Transepithelial Impedance Analysis of Chloride Secretion

Singh, Ankita; Singh, Sangeeta; Devor, Daniel C.; Frizzell, Raymond A.; Driessche, Willy Van; Bridges, Robert J.

doi:10.1385/1-59259-187-6:129

Cited by 20 publications

(42 citation statements)

References 18 publications

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“…It is also important to note that the effect of RS25344 on ADO stimulation of CFTR in cell-attached patches appeared more dramatic than the modest shift induced in the adenosine CFTR I sc doseresponse relation. The Calu-3 I sc is a composite of multiple electrochemical driving forces and does not directly measure apical conductance as described previously (34). Plausibly, stimulation of apical anion conductance due to PDE inhibition would not dominate I sc under these conditions.…”

Section: Resultsmentioning

confidence: 99%

Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium

Barnes

Livera

Huang

et al. 2005

Journal of Biological Chemistry

101

100

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We demonstrated previously that Calu-3 airway epithelial cells sense adenosine on their luminal surface through adenosine A2B receptors coupled to adenylyl cyclase. Occupancy of these receptors leads to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel through protein kinase A (PKA) anchored at the apical membrane. Because luminal A2B receptor activation does not raise total cellular cAMP levels, we hypothesized that activation of phosphodiesterases (PDEs) confines cAMP generated by apical A2B receptors to a microdomain that includes the CFTR channel. Using reverse transcription-PCR, Western blotting, and activity measurements, PDE4D was identified as the major PDE species in airway epithelia. Consistent with these results, inhibitors of PDE4, but not PDE3, selectively abolished the lateral confinement of cAMP signaling in apical membrane patches during cell-attached recordings. Furthermore, stimulation of the CFTR in excised apical patches by rolipram and RS25344 indicated that PDE4 is localized in close proximity to the CFTR channel. Indeed, immunohistochemistry of human airway sections revealed that PDE4D is localized in the apical domain of the cell. PDE4 was activated after luminal adenosine exposure in a PKA-dependent manner. Because PDE4 activity is positively regulated by PKA, our results support a model whereby the PDE diffusion barrier is proportional to the degree of receptor stimulation. These findings underscore the concept that subcellular localization of individual PDE isozymes is an important mechanism for confining cAMP signaling to functional domains within cells.

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Section: Resultsmentioning

confidence: 99%

Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium

Barnes

Livera

Huang

et al. 2005

Journal of Biological Chemistry

101

100

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“…4). Various modulators, such as forskolin and prostaglandin E 2 , that increased intracellular cAMP levels were previously shown to similarly reduce the resistance of both apical and basolateral membranes (40,44,50). This phenomenon was usually explained by the increased opening of ion channels or increased activities of transcellular ion transporters (40).…”

Section: Discussionmentioning

confidence: 99%

“…Gt and Isc values were obtained during PTH 1-34 or forskolin stimulation (50). Since Ra, Rb, Ca, and Cb from impedance analysis were consid- Table 1.…”

Section: Methodsmentioning

confidence: 99%

“…Since PTH has been reported to elevate intracellular cAMP levels (35), it was possible that PTH induced cAMP-dependent CFTR activation, thereby stimulating anion secretion in intestinal epithelial cells. By using alternating current (AC) impedance spectroscopy, one could demonstrate whether PTH induced CFTR opening and/or fusion of CFTR-rich vesicles to the apical membrane, because these processes usually affect the resistive and capacitive properties of the plasma membrane, respectively (12,40,44,50).…”

Section: Hcomentioning

confidence: 99%

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Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO₃⁻ secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH

Laohapitakworn

Thongbunchoo

Nakkrasae

et al. 2011

American Journal of Physiology-Cell Physiology

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“…Transepithelial impedance analysis. Impedance was measured by published methods using the same Ussing chambers and electrodes employed during flux studies (52). Total epithelial capacitance was measured at five selectable frequencies (2, 4.1, 8.2, 11.0, and 16.5 kHz) in response to 99 computerized sine waves, whereas the mean voltage across the tissue (V t) was clamped to 0 V. The fundamental frequency was 1 Hz, and the frequency range was 1 Hz-22 kHz.…”

Section: Methodsmentioning

confidence: 99%

Electrogenic bicarbonate secretion by prairie dog gallbladder

Moser¹,

Gangopadhyay²,

Bradbury³

et al. 2007

American Journal of Physiology-Gastrointestinal and Liver Physi

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.-Pathological rates of gallbladder salt and water transport may promote the formation of cholesterol gallstones. Because prairie dogs are widely used as a model of this event, we characterized gallbladder ion transport in animals fed control chow by using electrophysiology, ion substitution, pharmacology, isotopic fluxes, impedance analysis, and molecular biology. In contrast to the electroneutral properties of rabbit and Necturus gallbladders, prairie dog gallbladders generated significant short-circuit current (I sc; 171 Ϯ 21 A/cm 2 ) and lumen-negative potential difference (Ϫ10.1 Ϯ 1.2 mV) under basal conditions. Unidirectional radioisotopic fluxes demonstrated electroneutral NaCl absorption, whereas the residual net ion flux corresponded to I sc. In response to 2 M forskolin, Isc exceeded 270 A/cm 2 , and impedance estimates of the apical membrane resistance decreased from 200 ⍀ ⅐ cm 2 to 13 ⍀ ⅐ cm 2 . The forskolin-induced Isc was dependent on extracellular HCO 3 Ϫ and was blocked by serosal 4,4Ј-dinitrostilben-2,2Ј-disulfonic acid (DNDS) and acetazolamide, whereas serosal bumetanide and Cl Ϫ ion substitution had little effect. Serosal trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman and Ba 2ϩ reduced Isc, consistent with the inhibition of cAMP-dependent K ϩ channels. Immunoprecipitation and confocal microscopy localized cystic fibrosis transmembrane conductance regulator protein (CFTR) to the apical membrane and subapical vesicles. Consistent with serosal DNDS sensitivity, pancreatic sodium-bicarbonate cotransporter protein pNBC1 expression was localized to the basolateral membrane. We conclude that prairie dog gallbladders secrete bicarbonate through cAMP-dependent apical CFTR anion channels. Basolateral HCO 3 Ϫ entry is mediated by DNDS-sensitive pNBC1, and the driving force for apical anion secretion is provided by K ϩ channel activation.disease; ion transport; cystic fibrosis conductance membrane regulator; sodium-bicarbonate symporters ALTHOUGH GALLBLADDER DISEASE is the most frequent indication for abdominal surgery in the United States (19), the factors that confer susceptibility to gallstones remain poorly understood. Prairie dogs have been widely studied as an experimental model of human cholelithiasis due to their unique propensity for developing gallstones on high-cholesterol chow (7). Prior studies in cholesterol-fed prairie dogs (4) have demonstrated that gallbladder salt and water transport are altered before gallstones form, a phenomenon that may cause cholesterol to precipitate in the gallbladder lumen. The potential for targeting gallbladder ion transport as a strategy for preventing cholelithiasis is underscored by data showing that amiloride inhibits the formation of gallstones in cholesterol-fed prairie dogs (56).To use the prairie dog as a model to investigate this event, we performed a detailed characterization of gallbladder ion transport in animals fed control chow. Classic studies in Necturus and rabbit gallbladders established the paradigm for electr...

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Transepithelial Impedance Analysis of Chloride Secretion

Cited by 20 publications

References 18 publications

Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium

Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium

Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO₃⁻ secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH

Electrogenic bicarbonate secretion by prairie dog gallbladder

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Transepithelial Impedance Analysis of Chloride Secretion

Cited by 20 publications

References 18 publications

Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium

Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium

Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO3− secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH

Electrogenic bicarbonate secretion by prairie dog gallbladder

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Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO₃⁻ secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH