Oxidants and regulation of K⁺-Cl⁻cotransport in equine red blood cells

Abstract

The effect of oxidants on K(+)-Cl(-) cotransport (KCC) was investigated in equine red blood cells. Carbon monoxide mimicked O(2). The substituted benzaldehyde, 12C79 (5 mM), markedly increased O(2) affinity. In N(2), however, O(2) saturation was low (<10%) but KCC remained active. Nitrite (NO(2)(-)) oxidized heme to methemoglobin (metHb). High concentrations of NO(2)(-) (1 and 5 mM vs. 0.5 mM) increased KCC activity above control levels; it became O(2) independent but remained sensitive to other stimuli. 1-Chloro-2, 4-dinitrobenzene (1-3 mM) depleted reduced glutathione (GSH). Prolonged exposure (60-120 min, 1 mM) or high concentrations (3 mM) stimulated an O(2)-independent KCC activity; short exposures and low concentrations (30 min, 0.5 or 1 mM) did not. The effect of these manipulations was correlated with changes in GSH and metHb concentrations. An oxy conformation of Hb was necessary for KCC activation. An increase in its activity over the level found in oxygenated control cells required both accumulation of metHb and depletion of GSH. Findings are relevant to understanding the physiology and pathology of regulation of KCC.

Oxidative stress and phosphatidylserine exposure in red cells from patients with sickle cell anaemia

“…Initially, three oxidants were selected to represent a range of different oxidant challenges: XO/HO (2 mmol/l) mixtures to generate extracellular ROS including SOA and hydrogen peroxide (Baskurt et al , 1998; Rogers et al , 2009), PMS to generate SOA intracellularly (Nishikimi et al , 1972; Maridonneau et al , 1983) and NO 2 which promotes formation of methaemoglobin (metHb) (Muzyamba et al , 2000). …”

“…These particular oxidants were chosen because of the qualitative differences in oxidative challenge that they present to the red cell. XO/HO mixtures generate SOA and hydrogen peroxide extracellularly (Baskurt et al , 1998; Rogers et al , 2009), whilst PMS generates SOA intracellularly (Nishikimi et al , 1972; Maridonneau et al , 1983); NO 2 oxidises Hb to metHb (Muzyamba et al , 2000); t BHP increases generation of peroxyl and alkoxyl free radicals (Davies, 1989); whilst HOCl produced by myeloperoxidase released from neutorphils may also oxidise membrane thiols (Vissers et al , 1994; Gorudko et al , 2016). Red cell PS exposure is most reliably stimulated by elevation of intracellular Ca 2+ (Bevers & Williamson, 2010) whilst a number of oxidants have previously been shown to increase red cell cation permeability (Gibson & Muzyamba, 2003a,2003b).…”

Oxygen dependence of K+–Cl− cotransport in human red cell ghosts and sickle cells

“…Haemoglobin is the major protein of red cells and also reacts reversibly with O 2 making it an obvious candidate [6,7]. A number of observations support a role for Hb, such as the sigmoidal response of some transporters to O 2 tension and the inability of deoxygenation to influence transporter activity following carbon monoxide or nitrite treatment [6,8,9]. In addition, it has been suggested that in sickle cell disease, HbS polymerisation at low O 2 tensions subverts the normal response of transporters such as the K + -Cl À cotransporter (KCC), e.g.…”

Interaction of Deoxyhemoglobin with the Cytoplasmic Domain of Murine Erythrocyte Band 3

“…Importantly, the K + /Cl − cotransporter (KCC) in human erythrocytes increases in activity ~20-fold during erythrocyte oxygenation (23). Moreover, this O 2 -dependent regulation occurs only in whole cells and Hb-containing ghosts, but not in white ghosts or whole cells treated with CO to block O 2 binding (24–25). Together with data showing a sigmoidal dependence of K + /Cl − cotransport on O 2 pressure (i.e.…”