bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat Shock Induces Rapid Dephosphorylation of τ in Both Female and Male Rats Followed by Hyperphosphorylation Only in Female Rats: Implications for Alzheimer's Disease
Authors: Papasozomenos Sozos Ch.
Published: July 2010

Abstract: Female and male 2–3‐month‐old Sprague‐Dawley rats were heat shocked at 42°C for 15 min. At 0, 3, 6, 12, and 24 h after heat shock, qualitative and quantitative immunoblot analysis of cerebral extracts and immunohistochemistry were performed using monoclonal anti‐τ antibodies that recognize nonphosphorylated (Tau‐1), phosphorylated (PHF‐1), and phosphate‐independent (Tau‐5 and Tau‐46) epitopes. At 0 h after heat shock, there was dephosphorylation of τ in both female and male rats as evidenced by (1) accentuation and attenuation of τ isoforms recognized by Tau‐1 and PHF‐1, respectively, and recognition of additional τ polypeptides by Tau‐1, Tau‐5, and Tau‐46 but not PHF‐1; (2) significant increase in the nonphosphorylated Tau‐1 epitope with resultant decrease in the ratio of total (phosphorylated plus nonphosphorylated) τ to nonphosphorylated τ; and (3) dephosphorylation of the Tau‐1 epitope in the somatodendritic compartment. By 6 h after heat shock, there was progressive hyperphosphorylation of τ in female but not male rats exemplified by (1) upward gel mobility shift recognized by PHF‐1, Tau‐5, and Tau‐46, and by Tau‐1 after dephosphorylation; (2) significant increase in the ratio of total τ to nonphosphorylated τ; and (3) rephosphorylation of the Tau‐1 epitope in the somatodendritic compartment. Two‐dimensional electrophoresis showed shifts to basic and acidic τ polypeptides at 0 and 6 h after heat shock, respectively. Hyperphosphorylation of τ also occurred after multiple heat‐shock episodes. Microtubules were present at 6 h after heat shock. There were no differences between control and heat‐shocked rats in extracts from peripheral nerves. Thus, we now have a simple rat model to study within 6 h the processes of dephosphorylation and hyperphosphorylation of τ, which are altered in Alzheimer's disease.
Effect of Dopamine, Dimethoxyphenylethylamine, Papaverine, and Related Compounds on Mitochondrial Respiration and Complex I Activity
Authors: Morikawa Nami , Nakagawa‐Hattori Yuko , Mizuno Yoshikuni
Published: July 2010

Abstract: We report the effect of papaverine, tetrahydropapaverine, laudanosine, dimethoxyphenylethylamine, dopamine, and its metabolites on mitochondrial respiration and activities of the enzymes in the electron transfer complexes, as mitochondrial toxins may be implicated in the etiology and the pathogenesis of Parkinson's disease. Papaverine was the most potent inhibitor of complex I and NADH‐linked mitochondrial respiration among the compounds tested next to rotenone. Tetrahydropapaverine, dimethoxyphenylethylamine, and laudanosine also inhibited NADH‐linked mitochondrial respiration and complex I activity in this order. Dopamine and its metabolites showed either no inhibition or only very weak inhibition. Compounds with dimethoxy residues in the phenyl ring were associated with more potent inhibition of complex I than those without. Our results warrant further studies on these and some related compounds as candidate neurotoxins causing Parkinson's disease.
Osmotic Swelling Stimulates Ascorbate Efflux from Cerebral Astrocytes
Authors: Siushansian Ramin , Dixon S. Jeffrey , Wilson John X.
Published: July 2010

Abstract: Ascorbate (reduced vitamin C) is an important enzyme cofactor, neuromodulator, and antioxidant that is stored at millimolar concentrations in the cytosol of cerebral astrocytes. Because these cells swell during hyponatremia, cerebral ischemia, and trauma, we investigated the effects of osmotic stress on astrocytic transport of ascorbate. Ascorbate efflux from primary cultures of rat astrocytes was rapidly (within 1 min) increased by incubation in hypotonic medium. Efflux also increased when astrocytes, which had been adapted to a hypertonic environment, were swollen by transfer to isotonic medium. Swelling‐induced ascorbate efflux was inhibited by the anion‐transport inhibitors 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS) and 4,4′‐dinitrostilbene‐2,2′‐disulfonic acid (DNDS). The pathway that mediates ascorbate efflux was found to be selective because a larger anion, 2′,7′‐bis(carboxyethyl)‐5‐(or ‐6)‐carboxyfluorescein (BCECF), was retained in the swollen astrocytes. Na+‐dependent ascorbate uptake into astrocytes was inhibited slightly during the first minute of hypotonic stress, indicating that the sodium ascorbate cotransporter does not mediate swelling‐induced efflux. Cell concentration of authentic ascorbate was measured by HPLC with electrochemical detection. When astrocytes were incubated in ascorbate‐free medium, hypotonicity decreased cell ascorbate concentration by 50% within 3 min. When astrocytes were incubated in ascorbate‐supplemented hypotonic medium, intracellular ascorbate concentration was restored within 10 min because uptake remained effective. Many pathological conditions cause brain cell swelling and formation of reactive oxygen species. Ascorbate release during astrocytic swelling may contribute to cellular osmoregulation in the short‐term and the scavenging of reactive oxygen species.
Partially Purified RhoA‐Stimulated Phospholipase D Activity Specifically Binds to Phosphatidylinositol 4,5‐Bisphosphate
Authors: Yokozeki T. , Kuribara H. , Katada T. , Touhara K. , Kanaho Y.
Published: July 2010

Abstract: Phosphatidylinositol 4,5‐bisphosphate (PIP2) is absolutely required for the ADP‐ribosylation factor‐stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP2, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP2 for the RhoA‐stimulated PLD activation, too. It is interesting that the RhoA‐dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP2. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP2 that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA‐dependent PLD binds to PE/PC vesicles with PIP2. The degree of binding of the RhoA‐dependent PLD activity to the vesicles was totally dependent on the amount of PIP2 in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β‐adrenergic receptor kinase fused to glutathione S‐transferase, which specifically binds to PIP2, inhibited the PIP2‐stimulated, RhoA‐dependent PLD activity in a concentration‐dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP2, owing to its specific interaction with PIP2, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP2 effectively extracted the RhoA‐dependent PLD activity from rat brain membranes. This indicates that PIP2 serves as an anchor to localize PLD on plasma membranes in vivo.
Secretogranin II: Relative Amounts and Processing to Secretoneurin in Various Rat Tissues
Authors: Leitner Bernd , Fischer‐Colbrie Reiner , Scherzer Gerhard , Winkler Hans
Published: July 2010

Abstract: Secretoneurin is a 33‐amino‐acid peptide produced in vivo from secretogranin II. An antiserum raised against this peptide recognizes both the free peptide and its precursors. By HPLC and radioimmunoassay we characterized the immunoreactive molecules and determined the levels of immunoreactivity in various rat organs. In adrenal medulla and to a lesser degree in the anterior pituitary processing of secretogranin II to secretoneurin was very limited, whereas in all other organs studied (brain, intestine, endocrine pancreas, thyroid gland, and posterior pituitary) a high degree of processing was apparent. Thus, practically all of the immunoreactivity was present as free secretoneurin. This was also true for serum. When the total amount of secretoneurin immunoreactivity was calculated for the various organs, the largest pools in descending order were in the intestine, CNS, anterior pituitary, pancreas, and adrenal gland. This makes it likely that secretoneurin in serum is mainly derived from the intestine. The high degree of processing of secretogranin II in most organs is consistent with the concept that this protein acts as a precursor of a functional peptide, i.e., secretoneurin.
Microtubule Depolymerization Inhibits Ethanol‐Induced Enhancement of GABAA Responses in Stably Transfected Cells
Authors: Whatley Valerie J. , Brozowski Susan J. , Hadingham Karen L. , Whiting Paul J. , Harris R. Adron
Published: July 2010

Abstract: We studied whether microtubule organization is important for actions of ethanol on GABAA ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk−) cells stably transfected with GABAA receptor α1β1γ2L subunits. The microtubule‐disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol‐induced enhancement of muscimol‐stimulated chloride uptake. β‐Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicine did not alter the potentiating actions of flunitrazepam or pentobarbital on muscimol‐stimulated chloride uptake. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol‐induced enhancement of GABAA responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.
Distribution of Three Nicotinic Receptor α4 mRNA Transcripts in Rat Brain: Selective Regulation by Nicotine Administration
Authors: Yu Z. Jian , Morgan David G. , Wecker Lynn
Published: July 2010

Abstract: Northern blot analysis determined whether multiple α4 transcripts for neuronal nicotinic receptors in rat brain could be detected as distinct bands. When poly(A)+ RNA was isolated from brain regions and hybridized with a Hinfl fragment of α4‐1 cDNA containing a sequence shared by both α4‐1 and α4‐2, but little homology with other α or β subunits, bands at 6.0, 4.6, and 2.6 kb were obtained. When a Taql fragment with selectivity for α4‐1 was used, a single band was present at 6.0 kb. The 6.0‐kb band was least abundant in all brain regions; the 2.6‐kb band was most abundant in frontal cortex, hippocampus, striatum, basal forebrain, and thalamus, whereas the 4.6‐kb band was most abundant in midbrain and cerebellum. Nicotine (3.6 µmol/kg, s.c., twice daily) increased the abundance of the 4.6‐kb transcript in frontal cortex significantly by 28% following 2.5 days of injections; the 6.0‐ and 2.6‐kb transcripts were unchanged. Nicotine did not affect α4 transcripts in other brain regions. Results suggest that increased mRNA levels may mediate the nicotine‐induced up‐regulation of receptors in cerebral cortex.
Role of External and Internal Calcium on Heterocarrier‐Mediated Transmitter Release
Authors: Fassio A. , Bonanno G. , Fontana G. , Usai C. , Marchi M. , Raiteri M.
Published: July 2010

Abstract: Release‐regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [3H]acetylcholine and [3H]noradrenaline from rat hippocampal synaptosomes and of [3H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca2+ or addition of tetrodotoxin selectively abolished the GABA‐evoked release of [3H]noradrenaline, leaving unaffected that of [3H]acetylcholine or [3H]dopamine. 1,2‐Bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM) or vesamicol attenuated the release of [3H]acetylcholine elicited by GABA. Reserpine, but not BAPTA‐AM, prevented the effect of GABA on [3H]dopamine release. Autoreceptor activation inhibited the GABA‐evoked release of [3H]noradrenaline but not that of [3H]acetylcholine or [3H]dopamine. It is concluded that (a) the release of [3H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca2+]‐independent releases of [3H]acetylcholine and [3H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca2+, and (c) autoreceptor activation only affects heterocarrier‐mediated vesicular release linked to entry of extracellular Ca2+.
Visualization of D1 Dopamine Receptors on Living Nucleus Accumbens Neurons and Their Colocalization with D2 Receptors
Authors: Shetreat Maya E. , Lin Ling , Wong Andrew C. , Rayport Stephen
Published: July 2010

Abstract: To examine the substrate for dopamine (DA) synaptic action in the nucleus accumbens (nAcc), we visualized the cellular and subcellular distribution of DA receptors on postnatal nAcc neurons in culture using fluoroprobe derivatives of DA receptor ligands. Previously, we have shown that rhodamine‐N‐(p‐aminophenethyl)‐spiperone (NAPS) (10 nM), a derivative of the D2 antagonist spiperone, labels D2‐like receptors on living nAcc neurons. We now show that rhodamine‐Sch‐23390 (30 nM), a derivative of the D1 antagonist, labels D1‐like receptors. Putative specific membrane labeling reached a plateau after about 20 min. Labeling was stereospecific, as it was unaffected by competition with (−)‐butaclamol, but blocked with (+)‐butaclamol. We found that 52 ± 7% of nAcc medium‐sized neurons showed D1 labeling, which extended onto the dendrites. Labeling was also seen on presynaptic terminals, often abutting D1‐positive and D1‐negative cell bodies, consistent with a presynaptic modulatory role for D1 receptors. Larger neurons, which may be GABAergic or cholinergic interneurons, were also labeled. By sequential labeling first with rhodamine‐Sch‐23390 and then rhodamine‐NAPS, we found that 38 ± 6% of medium‐sized neurons express both D1‐ and D2‐like receptors, indicating that D1–D2 interactions may occur at the level of single postsynaptic neurons.
Interleukin‐1β Uses Common and Distinct Signaling Pathways for Induction of the Interleukin‐6 and Tumor Necrosis Factor α Genes in the Human Astrocytoma Cell Line U373
Authors: Lieb Klaus , Kaltschmidt Christian , Kaltschmidt Barbara , Baeuerle Patrick A. , Berger Mathias , Bauer Joachim , Fiebich Bernd L.
Published: July 2010

Abstract: Cytokines are involved in the etiology of different disorders of the CNS. For a better understanding of their pathogenic role, we analyzed signal transduction pathways mediating the interleukin (IL)‐1β‐induced synthesis of IL‐6 and tumor necrosis factor α (TNFα) in the human astrocytoma cell line U373 MG. Both protein kinase C and reactive oxygen intermediates (ROIs) were involved in IL‐6 and TNFα gene expression by IL‐1β. In contrast, protein tyrosine kinases were only necessary for expression of the IL‐6 gene. Whereas activation of protein kinase A was able to induce expression of the IL‐6 gene, it did not induce TNFα gene expression and was not involved in IL‐1β‐induced IL‐6 and TNFα gene expression. Activation of the transcription factor nuclear factor‐κB by IL‐1β involved ROIs, whereas the IL‐1β‐induced activation of the transcription factor AP‐1 was mediated via protein kinase C. Our findings provide the basis for the development of specific drugs for the treatment of disorders of the CNS in which cytokines play a pathogenic role.
In Vivo Expression of Inducible Nitric Oxide Synthase in Cerebellar Neurons
Authors: Minc‐Golomb Dahlia , Yadid Gal , Tsarfaty Ilan , Resau James H. , Schwartz Joan P.
Published: July 2010

Abstract: In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS—both the neuronal and the endothelial types. The other class of NOS—the inducible NOS (iNOS)—is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon‐γ) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser‐scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults.
Identification and Determination of 3,4‐Dihydroxyphenylacetaldehyde, the Dopamine Metabolite, in In Vivo Dialysate from Rat Striatum
Authors: Colzi A. , Musolino A. , Iuliano A. , Fornai F. , Bonuccelli U. , Corsini G. U.
Published: July 2010

Abstract: 3,4‐Dihydroxyphenylacetic acid (DOPAC) is commonly considered to be the main dopamine (DA) metabolite produced by monoamine oxidase (MAO); however, the initial product of DA oxidation is 3,4‐dihydroxyphenylacetaldehyde (DOPALD). Owing to technical difficulties in detecting DOPALD from a biological matrix, no studies have so far been performed to measure brain levels of this aldehyde in vivo. In this work, using transstriatal microdialysis in freely moving rats, we identified DOPALD by HPLC coupled to a coulometric detector. In chromatograms obtained from microdialysis samples, DOPALD appeared as a peak with a retention time coincident with that of the standards obtained via enzymatic and chemical synthesis. On the other hand, DOPALD was undetectable ex vivo from rat striatal homogenates. This discrepancy is probably due to the preferential extraneuronal localization together with the high reactivity of the aldehyde, which is rapidly removed by the dialysis probe, whereas the ex vivo procedure allows its condensation and enzymatic conversion. Measurement of DOPALD levels as a routine procedure might represent a reliable tool to evaluate DA oxidative metabolism directly, in vivo. Moreover, parallel detection of DOPALD and DOPAC levels in brain dialysate may make it possible to distinguish between the activity of MAO and aldehyde dehydrogenase. DOPALD, like many endogenous aldehydes, has been shown to be toxic to the cell in which it is formed. Therefore, in vivo measurement of DOPALD levels could highlight new aspects in the molecular mechanisms underlying both acute neurological insults and neurodegenerative diseases.
β‐Dystroglycan: Subcellular Localisation in Rat Brain and Detection of a Novel Immunologically Related, Postsynaptic Density‐Enriched Protein
Authors: Mummery Rosemary , Sessay Abdul , Lai F. Anthony , Beesley Philip W.
Published: July 2010

Abstract: The distribution of a glycoprotein component of the muscle dystrophin complex, β‐dystroglycan, has been determined in subcellular fractions of adult rat forebrain. The results show that β‐dystroglycan is enriched in several membrane fractions, including synaptic membranes, but in marked contrast to dystrophin is not detectable in the postsynaptic density fraction. The antiserum also recognises a second molecular species of apparent molecular mass of 164 kDa which is highly enriched in the postsynaptic density fraction. Preabsorption of the antiserum with the antigen (a 22‐mer peptide corresponding to the C‐terminal sequence of rabbit skeletal muscle β‐dystroglycan) abolished reactivity against both β‐dystroglycan and the 164‐kDa postsynaptic density‐enriched protein, confirming that the two species are immunologically related. Enzymatic removal of N‐linked oligosaccharide lowered the apparent molecular mass of β‐dystroglycan by 3 kDa but did not alter the mass of the 164‐kDa species.
Amyloid β Protein Primes Cultured Rat Microglial Cells for an Enhanced Phorbol 12‐Myristate 13‐Acetate‐Induced Respiratory Burst Activity
Authors: Van Muiswinkel F. L. , Veerhuis R. , Eikelenboom P.
Published: July 2010

Abstract: Activated microglia, often associated with neuritic amyloid plaques in the Alzheimer's disease brain, are likely to contribute to the progression of the disease process, e.g., by releasing neurotoxic reactive oxygen and/or nitrogen intermediates. In the present study, whether the amyloid β peptide (Aβ), the principal constituent of amyloid plaques, can stimulate microglial respiratory burst activity and/or microglial production of nitric oxide was examined. Using neonatal rat microglial cultures as a model, it was found that neither the spontaneous release of nitric oxide nor the lipopolysaccharide‐induced production of nitric oxide was altered in cultures previously incubated with synthetic Aβ(1–40). for 24 h. In addition, no direct stimulatory effect of Aβ(1–40) on the respiratory burst activity was observed. Nevertheless, concomitant with an increase in the number of responsive cells, a profound priming of the phorbol 12‐myristate 13‐acetate‐evoked production of superoxide anion was observed in Aβ(1–40)‐treated cultures. Thus, both the maximal rate and the total phorbol 12‐myristate 13‐acetate‐induced production of superoxide appeared to be statistically significantly higher as compared with untreated cultures. It is concluded that, as far as activation of the microglial respiratory burst is concerned, Aβ(1–40) may merely act as a priming rather than a triggering stimulus.
Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors
Authors: Joseph Joan , Cruz‐Sánchez Félix F. , Carreras José
Published: July 2010

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα‐, αγ‐, and γγ‐enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα‐enolase was the predominant isoenzyme, and αγ‐enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα‐enolase and a lower proportion of γγ‐enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα‐enolase and lowest proportion of γγ‐enolase.
Interleukin‐1β Induces Prostaglandin G/H Synthase‐2 (Cyclooxygenase‐2) in Primary Murine Astrocyte Cultures
Authors: O'Banion M. Kerry , Miller Janice C. , Chang Julia W. , Kaplan Mitchell D. , Coleman Paul D.
Published: July 2010

Abstract: Activation of glial cells and the consequent release of cytokines, proteins, and other intercellular signaling molecules is a well‐recognized phenomenon in brain injury and neurodegenerative disease. We and others have previously described an inducible prostaglandin G/H synthase, known as PGHS‐2 or cyclooxygenase‐2, that is up‐regulated in many cell systems by cytokines and growth factors and down‐regulated by glucocorticoid hormones. In cultured mouse astrocytes we observed increased production of prostaglandin E2 (PGE2) after stimulation with either interleukin‐1β (IL‐1β) or the protein kinase C activator phorbol 12‐myristate 13‐acetate (TPA). This increase in PGE2 content was blocked by pretreatment with dexamethasone and correlated with increases in cyclooxygenase activity measured at 4 h. Northern blots revealed concomitant increases in PGHS‐2 mRNA levels that peaked at 2 h and were dependent on the dosage of IL‐1β. Dexamethasone inhibited this induction of PGHS‐2 mRNA by IL‐1β. TPA, basic fibroblast growth factor, and the proinflammatory factors tumor necrosis factor α and lipopolysaccharide, but not interleukin‐6, also stimulated PGHS‐2 mRNA expression. Relative to IL‐1β, the greater increases in PGE2 production and cyclooxygenase activity caused by TPA correlated with a greater induction of PGHS‐2 mRNA. Furthermore, NS‐398, a specific inhibitor of cyclooxygenase‐2, blocked >80% of the cyclooxygenase activity in TPA‐treated astrocytes. These findings indicate that increased expression of PGHS‐2 contributes to prostaglandin production in cultured astrocytes exposed to cytokines and other factors.
Regulation of GTP Cyclohydrolase I Gene Expression and Tetrahydrobiopterin Content in Cultured Sympathetic Neurons by Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor
Authors: Stegenga Susan L. , Hirayama Kei , Kapatos Gregory
Published: July 2010

Abstract: Cultures of neonatal rat superior cervical ganglia (SCG) were used to test the hypothesis that the cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) control GTP cyclohydrolase I (GTPCH) gene expression and 5,6,7,8‐tetrahydrobiopterin (BH4) content as traits of the noradrenergic phenotype. Treatment for 7 days with 1 ng/ml of LIF was found to produce the characteristic switch in the SCG neurotransmitter phenotype reported by others, as evidenced by a 60% decline in tyrosine hydroxylase (TH) activity and a 75% increase in choline acetyltransferase activity. This LIF treatment paradigm decreased BH4 levels in a concentration‐dependent manner, with a maximal decline of 60% observed at 1 ng/ml. Analysis of the time course of this response indicated that LIF decreased BH4 levels by 60% following 3–7 days of treatment. Treatment of cultures with CNTF (2 ng/ml) resulted in a decline in BH4 levels that was of equal magnitude and followed the same time course as that produced by LIF. The LIF‐dependent decline in BH4 levels resulted from a reduction in GTPCH enzyme activity, which decreased by 75% following 7 days of treatment. Nuclease protection assays of RNA extracted from cells treated for 7 days with 2 ng/ml of LIF or CNTF detected a 78–96% reduction in GTPCH mRNA content relative to β‐actin mRNA content. Concomitant decreases in TH and GTPCH gene expression in response to LIF or CNTF demonstrate a coordinated regulation of gene expression for this BH4‐dependent enzyme and the rate‐limiting enzyme in the synthesis of its essential cofactor, BH4. Moreover, these results indicate that GTPCH gene expression in SCG neurons should be regarded as a trait of the noradrenergic phenotype.
Effect of Hypoxia on Na+‐K+‐Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat
Authors: Kawai Nobutoshi , McCarron Richard M. , Spatz Maria
Published: July 2010

Abstract: The effect of hypoxia on Na+,K+‐ATPase and Na+‐K+‐Cl− cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring 86Rb+ uptake as a tracer for K+. RBECs expressed both Na+,K+‐ATPase and Na+‐K+‐Cl− cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na+,K+‐ATPase activity by 38.9%, whereas it significantly increased Na+‐K+‐Cl− cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin‐induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time‐dependent decrease of cellular ATP content (by ∼65%) along with a complete inhibition of Na+,K+‐ATPase and a coordinated increase of Na+‐K+‐Cl− cotransport activity (up to 100% above control values). Oligomycin augmentation of Na+‐K+‐Cl− cotransport activity was not observed in the presence of 2‐deoxy‐d‐glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na+,K+‐ATPase activity is reduced, RBECs have the ability to increase K+ uptake through Na+‐K+‐Cl− cotransport.
Allelic Variation of Human Serotonin Transporter Gene Expression
Authors: Heils Armin , Teufel Andreas , Petri Susanne , Stöber Gerald , Riederer Peter , Bengel Dietmar , Lesch K. Peter
Published: July 2010

Abstract: Mood, emotion, cognition, and motor functions as well as circadian and neuroendocrine rhythms, including food intake, sleep, and reproductive activity, are modulated by the midbrain raphe serotonin (5‐HT) system. By directing the magnitude and duration of postsynaptic responses, carrier‐facilitated 5‐HT transport into and release from the presynaptic neuron are essential for the fine tuning of serotonergic neurotransmission. Interest in the mechanism of environmental factor‐, disease‐, and therapy‐induced modification of 5‐HT transporter (5‐HTT) function and its impact on early brain development, event‐related synaptic plasticity, and neurodegeneration is widespread and intensifying. We have recently characterized the human and murine 5‐HTT genes and performed functional analyses of their 5′‐flanking regulatory regions. A tandemly repeated sequence associated with the transcriptional apparatus of the human 5‐HTT gene displays a complex secondary structure, represses promoter activity in nonserotonergic neuronal cells, and contains positive regulatory components. We now report a novel polymorphism of this repetitive element and provide evidence for allele‐dependent differential 5‐HTT promoter activity. Allelic variation in 5‐HTT‐related functions may play a role in the expression and modulation of complex traits and behavior.
New Species of Human Tyrosine Hydroxylase mRNA Are Produced in Variable Amounts in Adrenal Medulla and Are Overexpressed in Progressive Supranuclear Palsy
Authors: Dumas Sylvie , Le Hir Hervé , Bodeau‐Péan Sylvie , Hirsch Etienne , Thermes Claude , Mallet Jacques
Published: July 2010

Abstract: Alternative splicing of human tyrosine hydroxylase (TH) pre‐mRNA produces four mRNAs leading to four different TH isoforms and is thought to have important regulatory functions. We show that the diversity of TH mRNAs is greater than previously described in the autonomous nervous system: New splice junctions corresponding to the skipping of exon 3 were identified by amplification of cDNA synthesized from pheochromocytoma RNA. In all cases the reading frame was maintained. These species were assayed by RNase protection experiments; their abundance (4–6%) was comparable to that of the previously identified human TH‐3 and ‐4 species in normal adrenal medulla. However, higher levels (11–34%) of these species were found in adrenal medullas of patients suffering from progressive supranuclear palsy. Whether such changes are specific to the disease or the consequences of the stress associated with this severe neurodegeneration remains to be established.
The C‐Terminal Domain of the mGluR1 Metabotropic Glutamate Receptor Affects Sensitivity to Agonists
Authors: Flor P. J. , Gomeza J. , Tones M. A. , Kuhn R. , Pin J.‐P. , Knöpfel T.
Published: July 2010

Abstract: The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (−1a, −1b, and −1c) generated by alternative splicing at the C‐terminal domain. Fluorometric Ca2+ measurements were used to compare the concentration dependency of agonist‐induced rises in intracellular free Ca2+ concentration ([Ca2+]i) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate ≫ (2S,1′S,2′S)‐2‐(carboxycyclopropyl)glycine (L‐CCG‐I) > (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid [(1S,3R)‐ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C‐terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca2+]i in response to agonists. The potency of agonists was higher at the chimera having the C‐terminus of mGluR1a as compared with those having the mGluR1c C‐terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L‐CCG‐I ≫ (1S,3R)‐ACPD ∼ quisqualate. These data support the hypothesis that the C‐terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR‐mediated functional responses.
Adenosine Release and Uptake in Cerebellar Granule Neurons Both Occur via an Equilibrative Nucleoside Carrier that Is Modulated by G Proteins
Authors: Sweeney M. I.
Published: July 2010

Abstract: There is debate about the mechanisms mediating adenosine release from neurons. In this study, the release of adenosine evoked by depolarizing cultured cerebellar granule neurons with 50 mM K+ was inhibited by 49 ± 7% in Ca2+‐free medium. The remaining release was blocked by dipyridamole (IC50 = 6.4 × 10−8M) and nitrobenzylthioinosine (IC50 = 3.6 × 10−8M), inhibitors of adenosine uptake. Ca2+‐dependent release was reduced by 78 ± 9% following a 21‐h pretreatment of the cells with pertussis toxin, which ADP‐ribosylates Gi/Go G proteins, thereby preventing their dissociation. The nucleoside transporter‐mediated component of K+‐induced adenosine release also was inhibited by 62 ± 8% by pertussis toxin and was potentiated by 78 ± 11% following cholera toxin treatment, which permanently activates Gs. Uptake of [3H]adenosine into cultured cerebellar granule neurons over a 10‐min period was not dependent on extracellular Na+ but was reduced by dipyridamole (IC50 = 3.2 × 10−8M) and nitrobenzylthioinosine (IC50 = 2.6 × 10−8M). Thus, adenosine uptake likely occurs via the same transporter mediating Ca2+‐independent adenosine release. Adenosine uptake was potentiated by cholera toxin pretreatment (152 ± 15% of control), but pertussis toxin had no statistically significant effect. It is possible that Gs, Gi/Go, or free Gβγ dimer modulate the equilibrative, inhibitor‐sensitive nucleoside carrier to enhance adenosine transport.
Nerve Growth Factor and Ras Regulate β‐Amyloid Precursor Protein Gene Expression in PC12 Cells
Authors: Cosgaya José Miguel , Latasa Maria Jesús , Pascual Angel
Published: July 2010

Abstract: The β‐amyloid protein, the major component of the vascular and plaque amyloid deposits that characterize Alzheimer's disease, derives from a larger β‐amyloid precursor protein (APP) that is expressed in both neural and nonneural cells. An increased expression of APP might actively contribute to the development of the pathology; however, the mechanisms involved in the regulation of APP gene expression are not yet well understood. In PC12 cells, a rat pheochromocytoma cell line, we have demonstrated that nerve growth factor (NGF) induces the APP gene expression and increases APP mRNA levels in the presence of 0.5 or 15% serum. Expression of activated ras in the PC12 cell subline UR61 also leads to a significant increase in content of APP transcripts, and a dominant negative mutant of ras blocks the NGF‐induced response. Other ligands of tyrosine kinase receptors, such as fibroblast growth factor, which causes morphological differentiation, or epidermal growth factor, which induces cell growth, also increase APP mRNA levels in PC12 cells. These results suggest that ras mediates the induction of APP gene expression by NGF and other ligands of tyrosine kinase receptors.
Adenylyl Cyclases: mRNA and Characteristics of Enzyme Activity in Three Areas of Brain
Authors: Hellevuo Kaisa , Hoffman Paula L. , Tabakoff Boris
Published: July 2010

Abstract: RNase protection assays were used in a comparative analysis of the quantities of mRNA for five “calcium‐sensitive” (types I, III, V, VI, and VIII) adenylyl cyclases and one “calcium‐insensitive” (type II) adenylyl cyclase in mouse cerebral cortex, cerebellum, and nucleus accumbens. The mRNA levels for type V adenylyl cyclase were dominant in the nucleus accumbens. Type V adenylyl cyclase mRNA was also found in the cerebral cortex and at low levels in the cerebellum. Type I adenylyl cyclase mRNA was the major form in the cerebellum with 15–50‐fold higher levels compared with other adenylyl cyclase mRNAs. Type I adenylyl cyclase mRNA was also the most prominent adenylyl cyclase mRNA in the cerebral cortex, although the mRNA levels of other adenylyl cyclase forms were more comparable to those of the type I enzyme in this brain area. The mRNA levels for adenylyl cyclase types II, III, VI, and VIII were intermediate to low depending on the brain area. Cell membranes from the nucleus accumbens demonstrated adenylyl cyclase activity that was synergistically activated by concomitant addition of GTP and forskolin to assay mixtures, reflecting a characteristic of type V adenylyl cyclase protein. Calcium/calmodulin stimulated adenylyl cyclase activity in membranes from all three brain areas. However, synergistic activation of adenylyl cyclase activity by GTP and calcium/calmodulin was noted only with cortical membranes, and this characteristic may reflect the presence of type VIII adenylyl cyclase mRNA in the cortex. Although mRNA for type VIII adenylyl cyclase was almost equivalent in the cortex and cerebellum, the lack of a synergistic effect of GTP plus calcium/calmodulin on the cerebellar enzyme activity may be a result of the significant dominance of type I adenylyl cyclase mRNA (and protein) in the cerebellum. In general, the mRNA levels for the various adenylyl cyclases were predictive of the regulatory characteristics of adenylyl cyclase activity in membranes of the brain areas studied.
Presence of Pituitary Adenylate Cyclase‐Activating Polypeptide Receptors in Y‐79 Human Retinoblastoma Cells
Authors: Olianas M. C. , Ennas M. G. , Lampis G. , Onali P.
Published: July 2010

Abstract: Cytochemical analysis demonstrated that a high percentage of human Y‐79 retinoblastoma cells displayed a specific labeling by the biotinyl derivative of pituitary adenylate cyclase‐activating polypeptide (PACAP), a novel neuropeptide of the secretin‐vasoactive intestinal peptide (VIP) family of peptides. In cell membranes, the two molecular forms of PACAP, the one with 38 (PACAP 38) and the other with 27 (PACAP 27) amino acids, displaced the binding of 125I‐PACAP 27 with IC50 values in the picomolar range and increased adenylyl cyclase activity by 100‐fold with EC50 values of 27 and 180 pM, respectively. VIP, human peptide histidine‐isoleucine, glucagon, and secretin were much less effective and potent in both receptor assays. The PACAP receptor antagonists PACAP 6–27 and PACAP 6–38 and an antiserum directed against the stimulatory G protein Gs inhibited the PACAP stimulation of adenylyl cyclase. In intact cells, both PACAPs and VIP failed to stimulate the phosphoinositide hydrolysis, whereas in cell membranes PACAP 38, but not the other peptides, produced a modest increase (40%) of inositol phosphate formation with an EC50 value of 22 nM. However, this effect was not antagonized by either PACAP 6–38 or PACAP 6–27. These data demonstrate the presence in human Y‐79 retinoblastoma cells of specific PACAP receptors and provide further evidence that PACAP may act as a neurotransmitter/neuromodulator in mammalian retina.
The Dual Peptidase Inhibitor RB101 Induces a Long‐Lasting Increase in the Extracellular Level of Met‐Enkephalin‐Like Material in the Nucleus Accumbens of Freely Moving Rats
Authors: Daugé Valérie , Mauborgne Annie , Cesselin François , Fournié‐Zaluski Marie‐Claude , Roques Bernard‐Pierre
Published: July 2010

Abstract: This study was carried out to analyze the extracellular levels of Met‐enkephalin‐like material in the nucleus accumbens, a brain structure involved in the effects of opioids on motor activity and reward processes, using microdialysis in awake and freely moving rats, combined with a sensitive radioimmunoassay. The levels of Met‐enkephalin‐like material were measured after administration of a dual inhibitor of enkephalin‐degrading enzymes, RB101, to evaluate its in vivo protecting effects. The basal levels of Met‐enkephalin‐like immunoreactivity in the nucleus accumbens were ∼1.2 pg/30 min or 2.2 fmol/30 min (37 pM). Perfusion of KCl (100 mM) produced a 17‐fold increase in the level of Met‐enkephalin‐like material in this structure. During the 8‐h perfusion, which started at 9 a.m., a spontaneous increase of the basal level of Met‐enkephalin‐like material in the nucleus accumbens occurred between 4 and 4:30 p.m., suggesting the existence of variation in opioid peptide secretion, at least in this structure. Intraperitoneal injection of RB101 induced a dose‐dependent and long‐lasting (210‐min) increase in the extracellular levels of Met‐enkephalin‐like material. A prolonged effect was also observed in the behavioral studies in which the inhibitor increased global motor activity of rats 210 min after injection. These data represent the first direct evidence that dual inhibitors of enkephalin‐degrading enzymes increase in vivo the extracellular levels of Met‐enkephalin‐like material in awake and freely moving rats.
Time‐Dependent Increase in Release of Arachidonic Acid Following Passive Avoidance Training in the Day‐Old Chick
Authors: Clements M. P. , Rose S. P. R.
Published: July 2010

Abstract: We have investigated the role of arachidonic acid, a putative retrograde messenger, in a one‐trial aversive learning task in the day‐old chick. The left and right intermediate medial hyperstriatum ventrale (IMHV) in the chick forebrain have previously been implicated in the formation of memory for this task. Using an ex vivo technique we have determined the concentrations of various fatty acids liberated from prisms prepared from these brain regions at different time points up to 24 h following passive avoidance training. At 30, 60, and 75 min post‐training the concentration of arachidonic acid, but not of other fatty acids, in prisms prepared from the left IMHV, but not the right IMHV, was enhanced compared with that in chicks trained on a nonaversive water‐coated bead. To test whether arachidonic acid liberation from the left IMHV was receptor‐stimulated we showed that (a) liberation of endogenous arachidonic acid from homogenate prepared from the left and right IMHV of untrained chicks was stimulated by depolarization with KCl (50 mM) and that (b) glutamate agonists of the NMDA and metabotropic subtypes of glutamate receptor stimulated release of preloaded [14C]arachidonic acid from prisms prepared from the left IMHV but not the right IMHV. These results indicate that arachidonic acid is liberated from the left IMHV following passive avoidance training in the day‐old chick and may play a role as a retrograde messenger in this memory task.
Brain S‐Adenosylmethionine Levels Are Severely Decreased in Alzheimer's Disease
Authors: Morrison Lesley D. , Smith David D. , Kish Stephen J.
Published: July 2010

Abstract: S‐Adenosylmethionine is an essential ubiquitous metabolite central to many biochemical pathways, including transmethylation and polyamine biosynthesis. Reduced CSF S‐adenosylmethionine levels in Alzheimer's disease have been reported; however, no information is available regarding the status of S‐adenosylmethionine or S‐adenosylmethionine‐dependent methylation in the brain of patients with this disorder. S‐Adenosylmethionine concentrations were measured in postmortem brain of 11 patients with Alzheimer's disease. We found decreased levels of S‐adenosylmethionine (−67 to −85%) and its demethylated product S‐adenosylhomocysteine (−56 to −79%) in all brain areas examined (cerebral cortical subdivisions, hippocampus, and putamen) as compared with matched controls (n = 14). S‐Adenosylmethionine and S‐adenosylhomocysteine levels were normal in occipital cortex of patients with idiopathic Parkinson's disease (n = 10), suggesting that the decreased S‐adenosylmethionine levels in Alzheimer's disease are not simply a consequence of a chronic, neurodegenerative condition. Reduced S‐adenosylmethionine levels could be due to excessive utilization in polyamine biosynthesis. The severe reduction in levels of this essential biochemical substrate would be expected to compromise seriously metabolism and brain function in patients with Alzheimer's disease and may provide the basis for the observations of improved cognition in some Alzheimer's patients following S‐adenosylmethionine therapy.
Role of Egr‐1 in Cholinergic Stimulation of Phenylethanolamine N‐Methyltransferase Promoter
Authors: Morita Kyoji , Wong Dona L.
Published: July 2010

Abstract: The effects of the cholinergic agonist carbachol on phenylethanolamine N‐methyltransferase promoter activity and Egr‐1 mRNA expression in PC12‐derived RS1 cells were examined to investigate the potential involvement of Egr‐1 in the neural regulation of phenylethanolamine N‐methyltransferase gene expression. Carbachol stimulated luciferase expression in cells transfected with a rat phenylethanolamine N‐methyltransferase promoter‐luciferase reporter gene construct and also elevated Egr‐1 mRNA levels in untransfected cells. Maximum induction of Egr‐1 mRNA by carbachol was rapid (0.5 h), whereas by comparison, peak luciferase activity was delayed (6 h). In addition, carbachol stimulation of both luciferase and Egr‐1 mRNA expression could be completely inhibited by atropine but not hexamethonium. Furthermore, bethanechol but not nicotine could mimic the effects of carbachol, indicating that carbachol activation was medicated through muscarinic cholinergic receptors. Finally, carbachol failed to stimulate luciferase expression in cells transfected with a mutant construct, in which the Egr‐1 binding element in the phenylethanolamine N‐methyltransferase promoter was mutated. These results suggest that carbachol activates the phenylethanolamine N‐methyltransferase promoter through stimulation of Egr‐1 expression, and are consistent with the potential involvement of Egr‐1 in the cholinergic activation of the phenylethanolamine N‐methyltransferase gene.
Interleukin‐6 Induces Expression of Peripherin and Cooperates with Trk Receptor Signaling to Promote Neuronal Differentiation in PC12 Cells
Authors: Sterneck Esta , Kaplan David R. , Johnson Peter F.
Published: July 2010

Abstract: In contrast to the intensively studied nerve growth factor (NGF)‐related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin‐6 (IL‐6). We have examined the mechanisms of IL‐6‐induced neuronal differentiation of the pheochromocytoma cell line PC12. IL‐6 independently induced the expression of peripherin, identifying this gene as the first neuronal‐specific target of IL‐6. However, IL‐6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL‐6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL‐6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk‐1 mitogen‐activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL‐6 receptor, providing another potential mechanism of cooperativity between NGF and IL‐6 signaling. We propose that IL‐6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor‐specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling activity by the IL‐6 receptor system.