bluuebunny/crossref_metadata_2025 huggingface dataset:
Guinea Pig Histamine H1 Receptor. I. Gene Cloning, Characterization, and Tissue Expression Revealed by In Situ Hybridization
Authors: Traiffort E. , Leurs R. , Arrang J. M. , Tardivel‐Lacombe J. , Diaz J. , Schwartz J.‐C. , Ruat M.
Published: July 2010

Abstract: An intronless DNA encoding the guinea pig H1 receptor was cloned from a genomic library using probes derived from the bovine H1 receptor. It encodes a protein of 488 amino acids with a calculated molecular mass of 55,619 daltons compared with a size of 56–68 kDa for the photoaffinity‐labeled receptor as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. The protein displays a 66% homology with the bovine receptor. Stable expression of the H1 receptor, characterized by the appearance of [3H]mepyramine binding sites with a pharmacology similar to that of the native H1 receptor, was obtained following transfection of Chinese hamster ovary cells. Southern blot analysis, using a variety of restriction enzymes, did not provide any evidence of multiple H1 isoreceptors. Northern blot analysis of a variety of guinea pig peripheral or cerebral tissues identified, in most cases, a single transcript of 3.3 kb, but also, in some tissues, a second transcript of 3.7 kb, possibly generated by the use of different promoter or polyadenylation sites or corresponding to a transcript from a distinct gene. In situ hybridization studies showed the highly contrasted cerebral expression of H1‐receptor gene transcripts, which was compared with autoradiographic receptor localization. This allowed the identification of some major cell populations expressing the H1 receptor, e.g., Purkinje cells in cerebellum or pyramidal cells in the hippocampal complex.
Propentofylline and Other Adenosine Transport Inhibitors Increase the Efflux of Adenosine Following Electrical or Metabolic Stimulation of Rat Hippocampal Slices
Authors: Fredholm Bertil B. , Lindström Karin , Wallman‐Johansson Agneta
Published: July 2010

Abstract: Propentofylline is a novel neuroprotective agent that has been shown to act as an adenosine transport inhibitor as well as an adenosine receptor antagonist. In the present series of experiments we have compared the effects of propentofylline with those of known adenosine transport inhibitors and receptor antagonists on the formation of adenosine in rat hippocampal slices. The ATP stores were labeled by incubating the slices with [3H]‐adenine. The total 3H overflow and the overflow of endogenous and 3H‐labeled adenosine, inosine, and hypoxanthine were measured. Adenosine release, secondary to ATP breakdown, was induced both by hypoxia/hypoglycemia and by electrical field stimulation. Propentofylline (20–500 µM) increased the release of endogenous and radiolabeled adenosine, without increasing the total release of purines. Thus, the drug altered the pattern of released purines, i.e., increasing adenosine and decreasing inosine and hypoxanthine. This pattern, which was observed when purine release was induced both by electrical field stimulation and by hypoxia/hypoglycemia, was shared by the nucleoside transport inhibitor dipyridamole (1 µM) and by mioflazine (1 µM) and nitrobenzylthioinosine (1 µM). By contrast, other xanthines, including theophylline (100 µM) and 8‐cyclopentyltheophylline (10 µM), enprofylline (100 µM), or torbafylline (300 µM), if anything, increased the total release of purines without alterations of the pattern of release. These results indicate that nucleoside transport inhibitors can decrease the release of purines from cells and at the same time increase the concentration of extracellular adenosine, possibly by preventing its uptake and subsequent metabolism. This change in purine metabolism may be beneficial with regard to cell damage after ischemia. The results also indicate that propentofylline behaves in such a potentially beneficial manner.
Effects of Chronic Nicotine Infusion on Kinetics of High‐Affinity Nicotine Binding
Authors: Bhat Ratan V. , Marks Michael J. , Collins Allan C.
Published: July 2010

Abstract: It is well established that chronic nicotine treatment produces a dose‐dependent increase in high‐affinity l‐[3H]nicotine binding. This increase may be due to chronic desensitization of the receptor. Sophisticated kinetic analyses of high‐affinity nicotine binding to rat brain have demonstrated that the association rate is biphasic; the fast phase may represent binding to a high‐affinity predesensitized state and the slow phase may represent binding to a lower affinity ground state that then isomerizes to form the high‐affinity binding site. This isomerization presumably leads to receptor desensitization. The studies reported here assessed whether binding to mouse brain nicotinic receptors shows these same properties and whether chronic intravenous infusion of nicotine results in changes in these kinetic properties. The results obtained indicate that mouse brain nicotine binding also shows biphasic association kinetics and uniphasic dissociation kinetics, which supports the assertion that the receptor exists in two interconvertible states. However, unlike other results obtained with rat brain, the rate of the slow association process did not change with ligand concentration. Chronic infusion resulted in a dose‐dependent increase in l‐[3H]‐nicotine binding, but the ratio of fast/slow phases of binding was not changed by these treatments. These results suggest that chronic infusion does not alter measurably the kinetics of nicotinic receptor binding when measured in vitro.
Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue
Authors: Ozaki Naoko , Moroi Kayoko , Kadota Tomoko , Suzuki Shiro , Kadota Ken
Published: July 2010

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D1 and D2 receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D1 receptor antagonist, [3H]SCH 23390, and a dopamine D2 receptor antagonist, [3H]‐spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant (KD) of 0.65 and 0.5 nM, respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [3H]SCH 23390 and [3H]spiroperidol in a stereoselective and concentration‐dependent manner with an appropriate rank order potency for dopamine D1 or D2 receptors. The regulations of the agonist binding by guanyl‐5‐ylimidodiphosphate were observed. ADP ribosylation of the CVs with [32P]NAD demonstrated predominant labeling of bands of Mr 47,000–52,000, 42,000–45,000, and 40,000‐39,000, which corresponded to the known molecular weights of the α subunits of Gs and Gi proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D2 receptor agonists, was present in the CVs. These findings suggest that the dopamine D1 and D2 receptors in the CVs couple with adenylate cyclase via Gs/Gi protein.
Changes in Intracellular Ca2+ and Energy Levels During In Vitro Ischemia in the Gerbil Hippocampal Slice
Authors: Mitani Akira , Takeyasu Shingo , Yanase Hisato , Nakamura Yoichi , Kataoka Kiyoshi
Published: July 2010

Abstract: The time course of the decline in energy levels during an in vitro ischemia‐like condition was compared with changes in intracellular Ca2+ concentration ([Ca2+]i) in subregions of the gerbil hippocampal slice [CA1, CA3, and the inner and outer portions of the dentate gyrus (DG)]. Hippocampal transverse slices were loaded with a fluorescent indicator, rhod‐2. During the on‐line monitoring of [Ca2+]i, the slices were perfused with an in vitro ischemia‐like medium (33°C). The slices were collected at several experimental time points, frozen, dried, and dissected into subregions. The contents of adenine nucleotides (ATP, ADP, and AMP) and phosphocreatine (PCr) were measured by HPLC methods. Region‐specific and acute [Ca2+]i elevations were observed in CA1 ∼4 min after onset of the in vitro ischemia‐like condition and also in the inner portion of the DG with a delay of 10–40 s. The change in ATP levels was related to the increase in [Ca2+]i. ATP levels in all subregions gradually decreased before the acute [Ca2+]i elevation. Concomitant with the acute [Ca2+]i elevation in CA1 and the inner portion of the DG, ATP levels in the subregions rapidly decreased, whereas declines in levels of high‐energy‐charge phosphates were gradual in CA3 and the outer portion of the DG, in which the remarkable [Ca2+]i elevation was not observed. These results suggest that ATP depletion observed in CA1 and the inner portion of the DG is due to the region‐specific increase in [Ca2+]i, which activates a Ca2+‐ATP‐driven pump and produces a subsequent fall in neuronal ATP content.
The Effects of Alzheimer's Disease, Other Dementias, and Premortem Course on β‐Amyloid Precursor Protein Messenger RNA in Frontal Cortex
Authors: Harrison Paul J. , Barton Amanda J. L. , Procter Andrew W. , Bowen David M. , Pearson R. Carl A.
Published: July 2010

Abstract: There are conflicting data regarding alterations in β‐amyloid precursor protein (APP) mRNAs in Alzheimer's disease (AD). This may be due partly to variables such as agonal state and choice of control group. We have used in situ hybridization histochemistry to study expression of APP mRNAs, with and without the domain encoding the Kunitz protease inhibitor, in a way that overcomes some of the limitations of the current data. Tissue from frontal cortex was collected at rapid autopsy from patients with AD or other cognitive impairments whose terminal phase was prospectively assessed. There were three main findings. Firstly, the amount of APP mRNAs correlated strongly with glutamate decarboxylase activity and was reduced in association with terminal pyrexia. These correlations suggest that agonal state affects APP mRNA and, therefore, that differences in premortem course may contribute to the varying changes in APP transcript abundance reported in AD. Secondly, a reduction of both forms of APP mRNA, normalized to polyadenylated mRNA, was found in AD compared with normal controls and with non‐AD dementias. This supports findings that the APP‐related pathology of AD is not due to overexpression of APP mRNA or an altered proportion of Kunitz protease inhibitor‐containing isoforms. Thirdly, the amount of APP mRNA correlated inversely with that of heat‐shock protein (hsx70) mRNA. This relationship was unexpected given current theories that APP expression occurs as part of a stress response, and suggests that other factors predominate in determining neocortical APP mRNA content in neurodegenerative disorders.
Influence of Region‐Specific Alterations of Neuropeptidase Content on the Catabolic Fates of Neuropeptides in Alzheimer's Disease
Authors: Ichai Carole , Chevallier Nathalie , Delaere Pia , Dournaud Pascal , Epelbaum Jacques , Hauw Jean‐Jacques , Vincent Jean‐Pierre , Checler Frédéric
Published: July 2010

Abstract: We established the cartography of 11 exo‐ and endopeptidases in the frontal and parietal cortices and in the cerebellum of brains of patients diagnosed with a senile dementia of the Alzheimer's type (SDAT). Comparison with those of four subjects who had died without known neurologic or psychiatric illness indicated that there existed a region‐specific alteration of the peptidase contents in the disease. In the frontal area of SDAT brains, postproline dipeptidyl aminopeptidase and aminopeptidase M activities were significantly reduced. In the parietal cortex of SDAT brain, activities of three additional endopeptidases—angiotensin‐converting enzyme, proline endopeptidase, and endopeptidase 24.15—were also drastically reduced. In contrast, the cerebellum displayed a set of proteolytic activities that remained unaffected in SDAT brain. The putative influence of the disease on the catabolic fates of neurotensin, neuropeptide Y, and somatostatin(1–14) was investigated. Neurotensin was catabolized at identical rates in the frontal and parietal cortices in nondemented and SDAT brains. In contrast, neuropeptide Y metabolism was slowed down in SDAT brains in the frontal but not in the parietal cortex. Finally, the degradation velocities of somatostatin(1–14) were lowered in both cortical areas of SDAT brains. It is interesting that, by means of specific peptidase inhibitors, we demonstrated that endopeptidase 24.15 participated in somatostatin(1–14) inactivation in the parietal but not in the frontal cortex. It is suggested that the lowering of the rate of somatostatin(1–14) inactivation in the parietal cortex of SDAT brains likely results from the depletion of endopeptidase 24.15 in this brain region.
Changes in Neurofilament Protein NF‐L and NF‐H Immunoreactivity Following Kainic Acid‐Induced Seizures
Authors: Wang S. , Hamberger A. , Yang Q. , Haglid K. G.
Published: July 2010

Abstract: Postlesion plasticity of neuronal processes might contribute to secondary spontaneous seizures after kainic acid administration. In this study, neurofilament (NF) proteins were examined following intraperitoneal injection of kainic acid, and special reference was given to temporal changes in quantity and quality of the NF light (NF‐L) and heavy (NF‐H) subunits. A pronounced decrease in phosphorylation‐related immunoreactivity of NF‐H occurred as early as 1 day after the injection in the amygdala/pyriform cortex, hippocampus, striatum, and dorsal cerebral cortex. A shift of NF‐H from the phosphorylated to nonphosphorylated form was evident in immunoblots, suggesting dephosphorylation contributed to the decrease. Decreases in NF‐L and phosphorylated NF‐H contents in the limbic structure at 3 days were correlated with the increasing kainic acid doses from 2.5 to 10 mg/kg. The degradation pattern in immunoblots with antibodies against NF‐L indicated that the decrease in NF‐L was probably due to calcium‐activated proteolysis. NF‐L and phosphorylated NF‐H contents secondarily increased from 9 days onward, with ∼20% above the control level of phosphorylated NF‐H immunoreactivity at 27 days in the amygdala/pyriform cortex and ventral hippocampus. Immunohistochemical examination of the hippocampus revealed that an increase of NF staining in the mossy fiber system may contribute to the NF recovery in this region. Furthermore, the temporal changes of NF‐L and phosphorylated NF‐H contents were positively correlated with those of the neuronal cell adhesion molecule, a neuritic growth cone marker, substantiating postlesion regenerative reactions of NF proteins. Functional consequences of the NF plasticity remain to be identified.
Cholinesterases Display Genuine Arylacylamidase Activity but Are Totally Devoid of Intrinsic Peptidase Activities
Authors: Checler Frédéric , Grassi Jacques , Vincent Jean‐Pierre
Published: July 2010

Abstract: The purpose of this article was to evaluate the intrinsic character of arylacylamidase and peptidase activities that are often detected along with cholinesterase activities. Various pools of commercial or affinity‐purified acetylcholinesterases (AChEs) were examined. Affinity‐purified AChE displays esterase‐ and amidase‐specific activities that are similarly enriched when compared with commercial AChE. By contrast, commercial AChE exhibits much higher tryptic‐like and carboxypeptidase‐specific activities than the affinity‐purified enzyme. The parallel enrichment in esterase and arylacylamidase suggests that these two activities are copurified, whereas peptidases do not seem to behave similarly. We show that trypsinolysis or spontaneous degradation of affinity‐purified AChE leads to the conversion of the 75‐kDa monomer protein into two fragments of 50 and 25 kDa after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. However, these modifications are without effect on the esterase, arylacylamidase, and peptidase activities. This clearly shows that AChE does not behave as a zymogen of peptidases that would have been activated on autolysis of AChE. Immunoprecipitation of AChEs with a purified monoclonal antibody directed toward electric eel AChE totally separated the esterase and arylacylamidase activities (pellet) from peptidase activities (supernatant). The immunoprecipitated AChEs could be dissociated from the interaction with IgGs. These resolubilized AChE preparations have kept the same percentage of initial esterase and arylacylamidase activities but were totally devoid of peptidase activities. These data clearly indicate that commercial and affinity‐purified AChEs from Electrophorus electricus bear an intrinsic arylacylamidase activity but that the peptidase activity detected in these preparations is not an integral property of the AChE molecule and most probably represents a contaminating activity. It appears therefore unlikely that AChE may participate to the processing of the β‐amyloid protein precursor (β‐APP) leading to the secretion of protease nexin II and therefore acts as an APP secretase, as was recently suggested. By a similar approach, we established that human butyrylcholinesterase recovered after immunoprecipitation retained its esterase activity but was no longer able to act as a peptidase.
Expression of Apolipoprotein A‐I in Porcine Brain Endothelium In Vitro
Authors: Möckel Babette , Zinke Holger , Flach Regina , Weiß Birgit , Weiler‐Güttler Hartmut , Gassen Hans G.
Published: July 2010

Abstract: Apolipoprotein (apo) A‐I is the major protein component of high‐density lipoproteins (HDLs), which are responsible for reverse cholesterol transport from peripheral tissues to the liver. A low level of plasma HDL is correlated with susceptibility to atherosclerosis and coronary heart disease. Mammalian apo A‐I synthesis has been attributed mainly to liver and intestine. Recently, apo A‐I expression has been shown in porcine brain capillaries, suggesting an independent lipid metabolism within the brain. In this study, protein synthesis and secretion were investigated in primary cultures of porcine brain microvascular endothelial cells and compared with those in large vessel endothelium. Active protein synthesis in vitro was demonstrated by metabolic labeling. Cerebral endothelial cells were shown to secrete apo A‐I into the culture supernatant, whereas aortic endothelial cells were negative for apo A‐I expression. Further studies of transcriptional regulation showed that cerebral endothelium was responsive to apo A‐I‐inducing agents, such as cholesterol, insulin, and retinoic acid, as previously shown in human hepatoma HepG2 cells. Thus, cultures of porcine cerebral endothelial cells may represent a suitable model for physiological studies of apo A‐I‐regulation with regard to brain lipid metabolism and blood‐brain barrier function. To investigate the interspecies conservation of regulatory elements, 178 bp of the 5′ flanking region of the porcine apo A‐I gene was cloned using PCR techniques. Alignments of the cDNA, of the deduced apo A‐I protein sequence, and of the 5′ promoter region with the corresponding genomic sequences of different species show a high degree of similarity between the porcine and the primate apo A‐I genes, thus indicating a similar function and possibly common regulatory mechanisms in those species. In contrast, the rodent and avian apolipoprotein A‐I promoter sequences differed significantly.
Splicing of the Muscle‐Specific Plasma Membrane Ca2+‐ATPase Isoform PMCA1c Is Associated with Cell Fusion in C2 Myocytes
Authors: Brandt Paul , Vanaman Thomas C.
Published: July 2010

Abstract: The regulation of intracellular calcium is essential for proper muscle function. Muscle cells have several mechanisms for dealing with the rapid and large changes in cytosolic calcium level that occur during contraction. Among these is the plasma membrane Ca2+‐ATPase (PMCA), which pumps calcium from the cytosol to the extracellular space. We have previously shown that in human fetal muscle the PMCA1 isoforms present are PMCA1a‐d, with PMCA1b and c predominating. Alternative splicing of mRNAs encoding proteins involved in muscle contraction is common in developing muscle. Therefore, we examined the expression of muscle‐specific PMCA mRNAs in pre‐ and postfusion mouse C2 myoblasts. The housekeeping form of the Ca2+‐ATPase, PMCA1b, was found at all times and under all conditions. However, the other predominating isoform found in muscle, PMCA1c, was expressed on myotube formation. Simple cell‐cell contact was not sufficient to induce PMCA1c expression, as cells plated at confluence but harvested before myotube formation did not express PMCA1c. The induction of this muscle‐specific Ca2+‐ATPase at myotube formation suggests that it may play an important role in muscle function.
Modulation of In Vivo Striatal Transmitter Release by Nitric Oxide and Cyclic GMP
Authors: Guevara‐Guzman Rosalinda , Emson Piers C. , Kendrick Keith M.
Published: July 2010

Abstract: The effects of nitric oxide (NO) and cyclic GMP on in vivo transmitter release in the rat striatum were investigated using microdialysis sampling in urethane‐anaesthetised animals. The NO release‐inducing substances S‐nitrosoacetylpenicillamine (SNAP), S‐nitrosoglutathione (SNOG), and sodium nitroprusside (SNP) increased extracellular concentrations of aspartate (Asp), glutamate (Glu), γ‐aminobutyric acid (GABA), taurine (Tau), acetylcholine (ACh), and serotonin (5‐HT). Dopamine (DA) concentrations were decreased by SNAP but were increased by SNOG and SNP. An NO scavenger, haemoglobin, blocked or reduced the effects of SNAP on transmitter release. However, the control carrier compounds for SNAP, SNOG, and SNP (penicillamine, glutathione, and potassium ferricyanide, respectively, which do not induce release of NO) also increased GABA, Tau, DA, and 5‐HT concentrations. When NO gas was given directly by dissolving it in degassed Ringer's solution, DA concentrations decreased significantly, and those of Asp, Glu, GABA, Tau, ACh, and 5‐HT increased. These effects of NO gas were all inhibited by coadministration of haemoglobin and for GABA, Tau, ACh, and DA showed some calcium dependency. The cyclic GMP agonists 8‐bromo‐cyclic GMP and dibutryl‐cyclic GMP stimulated dose‐dependent increases in Asp, Glu, GABA, Tau, ACh, DA, and 5‐HT concentrations. Increased striatal transmitter release in response to NO may therefore be mediated by its stimulatory action on cyclic GMP formation. NO inhibition of DA release may be mediated indirectly through its stimulation of local cholinergic and GABAergic neurones.
Quaternary Structure of the Native GABAA Receptor Determined by Electron Microscopic Image Analysis
Authors: Nayeem N. , Green T. P. , Martin I. L. , Barnard E. A.
Published: July 2010

Abstract: In the transmitter‐gated ion channel class of receptors, the members of which are all believed to be heterooligomers, the number and arrangement of the subunits are only known with any certainty for the nicotinic acetylcholine receptor from Torpedo electric fish. That receptor has been shown to possess a pentameric rosette structure, with five homologous subunits (α2βγδ) arranged to enclose the central ion channel. The data were obtained by electron image analysis of two‐dimensional receptor arrays, which form as a consequence of that receptor's exceptionally high abundance in the Torpedo membranes and are therefore not attainable for other receptors. We have applied another direct approach to determine the quaternary structure of native ionotropic GABA receptors. We have purified those receptors from porcine brain cortex and analysed the rotational symmetry of isolated receptors visualized by electron microscopy. The results show the receptor to have a pentameric structure with a central water‐filled pore, which can now be said to be characteristic of the entire superfamily.
Bovine Tyrosine Hydroxylase Gene‐Promoter Regions Involved in Basal and Angiotensin II‐Stimulated Expression in Nontransformed Adrenal Medullary Cells
Authors: Goc A. , Stachowiak M. K.
Published: July 2010

Abstract:The tyrosine hydroxylase gene is expressed specifically in catecholaminergic cells, and its activity is regulated by afferent stimuli. To characterize molecular mechanisms underlying those regulations, we have constructed chimeric genes consisting of bovine tyrosine hydroxylase gene promoters (wild‐type or deletion mutants) and a luciferase reporter gene. The basal expression of these genes and their regulation by angiotensin II were examined in cultured bovine adrenal medullary cells. Luciferase activity was normalized to the amount of transfected plasmid DNA. A pTHgoodLUC plasmid containing the ‐428/+21‐bp fragment of the tyrosine hydroxylase gene promoter expressed luciferase activity at severalfold higher levels than the promoterless pOLUC plasmid. Deletion of the ‐194/‐54‐bp promoter fragment containing POU/Oct, SP1, and other putative regulatory elements increased luciferase expression fivefold. An additional deletion further upstream (‐269/‐194 bp), including a 12‐O‐tetradecanoylphorbol 13‐acetate (TPA)‐responsive element (TRE)‐like site, reduced promoter activity. These results indicate the presence of negatively and positively acting regions in the bovine tyrosine hydroxylase gene promoter controlling basal promoter activity in adrenal medullary cells. Angiotensin II stimulated the expression of endogenous tyrosine hydroxylase gene and pTHgood‐LUC approximately threefold without affecting the expression of pOLUC. A comparable threefold stimulation was observed following the deletion of the ‐194/‐54‐bp promoter region, despite the increase in basal promoter activity. Additional deletion of the ‐269/‐194‐bp promoter fragment reduced stimulation by angiotensin II to 1.5‐fold. These results indicate that the angiotensin II receptor‐responsive element is located in the ‐269/‐194‐bp promoter region containing the TRE‐like site. Additional angiotensin II‐responsive site(s) may be present outside this region. Gel mobility shift assays demonstrated constitutive and angiotensin II‐induced protein binding to the tyrosine hydroxylase gene promoter. Some DNA‐protein complexes were displaced with c‐Fos antibodies. The results suggest that c‐Fos‐related antigens support basal promoter activity and mediate activation of tyrosine hydroxylase by angiotensin II receptor.
Monoclonal Antibody NM2 Recognizes the Protein Kinase C Phosphorylation Site in B‐50 (GAP‐43) and in Neurogranin (BICKS)
Authors: Oestreicher A. Beate , Hens Jacques J. H. , Marquart Arnoud , Mercken Marc , De Graan Pierre N. E. , Zwiers Henk , Gispen Willem Hendrik
Published: July 2010

Abstract: Mouse monoclonal B‐50 antibodies (Mabs) were screened to select a Mab that may interfere with suggested functions of B‐50 (GAP‐43), such as involvement in neurotransmitter release. Because the Mab NM2 reacted with peptide fragments of rat B‐50 containing the unique protein kinase C (PKC) phosphorylation site at serine‐41, it was selected and characterized in comparison with another Mab NM6 unreactive with these fragments. NM2, but not NM6, recognized neurogranin (BICKS), another PKC substrate, containing a homologous sequence to rat B‐50 (34–52). To narrow down the epitope domain, synthetic B‐50 peptides were tested in ELISAs. In contrast to NM6, NM2 immunoreacted with B‐50 (39–51) peptide, but not with B‐50 (43–51) peptide or a C‐terminal B‐50 peptide. Preabsorption by B‐50 (39–51) peptide of NM2 inhibited the binding of NM2 to rat B‐50 in contrast to NM6. NM2 selectively inhibited phosphorylation of B‐50 during endogenous phosphorylation of synaptosomal plasma membrane proteins. Preabsorption of NM2 by B‐50 (39–51) peptide abolished this inhibition. In conclusion, NM2 recognizes the QASFR peptide in B‐50 and neurogranin. Therefore, NM2 may be a useful tool in physiological studies of the role of PKC‐mediated phosphorylation and calmodulin binding of B‐50 and neurogranin.
Opposing Actions of D1and D2‐Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons
Authors: Schinelli Sergio , Paolillo Mayra , Corona Gian Luigi
Published: July 2010

Abstract:D1‐and D2‐dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D1or D2receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [3H]AA, D2‐receptor stimulation enhanced release of [3H]AA produced by application of the Ca2+ionophore A23187 or of the purinergic agonist ATP. By contrast, D1‐receptor stimulation inhibited [3H]AA release. This inhibitory effect of D1receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D2and D1receptors in striatum, potentiation and inhibition, respectively, of Ca2+‐evoked AA release.
Depolarization and Neurotransmitters Increase Neuronal Protein Tyrosine Phosphorylation
Authors: Siciliano Julio C. , Gelman Michèle , Girault Jean‐Antoine
Published: July 2010

Abstract:In rat hippocampal slices and in neurons in primary culture, K+‐induced depolarization increased markedly and rapidly tyrosine phosphorylation of a 110‐kDa protein (pp110) and, to a lesser degree, of a 120‐kDa protein (pp120), in a calcium‐dependent fashion. Qlutamate, 1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (an agonist of metabotropic glutamate receptors), and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (an agonist of ionotropic glutamate receptors) stimulated also tyrosine phosphorylation of pp110 and pp120. These effects were not observed in astrocytes in primary culture. In hippocampal slices tyrosine phosphorylation of pp110 and pp120 was stimulated by Ca2+‐ionophores and by phorbol esters and antagonized by a chelator of intracellular Ca2+and by drugs that inhibit protein kinase C. Stimulation of muscarinic and α1,‐adrenergic receptors increased also tyrosine phosphorylation of pp110 and pp120. These results demonstrate that membrane depolarization and stimulation of neurotransmitter receptors activate a tyrosine phosphorylation pathway in neurons. This pathway involves an increase in intracellular Ca2+concentrations and the activation of protein kinase C. It may provide a biochemical basis for some neurotrophic effects of electrical activity and neurotransmitters and may contribute to the role of tyrosine phosphorylation in long‐term potentiation.
Adenosine A2aReceptor‐Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release
Authors: Kirk Ian P. , Richardson Peter J.
Published: July 2010

Abstract:The ability of adenosine agonists to modulate K+‐evoked 4D†‐[3H]aminobutyric acid ([3H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A2areceptor‐selective agonist CGS 21680 inhibited Ca2+‐dependent [3H]GABA release evoked by 15 mMKCI with a maximal inhibition of 29 ± 4% (IC50of ∼4 ± 10−12M). The relative order of potency of three agonists was CGS 21680 ± 5′‐N‐ethylcarboxamidoadenosine > R‐phenylisopropyladenosine (R‐PIA), with the inhibition being blocked by A2areceptor‐selective antagonists (CP 66,713 and CGS 15943A) but not by the A1‐selective antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX). When release of [3H]GABA was evoked by 30mMKCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [3H]ACh evoked by 30 mMKCI, with a maximal stimulation of 26 ± 5% (IC50of ∼10−11M). This effect was blocked by CP 66,713 but not by DPCPX. The A1agonistR‐PIA inhibited [3H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A2areceptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively.Key Words: GABA—Acetylcholine—Adenosine receptors—Striatum.
Evidence for Protein Kinase C Involvement in the Short‐Term Activation by Prolactin of Tyrosine Hydroxylase in Tuberoinfundibular Dopaminergic Neurons
Authors: Pasqualini Catherine , Guibert Bernard , Frain Odile , Leviel Vincent
Published: July 2010

Abstract:The mechanism of the short‐term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4‐dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose‐dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting aK1(DA)value of 29.92 ± 0.49 μM, the other being × 15‐fold more sensitive to DA inhibition with aK1(DA)of 1.96 ± 0.09 μM, likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of lowK1(DA)remained unaffected, whereas theK1(DA)of the purported active form of TH increased to 62.6 ± 0.8 μM, suggesting an increase in the enzyme phosphorylation. This increase in theKI(DA)of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12‐O‐tetradecan‐oylphorbol 13‐acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.
A Comparison of the Effects of Ethanol and the Competitive Glycine Antagonist 7‐Chlorokynurenic Acid onN‐Methyl‐d‐Aspartic Acid‐Induced Neurotransmitter Release from Rat Hippocampal Slices
Authors: Woodward John J.
Published: July 2010

Abstract:N‐Methyl‐d‐aspartate (NMDA; 500 μM) stimulated the net release of preloaded tritiated norepinephrine from rat hippocampal slices. Both ethanol and the competitive glycine antagonist 7‐chlorokynurenic acid (7‐CK) dose‐dependently inhibited NMDA‐stimulated release without affecting basal, nonstimulated efflux. These inhibitory effects were readily reversed upon washout of the drugs. Over the concentration range tested (25–200 mM), ethanol inhibited ∼65% of NMDA‐stimulated release with an estimated IC50of ∼70 mM. In contrast, 7‐CK fully inhibited release (>95%) at a concentration of 30 μMwith half‐maximal inhibition occurring at ∼2 μM. The combination of 7‐CK (1–30 μM) and ethanol (25–100 mM) had an additive inhibitory effect on NMDA‐stimulated release but did not alter the inhibitory potency of 7‐CK. Calculated IC50values for 7‐CK in the presence of 25, 50, or 100 mMethanol were (mean × SEM; μM) 2.33 (0.11), 2.38 (0.23), and 1.99 (0.30), respectively. 7‐CK (3 μM) inhibited NMDA‐stimulated [3H]norepinephrine release by ∼50%. This inhibition was fully attenuated by the addition of the glycine agonistserine with complete reversal occurring at 30 μMd‐serine. Increasing the 7‐CK concentration to 10 μMshifted thed‐serine dose‐effect curve to the right in a parallel fashion as expected for a competitive antagonist. In contrast, the inhibitory effects of ethanol or the combination of 7‐CK (3 μM) and ethanol (25 or 50 mM) were not reversed by the addition ofd‐serine (0.1–1,000 μM). Together, these results suggest that ethanol's inhibition of NMDA‐stimulated [3H]norepinephrine release from hippocampal slices is not due to a simple competitive interaction with the glycine site on the NMDA receptor.
Characterization and Localization of Adenosine A2Receptors in Bovine Rod Outer Segments
Authors: McIntosh Helen H. , Blazynski Christine
Published: July 2010

Abstract:Previous work from this laboratory has shown that retinal adenosine A2binding sites are localized over outer and inner segments of photoreceptors in rabbit and mouse retinal sections. In the present study, adenosine receptor binding has been characterized and localized in membranes from bovine rod outer segments (ROS). Saturation studies with varying concentrations (10–150 nM) of 5′‐(N‐[2,8‐3H]ethylcarboxamido)adenosine ([3H]NECA) and 100 μg of ROS membrane protein show a single site with aKDof 103 nMand aBmaxof 1.3 pM/mg of protein. Cold Scatchards, which used nonradiolabeled NECA (concentrations ranging from 10 nMto 250 nM) in competition with a fixed amount of [3H]NECA (30 nM), demonstrated the presence of a low‐affinity site (KD, 50 μM) in addition to the high‐affinity site. To confirm the presence of A2abinding sites, saturation analyses with 2‐p‐(2‐[3H]‐carboxyethyl)phenylamino‐5′‐N‐ethylcarboxamido adenosine (0–80 nM) also revealed a single population of high‐affinity A2areceptors (KD, 9.4 nM). The binding sites labeled by [3H]NECA appear to be A2receptor sites because binding was displaced by increasing concentrations of 5′‐(N‐methylcarboxamido)adenosine and 2‐chloroadenosine. ROS were fractionated into plasma and disk membranes for localization studies. Receptor binding assays, used to determine specific binding, showed that the greatest concentration of A2receptors was on the plasma membranes. Therefore, adenosine A2receptors are in a position to respond to changes in the concentration of extracellular adenosine, which may exhibit a circadian rhythm.
Effects of Ischaemia on Neurotransmitter Release from the Isolated Retina
Authors: Neal Michael J. , Cunningham Jo R. , Hutson Peter H. , Hogg Joanne
Published: July 2010

Abstract:The effects of “ischaemia” (glucose‐free Krebs‐bicarbonate medium gassed with N2/CO2on the release of glutamate and other major neurotransmitters in the retina were examined using the isolated rat and rabbit retina. Amino acid transmitters, acetylcholine, and dopamine were measured by HPLC. The release of glutamate, aspartate, GABA, and glycine from ischaemic retinas was more than doubled after 30 min, and after 90 min of ischaemia the release of amino acids was ∼ 15–20‐fold that of control values. Ischaemia also produced large increases in the release of dopamine from both the rat and especially the rabbit retina. In contrast, the release of acetylcholine from the rat retina was significantly decreased by ischaemia, although the release of choline was increased. Because the ischaemia‐induced release of glutamate, aspartate, and GABA from the rat retina was completely Ca independent, and exposure of the retina to high K (50 mM) did not stimulate amino acid release, it is concluded that the mechanisms underlying the ischaemia‐induced release do not involve an initial release of K or an influx of calcium.
Iron and Other Metals in Neuromelanin, Substantia Nigra, and Putamen of Human Brain
Authors: Zecca Luigi , Pietra Romano , Goj Carlo , Mecacci Claudio , Radice Davide , Sabbioni Enrico
Published: July 2010

Abstract: Radiochemical neutron activation analysis has been used to determine the concentration of 36 elements in neuromelanin, 22 elements in substantia nigra, and 32 elements in putamen of healthy subjects without signs of neurological disorders. Substantia nigra and putamen tissues were carefully dissected from the brain using special surgical instruments and tools as well as an adequate sampling procedure to avoid the risk of metal contamination during sampling. Neuromelanin was isolated from putamen by a multiple‐step procedure (extraction with phosphate buffer, lipid and protein elimination by methanol extraction, and sodium dodecyl sulfate‐proteinase). The isolated pigment as well as substantia nigra and putamen underwent neutron activation analysis involving irradiation in a high‐neutron‐flux reactor, radiochemical separations, and counting of the induced radionuclides by computer‐based γ‐ray spectrometry. Iron was the element present in the highest concentration in all analyzed samples. The amount of iron was similar in substantia nigra and putamen (3,000 and 3,830 ng/mg wet weight, respectively) and 10 times higher in neuromelanin (30,800 ng/mg dry weight). Zinc was also present at high levels in three samples, ranging from 16.8 (substantia nigra) to 1,500 ng/mg (neuromelanin). Elements such as Zn, Cr, Se, Sr, Co, Sb, Ni, Hg, Ce, Au, Ag, Ta, and Sc were present in neuromelanin at much higher concentrations than in substantia nigra and putamen. These findings indicate that substantia nigra and putamen contain metals at higher concentrations than observed in blood and that neuromelanin has a particular affinity for metals.
In Vivo High Intrinsic Efficacy of Triazolam: A Positron Emission Tomography Study in Nonhuman Primates
Authors: Bottlaender Michel , Brouillet Emmanuel , Varastet Marina , Le Breton Christine , Schmid Lorenz , Fuseau Chantal , Sitbon René , Crouzel Christian , Mazière Mariannick
Published: July 2010

Abstract:The triazolobenzodiazepine triazolam is a central‐type benzodiazepine receptor (BZR) ligand that is widely prescribed as a hypnotic agent. Triazolam produces its effects through potentiation of γ‐aminobutyric acid‐mediated neurotransmission. Findings reported from in vitro binding studies showed some discrepancies concerning the pharmacological characteristics of triazolam. The present study aims to characterize in vivo the biochemical properties of triazolam, i.e., cerebral pharmacokinetics, interaction with BZR, potency, and intrinsic efficacy. Triazolam was studied in living nonhuman primates using positron emission tomography. Two different studies were carried out: (a) a direct study using [11C]triazolam and (b) an indirect competition study using the radiolabeled BZR antagonist [11C]flumazenil. Results showed that, in the brain in vivo, triazolam binds specifically and competitively to the BZR. Its rapid cerebral kinetics is consistent with a hypnotic profile (maximal binding after 23 min, elimination half‐life of 202 min). Triazolam is very potent in displacing [11C]flumazenil (ID50= 28 ± 6 μg/kg). Hill analysis of the displacement curve does not show obvious binding‐site heterogeneity. Triazolam is 20 times more potent in displacing [11C]flumazenil and 50 times more potent in inhibiting pentylenetetrazol‐induced paroxysmal activity than the full benzodiazepine agonist diazepam. Interestingly, the simultaneous use of positron emission tomography and EEG recording allowed us to show that triazolam‐positive intrinsic efficacy is slightly higher (20%) than that of diazepam. An attractive hypothesis proposes that the severity of side effects of BZR ligands is proportional to their intrinsic efficacy. Therefore, our study shows that triazolam side effects, as for other benzodiazepines, may be related to its high intrinsic efficacy in vivo.
Typical and Atypical Neuroleptics Induce Alteration in Blood‐Brain Barrier and Brain 59FeCl3 Uptake
Authors: Ben‐Shachar Dorit , Livne Erella , Spanier Liana , Leenders K. L. , Youdim Moussa B. H.
Published: July 2010

Abstract: Long‐term neuroleptic medication of schizophrenic patients induces extrapyramidal motor side effects, of which tardive dyskinesia (TD) is the most severe. The etiology of TD is still obscure. Recently, it was suggested that abnormal iron metabolism may play a crucial role in neuroleptic‐induced dopamine D2 receptor super‐sensitivity. The apparent relationship between neuroleptics and iron is further supported by the increase of iron in the basal ganglia of patients with TD. We now report on the ability of neuroleptics to alter the blood‐brain barrier in the rat and to potentiate the normally limited iron transport into the brain. Thus, chronic treatment of rats with chlorpromazine and haloperidol facilitated 59Fe3+ uptake into brain cells. In contrast, clozapine, an atypical antipsychotic neuroleptic with little extrapyramidal motor side effects, caused iron sedimentation in brain blood vessels with no sign of detectable iron in the cells. Moreover, chronic treatment with chlorpromazine and haloperidol caused a 43% and 24% reduction, respectively, in liver nonheme iron, whereas clozapine induced an 81% increase. The apparent different potentials of chlorpromazine, haloperidol, and clozapine to increase iron transport into the brain from its peripheral stores may be linked to the severity of extrapyramidal motor side effects they induce and to the pathophysiology of TD.
Differential Distribution of Purine Metabolizing Enzymes Between Glia and Neurons
Authors: Ceballos Guillermo , Tuttle Jeremy B. , Rubio Rafael
Published: July 2010

Abstract: Previous studies showed that in cultured chick ciliary ganglion neurons and CNS glia, adenosine can be synthesized by hydrolysis of 5′‐AMP and that the accumulation of the adenosine degradative products inosine and hypoxanthine was significantly greater in glial than in neuronal cultures. Furthermore, previous immunochemical and histochemical studies in brain showed that adenosine deaminase and nucleoside phosphorylase are localized in endothelial and glial cells but are absent in neurons; however, adenosine deaminase may be found in a few neurons in discrete brain regions. These results suggested that adenosine degradative pathways may be more active in glia. Thus, we have determined if there is a differential distribution of adenosine deaminase, nucleoside phosphorylase, and xanthlne oxidase enzyme fluxes in glia, comparing primary cultures of central and ciliary ganglion neurons and glial cells from chick embryos. Hypoxanthine‐guanine phosphoribosyltransferase and production of adenosine by S‐adenosylhomocysteine hydrolase activity were also examined. Our results show that there is a distinct profile of purine metabolizing enzymes for glia and neurons in culture. Both cell types have an S‐adenosylhomocysteine hydrolase, but it was more active in neurons than in glia. In contrast, in glia the enzymatic activities of xanthine oxidase (443 ± 61 pmol/min/107 cells), nucleoside phosphorylase (187 ± B pmol/min/107 cells), and adenosine deaminase (233 ± 32 pmol/min/107 cells) were more active at least 100, 20, and five times, respectively, than in ciliary ganglion neurons and 100, 100, and nine times, respectively, than in central neurons.
B1and B2Bradykinin Receptors Encoded by Distinct mRNAs
Authors: Webb Michael , McIntyre Peter , Phillips Elsa
Published: July 2010

Abstract:Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B1and B2. The cDNAs encoding functional B2receptors have recently been cloned, but no molecular information exists at present on the B1receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B1and B2types of receptor. We showed previously that the Human fibroblast cell line W138 expresses both B1and B2receptors. In this report, we describe oocyte expression experiments showing that the B1receptor in W138 human fibroblast cells is encoded by a distinct mRNA ∼2 kb shorter than that encoding the B2receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B2sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B1‐type pharmacology may arise from two independent molecular mechanisms.