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V L Lew

Publications and source records attributed to V L Lew.

At least 73 records · Page 4Linked to original sources
PubMed1984

Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells.

Calcium chelators which can be incorporated inside small cells without disruption have become useful tools to investigate the role of intracellular ionized calcium in the processes of cell activation and signal-effect mediation. In experiments designed to investigate further Ca2+ pump function in chelator-loaded human red cells we found that the chelator-loading procedure itself caused delayed Ca2+-pump inhibition when pump function was explored by increasing the intracellular Ca2+ levels with the aid of the divalent cation ionophore A23187. Ca2+-pump inhibition was found to be secondary to ATP-depletion, and ATP-depletion, in turn, could be attributed to formaldehyde, which was released during the hydrolytic incorporation of free chelator, from the cleavage of the four ester groups which anchor it to cell membranes on addition to cell suspensions. The evidence suggests that the formaldehyde released stays largely within the cells. Formaldehyde, in concentrations of up to 20 mmol/l cells had no direct effects on Ca2+ transport in red cells, other than through ATP depletion. Procedures to circumvent the difficulties arising from the formaldehyde effects are outlined and discussed.

Adenosine Triphosphate↗
PubMed1984

Dehydration and delayed proton equilibria of red blood cells suspended in isosmotic phosphate buffers. Implications for studies of sickled cells.

PO4 buffers isosmotic with plasma or phosphate-buffered saline solution with a substantial proportion of PO4 are often used to wash and suspend red blood cells in studies of respiratory or sickling behavior. Measurements of sequential changes in mean cell hemoglobin concentration, pH, and ion content of red blood cells suspended in 295 mOsm Na-phosphate, pH 7.4, at 23 degrees or 37 degrees C, showed (1) rapid, persistent cell dehydration (mean cell hemoglobin concentration greater than 40 gm/dl) caused initially by Cl- efflux and later by replacement of monovalent Cl- by divalent HPO=4; and (2) temporary reversal of membrane pH gradients with normalization time (30 to 120 minutes) dependent on factors controlling the rate of phosphate-chloride exchange. Sequential equilibration of red blood cells in isosmotic citrate (impermeable) followed by PO4 demonstrated the two stages of the observed shifts in PO4 alone, and red blood cells suspended in 0.15 mol/L 32PO4 at 37 degrees C showed PO4 influx consistent with pH equilibrium kinetics. Sickle trait red blood cells deoxygenated at 37 degrees C, pH 7.4, in plasma or 10 mmol/L HEPES-buffered saline solution showed only 6% to 20% sickling. In isosmotic PO4, mean cell hemoglobin concentration was 40 to 41 gm/dl with approximately 80% sickling. In phosphate-buffered saline solution containing 70 mmol/L PO4, red blood cells showed smaller, similar changes (mean cell hemoglobin concentration approximately 38 gm/dl) with a longer equilibration period and deoxygenated sickle trait cells showed 40% sickling. The altered properties of red blood cells suspended in PO4 or phosphate-buffered saline solution were neither intended nor appropriate for many studies using these media, particularly with hemoglobin S-containing red blood cells, and interpretations of reported results must be reassessed in light of these findings.

Anemia, Sickle Cell↗
PubMed1983

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

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+]i) was controlled using the ionophore A23187. In normal cells, ouabain-sensitive 24Na efflux was inhibited with an apparent affinity for [Ca2+]i which depended on the concentration of A23187; 50% inhibition required 20-40 microM and 160-300 microM-cytoplasmic Ca2+ with 10 microM and 0.63 microM-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 microM and 110-170 microM-cytoplasmic Ca2+ at 10 microM and 0.63 microM-A23187 respectively. Removal of Ca from the cells failed to reverse either the Na pump inhibition or the fall in cell ATP. In ATP-enriched cells cytoplasmic Ca caused inhibition of ouabain-sensitive 24Na efflux in an A23187-dependent manner with apparent affinities for [Ca2+]i similar to those observed in the normal cells. Inhibition was complete at high [Ca2+]i. 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. 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 microM-Ca2+ and in the latter case complete at below 400 microM-Ca2+. 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. The data for Na pump inhibition by cytoplasmic Ca in the intact cells were well fitted by several kinetic models involving either [Ca2+]i or CaATP as the inhibitory species and a low affinity dependence of pump activity on MgATP or total ATP. However, for any model, the apparent affinities for CaATP or for Ca2+ required to fit the ATPase data were 2.5-10 times higher than those required to fit the data for Na efflux.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1982

Uniform ionophore A23187 distribution and cytoplasmic calcium buffering in intact human red cells.

The divalent cation-selective ionophore A23187 has been used to characterize cytoplasmic Ca and Mg buffering, Ca2+-pump parameters and the properties of a Ca2+-activated K+-channel in intact red cells. A critical assumption in these studies has been that the ionophore causes a uniform increase in divalent cation-permeability in all the cells. This has now been tested directly in ATP-depleted human red cells by analysing the kinetics of ionophore-induced 45Ca-tracer and net Ca2+ fluxes. The experimental curves were all adequately fitted by single-exponentials at all ionophore concentrations tested. Moreover, statistical analysis of 61 individual tracer influx curves and of pooled data showed no trend towards fast second exponential components. These results demonstrate uniformity of ionophore distribution, ionophore-induced Ca2+-permeability, and cytoplasmic Ca-buffering among all the cells. Experiments involving mixing of cell suspensions with high and low original ionophore content, and involving ionophore extraction by albumin, demonstrate a rapid redistribution of ionophore among the cells, indicating that homogeneity of ionophoric effects is achieved through dynamic ionophore redistribution.

Anti-Bacterial Agents↗
PubMed1982

Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator.

The physiological actions of Ca2+ as a trigger and second messenger depend on the maintenance of large inward resting Ca2+ gradients across the cell plasma membrane. An ATP-fuelled Ca-pump, originally discovered and still best characterized in human red cells, is now believed to mediate resting Ca2+ extrusion in most animal cells. However, even in red cells, the truly physiological pump-leak turnover rate and cytoplasmic free Ca2+ level are unknown. Previous estimates were only very imprecise upper limits because normal intact red cells have a minute total pool of exchangeable Ca of less than 1 mumol 1 cells; Ca fluxes could not be measured without artificially increasing that pool with ionophores or disrupting the membrane to incorporate Ca buffers. Both procedures leave the membrane considerably leakier than in intact cells. Here, we have increased the exchangeable Ca pool by non-disruptively loading a Ca-chelator into intact cells, using intracellular hydrolysis of a membrane-permeant ester. The trapped chelator made the free cytoplasmic calcium concentration, [Ca2+]i, an easily defined function of directly measurable total cell Ca. We were then able to establish the physiological steady-state [Ca2+]i and pump-leak turnover rate of fresh cells suspended in their own plasma. If [Ca2+]i was lowered below the normal resting level, the Ca pump rate decreased according to the square of [Ca2+]i, and the inward Ca leak increased. The increase in leak did not develop if the cells were depleted of ATP and ADP.

Adenosine Triphosphate↗
PubMed1981

Effect of a 'sickling pulse' on calcium and potassium transport in sickle cell trait red cells.

1. To trace the early development of the extensive functional membrane abnormalities found in sickle cell anaemia red cells which result from polymerization of haemoglobin S, we followed the effects on Ca and K transport of an in vitro sickling pulse in sickle cell trait (SA) red cells, whose membranes are initially normal.2. Sickling induced a progressively larger uptake of Ca in fed, starved and ATP-depleted SA cells, always substantially higher than that in normal (AA) red cells under comparable conditions. The fraction of ionized Ca within the SA cells, estimated from the equilibrium distribution of (45)Ca induced by the ionophore A23187 was about 0.4 of the total Ca content and similar in SA and AA cells.3. With ATP-depleted SA cells, Ca uptake (representing Ca permeability) was maximal during sickling and was only partially reduced towards normal after desickling. Net Ca uptake during sickling of fed or starved SA cells reverted to net Ca loss upon reoxygenation, irrespective of the Ca gradient, indicating full restoration of the low Ca permeability of the control conditions.4. Following desickling of both fed and starved SA cells, the rates of uphill extrusion of Ca gained during sickling were much smaller than those expected with normal Ca pumps operating at similar internal Ca concentrations.5. After 2 hr sickling ATP levels in starved SA cells were reduced by 50% regardless of the presence or absence of Ca in the medium; therefore sickling-induced Ca uptake was associated with no measurable consumption of ATP due to Ca-pump activity.6. With ATP-depleted SA cells, a Ca uptake of 2-3 mumole/l. cells elicited a maximal response of the K permeability system resulting in full equilibration of the K pools in the cell suspensions. Sickling of fed and starved SA cells produced a small increase in K permeability which was entirely independent of the presence or absence of Ca.7. Sickled forms persisted after reoxygenation only with ATP-depleted SA cells and were more frequent after sickling in the presence of Ca (about 20%) than in a Ca-free medium (about 4%).8. These findings show that initial sickling produces an increased Ca permeability whose extent and reversibility depends on the metabolic state of the cells, and a partial Ca-pump failure, which appears to be irreversible. We confirm a small sickling-related, reversible increase in K permeability but a Ca-dependent increase in K permeability does not occur unless the cells are fully depleted of ATP. The implications for sequential development of related abnormalities in SS cells are discussed.

Adenosine Triphosphate↗
PubMed1981

The magnesium dependence of sodium-pump-mediated sodium-potassium and sodium-sodium exchange in intact human red cells.

1. The magnesium content of human red blood cells was controlled by varying the magnesium concentration in the medium in the presence of the ionophore A23187. The new magnesium levels attained were very stable, which allowed the magnesium dependence of the sodium pump to be investigated.2. The effects of magnesium were shown to occur at the inner surface of the red cell membrane for the range of magnesium concentrations tested (10(-7) to 6 x 10(-3)m).3. At intracellular ionized magnesium concentrations below 0.8 mm the activation of ouabain-sensitive sodium-potassium exchange by internal ionized magnesium could be resolved into two or three components: (a) a small component, about 5% of the maximum flux, which is apparently independent of the ionized magnesium concentration below 2 mum, (b) a saturating component with a K((1/2)) of between 30 and 45 mum, and possibly (c) a component which increases linearly with ionized magnesium concentration and which only becomes apparent at concentrations above 0.1 mm.4. At intracellular ionized magnesium concentrations below 0.8 mm, activation of ouabain-sensitive sodium-sodium exchange by internal ionized magnesium could be resolved into two components: (a) a small component, about 6% of the maximal flux, which is apparently independent of the ionized magnesium concentration below 2 mum, and (b) a saturating component with a K((1/2)) of about 9 mum. At ionized magnesium concentrations between about 0.2 and 0.8 mm the rate of sodium-sodium exchange remained constant at the maximal level.5. The intracellular concentration of ATP decreased and the ADP concentration increased as the magnesium content of the cells was reduced from the normal level. A small increase in ATP and a small decrease in ADP was seen when the magnesium content was increased above the normal level. The variation in the ATP: ADP ratio from 2.5 at very low magnesium levels to about 6 at normal magnesium levels can account, at least in part, for the different K((1/2)) values of sodium-potassium and sodium-sodium exchange.6. When the concentration of ionized magnesium was increased above about 0.8 mm both sodium-potassium and sodium-sodium exchange were inhibited. Sodium-sodium exchange was more strongly inhibited than sodium-potassium exchange.7. The possible sites of action of magnesium in the sodium pump cycle are discussed.

Adenosine Diphosphate↗
PubMed1980

A Ca2+-refractory state of the Ca-sensitive K+ permeability mechanism in sickle cell anaemia red cells.

Simultaneous measurements of Ca content and 42K+ influx in sickle cell anaemia red cells confirm predictions from earlier data in the literature that the increased Ca content of sickle cell anaemia cells which are not metabolically depleted does not cause a quinine-sensitive increase in K+ permeability. It is shown that the ionophore, A23187, can cause the Ca contained inside sickle cell anaemia cells to activate the quinine-sensitive K+-permeability mechanism. This demonstrates the existence of a Ca2+-refractory state of the K+ channel in sickle cell anaemia cells and a direct stimulatory effect of the ionophore A23187 on its Ca sensitivity.

Anemia, Sickle Cell↗
PubMed1980

Progressive inhibition of the Ca pump and Ca:Ca exchange in sickle red cells.

Sickle cell anaemia red cells (SS) were reported to have a high Ca content and an increased Ca uptake on deoxygenation, but their Ca-pump activity was described as normal. This seemed puzzling because the saturated Ca-extrusion rate of the normal, high Ca-affinity Ca pump is about 10 mmol per 1 cells per h (refs 3, 4) and the highest sickling-induced Ca influx reported in SS cells and observed in ATP-depleted sickle-trait (SA) red cells never exceeded 0.2 mmol per 1 cells per h. Normal pump performance is, therefore, incompatible with Ca accumulation unless SS cells have abnormally high Ca-binding capacity. We provide here evidence which suggests that SS cells have normal Ca-buffering capacity and probably genetically normal Ca pumps, but that the sickling process causes progressive Ca-pump failure and a marked reduction in Ca:Ca exchange.

Anemia, Sickle Cell↗
PubMed1980

Magnesium buffering in intact human red blood cells measured using the ionophore A23187.

1. A method was developed for measuring the cytoplasmic magnesium buffering of intact red cells using the divalent cation selective ionophore A23187. Addition of A23187 to a suspension of red cells induces rapid equilibration of ionized magnesium across the cell membrane. 2. Entry of magnesium into red cells is associated with cell swelling and depolarization of the membrane potential. 3. At an external ionized magnesium concentration of about 0.15 mM corresponding to an internal ionized concentration of 0.4 mM the addition of A23187 did not produce a change in the magnesium content of the cells. This indicates that the normal ionized magnesium concentration inside the oxygenated red cell is about 0.4 mM. 4. The magnesium buffering curve for oxygenated, inosine-fed human red blood cells is adequately described by the existence of three buffer systems of increasing capacity and decreasing affinity. These are 0.15 mM with a Km < 10(-7) M, probably structural magnesium bound within the cell proteins; 1.6 mM with a Km approximately equal to 0.08 mM, mainly ATP and other nucleotides; and about 21-25 mM with a Km approximately equal to 3.6 mM, a major portion of this being organic phosphates. It is suggested that the contribution of 2,3-DPG to the low affinity site involves each phosphate group acting as an independent binding site for magnesium.

Anti-Bacterial Agents↗
PubMed1980

Excess magnesium converts red cell (sodium+potassium) ATPase to the potassium phosphatase.

1. The ATPase and phosphatase activities of red cell membranes were measured simultaneously as a function of the magnesium content of the medium. 2. It was found that when the magnesium concentration was greater than that of ATP, magnesium inhibited the ATPase and simultaneously stimulated the phosphatase. The concentrations of magnesium needed for half-maximal stimulation of the phosphatase and half-maximal inhibition of the ATPase were similar. 3. It is suggested that increasing the concentration of magnesium directly causes a change in the conformation of the enzyme from one which favours ATPase activity to one which favours phosphatase activity.

Biological Transport, Active↗
PubMed1979

The behaviour of transporting epithelial cells. I. Computer analysis of a basic model.

We analyse the non-steady state behaviour of a computer model representing functional epithelial cells. The results show that a simple model of an epithelium, containing the essential ion transport asymmetries of the original Koefoed-Johnsen-Ussing model, predicts much of the observed behaviour of 'tight-type' epithelia under various well characterized experimental conditions.

Amiloride↗