Electrogenic bicarbonate secretion by prairie dog gallbladder

Abstract

.-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...

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

“…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).…”

“…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). Although some cAMP-dependent mediators, such as prostaglandin E 2 , were capable of altering the paracellular permeability (47), the absence of changes in R p and transepithelial mannitol transport suggested that paracellular pathway was not involved.…”

“…Ra, Rb, Ca, and Cb were important parameters obtained from Zplot/Zview. Determination of R p or paracellular conductance (Gp; Gp ϭ 1/Rp) was required before analyses of Ra, Rb, Ca, and Cb since Rp was an initial parameter essential for curve fitting by Zplot/Zview (40). According to the method of Wills et al (60), G p was the y-intercept in a linear plot between transepithelial conductance (Gt; Gt ϭ 1/Rt) and Isc, as follows:…”

“…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).…”

Substance P stimulates CFTR-dependent fluid secretion by mouse tracheal submucosal glands

“…b Mean secretion rates by CFTR−/− glands (n=6 tracheas) and wild-type littermates (n=4 tracheas). Asterisks indicate significant differences between groups (Student's t test, p<0.05) Substance-P-stimulated secretion also requires Ca 2+ -activated K + channels Transepithelial fluid and electrolyte secretion depend on apical Cl − channels and also on the activity of a variety of basolateral K + channels, including small conductance Ca 2+ -activated K + (SK) channels, which hyperpolarize the cell and increase the apical driving force for anion efflux [34][35][36][37][38][39]. Thus, inhibiting basolateral SK channels with clotrimazole blocks transepithelial anion transport in Calu-3 cells [37], a widely used model for submucosal gland serous cells.…”