Article date: July 1994
By: Patrick S.K. Wong, Paul L. Barclay, Michael J. Newman, Edward J. Johns, in Volume 112, Issue 3, pages 881-886
This investigation set out to use 23Na n.m.r. spectroscopy to measure changes in intracellular levels of sodium in isolated suspensions of rat proximal tubules. The effects of temperature, an inhibitor of the sodium pump and known natriuretic drugs on intracellular sodium content of such tubular preparations were measured and compared with calcium channel antagonists where action at this level is unclear.
Rat kidneys were perfused with collagenase, roughly chopped, subjected to mechanical dispersion and washed to remove all traces of the enzyme. The proximal tubules were then purified and concentrated by Percoll density gradient centrifugation and then resuspended in buffer containing dysprosium tripolyphosphate shift reagent.
Distinct peaks corresponding to intracellular and extracellular sodium signals were observed when the tubules were placed into the n.m.r. spectrometer. As the temperature of the suspension rose to 37°C, there was an exponential decrease in sodium content, with a decay constant of 0.15 ± 0.02 min−1, which reached a stable level within 20 to 25 min. Addition of ouabain, 10−3m, resulted in a significant (P < 0.01) 30% increase in intracellular sodium content within 5 min which peaked at 70% 20 min later. Although acetazolamide (10−3m) significantly (P < 0.01) increased intracellular sodium content by 45%, amlodipine (10−4m) had no effect.
These data show that changes in the activity of the Na+/K+/ATPase have a considerable influence on the intracellular levels of sodium in proximal tubule cells. Inhibition of carbonic anhydrase activity resulted in a rise in intracellular sodium content which is compatible with its action to reduce the turnover rate of the Na+/(HCO3−)3 symporter. The lack of effect of amlodipine was consistent with the suggestion that it does not have a direct action on the sodium handling processes at the level of the proximal tubule.
This investigation set out to use 23Na n.m.r. spectroscopy to measure changes in intracellular levels of sodium in isolated suspensions of rat proximal tubules. The effects of temperature, an inhibitor of the sodium pump and known natriuretic drugs on intracellular sodium content of such tubular preparations were measured and compared with calcium channel antagonists where action at this level is unclear.
Rat kidneys were perfused with collagenase, roughly chopped, subjected to mechanical dispersion and washed to remove all traces of the enzyme. The proximal tubules were then purified and concentrated by Percoll density gradient centrifugation and then resuspended in buffer containing dysprosium tripolyphosphate shift reagent.
Distinct peaks corresponding to intracellular and extracellular sodium signals were observed when the tubules were placed into the n.m.r. spectrometer. As the temperature of the suspension rose to 37°C, there was an exponential decrease in sodium content, with a decay constant of 0.15 ± 0.02 min−1, which reached a stable level within 20 to 25 min. Addition of ouabain, 10−3m, resulted in a significant (P < 0.01) 30% increase in intracellular sodium content within 5 min which peaked at 70% 20 min later. Although acetazolamide (10−3m) significantly (P < 0.01) increased intracellular sodium content by 45%, amlodipine (10−4m) had no effect.
These data show that changes in the activity of the Na+/K+/ATPase have a considerable influence on the intracellular levels of sodium in proximal tubule cells. Inhibition of carbonic anhydrase activity resulted in a rise in intracellular sodium content which is compatible with its action to reduce the turnover rate of the Na+/(HCO3−)3 symporter. The lack of effect of amlodipine was consistent with the suggestion that it does not have a direct action on the sodium handling processes at the level of the proximal tubule.
DOI: 10.1111/j.1476-5381.1994.tb13162.x
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