Alterations in calcium-handling of erythrocytes in spontaneously hypertensive rats. Calcium-induced changes in the osmotic fragility of erythrocytes in hypertension

To investigate the sensitivity to calcium of erythrocytes in hypertension, changes in the osmotic fragility of erythrocytes following Ca-loading were observed. Washed erythrocytes were obtained from spontaneously hypertensive rats (SHR, Okamoto and Aoki) and age-matched normotensive Wistar Kyoto rats (WKY). Treatment of erythrocytes with Ca-ionophore A23187 and Ca in the medium caused a reduction in the osmotic fragility which correlated with the Ca-concentration. The degree of alteration in the osmotic fragility of erythrocytes was greater in SHR than in WKY. Oral administration of hydralazine to SHR significantly reduced the blood pressure. However, the alterations in the osmotic fragility of erythrocytes secondary to Ca-loading were not different between hydralazine-treated and untreated SHR. In the presence of a Ca-antagonist (verapamil or diltiazem) in the medium, the reduction of the osmotic fragility of erythrocytes caused by Ca-loading was inhibited, and the differences between SHR and WKY were abolished by Ca-antagonists. These results suggest that the greater changes in osmotic fragility of erythrocytes caused by Ca-loading in SHR could be due to a genetic abnormality of Ca-handling by the cell membranes, and that this abnormality might cause an increase in intracellular Ca, which contributes, in part, to the pathogenesis of hypertension.     

Age-related changes in membrane fluidity of erythrocytes in essential hypertension

In the present study, age- and calcium-related changes in the membrane fluidity of erythrocytes were examined in patients with essential hypertension by use of electron spin resonance method (ESR). The erythrocytes were obtained from patients with essential hypertension. We examined the ESR spectra for a fatty acid spin label agent (5-nitroxy stearate) incorporated into the erythrocyte membranes. The values of outer hyperfine splitting and order parameter (S) were significantly higher in subjects with essential hypertension than in the normotensive subjects. This finding indicates that the membrane fluidity of erythrocytes was lower in essential hypertension. Calcium-loading of erythrocytes with the Ca-ionophore A23187 decreased the membrane fluidity (S value was increased) more strongly in essential hypertension than in the normotensive subjects. Furthermore, this Ca-induced change in membrane fluidity was significantly correlated with age in essential hypertension. These results demonstrate that the membrane fluidity of erythrocytes is markedly decreased by calcium, especially in essential hypertension in older patients. This suggests an increased calcium-sensitivity of cell membranes in the aged hypertensive patient.     

Continuous flow method for determination of erythrocyte osmotic fragility

A simple and accurate micromethod for the determination of erythrocyte osmotic fragility is introduced. The method uses a laminar parabolic flow pattern, together with gravity, to retain cells in a long, small-diameter tube while a solution with decreasing osmolarity is passed through the tube. As the cells hemolyze, hemoglobin released from the cells is quickly removed by the axial flow pattern and monitored with a 547 nm optical detector for recording the hemolysis curve. Consequently, a continuous curve is obtained, with a peak occurring at the salt concentration that produces the maximum hemolysis rate. The advantages of this method are simplicity, accuracy, and small sample size (2 microliters of whole blood). The small sample size is of particular importance for infants. A comparison is made with the Parpart method using samples from 18 normal adults. Results are also given for a few abnormal adults and for a series of 26 normal newborns.     

A method for the determination of the osmotic fragility of erythrocytes

A method for registration of the haemolysis of erythrocytes as a measure for their osmotic resistance is presented. The technical realization is performed by means of a cuvette head-piece for holding a filter, which separates the investigation material from water. A cuvette insertion guarantees the regular construction of a haemoglobin gradient, serving as a measure for haemolysis, which is photometrically measured in the Spekol (VEB Carl Zeiss JENA) and registered by means of the compensation tape writer G 1 B 1 (VEB Carl Zeiss JENA). 0.3-0.4 ml investigation material are used. The expenditure of time amounts to about 20 minutes.     

The osmotic fragility of erythrocytes after prolonged liquid storage and after reinfusion

Although it is recognized that red cells lose membrane during storage, estimation of the osmotic fragility of erythrocytes has not previously proven to be a useful measurement of the storage lesion. Erythrocytes from blood stored in CPD-A2 were found to have a markedly increased osmotic fragility. A major portion of this increase was found to be due to accumulation of lactate, which is only slowly transported from within erythrocytes and which therefore exerts a strong osmotic effect in the usual osmotic fragility test. After an hour's incubation in a large volume of iso-osmotic buffer, the osmotic fragility curve of stored erythrocytes was much more nearly normal. Such cells were found to have a volume 5%--8% greater than that of normal cells, indicating that even after removal of lactate more osmotically active material was present in the stored erythrocytes than in fresh cells. Most of this differences can be accounted for by substitution of chloride ion for 2,3-DPG, since chloride exerts approximately 3.7 times the osmotic effect of 2,3-DPG per unit charge. In addition to the shift in osmotic fragility produced by the increased intracellular osmotically active material, a "fragile tail" of red cells was also present. Stored erythrocytes were labeled with 51Cr and reinfused into the volunteer donors. The osmotic fragility of the reinfused cells was estimated using a technique of sequential osmotic hemolysis that permitted accurate estimation of osmotic fragility of transfused cells using very small amounts of 51Cr. The osmotic fragility of the reinfused cells became less than those of fresh cells after 24 hr and was exactly the same as those of fresh cells after 4 days. The fragile tail disappeared at a rate that approximated the rate of loss of nonviable erythrocytes from the circulation as measured by 51Cr. These findings are consistent with the preferential destruction of a subpopulation of red cells with a diminished surface area.     

Potassium influx into erythrocytes in essential hypertension

Sodium plus potassium (Na+K) cotransport in erythrocytes of patients with essential hypertension has mainly been studied elsewhere by measuring net outward frusemide-sensitive Na and K movements. We compared K influx (tracer 86 Rubidium) in control subjects and hypertensives who had never previously been treated for hypertension. The cotransport K influx rates in controls and hypertensives were 0.44 +/- 0.02 (mumol/ml cells/h; mean +/- s.e.; n = 20) and 0.67 +/- 0.06 (mumol/ml cells/h; mean +/- s.e.; n = 23) respectively. The active influxes were 1.03 +/- 0.03 and 2.06 +/- 0.13 (mumol/ml cells/h; mean +/- s.e.) respectively. The variance of both parameters was significantly greater in the hypertensive group but, taking this into account, the differences were significant (P less than 0.01).     

The effect of EDTA as an anticoagulant on the osmotic fragility of erythrocytes

The osmotic fragility test is used to determine the extent of red blood cell haemolysis produced by osmotic stress. Since the quality of this test may easily be influenced by environmental and technical factors we have determined osmotic fragility reference values in our own conditions. The results show significantly increased osmotic resistance of erythrocytes in our conditions vs the published values for blood samples anticoagulated with heparin. Furthermore, the use of EDTA as an anticoagulant increased the osmotic fragility of red blood cells as compared with heparin. We conclude that EDTA can be used as an anticoagulant for the osmotic fragility test in order to simplify routine procedures. However, every laboratory should determine its own reference values which would reflect the local environmental and technical factors.     

Increased erythrocyte magnesium in never treated essential hypertension

In the present study we aimed at evaluating the intracellular concentrations of magnesium, potassium and sodium in 50-year-old, otherwise healthy white men with never treated, essential hypertension (n = 12) and in normotensive control subjects (n = 12) matched for age, sex, race, height, weight and smoking habits. Intraerythrocyte magnesium was significantly increased in the hypertensive group (P less than .001) and correlated positively and significantly to blood pressure in the total group (P less than .01). The intracellular potassium to sodium ratio tended to be lower in the hypertensive group (P less than .05). Thus, the present study supports increased intracellular magnesium probably unrelated to intracellular potassium-sodium imbalance in never treated, essential hypertension.     

Diminished osmotic fragility of human erythrocytes following the membrane insertion of oxygenated sterol compounds

Oxygenated sterol compounds (OSC), when incubated for 1 hr with human erythrocytes in lipoprotein-depleted medium at concentrations of 0.625- 5 X 10(-5) M, are inserted into the cell membrane and remain there despite subsequent washing of the cells. The insertion results in expansion of the surface area of the red cell ghost membrane, an increase in critical hemolytic volume, and as a consequence, in diminished osmotic fragility of the erythrocytes. This effect is seen with echinocyte-forming as well as with non-echinocyte-forming OSC. Erythrocytes treated with OSC do not differ from control cells with respect to their mean cell volume (MCV) in isotonic solution, water content, ion fluxes, and filterability through polycarbonate filters. The shift of the osmotic fragility curve toward lower NaCl concentrations is proportional to the amount of OSC inserted into the red cell membrane. 7 beta-Hydroxycholesterol, 22-ketocholesterol, and 20 alpha-hydroxycholesterol are the most potent inhibitors of osmotic lysis. The effect of OSC on osmotic fragility is diminished if the erythrocytes are incubated in a lipoprotein-containing medium; free cholesterol, however, does not change this effect. Various progesterones also protect red cell from osmotic lysis, but only if the erythrocytes are directly exposed to the compounds present in the hypotonic NaCl solutions used for measurement of their osmotic fragility. Progesterones do not remain in the membrane after the cells have been washed. The OSC are also capable of correcting the osmotic fragility curve of red cells from patients with hereditary spherocytosis. These experiments may suggest an approach to the pharmacologic treatment of hereditary spherocytosis.     

Adrenomedullin and membrane fluidity of erythrocytes in mild essential hypertension

OBJECTIVE: Adrenomedullin is a newly discovered 52 amino acid peptide that has a potent vasodilating action. The present study was undertaken to investigate the role of adrenomedullin in the regulation of membrane fluidity of erythrocytes in patients with essential hypertension. METHODS AND RESULTS: We used an electron paramagnetic resonance and spin-labeling method. Adrenomedullin significantly decreased the order parameter for 5-nitroxide stearate and peak height ratio for 16-nitroxide stearate obtained from electron paramagnetic resonance spectra of erythrocyte membranes in normotensive volunteers (mean +/- SEM order parameter value: control, 0.718 +/- 0.003, n = 16; adrenomedullin at 10(-9) mol/l, 0.692 +/- 0.004, n = 16, P < 0.05; adrenomedullin at 10(-8) mol/l, 0.690 +/- 0.004, n = 16, P < 0.05; adrenomedullin at 10(-7) mol/l, 0.683 +/- 0.004, n = 16, P < 0.05). The findings showed that adrenomedullin increased the membrane fluidity of erythrocytes. In addition, the effect of adrenomedullin was significantly potentiated by prostaglandin E1 and dibutyryl cyclic AMP. In contrast, the calcium ionophore A23187 counteracted the actions of adrenomedullin. In patients with essential hypertension, who had higher order parameter values, the membrane fluidity of erythrocytes was significantly lower than in the normotensive control subjects (order parameter: 0.728 +/- 0.004 in hypertensives, n = 20; 0.692 +/- 0.002 in normotensives, n = 36, P < 0.01). The effect of adrenomedullin on membrane fluidity was more pronounced in the erythrocytes of essential hypertensive than in the erythrocytes of normotensive subjects (change in the order parameter with adrenomedullin at 10(-9) mol/l: -4.2 +/- 0.3% in hypertensives, n = 20; -1.8 +/- 0.2% in normotensives, n = 20, P < 0.05; adrenomedullin at 10(-8) mol/l: -4.5 +/- 0.3% in hypertensives, n = 20; -1.8 +/- 0.2% in normotensives, n = 36, P < 0.05). CONCLUSIONS: The results of the present study demonstrate that adrenomedullin significantly increased the membrane fluidity of erythrocytes. The mechanisms were partially mediated by a prostaglandin E1- and cyclic AMP-dependent pathway which might be linked to changes in intracellular calcium kinetics. The greater effect of adrenomedullin in patients with essential hypertension suggests that the peptide might actively participate in the regulation of membrane functions in hypertension.     

Sodium and calcium cation transport of erythrocytes in essential hypertension

The sodium and calcium transport of erythrocyte and the influencing factors were studied in essential hypertensive (EH) subjects. The result showed that plasma endogenous digitalis-like compound (EDLC) increased and sodium pump depressed in some EH patients, but there were no parallel correlation between EDLC and sodium pump. The patients with normal sodium pump mainly showed their maximal Ca2+ pump activity and decreased calmodulin (CaM) content of erythrocyte. Thus there may be different types of ion transport defect in EH, and the abnormalities of these cation transports have an important role in the pathogenesis of EH.