pH dependence of Na+/myo-inositol cotransporters in rat thick limb cells.
BACKGROUND
To balance medullary interstitium hypertonicity generated by transepithelial NaCl absorption, medullary thick ascending limb (MTAL) cells accumulate myo-inositol (MI). Expression of Na+-MI cotransporter (SMIT) mRNA in TAL is correlated with the NaCl absorption rate. Our present study aimed to determine the plasma membrane location and functional properties of the Na+-MI cotransporter in MTAL cells.


METHODS
Preparation of basolateral (BLMV) and luminal (LMV) membrane vesicles were simultaneously isolated from purified rat MTAL suspension, and uptake of [3H]myo-inositol ([3H]MI) was used to assess Na+-MI cotransport activity.


RESULTS
In the presence of an inside-negative membrane potential, imposing an inwardly-directed Na+-gradient versus tetramethylammonium (TMA) stimulated the initial [3H]MI uptake in BLMV and LMV. Phlorizin inhibited Na+ gradient-dependent initial [3H]MI uptake in both preparations, with IC50 values of 565 and 29 micromol/L in BLMV and LMV, respectively. 2-0,C-methylene myo-inositol (MMI), a competitive inhibitor of MI transport, only inhibited the BLMV Na+-MI cotransporter. Phlorizin-sensitive Na+ gradient-dependent initial [3H]MI uptake showed Michaelis-Menten kinetics in both preparations, with similar Vmax but different Km values of 51 and 107 micromol/L in BLMV and LMV, respectively. Finally, BLMV but not LMV Na+-MI cotransporter exhibited a marked pH dependence with sigmoidal patterns of activation, as intravesicular pH (pHi) was decreased from 8.0 to 6.0 at extravesicular pH (pHe) 8.0, and as pHe was increased from 6.0 to 8.0 at pHi 6.0. Maximal activation was observed at pHi 6.5 and pHe 7.5.


CONCLUSIONS
In rat MTAL cells, Na+-MI cotransporter activity is present in both BLM and LM, and has markedly different functional properties, indicating the presence of distinct transporters. Basolateral Na+-MI cotransporter activity is maximal at physiological pH values of MTAL cells and interstitium, and a powerful modulation of the transporter activity may be exerted by pHe and pHi.
Eladari
Dominique
D
Institut National de la Santé et de la Recherche Médicale, Unité 356, Université Pierre et Marie Curie, Institut Fédératif de Recherche 58 and Hôpital Européen Georges Pompidou, Assistance-Publique, Hôpitaux de Paris, Paris, France.
Chambrey
Régine
R
Pezy
Françoise
F
Podevin
René-Alexandre
RA
Paillard
Michel
M
Leviel
Françoise
F
D000136:Acid-Base Equilibrium / Q000502:physiology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000493:pharmacokinetics; D007679:Kidney Medulla / Q000378:metabolism; D008138:Loop of Henle / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins; D010695:Phlorhizin / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000493:pharmacokinetics; D027981:Symporters / Q000378:metabolism*; D014316:Tritium
D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D027981:Symporters; D014316:Tritium; C073884:SLC5A3 protein, human; D007294:Inositol; D012964:Sodium; D010695:Phlorhizin