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  • The lack of inhibition of K efflux in


    The lack of inhibition of K+ efflux in normal red cells reported by Fathallah et al. [16] was attributed by the authors to a lower level (about half) of membrane PKCα activity. In our experiments, single channel recording probably overcame this difficulty because of its molecular detection level. Our results clearly indicate that PKC and PKA promote regulatory mechanisms of Gardos channels. Their actions may integrate signals mediated through these parallel pathways, determining the channel opening frequency. The results of concurring activations of PKC and PKA reveal that neither of the two have any precedence in order to determine the effect induced by the other, as in the reported convergent regulation of sodium channels [34]. However, while the level of activation of the Gardos channel by PKA is enhanced by the increase of [Ca2+]i[13], the inhibition by PKC is progressively reduced by the same factor. Thus, if concurrently stimulated, the two signal transduction pathways are expected to balance their effects at a basal or low [Ca2+]i, whereas at high [Ca2+]i the activation by PKA should prevail. This unbalance might be critical in determining elimination of senescent red blood cells, in which an age-dependent decrease of PKC activity has been reported [35].
    Introduction Sickle cell disease (SCD) is one of the commonest severe inherited disorders affecting millions of people worldwide (Piel et al., 2013). Complications of the disease arise from the presence in patients' red cells of the abnormal haemoglobin (Hb), HbS, which has a single amino KU-57788 substitution compared to normal adult Hb, HbA. In HbS, valine replaces glutamic acid in the 6th codon of the β chain, with loss of a negative charge (Bunn and Forget, 1986). On deoxygenation, this substitution allows neighbouring molecules of HbS to adhere, forming rigid polymers which distort the shape of the red cell. The complications of SCD all follow from polymerisation of HbS, although in many cases details of the pathogenesis remain unclear. About two-thirds of SCD patients are homozygous for HbS (HbSS genotype, or disease, sometimes referred to as sickle cell anaemia, SCA) (Rees et al., 2010). Co-inheritance of a second abnormal Hb, HbC, in which lysine replaces glutamic acid at the same position of the β chain, along with HbS produces the heterologous HbSC genotype (HbSC disease) (Nagel and Lawrence, 1991, Nagel and Steinberg, 2001). HbSC individuals account for about one-third of SCD patients (Nagel and Steinberg, 2001) and thereby represent a sizeable patient cohort. The vast majority of laboratory and clinical studies on SCD, however, including those on red cell cation homeostasis, have been carried out on HbSS patients, with HbSC patients being largely and unjustifiably neglected. Both HbSS and HbSC disease have profound clinical impact, although those homozygous for HbC (HbCC) are largely asymptomatic (Nagel and Steinberg, 2001). Complications are multiple including chronic anaemia, pain and organ dysfunction with signs dependent on the identity of the affected organ - stroke, acute chest syndrome, nephropathy, osteonecrosis, dactylitis, etc. (Rees et al., 2010, Steinberg et al., 2001, Nagel and Platt, 2001). Between individuals, clinical severity is markedly heterogeneous, with the health of some patients being severely compromised, whilst others present with a less severe disease or even a subclinical course. In many cases it is not understood why. Generally, HbSC disease is milder, though it still presents with significant morbidity (Platt et al., 1991, Nagel et al., 2003). For example, life expectancy of HbSC individuals is markedly reduced (Platt et al., 1994), and some complications of SCD, like proliferative retinopathy (Condon and Serjeant, 1970), are over-represented in HbSC patients. Intracellular cation homeostasis in red cells is maintained mainly by active movement of Na+ and K+ via the ATP-driven Na+/K+-pump coupled with a relatively low passive permeability through various transport pathways. Together these set intracellular [K+] at about 100mM and Na+ at about 15mM (Joyce, 1958). A major feature of red cells from SCD patients, however, is their abnormally high cation permeability. This characteristic is important, as it causes red cells to lose intracellular solutes and shrink, thus elevating the intracellular concentration of HbS ([HbS]). As the lag time to polymerisation of deoxygenated HbS is inversely proportional to a very high power of [HbS] (Eaton and Hofrichter, 1987), any shrinkage markedly increases the likelihood of polymerisation as red cells traverse hypoxic regions of the circulation. Considerable effort has been expended on investigating this high cation permeability (Tosteson et al., 1952, Joiner et al., 1993, Gibson and Ellory, 2002, Lew and Bookchin, 2005), and designing potential inhibitors (eg Stocker et al., 2003), but studies are restricted almost exclusively to red cells from HbSS patients.