Sodium-lithium exchange and sodium-potassium cotransport in human erythrocytes. Part 1: Evaluation of a simple uptake test to assess the activity of the two transport systems.

Abstract

of inward to outward transport is examined for each of the transport systems. The accompanying paper 14 reports on the results obtained when these tests were applied to erythrocytes of hypertensive patients. Materials and MethodBlood of normotensive, apparently healthy, individuals who were not taking medication was drawn into heparin. Plasma and buffy coat were removed after centrifugation (4500 x g). The erythrocytes were washed three times in 10 volumes of 150 mM choline chloride. In addition to the components listed, all solutions (305-315 mOsm»kg H 2 O"') contained 5 mM glucose, 1 mM inorganic phosphate, 10 mM morpholinopropane sulfonic acid, titrated to pH 7.4 at 37°C with tris-(hydroxymethyl)-aminomethane (Tris).Uptake of rubidium (Rb + ) and Li + was initiated by adding washed erythrocytes to 25 ml of incubation medium containing 0.2 mM ouabain (37°C, hematocrit 0.5% to 2.5%). When not otherwise stated, the media were 150 mM in chloride, sucrose being used to maintain tonicity in the magnesium (Mg 2+ ) media. Furosemide and phloretin were dissolved in dimethylsulfoxide and ethanol, respectively. The amount of the solvents present in the cell suspensions (0.1% to 0.2%, vol/vol) did not alter cotransport or countertransport. The final concentrations of furosemide and phloretin were 0.5 and 0.1 mmolHiter suspension" 1 respectively.The influx period was generally terminated after 1 hour of incubation by transferring the suspensions into an ice bath and centrifugation at 0°C (10,000 X g). The cells were then washed three times with a 7-to 25-fold excess of cold choline chloride. Following the washes the cells were lysed in a 5-to 25-fold excess of distilled water containing 6% 1-butanol (vol/vol).In Li + release experiments, the erythrocytes were preloaded for 3 hours in 150 mM LiCl, washed six times with cold Mg 2+ -sucrose medium (75 mM MgCl 2 , 85 mM sucrose), and resuspended in Mg 2+ or Na + media (hematocrit 5%, 37°C, pH 7.4). Li + was analyzed in the supernatants obtained after each 5, 25 and 45 minutes of incubation. Release rates (/nmole • ml cells" 1 • hr" 1 ) were calculated with the formula:where s is the slope of the linear increase in external Li + concentration over time (r > 0.99), MCHC is the mean cellular hemoglobin content (calculated from hemoglobin and hematocrit measurements in fresh blood), and Hb, is the hemoglobin content of the cell suspension (hemoglobin was determined as cyanmethemoglobin). This technique is similar to that described by Canessa et al.

Racial differences in erythrocyte cation transport.

“…Such a dissociation has no obvious mechanistic basis since the cotransport system in human erythrocytes is thought to operate with tight 1:1 stoichiometry for sodium and potassium. However, controversy exists because some workers have 27 while others have not 23 demonstrated furosemide-sensitive, ouabain-insensitive fluxes of potassium in the absence of sodium or lithium ions. The possibility that blacks possess a qualitatively different furosemide-sensitive potassium efflux system is currently under investigation in this laboratory.…”

Red cell sodium countertransport and cotransport in normotensive and hypertensive blacks.

“…Unfortunately, some investigators have used flux assays under inadequate kinetic conditions to isolate a single transport pathway at its maximal rate. Li influx 39 or "Na influx 40 into fresh cells is dependent on intracellular Na and does not measure the V,^ of the Na exchange system; it also comprises an inward diffusional leak pathway and a small fraction of the Na-K cotransport. 39 -40 Furosemide-sensitive Na efflux from fresh cells, being sigmoidally dependent on intracellular Na,' 4 cannot discriminate whether the change in rate is caused by different cell Na, different affinity, or different maximal rate of the Na-K cotransport.…”

Sodium and potassium ion transport accelerations in erythrocytes of DOC, DOC-salt, two-kidney, one clip, and spontaneously hypertensive rats. Role of hypokalemia and cell volume.

“…39 Moreover, human erythrocytes do not lose K + nor do they shrink during K + depletion to an extent comparable to rat erythrocytes. 32 - 45 Thus, it is not to be expected that K + deficiency induces accelerations of furosemide-sensitive transport in human erythrocytes similar to those seen in rat red blood cells.…”