VIDEO-IMAGING MICRO-FLUOROMETRIC ASSESSMENT OF LUMINAL CHLORIDE BICARBONATE EXCHANGE ACTIVITY IN MADIN-DARBY CANINE KIDNEY-CELLS - INFLUENCE OF CELL-DENSITY, 4,4'-DIISOTHIOCYANO-2,2'-DISULFONIC STILBENE AND ACETAZOLAMIDE/

To investigate whether or not MDCK cells may be used as a model for be ta-intercalated cells, we studied: (1) The effect of luminal [Cl-](0) changes on pH(i) measured by video-imaging micro-fluorometry, (2) the influence of the inhibitor 4,4'-diisothiocyano-2,2'-disulfonic stilben e (DIDS) on anion-exchange activity, and (3) the effect of acetazolami de on intracellular pH-indicator (c-SNAFL-2) accumulation and anion-ex change activity. At least three different modes of fluorescence accumu lation were found in confluent monolayers: cells with high, low or und etectable fluorescence. Highly fluorescent cells responded to a rise o f [Cl-](0) (30-140 mM) with a proportional decrease of pH(i) (7.6-6.4) . Acetazolamide (10(-4) M) completely blocked the acidifying effects o f the increased [Cl-](0) indicating that HCO3- is the intracellular io n exchanged for extra-cellular Cl-. Acetazolamide caused a reduction o f SNAFL-2 fluorescence suggesting that carbonic anhydrase activity con tributes to indicator accumulation. The high DIDS concentration (50 mu M) required to prevent intracellular acidification suggests that the exchanger involved is identical to that present in beta-intercalated c ells. All cells of non-confluent monolayers were highly fluorescent an d expressed Cl-/HCO3--exchanger activity. In conclusion, highly fluore scent MDCK cells in confluent monolayers have a luminal DIDS inhibitab le, carbonic anhydrase dependent Cl-/HCO3--exchanger, and may therefor e be used as a model for beta-intercalated cells.