Differential oxygen sensitivity of the K+-Cl- cotransporter in normal and sickle human red blood cells

Abstract

1. K+ influx and efflux were measured in normal (HbA) and sickle (HbS) red blood cells to investigate the interaction of swelling, H+ ions and urea with O2 (0 to 150 mmHg O2) in the presence of ouabain and bumetanide (both 100 microM). 2. In HbA cells, K+-C1- cotransport was O2 dependent. At low oxygen tensions (PO2s) the transporter was inactive and refractory to low pH, swelling or urea. 3. C1--independent K+ influxes in sickle cells were elevated at low PO2s, as previously reported. C1--dependent K+ influxes were large at both high and low PO2s, whether stimulated by swelling, H+ ions or urea. In the absence of O2, C1--dependent K+ influxes were similar in magnitude to those measured at high PO2s. The minimum for C1--dependent K+ influx was observed at PO2s of about 40-70 mmHg. 4. K+ efflux from HbS cells was stimulated by the addition of urea (500 mM). The rate constants were of similar magnitude whether measured at high PO2 or in the absence of O2, and were predominantly C1- dependent under both conditions. 5. In HbS red blood cells, reduction of extracellular Ca2+, addition of 1 mM Mg2+ or nitrendipine (10 microM) to the saline had no effect. Inhibitors of K+-C1- cotransport, [(dihydroindenyl)oxy] alkanoic acid (DIOA; 100 microM) or calyculin A (0.1 microM), inhibited influxes by a similar magnitude to C1- substitution. 6. Results are significant for the pathophysiology of sickle cell disease. Low pH and urea are able to stimulate KC1 loss from sickle cells, leading to cellular dehydration, even in regions of low PO2.

Figure 1. The effect of oxygen tension on swelling/H⁺-stimulated K⁺ influx in normal human red blood cells

K⁺ influx (mmol (l cells)⁻¹ h⁻¹) was measured in the presence and absence of Cl⁻ (substituted with NO₃⁻), 260 mosmol kg⁻¹, pH 7.0. Cl⁻-dependent K⁺ influx was calculated as the difference in influx ± Cl⁻. Points represent the means ±s.e.m. for 6 samples from different individuals.

Figure 2. The effect of oxygen tension on urea-stimulated K⁺ influx in normal human red blood cells

K⁺ influx (mmol (l cells)⁻¹ h⁻¹) was measured in the presence and absence of Cl⁻ (substituted with NO₃⁻), pH 7.4, with 500 mm urea. Cl⁻-dependent K⁺ influx was calculated as the difference in influx ± Cl⁻. Points represent the means ±s.e.m. for 5 samples from different individuals.

Figure 3. The effect of oxygen tension on swelling-stimulated K⁺ influx in sickle human red blood cells

K⁺ influx (mmol (l cells)⁻¹ h⁻¹) was measured in the presence and absence of Cl⁻ (substituted with NO₃⁻) in samples swollen anisosmotically by 10 %, pH 7.4. Cl⁻-dependent K⁺ influx was calculated as the difference in influx ± Cl⁻. Points represent the means ±s.d. for triplicate determinations on a single sample, representative of 3 other experiments.

Figure 4. The effect of oxygen tension on H⁺-stimulated K⁺ influx in human sickle red blood cells

K⁺ influx (mmol (l cells)⁻¹ h⁻¹) was measured in the presence and absence of Cl⁻ (substituted with NO₃⁻), at pH 7.0. Cl⁻-dependent K⁺ influx was calculated as the difference in influx ± Cl⁻. Points represent the means ±s.d. for triplicate determinations on a single sample, representative of 3 other experiments.

Figure 5. The effect of oxygen tension on urea-stimulated K⁺ influx in human sickle red blood cells

K⁺ influx (mmol (l cells)⁻¹ h⁻¹) was measured in the presence and absence of Cl⁻ (substituted with NO₃⁻), at pH 7.4 with 500 mm urea. Cl⁻-dependent K⁺ influx was calculated as the difference in influx ± Cl⁻. Points represent the means ±s.d. for triplicate determinations on a single sample, representative of 3 other experiments.

Figure 6. Reversibility of the effect of urea on K⁺ influx in human sickle cells

The cell sample was divided into three; two aliquots were pre-incubated for 60 min at 37 °C in the presence of urea (500 mm), one in its absence (control). The three aliquots were then washed, leaving urea present in only one of the urea-treated aliquots. K⁺ influx (mmol (l cells)⁻¹ h⁻¹) was then measured simultaneously in all aliquots. Histograms represent means ±s.d. for triplicate determinations on a single sample, representative of 2 others.