Calcium Homeostasis In Erythrocytes Research Articles

Regulatory role of nitric oxide in the reduced survival of erythrocytes in visceral leishmaniasis

Background Nitric oxide (NO) plays a vital role in maintaining the survivability of circulating erythrocytes. Here we have investigated whether NO depletion associated with visceral leishmaniasis (VL) is responsible for the reduced survival of erythrocytes observed during the disease. Methods Infected hamsters were treated with standard anti-leishmanial sodium stibogluconate (SAG) and NO donor isosorbide dinitrate (ISD). Erythrophagocytosis by macrophages was determined by labelling the cells with FITC followed by flow cytometry. Aggregation of band3 was estimated from band3 associated EMA fluorescence. Caspase 3 activity was measured using immunosorbent assay kit. Phosphatidylserine (PS) externalization and cell shrinkage were determined using annexin V. Aminophspholipid translocase and scramblase activities were measured following NBD-PS and NBD-PC internalization, respectively. Results Impairment of both synthesis and uptake of NO resulted in decreased bioavailability of this signaling molecule in erythrocytes in VL. NO level was replenished after simultaneous treatment with ISD and SAG. Combination treatment decreased red cell apoptosis in infected animals by deactivating caspase 3 through s-nitrosylation. Drug treatment prevented infection-mediated ATP depletion and altered calcium homeostasis in erythrocytes. Improved metabolic environment effectively amended dysregulation of aminophospholipid translocase and scramblase, which in turn reduced cell shrinkage, and exposure of phosphatidylserine on the cell surface under the diseased condition. Conclusion and general significance In this study, we have identified NO depletion to be an important factor in promoting premature hemolysis with the progress of leishmanial infection. The study implicates NO to be a possible target for future drug development towards the promotion of erythrocyte survival in VL.

Disturbed erythrocyte calcium homeostasis and adenine nucleotide dysregulation in canine phosphofructokinase deficiency

Muscle-type phosphofructokinase deficiency (PFKD) causes a hemolytic disorder and exertional myopathy in humans and dogs. In humans, PFKD is accompanied by a disturbed calcium homeostasis and associated adenine nucleotide dysregulation, which may potentiate the erythroenzymopathy associated with this inherited disorder. This study shows that canine PFKD also manifests these erythrocyte abnormalities. Compared to normal, healthy red cells, PFK-deficient erythrocytes contain lower concentrations of ATP and higher concentrations of IMP and calcium, the latter as per a calcium indicator dye. Adenosine monophosphate deaminase (AMPD) enriched 5000-fold from canine erythrocytes adsorbs to immobilized calcium–calmodulin and the interaction between these two proteins activates AMPD through a Kmapp effect. This behavior is similar to that of the human erythrocyte enzyme and provides a potential contributing mechanism for accelerated adenine nucleotide turnover in canine PFKD. We propose that adenine nucleotide replacement strategies could benefit the erythroenzymopathy in human and canine PFKD and that the dog model of this disorder is an appropriate vehicle for further elucidating this hypothesis.

Ca2+-Mg2+-dependent ATP-ase activity and calcium homeostasis in children with chronic kidney disease

Extracellular calcium concentrations in humans are thousands times higher than within cells. Maintenance of such gradient requires specific regulation including intracellular stores, Ca binding proteins and transmembrane protein systems. The aim of the study was to estimate PMCA (plasma membrane Ca-transporting adenosine triphosphatase; ATPase 3.6.1.38) activity and calcium homeostasis in erythrocytes of children with chronic kidney disease (CKD). Twenty-one children wth CKD stages 1-3 (group I) and 18 healthy children (group II) were examined. Group I was divided into two subgroups: Ia (8 patients with normal intact parathyroid hormone, iPTH, serum levels) and Ib (13 patients with increased iPTH). iPTH, urea, creatinine, inorganic phosphorus, cytosolic Ca2+ in red blood cells (R-Ca), and PMCA were determined. Significantly elevated R-Ca levels were observed in children from subgroup Ib in comparison with group II and subgroup Ia. The lowest activity of PMCA was found in subgroup Ia and Ib in comparison with group II. There was a negative correlation between PMCA and R-Ca in group Ia and Ib (r=-0.8, r=-0.9, respectively). In children with CKD treated conservatively, activity of PMCA in erythrocytes is disturbed. An increase in R-Ca and decrease in PMCA activity are also observed.

Familial phosphofructokinase deficiency is associated with a disturbed calcium homeostasis in erythrocytes.

To critically evaluate whether an altered calcium homeostasis in erythrocytes could be contributing to the symptomatology of the Tarui's disease, which is an inherited phosphofructokinase (PFK) deficiency of the muscle isoenzyme. PFK is a tetrameric enzyme with three different isoenzymes, muscle (M), liver (L), and platelet (P). Erythrocytes contain a 50 : 50 hybrid of M and L type. The deficiency of the muscle isoenzyme displays a symptomatology which is mainly characterized by myopathy, and a compensated haemolytic anaemia. Erythrocyte deformability was assessed before and after autoincubation. Energy related metabolites and energy charge was determined in erythrocytes under various experimental conditions. The clinical part of the study was performed at the Departments of Cardiology and Clinical Chemistry, Umeå University Hospital, and the experimental investigation was carried out at the Department of Clinical Chemistry of the University Hospital of Uppsala, Sweden. Four family members with Tarui's disease participated in the study: the proband (patient 1), a 39-year-old male and two siblings, patient 2 (male, aged 46 years) and patient 3 (female, 30 years). Patient 4 (male, 16 years) was the son of the patient 2. Five healthy persons served as controls (controls 1-5). None. Cell-physiological variables were determined after autoincubation of erythrocytes (i.e. incubation in their own plasma at 37 degrees C) and after incubation in a composite buffered medium. Erythrocyte deformability as assessed by the erythrocyte fluidity was substantially decreased in patients compared to the moderate decrease in the control after 24 h of autoincubation, in presence of endogenous Ca2+ (heparin plasma). Moreover, autoincubation of erythrocytes shows that the patient's erythrocytes, although being moderately deficient in PFK activity, exhibit a normal (or slightly increased) lactate production compared to controls. Despite this, we show an increased ATP turnover with an Ca2+-induced AMP deaminase (and 5'-nucleotidase) activation leading to an increase in hypoxanthine content in patients' erythrocytes of about 100% after 24 h of autoincubation in heparin plasma, when compared to controls. A loss of volume in patient's erythrocytes after 24 h of autoincubation (in presence of Ca2+), as revealed by a diminished MCV, was consistent with an increased metabolic pool of intracellular calcium ions with a selective loss of K+ due to the activation of the K+ channel by intracellular Ca2+ (Gardos-effect). We conclude that the different calcium ion-induced effects on energy metabolism, structure and function of patients' erythrocytes are due to an augmented membrane leakage of Ca2+ and therefore an accumulated intracellular Ca2+ pool. This will result in an increased energy demand by the Ca2+-stimulated ATPase (calcium pump) to compensate for the dissipated Ca2+ gradient across the plasma membrane. The concomitant haemolysis may be explained by a diminished erythrocyte deformability due to Ca2+ overload.

Characterization of Erythrocyte Compounds in Asphyxiated Newborns

Perinatal hypoxic–ischemic damage remains a major cause of acute mortality in infants. In our study we have shown that ATP-powered calcium pump was degraded in asphyxiated erythrocyte membranes. Moreover, the activity of Ca2+-ATPase, the enzyme that is solely responsible for maintenance of calcium homeostasis in erythrocytes, was reduced by 50% compared to healthy newborns. We have also detected the enhanced lipid peroxidation in asphyxiated erythrocyte ghosts. To elucidate the potential mechanisms of the calcium pump damage, we have examined the effect of peroxynitrite on Ca2+-ATPase purified from adult human erythrocyte membranes. We have concluded that calcium pump is a direct target for peroxynitrite action in vitro. Our results indicate that erythrocyte membrane compounds could be a primary target for asphyxia-induced damage, and the impairment of the plasma membrane Ca2+-ATPase function could be, in part, mediated by reactive oxygen species.

Abnormal erythrocyte calcium homeostasis in oxidant-induced hemolytic disease

The processes leading to red cell destruction in oxidant-induced hemolytic disease are not yet fully known. Oxidant damage to hemoglobin per se may be insufficient to explain the process, and the involvement of membrane damage has been suggested. We now report that at least one crucial membrane function-exclusion of calcium-is disrupted by the potent oxidant phenylhydrazine. Phenylhydrazine, both in vitro and when administered to mice in hemolytic doses, causes profound inhibition of red cell calcium ATPase. Coincident with this, red cell calcium content increases very rapidly in mice given phenylhydrazine. These observations suggest that disordered red cell calcium homeostasis may be an important element in oxidant-induced red cell destruction.