Cultured Rat Cortical Cells Research Articles

The presence of calbindin in rat cortical neurons protects in vitro from oxydative stress

Free radicals are highly reactive chemicals containing an unpaired electron and are normally produced by the cellular metabolism. The oxydative stress is defined as a lack of balance between the production of free radicals and the activity of antioxydant metabolites. It induces cellular damages to lipids, proteins and membranes. Abnormal calcium metabolism can be a consequence of oxydative stress leading to increased intracellular concentrations. Calbindin D 28K is a calcium binding protein which could have a neuroprotective action against various cellular insults. In this study rat cortical cell cultures were exposed during various times and at different concentrations to the couple Xanthine/Xanthine oxydase (XA/XO), which produces the superoxyde radical O 2 −. Neuronal survival revealed that XA/XO is toxic for cortical cell cultures. The Calbindin D 28K immunocytochemical study shows that the percentages of Calbindin positive cells are greater in surviving neurons following the XA/XO exposure compared to controls. There is a time-dependent and a dose-dependent relation between the number of surviving neurons and the percentage of Calbindin positive neurons. These results suggest that the presence of cytosolic neuronal Calbindin D 28k is associated with a greater resistance to oxydative stress.

Phosphorylated neurofilament expression and resistance to kainate toxicity.

Antibodies directed against phosphorylated neurofilaments, which are major proteins of the neuronal cytoskeleton, usually do not label neuronal cell bodies except in some neurological diseases. In the present study, we show that in rat cortical cell cultures exposed to kainate there is an inverse relation between neuronal survival and the proportion of neuronal cell bodies stained by a monoclonal antibody (clone SMI31) that recognizes extensively phosphorylated neurofilament proteins (150 kDa and 200 kDa). The immunoblot analysis also revealed an increase in 150-kDa phosphorylated neurofilament expression in kainate-treated cell cultures. Furthermore, the direct quantification of viable neurons SMI31-immunopositive or immunonegative in perikarya showed that the majority of neurons resistant to kainate toxicity expressed phosphorylated neurofilaments in their cell bodies. The percentage of viable neurons displaying SMI31-immunoreactivity in their cell bodies increased from 14.7% in control cultures to 30.0% in cultures treated with 10 microM kainate. These data suggest that phosphorylated neurofilament expression is associated with a reduced cell vulnerability to excitotoxicity induced by kainate.

Energetics of drug efficacy at G protein-coupled receptors

Acori graminei Rhizoma (AGR) is shown to exhibit a number of pharmacological actions including sedation and anticonvulsive action. To further characterize its actions in the CNS, the present study evaluated the effects of essential oils (EO) from AGR on the excitotoxic neuronal cell death induced in primary rat cortical cell cultures. EO inhibited the glutamate-induced excitotoxicity in a concentration-dependent manner, with the IC50 of 0.241mg/ml. EO exerted more potent neuroprotection against the toxicity induced by NMDA (IC50 = 0.139 mg/ml). In contrast, the AMPA-induced toxicity was not inhibited by EO. Receptor-ligand binding studies were performed to investigate the neuroprotective action mechanism. EO dramatically inhibited the specific bindings of a use-dependent NMDA receptor-ion channel blocker [3H]MK-801, indicating an NMDA receptor antagonist-like action. However, the bindings of [3H]MDL 105,519, a ligand selective for the glycine binding site of NMDA receptor, were not considerably inhibited. These results demonstrated that EO extracted from AGR exhibited neuroprotective effects on cultured cortical neurons through the blockade of NMDA receptor activity, and that the glycine binding site appeared not to be the major site of action.

Inhibition of l-carnitine uptake into primary rat cortical cell cultures by gaba and gaba uptake blockers

l-carnitine plays a central role in mitochondrial function and is found to be differentially distributed in the brain. We have shown before that the uptake of l-carnitine into cultured rat cortical neurones was temperature-dependent, as well as potently inhibited by factors affecting the sodium gradient as well as by molecules resembling its structure, e.g. d-carnitine, acetyl- l-carnitine and γ-aminobutyric acid (GABA). GABA was the most potent inhibitor of l-carnitine uptake. In the present study we have found that specific GABA uptake blockers, nipecotic acid, cis-4-hydroxynipecotic (HNA), guvacine, 2,4-diaminobutyric acid (DABA) and NO 711 inhibit l-carnitine uptake even more potently than GABA. However, apart from NO 711, they caused about the same maximal inhibition, 67.4% at 50 μM for guvacine, compared to 60.5% by GABA. NO 711 was extremely potent and blocked 80.5% of the l-carnitine uptake. In contrast, the GABA A receptor agonists, isonipecotic acid and isoguvacine, or the antagonist bicuculline, at similar concentrations (50 μM) did not significantly inhibit the uptake of the l-carnitine. However, bicuculline at relatively high concentration (500 μM) was inhibitory (38%). The GABA B receptor agonist, baclofen, or antagonist, phaclofen, were ineffective, although 5-aminovaleric acid did significantly inhibit uptake at both 50 and 500 μM) causing 22 and 48% inhibition respectively. Like bicuculline, it was not as effective as GABA or the specific GABA uptake blockers. The results indicate that the uptake of l-carnitine by rat cortical neurones occurs in part by a process that can be potently inhibited by GABA and GABA uptake blockers.

Ethanol and glucose-deprivation neurotoxicity in cortical cell cultures

The toxic effects of ethanol on rat cortical cell cultures were compared with neuronal damage induced by glucose deprivation. Exposure to decreased glucose concentrations produced dose-dependent neuronal injury, as indicated by the release of lactate dehydrogenase (LDH) into the culture medium. Complete glucose deprivation resulted in mean LDH release that was more than 60% greater than that from sister cultures incubated in the presence of 5.5 mmol/L glucose. Exposure to ethanol (25, 50, or 100 mmol/L) similarly resulted in dose-related LDH release. The degree of injury resulting from complete glucose deprivation or 100 mmol/L ethanol approximated that produced by exposure to 100 mmol/L glutamic acid. Ethanol did not significantly alter LDH release from cultures consisting of only astrocytes. Both effects were inhibited by the N-methyl- d-aspartate (NMDA) receptor antagonist, d,l-2-amino-5-phosphonovaleric acid (APV). Glutamate levels were increased in the culture medium to 191% ± 8% of the control value after glucose deprivation ( P < .001) and to 186% ± 16% after exposure to 100 mmol/L ethanol ( P < .01). 3H-glutamate uptake by cultured astrocytes was reduced by glucose deprivation and by ethanol. This range of ethanol concentrations has previously been shown to inhibit hexose uptake by cultured astrocytes. The present results suggest that decreased glucose uptake by astrocytes in the presence of ethanol impairs their uptake of glutamate, which contributes to excitotoxic neuronal injury.

Stimulation of GABAergic neuron differentiation by NT-4/5 in cultures of rat cerebral cortex

Primary cultures of fetal rat cortical cells were used to investigate trophic effects of neurotrophin-4/5 (NT-4/5) on GABAergic neurons. Chronic administration of NT-4/5 resulted in a significant increase in high-affinity GABA uptake and in a stronger immunohistochemical staining pattern and bigger cell size of neurons visualized by GABA immunohistochemistry. Brain-derived neurotrophic factor (BDNF) exerted similar actions. These effects were observed in cultures prepared from rat fetuses from embryonic day (ED) 15 and ED18. The total number of cells was marginally increased in ED15 but not ED18 cultures. NT-4/5 did not elevate uptake of excitatory amino acids. Administration of NT-4/5 and BDNF resulted in higher levels of the calcium binding protein, calbindin, in these cultures. Our findings suggest that subpopulations of cortical GABAergic and calbindin-expressing neurons respond to NT-4/5 during development.

Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl- d-aspartate neurotoxicity

We examined the role of nitric oxide in N-methyl- d-aspartate (NMDA) receptor-mediated neurotoxicity in rat and mouse primary cortical cell cultures. In rat and mouse cultures, the NO synthase inhibitor, N G-Nitro- l-arginine, blocked cGMP formation but not neuronal cell death following a 5–10 min exposure to 300–500 μM NMDA. N G-Monomethyl- l-arginine was also unable to prevent neuronal death. In contrast, the non-competitive NMDA receptor antagonist, dextrophan, prevented both cGMP formation and cell death. While other data suggest that the synthesis of nitric oxide can mediate NMDA receptor-mediated neurotoxicity, present results suggest that such synthesis is not necessarily required.

Cytoprotective effect of NMDA receptor antagonists on prion protein (Prion Sc)-induced toxicity in rat cortical cell cultures

Rat cortical cells were incubated with the Scrapie prion protein, Prion Sc. At concentrations of 3 ng/ml of Prion Sc and higher, the viability of the cells decreased significantly after a 12-h incubation period. Simultaneously, the degree of DNA fragmentation increased. In control experiments with antibodies against Prion Sc, Prion Sc lost its deleterious effect on neurons. Prion Sc did not affect the viability of astrocytes. Drugs known to block NMDA receptor channels, such as memantine (1-amino-3,5-dimethyl-adamantane) (Mem), its analogue 1-N-methylamino-3,5-dimethyl-adamantane as well as (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) prevented the effect of Prion Sc. Production of Prion Sc in the Scrapie prion-infected subclone of N 2a cells (ScN 2a cells) was not affected by memantine. We conclude that antagonists of the NMDA receptor-channel complex (i) abolish the Prion Sc-induced neuronal injury in vitro and/or the processing of Prion Sc.

HIV-1 Tat alters normal organization of neurons and astrocytes in primary rodent brain cell cultures: RGD sequence dependence.

The HIV-1 trans-activator protein Tat has been implicated as a mediator of neuronal dysfunction in several model systems. To explore the possibility that Tat can affect primary brain cells, we examined the effect of recombinant Tat protein on rat cortical brain cell cultures. Tat induced marked aggregation of neurons and astrocytes in developing cultures and caused the neuritic processes to coalesce into fascicles. Cell death was not seen and brain macrophages were not affected. These effects mapped to a different region from the trans-activation domain of Tat, as mutating the RGD (arginine-glycine-aspartic acid) sequence within the second exon abrogated aggregation and fascicle formation without affecting trans-activation capacity. Such effects on primary neurons and astrocytes may reflect specific interactions of Tat with uninfected cells within the CNS in vivo.

31 gp120 of HIV-1 induces apoptosis in rat cortical cell cultures: Prevention by memantine: W.E.G. Müller °, H.C. Schröder ° and H. Ushijima +° Institut für Physiologische Chemie, Abteilung “Angewandte Molekularbiologie”, Universität, Duesbergweg 6, D-6500 Mainz, Germany and + Division of AIDS Virus, AIDS Research Center, National Institute of Health, Gakuen 4-7-1, Tokyo 208, Japan

31 gp120 of HIV-1 induces apoptosis in rat cortical cell cultures: Prevention by memantine: W.E.G. Müller °, H.C. Schröder ° and H. Ushijima +° Institut für Physiologische Chemie, Abteilung “Angewandte Molekularbiologie”, Universität, Duesbergweg 6, D-6500 Mainz, Germany and + Division of AIDS Virus, AIDS Research Center, National Institute of Health, Gakuen 4-7-1, Tokyo 208, Japan

Effects of free fatty acids on somatostatin secretion, content and mRNA levels in cortical and hypothalamic fetal rat neurones in monolayer culture.

The effects of free fatty acids on somatostatin secretion, content and mRNA levels in fetal rat hypothalamic and cortical cell cultures were investigated. Somatostatin secretion and content were quantified by radioimmunoassay. Somatostatin mRNA levels were measured by Northern blot hybridization using a cRNA probe. Treatment with either caprylic acid (5 x 10(-3) M) or oleic acid (5 x 10(-5) M) for 90 min inhibited basal somatostatin secretion in both hypothalamic and cortical cell cultures. In addition, the increase in somatostatin secretion induced by incubation with veratridine (10(-4) M) or carbachol (10(-4) M) for 90 min was significantly reduced by the addition of caprylic acid, but somatostatin release stimulated by 5.6 x 10(-2) M KCl was not affected. Furthermore, treatment with these free fatty acids for 90 min markedly decreased somatostatin mRNA levels in both types of neurone culture. These inhibitory effects were transient, being observed after 90 min, but not after 5 h. These results support the probability that there is a role for free fatty acids in the regulation of somatostatin mRNA levels and somatostatin secretion in both hypothalamic and cortical cell cultures.

Effect of varying levels of glucose on oxygen deprivation-induced delayed neuronal cell death in primary cerebrocortical cultures

Calcium accumulation and neuronal injury of rat cortical cell cultures in vitro were examined following oxygen deprivation under conditions of normal and low glucose. 45Ca 2+ uptake and lactate dehydrogense (LDH) release, measured at 12 and 24 h after oxygen deprivation, were significantly elevated in cultures exposed to combined oxygen deprivation and low glucose (1.7 or 0.6 mM). Although those cultures deprived of oxygen combined with no glucose displayed delayed increases in 45Ca 2+ influx, no significant elevation in LDH release at 24 h was observed.

Nitric oxide synthase induction in glial cells: Effect on neuronal survival

In primary rat cortical glial cell cultures lipopolysaccharide (LPS) induced a dose- and time-dependent increase of intracellular cyclic GMP concentration associated with a release of nitrite. The LPS-induced cyclic GMP and nitrite increase was enhanced by interferon-γ and was prevented by L-N G- nitroarginine, dexamethasone and cycloheximide. Thus indicates that LPS effect occured via the production of nitric oxide (NO) and involved new protein synthesis suggesting the induction of NO syntahse in these cells. Furthermore this induction was Ca 2+-independent and was blocked by an inhibitor of the synthesis of tetrahydrobiopterin. The inducible NO synthase was also expressed by C6 glioma cells. In primary mixed cultures containing both neuronal and glial cells, the effects of LPS were less important than in primary glial cell cultures suggesting that glial cells rather than neurons expressed the inducible form of NO synthase. On the other hand no change on neuronal viability was observed after NO synthase induction by LPS in this culture type. This study indicates that glial cells are able to induce NO synthase without affecting neuronal survival.

Gp120 of HIV-1 induces apoptosis in rat cortical cell cultures: prevention by memantine

After incubation of rat cortical cell cultures with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 for 12 h, cells showed fragmentation of DNA at internucleosomal linkers, the characteristic feature of apoptosis. In a quantitative approach, it was determined that the percentage of DNA fragmentation increased from 7%, in the absence of gp120, to 62% following incubation with 24 ng/ml of gp120. Simultaneously, the percentage of viable cells decreased from 94% to 33%. Memantine (1-amino-3,5-dimethyladamantane), a drug currently used in the therapy of spacticity and Parkinson's diseases as well as the NMDA antagonist MK-801 both prevented the effects of gp120 at micromolar concentrations. In human cultured astrocytes, gp120 was ineffective with respect to DNA fragmentation and cell toxicity. From these data, we conclude that the gp120-induced apoptosis may contribute to the neurological complications frequently associated with the immunodeficiency syndrome. The cytoprotective effect of memantine in cortical cell cultures may qualify the drug for the treatment of AIDS-related dementia.

Extracellular pH modulates N-methyl- d-aspartate receptor-mediated neurotoxicity and calcium accumulation in rat cortical cultures

The effect of extracellular pH (pHo) on the excitotoxicity of N-methyl- d-aspartate (NMDA) in cultured rat cortical cells was studied. Treatment of cells with 500 μM NMDA for 15 min at various pH's in a range from 6.5 to 8.0 progressively enhanced staining with Trypan blue and release of lactate dehydrogenase with increased pH after 18 h of culture following treatment. The cytotoxic effect of high concentration of K + (40 mM) or veratridine (10 μM) was also directly related to the increase in pHo. Free calcium accumulation in cells on addition of NMDA increased parallel to pHo. Changes in intracellular pH were estimated to be minor compared with extracellular changes. Specific NMDA antagonists could block both the NMDA- and membrane depolarization-induced neurotoxicity and calcium accumulation completely. These result suggest that the proton concentration outside of cells attenuates NMDA-induced neurotoxicity by blocking calcium accumulation.

Quinolinic acid stimulates somatostatin gene expression in cultured rat cortical neurons.

Striatal atrophy in Huntington's disease (HD) is characterized by selective preservation of a subclass of neurons colocalizing NADPH-diaphorase (NADPH-d), somatostatin (SS), and neuropeptide Y (NPY), which have been reported to show three- to fivefold increases in SS-like immunoreactivity (SSLI) and NPY content. Since HD brain is capable of producing excessive quantities of the excitotoxin quinolinic acid (Quin), an N-methyl-D-aspartate (NMDA) receptor agonist, and since experimental Quin lesions show neuronal loss with sparing of NADPH-d/SS/NPY neurons, it has been suggested that Quin may be important in the pathogenesis of HD. In the present study we determined whether Quin stimulates SS gene function in cultured cortical cells known to be rich in NADPH-d/SS/NPY neurons. Cultures of dispersed fetal rat cortical cells were exposed to Quin (1 and 10 mM) with or without (-)-2-amino-5-phosphonovaleric acid (APV; 0.5 mM), an NMDA receptor antagonist, NMDA (0.2 and 0.5 mM), and glutamate (Glu; 0.5 mM). Medium and cellular SSLI was determined by radioimmunoassay and SS mRNA by Northern analysis with a cRNA probe. Quin induced significant (p less than 0.01) 1.6- and 2.5-4 fold increases in SSLI and SS mRNA accumulation, respectively, which were abolished by APV. Release of SSLI into the culture medium was stimulated two- to fivefold by Quin over a 2- to 20-h period. The increase in SS mRNA produced by Quin was time and dose dependent. A similar dose-dependent increase in SS mRNA comparable with that observed with Quin was induced by NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)

5,7-Dichlorokynurenic acid, a potent and selective competitive antagonist of the glycine site on NMDA receptors

Fourteen substituted derivatives of kynurenic acid were compared for their ability to block ionic currents evoked by N- methyl- d-aspartate (NMDA) plus glycine, or kainate, in voltage-clamped Xenopus oocytes injected with rat brain messenger RNA. Among these analogues there was an excellent correlation between the K i for displacing [ 3H]glycine binding to rat brain membranes, and the ability to inhibit ionic currents evoked by glycine/NMDA in Xenopus oocytes. In the oocyte 5,7-dichlorokynurenic acid (5,7-DCK) was a competitive blocker of the glycine recognition site on NMDA receptors, and was more potent ( K B 65 nM in Schild analysis) and selective (509-fold more potent vs glycine than kainate) than the prototype glycine antagonist, 7-chlorokynurenic acid. 5,7-DCK also reduced NMDA-induced neuron injury in rat cortical cell cultures.

The effect of NMDA receptor glycine site antagonists on hypoxia-induced neurodegeneration of rat cortical cell cultures

The neuroprotective potential of an antagonist (7-chlorokynurenic acid (7-CIKYNA)) and a low efficacy partial agonist (HA-966) for the glycine modulatory site on the N-methyl- d-aspartate (NMDA) receptor complex has been examined using a neuronal cell culture/hypoxia model of neurodegeneration. Their effects were compared to those of the potent uncompetitive NMDA antagonist, MK-801. Hypoxic cell injury was assessed visually and quantified by measuring the appearance of two cytosolic enzymes, lactate dehydrogenase (LDH) and neurone specific enolase (NSE), in the culture medium. MK-801 prevented the hypoxia-induced cell mortality in a concentration-related manner with an IC 50 of 15 nM against increases in LDH levels. HA-966 and 7-CIKYNA also produced concentration-related protective effects with IC 50s of 175 and 18 μM, respectively. Although both glycine antagonists were considerably weaker than MK-801 their maximum neuroprotective effects were comparable to that produced by MK-801, i.e. complete protection. This indicates that the level of NMDA receptor activation which can take place in the presence of the partial agonist HA-966 is insufficient to cause permanent neuronal damage. Concentration-effect curves were similar when NSE was used as the marker enzyme, supporting previous observations that the increases in LDH levels accurately and specifically reflect neuronal cell death. These results provide further evidence that hypoxia-induced injury to cortical neuronal cultures is mediated by an excessive stimulation of NMDA receptors and that glycine-site antagonists and partial agonists may have therapeutic potential in conditions where pathologically high levels of NMDA receptor activation are thought to occur.

Studies of structure-function relationship of human parathyroid hormone using rat and human renal cortical cells in culture.

The structure-function relationship of human PTH was studied by testing the effects of its synthetic analogues on cAMP production in cultures of rat and human renal cortical cells. The human cell system was by two orders more sensitive to an agonist, hPTH1-34, than the rat cell system. In both culture systems, Tyr34hPTH3-34NH2, which by itself had no influence on adenylate cyclase, caused approximately 90% inhibition of the hPTH 1-34-induced cAMP production at a molar ratio of 1000:1. On the other hand, Tyr34hPTH5-34NH2, Tyr34hPTH7-34NH2, Tyr34hPTH8-34NH2 and Tyr34hPTH9-34NH2 had weak but distinct agonist activity. When these four analogues were tested in the presence of hPTH1-34, they acted as antagonists in the human cell system, whereas they still showed agonist activity in the rat cell system which was additive to that of hPTH1-34. These data indicate that the amino acids at positions 1 and 2 of hPTH are important for its biological activity and that those in the sequence 9-34 also participate in the activation of adenylate cyclase. The higher sensitivity to hPTH1-34 and the different response to some of the analogues of human renal cortical cells compared with rat renal cortical cells emphasize the importance of employing human cell systems in evaluating the structure-function relationship of hPTH.