The Use of Radioisotopes to Characterise the Abnormal Permeability of Red Blood Cells from Sickle Cell Patients

Hannemann, Anke; Cytlak, Urszula; Wilkins, Robert J.; Ellory, J. Clive; Rees, David C.; Gibson, John S.

doi:10.5772/24426

Cited by 6 publications

(6 citation statements)

References 91 publications

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“…To determine the activity of the K + transport pathways, K + influx was measured at 37°C using 86 Rb + as a congener for K + (Dunham & Ellory, ; Hannemann et al . ). RBCs washed in N‐MBS were equilibrated in tonometers for 20 min then diluted 10‐fold into saline, pre‐equilibrated at the appropriate oxygen tension, at 260 mosmol kg −1 and pH 7.…”

Section: Methodsmentioning

confidence: 97%

Effects of 5‐hydroxymethyl‐2‐furfural on the volume and membrane permeability of red blood cells from patients with sickle cell disease

Hannemann

Cytlak

Rees

et al. 2014

The Journal of Physiology

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Key pointsr We addressed the hypothesis that the heterocyclic aldehyde 5-hydroxymethyl-2-furfural (5HMF) may act synergistically to ameliorate the complications of sickle cell disease through effects on red blood cell (RBC) membrane transport, in addition to its well-known action of increasing the oxygen affinity of the abnormal form of haemoglobin, HbS.r 5HMF was found to reduce deoxygenation-induced dehydration of RBCs, whether in response to maintained deoxygenation or cyclical deoxygenation/re-oxygenation.r Acting at low millimolar concentrations, 5HMF reduced the activity of deoxygenation-induced cation conductance (sometimes termed P sickle ), an effect which correlated with reduction in sickling. 5HMF similarly inhibited deoxygenation-induced activation of the Ca 2+ -activated K + channel (or Gardos channel), an effect not seen following pharmacologically mediated increases in intracellular Ca 2+ via the ionophore A23187. Deoxygenation-induced phosphatidylserine exposure, which is associated with Ca 2+ entry via P sickle , was also inhibited by 5HMF.r By contrast, effects of 5HMF on the K + -Cl − cotransporter (KCC) were modest, with slight inhibition following treatment with N-ethylmaleimide (NEM) to abolish activity of its regulatory protein kinases, but stimulation in RBCs untreated with NEM.r It would therefore appear that an important beneficial action of 5HMF, in addition to effects on HbS oxygen affinity, is reduction in P sickle -mediated Ca 2+ entry following RBC sickling, thereby inhibiting the deleterious sequelae of Gardos channel activation, RBC dehydration and also lipid scrambling. AbstractThe heterocyclic aldehyde 5-hydroxymethyl-2-furfural (5HMF) interacts allosterically with the abnormal form of haemoglobin (Hb), HbS, in red blood cells (RBCs) from patients with sickle cell disease (SCD), thereby increasing oxygen affinity and decreasing HbS polymerization and RBC sickling during hypoxia. We hypothesized that should 5HMF also inhibit the main cation pathways implicated in the dehydration of RBCs from SCD patients -the deoxygenation-induced cation pathway (P sickle ), the Ca 2+ -activated K + channel (the Gardos channel) and the K + -Cl − cotransporter (KCC) -it would have a synergistic effect in protection against sickling, directly through interacting with HbS, and indirectly through maintaining hydration and reducing [HbS]. This study was therefore designed to investigate the effects of 5HMF on RBC volume and K + permeability in vitro. 5HMF markedly reduced the deoxygenation-induced dehydration of RBCs whether in response to maintained deoxygenation or to cyclical deoxygenation/re-oxygenation. 5HMF was found to inhibit P sickle , an effect which correlated with its effects on sickling. Deoxygenation-induced activation of the Gardos channel and exposure of phosphatidylserine were also inhibited, probably indirectly via reduced entry of Ca 2+ through the P sickle pathway. Effects of 5HMF on KCC were more modest with a slight inhibition in N-ethylmaleimide (NEM, 1 mM)-treated RBCs and sti...

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Section: Methodsmentioning

confidence: 97%

Effects of 5‐hydroxymethyl‐2‐furfural on the volume and membrane permeability of red blood cells from patients with sickle cell disease

Hannemann

Cytlak

Rees

et al. 2014

The Journal of Physiology

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“…To determine the activity of the K + transport pathways, K + influx was measured at 37°C using 86 Rb + as a congener for K + (Dunham and Ellory ; Hannemann et al. ). RBCs washed in N‐MBS, preincubated without or with GBT1118 for 45 min, equilibrated in tonometers for 20 min, and then diluted 10‐fold into saline, also preequilibrated at the appropriate O 2 tension, at 260 mOsm·kg −1 and pH 7.…”

Section: Methodsmentioning

confidence: 99%

The effect of the antisickling compoundGBT1118 on the permeability of red blood cells from patients with sickle cell anemia

et al. 2019

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Sickle cell anemia ( SCA ) is one of the commonest severe inherited disorders. Nevertheless, effective treatments remain inadequate and novel ones are avidly sought. A promising advance has been the design of novel compounds which react with hemoglobin S (HbS) to increase oxygen (O 2 ) affinity and reduce sickling. One of these, voxelotor ( GBT 440), is currently in advanced clinical trials. A structural analogue, GBT 1118, was investigated in the current work. As RBC dehydration is important in pathogenesis of SCA , the effect of GBT 1118 on RBC cation permeability was also studied. Activities of P sickle , the Gardos channel and the KC l cotransporter ( KCC ) were all reduced. Gardos channel and KCC activities were also inhibited in RBC s treated with Ca 2+ ionophore or the thiol reagent N ‐ethylmaleimide, indicative of direct effects on these two transport systems. Consistent with its action on RBC membrane transporters, GBT 1118 significantly increased RBC hydration. RBC hemolysis was reduced in a nonelectrolyte lysis assay. Further to its direct effects on O 2 affinity, GBT 1118 was therefore found to reduce RBC shrinkage and fragility. Findings reveal important effects of GBT 1118 on protecting sickle cells and suggest that this is approach may represent a useful therapy for amelioration of the clinical complications of SCA .

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“…To determine the activity of the K + transport pathways, K + influx was measured using 86 Rb + as a congener for K + (Dunham and Ellory, 1981; Hannemann et al, 2011) at 37°C. Red cells were taken from tonometers and diluted 10-fold into saline, pre-equilibrated at the appropriate O 2 tension at 260 mOsm.kg −1 (with the addition of 10% water to the appropriate standard MBSs) and pH 7 (conditions used to stimulate KCC activity).…”

Section: Methodsmentioning

confidence: 99%

The Effect of Antioxidants on the Properties of Red Blood Cells From Patients With Sickle Cell Anemia

et al. 2019

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Oxidative damage to red blood cells (RBCs) may contribute to pathogenesis of sickle cell anemia. Reducing the deleterious effects of oxidants by exposing RBCs to a number of antioxidants has been shown to have protective effects against lipid and protein peroxidation. We hypothesize that antioxidants may also have beneficial effects on the abnormal membrane permeability of sickle cells. Increased cation permeability of these cells encourages HbS polymerization by causing RBC dehydration and also leads to externalization of the prothrombotic aminophospholipid phosphatidylserine (PS). Three antioxidants with different mechanisms of action were investigated – dithiothreitol, N -acetylcysteine, and quercetin. All three were found to inhibit the main cation pathways responsible for dehydration – the deoxygenation-induced cation conductance (or P sickle ), the Ca 2+ -activated K + channel (or Gardos channel), and the K + -Cl − cotransporter. They also reduced Ca 2+ -induced PS exposure and hemolysis. Findings provide evidence for additional beneficial actions of antioxidants in maintenance of rheology and reducing vascular adhesion and further inform the rationale for their clinical use.

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The Use of Radioisotopes to Characterise the Abnormal Permeability of Red Blood Cells from Sickle Cell Patients

Cited by 6 publications

References 91 publications

Effects of 5‐hydroxymethyl‐2‐furfural on the volume and membrane permeability of red blood cells from patients with sickle cell disease

Effects of 5‐hydroxymethyl‐2‐furfural on the volume and membrane permeability of red blood cells from patients with sickle cell disease

The effect of the antisickling compoundGBT1118 on the permeability of red blood cells from patients with sickle cell anemia

The Effect of Antioxidants on the Properties of Red Blood Cells From Patients With Sickle Cell Anemia

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