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

Abstract

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 μM). In HbA cells, K+‐Cl− cotransport was O2 dependent. At low oxygen tensions (PO2s) the transporter was inactive and refractory to low pH, swelling or urea. Cl−‐independent K+ influxes in sickle cells were elevated at low PO2s, as previously reported. Cl−‐dependent K+ influxes were large at both high and low PO2s, whether stimulated by swelling, H+ ions or urea. In the absence of O2, Cl−‐dependent K+ influxes were similar in magnitude to those measured at high PO2s. The minimum for Cl−‐dependent K+ influx was observed at PO2s of about 40‐70 mmHg. 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 Cl− dependent under both conditions. In HbS red blood cells, reduction of extracellular Ca2+, addition of 1 mM Mg2+ or nitrendipine (10 μM) to the saline had no effect. Inhibitors of K+‐Cl− cotransport, [(dihydroindenyl)oxy] alkanoic acid (DIOA; 100 μM) or calyculin A (0·1 μM), inhibited influxes by a similar magnitude to Cl− substitution. Results are significant for the pathophysiology of sickle cell disease. Low pH and urea are able to stimulate KCl loss from sickle cells, leading to cellular dehydration, even in regions of low PO2.

Regulation of Na+‐K+‐2Cl− cotransport in turkey red cells: the role of oxygen tension and protein phosphorylation

“…NKCC in turkey: Palfrey & Greengard, 1981; â_NHE in trout: Motais et al 1987;Nikinmaa et al 1987b;and KCC in trout: Borgese et al 1991). The response of KCC to Oµ has been well characterized in trout (Nielsen et al 1992;Cossins et al 1994) and other species (carp: Jensen, 1992; horse: Gibson et al 1993;sheep: Campbell & Gibson, 1998; human HbA and HbS red cells: Gibson et al 1998). Physiological POµ levels activate the cotransporter (Nielsen et al 1992;Honess et al 1996; and are required before it can respond to other stimuli such as swelling, acid or urea.…”

Investigation of Some Biochemical Parameters in Subject with HbSC and HbSS in Saki West Local Government - a Rural Community in Nigeria

“…When serum creatinine is elevated the disease has reached an advanced stage and lead to renal failure. Deoxygenation of sickle cell is known to increase cation permeability of sodium (Na + ), potassium (K + ) and calcium (Ca 2+ ) (Vitoux et al, 1989;Rhoda et al, 1990;Gibson et al, 1998). Urea, an end product of protein metabolism, is excreted by the kidney.…”

Ion Transport

“…This can be achieved by a decrease in cell volume mediated by the KCl cotransporter (KCC l). Unlike in normal human red cells, KCC1 in SCs remains active at low O 2 tensions (Po,s) (Gibson et al 1998). Thus KCC1 in SCs can be stimulated-by H+ ions and urea, even in hypoxic tissues, e.g.…”

“…RBC samples were handled as described previously, with an additional filtration through glass wool to ensure removal of nucleated cells. KCC activity was measured at 37°C under various conditions (saline osmolality 290 mosmol kg-1 at pH 7-4, unless indicated otherwise; see Gibson et al 1998 We conclude that KCC in RBCs from human cord blood has very similar properties to that seen in adult blood samples.…”

“…We used various manipulations (CO, CDNB, nitrite) to investigate the relationship between GSH, Hb conformation and activity of KCC in equine RBCs , a system whose O 2 response has been well characterised. K+ influx was used as a measure of KCC (Gibson et al 1998); metHb and GSH levels were determined using standard methods. Unless otherwise stated, values are given as means ± S.D.…”