SUMMARY1. 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 0f2 mm DIDS, membrane voltages hyperpolarized from -44 0 + 1'8 to -7341 + 1P9 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 1P5 kg) 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 + 1P9 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 /lM) 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 (0h1-10 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 MIS 2009 618 F. WEHNER, S. ROSIN-STEINER, G. BEETZ AND H. SAUER 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°C and which was completely blocked by 0 5 mm BSP.11. In conclusion, DIDS hyperpolarizes rat hepatocytes by increasing cell membrane K+ conductance. In addition, cell Cl-conductance is decreased. The increase in K+ conductance is mediated via uptake of the compound through the bilirubin-BSP pathway.

Rat hepatocytes in confluent primary cultures were impaled with conventional microelectrodes. Reducing extracellular osmolarity by 80 mosmol/l leads to a transient hyperpolarization of cell membranes (maximum after 5 min) from -40 +/- 4 to -51 +/- 2 mV (n = 7). This hyperpolarization is blocked by 1 mmol/l Ba2+ and 0.5 mmol/l quinine. In ion substitution experiments, increasing K+ 10-fold (from 2.7 to 27 mmol/l) depolarizes membrane voltage by 9 +/- 2 mV in normosmotic solutions. In hyposmotic solutions this depolarization is increased to 20 +/- 1 mV at the time of maximum hyperpolarization and decreases thereafter to 8 +/- 2 mV (n = 4). Cable analysis reveals a transient decrease of specific membrane resistance that exactly parallels the increase in membrane voltage response to high K+. In addition, electrical coupling between cells continuously decreases under hyposmotic conditions, indicating that intercellular communication is affected. Reducing Cl- 100-fold (from 116.5 to 1.2 mmol/l; HCO(3-)-free solutions) depolarizes hepatocytes by 24 +/- 3 mV under normosmotic conditions. In hyposmotic solutions, this effect is increased to 39 +/- 4 mV at maximum hyperpolarization and decreases again to 26 +/- 3 mV (n = 8). This transient increase in the voltage response to Cl- removal is abolished by 0.5 mmol/l quinine (n = 5) and 1 mmol/l Ba2+ (n = 5), suggesting that it is indirect via changes in K+ conductance. This concept is corroborated by ion substitution experiments (HCO(3-)-free conditions), which show that under hyposmotic conditions voltage response to high K+ is considerably decreased in low Cl- solutions.(ABSTRACT TRUNCATED AT 250 WORDS)