The anion transport inhibitor DIDS increases rat hepatocyte K+ conductance via uptake through the bilirubin pathway.
1. In confluent primary cultures of rat hepatocytes, membrane effects of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) were recorded with conventional microelectrodes. In addition, cell pH and cell Ca2+ were monitored by use of the fluorescent dyes BCECF and fluo-3, respectively. Uptake of DIDS was determined by measuring intracellular DIDS fluorescence between 470 and 520 nm (excitation wavelength 390 nm). 2. In the presence of 0.2 mM DIDS, membrane voltages hyperpolarized from -44.0 +/- 1.8 to -73.1 +/- 1.9 mV (n = 16). This change was monophasic and occurred with a time constant of 170 +/- 25 s. The effect was only partly reversible. 3. Cable analysis revealed a concomitant decrease in the specific cell membrane resistance from 3.2 to 1.5 k omega cm2. 4. In ion substitution experiments, a 10-fold elevation of external K+ (from 2.5 to 25 mM) depolarized cell membranes by 6.2 +/- 1.5 mV (n = 5). In the presence of 0.2 mM DIDS, this membrane response was increased 5-fold to 32.2 +/- 4.1 mV. 5. Replacement of Cl- by 99% with gluconate depolarized the cells by 9.3 +/- 1.9 mV. In contrast, with 0.2 mM DIDS present, Cl- removal led to a membrane hyperpolarization of 5.9 +/- 0.9 mV (n = 4). 6. DIDS had no effect on cytosolic pH or Ca2+. 7. To determine the sidedness of the DIDS effect, i.e. to analyse if the increase in K+ conductance is mediated by uptake of the compound, DIDS was added in the presence of different substrates of hepatocellular anion transport. Taurocholate (50 microM) and frusemide (0.5 mM), which are both taken up via the sinusoidal multi-specific bile acid transporter, did not change DIDS-induced membrane hyperpolarization. 8. In contrast, 0.5 mM bromosulphthalein (BSP), a substrate of the bilirubin transporter, competitively inhibited the membrane hyperpolarization elicited by various concentrations of DIDS (0.1-1.0 mM). 9. Hepatocellular uptake of BSP is known to be, in part, Cl- dependent and to be competitively inhibited by Indocyanine Green. When 0.2 mM DIDS was added to a superfusate, in which 99% of Cl- had been exchanged by gluconate, the velocity of membrane hyperpolarization was decreased by 45%. In the presence of Indocyanine Green (0.1 mM) DIDS-induced membrane hyperpolarization was reduced to approximately 20%. 10. Addition of 0.2 mM DIDS to hepatocyte monolayers led to a time-dependent increase in cell fluorescence which was absent at 4 degrees C and which was completely blocked by 0.5 mM BSP.(ABSTRACT TRUNCATED AT 400 WORDS)
Wehner
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F
Max-Planck-Institut für molekulare Physiologie, Dortmund, FRG.
Rosin-Steiner
S
S
Beetz
G
G
Sauer
H
H
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D001663:Bilirubin / Q000378:metabolism*; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004553:Electric Conductivity / Q000187:drug effects / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D013448:Sulfobromophthalein / Q000494:pharmacology
D013448:Sulfobromophthalein; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D001663:Bilirubin; D011188:Potassium; D002118:Calcium