title: Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.
abstract: The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues.
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK 5230 Odense M, Denmark.
authors.forename: Rune Nørgaard
authors.initials: RN
authors.lastname: Rasmussen
authors.affiliation: Drug Transporters in ADME, Department of Pharmacy, Faculty of Health and Medical Sciences, Universitetsparken 2, DK 2100, Copenhagen, Denmark.
authors.forename: Candela
authors.initials: C
authors.lastname: Lagunas
authors.affiliation: Drug Transporters in ADME, Department of Pharmacy, Faculty of Health and Medical Sciences, Universitetsparken 2, DK 2100, Copenhagen, Denmark.
authors.forename: Jakob
authors.initials: J
authors.lastname: Plum
authors.affiliation: Pharmaceutical Science and CMC Biologics, H. Lundbeck A/S, DK 2500, Valby, Denmark.
authors.forename: René
authors.initials: R
authors.lastname: Holm
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK 5230 Odense M, Denmark. Electronic address: cun@sdu.dk.
authors.forename: Carsten Uhd
authors.initials: CU
authors.lastname: Nielsen
mesh_terms: D000622:Aminolevulinic Acid; D000818:Animals; D018938:Caco-2 Cells; D002460:Cell Line; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D018374:LLC-PK1 Cells; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009539:Nicotinic Acids / Q000494:pharmacology; D009557:Nipecotic Acids / Q000494:pharmacology; D009994:Osmolar Concentration; D009995:Osmosis; D011392:Proline / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013552:Swine; D013654:Taurine / Q000494:pharmacology; D020888:Vigabatrin / Q000494:pharmacology; D015091:beta-Alanine / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology; D000099247:Taurine Transporters
chemical_list: D000622:Aminolevulinic Acid; D007555:Isoxazoles; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009539:Nicotinic Acids; D009557:Nipecotic Acids; D011392:Proline; D012333:RNA, Messenger; D013654:Taurine; D020888:Vigabatrin; D015091:beta-Alanine; D005680:gamma-Aminobutyric Acid; D000099247:Taurine Transporters; C030278:nipecotic acid; C015910:guvacine; C015542:gaboxadol
keywords: GABA; GABA-mimetics; SKPT cells; TauT; Taurine transporter; Vigabatrin