4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Colonic-Neoplasms

We studied the consequences of cystic fibrosis transmembrane conductance regulator (CFTR) expression in NIH-3T3 fibroblasts as a model for the effects of virally transduced CFTR expression in non-epithelial cells. Fibroblasts were infected with a retrovirus vector that contained the human CFTR and neor cDNAs. We selected and expanded G418-resistant clones that encompassed a range of CFTR expression. CFTR-mediated Cl-conductance function was measured as whole cell current, and CFTR protein was quantitated by immunoblot analysis. Overall, there was a good relationship between CFTR protein levels and CFTR-mediated Cl- conductance. Some clones had consistently high basal levels of CFTR-mediated Cl- conductance. This variation in function was partially explained by CFTR protein levels and was not due to clonal variation in cAMP metabolism. High levels of CFTR expression were associated with depolarization of fibroblast membrane potential. The CFTR-expressing clones with the largest basally active CFTR Cl- conductances and the most depolarized membrane potentials also exhibited slower growth rates. These results suggest that potential side effects of gene replacement therapy for cystic fibrosis include functional consequences of CFTR expression in non-epithelial cells.

Topics: 3T3 Cells; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Blotting, Western; Chloride Channels; Chlorides; Clone Cells; Colforsin; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Fibroblasts; Humans; Ion Channels; Membrane Potentials; Membrane Proteins; Mice; Recombinant Proteins; Transfection; Tumor Cells, Cultured

The outwardly rectified chloride channel of secretory epithelial cells is inhibited by disulfonic stilbene (DS) compounds such as 4,4'-diisothiostilbene-2,2'-disulfonic acid (DIDS) [R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256 (Cell Physiol. 25): C902-C912, 1989]. A 13-amino acid peptide (P49) corresponding to the putative DS binding site region of the murine anion exchange protein was synthesized, and polyclonal antibodies were generated against it and then purified over a P49 affinity column. The resulting monospecific antibodies reacted on Western blots with a 95- to 100-kDa protein from human erythrocytes and a 55- to 60-kDa protein from the human colonic tumor cell line, T84. The reaction with T84 protein did not appear to represent recognition of an anion exchanger because anion efflux from T84 cells was independent of external Cl-. In addition, monoclonal antibodies raised against human band 3 recognized the band 3 protein in human red cell ghost preparations but recognized nothing in T84 cell membrane preparations. In T84 cells, DIDS protected the 60-kDa protein from antibody binding. The anti-P49 antibody blocked outwardly rectified Cl- channels incorporated into planar lipid bilayer membranes from rat colon. Immunocytochemical data reveal specific binding of the anti-P49 antibody to perinuclear cytoplasmic vesicles. Forskolin caused these antibody-labeled vesicles to migrate from the perinuclear region to the plasma membrane under conditions and with a time course identical to that seen for stimulation of Cl- transport in these cells. Our results suggest that the protein may be a part of a chloride channel complex of secretory epithelial cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Antibodies; Binding Sites; Chlorides; Colforsin; Colonic Neoplasms; Cyclic AMP; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Immunoblotting; Iodine; Iodine Radioisotopes; Membrane Proteins; Peptides; Tumor Cells, Cultured

The factors which influence the exocytosis of mucins are not well characterized. Since the physical properties of mucins may be affected significantly by the co-secretion of electrolytes and water, we studied the relationship between ion movement and mucin secretion in T84 cells, a human colonic adenocarcinoma cell line which has been well characterized with respect to apical chloride secretion. Secretion of mucin was assessed by immunoassay of mucin appearing in the medium within 30 min of stimulation. Cells were grown on plastic in DMEM/Ham's F12 medium and experiments were carried out at 70% confluence. Mucin secretion was stimulated by the calcium ionophore A23187, or A23187 plus vasoactive intestinal polypeptide. Stimulated mucin secretion was not affected by loop diuretics (furosemide (1 x 10(-3) M) or bumetanide (1 x 10(-4) M)), with or without the addition of ouabain (5 x 10(-5) M) and amiloride (1 x 10(-5) M), making it unlikely that transcellular chloride movements in necessary for mucin secretion. However, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; (1 x 10(-5) and 5 x 10(-5) M) and three potassium channel blockers BaCl2 (1 x 10(-3) and 5 x 10(-3) M), tetraethylammonium chloride (1 x 10(-2) M) and quinine (5 x 10(-4) M) inhibited mucin secretion. A DIDS-sensitive chloride channel or chloride/bicarbonate exchanger and a Ca2(+)-dependent potassium channel may play important roles in mucin secretion. Since plasma membranes are sparingly permeable to DIDS, the DIDS-sensitive site is likely to be on the apical plasma membrane, perhaps at an initiation locus for exocytosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenocarcinoma; Amiloride; Barium; Barium Compounds; Calcimycin; Cell Line; Chlorides; Colonic Neoplasms; Furosemide; Humans; Kinetics; Mucins; Ouabain; Potassium Channels; Quinine; Stilbenes; Tetraethylammonium; Tetraethylammonium Compounds; Tumor Cells, Cultured; Vasoactive Intestinal Peptide