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Enhancement of in vitro binding and some of the pharmacological properties of diazepam by a novel anthelmintic agent, avermectin B1a
European Journal of Pharmacology, 56 (1979) 273--276
273
© Elsevier/North-Holland Biomedical Press Short communication
ENHANCEMENT O F IN V I T R O B I N D I N G A N D S O M E O F T H E P H A R M A C O L O G I C A L PROPERTIES OF DIAZEPAM BY A NOVEL ANTHELMINTIC AGENT, AVERMECTIN
B1a
MICHAEL WILLIAMS and GEORGE G. YARBROUGH Merck Institute for Therapeutic Research, West Point, Pennsylvania 19486, U.S.A.
Received 10 April 1979, accepted 18 April 1979 M. WILLIAMS and G.G. YARBROUGH, Enhancement of in vitro binding and some of the pharmacological properties of diazepam by a novel anthelmintic agent, Avermectin B l a , European J. Pharmacol. 56 (1979) 273-276. A novel macrocyclic lactone disaccharide anthelmintic agent, &vermectin B la (AVM) has been found to cause a concentration-dependent increase in the in vitro binding of 3H-diazepam to rat and mouse brain membranes. The increase in binding is manifested as both an increase in the affinity and number of binding sites for 3H-diazepam. Preliminary in vivo studies demonstrate that AVM can also enhance some of the pharmacological actions of diazepam. 3H-Diazepam binding
Anthelmintic
Diazepam
1. I n t r o d u c t i o n A high a f f i n i t y , specific binding site for a H - d i a z e p a m has b e e n described in m a m m a lian brain. F r o m correlations o f t h e pharmacological actions o f various b e n z o d i a z e p i n e s and t h e i r e f f e c t s t o displace 3H~liazepam binding, this site has b e e n designated as an " a n x i o l y t i c r e c e p t o r " (Squires and Braestrup, 1 9 7 7 ; M o h l e r and O k a d a , 1 9 7 7 ) . Several diverse c o m p o u n d s , a m o n g the Ni 2÷ (Mackerer a n d K o c h m a n , 1 9 7 8 ) , 7 - a m i n o b u t y r i c acid ( G A B A ) and m u s c i m o l (Tallman et al., 1 9 7 8 ; Williams and Risley, 1 9 7 9 ) and a novel a n x i o l y t i c , SQ 2 0 0 0 9 (Williams and Risley, 1 9 7 9 ) have b e e n s h o w n to e n h a n c e the binding o f aH-diazepam t o rat brain m e m branes. T h e significance o f these findings is * Avermectin Bla = (2aE,4E,5'S,6S,6'R,7S,8E,11R, 13R,15S,17aR,20R,20aR,20bS)-6'- [ (R)-sec-butyl ] -7[ [ 2,6-dideoxy-4-O-( 2,6-dideoxy-3-O-methyl-(~-L-arabino-hexopyranosyl)-3-O-methyl-(~-L-arabino-hexopyranosyl] oxy ]-5t,6,6t,7,10,11,14,15,17a,20,20a,20b o dodecahydro-20,20b-dihydroxy-5P,6,8,19-tetramethylspiro-[ 11,15-methano-2H,13H,17H-furo-[4,3,2-pq] [ 2, 6 ] benzodioxyacyclo-octadecin°l3,2'- [ 2H ] pyran ] -17one.
n o t e n t i r e l y clear a l t h o u g h t h e m o d u l a t i o n of 3H-diazepam binding b y GABAergic agents appears c o m p a t i b l e w i t h the w e l l - k n o w n in vivo i n t e r a c t i o n s o f b e n z o d i a z e p i n e s and G A B A ( H a e f l e y et al., 1 9 7 5 ) . A d d i t i o n a l l y , the i n t e r a c t i o n s o f SQ 2 0 0 0 9 with d i a z e p a m binding m a y have some relevance to t h e purp o r t e d a n x i o l y t i c actions o f this c o m p o u n d (Beer et al., 1 9 7 2 ) . In t h e present studies, we have e x a m i n e d t h e i n t e r a c t i o n o f a novel m a c r o c y c l i c l a c t o n e disaccharide a n t h e l m i n t i c agent, A v e r m e c t i n S l a * (AVM: Fritz et al., 1 9 7 9 ) with aH-diazepam binding and with some o f t h e p h a r m a c o l o g i c a l actions o f diazepam.
2. Materials and m e t h o d s 2.1. aH-diazepam b i n d i n g
Binding o f aH-diazepam t o c r u d e s y n a p t i c m e m b r a n e s p r e p a r e d f r o m rat brain was m e a s u r e d b y a m o d i f i c a t i o n o f previously published m e t h o d s (Squires and Braestrup, 1 9 7 7 ; Mohler and O k a d a , 1 9 7 7 ) . T h e binding assay r o u t i n e l y consisted o f triplicate t u b e s to
274 which were added: 3H-diazepam (NEN; specific activity 39.1 Ci/mmol) to a final c o n cen tr atio n o f 1.5 nM; 1 ml of P2 protein suspension (1 mg/ml) and 50 mM Tris HC1 pH 7.4 to a final volume of 2 ml. Experimental c o m p o u n d s when used were diluted in 50 mM Tris HC1 pH 7.4 and added to the incubation m i x t ur e in a volume o f 0.1 ml to t he final concentrations indicated. The reaction was initiated by the addition of m e m b r a n e protein to tubes maintained at 4°C in an ice water bath and continued for 15 min at which time equilibrium was reached, and terminated by filtration u n d er vacuum through Whatman G F / B filters. Th e filters were washed twice with 5 ml of ice-cold 50 mM Tris HC1 pH 7.4, transferred to scintillation vials and 10 ml o f Aquasol 2 (NEN) added. After shaking at r o o m t e m p e r a t u r e for 30 min, radioactivity was measured in a Packard Model 3255 Scintillation S p e c t r o m e t e r at an efficiency of 38%. Specific binding was det er m i ned as the difference in the radioactivity b o u n d in the absence and presence of 1 pM unlabelled diazepam and was a p p r o x i m a t e l y 90% of the total radioactivity bound. Scatchard analysis o f the changes in 3H-diazepam binding was determined over a c o n c e n t r a t i o n range of 0.625--10 nM.
2.2. Behavioral studies Carworth, CF1, female mice weighing 18-24 g were used in all studies. "Muscle p o w e r " was assessed through the use of a fixed wire mesh platform attached to a calibrated linear differential tr an s f or m e r which was c o n n e c t e d electrically to a Servo pen recorder. Mice placed on the p lat f or m and restrained by t he tail exerted considerable force in attempting to grip the mesh when pulled across it by t he tail. This force was r e c or ded as spaces of pen deflection. L o c o m o t o r activity was assessed with " d o n u t " shaped photocell chambers consisting o f a circular r unw a y with a central light source and six photocells equally spaced around th e o u ter perimeter of the runway. Counts resulting f r om i n t e r r u p t i o n of the
M. WILLIAMS, G.G. YARBROUGH light beams were recorded on Sodeco counters. Animals were placed (one mouse per chamber) in a particular chamber on a randomized basis, and treated and cont rol animals were tested concurrently.
3. Results
3.1. 3H-diazepam binding AVM caused a c o n c e n t r a t i o n - d e p e n d e n t increase in t he binding o f 3H-diazepam to rat brain membranes (table 1) with a near maximal effect observed at 10 pM. Examination of the effects of AVM on 3H-diazepam binding by kinetic analysis showed that the increase in binding was reflected as an increase in bot h the affinity (decrease in KD) and n u m b e r of binding sites (table 1). The effects of AVM on 3H-diazepam binding to mouse brain membranes yielded similar data to t hat observed in rats {data n o t presented).
TABLE 1 AVM enhancement of 3H-diazepam binding to rat brain membranes. (A) Concentration--responses of AVM to enhance 3Hdiazepam binding AVM concentration (pM)
% Increase in binding (mean _+S.D.; n = 4)
0.1 1 10
19 -+ 9 44_+7 65 _+3
(B) The effect of 500 nM AVM on affinity and number of 3H-diazepam binding sites Control +500 nM % Change (mean + S.D.; AVM (signifin = 4) (mean -+S.D.; cance) n=4) KD
4.56-+ 0.46
3.65 -+ 0.32 --20
n (pmoles/ m g protein)
1.46 -+ 0.07
1.64 + 0.06
(P < 0.02) +12 (P < 0.01)
AVM ENHANCEMENT OF DIAZEPAM BINDING To the extent tested the action of AVM on 3 H - d i a z e p a m b i n d i n g a p p e a r e d specific i n s o f a r as it e x h i b i t e d n o activity at c o n c e n t r a t i o n s u p t o 50 pM o n t h e b i n d i n g o f 3H-WB 4 1 0 1 (a-adrenergic receptor), 3H-GABA (GABA r e c e p t o r ) , 3H-spiroperidol ( d o p a m i n e recept o r ) , 3H-quinuclidinyl b e n z i l a t e ( m u s c a r i n i c cholinergic r e c e p t o r ) , 3H-kainic acid (glutamate receptor) and 3H.LSD (serotonin receptor).
3.2. Behavioral studies P r e t r e a t m e n t w i t h A V M (5 m g / k g i.p.) e n h a n c e d t h e r e d u c t i o n in " m u s c l e p o w e r " i n d u c e d b y d i a z e p a m ( t a b l e 2). This d o s e o f A V M did n o t a l t e r t h e m u s c l e r e l a x a n t p r o p e r t i e s o f c y c l o b e n z a p r i n e assessed b y this test procedure. Furthermore, diazepam caused a d o s e - r e l a t e d d e c r e a s e in s p o n t a n e o u s m o t o r a c t i v i t y w h i c h was e n h a n c e d at all d o s e levels b y p r e t r e a t m e n t ( 3 0 m i n p r i o r t o receiving d i a z e p a m ) w i t h 5 m g / k g i.p. o f A V M
TABLE 2 Enhancement by AVM of the decrease in 'muscle power' induced by diazepam 1 Pretreatment 2 (mg/kg i.p.)
Treatment 3 (mg/kg i.p.)
Units of pen deflection 4 (X -+ S.E.M.)
Methocel Methocel Methocel Methocel AVM (5) AVM (5) AVM (5) AVM (5)
Methocel Diazepam Diazepam Diazepam Diazepam Diazepam Diazepam Methocel
9.4 7.5 7.1 6.3 5.3 4.8 4.0 9.0
+ + + + + + + +
(5) (10) (20) (5) (10) (20)
+ 0.2 + 0.4 + 0.4 -+ 0.5 + 0.5 -+ 0.4 -+ 0.4 -+ 0.2
s s s 6 6 6
1 N = 20 mice/treatment. 2 30 min before treatment. 3 30 rain before test. 4 10 units of pen deflection = 100 g. s Significantly different (P < 0.05) from methocel + methocel (Duncan's multiple range test). 6 Significantly different (P < 0.05) from methocel + corresponding doses of diazepam (Duncan's multiple range test).
275 {data n o t s h o w n ) . This d o s e o f A V M did n o t significantly a f f e c t m o t o r a c t i v i t y b y itself. 4. Discussion AVM enhances the binding of 3H-diazepam t o rat brain m e m b r a n e s b y increasing b o t h t h e a f f i n i t y and n u m b e r o f b i n d i n g sites f o r t h e b e n z o d i a z e p i n e . T o o u r k n o w l e d g e this is t h e first s u b s t a n c e s h o w n t o increase t h e n u m b e r o f b i n d i n g sites available t o t h e r a d i o l i g a n d , Ni 2÷, G A B A , m u s c i m o l a n d S Q 2 0 0 0 9 prod u c i n g t h e i r e f f e c t s solely via a c h a n g e in t h e a f f i n i t y o f t h e b i n d i n g site. T o t h e e x t e n t t e s t e d , this e f f e c t o f A V M a p p e a r s specific to t h e d i a z e p a m r e c e p t o r . F u r t h e r m o r e , t h e observed in vivo e n h a n c e m e n t b y A V M o f d i a z e p a m i n d u c e d decreases in m o t o r a c t i v i t y a n d m u s c l e p o w e r w o u l d a p p e a r t o indicate a degree o f p h a r m a c o l o g i c a l relevance f o r t h e e n h a n c e m e n t o f binding o b s e r v e d in vitro. Clearly, f u r t h e r studies are r e q u i r e d t o determ i n e t h e specificity o f t h e p h a r m a c o l o g i c a l i n t e r a c t i o n b e t w e e n A V M a n d d i a z e p a m in vivo. F u r t h e r m o r e , while it is possible t h a t this i n t e r a c t i o n m a y have relevance to t h e n o v e l a n t h e l m i n t i c a c t i o n s o f this m a c r o l i d e a n d its r e p o r t e d e f f e c t s on chloride c h a n n e l s in i n v e r t e b r a t e s (Fritz et al., 1 9 7 9 ) , t h e lack o f d i a z e p a m b i n d i n g sites in i n v e r t e b r a t e s (Neilsen et al., 1 9 7 8 ) w o u l d q u e s t i o n such an e x t r a p o l a t i o n . I t m a y be n o t e d , h o w e v e r , that a relationship between the diazepam r e c e p t o r a n d chloride a n i o n c h a n n e l s in m a m m a l i a n brain m e m b r a n e s has b e e n i n f e r r e d (Costa et al., 1979). Acknowledgements The authors are most appreciative of the expert technical assistance provided by J. McGuffin, E.A. Risley and J.A. Totaro during these studies. References
Beer, B., M. Chasin, D.E. Clody, J.R. Vogel and Z.P. Horovitz, 1972, Cyclic adenosine monophosphate
276 phosphodiesterase in brain: Effect on anxiety, Science 176,428. Costa, T., D. Rodbard and C.B. Pert, 1979, Is the benzodiazepine receptor coupled to a chloride anion channel?, Nature London 277,315. Fritz, L.C., C.C. Wang and A. Gorio, 1979, Avermectin Bla blocks inhibitory and excitatory postsynaptic potentials at the lobster neuromuscular junction, Proc. Nat. Acad. Sci. U.S.A. (in press). Haefley, W., A. Kulscar, H. Mohler, L. Pieri, P. Polc and R. Schaffner, 1975, Possible involvement of GABA in the central actions of benzodiazepines, Advan. Biochem. Psychopharmacol. 14,131. Mackerer, C.R. and R.L. Kochman, 1978, Effects of cations and anions on the binding of 3H-diazepam to rat brain, Proc. Soc. Exptl. Biol. Med. 158, 393. Mohler, H. and T. Okada, 1977, Properties of 3H-
M. WILLIAMS, G.G. YARBROUGH diazepam binding to benzodiazepine receptor in the rat cerebral cortex, Life Sci. 20, 2102. Neilsen, M., C. Braestrup and R.F. Squires, 1978, Evidence for a late evolutionary appearance of brain-specific benzodiazepine receptors: an investigation of 18 vertebrate and 5 invertebrate species, Brain Res. 141,342. Squires, R.F. and C. Braestrup, 1977, Benzodiazepine receptors in rat brain, Nature London 266, 732. Tallman, J.F., J.W. Thomas and D.W. Gallager, 1978, GABAergic modulation of benzodiazepine binding site sensitivity, Nature London 274,383. Williams, M. and E.A. Risley, 1979, Enhancement of the binding of 3H-diazepam to rat brain membranes in vitro by SQ 20009, a novel anxiolytic, 7-aminobutyric acid (GABA) and muscimot, Life Sci. 24 (in press).
273
© Elsevier/North-Holland Biomedical Press Short communication
ENHANCEMENT O F IN V I T R O B I N D I N G A N D S O M E O F T H E P H A R M A C O L O G I C A L PROPERTIES OF DIAZEPAM BY A NOVEL ANTHELMINTIC AGENT, AVERMECTIN
B1a
MICHAEL WILLIAMS and GEORGE G. YARBROUGH Merck Institute for Therapeutic Research, West Point, Pennsylvania 19486, U.S.A.
Received 10 April 1979, accepted 18 April 1979 M. WILLIAMS and G.G. YARBROUGH, Enhancement of in vitro binding and some of the pharmacological properties of diazepam by a novel anthelmintic agent, Avermectin B l a , European J. Pharmacol. 56 (1979) 273-276. A novel macrocyclic lactone disaccharide anthelmintic agent, &vermectin B la (AVM) has been found to cause a concentration-dependent increase in the in vitro binding of 3H-diazepam to rat and mouse brain membranes. The increase in binding is manifested as both an increase in the affinity and number of binding sites for 3H-diazepam. Preliminary in vivo studies demonstrate that AVM can also enhance some of the pharmacological actions of diazepam. 3H-Diazepam binding
Anthelmintic
Diazepam
1. I n t r o d u c t i o n A high a f f i n i t y , specific binding site for a H - d i a z e p a m has b e e n described in m a m m a lian brain. F r o m correlations o f t h e pharmacological actions o f various b e n z o d i a z e p i n e s and t h e i r e f f e c t s t o displace 3H~liazepam binding, this site has b e e n designated as an " a n x i o l y t i c r e c e p t o r " (Squires and Braestrup, 1 9 7 7 ; M o h l e r and O k a d a , 1 9 7 7 ) . Several diverse c o m p o u n d s , a m o n g the Ni 2÷ (Mackerer a n d K o c h m a n , 1 9 7 8 ) , 7 - a m i n o b u t y r i c acid ( G A B A ) and m u s c i m o l (Tallman et al., 1 9 7 8 ; Williams and Risley, 1 9 7 9 ) and a novel a n x i o l y t i c , SQ 2 0 0 0 9 (Williams and Risley, 1 9 7 9 ) have b e e n s h o w n to e n h a n c e the binding o f aH-diazepam t o rat brain m e m branes. T h e significance o f these findings is * Avermectin Bla = (2aE,4E,5'S,6S,6'R,7S,8E,11R, 13R,15S,17aR,20R,20aR,20bS)-6'- [ (R)-sec-butyl ] -7[ [ 2,6-dideoxy-4-O-( 2,6-dideoxy-3-O-methyl-(~-L-arabino-hexopyranosyl)-3-O-methyl-(~-L-arabino-hexopyranosyl] oxy ]-5t,6,6t,7,10,11,14,15,17a,20,20a,20b o dodecahydro-20,20b-dihydroxy-5P,6,8,19-tetramethylspiro-[ 11,15-methano-2H,13H,17H-furo-[4,3,2-pq] [ 2, 6 ] benzodioxyacyclo-octadecin°l3,2'- [ 2H ] pyran ] -17one.
n o t e n t i r e l y clear a l t h o u g h t h e m o d u l a t i o n of 3H-diazepam binding b y GABAergic agents appears c o m p a t i b l e w i t h the w e l l - k n o w n in vivo i n t e r a c t i o n s o f b e n z o d i a z e p i n e s and G A B A ( H a e f l e y et al., 1 9 7 5 ) . A d d i t i o n a l l y , the i n t e r a c t i o n s o f SQ 2 0 0 0 9 with d i a z e p a m binding m a y have some relevance to t h e purp o r t e d a n x i o l y t i c actions o f this c o m p o u n d (Beer et al., 1 9 7 2 ) . In t h e present studies, we have e x a m i n e d t h e i n t e r a c t i o n o f a novel m a c r o c y c l i c l a c t o n e disaccharide a n t h e l m i n t i c agent, A v e r m e c t i n S l a * (AVM: Fritz et al., 1 9 7 9 ) with aH-diazepam binding and with some o f t h e p h a r m a c o l o g i c a l actions o f diazepam.
2. Materials and m e t h o d s 2.1. aH-diazepam b i n d i n g
Binding o f aH-diazepam t o c r u d e s y n a p t i c m e m b r a n e s p r e p a r e d f r o m rat brain was m e a s u r e d b y a m o d i f i c a t i o n o f previously published m e t h o d s (Squires and Braestrup, 1 9 7 7 ; Mohler and O k a d a , 1 9 7 7 ) . T h e binding assay r o u t i n e l y consisted o f triplicate t u b e s to
274 which were added: 3H-diazepam (NEN; specific activity 39.1 Ci/mmol) to a final c o n cen tr atio n o f 1.5 nM; 1 ml of P2 protein suspension (1 mg/ml) and 50 mM Tris HC1 pH 7.4 to a final volume of 2 ml. Experimental c o m p o u n d s when used were diluted in 50 mM Tris HC1 pH 7.4 and added to the incubation m i x t ur e in a volume o f 0.1 ml to t he final concentrations indicated. The reaction was initiated by the addition of m e m b r a n e protein to tubes maintained at 4°C in an ice water bath and continued for 15 min at which time equilibrium was reached, and terminated by filtration u n d er vacuum through Whatman G F / B filters. Th e filters were washed twice with 5 ml of ice-cold 50 mM Tris HC1 pH 7.4, transferred to scintillation vials and 10 ml o f Aquasol 2 (NEN) added. After shaking at r o o m t e m p e r a t u r e for 30 min, radioactivity was measured in a Packard Model 3255 Scintillation S p e c t r o m e t e r at an efficiency of 38%. Specific binding was det er m i ned as the difference in the radioactivity b o u n d in the absence and presence of 1 pM unlabelled diazepam and was a p p r o x i m a t e l y 90% of the total radioactivity bound. Scatchard analysis o f the changes in 3H-diazepam binding was determined over a c o n c e n t r a t i o n range of 0.625--10 nM.
2.2. Behavioral studies Carworth, CF1, female mice weighing 18-24 g were used in all studies. "Muscle p o w e r " was assessed through the use of a fixed wire mesh platform attached to a calibrated linear differential tr an s f or m e r which was c o n n e c t e d electrically to a Servo pen recorder. Mice placed on the p lat f or m and restrained by t he tail exerted considerable force in attempting to grip the mesh when pulled across it by t he tail. This force was r e c or ded as spaces of pen deflection. L o c o m o t o r activity was assessed with " d o n u t " shaped photocell chambers consisting o f a circular r unw a y with a central light source and six photocells equally spaced around th e o u ter perimeter of the runway. Counts resulting f r om i n t e r r u p t i o n of the
M. WILLIAMS, G.G. YARBROUGH light beams were recorded on Sodeco counters. Animals were placed (one mouse per chamber) in a particular chamber on a randomized basis, and treated and cont rol animals were tested concurrently.
3. Results
3.1. 3H-diazepam binding AVM caused a c o n c e n t r a t i o n - d e p e n d e n t increase in t he binding o f 3H-diazepam to rat brain membranes (table 1) with a near maximal effect observed at 10 pM. Examination of the effects of AVM on 3H-diazepam binding by kinetic analysis showed that the increase in binding was reflected as an increase in bot h the affinity (decrease in KD) and n u m b e r of binding sites (table 1). The effects of AVM on 3H-diazepam binding to mouse brain membranes yielded similar data to t hat observed in rats {data n o t presented).
TABLE 1 AVM enhancement of 3H-diazepam binding to rat brain membranes. (A) Concentration--responses of AVM to enhance 3Hdiazepam binding AVM concentration (pM)
% Increase in binding (mean _+S.D.; n = 4)
0.1 1 10
19 -+ 9 44_+7 65 _+3
(B) The effect of 500 nM AVM on affinity and number of 3H-diazepam binding sites Control +500 nM % Change (mean + S.D.; AVM (signifin = 4) (mean -+S.D.; cance) n=4) KD
4.56-+ 0.46
3.65 -+ 0.32 --20
n (pmoles/ m g protein)
1.46 -+ 0.07
1.64 + 0.06
(P < 0.02) +12 (P < 0.01)
AVM ENHANCEMENT OF DIAZEPAM BINDING To the extent tested the action of AVM on 3 H - d i a z e p a m b i n d i n g a p p e a r e d specific i n s o f a r as it e x h i b i t e d n o activity at c o n c e n t r a t i o n s u p t o 50 pM o n t h e b i n d i n g o f 3H-WB 4 1 0 1 (a-adrenergic receptor), 3H-GABA (GABA r e c e p t o r ) , 3H-spiroperidol ( d o p a m i n e recept o r ) , 3H-quinuclidinyl b e n z i l a t e ( m u s c a r i n i c cholinergic r e c e p t o r ) , 3H-kainic acid (glutamate receptor) and 3H.LSD (serotonin receptor).
3.2. Behavioral studies P r e t r e a t m e n t w i t h A V M (5 m g / k g i.p.) e n h a n c e d t h e r e d u c t i o n in " m u s c l e p o w e r " i n d u c e d b y d i a z e p a m ( t a b l e 2). This d o s e o f A V M did n o t a l t e r t h e m u s c l e r e l a x a n t p r o p e r t i e s o f c y c l o b e n z a p r i n e assessed b y this test procedure. Furthermore, diazepam caused a d o s e - r e l a t e d d e c r e a s e in s p o n t a n e o u s m o t o r a c t i v i t y w h i c h was e n h a n c e d at all d o s e levels b y p r e t r e a t m e n t ( 3 0 m i n p r i o r t o receiving d i a z e p a m ) w i t h 5 m g / k g i.p. o f A V M
TABLE 2 Enhancement by AVM of the decrease in 'muscle power' induced by diazepam 1 Pretreatment 2 (mg/kg i.p.)
Treatment 3 (mg/kg i.p.)
Units of pen deflection 4 (X -+ S.E.M.)
Methocel Methocel Methocel Methocel AVM (5) AVM (5) AVM (5) AVM (5)
Methocel Diazepam Diazepam Diazepam Diazepam Diazepam Diazepam Methocel
9.4 7.5 7.1 6.3 5.3 4.8 4.0 9.0
+ + + + + + + +
(5) (10) (20) (5) (10) (20)
+ 0.2 + 0.4 + 0.4 -+ 0.5 + 0.5 -+ 0.4 -+ 0.4 -+ 0.2
s s s 6 6 6
1 N = 20 mice/treatment. 2 30 min before treatment. 3 30 rain before test. 4 10 units of pen deflection = 100 g. s Significantly different (P < 0.05) from methocel + methocel (Duncan's multiple range test). 6 Significantly different (P < 0.05) from methocel + corresponding doses of diazepam (Duncan's multiple range test).
275 {data n o t s h o w n ) . This d o s e o f A V M did n o t significantly a f f e c t m o t o r a c t i v i t y b y itself. 4. Discussion AVM enhances the binding of 3H-diazepam t o rat brain m e m b r a n e s b y increasing b o t h t h e a f f i n i t y and n u m b e r o f b i n d i n g sites f o r t h e b e n z o d i a z e p i n e . T o o u r k n o w l e d g e this is t h e first s u b s t a n c e s h o w n t o increase t h e n u m b e r o f b i n d i n g sites available t o t h e r a d i o l i g a n d , Ni 2÷, G A B A , m u s c i m o l a n d S Q 2 0 0 0 9 prod u c i n g t h e i r e f f e c t s solely via a c h a n g e in t h e a f f i n i t y o f t h e b i n d i n g site. T o t h e e x t e n t t e s t e d , this e f f e c t o f A V M a p p e a r s specific to t h e d i a z e p a m r e c e p t o r . F u r t h e r m o r e , t h e observed in vivo e n h a n c e m e n t b y A V M o f d i a z e p a m i n d u c e d decreases in m o t o r a c t i v i t y a n d m u s c l e p o w e r w o u l d a p p e a r t o indicate a degree o f p h a r m a c o l o g i c a l relevance f o r t h e e n h a n c e m e n t o f binding o b s e r v e d in vitro. Clearly, f u r t h e r studies are r e q u i r e d t o determ i n e t h e specificity o f t h e p h a r m a c o l o g i c a l i n t e r a c t i o n b e t w e e n A V M a n d d i a z e p a m in vivo. F u r t h e r m o r e , while it is possible t h a t this i n t e r a c t i o n m a y have relevance to t h e n o v e l a n t h e l m i n t i c a c t i o n s o f this m a c r o l i d e a n d its r e p o r t e d e f f e c t s on chloride c h a n n e l s in i n v e r t e b r a t e s (Fritz et al., 1 9 7 9 ) , t h e lack o f d i a z e p a m b i n d i n g sites in i n v e r t e b r a t e s (Neilsen et al., 1 9 7 8 ) w o u l d q u e s t i o n such an e x t r a p o l a t i o n . I t m a y be n o t e d , h o w e v e r , that a relationship between the diazepam r e c e p t o r a n d chloride a n i o n c h a n n e l s in m a m m a l i a n brain m e m b r a n e s has b e e n i n f e r r e d (Costa et al., 1979). Acknowledgements The authors are most appreciative of the expert technical assistance provided by J. McGuffin, E.A. Risley and J.A. Totaro during these studies. References
Beer, B., M. Chasin, D.E. Clody, J.R. Vogel and Z.P. Horovitz, 1972, Cyclic adenosine monophosphate
276 phosphodiesterase in brain: Effect on anxiety, Science 176,428. Costa, T., D. Rodbard and C.B. Pert, 1979, Is the benzodiazepine receptor coupled to a chloride anion channel?, Nature London 277,315. Fritz, L.C., C.C. Wang and A. Gorio, 1979, Avermectin Bla blocks inhibitory and excitatory postsynaptic potentials at the lobster neuromuscular junction, Proc. Nat. Acad. Sci. U.S.A. (in press). Haefley, W., A. Kulscar, H. Mohler, L. Pieri, P. Polc and R. Schaffner, 1975, Possible involvement of GABA in the central actions of benzodiazepines, Advan. Biochem. Psychopharmacol. 14,131. Mackerer, C.R. and R.L. Kochman, 1978, Effects of cations and anions on the binding of 3H-diazepam to rat brain, Proc. Soc. Exptl. Biol. Med. 158, 393. Mohler, H. and T. Okada, 1977, Properties of 3H-
M. WILLIAMS, G.G. YARBROUGH diazepam binding to benzodiazepine receptor in the rat cerebral cortex, Life Sci. 20, 2102. Neilsen, M., C. Braestrup and R.F. Squires, 1978, Evidence for a late evolutionary appearance of brain-specific benzodiazepine receptors: an investigation of 18 vertebrate and 5 invertebrate species, Brain Res. 141,342. Squires, R.F. and C. Braestrup, 1977, Benzodiazepine receptors in rat brain, Nature London 266, 732. Tallman, J.F., J.W. Thomas and D.W. Gallager, 1978, GABAergic modulation of benzodiazepine binding site sensitivity, Nature London 274,383. Williams, M. and E.A. Risley, 1979, Enhancement of the binding of 3H-diazepam to rat brain membranes in vitro by SQ 20009, a novel anxiolytic, 7-aminobutyric acid (GABA) and muscimot, Life Sci. 24 (in press).