ABSTRACT
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.
Subject(s)
Acid-Base Equilibrium/physiology , Heat-Shock Proteins/metabolism , Hydrogen-Ion Concentration , Loop of Henle/metabolism , Membrane Proteins , Symporters/metabolism , Animals , Biological Transport/drug effects , Biological Transport/physiology , Inositol/pharmacokinetics , Kidney Medulla/metabolism , Male , Phlorhizin/pharmacology , Rats , Rats, Sprague-Dawley , Sodium/pharmacokinetics , TritiumABSTRACT
In the proximal tubule, the apical Na(+)/H(+) exchanger identified as NHE3 mediates most NaCl and NaHCO(3) absorption. The purpose of this study was to analyze the long-term regulation of NHE3 during alkalosis induced by dietary NaHCO(3) loading and changes in NaCl intake. Sprague-Dawley rats exposed to a low-NaCl, high-NaCl, or NaHCO(3) diet for 6 days were studied. Renal cortical apical membrane vesicles (AMV) were prepared from treated and normal rats. Na(+)/H(+) exchange was assayed as the initial rate of (22)Na(+) uptake in the presence of an outward H(+) gradient. (22)Na(+) uptake measured in the presence of high-dose 5-(N-ethyl-N-isopropyl) amiloride was not different among models. Changes in NaCl intake did not affect NHE3 activity, whereas NaHCO(3) loading inhibited (22)Na(+) uptake by 30%. AMV NHE3 protein abundance assessed by Western blot analysis was unaffected during changes in NaCl intake. During NaHCO(3) loading, NHE3 protein abundance was decreased by 65%. We conclude that proximal NHE3 adapts to chronic metabolic acid-base disorders but not to changes in dietary NaCl intake.
