bluuebunny/crossref_metadata_2025 huggingface dataset:
Potent Neurotoxic Fluorinated 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Analogs as Potential Probes in Models of Parkinson Disease
Authors: Riachi Naji J. , Arora Pramod K. , Sayre Lawrence M. , Harik Sami I.
Published: October 2006

Abstract: We synthesized a number of fluorinated analogs of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), and tested their suitability as substrates for monoamine oxidase B in vitro and their dopaminergic neurotoxicity in vivo. Two of the compounds tested, 2′‐F‐MPTP and 2′‐CF3‐MPTP, were better enzyme substrates and possessed more potent neurotoxicity for nigrostriatal dopamine neurons than MPTP, especially 2′‐F‐MPTP. The results of the in vivo neurotoxicity experiments correlated well with the suitability of the compounds as substrates for monoamine oxidase. These findings could serve as a basis for the use of 18F‐labeled analogs of MPTP for positron emission tomography studies of nonhuman primates for better understanding of the factors underlying MPTP toxicity. Furthermore, the discovery of two MPTP analogs with enhanced selective neurotoxicity to dopaminergic neurons may be an important clue in the continuing efforts to define the chemical structure‐activity factors governing MPTP neurotoxic activation mechanisms.
Down's Syndrome Individuals Begin Life with Normal Levels of Brain Cholinergic Markers
Authors: Kish Stephen , Karlinsky Harry , Becker Lawrence , Gilbert Joseph , Rebbetoy Michelle , Chang Li‐Jan , DiStefano Linda , Hornykiewicz Oleh
Published: October 2006

Abstract We measured the activities of the cholinergic marker enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in autopsied brains of seven infants (age range 3 months to 1 year) with Down's syndrome (DS), a disorder in which virtually all individuals will develop by middle age the neuropathological changes of Alzheimer's disease accompanied by a marked brain cholinergic reduction. When compared with age‐matched controls cholinergic enzyme activity was normal in all brain regions of the individuals with infant DS with the exception of above‐normal activity in the putamen (ChAT) and the occipital cortex (AChE). Our neurochemical observations suggest that DS individuals begin life with a normal complement of brain cholinergic neurons. This opens the possibility of early therapeutic intervention to prevent the development of brain cholinergic changes in patients with DS.
Endogenous Amino Acid Release from Cultured Cerebellar Neuronal Cells: Effect of Tetanus Toxin on Glutamate Release
Authors: Van Vliet Bernard J. , Sebben Michèle , Dumuis Aline , Gabrion Jacqueline , Bockaert Joël , Pin Jean‐Philippe
Published: October 2006

Abstract Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron‐microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 m M K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, γ‐aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+‐dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+‐dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 μ/ml) for 24 h resulted in a total inhibition of the Ca2+‐dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.
Cyclic AMP‐Dependent Protein Phosphorylation in Isolated Neuronal Growth Cones from Developing Rat Forebrain
Authors: Lockerbie R. O. , Eddé B. , Prochiantz A.
Published: October 2006

Abstract: We have shown recently that neuronal growth cones isolated from developing rat forebrain possess an appreciable activity of adenylate cyclase, which produces cyclic AMP and can be stimulated by various neurotransmitter receptor agonists and by forskolin. To investigate cyclic AMP‐mediated biochemical mechanisms in isolated growth cones, we have centered the present study on cyclic AMP‐dependent protein phosphorylation. One‐dimensional gel electrophoretic analysis showed that cyclic AMP analogs increased incorporation of 32P into several phosphoproteins in molecular mass ranges of 50–58 and 76–82 kilodaltons, including those of 82, 76, and 51 kilodaltons. Two‐dimensional electrophoresis, using isoelectric focusing in the first dimension, resolved phosphorylated α‐ and β‐tubulin species, actin, a very acidic protein (isoelectric point 4.0) with a molecular mass of 93 kilodaltons, and two proteins (x and x′) closely neighboring β‐tubulin. Two other phosphoproteins seen in the gels had molecular masses of 56 and 51 kilodaltons (respective isoelectric points, 4.5 and 4.4) and, along with the 93‐kilodalton phosphoprotein, were highly enriched in the isolated growth cones. Only the tubulin and actin species were major proteins in the isolated growth cones. Cyclic AMP analogs enhanced incorporation of 32P into phosphoproteins x and x′, and, as assessed by immunoprecipitation, into β‐tubulin. Peptide digest experiments suggested that phosphoproteins x and x′ are unrelated to β‐tubulin. Nonequilibrium two‐dimensional electrophoresis resolved many phosphoproteins, of which a 79‐ and 75‐kilodalton doublet, a 74‐kilodalton species, and a 58‐kilodalton doublet showed enhanced incorporation of 32P in the presence of cyclic AMP.
Differential Localization of Glutathione‐S‐Transferase Yp and Yb Subunits in Oligodendrocytes and Astrocytes of Rat Brain
Authors: Cammer Wendy , Tansey Francine , Abramovitz Mark , Ishigaki Seishi , Listowsky Irving
Published: October 2006

Abstract: Glutathione‐S‐transferase Yb subunits were recently identified in rat brain and localized to astrocytes, ependymal cells lining the ventricles, subventricular zone cells, and tanycytes. Another isoform, Yp (π family), was detected in rat brain by immunoblotting, and its mRNA was detected by Northern hybridizations. Double immunofluorescence localized Yb and Yp in different glial cells. The strongly Yp‐positive cells were identified as oligodendrocytes by virtue of their arrangement in rows in white‐matter tracts, colocalization in strongly carbonic anhydrase‐positive cells, and association with myelinated tracts in the corpus striatum. Ependymal cells in the choroid plexus and ventricular lining were also strongly Yp positive, whereas Yb was not detected in the choroid plexus. The occurrence of Yp at low levels in astrocytes was indicated after immunostaining by a sensitive peroxidase‐antiperoxidase method, which revealed weak staining of those cells in the molecular layer of the cortex. The data suggest that Yb and Yp subunits are primarily localized to astrocytes and oligodendrocytes, respectively, and that both are absent from neurons. The glutathione‐S‐transferase in oligodendrocytes may participate in the removal of toxins from the vicinity of the myelin sheath. The finding of glutathione‐S‐transferases in ependymal cells and astrocytes in the brain also suggests that this enzyme could be a first line of defense against toxic substances.
Tellurium‐Induced Neuropathy: Metabolic Alterations Associated with Demyelination and Remyelination in Rat Sciatic Nerve
Authors: Harry G. J. , Goodrum J. F. , Bouldin T. W. , Wagner‐Recio M. , Toews A. D. , Morell P.
Published: October 2006

Abstract:Rats fed a diet containing 1.25% elemental tellurium initiated on postnatal day 20 undergo a transient neuropathy characterized by synchronous demyelination of peripheral nerves. In sciatic nerve, the extent of demyelination was maximal after 5 days of tellurium exposure; there was a loss of 25% of the myelin, as assayed by concentration of myelin‐specific P0protein. Tellurium‐induced alterations in the metabolic capacity of Schwann cells were examined by measuring the synthesis of myelin lipids in vitro in isolated sciatic nerve segments. Exposure to tellurium resulted in an early marked decrease of ∼50% in overall incorporation of [14C]acetate into lipids, with a preferential depression in synthesis of cerebrosides, cholesterol, and ethanolamine plasmalogens (components enriched in myelin). Most dramatically, within 1 day of initiation of tellurium exposure, there was a profound increase in [14C]acetate‐derived radioactivity in squalene; 23% of incorporated label was in this intermediate of cholesterol biosynthesis, compared to < 0.5% in controls. In association with the remyelinating phase seen after 5 days of tellurium exposure, synthesis of myelin components gradually returned to normal levels. After 30 days, metabolic and morphologic alterations were no longer apparent. We suggest that the sequence of metabolic events in sciatic nerve following tellurium treatment initially involves inhibition of the conversion of squalene to 2,3‐epoxysqualene, and that this block in the cholesterol biosynthesis pathway results, either directly or indirectly, in the inhibition of the synthesis of myelin components and breakdown of myelin.
Determination of Brain Interstitial Concentrations by Microdialysis
Authors: Benveniste Helene , Hansen Anker Jon , Ottosen Niels Saabye
Published: October 2006

Abstract: Microdialysis is an extensively used technique for the study of solutes in brain interstitial space. The method is based on collection of substances by diffusion across a dialysis membrane positioned in the brain. The outflow concentration reflects the interstitial concentration of the substance of interest, but the relationship between these two entities is at present unclear. So far, most evaluations have been based solely on calibrations in saline. This procedure is misleading, because the ease by which molecules in saline diffuse into the probe is different from that of tissue. We describe here a mathematical analysis of mass transport into the dialysis probe in tissue based on diffusion equations in complex media. The main finding is that diffusion characteristics of a given substance have to be included in the formula. These include the tortuosity factor (λ) and the extracellular volume fraction (α). We have substantiated this by studies in a welldefined complex medium (red blood cell suspensions) as well as in brain. We conclude that the traditional calculation procedure results in interstitial concentrations that are too low by a factor of λ2/α for a given compound.
Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion
Authors: Ahnert‐Hilger Gudrun , Bader Marie‐France , Bhakdi Sucharit , Gratzl Manfred
Published: October 2006

Abstract: Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO‐permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+‐dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+‐induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.
In Vivo Measurement of Extracellular 5‐Hydroxytryptamine in Hippocampus of the Anaesthetized Rat Using Microdialysis: Changes in Relation to 5‐Hydroxytryptaminergic Neuronal Activity
Authors: Sharp Trevor , Bramwell Steven R. , Clark David , Grahame‐Smith David G.
Published: October 2006

Abstract: The effect of manipulating the activity of central 5‐hydroxytryptamine (5‐HT) neurones on extracellular 5‐HT in ventral hippocampus of the chloral hydrate‐anaesthetized rat was studied using the brain perfusion method, microdialysis. Basal levels of 5‐HT in the dialysates were close to the detection limits of our assay using HPLC with electrochemical detection. However, addition of the selective 5‐HT reuptake inhibitor citalopram (10−6M) to the perfusion medium produced readily measurable amounts of dialysate 5‐HT. Citalopram, therefore, was used throughout our experiments. Hippocampal dialysate levels of 5‐HT sharply declined over the first hour after dialysis probe implantation, but then became constant. This stable output of 5‐HT was reduced by 57% in rats treated 14 days previously with intracerebroven‐tricular injections of the 5‐HT neurotoxin 5,7‐dihydroxy‐tryptamine. Electrical stimulation (1‐ms pulse width, 300 μA, 2–20 Hz) of the dorsal raphe nucleus for 20 min caused a rapid rise in hippocampal 5‐HT output, which immediately declined on cessation of the stimulus and was frequency‐dependent. Addition of tetrodotoxin (10−6M) to the perfusion medium reduced 5‐HT levels to 75% of predrug values. Injection of the 5‐HT1A agonist 8‐hydroxy‐2‐(di‐n‐propylami‐no)tetralin (0.5 and 2.5 μg) into the dorsal raphe nucleus caused a dose‐related fall in hippocampal output of 5‐HT compared to saline‐injected controls. We conclude from these data that the spontaneous output of endogenous 5‐HT into hippocampal dialysates, measured under our experimental conditions, predominantly originates from central 5‐HT neurones and changes in accordance with their electrical activity.
Ontogenesis of α2‐Adrenoceptor Coupling with GTP‐Binding Proteins in the Rat Telencephalon
Authors: Kitamura Yoshihisa , Mochii Makoto , Kodama Ryuji , Agata Kiyokazu , Watanabe Kenji , Eguchi Goro , Nomura Yasuyuki
Published: October 2006

Abstract: The ontogenesis of α2‐adrenoceptors and GTP‐binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese‐induced elevation of [3H]clonidine binding was increased in an age‐dependent manner but the guanosine 5′‐O‐(3‐thio)triphosphate‐induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age‐dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP‐ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39‐kDa proteins ADP‐ribosylated by pertussis toxin (Giα+ Goα) were increased with age and reached the adult level at day 12, whereas the 46‐kDa protein (Gsα) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against α‐ and β‐subunit of GTP‐binding proteins) demonstrated that the 39‐kDa α‐subunit of (Goα) and the 36‐kDa δ‐subunit of GTP‐binding protein (δ36) increased with postnatal age. In contrast, 35‐kDa δ‐subunit (δ35) did not change. From these results, it is suggested that the coupling activity of α‐adrenoceptor with GTP‐binding protein gradually develops in a manner parallel with the increase of α2‐adrenoceptor and pertussis toxin sensitive GTP‐binding proteins, Gi, and that α39β36γ may be related to the differentiation and/or growth of nerve cells in rat telencephalon.
4‐[18F]Fluoro‐L‐m‐Tyrosine: An L‐3,4‐Dihydroxyphenylalanine Analog for Probing Presynaptic Dopaminergic Function with Positron Emission Tomography
Authors: Melega William P. , Perlmutter Milton M. , Luxen Andre , Nissenson Charna H. K. , Grafton Scott T. , Huang Sung‐Cheng , Phelps Michael E. , Barrio Jorge R.
Published: October 2006

Abstract: 4‐[18F]Fluoro‐L‐m‐tyrosine (FMT), a biochemical probe of striatal dopaminergic function, has been synthesized as an L‐3,4‐di‐hydroxyphenylalanine analog for positron emission tomography. Biochemical characterization of this compound in the rat 30 min after intrajugular administration indicated that in the brain, selective decarboxylation occurred in the striatum, with the formation of 4‐fluoro‐3‐hydroxyphenylethylamine and its metabolites. Positron emission tomography analysis of brain tissue in monkeys (Macaca nemestrina) after intravenous injection of FMT revealed a true time‐dependent, specific accumulation of radioactivity in striatum, with a striatum/cerebellum (nonspecific) ratio of 4 at 180 min. Peripheral metabolism accounted for <40% of the total radioactivity in arterial blood samples after 120 min. The amino acid remained as the major component throughout the period of investigation (n = 3; 5 min, 95%; 10 min, 85%; 30 min, 67%; 60 min, 62%; 120 min, 60%), with a plasma clearance tl/2 of 112 min. 3‐O‐Methylated metabolites were not observed. The substrate specificity of FMT, coupled with its limited in vivo peripheral metabolism, makes it a useful, new biochemical probe for in vivo, noninvasive evaluation of central dopaminergic mechanisms.
Detection of a Novel Serotonin Receptor Subtype (5‐HT1E) in Human Brain: Interaction with a GTP‐Binding Protein
Authors: Leonhardt Sigrun , Herrick‐Davis Katharine , Titeler Milt
Published: October 2006

Abstract: [3H]Serotonin (5‐hydroxytryptamine, [3H]5‐HT) was used as a radioligand probe of brain 5‐HT receptors in homogenates of human cortical tissue. Two binding sites were detected in the presence of 1 μM pindolol (to block 5‐HT1Aand 5‐HT1B receptors), and 100 nM mesulergine (to block 5‐HT1c and 5‐HT2 receptors). One of these sites demonstrated high affinity for 5‐carboxyamidotryptamine (5‐CT) and er‐gotamine, consistent with the known pharmacology of the 5‐HT1D receptor; the second site demonstrated low affinity for 5‐CT and ergotamine. Computer‐assisted analyses indicated that both drugs displayed high affinities (Ki values of 1.1 nM and 0.3 nM for 5‐CT and ergotamine, respectively) for 55% of the sites and low affinities (Ki values of 910 nM and 155 nM for 5‐CT and ergotamine, respectively) for 45% of the sites. To investigate the non‐5‐HT1D component of the binding, 100 nM 5‐CT (to block 5‐HT1A, 5TTT1B, and 5‐HT1D receptors) was coincubated with [3H]5‐HT, membranes, and mesulergine. The remaining [3H]5‐HT binding (hereafter referred to as “5‐HT1E”) displayed high affinity and saturability (KD, 5.3 nM; Bmax, 83 fmol/mg) in human cortical tissue. Competition studies with nonradioactive drugs indicated that, of the drugs tested, 5‐CT and ergotamine displayed the highest selectivity for the 5‐HT1D site versus the 5‐HT1E site. The interaction of the 5‐HT1E site with a GTP‐binding protein was demonstrated; the nonhydrolyzable derivatives of GTP, guanosine 5′‐0‐(3‐thiotriphosphate) (GTPγS) and 5′‐guanylylimidodiphosphate [Gpp(NH)p], potently inhibited binding of [3H]5‐HT to the 5‐HT1E site (IC50 values of 16 and 172 nM, respectively) while adenosine 5′‐0‐(3‐thiotriphosphate) (ATPγS) and 5′‐adenylylimidodi‐phosphate [App(NH)p] were without effect. The high affinity of 5‐HT for the site, the unique pharmacological profile of the site, and the interaction of the site with a GTP‐binding protein indicate that this site represents a unique 5‐HT receptor subtype heretofore undescribed, and which we propose to call 5‐HT1E in keeping with the current system of nomenclature in the 5‐HT receptor field.
Ischemic Modification of Cerebrocortical Membranes: 5‐Hydroxytryptamine Receptors, Fluidity, and Inducible In Vitro Lipid Peroxidation
Authors: Villacara A. , Kumami K. , Yamamoto T. , Mrsulja B. B. , Spatz M.
Published: October 2006

Abstract: The effect of ischemia on the properties of 5‐hydroxytryptamine1A+B (5‐HT1A+B) and 5‐hydroxytrypt‐amine1B5‐HT1B) binding sites, physical‐state “fluidity” of the membrane, and its susceptibility to peroxidation in vitro was investigated in the cerebral cortex of gerbils. Ischemia was induced by bilateral carotid artery occlusion for 15 min alone or with release for 1 h. Ischemia both with and without reflow decreased the number of 5‐HT1A+B and 5‐HT1B binding sites, whereas ischemia and reflow altered the affinity for 5‐HT1B binding sites. Resistance to the temperature‐dependent increase in “fluidity” of the membrane was detected (by fluorescence anisotropy using l,6‐diphenyl‐l,3,5‐hexatriene as a probe) after ischemia and reflow but not in ischemia alone. Susceptibility of the membranes to Fe2+‐ and ascorbic acid‐stimulated lipid peroxidation in vitro was decreased following ischemia and recirculation only. These findings strongly suggest that the composition and the function of the membrane are markedly disturbed during recirculation after ischemia.
Identification and Characterization of Calcium‐Dependent Metalloproteases in Rat Brain
Authors: Nelson Robert B. , Siman Robert
Published: October 2006

Abstract: We have begun to identify and characterize brain protease activities separated by and assayed in substrate‐con‐taining polyacrylamide gels. In the present report, we focus on four proteolytic activities identified from rat brain that are dependent on micromolar and millimolar Ca2+ concentrations for activity. In contrast to the previously described Ca2+‐dependent neutral cysteine proteases (calpains), all four activities appear to be metalloproteases based on their inhibition by EDTA, EGTA, and 1,10‐o‐phenanthroline, but not by blockers of serine, cysteine, or aspartic proteases. In the presence of excess Ca2+ and the Zn2+‐chelating inhibitor 1,10‐o‐phenanthroline, activity of the enzymes was reconstituted by addition of lower concentrations of Zn2+, and inhibited by higher Zn2+ concentrations. The four metalloproteases were designated MP‐112, MP‐92, MP‐70, and MP‐65 on the basis of their apparent molecular masses in kilodaltons. MP‐70, the major activity detected, had an apparent Kact for Ca2+ > 100 μM versus 10‐25 μM for MP‐65 and 50‐100 μM for MP‐92. MP‐112 was a minor activity for which Ca2+ activation levels were not determined. MP‐112, MP‐70, and MP‐65 were similar in being most active in the soluble fraction of 7‐day neonate forebrain. In contrast, MP‐92 activity was highest in the particulate fraction of adult forebrain. About half of the MP‐92 activity and lower levels of the other three activities were still detectable in particulate fractions after detergent extraction of membrane, suggesting an association with cytoskeletal or other structural proteins. These results indicate the presence in CNS of a class of Ca2+‐dependent proteases that are distinct from the previously described cal‐pains, and that may be important in Ca2+‐mediated neural events.
Separate Binding and Functional Sites for ω co‐Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells
Authors: Ballesta J. J. , Palmero M. , Hidalgo M. J. , Gutierrez L. M. , Reig J. A. , Viniegra S. , Garcia A. G.
Published: October 2006

Abstract: Purified adrenomedullary plasma membranes contain two high‐affinity binding sites for l25I‐ω‐conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin‐receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled ω‐conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two‐site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cho‐linoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration‐dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmaxof 129 fmol/mg of protein. At 0.25 μM, co‐conotoxin was notable to block depolarization‐evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mMK+for 30 s, whereas under the same conditions, 1 μM nitrendipine inhibited uptake by ∼60%. When cells were hyper‐polarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mMK+, ω‐conotoxin partially inhibited Ca2+ uptake in a concentration‐dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of ω‐conotoxin binding sites remains to be determined.
Characterization and Regional Distribution of Peptides Derived from the Vasoactive Intestinal Peptide Precursor in the Normal Human Brain
Authors: Fahrenkrug Janj , Emson Piers C.
Published: October 2006

Abstract: To study the biosynthetic processing of the precursor for vasoactive intestinal peptide (prepro‐VIP) in the human brain, we have developed antiseraj against the five functional domains of the precursor molecule: prepro‐VIP 22‐79, peptide histidine methionine (PHM), prepro‐VIP 111‐122, VIP, and prepro‐VIP 156‐170. The antisera were used in radioimmunoassays in combinatioin with HPLC to identify and quantify the peptides in regions of the human brain. All five peptides were expressed, but mainly in non‐equimolar ratios. In only three regions were the same amounts of VIP and PHM found; in the remaining areas the concentration of PHM was two‐thirds that of VIP. The concentrations of prepro‐VIP 22‐79, prepro‐VIP 111‐122, and prepro‐VIP 156‐170 were considerably lower than the corresponding VIP concentrations, and the relative concentration of prepro‐VIP 111‐122 differed between cortical and subcortical areas. A small proportion of the VIP precursor followed a pathway in which the dibasic conversion site after PHM is not cleaved, as evidenced by the presence of a C‐terminally extended form of PHM. Finally, it was found that the C‐terminal lysine residue of prepro‐VIP is not removed during processing. The findings indicate that differences in the posttranslational processing of prepro‐VIP exist in subpopulations of neurons in the human brain.
Muscarinic Receptor‐Stimulated Phosphoinositide Turnover in Human SK‐N‐SH Neuroblastoma Cells: Differential Inhibition by Agents that Elevate Cyclic AMP
Authors: Akil Mayada , Fisher Stephen K.
Published: October 2006

Abstract: The possibility that an increased intracellular concentration of cyclic AMP (cAMP) can regulate the extent of muscarinic receptor‐stimulated phosphoinositide (PPI) turnover in the human neuroblastoma cell line SK‐N‐SH was examined. Addition of either forskolin (or its water‐soluble nalog, L‐85,8051), theophylline, isobutylmethylxanthine, or Liolera toxin, agents that interact with either the catalytic unit of adenylate cyclase, cAMP phosphodiesterase, or the guanine nucleotide binding protein linked to adenylate cyclase activation, resulted in a 45–181% increase in cAMP concentration and a 27–70% inhibition of carbachol‐stimu‐lated inositol phosphate release. Through the use of digitonin‐permeabilized cells, the site of inhibition was localized to a step at, or distal to, the guanine nucleotide binding protein that regulates phospholipase C activity. In contrast, when intact SK‐N‐SH cells were exposed to prostaglandin E1, the ensuing increases in cAMP were not accompanied by an inhibition of stimulated PPI turnover. These differential effects of increased cAMP concentrations on stimulated PPI turnover may reflect the compartmentation of cAMP within SK‐N‐SH cells.
Introduction of Impermeant Molecules into Synaptosomes Using Freeze/Thaw Permeabilization
Authors: Nichols Robert A. , Wu Wilson C.‐S. , Haycock John W. , Greengard Paul
Published: October 2006

Abstract: Brief freezing as a means of transiently permeabilizing synaptosomes was explored. Rat brain synaptosomes frozen and thawed in the presence of 5% dimethyl sulfoxide, a cryoprotectant, were shown to release, in a calcium‐dependent manner, previously accumulated [3H]norepinephrine and [14C]acetylcholine in response to elevated [K+]o. In addition, synaptosomes subjected to freeze/thaw were shown to retain their ability to exhibit resting protein phosphorylation, as well as stimulated protein phosphorylation occurring in response to calcium influx. Brief freezing of synaptosomes in the presence of [γ‐32P]ATP and either the catalytic subunit of cyclic AMP‐dependent protein kinase or calcium/calmodulin‐dependent protein kinase II rendered the synaptosomal interior accessible to these agents, as reflected by the phosphorylation of substrate proteins, such as synapsin I, which reside within the nerve terminal. Inclusion of inhibitors of these protein kinases during freeze/thaw blocked synaptosomal protein phosphorylation, indicating that the inhibitors were also introduced. After freezing, the synaptosomes resealed rapidly and spontaneously, as shown by the inability of any of the agents to elicit an effect on phosphorylation when added at the end of the freezing period. The permeabilization procedure should contribute to an understanding of the functional roles of phosphoproteins, and of their associated protein kinases and protein phosphatases, in nerve terminals.
Translocation and Activation of Protein Kinase C in Striatal Neurons in Primary Culture: Relationship to Phorbol Dibutyrate Actions on the Inositol Phosphate Generating System and Neurotransmitter Release
Authors: Weiss Samuel , Ellis John , Hendley Daniel D. , Lenox Robert H.
Published: October 2006

Abstract: The actions of the tumor‐promoting phorbol ester phorbol dibutyrate were examined, under identical physiological conditions, on three distinct cellular processes in striatal neurons: the distribution of protein kinase C, the carbachol‐stimulated generation of [3H]inositol monophosphate, and the KCl‐evoked release of γ‐[3H]aminobutyric acid ([3H]GABA). Phorbol dibutyrate induced a rapid (complete in 5 min), dose‐dependent, entirely reversible (t0.5= 15 min) translocation of protein kinase C from cytosol to membrane. On longer exposure to phorbol dibutyrate, membrane‐associated protein kinase C returned toward the control level, and total cellular enzyme activity declined markedly. Phorbol dibutyrate also induced the dose‐dependent attenuation of carbachol‐stimulated [3H]inositol monophosphate production and potentiation of KCl‐evoked release of [3H]GABA. The translocation of protein kinase C and the potentiation of KCl‐evoked [3H]GABA release were both rapidly reversed following washout of phorbol dibutyrate. In addition, for both processes, the effect of a 1‐h exposure to phorbol dibutyrate was markedly less than that observed following a 5‐min exposure to the agent. In direct contrast, inhibition of carbachol‐stimulated [3H]inositol monophosphate production was not rapidly reversed following washout of phorbol dibutyrate and was actually more pronounced following a 1‐h exposure, compared with a 5‐min exposure. These findings indicate that some, but not all, of the actions of phorbol dibutyrate are closely associated with the translocation of protein kinase C in striatal neurons in primary culture.
Effects of the Histamine H3‐Agonist (R)‐α‐Methylhistamine and the Antagonist Thioperamide on Histamine Metabolism in the Mouse and Rat Brain
Authors: Oishi Ryozo , Itoh Yoshinori , Nishibori Masahiro , Saeki Kiyomi
Published: October 2006

Abstract: To study the feedback control by histamine (HA) H3‐receptors on the synthesis and release of HA at nerve endings in the brain, the effects of a potent and selective H3‐agonist, (R)‐α‐methylhistamine, and an H3‐antagonist, thioperamide, on the pargyline‐induced accumulation of tele‐methylhistamine (t‐MH) in the brain of mice and rats were examined in vivo. (R)‐α‐Methylhistamine dihydrochloride (6.3 mg free base/kg, i.p.) and thioperamide (2 mg/kg, i.p.), respectively, significantly decreased and increased the steady‐state t‐MH level in the mouse brain, whereas these compounds produced no significant changes in the HA level. When administered to mice immediately after pargyline (65 mg/kg, i.p.), (R)‐α‐methylhistamine (3.2 mg/kg, i.p.) inhibited the pargyline‐induced increase in the t‐MH level almost completely during the first 2 h after treatment. Thioperamide (2 mg/kg, i.p.) enhanced the pargyline‐induced t‐MH accumulation by ∼70% 1 and 2 h after treatment. Lower doses of (R)‐α‐methylhistamine (1.3 mg/kg) and thioperamide (1 mg/kg) induced significant changes in the pargyline‐induced t‐MH accumulation in the mouse brain. In the rat, (R)‐α‐methylhistamine (3.2 mg/kg, i.p.) and thioperamide (2 mg/kg, i.p.) also affected the pargyline‐induced t‐MH accumulation in eight brain regions and the effects were especially marked in the cerebral cortex and amygdala. These results indicate that these compounds have potent effects on HA turnover in vivo in the brain.
Delayed Neurologic Deterioration Following Anoxia: Brain Mitochondrial and Metabolic Correlates
Authors: Wagner Kenneth R. , Kleinholz Marla , Myers Ronald E.
Published: October 2006

Abstract: Hyper‐ but not normoglycemic cats exposed to 8 min of anoxia show neurologic signs (fasciculations, myoclonic jerks, seizures) that develop after a symptom‐free period. We examined brain mitochondrial function and metabolite concentrations at 0, 1, 3, and 5 h following exposure to anoxia, to correlate biochemical findings with the presence (“symptomatic”) or absence (“presymptomatic”) of neurologic signs. Brain mitochondria isolated postexposure only from symptomatic cats showed markedly decreased (‐50%), state 3 (ADP‐stimulated), and uncoupler‐stimulated respiration rates with NAD‐ and FAD‐linked substrates. Respiratory control and ADP/oxygen (ADP/O) ratios remained unchanged, indicating, respectively, that coupling and efficiency of ATP synthesis were preserved. Thus, inhibition of electron transport chain function, not phosphorylative activity, may be rate limiting for respiration. During anoxia, hyperglycemic cats showed higher brain lactate levels (26 versus 20 μmol/g), but similar ATP and phosphocreatine concentrations, compared with normoglycemic cats. After exposure, in all animals lactate and phosphocreatine were restored to control levels, whereas ATP remained at 85%. Cats that became symptomatic demonstrated four‐ to sixfold increases in lactate and 50% reductions in phosphocreatine. At 3 and 5 h postexposure, symptomatic animals showed significant reductions in ATP concentrations. We conclude that although initially asymptomatic, hyperglycemic cats exposed to anoxia undergo a neurologic deterioration over several hours following reoxygenation that is correlated with inhibition of mitochondrial respiration, increases in tissue lactate, and decreases in energy state.
Identification and Characterisation of 5‐Hydroxytryptamine3 Recognition Sites in Human Brain Tissue
Authors: Barnes J. M. , Barnes N. M. , Costall B. , Ironside J. W. , Naylor R. J.
Published: October 2006

Abstract: [3H]Zacopride displayed regional saturable specific binding to homogenates of human brain tissues, as defined by the inclusion of BRL43694 [endo‐N‐(9‐methyl‐9‐azabicyclo[3.3.1]non‐3‐yl)‐l‐methylindazole‐3‐carboxamide] in the incubation media. Scatchard analysis of the saturation data obtained from amygdaloid and hippocampal tissues identified the binding as being of high affinity and to a homogeneous population of binding sites (KD= 2.64 ± 0.75 and 2.93 ± 0.41 nmol/L and Bmax= 55 ± 7 and 44 ± 9 fmol/ mg of protein in the amygdala and hippocampus, respectively). 5‐Hydroxytryptamine3 (5‐HT3) receptor agonists and antagonists competed for the [3H]zacopride binding site, competing with up to 40% of total binding with a similar rank order of affinity in both tissues; agents acting on various other neurotransmitter receptors failed to inhibit binding. Kinetic data revealed a fast association that was fully reversible (k+1= 6.61 × 105 and 7.65 × 105/mol/L/s and k‐1= 3.68 × 10−3 and 3.45 × 10−3/s in the amygdala and hippocampus, respectively). It is concluded that [3H]zacopride selectively labels with high affinity 5‐HT3 recognition sites in human amygdala and hippocampus and, if these binding domains represent 5‐HT3 receptors, may provide the opportunity for 5‐HT3 receptor antagonists to modify 5‐HT function in the human brain
Determination of Changes in the Phosphorylation State of the Neuron‐Specific Protein Kinase C Substrate B‐50 (GAP43) by Quantitative Immunoprecipitation
Authors: De Graan P. N. E. , Dekker L. V. , Oestreicher A. B. , Van der Voorn L. , Gispen W. H.
Published: October 2006

Abstract: To determine changes in the degree of phosphorylation of the protein kinase C substrate B‐50 in vivo, a quantitative immunoprecipitation assay for B‐50 (GAP43, F1, pp46) was developed. B‐50 was phosphorylated in intact hippocampal slices with 32Pi or in synaptosomal plasma membranes with [γ‐32P]ATP. Phosphorylated B‐50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti–B‐50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and the incorporation of 32P into B‐50 was quantified by densitometric scanning of the autoradiogram. Only a single 48‐kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B‐50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified 32P‐labelled B‐50 added to slice homogenates or synaptosomal plasma membranes was >95%; and (2) modulation of B‐50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50 values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4β‐phorbol 12,13‐dibutyrate stimulated B‐50 phosphorylation, whereas 4α‐phorbol 12,13‐didecanoate was inactive. Thus, we conclude that the B‐50 immunoprecipitation assay is suitable to monitor changes in B‐50 phosphorylation in intact neuronal tissue.
Effects of D‐2 Antagonists on Frequency‐Dependent Stimulated Dopamine Overflow in Nucleus Accumbens and Caudate‐Putamen
Authors: May Leslie J. , Wightman R. Mark
Published: October 2006

Stimulated dopamine overflow has been measured with in vivo voltammetry in the caudate‐putamen and nucleus accumbens. Overflow was induced by electrical stimulation of the medial forebrain bundle with 120 1‐ms, 300‐μA, biphasic pulses at frequencies between 10 and 60 Hz. Overflow was measured with a Nafion‐coated, carbon‐fiber electrode used with fast‐scan voltammetry (300 V s‐1). Quantification and identification of dopamine concentrations down to 100 nM in vivo is possible with this technique. The overflow curves were fit to a kinetic model that describes the measured response as a function of uptake (characterized by a Vmax and Km) and release (characterized by the concentration of dopamine released per stimulus pulse). Overflow curves in both regions could be described with similar kinetic parameters except for the Vmax, which in the nucleus accum bens was only 60% of that measured in the caudate‐putamen. Uptake inhibition by nomifensine (20 mg kg‐1) caused an apparent 15‐fold change in the value of Km in the nucleus accumbens, similar to results previously reported in the caudate‐putamen. In contrast, metoclopramide (10 mg kg‐1) and sulpiride (100 mg kg‐1) altered the apparent amount of dopamine released per stimulus pulse without a change in the uptake kinetics.
In Vivo Administration of Ethanol Enhances the Function of the γ‐Aminobutyric Acid‐Dependent Chloride Channel in the Rat Cerebral Cortex
Authors: Sanna E. , Concas A. , Serra M. , Biggio G.
Published: October 2006

The effect of in vivo administration of ethanol on the γ‐aminobutyric acidA (GABAA) receptor‐coupled chloride channel was studied by measuring ex vivo t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding in the rat cerebral cortex. Intragastric administration of ethanol (0.5–1 g/kg) elicited in 40 min a significant decrease of [35S]TBPS binding to unwashed cortical membrane preparations, an effect mimicked by diazepam (0.5–1 mg/kg, i.p.). However, Scatchard plot analysis indicated that, unlike the case with diazepam, the decrease was entirely due to a reduction in the apparent affinity of [35S]TBPS receptors with no change in the total number of binding sites. Moreover, ethanol, like diazepam, reduced the increase of [35S]TBPS binding elicited by isoniazid (350 mg/kg, s.c.), an inhibitor of the GABAergic transmission. Finally, ethanol markedly potentiated the inhibitory action of diazepam on [35S]TBPS binding. The results suggest that ethanol, like benzodiazepines, enhances the function of the GABAA‐coupled chloride channel.
In Vivo Measurements of Ethanol Concentration in Rabbit Brain by 1H Magnetic Resonance Spectroscopy
Authors: Petroff Ognen A. C. , Novotny Edward J. , Ogino Takashi , Avison Malcolm , Prichard James W.
Published: October 2006

In vivo 1H magnetic resonance spectroscopy was used to measure the cerebral ethanol concentration in the rabbit after both intraarterial and intragastric administration. There was good agreement between cerebral and blood ethanol concentrations at all times after administration by either route. Cerebral ethanol levels, measured using in vivo 1H spectroscopy, agreed well with those measured in perchloric acid extracts of brain, analyzed by both high‐resolution 1H spectroscopy and gas chromatography. Ethanol may be useful as an indicator to measure cerebral blood flow by 1H spectroscopy and chemical shift‐selective magnetic resonance imaging.
Enhancement of Dopamine Release In Vivo from the Rat Striatum by Dialytic Perfusion of 6R‐l‐erythro‐5,6,7,8‐Tetrahydrobiopterin
Authors: Koshimura Kunio , Miwa Soichi , Lee Ken , Fujiwara Motohatsu , Watanabe Yasuyoshi
Published: October 2006

We have previously reported that intracerebroventricular administration of 6R‐L‐erythro‐5,6,7,8‐tetrahydrobiopterin (6R‐BH4), a cofactor for tyrosine hydroxylase, enhances biosynthesis of 3,4‐dihydroxyphenylethylamine (dopamine) in the rat brain. In the present study, we have more precisely examined the effects of 6R‐BH4 on dopamine release in vivo from the rat striatum using brain microdialysis. The amount of dopamine collected in striatal dialysates was determined using HPLC with electrochemical detection after purification with an alumina batch method. When the striatum was dialyzed with Ringer solution containing various concentrations of 6R‐BH4 (0.25,0.5, and 1.0 mM), dopamine levels in striatal dialysates increased in a concentration‐dependent manner. Biopterin had little effect on dopamine levels in dialysates. The 6R‐BH4‐induced increase in dopamine levels in dialysates was abolished after pretreatment with tetrodotoxin (50 μM) added to the perfusion fluid, but after pretreatment with nomifensine (100 mg/kg, intraperitoneal injection), an inhibitor of dopamine uptake mechanism, a larger increase was observed. After inhibition of tyrosine hydroxylase by pretreatment with α‐methyl‐p‐tyrosine (250 mg/kg, intraperitoneal injection), most of the increase persisted. These results suggest that 6R‐BH4 has a dopamine‐releasing action, which is not dependent on biosynthesis of dopamine.
Evidence for δ‐Opioid Binding and GTP‐Regulatory Proteins in 5‐Day‐Old Rat Brain Membranes
Authors: Szücs Maria , Coscia Carmine J.
Published: October 2006

The availability of the bispenicillamine enkephalin [3H][d‐Pen2,d‐Pen5]enkephalin ([3H]DPDPE) a highly selective ligand for δ‐opioid receptors, has made possible a more definitive examination of the ontogeny of this receptor subtype. In this report, the binding characteristics of [3H]DPDPE in 5‐day‐old neonatal (P‐5) and adult rat brain are compared. Analysis of saturation curves as well as homologous displacement data revealed no significant difference in the binding affinity of [3H]DPDPE between P‐5 animals and adults. Conversely, the binding capacity increased fivefold during this period. The δ‐specificity of the sites was further proven by competition experiments with μ‐ and δ‐selective ligands. Mn2+ (0.5 mM) elevated [3H]DPDPE specific binding by lowering the Kd, whereas 50 μM 5′‐guanylylimidodiphosphate inhibited it by decreasing the total number of high‐affinity binding sites in both P‐5 animals and adults. Pertussis toxincatalyzed ADP ribosylation experiments revealed the presence of 40‐kDa proteins, with a molecular mass corresponding to G protein subunits αi/αo, as early as 1 h after birth. There was a low, but detectable, basal low‐Km GTPase activity in P‐5 animals, which increased fivefold during postnatal development. The present report establishes the existence of high‐affinity [3H]DPDPE binding as well as GTP‐regulatory proteins 5 days after birth. Yet, heterologous competition studies and ionic effects suggest that neonatal binding sites differ from adult receptors. Whether the neonatal sites are newly synthesized, incompletely processed sites or a developmentally programmed isoform remains to be determined.
Turnover of Dopamine and Serotonin and Their Metabolites in the Striatum of Aged Rats
Authors: Venero J. L. , Machado A. , Cano J.
Published: October 2006

Abstract: Turnover of dopamine (DA), serotonin [5‐hydroxytryptamine (5‐HT)], and their metabolites has been measured in adult and aged rats. Turnover rates of 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5‐hydroxy‐3‐indoleacetic acid (5‐HIAA) have been assayed from the disappearance rates after blocking by pargyline inhibition of monoamine oxidase (MAO) and from the accumulation rates by probenecid inhibition of the probenecid‐sensitive transport system. DA and 5‐HT turnover rates have been measured as accumulation rates of 3,4‐dihydroxyphenylalanine and 5‐hydroxytryptophan, respectively, after central decarboxylase inhibition by 3‐hydroxybenzylhydrazine (NSD‐1015) and as accumulation rates of DA and 5‐HT after pargyline inhibition of MAO. The DA turnover rate after NSD‐1015 was 23.9% lower in aged rats than in adults, whereas after pargyline there was no significant difference between the two age groups. The HVA fractional rate constant and turnover after pargyline were lower in aged rats than in adults, and HVA turnover after probenecid was higher in aged rats than in adults. The DOPAC‐HVA pathway seems to be reinforced at the expense of DOPAC conjugation. In aged and adult rats whose 5‐HT steady‐state levels were not statistically different, the 5‐HT turnover rate after pargyline and NSD‐1015 treatment was lower in aged rats than in adults. An increase of 5‐HIAA levels after pargyline and probenecid treatment in aged rats could be due to the handling stress.