Role of chloride and intracellular pH on the activity of the rat hepatocyte organic anion transporter.

Min, Albert D.; Johansen, Kirsten L.; Campbell, Celeste G.; Wolkoff, Allan W.

doi:10.1172/jci115159

Cited by 49 publications

(63 citation statements)

References 45 publications

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“…(i) While substrates of Na+-dependent bile acid transport were ineffective, BSP competitively inhibited hyperpolarization of membrane voltages; the K1 of (0 5 mM) BSP in this process was 50 AM, which is in good agreement with Km values of BSP uptake of rat liver in situ (46 /sM; Scharschmidt et al 1975) and of isolated perfused guinea-pig liver (18 /M; Rutishauser, 1983). (ii) Cl-substitution by gluconate decreased the rate of voltage changes by some 45 %, which exactly matches the decrease of [35S]BSP uptake by short-term cultured rat hepatocytes (Min et al 1991) and isolated perfused rat liver (Wolkoff et al 1987). (iii) The DIDS effect was significantly reduced by Indocyanine Green, which is another competitive inhibitor of BSP (bilirubin) transport (Scharschmidt et al 1975).…”

Section: Fluorimetric Determination Of Dids Uptakesupporting

confidence: 83%

The anion transport inhibitor DIDS increases rat hepatocyte K+ conductance via uptake through the bilirubin pathway.

Wehner

Rosin-Steiner

Beetz

et al. 1993

The Journal of Physiology

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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.

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Section: Fluorimetric Determination Of Dids Uptakesupporting

confidence: 83%

The anion transport inhibitor DIDS increases rat hepatocyte K+ conductance via uptake through the bilirubin pathway.

Wehner

Rosin-Steiner

Beetz

et al. 1993

The Journal of Physiology

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“…These data thus strongly suggest that Cl Ϫ does not directly modulate transport activity of oatp. Some studies (5,20) have indicated that although Cl Ϫ binds to albumin (21), it does not influence binding of organic anions to albumin. The mechanism resulting in Cl Ϫ -dependent kinetics that has been described in cultured hepatocytes (4,5,17) and perfused rat liver (4) thus requires further clarification.…”

Section: Discussionmentioning

confidence: 99%

“…These organic anions circulate tightly bound to albumin from which they are rapidly extracted by hepatocytes (2,3). Previous studies performed in short term cultured rat hepatocytes demonstrated a high affinity low capacity organic anion transporter (4,5). This transporter extracted BSP from albumin, was electroneutral and temperature-dependent, and was inhibited after depletion of cellular ATP.…”

mentioning

confidence: 98%

Stable Inducible Expression of a Functional Rat Liver Organic Anion Transport Protein in HeLa Cells

Shi

Bai

Ford

et al. 1995

Journal of Biological Chemistry

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“…As recently demonstrated, a characteristic feature of hepatocellular BSP uptake is its dependence on extracellular chloride (3,4,7). In cultured rat hepatocytes this chloride-dependent uptake system has an exceptionally high affinity for BSP (Km -0.3 AM), resulting in efficient extraction of BSP from its binding sites on albumin (4,7).…”

mentioning

confidence: 98%

“…In cultured rat hepatocytes this chloride-dependent uptake system has an exceptionally high affinity for BSP (Km -0.3 AM), resulting in efficient extraction of BSP from its binding sites on albumin (4,7). Because none of the putative BSP-transporting polypeptides (see above) has been identified as mediating chloride-dependent BSP uptake, we adopted a functional expression cloning strategy to isolate and characterize the full length cDNA encoding this important hepatocytic transport system.…”

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confidence: 99%

Expression of the hepatocellular chloride-dependent sulfobromophthalein uptake system in Xenopus laevis oocytes.

et al. 1991

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The expression of the basolateral chloride-activated organic anion uptake system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of oocytes with rat liver poly(A)+RNA resulted in the functional expression of chloridedependent sulfobromophthalein (BSP) uptake within 3-5 d. This expressed chloride-dependent BSP uptake system exhibited saturation kinetics (apparent K, 6.2 MM) and efficiently extracted BSP from its binding sites on BSA. Furthermore, the chloride-activated portion of BSP uptake was inhibited by bilirubin (10 gM; inhibition 53%), 4,4'-diisothiocyano-2,2-disulfonic acid stilbene (DIDS, 100 MLM; 80%), taurocholate (100 ,MM; 80%), and cholate (200 ,M; 95%). In contrast to results with total rat liver mRNA, injection of mRNA derived from the Na+/bile acid cotransporter cDNA (Hagenbuch, B., B. Stieger, M. Foguet, H. Lubbert, and P. J. Meier. 1991. Proc. Nati. Acad. Sci. USA. In press.) had no effect on BSP uptake into oocytes. Size fractionation of total rat liver mRNA revealed that a 2.0-to 3.5-kb size-class mRNA was sufficient to express the hepatic chloride-dependent BSP uptake system. These data indicate that "expression cloning" in oocytes represents a promising approach to ultimately clone the cDNA coding for the hepatocyte high affinity, chloride-dependent organic anion uptake system. Furthermore, the results confirm that the Na+/ bile acid cotransport system does not mediate BSP uptake. (J.

show abstract

Role of chloride and intracellular pH on the activity of the rat hepatocyte organic anion transporter.

Cited by 49 publications

References 45 publications

The anion transport inhibitor DIDS increases rat hepatocyte K+ conductance via uptake through the bilirubin pathway.

The anion transport inhibitor DIDS increases rat hepatocyte K+ conductance via uptake through the bilirubin pathway.

Stable Inducible Expression of a Functional Rat Liver Organic Anion Transport Protein in HeLa Cells

Expression of the hepatocellular chloride-dependent sulfobromophthalein uptake system in Xenopus laevis oocytes.

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