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Localization and functional characterization of the rat Oatp4c1 transporter in an in vitro cell system and rat tissues.

ABSTRACT: The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our in vitro studies with MDCKII cells showed that upon transfection rat Oatp4c1 polarizes to the apical membrane. In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature.

SUBMITTER: Kuo KL

PROVIDER: S-EPMC3387246 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Localization and functional characterization of the rat Oatp4c1 transporter in an in vitro cell system and rat tissues.

Kuo Kuei-Ling KL Zhu Haining H McNamara Patrick J PJ Leggas Markos M

PloS one 20120629 6

The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our in vitro studies with MDCKII cells showed that upo ...[more]

PMID: 22768102

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Project description:Previous work has demonstrated that human skeletal muscle and adipose tissue both express the GLUT5 fructose transporter, but to date the issue of whether this protein is also expressed in skeletal muscle and adipose tissue of rodents has remained unresolved. In the present study we have used a combination of biochemical and molecular approaches to ascertain whether rat skeletal muscle expresses GLUT5 protein and, if so, whether it possesses the capacity to transport fructose. An isoform-specific antibody against rat GLUT5 reacted positively with crude membranes prepared from rat skeletal muscle. A single immunoreactive band of approx. 50 kDa was visualized on immunoblots which was lost when using anti-(rat GLUT5) serum that had been pre-adsorbed with the antigenic peptide. Subcellular fractionation of skeletal muscle localized this immunoreactivity to a single membrane fraction that was enriched with sarcolemma. Plasma membranes, but not low-density microsomes, from rat adipose tissue also displayed a single protein band of equivalent molecular mass to that seen in muscle. Reverse transcription-PCR analyses, using rat-specific GLUT5 primers, of muscle and jejunal RNA revealed a single PCR fragment of the expected size in jejunum and in four different skeletal muscle types. Sarcolemmal vesicles from rat muscle displayed fructose and glucose uptake. Vesicular uptake of glucose was inhibited by nearly 90% in the presence of cytochalasin B, whereas that of fructose was unaffected. To determine whether fructose could regulate GLUT5 expression in skeletal muscle, rats were maintained on a fructose-enriched diet for 4 days. This procedure increased jejunal and renal GLUT5 protein expression by approx. 4- and 2-fold respectively, but had no detectable effects on the abundance of GLUT5 protein in skeletal muscle or on fructose uptake in rat adipocytes. The present results show that GLUT5 is expressed in the sarcolemma of rat skeletal muscle and that it is likely to mediate fructose uptake in this tissue. Furthermore, unlike the situation in absorptive and re-absorptive epithelia, GLUT5 expression in insulin-sensitive tissues is not regulated by increased substrate supply.

Dataset Information

Localization and functional characterization of the rat Oatp4c1 transporter in an in vitro cell system and rat tissues.

Publications

Localization and functional characterization of the rat Oatp4c1 transporter in an in vitro cell system and rat tissues.

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