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.
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D006096:Gramicidin / Q000494:pharmacology; D006168:Guinea Pigs; D007473:Ion Channels / Q000378:metabolism; D007668:Kidney / Q000032:analysis / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D009682:Magnetic Resonance Spectroscopy; D008871:Microvilli / Q000032:analysis / Q000201:enzymology / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D012964:Sodium / Q000032:analysis / Q000378:metabolism*
D007473:Ion Channels; D010710:Phosphates; D006096:Gramicidin; D012964:Sodium; D000469:Alkaline Phosphatase