pH dependence of Na+/myo-inositol cotransporters in rat thick limb cells

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 [ 3 H] myo- inositol ([ 3 H]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 [ 3 H]MI uptake in BLMV and LMV. Phlorizin inhibited Na + gradient-dependent initial [ 3 H]MI uptake in both preparations, with IC 50 values of 565 and 29 μmol/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 [ 3 H]MI uptake showed Michaelis-Menten kinetics in both preparations, with similar V max but different K m values of 51 and 107 μmol/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 (pH i ) was decreased from 8.0 to 6.0 at extravesicular pH (pH e ) 8.0, and as pH e was increased from 6.0 to 8.0 at pH i 6.0. Maximal activation was observed at pH i 6.5 and pH e 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 pH e and pH i .