Regulatory Role of the GTP-Binding Protein, Go, in the Mechanism of Exocytosis in Adrenal Chromaffin Cells

To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Loss and Ca2+-Dependent Retention of Scinderin in Digitonin-Permeabilized Chromaffin Cells: Correlation with Ca2+-Evoked Catecholamine Release

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.     

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.     

Regulation of Drosophila Ca2+/Calmodulin-Dependent Protein Kinase II by Autophosphorylation Analyzed by Site-Directed Mutagenesis

Abstract: In this study we demonstrate that Drosophila calcium/calmodulin-dependent protein kinase II (CaMKII) is capable of complex regulation by autophosphorylation of the three threonines within its regulatory domain. Specifically, we show that autophosphorylation of threonine-287 in Drosophila CaMKII is equivalent to phosphorylation of threonine-286 in rat α CaMKII both in its ability to confer calcium independence on the enzyme and in the mechanistic details of how it becomes phosphorylated. Autophosphorylation of this residue occurs only within the holoenzyme structure and requires calmodulin (CaM) to be bound to the substrate subunit. Phosphorylation of threonine-306 and threonine-307 in the CaM binding domain of the Drosophila kinase occurs only in the absence of CaM, and this phosphorylation is capable of inhibiting further CaM binding. Additionally, our findings suggest that phosphorylation of threonine-306 and threonine-307 does not mimic bound CaM to alleviate the requirement for CaM binding to the substrate subunit for intermolecular threonine-287 phosphorylation. These results demonstrate that the mechanism of regulatory autophosphorylation of this kinase predates the split between invertebrates and vertebrates.     

Regulation of Calcium Concentrations in Synaptosomes: α2-Adrenoceptors Reduce Free Ca2+ by Closure of N-Type Ca2+ Channels

The relationship between intrasynaptosomal total (CaT) and free ([Ca2+]i) calcium and 45Ca accumulation was studied under physiological and K(+)-depolarised conditions in rat cortical synaptosomes. Under physiological conditions, CaT (10.7 mM) was approximately 10,000 times higher than [Ca2+]i (118 nM), showing that there is a large reservoir of sequestered calcium in synaptosomes. 45Ca accumulation was rapid (initial rate, 3.4 nmol/mg protein/min), substantial (7 nmol/mg protein in 2 min), and depolarisation dependent, and reached equilibrium after 5 min. At equilibrium, only 10% of CaT was freely exchangeable. This pool was much larger than the free Ca2+ pool. CaT, [Ca2+]i, and 45Ca accumulations were directly related to the Ca2+ concentration in the buffer, suggesting that [Ca2+]i is not highly conserved but is maintained by simple equilibria between the various pools. Clonidine reduced 45Ca accumulation in a time- and dose-dependent manner. Maximum inhibition (40% at 100 microM) occurred at 2 min and the IC50 was 80 nM. The reduction caused by clonidine (1 microM) reached equilibrium after 5 min, but this equilibrium value was lower than in controls, suggesting that clonidine changes the exchangeable Ca2+ pool size. The effects of clonidine (1 microM) on [Ca2+]i (26% reduction) and on 45Ca accumulation (24% reduction) were most apparent under physiological conditions. However, while it was not dependent on depolarisation, it did not occur in physiological buffer containing low K+ concentration (0.1-1 mM). The inhibitory effect of clonidine on 45Ca accumulation is receptor mediated as it was antagonised by idazoxan (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Ca2+/Calmodulin-Dependent Protein Kinase II by Brain Gangliosides

Abstract: Purified rat brain Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca²⁺/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 μ M . Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca²⁺-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 μ M ) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis, CaMK 281-309 strongly inhibited kinase activity (IC₅₀=0.2 μ M ). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca²⁺/CaM on native CaM-kinase II and on peptide CaMK 281-309.     

Transmitter Glutamate Release from Isolated Nerve Terminals: Evidence for Biphasic Release and Triggering by Localized Ca2+

The kinetics of Ca2(+)-dependent release of glutamate from guinea-pig cerebrocortical synaptosomes evoked by KCl or 4-aminopyridine are investigated using a continuous fluorimetric assay. Release by both agents is biphasic, with a rapid phase complete within 2 s followed by a more extensive slow phase with a half-maximal release in 52 s for KCl-evoked release and greater than 120 s for 4-aminopyridine-evoked release. The two phases of glutamate release may reflect a dual localization of releasable vesicles at the active zone and in the bulk cytoplasm. Decreasing depolarization depresses the extent rather than increasing the time for half-maximal Ca2(+)-dependent release. Both the fast and the slow phases of glutamate release require external Ca2+ and cytoplasmic ATP. KCl depolarization produces a transient "spike" of cytoplasmic free Ca2+ [( Ca2+]c), which recovers to a plateau; the major component of glutamate release occurs during this plateau. Predepolarization in the absence of added external Ca2+, to inhibit transient Ca2+ channels, does not affect the subsequent glutamate release evoked by Ca2+ readdition. Thus, release involves primarily noninactivating Ca2+ channels. For a given increase in [Ca2+]c, KCl and 4-aminopyridine cause equal release of glutamate, while ionomycin releases much less glutamate. This lowered efficiency is not due to ATP depletion. It is concluded that glutamate exocytosis is evoked by localized Ca2+ entering through noninactivating voltage-dependent Ca2+ channels and that nonlocalized Ca2+ entry with ionomycin is inefficient.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Different Patterns of Agonist-Stimulated Increases of 3H-Inositol Phosphate Isomers and Cytosolic Ca2+ in Bovine Adrenal Chromaffin Cells: Comparison of the Effects of Histamine and Angiotensin II

Abstract: Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP₁, InsP₂, and InsP₃, respectively) isomers, intracellular free Ca²⁺ ([Ca²⁺]_i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca²⁺]_i by 200 nM 3–4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P₃ that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P₃ formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P₃ by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P₂ and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P₃, Ins(1,3)P₂, and Ins(3,4)P₂, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P₂ was the only other InsP₂ besides Ins(1,4)P₂ to accumulate within 1 min of agonist treatment [Ins(3,4)P₂ did not increase]. These results support a correlation between the time course of Ins(1,4,5)P₃ formation and the time course of [Ca²⁺]_i transients and illustrate that Ca²⁺-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca²⁺], changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

Ca2+ Mobilized by Caffeine from the Inositol 1,4,5-Trisphosphate-Insensitive Pool of Ca2+ in Somatic Regions of Sympathetic Neurons Does Not Evoke [3H]Norepinephrine Release

The effects of electrical stimulation, muscarinic and serotonergic agonists, and caffeine on [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) content, intracellular free Ca2+ concentration ([Ca2+]i), and release of [3H]norepinephrine ([3H]NE) were studied in cultured sympathetic neurons. Neuronal cell body [Ca2+]i was unaffected by muscarinic or serotonergic receptor stimulation, which significantly increased [3H]Ins(1,4,5)P3 content. Stimulation at 2 Hz and caffeine had no effect on [3H]Ins(1,4,5)P3, but caused greater than two-fold increase in [Ca2+]i. Only 2-Hz stimulation released [3H]NE. Caffeine had no effect on the release. When [Ca2+]i was measured in growth cones, only electrical stimulation produced an increase in [Ca2+]i. The other agents had no effect on Ca2+ at the terminal regions of the neurons. We conclude that Ins(1,4,5)P3-insensitive, but caffeine-sensitive Ca2+ stores in sympathetic neurons are located only in the cell body and are not coupled to [3H]NE release.     

The requirement for Mn2+ and Ca2+ in nitrogen fixation by the photosynthetic bacterium Rhodospirillum rubrum

Abstract: The role of Ca²⁺ and Mn²⁺ in Rhodospirillum rubrum grown under different conditions with respect to nitrogen source has been studied. The results show that this phototroph does not have an absolute requirement for these cations. In vitro studies of one of the enzymes operative in the metabolic regulation of nitrogenase in Rsp. rubrum have shown that Mn²⁺ or Fe²⁺ is required for activity. This investigation indicates that Mn²⁺ is not required in vivo for the function of this enzyme, suggesting that either Fe²⁺ is functional or that the enzyme has other properties when active in the cell.     

Ca2+ Sensitivity of Ca2+-Dependent Protein Kinase Activities Toward Intrinsic Proteins in Synaptosomal Membrane Fragments from Rat Cerebral Tissue

The Ca2+ and calmodulin sensitivity of endogenous protein kinase activity in synaptosomal membrane fragments from rat brain was studied in medium containing Ca2+ plus EGTA using a modified computer programme to calculate free Ca2+ concentrations that took into account the effect of all competing cations and chelators. The Ca2+-dependent phosphorylation of 10 major polypeptide acceptors with Mr values ranging from 50 to 360 kilodaltons required calmodulin in reactions that were all equally sensitive to Ca2+; half-maximal phosphorylation required a free Ca2+ concentration of 45 nM and maximal phosphorylation approximately 110 nM. The significance of these values in relation to published data on the intracellular concentration of free Ca2+ in the nervous system is discussed. One acceptor of 45 kilodaltons was phosphorylated in a Ca2+-dependent reaction that did not require calmodulin. This polypeptide appeared to correspond to the B-50 protein, an established substrate of the lipid-dependent protein kinase C. Further study of this phosphorylating system showed that the reaction was only independent of calmodulin at saturating concentrations of Ca2+; at subsaturating concentrations (in the range 50-130 nM), a small but significant stimulation of the enzyme by calmodulin was demonstrated. The possible significance of this finding is discussed.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

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.     

Functional Heterogeneity of Alternatively Spliced Isoforms of Drosophila Ca2+/Calmodulin-Dependent Protein Kinase II

Abstract: The gene for Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate up to 18 different proteins that vary only in a region analogous to the point where mammalian α, β, γ, and δ isozymes show the greatest divergence from each other. To investigate the function of this variable region, we have characterized the catalytic and structural properties of six of the Drosophila isoforms. By several criteria (domain organization, low affinity for calmodulin, holoenzyme structure, and ability to autophosphorylate and become independent of calcium), these proteins are functional homologues of the mammalian calcium/calmodulin-dependent protein kinase II. Two major isoform-specific catalytic differences were observed. First, the R3A isoform was found to have a significantly higher K _act for calmodulin than the other isoforms. This indicates that the variable region, which is located distal to the calmodulin-binding domain, may play a role in activation of the enzyme by calmodulin. Decreased sensitivity to calmodulin may be biologically important if free calmodulin is limiting within the neuron. The second catalytic difference noted was that the R6 isoform had a significantly lower K _m for the peptide substrate used in this study. Although the variable region is not in the catalytic part of the enzyme, it may have an indirect function in substrate selectivity.     

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.     

Ischemia-Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (⁴⁵Ca²⁺) accumulation in the microsomes mediated by Mg²⁺/Ca²⁺ ATPase. Ischemia caused a statistically significant inhibition of presteady-state and steady-state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia-induced inhibition of endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase.