Correction for the adverse influence of sodium-potassium cotransport on apparent sodium-lithium countertransport activity in human erythrocytes.

INTRODUCTION: 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). DISCUSSION: 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.

The apolipoprotein E2 allele modulates activity and maximal velocity of the sodium-lithium countertransporter.

BACKGROUND: Alterations in erythrocyte sodium-lithium countertransport (SLC) activity and its maximal velocity (Vmax) are associated with hypertension and hypertriglyceridemia. The presence of apolipoprotein (apo) E variants is associated with hypertriglyceridemia. This study investigated the relationship between apoE phenotype and SLC kinetics. METHODS: Cardiovascular risk factors and SLC kinetics were measured in 171 subjects and 69 controls. Apolipoprotein E phenotypes were determined by Western blotting. RESULTS: Patients were 51% male, aged 56+/-13 years, with a blood pressure (BP) of 134+/-22/81+/-11 mm Hg, total cholesterol of 6.71+/-1.57 (256+/-61 mg/dL); median triglycerides 1.65 mmol/L (146 mg/dL) (range, 0.31 to 9.85 mmol/L; 27 to 872 mg/dL) and high-density lipoprotein (HDL) 1.39+/-0.43 mmol/L (54+/-16.6 mg/dL); fasting glucose 4.91+/-0.61 mmol/L (88.5+/-11.0 mg/dL); median insulin 11.7 IU/L (range, 3.7 to 39.8 IU/L). Phenotype frequencies were E3/E3 56%, E2/E3 14%, E2/E2 1%, E3/E4 27%, and E4/E4 2%. The SLC activity, Vmax, and sodium affinity (Km) were not significantly different with respect to apoE phenotype in simple analysis by Kruskal Wallis test. However, in multiple regression analysis after exclusion of BP, a strong co-correlate of SLC activity, the presence of an apoE2 allele was associated reduced activity (beta = -0.061; P = .01) along with HDL:apoA1 ratio (beta = -0.170; P < .001), whereas for the kinetic parameter Vmax, associations were found with triglyceride (beta = 0.029; P = .04), HDL:apoA1 ratio (beta = -0.186; P = .03) and the presence of an apoE2 allele (beta = -0.089; P = .04). CONCLUSIONS: These findings suggest that the apoE phenotype may modulate SLC activity and that the presence of an apoE2 allele phenotype is associated with lower SLC activity and Vmax.