Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC191

Poster Communications

Oxygen dependence of membrane transport in red blood cells from HbSC sickle cell patients

A. Hannemann1, D. C. Rees2, J. S. Gibson1

1. Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom. 2. Molecular Haematology, King's College Hospital, London, United Kingdom.

  • Figure 1: Membrane transport in blood cells from HbSC patients at different oxygen tensions. Symbols represent means±S.E.M (n≥3). Full deoxygenation (0mmHg vs. 100mmHg(normoxia)) significantly changed all components of K+ influx and cell sickling (p<0.02, Student's t-test).

Sickle cell disease (SCD) represents one of the commonest severe inherited disorders with millions of sufferers worldwide. Although 1/3rd SCD patients are heterozygous for HbS and HbC (HbSC genotype), this group of patients has been relatively poorly studied. Differences are apparent when the clinical condition of HbSC patients is compared with that of HbSS homozygotes. Pathogenesis in both may involve dehydration and sickling of their red blood cells (RBCs) and understanding their behaviour is therefore important. Notwithstanding, little information is available pertaining to the characteristics of RBCs in HbSC patients (1, 2). Here we report the activity of the most important cation permeabilities across the full range of physiological O2 tensions. Routine discarded blood samples were obtained from patients with HbSC disease using EDTA as an anticoagulant. RBCs were washed and incubated in saline comprising (in mM): 145 NaNO3, 7.5 KNO3, 1.1 CaCl2, 10 MOPS and 5 glucose (pH 7.4, 37°C), or saline in which Cl- replaced NO3-. RBCs were pre-equilibrated at 20% haematocrit at the required O2 (using a Wösthoff gas mixing pump) in Eschweiler tonometers for 15min, before diluting 10-fold into saline at the same O2 tension (3). 86Rb+ was added as a K+ congener to measure activity over 10min of the K+-Cl- cotransporter (KCC, as Cl- dependent K+ influx), the Ca2+-activated K+ channel (Gardos channel, as 5µM clotrimazole-sensitive K+ influx) and the deoxygenation-induced cation channel (Psickle, as Cl--independent K+ influx) in cells swollen anisotonically by 10% at pH7. Ouabain (100µM) and bumetanide (10µM) were present to inhibit the Na+/K+ pump and the Na+-K+-2Cl- cotransporter. RBC samples were also fixed in saline containing 0.3% glutaraldehyde for analysis of cell shape. When fully oxygenated, mean KCC activity was 2.0±0.34 mmol.(l cells.h)-1 (range 0.37-3.6, n=10) and few RBCs showed evidence of sickling 0.2±0.1% (n=10). Upon deoxygenation KCC activity declined to 0.32±0.15 whilst Gardos channel activity and that of Psickle increased to 1.55±0.4 (range 0.36-3.55, n=10) and 0.58±0.09 (range 0.23-1.21, n=10), respectively. Inactivation of KCC, activation of the Gardos channel and Psickle, and onset of sickling all occurred at around the P50 for O2 saturation of haemoglobin (Fig. 1). During slow deoxygenation HbSC RBCs sickled with a t½ of 31±3.4min (n=5) compared to 19±3min (n=4) for HbSS RBCs. Although the behaviour of the Gardos channel and Psickle in HbSC cells is similar to that seen in RBCs from HbSS patients, strong inhibition of KCC on haemoglobin deoxygenation is only seen in HbSC samples whilst onset of sickling upon deoxygenation was slower. These findings illustrate important differences in the permeability of RBCs from HbSC patients which may have clinical relevance.

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