Novel dimeric acetylcholinesterase inhibitor bis7-tacrine, but not donepezil, prevents glutamate-induced neuronal apoptosis by blocking N-methyl-D-aspartate receptors

The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 mum glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 mum) on CGNs markedly reduced glutamate-induced apoptosis in dose- and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 mum bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-beta-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-d-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca(2+) imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca(2+) increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [(3)H]MK-801 with an IC(50) value of 0.763 mum in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.     

Divergent role of calcium on Abeta- and MPTP-induced cell death in SK-N-SH neuroblastoma

We attempted to clarify the role of Ca2+ in cell death caused by beta-amyloid protein (Abeta) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in SK-N-SH neuroblastoma, respectively. Two insults both reduced cell viability in a concentration-dependent manner and induced equal cytotoxicity in the presence of 20 microM Abeta and 0.4 mM MPTP for 72 h, respectively (68+/-7 vs. 64+/-6% viability). Time-related study showed that Abeta evoked cell death occurred quickly at 24 h. Relatively, MPTP exhibited a delayed cell death significantly after 72 h of culture. Pretreating the cells with nimodipine and chelating of Ca2+ by EGTA plus 1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) successfully rescued Abeta-induced cell death but failed to prevent MPTP toxicity. ELISA determination of mono/oligonucleosomes accumulation showed the mode of cell death evoked by MPTP was presumably apoptosis while by Abeta was necrosis. SK-N-SH cells constitutively expressed the alpha(1C) subunit of L-type Ca2+ channel and exposure to Abeta or MPTP for 96 h did not further modify its expression. By contrast, alpha(1D) subunit was undetectable or low level expressed in basal condition, but was induced to express after Abeta and MPTP stimulation in a time-dependent manner. Functional assay revealed that KCl-evoked [Ca2+]i rise was significantly greater in Abeta-, but not in MPTP-treated cells when compared with control. Taken together, these results showed that Abeta and MPTP elicited different mode of cell death in SK-N-SH. Nevertheless, Ca2+ overload seems to solely display a crucial role in Abeta-induced cytotoxicity and over-expressed alpha(1D) may contribute to the disruption of cellular Ca2+ homeostasis.     

Amyloid β Protein Potentiates Ca2+ Influx Through L-Type Voltage-Sensitive Ca2+ Channels: A Possible Involvement of Free Radicals

Abstract: Amyloid β protein (Aβ), the central constituent of senile plaques in Alzheimer's disease (AD) brain, is known to exert toxic effects on cultured neurons. The role of the voltage-sensitive Ca²⁺ channel (VSCC) in β(25–35) neurotoxicity was examined using rat cultured cortical and hippocampal neurons. When L-type VSCCs were blocked by application of nimodipine, β(25–35) neurotoxicity was attenuated, whereas application of ω-conotoxin GVIA (ω-CgTX-GVIA) or ω-agatoxin IVA (ω-Aga-IVA), the blocker for N- or P/Q-type VSCCs, had no effects. Whole-cell patch-clamp studies indicated that the Ca²⁺ current density of β(25–35)-treated neurons is about twofold higher than that of control neurons. Also, β(25–35) increased Ca²⁺ uptake, which was sensitive to nimodipine. The 2',7'-dichlorofluorescin diacetate assay showed the ability of β(25–35) to produce reactive oxygen species. Nimodipine had no effect on the level of free radicals. In contrast, vitamin E, a radical scavenger, reduced the level of free radicals, neurotoxicity, and Ca²⁺ uptake. These results suggest that β(25–35) generates free radicals, which in turn, increase Ca²⁺ influx via the L-type VSCC, thereby inducing neurotoxicity.     

Human group IIA secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels in cultured rat cortical neurons

Mammalian group IIA secretory phospholipase A2 (sPLA2-IIA) generates prostaglandin D2 (PGD2) and triggers apoptosis in cortical neurons. However, mechanisms of PGD2 generation and apoptosis have not yet been established. Therefore, we examined how second messengers are involved in the sPLA2-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons. sPLA2-IIA potentiated a marked influx of Ca2+ into neurons before apoptosis. A calcium chelator and a blocker of the L-type voltage-sensitive Ca2+ channel (L-VSCC) prevented neurons from sPLA2-IIA-induced neuronal cell death in a concentration-dependent manner. Furthermore, the L-VSCC blocker ameliorated sPLA2-IIA-induced morphologic alterations and apoptotic features such as condensed chromatin and fragmented DNA. Other blockers of VSCCs such as N type and P/Q types did not affect the neurotoxicity of sPLA2-IIA. Blockers of L-VSCC significantly suppressed sPLA2-IIA-enhanced Ca2+ influx into neurons. Moreover, reactive oxygen species (ROS) were generated prior to apoptosis. Radical scavengers reduced not only ROS generation, but also the sPLA2-IIA-induced Ca2+ influx and apoptosis. In conclusion, we demonstrated that sPLA2-IIA potentiates the influx of Ca2+ into neurons via L-VSCC. Furthermore, the present study suggested that eicosanoids and ROS generated during arachidonic acid oxidative metabolism are involved in sPLA2-IIA-induced apoptosis in cooperation with Ca2+.     

Peroxynitrite affects Ca2+ influx through voltage-dependent calcium channels

The effect of peroxynitrite (OONO-) on voltage-dependent Ca2+ channels (VDCCs) was examined by measuring [45Ca2+] influx into mouse cerebral cortical neurones. OONO- time- and dose-dependently increased [45Ca2+] influx and this increase was abolished by manganese (III) tetrakis (4-benzoic acid) porphyrin, a scavenger for OONO-. Inhibition of cyclic GMP (cGMP) formation did not alter the OONO(-)-induced [45Ca2+] influx. OONO-, as well as 30 mm KCl, significantly increased fluorescence intensity of cell-associated bis-(1,3-dibutylbarbituric acid) trimethine oxonol (bis-oxonol). Tetrodotoxin and membrane stabilizers such as lidocaine dose-dependently suppressed OONO(-)-induced [45Ca2+] influx. Although each of 1 microM nifedipine and 1 microM omega-agatoxin VIA (omega-ATX) significantly inhibited the OONO(-)-induced [45Ca2+] influx and the concomitant presence of these agents completely abolished the influx, 1 microM omega-conotoxin GVIA (omega-CTX) showed no effect on the influx. On the other hand, OONO- itself reduced 30 mM KCl-induced [45Ca2+] influx to the level of [45Ca2+] influx induced by OONO- alone, and the magnitude of this reduction was as same as that of KCl-induced [45Ca2+] influx by omega-CTX. These results indicate that OONO- increases [45Ca2+] influx into the neurones through opening P/Q- and L-type VDCCs subsequent to depolarization, and inhibits the influx through N-type VDCCs.     

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.     

Acute effect of beta amyloid on synchronized spontaneous Ca2+ oscillations in cultured hippocampal networks

The effects of beta amyloid (Abeta) on cytoplasmic Ca(2+) ([Ca(2+)](c)) have been studied extensively, but the current literature on this aspect is confusing. We reported that 20 microM Abeta(25-35) significantly inhibited the synchronized spontaneous cytoplasmic Ca(2+) transients immediately after application, whereas it had little effect on the baseline [Ca(2+)](c) concentration in neurons. Abeta(1-42) had a similar effect on the Ca(2+) transients as Abeta(25-35), while it increased baseline [Ca(2+)](c) concentration gradually. However, Abeta(1-40) had little effect on either Ca(2+) transients or baseline [Ca(2+)](c). Such differential effects of Abeta on Ca(2+) signals might explain, at least partially, the confusing observations from the previous studies and provide important therapeutic implications for preventing or reversing early neuron damage in Alzheimer's disease.     

Blockage of amyloid beta peptide-induced cytosolic free calcium by fullerenol-1, carboxylate C60 in PC12 cells

Amyloid beta protein (Abeta) alters signal transduction systems, including increases in the cytosolic free calcium ([Ca2+]i) response which have pathophysiological significance in Alzheimer's disease (AD). The purposes of this study were to elucidate the mechanism involved in Abeta's effect on the Ca2+ signal and to evaluate the effect of fullerenol-1, a water-soluble hydroxyl and superoxide radical scavenger, on the Abeta-induced Ca2+ response. Both Abeta and bradykinin (BK) dose-dependently elevated [Ca2+]i in PC12 cells. Fullerenol-1, at a concentration range between 100 nM and 1 microM, dose-dependently reduced the Abeta-induced [Ca2+]i response, but did not alter the subsequent BK-mediated process. Thapsigargin, an inhibitor of Ca2+-ATPase, released Ca2+ from the internal store and diminished the BK-mediated calcium spike but did not affect the Abeta-induced Ca2+ response. In the absence of extracellular calcium, the Abeta-induced, but not BK-induced, calcium spike was completely abolished. The Ca induced by Abeta did not enter through the voltage-dependent calcium channels or ligand gated calcium channels, because the peak of Abeta-evoked Ca2+ was not significantly altered by various Ca2+ channel blockers or a NMDA receptor antagonist MK801. In addition, neither cholera toxin nor pertussis toxin altered the Abeta-induced Ca response. The results demonstrated that Abeta-stimulated [Ca2+]i increase is due to Ca influx from an extracellular source rather than from the intracellular store. Alteration of the membrane lipid structure and permeability by free radicals generated by Abeta may be a major cause of Ca -influx. Furthermore, fullerenol-1, a novel antioxidant, may provide therapeutic benefits in neurodegenerative diseases such as AD.     

Characterization and Ca2+ Requirement of Histamine-Induced Catecholamine Secretion in Cultured Bovine Chromaffin Cells

The stimulation of cultured bovine chromaffin cells with histamine induced a continuous catecholamine secretion (EC50 = 3 x 10(-7) M) via the H1 receptor, in addition to an initial catecholamine burst due to a nonspecific stimulatory effect at higher doses (greater than or equal to 10(-4) M). The continuous secretion showed little desensitization and lasted for more than 1 h. In fura-2-loaded cells, the stimulation with histamine evoked a transient rise of intracellular free Ca2+ concentration ([Ca2+]i) which lasted only for a few minutes and was followed by a sustained [Ca2+]i rise which continued for more than 20 min. The addition of an activator for the L-type voltage-sensitive Ca2+ channel, i.e., Bay K 8644 (1 microM), facilitated the sustained [Ca2+]i rise, as well as the secretion, whereas the addition of relatively high concentrations of Ca(2+)-channel blockers (10 microM) suppressed the sustained [Ca2+]i rise and part of the secretion. Removal of extracellular Ca2+ completely abolished continuous secretion and sustained [Ca2+]i rise. When the external Ca2+ level was elevated, both sustained [Ca2+]i rise and continuous secretion were enhanced in a similar Ca(2+)-dependent manner, showing saturation with around 1-3 mM Ca2+. This Ca2+ dependence was clearly different from that observed with high K+ and nicotine, which is mediated by the L-type Ca2+ channel, in which the responses showed little or no saturation when the Ca2+ level was increased. The results indicate that stimulation with histamine induces a continuous secretion via the H1 receptor, in addition to a transient and nonspecific secretion at higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells

Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age-induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage-sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10-100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N-methyl-D-aspartate receptor activation. Amlodipine also inhibited free radical-induced damage to lipid constituents of the membrane in a dose-dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose- and time-dependent manners by antioxidants (U-78439G, alpha-tocopherol, and melatonin), nitric oxide synthase inhibitors (N-nitro-L-arginine and N-nitro-D-arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage-sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.     

State-dependent inhibition of L-type calcium channels: cell-based assay in high-throughput format

The current studies describe a new, robust cell-based functional assay useful to characterize L-type voltage-dependent calcium channels and their antagonists. The basis of this assay is measurement in plate format of Ca2+ influx through the L-type Ca2+ channel complex (alpha1C, alpha2delta, and beta2a subunits) in response to potassium-mediated depolarization; EC(50)=11 mM [K+](o). The Ca2+ influx was inhibited by the L-type Ca2+ channel antagonist, nimodipine; IC(50)=59 nM. These cells were also transfected with the Kir2.3 inward rectifier K(+) channel, which allows for changing the cell membrane potential by modulation of extracellular [K](o); -65 mV in physiological [K](o) and -28 mV in 30 mM [K](o) containing buffer. The conformational state of the voltage-sensitive Ca2+ channel is altered under these different conditions. Under the depolarized condition, nimodipine was a more potent antagonist, inhibiting Ca2+ influx with an IC(50) value of 3 nM. The results demonstrate that the interaction of nimodipine and other antagonists with the channel is modulated by changes in membrane potential and thus the state of the channel. Overall, this novel assay can be used to identify state-dependent calcium channel antagonists and should be useful for evaluating state-dependent inhibitory potency of a large number of samples.     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca2+ sparks are the elementary events of intracellular Ca2+ release from the sar-coplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca2+ channel activation contributes to the genesis of spontaneous Ca2+ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca2+ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI2 which inhibits spontaneous L-type Ca2+ channel activation, the rate of occurrence of spontaneous Ca2+ sparks was halved from 4.20 to 2.04 events/(100 μm·s), with temporal and spatial properties of individual Ca2+ sparks unchanged. Analysis of the Cd2+-sensitive spark production revealed an open probability of-10-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca2+ channels in resting heart cells contribute significantly to the production of spontaneous Ca2+ sparks.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

The role of Ca2+ channel modulation in the neuroprotective actions of estrogen in beta-amyloid protein and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) cytotoxic models

Physiologically relevant concentrations of 17beta-estradiol (E2) are neuroprotective in both beta-amyloid protein 25-35 (Abeta) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced cytotoxicity in SK-N-SH cells. MPTP, but not Abeta, induces apoptosis in this cell line. The L-type calcium channel blocker nifedipine or decreased extracellular Ca(2+) concentration blocked Abeta-induced cell death, but not MPTP-induced cell death. Other blockers selective for different Ca(2+) channel subtypes had no effects on either Abeta or MPTP induced death. Western blot analysis for L-type Ca(2+) channel alpha(1)-subunits demonstrated that Abeta increases the expression of the neuronal alpha(1C) and alpha(1D) subunits of L-type channels. Both E2 and nifedipine inhibit the increase in expression of these by Abeta. MPTP also increases expression of alpha(1C) and alpha(1D), but the increases were not influenced by E2 or nifedipine. These observations suggested that Abeta cytotoxicity in SK-N-SH cells may involve increased availability of calcium to cells, whereas MPTP induced cytotoxicity does not require extracellular Ca(2+). Both cytotoxic models were associated with increased expression of Ca(2+) channel alpha(1) subunits, and neuroprotection associated with inhibition of that increase. These studies reveal that nifedipine, in addition to its direct action of nifedipine on Ca(2+) channels, may also protect neurons from Abeta toxicity through the suppression of the channel protein overexpression. A new action of dihydropyridines (DHPs) may be considered in the regulation of calcium homeostasis.     

Neuroprotective Effects of Bis(7)-tacrine in a Rat Model of Pressure-Induced Retinal Ischemia

The retinal ischemia–reperfusion model has been studied extensively and is an ideal animal model for studying clinical situations such as acute glaucoma and optic neuropathy. Our previous reports showed that bis(7)-tacrine had neuroprotective effects against glutamate-induced retinal ganglion cells damage through the drug’s anti-NMDA receptor effects. Here, we investigated whether bis(7)-tacrine protects the retina from ischemic injury in a rat model. Retinal ischemia was induced by raising the intraocular pressure to 120 mmHg for 90 min. Rats received intraperitoneal injections of 0.2 mg/kg bis(7)-tacrine or saline at 30 min before ischemia, and then twice a day after retinal ischemia. Morphometric evaluation showed that bis(7)-tacrine dramatically reduced the retinal damage compared with the control group. Moreover, bis(7)-tacrine suppressed ischemia-induced reductions in a- and b-wave amplitudes of electroretinography. Protein levels of p53, the tumor suppressor gene known to induce apoptosis, were increased after ischemic injury, and treatment with bis(7)-tacrine reduced the expression of the protein. Our results suggest that bis(7)-tacrine has a neuroprotective effect against ischemic injury in the rat retina, possibly through the drug’s anti-apoptotic effects. Bis(7)-tacrine may potentially be useful as a therapeutic drug in the management of ischemic retinal diseases.     

Excitotoxic Death of a Subset of Embryonic Rat Motor Neurons In Vitro

Abstract : We have used cultures of purified embryonic rat spinal cord motor neurons to study the neurotoxic effects of prolonged ionotropic glutamate receptor activation. NMDA and non-NMDA glutamate receptor agonists kill a maximum of 40% of the motor neurons in a concentration- and time-dependent manner, which can be blocked by receptor subtype-specific antagonists. subunit-specific antibodies stain all of the motor neurons with approximately the same intensity and for the same repertoire of subunits, suggesting that the survival of the nonvulnerable population is unlikely to be due to the lack of glutamate receptor expression. Extracellular Ca²⁺ is required for excitotoxicity, and the route of entry initiated by activation of non-NMDA, but not NMDA, receptors is L-type Ca²⁺ channels. Ca²⁺ imaging of motor neurons after application of specific glutamate receptor agonists reveals a sustained rise in intracellular Ca²⁺ that is present to a similar degree in most motor neurons, and can be blocked by appropriate receptor/channel antagonists. Although the lethal effects of glutamate receptor agonists are seen in only a subset of cultured motor neurons, the basis of this selectivity is unlikely to be simply the glutamate receptor phenotype or the level/pattern of rise in agonist-evoked intracellular Ca²⁺.     

Novel dimeric bis(7)-tacrine proton-dependently inhibits NMDA-activated currents

Bis(7)-tacrine has been shown to prevent glutamate-induced neuronal apoptosis by blocking NMDA receptors. However, the characteristics of the inhibition have not been fully elucidated. In this study, we further characterize the features of bis(7)-tacrine inhibition of NMDA-activated current in cultured rat hippocampal neurons. The results show that with the increase of extracellular pH, the inhibitory effect decreases dramatically. At pH 8.0, the concentration-response curve of bis(7)-tacrine is shifted rightwards with the IC(50) value increased from 0.19+/-0.03 microM to 0.41+/-0.04 microM. In addition, bis(7)-tacrine shifts the proton inhibition curve rightwards. Furthermore, the inhibitory effect of bis(7)-tacrine is not altered by the presence of the NMDA receptor proton sensor shield spermidine. These results indicate that bis(7)-tacrine inhibits NMDA-activated current in a pH-dependent manner by sensitizing NMDA receptors to proton inhibition, rendering it potentially beneficial therapeutic effects under acidic conditions associated with stroke and ischemia.     

Release of acetylcholinesterase (AChE) from beta-amyloid plaques assemblies improves the spatial memory impairments in APP-transgenic mice

The major protein constituent of amyloid deposits in Alzheimer's disease (AD) is the amyloid-beta-peptide (Abeta). Amyloid deposits contain "chaperone molecules" which play critical roles in amyloid formation and toxicity. In the present work, we test an analog of hyperforin (IDN 5706) which releases the AChE from both the Abeta fibrils and the AChE-Abeta burdens in transgenic mice. Hyperforin is an acylphloroglucinol compound isolated from Hypericum perforatum (St. John's Wort), which is able to prevent the Abeta-induced spatial memory impairments and Abeta neurotoxicity. Altogether this gathered evidence indicates the important role of AChE in the neurotoxicity of Abeta plaques and finding new compounds which decrease the AChE-Abeta interaction may be a putative therapeutic agent to fight the disease.     

The Amyloid β-Protein of Alzheimer's Disease Increases Acetylcholinesterase Expression by Increasing Intracellular Calcium in Embryonal Carcinoma P19 Cells

Abstract: One of the characteristic changes that occurs in Alzheimer's disease is the loss of acetylcholinesterase (AChE) from both cholinergic and noncholinergic neurons of the brain. However, AChE activity is increased around amyloid plaques. This increase in AChE may be of significance for therapeutic strategies using AChE inhibitors. The aim of this study was to examine the effect of amyloid β-protein (Aβ), the major component of amyloid plaques, on AChE expression. Aβ peptides spanning residues 1–40 or 25–35 increased AChE activity in P19 embryonal carcinoma cells. A peptide containing a scrambled Aβ_25–35 sequence did not stimulate AChE expression. To examine the possibility that the increase in AChE expression was mediated by an influx of calcium through voltage-dependent calcium channels (VDCCs), drugs acting on VDCCs were tested for their effects. Inhibitors of L-type VDCCs (diltiazem, nifedipine, and verapamil), but not N- or P- or Q-type VDCCs, resulted in a decrease in AChE expression. Agonists of L-type VDCCs (maitotoxin and S (−)-Bay K 8644) increased AChE expression. As L-type VDCCs are known to be modulated by cyclic AMP-dependent protein kinase, the effect of the adenylate cyclase activator forskolin was also examined. Forskolin stimulated AChE expression, an action that was blocked by the L-type VDCC antagonist nifedipine. The Aβ_25–35-induced increase in AChE expression was mediated by an L-type VDCC, as the effect was also blocked by nifedipine. The results suggest that the increase in AChE expression around amyloid plaques could be due to a disturbance in calcium homeostasis involving the opening of L-type VDCCs.