Amyloid-β proteins activate Ca2+-permeable channels through calcium-sensing receptors

The amyloid-beta peptides (Aβ) are produced in excess in Alzheimer's disease (AD) and may contribute to neuronal dysfunction and degeneration. This study provides strong evidence for a novel cellular target for the actions of Aβ, the phospholipase C-coupled, extracellular Ca²⁺-sensing receptor (CaR). We demonstrate that Aβs produce a CaR-mediated activation of a Ca²⁺-permeable, nonselective cation channel (NCC), probably via elevation in cytosolic Ca²⁺ (Ca_i), in cultured hippocampal pyramidal neurons from normal rats and from wild type mice but not those from mice with targeted disruption of the CaR gene (CaR −/−). Aβs also activate NCC in CaR-transfected but not in nontransfected human embryonic kidney (HEK293) cells. Thus aggregates of Aβ deposited on hippocampal neurons in AD could inappropriately activate the CaR, stimulating Ca²⁺-permeable channels and causing sustained elevation of Ca_i with resultant neuronal dysfunction. © 1997 Wiley-Liss Inc.     

细胞外钙离子浓度对海马神经元兴奋性的影响

目的 在不同的生理、病理情况下,中枢神经系统细胞外钙离子浓度([Ca2 ]o)下降,导致神经元兴奋性增高.本研究的主要目的是探讨低钙条件下神经元兴奋性增高的机制,从而为临床治疗神经元过度兴奋性疾病探索新的治疗途径.方法 应用穿孔膜片钳及细胞培养技术,记录不同细胞外钙离子浓度对海马神经元兴奋性的影响.结果 低钙环境使神经元兴奋性显著增高,阈电位水平显著降低,动作电位幅度显著增高.并且出乎意料的是,mAHP拮抗剂apamin及sAHP拮抗剂Iso对海马神经元兴奋性的影响没有统计学的意义.作为INaP拮抗剂,低浓度的河豚毒(TTX)虽然阻断了低钙环境中神经元兴奋性的增加,但同时也阻断了正常钙离子浓度下的动作电位的发放.结论 低钙环境中海马神经元阈电位的显著下降可能是导致神经元兴奋性增高的主要原因.     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca²⁺-activated potassium (BK_Ca) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK_Ca channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K⁺ in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P_o) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P_o was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca²⁺]_i required to half activate BK_Ca channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca²⁺]_i sensitivity after ischemia; and (4) BK_Ca channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK_Ca channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

Gi/o protein-coupled receptors inhibit neurons but activate astrocytes and stimulate gliotransmission

G protein-coupled receptors (GPCRs) play key roles in intercellular signaling in the brain. Their effects on cellular function have been largely studied in neurons, but their functional consequences on astrocytes are less known. Using both endogenous and chemogenetic approaches with DREADDs, we have investigated the effects of G_q and G_i/o GPCR activation on astroglial Ca²⁺-based activity, gliotransmitter release, and the functional consequences on neuronal electrical activity. We found that while G_qGPCR activation led to cellular activation in both neurons and astrocytes, G_i/oGPCR activation led to cellular inhibition in neurons and cellular activation in astrocytes. Astroglial activation by either G_q or G_i/o protein-mediated signaling stimulated gliotransmitter release, which increased neuronal excitability. Additionally, activation of G_q and G_i/o DREADDs in vivo increased astrocyte Ca²⁺ activity and modified neuronal network electrical activity. Present results reveal additional complexity of the signaling consequences of excitatory and inhibitory neurotransmitters in astroglia-neuron network operation and brain function.     

Store-operated calcium entry modulates neuronal network activity in a model of chronic epilepsy

Store-operated Ca²⁺ entry (SOCE) over the plasma membrane is activated by depletion of intracellular Ca²⁺ stores and has only recently been shown to play a role in CNS processes like synaptic plasticity. However, the direct effect of SOCE on the excitability of neuronal networks in vitro and in vivo has never been determined. We confirmed the presence of SOCE and the expression of the calcium sensors STIM1 and STIM2, which convey information about the calcium load of the stores to channel proteins at the plasma membrane, in neurons and astrocytes. Inhibition of SOCE by pharmacological agents 2-APB and ML-9 reduced the steady-state neuronal Ca²⁺ concentration, reduced network activity, and increased synchrony of primary neuronal cultures grown on multi-electrode arrays, which prompted us to elucidate the relative expression of STIM proteins in conditions of pathologic excitability. Both proteins were increased in brains of chronic epileptic rodents and strongly expressed in hippocampal specimens from medial temporal lobe epilepsy patients. Pharmacologic inhibition of SOCE in chronic epileptic hippocampal slices suppressed interictal spikes and rhythmized epileptic burst activity. Our results indicate that SOCE modulates the activity of neuronal networks in vitro and in vivo and delineates SOCE as a potential drug target.     

BDNF potentiates spontaneous Ca oscillations in cultured hippocampal neurons

Brain-derived neurotrophic factor (BDNF) is thought to regulate neuronal plasticity in developing and matured neurons, although the molecular mechanisms are less well characterized. We monitored changes in the intracellular calcium (Ca²⁺) levels induced by BDNF using a fluorescence Ca²⁺ indicator (Fluo-3) by means of confocal laser microscopy in rat cultured hippocampal neurons. BDNF acutely potentiated spontaneous Ca²⁺ oscillations in dendrites and also in the soma of several neurons, although it increased intracellular Ca²⁺ in only selective proportion of resting neurons without Ca²⁺ oscillations. The potentiation was observed both in the frequency and the amplitude of Ca²⁺ oscillations, completely blocked by K-252a, and significantly reduced by 2-aminophosphonovaleric acid. These findings suggest that BDNF increases glutamate release and N-methyl-

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-aspartate (NMDA) channel-gated Ca²⁺ influx via TrkB and regulates the frequency and the amplitude of Ca²⁺ oscillations. BDNF may have the potential to modulate spontaneous Ca²⁺ oscillations to regulate neuronal plasticity in developing hippocampal neurons.     

Synaptic impairment induced by paroxysmal ionic conditions in neocortex

Purpose: Seizures are associated with a reduction in extracellular Ca²⁺ concentration ([Ca²⁺]_o) and an increase in extracellular K⁺ concentration ([K⁺]_o). The long‐range synchrony observed between distant electrodes during seizures is weak. We hypothesized that changes in extracellular ionic conditions during seizures are sufficient to alter synaptic neuronal responses and synchrony in the neocortex. Methods: We obtained in vivo and in vitro electrophysiologic recordings combined with microstimulation from cat/rat neocortical neurons during seizures and seizure‐like ionic conditions. In vitro the [K⁺]_o was 2.8, 6.25, 8.0, and 12 mm and the [Ca²⁺]_o was 1.2 and 0.6 mm . Key Findings: During seizures recorded in vivo, we observed abolition of evoked synaptic responses. In vitro, the membrane potential of both regular‐spiking and fast‐spiking neurons was depolarized in high [K⁺]_o conditions and hyperpolarized in high [Ca²⁺]_o conditions. During high [K⁺]_o conditions, changes in [Ca²⁺]_o did not affect membrane potential. The synaptic responsiveness of both regular‐spiking and fast‐spiking neurons was reduced during seizure‐like ionic conditions. A reduction in [Ca²⁺]_o to 0.6 mm increased failure rates but did not abolish responses. However, an increase in [K⁺]_o to 12 mm abolished postsynaptic responses, which depended on a blockade in axonal spike propagation. Significance: We conclude that concomitant changes in [K⁺]_o and [Ca²⁺]_o observed during seizures contribute largely to the alterations of synaptic neuronal responses and to the decrease in long‐range synchrony during neocortical seizures.     

GABA and glutamate receptors have different effects on excitability and are differentially regulated by calcium in spider mechanosensory neurons

GABA and glutamate receptors belonging to the ligand‐gated chloride‐channel family are primary targets of insecticides and antiparasitics, so their molecular structure, pharmacology and biophysical properties have attracted significant attention. However, little is known about the physiological roles of these channels or how they regulate neuronal excitability and animal behavior. Mechanosensory neurons of VS‐3 slit sensilla in the patella of the tropical wandering spider, Cupiennius salei, react to the GABA_A‐receptor agonists, GABA and muscimol, with depolarization and an increase in intracellular [Ca²⁺] and, during random noise stimulation, with a mixed inhibitory–excitatory response. We established that the GABA_A‐receptors in all VS‐3 neurons are identical, but there are at least two types of glutamate receptors and some neurons do not respond to glutamate at all. Immunohistochemistry with antibodies against Drosophila inhibitory glutamate receptor (GluCls) α‐subunit suggests that in addition to VS‐3 neurons, these receptors may also be present in the efferent neurons surrounding the sensory neurons. Most VS‐3 neurons were inhibited but not depolarized by glutamate during random stimulation, but some depolarized and had a similar excitatory–inhibitory response to glutamate as to muscimol. The membrane‐permeable Ca²⁺‐chelator BAPTA‐AM abolished muscimol effects but potentiated glutamate effects, indicating that GABA and glutamate receptors are differentially modulated by Ca²⁺, leading to diverse regulation of neuronal excitability. We hypothesize that this could be achieved by different Ca²⁺‐triggered phosphorylation processes at each receptor type. These findings are important for understanding the significance of Ca²⁺‐mediated regulation of transmitter receptor molecules and its role in controlling excitability.     

Transient Brain Ischaemia Provokes Ca2+, PIP2 and Calpain Responses Prior to Delayed Neuronal Death in Monkeys

To clarify the mechanism of postischaemic delayed cornu Ammonis (CA)-1 neuronal death, we studied correlations among calpain activation and its subcellular localization, the immunoreactivity of phosphatidylinositol 4,5-bisphosphate (PIP₂) and Ca²⁺ mobilization in the monkey hippocampus by two independent experimental approaches: in vivo transient brain ischaemia and in vitro hypoxia-hypoglycaemia of hippocampal acute slices. The CA-1 sector undergoing 20 min of ischaemia in vivo showed microscopically a small number of neuronal deaths on day 1 and almost global neuronal loss on day 5 after ischaemia. Immediately after ischaemia, CA-1 neurons ultrastructurally showed vacuolation and/or disruption of the lysosomes. Western blotting using antibodies against inactivated or activated μ-calpain demonstrated μ-calpain activation specifically in the CA-1 sector immediately after ischaemia. This finding was confirmed in the perikarya of CA-1 neurons by immunohistochemistry. CA-1 neurons on day 1 showed sustained activation of μ-calpain, and increased immunostaining for inactivated and activated forms of μ- and m-calpains and for PIP₂. Activated μ-calpain and PIP₂ were found to be localized at the vacuolated lysosomal membrane or endoplasmic reticulum and mitochondrial membrane respectively, by immunoelectron microscopy. Calcium imaging data using hippocampal acute slices showed that hypoxia-hypoglycaemia in vitro provoked intense Ca²⁺ mobilization with increased PIP₂ immunostaining specifically in CA-1 neurons. These data suggest that transient brain ischaemia increases intracellular Ca²⁺ and PIP₂ breakdown, which will activate calpain proteolytic activity. Therefore, we suggest that activated calpain at the lysosomal membrane, with the possible release of biodegrading enzyme, will cause postischaemic CA-1 neuronal death.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca concentration in hippocampal neurons

The short-term effect of bFGF on intracellular Ca²⁺ concentration ([Ca²⁺]_i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca²⁺]_i were measured by microfluorometrically monitoring the fluorescence intesities from indivudual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca²⁺]_i in hippocampal neurons, but clearly enhanced the [Ca²⁺]_i increase induced by NMDA. Quisqualate or KCl-induced [Ca²⁺]_i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.     

Alterations in Intracellular Calcium Ion Concentrations in Cerebellar Granule Cells of the CACNA1A Mutant Mouse, Leaner, During Postnatal Development

Maintaining calcium ion (Ca²⁺) homeostasis is crucial for normal neuronal function. Altered Ca²⁺ homeostasis interferes with Ca²⁺ signaling processes and affects neuronal survival. In this study, we used homozygous leaner and tottering mutant mice, which carry autosomal recessive mutations in the gene coding for the α_1A pore forming subunit of Ca_V2.1 (P/Q-type) voltage-gated calcium channels (VGCC). Leaner mice show severe ataxia and epilepsy, while tottering mice are less severely affected. Leaner cerebellar granule cells (CGC) show extensive apoptotic cell death that peaks at postnatal (P) day 20 and continues into adulthood. Intracellular Ca²⁺ ([Ca²⁺]_i) concentrations in leaner and tottering mouse Purkinje cells have been described, but [Ca²⁺]_i concentrations have not been reported for granule cells, the largest neuronal population of the cerebellum. Using the ratiometric dye, Fura-2 AM, we investigated the role of Ca²⁺ homeostasis in CGC death during postnatal development by demonstrating basal [Ca²⁺]_i, depolarization induced Ca²⁺ transients, and Ca²⁺ transients after completely blocking Ca_V2.1 VGCC. From P20 onward, basal [Ca²⁺]_i levels in leaner CGC were significantly lower compared to age-matched wild-type CGC. We also compared basal [Ca²⁺]_i levels in leaner and wild-type CGC to basal [Ca²⁺]_i in tottering CGC. Potassium chloride induced depolarization revealed no significant difference in Ca²⁺ transients between leaner and wild-type CGC, indicating that even though leaner CGC have dysfunctional P/Q-type VGCC, Ca²⁺ transients after depolarization are the same. This suggests that other VGCC are compensating for the dysfunctional P/Q channels. This finding was further confirmed by completely blocking Ca_V2.1 VGCC using ω-Agatoxin IV-A.     

Deletion of calcineurin from GFAP-expressing astrocytes impairs excitability of cerebellar and hippocampal neurons through astroglial Na+/K+ ATPase

Astrocytes perform important housekeeping functions in the nervous system including maintenance of adequate neuronal excitability, although the regulatory mechanisms are currently poorly understood. The astrocytic Ca²⁺/calmodulin-activated phosphatase calcineurin (CaN) is implicated in the development of reactive gliosis and neuroinflammation, but its roles, including the control of neuronal excitability, in healthy brain is unknown. We have generated a mouse line with conditional knockout (KO) of CaN B1 (CaNB1) in glial fibrillary acidic protein-expressing astrocytes (a stroglial c alcin eurin KO [ACN-KO]). Here, we report that postnatal and astrocyte-specific ablation of CaNB1 did not alter normal growth and development as well as adult neurogenesis. Yet, we found that specific deletion of astrocytic CaN selectively impairs intrinsic neuronal excitability in hippocampal CA1 pyramidal neurons and cerebellar granule cells (CGCs). This impairment was associated with a decrease in after hyperpolarization in CGC, while passive properties were unchanged, suggesting impairment of K⁺ homeostasis. Indeed, blockade of Na⁺/K⁺-ATPase (NKA) with ouabain phenocopied the electrophysiological alterations observed in ACN-KO CGCs. In addition, NKA activity was significantly lower in cerebellar and hippocampal lysates and in pure astrocytic cultures from ACN-KO mice. While no changes were found in protein levels, NKA activity was inhibited by the specific CaN inhibitor FK506 in both cerebellar lysates and primary astroglia from control mice, suggesting that CaN directly modulates NKA activity and in this manner controls neuronal excitability. In summary, our data provide formal evidence for the notion that astroglia is fundamental for controlling basic neuronal functions and place CaN center-stage as an astrocytic Ca²⁺-sensitive switch.     

Taxol stabilizes [Ca]i and protects hippocampal neurons against excitotoxicity

Elevation of intracellular calcium levels [Ca²⁺]_i induces microtubule depolymerization, a process which plays roles in regulation of cell motility and axonal transport. However, excessive Ca²⁺ influx, as occurs in neurons subjected to excitotoxic conditions, can kill neurons. We now provide evidence that the polymerization state of microtubules influences neuronal [Ca²⁺]_i homeostasis and vulnerability to excitotoxicity. The microtubule-stabilizing agent taxol significantly attenuated glutamate neurotoxicity in cultured rat hippocampal neurons. Experiments in which [Ca²⁺]_i was monitored using the Ca²⁺ indicator dye fura-2 showed that the elevation of [Ca²⁺]_i induced by glutamate was significantly attenuated in neurons pretreated with taxol. Experiments using selective glutamate receptor agonists suggested that taxol suppressed Ca²⁺ influx through α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, but not through N-methyl-D-aspartate (NMDA) receptors. Taxol attenuated the neurotoxicity of the microtubule-depolymerizing agent colchicine; colchicine neurotoxicity was, in part, dependent on Ca²⁺ influx. These findings suggest that microtobules play a role in the mechanism of excitotoxicity and suggest that taxol and related compounds may be useful as antiexcitotoxic agents.     

Neuroprotective properties of<Emphasis Type="Italic">Valeriana officinalis</Emphasis> extracts

Valeriana officinalis have been used in traditional medicine for its sedative, hypnotic, and anticonvulsant effects. There are several eports in the literature supporting a GABAergic mechanism of action for valerian. The rationale of the present work is based on the concept that by decreasing neuronal network excitability valerian consumption may contribute to neuroprotection. The aim of our investigation was to evaluate the neuroprotective effects ofV. officinalis against the toxicity induced by amyloid beta peptide 25–35 [Aβ_(25–35)]. Cultured rat hip-pocampal neurons were exposed to Aβ_(25–35)(25 μM) for 24–48 h,after which morphological and biochemical properties were evaluated. The neuronal injury evoked by Aβ, which includes a decrease in cell educing capacity and associated neuronal degeneration, was prevented by valerian extract. Analysis of intracellular free calcium ([Ca²⁺]_i)indicated that the neuroprotective mechanisms may involve the inhibition of excess influx of Ca²⁺ following neuronal injury. Moreover, membrane peroxidation in rat hippocampal synaptosomes was evaluated, and our data indicate that valerian extract partially inhibited ascorbate/iron-induced peroxidation. In conclusion we show evidence that the signalling pathways involving [Ca²⁺]_i and the redox state of the cells may play a central ole in the neuroprotective properties ofV. officinalis extract against Aβ toxicity. The novelty of the findings of the present work, indicating neuroprotective properties of valerian against Aβ toxicity may, at the long-term, contribute to introduction of a new elevant use of valerian alcoholic extract to prevent neuronal degeneration in aging or neurodegenerative disorders.     

Formaldehyde increases intracellular calcium concentration in primary cultured hippocampal neurons partly through NMDA receptors and T-type calcium channels

Objective Formaldehyde at high concentrations is a contributor to air pollution.It is also an endogenous metabolic product in cells,and when beyond physiological concentrations,has pathological effects on neurons.Formaldehyde induces mis-folding and aggregation of neuronal tau protein,hippocampal neuronal apoptosis,cognitive impairment and loss of memory functions,as well as excitation of peripheral nociceptive neurons in cancer pain models.Intracellular calcium([Ca²⁺]_i) is an important intracellular messenger,and plays a key role in many pathological processes.The present study aimed to investigate the effect of formaldehyde on[Ca²⁺]_i and the possible involvement of N-methyl-Z)-aspartate receptors （NMDARs） and T-type Ca²⁺ channels on the cell membrane.Methods Using primary cultured hippocampal neurons as a model,changes of[Ca²⁺]_i in the presence of formaldehyde at a low concentration were detected by confocal laser scanning microscopy.Results Formaldehyde at 1 mmol/L approximately doubled[Ca²⁺]_i.（2R）-amino-5-phosphonopentanoate （AP5,25μmol/L,an NMDAR antagonist） and mibefradil(MIB,1 umol/L,a T-type Ca²⁺channel blocker),given 5 min after formaldehyde perfusion,each partly inhibited the formaldehyde-induced increase of[Ca²⁺]_i,and this inhibitory effect was reinforced by combined application of AP5 and MIB.When applied 3 min before formaldehyde perfusion,AP5 （even at 50μmol/L） did not inhibit the formaldehyde-induced increase of[Ca²⁺]_i but MIB（1 umol/L） significantly inhibited this increase by 70%.Conclusion These results suggest that formaldehyde at a low concentration increases[Ca²⁺]_i in cultured hippocampal neurons;NMDARs and T-type Ca²⁺ channels may be involved in this process.     

Regulation of T-type Ca<Superscript>2+</Superscript> channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca²⁺ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca²⁺ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca²⁺ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca²⁺ current densities. Increased T-type Ca²⁺ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca²⁺ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca²⁺ channels is mediated by several factors, including decreased expression of Ca_v3.2 T-type Ca²⁺ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca²⁺ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca²⁺ channels, which can results in morphological and functional changes in electrical excitability.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

Relationship between resting cytosolic Ca and responses induced byN-methyl-d-aspartate in hippocampal neurons

Cytosolic calcium concentrations ([Ca²⁺]_i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca²⁺]_i showed greater [Ca²⁺]_i responses toN-methyl-d-aspartate (NMDA) and K⁺ depolarization. There was a strong relationship between resting [Ca²⁺]_i and the maximal changes in [Ca²⁺]_i (Δ[Ca²⁺]_i), which fit the our proposed equation to describe this relationship.