Potassium transport and nucleoside metabolism in human red cells

Self Cite

SUMMARY1. The synthesis of ATP has been studied in human erythrocytes. Fresh cells showed no net synthesis of ATP when incubated with adenine or adenosine, although labelled adenine was incorporated into ATP in small amounts.2. Cold-stored cells (3-6 weeks old) became progressively depleted of adenine nucleotides but incubation with adenosine or adenine plus inosine restored the ATP concentration to normal within 4 hr. Incorporation of labelled adenine or adenosine into the ATP of incubated stored cells corresponded to net ATP synthesis by these cells.

Section: Resultssupporting

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Some aspects of adenosine triphosphate synthesis from adenine and adenosine in human red blood cells

Whittam¹,

Wiley²

1968

Self Cite

“…at saturating levels of K is between 100 and 800 /M (e.g. Dunham & Glynn, 1961;Robinson, 1967Robinson, , 1974Robinson, , 1976Whittam & Wiley, 1967;Garrahan & Glynn, 1967b;Karlish & Glynn, 1974). Under normal conditions, therefore, cell ATP may be a rate limiting factor in Na pump turnover (Post, Hegyvary & Kume, 1972;Karlish, Yates & Glynn, 1978 An additional point to note is that at 10 min after A23187 addition in cells incubated with both A23187 concentrations, there appears to be a minimum level to which cell ATP falls (about 200 ,umole/l.…”

Section: Calculationsmentioning

confidence: 99%

The effect of intracellular calcium on the sodium pump of human red cells.

Brown¹,

Lew²

1983

SUMMARY1. The inhibitory effect of cytoplasmic Ca on Na-pump-mediated Na-K exchange was investigated in intact red cells under conditions of constant cell volume, membrane potential and inorganic ion composition. The ionized cytoplasmic Ca concentration ([Ca2+]1) was controlled using the ionophore A23187.2. In normal cells, ouabain-sensitive 24Na efflux was inhibited with an apparent affinity for [Ca2+], which depended on the concentration of A23187; 50 % inhibition required 20-40 fM and 160-300 LM-cytoplasmic Ca2+ with 10 /tM and 0-63 SM-A23187 respectively. Cytoplasmic Ca also affected cell ATP content which fell rapidly on addition of A23187 and subsequently increased, steadied or continued to fall more slowly depending on the Ca and A23187 concentrations. Half-maximal fall required 5-15 AM and 110-170 /tM-cytoplasmic Ca2+ at 10/M and 0-63 /M-A23187 respectively.Removal of Ca from the cells failed to reverse either the Na pump inhibition or the fall in cell ATP.3. In ATP-enriched cells cytoplasmic Ca caused inhibition of ouabain-sensitive 24Na efflux in an A23187-dependent manner with apparent affinities for [Ca2+], similar to those observed in the normal cells. Inhibition was complete at high [Ca2+],. As in the normal cells, the ATP content of the cells fell in the presence of cytoplasmic Ca, but always remained above 1-2 m-mole/l. cells. This was higher than the ATP content of Ca-free normal intact cells. 4. A23187 had no effect on the inhibition by Ca of ouabain-sensitive ATPase activity in isolated red cell membrane preparations. Both under conditions near optimal for Na-K-ATPase activity and under conditions resembling those in the cytoplasm, inhibition was half-maximal at about 25 um-Ca2+ and in the latter case complete at below 400 /tM-Ca2 .5. The apparent ATP-dependence of ouabain-sensitive Na efflux in the presence of cytoplasmic Ca was distinctly different in the normal and ATP-enriched cells but in both groups of cells it was similar for data obtained with high and low concentrations of A23187.6. The data for Na pump inhibition by cytoplasmic Ca in the intact cells were well fitted by several kinetic models involving either [Ca2+], or CaATP as the inhibitory species and a low affinity dependence of pump activity on MgATP or total ATP. A. M. BROWN AND V. L. LEWHowever, for any model, the apparent affinities for CaATP or for Ca2+ required to fit the ATPase data were 25-10 times higher than those required to fit the data for Na efflux. 7. From the kinetic models fitted to the Na efflux data, it is predicted that physiological (i.e. 1-10 #m) fluctuations in cell [Ca2+], will inhibit the Na pump by less than 35 %.

“…To investigate this possibility, the ATP content of the original cells was altered by incubating either without metabolites or with adenine plus inosine in the presence or absence of inorganic phosphate (Pi). ATP synthesis is markedly stimulated by Pi since this ion is required at the first step of inosine metabolism (Whittam & Wiley, 1967). In this way, cells containing low, moderate and high ATP concentrations were obtained, the AT Pcontent being about 9, 40 and 1,600 jutM, respectively.…”

Section: Preparation Of Ghostsmentioning

confidence: 84%

“…Ghosts containing different ATP concentrations were prepared from cells incubated in a Na-medium at 37°C either for 30 min with 15 mm inosine plus 5 mm adenine, in the presence or absence of 10 mm-Na phosphate (Whittam & Wiley, 1967) or 3-14 hr in the presence of 10 mM-KCl without added metabolites. In the latter case, 1 jug/ml.…”

Section: Preparation Of Ghostsmentioning

confidence: 99%

The alteration by ouabain of calcium movements in human red cell ghosts.

Isern¹,

Romero²

1977

SUMMARY1. The influence of ouabain on net Ca movements was studied in human erythrocyte ghosts by atomic absorption spectrophotometry.2. Ouabain (10-5 M) showed a dual effect, altering both entry and exit of Ca from K-rich ghosts incubated in a high-Na medium in the presence of 10 mm-Ca.3. Stimulation of Ca entry was observed in the first 15 min at 370 C, whereas during the subsequent 15 min incubation ouabain elicited Ca extrusion. This latter effect was eliminated when the ouabain concentration was raised to 1 mM.4. Ouabain-dependent Ca movements were abolished by replacing both internal K and external Na with choline. They were also absent from ghosts prepared at a high lytic ratio (1:100) or obtained from ATPdepleted cells.5. A moderate increase in cell ATP enhanced the effect of ouabain on Ca efflux whilst it was eliminated at higher ATP levels.6. The actions of ouabain markedly depended on the initial ADP/ATP ratio in ghosts, being optimal at about 2-5.7. The results suggest that the effects of ouabain on Ca movements are mediated through the Na pump. Reversal of this pump in Na-rich K-free medium may provide the energy for active Ca transport.