John S. Gibson

Deoxygenation-induced and Ca2+ dependent phosphatidylserine externalisation in red blood cells from normal individuals and sickle cell patients

Phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell (RBC) membrane. It may become externalised in various conditions, however, notably in RBCs from patients with sickle cell disease (SCD) where exposed PS may contribute to anaemic and ischaemic complications. PS externalisation requires both inhibition of the aminophospholipid translocase (or flippase) and activation of the scramblase. Both may follow from elevation of intracellular Ca(2+). Flippase inhibition occurs at low [Ca(2+)](i), about 1μM, but [Ca(2+)](i) required for scrambling is reported to be much higher (around 100μM). In this work, FITC-labelled lactadherin and FACS were used to measure externalised PS, with [Ca(2+)](i) altered using bromo-A23187 and EGTA/Ca(2+) mixtures. Two components of Ca(2+)-induced scrambling were apparent, of high (EC(50) 1.8±0.3μM) and low (306±123μM) affinity, in RBCs from normal individuals and the commonest SCD genotypes, HbSS and HbSC. The high affinity component was lost in the presence of unphysiologically high [Mg(2+)] but was unaffected by high K(+) (90mM) or vanadate (1mM). The high affinity component accounted for PS scrambling in ≥2/3rd RBCs. It is likely to be most significant in vivo and may be involved in the pathophysiology of SCD or other conditions involving eryptosis.

Oxygen and reactive oxygen species in articular cartilage: modulators of ionic homeostasis

Articular cartilage is an avascular tissue dependent on diffusion mainly from synovial fluid to service its metabolic requirements. Levels of oxygen (O2) in the tissue are low, with estimates of between 1 and 6%. Metabolism is largely, if not entirely, glycolytic, with little capacity for oxidative phosphorylation. Notwithstanding, the tissue requires O2 and consumes it, albeit at low rates. Changes in O2 tension also have profound effects on chondrocytes affecting phenotype, gene expression, and morphology, as well as response to, and production of, cytokines. Although chondrocytes can survive prolonged anoxia, low O2 levels have significant metabolic effects, inhibiting glycolysis (the negative Pasteur effect), and also notably matrix production. Why this tissue should respond so markedly to reduction in O2 tension remains a paradox. Ion homeostasis in articular chondrocytes is also markedly affected by the extracellular matrix in which the cells reside. Recent work has shown that ion homeostasis also responds to changes in O2 tension, in such a way as to produce significant effects on cell function. For this purpose, O2 probably acts via alteration in levels of reactive oxygen species. We discuss the possibility that O2 consumption by this tissue is required to maintain levels of ROS, which are then used physiologically as an intracellular signalling device. This postulate may go some way towards explaining why the tissue is dependent on O2 and why its removal has such marked effects. Understanding the role of oxygen has implications for disease states in which O2 or ROS levels may be perturbed.

Biomarkers in sickle cell disease

More than 100 different blood and urine biomarkers have been described in sickle cell disease (SCD), with the number increasing rapidly as analytical techniques develop. Nearly all of these biomarkers are abnormal in the steady state, and become more so during complications. The range of abnormalities demonstrates the multisystem nature of SCD and the complex pathophysiology. Some biomarkers indicate damage to specific organs, such as urine albumin:creatinine ratio in nephropathy, whereas others indicate more systemic processes. Biomarkers have been useful in identifying various interrelated pathological mechanisms, including haemolysis, inflammation, hypercoagulability, oxidative stress, reperfusion injury, vasculopathy and endothelial dysfunction. However, most biomarkers correlate closely with other more routine measurements, and also with each other. It is not clear that any provide specific prognostic or clinical information beyond that given by the simple measurement of haemoglobin concentration. The identification of prognostically validated biomarkers in prospective clinical trials would be useful.

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

1 Na+‐K+‐2Cl− cotransport (NKCC) was studied in turkey red cells using Na+ dependence or bumetanide sensitivity of 86Rb+ influx to monitor activity of the transporter. 2 Deoxygenation was the major physiological stimulus for NKCC activity: oxygen tensions (PO2) over the physiological range modulated the transporter, with a PO2 for half‐maximal activation of about 41 mmHg (n= 3). In air, activity of NKCC was also stimulated by shrinkage and isoproteronol (isoprenaline, 5 μm). By contrast, in deoxygenated cells, although the transporter activity was markedly elevated, it was no longer sensitive to volume or β‐adrenergic stimulation. 3 Calyculin A, a protein phosphatase inhibitor, stimulated cotransport with a lag of about 5 min. N‐Ethylmaleimide (NEM) inhibited cotransport and also blocked the stimulatory effect of calyculin A if administered before calyculin A. Stimulation by calyculin A and deoxygenation were not additive. Staurosporine (2 μm) inhibited deoxygenated‐stimulated K+ influxes, but not those stimulated by calyculin A. NEM added during calyculin A stimulation, i.e. during the 5 min lag, caused transport activity to be clamped at levels intermediate between maximal (calyculin A alone) and control. Cells treated with calyculin A alone or with calyculin A followed by NEM were no longer sensitive to volume, isoproteronol or PO2. 4 The results have characterized the interaction between deoxygenation and other stimuli of NKCC activity. They have also shown that it is possible to manipulate the transporter in a reciprocal way to that shown previously for K+‐Cl− cotransport.

A comparison in normal individuals and sickle cell patients of reduced glutathione precursors and their transport between plasma and red cells.

INTRODUCTION Reduced glutathione is an important antioxidant in red cells whose depletion may contribute to the pathophysiology of sickle cell disease. The current study was designed to examine the availability of reduced glutathione precursors (glutamate, cysteine, glycine and possibly glutamine) together with the activity of the main transport pathways for their uptake (system ASC for cysteine and glycine; system gly for glycine). MATERIALS AND METHODS Blood samples were obtained from normal (HbAA, HbA cells) and sickle cell disease patients (HbSS, HbS cells); amino acids were measured by HPLC; and transporter activity was measured by radioactive tracer fluxes (using serine and glycine for activity of system ASC; and glycine for that of system gly). RESULTS Plasma concentrations of cysteine and glycine were increased and concentrations of all amino acids were elevated in HbS cells. The activity of system ASC was increased in HbS cells (both transport capacity and affinity were elevated for serine transport; transport capacity only for glycine). Activity of system gly was also increased (twofold increase in V(max) for glycine flux), though not significantly. Oxygenation also increased the activity of both transporters in normal and HbS cells. CO prevented deoxy-inhibition of glycine transport. Staurosporine (5 microM) inhibited O(2)-stimulated glycine transport through system ASC. It also inhibited the absolute magnitude of transport through system gly, but the O(2)-dependent flux was unaffected. CONCLUSION Low reduced glutathione levels in HbS cells were not due to decreased substrate availability and O(2) stimulated transport of reduced glutathione precursors in both normal and HbS cells, through a mechanism that is likely to involve Hb and possibly protein phosphorylation.

Modulation of K(+)‐Cl‐ cotransport in equine red blood cells

Potassium transport was measured in equine red blood cells, using 86Rb+ influx as a convenient assay. A significant component of volume‐ and pH‐sensitive K(+)‐Cl‐ cotransport to the overall K+ flux was observed in all blood samples studied, although fluxes were variable between animals, and within individuals when measured at intervals over a period of weeks. The aryloxyacetic acid [(dihydroindenyl)oxy]alkanoic acid (DIOA), at a final concentration of 100 microM, inhibited most (> 95%) of the Cl(‐)‐dependent K+ flux, and DIOA sensitivity was therefore used to define the activity of the K(+)‐Cl‐ cotransport. K(+)‐Cl‐ cotransport was also sensitive to protein phosphatase inhibition with calyculin A or okadaic acid, with inhibition constants of 9 +/− 1 nM for calyculin and about 100 nM for okadaic acid. Peak fluxes were observed at an external pH of 6.7–7.0, with inhibition at higher and lower values. Volume‐sensitive K+ fluxes assayed in autologous plasma, controlled for osmolaity, pH and potassium concentration, were significantly lower (28 +/− 8% of control values, n = 6) than those measured in saline. This inhibition was mimicked by the culture medium RPMI, but disappeared following dialysis of the plasma. Phosphate (5.6 mM) inhibited volume‐sensitive K+ fluxes by 48 +/− 2%, n = 3; no significant effect was observed by increasing external magnesium concentrations to 0.5 or 2 mM. Thus, inhibition by RPMI, but not that by plasma, may be due to phosphate. Finally, volume‐ and pH‐sensitive K+ fluxes were sensitive to oxygen tension and were abolished reversibly by equilibrating solutions with nitrogen, as opposed to air. Use of solutions equilibrated with different values of Po2 may account for some of the variability in equine red blood cell KCl fluxes. The importance of these observations to equine red blood cell homeostasis and haemodynamics is discussed.

The conductance of red blood cells from sickle cell patients: ion selectivity and inhibitors.

Key points  •  The high cation permeability in red blood cells (RBCs) from patients with sickle cell disease (SCD) is central to pathogenesis and includes a deoxygenation‐induced pathway termed Psickle. •  Here whole‐cell patch clamp configuration was used to record from RBCs of SCD patients and showed a conductance reversibly induced upon deoxygenation, permeable to univalent (Na+, K+, Rb+) and divalent (Ca2+, Mg2+) cations, and sensitive to tarantula spider toxin GsMTx‐4, Mn2+ and o‐vanillin. •  Divalent cation permeability is particularly important as entry of Ca2+ stimulates the Gardos channel whilst Mg2+ loss will stimulate KCl cotransport. •  In oxygenated RBCs, the conductance was pH sensitive, increasing as pH fell from 7.4 to 6, but unaffected when pH was raised from 7.4 to 8. •  Results show a conductance that shares many features with the Psickle flux pathway, and indicating its possible identity as a stretch‐activated channel with activation requiring sickle cell haemoglobin (HbS) polymerisation.

Deoxygenation-Induced Non-Electrolyte Pathway in Red Cells from Sickle Cell Patients

Red cells from patients with sickle cell disease contain HbS rather than the normal HbA (here termed HbS cells). On deoxygenation, HbS cells exhibit a distinctive solute permeability pathway, Psickle, activated stochastically, and partially inhibited by DIDS and dipyridamole. It is often referred to as a cation channel although its permeability characteristics remain vague and its molecular identity is unknown. We show that, in contrast to normal red cells, a proportion of HbS cells underwent haemolysis when deoxygenated in isosmotic non-electrolyte solutions. Haemolysis was stochastic: cells unlysed after an initial deoxygenation pulse showed lysis when harvested, reoxygenated and subsequently exposed to a second period of deoxygenation. O2 dependence of haemolysis was similar to that of Psickle activation. Haemolysis was accompanied by high rates of sucrose influx, and both haemolysis and sucrose influx were inhibited by DIDS and dipyridamole. Sucrose influx was only detected as ionic strength was reduced below 80 mM. These findings are consistent with the postulate that deoxygenation of HbS cells, under certain conditions, activates a novel non-electrolyte pathway. Their significance lies in understanding the nature of the deoxygenation-induced permeability in HbS cells, together with its relationship with novel pathways induced by a variety of manipulations in normal red cells.

Effect of Intracellular Magnesium and Oxygen Tension on K + -Cl - Cotransport in Normal and Sickle Human Red Cells

In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb

Effect of 1-chloro-2,4-dinitrobenzene on K + transport in normal and sickle human red blood cells

1‐Chloro‐2,4‐dinitrobenzene (CDNB), which causes oxidative stress through depletion of reduced glutathione (GSH), increases the passive K+ permeability of red cells. In this paper, we investigated the effects of CDNB (1 mm) on the activities of the K+−Cl− cotransporter (KCC; measured as Cl−‐dependent K+ influx) and the Gardos channel (taken as clotrimazole‐sensitive K+ influx, 5 μm) in human red cells, using 86Rb+ as a K+ congener. 45Ca2+ was used to study passive Ca2+ entry and active Ca2+ efflux via the plasma membrane Ca2+ pump. Both the Gardos channel and KCC were stimulated in both normal and sickle red cells. In sickle cells, stimulation of KCC was similar in oxygenated and deoxygenated cells; that of the Gardos channel was greater in deoxygenated cells. In normal red cells, stimulation of both pathways was greater in oxygenated cells (by 4 ± 1‐fold; all means ±s.e.m., n= 3). The effects on the Gardos channel were dependent on extracellular Ca2+ and were associated with inhibition of the plasma membrane Ca2+ pump (by 29 ± 3 %, P < 0.01) and increased Ca2+ sensitivity of the channel (EC50 for [Ca2+]i reduced from 260 ± 26 to 175 ± 15 nm; P < 0.05). Cell volume, pHi, ATP levels and passive Ca2+ entry were not affected by CDNB. The effects on KCC were inhibited (93 ± 6 %) by prior treatment with the protein phosphatase inhibitor calyculin A (100 nm) and were not additive with stimulation by N‐ethylmaleimide (1 mm), regardless of the order of addition. These findings are therefore consistent with inhibition of a regulatory protein kinase, although stimulation of the conjugate protein phosphatase(s) may also occur. KCC stimulation was also Ca2+ dependent. These findings are important for understanding how GSH depletion alters membrane permeability and how to protect against red cell dehydration.