Correction for the adverse influence of sodium-potassium cotransport on apparent sodium-lithium countertransport activity in human erythrocytes.
BACKGROUND
Erythrocyte sodium-lithium countertransporter (SLC) has traditionally been characterised as the sodium-stimulated lithium efflux from lithium-loaded erythrocytes. Concurrent activity of the sodium-potassium cotransporter (NKCC) can be expected to lead to imprecise estimates of the activity of the SLC. In the present study, we have characterised this methodological problem and have shown that it can be corrected with the inclusion of bumetanide in the physiological salt solution.


METHODS
Lithium efflux was studied in lithium-loaded erythrocytes from 35 healthy, normotensive subjects. Erythrocytes were divided into two identical samples (A and B) and lithium efflux characteristics in both samples studied simultaneously by incubating aliquots from each in 10 media of differing external sodium concentrations. Efflux media employed for A and B were the same except for 0.02 mM bumetanide in the media used in B.


RESULTS
Increased external sodium was associated with increasing lithium efflux both in the absence and presence of bumetanide; efflux rates were consistently lower in media containing bumetanide (P<.05 in all cases). As external sodium increased, bumetanide-sensitive lithium efflux decreased in a manner that correlated inversely with external sodium concentration (r=-.77, P<.01). A small, nonsignificant increase in SLC activity was observed between measurements made under control conditions (median [range] in mmol Li/l RBC h; 0.272 [0.098-0.491]) and those made in the presence of bumetanide (0.286 [0.135-0.650]; P=.064). Bumetanide did not influence maximal rate of turnover or the affinity constant for external sodium. In contrast, the ratio of these variables was lower when determined in the absence than in the presence of bumetanide (5.5 [1.5-14.6] vs. 6.9 [2.8-24.2], respectively; P<.05).


CONCLUSIONS
This work shows that a component of lithium efflux mediated by the NKCC changes substantially with alterations in external sodium, resulting in a variable contribution of this second transport pathway to apparent SLC activity. To eliminate this variability, bumetanide should be included in all media when studying SLC, and the relationship to external sodium concentration determined.
Hardman
Timothy C
TC
Niche Science and Technology Ltd., 4B Lonsdale Road, NW6 6RD, London, UK. niche@dnet.co.uk
Morrish
Zoë
Z
Patel
Mayur
M
Chalkley
Sylvia
S
Noble
Mark I M
MI
D000328:Adult; D017920:Antiporters / Q000378:metabolism*; D002034:Bumetanide / Q000494:pharmacology; D002478:Cells, Cultured; D004232:Diuretics / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006405:Hematology / Q000379:methods*; D006801:Humans; D008094:Lithium / Q000378:metabolism*; D008297:Male; D015203:Reproducibility of Results; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*
D017920:Antiporters; D004232:Diuretics; D028021:Sodium-Potassium-Chloride Symporters; C045885:sodium-lithium countertransporter; D002034:Bumetanide; D008094:Lithium; D012964:Sodium