Scand J Haematol(l977) 18, 353-357
Increased Potassium Permeability by Calcium
in Hypochromic Red Blood Cells LUCIANO VETTORE, MARIACONCETTA DE MATTEIS 8r GIANCARLO FALEZZA Teaching of Haematology, University of Trieste, Italy arid Institute oi Medical Pathology in Verona, University of Padua, Italy
The red blood cell (RBC) content of Na' and K+ were measured both on fresh cells from normal, heterozygous P-thalassaemic and iron-deficiency-anaemic subjects, and on the same cells incubated for 24 h, at 37O C, either in presence or in absence of Calcium (Caz+). Caz+ did not increase membrane permeability to Na+, but increased the K loss, both from normal cells and t o a greater degree much more from hypochromic cells. Glucose largely prevented the K' loss from hypochromic cells incubated either in absence or in presence of Caz+, probably maintaining an adequate level of ATP during the incubation. EDTA only partially decreased the permeability to K' in hypochromic cells incubated for 24 h at 37O C, possibly removing CaZ+bound to the cell membrane. The results suggest that Caz+ does not represent the primary cause of K leak in hypochromic cells, but it is able t o enhance a pre-existing peculiar abnormality of the cell membrane when the ATP level slows down. K e y words: calcium - erythrocyte Na' and K+ - hypochromic red cells
Accepted for publication December 15, 1976 Correspondence to: Dr. Lucian0 Vettore, 1st. Patologia Medica 2 O dell'Universiti di Padova, Policlinico di Borgo Roma, 37100 Verona, Italy
It has been shown that red blood cells (RBC) from patients heterozygous for pthalassaemia (Th.) or affected by iron deficiency anaemia (I.D.A.) have a selective increase in membrane permeability to potassium (K+) when they are incubated at 37' C for 24 h in autologous plasma with-
out addition of glucose (Vettore et a1 1971, Gunn et a1 1972, Chapman et a1 1973, Vettore et a1 1974). Calcium (Caz+) can increase the permeability to K' of normal RBCs, provided that they are either previously depleted of energy (Kregenow & Hoffman 1972) or altered by
This work was supported by a grant fro'm Consiglio Nazionale delle Ricerche, Italy. Scand J Haematol (1977) 18
23
354
L.VETTORE, M. C. D E MATTEIS & G. C. FALEZZA
various substances such as fluoride or iodoacetic acid (Gardos 1958), propranolol (Manninen 1970), triose reductone (Passow 1961) etc. In each case the underlying mechanism is probably different. Gunn et a1 (1972) proposed that Ca2+ could play a role in the K leak shown by thalassaemic red cells. In the present paper we describe the effects of Ca2+on Na' and K content of normal and hypochromic red cells, both from heterozygous p-Th. and from I.D.A. patients. MATERIALS AND METHODS The study was carried out on 9 normal individuals, on 15 heterozygous P-thalassaemics and on 13 subjects with I.D.A. The RBC content of Nat and K+ was determined both on freshly drawn erythrocytes and on the same cells after incubation at 37O C for 24 h in the following solutions: 1. patients' own plasma 2. isotonic buffer pH 7.4, containing 140 mM NaCI, 20 mM Tris-HC1 and 1 mM CaC1z 3. the same buffer as above, containing 1 mM EDTA instead of Ca2+. Each cell suspension was previously diluted with the corresponding solution for incubation to a haematocrit value between 35 and 45 Yo. In four experiments hypochromic cells were incubated at 37O C for 24 h in a buffer containing 35 mM glucose together with Ca2+ (Figure 1). In one experiment the effect of 1 mM EGTA was compared with the effect of 1 mM EDTA on the K+ leakage. Before cation measurement, fresh and incubated cells were washed three times with 120 mM MgC12, and then 0.2 ml of packed cells were lysed in 9.8 ml of 0.1 % Triton-X 100 in distilled water. K* and Na+ concentrations were measured on haemolysate by flame photometry and referred to a constant number of RBCs (1013), as calculated from the MCH of the original blood and the haemoglobin concentration of each haemol ysate.
RESULTS AND DISCUSSION
Table 1 shows the mean content (f standard deviation) of Na' in fresh RBCs and in the same cells after incubation in different media, for the 3 groups of subjects (normals, Th. and I.D.A.). Ca2' did not increase the RBC membrane permeability to Na' both in normal and in hypochromic cells. The incubation in buffer containing EDTA increased the Na' content of hypochromic cells more than the incubation in plasma, possibly because the former did not contain glucose. Table 2 gives the mean content (f SD) of RBC K in the same experimental conditions described above. The results for Th. and I.D.A. cells, owing to their similarity, will be described together in the text. Previously presented data (Vettore et a1 1974) are here confirmed: as a consequence of incubation in plasma at 37O C for 24 h, hypochromic RBCs lost more K (- 27 %, compared with fresh cell content) than normal ones (- 10 %). The presence of CaZ+ in the incubation medium significantly increased (p < 0.001) the K+ loss from the cells of normal and hypochromic subjects; this loss was of 27 % in normal cells and of 51 % in hypochromic ones. Moreover the cells exhibiting the greater leak to K+ during the incubation in plasma lost much more K when they were incubated in buffer plus Ca2+,with a signscant positive correlation between the percentage decrease of cation content following the incubation without and with Ca2' (for normal and Th. cells p
Increased Potassium Permeability by Calcium
in Hypochromic Red Blood Cells LUCIANO VETTORE, MARIACONCETTA DE MATTEIS 8r GIANCARLO FALEZZA Teaching of Haematology, University of Trieste, Italy arid Institute oi Medical Pathology in Verona, University of Padua, Italy
The red blood cell (RBC) content of Na' and K+ were measured both on fresh cells from normal, heterozygous P-thalassaemic and iron-deficiency-anaemic subjects, and on the same cells incubated for 24 h, at 37O C, either in presence or in absence of Calcium (Caz+). Caz+ did not increase membrane permeability to Na+, but increased the K loss, both from normal cells and t o a greater degree much more from hypochromic cells. Glucose largely prevented the K' loss from hypochromic cells incubated either in absence or in presence of Caz+, probably maintaining an adequate level of ATP during the incubation. EDTA only partially decreased the permeability to K' in hypochromic cells incubated for 24 h at 37O C, possibly removing CaZ+bound to the cell membrane. The results suggest that Caz+ does not represent the primary cause of K leak in hypochromic cells, but it is able t o enhance a pre-existing peculiar abnormality of the cell membrane when the ATP level slows down. K e y words: calcium - erythrocyte Na' and K+ - hypochromic red cells
Accepted for publication December 15, 1976 Correspondence to: Dr. Lucian0 Vettore, 1st. Patologia Medica 2 O dell'Universiti di Padova, Policlinico di Borgo Roma, 37100 Verona, Italy
It has been shown that red blood cells (RBC) from patients heterozygous for pthalassaemia (Th.) or affected by iron deficiency anaemia (I.D.A.) have a selective increase in membrane permeability to potassium (K+) when they are incubated at 37' C for 24 h in autologous plasma with-
out addition of glucose (Vettore et a1 1971, Gunn et a1 1972, Chapman et a1 1973, Vettore et a1 1974). Calcium (Caz+) can increase the permeability to K' of normal RBCs, provided that they are either previously depleted of energy (Kregenow & Hoffman 1972) or altered by
This work was supported by a grant fro'm Consiglio Nazionale delle Ricerche, Italy. Scand J Haematol (1977) 18
23
354
L.VETTORE, M. C. D E MATTEIS & G. C. FALEZZA
various substances such as fluoride or iodoacetic acid (Gardos 1958), propranolol (Manninen 1970), triose reductone (Passow 1961) etc. In each case the underlying mechanism is probably different. Gunn et a1 (1972) proposed that Ca2+ could play a role in the K leak shown by thalassaemic red cells. In the present paper we describe the effects of Ca2+on Na' and K content of normal and hypochromic red cells, both from heterozygous p-Th. and from I.D.A. patients. MATERIALS AND METHODS The study was carried out on 9 normal individuals, on 15 heterozygous P-thalassaemics and on 13 subjects with I.D.A. The RBC content of Nat and K+ was determined both on freshly drawn erythrocytes and on the same cells after incubation at 37O C for 24 h in the following solutions: 1. patients' own plasma 2. isotonic buffer pH 7.4, containing 140 mM NaCI, 20 mM Tris-HC1 and 1 mM CaC1z 3. the same buffer as above, containing 1 mM EDTA instead of Ca2+. Each cell suspension was previously diluted with the corresponding solution for incubation to a haematocrit value between 35 and 45 Yo. In four experiments hypochromic cells were incubated at 37O C for 24 h in a buffer containing 35 mM glucose together with Ca2+ (Figure 1). In one experiment the effect of 1 mM EGTA was compared with the effect of 1 mM EDTA on the K+ leakage. Before cation measurement, fresh and incubated cells were washed three times with 120 mM MgC12, and then 0.2 ml of packed cells were lysed in 9.8 ml of 0.1 % Triton-X 100 in distilled water. K* and Na+ concentrations were measured on haemolysate by flame photometry and referred to a constant number of RBCs (1013), as calculated from the MCH of the original blood and the haemoglobin concentration of each haemol ysate.
RESULTS AND DISCUSSION
Table 1 shows the mean content (f standard deviation) of Na' in fresh RBCs and in the same cells after incubation in different media, for the 3 groups of subjects (normals, Th. and I.D.A.). Ca2' did not increase the RBC membrane permeability to Na' both in normal and in hypochromic cells. The incubation in buffer containing EDTA increased the Na' content of hypochromic cells more than the incubation in plasma, possibly because the former did not contain glucose. Table 2 gives the mean content (f SD) of RBC K in the same experimental conditions described above. The results for Th. and I.D.A. cells, owing to their similarity, will be described together in the text. Previously presented data (Vettore et a1 1974) are here confirmed: as a consequence of incubation in plasma at 37O C for 24 h, hypochromic RBCs lost more K (- 27 %, compared with fresh cell content) than normal ones (- 10 %). The presence of CaZ+ in the incubation medium significantly increased (p < 0.001) the K+ loss from the cells of normal and hypochromic subjects; this loss was of 27 % in normal cells and of 51 % in hypochromic ones. Moreover the cells exhibiting the greater leak to K+ during the incubation in plasma lost much more K when they were incubated in buffer plus Ca2+,with a signscant positive correlation between the percentage decrease of cation content following the incubation without and with Ca2' (for normal and Th. cells p
