bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevation of Basal Protein Kinase C Activity Increases Ethanol Sensitivity of GABAA Receptors in Rat Hippocampal CA1 Pyramidal Neurons
Authors: Weiner J. L. , Valenzuela C. F. , Watson P. L. , Frazier C. J. , Dunwiddie T. V.
Published: July 2010

Abstract: The ability of ethanol to enhance GABAA receptor function remains controversial; conflicting observations have been made even in the same brain region, and when using apparently similar methodologies. In this study we characterized a single protocol variable, the initial incubation temperature of brain slices, that had dramatic effects on the ethanol sensitivity of GABAA inhibitory postsynaptic currents (IPSCs) recorded from rat hippocampal CA1 pyramidal neurons. Incubation of hippocampal slices at relatively low temperatures (11–15°C) immediately after slice preparation significantly affected a number of physiological and biochemical parameters. Such slices showed a decrease in extracellular inhibitory postsynaptic potential amplitude, a significant increase in the ethanol sensitivity of GABAA IPSCs in CA1 pyramidal neurons, no change in pentobarbital or flunitrazepam potentiation of IPSCs, and an increase in basal protein kinase C (PKC) activity relative to slices incubated at 31–33°C. In addition, the increase in ethanol sensitivity of GABAA IPSCs was blocked by chelerythrine, a selective inhibitor of PKC. These results suggest that differences in hippocampal slice incubation protocols may have contributed to the disparate results of previous investigations of ethanol modulation of GABAA receptor‐mediated synaptic transmission in the rat hippocampus. In addition, these findings provide further evidence that PKC activity positively modulates the interaction between ethanol and GABAA receptors in the mammalian brain.
Modeling of the Interaction of Na+ and K+ with the Binding of the Cocaine Analogue 3β‐(4‐[125I]Iodophenyl)tropane‐2β‐Carboxylic Acid Isopropyl Ester to the Dopamine Transporter
Authors: Chen Nian‐Hang , Ding Jian‐Hua , Wang You‐Lin , Reith Maarten E. A.
Published: July 2010

Abstract: The present study examines the interaction of Na+ and K+ with the binding of the cocaine analogue 3β‐(4‐[125I]iodophenyl)tropane‐2β‐carboxylic acid isopropyl ester to dopamine transporters (DATs) in rat striatal synaptosomal membranes at 37°C. The binding increases with [Na+] from 10 to 100 mM and decreases with higher [Na+]. The presence of K+ reduces the maximal stimulatory effect of Na+ and causes a nonlinear EC50 shift for Na+. K+ strongly inhibits the binding at low [Na+]. Increasing [Na+] produces a linear IC50 shift for K+. Saturation analysis indicates a single binding site changing its affinity for the radioligand depending on [K+]/[Na+] ratio in the assay buffer. A reduced Bmax was observed in the presence of 10 mM Na+ and 30 mM K+. Both high [Na+] and high [K+] accelerate the dissociation of the binding, and K+‐induced acceleration was abolished by increasing [Na+]. Least squares model fitting of equilibrium data and kinetic analysis of dissociation rates reveal competitive interactions between Na+ and K+ at two sites allosterically linked on the DAT: One site mediates the stimulatory effect of Na+, and the other site involves the radioligand binding and the inhibitory effect of cations on the binding. Various uptake blockers and substrates, dopamine in particular, display reduced potency in inhibiting the binding at a higher [K+]/[Na+] ratio.
Identification of an Endogenous 5‐Hydroxytryptamine2A Receptor in NIH‐3T3 Cells: Agonist‐Induced Down‐Regulation Involves Decreases in Receptor RNA and Number
Authors: Saucier Caroline , Albert Paul R.
Published: July 2010

Abstract: NIH‐3T3 cells, a nontransformed murine fibroblast cell line previously found to be unresponsive to 5‐hydroxytryptamine (5‐HT) when cultured in 5‐HT‐free medium, became responsive to 5‐HT, which induced an increase in intracellular calcium concentration. Pharmacological and ligand binding studies showed that NIH‐3T3 cells endogenously express a 5‐HT2A receptor that, when activated, mobilizes calcium from ionomycin‐sensitive intracellular stores via coupling to a pertussis toxin‐insensitive pathway. Using reverse transcriptase‐PCR cloning and northern blot analysis, the presence of 5‐HT2A receptor RNA with a similar nucleotide sequence (99% identity) and molecular size to that of murine brain was detected in NIH‐3T3 cells. Responsiveness of the endogenous 5‐HT2A receptor in nontransfected cells was completely desensitized after chronic treatment (half‐time = 2 h) with 1 µM 5‐HT and resensitized on removal of 5‐HT. In contrast to NIH‐3T3 cells transfected with 5‐HT2A receptor cDNA under control of a viral promoter, the long‐term agonist‐induced functional desensitization in nontransfected NIH‐3T3 cells was paralleled by a decrease in both 5‐HT2A receptor density and RNA level. These results show that NIH‐3T3 cells express an endogenous 5‐HT2A receptor that is desensitized by agonist via down‐regulation of both receptor number and mRNA. The NIH‐3T3 cells provide a novel system for understanding 5‐HT2A receptor regulation.
An Assessment of Oxidative Damage to Proteins, Lipids, and DNA in Brain from Patients with Alzheimer's Disease
Authors: Lyras Leonidas , Cairns Nigel J. , Jenner Andrew , Jenner Peter , Halliwell Barry
Published: July 2010

Abstract: Oxidative stress may contribute to neuronal loss in Alzheimer's disease (AD). The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases from seven different brain areas of AD and matched control tissues by using a range of techniques. No differences in levels of lipid peroxidation were found in any of the brain regions by using two different assay systems. Overall, there was a trend for protein carbonyl levels to be increased in AD in frontal, occipital, parietal, and temporal lobe, middle temporal gyrus, and hippocampus, but a significant difference was found only in the parietal lobe. Gas chromatography‐mass spectrometry was used to measure products of damage to all four DNA bases. Increased levels of some (8‐hydroxyadenine, 8‐hydroxyguanine, thymine glycol, Fapy‐guanine, 5‐hydroxyuracil, and Fapy‐adenine), but not all, oxidized DNA bases were observed in parietal, temporal, occipital, and frontal lobe, superior temporal gyrus, and hippocampus. The baseline level of oxidative DNA damage in the temporal lobe was higher than in other brain regions in both control and AD brain. The finding of increased oxidative damage to protein and DNA strengthens the possibility that oxidative damage may play a role in the pathogenesis of AD in at least some key brain regions.
4‐Hydroxynonenal‐Derived Advanced Lipid Peroxidation End Products Are Increased in Alzheimer's Disease
Authors: Sayre Lawrence M. , Zelasko Dawn A. , Harris Peggy L. R. , Perry George , Salomon Robert G. , Smith Mark A.
Published: July 2010

Abstract: Recent studies have demonstrated oxidative damage is one of the salient features of Alzheimer's disease (AD). In these studies, glycoxidation adduction to and direct oxidation of amino acid side chains have been demonstrated in the lesions and neurons of AD. To address whether lipid damage may also play an important pathogenic role, we raised rabbit antisera specific for the lysine‐derived pyrrole adducts formed by lipid peroxidation‐derived 4‐hydroxynonenal (HNE). These antibodies were used in immunocytochemical evaluation of brain tissue from AD and age‐matched control patients. HNE‐pyrrole immunoreactivity not only was identified in about half of all neurofibrillary tangles, but was also evident in neurons lacking neurofibrillary tangles in the AD cases. In contrast, few senile plaques were labeled, and then only the dystrophic neurites were weakly stained, whereas the amyloid‐β deposits were unlabeled. Age‐matched controls showed only background HNE‐pyrrole immunoreactivity in hippocampal or cortical neurons. In addition to providing further evidence that oxidative stress‐related protein modification is a pervasive factor in AD, the known neurotoxicity of HNE suggests that lipid peroxidation may also play a role in the neuronal death in AD that underlies cognitive deficits.
The Neuronal Calcium‐Sensor Protein VILIP Modulates Cyclic AMP Accumulation in Stably Transfected C6 Glioma Cells: Amino‐Terminal Myristoylation Determines Functional Activity
Authors: Braunewell Karl‐Heinz , Spilker Christina , Behnisch Thomas , Gundelfinger Eckart D.
Published: July 2010

Abstract: VILIP ({ulbar|vi}sinin‐{ulbar|li}ke {ulbar|p}rotein) is a member of the neuronal subfamily of EF‐hand calcium sensor proteins. Members of this family are involved in the calcium‐dependent regulation of the desensitization of signal cascades in retinal photoreceptors. To gain insight into the function of VILIP in cell signaling, we have transfected wild‐type VILIP and mutant VILIP lacking the myristoylation consensus sequence into C6 glioma cells. Expression of wild‐type VILIP did not significantly influence the desensitization of β‐adrenergic receptors, which are coupled to adenylyl cyclase in C6 cells. However, VILIP expression increased the β‐adrenergic receptor‐stimulated cyclic AMP (cAMP) level in these cells severalfold. The stimulatory effect was also observed after direct stimulation of the adenylyl cyclase with forskolin, indicating that VILIP acts downstream of receptor and G protein in the β‐adrenergic signaling pathway in C6 cells. In contrast, the nonmyristoylated mutant of VILIP reduced cellular cAMP levels in C6 cells. Myristoylated wild‐type VILIP was associated in a calcium‐dependent manner with membrane fractions during subcellular fractionation, presumably owing to a calcium‐myristoyl switch. In contrast, association of non‐myristoylated mutant VILIP with membranes was strongly reduced. Thus, myristoylation and most likely the calcium‐dependent membrane association of VILIP are important prerequisites for the activating effect of wild‐type VILIP on cAMP accumulation in C6 cells. These results suggest that VILIP acts as a calcium sensor molecule that modulates cell signaling cascades, possibly by direct or indirect regulation of adenylyl cyclase activity.
Identification of a Strand‐Specific Egr Response Element Binding Complex Enriched in Rat Brain
Authors: Taira Eiichi , Baraban Jay M.
Published: July 2010

Abstract: Multiple members of the Egr family of transcription regulatory factors are rapidly induced in response to neuronal stimulation and share a common double‐stranded DNA binding consensus sequence, referred to as the Egr response element. Recent studies have identified transcription regulatory factors that bind preferentially to short segments of single‐stranded DNA, rather than the conventional double‐stranded versions of regulatory elements. Accordingly, in the present study, we have investigated whether the Egr response element may also be regulated by trans factors that bind to single‐stranded versions of this cis element. Using gel‐shift studies, we have identified a protein complex that binds selectively to the G‐rich strand of the Egr response element. In competition studies, an RNA oligonucleotide containing the corresponding G‐rich sequence is ∼25‐fold less potent than its DNA counterpart. This DNA binding complex, referred to as GS1, is present in several regions of the rat brain with highest levels in cerebellum; negligible binding activity was detected in multiple peripheral tissues surveyed. UV cross‐linking studies revealed two major protein bands with estimated molecular masses of 36 and 30 kDa. The highly restricted tissue distribution of this complex and its sequence‐specific binding properties indicate that GS1 may be involved in regulating transcription directed by the Egr response element in brain.
Cholecystokinin‐Induced Desensitization, Receptor Phosphorylation, and Internalization in the CHP212 Neuroblastoma Cell Line
Authors: Rao Rammohan V. , Hadac Elizabeth M. , Roettger Belinda F. , Miller Laurence J.
Published: July 2010

Abstract: Agonist stimulation of cells often results in desensitization of the response, to protect the cell from overstimulation. We have previously shown that the type A cholecystokinin (CCK) receptor on the pancreatic acinar cell and on the model CHO‐CCKR cell line undergoes desensitization in response to CCK, with receptor phosphorylation and internalization playing key roles. Although these mechanisms contribute in a cell‐specific manner, no analogous information exists for the CCK receptor expressed on neuronal cells, where in vivo data demonstrate a particularly sensitive response to CCK. The present study was designed to explore CCK receptor desensitization in the CHP212 neuroblastoma cell line, focusing on inositol 1,4,5‐trisphosphate (IP3) responses to CCK and on recognized molecular and cellular mechanisms of desensitization. CCK promptly stimulated IP3 responses in these cells, with hormonal responsiveness rapidly and completely desensitized. Both receptor phosphorylation and internalization were observed to occur, with the former occurring most rapidly and likely being responsible for the earliest desensitization observed. Although the time course of receptor phosphorylation and dephosphorylation, and the groups of kinases involved in the neuroblastoma cell line, were most similar to those in the pancreatic cell, the movement of the agonist‐bound receptor in these cells was quite different from that in the pancreatic cell and most similar to that in the CHO‐CCKR cell line. This hybrid response supports the cell‐specific nature of CCK receptor regulation and provides an important system to explore the molecular determinants of these processes.
Protection of Flupirtine on β‐Amyloid‐Induced Apoptosis in Neuronal Cells In Vitro: Prevention of Amyloid‐Induced Glutathione Depletion
Authors: Müller Werner E.G. , Romero Francisco J. , Perovic Sanja , Pergande Gabriela , Pialoglou Periklis
Published: July 2010

Abstract: Effective drugs are not available to protect against β‐amyloid peptide (Aβ)‐induced neurotoxicity. Cortical neurons from rat embryos were treated with the toxic fragment Aβ25‐35 at 1 µM in the presence or absence of flupirtine, a triaminopyridine, successfully applied clinically as a nonopiate analgesic drug. Five days later 1 µM Aβ25‐35 caused reduction of cell viability to 31.1%. Preincubation of cells with flupirtine (1 or 5 µg/ml) resulted in a significant increase of the percentage of viable cells (74.6 and 65.4%, respectively). During incubation with Aβ25‐35 the neurons undergo apoptosis as determined by appearance of the characteristic stepladder‐like DNA fragmentation pattern and by the TUNEL technique. Aβ25‐35‐induced DNA fragmentation could be abolished by preincubation of the cells with 1 µg/ml flupirtine. Incubation with Aβ25‐35 reduces the intraneuronal level of GSH from 21.4 to 7.4 nmol/106 cells. This depletion could be partially prevented by preincubation of the cells with flupirtine. Thus, flupirtine may be adequate for the treatment of the neuronal loss in Alzheimer's disease (where Aβ accumulates in senile plaques) and probably other neurological diseases such as amyotrophic lateral sclerosis.
4‐Hydroxynonenal, a Lipid Peroxidation Product, Rapidly Accumulates Following Traumatic Spinal Cord Injury and Inhibits Glutamate Uptake
Authors: Springer Joe E. , Azbill Robert D. , Mark Robert J. , Begley James G. , Waeg Georg , Mattson Mark P.
Published: July 2010

Abstract: Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult. One such event is the accumulation of free radicals that damage lipids, proteins, and nucleic acids. A major reactive product formed following lipid peroxidation is the aldehyde, 4‐hydroxynonenal (HNE), which cross‐links to side chain amino acids and inhibits the function of several key metabolic enzymes. In the present study, we used immunocytochemical and immunoblotting techniques to examine the accumulation of protein‐bound HNE, and synaptosomal preparations to study the effects of spinal cord injury and HNE formation on glutamate uptake. Protein‐bound HNE increased in content in the damaged spinal cord at early times following injury (1–24 h) and was found to accumulate in myelinated fibers distant to the site of injury. Immunoblots revealed that protein‐bound HNE levels increased dramatically over the same postinjury interval. Glutamate uptake in synaptosomal preparations from injured spinal cords was decreased by 65% at 24 h following injury. Treatment of control spinal cord synaptosomes with HNE was found to decrease significantly, in a dose‐dependent fashion, glutamate uptake, an effect that was mimicked by inducers of lipid peroxidation. Taken together, these findings demonstrate that the lipid peroxidation product HNE rapidly accumulates in the spinal cord following injury and that a major consequence of HNE accumulation is a decrease in glutamate uptake, which may potentiate neuronal cell dysfunction and death through excitotoxic mechanisms.
Koningic Acid (a Potent Glyceraldehyde‐3‐Phosphate Dehydrogenase Inhibitor)‐Induced Fragmentation and Condensation of DNA in NG108‐15 Cells
Authors: Nakazawa Minako , Uehara Takashi , Nomura Yasuyuki
Published: July 2010

Abstract: We examined nitric oxide (NO)‐induced cell death in NG108‐15 cells using NO donors. Both sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine caused lactate dehydrogenase (LDH) leakage from NG108‐15 cells. NO is known to increase the amount of radioisotopic labeled glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) in the presence of [32P]NAD and to inhibit the enzyme activity. To clarify the relationship between the NO‐induced inhibition of GAPDH activity and cell death, we studied the effect of koningic acid (KA), a potent selective inhibitor of GAPDH. Both SNP and KA elicited LDH leakage, chromosomal condensation, and fragmentation of nuclei in NG108‐15 cells. Gel electrophoretic analysis of cellular DNA extracted from SNP‐ and KA‐treated cells revealed the internucleosomal DNA fragmentation typical of apoptosis in these cultures. The results suggested that in NG108‐15 cells, (a) the inhibition of GAPDH activity results in apoptosis and (b) SNP‐induced cell death is partly due to the NO‐induced inhibition of GAPDH, perhaps by stimulating the binding of NAD to GAPDH.
Down‐Regulation of 5‐Hydroxytryptamine7 Receptors by Dexamethasone in Rat Frontocortical Astrocytes
Authors: Shimizu Masami , Nishida Akira , Zensho Hidenobu , Miyata Masahiko , Yamawaki Shigeto
Published: July 2010

Abstract: Astrocytes derived from rat frontal cortex contain 5‐hydroxytryptamine7 (5‐HT)7 receptors positively coupled to adenylyl cyclase. In the present study, we investigated the effects of glucocorticoids on adenylyl cyclase activity and 5‐HT7 receptor gene expression in astrocytes. Addition of dexamethasone (0.01–100 nM, 12–72 h) to the culture medium decreased cyclic AMP formation induced by 5‐HT in a time‐ and concentration‐dependent manner. Dexamethasone treatment (10 nM, 48 h) reduced maximum responses of cyclic AMP formation induced by 5‐HT and 5‐carboxamidotryptamine without alterations in the EC50 value. In contrast, treatment with the mineralocorticoid aldosterone (48 h) had no significant effects on 5‐HT‐induced cyclic AMP formation with concentrations up to 10 nM. It was observed that dexamethasone treatment (1–100 nM, 3–72 h) also decreased the expression of 5‐HT7 receptor mRNA, using reverse transcription and polymerase chain reaction analysis. A significant reduction in expression of 5‐HT7 mRNA appeared at 3 h of dexamethasone treatment and reached a maximum at 6 h of treatment. On the other hand, dexamethasone treatment (10 nM, 48 h) did not affect basal levels of cyclic AMP and cyclic AMP synthesis stimulated by isoproterenol, N‐ethylcarboxamido‐adenosine, cholera toxin, and forskolin. These results suggest that dexamethasone decreases the expression of the 5‐HT7 receptor gene and, consequently, 5‐HT7 receptor‐mediated signal transduction in frontocortical astrocytes.
Novel Isoforms of Rat Brain Fructose 6‐Phosphate 2‐Kinase/Fructose 2,6‐Bisphosphatase Are Generated by Tissue‐Specific Alternative Splicing
Authors: Watanabe Fusao , Sakai Akiko , Furuya Eisuke
Published: July 2010

Abstract: Fructose 6‐phosphate 2‐kinase/fructose 2,6‐bisphosphatase catalyzes the synthesis and degradation of fructose 2,6‐bisphosphate, which is the most potent activator of glycolysis. We have shown previously the occurrence of a partial cDNA (RB7) encoding the catalytic core domain of a novel brain‐type isozyme. To elucidate the full‐length sequence of RB7 cDNA, we have carried out cDNA cloning using the 3′‐ and 5′‐rapid amplification cDNA ends method and have isolated eight isoforms from rat brain. The cDNA sequences encoding the 5′‐untranslated region, the amino‐terminal domain, and the catalytic core domain were identical among all the isoforms. However, heterogeneity of the carboxyl‐terminus was found by sequence analysis. This heterogeneity was shown not to have resulted from transcription of multiple genes, as Southern blot and genomic sequence analysis revealed that the gene was a single copy in the rat genome. It is likely that these transcripts represent splice variants of the gene. High‐level expression of the gene was also observed by northern blot analysis in skeletal muscle. However, the pattern of alternative splicing was different from that of brain, and only four isoforms were detected in skeletal muscle.
Basic Fibroblast Growth Factor Stimulation of Glial Cells Protects Dopamine Neurons from 6‐Hydroxydopamine Toxicity: Involvement of the Glutathione System
Authors: Hou Jyh‐Gong Gabriel , Cohen Gerald , Mytilineou Catherine
Published: July 2010

Abstract: Neurotrophic factors have been shown to support the survival and promote the recovery of injured neurons both in vivo and in vitro. Here, we investigated whether glial cell line‐derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) could modify the damage to dopamine (DA) neurons in mesencephalic cultures caused by the neurotoxin 6‐hydroxydopamine (6‐OHDA). The data show that bFGF, but not GDNF, effectively protected DA neurons from 6‐OHDA toxicity. Because bFGF is a glial mitogen, whereas GDNF is not, we tested whether glial cells participated in bFGF neuroprotection. Inhibition of glial cell proliferation completely prevented the protective effect of bFGF. Because oxidative events have been associated with 6‐OHDA‐induced damage, we examined the levels of glutathione (GSH) in control and bFGF‐treated cultures. Cultures treated with bFGF had higher levels of GSH, which increased even further in response to 6‐OHDA exposure. Control cultures failed to up‐regulate GSH levels after 6‐OHDA, suggesting a relationship between increased GSH levels and protection from 6‐OHDA. Inhibition of glial cell proliferation prevented the rise in GSH in bFGF‐treated cultures and abolished the increase after 6‐OHDA treatment. Protection from 6‐OHDA by bFGF was also diminished when GSH levels were decreased by the GSH synthesis inhibitor l‐buthionine sulfoximine. Our study shows that stimulation of glial cells by bFGF allows the up‐regulation of antioxidant defenses and supports cell survival during oxidative stress.
Expression of the Na+‐d‐Glucose Cotransporter SGLT1 in Neurons
Authors: Poppe Robert , Karbach Ulrich , Gambaryan Stepan , Wiesinger Heinrich , Lutzenburg Michael , Kraemer Matthias , Witte Otto W. , Koepsell Hermann
Published: July 2010

Abstract: In brains of the rabbit, pig, and human, expression of the high‐affinity Na+‐d‐glucose cotransporter SGLT1 and of the protein RS1, which alters the activity of SGLT1, was demonstrated. In situ hybridization showed that SGLT1 and RS1 are transcribed in pyramidal cells of brain cortex and hippocampus and in Purkinje cells of cerebellum. In neurons of pig brain SGLT1 protein was demonstrated by western blotting with synaptosomal membranes and by immunohistochemistry, which showed SGLT1 in pyramidal and Purkinje cells. To test whether SGLT1 in neurons may be activated during increased d‐glucose consumption, an epileptic seizure was induced in rat brain, and the uptake of specific nonmetabolized substrates of SGLT1 {[14C]methyl‐α‐d‐glucopyranoside ([14C]AMG)} and of Na+‐independent transporters {2‐deoxy‐d‐[14C]glucose([14C]2‐DG)} was analyzed by autoradiography. During the seizure the uptake of AMG and 2‐DG was increased in the focus. Within two hours after the seizure 2‐DG uptake in the focus returned to normal. In contrast, the AMG uptake in the focus area was still increased 1 day later. The data show that the high‐affinity Na+‐d‐glucose cotransporter SGLT1 is expressed in neurons and can be up‐regulated.
Nitrite and Nitrate Levels in Individual Molluscan Neurons: Single‐Cell Capillary Electrophoresis Analysis
Authors: Cruz LouAnn , Moroz Leonid L. , Gillette Rhanor , Sweedler Jonathan V.
Published: July 2010

Abstract: Cell and tissue concentrations of NO2− and NO3− are important indicators of nitric oxide synthase activity and crucial in the regulation of many metabolic functions, as well as in nonenzymatic nitric oxide release. We adapted the capillary electrophoresis technique to quantify NO2− and NO3− levels in single identified buccal neurons and ganglia in the opisthobranch mollusc Pleurobranchaea californica, a model system for the study of the chemistry of neuron function. Neurons were injected into a 75‐µm separation capillary and the NO2− and NO3− were separated electrophoretically from other anions and detected by direct ultraviolet absorbance. The limits of detection for NO2− and NO3− were <200 fmol (<4 µM in the neurons under study). The NO2− and NO3− levels in individual neurons varied from 2 mM (NO2−) and 12 mM (NO3−) in neurons histochemically positive for NADPH‐diaphorase activity down to undetectable levels in many NADPH‐diaphorase‐negative cells. These results affirm the correspondence of histochemical NADPH‐diaphorase activity and nitric oxide synthase in molluscan neurons. NO2− was not detected in whole ganglion homogenates or in hemolymph, whereas hemolymph NO3− averaged 1.8 ± 0.2 × 10−3M. Hemolymph NO3− in Pleurobranchaea was appreciably higher than values measured for the freshwater pulmonate Lymnaea stagnalis (3.2 ± 0.2 × 10−5M) and for another opisthobranch, Aplysia californica (3.6 ± 0.7 × 10−4M). Capillary electrophoresis methods provide utility and convenience for monitoring NO2−/NO3− levels in single cells and small amounts of tissue.
Bidirectional Alterations of GABAA Receptor Subunit Peptide Levels in Rat Cortex During Chronic Ethanol Consumption and Withdrawal
Authors: Devaud Leslie L. , Fritschy Jean‐Marc , Sieghart Werner , Morrow A. Leslie
Published: July 2010

Abstract: The pharmacological properties of γ‐aminobutyric acidA (GABAA) receptors are altered by prolonged exposure to ethanol both in vivo and in vitro. We have shown previously that prolonged ethanol exposure elicits selective alterations in various GABAA receptor subunit mRNA levels in rat cerebral cortex. Some of these effects are rapidly reversed during ethanol withdrawal. The present study was conducted to determine the effects of prolonged ethanol exposure (dependence) and ethanol withdrawal on cerebral cortical peptide expression for several subunits. GABAA receptor α1 subunit peptide levels were decreased by nearly 40%, whereas α4 subunit peptide levels were increased by 27% in both ethanol‐dependent and withdrawn rats. These changes correlate well with observed alterations in mRNA levels following prolonged ethanol exposure in dependent rats, but do not match the effects on mRNA levels during ethanol withdrawal. β2/3 subunit peptide levels increased by ∼32% in both ethanol‐dependent rats and rats undergoing ethanol withdrawal. We observed a 30–60% increase in γ1 subunit peptide levels in both dependent rats and those undergoing withdrawal, also correlating with the previous report on ethanol‐induced alterations in mRNA levels. Peptide levels for γ2 subunits did not differ from control values in either condition. These findings show that specific alterations in GABAA receptor subunit peptide levels are associated with ethanol dependence in rats. GABAA receptor subunit peptide expression is more stable than mRNA expression, and mRNA levels are not representative of peptide expression during ethanol withdrawal. These findings are consistent with the suggestion that alterations in GABAA receptor gene expression underlie the functional properties of GABAA receptors in ethanol‐dependent rats and those undergoing ethanol withdrawal.
Importance of the Rab3a‐GTP Binding Domain for the Intracellular Stability and Function of Rabphilin3a in Secretion
Authors: Chung Sul‐Hee , Stabila Paul , Macara Ian G. , Holz Ronald W.
Published: July 2010

Abstract: We had previously demonstrated that Rab3a‐GTP inhibits and the Rab3a‐binding protein Rabphilin3a enhances secretion in bovine chromaffin cells. In this study, we investigated the role of Rab3a‐GTP binding in the intracellular expression and the function of Rabphilin3a in regulated exocytosis in bovine chromaffin cells. Using transient transfections, we found that a minimal domain, Rp(51–190), that inhibits secretion coincides with a minimal domain that effectively binds Rab3a‐GTP and allows intracellular stability of the construct. This domain includes a cysteine‐rich, Zn2+‐binding domain whose integrity is also required for Rab3a‐GTP binding and the ability to inhibit secretion. A Rabphilin3a mutant, containing both C2 domains but defective in Rab3a‐GTP, and wild‐type Rabphilin3a both localized to chromaffin granules and stimulated secretion similarly despite lessened intracellular expression of the mutant protein. The data are consistent with a sequence of events in which a Rab3a‐GTP · Rabphilin3a complex forms on the secretory granule as a precursor in a pathway that enhances secretion. The complex dissociates (perhaps because of GTP hydrolysis) to permit the enhancement of secretion by Rabphilin3a.
N‐Acetylaspartylglutamate Selectively Activates mGluR3 Receptors in Transfected Cells
Authors: Wroblewska Barbara , Wroblewski Jarda T. , Pshenichkin Sergei , Surin Alexander , Sullivan Suzanne E. , Neale Joseph H.
Published: July 2010

Abstract: In previous studies, we demonstrated that the neuropeptide, N‐acetylaspartylglutamate (NAAG), meets the traditional criteria for a neurotransmitter and selectively activates metabotropic glutamate receptor mGluR2 or mGluR3 in cultured cerebellar granule cells and glia. Sequence homology and pharmacological data suggest that these two receptors are highly related structurally and functionally. To define more rigorously the receptor specificity of NAAG, cloned rat cDNAs for mGluR1–6 were transiently or stably transfected into Chinese hamster ovary cells and human embryonic kidney cells and assayed for their second messenger responses to the two endogenous neurotransmitters, glutamate and NAAG, as well as to metabotropic receptor agonists, trans‐1‐aminocyclopentane‐1,3‐dicarboxylate (trans‐ACPD) and l‐2‐amino‐4‐phosphonobutyrate (l‐AP4). Despite the high degree of relatedness of mGluR2 and mGluR3, NAAG selectively activated the mGluR3 receptor. NAAG activated neither mGluR2 nor mGluR1, mGluR4, mGluR5, or mGluR6. The mGluR agonist, trans‐ACPD, activated each of the transfected receptors, whereas l‐AP4 activated mGluR4 and mGluR6, consistent with the published selectivity of these agonists. Hybrid cDNA constructs of the extracellular domains of mGluR2 and mGluR3 were independently fused with the transmembrane and cytoplasmic domain of mGluR1a. This latter receptor domain is coupled to phosphoinositol turnover, and its activation increases intracellular calcium. The cells transfected with these chimeric receptors responded to activation by glutamate and trans‐ACPD with increases in intracellular calcium. NAAG activated the chimeric receptor that contained the extracellular domain of mGluR3 and did not activate the mGluR2 chimera.
Demyelinating Antibodies to Myelin Oligodendrocyte Glycoprotein and Galactocerebroside Induce Degradation of Myelin Basic Protein in Isolated Human Myelin
Authors: Menon Krishna Kumar , Piddlesden Sara J. , Bernard Claude C. A.
Published: July 2010

Abstract: Although the specificity of multiple sclerosis (MS) brain immunoglobulins (lgs) remains unknown, the incubation of these lgs with human myelin can lead to myelin basic protein (MBP) degradation mediated by neutral proteases. In this study, we demonstrate that monoclonal antibodies (mAbs) specific to myelin components such as the CNS‐specific myelin oligodendrocyte glycoprotein (MOG) and galactocerebroside (GalC) are found to induce a significant loss of MBP mediated by neutral proteases in myelin. By contrast, antibodies to periaxonal and structural components of myelin, such as MBP and myelin‐associated glycoprotein, are ineffective in inducing such MBP degradation. Among the 11 different anti‐MOG mAbs directed to externally located epitopes of MOG, only two were found to induce a significant degradation of MBP, suggesting that antibody‐induced MBP degradation is not only antigen specific but also epitope specific. Based on the inhibition of MBP degradation in the presence of EGTA and the analysis of the degradation products obtained following incubation of myelin with mAbs to GalC and MOG (8‐18C5), the neutral protease involved in this antibody‐induced degradation of MBP could be calcium‐activated neutral protease. Taken together, these results suggest that antibodies to GalC and MOG can play a major role in destabilizing myelin through MBP breakdown mediated by neutral proteases and thus have an important role to play in the pathogenesis of MS.
Aggregation of β‐Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain
Authors: Klunk William E. , Xu Chong‐Jun , McClure Richard J. , Panchalingam Kanagasabai , Stanley Jeff A. , Pettegrew Jay W.
Published: July 2010

Abstract: Increased amounts of β‐amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross‐β‐pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α‐glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol‐1‐phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.
l‐Aspartate but Not the d Form Is Secreted Through Microvesicle‐Mediated Exocytosis and Is Sequestered Through Na+‐Dependent Transporter in Rat Pinealocytes
Authors: Yatsushiro Shouki , Yamada Hiroshi , Kozaki Shunji , Kumon Hiromi , Michibata Hitoshi , Yamamoto Akitsugu , Moriyama Yoshinori
Published: July 2010

Abstract: Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine‐stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca2+ ionophore) in the presence of Ca2+ and was proportional to the amount of l‐glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L‐ or N‐type voltage‐gated Ca2+ channel blockers. Bay K 8644, an agonist for the L‐type Ca2+ channel, stimulated the release of aspartate 2.1‐fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the l form, although pinealocytes also contain the d form in their cytoplasm, corresponding to ∼30% of the total free aspartate. Radiolabeled l‐aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na+‐dependent manner. The Na+‐dependent uptake of l‐aspartate was strongly inhibited by l‐cysteine sulfinate, β‐hydroxyaspartate, and l‐serine‐O‐sulfate, inhibitors for the Na+‐dependent glutamate/aspartate transporter on the plasma membrane. Na+‐dependent sequestration of l‐aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that l‐aspartate is released through microvesicle‐mediated exocytosis from pinealocytes and is taken up again through the Na+‐dependent transporter at the plasma membrane. The possible role of l‐aspartate as an intercellular chemical transmitter in the pineal gland is discussed.
Lesioning of Deep Prepiriform Cortex Protects Against Ischemic Neuronal Necrosis by Attenuating Extracellular Glutamate Concentrations
Authors: Kawaguchi Kenji , Huerbin Michelle , Simon Roger P.
Published: July 2010

Abstract: An area of the deep prepiriform cortex is a controlling site for limbic seizures. Focal pharmacologic blockade of NMDA receptors in the deep prepiriform cortex protects against hippocampal cell injury during limbic seizures induced by intravenous kainate and during the excitotoxicity of global ischemia. In the current study, the deep prepiriform cortex was lesioned bilaterally by microinjection of kainate, 3 days before 10 min of global ischemia induced by four‐vessel occlusion. Extracellular glutamate concentrations in the hippocampus were measured before, during, and after global ischemia by using in vivo microdialysis technique. Surviving hippocampal neurons were counted 7 days after ischemia. Lesioned animals showed significantly greater numbers of surviving neurons and significantly lower ischemia‐induced elevations of extracellular glutamate concentrations than non‐lesioned animals. During seizures induced from the deep prepiriform cortex, the immediate early gene cox‐2 is expressed in the hippocampus. These results indicate that deep prepiriform cortex can be a modulatory site for ischemic hippocampal injury.
Cloning, Characterization, and Expression of a Calcitonin Receptor from Guinea Pig Brain
Authors: Sarkar Atom , Dickerson Ian M.
Published: July 2010

Abstract: A calcitonin receptor was cloned from guinea pig brain by using a degenerate reverse transcription‐polymerase chain reaction (RT‐PCR) strategy. When the cloned guinea pig calcitonin receptor was transfected into COS 1 cells, salmon calcitonin stimulated intracellular cyclic AMP accumulation with an EC50 of 0.1 nM, whereas human calcitonin was >250‐fold less potent (EC50 27.6 nM). Related neuropeptides rat αCGRP and rat amylin did not activate the guinea pig calcitonin receptor at physiologic concentrations. Stimulation of the transfected guinea pig calcitonin receptor by salmon calcitonin also resulted in phosphatidylinositol hydrolysis with an EC50 of 2.5 nM. Expression of the calcitonin receptor was mapped by a combination of RT‐PCR, northern analysis, and expression in Xenopus oocytes. The guinea pig calcitonin receptor was most highly expressed in diencephalon and a single subtype was detected.
Polysialic Acid Synthase (ST8Sia II/STX) mRNA Expression in the Developing Mouse Central Nervous System
Authors: Kurosawa Nobuyuki , Yoshida Yukiko , Kojima Naoya , Tsuji Shuichi
Published: July 2010

Abstract: A comparative study was undertaken to correlate the immunohistochemical localization of polysialic acid (PSA) and the in situ localization of ST8Sia II mRNA. In situ hybridization of postnatal day 3 mouse brain showed high levels of ST8Sia II mRNA expression in the cerebral neocortex, striatum, hippocampus, subiculum, medial habenular nucleus, thalamus, pontine nuclei, and inferior colliculus; intermediate‐level expression in the olfactory bulb, hypothalamus, superior colliculus, and cerebellum; and low‐level expression in other regions. The distribution of ST8Sia II mRNA in the neocortex and cerebellum coincided with the immunohistochemical localization of PSA. During brain development, ST8Sia II mRNA started decreasing and had almost disappeared by postnatal day 14. Comparison between ST8Sia II and IV mRNA expression was also undertaken by northern blot analysis and competitive PCR analysis. During the late embryonic to early postnatal stages of the mouse CNS, the ST8Sia II mRNA showed abundant mRNA expression compared with the ST8Sia IV mRNA. Competitive PCR analysis of the adult mouse CNS showed weak expression of the two genes in the olfactory bulb, thalamus, hippocampus, and eyes. The regional and transient expression of ST8Sia II mRNA coincides with that of PSA, suggesting that ST8Sia II is closely involved in the biosynthesis and expression of PSA in the developing mouse CNS.
BCL‐2‐Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells
Authors: Maroto Rosario , Perez‐Polo J. Regino
Published: July 2010

Abstract: Expression of the BCL‐2 protein family members, BAX, BAK, BAD, BCL‐xL, BCL‐xS, and BCL‐2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H2O2 treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H2O2‐induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose‐dependent increase in BAX, (b) a dose‐independent increase in BAK, and (c) a dose‐dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time‐dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis‐sparing medium (i.e., NGF‐supplemented serum‐free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL‐xL expression was increased by H2O2 but unaffected by serum deprivation or long‐term NGF treatment. Neither BCL‐2 nor BCL‐xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL‐xL is altered in a stimulus‐dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL‐2‐related proteins induced by H2O2 exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.
Two Waves of Cyclin B and Proliferating Cell Nuclear Antigen Expression During Dopamine‐Triggered Neuronal Apoptosis
Authors: Shirvan Anat , Ziv Ilan , Machlin Tatyana , Zilkha‐Falb Rina , Melamed Eldad , Barzilai Ari
Published: July 2010

Abstract: The neurotransmitter dopamine is capable of inducing apoptosis in postmitotic sympathetic neurons via its oxidative metabolites. To detect genes whose expression is transcriptionally regulated during the early stages of dopamine‐triggered apoptosis, we applied the differential display method to cultured sympathetic neurons. One of the up‐regulated genes was identified as cyclin B2, which exhibited two waves of induction and destruction, both at the mRNA and protein levels, resembling the sequential oscillations typical of two successive mitotic events in proliferating cells. The time window between the two waves was characterized by a change in expression of other cell‐cycle stage‐specific genes, and oscillations in proliferating cell nuclear antigen and alterations in cyclin A were observed. Cyclin D1 and cyclin‐dependent kinases were undetected and no sign of active DNA synthesis could be observed, indicating that activation of cell‐cycle components is incomplete. In comparison with a normal cell cycle, temporal expression profile of these mediators was unsynchronized. Whereas the first wave of cell‐cycle changes occurred prior to the commitment of the cells to the death process and could be tolerated by the cells, the second wave of changes coincided with the death commitment point. Our findings indicate that inappropriate and incomplete activation of some cell cycle‐related genes in postmitotic neurons occurs during dopamine‐triggered neuronal apoptosis.
Apoptosis Induced via AMPA‐Selective Glutamate Receptors in Cultured Murine Cortical Neurons
Authors: Larm Jari A. , Cheung Nam S. , Beart Philip M.
Published: July 2010

Abstract: We have investigated the mechanisms of cell death induced by long‐term exposure to the glutamate receptor agonist (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate [(S)‐AMPA]. Using primary cultures of pure neurons (95%) grown in serum‐free conditions, we found that 24‐h exposure to (S)‐AMPA (0.01–1,000 µM) induced concentration‐dependent neuronal cell death (EC50 = 3 ± 0.5 µM) with cellular changes including neurite blebbing, chromatin condensation, and DNA fragmentation, indicative of apoptosis. (S)‐AMPA induced a delayed cell death with DNA fragmentation occurring in ∼50% of cells at concentrations between 100 and 300 µM detected using terminal transferase‐mediated dUTP nick end‐labeling (TUNEL) and agarose gel electrophoresis. Apoptotic chromatin condensation was detected using 4,6‐diamidino‐2‐phenylindole, a fluorescent DNA binding dye. Cell death induced by (S)‐AMPA was attenuated by the AMPA receptor‐selective antagonist LY293558 (10 µM) and the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 50 µM), yielding EC50 values of 73 ± 5 and 265 ± 8 µM, respectively, and was unaffected by the NMDA receptor antagonist MK‐801 (10 µM). The number of apoptotic nuclei induced by 300 µM (S)‐AMPA (57%) was also reduced substantially by the antagonists LY293558 and CNQX, with only 20% and 18% of neurons, respectively, staining TUNEL‐positive at 24 h. In addition, cycloheximide (0.5 µg/ml) also inhibited (S)‐AMPA‐induced DNA fragmentation and cell death. Our results show that long‐term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons, suggesting a wide involvement of AMPA‐sensitive glutamate receptors in excitotoxic injury and neurodegenerative pathologies.