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Effects of divalent cations and nucleotides on the binding of [3H]8-OH-DPAT in human brain
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P Poster Presentations deleted receptor. In view of these results, we propose that the activation of phospholipase C does not determine the rate of receptor internalization, but that a biochemical interaction of the receptor with a G-protein is required for the control of this cellular regulation process.
[2] Kolston J, Osen KK, Hackney eM, Ottersen OP, Storm-Mathisen J, Anal Embryol 186 (1992) 443-465. [31 Oertel 0 , SHWu, J. CompoNeurol. 283 (1 989) 228-247.
I P-20-11 I Effects of Divalent Cations andNucleotides on the
IP-20-9 1Immunohistochemical Localization of Neurotrophin Receptors in the Adult RatVentral Cochlear Nucleus A. Burette, I. Jalengues, R. Romand. Lab. Neurobiology, Blaise Pascal
Univ., Aubiere, France Neurotrophins mediate theireffects through interactions withhigh-affinity tyrosine kinase receptors (Trk). Recent observations [I j of a large distribution of high affinity ,BNGF biding sites and trkA mRNA in adult cochlear nuclei suggest that ,BNGF may playa role in this structure. The present study employed polyclonal antibodies (Santa Cruz Biotechnology) to characterize and map the distribution of the three Trk-irnmunoreactive cells within the adult rat ventral cochlear nucleus (VCN). A large number of Trk immunopositive neurons is observed within all parts of the VCN; howeverthere are specific labelled variations according to cell types. Our experiments lead us to classify Trks-labelled cells in three groups. The first one is a cell population stained by both TrkA and TrkB. This population regroups major part of Golgi cells, small and small round neurons and fusiform cells. The second group corresponds to TrkC labelled cells, i. e. spherical, globular and granule cells. Lastly, the third one regroups various proportions of immunopositive cells with the three Trk antibodies: multipolars, giant, and octopus cells. These observations indicate a possible correlation between Trk expression and cell functional properties. TrkA and TrkB immunoreactive cells generally demonstrate inhibitory properties [2] while cells stained by TrkC display excitatory ones [3]. The abundant and widespread neuronaldistribution of signal-transducing forms of TrkA, TrkB, TrkC predicts that their cognate ligans, ,BNGF, BDNF, NT-4/5 and NT-3, may exert significant effects on a large proportion of neurons within the mature VCN. [I) Gibbs RB, PfaffDW, 1. CompoNeurol, 284 (1994) 187-204. [2] Kolston J, Osen KK, Hackney CM, Otterson OP. Storm-Mathisen J, Anat Ernbryol 186 (1992) 443-465. [3] Rhode WS, in: Neurobiology of Hearing (199 1) pp 41 - 77.
IP-20-10 I Distribution of TRK Receptors in the Adult Dorsal Cochlear Nucleus of the Rat
A. Burette, I. Jalengues, R. Romand. Lab. Neurobiology, Blaise Pascal Univ., Aubiere, France A family of tyrosine receptor kinases known collectively as Trk receptors plays an essential role in signal transduction induced by nerve growth factor and related neurotrophins. Recent observations [I] of a large distribution of high affinity ,BNGF biding site and trkA mRNA in adult cochlear nuclei suggest that NGF may playa role in this structure. The purpose of our immunohistochemical study was to determine and mappe the spacial expression of the three Trk receptors in the adult rat dorsal cochlear nucleus (DCN), as revealed by antibodies (Santa Cruz Biotechnology) against the full-Trk proteins. A large number of Trk immunoreactive neurons is observed within all parts of the DCN; however we observe specific labelling variations according to cell types. Our experiments lead us to classify Trk-Iabelled cells in two groups. The first one is a cell population stained by both TrkA and TrkB. This population regroups cartwheel and tuberculoventral cells as well as longitudinal and horizontal small neurons. The second group corresponds to TrkC immunolabelled cells i, e. fusiform, giant and granule cells. These observations indicate a possible correlation between Trk expression and cell functional properties. TrkA and TrkB immunoreactive cells generally demonstrate inhibitory properties [2] while cells stained by TrkC display excitatoryones [3]. The abundantand widespread neuronal distribution of signal-transducing forms of TrkA, TrkB, TrkC predict that their cognate ligans, ,BNGF, BDNF, NT-4/5 and NT-3, may exert significanteffects on a large proportion of neurons within the mature DCN. [I] Gibbs RB, PfaffDW, 1. Compo Neurol. 284(1 994) 187-204.
Binding of [3H]8-0H.DPAT in Human Brain L. Palego I , A, Rossi 2, A. Batistini I , M. Fancelli I , F. Vergata ), G. Giannaccini 2, D. Marazziti ' . I Institute of Psychiatry; University of Pisa, Pisa, Italy; 2 "lstituto Policattedra Discipline Biologiche", University of Pisa, Pisa. Italy
The effects of divalentcations (Mn2+ and Mg2+) and nucleotides (GTPy S, GMP and AMP) on the binding of the serotonin-IA (5-HTIA) receptor agonist [3Hj8-hydroxy-2(di-N-propylamino)tetralin ([3H]8-0H-DPAT), were evaluated and comparedin the frontal cortex and hippocampus from 5 autopsy subjects (age range: 45-78 years; postmortem delay: 18-38 hours). Membranes were incubated with 2.5 nM [3H]8-0H-DPAT in the presence or absence of I mM divalent cations and 0.1 mM nucletides, according to Hall et al. [I]. The results revealed that divalent cations significantly enhanced the [3H] 8-0H-DPAT binding in the hippocampus and at a lesser extent in the cortex. Conversely. GTPyS inhibited in a similar fashion the [3H]8OH-DPAT binding in the 2 regions. GMP and AMP failed to modulate the r> Hj8-0 H-DPAT binding. These findings suggest that the modulatory parameters of the serotonin- IA receptor binding are detectable in postmortem brain tissues. Moreover, area-dependent differences on r3Hj8-0 H-DPAT binding stimulation by divalent cations should be further investigated in a greater number of autopsy subjects and assessed by functional studies. [1] Hall M.D.. EJ Mestikawy S.. Emerir M.B., Pichat L., Hamon M. andGozlan H., J. Neurochem.. 44(1985) 1685-1696.
IP-20-12I The Binding of some Psychotropic Drugs at the MolecUlarly Cloned Human Histamine H1 Receptor Expressed by CHOCells M. Yamada, S. Martinez, Mi Yamada, E. Richelson. Department of Psychiatry and Pharmacology, Mayo Clinic Jacksonville, Jacksonville, Florida, USA Many drugs, including antidepressants and neuroleptics, may exert some of their therapeutic effects and side effects by blocking the HI receptor in brain. Most notable effects are sedation and weight gain. The affinities of these drugs for the HI-receptor are predictiveof the likelihood of their causing these clinical effects. Recently, a cDNA encoding the human HI-receptor was isolated and sequenced (Moguilevsky et aI., Eur. J. Biochem. 1994;224:489-495). This protein shows characteristic features of G-protein-coupled receptors. We directionally ligated the receptor cDNA into the pRc/CMVexpression vectorand transfectedCHO cells by the calcium phosphate method. The receptor protein expressed on CHO cell membranes specifically bound [3Hlpyrilamine with an Kd of 2 nM. The expression of this receptor in stably transfected CHO cells allowed the pharmacological characterization of antidepressants and neuroleptics without using postmortem human brains. In our preliminary studies, we are testing newer, second generation antidepressants, atypical neuroleptics, and some of their metabolites. All the data were analyzed with the LIGAND program. Hill coefficients for all compounds tested thus far were essentially equal to unity, showing that binding of these drugs obeyed the law of mass action. With the information obtained in these experiments, we can investigate further the structure-activity relationships among psychotropic drugs at the human histamine HI receptor. This work is supported by Mayo Foundation for Medical Education and Research
I P-20-13 ! Receptors of Chromatin for Drug Discovery B.A. Reikhardt, O.G. Kulikova, N.S. Sapronov. Departmentof Neuropharmacology, Institute of Experimental Medicine, St. Petersburg, Russia The number of neurological diseases caused by changes of gene ex-
P Poster Presentations deleted receptor. In view of these results, we propose that the activation of phospholipase C does not determine the rate of receptor internalization, but that a biochemical interaction of the receptor with a G-protein is required for the control of this cellular regulation process.
[2] Kolston J, Osen KK, Hackney eM, Ottersen OP, Storm-Mathisen J, Anal Embryol 186 (1992) 443-465. [31 Oertel 0 , SHWu, J. CompoNeurol. 283 (1 989) 228-247.
I P-20-11 I Effects of Divalent Cations andNucleotides on the
IP-20-9 1Immunohistochemical Localization of Neurotrophin Receptors in the Adult RatVentral Cochlear Nucleus A. Burette, I. Jalengues, R. Romand. Lab. Neurobiology, Blaise Pascal
Univ., Aubiere, France Neurotrophins mediate theireffects through interactions withhigh-affinity tyrosine kinase receptors (Trk). Recent observations [I j of a large distribution of high affinity ,BNGF biding sites and trkA mRNA in adult cochlear nuclei suggest that ,BNGF may playa role in this structure. The present study employed polyclonal antibodies (Santa Cruz Biotechnology) to characterize and map the distribution of the three Trk-irnmunoreactive cells within the adult rat ventral cochlear nucleus (VCN). A large number of Trk immunopositive neurons is observed within all parts of the VCN; howeverthere are specific labelled variations according to cell types. Our experiments lead us to classify Trks-labelled cells in three groups. The first one is a cell population stained by both TrkA and TrkB. This population regroups major part of Golgi cells, small and small round neurons and fusiform cells. The second group corresponds to TrkC labelled cells, i. e. spherical, globular and granule cells. Lastly, the third one regroups various proportions of immunopositive cells with the three Trk antibodies: multipolars, giant, and octopus cells. These observations indicate a possible correlation between Trk expression and cell functional properties. TrkA and TrkB immunoreactive cells generally demonstrate inhibitory properties [2] while cells stained by TrkC display excitatory ones [3]. The abundant and widespread neuronaldistribution of signal-transducing forms of TrkA, TrkB, TrkC predicts that their cognate ligans, ,BNGF, BDNF, NT-4/5 and NT-3, may exert significant effects on a large proportion of neurons within the mature VCN. [I) Gibbs RB, PfaffDW, 1. CompoNeurol, 284 (1994) 187-204. [2] Kolston J, Osen KK, Hackney CM, Otterson OP. Storm-Mathisen J, Anat Ernbryol 186 (1992) 443-465. [3] Rhode WS, in: Neurobiology of Hearing (199 1) pp 41 - 77.
IP-20-10 I Distribution of TRK Receptors in the Adult Dorsal Cochlear Nucleus of the Rat
A. Burette, I. Jalengues, R. Romand. Lab. Neurobiology, Blaise Pascal Univ., Aubiere, France A family of tyrosine receptor kinases known collectively as Trk receptors plays an essential role in signal transduction induced by nerve growth factor and related neurotrophins. Recent observations [I] of a large distribution of high affinity ,BNGF biding site and trkA mRNA in adult cochlear nuclei suggest that NGF may playa role in this structure. The purpose of our immunohistochemical study was to determine and mappe the spacial expression of the three Trk receptors in the adult rat dorsal cochlear nucleus (DCN), as revealed by antibodies (Santa Cruz Biotechnology) against the full-Trk proteins. A large number of Trk immunoreactive neurons is observed within all parts of the DCN; however we observe specific labelling variations according to cell types. Our experiments lead us to classify Trk-Iabelled cells in two groups. The first one is a cell population stained by both TrkA and TrkB. This population regroups cartwheel and tuberculoventral cells as well as longitudinal and horizontal small neurons. The second group corresponds to TrkC immunolabelled cells i, e. fusiform, giant and granule cells. These observations indicate a possible correlation between Trk expression and cell functional properties. TrkA and TrkB immunoreactive cells generally demonstrate inhibitory properties [2] while cells stained by TrkC display excitatoryones [3]. The abundantand widespread neuronal distribution of signal-transducing forms of TrkA, TrkB, TrkC predict that their cognate ligans, ,BNGF, BDNF, NT-4/5 and NT-3, may exert significanteffects on a large proportion of neurons within the mature DCN. [I] Gibbs RB, PfaffDW, 1. Compo Neurol. 284(1 994) 187-204.
Binding of [3H]8-0H.DPAT in Human Brain L. Palego I , A, Rossi 2, A. Batistini I , M. Fancelli I , F. Vergata ), G. Giannaccini 2, D. Marazziti ' . I Institute of Psychiatry; University of Pisa, Pisa, Italy; 2 "lstituto Policattedra Discipline Biologiche", University of Pisa, Pisa. Italy
The effects of divalentcations (Mn2+ and Mg2+) and nucleotides (GTPy S, GMP and AMP) on the binding of the serotonin-IA (5-HTIA) receptor agonist [3Hj8-hydroxy-2(di-N-propylamino)tetralin ([3H]8-0H-DPAT), were evaluated and comparedin the frontal cortex and hippocampus from 5 autopsy subjects (age range: 45-78 years; postmortem delay: 18-38 hours). Membranes were incubated with 2.5 nM [3H]8-0H-DPAT in the presence or absence of I mM divalent cations and 0.1 mM nucletides, according to Hall et al. [I]. The results revealed that divalent cations significantly enhanced the [3H] 8-0H-DPAT binding in the hippocampus and at a lesser extent in the cortex. Conversely. GTPyS inhibited in a similar fashion the [3H]8OH-DPAT binding in the 2 regions. GMP and AMP failed to modulate the r> Hj8-0 H-DPAT binding. These findings suggest that the modulatory parameters of the serotonin- IA receptor binding are detectable in postmortem brain tissues. Moreover, area-dependent differences on r3Hj8-0 H-DPAT binding stimulation by divalent cations should be further investigated in a greater number of autopsy subjects and assessed by functional studies. [1] Hall M.D.. EJ Mestikawy S.. Emerir M.B., Pichat L., Hamon M. andGozlan H., J. Neurochem.. 44(1985) 1685-1696.
IP-20-12I The Binding of some Psychotropic Drugs at the MolecUlarly Cloned Human Histamine H1 Receptor Expressed by CHOCells M. Yamada, S. Martinez, Mi Yamada, E. Richelson. Department of Psychiatry and Pharmacology, Mayo Clinic Jacksonville, Jacksonville, Florida, USA Many drugs, including antidepressants and neuroleptics, may exert some of their therapeutic effects and side effects by blocking the HI receptor in brain. Most notable effects are sedation and weight gain. The affinities of these drugs for the HI-receptor are predictiveof the likelihood of their causing these clinical effects. Recently, a cDNA encoding the human HI-receptor was isolated and sequenced (Moguilevsky et aI., Eur. J. Biochem. 1994;224:489-495). This protein shows characteristic features of G-protein-coupled receptors. We directionally ligated the receptor cDNA into the pRc/CMVexpression vectorand transfectedCHO cells by the calcium phosphate method. The receptor protein expressed on CHO cell membranes specifically bound [3Hlpyrilamine with an Kd of 2 nM. The expression of this receptor in stably transfected CHO cells allowed the pharmacological characterization of antidepressants and neuroleptics without using postmortem human brains. In our preliminary studies, we are testing newer, second generation antidepressants, atypical neuroleptics, and some of their metabolites. All the data were analyzed with the LIGAND program. Hill coefficients for all compounds tested thus far were essentially equal to unity, showing that binding of these drugs obeyed the law of mass action. With the information obtained in these experiments, we can investigate further the structure-activity relationships among psychotropic drugs at the human histamine HI receptor. This work is supported by Mayo Foundation for Medical Education and Research
I P-20-13 ! Receptors of Chromatin for Drug Discovery B.A. Reikhardt, O.G. Kulikova, N.S. Sapronov. Departmentof Neuropharmacology, Institute of Experimental Medicine, St. Petersburg, Russia The number of neurological diseases caused by changes of gene ex-