A method for measuring Cl efflux from dispersed cells of airway epithelium.
This paper describes a method for measuring the increase in halide permeability of isolated airway epithelial cells induced by adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of isolated cells, known to contain the cystic fibrosis transmembrane conductance regulator (CFTR), were placed in the upper part of a Swinnex filter holder containing a filter with pores of 0.65 micron diameter. Medium was perfused over the cells at room temperature and collected at minute intervals following its passage through the filter. Experiments were performed on Calu-3 and T84 cells (human lung and colonic epithelial cell lines), primary cultures of dog and human tracheal epithelium, and Swiss 3T3 fibroblasts stably transfected with CFTR. In all cell types, addition of agents that elevate cAMP led to increases in the rates of loss of 36Cl and 125I. However, in human tracheal epithelial cells, warming the medium from room temperature to 37 degrees C was a more effective way of stimulating tracer efflux. Increases in efflux in response to either temperature or cAMP-elevating agents were inhibited by diphenylamine-2-carboxylate, a blocker of CFTR. Reproducible increases in tracer efflux were seen with as few as 10(6) cells. Cells that had been trypsinized off their culture dishes responded better than cells that had been scraped off, although treatment of scraped cells with trypsin enhanced their responsiveness to cAMP-elevating agents. Cystic fibrosis is characterized by the lack of a cAMP-activated Cl conductance in the apical membrane of airway epithlia.(ABSTRACT TRUNCATED AT 250 WORDS)
Ohrui
T
T
Cystic Fibrosis Research Center, University of California, San Francisco 94143, USA.
Shen
B Q
BQ
Mrsny
R J
RJ
Widdicombe
J H
JH
D000818:Animals; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000378:metabolism*; D002713:Chlorine; D000242:Cyclic AMP / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004285:Dogs; D004594:Electrophysiology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000378:metabolism; D010827:Physiology / Q000295:instrumentation / Q000379:methods*; D011868:Radioisotopes; D014132:Trachea / Q000166:cytology / Q000378:metabolism* / Q000502:physiology
C505032:CFTR protein, human; D002712:Chlorides; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011868:Radioisotopes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002713:Chlorine; D000242:Cyclic AMP