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 beta-intercalated cells, we studied: (1) The effect of luminal [Cl-]0 changes on pHi measured by video-imaging micro-fluorometry, (2) the influence of the inhibitor 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) on anion-exchange activity, and (3) the effect of acetazolamide on intracellular pH-indicator (c-SNAFL-2) accumulation and anion-exchange activity. At least three different modes of fluorescence accumulation were found in confluent monolayers: cells with high, low or undetectable fluorescence. Highly fluorescent cells responded to a rise of [Cl-]0 (30-140 mM) with a proportional decrease of pHi (7.6-6.4). Acetazolamide (10(-4) M) completely blocked the acidifying effects of the increased [Cl-]0, indicating that HCO3- is the intracellular ion exchanged for extra-cellular Cl-. Acetazolamide caused a reduction of SNAFL-2 fluorescence suggesting that carbonic anhydrase activity contributes to indicator accumulation. The high DIDS concentration (50 microM) required to prevent intracellular acidification suggests that the exchanger involved is identical to that present in beta-intercalated cells. All cells of non-confluent monolayers were highly fluorescent and expressed Cl-/ HCO3(-)-exchanger activity. In conclusion, highly fluorescent MDCK cells in confluent monolayers have a luminal DIDS inhibitable, carbonic anhydrase dependent Cl-/HCO3(-)-exchanger, and may therefore be used as a model for beta-intercalated cells.
Ebner
S
S
Physiologisches Institut der Universität, München, Germany.
Marin-Grez
M
M
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D000086:Acetazolamide / Q000494:pharmacology*; D000818:Animals; D001581:Benzothiadiazines; D001639:Bicarbonates / Q000378:metabolism*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D003592:Cytophotometry; D004232:Diuretics; D004285:Dogs; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018715:Microscopy, Video; D009550:Nigericin / Q000494:pharmacology; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology*
D001581:Benzothiadiazines; D001639:Bicarbonates; D002712:Chlorides; D004232:Diuretics; D005456:Fluorescent Dyes; D007476:Ionophores; D049993:Sodium Chloride Symporter Inhibitors; D000086:Acetazolamide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D009550:Nigericin