Bicuculline and GABA-metabolising enzymes

226

SHORT COMMUNICATION5

Bicuculline and GABA-metabolising enzymes The inhibitory effect of ~,-amlnobutyric acid (GABA) on the firing of neurones in mammals 2,4, amphibians 6,10 and invertebrates 8 is antagonlsed by the convulsant alkaloid blcuculhne a Examination of molecular models of bicuculhne, G A B A and related G A B A agonlsts suggests that all these substances may interact with the same postsynaptic receptors However, bicuculhne does not antagomse all interactions of G A B A with macromolecules smce the active uptake of G A B A into brain slices is not influenced by this alkaloid z We have studied the effect of blcuculhne on the activity of some GABA-metabohsmg enzymes in order to investigate other G A B A - m a c r o molecule interactions Four enzymic activities were studied L-glutamate 1-carboxy-lyase (E C 4 1.1 15) from Eschertchta colt (bacterial G A D , Worthington Biochemical Corp ) and from rat cerebral grey matter (bram G A D I), and GABA-2-oxoglutarate aminotransferase (E C 2 6 1 19) from Pseudomonasfluorescens (bacterial GABA-T, Sigma Chemical C o m p , type III) and from rat cerebral mitochondria (brain GABA-T) These activities were assayed by radiochemIcal procedures similar to those described by Kravitz and co-workers ~,7, measuring the formation of G A B A from L-glutamate by the carboxy-lyases and the coupled formation of succinate from GABA by the transamlnases Both assays were sensitive to small pH changes and care was taken to ensure that blcuculhne did not alter the pH of the reaction mixtures This limited the maximum concentration of bicuculhne hydrochlorlde that could be dissolved m the reaction mixtures to 10-4 M The convulsive dose of bicuculhne in mammals (approx 0 2 mg of the hydrochlorlde/kg body weight 9) is equivalent to approx l0 -6 m ~ if uniformly distributed in the body fluids Table I shows that bicuculhne had no significant effects on the enzymic activities studied This is in agreement with histological studies on GABA-T actlvlty in the

TABLE I BICUCULLINE AND GABA-METABOLISING ENZYMES

The enzyme preparations were premcubated wah or without bmucullme for 5 mm at 37°C before mitmtlon of the reaction, and mcubation continued for 30 mm m the case of the brain extracts and 10 mm for the bacterial extracts All values are means :[: S E M of results flora the number of experiments shown m brackets The mean control actwltms were as follows brain GAD I, 16 35 /*moles of GABA produced/h/g wet bram, bacterial GAD, t 57/~moles/mm/mg acetone powder, brain GABA-T, 3 26 #moles of succmate produced/h/g wet bram, bacterial GABA-T, 2 02/tmoles/ /mm/g acetone powder Preparation

Brain GAD I Bacterial GAD Brain GABA-T Bacterial GABA-T

Bram Research, 38 (1972) 226-227

Enzymic actwtty as % mean control

Control

+lO-4Mbwucullme

100 d_ 2 (4) 100 ± 3 (4) 100 ~ 4 (3) 100 :t: 5 (6)

100 ± 1 (4) 101 ± 2 (4) 95 d: 3 (4) 100 4- 3 (6)

SHORT COMMUNICATIONS

227

guinea pig b r a i n stem, using b l c u c u l h n e as a s o l u t i o n o f the free base (10 -3 M ) m aqueous dlmethylsulphoxldea M a n y l n h l b l t o r s o f b r a i n G A D I are c o n v u l s a n t s (e g ref l) the p r e s e n t study shows t h a t b l c u c u l h n e - l n d u c e d c o n v u l s i o n s are u n h k e l y to be the result o f l n h l b m o n o f this enzymic activity B l c u c u l h n e - G A B A a n t a g o m s m a p p e a r s to be confined to p a r t i c u l a r p o s t s y n a p tic r e c e p t o r s Th~s selective a n t a g o m s m m a y p r o v i d e useful i n f o r m a t i o n r e g a r d i n g m o l e c u l a r aspects o f the i n t e r a c t i o n o f G A B A with these receptors, a n d m a y md in their ~solat~on a n d c h a r a c t e n s a t l o n Department of Physiology, Austrahan Natwnal Umverstty, Canberra ( Austraha)

P M BEART G A R JOHNSTON

I ALBERICI,M , ARNAIZ,G R DE L , AND DE ROBERTIS,E , Glutarmc acid decarboxylase lrlhibl= tlon and ultrastructural changes by the convulsant drug allylglycme, Bwchem Pharmacol, 18 (1969) 137-143 2 CURTIS, D R , DUGOAN, A W , FELIX)D , AND JOHNSTON, G A R , GABA, blcuculhne and central mhlbmon, Nature (Lond), 226 (1970) 1222-1224 3 DAVIES,W E , AND COMIS, S D , Blcuculhne and its effect on 7-ammobutync acid transammase m the guinea-pig braan stem, Nature (Lond), 231 (1971) 156-157 4 DE GROAT, W C , ].,ALLEY,P M , AND BLOCK, M , The effects of blcuculhne and GABA on the superior cervical ganghon of the cat, Brain Research, 25 (1970) 665-668 5 HALL,Z W , ANDKRAVITZ,E A , The metabohsm ofT-armnobutync aod (GABA) in the lobster nervous system 1 GABA-glutamate transammase, J Neurochem, 14 (1967) 45-54 6 MCLENNAN,H , Bzcuculhne and mhlbmon of crayfish stretch ieceptor neurones, Nature (Lond), 228 (1970) 674-675 7 MOLINOFF,P B , AND KRAVITZ, E A , The metabohsm of 7-ammobutync acid (GABA) m the lobster nervous system-glutamlc decarboxylase, J Neurochem, 15 (1968) 391-409 8 WALKER)R J , CROSSMAN,A R , WOODRUFF,G N ,ANDKERKUT,G A ,Theeffectofblcuculhne on the gamma-amlnobutync acid (GABA) receptors of neurones of Pertplanetu amerwana and Hehx aspersa, Bram Research, 33 (1971) 75-82 9 WELCH, A D , AND HENDERSON,V E , A comparative study of hydrastme, btcuculhne and adlumme, J Pharmacol exp Ther , 51 (1934) 482491 10 WOODWARD,D J , HOFFER,B J , SIC,GINS, G, R , AND OLIVER,A P , Inhlbmon of Purkmje cells m the frog cerebellum II Ewdence for GABA as the inhibitory transmitter, Brain Research, 33 (1971) 91-100 Accepted December 10th, 1971)

Brain Research, 38 (1972) 226-227