NMR measurements of intra- and extravesicular sodium in renal microvilli

Previous attempts to separate the nuclear magnetic resonances of intra- and extravesicular Na⁺ in brush-border membrane vesicles (BBMV) were unsuccessful and led to the proposal of rapid exchange of Na⁺ via sodium channels in BBMV. However, passive conductance of Na⁺ in this membrane has been found to be relatively small. This inconsistency prompted us to use a different shift reagent to reassess the issue. In guinea pig renal BBMV (15–30 mg protein/ml) equilibrated with Na⁺ (130 mequiv. 1), using the impermeant Na⁺ shift reagent dysprosium tripolyphosphate (3 mM), the resonances of intra- (3.3%) and extravesicular (96.7%) Na⁺ were resolved by 6 ppm. Increases in Na⁺ conductance induced by gramicidin D did not alter the characteristics of intra- and extravesicular Na⁺ resonances. By contrast, addition of glucose caused a transient increase in the area of the intravesicular Na⁺ resonance. The clear separation between the intra- and the extravesicular Na⁺ resonances allowed us to measure the relaxation times of Na⁺, which depend on its interactions with its immediate environment. The longitudinal relaxation time of intravesicular Na⁺ (13 ± 1 ms) was much shorter than that of the extravesicular Na⁺ (44.0 ± 0.4 ms). Thus, in intact renal BBMV, as well as in membranes treated with the cationophore gramicidin D, the exchange of Na⁺ between the intra- and the extravesicular compartments is slow on the NMR time scale, consistent with the low Na⁺ channel density of this membrane. In contrast, the increase in intravesicular Na⁺ induced by glucose, is consistent with a significant contribution of the glucose cotransport pathway to Na⁺ flux across these membranes. The short longitudinal relaxation time of Na⁺ in the intravesicular space indicates interaction of Na⁺ with BBMV binding sites or ordering of these ions in the intravesicular compartment.