Relation of Acetylcholine Release to Ca2+ Uptake and Intraterminal Ca2+ Concentration in Guinea-Pig Cortex Synaptosomes

[14C]Acetylcholine (ACh) release and parallel alterations in 45Ca2+ uptake and intrasynaptosomal free CA2+ concentration ([Ca2+]i) were measured in guinea-pig brain cortex synaptosomes. Depolarization by high K+ concentrations caused a rapid transient increase in Ca2+ uptake, terminating within 60 s (rate constant = 0.060 s-1; t1/2 = 11.6 s). This resulted in a rapid increase (within 1 s) in [Ca2+1]i, which then fell to a maintained but still-elevated plateau level (t1/2 for the decline was 15 s). Peaks of [Ca2+]i showed a sigmoidal dependence on depolarization, contrasting with the simple linear dependence of plateau levels of [Ca2+]i. The K+-evoked ACh release also had two phases: a fast initial increase (t1/2 = 11.3 s), which terminated within 60 s, was followed by a slow additional increase during sustained depolarizations of up to 10 min. Depolarization by veratridine led to a slow gradual increase in Ca2+ uptake (t1/2 = 130 s) over a 10-min incubation period, whereas an elevated plateau level of [Ca2+]i was achieved within 2 min (without a rapid peak elevation). The Ca2+-dependent fraction of the veratridine-evoked ACh release correlated with the increase in [Ca2+]i rather than with Ca2+ uptake. Using two different methods of depolarization partially circumvented the time limitations imposed by a buffering Ca2+ indicator and we suggest that, in the main, ACh is released in bursts associated with [Ca2+]i transients.     

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.     

Possible Regulation of Caffeine-Induced Intracellular Ca2+ Mobilization by Intracellular Free Na+

To gain some understanding of the regulatory mechanism involved in caffeine-induced Ca2+ release in adrenal chromaffin cells, we took advantage of the paradoxical observation that removal of divalent cations potentiated the secretory response to caffeine. We measured the concentration of cytosolic free Ca2+ ([Ca]in) in isolated cat chromaffin cells, by fura-2 microfluorometry, to see whether there was any correlation between the secretory response and the rise in [Ca]in. The caffeine-induced [Ca]in rise and catecholamine secretion were increased by treatment of cells with a divalent cation-deficient solution. These potentiated responses were strongly inhibited either by pretreatment with ryanodine, by the reduction of the external Na+ concentration, or by the addition of Ca2+ channel blockers. Removal of divalent cations caused a large rise in the cytosolic free Na+ concentration ([Na]in), which was measured using SBFI microfluorometry. This rise in [Na]in was reduced either by adding Ca2+ channel blockers or by reducing the external Na+ concentration. These results show a good correlation between caffeine-induced Ca2+ release and [Na]in at the time of stimulation, suggesting that caffeine-induced Ca2+ release is regulated by [Na]in.     

Neutral Amino Acid Transport in Astrocytes: Characterization of Na+-Dependent and Na+-Independent Components of α-Aminoisobutyric Acid Uptake

Neutral amino acid transport is largely unexplored in astrocytes, although a role for these cells in blood-brain barrier function is suggested by their close apposition to cerebrovascular endothelium. This study examined the uptake into mouse astrocyte cultures of alpha-aminoisobutyric acid (AIB), a synthetic model substrate for Na+-dependent system A transport. Na+-dependent uptake of AIB was characteristic of system A in its pH sensitivity, kinetic properties, regulatory control, and pattern of analog inhibition. The rate of system A transport declined markedly with increasing age of the astrocyte cultures. There was an unexpectedly active Na+-independent component of AIB uptake that declined less markedly than system A transport as culture age increased. Although the saturability of the Na+-independent component and its pattern of analog inhibition were consistent with system L transport, the following properties deviated: (1) virtually complete inhibition of Na+-independent AIB uptake by characteristic L system substrates, suggesting unusually high affinity of the transporter; (2) apparent absence of trans-stimulation of AIB influx; (3) unusually concentrative uptake at steady state (the estimated distribution ratio for 0.2 mM AIB was 55); and (4) susceptibility to inhibition by N-ethylmaleimide. Direct study of the uptake of system L substrates in astrocytes is needed to confirm the present indications of high affinity and concentrative Na+-independent transport.     

Na+-Dependent Uptake and Release of Taurine by Neuroblastoma × Glioma Hybrid Cells

Abstract In neuroblastoma × glioma hybrid cells, a cell line of neuronal character, a saturable uptake system for taurine is found which displays high affinity and high capacity ( K _m= 38 μ m , V = 1.25 nmol mg⁻¹ min⁻¹)- Only the closely related structural analogues hypotaurine and β-alanine are able to inhibit the transport of radioactively labeled taurine. Imipramine or haloperidol at 100 μ m effectively blocks taurine uptake. High-affinity taurine uptake shows an absolute and highly specific requirement for Na⁺. The hybrid cells internalize taurine very slowly and, with 1 m m extracellular taurine, attain a plateau only after more than 20 h, at which time approximately 34 m m labeled taurine has accumulated in the cytosol. Generally there is hardly any spontaneous release of accumulated taurine. Efflux can, however, be induced by increasing the intracellular Na⁺ content and is then accelerated by lowering the extracellular Na⁺ concentration. The hypothesis is forwarded that taurine may exert its function by driving the extrusion of Na⁺ in emergency situations.     

Effects of Na+, Ca2+, and Acetylcholine on Phosphoinositide- and ATP-Phosphate Turnover in 32P-Labeled Rabbit Iris Smooth Muscle

Parallel studies were carried out in the rabbit iris on (a) the effects of Na+ and/or Ca2+ on the acetylcholine-stimulated 32P labeling of phosphatidic acid (PA) and phosphatidylinositol (PI) and the breakdown of polyphosphoinositides (poly PI), and (b) the effects of these cations on the specific radioactivity of [gamma-32P]ATP. Incorporation of 32P1 into ATP and phosphoinositides is time-dependent, and it is remarkably dependent upon Na+ concentration in the incubation medium. The Na+ effect is reversible. Calcium ion, in the absence of Na+, had no effect on the specific radioactivity of ATP in 32P-labeled iris muscle; however, it moderately stimulated the 32P labeling of PA and PI and the breakdown of poly PI. In contrast, the addition of Na+, in the presence or absence of Ca2+, significantly reduced the specific radioactivity of ATP and 32P labeling of phospholipids in the 32P-labeled iris muscle. Acetylcholine had no measurable effect on the specific radioactivity of ATP. Furthermore, the neurotransmitter stimulated the 32P labeling of PA and PI and the breakdown of poly PI in the 32P-labeled muscle only in the presence of both Na+ and Ca2+. These data provide additional support for the concept that in the rabbit iris receptor-activated Ca2+ fluxes mediate or precede the effects of alpha-adrenergic and cholinergic muscarinic agents on phosphoinositide breakdown into 1,2-diacylglycerol and inositol phosphates and that restoration of the polar head groups to the 1,2-diacylglycerol (i.e., the recovery stage) is probably associated with Na+ outflux, via the Na+ -pump mechanism.     

Studies on α-Latrotoxin Receptors in Rat Brain Synaptosomes: Correlation Between Toxin Binding and Stimulation of Transmitter Release

Abstract: α-Latrotoxin (α-LT), the major component of black widow spider venom, is a high-molecular-weight protein that acts presynaptically by stimulating the release of stored neurotransmitters. The purified toxin was iodinated to high specific radioactivity by the Bolton-Hunter procedure, without appreciable loss of biological activity. By the use of the ¹²⁵I-toxin, specific receptors were revealed in synaptosome fractions isolated from various regions of the rat brain, but not in nonneural tissues. The density of α-LT receptors [which are probably composed of, or include, membrane protein(s)] varies between 0.6 and 0.88 pmol/mg of synaptosome protein, their affinity is very high ( K _A of the order of 10¹⁰ M ⁻¹), their association rate is fast, and their dissociation rate slow. They might belong to a single, homogeneous class. This last conclusion, however, is still uncertain, because results suggesting a possible heterogeneity were obtained by studying the dissociation of the toxin from synaptosomes incubated in high-salt buffer. Experiments in which the binding of α-LT and its dopamine release activity in striatal synaptosomes were investigated in parallel in a variety of experimental conditions support the hypothesis that occupation of the high-affinity receptors is the initial step in the α-LT activation of the presynaptic response.     

Behavior of Na+/K+-ATPase α-subunit alleles in sea urchin eggs upon fertilization

The allelic division of the Na⁺/K⁺-ATPase α-subunit gene was found in eggs of the sea urchin Hemicentrotus pulcherrimus by polymerase chain reaction (PCR). Two PCR products of different lengths were detected from a genome in one embryo derived from a fertilized egg, although only one product in one embryo derived from an artificially activated egg by parthenogenesis was detected, indicating one copy of the gene in a haploid genome. One of the two PCR products from each fertilized egg was identical in size to the product from an artificially activated egg in the same batch. The other PCR product was the same in length as one of the products from the sperm with which the eggs were fertilized. These results indicate that recombination of the heteromeric alleles of the Na⁺/K⁺-ATPase α-subunit gene occurs in the sea urchin egg due to meiosis and fertilization. The sequencing of these products demonstrated that their exon sequences were identical and that the intron, inserted in the PCR products, generated polymorphism in length due to the frequency of the repeating 53 bp sequence and insertion/deletion of other two segments.     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

The Na+ and K+ Content of Isolated Torpedo Synaptosomes and Its Effect on Choline Uptake

Abstract: The Na⁺ and K⁺ concentrations in isolated Torpedo marmorata synaptosomes were determined. Synaptosomes made according to the method of Israël et al. have high internal Na⁺ (290 MM) and low internal K⁺ (30 mM) concentrations. Modification of the homogenisation media permitted the isolation of synaptosomes which could maintain transmembrane ion gradients (internal Na⁺, 96 mM; K⁺, 81 mM); 0.1 mM-ouabain abolished these gradients. The trans-membrane Na⁺ gradient started to dissipate after 15 min at 20°C. Inclusion of ATP in the homogenisation medium enabled the synaptosomes to maintain the Na⁺ gradient for about 90 min. The presence of these transmembrane ion gradients stimulated choline uptake sevenfold. It is concluded that (a) by selecting the isolation media, Torpedo synaptosomes can be prepared with transmembrane ion gradients; (b) these gradients are ouabain-sensitive and stimulate choline uptake: (c) the synaptosomes require additional ATP to maintain the ion gradients.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.     

Interactions Between α-Latrotoxin and Trivalent Cations in Rat Striatal Synaptosomal Preparations

The interactions between alpha-latrotoxin (alpha-LTx), a neurosecretagogue purified from the venom of the black widow spider, and the trivalent cations Al3+, Y3+, La3+, Gd3+, and Yb3+ were investigated in rat striatal synaptosomal preparations. All trivalent cations tested were inhibitors of alpha-LTx-induced [3H]dopamine [( 3H]DA) release (order of potency: Yb3+ greater than Gd3+ approximately Y3+ greater than La3+ greater than Al3+). Only with Al3+ could inhibition of [3H]DA release be attributed to a block of 125I-alpha-LTx specific binding to synaptosomal preparations. The inhibitory effect of trivalent ions was reversible provided synaptosomes were washed with buffer containing EDTA. Trivalent ions also inhibited alpha-LTx-induced [3H]DA release at times when alpha-LTx-stimulated release was already evident. alpha-LTx-induced synaptosomal membrane depolarization was blocked by La3+, but not affected by Gd3+, Y3+, and Yb3+. alpha-LTx-stimulated uptake of 45Ca2+ was inhibited by all trivalent cations tested. These results demonstrate that there exist at least three means by which trivalent cations can inhibit alpha-LTx action in rat striatal synaptosomal preparations: (1) inhibition of alpha-LTx binding (Al3+); (2) inhibition of alpha-LTx-induced depolarization (La3+); and (3) inhibition of alpha-LTx-induced 45Ca2+ uptake (Gd3+, Y3+, Yb3+, La3+).     

Acetylcholine Release in the Rat Prefrontal Cortex In Vivo: Modulation by α2-Adrenoceptor Agonists and Antagonists

Abstract: We have previously shown that the release of acetylcholine (ACh) in the medial prefrontal cortex of the conscious rat, as measured by microdialysis, is increased following intraperitoneal injection of the selective α₂-adrenoceptor antagonist (+)-efaroxan. To characterize further the receptor pharmacology of this response, the effects of other selective α₂-adrenoceptor ligands were examined. The α₂-adrenoceptor antagonists idazoxan (2.5 and 20 mg/kg), atipamezole (2.5 mg/kg), and fluparoxan (10 mg/kg) increased ACh outflow by up to 250–325% of basal levels over a 3-h period following intraperitoneal injection. The α₂-adrenoceptor agonists UK-14304 (2.5 mg/kg) and guanabenz (2.5 mg/kg) reduced ACh outflow by 80 and 60%, respectively. Clonidine (0.00063–0.16 mg/kg) had no significant depressant effect and at 2.5 mg/kg increased ACh outflow to 233% of basal levels. These results indicate a modulatory role for α₂-adrenoceptors on the release of ACh in the rat prefrontal cortex in vivo. Based on the facilitatory effects produced by the antagonists alone, this α₂-adrenoceptor modulation appears to be tonic and inhibitory. The ability of α₂-adrenoceptor antagonists to enhance ACh outflow suggests a therapeutic usefulness in disorders where cortical ACh release deficits have been implicated.     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.     

α-Latrotoxin: preparation and effects on calcium fluxes

Abstract A toxin that causes a massive presynaptic activation of transmitter release from nerve terminals is α-latrotoxin, isolated from Latrodectus tredecimguttatus spider venom. This toxin has been highly purified, utilizing as a biological assay a toxin-dependent increase in ⁴⁵Ca²⁺-accumulation by PC12 cells. The purification protocol includes an ion-exchange step and a gel-filtration column, by fast-flow liquid chromatography. The resulting toxin is a polypeptide of about 125 kDa in molecular mass. At nmol concentrations it specifically activates calcium influx and transmitter secretion after interacting with neuronal acceptors of the presynaptic membrane. The inhibitory effect of trivalent ions (which may develop as degradation product of ⁴⁵Ca²⁺) on toxin-dependent calcium accumulation by PC12 cells is described. The results obtained suggest that calcium fluxes directly involved in the neurosecretory event, may occur through newly formed toxin-dependent channels.     

Distribution of Na+, K+-ATPase α-Subunit Isoforms in Rat Pituitary

The distributions of alpha-subunit isoforms of the Na+,K(+)-ATPase in rat pituitary were determined by immunoblotting and immunohistochemistry. Immunoreactivity for all three forms is present in the neural lobe, whereas the anterior lobe contains only alpha 1 and alpha 2. Most areas of the intermediate lobe exhibit faint immunoreactivity for only alpha 1, but thin strands of cells which stain strongly for all three isoforms are also present in this lobe. The previously reported ouabain inhibitable Na+,K(+)-ATPase activity in the neural lobe is consistent with the presence of both alpha 2 and alpha 3 subunits.     

Amyloid β Toxicity Consists of a Ca2+-Independent Early Phase and a Ca2+-Dependent Late Phase

Abstract: Amyloid β protein (Aβ), which accumulates in the senile plaques in the brain of Alzheimer's patients, is cytotoxic to neurons. A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in which a yellow redox dye, MTT, is reduced to purple formazan, is very sensitive to the effect of Aβ. In primary hippocampal cultures, inhibition of MTT reduction starts within 2 h after the addition of low concentrations of Aβ and reaches a plateau in 12 h. This effect of Aβ is not blocked by Ca²⁺ channel blockers or in Ca²⁺-free medium. In contrast, lactate dehydrogenase (LDH) release and trypan blue exclusion, which are indices of cell death, start 3 days after exposure to high concentrations of Aβ and are blocked by Ca²⁺ channel blockers such as Co²⁺, nicardipine, and diltiazem. When Aβ was washed out from the medium after 12 h, MTT reduction recovers and LDH release does not occur, suggesting that a long-lasting inhibition of the cellular redox system may be required to induce cell death. These observations demonstrate that Aβ toxicity consists of two phases—a Ca²⁺-independent early phase and a Ca²⁺-dependent late phase—and that the early phase may be required to induce the late phase.