NMR measurements of intracellular sodium in the rabbit proximal tubule
Gullans, S.R.; Avison, M.J.; Ogino, T.; Giebisch, G.; Shulman, R.G.
American Journal of Physiology 249(1 Pt 2): F160-F168
1985
ISSN/ISBN: 0002-9513 PMID: 2409818 Document Number: 253808
The present study evaluated the use of nuclear magnetic resonance (NMR) spectroscopy to minotor directly and continuously intracellular Na levels in rabbit renal cortical tubule suspensions. When the paramagnetic shift reagent dysprosium tripolyphosphate was added to the extracellular medium, it was possible to resolve signals from intracellular and extracellular Na without adversely affecting cellular viability. An efflux of intracellular Na against a significant concentration gradient was observed when Na loaded cells were warmed from 4.degree.-37.degree. C. At 37.degree. C in steady state, inhibition of Na+-K+-ATPase activity by ouabain increased intracellular Na content in a dose-dependent and time-dependent manner. A biphasic time course of increased intracellular Na following ouabain (10-3 M) suggested that the sodium permeability of the plasma membrane may decrease following pump inhibition, thus limiting Na influx. Nystatin, an agent known to facilitate Na entry across cell membranes, increased intracellular Na 5-fold. In another series of experiments several maneuvers were performed to ascertain the fraction of intracellular Na that was NMR visible. Quantitative assessment of either an efflux or influx of Na indicated that the NMR visibility of the transported sodium was 100%. Disruption of the cell membranes with Triton X-100 showed that the entire pool of intracellular Na was 100% NMR visible. Based on a comparison of intracellular and extracellular signal intensities, intracellular Na content was 185 .+-. 10 nmol/mg protein, which, using an estimate of intracellular volume (4.2 .mu.l/mg protein), is .apprx. 44 mM.