bluuebunny/crossref_metadata_2025 huggingface dataset:
Lithium Enhances Muscarinic Receptor–Stimulated CDP‐Diacylglycerol Formation in Inositol‐Depleted SK‐N‐SH Neuroblastoma Cells
Authors: Stubbs Evan B. , Agranoff Bernard W.
Published: October 2006

Abstract: The psychotherapeutic action of Li+ in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase‐mediated signal transduction in affected cells. In the present study, the effect of Li+ on muscarinic receptor–stimulated formation of the immediate precursor of phosphatidylinositol, CDP‐diacylglycerol (CDP‐DAG), has been examined in human SK‐N‐SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo‐inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 ± 4 nmol/mg of protein. The addition of carbachol to [3H]cytidine‐prelabeled cells elicited a four‐ to fivefold increase in the accumulation of labeled CDP‐DAG. This stimulated formation of [3H]CDP‐DAG was completely blocked by the addition of 10 μM atropine, was not dependent on the presence of Li+, nor was it affected by co‐incubation with myo‐inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol‐stimulated formation of [3H]CDP‐DAG was potentiated ∼ 10‐fold by Li+ and substantially reduced by coincubation with inositol. The formation of [3H]CDP‐DAG in labeled SK‐N‐SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [3H]‐CDP‐DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol ≥ oxotremorine‐M > bethanecol ≥ arecoline > oxotremorine > pilocarpine. Extended culture of the SK‐N‐SH cells in an inositol‐free chemically defined growth medium progressively reduced the intracellular inositol content to <5 nmol/mg of protein, a level comparable with that seen in cortical slices. In these inositol‐depleted cells, Li+ potentiated carbachol‐stimulated [3H]CDP‐DAG formation, and this effect was completely reversed by coincubation with inositol (EC50 0.2 mM). The present study thus demonstrates, in the same cultured cell line, the effects of normal and reduced intracellular inositol levels on the ability of Li+ to attenuate phosphoinositide resynthesis, as inferred from [3H]CDP‐DAG accumulation. The results indicate that Li+ can lead to a slowing of stimulated phosphoinositide turnover in neuroblastoma cells, provided that the intracellular inositol content has been significantly reduced.
Determination of Amino Acids and Monoamine Neurotransmitters in Caudate Nucleus of Seizure‐Resistant and Seizure‐Prone BALB/c Mice
Authors: Vriend Jerry , Alexiuk Nancy A. M. , Green‐Johnson Julia , Ryan Elizabeth
Published: October 2006

Abstract: Amino acid and monoamine concentrations were examined in tissue extracts of caudate nucleus of genetic substrains of BALB/c mice susceptible or resistant to audiogenic seizures. Amino acids [aspartate, glutamate, glycine, taurine, serine, γ‐aminobutyric acid (GABA)], monoamines, and related metabolites were separated by isocratic reverse‐phase chromatography and detected by a coulometric electrode array system. In situ activity of tyrosine hydroxylase and tryptophan hydroxylase were determined by measuring the accumulation of L‐DOPA and 5‐hydroxytryptophan after administration of the decarboxylase inhibitor NSD‐1015. Highly significant decreases in concentrations of both excitatory (glutamate and aspartate) and inhibitory amino acids (GABA and taurine) were observed in extracts of caudate nucleus of seizure‐prone mice. Substantial decreases in concentrations of dopamine (DA) and its metabolites, 3,4‐dihydroxyphenylacetic acid and homovanillic acid, were also noted. Decreased accumulation of L‐DOPA after NSD‐1015 administration provided evidence for decreased tyrosine hydroxylase activity and decreased DA synthesis in striatum of seizure‐prone mice compared with seizure‐resistant mice. Decreased concentrations of the DA metabolite 3‐methoxytyramine (after NSD‐1015 administration) suggested that DA release was also compromised in seizure‐prone mice. No significant difference in 5‐hydroxytryptophan accumulation in striatum of seizure‐prone and seizure‐resistant mice suggested that tryptophan hydroxylase activity and serotonin synthesis were not affected. The data suggest that seizure‐prone BALB/c mice have a deficiency in intracellular content of both excitatory and inhibitory amino acids. The data also raise the issue of whether GABAergic interactions with the nigrostriatal DA system are important in the regulation of audiogenic seizure susceptibility.
Deletions of the Synenkephalin Domain Which Do Not Alter Cell‐Specific Proteolytic Processing or Secretory Targeting of Human Proenkephalin
Authors: Albert Paul R. , Liston Dane
Published: October 2006

Abstract: To identify signals that direct the proteolytic processing and regulated secretion of human proenkephalin (hPE), we have transfected the hPE gene or minigene constructs into pituitary tumor cells, either rat GH4C1 cells or mouse AtT‐20 cells. Cells transfected with either the hPE gene or minigene contained similar levels of methionine‐enkephalin (ME)‐containing peptides and hPE mRNA. In the GH4C1 clones, ME was present predominantly in high‐molecular‐mass forms (5–25 kDa). In contrast, the AtT‐20 clones contained almost exclusively free ME and low‐molecular‐mass forms (<5 kDa), with very little high‐molecular‐mass species present. Thus, among pituitary cells, corticotroph‐derived cells appear better equipped to process hPE than lactotroph‐derived cells. Despite limited proteolytic processing, GH4C1 clones secreted large amounts of unprocessed (>20 kDa) hPE into the medium, making up to 10% of endogenous rat prolactin secretion. Both precursor and processed forms of ME were cosecreted acutely (<1 h) with rat prolactin, and release of both polypeptides was stimulated up to 12‐fold by secretagogues. Thus, complete proteolytic processing was not required for accurate targeting of hPE to the regulated secretory pathway. When transfected with constructs bearing deletions of amino‐terminal amino acids 2–43 or 2–67, i.e., part or nearly all of the synenkephalin moiety, GH4C1 cells handled the modified protein much like cells expressing the complete protein. They did not process the modified hPE extensively, but the protein was correctly targeted to the regulated secretory pathway. AtT‐20 cells transfected with truncated hPE cDNA constructs expressed and processed the protein as efficiently as cells expressing unmodified hPE and expressed predominantly low‐molecular‐mass forms of ME. Therefore, the structural features required for correct targeting and processing are not present in the cysteine‐rich amino‐terminal third of the prohormone. It is interesting that the deletions did not include the SHLL peptide motif in synenkephalin, a motif that has been proposed as a sorting signal.
Regulation of β1‐Adrenergic Receptor mRNA and Ligand Binding by Antidepressant Treatments and Norepinephrine Depletion in Rat Frontal Cortex
Authors: Hosoda Kohkichi , Duman Ronald S.
Published: October 2006

Abstract: The number of β1‐adrenergic receptor (β1AR) binding sites is decreased by chronic antidepressant treatments, including electroconvulsive seizure (ECS) and imipramine, whereas administration of agents that deplete norepinephrine (NE) increases the number of β1AR binding sites in cerebral cortex. The present study was carried out to examine the influence of these treatments on levels of β1 AR mRNA in frontal cortex to study the molecular mechanisms that underlie the regulation of β1 ARs in brain. Levels of β1 AR mRNA were measured by RNase protection analysis using a riboprobe derived from rat β1AR cDNA, and the levels of βAR binding were measured using the nonselective ligand [3H]CGP‐12177. Studies to verify the specificity of the RNase protection assay revealed that the distribution of β1AR mRNA was in agreement with the reported distribution of β1AR ligand binding: Levels of β1AR mRNA were highest in cerebral cortex or frontal cortex, intermediate in neostriatum, hippocampus, lung, and heart, and lowest in cerebellum, kidney, and liver. Chronic ECS treatment (once daily for 10 days) significantly decreased levels of βAR ligand binding and resulted in a corresponding, time‐dependent down‐regulation of β1AR mRNA levels in frontal cortex. However, imipramine administration regulated levels of β1AR mRNA in a biphasic manner, with treatments for 7–14 days increasing and treatments for 18–21 days decreasing levels of β1AR mRNA in frontal cortex. In contrast, levels of [3H]CGP‐12177 ligand binding were decreased at all time points examined (3–21 days). The influence of NE depletion, using the neurotoxin 6‐hydroxy‐dopamine (6‐OHDA), on levels of β1AR mRNA was also examined. Three days after 6‐OHDA treatment, levels of [3H]CGP‐12177 ligand binding were not altered, but 7–14 days after neurotoxin treatment, levels of ligand binding were significantly increased. In contrast, 3–9 days after 6‐OHDA treatment, levels of β1AR mRNA were significantly decreased, and 14 days after treatment, levels of β1AR mRNA returned to control values. The results demonstrate that β1AR mRNA and ligand binding are regulated in parallel by ECS treatment but that levels of receptor mRNA are regulated in a complex manner by imipramine or 6‐OHDA treatments, not predicted by changes in ligand binding.
Differential Glycosylation of the 5A11/HT7 Antigen by Neural Retina and Epithelial Tissues in the Chicken
Authors: Fadool James M. , Linser Paul J.
Published: October 2006

Abstract: The 5A11/HT7 antigen, a member of the immunoglobulin supergene family, has been implicated in heterotypic cell‐cell interactions during retina development. Immunopurified 5A11 antigen isolated from Nonidet P‐40‐solubilized retina membranes had two components as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), a 45.5‐kDa doublet and a 69‐kDa polypeptide. Immunoreactive bands of 46‐50 kDa were recognized following SDS‐PAGE of detergent‐solubilized membrane proteins from liver, kidney, and erythrocytes. Treatment with N‐glycosidase F (EC 3.2.2.18) converted the 45.5–50‐kDa immunoreactive polypeptides from all tissues to 32 kDa, indicating that the observed differences in molecular mass were due to differences in glycosylation. N‐Glycosidase Ftreatment also converted the 69‐kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32‐kDa species. Treatment with endo‐β‐N‐acetylglucosaminidase H (EC 3.2.1.96) resulted in modest increases in electrophoretic mobility due to hydrolysis of high mannose or hybrid oligosaccharides and lack of hydrolysis of complex oligosaccharides resistant to endo‐β‐N‐acetylglucosaminidase H digestion. Immunoreactivity was retained after deglycosylation. Much of the difference in molecular weight could be attributed to variations in sialylation. The higher molecular mass species of the 45.5‐kDa doublet from retina and the polypeptides from other tissues were susceptible to neuraminidase (EC 3.2.1.18) and O‐glycosidase (endo‐α‐N‐acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for α2, 6‐linked sialic acid) was confined to the higher molecular mass species of the 45.5‐kDa doublet and was considerably greater in antigen derived from epithelia rather than neural retina. In paraffin sections of chick retina, elderberry bark lectin staining was confined to the retinal pigmented epithelium, photoreceptor cells, and bipolar cells with no staining of the Müller cells, which bear the bulk of the 5A11 antigen. These results indicate tissue‐specific posttranslational modifications, particularly differences in sialylation of antigen‐bearing polypeptides.
Neurofilament Monoclonal Antibodies RT97 and 8D8 Recognize Different Modified Epitopes in Paired Helical Filament‐τ in Alzheimer's Disease
Authors: Brion Jean‐Pierre , Couck Anne‐Marie , Robertson Janice , Loviny Thérèse L. F. , Anderton Brian H.
Published: October 2006

Abstract: Neurofibrillary tangles in Alzheimer's disease have been previously found to be labeled by some neurofilament antibodies that also recognize τ proteins. We have studied the reactivity of two such monoclonal antibodies, RT97 and 8D8, and of an anti‐ubiquitin serum with the abnormal paired helical filaments (PHF)‐τ (A68) polypeptides known to be the main component of the PHFs constituting the neurofibrillary tangles. 8D8 recognized the three major PHF‐τ polypeptides, but RT97 reacted only with the two larger PHF‐τ species. PHF‐τ polypeptides were labeled by 8D8 and RT97 much more strongly than normal human τ and this labeling was decreased after alkaline phosphatase treatment. Anti‐ubiquitin and anti‐phosphotyrosine antibodies did not label PHF‐τ polypeptides. The immunoreactivity of proteolytic fragments of PHF‐τ polypeptides was studied with RT97, 8D8, and a panel of τ antibodies. The epitope for 8D8 on PHF‐τ was localized between amino acids 222 and 427 in the carboxyl half of τ. The RT97 epitope on PHF‐τ was localized in the amino domain of τ, probably in the 29‐amino‐acid insertion (insert 1) found towards the amino terminus of some τ isoforms. These results show that the basis for the labeling of neurofibrillary tangles by antibodies 8D8 and RT97 to neurofilament is their ability to react with PHF‐τ polypeptides by recognizing sites specifically modified on PHF‐τ, including a site specific to some τ isoforms.
Antibodies Specific for α‐Subunit Subtypes of GABAA Receptors Reveal Brain Regional Heterogeneity
Authors: Endo Shuichi , Olsen Richard W.
Published: October 2006

Abstract: Antisera were produced in rabbits against synthetic peptides based on subtype‐specific regions of the cDNA sequences of the α1, α2, α3, and α4 (also termed α5) subunits of mammalian GABAA receptors. The antigen peptides were chosen from the putative cytoplasmic loop between the proposed third and fourth membrane spanning helices; they were not only subtype‐specific sequences, but also their hydrophilicity and predicted secondary structures suggested high potential antigenicity. In all cases, antipeptide antisera recognized on western blots the corresponding α‐subunit polypeptide of the GABAA receptors purified from bovine brain by benzodiazepine‐affinity chromatography, and were able to immunoprecipitate binding activity from detergent‐solubilized purified receptors. The four antisera each recognized a unique polypeptide, and only one, in the purified receptor, with α1, α2, α3, and α4 identified at 51, 52, 56, and 57 kDa, respectively. This represents the first identification of the α4 gene product on a gel. Both the relative amount of staining in immunoblots and the fraction of receptor binding that could be immunoprecipitated by saturating concentrations of each of the four subtypespecific antibodies varied in a consistent manner between receptors purified from different brain regions. Thus, cerebral cortex receptor contained all four α polypeptides on western blots, and significant activity could be precipitated by all four. Hippocampal receptor lacked α3 immunoreactivity on blotting and by immunoprecipitation; α1 was less, whereas both α2 and α4 were more abundant in hippocampus than in cortex by both techniques. Cerebellum receptor contained only α1 of the four α subunits tested, and the anti‐α1 antibodies immunoprecipitated >90% of the binding activity. The variable amounts of staining and immunoprecipitation from the three brain areas by the four antisera demonstrate the presence of heterooligomeric receptor complexes with different α‐subunit constituents in cortex, hippocampus, and cerebellum. The sum of cortical receptor activity precipitated individually by the four anti‐α antisera was > 150%, indicating that some heterooligomers are likely to contain more than one class of α subtype, although most receptor complexes probably contain only one α subtype. These α‐subunit subtype‐specific antibodies should be useful in analyzing structure, function, and localization of GABAA/benzodiazepine receptors in mammalian brain.
Calcium Potentiates Cyclic AMP Stimulation of Pineal Arylalkylamine N‐Acetyltransferase
Authors: Yu Li , Schaad Nicolas C. , Klein David C.
Published: October 2006

Abstract: Pineal arylalkylamine N‐acetyltransferase (N‐acetyltransferase) controls large daily changes in melatonin production. It is generally thought that the activity of this enzyme is controlled by norepinephrine acting exclusively via elevation of cyclic AMP. However, norepinephrine also elevates pineal intracellular Ca2+ concentration ([Ca2+]i), and it is not known whether Ca2+ is involved in regulating N‐acetyltransferase activity other than through its established role in cyclic AMP production. In this study, the issue of whether Ca2+ enhances the effects of cyclic AMP on N‐acetyltransferase activity was investigated. The effects of cyclic AMP protagonists (isobutylmethylxanthine, N6, 2′‐O‐dibutyryladenosine 3′,5′‐cyclic monophosphate, 8‐bromoadenosine 3′,5′‐cyclic monophosphate, and adenosine 3′,5′‐cyclic monophosphothioate, Sp‐diastereomer) were examined in combination with [Ca2+]i protagonists (A23187, ionomycin, and phenylephrine). All [Ca2+]i protagonists potentiated the effects of cyclic AMP protagonists. For example, ionomycin potentiated the effects of low concentrations of 8‐bromoadenosine 3′,5′‐cyclic monophosphate, and A23187 potentiated the effects of isobutylmethylxanthine without altering cyclic AMP accumulation. These findings indicate that Ca2+ and cyclic AMP probably act physiologically in a coordinated manner to stimulate N‐acetyltransferase activity; these second messengers could act directly at one or more sites or through indirect actions mediated by kinases.
Modulation of Adhesion Molecule Expression on Rat Cortical Astrocytes During Maturation
Authors: Smith George M. , Jacobberger James W. , Miller Robert H.
Published: October 2006

Abstract: During development of the vertebrate CNS the functional properties of astrocytes change significantly. Many of these functional changes result from modifications in the expression of cell surface adhesion molecules on astrocytes that mediate the interactions of astrocytes with other astrocytes, neurons, and growing axons. In this study we have compared the cell surface expression of HNK‐1, NCAM, and laminin on rat cortical type‐I‐like astrocytes during maturation in vitro and in vivo. Both the proportion of immunoreactive cells and the relative levels of expression of these antigens on different aged astrocyte populations were assayed by flow cytometry. At birth, most cortical type‐I astrocytes express high levels of HNK‐1 and NCAM, while ∼50% of the cells express laminin. During maturation in vitro, the proportion of cortical astrocytes that expressed these surface molecules decreased over a period of 28 days, even though cell size and glial fibrillary acidic protein content increased. During maturation in vivo, a qualitatively and temporally similar decrease in antigen expression on astrocytes was observed. This reduction in the expression of specific cell surface molecules on maturing astrocytes results from maturation of a single population of astrocytes and not differential proliferation of a nonexpressing subpopulation of astrocytes, as shown by cell cycle analysis of both immunoreactive and nonimmunoreactive cell populations. These data indicate that during maturation of rat cortical type‐I‐like astrocytes, the expression of cell surface adhesion molecules is regulated. Furthermore, this regulation appears to be cell autonomous and not dependent on environmental factors. Such regulation of adhesion molecule expression may have profound consequences for the functional properties of astrocytes during CNS maturation.
Glutathione Depletion Induces Heme Oxygenase‐1 (HSP32) mRNA and Protein in Rat Brain
Authors: Ewing James F. , Maines Mahin D.
Published: October 2006

Abstract: In mammalian systems, the heme oxygenase (HO) isozymes HO‐1 (HSP32) and HO‐2 oxidatively cleave the heme molecule to produce bile pigments and carbon monoxide. Although HO‐1 is inducible by various chemicals in systemic organs and cell culture systems, this communication reports for the first time the induction of this stress protein and its transcript by a chemical in the brain. In addition, this study demonstrates expression of HO‐1 in select populations of cells in the brain in response to GSH depletion. Specifically, treatment of adult rats with diethyl maleate (DEM; 4.7 mmol/kg) caused a pronounced decrease in brain GSH content within 1 h. GSH levels remained significantly depressed for at least 24 h postinjection. Northern blot analysis of brain poly(A)+ mRNA following DEM treatment revealed on the average a sixfold increase in the 1.8‐kb HO‐1 mRNA level compared with that of controls; concomitant with this change was a decrease in GSH levels. Total brain HO activity was not significantly altered along with the increase in HO‐1 mRNA level. The increase in transcription of HO‐1 was a direct response to GSH depletion, as judged by the observation that treatment of neonatal rats with L‐buthionine‐(S,R)‐sulfoximine (BSO) (3 mmol/kg, twice daily, for 2 days), a selective inhibitor of GSH synthesis, caused a marked depression in total brain GSH level and a concomitant increase in brain 1.8‐kb HO‐1 mRNA content. The magnitude of the increase was up to ∼ 11.5‐fold that of the control level, as evidenced by northern blot analysis. In contrast, the level of two homologous HO‐2 transcripts (1.3 and 1.9 kb) did not increase in response to either DEM or BSO treatment. Analysis of brain HO‐1‐immunoreactive protein following DEM treatment for 9 h indicated induction of HO‐1 protein in only select nonneuronal cell populations. In particular, the ependymal cells lining ventricles throughout brain, Bergmann glia of cerebellum, and leptomeninges lining brain and glia throughout brain responded to treatment by increasing the level of HO‐1‐like protein. We suggest that when GSH is depleted, an increase in HO‐1 protein content, resulting in increased capacity to form bile pigments, may be of significance to cells with compromised antioxidant capability. Bile pigments are potent antioxidants in biological systems.
Chronic Ethanol Exposure Potentiates NMDA Excitotoxicity in Cerebral Cortical Neurons
Authors: Chandler L. Judson , Newsom Hunter , Sumners Colin , Crews Fulton
Published: October 2006

Abstract: The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined. Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA. Addition of NMDA (25–100 μM) to the culture dishes for 25 min in Mg2+‐free buffer resulted in a dose‐dependent increase in LDH accumulation. Phase‐contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes. Chronic exposure of neuronal cultures to ethanol (100 mM) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA‐stimulated LDH release by 36 and 22% in response to 25 μM and 50 μM NMDA, respectively. Neither basal LDH release nor that in response to maximal NMDA (100 μM) stimulation was altered by chronic alcohol exposure. In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 mM) only during the NMDA exposure period significantly reduced LDH release by ∼ 50% in both control and chronically treated dishes. This reduction by acute ethanol was also observed under phase‐contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes. These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.
Enhancement of the responsiveness of cortical adrenergic receptors by chronic administration of the 5‐hydroxytryptamine uptake inhibitor citalopram.
Authors: Nalepa Irena , Vetulani Jerzy
Published: October 2006

Abstract: The aim of this study was to evaluate the effect of citalopram, a second generation antidepressant agent producing no β‐down‐regulation, on the receptors and second messenger systems related to noradrenergic transmission in the cerebral cortex of the rat. We confirmed that citalopram does not bind to α1‐, α2‐, and β2‐adrenoceptors, but we found that it attenuates the inhibitory action of the protein kinase C activator, 12‐O‐tetradecanoylphorbol 13‐acetate, on the noradrenergic response from α1‐adrenoceptor. In contrast to most antidepressants, chronic treatment with citalopram does not produce β‐down‐regulation, but increases the responses to noradrenaline from β‐adrenoceptors without increasing the β1,‐adrenoceptor density. Chronic treatment with citalopram also increases the maximal response from α1‐adrenoceptor. The results indicate that β‐down‐regulation is not a necessary characteristic of an efficient antidepressant drug.
Noradrenaline Stimulation Unbalances the Phosphoinositide Cycle in Rat Cerebral Cortical Slices
Authors: Claro Enrique , Fain John N. , Picatoste Fernando
Published: October 2006

Abstract: Muscarinic cholinergic and α1‐adrenoceptor‐mediated stimulation of phosphoinositide hydrolysis in rat cerebral cortex were compared by measuring carbachol‐ and noradrenaline‐induced accumulation of various intermediates of the phosphoinositide cycle. Unlike carbachol, noradrenaline in the presence of guanosine 5′‐O‐(3‐thiotriphosphate) did not stimulate phospholipase C activity in brain cortical membranes. In cortical slices, the efficacy of noradrenaline to stimulate accumulation of 3H‐inositol phosphates and [32P]phosphatidic acid was 2.5 to threefold that of carbachol. However, noradrenaline was less effective than carbachol in stimulating accumulation of [3H]CDP‐diacylglycerol and resynthesis of phosphatidylinositol. This was not due to calcium inhibition of CTP:phosphatidate cytidyltransferase or to different lithium requirements for carbachol‐ and noradrenaline‐stimulated accumulation of [3H]CDP‐diacylglycerol. The noradrenaline‐induced unbalance of the phosphoinositide cycle, which was most apparent at relatively high concentrations of calcium (2.5 mM) in the incubation buffer, was qualitatively reproduced with ionomycin. The use of the α1a‐subtype‐selective adrenoceptor antagonists WB4101 and 5‐methylurapidil revealed a single α1a‐like component mediating the effects of noradrenaline. Our results suggest that the primary mechanism for phospholipase C activation by brain α1 adrenoceptors involves an increase in intracellular calcium concentration.
Developmentally Regulated Secreted Factors Control Expression of Muscarinic Receptor Subtypes in Embryonic Chick Retina
Authors: Skorupa A. F. , Klein W. L.
Published: October 2006

Abstract: Two molecular mass subtypes of muscarinic receptor are expressed by the chick retina (72 and 86 kDa). During development, the ratio of subtypes changes, with the 72‐kDa form becoming predominant. We have found that subtype switch can occur in retina cell culture, and have investigated factors that influence this in vitro increase in the 72‐kDa receptor. Increases similar to those in vivo occurred when cells were cultured at 105 cells/cm2, but not at 10‐fold lower density. High‐density cultures, maintained on coverslips, showed no receptor development when transferred to large volumes of fresh medium, indicating that cell‐cell contact alone was not responsible for induction. However, replacement of fresh medium with conditioned medium (from high‐density cultures) resulted in normal induction. There were no morphological differences between cultures with high and low levels of the 72‐kDa receptor. Conditioned medium also induced 72‐kDa receptors in low‐density cultures, consistent with a minimal role for cell‐cell contact. Efficacy of conditioned medium was markedly dependent on age. Media from cells cultured 1–4 days had no effect, but media from cells cultured 5–8 and 1–8 days elicited 1.6‐fold and fourfold increases in the 72‐kDa subtype, respectively. The data indicate that maturing retina cells secrete developmentally regulated factors that are necessary for abundant expression of the 72‐kDa muscarinic receptor subtype.
Synaptic Neurochemistry of Human Striatum During Development: Changes in Sudden Infant Death Syndrome
Authors: Kalaria R. N. , Fiedler C. , Hunsaker J. C. , Sparks D. L.
Published: October 2006

Abstract: There is evidence of abnormalities in the brain‐stem monoamine‐containing neurons in infants with sudden infant death syndrome (SIDS). By taking advantage of the rich innervation of the human basal ganglia by monoam‐inergic afferents from cell bodies in the brainstem, we studied the synaptic chemistry of catecholamine and associated neurons of the putamen obtained postmortem from 14 SIDS infants, eight age‐matched control infants, and older control subjects of various ages. We found significantly lower concentrations of dopamine and higher homovanillic acid/DA ratios in samples from SIDS infants compared with age‐matched control infants. Noradrenaline and 5‐hydroxytryptamine were lower in SIDS compared with control subjects, but the difference did not reach statistical significance. There was no clear evidence that dihydroxyphe‐nylacetic acid and 5‐hydroxyindoleacetic acid were altered. Immunoblot analysis of striatal tissue showed that samples from infants with SIDS, which exhibited lower DA, also had lower tyrosine hydroxylase protein. Other transmitter‐specific neuronal markers were also assessed, including enzymes associated with cholinergic and GABA‐containing neurons. We found significantly decreased choline acetyltransferase activities. However, GABA, glutamate, or somatostatin concentrations or monoamine oxidase activities were unchanged in SIDS. We also noted age‐dependent changes in brain weights and some synaptic markers by comparing the age‐matched infants with older control subjects. Analysis of variance revealed that homovanillic acid, dihydroxyphenylacetic acid, and monoamine oxidase B activities were increased with age. DA and choline acetyltransferase were also found to be positively correlated in putamen. Our findings suggest developmental changes in some transmitter‐specific neurons in SIDS that may result from apneic episodes or chronic hypoxia induced before death.
Rapid Phosphorylation of Phospholipase Cγ 1 by Brain‐Derived Neurotrophic Factor and Neurotrophin‐3 in Cultures of Embryonic Rat Cortical Neurons
Authors: Widmer Hans R. , Kaplan David R. , Rabin Stuart J. , Beck Klaus D. , Hefti Franz , Knüsel Beat
Published: October 2006

Abstract: Phospholipase Cγ1 (PLC‐γ1) is involved at an early step in signal transduction of many hormones and growth factors and catalyzes the hydrolysis of phosphatidylinositol (PI) 4,5‐bisphosphate to diacylglycerol and inositol trisphosphate, two potent intracellular second messenger molecules. The transformation of PC12 cells into neuron‐like cells induced by nerve growth factor is preceded by a rapid stimulation of PLC‐γ1 phosphorylation and PI hydrolysis. The present study analyzed the effects of brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) on phosphorylation of PLC‐γ1 in primary cultures of embryonic rat brain cells. BDNF and NT‐3 stimulated the phosphorylation of PLC‐γ1, followed by hydrolysis of PI. The stimulation of PLC‐γ1 phosphorylation occurred within 20 s after addition of BDNF or NT‐3 and lasted up to 30 min, with a peak after 4 min. ED50 values were similar for BDNF and NT‐3, with τ25 ng/ml. Phosphorylation of PLC‐γ1 by BDNF and NT‐3 was found in cultures from all major brain areas. K‐252b, a compound known to inhibit selectively neurotrophin actions by interfering with the phosphorylation of trk‐type neurotrophin receptors, prevented the BDNF‐ and NT‐3‐stimulated phosphorylation of PLC‐γ1. Receptors of the trk type were coprecipitated with anti‐PLC‐γ1 antibodies. The presence of trkB mRNA in the cultures was substantiated by northern blot analysis. The action of BDNF and NT‐3 seems to be neuron specific because no phosphorylation of PLC‐γ1 was observed in cultures of nonneuronal brain cells. The results provide evidence that developing neurons of the cerebral cortex and other brain areas are responsive to BDNF and NT‐3, and they indicate that the transduction mechanism of BDNF and NT‐3 in the brain involves rapid phosphorylation of PLC‐γ1 followed by PI hydrolysis.
Nicotinic Receptor‐Mediated Release of Noradrenaline in the Human Neuroblastoma SH‐SY5Y
Authors: Vaughan Peter F. T. , Kaye David F. , Reeve Helen L. , Ball Stephen G. , Peers Chris
Published: October 2006

Abstract: Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH‐SY5Y cells that have been pretreated with 12‐O‐tetradecanoylphorbol 13‐acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 μM mecamylamine but not by 1 μM atropine, which suggests that SH‐SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide‐evoked release was enhanced by 5 μM Bay K 8644 (an L‐type calcium agonist) and inhibited by 1 μM nifedipine. Dimethylphenylpiperazinium iodide depolarised SH‐SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 μM desmethylimipramine. The results of this study show that nicotinic receptors in SH‐SY5Y cells stimulate noradrenaline release by activation of L‐type calcium channels.
S(‐)‐Nornicotine Increases Dopamine Release in a Calcium‐Dependent Manner from Superfused Rat Striatal Slices
Authors: Dwoskin Linda P. , Buxton Susan T. , Jewell Ann L. , Crooks Peter A.
Published: October 2006

Abstract: The present study demonstrates that S(‐)‐nornicotine evoked a concentration‐dependent increase in dopamine (DA) release from superfused rat striatal slices. The increase in DA release was indicated by an S(‐)‐nornicotine‐induced overflow of endogenous 3,4‐dihydroxyphenyl‐acetic acid (DOPAC) in the striatal superfusate and by an S(‐)‐nornicotine‐induced increase in tritium overflow from striatal slices preloaded with [3H]DA. Low concentrations (0.01–1.0 μM) of S(‐)‐nornicotine, which did not evoke endogenous DOPAC overflow, also were unable to modulate electrically evoked DOPAC overflow. The increase in DOPAC overflow induced by S(‐)‐nornicotine was compared with that produced by S(‐)‐nicotine. Comparing equimolar concentrations (0.1‐100 μM) of S(‐)‐nornicotine and S(‐)‐nicotine, superfusion with S(‐)‐nornicotine resulted in a significantly greater DOPAC overflow. In contrast to the effect of S(‐)‐nicotine, S(‐)‐nornicotine evoked a sustained increase in DOPAC over‐flow for the entire period of S(‐)‐nornicotine exposure. Furthermore, DOPAC overflow evoked by S(‐)‐nornicotine in control Krebs buffer was inhibited by superfusion with a low‐calcium buffer. Moreover, in the low‐calcium buffer, DOPAC overflow induced by 30 and 100 μM S(‐)‐nornicotine was not different from that with no S(‐)‐nornicotine. The results indicate that S(‐)‐nornicotine, a constituent of tobacco products and a known metabolite of S(‐)‐nicotine, increases DA release in a calcium‐dependent manner in superfused rat striatal slices. It is interesting that unlike S(‐)‐nicotine, there does not appear to be desensitization to this effect of S(‐)‐nornicotine.
Phosphorylation of Neuronal Kinesin Heavy and Light Chains In Vivo
Authors: Hollenbeck Peter J.
Published: October 2006

Abstract: The microtubule‐based motor protein kinesin is thought to drive anterograde organelle transport in axons, but nothing is known about how its force‐generating activity or organelle‐binding properties are regulated. Studies in other motility systems suggest that protein phosphorylation is a reasonable candidate for this function. I report here that the kinesin heavy chain (HC) and light chain (LC), as well as the 160‐kDa kinesin‐associated protein kinectin, are phosphorylated in vivo in cultures of chick sympathetic neurons and PC12 cells labeled metabolically with 32P. In neurons, both kinesin chains are phosphorylated exclusively on serine residues, and limiting tryptic digestion demonstrated that the phosphorylation sites are clustered in a region of ˜5 kDa for the HC and ˜14 kDa for the LC. Partial tryptic digestion of 32P‐labeled HC followed by immunoblotting with SUK4 monoclonal anti‐HC and fluorography showed that the sites of HC phosphorylation are outside the globular N‐terminal head region where kinesin's microtubulebinding and mechanochemical activities reside. Treatment of metabolically labeled neurons with forskolin, phorbol esters, or calcium ionophore did not alter the extent of phosphorylation, the phosphoamino acid composition, or the V8 protease phosphopeptide maps of the HC, LC, and 160‐kDa protein, with one exception: treatment with calcium ionophore reduced the specific activity of the LC. In addition, when kinesin from PC12 cells was compared with that from PC12‐derived cell lines lacking protein kinase A activity, neither the extent of phosphorylation nor the phosphopeptide maps were altered for either chain. Phosphopeptide mapping experiments also showed that postlysis kinase activity can phosphorylate both the neuronal HC and LC at sites not phosphorylated in vivo.
Effects of Physostigmine and Some Nitric Oxide‐Cyclic GMP‐Related Compounds on Muscarinic Receptor‐Mediated Autoinhibition of Hippocampal Acetylcholine Release
Authors: Suzuki Takeshi , Nonaka Hikaru , Fujimoto Kazuko , Kawashima Koichiro
Published: October 2006

Abstract:We have investigated the effects of (a) the cholinesterase inhibitor physostigmine and (b) drugs that are known to change intracellular cyclic GMP levels on the autoinhibition of acetylcholine release from rat hippocampal slices. Autoinhibition was triggered by submaximal electrical stimulation in both the absence and presence of physostigmine. The results obtained indicate that an unusual increase in the extracellular acetylcholine content, such as that induced by cholinesterase inhibition, is not essential for autoinhibition triggering. Dibutyryl cyclic GMP reduced significantly the stimulation‐evoked acetylcholine release in the presence, but not in the absence, of atropine. Neither sodium nitroprusside nor glyceryl trinitrate exerted a dibutyryl cyclic GMP‐like effect.NG‐Nitro‐L‐arginine did not lessen the autoinhibition. These results indicate that an increase in the intracellular cyclic GMP level reduces acetylcholine release, and that the muscarinic receptor stimulation‐nitric oxide synthesis‐(soluble) guanylyl cyclase activation pathway is not involved in the cholinergic autoinhibition process.
Evidence for Cyclic AMP‐Mediated Increase of Aromatic L‐Amino Acid Decarboxylase Activity in the Striatum and Midbrain
Authors: Young E. A. , Neff N. H. , Hadjiconstantinou M.
Published: October 2006

Abstract: Aromatic L‐amino acid decarboxylase (AAAD) activity of mouse striatum and midbrain increased after an intracerebro‐ventricular injection of either forskolin or 8‐bromo‐cyclic AMP. The increase was transient, peaking between 15 and 30 min and returning to baseline by ˜90 min. The increase of AAAD activity after forskolin was not affected by pretreatment with cycloheximide. Kinetic studies indicated an apparent increase of Vmax with little change of the Km for L‐DOPA or pyridoxal 5′‐phosphate. We conclude that AAAD activity of striatum and midbrain can be modulated by a cyclic AMP‐dependent process.
A Comparison of Two Alternatively Spliced Forms of a Metabotropic Glutamate Receptor Coupled to Phosphoinositide Turnover
Authors: Pickering D. S. , Thomsen C. , Suzdak P. D. , Fletcher E. J. , Robitaille R. , Salter M. W. , MacDonald J. F. , Huang X.‐P. , Hampson D. R.
Published: October 2006

Abstract: A comparison of the pharmacological and physiological properties of the metabotropic glutamate 1α and 1β receptors (mGluR1α and mGluR1β) expressed in baby hamster kidney (BHK 570) cells was performed. The mGluR1β receptor is an alternatively spliced form of mGluR1α with a modified carboxy terminus. Immunoblots of membranes from the two cell lines probed with receptor‐specific antipeptide antibodies showed that mGluRIa migrated with an Mr= 154, 000, whereas mGluR1β migrated with an Mr= 96, 000. Immunofluorescence imaging of receptors expressed in BHK 570 cells revealed that the mGluR1α receptor was localized to patches along the plasmalemma and on intracellular membranes surrounding the nucleus, whereas mGluR1β was distributed diffusely throughout the cell. Agonist activation of the mGluR1α and the mGluR1β receptors stimulated phosphoinositide hydrolysis. At both receptors, glutamate, quisqualate, and ibotenate were full agonists, whereas trans‐(+)‐1‐aminocyclopentane‐1, 3‐dicarboxylate appeared to act as a partial agonist. The stimulation of phosphoinositide hydrolysis by mGluR1α showed pertussis toxin‐sensitive and insensitive components, whereas the mGluR1β response displayed only the toxin‐insensitive component. The mGluR1α and mGluR1β receptors also increased intracellular calcium levels by inducing release from intracellular stores. These results indicate that the different carboxy terminal sequences of the two receptors directly influences G protein coupling and subcellular deposition of the receptor polypeptides and suggest that the two receptors may subserve different roles in the nervous system.
Adrenergic Stimulation of Cyclic GMP Formation Requires NO‐Dependent Activation of Cytosolic Guanylate Cyclase in Rat Pinealocytes
Authors: Spessert Rainer , Layes Elisabeth , Vollrath Lutz
Published: October 2006

Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving β‐and α1‐adrenergic receptors. The effects of N‐monomethyl‐l‐arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO‐mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited β‐adrenergic stimulation of cGMP formation as well as α1‐adrenergic potentiation of β‐adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of l‐arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.
Metabolism of Arachidonic Acid to Epoxyeicosatrienoic Acids. Hydroxyeicosatetraenoic Acids, and Prostaglandins in Cultured Rat Hippocampal Astrocytes
Authors: Amruthesh Shivachar C. , Boerschel Markus F. , McKinney Jerry S. , Willoughby Karen A. , Ellis Earl F.
Published: October 2006

Abstract: We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxy‐genase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [3H]‐arachidonic acid, formed products that eluted in four major groups designated as R17–30, R42–50, R51–82, and R83–90 based on their retention times in reverse‐phase HPLC. These fractions were further segregated into as many as 13 peaks by normal‐phase HPLC and a second reverse‐phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact‐mass spectrometry. Based on HPLC retention times and gas chromatography/electron impactmass spectrometry analysis, the more polar fractions (R17–30) contained prostaglandin D2 as the major cyclooxygenase product. Minor products included 6‐keto prostaglandin F1α, prostaglandin E2, prostaglandin F2α, and thromboxane B2. Fractions R42–50, R51–82. and R83–90 contained epoxygenase and lipoxygenase‐like products. The major metabolite in fractions R83–90 was 5, 6‐epoxyeicosatrienoic acid (EET). Fractions R51–82 contained 14, 15‐and 8, 9‐EETs, 12‐and 5‐hydroxyeicosatetraenoic acids, and 8, 9‐and 5, 6‐dihydroxyeicosatrienoic acids (DHETs). In fractions R42–50, 14, 15‐DHET was the major product. When radiolabeled [3H]14, 15‐EET was incubated with astrocyte homogenate, it was rapidly metabolized to [3H]14, 15‐DHET. The metabolism was inhibited by submicromolar concentration of 4‐phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxyalcohols from 14, 15‐DHET was not observed. In conclusion, astro‐cytes readily metabolize arachidonic acid to 14, 15‐EET, 5, 6‐EET, and their vicinal‐diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.
Analysis of a Segment of the Human Glial Fibrillary Acidic Protein Gene That Directs Astrocyte‐Specific Transcription
Authors: Masood Khalid , Besnard François , Su Yuan , Brenner Michael
Published: October 2006

Abstract: To understand astrocyte‐specific transcription, we have been studying the human gfa gene. This gene encodes glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed primarily in astrocytes. A survey of the gfa 5′ flanking region showed it to contain several segments that contribute to expression of a chloramphenicol acetyltransferase reporter gene in transfected cells. The most active of these was the 124‐bp B region, which spans bp ‐1612 to ‐1489. We have now used site‐ directed mutagenesis to analyze this region in greater detail, and show that the B region itself contains several important elements. The most crucial of these is a consensus AP‐1 sequence, the binding site for the Fos and Sun families of transcription factors. The presence of members of both these families in the glial fibrillary acidic protein‐ expressing U251 cell line used for our transfection studies was verified by gel mobility‐shift experiments. This is the first demonstration of the functioning of a specific transcription factor site for astrocytes, and provides a focus for future studies of glial fibrillary acidic protein regulation during development and reactive gliosis.
Extracellular Neuroactive Amino Acids in the Rat Striatum During Ischaemia: Comparison Between Penumbral Conditions and Ischaemia with Sustained Anoxic Depolarisation
Authors: Obrenovitch Tihomir Paul , Urenjak Jutta , Richards Douglas Arthur , Ueda Yukihiko , Curzon Gerald , Symon Lindsay
Published: October 2006

Abstract: Changes in the extracellular levels of excitatory and inhibitory amino acid transmitters were studied in the rat striatum during penumbral ischaemia using intracerebral microdialysis. Effects of penumbral forebrain ischaemia were compared with those of ischaemia with sustained anoxic depolarisation and K+ (100 mM). Comparisons were also made between different groups of animals at 2 and 24 h after dialysis probe implantation. The K+ stimulus did not provoke any release of excitatory amino acids in the 24‐h group, probably reflecting a decrease of functional synapses adjacent to the probe. During 30 min of penumbral ischaemia, excitatory amino acids did not reach critical concentrations in the extracellular fluid, and increases in levels of inhibitory/modulatory amino acids were similar. On the other hand, severe transient ischaemia resulted in massive synchronous release of many neuroactive excitatory and inhibitory compounds, in both the 2‐ and 24‐h groups. These and other data suggest that changes during severe ischaemia may arise from both neurotransmitter and metabolic pools. It is concluded that is‐ chaemic damage in the penumbra may not be related to extracellular neuroactive amino acid changes generated within this region.