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Molecular cloning of the cDNA for an MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is regulated by hypertonicity.

ABSTRACT: Cells in the hypertonic renal medulla maintain their intracellular ion concentration at isotonic levels, despite much higher concentrations of extracellular electrolytes, by accumulating high concentrations of nonperturbing small organic solutes termed osmolytes. Taurine has been identified as a nonperturbing osmolyte in the renal medulla and Madin-Darby canine kidney (MDCK) cells. In hypertonic medium, the increased accumulation of taurine in MDCK cells is the result of increased activity of a Na(+)- and Cl(-)-dependent taurine transporter. We have isolated a cDNA encoding a Na(+)- and Cl(-)-dependent taurine transporter, whose sequence corresponds to a protein of 655 amino acids with significant amino acid sequence similarity to previously cloned Na(+)- and Cl(-)-dependent transporters, including the MDCK cell betaine/gamma-aminobutyric acid transporter and several brain neurotransmitter transporters. Northern hybridization indicates that mRNA for the taurine transporter is present in renal cortex and medulla, ileal mucosa, brain, liver, and heart. The abundance of mRNA for the taurine transporter is increased in MDCK cells cultured in hypertonic medium, suggesting that regulation of transport activity by medium hypertonicity occurs at the level of mRNA accumulation.

SUBMITTER: Uchida S

PROVIDER: S-EPMC49891 | biostudies-other | 1992 Sep

REPOSITORIES: biostudies-other

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Molecular cloning of the cDNA for an MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is regulated by hypertonicity.

Uchida S S Kwon H M HM Yamauchi A A Preston A S AS Marumo F F Handler J S JS

Proceedings of the National Academy of Sciences of the United States of America 19920901 17

Cells in the hypertonic renal medulla maintain their intracellular ion concentration at isotonic levels, despite much higher concentrations of extracellular electrolytes, by accumulating high concentrations of nonperturbing small organic solutes termed osmolytes. Taurine has been identified as a nonperturbing osmolyte in the renal medulla and Madin-Darby canine kidney (MDCK) cells. In hypertonic medium, the increased accumulation of taurine in MDCK cells is the result of increased activity of a ...[more]

PMID: 1518851

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Project description:Our previous studies with a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) transfected with FLAG-labeled alpha, beta, and gamma subunits of epithelial Na(+) channel (ENaC) showed that, although most of the short-circuit current (I (sc)) was amiloride sensitive (AS-I (sc)), there was also an amiloride-insensitive component (NS-I (sc)) due to Cl(-) secretion (Morris and Schafer, J Gen Physiol 120:71-85, 2002). In the present studies, we observed a progressive increase in NS-I (sc) and a corresponding decrease in AS-I (sc) during experiments. There was a significant negative correlation between AS-I (sc) and NS-I (sc) both in the presence and absence of treatment with cyclic adenosine monophosphate (cAMP). NS-I (sc) could be attributed to both cystic fibrosis transmembrane conductance regulator (CFTR) and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive Ca(2+)-activated Cl(-) channel (CaCC). Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions, or addition of hexokinase (6 U/ml), prevented the time-dependent changes and increased AS-I (sc) by 40-60%, with a proportional decrease in NS-I (sc). Addition of 100 muM adenosine triphosphate (ATP) in the presence of luminal amiloride produced a transient four-fold increase in NS-I (sc) that was followed by a sustained increase of 50-60% above the basal level. ATP release from the monolayers, measured by bioluminescence, was found to occur across the apical but not the basolateral membrane, and the apical release was tripled by cAMP treatment. These data show that constitutive apical ATP release, which occurs under both basal and cAMP-stimulated conditions, underlies the time-dependent rise in Cl(-) secretion and the proportional fall in ENaC-mediated Na(+) absorption in FL-MDCK cells. Thus, endogenous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells, and it may underlie at least some of the observed interaction between Cl(-) secretion and Na(+) absorption.

Dataset Information

Molecular cloning of the cDNA for an MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is regulated by hypertonicity.

Publications

Molecular cloning of the cDNA for an MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is regulated by hypertonicity.

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