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 Turnover of eicosanoid precursor fatty acids among phospholipid classes and subclasses of cultured human umbilical vein endothelial cells

1989 ◽  
Vol 258 (2) ◽  
pp. 427-434 ◽  
Author(s):  
H Takayama ◽  
M H Kroll ◽  
M A Gimbrone ◽  
A I Schafer
Keyword(s):  
Fatty Acids ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Ionophore A23187 ◽  
Human Umbilical Vein ◽  
Phospholipid Classes ◽  
Specific Increase ◽  
Umbilical Vein Endothelial Cells ◽  
Relationship Of ◽  
The Relationship

Using cultured human umbilical vein endothelial cells, in which phosphatidylcholine (PC) is equally pulse-labelled by various eicosanoid precursor fatty acids (EPFAs), we have studied the remodelling of EPFAs among the phospholipid classes and subclasses with and without activation, and the relationship of this remodelling process to the selective release of arachidonic acid (AA) by phospholipase A2-mediated cell stimulation. When endothelial cells are pulse-incubated with radiolabelled EPFA for 15 min, greater than 80% of cell-associated radioactivity is present in phospholipids, among which greater than 60% is found in 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl PC). After removing unincorporated radioactivity, reincubation of the pulse-labelled cells for up to 6 h results in progressive decrease in EPFA-labelled diacyl PC, increase in AA- or eicosapentaenoic acid (EPA)-labelled 1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmalogen PE) and increase only in AA-labelled 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl PC). This redistribution of radiolabelled phospholipids is not altered by the presence of excess non-radiolabelled EPFAs. When aspirin-treated EPFA-labelled endothelial cells are stimulated with ionophore A23187, a very selective release of AA is noted in comparison with eicosatrienoate (ETA) or EPA, accompanied by an equivalent decrease in AA-labelled diacyl PC and specific increase in AA-labelled plasmalogen PE and alkyl PC. These selective changes in AA radioactivity induced by A23187 are enhanced 2-fold by pretreating the AA-labelled cells with phorbol 12-myristate 13-acetate, which by itself induces no changes. The changes in radioactivity induced by A23187 without and with phorbol ester among the released AA, the diacyl PC and the plasmalogen PE are significantly correlated with each other. These results indicate that human endothelial cells incorporate EPFAs (AA, ETA, EPA) equally into diacyl PC but selectively release AA esterified into diacyl PC with specific remodelling into plasmalogen PE and alkyl PC.

The relationship of NADH-dependent diaphorase activity and methemoglobin reduction in human erythrocytes

1968 ◽  
Vol 19 (3) ◽  
pp. 524-526 ◽  
Author(s):  
Yasunori Kanazawa ◽  
Masao Hattori ◽  
Kinori Kosaka ◽  
Kiku Nakao
Keyword(s):  
Human Erythrocytes ◽  
Diaphorase Activity ◽  
Relationship Of ◽  
The Relationship

Ki+-Na+o Countertransport in Erythrocytes of Patients with Essential Hypertension

1981 ◽  
Vol 61 (s7) ◽  
pp. 11s-12s ◽  
Author(s):  
N. Adragna ◽  
M. Canessa ◽  
I. Bize ◽  
H. Solomon ◽  
D. C. Tosteson
Keyword(s):  
Essential Hypertension ◽  
Intracellular Calcium ◽  
Normal Subjects ◽  
Human Erythrocytes ◽  
Hypertensive Patients ◽  
Tentative Hypothesis ◽  
Relationship Of ◽  
The Relationship ◽  
In Cells

1. We describe in this paper a new ouabain-insensitive pathway for Na+ and K+ in human erythrocytes. K+ efflux was measured in cells loaded by the p-chloromercuribenzene-sulphonate (PCMBS) procedure to contain approximately equal amounts of Na+ and K+. K+ efflux was stimulated by external Na+ in the presence of ouabain and frusemide. Na+-stimulated K+ efflux was 0.35 ± 0.12 (mmol h−1 l−1 of cells) in eight normal subjects and 0.64 ± 0.13 in 13 patients with essential hypertension. 2. The Na+-stimulated K+ efflux was not observed in cells loaded in the presence of EGTA. This inhibition by EGTA suggests that K+ efflux is dependent on intracellular calcium. The Ki+-Nao+ countertransport of hypertensive patients was also inhibited by EGTA. The elevated K+—Na+ countertransport of hypertensive patients could be due to a higher intracellular Ca2+ content (Cai2+) or to an increased affinity for Cai2+. The relationship of this pathway to the Gardos effect is not clear since Na+-stimulated K+ efflux occurs without metabolic depletion or inhibition of the Ca2+ pump. As a tentative hypothesis, we relate the Ca2+-dependent downhill movement of K+ and Na+ to the Ca2+-dependent channels described in muscle and nerve, but other hypotheses cannot be excluded at this stage of our research.

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+.

A SEROLOGICAL STUDY OF CELL WALLS OF CERTAIN LACTIC ACID BACTERIA

1962 ◽  
Vol 8 (5) ◽  
pp. 629-637
Author(s):  
K. L. Chung ◽  
Roma Z. Hawirko
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Intact Cell ◽  
Synthetic Medium ◽  
Specific Cell ◽  
Intact Cells ◽  
Common Antigens ◽  
Species Specific ◽  
Relationship Of ◽  
The Relationship

From three species of Lactobacillus and three species of Streptococcus, cultured in a synthetic medium, cell walls were isolated following sonic disintegration and purified by washing. Sera against each species were prepared by injecting three rabbits with cell walls, and three with intact cells. Reciprocal agglutination tests were carried out with unabsorbed and absorbed antisera. More kinds of antibodies were detected with cell-wall antisera than with intact-cell antisera. Many species in the two genera shared common antigens. S. faecalis was the exception. Certain antigens believed to be complex haptens in nature reacted with heterologous antisera. Haemagglutination of tanned erythrocytes sensitized with a particulate cell-wall suspension showed fewer cross reactions than agglutination of intact-cell suspensions.The evidence presented shows the possibility of using antisera against species-specific cell-wall antigens for the identification of these species. The relationship of these species is discussed.

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.

Relationship of the Escherichia coli TrkA System of Potassium Ion Uptake with the F0F1-ATPase Under Growth Conditions Without Anaerobic or Aerobic Respiration

1998 ◽  
Vol 18 (3) ◽  
pp. 143-154 ◽  
Author(s):  
Armen Trchounian ◽  
Yelena Ohanjanyan ◽  
Karine Bagramyan ◽  
Vitya Vardanian ◽  
Eleonora Zakharyan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Transport Systems ◽  
Aerobic Respiration ◽  
Anaerobic Conditions ◽  
Intact Cells ◽  
Potassium Ion Uptake ◽  
Relationship Of ◽  
The Relationship

K+ uptake by the Escherichia coli TrkA system is unusual in that it requires both ATP and ΔμH; a relation withH+ circulation through the membrane is thereforesuggested. The relationship of this system with the F0F1-ATPase was studied in intact cells grownunder different conditions. A significant increase of the N,N'-dicyclohexylcarbodiimide(DCCD)-inhibitedH+ efflux through the F0F1 by 5 mMK+, but not by Na+ added into thepotassium-free medium was revealed only in fermenting wild-type orparent cells, that were grown under anaerobic conditions withoutanaerobic or aerobic respiration and with the production of H2. Such an increase disappeared in the Δunc or the trkA mutants that have alteredF0F1 or defective TrkA, respectively. This finding indicates a closed relationship between TrkA andF0F1, with these transport systems beingassociated in a single mechanism that functions as an ATP-driven H+–K+-exchanging pump. ADCCD-inhibited H+–K+-exchangethrough these systems with the fixed stoichiometry of H+and K+ fluxes(2H+/K+) and a higherK+ gradient between the cytoplasm and the externalmedium were also found in these bacteria. They were not observed incells cultured under anaerobic conditions in the presence of nitrate orunder aerobic conditions with respiration and without production of H2. The role of anaerobic or aerobic respiration as adeterminant of the relationship of the TrkA with the F0F1 is postulated. Moreover, an increase of DCCD-inhibited H+ efflux by added K+, aswell as the characteristics of DCCD-sensitiveH+–K+-exchange found in a parentstrain, were lost in the arcA mutant with a defective Arc system, suggesting a repression of enzymes in respiratorypathways. In addition, K+ influx in the latest mutantwas not markedly changed by valinomycin or with temperature. The arcA gene product or the Arc system is proposed to beimplicated in the regulation of the relationship between TrkA and F0F1.

scholarly journals The Discocyte-Echinocyte Transformation: Comparison of Normal and ATP-enriched Human Erythrocytes

Blood ◽  
1974 ◽  
Vol 44 (5) ◽  
pp. 639-647 ◽  
Author(s):  
Claude J. Féo ◽  
Pierre F. Leblond
Keyword(s):  
Inorganic Phosphate ◽  
Complete Recovery ◽  
Similar Rate ◽  
Atp Depletion ◽  
Temperature Dependent ◽  
Intracellular Atp ◽  
Human Red Blood Cells ◽  
Atp Levels ◽  
Intrinsic Mechanism ◽  
The Relationship

Abstract This work studies the relationship existing between intracellular ATP levels and the discocyte-echinocyte shape equilibrium in normal as well as ATP-enriched human red blood cells. Fresh erythrocytes metabolically depleted by incubation in a glucosefree buffer undergo echinocytic transformation (crenation) as intracellular ATP levels decline below 50% of their original value. When ATP is regenerated by further incubation of the same cells in the presence of glucose, inosine, and adenine (GIA), concentrations required for the complete recovery of the discocytic shape are lower than those which were necessary to maintain this shape in the first place. Addition of inorganic phosphate (Pi) to the GIA medium results in elevation of fresh cell ATP to supranormal levels. When such ATP-enriched cells are then depleted in the presence of the same concentration of inorganic phosphate, echinocytic transformation occurs much more rapidly than in normal fresh cells not previously incubated with Pi despite a similar rate of ATP depletion in both cases. It is suggested that the intrinsic mechanism responsible for shifting the discocyte-echinocyte equilibrium in the human erythrocyte is dependent on one or more intracellular or intramembrane factors occurring in conjunction with ATP depletion or repletion rather than to the absolute concentration of this nucleotide within the cell. This additional factor(s) appears to be temperature dependent and is influenced by the concentration of Pi in the medium.

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