Vanadate inhibition of the Ca2+-ATPase from human red cell membranes

(1) VO3(-) combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state of the Ca2+-dependent phosphoenzyme. (2) VO3(-) blocks hydrolysis of ATP at the catalytic site. The sites for VO3(-) also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase. (3) The sites for VO39-) show positive interaactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3(-). Although, with less effectiveness, Na2+ and K+ substitutes for K+ whereas Li+ does not. The apparent affinites for Mg24 and K+ for inhibiton by VO3(-) seem to be less than those for activation of the Ca2+-ATPase. (4) Inhibition by VO3(-) is independent of Ca2+ at concentrations up to 50 microM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitiory effect of VO3(-).     

Trifluoperazine inhibition of calmodulin-sensitive Ca2+ -ATPase and calmodulin insensitive (Na+ +K+)- and Mg2+ -ATPase activities of human and rat red blood cells

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca2+ -ATPase and calmodulin-insensitive (Na+ +K+)- and Mg2+ -ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca2+ -ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca2+ -ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na+ +K+)-ATPase and Mg2+ -ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.     

Reincorporated plasma membrane Ca2+-ATPase can mediate B-Type Ca2+ channels observed in native membrane of human red blood cells

Recently, we reported indirect evidence that plasma membrane Ca2+-ATPase (PMCA) can mediate B-type Ca2+ channels of cardiac myocytes. In the present study, in order to bring more direct evidence, purified PMCA from human red blood cells (RBC) was reconstituted into giant azolectin liposomes amenable to the patch-clamp technique. Purified RBC PMCA was used because it is available pure in larger quantity than cardiac PMCA. The presence of B-type Ca2+ channels was first investigated in native membranes of human RBC. They were detected and share the characteristics of cardiac myocytes. They spontaneously appeared in scarce short bursts of activity, they were activated by chlorpromazine (CPZ) with an EC50 of 149 mmole/l or 1 mmole/l vanadate, and then switched off by 10 mmole/l eosin or dose-dependently blocked by 1-5 mmole/l ATP. Independent of membrane potential, the channel gating exhibited complex patterns of many conductance levels, with three most often observed conductance levels of 22, 47 and 80 pS. The activation by vanadate suggests that these channels could play a role in the influx of extracellular Ca2+ involved in the vanadate-induced Gardos effect. In PMCA-reconstituted proteoliposomes, nearly half of the ATPase activity was retained and clear "channel-like" openings of Ba2+- or Ca2+-conducting channels were detected. Channel activity could be spontaneously present, lasting the patch lifetime or, when previously quiescent, activity could be induced by application of 50 mmole/l CPZ only in presence of 25 U/ml calmodulin (CaM), or by application of 1 mmole/l vanadate alone. Eosin (10 mmole/l) and ATP (5 mmole/l) significantly reduced spontaneous activity. Channel gating characteristics were similar to those of RBC, with main conductance levels of 21, 40 and 72 pS. The lack of direct activation by CPZ alone might be attributed to a purification-induced modification or absence of unidentified regulatory component(s) of PMCA. Despite a few differences in results between RBC and reincorporated PMCA, most probably attributable to the decrease in ATPase activity following the procedure of reincorporation, the present experimental conditions appear to reveal a channel-mode of the PMCA that shares many similarities with the B-type Ca2+ channel.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+ -transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca2+ + Mg2+)-ATPase activity and in Ca2+ efflux at 37 degrees C, depending on the side of the membrane at which the monovalent cations K+ and Na+ were placed. Under the conditions used, (Ca2+ + Mg2+)-ATPase activity and Ca2+ efflux was highest when K+ (35 +/- 0.5 mM (+/- S.E.), mean of four experiments) was at the inside and Na+ (130 mM) at the outside of the ghost membrane.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

Pre-steady-state phosphorylation of the human red cell Ca2+-ATPase

The pre-steady-state kinetics of phosphorylation of the Ca2+-ATPase by ATP was studied at 37 degrees C and in intact red cell membranes to approach physiological conditions. ATP and Ca2+ activate with K0.5 of 4.9 and 26.4 microM, respectively. Preincubation with Ca2+ did not change the K0.5 for ATP. Preincubation with ATP did not alter the initial velocity of phosphorylation suggesting that binding of ATP was not rate-limiting. Mg2+ added at the start of the reaction increased the initial rate of phosphorylation from 4 to 8 pmol/mg/s. With 30 microM Ca2+, the K0.5 for Mg2+ was 60 microM. Mg2+ and Ca2+ added together beforehand accelerated phosphorylation to 70 pmol/mg/s. Phosphorylation of calmodulin-bound membranes was the fastest (280 pmol/mg/s), and its time course showed a neat overshoot before steady state. The results suggest that either preincubation with Ca2+ plus Mg2+ or calmodulin accelerated phosphorylation shifting toward E1 the equilibrium between the E1 and E2 conformers of the enzyme. K+ had no effect on the initial rate of phosphorylation and lowered by 40% the steady-state level of phosphoenzyme in the absence of Mg2+. Phosphorylation is not rate-limiting for the overall reaction since its initial rate was always higher than ATPase activity. In the absence of K+, the turnover of the phosphoenzyme was 2000 min-1, which is close to the values for other transport ATPases.     

In vitro stimulation of human red blood cell Ca2+-ATPase by thyroid hormone

Ca²⁺-ATPase activity in human erythrocyte ghosts previously washed to remove endogenous thyroid hormone is stimulated $\text{in}\text{vitro}$ by physiologic concentrations of thyroxine (T₄) and triiodothyronine (T₃). Two- to three-fold increases (P <0.005) in Ca²⁺-ATPase activity occurred after 60–120 minutes' exposure of membranes to iodothyronines at concentrations of T₄ and T₃ of 10^?8 M to 10^?12 M. T₄ was more active than T₃ and its activity did not depend upon prior conversion to T₃. The Ca²⁺-ATPase effect represents an extranuclear action of thyroid hormone in a human cell model.     

(Ca2+ + Mg2+)-ATPase activator: its presence in human red blood cells and rabbit skeletal muscle.

We find that both human red blood cells and rabbit skeletal muscle contain a soluble activator which can stimulate (Ca2+ + Mg2+)-ATPase activity. The activator protein from either source can enhance the (Ca2+ + Mg2+)-ATPase of both the red blood cell membrane and the microsomal fraction from skeletal muscle. The data suggest that they are members of the class of Ca2+-binding modulator proteins. A possible physiological role for the skeletal muscle activator protein in the contractile process is discussed.     

Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.     

Sensitivity and reversibility of Ca-dependent inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na⁺ + K⁺)-ATPase to inhibition by Ca was increased 30-fold by a partially purified extract of human red cell hemolysate. The hemolysate fraction reduced the concentration of free Ca required for 50% inhibition from 30 μM to approx. 1 μM. Ca-dependent inhibition of the (Na⁺ + K⁺)-ATPase in the presence and absence of the hemolysate fraction was completely reversible. The hemolysate fraction also stimulated the Ca²⁺-ATPase and increased its affinity for Ca. In the presence of the hemolysate fraction, the concentration of free Ca that inhibited the (Na⁺ + K⁺)-ATPase by 50% was similar to that which half-maximally stimulated the Ca²⁺-ATPase. Boiling the fraction destroyed its effect on the (Na⁺ + K⁺)-ATPase, but did not impair its stimulation of the Ca²⁺-ATPase.     

Two modes of inhibition of the Ca2+ pump in red cells by Ca2+

Two different and independent modes of inhibition of the Ca2+ pump by Ca2+ can be detected measuring active Ca2+ extrusion from resealed ghosts of human red cells: one requires extracellular and the other requires intracellular Ca2+. Ki for inhibition by extracellular Ca2+ is about 10 mM. Extracellular Mg2+ replaces Ca2+ in inhibiting Ca2+ transport but with an apparent affinity for inhibition about 3-times less than that for Ca2+. Inhibition by external Ca2+ is not affected by Na+ or K+ at both surfaces of the cell membrane, external EGTA, internal Ca2+ or ATP. The apparent affinity for external Ca2+ progressively raises as pH increases. The effects of extracellular Ca2+ and Mg2+ are consistent with the idea that for Ca2+ pumping to proceed, external sites in the pump must be protonated and not occupied by extracellular Ca2+ or Mg2+. Inhibition by intracellular Ca2+ takes place with a Ki of about 1 mM and is independent of external Ca2+. The inhibitory effects of intracellular Ca2+ can be accounted for if Ca2+ and CaATP were competitive inhibitors of the activation of the pump by Mg2+ and MgATP, respectively.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Activation of partial reactions of the Ca2+-ATPase from human red cells by Mg2+ and ATP.

(1) At ATP concentrations up to 30 micrometer addition of 0.5 mM MgCl2 in the reaction mixture increases both the rate of formation and the steady-state level of the phosphoenzyme of the Ca2+-ATPase from human red cell membranes. Under these conditions Mg2+ has no effect on the rate of dephosphorylation, which remains slow. (2) In the presence of Mg2+ the rate of dephosphorylation is increased 5 to 10 times by high (1 mM) concentrations of ATP. (3) Provided Mg2+ has reacted with the phosphoenzyme, acceleration of dephosphorylation by ATP takes place in the absence of Mg2+. This suggests that the role of Mg2+ on dephosphorylation is to convert the phosphoenzyme into a form that, after combination with ATP, reacts rapidly with water. (4) The results are consistent with the idea that combination of ATP at a non-catalytic site is needed for rapid dephosphorylation of the Ca2+-ATPase.     

Elevated intracellular Ca2+ affects Lii-Nao countertransport in human red blood cells

Changes in cytoplasmic Ca2+ concentration and in Lii-Nao countertransport activity have been shown to be associated with essential hypertension. Elevated intracellular free [Ca2+], as well as abnormalities of Ca2+ binding and transport have been reported in cells from different tissues of hypertensive laboratory animals and essential hypertensive patients. Similarly, enhanced rates of Lii-Nao countertransport and the modified pattern of the temperature dependence of this activity in red blood cells from essential hypertensive patients have been previously demonstrated. The aim of the present study was to investigate possible interaction between changes in intracellular free [Ca2+] and the Lii-Nao exchange in human red blood cells. The ionophore ionomycin was used to allow Ca2+ incorporation into the cells in a dose-dependent manner. The elevation of intracellular [Ca2+], in turn, resulted in enhanced Li+ efflux from the cells. At 3 microM, ionomycin selectively and significantly enhanced the Lii-Nao countertransport but not Li+ leakage from the cells. EGTA totally abolished the effect of ionomycin, indicating that the effect is directly related to Ca2+. As low as 0.4 microM Ca2+ caused a statistically significant effect. The maximal effect of Ca2+ on the Lii-Nao countertransport was achieved around the external pH range of 6.8-7.5. In contrast, the leakage of Li+ was significantly enhanced by Ca2+ at a pH of 7.4 and above. Ca2+ did not affect the Km of the Lii-Nao countertransport for Li+. Amiloride, which inhibits Na+/H+ exchange, inhibited only 10% of the Ca2+-enhanced countertransport. It is concluded that Ca2+ may play a role in the regulation of Lii-Nao countertransport in erythrocytes.     

The plasma membrane Ca2+-ATPase protein from red blood cells is not modified in preeclampsia

Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.     

Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

Red blood cells (RBCs) are a major component of blood clots, which form physiologically as a response to injury or pathologically in thrombosis. The active participation of RBCs in thrombus solidification has been previously proposed but not yet experimentally proven. Holographic optical tweezers and single-cell force spectroscopy were used to study potential cell-cell adhesion between RBCs. Irreversible intercellular adhesion of RBCs could be induced by stimulation with lysophosphatidic acid (LPA), a compound known to be released by activated platelets. We identified Ca²⁺ as an essential player in the signaling cascade by directly inducing Ca²⁺ influx using A23187. Elevation of the internal Ca²⁺ concentration leads to an intercellular adhesion of RBCs similar to that induced by LPA stimulation. Using single-cell force spectroscopy, the adhesion of the RBCs was identified to be approximately 100 pN, a value large enough to be of significance inside a blood clot or in pathological situations like the vasco-occlusive crisis in sickle cell disease patients.     

Some properties of the purified (Ca2+ + Mg2+-ATPase from human red cell membranes

The (Ca²⁺ + Mg²⁺-ATPase from red cell membranes, purified by means of a calmodulin-containing affinity column according to the method of Gietzen et al. (Gietzen, K., Tej?ka, M. and Wolf, H.U. (1980) Biochem. J. 189, 81–88) with either phosphatidylcholine or phosphatidylserine as phospholipid is characterized. The phosphatidylcholine preparation can be activated by calmodulin, while the phosphatidylserine preparation is fully activated without calmodulin. The enzyme shows a biphasic ATP dependence with two K_m values of 3.5 and 120 μM. The enzyme is phosphorylated by ATP in the presence of Ca²⁺ only.     

The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+

Media prepared with CDTA and low concentrations of Ca2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na+ +K+)-ATPase preparations are free of contaminant Mg2+. In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. In the absence of Mg2+ the rate of phosphorylation in the presence of 1 microM Ca2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca2+ is increased to 5 mM. The KCa for phosphorylation is 2 microM in the presence and 60 microM in the absence of Mg2+. Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca2+ at the transport site and Mg2+ at an activating site and that Ca2+ replaces Mg2+ at this site. Under conditions in which it increases the rate of phosphorylation, Ca2+ is without effect on the Ca2+-ATPase activity in the absence of Mg2+ suggesting that to stimulate ATP hydrolysis Mg2+ accelerates a reaction other than phosphorylation. Activation of the E1P----E2P reaction by Mg2+ is prevented by Ca2+ after but not before the synthesis of E1P from E1 and ATP, suggesting that Mg2+ stabilizes E1 in a state from which Mg2+ cannot be removed by Ca2+ and that Ca2+ stabilizes E1P in a state insensitive to Mg2+. The response of the Ca2+-ATPase activity to Mg2+ concentration is biphasic, activation with a KMg = 88 microM is followed by inhibition with a Ki = 9.2 mM. Ca2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg2+, and Mg2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca2+ at the transport site of the Ca2+-ATPase.     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.