Primary Cultures Of Cerebellar Granule Cells Research Articles

Neuroprotective Effect of a Pharmaceutical Extract of Cannabis with High Content on CBD Against Rotenone in Primary Cerebellar Granule Cell Cultures and the Relevance of Formulations.

Introduction: Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e.g., epilepsy); however, their neuroprotective potential has not been widely investigated. Materials and Methods: Using primary cultures of cerebellar granule cells, we evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, trace levels of Δ9-tetrahydrocannabinol, and the acid form of CBD. We determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). Results: The results revealed that EPI induced a significant reduction in the rotenone-induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se. EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions between individual substances present in EPI occurred. In contrast, CBD did show a different profile to EPI and XAL because a neurotoxic effect per se was observed at higher concentrations assayed. Medium-chain triglyceride oil used in EPI formulation could explain this difference. Conclusion: Our data support a neuroprotective effect of EPI that may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based products that could be critical to avoid neurotoxicity at very high doses.

Activation of voltage-gated sodium channels by BmK NT1 augments NMDA receptor function through Src family kinase signaling pathway in primary cerebellar granule cell cultures

Voltage-gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in excitable cells and are the molecular targets of an array of neurotoxins. BmK NT1, an α-scorpion toxin obtained from the scorpion Buthus martensii Karsch (BmK), produces neurotoxicity that is associated with extracellular Ca2+ influx through Na+-Ca2+ exchangers, N-methyl-d-aspartic acid (NMDA) receptors, and L-type Ca2+ channels in cultured cerebellar granule cells (CGCs). In the present study, we demonstrated that BmK NT1 triggered concentration-dependent release of excitatory neurotransmitters, glutamate and aspartate; both effects were eliminated by VGSC blocker, tetrodotoxin. More importantly, we demonstrated that a threshold concentration of BmK NT1 that produced marginal Ca2+ influx and neuronal death augmented glutamate-induced Ca2+ elevation and neuronal death in CGCs. BmK NT1-augmented glutamate-induced Ca2+ influx and neuronal death were suppressed by tetrodotoxin and MK-801 suggesting that the augmentation was through activation of VGSCs and NMDA receptors. Consistently, BmK NT1 also enhanced NMDA-induced Ca2+ influx. Further mechanistic investigations demonstrated that BmK NT1 increased the expression level of NMDA receptors on the plasma membrane and increased the phosphorylation level of NR2B at Tyr1472. Src family kinase inhibitor, 1-tert-butyl-3-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidin-4-yl]amine (PP2), but not the inactive analogue, 4-amino-1-phenylpyrazolo[3,4-d]pyrimidine (PP3), eliminated BmK NT1-triggered NR2B phosphorylation, NMDA receptor trafficking, as well as BmK NT1-augmented NMDA Ca2+ response and neuronal death. Considered together, these data demonstrated that both presynaptic (excitatory amino acid release) and postsynaptic mechanisms (augmentation of NMDA receptor function) are critical for VGSC activation-induced neurotoxicity in primary CGC cultures.

Cerebrospinal fluids from Alzheimer's disease patients exhibit neurotoxic effects on neuronal cell cultures.

A growing number of studies suggest amyloid-β and tau present in cerebrospinal fluid (CSF) and blood as putative biomarkers for Alzheimer's disease (AD). However, there is a question whether these compounds present in patients' bodily fluids can directly cause neurotoxic effects. We investigated effects of AD and other dementia (OD) patients' blood serum and CSF on viability of cells in primary cerebellar granule cell cultures. Overall, 59 individuals participated in the study from whom 55 samples of biological fluids were taken. Participants were classified into early (E-AD) and middle (M-AD) stages of AD, cognitively healthy control (HC) and OD groups. We found that concentrations of total and phosphorylated tau were higher in CSF from AD patients, while amyloid-β42 and amyloid-β40 in the serum was lower compared to HC. The most cytotoxic effects were induced by CSFs from M-AD patients which caused neuronal necrosis and suppressed microglial proliferation, whereas CSFs from the groups of other patients did not kill neurons. Serum and CSF from the E-AD group caused a reduction of neuronal numbers in cultures. There were no significant differences in levels of CSF biomarkers between the AD groups although both tau species in CSFs from M-AD patients were found to be significantly elevated compared to HC. Our data suggest that biological fluids from E-AD induce neuronal loss, whereas effects of CSF on the reduction in neuronal viability can serve as an indicator of M-AD and may be associated with extracellular tau.

Reduced serine racemase expression in aging rat cerebellum is associated with oxidative DNA stress and hypermethylation in the promoter

Regulation of serine racemase (SR) occurs at transcriptional and translational levels; post-translational modification, cytosolic distribution as well as allosteric effect regulate SR activity. In this study, we report a new route of SR regulation, i.e. oxidative stress and hypermethylation of the srr (gene of SR) promoter correlate with its reduced transcription in aging rat cerebella. We first showed that the mRNA and protein level of srr were decreased in the homogenates of rat cerebellum at age 12 months compared with the counterparts from age 20 days. The reduction of SR protein level in aging cerebella was evidenced by decreased immunostaining observed in the cell body of granule cells or Purkinje cells. Staining for 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker for oxidative stress to DNA, was much stronger in granule cell or Purkinje cell nuclei from rat cerebella at 12 months compared with staining at 20 days. We further detected srr promoter hypermethylation at 12 months compared with that at 20 days by use of bisulfite sequencing PCR, coinciding with elevated protein levels of DNA methyltransferase 1 (DNMT1) in homogenates of aging cerebella. In vitro, we demonstrated that chronic treatment with the oxidant, menadione (VK3), reduced srr mRNA levels, which was reversed by the DNA demethylating agent 5-Aza-dC-2′-deoxycytidine (5-Aza-dC) in primary cerebellar granule cell cultures. Together, the in vivo and ex vivo results suggest that oxidative DNA stress and srr promoter hypermethylation are associated with reduced srr gene transcription and corresponding reduced protein expression in aging cerebella.

Altered sensitivity to excitotoxic cell death and glutamate receptor expression between two commonly studied mouse strains

Alterations in glutamatergic synapse function have been implicated in the pathogenesis of many different neurological disorders, including ischemia, epilepsy, Parkinson's disease, Alzheimer's disease, and Huntington's disease. While studying glutamate receptor function in juvenile Batten disease on the C57BL/6J and 129S6/S(v)E(v) mouse backgrounds, we noticed differences unlikely to be due to mutation difference alone. We report here that primary cerebellar granule cell cultures from C57BL/6J mice are more sensitive to N-methyl-D-aspartate (NMDA)-mediated cell death. Moreover, sensitivity to AMPA-mediated excitotoxicity is more variable and is dependent on the treatment conditions and age of the cultures. Glutamate receptor surface expression levels examined in vitro by in situ ELISA and in vivo by Western blot in surface cross-linked cerebellar samples indicated that these differences in sensitivity likely are due to strain-dependent differences in cell surface receptor expression levels. We propose that differences in glutamate receptor expression and in excitotoxic vulnerability should be taken into consideration in the context of characterizing disease models on the C57BL/6J and 129S6/S(v)E(v) mouse backgrounds.

Long‐term exposure to dieldrin reduces γ‐aminobutyric acid type A and N‐methyl‐D‐aspartate receptor function in primary cultures of mouse cerebellar granule cells

The organochlorine pesticide dieldrin is a persistent organic pollutant that accumulates in the fatty tissue of living organisms. In mammals, it antagonizes the GABA(A) receptor, producing convulsions after acute exposure. Although accumulation in human brain has been reported, little is known about the effects of long-term exposure to dieldrin in the nervous system. Homeostatic control of the balance between excitation and inhibition has been reported when neuronal activity is chronically altered. We hypothesized that noncytotoxic concentrations of dieldrin could decrease glutamatergic neurotransmission as a consequence of a prolonged reduction in GABA(A) receptor function. Long-term exposure of primary cerebellar granule cell cultures to 3 microM dieldrin reduced the GABA(A) receptor function to 55% of control, as measured by the GABA-induced (36)Cl(-) uptake. This exposure produced a significant reduction (approximately 35%) of the NMDA-induced increase in [Ca(2+)](i) and of the [(3)H]MK-801 binding, which was not accompanied by a reduction in the NMDA receptor subunit NR1, as determined by Western blot. Consistent with the decreased NMDA receptor function, dieldrin-treated cultures were insensitive to an excitotoxic stimulus induced by exposure to high potassium. In summary, we report that the chronic reduction of GABA(A) receptor function induced by dieldrin decreases the number of functional NMDA receptors, which may be attributable to a mechanism of synaptic scaling. These effects could underlie neural mechanisms involved in cognitive impairment produced by low-level exposure to dieldrin.

Effects of the marine phycotoxin palytoxin on neuronal pH in primary cultures of cerebellar granule cells

Palytoxin (PTX) is a potent marine phycotoxin that binds to the Na,K-ATPase, converting this pump into an open channel. We have recently shown (Vale et al., 2006) that PTX causes an irreversible increase in the cytosolic calcium concentration ([Ca(2+)](c)) in primary cultures of cerebellar granule cells (CGC). In this work, we investigated the effect of PTX on the intracellular pH (pH(i)) in the same cellular model. PTX-induced changes in pH(i) were studied in CGC by using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). PTX caused an irreversible intracellular acidification of CGC. This acidification was due to an influx of extracellular calcium, inasmuch as it was completely abolished by the use of Ca(2+)-free medium. Different mechanisms that could be involved in the PTX-induced pH(i) decrease such as displacement of H(+) by Ca(2+) from a common intracellular binding site, PTX-induced alteration of pH(i) regulation mechanisms, and a possible acidification caused by an increase of mitochondrial Ca(2+) uptake by PTX were excluded. PTX-induced intracellular acidification was completely prevented by several inhibitors of the plasma membrane calcium ATPase (PMCA), including orthovanadate, lanthanum, high extracellular pH, and caloxin 2A1. Our results indicate that the PMCA is involved in the PTX-induced intracellular acidification in primary cultures of CGC. The PTX-evoked increase in [Ca(2+)](c) will activate the calcium extrusion mechanisms through the PMCA, which, in turn, will decrease pH(i) by countertransport of H(+) ions. The effect of PTX on neuronal pH could be a potential factor to contribute to the high cytotoxicity of this toxin in cultured cerebellar neurons.

Role of the plasma membrane calcium adenosine triphosphatase on domoate-induced intracellular acidification in primary cultures of cerebelar granule cells

Changes in intracellular pH (pH(i)) and cytosolic calcium concentration ([Ca(2+)](c)) caused by the glutamate agonist domoate (DOM) were studied in single cultured mouse cerebellar granule cells (CGC) by using the fluorescent probes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and simultaneous evaluation of cytosolic calcium concentration with the fluorescent dye Fura-2 acetoxymethyl ester (Fura-2 AM). DOM caused a concentration-dependent increase in [Ca(2+)](c) and a concentration-dependent intracellular acidification of CGC. DOM-induced intracellular acidification was completely abolished by the use of Ca(2+)-free medium, suggesting that it was due mostly to an influx of extracellular calcium. The pH(i) decrease caused by DOM was also completely blocked in the presence of the AMPA/kainate receptor antagonist CNQX, indicating that the DOM-induced intracellular acidification was caused by DOM activation of the AMPA/kainate subtype of glutamate receptors. Different mechanisms that could be involved in DOM-induced pH(i) decrease, such as displacement of H(+) by Ca(2+) from a common intracellular binding site, DOM-induced alteration of pH(i) regulation mechanisms, and a possible acidification caused by DOM-induced increase of mitochondrial Ca(2+) uptake, were excluded. DOM-induced intracellular acidification was completely prevented by inhibitors of the plasma membrane calcium adenosine triphosphatase (ATPase) (PMCA), including orthovanadate, lanthanum extracellular pH of 8.5, and the specific PMCA inhibitor caloxin 2A1. Our results therefore indicate that PMCA is involved in DOM-induced intracellular acidification in primary cultures of CGC. Simultaneous recording of [Ca(2+)](c) and pH(i) indicates that the increase in intracellular calcium evoked by DOM will activate the calcium extrusion mechanisms through the calcium pump, which, in turn, will decrease intracellular pH by countertransport of H(+) ions.

Nitric oxide negatively regulates proliferation and promotes neuronal differentiation through N-Myc downregulation.

Nitric oxide (NO) has been found to act as an important negative regulator of cell proliferation in several systems. We report here that NO negatively regulates proliferation of neuronal cell precursors and promotes their differentiation by downregulating the oncogene N-Myc. We have studied this regulatory function of NO in neuroblastoma cell lines (SK-N-BE) and in primary cerebellar granule cell cultures. In a neuronal NO synthase (nNOS) overexpressing neuroblastoma cell line exposed to the differentiative action of retinoic acid, NO slowed down proliferation and accelerated differentiation towards a neuronal phenotype. This effect was accompanied by a parallel decrease of N-Myc expression. Similar results could be obtained in parental SK-N-BE cells by providing an exogenous source of NO. Pharmacological controls demonstrated that NO's regulatory actions on cell proliferation and N-Myc expression were mediated by cGMP as an intermediate messenger. Furthermore, NO was found to modulate the transcriptional activity of N-Myc gene promoter by acting on the E2F regulatory region, possibly through the control of Rb phosphorylation state, that we found to be negatively regulated by NO. In cerebellar granule cell cultures, NOS inhibition increased the division rate of neuronal precursors, in parallel with augmented N-Myc expression. Because a high N-Myc expression level is essential for neuroblastoma progression as well as for proliferation of neuronal precursors, its negative regulation by NO highlights a novel physiopathological function of this important messenger molecule.

FGF-2, NGF and IGF-1, but not BDNF, utilize a nitric oxide pathway to signal neurotrophic and neuroprotective effects against alcohol toxicity in cerebellar granule cell cultures

Neuronal death is a prominent neuropathological component of fetal alcohol syndrome (FAS). Identification of molecular agents and pathways that can ameliorate alcohol-induced cell loss offers possible therapeutic strategies for FAS and potential insight into its pathogenesis. This study investigated the effects of growth factors on cellular survival in alcohol-exposed cerebellar granule cell (CGC) cultures and examined the role of the nitric oxide (NO)-cGMP-PKG (cGMP-dependent protein kinase) pathway in the cell survival-promoting effects of these growth factors. Primary CGC cultures were exposed to 0 or 400 mg/dl ethanol, accompanied by either no growth factor or 30 ng/ml fibroblast growth factor-2 (FGF-2), nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF) or epidermal growth factor (EGF). Viable neurons were quantified after 1 day of exposure. Two distinct types of cell survival-promoting effects of growth factors were detectable: (1) a neurotrophic effect, in which the growth factors diminished the background death of neurons that occurred in alcohol-free cultures; and (2) a neuroprotective effect, in which the growth factors diminished alcohol-induced cell death. The various growth factors differed markedly in their patterns of cell survival promotion. While BDNF and FGF-2 exerted both a neurotrophic and a neuroprotective effect, IGF-1 had only a neurotrophic effect and did not protect against alcohol toxicity, and NGF had only a neuroprotective effect and did not diminish background cell death. EGF had neither a neurotrophic nor a neuroprotective effect. In order to determine the role of the NO-cGMP-PKG pathway in the cell survival-promoting effects mediated by growth factors, cultures were exposed to one of several pharmacological inhibitors of the pathway, including NAME, LY83583 and PKG inhibitor. The cell survival-promoting effects of FGF-2, NGF and IGF-1 were all substantially reduced by each of the pathway inhibitors. In contrast, neither the neurotrophic nor the neuroprotective effects of BDNF were altered by any of the pathway inhibitors. Thus, growth factors differ in their patterns of neurotrophic and neuroprotective effects, and they differ in their reliance on the NO-cGMP-PKG pathway. While FGF-2, NGF and IGF-1 all signal their survival-promoting effects through the NO-cGMP-PKG pathway, BDNF does not rely upon this pathway for signal transduction in CGC cultures.

Relationship between Availability of NMDA Receptor Subunits and Their Expression at the Synapse

The effect of increasing the expression of NMDA subunits in cerebellar granule cells (CGCs) by transfection was studied to determine how the availability of various NMDA subunits controls both the total pool of functional receptors and the synaptic pool. Overexpression of either NR2A or NR2B, but not splice variants of NR1, by transfection caused a significant increase in the total number of functional NMDA receptors and in surface NR1 subunit cluster density in CGCs in primary culture. These data solidify the central role of NR2 subunit availability in determining the number of cell surface receptors. Overexpression of either NR2A or NR2B significantly altered the deactivation kinetics of NMDA-mediated miniature EPSCs (NMDA-mEPSCs). However, there was no significant effect of NR2 subunit overexpression on the mEPSC amplitude or single-channel conductance. NR2 subunit overexpression did not change the rate of block by MK-801 of NMDA-mediated currents in excised patches from CGCs, indicating that subunit composition does not regulate peak open probability of the channel in CGCs. With the overexpression of a mutant of NR2B lacking the PDZ binding domain, there was an increase in the total number of NMDA receptors without a change in mEPSC kinetics. Therefore, the entry of NMDA receptors into the synapse requires a PDZ binding domain and is limited by means other than receptor subunit availability.

Neuronal Expression of Synaptotagmin-Related Gene 1 Is Regulated by Thyroid Hormone during Cerebellar Development

Thyroid hormone (TH) is essential for proper brain development, acting through nuclear receptors that modulate the expression of specific genes in response to hormone binding. In a screen for genes regulated by TH in the rat cerebellum, we recently identified a novel gene, synaptotagmin-related gene 1 (Srg1). The Srg1 protein is structurally similar to synaptotagmins, a family of proteins involved in regulating neurotransmission. To elucidate a potential role of Srg1 in brain development, we have investigated the developmental and TH-regulated expression of Srg1 in the neonatal rat brain. We show that expression of both Srg1 RNA and protein is detected only in the brain and specifically in neurons. Srg1 mRNA and protein levels increase postnatally, nearing adult levels after the third postnatal week. Neonatal TH deficiency results in a significant reduction and delay in expression of both Srg1 RNA and protein. Using immunohistochemistry, we were able to detect Srg1 protein in numerous brain regions. In the cerebellum, Srg1 protein is localized to the molecular layer, indicating that it is highly expressed in granule cell axons. To further examine Srg1 expression in cerebellar granule cells (CGCs), we used an in vitro cell culture model. In primary cultures of CGCs, Srg1 expression is significantly reduced in the absence of TH. Srg1 mRNA is rapidly upregulated in cultured CGCs, suggesting a direct response to TH. Neuronal and TH-regulated expression of Srg1, together with its localization to neurites, implicates Srg1 as an important component of the program of gene expression induced by TH in the developing brain.

Do nuclear condensation or fragmentation and DNA fragmentation reflect the mode of neuronal death?

It is generally believed that nuclear condensation and fragmentation as well as DNA fragmentation reflect the events related to the neuronal apoptosis. Our report demonstrates that severe oxygen-glucose deprivation (OGD) induced condensation and fragmentation of nuclear chromatin of neurones in primary cultures of cerebellar granule cells without intemucleosomal DNA fragmentation. DNA fragmentation detected by TUNEL assay was seen only after mild OGD or after addition of colchicine but not after severe OGD. Thus, at least in primary cerebellar granule cell cultures, the chromatin condensation and fragmentation cannot be considered as a hallmark of apoptosis but rather reflect the neuronal death despite of its form.

D1 dopamine receptor activity of anti-parkinsonian drugs.

Clinical and preclinical investigations suggest that stimulation of D1 dopamine receptors may be responsible for dyskinesias induced by dopamine agonist treatment of Parkinson's Disease (PD), and that these dyskinesias may be decreased by treatment with a D1 antagonist (clozapine). Therefore, the effects of dopamine agonists and antagonists have been investigated in a primary cerebellar granule cell model of cAMP formation that seems to be highly responsive to the D1 receptors. SKF 38393, lisuride, apomorphine, pergolide, dopamine, bromocriptine and 7-OH-DPAT showed concentration-dependent increases in cAMP formation, with EC50s (in microM) of 0.013, 0.053, 0.25, 1.04, 2.18, 50.9 and 54.4, respectively. SKF 38393, apomorphine, dopamine and pergolide had similar intrinsic activity (100%), while the intrinsic activities of 7-OH-DPAT, bromocriptine and lisuride were 28.0%, 20.7% and 17.2%, respectively. SCH 23390, a selective D1 dopamine receptor antagonist, blocked an increase in cAMP formation produced by EC50 concentrations of all of the dopamine agonists investigated in this study. Clozapine concentration-dependently blocked pergolide-induced increases in cAMP and was approximately 1700-fold less potent than SCH 23390 (IC50: 0.97 microM and 0.56 nM, respectively). U-95666A (1-1000 microM), selective for the D2 receptors, showed no significant effect on cAMP, while pramipexole (0.1-100 microM), a D3 preferring agonist, did not elevate cAMP. These data suggest that primary cerebellar granule cell cultures are an excellent model for measuring D1 dopamine receptor-mediated changes in cellular cAMP. The results are discussed with reference to the relationship between the D1 receptor-stimulated increase in cAMP formation and the induction of dyskinesia in humans by these anti-parkinsonian drugs.

D 2 and D 1 dopaminergic activity of 7-OH-DPAT

The D 1 and D 2 dopamine receptor agonist properties of 7-hydroxy-2-( N,N-di- n-propylamino) tetraline (7-OH-DPAT) was determined by investigating the effect of this compound on rat striatal acetylcholine (ACh) concentration and increase in cAMP formation in primary cerebellar granule cell cultures. 7-OH-DPAT at low doses (0.01 to 0.1 μmol/kg) had no significant effect, and at high doses (0.3 to 30 μmol/kg) significantly ( P < 0.01) increased striatal ACh levels. Likewise, quinpirole was found to significantly elevate ACh content. Pretreatment with haloperidol, a non-selective antagonist of the D2 family of receptors, significantly ( P < 0.01) blocked 7-OH-DPAT- and quinpirole-induced increases in ACh. U-99194A, a D 3 selective dopamine antagonist, had no significant effect on 7-OH-DPAT-induced increases in striatal ACh. However, raclopride, a D 2 selective dopamine antagonist, completely blocked 7-OH-DPAT-induced elevations in ACh. 7-OH-DPAT in the μmolar range increased cAMP formation in granule cell cultures, and this effect was antagonized by SCH 23390, a D 1 selective dopamine antagonist. The neurochemical study indicates that, at high doses, 7-OH-DPAT has both D 1 and D 2 agonist activities.

U-19451A: A selective inducible nitric oxide synthase inhibitor

Drugs with high selectivity for iNOS inhibition may be useful for treatment of neurodegenerative disorders, chronic inflammatory diseases, and septic shock. Therefore, U-19451A (2-benzyl-2-thio-pseudourea hydrochloride), a potential NOS inhibitor, has been investigated for its selectivity for iNOS using tissues, primary cerebellar granule cell cultures and glial cell cultures. Lungs isolated from rats treated with intravenous injection of E coli lipopolysaccharide and glial cell cultures treated with the same bacterial toxin plus γ-interferon were used for iNOS activity. Rat cerebellum and primary cerebellar granule cell cultures were utilized for neuronal NOS (nNOS) activity. S-methylthiourea (SMT) and L-nitroarginine methyl ester (L-NAME), selective iNOS and nNOS inhibitors, respectively, were chosen as standards. Both U-19451A and SMT were 4-times more selective for iNOS as compared to nNOS in tissues. U-19451A was more selective than SMT for iNOS inhibition using cultures. L-NAME was 16–31 times more selective for inhibiting nNOS activity. Based on the selectivity of U-19451A for iNOS inhibition, this drug would be expected to be effective in the treatment of diseases with inflammatory pathology without producing side effects associated with nNOS inhibition.

5-( N-ethyl-N-isopropyl)amiloride and mild acidosis protect cultured cerebellar granule cells against glutamate-induced delayed neuronal death

In the experiments on the primary cerebellar granule cell cultures, delayed neuronal death was induced by 15 min treatment of the cells with 50 μ M glutamate. 5-( N-ethyl-N-isopropyl)amiloride (10 μ M) known as a potent inhibitor of the Na +/H + exchanger, when added to the glutamate-containing Mg 2+-free solution caused a considerable (approximately by 40%) decrease in the number of dead cells counted 4 h after the termination of glutamate treatment. Patch-clamp experiments with freshly isolated rat hippocampal neurons have shown that the neuroprotective effect of 5-( N-ethyl-N-isopropyl)amiloride can be explained by its ability to block N-methyl- d-asparate channels (receptors) at micromolar concentrations. A similar mechanism apparently underlies neuroprotective effect of external acidosis (reduction of pH from 7.6–7.8 to 6.7–6.8) during glutamate application. 5-( N-ethyl-N-isopropyl)amiloride (10 μM) and low pH (6.7) also proved capable of exhibiting neuroprotective effects upon application during the post-glutamate period. In this instance, however, the number of dead cells was decreased by no more than 20%. This neuroprotective effect of 5-( N-ethyl-N-isopropyl)amiloride and low pH is interpreted as resulting from inhibition of Na +/H + exchange, since a direct blockade of N-methyl- d-aspartate receptors by 1 mM dl-2-amino-5-phosphonovalerate after termination of glutamate treatment did not attenuate the delayed neuronal death. Finally, we have established that the addition of 10 μ M 5-( N-ethyl-N-isopropyl)amiloride to the cultures both during glutamate treatment and after its termination results in a complete protection of cultured cerebellar granule cells.

Paradoxical potentiation by low extracellular Ca2+ of acute chemical anoxic neuronal injury in cerebellar granule cell culture

Acute chemical anoxic injury was produced in primary cerebellar granule cell cultures incubated with iodoacetate (IAA) alone or IAA combined with potassium cyanide (KCN). Cytotoxicity was assessed using Trypan blue exclusion or LDH release. Four millimolars of KCN induced approx 30% neuron death at 3 h, whereas greater than 50% cell death was produced by 0.2 mM IAA. No potentiation of cytotoxicity was observed by IAA + KCN. A total of 0.2 mM IAA produced an early major reduction of intracellular ATP prior to the onset of neuron injury or reduction in intracellular glutathione (GSH). Medium Na+ replacement by choline, K+, or methylglucamine protected against IAA-induced neuronal injury, reduced the rate of decline of intracellular ATP but had no effect on intracellular GSH. Some 80% neuronal survival was obtained when Na+ was deleted from the medium even after the intracellular ATP had been reduced to less than 10% of control. Removal of Ca2+ from the medium had no effect on control culture, Trypan blue exclusion, GSH, or ATP, but potentiated the onset and magnitude of IAA-induced cytotoxicity. ATP and GSH decline. Loading of granule cells with the Ca2+ chelator Fura-2 did not influence IAA-induced cytotoxicity in control or low Ca2+ media. Addition of 50 microM glutamate had a minimal cytotoxic effect over 3 h and the combined addition of 0.2 mM IAA plus 50 microM glutamate did not potentiate IAA-induced injury. The glutamate receptor antagonists, D-2-amino-5-phosphonovaleric acid (APV) or kynurenate did not block IAA-induced injury in control medium but inhibited the potentiation of toxicity seen in the low Ca2+ medium. This study suggests the use of IAA as a chemical anoxic agent in cerebellar granule cell culture. The early, dose-dependent decline in ATP may be dissociated from GSH change. Acute IAA-induced injury is Na+/Cl- dependent but paradoxically potentiated in low Ca2+ medium. The low Ca2+ potentiated component was sensitive to glutamate/NMDA receptor antagonists and associated with reduction of intracellular GSH.

Inhibition of Na +/Ca 2+ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures

Experiments have been carried out on the primary cerebellar granule cell cultures from 7- to 8-day-old Wistar rats. To study a possible contribution of Na +/Ca 2+ exchange to the toxic effect of glutamate, two amiloride derivatives, 3′,4′-dichlorobenzamil (DCB) and 5-( N-4-chlorobenzyl)-2′,4′-dimethylbenzamil (CBDMB), known to be the potent inhibitors of this exchange system, were used. Addition of DCB or CBDMB (at 30 and 10 μM, respectively) to a 25 μM glutamate solution dramatically enhanced the delayed neuronal death observed during the 4 h after termination of glutamate treatment. Similar but insignificantly smaller effects were obtained when these agents were added to the cultures in the post-glutamate period. Removal of Na + (by substituting for choline chloride) from the external Mg 2+ - free solution in the post-glutamate period also enhanced a delayed neuronal damage. The data obtained suggest that Na +/Ca 2+ exchanger does not constitute the route for Ca 2+ entry during the post-glutamate period but, on the contrary, attenuates glutamate neurotoxicity providing Ca 2+ extrusion from the cells under the conditions of a sustained Ca 2+ influx.