Polarized expression of cAMP-activated chloride channels in isolated epithelial cells.
We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.
Torres
R J
RJ
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641, USA.
Altenberg
G A
GA
Cohn
J A
JA
Reuss
L
L
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000276:immunology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000276:immunology / Q000502:physiology*; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology; D005704:Gallbladder / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008839:Microelectrodes; D009337:Necturus; D018408:Patch-Clamp Techniques; D011817:Rabbits
D000906:Antibodies; C505032:CFTR protein, human; D018118:Chloride Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000242:Cyclic AMP