Hepatic uptake of organic cations is essential for the metabolism and secretion of numerous endobiotics and drugs. Several hepatic organic cation transporters have been kinetically defined, yet have not been isolated or cloned. We have isolated a complementary DNA (cDNA) from both murine liver and kidney cDNA libraries (mOct1/ Slc22a1), and have functionally expressed it in Xenopus laevis oocytes. Although mOct1/Slc22a1 is homologous to previously cloned rat and human organic cation transporters, organic cation transport kinetics differed markedly. The liver and kidneys have critical roles in the secretion of drugs and endobiotics into the bile and urine. Hepatic elimination of many organic cationic drugs involves uptake across the basolateral surface of the hepatocyte, hepatic biotransformation, and canalicular secretion. 1 Previous studies in the intact rat, isolated perfused rat liver, and isolated hepatocytes show that the hepatobiliary transport of organic cations is carrier-mediated. [2][3][4] In addition, the organic cation choline is an essential nutrient and precursor for the synthesis of phospholipids. Hepatic choline levels are approximately 100-fold greater than blood concentrations, and de novo choline synthesis is minimal, suggesting the existence of a transport mechanism for hepatic choline uptake. 5,6 Several organic cation transporters have been kinetically defined on the plasma membrane of hepatocytes and in lysosomes. Functional studies in rat liver plasma membrane vesicles have shown at least two distinct organic cation:H ϩ exchangers, and two electrogenic organic cation transporters. [7][8][9][10][11][12][13] The complementary DNA (cDNA) encoding for the rat electrogenic polyspecific transporter (OCT1) has been isolated using functional expression cloning in Xenopus laevis oocytes, and assaying n-methyl-nicotinamide (NMN)-inhibitable uptake of the model quaternary organic cation tetraethylammonium (TEA). 14 A human homologue of rat OCT1 was recently cloned that transports 1-methyl-4-phenylpyridinium (MPP ϩ ), and uptake is also inhibited by the organic cation NMN. 15 In this study, we have isolated a murine cDNA (mOct1/ Slc22a1) from both liver and kidney libraries, and have functionally expressed it in X. laevis oocytes. Although mOct1/Slc22a1 is homologous with OCT1, transport kinetics differed markedly. mOct1/Slc22a1 transports several organic cations, but uptake is increased by inside:outside proton gradients, is unaffected by membrane potential differences, and is not inhibited by NMN. These data indicate that this cloned cDNA is a member of the hepatic and renal organic cation transporter family.
It has been proposed that the neurotoxicity observed in severely jaundiced infants results from the binding of unconjugated bilirubin to nerve cell membranes. However, despite potentially important clinical ramifications, there remains significant controversy regarding the physical nature of bilirubin-membrane interactions. We used the technique of parallax analysis of fluorescence quenching (Chattopadhyay, A., and E. London. 1987. Biochemistry. 26: 39-45) to measure the depth of penetration of bilirubin in model phospholipid bilayers. The localization of unconjugated bilirubin and ditaurobilirubin within small unilamellar vesicles composed of dioleoylphosphatidylcholine was determined through an analysis of the quenching of bilirubin fluorescence by spin-labeled phospholipids, and by bilirubin-mediated quenching of a series of anthroyloxy fatty acid probes at various depths within the membrane bilayer. Findings were further verified with potassium iodide as an aqueous quencher. Our results indicate that, at pH 10, unconjugated bilirubin localizes approximately 20 Å from the bilayer center, in the region of the polar head groups. Further analyses suggest a modest influence of pH, membrane cholesterol content, and vesicle diameter on the bilirubin penetration depth. Taken together, these data support that, under physiologic conditions, bilirubin localizes to the polar region of phospholipid bilayers, near the membrane-water interface. -Zucker, S. D., W. Goessling, E. J. Bootle, and C. Sterritt. Localization of bilirubin in phospholipid bilayers by parallax analysis of fluorescence quenching.
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