Effect of Phenazine Methosulphate on K⁺ Transport in Human red Cells

Abstract

The effect of phenazine methosulphate (PMS; 1 mM) on (⁸⁶Rb⁺) K⁺ transport in human red cells was investigated to ascertain its action on the K⁺-Cl^- cotransporter (KCC; defined as the Cl^- dependent component of K⁺ flux measured in the presence of ouabain and bumetanide) and the Ca²⁺-activated K⁺ channel (Gardos channel; defined as the clotrimazole, 5 µM, -sensitive K⁺ flux). In the presence of Ca²⁺, both transport pathways were stimulated but effects were markedly greater under deoxygenated conditions (5-fold for KCC; 20-fold for the Gardos channel). KCC activation was inhibited by prior treatment with calyculin A (100nM), implying action via protein dephosphorylation. Activation of the Gardos channel correlated with 28 ± 3% inhibition of the plasma membrane Ca²⁺ pump, with maximal activity reduced from 7.7 ± 1.1 to 2.7 ± 0.7 µmol.(l cells.h) ^-1 (all means ± S.E.M. for n = 3), and a 3-fold increase in sensitivity of the channel to Ca²⁺ (EC₅₀ reduced from 437 ± 156 to 152 ± 57 nM). Increased availability of NADH in deoxygenated conditions, resulting in increased free radical generation by PMS, may be responsible. We speculate that the similarity of the K⁺ transport phenotype produced by PMS to that seen in deoxygenated sickle cells is relevant to the pathophysiology of sickle cell disease.

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

“…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). We therefore hypothesised that oxidants may act synergistically with intracellular Ca 2+ to increase PS exposure and alter its Ca 2+ affinity, thereby accounting for the raised percentage of PS‐positive red cells in SCA patients.…”

“…Oxidative damage has also been associated with deleterious effects on red cell cation balance. It causes inhibition of the plasma membrane Ca 2+ pump (Shalev et al , 1981; Zaidi et al , 2003), which maintains intracellular Ca 2+ concentrations at low levels, perhaps via interaction with calmodulin (Gao et al , 2001), and also has marked effects on red cell potassium permeability (Gibson & Muzyamba, 2003a,2003b). A randomized controlled trial has also recently shown that l ‐glutamine, believed to act predominantly as an antioxidant, reduced the frequency of acute pain in SCA (Niihara et al , 2016).…”

Arachidonic Acid Activates K+-Cl--cotransport in HepG2 Human Hepatoblastoma Cells

“…Other studies have also implicated that ROS are involved in the regulation of KCC activity in red blood cells (Muzyamba et al, 2000;Gibson et al, 2003). NEM appeared to generate ROS by activation of the NADPH oxidase which is known to exist in HepG2 cells (Ehleben et al, 1997;Cool et al, 1998).…”

The effect of xanthine oxidase and hypoxanthine on the permeability of red cells from patients with sickle cell anemia

“…A number of oxidants including nitrite (Muzyamba et al. ), phenazine methosulfate (Gibson and Muzyamba ,b), 1‐chloro‐2,4‐dinitrobenzene (Gibson and Muzyamba ,b), and peroxynitrite (Kucherenko et al. ) have previously been found to have significant stimulatory effects on K + transport systems in red cells from normal individuals and from other species, and may therefore mediate solute loss and red cell dehydration.…”