G. Gárdos

Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells.

Ca2+-transport and its energy consumption were studied in intact human red cells loaded with Ca2+ by the aid of the ionophore A23187. After the complete elimination of the ionophore the passive Ca2+-permeability of the membrane returned to its normal low value, except when the intracellular Ca2+-concentration was higher than 3 mM or the ATP level fell below 100 muM. Within these limits the rate of Ca2+-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca2+]i and reached a plateau at about 1 mM intracellular Ca2+-concentration. The maximum rate of Ca2+-efflux was about 85 mumol/l of cells per min at 37 degrees C, pH 7.4. The activation energy of active Ca2+-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7. Ca2+-efflux was not connected with the counter-transport of cations. The Ca2+-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, N-ethylmaleimide and p-chloromercuribenzoate or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca2+-extrusion was brought about by 50-250 muM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca2+-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane. To examine the energy consumption of the Ca2+-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca2+-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na+-tetrathionate. Ca2+-activated ATPase activity connected with the Ca2+-pump was distinguished from other Ca2+-ATPases by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca2+-transported per ATP split was found to be 2 : 1.

Molecular properties of the red cell calcium pump: I. Effects of calmodulin, proteolytic digestion and drugs on the kinetics of active calcium uptake in inside-out red cell membrane vesicles

Abstract In calmodulin-stripped inside-out human red cell membrane vesicles /IOV/ ATP + Mg2+-dependent active calcium uptake is stimulated by the addition of calmodulin. Calmodulin increases the maximum calcium transport rate /Vmax/, decreases KCa, and does not affect KATP of calcium uptake. The action of both membrane bound and external calmodulin is competitively inhibited by phenothiazines. Drugs reacting with SH groups of proteins reversibly inhibit calcium pumping by decreasing Vmax and not affecting KCa and KATP. The relative magnitude of calmodulin stimulation of calcium transport is unaltered by SH reagents. Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters — that is converts the system to a “high calcium-affinity” state. Proteolysis eliminates calcium-dependent calmodulin binding to IOV membranes and any further stimulation of calcium uptake by calmodulin. Based on these results the presence of a calmodulin-binding regulatory subunit of the red cell calcium pump at the internal membrane surface is postulated.

Effects of lanthanum on calcium-dependent phenomena in human red cells.

Lanthanum (0.25 mM) does not penetrate into fresh or Mg2+-depleted cells, whereas it does into ATP-depleted or ATP + 2,3-diphosphoglycerate-depleted cells, into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca2+-efflux completely and inhibits (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg2+-depleted cells Ca2+-Ca2+ exchange is inhibited by lanthanum. Ca2+-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca2+-leak +/ S.D. amounts to 0.28 +/ 0.08 mumol/1 of cells per min, whereas in KC1 medium to 0.15 +/ 0.04 mumol/1 of cells per min at 2.5 mM [Ca2+]e and 0.25 mM [La3+]e pH 7.1. Lanthanum inhibits Ca2+-dependent rapid K+ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K+ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol. Lanthanum at 0.2--0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca2+-loaded cells, however, is blocked by lanthanum owing to Ca2+-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca2+ concentrations.

Characterization of the inositol trisphosphate-sensitive and insensitive calcium stores by selective inhibition of the endoplasmic reticulum-type calcium pump isoforms in isolated platelet membrane vesicles

In mixed platelet membrane vesicles the presence of two distinct endoplasmic reticulum-type calcium pump enzymes of 100 and 97 kD molecular mass has been demonstrated. We have previously shown that both calcium pumps were recognized by polyclonal anti-sarcoplasmic reticulum calcium pump antisera [11]. In the present work we studied the effects of several calcium pump inhibitors on active calcium transport and inositol trisphosphate-induced calcium release in these vesicles in an attempt to assign the two calcium pump isoenzymes to specific calcium pools. The effect of the PL/IM 430 inhibitory anti-calcium pump antibody was compared to that of other calcium pump inhibitors acting predominantly on the 100 and the 97 kD calcium pump isoforms, respectively. The PL/IM 430 antibody, which recognized the 97 kD pump on Western blots and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, which inhibited phosphoenzyme formation of the same pump isoform, inhibited calcium accumulation predominantly into an inositol trisphosphate-releasable calcium pool. On the other hand, low concentration of thapsigargin, which inhibited phosphoenzyme formation mainly of the 100 kD pump isozyme, had a more pronounced effect on calcium uptake into an inositol trisphosphate-resistant pool. These data suggest that in platelets the 97 kD calcium pump isoform is likely to be associated with the inositol trisphosphate-sensitive calcium storage organelle.

Mechanism of Ca2+-dependent selective rapid K+-transport induced by propranolol in red cells.

SummaryPassive Ca2+ influx is gradually enhanced by 0.5 to 5mm propranolol in fresh and phosphate ester-depleted human red cells. In fresh cells the active Ca2+ efflux tends to counteract Ca2+ uptake. Membrane hyperpolarization, induced by the K+ transport that accompanies Ca2+ uptake, further enhances the rate of Ca2 uptake. The dissociated, positively charged form of propranolol seems to be crucial in the increase of passive Ca2+ influx caused by the drug. The effect can be attributed to the release of structural Ca2+ from the membrane (lipids).The release of structural Ca2+ promotes the formation of the selectively K+-permeable membrane structure as well. The transitions of lipid structure responsible for the opening of the passive Ca2+ and K+ pathways, however, are not identical. The opening of the K+ pathways is prevented by certain highly lipid-soluble substances (chlorobutanol, heptanol, oligomycin, etc.), whereas the formation of the Ca2+ pathways is unaffected. Passive K+ transport is inhibited by high propranolol concentrations (more intensively at alkaline pH), whereas Ca2+ transport is promoted. A further difference between the passive K+ and Ca2+ pathways is that SH-proteins also seem to be involved in the formation of the K+ pathways, whereas they do not play a specific role in the opening of the passive Ca2+ channels. The additional Ca2+ binding that triggers the formation of the K+ pathways also seems to occur in the protein area of the inner membrane surface.

Molecular properties of the red cell calcium pump: II. Effects of calmodulin, proteolytic digestion and drugs on the calcium-induced membrane phosphorylation by ATP in inside-out red cell membrane vesicles

In inside-out human red cell membrane vesicles /IOV/, in the absence of Mg2+, the only calcium-induced labelling by γ32P-ATP occurs in a 140–150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated intermediate /EP/ of the calcium pump. In the presence of Mg2+ calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accelerates EP formation both in the absence and presence of Mg2+. Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVs, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of EP. In trypsin-digested IOVs the molecular weight of the 32P-labelled EP is shifted to lower values /110–120 000/ We suggest that trypsin digestion cleaves off a 20–40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.

Mechanism of various drug effects on the Ca2+-dependent K+-efflux from human red blood cells

If ATP is reduced to a minimum in red blood cells (RBC) and Ca2+ is present in the medium a rapid K’efflux sets in unaccompanied by equimolar N

Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts

-uptake [ 11. Propranolol and pronethalol induce this phenomenon even in cells containing normal ATP levels [ 21. This work reports a study of the mechanism of these drug actions By obtaining different results in ATP-containing and ATP-depleted cells propranolol, pronethalol and tetraCaine were shown to interfere with ATP-membrane interactions. Various Ca*‘-membrane interactions (responsible for the Ca’+-penetration, rapid K?-transport, Ca-ATPase function and maintenance of the biconcave shape) were also affected. These effects are compared with effects of other membrane active drugs (e.g. histamine, antihistamines, chlorpromazine). Data are presented concerning the relationship between the Ic’ channels of RBC and the excitable membranes. Lew [3] classified the regulating factors of the rapid r-transport into four groups: 1) ATP depletion; 2) Ca2+ entry; 3) interaction between intracellular Ca2+ and the K+ channel or carrier; 4) movement of r through the K’ channel or carrier. From our results we introduced slight modifications into this classification and present our results within this frame.

Thrombin-induced activation of calcium transport pathways and their role in platelet functions

SummaryJurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30–50 μm) of indo-1 and with high concentrations (3.5–4.5mm) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca2+]i) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases, aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin-2 overloaded cells, a significant initial rise in [Ca2+]i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells.

Characterization of membrane calcium pumps by simultaneous immunoblotting and 32P radiography

In human platelets thrombin-induced calcium release from intracellular stores, the consequent influx of extracellular calcium, as well as their role in the aggregation and ATP-secretion reactions were examined. In indo-1-loaded platelets intracellular calcium release was studied in the presence of excess EGTA in the incubation medium, while calcium influx was followed after a rapid repletion of external calcium. After thrombin-stimulation both calcium release and calcium influx produced about the same peak levels of cytoplasmic free calcium but in the first case it was only a transient response, while in the latter one a sustained calcium signal was observed. Increased calcium influx could be evoked for several minutes after the addition of thrombin, it was selectively inhibited by Mg2+ (20 mM) and Ni2+ (1 mM) ions, by neomycin and by PCMB, a non-penetrating SH-group reagent. This calcium influx was practically insensitive to organic calcium channel blockers. Thrombin-induced platelet aggregation was only partial in the absence of external calcium, even if excess magnesium was present in the media, while the aggregation response became complete if external calcium was repleted. A significantly reduced aggregation could be seen in calcium-containing media if calcium influx was selectively inhibited. Platelet ATP-secretion under the same conditions did not depend on external calcium or on calcium influx. These data indicate that in thrombin-stimulated platelets the opening of specific plasma membrane calcium channels can be selectively modulated and these channels play a major role in the development of a full-scale aggregation.