scholarly journals The effects of Ca2+ and Sr2+ on Ca2+-sensitive biochemical changes in human erythrocytes and their membranes

1981 ◽  
Vol 198 (3) ◽  
pp. 441-445 ◽  
Author(s):  
D Allan ◽  
P Thomas
Keyword(s):  
Direct Interaction ◽  
Human Erythrocytes ◽  
Biochemical Changes ◽  
Intracellular Concentration ◽  
Ionophore A23187 ◽  
Erythrocyte Ghosts ◽  
High Concentration ◽  
Intact Cells ◽  
Whole Cells

1. The Ca2+-dependency of K+ efflux, microvesiculation and breakdown of polyphosphoinositides and of ankyrin have been measured in intact human erythrocytes exposed to ionophore A23187 and HEDTA [N'-(2-hydroxyethyl)ethylenediamine NNN'-triacetate]-Ca2+ buffers. Half-maximal responses were observed at pCa values of 6.4, 4.1, 5.0 and 4.8 respectively. 2. The Ca2+ dependencies of K+ efflux and breakdown of polyphosphoinositides and ankyrin measured in erythrocyte ghosts without addition of ionophore showed almost identical values with those seen in whole cells treated with ionophore. 3. We conclude that ionophore A23187 is able to cause rapid equilibration of extracellular and intracellular [Ca2+] in intact cells and that in the presence of a suitable Ca2+ buffer, ionophore A23187 can be used to precisely fix the intracellular concentration of Ca2+ in erythrocytes. 4. The relatively high concentration of Ca2+ required to produce microvesiculation in intact cells may indicate that microvesiculation could be at least partly dependent on a direct interaction of Ca2+ with phospholipid. 5. Results obtained with Sr2+ paralleled those with Ca2+, although higher Sr2+ concentrations were required to achieve the same effects as Ca2+. Mg2+ produced none of the changes seen with Ca2+ or Sr2+.

scholarly journals Evidence that the uptake of tri-iodo-l-thyronine by human erythrocytes is carrier-mediated but not energy-dependent

1982 ◽  
Vol 208 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Roelof Docter ◽  
Eric P. Krenning ◽  
Greetje Bos ◽  
Durk F. Fekkes ◽  
George Hennemann
Keyword(s):  
Contrast Agents ◽  
Rat Hepatocytes ◽  
Human Erythrocytes ◽  
Uptake System ◽  
Erythrocyte Ghosts ◽  
Intact Cells ◽  
X Ray ◽  
Dose Dependent Fashion ◽  
Uptake Studies ◽  
Energy Dependent

We investigated 3,3′,5-tri-iodo-l-thyronine transport by human erythrocytes and by ‘ghosts’ prepared from these cells. Uptake of tri-iodothyronine by erythrocytes at 37°C was time-dependent with a maximum reached after 60min. Tracer analysis after incubation for 1min revealed only one saturable binding site, with Km 128±19nm (mean±s.e.m.; n=7) and Vmax. 4.6±0.7pmol of tri-iodothyronine/min per 6×107 cells. After 10min incubation Km 100±16nm (n=10) was found with Vmax. 7.7±1.2pmol of tri-iodothyronine/10min per 6×107 cells. At 0°C the uptake system is still active, with Km 132±26nm and Vmax. 1.8±0.3pmol of tri-iodothyronine/10min per 6×107 cells. The Vmax. with intact cells is 5-fold greater than the Vmax. with membranes derived from the same amount of cells when uptake studies are performed in media with similar free tri-iodothyronine concentrations. This indicates that at least 80% of tri-iodothyronine taken up by the intact erythrocytes enters the cell. This saturable uptake system can be inhibited by X-ray-contrast agents in a dose-dependent fashion. (±)-Propranolol, but not atenolol, has the same effect, indicating that the membrane-stabilizing properties of (±)-propranolol are involved. Furthermore, there is no inhibition by ouabain or vanadate, which indicates that tri-iodothyronine uptake is not dependent on the activity of Na++K+-dependent adenosine triphosphatase. We have prepared erythrocyte ‘ghosts‘, resealed after 2.5min with 0mm-, 2mm- or 4mm-ATP inside. Inclusion of ATP and integrity of the membrane of the erythrocyte ‘ghosts’ were verified on the basis of an ATP-concentration-dependent functioning of the Ca2+ pump. No difference was found in the uptake of tri-iodothyronine by erythrocyte ‘ghosts’ with or without ATP included, indicating that uptake of tri-iodothyronine is not ATP-dependent. The following conclusions are drawn. (1) Tri-iodothyronine enters human erythrocytes. (2) There is only one saturable uptake system present for tri-iodothyronine, which is neither energy (i.e. ATP)-dependent nor influenced by the absence of an Na+ gradient across the plasma membrane. This mode of uptake of tri-iodothyronine by human erythrocytes is in sharp contrast with that of rat hepatocytes, which uptake system is energy-dependent and ouabain-sensitive [Krenning, Docter, Bernard, Visser & Hennemann (1978) FEBS Lett.91, 113–116; Krenning, Docter, Bernard, Visser & Hennemann (1980) FEBS Lett.119, 279–282]. (3) X-ray-contrast agents inhibit tri-iodothyronine uptake by erythrocytes in a similar fashion to that by which they inhibit the uptake of tri-iodothyronine by rat hepatocytes [Krenning, Docter, Bernard, Visser & Hennemann (1982) FEBS Lett.140, 229–233].

scholarly journals Inhibition by calcium ions of adenosine cyclic monophosphate formation in sealed pigeon erythrocyte ‘ghosts’. A study using the photoprotein obelin

1978 ◽  
Vol 176 (1) ◽  
pp. 53-66 ◽  
Author(s):  
A K Campbell ◽  
R L Dormer
Keyword(s):  
Cyclic Amp ◽  
Initial Rate ◽  
Ionophore A23187 ◽  
Erythrocyte Ghosts ◽  
Adrenergic Agonists ◽  
Intact Cells ◽  
Beta Adrenergic ◽  
Beta Adrenergic Agonists ◽  
The Relationship ◽  
Stimulation Of

1. Sealed pigeon erythrocyte ‘ghosts’ were prepared containing ATP and the Ca2+-activated photoprotein obelin to investigate the relationship cyclic AMP formation and internal free Ca2+. 2. The ‘ghosts’ were characterized by (a) morphology (optical and electron microscopy), (b) composition (haemoglobin, K+, Na+, Mg2+, ATP, obelin), (c) permeability to Ca2+, assessed by obelin luminescence and (d) hormone sensitivity (the effect of beta-adrenergic agonists and antagonists on cyclic AMP formation). 3. The effect of osmolarity at haemolysis and ATP at resealing on these parameters was investigated. 4. Sealed ‘ghosts’, containing approx. 2% of original haemoglobin, 150mM-K+, 0.5MM-ATP, 10(3)–10(4) obelin luminescence counts/10(6) ‘ghosts’, which were relatively impermeable to Ca2+ and in which cyclic AMP formation was stimulated by beta-adrenergic agonists over a concentration range similar to that for intact cells, could be prepared after haemolysis in 6mM-NaCl3mM-MgCl2/50mM-Tes, pH7, and resealing for 30min at 37 degrees C in the presence of ATP and 150mM-KCl. 5. The initial rate of adrenaline-stimulated cyclic AMP formation in these ‘ghosts’ was 30–50% of that in intact cells and was inhibited by the addition of extracellular Ca2+. Addition of Ca2+ to the ‘ghosts’ resulted in a stimulation of obelin luminescence, indicating an increase in internal free Ca2+ under these conditions. 6. The ionophore A23187 increased the rate of obelin luminescence in the ‘ghosts’ and also inhibited the adrenaline-stimulated increase in cyclic AMP. 7. The effect of ionophore A23187 on obelin luminescence and on cyclic AMP formation in the ‘ghosts’ was markedly decreased by sealing EGTA inside the ‘ghosts’. 8. It was concluded that cyclic AMP formation inside sealed pigeon erythrocyte ‘ghosts’ could be inhibited by more than 50% by free Ca2+ concentrations in the range 1–10 micrometer.

scholarly journals Ca2+-induced biochemical changes in human erythrocytes and their relation to microvesiculation

1981 ◽  
Vol 198 (3) ◽  
pp. 433-440 ◽  
Author(s):  
D Allan ◽  
P Thomas
Keyword(s):  
Human Erythrocytes ◽  
Biochemical Changes ◽  
Cross Linking ◽  
Ionophore A23187 ◽  
Cell Shrinkage ◽  
Polypeptide Pattern ◽  
Intracellular Concentrations ◽  
Protein Cross Linking

1. Human erythrocytes were treated with Ca2+ and ionophore A23187 and measurements were made of K+ efflux, polyphosphoinositide breakdown, 1,2-diacylglycerol accumulation, phosphatidate synthesis, changes in membrane polypeptide pattern and release of microvesicles. 2. It was shown that neither transamidase-mediated protein cross-linking, proteolysis of polypeptides 2.1 (ankyrin) or 4.1, nor accumulation of diacylglycerol or phosphatidate appeared to be necessary for microvesiculation to occur. 3. Microvesicles were released only under conditions where KCl efflux leading to cell shrinkage occurred and where polyphosphoinositides were broken down. These circumstances were sufficient to cause microvesiculation only in the presence of increased intracellular concentrations of Ca2+.

Calcium-dependent hydrolyses of polyphosphoinositides in human erythrocyte membranes

1984 ◽  
Vol 62 (6) ◽  
pp. 363-368 ◽  
Author(s):  
R. Blaine Moore ◽  
Stanley H. Appel
Keyword(s):  
Time Course ◽  
Erythrocyte Membranes ◽  
Ionophore A23187 ◽  
Erythrocyte Ghosts ◽  
Calcium Activation ◽  
Activation Curve ◽  
Intact Cells ◽  
Calcium Dependent ◽  
Human Erythrocyte Membranes ◽  
Phosphatidylinositol 4

Incubation of erythrocytes with [32P]phosphate resulted in a linear incorporation of the label into PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), PtdIns4P (phosphatidylinositol 4-monophosphate), and PA (phosphatidic acid) over a period of 2 h at 37 °C. Exposure of 32P-labelled erythrocyte ghosts to calcium caused a loss of label from PtdIns(4,5)P2 and PtdIns4P, but not PA. The concentration of calcium required for half-maximal hydrolyses of both polyphosphoinositides was about 1 μM. Strontium, at higher concentrations, stimulated the hydrolyses of both polyphosphoinositides but barium, up to 1 mM, had little effect. Intact erythrocytes incubated in the presence of Ca–EGTA buffers and the ionophore A23187 did not show marked losses of [32P]PtdIns(4,5)P2 or [32P]PtdIns4P, but rather exhibited a dramatic increase in the level of [32P]PA. In contrast, cells which had been depleted of their ATP lost significant amounts of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P and had less change in their levels of [32P]PA relative to intact cells. The calcium activation curve and the time course for [32P]PA synthesis in intact cells were similar to the calcium activation curve and the time course for the hydrolyses of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P in ATP-depleted erythrocytes. These results strongly support a link between Ca2+-dependent polyphosphoinositide breakdown and PA synthesis in human erythrocytes.

Effect of neomycin and ionophore A23187 on ATP levels and turnover of polyphosphoinositides in human erythrocytes

1977 ◽  
Vol 55 (9) ◽  
pp. 1007-1012 ◽  
Author(s):  
Verna Lang ◽  
Glen Pryhitka ◽  
J. Thomas Buckley
Keyword(s):  
Energy Charge ◽  
Human Erythrocytes ◽  
Metabolic Inhibitors ◽  
Ionophore A23187 ◽  
Intact Cells ◽  
Intracellular Atp ◽  
Atp Levels ◽  
Relationship Of ◽  
The Relationship ◽  
Polyphosphoinositide Metabolism

The relationship of polyphosphoinositide metabolism to erythrocyte ATP levels was examined. Turnover of polyphosphoinositides was not as closely dependent on ATP as it is reported to be in yeast. Neomycin increased 32P incorporation into diphosphoinositide and to a lesser extent into triphosphoinositide without affecting intracellular ATP. Treatment of intact cells with ionophore A23187 resulted in a decrease of at least 80% in polyphosphoinositide levels which followed a decrease in cellular ATP and an increase in phosphatidate. The results indicate that polyphosphoinositide turnover does not regulate energy charge in the erythrocyte. However some of the events which follow treatment of erythrocytes with metabolic inhibitors or calcium and ionophore may be related to the accompanying decrease in polyphosphoinositide levels.

scholarly journals THE ELECTROPHORETIC MOBILITY OF HUMAN ERYTHROCYTES—WHOLE CELLS, GHOSTS, AND FRAGMENTS

1938 ◽  
Vol 22 (1) ◽  
pp. 1-5 ◽  
Author(s):  
W. H. Byler ◽  
H. M. Rozendaal
Keyword(s):  
Cell Surface ◽  
Electrophoretic Mobility ◽  
Human Erythrocytes ◽  
Red Cell ◽  
Chicken Serum ◽  
Whole Cells ◽  
Slow Change ◽  
Foreign Substance ◽  
Pure Salt

The electrophoretic mobility of human red cell ghosts decreases in the presence of chicken serum. The decrease is not directly due to the presence of adsorbed material but to a change which is catalyzed by the foreign substance. It is suggested that abnormal serum materials resulting from disease may serve as catalysts. Fragments of broken cells have the same mobility as whole cells at first, then decrease even in pure salt suspension, while the whole cells remain essentially unchanged for hours. The results suggest that the slow change of whole cells, the change of ghosts in the presence of foreign serum, and the change of fragments are all manifestations of the same modification of structure or composition of the cell surface.

scholarly journals Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase

1988 ◽  
Vol 250 (2) ◽  
pp. 343-348 ◽  
Author(s):  
T Matsumoto ◽  
W Tao ◽  
R I Sha'afi
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Phospholipase A2 ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Membrane Preparation ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Intact Cells

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.

Lead and calcium transport in human erythrocyte

1999 ◽  
Vol 18 (5) ◽  
pp. 327-332 ◽  
Author(s):  
J V Calderón-Salinas ◽  
M A Quintanar-Escorcia ◽  
M T González-Martínez ◽  
C E Hernández-Luna
Keyword(s):  
Transport System ◽  
Human Erythrocyte ◽  
Calcium Transport ◽  
Human Erythrocytes ◽  
The Other ◽  
Erythrocyte Ghosts ◽  
Passive Transport ◽  
Ca Uptake

In this paper we report the lead (Pb) and calcium (Ca) uptake by erythrocyte ghosts. In both cases the transport was carried out by a passive transport system with two kinetic components (Michaelis-Menten and Hill). Pb and Ca were capable of inhibiting the transport of the other metal in a non-competitive way. Under hyperpolarization, the uptakes of Ca and Pb were enhanced and the Michaelis-Menten component prevailed. Both Ca and Pb uptakes were inhibited by N-ethyl-maleimide to the same extent. These results indicate that Pb and Ca share the same permeability pathway in human erythrocytes and that this transport system is electrogenic.

scholarly journals In Vitro and In Vivo Neutralization of the Relapsing Fever Agent Borrelia hermsii with Serotype-Specific Immunoglobulin M Antibodies

2001 ◽  
Vol 69 (2) ◽  
pp. 1009-1015 ◽  
Author(s):  
Alan G. Barbour ◽  
Virgilio Bundoc
Keyword(s):  
Monoclonal Antibodies ◽  
Antibody Response ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Intact Cells ◽  
Whole Cells

ABSTRACT The antigenic variation of the relapsing fever agent Borrelia hermsii is associated with changes in the expression of the Vlp and Vsp outer membrane lipoproteins. To investigate whether these serotype-defining proteins are the target of a neutralizing and protective antibody response, monoclonal antibodies were produced from spleens of infected mice just after clearance of serotype 7 cells from the blood. Two immunoglobulin M monoclonal antibodies, H7-7 and H7-12, were studied in detail. Both antibodies specifically agglutinated serotype 7 cells and inhibited their growth in vitro. Administered to mice before or after infection, both antibodies provided protection against infection or substantially reduced the number of spirochetes in the blood of mice after infection. Whereas antibody H7-12 bound to Vlp7 in Western blotting, enzyme-linked immunosorbent assay, and immunoprecipitation assays, as well as to whole cells in other immunoassays, antibody H7-7 only bound to wet, intact cells of serotype 7. Antibody H7-7 selected against cells expressing Vlp7 in vitro and in vivo, an indication that Vlp7 was a conformation-sensitive antigen for the antibody. Vaccination of mice with recombinant Vlp7 with adjuvant elicited antibodies that bound to fixed whole cells of serotype 7 and to Vlp7 in Western blots, but these antibodies did not inhibit the growth of serotype 7 in vitro and did not provide protection against an infectious challenge with serotype 7. The study established that a Vlp protein was the target of a neutralizing antibody response, and it also indicated that the conformation and/or the native topology of Vlp were important for eliciting that immunity.

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