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The actions of barbiturates on release of noradrenaline from rat hippocampal synaptosomes
Neuropharmacology Vol. 23, No 9, pp. I 113-l 116. 1984 Printedin Great Britain
0028-3908184 $3.00+ 0.00 PergamonPress Ltd
THE ACTIONS OF BARBITURATES ON HIPWCAMPALSYNAPTOSOMES
RELEASE OF
NORADRENALINE FROM RAT
SEK-CHUNGFUNG* and MARIANNEFILLENZ
UniversityI.&oratoryof Fhysiolqy, Parks F&ad,OxfordOX1 3PT, tigland.
(Accepkd
15 Augubt
1984)
Summary: Barbiturates are believed to work both by augmenting GABA action and independently perhaps by decreasing neurotransmitter release. By studying tQe effect of pentobarbitates on the release of H noradrenalinefrom rat hippo ampal syn ptosomes it was found that pantobarbitone $lOs M and 10-!M) augmented-$? GABA evoked release of H noradrenaline but 10 M depressedthis GABA effect. This latterconcentrationf pentobarbitone also depressedthe I<'-evoked releaseof % noradrenaline by a Ca++ dependent but picrotoxin insensitive mechanism. Depolarisation of synapto2Tmes increases voltage-dependentCa2+ conductance: triggers release of transmitter. the entry of Ca Experimentally such depolarisation is usually produced either by raising extracellular [Kfl or by adding the drug veratridine, which increases Na conductance. However, GABA, by opening Cl- chanels,has also been shown to cause membrane depolarisaticn in primary aff rent neurones (Ga lagher, 1978). We have shown that Ca*-dependentreleaseof Q d-noradrenaline (3H-NA)from rat hippocampalsynaptosonescan be triggeredby GABA. This effect is antagonised by picrotoxin(Fungand Fillenz,1983). Barbituratespotentiatethe action oE GABA by an action at a regulatory site which is part of the G%BA-receptor multimolecularcomplex (Olsen,1982): this effect-of barbiturates is antagonised by picrotoxin. However, pentobarbital has also been shown to depress the release by electrical stimulationof endqenous amino acids (Collins1980). In the presentstudy we have examined the effect of pentobarbital on the releaseof 3H-NA from rat hippccsnpalsynaptcsones. Materials_and Methods Hippocampi from male Sprague-Dawley rats were homogenised in 0.32 Y sucrose and a crude synaptosomal pellet was isolated by two successive centrifugationsat 1000 x g 10 min and 15000 x g for 20 min. The P pellet was resuspendedin oxygenatedHEPES-bufferedincubationmedium pH 7.i of the following composition in NM: NaCl144, KC15, HEPES 20, NaH2P04 1.2,CaC121, MgS041,glucose 10, pargyline 0.2and ascorbate 0.1. The synaptosomes w re s H) preincubated at 37O for 20 min andthenpre-labelled with100 nM l-(7-8noradrenaline (Amersham,Englandspecificactivity36 Ci/mmole)for a further 15 min. They were then loadedonto milli,por? filtersin a supecfusionsystem with a Elow rate of O.Sml/min. Release of H-NA was measured as described previously(Fung and Filenz, 1983).
* Present
address:
Cullen Eye Institute, Houston, Texas 77030,
Baylor USA.
1113
College
of
Medicine,
1114
Preliminary
Notes
Results The addition of 9 4 min pulse of 10-4M GABA to the superfusionmedium caused a release of H-NA; the release was expressed as a % of the total tissue radioactivityand in the absence of drugs was 1.12+0.06 (n=8). The effectof adding pentobaritalto the superfusionm ium varl'eawith the dose and is shown in Fig. 1.: the GABA-evokedreleaseofe!I H-NA was enhancedby 44%
*
*
n:4
1-4
-
-
1 n=8
lO@l GABA 10-7
M
lo-6M
lo-5M
lo-4M
PB
F'gure 1. EffectoE pentobarbital (PB)on GABA-evoked release of LAINA from rat hiboocamwl svnantosomes.A 6-min pulse of 10V5M. GABA was added to the super*fusion fluid. The effect of pentobarbital was studiedby addingdifferentconcentations of this drug 2 min before and during the GABApulse. Mean2S.E.M. * PC 0.002,** p C 0.05,compared to control.n = number of separate determinations. and 41.7% by low6 and 10-5M pentobarbital respectively;this reversed t a deo ession of 33. 9% at 10e4M pent arbital. Pentobarbital alone (10-8 _ --;F 10 M) had no effect on the efflux of -NA. Next we looked at the effect on K+-evoked release of 3H-NA. Table 1 shows that pentobarbital produced a depression of release; this was dosedependentand was independent of GABA since it occurredin the absenceof GABA and was not blockedby picrotoxinin.The size of tp+depression,hoy$ver,"$s sensitiveto the extracellular concentration of Ca : at 1 mM [Ca lo 10 M pentobarbitonedepressed K+-evoked release by 19.7+ 4.6% (n = 31, whereas with O.lmM [Ca2+lothe release was depressedby 42.224.2% (n=3).
The present results confirm that barbiturates have at least two distinctactionson excitablemembranes. Willow and John&on (1983)in their exhaustive review conclude that the main actions of barbiturates are a depression of transmitter release and a potentiationof GABA action by an is.G2E+stein and Ftor (1974)showed increasein the affinityof CXBA recepto into depolarlsedsynaptosmnes. that barbituratesinhibitthe entry of
Prel imi nary Notes
7115
1116
Preliminary
Notes
Haycock,J.W.,Javy, W.B.and Cotman, C.W.,1977,Pentobarbitaldepressionof stimulus-secretion coupling in brain-selective inhibitionof depolarisationinducedcalcium-dependent release,Biochem.Phannacol.26, 159-161. MacDonald,R.L.and Barker,J.L.,1978, Different actions of anticonvulsant and anaestheticbarbituratesrevealedby use of culturedmammalian neurons, Science 200, 775-777. Yathers,ISA.and Barker,J.L.,1980, (-1pentobarbital opens ion channelsof long durationin culturedmouse spinal neurons,Science209, 507-509. Olsen, RW., 1982, Drug interactionat @BA receptor-ionophore complex,Ann. Rev. Pharmacol.Toxicol.22, 245-277. Olsen, R.W.,Snowman, A. and Leeb-Lundberg,F., 1981, Barbituratesenhance GABAreceptor binding inmammalian brain membranes in vitro, Fed. Proc. 40, 309. Willow, M. and Johnston, G.A.R.,1981, Enhancement by anaesthetic and convulsantbarbiturates of GABA birxlirqto rat brain synaptoscmalmembranes, J. Neurosci.1, 364-367. Willow, M. and Johnston, G.A.R, 1983, Pharmacology of Barbiturates: Electrophysiolcqical and ~~eurochemical Studies,Int. Rev. Neurobiol.24, 1550.
0028-3908184 $3.00+ 0.00 PergamonPress Ltd
THE ACTIONS OF BARBITURATES ON HIPWCAMPALSYNAPTOSOMES
RELEASE OF
NORADRENALINE FROM RAT
SEK-CHUNGFUNG* and MARIANNEFILLENZ
UniversityI.&oratoryof Fhysiolqy, Parks F&ad,OxfordOX1 3PT, tigland.
(Accepkd
15 Augubt
1984)
Summary: Barbiturates are believed to work both by augmenting GABA action and independently perhaps by decreasing neurotransmitter release. By studying tQe effect of pentobarbitates on the release of H noradrenalinefrom rat hippo ampal syn ptosomes it was found that pantobarbitone $lOs M and 10-!M) augmented-$? GABA evoked release of H noradrenaline but 10 M depressedthis GABA effect. This latterconcentrationf pentobarbitone also depressedthe I<'-evoked releaseof % noradrenaline by a Ca++ dependent but picrotoxin insensitive mechanism. Depolarisation of synapto2Tmes increases voltage-dependentCa2+ conductance: triggers release of transmitter. the entry of Ca Experimentally such depolarisation is usually produced either by raising extracellular [Kfl or by adding the drug veratridine, which increases Na conductance. However, GABA, by opening Cl- chanels,has also been shown to cause membrane depolarisaticn in primary aff rent neurones (Ga lagher, 1978). We have shown that Ca*-dependentreleaseof Q d-noradrenaline (3H-NA)from rat hippocampalsynaptosonescan be triggeredby GABA. This effect is antagonised by picrotoxin(Fungand Fillenz,1983). Barbituratespotentiatethe action oE GABA by an action at a regulatory site which is part of the G%BA-receptor multimolecularcomplex (Olsen,1982): this effect-of barbiturates is antagonised by picrotoxin. However, pentobarbital has also been shown to depress the release by electrical stimulationof endqenous amino acids (Collins1980). In the presentstudy we have examined the effect of pentobarbital on the releaseof 3H-NA from rat hippccsnpalsynaptcsones. Materials_and Methods Hippocampi from male Sprague-Dawley rats were homogenised in 0.32 Y sucrose and a crude synaptosomal pellet was isolated by two successive centrifugationsat 1000 x g 10 min and 15000 x g for 20 min. The P pellet was resuspendedin oxygenatedHEPES-bufferedincubationmedium pH 7.i of the following composition in NM: NaCl144, KC15, HEPES 20, NaH2P04 1.2,CaC121, MgS041,glucose 10, pargyline 0.2and ascorbate 0.1. The synaptosomes w re s H) preincubated at 37O for 20 min andthenpre-labelled with100 nM l-(7-8noradrenaline (Amersham,Englandspecificactivity36 Ci/mmole)for a further 15 min. They were then loadedonto milli,por? filtersin a supecfusionsystem with a Elow rate of O.Sml/min. Release of H-NA was measured as described previously(Fung and Filenz, 1983).
* Present
address:
Cullen Eye Institute, Houston, Texas 77030,
Baylor USA.
1113
College
of
Medicine,
1114
Preliminary
Notes
Results The addition of 9 4 min pulse of 10-4M GABA to the superfusionmedium caused a release of H-NA; the release was expressed as a % of the total tissue radioactivityand in the absence of drugs was 1.12+0.06 (n=8). The effectof adding pentobaritalto the superfusionm ium varl'eawith the dose and is shown in Fig. 1.: the GABA-evokedreleaseofe!I H-NA was enhancedby 44%
*
*
n:4
1-4
-
-
1 n=8
lO@l GABA 10-7
M
lo-6M
lo-5M
lo-4M
PB
F'gure 1. EffectoE pentobarbital (PB)on GABA-evoked release of LAINA from rat hiboocamwl svnantosomes.A 6-min pulse of 10V5M. GABA was added to the super*fusion fluid. The effect of pentobarbital was studiedby addingdifferentconcentations of this drug 2 min before and during the GABApulse. Mean2S.E.M. * PC 0.002,** p C 0.05,compared to control.n = number of separate determinations. and 41.7% by low6 and 10-5M pentobarbital respectively;this reversed t a deo ession of 33. 9% at 10e4M pent arbital. Pentobarbital alone (10-8 _ --;F 10 M) had no effect on the efflux of -NA. Next we looked at the effect on K+-evoked release of 3H-NA. Table 1 shows that pentobarbital produced a depression of release; this was dosedependentand was independent of GABA since it occurredin the absenceof GABA and was not blockedby picrotoxinin.The size of tp+depression,hoy$ver,"$s sensitiveto the extracellular concentration of Ca : at 1 mM [Ca lo 10 M pentobarbitonedepressed K+-evoked release by 19.7+ 4.6% (n = 31, whereas with O.lmM [Ca2+lothe release was depressedby 42.224.2% (n=3).
The present results confirm that barbiturates have at least two distinctactionson excitablemembranes. Willow and John&on (1983)in their exhaustive review conclude that the main actions of barbiturates are a depression of transmitter release and a potentiationof GABA action by an is.G2E+stein and Ftor (1974)showed increasein the affinityof CXBA recepto into depolarlsedsynaptosmnes. that barbituratesinhibitthe entry of
Prel imi nary Notes
7115
1116
Preliminary
Notes
Haycock,J.W.,Javy, W.B.and Cotman, C.W.,1977,Pentobarbitaldepressionof stimulus-secretion coupling in brain-selective inhibitionof depolarisationinducedcalcium-dependent release,Biochem.Phannacol.26, 159-161. MacDonald,R.L.and Barker,J.L.,1978, Different actions of anticonvulsant and anaestheticbarbituratesrevealedby use of culturedmammalian neurons, Science 200, 775-777. Yathers,ISA.and Barker,J.L.,1980, (-1pentobarbital opens ion channelsof long durationin culturedmouse spinal neurons,Science209, 507-509. Olsen, RW., 1982, Drug interactionat @BA receptor-ionophore complex,Ann. Rev. Pharmacol.Toxicol.22, 245-277. Olsen, R.W.,Snowman, A. and Leeb-Lundberg,F., 1981, Barbituratesenhance GABAreceptor binding inmammalian brain membranes in vitro, Fed. Proc. 40, 309. Willow, M. and Johnston, G.A.R.,1981, Enhancement by anaesthetic and convulsantbarbiturates of GABA birxlirqto rat brain synaptoscmalmembranes, J. Neurosci.1, 364-367. Willow, M. and Johnston, G.A.R, 1983, Pharmacology of Barbiturates: Electrophysiolcqical and ~~eurochemical Studies,Int. Rev. Neurobiol.24, 1550.