland serous cell line Calu-3. This increase was almost entirely abolished by the addition of diphenylamine-2-carboxylate (DPC), implicating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl _ channel in the response. The response was also reduced by inhibitors of basolateral K + channels. Studies of electrically isolated apical and basolateral membranes revealed that H 2 O 2 stimulated both apical Cl _ and basolateral K + conductances (G Cl and G K). Apical G Cl was sensitive to DPC, but unaffected by 4,4‚-diisothiocyanatostilbene-2,2‚-disulfonic acid (DIDS), suggesting that CFTR is the major anion conduction pathway mediating the response to H 2 O 2. Additionally, H 2 O 2 had no effect on G Cl in the presence of the adenylate cyclase inhibitor SQ22536 or following maximal stimulation of G Cl with forskolin, implicating the cAMP-dependent protein kinase pathway in the apical response to H 2 O 2. Basolateral G K was reduced by the K + channel inhibitors clotrimazole and clofilium, indicating roles for KCNN4 and KCNQ1 in the H 2 O 2-stimulated response. We propose that ROS-stimulated anion secretion from serous cells plays an important role in keeping the airways clear from damaging radicals that could potentially initiate tissue destruction. Our finding that this response is CFTR dependent suggests that an important host defence mechanism would be dysfunctional in the cystic fibrosis (CF) lung. Loss of this compensatory protective mechanism could expose the CF lung to ROS for extended periods, which could be important in the pathogenesis of CF lung disease.