bluuebunny/crossref_metadata_2025 huggingface dataset:
Visualizing Dopamine and Serotonin Transporters in the Human Brain with the Potent Cocaine Analogue [125I]RTI‐55: In Vitro Binding and Autoradiographic Characterization
Authors: Staley Julie K. , Basile Margaret , Flynn Donna D. , Mash Deborah C.
Published: July 2010

Abstract: The cocaine analogue RTI‐55 was evaluated as a probe for in vitro labeling and localization of dopamine and serotonin transporters after death in the human brain. Kinetic, saturation, and competition binding experiments indicated complex interactions of the radioligand with the identification of multiple recognition sites. In membrane binding assays, the association of [125I]RTI‐55 at 25°C to putamen membranes was monophasic. In contrast, dissociation of [125I]RTI‐55 occurred in two phases with t1/2 values of 9.4 and 36.5 min, respectively. Saturation analysis of [125I]RTI‐55 binding demonstrated two binding sites in the human putamen with KD values of 0.10 ± 0.02 and 1.81 ± 0.46 nM. The binding of [125I]RTI‐55 was displaced by a wide range of cocaine analogues and monoamine uptake inhibitors. The rank order of potency demonstrated in competition assays with human putamen membranes indicates that the radioligand labels cocaine recognition sites on the dopamine transporter (mazindol > GBR 12909 > GBR 12935 > paroxetine > nisoxetine > desipramine ≥ fluoxetine > citalopram). In the human occipital cortex, [125I]RTI‐55 recognized multiple binding sites with KD values of 0.02 ± 0.01 and 4.18 ± 0.46 nM. The rank order of potency for inhibition of [125I]RTI‐55 binding to cerebral cortex membranes (paroxetine > citalopram > GBR 12909 ≥ mazindol ≥ nisoxetine > benztropine) suggests that [125I]RTI‐55 labels the serotonin transporter in the human occipital cortex. Autoradiographic mapping of [125I]RTI‐55 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderately elevated densities of [125I]RTI‐55 binding sites were also seen throughout the thalamus, hypothalamus, and substantia nigra. [125I]RTI‐55 binding sites were prevalent throughout the cerebral cortex and amygdala. In autoradiographic studies, the addition of the selective serotonin transport blocker citalopram completely prevented [125I]RTI‐55 labeling in the thalamus, hypothalamus, and throughout most of the cerebral cortex. In the presence of citalopram, [125I]RTI‐55 binding site densities remained elevated over the striatum and substantia nigra, with selective residual labeling also seen in the external segment of the globus pallidus and the lateral nucleus of the amygdala. These results demonstrate that in the human brain, [125I]RTI‐55 labels multiple recognition sites on dopamine and serotonin transporters.
Increase in the Extracellular Histamine Concentration in the Rat Striatum by μ‐Opioid Receptor Activation
Authors: Chikai Takashi , Oishi Ryozo , Saeki Kiyomi
Published: July 2010

Abstract: The effects of morphine and selective ligands for μ‐, κ‐, and δ‐opioid receptors on the extracellular histamine (HA) concentration in the striatum of freely moving rats were examined by in vivo microdialysis. On the day after implantation of the dialysis probe, the HA output per 30‐min period was measured using HPLC‐fluorometry. Morphine (3.8 mg/kg, s.c.) significantly increased the HA output by ∼200% 1–3 h after treatment. This effect was completely antagonized by naltrexone (1.6 mg/kg, s.c.). The HA output decreased to a level below 10% of the basal value by 4 h after treatment with (S)‐α‐fluoromethylhistidine (77 mg/kg, s.c.). In such animals, morphine (3.8 mg/kg, s.c.) had no influence on the HA output. [d‐Ala2,MePhe4,Gly(ol)5]Enkephalin (DAGO; 0.2 µg, i.c.v.), a selective μ‐agonist, significantly increased the HA output by ∼150% 0.5–1.5 h after treatment, and this effect was also completely blocked by naltrexone. A selective κ‐agonist, U‐50,488 (3.8 and 7.6 mg/kg, s.c.), and a selective δ‐agonist, [d‐Pen2,d‐Pen5]enkephalin (0.5 and 2 µg, i.c.v.), had no effect on the HA output. These findings suggest that the stimulation of μ‐opioid receptors by morphine and DAGO increases the extracellular HA concentration by accelerating HA release from nerve endings.
Polyclonal Antibodies Directed Against an Epitope Specific for the α4‐Subunit of GABAA Receptors Identify a 67‐kDa Protein in Rat Brain Membranes
Authors: Kern W. , Sieghart W.
Published: July 2010

Abstract: Polyclonal antibodies were raised to the C‐terminal part of the γ‐aminobutyric acidA (GABAA) receptor α4‐subunit. These anti‐peptide α4 (517–523) antibodies specifically identified a protein with apparent molecular mass 67 kDa in rat brain membranes. This protein was enriched by immunoaffinity chromatography of brain membrane extracts on Affigel 10 coupled to the anti‐peptide α4 (517–523) antibodies and could then be identified by the anti‐α4‐antibodies as well as by the GABAA receptor subunit‐specific monoclonal antibody bd‐28. This appears to indicate that the 67‐kDa protein is the α4‐subunit of GABAA receptors. Intact GABAA receptors appeared to be retained by the immunoaffinity column because other GABAA receptor subunit proteins like the β2/β3‐subunits and the γ2‐subunit were detected in the immunoaffinity column eluate. Furthermore, in addition to the 67‐kDa protein, a 51‐kDa protein could be detected by the antibody bd‐28 and the anti‐peptide α4 (517–523) antibody in the immunoaffinity column eluate. A protein with similar apparent molecular mass was identified by the α1‐subunit‐specific anti‐peptide α1 (1–9) antibody. In contrast to the α1‐subunit, the 51‐kDa protein identified by the anti‐α4 antibody could not be deglycosylated by N‐Glycanase. The identity of the 51‐kDa protein identified by the anti‐α4‐antibodies thus must be further investigated.
Production and Characterization of Recombinant Mouse Brain‐Derived Neurotrophic Factor and Rat Neurotrophin‐3 Expressed in Insect Cells
Authors: Meyer Sheryl L. , Lang Diane M. , Forbes M. Elizabeth , Knight Ernest , Hirsch James D. , Trusko Stephen P. , Scott Richard W.
Published: July 2010

Abstract: Bioactive brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 were produced using the baculovirus expression system and purified to homogeneity using ion‐exchange and reversed‐phase chromatography. Yields of purified neurotrophin‐3 (300–500 μg/L) were similar to levels reported for baculovirus‐expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20–50 μg/L). Improved production of BDNF (150–200 μg/L) was achieved by expressing BDNF from a chimeric prepro‐NGF/mature BDNF construct using the Trichoplusia ni insect cell line, Tn‐5B1‐4. Examination of the distribution of BDNF protein from both the nonchimeric prepro‐BDNF and the chimeric prepro‐NGF/mature BDNF viruses in Sf‐21‐and Tn‐5B1‐4‐infected cells suggests a specific deficiency in the Tn‐5B1‐4 cells in processing the nonchimeric precursor. In addition, the vast majority of the BDNF protein at 2 days after infection was intracellular and insoluble. N‐terminal amino acid sequencing of purified recombinant BDNF and neurotrophin‐3 demonstrated that the insect cells processed their precursors to the correct N‐terminus expected for the mature protein. Bioactivity was characterized in vitro on primary neuronal cultures from the CNS and PNS.
Increased Cell‐Cell Contact Stimulates the Transcription Rate of the Tyrosine Hydroxylase Gene in Rat Pheochromocytoma PC 18 Cells
Authors: Carlson Christopher D. , Tank A. William
Published: July 2010

Abstract:Cell aggregation is one of several environmental cues that influence the expression of neurotransmitter phenotype during development. The expression of the catecholaminergic phenotype is increased in rat pheochromocytoma cells cultured at high density. In the present study we have investigated whether this cell density‐mediated effect on the catecholaminergic phenotype is due to the stimulation of the tyrosine hydroxylase gene. When rat pheochromocytoma PC 18 cells are cultured at high density (2 ± 105cells/cm2), tyrosine hydroxylase enzymatic activity and tyrosine hydroxylase protein increase two‐to threefold over that observed in cells cultured at low density (1 ± 104cells/cm2). This increase in tyrosine hydroxylase protein observed in high‐density cultures is fully accounted for by a preceding increase in tyrosine hydroxylase mRNA levels. The relative transcription rate of the tyrosine hydroxylase gene, measured using a nuclear run‐on assay, is two‐to threefold greater in PC 18 cells cultured at high density than in cells cultured at low density. Using flow cytometry, we have determined that in high‐density cultures, there are approximately twice as many cells in the G0‐G1phases of the cell cycle compared with the number of G0‐G1cells observed in low‐density cultures. However, when G0‐G1cells are isolated by cellular elutriation, tyrosine hydroxylase gene transcription rate remains two‐to threefold greater in G0‐G1cells from high‐density cultures than in G0‐G1cells from low‐density cultures. These results indicate that increased cell‐cell contact stimulates the transcription rate of the tyrosine hydroxylase gene, resulting in the subsequent increased expression of tyrosine hydroxylase mRNA and protein.
High‐Resolution Mapping of GenS3 and B11F7 Epitopes on Myelin‐Associated Glycoprotein by Expression PCR
Authors: Tropak Michael B. , Roder John C.
Published: July 2010

Abstract: The GenS3 and B11F7 monoclonal antibodies (MAbs) have been widely used for biochemical and immunocytochemical experiments on myelin‐associated glycoprotein (MAG), a cell adhesion molecule mediating the interaction between myelinating glia and axons. We have mapped the epitopes to within several amino acids on Ig domain 2 (D2) (amino acids 167–77) and domain 4 (D4) (amino acids 375–388) for GenS3 and B11F7, respectively. Domain deletion and substitution mutants of the MAG cDNA were first used to map the epitopes to a given domain. In the cases of GenS3, insertion mutants were used to resolve the epitope to a small region of D2. For the B11F7 epitope, a novel technique combining PCR and in vitro transcription and translation was used to generate small C‐terminal deletions and map the epitope to 13 amino acids. Then, inhibition by peptides corresponding to the GenS3 (ELRPELSWLGHE; amino acids 167–177) and B11F7 (QLELPAVTPEDDGE; amino acids 375–388) epitopes was used to confirm the position of the epitopes based on the mutant data. Interestingly, the GenS3 epitope maps to a region predicted to be sequestered within the hydrophobic core of D2. This is consistent with the inability of GenS3 to recognize the epitope in native MAG; GenS3 epitope recognition occurs only in denatured MAG, where the epitope is more accessible. With the definition of the GenS3 and B11F7 epitopes, these antibodies will be useful for further structure‐function studies on MAG.
Antipeptide Antibodies Against a Torpedo Cysteine‐String Protein
Authors: Mastrogiacomo Alessandro , Evans Christopher J. , Gundersen Cameron B.
Published: July 2010

Abstract: An antipeptide antiserum was raised against the C‐terminal undecapeptide of a Torpedo cysteine‐string protein (csp), a putative subunit or modulator of presynaptic calcium channels. This antiserum was shown to identify selectively the 27‐kDa in vitro translation product of the csp cRNA both by immunoprecipitation and on immunoblots. When affinity‐purified anti‐csp antibodies were used to probe immunoblots of membrane proteins from Torpedo electric organ or liver, specific immunoreactivity was detected only in electric organ. This immunoreactivity was associated principally with a single protein species of about 34 kDa. These results indicate that csp immunoreactivity is detectably expressed in electroplax, a heavily innervated tissue, but not in liver, which should have an appreciably lower abundance of presynaptic calcium channel proteins. Moreover, the increased relative molecular mass of csp in electric organ (compared with in vitro translated material) implies that csp is posttranslationally modified. Finally, immunoblot analysis of either intact, alkali‐treated, or solubilized membrane fractions of electric organ reveals that csp is predominantly a membrane protein.
SK‐N‐BE: A Human Neuroblastoma Cell Line Containing Two Subtypes of δ‐Opioid Receptors
Authors: Polastron Jocelyne , Mur Mireille , Mazarguil Honoré , Puget Alain , Meunier Jean‐Claude , Jauzac Philippe
Published: July 2010

Abstract:A human neuroblastoma cell line, SK‐N‐BE, was shown to express a substantial amount of opioid receptors (200–300 fmol/mg of protein). A ligand binding profile of these receptors revealed that they could belong to two distinct subtypes of δ‐opioid receptors. Results from sucrose‐gradient sedimentation experiments were compared with similar data obtained with the μ‐opioid receptor of the rabbit cerebellum and the δ‐opioid receptor of the hybrid NG108–15 cell line and have shown that the opioid receptor of the SK‐N‐BE cell line behaved hydrodynamically as an intermediate between μ‐and δ‐opioid receptors. Taken together, pharmacological and hydrodynamic studies suggest that the opioid receptors present in the SK‐N‐BE cell membranes could belong to two δ‐opioid receptor subtypes interacting allosterically. Functional experiments suggest that at least one of these subtypes of δ‐opioid receptor is negatively coupled to the adenylate cyclase via a Giprotein and that the opiate receptors of the SK‐N‐BE neuroblastoma cell line undergo a rapid down‐regulation when preincubated in the presence of the high‐affinity opioid agonist, etorphine.
Epidermal Growth Factor Promotes Uncoupling from Adenylyl Cyclase of the Rat D2SReceptor Expressed in GH4C1 Cells
Authors: Missale Cristina , Boroni Flora , Sigala Sandra , Castelletti Laura , Falardeau Pierre , Dal Toso Roberto , Caron Marc G. , Spano PierFranco
Published: July 2010

Abstract:In anterior pituitary cells or when transfected into host cell lines, the D2dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH‐3 pituitary tumor cell line, which lacks functional D2receptors, responds to epidermal growth factor (EGF) by expressing a D2receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in α subunit of the G protein G13. In this study we have investigated the effects of EGF on the transduction mechanisms of D2receptors in GH4C1 cells transfected and permanently overexpressing the rat short D2receptor. Activation of D2receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP‐dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D2receptor to inhibit cyclic AMP‐dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of G13protein. These results suggest that the transmembrane signaling specificity of G protein‐coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.
Bidirectional Modulation of GABA‐Gated Chloride Channels by Divalent Cations: Inhibition by Ca2+and Enhancement by Mg2+
Authors: Schwartz Rochelle D. , Wagner Joseph P. , Yu Xiao , Martin David
Published: July 2010

Abstract:The effects of the divalent cations Ca2+, Sr2+, Ba2+, Mg2+, Mn2+, and Cd2+were studied on γ‐aminobutyric acidA(GABAA) responses in rat cerebral cortical synaptoneurosomes. The divalent cations produced bidirectional modulation of muscimol‐induced36Cl−uptake consistent with their ability to permeate and block Ca2+channels. The order of potency for inhibition of muscimol responses was Ca2+> Sr2+> Ba2+, similar to the order for permeation of Ca2+channels in neurons. The order of potency for enhancement of muscimol responses was Cd2+> Mn2+> Mg2+, similar to the order for blockade of Ca2+channels in neurons. Neither Ca2+nor Mg2+caused accumulation of GABA in the extravesicular space due to increased GABA release or decreased reuptake of GABA by the synaptoneurosomes. The inhibition of muscimol responses by Ca2+was most likely via an intracellular site of action because additional inhibition could be obtained in the presence of the Ca2+ionophore, A23187. This confirms electrophysiologic findings in cultured neurons from several species. In contrast, the effects of Cd2+, Mn2+, and Mg2+may be mediated via blockade of Ca2+channels or by intracellular sites, although the results of these studies do not distinguish between the two loci. The effects of Zn2+were also studied, because this divalent cation is reported to have widely divergent effects on GABAAresponses. In contrast to other studies, we demonstrate that Zn2+inhibits GABAAresponses in an adult neuronal preparation. Zn2+produced a concentration‐dependent inhibition (limited to 40%) of muscimol responses with an EC50of 60 μM. The inhibition of muscimol‐induced38Cl−uptake by Zn2+was noncompetitive. The effect of Zn2+was reduced in the presence of Mg2+in a competitive or allosteric manner. The portion of GABAAreceptors sensitive to Zn2+may reflect a specific subunit composition in cerebral cortex as previously observed for recombinant GABAAreceptors in several expression systems. The modulation of GABAAreceptor function by Ca2+and other divalent cations may play an important role in the development and/or attenuation of neuronal excitability associated with pathologic conditions such as seizure activity and cerebral ischemia.
pp60c‐src Enhances the Acetylcholine Receptor‐Dependent Catecholamine Release in Vaccinia Virus‐Infected Bovine Adrenal Chromaffin Cells
Authors: Ely Constance M. , Tomiak William M. , Allen Colleen M. , Thomas Laurel , Thomas Gary , Parsons Sarah J.
Published: July 2010

Abstract: Secretion of catecholamines by adrenal chromaffin cells is a highly regulated process that involves serine/threonine and tyrosine phosphorylations. The nonreceptor tyrosine kinase pp60c‐sre is expressed at high levels and localized to plasma membranes and secretory vesicle membranes in these cells, suggesting an interaction of this enzyme with components of the secretory process. To test the hypothesis that pp60c‐sic is involved in exocytosis, we transiently expressed exogenous c‐src cDNA using a vaccinia virus vector in primary cultures of bovine adrenomedullary chromaffin cells. Chromaffin cells infected with a c‐src recombinant virus restored the diminished secretory activity accompanying infection by wild type virus alone or a control recombinant virus. The level of enhanced catecholamine release correlated directly with the time and level of exogenous c‐src expression. These results could not be attributed to differences in cytopathic effects of wild type versus recombinant viruses as assessed by cell viability assays, nor to differences in norepinephrine uptake or basal release, suggesting that pp60c‐src is involved in stimulus‐secretion coupling in infected cells. Surprisingly, exogenous expression of an enzymatically inactive mutant c‐src also restored catecholamine release, indicating that regions of the introduced c‐src protein other than the kinase domain may affect catecholamine release. Secretory activity was elevated by both forms of c‐src in response to either nicotine or carbachol (which activate the nicotinic and the nicotinic/muscarinic receptors, respectively). In contrast, release of catecholamines upon membrane depolarization (as elicited by 55 mM K+) or by treatment with the calcium ionophore A23187 was unaffected by either vaccinia infection or increased levels of pp60c‐src. These results suggest that pp60c‐src affects secretory processes in vaccinia‐infected cells that are activated through ligand‐gated, but not voltage‐gated, ion channels.
Methyl 3β‐(4‐[125I]Iodophenyl)Tropane‐2β‐Carboxylate In Vitro Binding to Dopamine and Serotonin Transporters Under “Physiological” Conditions
Authors: Laruelle Marc , Giddings Suzanne S. , Zea‐Ponce Yolanda , Charney Dennis S. , Neumeyer John L. , Baldwin Ronald M. , Innis Robert B.
Published: July 2010

Abstract:Methyl 3β‐(4‐[125I]iodophenyl)tropane‐2β‐carboxylate ([123I]β‐CIT) is a single photon emission computed tomographic radiotracer for in vivo labeling of dopamine (DA) and serotonin (5‐HT) transporters. Single photon emission computed tomographic experiments in nonhuman primates showed that [123I]β‐CIT in vivo binding to DA transporters had a much slower washout than binding to 5‐HT transporters. This observation was not predicted from previously published in vitro studies. These studies, performed at 22°C in nonphysiological buffer, reported similar affinity of [125I]β‐CIT for DA and 5‐HT transporters. We now report [125I]β‐CIT binding parameters to fresh rat membranes at 22°C and 37°C, in a buffer mimicking the composition of cerebrospinal fluid. At both temperatures, binding to DA transporters was best fit by a twosite model, whereas binding to 5‐HT transporters was compatible with one population of sites. At 22°C, [125I]β‐CIT showed similar affinity to high‐affinity DA (0.39 nM) and 5‐HT transporter sites (0.47 nM). Increasing the incubation temperature from 22°C to 37°C reduced binding to DA transporters by 60%, whereas binding to 5‐HT transporters was only marginally affected. In vitro kinetic experiments failed to detect significant differences in on or off rates that could explain the observed in vivo kinetics. These experiments thus failed to explain [123I]β‐CIT in vivo uptake kinetics, suggesting the existence of specific factors affecting the in vivo situation.
Striatal Transporter for Dopamine: Catechol Structure‐Activity Studies and Susceptibility to Chemical Modification
Authors: Meiergerd Susan M. , Schenk James O.
Published: July 2010

Abstract:The apparent second‐order association rate constant of dopamine binding to the striatal transporter (∼1 ± 106M−1s−1) as well as the transporter turnover number (∼1.5 s−1) was estimated using rotating disk electrode voltammetry to monitor apparent zero trans entry of dopamine into striatal suspensions. The substrate specificity of the transporter was also assessed using catechol derivatives. Dopamine and norepinephrine were transported, whereas epinephrine and the acidic metabolites of dopamine were not transported. The metabolite, 3‐meth‐oxytyramine, was transported with aKmseven times greater than and aVmaxclose to that for dopamine. 4‐Methoxytyramine was transported more facilely than the 3‐methoxy derivative.N‐Alkylation of the amine side chain of dopamine reduced transport dramatically. 4‐Ethylcatechol and 3,4‐dihydroxybenzylamine were transported with velocities 79 and 91 % less than that for dopamine, respectively. The rigid analogue 6,7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene was transported with a greater velocity than the 5,7‐dihydroxy derivative. Finally, the apparentKmvalues for 4‐ethylcatechol, 1‐amino‐2‐phenylethane, tyramine, andm‐tyramine as cosubstrates with dopamine were 1.1, 11, 17, and 2.6 μM, respectively. Pretreatments of striatal suspensions with chloroethylnorapomorphine,N‐ethylmaleimide, Hg2+, 4,5‐dihydroxy‐4,5‐dioxo‐1H‐pyrrolo[2,3‐f]quinoline‐2,7,9‐tricarboxylic acid (a redox modulator of receptors in neuronal as well as other tissues), and neuraminidase reduced the velocity of transport of dopamine, whereasN‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline had no effect. Thus, the dopamine transporter requires an intact catechol with a primary ethylamine side chain for optimal activity relative to shorter side chain derivatives (side chains longer than two carbons were not tested), the 3‐hydroxyl group of dopamine is the more critical hydroxyl group, and the β rotamer of the extended conformation of dopamine is transported preferentially. The catechol appears to mediate the recognition of the substrate, whereas the amine side chain apparently facilitates the conformational change of the transporter that results in movement of dopamine into or across the membrane. The transporter distinguishes between agents known to block dopamine recognition sites on dopamine receptors? appears to possess a reduction/oxidation modulatory site, and requires sulfhydryl groups andexternalglycosylation for optimal function.
μ‐Opioid Receptor Stimulation of Inositol (1,4,5)Trisphosphate Formation via a Pertussis Toxin‐Sensitive G Protein
Authors: Smart D. , Smith G. , Lambert D. G.
Published: July 2010

Abstract:The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH‐SY5Y human neuroblastoma cells (expressing both μ‐and δ‐opioid receptors), we demonstrated that fentanyl, a μ‐preferring opioid, caused a dose‐dependent (EC50= 16 nM) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1–2 min. This response was of similar magnitude (25.4 ± 0.8 pmol/mg of protein for 0.1 μMfentanyl) to that found in the plateau phase (5 min) following stimulation with 1 mMcarbachol (18.3 ± 1.4 pmol/mg of protein), and was naloxone‐, but not naltrindole‐(a δ antagonist), reversible. Further studies using [d‐Ala2, MePhe4, Gly(ol)5]enkephalin and [d‐Pen2,5]enkephalin confirmed that the response was specific for the μ receptor. Incubation with Ni2+(2.5 mM) or in Ca2+‐free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 μMfentanyl, 26.9 ± 1.5 pmol/mg of protein; pertussis‐treated plus 0.1 μMfentanyl, 5.1 ± 1.3 pmol/mg of protein). In summary, we have demonstrated a μ‐opioid receptor‐mediated activation of phospholipase C, via a pertussis toxin‐sensitive G protein, that is Ca2+‐dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate‐mediated rises in intracellular Ca2+, could play a part in the cellular mechanisms of opioid action.
Opioid Effects on [3H]Norepinephrine Release from Dissociated Embryonic Locus Coeruleus Cell Cultures
Authors: Raymon Heather K. , Leslie Frances M.
Published: July 2010

Abstract:The acute and chronic effects of opioid exposure on [3H]norepinephrine ([3H]NE) release were examined in cell cultures of embryonic rat locus coeruleus (LC). Initial morphological and biochemical characterization of the cultures indicated that the cells exhibited properties similar to those observed in situ. Specific [3H]NE uptake was saturable with aKmvalue of 222 ± 52 nM. [3H]NE accumulated by LC cells was released in response to 20 mMK+stimulation, in a calcium‐dependent manner. Both components of neurotransmitter release, spontaneous and K+evoked, were significantly inhibited by β‐endorphin, with the latter being maintained in the presence of tetrodotoxin. The pharmacology of the opioid effect was consistent with that of μ‐receptor activation. The effect of chronic exposure to the μ‐selective agonist fentanyl (1 μM) was examined following 4 days of drug treatment. Although there was no significant effect of fentanyl on K+‐evoked [3H]NE release, these cells were tolerant to the acute inhibitory effect of β‐endorphin. These results indicate that this is an appropriate system for examining the effects of acute and chronic opioid treatment on noradrenergic cells in vitro. In addition, this system may be useful as a CNS model for examining mechanisms that underlie tolerance and dependence following chronic opioid exposure.
Antioxidant Properties of Bromocriptine, a Dopamine Agonist
Authors: Yoshikawa Toshikazu , Minamiyama Yukiko , Naito Yuji , Kondo Motoharu
Published: July 2010

Abstract:It has been suggested that free radicals may adversely influence the pathogenesis of Parkinson's disease. We conducted this study to determine whether bro‐mocriptine, an agent widely used for treating parkinsonism, possesses antioxidant effects. Bromocriptine scavenged superoxide produced from a superoxide generating system (hypoxanthine‐xanthine oxidase) by the spin‐trapping method using electron spin resonance. Bromocriptine had a strong scavenging effect on the 5,5‐dimethyl‐1‐pyrroline‐N‐oxide hydroxide signal produced from Fenton's reaction. Bromocriptine also attenuated the stable free radical diphenyl‐p‐picrylhydrazyl signal. This drug inhibited the autooxidation of rat brain homogenates in a dose‐dependent manner in vitro. Autooxidation of brain homogenates collected from rats treated with bromocriptine (2.5 mg/kg, i.p., daily for 3 days) was significantly reduced as compared with values in untreated rat homogenates. These observations suggest that bromocriptine is a free radical scavenger and a potent antioxidant.
Membrane Lipids, Selectively Diminished in Alzheimer Brains, Suggest Synapse Loss as a Primary Event in Early‐Onset Form (Type I) and Demyelination in Late‐Onset Form (Type II)
Authors: Svennerholm Lars , Gottfries Carl‐Gerhard
Published: July 2010

Abstract:Major membrane lipids were quantified in frontal (Brodmann area 9) and temporal (Brodmann areas 21 and 22) cortices, caudate nucleus, hippocampus, and frontal white matter of 12 cases with Alzheimer's disease (AD) type I (early onset), 21 cases with AD type II (late onset), and 20 age‐matched controls. The concentration of gangliosides—a marker for axodendritic arborization—was reduced to 58–70% of the control concentration in all four gray areas (p < 0.0001) and to 81 % in frontal white matter (p < 0.01) of AD type I cases, whereas it was only significantly reduced in temporal cortex (p < 0.01), hippocampus (p < 0.05), and frontal white matter (p < 0.05) in AD type II cases. The concentration of phospholipids was also significantly reduced (p < 0.01–0.0001) in all four gray areas of AD type I cases but in no area of AD type II cases. The loss of cholesterol was only 50% of the corresponding phospholipid diminution in AD type I. These results suggested a pronounced loss of nerve endings in AD type 1. The characteristic membrane lipid disturbance in AD type II was a loss of myelin lipids. This is the first time a fundamental biochemical difference has been shown between the two major forms of AD.
Aging Diminishes Serotonin‐Stimulated Arachdonic Acid Uptake and Cholinergic Receptor‐Activated Arachidonic Acid Release in Rat Brain Cortex Membrane
Authors: Strosznajder J. , Samochocki M. , Duran M.
Published: July 2010

Abstract:Synaptoneurosomal and synaptosomal fractions from the brain cortex of adult (4‐month‐old) and aged (27‐month‐old) rats were used for studies on the uptake and subsequent release of [14C]arachidonic acid ([14C]AA) from brain lipids. The incorporation of AA and the pattern of its uptake into lipids of the aged brain cortex synapto‐neurosomes and synaptosomes were not significantly different when compared with those in the adult brain cortex fractions. Serotonin (5‐HT), at 10 μM to 1 μM in the presence of pargyline and the agonist of the 5‐HT1Areceptor, buspirone, stimulated AA uptake into membrane lipids, mainly into phosphatidylinositol, by about 40% exclusively in adult brain synaptoneurosomes. Aging significantly diminished the effect of 5‐HT on AA uptake. Synaptoneurosomal and synaptosomal fractions prelabeled with [14C]AA were used subsequently for investigation of voltage‐dependent, muscarinic and 5‐HT receptor‐mediated AA release. Aging diminished markedly carbachol‐stimulated Ca2+‐dependent AA liberation from membrane lipids of synaptoneurosomes and synaptosomes. Moreover, aging decreased voltage‐dependent and 5‐HT2receptor‐mediated AA release. These results show that aging affects receptor‐dependent AA uptake and pre‐and postsynaptic receptor‐mediated AA release. These modulations of AA incorporation and release in aged brain may be of patho‐physiological significance, in view of the importance of these processes for signal transmission in the brain. The changes of receptor‐dependent processes of deacylation and reacylation may be responsible for alteration in the function of neuronal cells and may affect learning and memory ability and brain plasticity during aging.
Changes in the Extracellular Concentrations of Amino Acids in the Rat Striatum During Transient Focal Cerebral Ischemia
Authors: Uchiyama‐Tsuyuki Yoshimi , Araki Hiroaki , Yae Tetsuji , Otomo Susumu
Published: July 2010

Abstract: Although considerable evidence supports a role for amino acids in transient global cerebral ischemia and permanent focal cerebral ischemia, effects of transient focal cerebral ischemia on the extracellular concentrations of amino acids have not been reported. Accordingly, our study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, GABA, taurine, glutamine, alanine, and phosphoethanolamine in the striatum of transient focal cerebral ischemia, as evidence to support their pathogenic roles. Focal ischemia was induced using the middle cerebral artery occlusion model, with no need for craniotomy. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. One hour of middle cerebral artery occlusion followed by recirculation caused neuronal damage that was common in the frontoparietal cortex and the lateral segment of the caudate nucleus. During 1 h of ischemia, the largest increase occurred for GABA and moderate increases were observed for taurine, glutamate, and aspartate. Alanine, which is a nonneuroactive amino acid, increased little. After recirculation, the levels of glutamate and aspartate reverted to normal baseline values right after reperfusion. Despite these rapid normalizations, neuronal damage occurred. Therefore, uptake of excitatory amino acids can still be restored after 1 h of middle cerebral artery occlusion, and tissue damage occurs even though high extracellular levels of glutamate are not maintained.
Adrenalectomy‐Induced Increase of Brain Protein Synthesis Is Antagonized by Corticosterone Replacements in Free‐Moving Rats
Authors: Grange Eric , Lepetit Patrick , Baubet Valérie , Giaume Murielle , Leclerc Michel , Gay Nadine , Gagnon Jean , Bobillier Pierre
Published: July 2010

Abstract: The autoradiographic method with l[35S]‐methionine was used to determine whether changes in glucocorticoid circulating levels were associated with changes in local rates of protein synthesis in rat brain. Chronic bilateral adrenalectomy induced an increase of me‐thionine incorporation rates into proteins in 60 of the 62 brain regions examined (mean effect, +50%). This effect was confirmed biochemically and quantified by correcting for the relative contribution of methionine derived from protein degradation to the precursor pool for protein synthesis in the whole brain. Acute or chronic administration of corticosterone, at doses that normalize basal levels of adrenocorticotrophic hormone, reversed or prevented the adrenalectomy‐induced increase of protein synthesis in most regions. However, in nearly all the regions studied (59 of 62), acute corticosterone administration to sham‐operated rats did not change the apparent rate of protein synthesis. These results demonstrate that glucocorticoids exert a generalized inhibitory action on brain protein synthesis, because the stimulatory and persistent effect of adrenalectomy on protein synthesis was antagonized by corticosterone replacements at physiological doses. Thus, the regulation of overall brain protein synthesis by glucocorticoids emphasizes the role of neuroendocrine events on long‐term neurochemical processes.
Localization of Drug‐Metabolizing Enzyme Activities to Blood‐Brain Interfaces and Circumventricular Organs
Authors: Ghersi‐Egea J. F. , Leninger‐Muller B. , Suleman G. , Siest G. , Minn A.
Published: July 2010

Abstract:The brain, with the exception of the choroid plexuses and Circumventricular organs, is partially protected from the invasion of blood‐borne chemicals by the specific morphological properties of the cerebral micro‐vessels, namely, the tight junctions of the blood‐brain barrier. Recently, several enzymes that are primarily involved in hepatic drug metabolism have been shown to exist in the brain, albeit at relatively low specific activities. In the present study, the hypothesis that these enzymes are located primarily at blood‐brain interfaces, where they form an “enzymatic barrier,” is tested. By using microdissection techniques or a gradient‐centrifugation isolation procedure, the activities of seven drug‐metabolizing enzymes in isolated microvessels, choroid plexuses, meningeal membranes, and tissue from three Circumventricular organs (the neural lobe of the hypophysis, pineal gland, and median eminence) were assayed. With two exceptions, the activities of these enzymes were higher in the three Circumventricular organs and cerebral microvessel than in the cortex. Very high membrane‐bound epoxide hydrolase and UDP‐glucuronosyltransferase activities (approaching those in liver) and somewhat high 7‐benzoxyre‐sorufin‐O‐dealkylase and NADPH‐cytochrome P‐450 reductase activities were determined in the choroid plexuses. The pia‐arachnoid membranes, but not the dura matter, displayed drug‐metabolizing enzyme activities, notably that of epoxide hydrolase: The drug‐metabolizing enzymes located at these nonparenchymal sites may function to protect brain tissue from harmful compounds.
Disaccharide Composition of Heparan Sulfates: Brain, Nervous Tissue Storage Organelles, Kidney, and Lung
Authors: Tekotte H. , Engel M. , Margolis R. U. , Margolis R. K.
Published: July 2010

Abstract: We have characterized the structural properties of heparan sulfates from brain and other tissues after de‐polymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum. The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards. In rat, rabbit, and bovine brain, 46–69% of the heparan sulfate disaccharides are N‐acetylated and unsulfated, and 17–21% contain a single sulfate residue in the form of a sulfoamino group. In rabbit, bovine, and 1‐day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N‐sulfate account for an additional 10–14%, together with smaller and approximately equall proportions (5–9%) of mono‐, di‐, and trisulfated disaccharides having sulfate at the 6‐position of the glucosamine residue. Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6‐sulfated N‐acetylglucosamine residues. In chromaffin granules, the catecholamine‐and peptide‐storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5–10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71 %) or monosulfated (18%). In sympathetic nerves, norepinephrine is stored in large densecored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules. However, in contrast to chromaffin granules, heparan sulfate accounts for ∼ 75% of the total glycosaminoglycans in large dense‐cored vesicles and more closely resembles heparin, insofar as it contains only 21 % unsulfated disaccharides, 10% mono‐and disulfated disaccharides, and 69% trisulfated disaccharides. Our results therefore reveal significant differences among heparan sulfates from different sources, supporting other evidence that structural variations in heparan sulfate may be related to specific biological functions, such as the switching in the neural response from fibroblast growth factor‐2 to fibro‐blast growth factor‐1 resulting from developmental changes in the glycosaminoglycan chains of a heparan sulfate proteoglycan.
Protein and Lipid Composition of Radial Component‐Enriched CNS Myelin
Authors: Karthigasan J. , Kosaras B. , Nguyen J. , Kirschner D. A.
Published: July 2010

Abstract: The radial component is a junctional complex that is believed to stabilize the apposition of myelin membranes in the internode of CNS myelin. Based on our previous finding that the radial component of compact myelin retains its structure in tissue treated with the detergent Triton X‐100, we have attempted to isolate the junctional complex from spinal cord myelin treated with this detergent. Using 0.5% Triton X‐100, our procedures yielded a fraction of isolated myelin that was enriched in well‐preserved radial component. This fraction that contained morphologically well‐defined radial component was examined by sodium dodecyl sulfate‐polyacrylamide gel electropho‐resis and immunoblotting, and TLC, and was found to be significantly and consistently enriched in the 21.5‐kDa and 17‐kDa isoforms of myelin basic protein, and in cerebro‐sides, hydroxy sulfatide, and sphingomyelin. In addition, the myelin‐associated enzyme 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase, tubulin, and actin tended to be resistant to Triton extraction. The fraction of isolated myelin that contained radial component was deficient in proteolipid protein and DM‐20, the 18.5‐and 14‐kDa isoforms of myelin basic proteins, and in the major phospholipids, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. Our data indicate that the radial component can be isolated and that certain myelin and cytoskeletal proteins and lipids are closely associated with it.
Primary Structure of the Precursor for the Anthozoan Neuropeptide Antho‐RFamide from Renilla köllikeri: Evidence for Unusual Processing Enzymes
Authors: Reinscheid Rainer K. , Grimmelikhuijzen Cornells J. P.
Published: July 2010

Abstract: Neuropeptides containing the C‐terminal sequence Arg‐Phe‐NH2 are an important group of hormones mediating or modulating neuronal communication. Arg‐Phe‐NH2 peptides are abundant in evolutionary “old” nervous systems such as those of coelenterates, the lowest animal group having a nervous system. Here, we have cloned the precursor protein for the anthozoan neuropep‐tide Antho‐RFamide (<Glu‐Gly‐Arg‐Phe‐NH2) from the sea pansy Renilla köllikeri. This precursor contains 36 copies of immature Antho‐RFamide (Gln‐Gly‐Arg‐Phe‐Gly) and two additional putative neuropeptide sequences, which are regularly distributed over the precursor protein. Of the 36 Antho‐RFamide sequences, 29 copies are separated by the five amino acid spacer sequence Arg‐Glu/Gly‐Asn/Ser/Asp‐Glu/Lys‐Glu. This implicates processing at single Arg and single Glu residues. Endoproteolytic cleavage at the C‐terminal side of paired or single basic residues is a well known initial step in the maturation of precursor proteins. Cleavage at the C‐terminal side of acidic residues, however, is unusual and must be catalyzed by a new type of processing enzyme. This processing enzyme is most likely to be an endoprotease, because the simplest way to generate Antho‐RFamide is by endoproteolytic cleavage at the C‐terminal side of Glu residues. The enzyme could also be an aminopeptidase, but in this case other proteases must be involved. As a possible alternative, one single “unspecific” aminopeptidase could cleave at Glu, Asp, Gly, Asn, Ser, and possibly also at other residues, and thus liberate all Antho‐RFamide sequences. The processing of one precursor molecule probably yields 38 neuropeptides. Thus, the Antho‐RFamide precursor from Renilla is one of the most productive precursor proteins known so far.
The Production of Arachidonic Acid Can Account for Calcium Channel Activation in the Second Messenger Pathway Underlying Neurite Outgrowth Stimulated by NCAM, N‐Cadherin, and L1
Authors: Williams Emma J. , Walsh Frank S. , Doherty Patrick
Published: July 2010

Abstract:We have used monolayers of control 3T3 fibroblasts and 3T3 fibroblasts expressing transfected cell adhesion molecules (CAMs)—NCAM, N‐cadherin, and L1—as a culture substrate for cerebellar neurones. The transfected CAMs promote neurite outgrowth by activating a second messenger pathway that culminates in calcium influx into neurones through N‐andl‐type calcium channels. We show that the same neurite outgrowth response can be directly induced by arachidonic acid (10 μM) and that this response can be inhibited by N‐andl‐type calcium channel antagonists. In cells, arachidonic acid can be generated by phospholipase A2or by the sequential activities of a phospholipase C (to generate diacylglycerol) and diacylglycerol lipase. In the present study we show the neurite outgrowth stimulated by CAMs (but not by various other agents) can be abolished by an inhibitor of diacylglycerol lipase acting at a site upstream from calcium channel activation. The results suggest that arachidonic acid and/or one of its metabolites is the second messenger that activates calcium channels in the CAM signalling pathway leading to axonal growth, and this is supported by recent evidence that shows the same concentrations of arachidonic acid can increase voltage‐dependent calcium currents in cardiac myocytes.
Antisense Strategy Unravels a Dopamine Receptor Distinct from the D2 Subtype, Uncoupled with Adenylyl Cyclase, Inhibiting Prolactin Release from Rat Pituitary Cells
Authors: Valeric Alessandra , Alberici Antonella , Tinti Cristina , Spano PierFranco , Memo Maurizio
Published: July 2010

The antisense strategy was used to unravel the functional contribution of the mRNAs encoding dopamine (DA) receptors to the multiple transduction mechanisms operated by DA in rat pituitary cells. An antisense oligonucleotide was designed to recognize seven nucleotides upstream and 11 nucleotides downstream from the initiation translation codon of the mRNA that encodes the DA D2 receptor. Addition of the antisense oligonucleotide for 7 days to primary culture of rat pituitary cells resulted in a decreased expression of DA D2 receptor as shown by (a) the virtual disappearance of [3H]spiroperidol binding sites and (b) the marked reduction in the levels of both the long and the short splice variant of the D2 receptor mRNAs. After this treatment, the DA D2 receptor agonist bromocriptine lost its capability both to inhibit adenylyl cyclase activity and to reduce prolactin mRNA levels. On the contrary, the inhibition of prolactin release induced by bromocriptine was affected minimally by the antisense oligonucleotide treatment. These data indicate that (a) translation of the mRNA encoding DA D2 receptors results in receptors that are negatively coupled with adenylyl cyclase and functionally linked to inhibition of prolactin synthesis; and (b) the release of prolactin might be regulated, at least in part, by a DA receptor that is encoded by mRNA species distinct from those encoding the D2 receptor.
Effect of Fish Oil Diet on Fatty Acid Composition of Phospholipids of Brain Membranes and on Kinetic Properties of Na+, K+‐ATPase Isoenzymes of Weaned and Adult Rats
Authors: Gerbi Alain , Zérouga Mustapha , Debray Marcel , Durand George , Chanez Claude , Bourre Jean‐Marie
Published: July 2010

Abstract:The influence of dietary (n‐3) fatty acids (such as eicosapentaenoic and docosahexaenoic acids) as found in fish oil on Na+sensitivity and ouabain affinity of Na+, K+‐ATPase isoenzymes (α1, α2, α3) was studied in whole brain membranes from weaned and adult rats fed diets for two generations. The long chain (n‐3) fatty acids supplied by fish oil decreased the fatty acids of the (n‐6) series compared with the standard diet, resulting in a decrease in the (n‐6)/(n‐3) molar ratio in both 21 ‐ and 60‐day‐ old rats. On the basis of ouabain titration, three inhibitory processes with markedly different affinities were associated with isoenzymes, i.e., low affinity (α1), high affinity (α2), and very high affinity (α3). It appears that the fish oil diet, in part via the modification of membrane fatty acid composition, altered the proportion and ouabain affinity of isoenzymes. Na+sensitivity is the best criterion of physiologic change induced by fish oil diet. We calculated the Na+activation for each isoenzyme and found one Na+sensitivity and two Na+sensitivities per isoenzyme in weanling and adult rats fed different diets, respectively. In contrast to α2 and α3, α1 appears insensitive to membrane change induced by fish oil diet. Fish oil diet, which is known to confer cardioprotection, induced significant modulation of Na+, K+‐ATPase isoenzymes at the brain level.