Triethyllead decreases central benzodiazepine receptor binding in rat cerebellum ex vivo

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European Journal of Pharmacology Enwronmental Toxicology and Pharmacology Section 293 (1995) 167-17]

environmental toxicology and pharmacology

Triethyllead decreases central benzodiazepine receptor binding in rat cerebellum ex vivo Hannu Komulainen a,b,* Arto Ker~inen c, Veijo Saano " a Department of Pharmacology and Toxtcology, Untverstty of Kuopto, P 0 B 1627, 70211 Kuopto, Finland b Nattonal Pubhc Health Instttute, Dwtston of Envtronmental Health, P 0 B 95, 70701 Kuopto, Finland c Department of Btochemzstry and Btotechnology, Umverszty ofKuopto, Kuopto, Fmland Recewed 15 August 1994, rewsed 2 January 1995, accepted 31 January 1995

Abstract

Effect of trlethyllead on the specific [3H]flunltrazepam binding was studied in rat cortical and cerebellar P2 fractions in vitro and In tissue homogenates of several rat brain regions ex VlVOafter 5 daily subcutaneous doses of 1 9 mg/kg trlethyllead acetate to rats Up to concentration of 100 /zM, trlethyllead did not affect significantly the specific [3H]flunltrazepam binding but attenuated marginally (14-18%) the G A B A A r e c e p t o r agonlst, musclmol-lnduced elevation of [3H]flunltrazepam binding in cerebellar tissue After the subacute treatment of rats with trlethyllead, the specific [3H]flunitrazepam binding was 27% lower in cerebellum compared to control animals In other brain regions the receptor binding was not changed. The data suggest that triethyllead modified the cerebellar G A B A A receptor complex causing decreased binding in the benzodiazeplne site Such an inhibitory effect in the GABA A receptor complex may decrease cerebellar inhibitory output and augment the trlethyllead induced convulsions and tremor

Keywords Trlethyllead, Benzodlazeplne receptor, GABA A receptor, Flunltrazepam, Muscimol, (Rat), (Brain) 1. Introduction

Triethyllead, the main m a m m a h a n metabohte of the gasohne anti-knock additive tetraethyllead (Bolanowska, 1968), is neurotoxlc in the central nervous system In animals triethyllead has elicited timeand dose-dependent alterations in sensory responsiveness and motor function (for review see Walsh and Tllson, 1984) It has an antlnoclceptlve effect In rats Typical clinical signs in rats include hyper- and hypoactlvity, hyperexcitablhty, aggressiveness, tremor and convulslons The changes in emotlonahty and reactivtty to environmental stimuli suggest that llmbic system and its associated cortical areas are primary targets of toxicity (Walsh and Tilson, 1984). Both hlstopathologlcal (Seawright et a l , 1984; Booze and Mactutus, 1990) and neurochemical evidence suggest that hippocampus is particularly vulnerable to tnethyllead

* Corresponding author At National Pubhc Health Insntute, DIvision of Environmental Health, P O B 95, 70701 Kuoplo, Finland Tel 358-71-201322, Fax 358-71-201265 0926-6917/95/$09 50 © 1995 Elsevier Soence SSDI 0 9 2 6 - 6 9 1 7 ( 9 5 ) 0 0 0 1 3 - 5

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Trtethyllead has a range of cellular effects, e g decreased energy production in mltochondria (A1drldge et al, 1977, Kaupplnen et al., 1988), Increase in free lntracellular calcium (Komulalnen and Bondy, 1987, Kaupplnen et a l , 1988), decreased myehnation (Grundt et al, 1974) and depolymerlzatlon of microtubules and mhlbitlon of their assembly ( Z l m m e r m a n n et al, 1988) but the exact neuronal pathways and the neurochemtcal correlates of the chnlcal signs have, however, not yet been uniformly characterized. Convulsions and tremor m trlethyllead-treated antmals reflect to imbalance between excitatory and inhibitory circuits m the brain. By lnhlbttIng GABAerglc transmission, the main inhibitory system in the brain, in critical sites trlethyllead would move the balance towards excitatory effects There is already some evidence that trtethyllead affects GABAerglc transmtsslon Trtethyllead lnhlbtts G A B A uptake in isolated nerve endings (Seldman and Verity, 1987), probably by preventing binding of G A B A into the uptake carrier (Seldman et al, 1987) a n d / o r by mcreaslng spontaneous release of G A B A (Minnema et al., 1991) On the other hand, the antinoclceptlve effect of trlethyllead

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H Komulamen et al /Eur J Pharmacol Enctron Toxtcol Pharmacol Secnon 293 (1995) 167-171

Male Wlstar rats (Wlstar Han, bred by the National Laboratory Animal Center, University of Kuoplo, Kuoplo, Finland) were used in these experiments The animals ( 6 - 7 weeks old) were housed in wire-mesh cages, 5 - 7 animals per cage, under standard animal facility atmosphere (temperature 20 + 0 5° C, relative humidity between 55 and 75%, h g h t / d a r k cycle 14 h / 1 0 h) The animals had free access to pelleted rodent chow (R3-Ewos-Alab, Sweden) and tap water During the subacute study with triethyllead the animals were housed in plastic Macrolon cages in groups of 4 animals

mln, 4 ° C) to obtain a P2 fraction The P2 fraction was suspended in 10 vols. of ice-cold Trls-HC1 buffer, pH 7 4 and sonlcated on ice (MSE Sonlprep, 30 s, poSltlOn 7) Allquots of fresh suspension (100 /xl, about 400 k~g protein) were incubated in duplicate with 3 nM [3H]flunltrazepam in 0 050 M Trls-HCl buffer pH 7 4 (1 9 ml) in the absence (total binding) and presence of 10/xM unlabelled flunltrazepam (non-specific binding) for 30 mln on ice Incubations were initiated by pipettlng the tissue last Tubes were mlxed with a vortex mixer and the incubation termlnated by rapid vacuum filtration on W h a t m a n G F / B glass-fibre filters on MlllIpore samphng manifold The filters were rinsed twice with 4 ml of ice-cold incubation buffer and their radioactivity counted after an overnight digestion m 5 ml of ACS scintillation hquid in a hquld scintillation counter (LKB Wallac 1216 Rackbeta) When the effect of trlethyllead on [~H]flunltrazepam binding was evaluated in vitro, trlethyllead was added in the incubation medium at final concentrations from 10 .9 M to 10 4 M In other experiments [3H]flunltrazepam binding was assayed in the presence of a G A B A agonlst musclmol, to evaluate whether or not trlethyllead affected the connectlon between the G A B A A binding site and the benzodlazeplne site of the receptor complex In those experiments P2 fraction was washed by repeating the homogenization and centrlfugatlon phases 3 times with ice-cold Trls-HCl buffer before sonlcatlon to remove endogenous G A B A Tissue allquots and 10 4 M trlethyllead were prelncubated first together for 10 rain on ice Then 3 txM or 3 0 / x M musclmol was added and 10 mln later 3 nM [3H]flunitrazepam without (to measure the total binding) or with 10 /xM unlabelled flunltrazepam (non-specific binding) The mixture was incubated further for 30 mln on ice and terminated by filtration as described above The effect of only musclmol or trlethyllead on [3H]flunltrazepam binding was evaluated with separate samples omitting trlethyllead or musclmol, respectively, from the incubation medium In all experiments the speclflC [3H]flunltrazepam blndlng was defined as a difference in total binding and non-specific binding and is presented as f m o l / m g protein

2 3 [3H]flumtrazepam b m d m g tn vttro

2 4 [3H]flumtrazepam b m d m g ex vwo

[3H]flunltrazepam binding was assayed essentially as described in Saano et al. (1981) Briefly, rats were decapitated and the occipital cortex and cerebellum were dissected out quickly on ice Tissue pieces were weighed and homogenized with a glass-teflon homogenizer in 20 volumes of ice cold 0.32 M buffered sucrose The homogenate was centrifuged at 1500 × g (15 mln, 4°C, Sorvall RC-5B, Dupont, rotor SM-24) and the resulting supernatant recentrlfuged (17500 × g, 30

For ex vivo experiments, trlethyllead acetate was administered dally (at about 1 p m ) subcutaneously (neck) at a dose of 1 9 m g / k g (in sterile 0 9% NaC1 solution, 1 m l / k g ) to a group of 5 animals for 5 consecutive days Control animals (n = 6) received 0 9% NaC1 solution. On the 6th day, 150 p.Cl of [3H]flunltrazepam (in 0 9% NaC1) were injected into the tall vein as a bolus to label the central benzodlazepine receptors as described in Saano et al (1981)

has a strong emotional component (Walsh et a l , 1986) which can be attenuated with chlordiazepoxlde (Hong et a l , 1983) Antlnociception was temporally associated with a transient decrease in hlppocampal benzodlazeplne receptor binding (Bondy et al, 1985) Since the central benzodlazepine receptor is a component of the G A B A A receptor complex, interference with either binding site would modulate signal transductlon through the G A B A A receptor In the present study effects of trlethyllead on the GABAA receptor complex were studied [3H]flunltrazepam was utlhzed as a central benzodlazepine receptor hgand and musclmol as a G A B A A agonlst Central benzodlazepine receptor binding was also measured in several brain regions after dosing triethyllead subacutely to rats In those studies central benzodlazeplne receptors were labelled in VlVO by injecting [3H]flunltrazepam intravenously to rats

2. Materials and methods 2 1 Chemtcals

[ 3 H ] f l u n l t r a z e p a m (the specific actlvlty 72 4 C1/mmol) was purchased from N E N Products (England), flunitrazepam was obtained from Hoffman-La Roche (Switzerland) and trlethyllead acetate from Alfa Products (England) 2 2 Ammals

H Komulamen et a l / E u r

J Pharmacol Ent tron Toxtcol Pharrnacol Sectton 293 (1995) 167-171

169

Table 1 Effect of tnethyllead on [3H]flumtrazepam binding m the absence and presence of a GABA receptor agomst musclmol m rat cerebellar and cortical P2 fractions m wtro Without musclmol

Musclmol (3/xM)

Musclmol (30 p.M)

Cerebellum Control Trtethyllead (104 M)

86 + 5 (100%) ~ 85 ± 4 (99 + 2%)

131 _+ 10 b (100%) 112 _+ 12 (82 ± 5%)

105 _+ 10 (100%) 91 _+ 9 (86 _+ 3%)

Cortex Control Tnethyllead (104 M)

152 +_ 14 (100%) a 152 ± 12 (101 _+ 2%)

201 _+ 26 (100%) 202 ± 23 (102 _+ 2%)

165 ± 20 (100%) 170 ± 20 (103 ± 6%)

Specific binding ( f m o l / m g protein) Mean +_ S E M of five independent experiments a Binding as percentage, binding w~thout tnethyllead = 100% b p < 0 01 compared to the respective control value, Duncan test c p < 0 05 compared to the respective value without musclmol

The animals were decapitated 2 mm later and the brams dissected immediately on ice m a cold room ( + 4 ° C ) according to Glowlnskl and Iversen (1966) Occ~pltal cortex, forebraln, hypothalamus, medulla/oblongata, hlppocampus and cerebellum were separated and immediately frozen on dry ice The frozen pteces were weighed and homogemzed in 25 volumes of ice cold 0 050 M TrIs-HC1 buffer, pH 7 4 Total binding of [~H]flunttrazepam was determmed by filtermg a 0 5 ml sample of the homogenate in duphcate lmmedmtely after homogenlzatton and the non-spectfic bmdlng by incubating 0 5 ml samples of the homogenate wtth 2 0 / x M unlabelled flunltrazepam on ice for 30 mln In each case the filters were rinsed twice with 4 ml of ice-cold buffer and the radtoacttvtty on filters counted as in in vitro expertments The specific [3H]flunltrazepam binding in ex VlVO experiments was defined as a difference m the total binding and the non-specific blndmg and is expressed as percentage of the total radioactivity in the tissue aliquot

2 5 Protem determmatton Protem in tissue homogenates was determined by the method of Lowry et al (1951) usmg bovine serum albumm as a reference

2 6 Stattsttcs The results of in vitro expertments were analyzed wtth A N O V A for trlethyllead and musctmol effects If significant effects were observed, comparisons between groups were made using Duncan test The receptor bmdmg data ex vtvo were analyzed with Student's t test The difference at P < 0 05 was regarded as slgntficant

3. Results

3 1 Effect of tnethyllead on [~H]flunttrazepam bmdmg in vitro Trlethyllead did n o t a f f e c t s i g n i f i c a n t l y [3H]flunltrazepam binding m rat cortical P2 fraction xn vitro at concentrations between 10 - 9 M and 10 - 4 M (data not shown) indicating that tt has no direct effect on the central benzodtazeplne receptor stte of the G A B A A receptor complex This result was confirmed m washed cortical and cerebellar P2 fractions at 100 IxM trlethyllead (Table 1) In washed cerebeltar P2 fractton musctmol stimulated [3H]flumtrazepam binding (ANOVA, P = 0 001),

Table 2 Effect of tnethyllead on [3H]flumtrazepam binding ex VlVO

Control (n = 6) Tnethyllead(n=5)

Specific binding (%) Cortex Forebram

Medulla/pons

Hypothalamus

Hlppocampus

Cerebellum

309 ± 2 6 259_+23

27 1 +_ 2 8 248_+11

34 1 + 2 1 323_+37

42 7_+ 24 407±42

35 6 + 3 3 259_+23a

249_+ 3 2 259_+08

Tnethyllead acetate was administered as 5 dally s c admmlstrahons of 1 9 m g / k g to rats On the next day, [3H]flunltrazepam (150 p, C0 was injected into the tall veto and the animals were killed 2 mln later A crude tissue homogenate was prepared from the indicated brain regions and the specific binding of [3H]flumtrazepam determined The figures are percent values of the total [3H]flumtrazepam content of the tissue (Mean + SEM) a p < 0 05 compared to control, Student's t test

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H Komulamen et al / E u r J Pharmacol Enctron Toxtcol Pharmacol Section 293 (1995) 167-171

by 52% ( P < 0.01) at 3 / x M and 22% at 3 0 / x M (Table 1) Although trlethyllead (100 /xM) did not affect [3H]flunltrazepam binding per se, it attenuated slightly but not statistically significantly the ability of m u s o m o l to stimulate [3H]flunitrazepam binding At the m u s o mol concentration of 3 /xM the decrease in the [~H]flunltrazepam binding was 18% and at 30 /xM 14% In washed cortical P2 fraction muscImol increased [3H]flunltrazepam binding (ANOVA, P = 0 048) but less than m cerebellum, l e by 32% at the concentration 3 /xM (Table 1). Triethyllead (100 /xM) did not modify the musclmol-lnduced increase in [3H]flumtrazepam binding in cortex at all (Table 1)

3 2 Effect of trtethyllead on [ ~H]flumtrazepam binding ex Ull'O

Trlethyllead treatment decreased shghtly the weight gain of the animals The mean terminal body weight was 11% lower ( P < 0 . 0 5 ) in the trlethyllead group compared to controls The specific [3H]flunltrazepam binding was highest m hlppocampus and decreased in other brain regions m the order of cerebellum, hypothalamus, cortex, m e d u l l a / p o n s and forebraln (Table 2) In the trlethyllead treated animals the specific [3H]flunltrazepam binding was 27% lower ( P < 0 05) in cerebellum than in controls In other brain regions the binding did not differ significantly between the groups (Table 2)

4. D i s c u s s i o n

Trlethyllead did n o t a f f e c t t h e s p e c i f i c [3H]flunltrazepam binding in cortical and cerebellar t~ssue m e m b r a n e s m vitro indicating that trlethyllead has no direct affinity to central benzodlazeplne receptors However, a slight decrease in the ability of musclmol, a G A B A A agonist, to stimulate the specific [3H]flunltrazepam binding in cerebellar m e m b r a n e s suggests that triethyllead may modify the connection between the G A B A site and the benzodiazeplne site of the receptor complex G A B A receptor agonlsts enhance the benzodlazeplne binding by Increasing the affinity of the receptor to benzodlazepines (Tallman et al, 1978, Brlley and Langer, 1978, Olsen, 1982) Ability to modulate GABA-agonlst-stimulated benzodlazeplne binding has been suggested to be the most sensitive and accurate biochemical indicator of the efficacy of the compound to influence benzodlazeplne receptors (Marangos et a l , 1981) Hence, trlethyllead might somehow restrict the muscimol-lnduced conformation change of the receptor complex resulting in decreased central benzodlazepine receptor binding Trlethyllead inhibits G A B A binding to G A B A uptake carrier in synaptosomes (Seldman et al, 1987) It is thus possible

that trlethyllead exerts similar effects on the G A B A recognition site in the uptake carrier and the receptor site Trlethyllead did not modify to any extent the musclmol response in the cerebral cortex Musclmol caused a more profound increase m the central benzodlazeplne receptor binding in cerebellar tissue than in cortex suggesting some difference in the receptor complexes G A B A A receptor has several, at present unknown, number of subtypes composed of more than 15 different subunlts (Burt and Kamatchl, 1991, DeLorey and Olsen, 1992, Sleghart, 1992) Along with different subunlts the sensitivity of the G A B A A receptor to trlethyllead may vary and regional heterogenlclty among the subtypes may cause different regional sensitivity to trlethyllead In concordance with the m vitro data, the specific cerebellar [~H]flunltrazepam binding was decreased in rats after subacute treatment with trlethyllead Because trlethyllead does not appear to affect the central benzodlazepine receptor binding per se, the critical effect had to be directed elsewhere in the receptor complex The conformation of the receptor complex might have changed unfavourably for central benzodlazeplne receptor binding or the total number of the receptor complexes had decreased A single dose of 7 9 m g / k g trlethyllead chloride has been reported to decrease [3H]flunltrazepam binding transiently in rat hippocampus but not in the cortex or striatum (Bondy et a l , 1985) The effect disappeared by 21 days after administration indicating that the damage was not permanent In the present study no change in the hlppocampal central benzodiazeplne receptor binding was observed but the dose, although given repeatedly, was lower With the similar administration schedule of trlethyllead as used in the present study a long-lasting increase m lipid peroxldahon was observed m rat frontal cortex but not in the cerebellum or hlppocampus after the end of the administration (All and Bondy, 1989) In spite of increased hpld peroxldatlon, [3H]dlazepam binding m the cortex was not changed, implying that hpld peroxidatlon did not destroy the central benzodlazeplne receptor sites In conclusion, methyllead decreases the central benzodiazeplne receptor binding in the rat cerebellum in vivo and attenuates slightly the GABAerglc shmulatlon of the benzodlazeplne binding m cerebellum, but not m cerebrum, in vitro This Inhibitory effect on the G A B A a receptors may diminish the inhibitory neural input and contribute to trlethyllead induced cerebellar symptoms, particularly tremor

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