Differentiated thick ascending limb (TAL) cultured cells derived from SV40 transgenic mice express functional apical NHE2 isoform: effect of nitric oxide.
Studying the apical Na/H exchanger NHE2 is difficult in the intact thick ascending limb (TAL) because of its weak expression and transport activity compared with the co-expressed NHE3. From a mouse transgenic for a recombinant plasmid adeno-SV(40) (PK4), we developed an immortalized TAL cell line, referred to as MKTAL, which selectively expresses NHE2 protein and activity. The immortalized cells retain the main properties of TAL cells. They have a stable homogeneous epithelial-like phenotype, express SV(40) T antigen and exhibit polarity with an apical domain bearing few microvilli and separated from lateral domains by typical epithelial-type junctional complexes expressing ZO1 protein. Tamm-Horsfall protein is present on the apical membrane. MKTAL cells express NHE2 and NHE1 proteins but not NHE3 and NHE4, whereby NHE2 protein is expressed selectively in the apical domain of the plasma membrane. NHE2 contributed about half of the total Na/H exchange activity. mRNAs for the Na-K-2Cl cotransporter-2 (NKCC2) and the anion exchangers AE2 and AE3 were also present. While acute exposure to NO donors did not alter NHE2 activity, chronic exposure inhibited NHE2 activity selectively and down-regulated NHE2 mRNA abundance. In conclusion, MKTAL cells retain structural and functional properties of their in vivo TAL counterparts and express functional NHE2 protein in the apical membrane, which may be inhibited by NO. Thus, MKTAL cells may be an appropriate model for studying the cellular mechanisms of NHE2 regulation.
Bourgeois
Soline
S
INSERM Unité 356, IFR 58, Université Paris VI, Institut des Cordeliers, 15-21 rue de l'Ecole de Médecine, 75270, Paris Cedex 6, France. bourgeoi@ccr.jussieu.fr
Rossignol
Patrick
P
Grelac
Françoise
F
Chalumeau
Cécile
C
Klein
Christophe
C
Laghmani
Kamel
K
Chambrey
Régine
R
Bruneval
Patrick
P
Duong
Jean-Paul
JP
Poggioli
Josiane
J
Houillier
Pascal
P
Paillard
Michel
M
Kellermann
Odile
O
Froissart
Marc
M
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001710:Biotin; D002478:Cells, Cultured; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D005260:Female; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D015151:Immunoblotting; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis* / Q000235:genetics; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D064466:Solute Carrier Family 12, Member 1; D019108:Tight Junctions / Q000378:metabolism / Q000648:ultrastructure
D000952:Antigens, Polyomavirus Transforming; D027963:Chloride-Bicarbonate Antiporters; D007527:Isoenzymes; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; C578715:Slc12a1 protein, mouse; C513111:Slc9a2 protein, mouse; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D009569:Nitric Oxide; D001710:Biotin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases