title: Role of H(2)O(2) on the kinetics of low-affinity high-capacity Na(+)-dependent alanine transport in SHR proximal tubular epithelial cells.
abstract: The presence of high and low sodium affinity states for the Na(+)-dependent [(14)C]-l-alanine uptake in immortalized renal proximal tubular epithelial (PTE) cells was previously reported (Am. J. Physiol. 293 (2007) R538-R547). This study evaluated the role of H(2)O(2) on the Na(+)-dependent [(14)C]-l-alanine uptake of ASCT2 in immortalized renal PTE cells from Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). Na(+) dependence of [(14)C]-l-alanine uptake was investigated replacing NaCl with an equimolar concentration of choline chloride in vehicle- and apocynin-treated cells. Na(+) removal from the uptake solution abolished transport activity in both WKY and SHR PTE cells. Decreases in H(2)O(2) levels in the extracellular medium significantly reduced Na(+)-K(m) and V(max) values of the low-affinity high-capacity component in SHR PTE cells, with no effect on the high-affinity low-capacity state of the Na(+)-dependent [(14)C]-l-alanine uptake. After removal of apocynin from the culture medium, H(2)O(2) levels returned to basal values within 1 to 3h in both WKY and SHR PTE cells and these were found stable for the next 24h. Under these experimental conditions, the Na(+)-K(m) and V(max) of the high-affinity low-capacity state were unaffected and the low-affinity high-capacity component remained significantly decreased 1day but not 4days after apocynin removal. In conclusion, H(2)O(2) in excess is required for the presence of a low-affinity high-capacity component for the Na(+)-dependent [(14)C]-l-alanine uptake in SHR PTE cells only. It is suggested that Na(+) binding in renal ASCT2 may be regulated by ROS in SHR PTE cells.
authors.affiliation: Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal.
authors.forename: Vanda
authors.initials: V
authors.lastname: Pinto
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Pinho
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Jose
authors.forename: Patrício
authors.initials: P
authors.lastname: Soares-da-Silva
mesh_terms: D000409:Alanine / Q000378:metabolism*; D026962:Amino Acid Transport System ASC / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D012964:Sodium / Q000378:metabolism*
chemical_list: D026962:Amino Acid Transport System ASC; D015778:Minor Histocompatibility Antigens; C498849:Slc1a5 protein, rat; D012964:Sodium; D006861:Hydrogen Peroxide; D000409:Alanine