Lysis of erythrocytes byTrichomonas vaginalis

The in vitro hemolytic activity of 4 isolates ofTrichomonas vaginalis was investigated. Repetitive hemolysis assays of any one isolate showed cyclical fluctuations in hemolytic activity, varying over 24 hr of continuous culture. Maximal hemolytic activity was detected using trichomonads in the lag phase of the growth cycle. Investigations showed that hemolysis was a contact-dependent phenomenon and microscopic investigation of samples showed a significant correlation between hemolysis and attachment of erythrocytes to the trichomonad surface. Quantitative data from cytoadherence assays using [⁵¹Cr]-labeled erythrocytes were consistent with these observations. It is suggested that hemolytic activity is dependent upon adherence of red blood cells to the surface ofT. vaginalis.     

The expression of optimum ATPase activities in human erythrocytes. A comparison of different lytic procedures.

The exposure of the Na⁺/K⁺/Mg²⁺- and Ca²⁺/Mg²⁺-stimulated ATPase activities in human erythrocytes through the use of several different lytic procedures revealed significant variations in the level of activity. Density (age)-separated as well as mixed-age human erythrocytes were subjected to hemolysis in isotonic buffer using saponin or ethylene glycol, to hemolysis in hypotonie buffer using low osmolarity buffers, or to freeze-thaw to allow potential accessibility to the ATPases. The results ranged from maximum exposure of both types of ATPases in saponin-treated cells, to little or no exposure of activity in ethylene glycol-treated cells, to variable responses in membranes derived by hypotonie hemolysis. The inability to elicit maximum exposure of ATPases in young cells by the freeze-thaw treatment was reversed by the use of saponin lysis in isotonic medium. These results illustrate the importance of the lytic conditions of membrane preparations on the recovery of as well as exposure to ATPase activities. It is concluded that saponin lysis in isotonic buffer medium is the preferred lytic technique for preparation of membranes retaining significant levels of the Na⁺/K⁺/Mg²⁺- and Ca²⁺/Mg²⁺-stimulated ATPases. These data are also discussed in reference to the degree of retention of the activator protein for the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase system.     

Bioimpedance spectroscopy parameters of suspensions of young and old erythrocytes

The bioimpedance spectroscopy (BIS) parameters of the suspensions of young and old erythrocytes were studied. The separation of the erythrocytes by age was made by density gradient. The BIS parameters: extracellular (Re) and intracellular (Ri) fluid resistance, characteristic frequency (Fchar), cell membranes capacitance (Cm) and Alpha parameter of concentrate suspensions of young and old erythrocytes were measured on the BIA analyzer ABC-01 "Medass" in the frequency range 5-500 kHz. It was found that Re (300.4 +/- 30.0 Ohm and 261.2 +/- 21.8 Ohm for old and young respectively, p < 0.05), Ri (86.6 +/- 9.1 Ohm and 73.4 +/- 7.3 Ohm for old and young respectively, p < 0.001) and Alpha (0.305 +/- 0.003 and 0.302 +/- 0.001 for old and young respectively, p < 0.05) of the old erythrocytes suspensions were higher, than of the young one, and Fchar (308.3 +/- 42.0 kHz and 347.4 +/- 48.0 kHz for old and young respectively, p <0.05) and Cm (99.3 +/- 10.1 pF and 112.8 +/- 6.3 pF for old and young respectively, p < 0.01) of the old erythrocytes were lower, than of the young one. The found differences between electrical properties of the suspensions of young and old erythrocytes were obviously determined by the alterations of the red blood cells during aging (growth of intracellular hemoglobin concentration, erythrocytes rapprochement because of diminishing of surface negative charge, increase of red blood cell sphericity and cell membrane permeability for ions). Thus the BIS parameters are related to the erythrocyte aging.     

Clostridium perfringens alpha-toxin-induced hemolysis of horse erythrocytes is dependent on Ca2+ uptake

Clostridium perfringens alpha-toxin is able to lyse various erythrocytes. Exposure of horse erythrocytes to alpha-toxin simultaneously induced hot-cold hemolysis and stimulated production of diacylglycerol and phosphorylcholine. When A23187-treated erythrocytes were treated with the toxin, these events were dependent on the concentration of extracellular Ca2+ . Incubation with the toxin of BAPTA-AM-treated horse erythrocytes caused no hemolysis or production of phosphorylcholine, but that of the BAPTA-treated erythrocytes did. When Quin 2-AM-treated erythrocytes were incubated with the toxin in the presence of 45Ca2+, the cells accumulated 45Ca2+ in a dose- and a time-dependent manner. These results suggest that the toxin-induced hemolysis and hydrolysis of phosphatidylcholine are closely related to the presence of Ca2+ in the cells. Flunarizine, a T-type Ca2+ channel blocker, and tetrandrine, an L- and T-type Ca2+ channel blocker, inhibited the toxin-induced hemolysis and Ca2+ uptake. However, L-type Ca2+ channel blockers, nifedipine, verpamil and diltiazem, an N-type blocker, omega-conotoxin SVIB, P-type blockers, omega-agatoxin TK and omega-agatoxin IVA, and a Q-type blocker, omega-conotoxin MVII C, had no such inhibitory effect. The observation suggests that Ca2+ taken up through T-type Ca2+ channels activated by the toxin plays an important role in hemolysis induced by the toxin.     

Plasmodium falciparum activates endogenous Cl(-) channels of human erythrocytes by membrane oxidation.

Intraerythrocytic survival of the malaria parasite Plasmodium falciparum requires that host cells supply nutrients and dispose of waste products. This solute transport is accomplished by infection-induced new permeability pathways (NPP) in the erythrocyte membrane. Here, whole-cell patch-clamp and hemolysis experiments were performed to define properties of the NPP. Parasitized but not control erythrocytes constitutively expressed two types of anion conductances, differing in voltage dependence and sensitivity to inhibitors. In addition, infected but not control cells hemolyzed in isosmotic sorbitol solution. Both conductances and hemolysis of infected cells were inhibited by reducing agents. Conversely, oxidation induced identical conductances and hemolysis in non-infected erythrocytes. In conclusion, P.falciparum activates endogenous erythrocyte channels by applying oxidative stress to the host cell membrane.     

Freezing injury to erythrocytes. I. Freezing patterns and post-thaw hemolysis.

The extent of hemolysis of human red blood cells suspended in different concentrations of glycerol and frozen at various cooling rates was investigated on the basis of morphological observation in the frozen state. Hemolysis of the cells in the absence of glycerol showed a V-shaped curve in terms of cooling rates. There was 70% hemolysis at an optimal cooling rate of approximately 10³ °C/min and 100% hemolysis at all other rates tested. Morphologically, a lower than optimal cooling rate resulted in cellular shrinkage, while a higher than optimal rate resulted in the formation of intracellular ice.The cryoprotective effect of glycerol was dependent upon its concentration and on the cooling rate. Samples frozen at 10³ and 10⁴ °C/min showed freezing patterns which differed from cell to cell. The size of intraand extracellular ice particles became smaller, and there was less shrinkage or deformation of cells as the rate of cooling and concentration of glycerol were increased.There was some correlation between the morphology of frozen cells and the extent of post-thaw hemolysis, but the minimum size of intracellular ice crystals which might cause hemolysis could not be estimated. As a cryotechnique for electron microscopy, the addition of 30% glycerol and ultrarapid freezing at 10⁵ °C/min are minimum requirements for the inhibition of ice formation and the prevention of the corresponding artifacts in erythrocytes.     

Interaction of dichlone with human erythrocytes. I. Changes in cell permeability

The uptake of the fungicide dichlone (2,3-dichloro-1,4-naphthoquinone) by human erythrocytes was extremely rapid, reaching a maximum within 5 min of treatment. Most of the dichlone taken up was present in the interior of the cell; only a small fraction of the pesticide (less than 5%) was bound to the cell membrane. Dichlone (3 · 10^?5M-10^?4M) induced a rapid loss of intracellular potassium from the erythrocytes; the leakage of K⁺ varied with the fungicide concentration as well as with cell concentration. Pretreatment of the cells with glutathione was able to reduce potassium loss. Cells exposed to dichlone showed increased osmotic fragility. Dichlone also inhibited Na⁺-K⁺ ATPase, which is associated with active ion transport. However, the leakage of potassium in dichlone-treated cells does not appear to be related to the interference with active ion transport. An extensive loss of potassium within a relatively short time after treatment suggests that dichlone produces its effect by increasing passive cation permeability, probably as a result of direct action on the membrane structure. Dichlone was able to induce hemolysis, but only at concentrations higher than those which resulted in K⁺ loss. The loss of hemoglobin appeared to be mainly due to osmotic swelling of the treated cells. Exposure of red cells to dichlone also resulted in a rapid and extensive formation of methemoglobin as well as a denaturation of hemoglobin. Thus, dichlone not only may be capable of lowering the capacity of erythrocytes to transport oxygen but also alters their permeability.     

Clostridium perfringens alpha-toxin activates the sphingomyelin metabolism system in sheep erythrocytes

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.     

Membrane perturbations induced by the interactions of zinc ions with band 3 in human erythrocytes

Of group 12 metals, zinc is an essential element to maintain our life, but other metals such as cadmium and mercury are toxic in cellular activities. Interactions of these metals with biomembranes are important to understand their effects on our living cells. Here, we describe the membrane perturbations induced by these metals in human erythrocytes. Of these metals, Zn²⁺ ions only induced the erythrocyte agglutination. Histidine residues in extracellular domains of band 3 participated in Zn²⁺-induced agglutination. Interestingly, it was found that band 3-cytoskeleton interactions play an important role in Zn²⁺-induced agglutination. In contrast with Hg²⁺ and Cd²⁺ ions, Zn²⁺ ions greatly suppressed pressure-induced hemolysis by cell agglutination. Such a suppression was removed upon dissociation of agglutinated erythrocytes by washing, indicating the reversible interactions of Zn²⁺ ions with erythrocyte membranes. Excimer fluorescence of pyrene indicated that spectrin is denatured by a pressure of 200 MPa irrespective of hemolysis suppression. Taken together, these results suggest that the agglutination of erythrocytes due to the interactions of Zn²⁺ ions with band 3 is stable under pressure, but spectrin, cytoskeletal protein, is denatured by pressure     

Lidocaine: An inhibitor in the free‐radical‐induced hemolysis of erythrocytes

Lidocaine was reported to protect erythrocytes from hemolysis induced by 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH). Since AAPH‐induced hemolysis was a convenient in vitro experimental system to mimic erythrocytes undergoing peroxyl radicals attack, the aim of this work was to investigate the antioxidant effect of lidocaine on AAPH‐induced hemolysis by chemical kinetics. As a result, one molecule of lidocaine can only trap 0.37 radical, much lower than melatonin. Meanwhile, lidocaine cannot protect erythrocytes from hemolysis induced by hemin, which the mechanism of hemolysis was due to the erythrocyte membrane destroyed by hemin. Accordingly, lidocaine protected erythrocytes by scavenging radicals preferentially rather than by stabilizing membrane. Moreover, the interactions of lidocaine with two radical species, including 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate) radical cation (ABTS^+?) and 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH), indicated that lidocaine can reduce ABTS^+? with 260 µM as the 50% inhibition concentration (IC₅₀) and cannot react with DPPH. Thus, lidocaine served as a reductant rather than a hydrogen donor to interact with radicals. Finally, the quantum calculation proved that, compared with the melatonin radical, the stabilization of N‐centered radical of lidocaine was higher than the amide‐type N‐centered radical but lower than the indole‐type N‐centered radical in melatonin. These results provided basic information for lidocaine to be an antiradical drug. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:81–86, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20267     

Differential resistance to calcium-induced bilirubin-dependent hemolysis in mammalian erythrocytes

Washed erythrocytes from human, buffalo, sheep and goat preincubated with different concentrations of calcium chloride (16.7–1830 μM) showed significantly different rates of hemolysis (up to 62%) after addition of bilirubin (72 μM). Goat erythrocytes displayed marked resistance to hemolysis with only 11% hemolysis observed at the highest calcium concentration. Similar trend in hemolysis was also observed when the concentration of CaCl₂ was fixed (330 μM) and bilirubin concentration varied (0–72 μM). (Ca²⁺–Mg²⁺)-ATPase levels were found significantly lower in goat and sheep erythrocyte membranes compared to human and buffalo erythrocyte membranes. This was correlated well with the observed hemolysis in various mammalian erythrocytes.     

Parameters of bioimpedance spectroscopy in young and old erythrocytes

The parameters of bioimpedance spectroscopy (BIS) were studied in suspensions of young and old erythrocytes. The separation of erythrocytes according to age in a density gradient was performed. The BIS parameters, including the extracellular (Re) and intracellular (Ri) fluid resistances, characteristic frequency (Fchar), cell membranes’ capacitance (Cm), and the Alpha parameter of concentrated suspensions of young and old erythrocytes (n = 6) were measured using an ABC-01 Medass bioimpedance analyzer in the frequency range 5–500 kHz. Re (300.4 ± 30.0 and 261.2 ± 21.8 Ω in old and young erythrocytes, respectively, p < 0.05), Ri (86.6 ± 9.1 Ω and 73.4 ± 7.3 Ω in old and young erythrocytes, respectively, p < 0.001), and Alpha (0.305 ± 0.003 and 0.302 ± 0.001 in old and young erythrocytes respectively, p < 0.05) were greater in the suspension of old erythrocytes than in the suspension of young erythrocytes; and Fchar (308.3 ± 42.0 kHz and 347.4 ± 48.0 kHz in old and young erythrocytes, respectively, p < 0.05) and Cm (99.3 ± 10.1 pF and 112.8 ± 6.3 pF in old and young erythrocytes, respectively, p < 0.01) were less in the suspension of old erythrocytes than in the suspension of young erythrocytes. These differences between the BIS parameters of old and young erythrocytes were possibly due to the structural change in erythrocytes during aging (an increase in the concentration of intracellular hemoglobin, the change in the shape of the erythrocyte, their converging due to the decrease in cellular negative surface charge, and an increase in membrane permeability to ions). Thus, the BIS parameters depend on the erythrocyte age composition.     

Effect of ethanol on the hemolytic stability of erythrocytes

The stability of rabbit erythrocytes to hemolysis induced by different compounds in the presence or absence of ethanol or acetaldehyde has been analyzed. Ethanol slightly reduced erythrocyte stability against acidic hemolysis only after long-term preincubation, but the effect of ethanol on stability to oxidative hemolysis manifested itself immediately after its addition to the cells. Ethanol decreased both stability of cells to oxidative damage and dispersion of the hemolytic curve. Comparison of the effects of ethanol and acetaldehyde showed that the destabilizing effect of ethanol might be caused by either its direct action or the effect of its metabolites formed during preincubation of ethanol with erythrocytes. Possible mechanisms of ethanol and acetaldehyde effects on erythrocyte stability are discussed.     

Acetylcholinesterase in aging of human erythrocytes]

Acetylcholinesterase possessed a different activity in the membranes of young, mature and old human erythrocytes - it was greatest in the mature and least in the old cells. In the young and mature erythrocytes the enzyme existed in the form of 3- and in the old ones in the form of 2-molecular components. The data obtained suggested that the changes in the structural organization of acetylcholinesterase in the red cell membrane had a direct relationship to the aging of these cells.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

Membrane alterations in cellular aging: Susceptibility of phospholipids in density (age)-separated human erythrocytes to phospholipase A2

Human erythrocytes were separated into four density (age) groups representing the top 10% (young), bottom 10% (old), and two middle fractions of 40% each (intermediary ages). When these erythrocytes of different age groups were treated with the low levels of a purified basic phospholipase A₂ from Agkistrodon halys blomhofii, under conditions where little or no hemolysis occurred, the optimum extent of phosphatidylcholine (PC) hydrolysis in all age groups was the same, but interestingly, the rate of its hydrolysis was two to three times faster in the older cells compared to younger erythrocytes. On the other hand, hydrolysis of phosphatidylethanolamine (PE) of younger erythrocytes by the phospholipase A₂ was negligible under the particular experimental conditions. However, in erythrocytes of older age groups, both the rate and extent of PE hydrolysis by the enzyme increased in a distinctive fashion. Concomitant with the above pattern of PC and PE hydrolysis, the shape changes in the erythrocytes also were different; whereas all older erythrocytes became echinocytic only two-thirds of the younger erythrocytes showed a similar shape change. These observations firmly establish that during in vivo aging of normal erythrocytes in circulation significant changes in the structural organization of membrane phospholipids take place. Importance of this phenomenon in membrane phospholipid asymmetry studies and in the elimination of senescent cells also is discussed.     

Hemolysis of human erythrocytes by paraquat in relation to superoxide dismutase activity.

Paraquat, a widely used herbicide, induced hemolysis of human erythrocytes in hypotonic saline solution. The degree of hemolysis depended on the intracellular superoxide dismutase level. Erythrocytes with higher enzyme activity were more sensitive to paraquat and those depleted of superoxide dismutase by diethyldithiocarbamate were more resistant. This apparent paradox was interpreted to be due to a rapid turnover of the enzymic dismutation reaction with a resultant increase in the generation of the reactive species responsible for hemolysis. Studies with various scavengers suggested that the hemolytic agent is singlet oxygen. No definite evidence for lipid peroxidation could be demonstrated in erythrocytes exposed to paraquat.     

Canine erythrocytes express the P2X7 receptor: greatly increased function compared with human erythrocytes

Over three decades ago, Parker and Snow (Am J Physiol 223: 888-893, 1972) demonstrated that canine erythrocytes undergo an increase in cation permeability when incubated with extracellular ATP. In this study we examined the expression and function of the channel/pore-forming P2X(7) receptor on canine erythrocytes. P2X(7) receptors were detected on canine erythrocytes by immunocytochemistry and immunoblotting. Extracellular ATP induced (86)Rb(+) (K(+)) efflux from canine erythrocytes that was 20 times greater than that from human erythrocytes. The P2X(7) agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-trisphosphate (BzATP) was more potent than ATP, and both stimulated (86)Rb(+) efflux from erythrocytes in a dose-dependent fashion with EC(50) values of approximately 7 and approximately 309 microM, respectively. 2-Methylthioadenosine 5'-triphosphate and adenosine 5'-O-(3-thiotriphosphate) induced a smaller (86)Rb(+) efflux from erythrocytes, whereas ADP, AMP, UTP, or adenosine had no effect. ATP-induced (86)Rb(+) efflux from erythrocytes was inhibited by oxidized ATP, KN-62, and Brilliant blue G, known P2X(7) antagonists. ATP also induced uptake of choline(+) into canine erythrocytes that was 60 times greater than that into human erythrocytes. Overnight incubation of canine erythrocytes with ATP and BzATP induced phosphatidylserine exposure in >80% of cells and caused up to 20% hemolysis. In contrast, <30% of human erythrocytes showed phosphatidylserine exposure after overnight incubation with ATP and BzATP, and hemolysis was negligible. Flow cytometric measurements of ATP-induced ethidium(+) uptake showed that P2X(7) function was three times lower in canine monocytes than in human monocytes. These data show that the massive cation permeability increase induced by extracellular ATP in canine erythrocytes results from activation and opening of the P2X(7) receptor channel/pore.     

Characteristics of the interaction of a treponemal hemolysin with rabbit erythrocytes

The mechanism of action on rabbit red cells of Treponema hyodysenteriae hemolysin was studied using volume analysis and release of hemoglobin. While fixation of the hemolysin on the erythrocytes is temperature independent, it appears that hemolysis is temperature dependent. The kinetics of hemolysis proceed according to a sigmoid curve characterized by a prelytic lag. The duration of the prelytic lag varies inversely with the quantity of hemolysin but the rate and the maximum value of hemolysis are directly proportional to the quantity of hemolysin. The effect of sucrose and trypan blue on the hemolysin and the red cells suggest that erythrocyte lysis is likely to be induced by the hemolysin in a way different from that known for other hemolytic agents.     

Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human

The plasma membrane Ca²⁺ ATPase in erythrocytes is vital for the maintenance of intracellular Ca²⁺ levels. Since the cytoplasmic Ca²⁺ concentration is elevated in older erythrocytes, the properties of the Ca²⁺ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca²⁺ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca²⁺ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca²⁺ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca²⁺ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca²⁺ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca²⁺ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4°C). The decrease in Ca²⁺ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.Abbreviations IOV Inside-Out Vesicles - Ey Erythrocytes enriched with young (less dense) cells - Eo Erythrocytes enriched with old (more dense) cells - ACEase Acetylcholinesterase