Fusion of rat erythrocytes by membrane-mobility agent A2C depends on membrane proteolysis by a cytoplasmic calpain

The membrane-mobility agent 2-(2-methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) promotes fusion of rat, but not of human, erythrocytes. The difference in fusibility was shown to be correlated with membrane proteolysis, a process induced by Ca2+ in the rat erythrocytes or hemolysate-loaded ghosts, but not in the human cell. Membrane proteolysis is necessary but not sufficient for fusion. Fusion requires both Ca2+ and A2C [Kosower, N. S., Glaser, T. and Kosower, E. M. (1983) Proc. Natl Acad. Sci USA 80, 7542-7546]. Membrane proteolysis (Ca2+-dependent) and fusion (Ca2+ and A2C-dependent) requires a Ca2+-activated cytoplasmic thiol protease, as shown by the following observations. In intact rat erythrocytes, proteolysis and fusion are prevented by thiol alkylation and by inhibitors of Ca2+-dependent thiol proteases. Inhibitors to other proteases have no effect. Erythrocyte ghosts undergo proteolysis and fusion only when loaded with non-inhibited hemolysate, irrespective of membrane status (native or alkylated membrane). A partially purified cytosolic enzyme, identified as calpain I, promotes proteolysis in rat erythrocyte ghosts. A2C induces fusion only in such calpain-treated ghosts.     

Activation of intracellular calcium-activated neutral proteinase in erythrocytes and its inhibition by exogenously added inhibitors

Intracellular calcium-activated neutral proteinase (CANP) in rabbit erythrocytes was activated by an influx of Ca2+ into the cells. The catalytic large subunit changed from the original 79 kDa from to the 77 kDa and 76 kDa forms on activation just in the same manner as occurs in the autolytic activation of purified CANP in vitro. The activation required both extracellular Ca2+ and A23187, and was accompanied by the degradation of some membrane proteins and morphological changes in erythrocyte shape from discocytes to echinodisks, echinocytes, and spherocytes. Exogenously added Cbz-Leu-Leu-Leu-aldehyde inhibited the activation of intracellular CANP as well as the degradation of membrane proteins and the morphological changes indicating that the latter two processes are due to the action of CANP. Leupeptin and E64d were without effect on intracellular CANP.     

Assessment of the role of endogenous regulators in the activation of Ca-ATPase in erythrocyte membranes

The contribution of calmodulin and protein kinases A or C to the activation of membrane Ca-ATPase was studied on saponin-permeabilized rat erythrocytes. In the presence of all endogenous regulators, the dependence of the Ca-ATPase activity of Ca2+ concentration was described by a bell-shaped curve with a maximum at 2-5 microM Ca2+; K0.5 = 0.43 microM Ca2+. Washing of erythrocyte membranes with 5-10 microM Ca2+ maintained up to 75% of the ATPase activity, while washing with EGTA (2 mM) decreased the activity, on the average, 5-fold, and increased K0.5 up to 0.54-0.6 microM Ca2+. An addition of an EGTA extract to washed membranes restored up to 75% of the original ATPase activity, while calmodulin restored about 40% of the original Ca-ATPase activity and decreased K0.5 to 0.23-0.3 microM Ca2+. The calmodulin inhibitor R24571 failed to alter the Ca-ATPase activity in permeabilized erythrocytes but slightly diminished it in reconstituted membranes. The protein kinase C inhibitors H7 and polymyxin increased the Ca-ATPase activity in permeabilized red cells and suppressed it in reconstituted membranes. The data obtained suggest that in native red cell membranes Ca-ATPase is activated by regulator(s) dependent on Ca2+ and protein kinase which are other than calmodulin.     

Calcium-dependent process in reduction of cell surface charge after x-irradiation.

The electrophoretic mobility (EPM) of rat erythrocytes and cultured melanoma cells decreased with time after X-irradiation in the presence of calcium at concentrations higher than 10 (-5) M. At 37 degrees C, the presence of calcium for the first 20 min of exposure was suffcient to induce the EPM reduction, and Ca 2+ administration subsequent to Ca 2+ -free incubation for 30 min following irradiation had no effect on EPM. At lower temperatures, from 10 down to 20 degrees C however, the effect of calcium on the reduction of EPM decreased drastically. If the cells were kept Ca 2+ -inonophore A23187 also induced to decrease in EPM only in the presence of Ca 2+. These results revealed the transitory existence of membrane condition reactive to extracellular Ca 2+ immediately after X-irradiation, which can be postponed at low temperatures. The reduction of EPM by Ca 2+ -ionophore might suggest that the influx of Ca 2+ is a step in the reduction of EPM after X-irradiation.     

mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

mu-Calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that mu-calpain becomes proteolytically active in the presence of 2-200 microM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; approximately 1 microM) than the levels at which mu-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 microM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for mu-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with approximately 15% autolysis observed after 1-min exposure to 2.5 microM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze mu-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% VO2 peak until fatigue, n = 3) on the amount of autolyzed mu-calpain or calpain-3 in human muscle. No significant autolysis of mu-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.     

Calcium ions and cell fusion. Effects of chemical fusogens on the permeability of erythrocytes to calcium and other ions.

1. Fusogenic and non-fusogenic chemicals were tesetd for their ability to allow 45Ca2+ and 3H2O to enter hen and human erythrocytes. 2. The ratio of 45Ca2+/3H2O in treated cells to that in untreated cells is referred to as the entry ratio. 3. Within 1 min at 37 degrees C both water-soluble and lipid-soluble fusogens increased the value of the entry ratio, which reached maximum values in 5--10 min. 4. Values of the entry ratio in the range of 4--12 were found under conditions that led to cell fusion. 5. Closely related but non-fusogenic chemicals did not significantly alter the entry ratio. 6. The entry ratios for 86Rb+, 22Na+ and 35SO42- were also significantly increased by both lipid-soluble and water-soluble fusogens, though the increases were not as large as those for 45Ca2+. 7. It is suggested that fusogenic compounds increase the permeability of biological membranes to ions, and that an increase in the concentration of intracellular Ca2+ initiates or facilitates events that lead to the chemically induced fusion of erythrocytes.     

Activation of protein kinase C with cardiolipin-containing liposomes in relation to membrane-protein interaction

Many cytoplasmic proteins, including Ca2+- and phospholipid-dependent protein kinase (protein kinase C) of polymorphonuclear leukocytes (PMNs) associate in Ca2+-dependent manner with phospholipid liposomes containing cardiolipin (CL), as in the case of phosphatidylserine (PS)-containing liposomes. A crude protein kinase C fraction was purified by association of the enzyme with CL-containing liposomes (flotation method). The partially purified protein kinase C from rat brain or guinea pig PMN was activated by the CL-containing liposomes in the presence of dioleoylglycerol (DG) and Ca2+. This activation was analogous to that of PS. The half maximum activity was obtained with 20 microM CL in the presence of 1 microM Ca2+ and 5 microM DG. Many of the cytoplasmic proteins which associate with CL-containing liposomes were preferentially phosphorylated by membrane-associated protein kinase C in the presence of DG and Ca2+. These results suggest that the association of cytoplasmic protein kinase C with the membrane has an important role in regulation of protein kinase C activity in relation to the association of other cytoplasmic proteins to the membrane.     

Isovalerylcarnitine is a specific activator of the high calcium requiring calpain forms

Isovalerylcarnitine, a product of the catabolism of L-leucine, is a potent activator of rat calpains isolated from erythrocytes, kidney, liver, skeletal and heart muscle. Only calpains II, but not calpains I, are activated by IVC, with the only exception of rat erythrocyte calpain I, the only species present in these cells which has a Ca2+ requirement higher than that of most calpain I isoenzymes. Activation by IVC involves a dual effect: 1) a ten fold increase in the affinity of calpain for Ca2+, and 2) an increase in the Vmax 1.3-1.6 fold above the values observed with the native enzymes at saturating [Ca2+] as well as with the autolyzed fully active calpain form at 5 microM Ca2+. The increased affinity for calcium results in an increased rate of autoproteolysis of calpain II. Activation by IVC is additive to that promoted by interaction (or association) to phospholipids vesicles. Together these results suggest that IVC may operate as a selective activator of calpain both in the cytosol and at the membrane level; in the latter case in synergism with the activation induced by association of the proteinase to the cell membrane.     

Extracellular calcium is required for the maintenance of plasma membrane integrity in nucleated cells

In contrast to rat and human erythrocytes, nucleated erythrocytes from two fish species (Cyprinus carpio and Salmo trutta) underwent almost complete haemolysis in 20 min of EDTA addition. Using Ca2+/Mg2+ EGTA-citrate buffer, we observed that half-maximal haemolysis of fish erythrocytes occurs at [Ca2+]o approximately 10 microM independently of extracellular Mg2+ concentration. Attenuation of [Ca2+]o with EGTA also decreased stability of the plasma membrane of vascular smooth muscle cells (VSMC) and HeLa cells, indicated by a three- to five-fold elevation of lactate dehydrogenase release and passive permeability of plasma membrane for Na+. In VSMC, EGTA lowered [Ca2+]i by approximately 20%. This effect was absent in VSMC-loaded with the intracellular Ca2+ chelator BAPTA. In contrast to EGTA, BAPTA did not affect haemoglobin release from fish erythrocytes and passive permeability for Na+ in VSMC. Viewed collectively, our data show that in nucleated cells, extracellular Ca2+ plays a crucial role in the maintenance of plasma membrane integrity.     

Lack of correlation between calcium mobilization and respiratory burst activation induced by chemotactic factors in rabbit polymorphonuclear leukocytes

Low concentrations of FMLP, partially purified rabbit C5a, leukotriene B4 and platelet activating factor induced a rapid rise of intracellular free Ca2+ in rabbit polymorphonuclear leukocytes. However, the four factors differed markedly in their ability to activate the respiratory burst. The peptides FMLP and C5a induced a single, strong chemiluminescence response whereas the lipids leukotriene B4 and platelet activating factor induced a markedly less intense response with a two-peak profile. Respiratory burst activation by the peptides was dependent on extracellular Ca2+ whereas the lipids required both Mg2+ and Ca2+. The results indicate that mobilization of intracellular Ca2+ is insufficient by itself to induce respiratory burst activation and that the intracellular pathways leading to activation differ depending on the nature of the stimulus.     

The effects of ionophore A23187 and concanavalin A on the membrane potential of human peripheral blood lymphocytes and rat thymocytes

Effects of the Ca2+-ionophore A23187 and concanavalin A on the membrane potential of human lymphocytes and rat thymocytes have been studied using the fluorescent potential probe diS-C3-(5). At concentrations of 10(-8) to 10(-6) M A23187 changes the membrane potential, inducing both hyper- and depolarization. Depending on concentrations of A23187 and the external Ca2+, and on the type of lymphocytes, one of these effects predominates. The hyperpolarization induced by A23187 is caused by activation of Ca2+-dependent K+ channels. It is blocked by quinine and high concentrations of extracellular K+. The dependence of Ca2+-activated K+ transport on extracellular Ca2+ and its sensitivity to calmodulin antagonists is different for human lymphocytes and for thymocytes. As distinct from lymphocytes, in thymocytes calmodulin is not involved in activation of Ca2+-dependent K+ transport. The depolarization induced in lymphocytes by A23187 is caused by an increase in Na+ permeability of the lymphocyte plasma membrane: it is eliminated in a low-Na+ medium. At mitogenic concentrations concanavalin A does not change the membrane potential of the lymphocytes. The results obtained permit elucidation of the relationship between two early events in lymphocyte activation, namely the increase in intracellular Ca2+ concentration and the increase in lymphocyte plasma membrane permeabilities to monovalent cations.     

A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. Dependence of an endogenous protein activator distinct from calmodulin

A Mg-dependent adenosine triphosphatase (ATPase) activated by submicromolar free Ca2+ was identified in detergent-dispersed rat liver plasma membranes after fractionation by concanavalin A-Ultrogel chromatography. Further resolution by DE-52 chromatography resulted in the separation of an activator from the enzyme. The activator, although sensitive to trypsin hydrolysis, was distinct from calmodulin for it was degraded by boiling for 2 min, and its action was not sensitive to trifluoperazine; in addition, calmodulin at concentrations ranging from 0.25 ng-25 micrograms/assay had no effect on enzyme activity. Ca2+ activation followed a cooperative mechanism (nH = 1.4), half-maximal activation occurring at 13 +/- 5 nM free Ca2+. ATP, ITP, GTP, CTP, UPT, and ADP displayed similar affinities for the enzyme; K0.5 for ATP was 21+/- 9 microM. However, the highest hydrolysis rate (20 mumol of Pi/mg of protein/10 min) was observed at 0.25 mM ATP. For all the substrates tested kinetic studies indicated that two interacting catalytic sites were involved. Half-maximal activity of the enzyme required less than 12 microM total Mg2+. This low requirement for Mg2+ of the high affinity (Ca2+-Mg2+)ATPase was probably the major kinetic difference between this activity and the nonspecific (Ca2+ or Mg2+)ATPase. In fact, definition of new assay conditions, i.e. a low ATP concentration (0.25 mM) and the absence of added Mg2+, allowed us to reveal the (Ca2+-Mg2+)ATPase activity in native rat liver plasma membranes. This enzyme belongs to the class of plasma membrane (Ca2+-Mg2+)ATPases dependent on submicromolar free Ca2+ probably responsible for extrusion of intracellular Ca2+.     

Calcium ion-dependent diacylglycerol accumulation in erythrocytes is associated with microvesiculation but not with efflux of potassium ions.

Erythrocytes from several different species were exposed to Ca2+ and the bivalent-cation ionophore A23187. The lipid composition, morphology and K+ permeability of the treated cells were investigated. Erythrocytes from human, rat, guinea pig and rabbit (a) showed an increased concentration of 1,2-diacyl-sn-glycerol and enhanced labelling of phosphatidate with 32P, (b) underwent echinocytosis and outward vesiculation, and (c) rapidly released much of their intracellular K+. Pig cells showed only the K+ loss, and ox and sheep (high-K+) cells showed none of these Ca2+-evoked effects. All of the cells underwent stomatocytosis and inward vesiculation when treated externally with Clostridium perfringens phospholipase C. These results support the idea that there is a correlation between the asymmetric insertion of diacylglycerol (or ceramide) into the membrane and the shape-changes leading to microvesiculation, but they indicate that Ca2+-triggered K+ efflux and diacylglycerol production are unrelated events. Erythrocytes of chicken and turkey showed no Ca2+-stimulated K+ efflux. They showed slight ionophore A23187-stimulated vesiculation, but this appeared to be associated with the appearance in the membrane of ceramide rather than of diacylglycerol. Phospholipase C treatment caused very similar changes in morphology and phosphatidate labelling to those seen in mammalian erythrocytes.     

Limited breakdown of cytoskeletal proteins by an endogenous protease controls Ca2+-induced membrane fusion events in chicken erythrocytes

The profound morphological changes which follow the treatment of chicken erythrocytes with the ionophore A23187 and Ca2+ are associated with a concomitant breakdown of certain membrane-associated proteins including alpha-spectrin, goblin and microtubule-associated proteins (MAPS) which undergo a limited proteolysis to give large, well-defined fragments. The Ca2+-sensitive protease responsible for these changes appears to be present in the soluble fraction of the cells. Treatment with TLCK or iodoacetamide inhibits both the major morphological changes and the proteolytic events but these agents do not prevent the dissociation of microtubules or the activation of endogenous sphingomyelinase which occur in cells with raised levels of intracellular Ca2+. It is suggested that the sphingomyelinase is activated as a consequence of a Ca2+-induced loss of phospholipid asymmetry in the plasma membrane.     

Peptide-biphenyl hybrids as calpain inhibitors

Calpain is a cysteine protease that is activated by Ca2+. The over-activation of calpain, which occurs on increasing Ca2+ concentration, causes a variety of diseases. This paper reports experimental results on the inhibition of calpain I (mu-calpain) by peptide-biphenyl hybrids. We have found that some peptide-biphenyl hybrids, with aromatic amino acids in the peptide chains, inhibit calpain with IC50 values in the nanomolar range. Since the peptide-biphenyl hybrids reported in the present paper do not possess a reactive electrophilic functionality, we hypothesize that they interfere with the activation of calpain by Ca2+, and present experimental and computational results on the binding of peptide-biphenyl hybrids to Ca2+.     

Ca2+- and inositol-1,4,5-triphosphate induced release of Ca2+ in the microsomal fraction of the rat brain

Using the fluorescent probes, Quin 2 and chlortetracycline, a comparative study of the Ca2+ and inositol-1.4.5-triphosphate (IP3)-induced Ca2+ release from rabbit skeletal muscle sarcoplasmic reticulum (SR) terminal cisterns and rat brain microsomal vesicles was carried out. It was shown that Ca2+ release from rat brain microsomal vesicles is induced both by IP3 and Ca2+, whereas that in SR terminal cisterns is induced only by Ca2+. Data from chlorotetracycline fluorescence analysis revealed that CaCl2 (50 microM) causes the release of 15-20% and 40-50% of the total Ca2+ pool accumulated in rat brain microsomal vesicles and rabbit SR terminal cisterns, respectively. Using Quin 2, it was found that IP3 used at the optimal concentration (1.5 mM) caused the release of 0.4-0.6 nmol of Ca2+ per mg microsomal protein, which makes up to 10-15% of the total Ca2+ pool. IP3 does not induce Ca2+ release in SR. Preliminary release of Ca2+ from brain microsomes induced by IP3 diminishes the liberation of this cation induced by Ca2+. It is suggested that brain microsomes contain a Ca2+ pool which is exhausted under the action of the both effectors, Ca2+ and IP3.     

Rearrangement of intramembranous particles and fusion promoted in chicken erythrocytes by intracellular Ca2+.

Ca2+ was introduced into fresh and ATP-depleted chicken erythrocytes through the aid of the inophore A23187. Intracellular Ca2+ (10-40 mM) induced fusion in ATP-depleted cells after 30-60 min incubation at 37 degrees C, but not in fresh cells. Fresh cells underwent a higher degree of haemolysis than ATP-depleted cells after accumulation of Ca2+. Uptake of Ca2+ was the same in these two systems. Intracellular Ca2+ induced rearrangement of intramembranous particles, as revealed by freeze-etching studies. The intramembranous particles in the protoplasmic face of fractured membranes obtained from fresh cells incubated with 1 mM of Ca2+ were more scattered and their density was lower than in control cells. Incubation with higher concentrations of Ca2+ (10-40 mM) induced transient changes in the intramembranous particles' density with the appearance of protrusions and depressions on the protoplasmic and exoplasmic faces of the fractured membranes, respectively. These effects were reversible upon removal of Ca2+ by washing the cells with ethyleneglycol bis(alpha-aminoethylether)-N,N'-tetraacetic acid; rearrangement of intramembranous particles was less evident after accumulation of Ca2+ in ATP-depleted cells, whose fractured membranes did not contain any protrusions or depressions. Transferring Ca2+-loaded cells to the cold caused the formation of large smooth areas devoid of intramembranous particles in the protoplasmic face of the fractured membranes. Cells containing Ca2+ appeared spherical, and removal of Ca2+ restored the normal oval shape of chicken erythrocytes.     

Changes in the distribution of intramembranous particles in hen erythrocytes during cell fusion induced by the bivalent-cation ionophore A23187.

Incubation of hen erythrocytes with Ca2+ and the bivalent-cation ionophore A23187 induced slight cell fusion in 1 h at 37 degrees C, and extensive fusion during a subsequent 15 min at 47 degrees C. Redistributions of intramembranous particles were observed, possibly involving interactions between Ca2+ and phospholipids, which are discussed in relation to molecular mechanimss of cell fusion.     

A Ca(2+)-ATPase from rat parotid gland plasma membranes has the characteristics of an ecto-ATPase.

A Ca(2+)-ATPase with an apparent Km for free Ca2+ = 0.23 microM and Vmax = 44 nmol Pi/mg/min was detected in a rat parotid plasma membrane-enriched fraction. This Ca(2+)-ATPase could be stimulated without added Mg2+. However, the enzyme may require submicromolar concentrations of Mg2+ for its activation in the presence of Ca2+. On the other hand, Mg2+ could substitute for Ca2+. The lack of a requirement for added Mg2+ distinguished this Ca(2+)-ATPase from the Ca(2+)-transporter ATPase in the plasma membranes and the mitochondrial Ca(2+)-ATPase. The enzyme was not inhibited by several ATPase inhibitors and was not stimulated by calmodulin. An antibody which was raised against the rat liver plasma membrane ecto-ATPase, was able to deplete this Ca(2+)-ATPase activity from detergent solubilized rat parotid plasma membranes, in an antibody concentration-dependent manner. Immunoblotting analysis of the pellet with the ecto-ATPase antibody revealed the presence of a 100,000 molecular weight protein band, in agreement with the reported ecto-ATPase relative molecular mass. These data demonstrate the presence of a Ca(2+)-ATPase, with high affinity for Ca2+, in the rat parotid gland plasma membranes. It is distinct from the Ca(2+)-transporter, and immunologically indistinguishable from the plasma membrane ecto-ATPase.     

Calcium regulation of porcine aortic myosin

Calcium regulation of actin-activated porcine aortic myosin MgATPase was studied. The MgATPase of the purified actomyosin was stimulated about 10-fold by 0.1 mM Ca2+. The 20,000 molecular weight light chain subunit (LC20) of myosin was phosphorylated by an endogenous kinase that required Ca2+. Half-maximal activation of both kinase and ATPase occurred at about 0.9 microM Ca2+. Phosphorylated and unphosphorylated myosins, free of actin, kinase, and phosphatase, were purified by gel filtration. The MgATPase of phosphorylated myosin was activated by rabbit skeletal muscle actin; unphosphorylated myosin was actin activated to a much lesser extent. Actin activation was maximal in the presence of Ca2+. Regulation of the aortic myosin MgATPase seems to involve both direct interaction of calcium with phosphorylated myosin and calcium activation of the myosin kinase. The MgATPase of trypsin-treated actomyosin did not require Ca2+ for full activity. The trypsin-treated actomyosin was devoid of LC20. When purified unphosphorylated aortic myosin was treated with trypsin, the LC20, was cleaved and the MgATPase, which was not appreciably actin activated before exposure to protease, was increased and was activated by skeletal muscle actin. After incubation of this light chain-depleted myosin with light chain from rabbit skeletal muscle myosin, the actin activation but not the increased activity, was abolished. Unphosphorylated LC20 seems to inhibit actin activation in this smooth muscle.