valinomycin and Cystic-Fibrosis

Various K(+) and Cl(-) channels are important in cell volume regulation and biliary secretion, but the specific role of cystic fibrosis transmembrane conductance regulator in cholangiocyte cell volume regulation is not known. The goal of this research was to study regulatory volume decrease (RVD) in bile duct cell clusters (BDCCs) from normal and cystic fibrosis (CF) mouse livers. Mouse BDCCs without an enclosed lumen were prepared as described (Cho, W. K. (2002) Am. J. Physiol. 283, G1320-G1327). The isotonic solution consisted of HEPES buffer with 40% of the NaCl replaced with isomolar amounts of sucrose, whereas hypotonic solution was the same as isotonic solution without sucrose. The cell volume changes were indirectly assessed by measuring cross-sectional area (CSA) changes of the BDCCs using quantitative videomicroscopy. Exposure to hypotonic solutions increased relative CSAs of normal BDCCs to 1.20 +/- 0.01 (mean +/- S.E., n = 50) in 10 min, followed by RVD to 1.07 +/- 0.01 by 40 min. Hypotonic challenge in CF mouse BDCCs also increased relative CSA to 1.20 +/- 0.01 (n = 53) in 10 min but without significant recovery. Coadministration of the K(+)-selective ionophore valinomycin restored RVD in CF mouse BDCCs, suggesting that the impaired RVD was likely from a defect in K(+) conductance. Moreover, this valinomycin-induced RVD in CF mice was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate, indicating that it is not from nonspecific effects. Neither cAMP nor calcium agonists could reverse the impaired RVD seen in CF cholangiocytes. Our conclusion is that CF mouse cholangiocytes have defective RVD from an impaired K(+) efflux pathway, which could not be reversed by cAMP nor calcium agonists.

Topics: 1-Methyl-3-isobutylxanthine; Angiogenesis Inhibitors; Animals; Bile Ducts; Buffers; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; HEPES; Immunohistochemistry; Ionophores; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Microscopy, Video; Nitrobenzoates; Potassium; Sodium Chloride; Time Factors; Valinomycin

It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl- conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl- fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl- channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl- flux in membrane vesicles and single Cl- channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl- channel inhibition cannot be decided at present.

Topics: Animals; Chloride Channels; Chlorides; Cystic Fibrosis; Cytosol; Electrophysiology; Epithelium; Humans; Membrane Proteins; Microvilli; Respiratory System; Swine; Tumor Cells, Cultured; Valinomycin

Recent studies have identified abnormalities in Cl- permeation across two target cystic fibrosis (CF) epithelia (sweat duct and respiratory epithelium). In the present study, anion conductances of red blood cells (RBCs) and peripheral blood lymphocytes (PBLs) from CF and normal subjects were estimated and compared. For RBCs, the valinomycin-induced rate constant for K+ loss (PK+) was taken as an index of PCl-. For PBLs, the secondary volume increase after gramicidin pretreatment and hypotonic (0.67 X isotonic) stress was used to estimate PCl-. The Cl- permeabilities of RBCs and PBLs from CF and control subjects were comparable. These findings suggest that the abnormality in PCl- reported for CF sweat ductal and respiratory epithelia is not expressed in circulating blood elements.

Topics: Adult; Cell Membrane Permeability; Chlorides; Cystic Fibrosis; Erythrocytes; Female; Humans; Lymphocytes; Male; Mathematics; Middle Aged; Potassium; Valinomycin

Normal values of mean cell volume (M.C.V.) and of distribution of single cell volumes (S.C.V.) have been observed in erythrocytes of patients with cystic fibrosis using an electronical particle-volume analyzer (sheath flow detector). From these results a strong defect in red cell salt transport seems improbable. Valinomucin induces shrinking (by increase of K+ permeability) and gramicidin D induces swelling (by increase of Na+ permeability) of normal erythrocytes. Addition of C.F. sweat to normal erythrocytes induces no volume change. From this result no influence of the "C.F. factor" on passive ion permeability is concluded.

Topics: Cystic Fibrosis; Erythrocytes; Gramicidin; Humans; Ion Exchange; Permeability; Potassium; Sodium; Sweat; Valinomycin