RESUMO
The mechanism of organic anion uptake by hepatocytes has kinetics that suggest facilitated diffusion, and carrier-mediated membrane transport has been postulated. In previous studies, we purified a 55,000-mol wt organic anion-binding protein (OABP) by affinity chromatography on sulfobromophthalein (BSP)-Sepharose of deoxycholate solubilized liver cell plasma membrane preparations. Using specific goat and rabbit antibodies to OABP, we have now investigated the distribution of this protein in liver fractions and other tissues by an enzyme-linked immunosorbent assay and by the immunoblot (Western blot) procedure. These studies indicated that OABP is present in significant amounts in all tissues examined except for blood. Although OABP has not as yet been isolated from each of these tissues and characterized, OABP in heart retained the ability to bind organic anions, and was purified by affinity chromatography on BSP-sepharose. In liver, OABP was membrane bound and remained so after extraction with 0.9 M NaCl, which suggests that it is an intrinsic membrane protein. OABP did not have a ubiquitous subcellular distribution within the hepatocyte. Preparation of subfractions of liver cell plasma membrane revealed that OABP is present in the sinusoidal and absent from the canalicular membrane. Immunofluorescence studies performed in short-term cultured hepatocytes suggest that OABP is associated with the surface of these cells and does not have a significant intracellular distribution.
Assuntos
Proteínas de Transporte/imunologia , Fígado/citologia , Animais , Proteínas de Transporte de Ânions , Membrana Celular/análise , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Masculino , Peso Molecular , Coelhos , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
Transport of 35S-labeled sulfobromophthalein [35S]BSP was studied in short-term cultured rat hepatocytes incubated in bovine serum albumin. At 37 degrees C, initial uptake of [35S]BSP was 5-10-fold that at 4 degrees C, linear for at least 15 min, saturable, inhibited by bilirubin, and reduced by greater than 70% after ATP depletion or isosmotic substitution of sucrose for NaCl in medium. Replacement of Na+ by K+ or Li+ did not alter uptake, whereas replacement of Cl- by HCO-3 or gluconate- reduced uptake by approximately 40%. Substitution of Cl- by the more permeant NO-3 enhanced initial BSP uptake by 30%. Efflux of [35S]BSP from cells to media was inhibited by 40% after ATP depletion or sucrose substitution. To confirm these results in a more physiologic system, transport of [3H]bilirubin was studied in isolated livers perfused with control medium or medium in which Cl- was replaced by gluconate-. Perfusion data analyzed by the model of Goresky, revealed 40-50% reductions in influx and efflux with gluconate- substitution. These results are consistent with existence of a Cl-/organic anion-exchange mechanism similar to that described by others in renal tubules.
Assuntos
Cloretos/farmacologia , Fígado/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Bilirrubina/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Cloretos/metabolismo , Lítio/metabolismo , Cloreto de Lítio , Fígado/metabolismo , Masculino , Perfusão , Cloreto de Potássio/metabolismo , Ratos , Ratos Endogâmicos , Cloreto de Sódio/metabolismo , Sulfobromoftaleína/metabolismoRESUMO
A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.
Assuntos
Proteínas de Transporte/imunologia , Fígado/metabolismo , ATPases Translocadoras de Prótons/imunologia , Marcadores de Afinidade , Animais , Sequência de Bases , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Clonagem Molecular , Reações Cruzadas , Imuno-Histoquímica , Microscopia Eletrônica , Mitocôndrias Hepáticas/imunologia , Mitocôndrias Hepáticas/metabolismo , Testes de Precipitina , Ratos , Proteínas Recombinantes de Fusão/imunologia , Mapeamento por RestriçãoRESUMO
Recent work has found evidence of deterioration in water quality in the Elbow River upstream from Calgary, Alberta, Canada. We sampled this basin to describe spatial and temporal trends and factors that could be contributing to this deterioration. Sources near Calgary generally contributed most of the total phosphorus (TP) entering the river, and most total suspended solids (TSS) during low to average flows. During high flows, a large influx of TSS occurred further upstream. Sources of TP could include runoff from residential developments and agriculture, and groundwater, while re-suspension of bed material, erosion, and storm sewers may contribute TSS. Increasing trends in dissolved phosphorus (TDP) and ammonia suggest that sources in this reach are also contributing dissolved nutrients. Appreciable loading of nitrate + nitrite also occurred near Calgary, with a significant increasing trend in nitrate + nitrite occurring at every Elbow River site and several tributaries. Fecal coliforms have increased significantly over time at a downstream site. Runoff from residential developments, agriculture, or contributions from groundwater could account for this trend. In the 2003 bacterial source tracking study, DNA markers from ruminant animals were found in samples from most locations sampled in this basin, even at some headwaters sites, but no human markers were found.
Assuntos
Rios , Poluentes da Água/análise , Alberta , Amônia/análise , Bacteroides/genética , Campylobacter/isolamento & purificação , Carbono/análise , DNA Bacteriano/análise , Enterobacteriaceae/isolamento & purificação , Concentração de Íons de Hidrogênio , Nitratos/análise , Nitritos/análise , Nitrogênio/análise , Oxigênio/análise , Fósforo/análise , Rios/química , Rios/microbiologia , Temperatura , Microbiologia da Água , Abastecimento de ÁguaRESUMO
Metabolism of 125I-sulfobromophthalein (BSP) prepared by the chloramine-T method was studied in rats. 125I-BSP is removed rapidly from the circulation. However, as compared to BSP, its plasma clearance and biliary excretion are delayed, and its accumulation in the liver is prolonged. Although BSP and 125I-BSP show similar binding to albumin in serum, their binding properties to liver cytosolic proteins and to the liver cell plasma membrane organic anion binding protein (OABP) differ. In contrast to the X-, Y- and Z-protein binding of BSP, 125I-BSP binds predominantly to a high molecular weight protein and only a small proportion of 125I-BSP binds to OABP.