bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunological Detection of the NMDAR1 Glutamate Receptor Subunit Expressed in Human Embryonic Kidney 293 Cells and in Rat Brain
Authors: Chazot Paul L. , Cik Miroslav , Stephenson F. Anne
Published: October 2006

Abstract: The rat NMDAR1 (N‐methyl‐d‐aspartate receptor) was expressed transiently in human embryonic kidney cells. Transfected cell homogenates showed saturable [3H]MK‐801 binding activity that was best fit by a single high‐affinity site with a KD of 9 nM and a 5max of 113 fmol of binding sites/mg of protein. Antibodies raised against the peptide sequence NMDAR1 (929–938) coupled to keyhole limpet haemocyanin specifically recognised a single band with Mr 117,000 in immunoblots from adult rat brain. In the transfected cells, the antibody recognised two bands: one with Mr 117,000, which was coincident with that from brain membranes, and one with Mr 97,000, which was identified as nonglycosylated NMDAR1 subunit. These results identify the NMDAR1 of rat brain and further show that the homooligomer binds MK‐801, albeit at low efficiency.
Multiple Forms and Distribution of Calcium/Calmodulin‐Stimulated Protein Kinase II in Brain
Authors: Rostas John A. P. , Dunkley Peter R.
Published: October 2006

In recent years, the enzyme Ca2+/calmodulin‐stimulated protein kinase II1 (CaM‐PK II) as attracted a great deal of interest. CaM‐PK II is the most abundant calmodulin‐stimulated protein kinase in brain, where it is particularly enriched in neurons (Ouimet et al., 1984; Erondu and Kennedy, 1985; Lin et al., 1987; Scholz et al., 1988). Neuronal CaM‐PK II has been suggested to be involved in several phenomena associated with synaptic plasticity (Lisman and Goldring, 1988; Kelly, 1992), including long‐term potentiation (Malinow et al., 1988; Malenka et al.,1989), neurotransmission (Nichols et al., 1990; Siekevitz, 1991), and learning (for review, see Rostas, 1991). This enzyme has also been postulated to be selectively vulnerable in several pathological condition, including epilepsy/kindling (Bronstein et al.,1990; Wu et al., 1990), cerebral ischemia (Taft et al., 1988), and organophosphorus toxicity (Abou‐Donia and Lapadula, 1990).
Evidence that the Loss of the Voltage‐Dependent Mg2+ Block at the N‐Methyl‐D‐Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism
Authors: Zeevalk Gail D. , Nicklas William J.
Published: October 2006

Abstract: In this study, the importance of the Mg2+ blockade of the N‐methyl‐D‐aspartate (NMDA) receptor during metabolic stress was examined in embryonic day 13 chick retina. Retina exposed to mild conditions of metabolic stress (i.e., blockade of glycolysis with 1 mM iodoacetate for 30 min) underwent acute histological somal and neuritic swelling and an increase in γ‐aminobutyric acid (GABA) release into the medium. These acute signs of metabolic stress were eliminated by NMDA antagonists present during pharmacological blockade of glycolysis, occurred in the absence of a net increase in extracellular glutamate or aspartate, and were not affected by the presence or absence of Ca2+ in the incubation medium. One possible explanation for the activation of NMDA receptors in the absence of an increase in extracellular ligand is that NMDA sensitivity during metabolic stress may be governed at the receptor level. Depolarization of membrane potential during metabolic stress may result in the loss of the Mg2+ blockade from the NMDA receptor channel, resulting in an increased potency for glutamate. To test this, the dose‐response characteristics for NMDA, glutamate, and kainate in the presence or absence of extracellular Mg2+ and the effects of Mg2+ on metabolic inhibition were examined. The potency for NMDA‐ or glutamate‐mediated acute toxicity was enhanced two‐ to fivefold in the absence of Mg2+. Omission of Mg2+ greatly decreased the minimal concentration of agonist needed to produce acute excitotoxicity; 25 versus 5 μM for NMDA and 300 versus 10 μM for glutamate in 1.2 or zero Mg2+, respectively. Elevating external Mg2+ to 20 mM completely protected against NMDA‐mediated acute toxic effects. In contrast, varying external Mg2+ had no effect on kainate‐induced toxicity. Acute toxicity caused by inhibition of metabolism was not potentiated in the absence of Mg2+ but was attenuated by elevating extracellular Mg2+. The protective effect of Mg2+ during metabolic inhibition was not additive with NMDA antagonists, suggesting that the action of Mg2+ was at the level of the NMDA receptor. These findings are consistent with the hypothesis that the Mg2+ block is lifted during metabolic inhibition and may be the primary event resulting in NMDA receptor activation.
Analogues of Somatostatin Bind Selectively to Brain Somatostatin Receptor Subtypes
Authors: Raynor Karen , Coy David C. , Reisine Terry
Published: October 2006

Abstract: Somatostatin (SRIF) is a neurotransmitter that produces its multiple effects in the CNS through interactions with membrane‐bound receptors. Subtypes of SRIF receptors are found in the CNS that are distinguished by their sensitivities to the cyclic hexapeptide MK‐678, such that SRIF, receptors are sensitive to MK‐678 and SRIF2 receptors are insensitive to MK‐678. In the present study, we further examined the selectivities of a series of structurally diverse SRIF analogues for SRIF receptor subtypes. SRIF receptors were labeled by 125I‐Tyr11 SRIF, which has indistinguishable affinities for SRIF receptor subtypes. The inhibition by MK‐678 was incomplete, indicating this peptide is highly selective for a subtype of SRIF receptor that we have termed the SRIF, receptor. The binding of 125I‐MK‐678 to SRIF, receptors was monophasically inhibited by SRIF, the octapeptides (such as SMS‐201–995), and the hexapeptides (such as MK‐678), consistent with the highly selective labeling of a subtype of SRIF receptor. In contrast, the smaller CGP‐23996‐like analogues did not inhibit 125I‐MK‐678 binding to SRIF, receptors. The binding of 125I‐CGP‐23996 to SRIF receptors was inhibited by SRIF and the octapeptides with Hill coefficients of < 1, indicating that 128I‐CGP‐23996 labels multiple SRIF receptor subtypes. The hexapeptides and CGP‐23996‐like compounds produced only partial inhibitions of 125I‐CGP‐23996 binding, which were additive, indicating selective interactions of these compounds with the different receptor subpopulations labeled by 125I‐CGP‐23996. 125I‐Tyr11‐SRIF binding and 125I‐CGP‐23996 binding to SRIF receptors were likewise only partially affected by 100 μM guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS), a concentration that completely abolishes specific 125I‐MK‐678 binding to SRIF, receptors. The component of 125I‐CGP‐23996 labeling that was sensitive to GTPγS was also MK‐678 sensitive. Thus, two subpopulations of SRIF receptors exist in the CNS. The SRIF, receptor is sensitive to cyclic hexapeptides such as MK‐678 and to GTPγS but insensitive to smaller CGP‐23996‐like compounds. The SRIF2 receptor is sensitive to the CGP‐23996‐like compounds and can be selectively labeled by 125I‐CGP‐23996 in the presence of high concentrations of the hexapeptides or GTPγS because, unlike the SRIF, receptor, the SRIF2 receptor is insensitive to these agents. The SRIF receptor subtype‐selective peptide analogues will be useful in the future characterization of the functions mediated by SRIF receptor subtypes in the CNS.
Effect of N,N‐Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore
Authors: Sbia Mohammed , Diebler Marie‐Francoise , Morel Nicolas , Israël Maurice
Published: October 2006

Abstract: The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15‐kDa proteolipid, presents a very high sequence homology with the N,N′‐dicyclohexylcarbodiimide (DCCD)‐binding proteolipid subunit of the vacuolar‐type H+‐ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium‐dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15‐kDa proteolipid subunit of the mediatophore. These results suggest that although the 15‐kDa proteolipid subunits of the mediatophore and the vacuolar H+‐ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium‐dependent ACh release and that the two proteins have a different membrane organization.
Stimulus Deprivation Increases Pineal Gsα and Gβ
Authors: Babila Tamar , Klein David C.
Published: October 2006

Abstract: Denervation and other forms of stimulus deprivation cause an increase in the magnitude of subsequent responses, a phenomenon commonly referred to as denervation supersensitivity. This has been well demonstrated with the cyclic AMP response to norepinephrine in the pineal gland. In the present report, we address the question of whether stimulus deprivation alters α and β subunits of the GTP binding regulatory protein that stimulates adenylyl cyclase activity (Gs). Stimulus deprivation of the pineal gland was produced by denervation (superior cervical ganglionectomy), decentralization of the superior cervical ganglia, or by exposure of the animal to continuous lighting. All increased both the α and β subunits of Gs (Gsα and Gβ) by up to fourfold, as estimated using semiquantitative western blot technology. These effects were detectable after 1 day of stimulus deprivation and were sustained for 2 weeks. The stimulatory effects of constant light‐induced stimulus deprivation were also apparent by measuring cholera toxin‐dependent ADP‐ribosylation of Gsα, which revealed a fourfold increase in the amount of labeled substrate. The results of in vivo studies were confirmed with in vitro studies, which demonstrated a spontaneous increase in both Gsα and Gβ during 72 h of organ culture. The constant light‐induced increases in both Gsα and Gβ were prevented by continuous administration of isoproterenol (0.3 mg/kg/day), supporting the suggestion that adrenergic stimulation controls the levels of Gsα and Gβ. These studies indicate that stimulus deprivation increases both Gsα and Gβ. It appears that such increases play a central role in generating supersensitivity in the pineal gland and may play a role in supersensitivity in other systems.
Regional and Subcellular Localization of Li+ and Other Cations in the Rat Brain Following Long‐Term Lithium Administration
Authors: Lam Henrik Rye , Christensen Sten
Published: October 2006

Abstract: Rats were given LiCl in their diet (40 mmol/kg dry weight) for at least 3 months to elucidate the regional and subcellular localization of Li+ in the brain as well as the effect of chronic lithium administration on the distribution of other cations. At steady‐state the mean concentrations of Li+ were 0.66 mmol/kg wet weight in the whole brain and 0.52 mM in plasma. The tissue/plasma concentration ratio exceeded unity in all anatomical regions. No region showed excessive accumulation of Li+. Whole brain or regional contents of Na+ or K+ were unaffected by lithium treatment. Subcellular Li+ localization was demonstrated in nuclear, crude mitochondrial, and microsomal fractions of whole brain homogenate. Subfractionation of the crude mitochondrial fraction revealed energy‐independent intrasynaptosomal and intramitochondrial Li+ and K+ localization at 0–4°C. Li+ administered in vivo disappeared within 10 min from synaptosomes incubated at 37°C. Li+ added in vitro at 1 mM attained a synaptosomal steady‐state concentration within 30 min at 37°C. In control rats, synaptosomal concentrations and synaptosomal/medium concentration gradients of cations paralleled their respective in vivo concentrations and gradients. Lithium treatment caused synaptosomal depletion of K+ and Mg2+ and hence probably partial membrane depolarization. Addition of 1 mM Li+ in vitro also caused synaptosomal Mg2+ depletion. The results indicate that Li+ is “accumulated” in brain sediments and synaptosomes following its long‐term treatment. The estimated intracellular and intrasynaptosomal Li+ concentrations are lower than predicted by passive distribution according to the Nernst equation, evidencing active extrusion of Li+.
m‐Sulfonate Benzene Diazonium Chloride: A Powerful Affinity Label for the γ‐Aminobutyric Acid Binding Site from Rat Brain
Authors: Bouchet Marie‐Jeanne , Jacques Patrice , Ilien Brigitte , Goeldner Maurice , Hirth Christian
Published: October 2006

Abstract: m‐Sulfonate benzene diazonium chloride (MSBD) was used to affinity‐label the γ‐aminobutyric acid (GABA) binding site from rat brain membranes. To assess the irreversibility of the labeling reaction, we used an efficient ligand dissociation procedure combined to a rapid [3H]muscimol binding assay, both steps being performed on filter‐adsorbed membranes. Inactivation of specific [3H]‐muscimol binding sites by MSBD and its prevention by GABA were both time‐ and concentration‐dependent. The time course of MSBD labeling was shortened as the pH of the incubation medium was increased from 6.2 to 8. These data suggest that MSBD can efficiently label the GABA binding site through alkylation of a residue having an apparent dissociation constant around neutrality.
Comparison of Rates of Local Cerebral Glucose Utilization Determined with Deoxy[l‐14C]glucose and Deoxy[6‐14C]glucose
Authors: Dienel Gerald A. , Cruz Nancy F. , Nakanishi Hajime , Melzer Peter , Moulis Penny , Sokoloff Louis
Published: October 2006

Abstract: The activity of the pentose phosphate shunt pathway in brain is thought to be linked to neurotransmitter metabolism, glutathione reduction, and synthetic pathways requiring NADPH. There is currently no method available to assess flux of glucose through the pentose phosphate pathway in localized regions of the brain of conscious animals in vivo. Because metabolites of deoxy[l‐14C]glucose are lost from brain when the experimental period of the deoxy[14C]glucose method exceeds 45 min, the possibility was considered that the loss reflected activity of this shunt pathway and that this hexose might be used to assay regional pentose phosphate shunt pathway activity in brain. Decarboxylation of deoxy[l‐14C]glucose by brain extracts was detected in vitro, and small quantities of 14C were recovered in the 6‐phosphodeoxygluconate fraction when deoxy[14C]glucose metabolites were isolated from freeze‐blown brains and separated by HPLC. Local rates of glucose utilization determined with deoxy[l‐14C]glucose and deoxy[6‐14C]glucose were, however, similar in 20 brain structures at 45,60,90, and 120 min after the pulse, indicating that the rate of loss of 14CO2 from deoxy[l‐14C]glucose‐6‐phosphate in normal adult rat brain is too low to permit assay pentose phosphate shunt activity in vivo. Further metabolism of deoxy[l‐14C]glucose‐6‐phosphate via this pathway does not interfere during routine use of the deoxyglu‐cose method or explain the progressive decrease in calculated metabolic rate when the experimental period exceeds 45 min.
Bradykinin and Phorbol Dibutyrate Activate Phospholipase D in PC12 Cells by Different Mechanisms
Authors: Horwitz Joel , Ricanati Steven
Published: October 2006

Abstract: Bradykinin is known to activate phospholipase D in PC12 cells. Because bradykinin may also activate protein kinase C in these cells, the possible role of this kinase in mediating the action of bradykinin was investigated. Phospholipase D activity in PC12 cells was assayed by measuring the formation of [3H]phosphatidylethanol in cells prelabeled with [3H]palmitic acid and incubated in the presence of ethanol. The phorbol ester phorbol dibutyrate mimicked the effect of bradykinin on [3H]phosphatidylethanol formation. The protein kinase C inhibitor staurosporine (1 μM) significantly attenuated the effect of phorbol dibutyrate (35–70%) but did not block bradykinin‐stimulated [3H]‐phosphatidylethanol formation. In addition, the effect of phorbol dibutyrate was additive with that of bradykinin. Prolonged treatment of PC12 cells with phorbol dibutyrate (24 h), which depletes cells of protein kinase C, greatly attenuated bradykinin‐stimulated [3H]phosphatidylethanol accumulation in intact cells. This treatment caused a 55% decrease in both fluoride‐stimulated [3H]phosphatidylethanol production in the intact cell and phospholipase D activity as assessed by an in vitro assay using an exogenous substrate. Therefore, the effect of prolonged phorbol dibutyrate pretreatment on bradykinin‐stimulated [3H]phosphatidylethanol production could not be attributed exclusively to the depletion of protein kinase C. Thus, although the data with phorbol ester suggest that activation of protein kinase C leads to an increase in phospholipase D activity, this kinase probably does not play a role in mediating the effect of bradykinin. Finally, although pretreatment with phorbol dibutyrate completely blocked bradykinin‐stimulated [3H]‐phosphatidylethanol production in the intact cell, it only partially (∼50%) inhibited bradykinin‐stimulated [3H]‐diacylglycerol formation. This suggests that bradykinin promotes diacylglycerol formation by phospholipase D independent as well as phospholipase D dependent mechanisms.
γ‐Aminobutyric Acid and Glycine Modulate Each Other's Release Through Heterocarriers Sited on the Releasing Axon Terminals of Rat CNS
Authors: Raiteri M. , Bonanno G. , Pende M.
Published: October 2006

Abstract: The ability of γ‐aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [3H]GABA or [3H]‐Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [3H]Gly (EC50, 20.8 μM) from spinal cord synaptosomes. Neither muscimol nor (‐)‐baclofen, up to 300 μM, mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N‐(4,4‐diphenyl‐3‐butenyl)nipecotic acid. Moreover, the GABA‐induced [3H]Gly release was Na+ dependent and disappeared when the medium contained 23 mM Na+. The effect of GABA was Ca2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [3H]‐GABA from rat spinal cord synaptosomes (EC50, 100.9 μM). This effect was insensitive to both strychnine and 7‐chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na+ dependent. Also, the Gly‐evoked [3H]‐GABA release was Ca2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [3H]Gly (EC50, 11.1 μM) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (—)‐baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na+ dependent. Gly increased [3H]GABA release from cerebellar synaptosomes (EC50, 110.7 μM) in a strychnine‐ and 7‐chlorokynurenic acid‐insensitive manner. This effect was Na+ dependent. The effects of GABA on [3H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes. However, in cortex, the effect of Gly on [3H]GABA release was much lower than in spinal cord or cerebellum, although it was partly Na+ dependent. No changes of [3H]Gly release were observed in hippocampal synaptosomes exposed to GABA. It is suggested that transporters specific for GABA or Gly are colocalized on the same nerve terminal in rat spinal cord, cerebellum, and cerebral cortex, but not in hippocampus. Moreover, GABA uptake modulates Gly release and, at least in spinal cord and cerebellum, Gly uptake modulates GABA release. These conclusions are compatible with the reported coexistence of GABA and Gly in spinal and cerebellar neurons.
Noncompetitive Inhibition of N‐Methyl‐D‐Aspartate by Conantokin‐G: Evidence for an Allosteric Interaction at Polyamine Sites
Authors: Skolnick Phil , Boje Kathleen , Miller Rachel , Pennington Micheal , Maccecchini Maria‐Luisa
Published: October 2006

Abstract: Conantokins T and G are polypeptide toxins present in snails of the genus Conus. These substances were recently reported to act as N‐methyl‐D‐aspartate (NMDA) antagonists. In the present study, we examined the possible mechanisms producing this antagonism. Conantokin‐G inhibited spermine‐ and spermidine‐stimulated [3H]MK‐801 binding to extensively washed rat forebrain membranes in a noncompetitive manner with IC50 values of ∼507 and ∼946 nM, respectively. In contrast, glutamate‐enhanced [3H]MK‐801 binding was unaffected by conantokin‐G concentrations of <20 μM. At concentrations >5 μM, conantokin‐G effected a modest, noncompetitive inhibition of glycine‐stimulated [3H]MK‐801 binding and also produced a small enhancement of basal [3H]MK‐801 binding. Conan tokin‐G reduced (IC50∼1.08 μM) the NMDA‐stimulated accumulation of cyclic GMP in cerebellar granule cell cultures to basal values, but did not affect kainate‐mediated increases in cyclic GMP. These findings indicate that conantokin‐G acts as a noncompetitive NMDA antagonist through an allosteric inhibition of polyamine responses. The neurochemical profile of this polypeptide is distinct from previously described noncompetitive NMDA antagonists.
Monoclonal Antibody Analysis of Phosphatidylserine and Protein Kinase C Localizations in Developing Rat Cerebellum
Authors: Miyazawa Atsuo , Inoue Hiroko , Yoshioka Tohru , Horikoshi Tetsuro , Yanagisawa Keiji , Umeda Masato , Inoue Keizo
Published: October 2006

Abstract: Understanding the topographical relationships between phosphatidylserine (PS) and protein kinase C (PKC) within neurons can provide clues about the mechanism of translocation and activation of PKC. For this purpose we applied monoclonal antibodies (Abs) of PS and PKC to sections of developing rat cerebellum. The anti‐PKC Ab immunohistochemical pattern showed homogeneous staining of Purkinje cells over various postnatal ages, whereas the anti‐PS Ab staining showed a heterogeneous localization over these ages. Purkinje cells did not stain well between postnatal day 14 (PND 14) and PND 21, suggesting that the PS was lost from the membrane during preparation of the sections during this period. These data imply that interactions between PS and PKC vary in Purkinje cells during postnatal development.
Endogenous Dopamine Facilitates Striatalln Vivo Acetylcholine Release by Acting on D1 Receptors Localized in the Striatum
Authors: Consolo Silvana , Girotti Prisca , Russi Giovanni , Chiara Gaetano Di
Published: October 2006

Abstract: Intrastriatal application of the D1 antagonist SCH 23390 by two procedures, reverse dialysis (20 μM) and local injection (0.45 nmol per striatum), elicited a reduction in acetylcholine (ACh) release superimposable on that induced by systemic administration. The novel selective D1 antagonist SCH 39166 produced a similar decreasing effect on striatal ACh release on local injection (0.45 nmol per striatum). On the other hand, local application of SCH 23390 into the frontal cortices (0.45 nmol per side) failed to alter striatal ACh overflow, indicating that the drug does not diffuse out of its injection site to any significant extent. The dopamine release inducer d‐amphetamine (2 mg/kg s.c.) and the dopamine uptake inhibitor cocaine raised ACh release like the D1 agonists. These effects were completely blocked by 10 μM SCH 23390 applied by reverse dialysis. The results suggest that D1 receptors regulating ACh release are located in the striatum.
Drosophila Cyclodiene Resistance Gene Shows Conserved Genomic Organization with Vertebrate γ‐Aminobutyric AcidA Receptors
Authors: Ffrench‐Constant Richard H. , Rocheleau Thomas
Published: October 2006

Abstract: Genomic clones from the Rdl locus of Drosophila, whose mutant phenotype is resistant to cyclodiene insecticides and picrotoxin, were characterized by restriction mapping and partial sequencing to determine intron/exon structure. The coding region of the gene comprises nine identified exons and spans >25 kb of genomic DNA. The structure of the Drosophila Rdl receptor subunit was compared with those of vertebrate γ‐aminobutyric acid subtype A (GABAA) receptors and nicotinic acetylcholine receptors (nAChRs). The first six introns in Rdl show positions similar to those in vertebrate GABAA receptors, whereas the last two differ. It is interesting that the last intron appears to be in a position similar to that in nAChRs. These results are examined in relation to the proposal, based on amino acid identities, that Rdl codes for a novel class of GABAA receptor subunit more closely related to glycine receptors, and the possible place ofRdl in the lineage of the receptor superfamily is discussed.
Activation of Protein Kinase C by Phorbol Esters and Arachidonic Acid Required for the Optimal Potentiation of Glutamate Exocytosis
Authors: Herrero Immaculada , Miras‐Portugal María Teresa , Sánchez‐Prieto José
Published: October 2006

Abstract: The effects of arachidonic acid and phorbol esters in the Ca2+‐dependent release of glutamate evoked by 4‐aminopyridine (4‐AP) in rat cerebrocortical synaptosomes were studied. In the absence of arachidonic acid, high concentrations (500 nM) of 4β‐phorbol dibutyrate (4β‐PDBu) were required to enhance the release of glutamate. However, in the presence of arachidonic acid, low concentrations of 4β‐PDBu (1–50 nM) were effective in potentiating glutamate exocytosis. This potentiation of glutamate release by phorbol esters was not observed with the methyl ester of arachidonic acid, which does not activate protein kinase C. Moreover, pretreatment of synaptosomes with the protein kinase inhibitor staurosporine also prevented the stimulatory effect by arachidonic acid and phorbol esters. These results suggest that the activation of protein kinase C by both arachidonic acid and phorbol esters may play a role in the potentiation of glutamate exocytosis.
Lan‐1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization
Authors: Fatatis A. , Bassi A. , Monsurrò M. R. , Sorrentino G. , Mita G. D. , Di Renzo G. F. , Annunziato L.
Published: October 2006

The LAN‐1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1 ‐1,000 μM) caused a dose‐dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to ∼70% but completely abolished the plateau phase. The muscarinic‐activated Ca2+ channel was gadolinium (Gd3+) blockable and nimodipine and ω‐conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh‐induced [Ca2+]i elevation was concentration‐dependently inhibited by pirenzepine and 4‐diphenylacetoxy‐N‐methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin‐sensitive guanine nucleotide‐binding protein or by the diacylglycerol‐protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate‐sensitive intracellular store. In addition, ryanodine did not prevent CCh‐induced [Ca2+]i mobilization, and, finally, LAN‐1 cells appear to lack caffeine‐sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 μM), or after thapsigargin.
Purification and Lectin‐Binding Properties of s‐Laminin, A Synaptic Isoform of the Laminin B1 Chain
Authors: Chiu Arlene Y. , Ugozolli Mildred , Meiri Karina , Ko Jan
Published: October 2006

The extracellular matrix (ECM) at the vertebrate neuromuscular junction is a repository of functionally important molecules, some of which can regulate the formation of synapses during regeneration. One candidate molecule is s‐laminin, a 185‐kDa homologue of the laminin B1 chain. Whereas several members of the laminin family are present throughout the ECM ensheathing muscle fibers, immunoreactivity for s‐laminin is found selectively at synaptic sites in adult and embryonic rats, and is detectable at a time when synaptogenesis is taking place during development. We have reported previously that a rat schwannoma cell line, D6P2T, produces and releases large amounts of s‐laminin in culture. We have now purified s‐laminin from medium conditioned by these cells by using a simple three‐step procedure. Serum‐free, conditioned medium is separated by ion‐exchange chromatography on DEAE‐Sephacel, followed by size‐exclusion chromatography on 500 HR‐Seph‐acryl. Finally, s‐laminin is dissociated from other ECM components by agarose gel electrophoresis under reducing conditions and recovered in solution by extracting slices of agarose gel. The purified preparation displays one silver‐stained band that is recognized by three monoclonal antibodies known to bind to different epitopes on s‐laminin. Lectin‐binding studies demonstrate that s‐laminin is a glycoprotein and bears many of the carbohydrate moieties present on the B1 and B2 chains of laminin. Thus, the three 185–220‐kDa members of the laminin family are related in both their protein and carbohydrate domains.
Metabolism of Lactate in the Rat Brain During the Early Neonatal Period
Authors: Vicario Carlos , Medina José M.
Published: October 2006

The metabolism of lactate in isolated cells from early neonatal rat brain has been studied. In these circumstances, lactate was mainly oxidized to CO2, although a significant portion was incorporated into lipids (78% sterols, 4% phosphatidylcholine, 2% phosphatidylethanolamine, and 1% phosphatidylserine). The rate of lactate incorporation into CO2 and lipids was higher than those found for glucose and 3‐hydroxybutyrate. Lactate strongly inhibited glucose oxidation through the pyruvate dehydrogenase‐catalyzed reaction and the tricarboxylic acid cycle while scarcely affecting glucose utilization by the pentose phosphate pathway. Lipogenesis from glucose was strongly inhibited by lactate without relevant changes in the rate of glycerol phosphate synthesis. These results suggest that lactate inhibits glucose utilization at the level of the pyruvate dehydrogenase‐catalyzed reaction, which may be a mechanism to spare glucose for glycerol and NADPH synthesis. The effect of 3‐hydroxybutyrate inhibiting lactate utilization only at high concentrations of 3‐hydroxybutyrate suggests that before ketogenesis becomes active, lactate may be the major fuel for the neonatal brain. (‐)‐Hydroxycitrate and aminooxyacetate markedly inhibited lipogenesis from lactate, suggesting that the transfer of lactate carbons through the mitochondrial membrane is accomplished by the translocation of both citrate and N‐acetylaspartate.
Dopamine Efflux from Striatum After Chronic Nicotine: Evidence for Autoreceptor Desensitization
Authors: Harsing Laszlo G. , Sershen Henry , Lajtha Abel
Published: October 2006

We examined the effect of chronic nicotine treatment on dopaminergic activity by measuring the effects of D1 and D2 dopamine (DA) receptor agonists and antagonists on tritium release from mouse striatum preloaded with [3H]DA. The radioactivity released during superfusion was separated on alumina columns and the distribution and efflux of [3H]DA and its main 3H‐labeled metabolites were quantified. After preloading by incubation with [3H]DA, the electrical stimulation‐evoked tritium overflow was higher in striatum prepared from nicotine‐treated mice, whereas in vitro addition of nicotine caused a similar increase in tritium release from striatum of untreated and chronic nicotine‐treated mice. The overflow of [3H]DA and its 3H‐metabolites exhibited similar distribution patterns in [3H]DA‐preloaded striatum dissected from untreated and chronic nicotine‐pretreated mice, indicating that repeated injections with nicotine did not alter the metabolism of [3H]DA taken up by the tissue. (−)‐Quinpirole, a selective agonist for D2 DA receptors, and apomorphine, a nonselective D1/D2 agonist, inhibited the electrical stimulation‐induced tritium efflux from striatum of untreated mice, whereas (±)‐sulpiride, a D2 DA receptor antagonist, enhanced the evoked release of tritium. These changes in tritium efflux effected by (−)‐quinpirole and (±)‐sulpiride reflected changes in [3H]DA release and not in DA metabolism, as shown by separation of the released radioactivity on alumina columns. The D1 receptor agonist (±)‐SKF‐38393 did not affect the tritium overflow, whereas the D1 receptor antagonist (+)‐SCH‐23390 exerted a stimulatory action but only at a high concentration. In contrast, neither the DA receptor agonists [(−)‐quinpirole, apomorphine, and (±)‐SKF‐38393] nor the antagonists [(±)‐sulpiride and (+)‐SCH‐23390] altered the stimulation‐evoked tritium release in striatum obtained from mice repeatedly treated with nicotine. It is concluded that chronic administration of nicotine produces an increased release of DA in the striatum with a resulting elevation of synaptic DA concentrations leading to development of D2 DA autoreceptor subsensitivity. As a consequence, chronic nicotine may attenuate autoinhibition of dopaminergic neurotransmission in the striatum.
Distinct Developmental Patterns of Expression of Rat α1, α5, γ2S, andα12Lγ‐Aminobutyric AcidA Receptor Subunit mRNAs In Vivo and In Vitro
Authors: Bovolin Patrizia , Santi Maria‐Rita , Memo Maurizio , Costa Erminio , Grayson Dennis R.
Published: October 2006

We have quantitated the α1, α5, γ2S, and γ2Lγ‐aminobutyric acidA (GABAA) receptor subunit mRNAs in the maturing cerebellum in vivo and in cerebellar granule neurons differentiating in vitro. Absolute amounts of mRNA were measured by reverse transcription and competitive polymerase chain reaction (PCR) analysis with appropriate internal standards. The α1 and γ2L mRNA content increased continuously during postnatal cerebellar maturation and their changes with time matched very closely those of the cerebellar granule cells differentiating in vitro. The γ2S subunit mRNA showed a relatively constant pattern of expression both in vivo and in vitro, with comparable absolute concentrations in both developmental paradigms. The α5 mRNA was initially high in vivo and decreased (eightfold) to adult levels as postnatal cerebellar development progressed. In vitro the amount of α5 GABAA receptor subunit mRNA was higher than in vivo at 3 days, increased by more than twofold by 8 days, and declined to approximately the initial values at 23 and 28 days in vitro. Collectively, the results indicate that the α1, α5, γ2S, and γ2L GABAA receptor subunit mRNAs are regulated differentially in a temporal manner during in vivo and in vitro maturation. Moreover, a comparison of the ontogenetic profiles of the γ2S and γ2L mRNAs indicates that alternative splicing of the γ2 primary RNA transcript is regulated developmentally during postnatal maturation of the rat cerebellum.
Development of an Antiserum to the Midportion of Substance P: Applications for Biochemical and Anatomical Studies of Substance P‐Related Peptide Species in CNS Tissues
Authors: Shimonaka Hiroyuki , Marchand James E. , Connelly Christopher S. , Kream Richard M.
Published: October 2006

This report describes the generation and biochemical characterization of a high‐affinity antiserum that recognizes an epitope contained in the midportion sequence of substance P, i.e., substance P4–10. Designated A47, this reagent bound a variety of related peptide species containing the substance P4–10 sequence with apparent equipotency. A double radioimmunoassay procedure was developed that utilized A47, in combination with a traditional high‐affinity COOH‐terminally directed anti‐substance P serum, to provide quantification of mature and immature forms of substance P in CNS tissues. Across most rat CNS areas, levels of substance P‐like immunoreactivity were consistently 15% higher when monitored by analyses using A47 versus antisubstance P serum. In the dorsal root ganglia, an apparent enhancement in levels of substance P‐like immunoreactivity of approximately 40%, when quantified by analyses using A47 versus anti‐substance P serum, was observed; this most likely reflected the presence of an active biosynthetic pool of intermediate processing forms of substance P in this tissue. Coordinated HPLC/radioimmunoassay analyses of extracted dorsal root ganglia tissues demonstrated multiple peaks of immunoreactivity corresponding to mature substance P and to several of its precursor forms found in the normal biosynthetic pathway. Of the total recovered HPLC‐fractionated immunoreactivities, that corresponding to the putative immediate precursor to substance P, i.e., substance P‐glycine, was the predominant peak. In an additional series of HPLC/radioimmunoassay analyses, selective decreases in immunoreactive peaks corresponding to precursor forms of substance P were observed in dorsal root ganglia tissues from rats treated with the neurotoxic agent capsaicin. These results indicated decreased turnover of substance P as a consequence of drug treatment. Finally, initial immunohisto‐chemical analyses employing affinity‐purified A47 produced an unusual pattern of labeling characterized by well defined punctate terminal elements within the superficial aspects of the dorsal horn of the spinal cord.
Brain‐Derived Neurotrophic Factor Protects Dopamine Neurons Against 6‐Hydroxydopamine and N‐Methyl‐4‐Phenylpyridinium Ion Toxicity: Involvement of the Glutathione System
Authors: Spina Mary Beth , Squinto Stephen P. , Miller James , Lindsay Ronald M. , Hyman Carolyn
Published: October 2006

Brain‐derived neurotrophic factor (BDNF) has recently been shown to enhance the survival of dopamine neurons in cultures derived from the embryonic rat mesencephalon. We now extend this study by demonstrating that, in addition to the effect of sustaining survival of dopaminergic neurons, BDNF also confers protection against the neurotoxic effects of 6‐hydroxydopamine (6‐OHDA) and N‐methyl‐4‐phenylpyridinium ion (MPP+). Exposure of mesencephalic cultures to either 6‐OHDA or MPP+ resulted in a loss of 70–80% of dopaminergic neurons, as determined by tyrosine hydroxylase (TH) immunocytochemistry. In BDNF‐treated cultures, loss of TH‐positive cells after exposure to either toxin was reduced to only 30%. To facilitate biochemical measurements, we studied SH‐SY5Y dopaminergic neuroblastoma cells. BDNF was found to protect these cells from the dopaminergic neurotoxins, 6‐OHDA and MPP+. Indicative of oxidative stress, treatment of SH‐SY5Y cells with 10 μM 6‐OHDA for 24 h caused a fivefold increase in the levels of oxidized glutathione (GSSG). Pre‐treatment with BDNF for 24 h completely prevented the rise in GSSG. Further examination revealed that BDNF increased the activity of the protective enzyme, glutathione reductase, by 100%. In contrast, BDNF had no effect on the activity of catalase. These results add further impetus to exploring the therapeutic potential of BDNF in animal models of Parkinson's disease.
Adult Rat Retina Interneurons Synthesize GD3: GD3 Expression by These Cells Is Regulated by Cell‐Cell Interactions
Authors: Daniotti J. L. , Landa C. A. , Rosner H. , Maccioni H. J. F.
Published: October 2006

GD3, a ganglioside of the lactosyl series, is prevalent in rat retina neuronal cells. We studied here whether rat retina neurons synthesize their own surface GD3 or if they acquire it from Müller glia cells. We analyzed the activity of GD3 synthase and the in vivo labeling of gangliosides from N‐[3H]acetylmannosamine in adult rat retinas after selective destruction of Müller glia cells with the gliotoxic α‐D,L‐aminoadipate (AAA). Immunostaining of rat retina sections and western blot analysis with an antivimentin antibody confirmed the gliotoxic effect of AAA. Neither GD3 synthase activity nor the in vivo labeling of GD3 and other gangliosides was significantly affected by AAA, indicating that neuronal cells synthesize their own GD3. We next analyzed the regulation of the expression of GD3 by these neurons in culture. About 80% of freshly dissociated cells from retina of 4‐day‐old rats (R4) immunoexpress surface GD3. After 3 days in dispersed cell culture conditions, GD3 expression was under the limit of detection in 80% of neuronal cells, indicating a failure of these cells to maintain the expression of surface GD3 in these experimental conditions. Most flat Miiller glia‐derived cells present in these cultures were GD3 positive. Surface GD3 was detected in ∼60% of neuronal cells dissociated from R4 tissue that was developed in vitro as an organ culture for 3 days. Likewise, ~50% of neurites that had grown out from R4 retinal ex‐plants within 3 days in culture and whose neuronal character was indicated by immunoexpression of growth‐associated protein GAP‐43 were GD3 positive. These findings suggest that the tissue organization and/or specific interactions modulate GD3 expression in neuronal cells. Under dispersed‐cell culture conditions, c‐pathway gangliosides (GQ1c and GT1c), which are built up from the sialylation of GD3 and later completion of the oligosaccharide backbone, were detected in ~60% of neuronal cells, suggesting a maintenance of production of GD3 as an intermediate for gangliotetraosyl gangliosides.
Toxic Effects of Iron for Cultured Mesencephalic Dopaminergic Neurons Derived from Rat Embryonic Brains
Authors: Michel Patrick P. , Vyas Sheela , Agid Yves
Published: October 2006

Iron, a transition metal possibly involved in the pathogenesis of Parkinson's disease, was tested for its toxic effects toward cultures of dissociated rat mesencephalic cells. When cultures were switched for 24 h to serum‐free conditions, the effective concentrations of ferrous iron (Fe2+) producing a loss of 50% of dopaminergic neurons, as quantified by tyrosine hydroxylase (TH) immunocytochem‐istry, TH mRNA in situ hybridization, and measurement of TH activity, were on the order of 200 μM. High‐affinity dopamine (DA) uptake, which reflects integrity and function of dopaminergic nerve terminals, was impaired at significantly lower concentrations (EC50= 67 μM). Toxic effects were not restricted to dopaminergic neurons inasmuch as trypan blue dye exclusion index and γ‐aminobutyric acid uptake, two parameters used to assess survival of other types of cells present in these cultures, were also affected. Protection against iron cytotoxicity was afforded by desferriox‐amine and apotransferrin, two ferric iron‐chelating agents. Normal supplementation of the culture medium by serum proteins during treatment was also effective, presumably vianonspecific sequestration. Potential interactions with DA were also investigated. Fe2+ at subtoxic concentrations and desferrioxamine in the absence of exogenous iron added to the cultures failed to potentiate or reduce DA cytotoxicity for mesencephalic cells, respectively. Transferrin, the glyco‐protein responsible for intracellular delivery of iron, was ineffective in initiating selective cytotoxic effects toward dopaminergic neurons preloaded with DA. Altogether, these results suggest (a) that ferrous iron is a potent neurotoxin for dopaminergic neurons as well as for other cell types in dissociated mesencephalic cultures, acting likely via autoxida‐tion into its ferric form, and (b) that the presence of intra‐and extracellular DA is not required for the observed toxic effects.
Glucocorticoids Exacerbate Hypoxic and Hypoglycemic Hippocampal Injury In Vitro: Biochemical Correlates and a Role for Astrocytes
Authors: Tombaugh Geoffrey C. , Yang Susan H. , Swanson Raymond A. , Sapolsky Robert M.
Published: October 2006

The acute secretion of glucocorticoids is critical for responding to physiological stress. Under normal circumstances these hormones do not cause acute neuronal injury, but they have been shown to enhance ischemic and seizure‐induced neuronal injury in the rat brain. Using fetal rat hippocampal cultures, we asked whether hypoxic and hypoglycemic cell damage in vitro could be exacerbated by direct exposure to corticosterone (CORT). Each of these insults alone damaged neuronal cells, whereas 4–6 h of hypoxic treatment could damage age‐matched astrocytes if glucose was reduced or omitted. Ischemic‐like injury to both cell types could be attenuated by pretreatment with high (30 mM) glucose. Exposure to 100 nM CORT did not affect cell viability under control conditions but enhanced both hypoxic and hypoglycemic neuronal injury. In both cases, pretreatment with high glucose abolished this CORT‐mediated synergy. In astrocyte cultures, CORT exacerbated both hypoxic and hypoglycemic injury and this effect was also attenuated by high‐glucose pretreatment. Identical 24‐h CORT treatment caused a 13% reduction in glucose uptake in astrocytes and a 38% reduction in glycogen content, without affecting the level of intracellular glucose. Thus, CORT could endanger both neurons and astrocytes in mixed hippocampal cultures and this effect emerged only under conditions of substrate depletion. The metabolic disruption in astrocytes by CORT further suggests that the ability of CORT to exacerbate neuronal injury may be due in part to impaired glial cell function.
Manipulation of Intracellular Calcium in NCB‐20 Cells
Authors: Garritsen Anja , Cooper Dermot M. F.
Published: October 2006

A number of lines of evidence indicate that the Ca2+ and cyclic AMP signalling systems interact in NCB‐20 cells. However, to date, the regulation of [Ca2+]i homeosta‐sis has not been studied in this cell line. The present study aimed to clarify our understanding of [Ca2+]i homeostasis in these cells and to evaluate tools that manipulate [Ca2+]i, independently of protein kinase C effects. Bradykinin, by a B2‐receptor, elevated [Ca2+]i by a pertussis‐toxin‐insensitive mechanism. The BK‐stimulated [Ca2+]i rise originated from intracellular sources, without a contribution from Ca2+ entry mechanisms. The effect of BK was precluded by pre‐treatment with thapsigargin and ionomycin—compounds that elevated [Ca2+]i independent of phospholipase C activation. Both compounds, however, exerted effects in addition to stimulating release of Ca2+ from BK‐sensitive stores; the BK‐sensitive Ca2+ pool was a subset of the thapsigargin‐sensitive pool; ionomycin strongly stimulates Ca2+ entry. Activation of protein kinases A and C attenuated the duration of the BK‐induced rise in [Ca2+]i, without affecting the peak [Ca2+]i, suggesting interference with the BK response at a step downstream of the activation of phospholipase C. Application of these approaches should enhance the delineation of the consequences of Ca2+ mobilization on cyclic AMP accumulation.