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Mechanism of anticomvulsant action of valproate (dipropylacetate)
ABSTRACTS
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SPECIFIC CEREBROVASCULAR LOCALIZATION OF GABA-RELATED RECEPTORS AND ENZYMES. KPBUSB. Diana N. Oivision of Neurosciences, City of Hope Naiional Medical Center, Ouarte, CA 91010
MODULATION OF THE G4BA SHUNT BASED ON THE KNOWLEDGE OF THE ENZYMES INVOLVEO. P. Mandel. M. Maitre, L. Ciesielski, J.M. Blindermann. S.Simler, C.Cash, G. Mack and E. Kempf. Centre de Neurochimie du CNRS, 11 rue Humann. 67085 Strasbourg cedex - France
A role for GABA in cerebra1 vascular function is supported by data indicating that specific GABA receptors and GABA-related enzymes we associated with cerebra1 blood vessels. [%]Huscimol, which labels GABA receptor sites in brain. was found to bind to a crude membrane fraction prepared from pial blood vessels. Specific [3H]nuscimo1 binding was saturable, of high affinity (Kg = 41 nH), and was xlectively inhibited by known GABA agonists. The relative potencies for Ihe agonists in competing for the GABA binding site correlated well with the effectiveness of these compounds in dilating pial arteries in vitro (see abstract by I. Edvinsson). No specific [3H]muscimol binding WIS detected in aorta and mesenteric arteries. An association of GABA with cerebral blood vessels 1s further indicated by the presence of a non-neuronal form of the enzyme which synthesizes GABA, glutamate decarboxylase (GAD), in relatively high concentrations in pial arteries as compared to peripheral vessels. GABA transaminase (GABA-r), the degraddtive enzyme for GABA. also has been demonstrated histochemically in large concentrations around brain vessels. but not around other, e.g. kidney. vessels. Biochemical and pharmacologic studies are in progress to determine whether or not the GABA systen in cerebra1 blood vessels plays 1 role in the indigenous control of vascular tone.
A224 Srms OF GRADIENTS
Current data suggest that like in other basic biological phenomena (e.g., transcription of the genetic code; regulation of enzyme biosynthesis and activities) control mechanisms in the CNS operate fundamentally though inhibition and disinhibition mxhanisms. u-Aminobutyric acid (GABA) appears to be a major inhibitory neurotransmitter in mammalian CNS and seems to be involved in several physiological events, in molecular rrechanismsof behavior, and in certain neurological diseases. Thus, extensive knovledge of the enzymes involved in the GABA shunt and of the mchanisms of action of substances that can modulate the GABA-ergic system may be of great help in neurology, neuropathology and therapeutics. We have purified GAD (rat and human). GABA-T (mouse, rat, beef, human). succinic semialdehyde dehydrogenase (rat and human) and SSA reductase (human). Knwledge concerning the kinetics of these enzymes and.the effects of branched-chain fattv acids has orovided oossibilities for adulating the GA&ergic sys&m. Some effects of branched-chain fatty acids on seizures, learning, and aggressive behavior will be discussed.
A227 ORIGIN “-NQ RoSTRocAUDAL OF CERBpRosPINAL FLUID
CONCENTRATION GAB* IN MAN.
James H. Wocd, Theodore A. Hare, S.J. Enna and N.V. Bala Man ain, Univ. of Penn., Phila., PA, Thomas Jefferson Univ., Phila., PA, Univ. of TexaD, Houston, TX, Vet. Admln. Med. 6 Reg. Office Cent., Wilmington, tE Central contamination of cerebrospinal fluid (CSF) with GABA from peripheral sources does not occur with intact blood-brain-CSF barriers. Although GASA in CSF appears to have a central origin, further refinement of its contributing sources in lumbar CSF is crucial for clinical interpretations. Sequential lumbar CSF aliquots CSF were obtained from naurologic patients. samples assayed by the radioreceptor method had 202t23, 243f37 and 298535 pmol/ml (SE) in the 8th. Simi24th and 40th ml fractions, respectively. larly, the GASA content in samples assayed by the ion-exchange/fluorotric method had a mean 2% increase per additional ml collected after the initial aliquot. Since low lumbar CSF C.AS* levels have been demonstrated in neuroloqic disorders associated with deficiencies in brain GABA, lumbar CSF GRBA appears to reflect brain GABA content. However, bulbar/spinal deqenerative discrders may also lower lumbar CSF GABA content.
RELATIONSHIP CELLS. F. A.
OF GABA AND DIAZEPAM
RECEPTORS IN
CEREBELLAR
PURKINJE
Hem and S. U. Han. Neurochemical Research Laboratories, Department of Psychiatry, University of Iowa, Iowa City, IA 522112. Receptor studies ape routinely carried out on crude hetercgeneous membrane fractions. have atfempted to prepare a fraction containing a single cell type and analyze the pmperties of various receptors on this preparation. The preparation we developed consists of isolated Pwkinje cell bodies prepared in ,e,reater than 90% purity. Using this preparation ELBA receptors and diazepam receptors were measured and their interaction was studied. An analysis of GAB* receptor binding using either (%) W\B* OD c3H) muscimol reveals two binding sites with Kd's of 1.U and 26.7 nM respectively. These two sites ape clearly present on Purkinje cells and do not represent two narphologically distinct receptors, such as a presynaptic and postsynaptic receptor. Diazepam binding studies carried OUT on These cells reveal specific binding with a Kd of 2.6 nM. The specific density of GABA receprors is 0.6 pmoles/mg for the higher affinity site and 1.0 poal/mg for the lower, while the diazepam sites have 0.55 pnaleslmg maximwn binding. This suggests that the recepTor densities of these compounds are nearly equivalent, with the combined GAB* sites being about 3 times mare prevalent than diazepam sites. The influence of GABA on diazepam binding and diazepam on GAB* binding will be presenred as will The effect of these compounds on Cl- permeability.
He
A228 A225 MECHANISM OF ANTICDN”“LSANT ACTION OF “ALPROATE (DIPROPYLACETATE). P. Maodd, M. Maitre, L. Ciesielski and S. Simler. Centre de
GABAPEGIC pROPEYRTX3 OF B-(p-CHLORPHISIYL) GASA AND SIXZOODLUEPINES INNYNO John L. Waddington. Division of Psychxatry, NRC Clinical Research Centw, W&ford Road, Harrow HA1 YJ, U.K.
Neurvchimie du C.N.R.S.. 11 rue Hunann, 67085 Strasbourg, France. After purification of GAB* transaminase (GAB&T), glutamate decarbovylase (GAD) and succinic semialdehyde dehydragenase from several sowces, we showed that Valpmate (VA; dipropylacefate) a sfructural analogue of GAB* is a competitive inhibitor of GAB&T and a non- or uncompetitive inhibitor of SSADH. VA does not affect GAD. If produces an increase of GAB* in whole brain which is particularly pronounced in some brain areas
We have proposed that the induction of contralateral rotational behwiour by unilateral Injections of GABAergic drugs axb a6 muscimol into the reticulata region of the ratlsubstantia nigra may constitute a GUM.-dependant rote,tionalbehaviour model. Baclofen (P-p-chlorphenyl GABA) is active in this model, in a manner weakly sensitiveto antagonism by picrotoxin. tbougb log dose-response curves for racemic baclofen end muscimol failed to shw parallelism and high doses of baclofen did not produce the same maximal respon.w as muscimol. Baclofen may therefore have partial but notlpure GABA agonist activity. a and &-baclofen uere equiactive in this model. This ie consistent ith the e&potency of the isomers in weakly displacing r,-GABA bound TV frozen/thawed but not tritontreated membrane preparations -in vitro (Waddington & Cross, Neurosci.Lett., in press) and further indi&tes the we& OABplmimetic action of baclofen at at leasteone class of, possibly low affinity, GABA receptqr sit*e. Further data with closely related structural analogues of baclofen will be presented. as will data with e series of benzodiazcpins compounds following our preliminary demonstration that fluramepam is active in this behavioural model in e manner consistent with GAEAergic activity.
705
A223
A226
SPECIFIC CEREBROVASCULAR LOCALIZATION OF GABA-RELATED RECEPTORS AND ENZYMES. KPBUSB. Diana N. Oivision of Neurosciences, City of Hope Naiional Medical Center, Ouarte, CA 91010
MODULATION OF THE G4BA SHUNT BASED ON THE KNOWLEDGE OF THE ENZYMES INVOLVEO. P. Mandel. M. Maitre, L. Ciesielski, J.M. Blindermann. S.Simler, C.Cash, G. Mack and E. Kempf. Centre de Neurochimie du CNRS, 11 rue Humann. 67085 Strasbourg cedex - France
A role for GABA in cerebra1 vascular function is supported by data indicating that specific GABA receptors and GABA-related enzymes we associated with cerebra1 blood vessels. [%]Huscimol, which labels GABA receptor sites in brain. was found to bind to a crude membrane fraction prepared from pial blood vessels. Specific [3H]nuscimo1 binding was saturable, of high affinity (Kg = 41 nH), and was xlectively inhibited by known GABA agonists. The relative potencies for Ihe agonists in competing for the GABA binding site correlated well with the effectiveness of these compounds in dilating pial arteries in vitro (see abstract by I. Edvinsson). No specific [3H]muscimol binding WIS detected in aorta and mesenteric arteries. An association of GABA with cerebral blood vessels 1s further indicated by the presence of a non-neuronal form of the enzyme which synthesizes GABA, glutamate decarboxylase (GAD), in relatively high concentrations in pial arteries as compared to peripheral vessels. GABA transaminase (GABA-r), the degraddtive enzyme for GABA. also has been demonstrated histochemically in large concentrations around brain vessels. but not around other, e.g. kidney. vessels. Biochemical and pharmacologic studies are in progress to determine whether or not the GABA systen in cerebra1 blood vessels plays 1 role in the indigenous control of vascular tone.
A224 Srms OF GRADIENTS
Current data suggest that like in other basic biological phenomena (e.g., transcription of the genetic code; regulation of enzyme biosynthesis and activities) control mechanisms in the CNS operate fundamentally though inhibition and disinhibition mxhanisms. u-Aminobutyric acid (GABA) appears to be a major inhibitory neurotransmitter in mammalian CNS and seems to be involved in several physiological events, in molecular rrechanismsof behavior, and in certain neurological diseases. Thus, extensive knovledge of the enzymes involved in the GABA shunt and of the mchanisms of action of substances that can modulate the GABA-ergic system may be of great help in neurology, neuropathology and therapeutics. We have purified GAD (rat and human). GABA-T (mouse, rat, beef, human). succinic semialdehyde dehydrogenase (rat and human) and SSA reductase (human). Knwledge concerning the kinetics of these enzymes and.the effects of branched-chain fattv acids has orovided oossibilities for adulating the GA&ergic sys&m. Some effects of branched-chain fatty acids on seizures, learning, and aggressive behavior will be discussed.
A227 ORIGIN “-NQ RoSTRocAUDAL OF CERBpRosPINAL FLUID
CONCENTRATION GAB* IN MAN.
James H. Wocd, Theodore A. Hare, S.J. Enna and N.V. Bala Man ain, Univ. of Penn., Phila., PA, Thomas Jefferson Univ., Phila., PA, Univ. of TexaD, Houston, TX, Vet. Admln. Med. 6 Reg. Office Cent., Wilmington, tE Central contamination of cerebrospinal fluid (CSF) with GABA from peripheral sources does not occur with intact blood-brain-CSF barriers. Although GASA in CSF appears to have a central origin, further refinement of its contributing sources in lumbar CSF is crucial for clinical interpretations. Sequential lumbar CSF aliquots CSF were obtained from naurologic patients. samples assayed by the radioreceptor method had 202t23, 243f37 and 298535 pmol/ml (SE) in the 8th. Simi24th and 40th ml fractions, respectively. larly, the GASA content in samples assayed by the ion-exchange/fluorotric method had a mean 2% increase per additional ml collected after the initial aliquot. Since low lumbar CSF C.AS* levels have been demonstrated in neuroloqic disorders associated with deficiencies in brain GABA, lumbar CSF GRBA appears to reflect brain GABA content. However, bulbar/spinal deqenerative discrders may also lower lumbar CSF GABA content.
RELATIONSHIP CELLS. F. A.
OF GABA AND DIAZEPAM
RECEPTORS IN
CEREBELLAR
PURKINJE
Hem and S. U. Han. Neurochemical Research Laboratories, Department of Psychiatry, University of Iowa, Iowa City, IA 522112. Receptor studies ape routinely carried out on crude hetercgeneous membrane fractions. have atfempted to prepare a fraction containing a single cell type and analyze the pmperties of various receptors on this preparation. The preparation we developed consists of isolated Pwkinje cell bodies prepared in ,e,reater than 90% purity. Using this preparation ELBA receptors and diazepam receptors were measured and their interaction was studied. An analysis of GAB* receptor binding using either (%) W\B* OD c3H) muscimol reveals two binding sites with Kd's of 1.U and 26.7 nM respectively. These two sites ape clearly present on Purkinje cells and do not represent two narphologically distinct receptors, such as a presynaptic and postsynaptic receptor. Diazepam binding studies carried OUT on These cells reveal specific binding with a Kd of 2.6 nM. The specific density of GABA receprors is 0.6 pmoles/mg for the higher affinity site and 1.0 poal/mg for the lower, while the diazepam sites have 0.55 pnaleslmg maximwn binding. This suggests that the recepTor densities of these compounds are nearly equivalent, with the combined GAB* sites being about 3 times mare prevalent than diazepam sites. The influence of GABA on diazepam binding and diazepam on GAB* binding will be presenred as will The effect of these compounds on Cl- permeability.
He
A228 A225 MECHANISM OF ANTICDN”“LSANT ACTION OF “ALPROATE (DIPROPYLACETATE). P. Maodd, M. Maitre, L. Ciesielski and S. Simler. Centre de
GABAPEGIC pROPEYRTX3 OF B-(p-CHLORPHISIYL) GASA AND SIXZOODLUEPINES INNYNO John L. Waddington. Division of Psychxatry, NRC Clinical Research Centw, W&ford Road, Harrow HA1 YJ, U.K.
Neurvchimie du C.N.R.S.. 11 rue Hunann, 67085 Strasbourg, France. After purification of GAB* transaminase (GAB&T), glutamate decarbovylase (GAD) and succinic semialdehyde dehydragenase from several sowces, we showed that Valpmate (VA; dipropylacefate) a sfructural analogue of GAB* is a competitive inhibitor of GAB&T and a non- or uncompetitive inhibitor of SSADH. VA does not affect GAD. If produces an increase of GAB* in whole brain which is particularly pronounced in some brain areas
We have proposed that the induction of contralateral rotational behwiour by unilateral Injections of GABAergic drugs axb a6 muscimol into the reticulata region of the ratlsubstantia nigra may constitute a GUM.-dependant rote,tionalbehaviour model. Baclofen (P-p-chlorphenyl GABA) is active in this model, in a manner weakly sensitiveto antagonism by picrotoxin. tbougb log dose-response curves for racemic baclofen end muscimol failed to shw parallelism and high doses of baclofen did not produce the same maximal respon.w as muscimol. Baclofen may therefore have partial but notlpure GABA agonist activity. a and &-baclofen uere equiactive in this model. This ie consistent ith the e&potency of the isomers in weakly displacing r,-GABA bound TV frozen/thawed but not tritontreated membrane preparations -in vitro (Waddington & Cross, Neurosci.Lett., in press) and further indi&tes the we& OABplmimetic action of baclofen at at leasteone class of, possibly low affinity, GABA receptqr sit*e. Further data with closely related structural analogues of baclofen will be presented. as will data with e series of benzodiazcpins compounds following our preliminary demonstration that fluramepam is active in this behavioural model in e manner consistent with GAEAergic activity.