TY - JOUR
T1 - Expression of the hepatocellular chloride-dependent sulfobromophthalein uptake system in Xenopus laevis oocytes
AU - Jacquemin, Emmanuel
AU - Hagenbuch, Bruno
AU - Stieger, Bruno
AU - Wolkoff, Allan W.
AU - Meier, Peter J.
PY - 1991
Y1 - 1991
N2 - 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 chloride-dependent sulfobromophthalein (BSP) uptake within 3-5 d. This expressed chloride-dependent BSP uptake system exhibited saturation kinetics (apparent Km ∼ 6.2 μM) and efficiently extracted BSP from its binding sites on BSA. Furthermore, the chloride-activated portion of BSP uptake was inhibited by bilirubin (10 μM; inhibition 53%), 4,4′-diisothiocyano-2,2-disulfonic acid stilbene (DIDS, 100 μM; 80%), taurocholate (100 μM; 80%), and cholate (200 μM; 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. Lübbert, and P. J. Meier. 1991. Proc. Natl. 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.
AB - 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 chloride-dependent sulfobromophthalein (BSP) uptake within 3-5 d. This expressed chloride-dependent BSP uptake system exhibited saturation kinetics (apparent Km ∼ 6.2 μM) and efficiently extracted BSP from its binding sites on BSA. Furthermore, the chloride-activated portion of BSP uptake was inhibited by bilirubin (10 μM; inhibition 53%), 4,4′-diisothiocyano-2,2-disulfonic acid stilbene (DIDS, 100 μM; 80%), taurocholate (100 μM; 80%), and cholate (200 μM; 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. Lübbert, and P. J. Meier. 1991. Proc. Natl. 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.
KW - Expression-cloning
KW - Hepatic organic anion uptake
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U2 - 10.1172/JCI115546
DO - 10.1172/JCI115546
M3 - Article
C2 - 1752967
AN - SCOPUS:0026331027
SN - 0021-9738
VL - 88
SP - 2146
EP - 2149
JO - Journal of Clinical Investigation
JF - Journal of Clinical Investigation
IS - 6
ER -