FLUORESCENCE-OPTICAL MEASUREMENTS OF CHLORIDE MOVEMENTS IN CELLS USING THE MEMBRANE-PERMEABLE DYE DIH-MEQ

Fluorescence-optical measurements of the intracellular chloride concen tration facilitate identification of chloride movements across the cel l membrane of Living cells. The two main dyes used for this purpose 6- methoxy-N-(3-sulfopropyl)quinolinium (SPQ) 6-methoxy-quinolyl acetoeth yl ester (MQAE). The use of both substances is impaired by their poor membrane permeability and therefore limited loading of the cells to be studied. Here we report the use of 6-methoxy-N-ethylquinolinium iodid e (MEQ), a chloride-sensitive dye for which a membrane-permeable form is easily prepared. This makes the loading procedure as easy as with t he acetoxymethyl (AM) forms of other dyes for sensing intracellular io ns. In addition, the original method, which described absolute concent ration Measurements of chloride in the cytosol, was modified in so far as only relative measurements were made. This avoids the known limita tions of single wavelength excitation and emission dyes with respect t o exact concentration measurements. Moreover to enhance the signal-to- noise ratio the driving force for chloride was considerably increased by changing the original direction of the anion flux In the cells unde r investigation. We verified the method by using fibroblasts and activ ating I-Cln, a putative chloride channel cloned from epithelial cells and of paramount importance in the regulatory volume decrease in these cells. In the presence of SCN- the MEQ quench measured in NIH 3T3 fib roblasts is dramatically enhanced in hypotonically challenged cells co mpared with cells under isotonic conditions. Antisense oligodeoxynucle otides sensing I-Cln considerably impeded the swelling-induced chlorid e current (I-Cl) in NIH 3T3 fibroblasts. Accordingly, the chloride mov ement measured by the SCN- quench of the MEQ signal was significantly reduced. Similar results can be obtained in the presence of 5-nitro-2- (3-phenylpropylamino)benzoic acid (NPPB) or 4,4'-diisothiocyanatostilb ene-2,2'-disulfonic acid (DIDS), two known blockers of chloride transp ort in the plasma membrane of a variety of cells. In conclusion, fluro scence-optical measurements using MEQ as the chloride-sensitive dye pr ovide a reliable and easy-to-use method for measuring changes of the c hloride nux across the cell membrane of Living cells.