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Effect of tetrahydrocannabinols on brain acetylcholine
Brain Research, 69 (1974) 375-378 ~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
375
Effect of tetrahydrocannabinols on brain acetylcholine
WILLIAM E. ASKEW, A. P. KIMBALL AND BENG T. HO* Texas Research Institute of Mental Sciences, Houston, Tex. 77025 and Department of Biophy~'ical Sciences, University of Houston, Houston, Tex. 77004 (U.S.A.)
(Accepted December 14th, 1973)
A tetrahydrocannabinol (THC), trans(--)-AS-THC, was reported to have an effect similar to that of anticholinergic agents in abolishing the behavioral inhibition of habituating experience in mice 1. It was then suggested that the tetrahydrocannabinol perhaps exerts its effect via an anticholinergic mechanism1; however, little biochemical evidence supports this postulation. We have investigated the effects of Ag-THC, AS-THC and its major metabolite, 11-hydroxy-AS-THC (11-HO-AS-THC) on brain acetylcholine (ACh) in the rat. This report describes the result of these findings. Male Sprague-Dawley rats, 200-250 g, were injected intravenously with various doses of tetrahydrocannabinols in 4% Tween-80 and saline suspension. Animals were sacrificed 1 h after the injection by exposure to microwave radiation in a commercial microwave oven for 30 sec (see ref. 9). ACh was extracted from brain tissue into a mixture of 90% acetone and 1 0 ~ 1 N formic acid (1:3, w/w) by a modified method of Saelens et al. s. ACh content in brain was determined by the method of Reid et al. 7, which involves the separation of ACh electrophoretically on Schlecher and Schnell paper strips, hydrolysis to choline, and incubation with choline kinase in the presence of [32P]ATP. The radioactive phosphorylcholine was then assayed by liquid scintillation spectrometry against a standard curve. Choline kinase was prepared for Ehrlich's ascites tumor fluid as described by Reid et al. 7. Synaptosomes were isolated by the procedure of Gray and Whittaker 4. For confirmation ACh was also assayed by the procedure of Goldberg and McCaman 3' ACh was extracted into a mixture of 85 % acetone and 15 ~/o I N formic acid (1 : 6.6, w/w) and in the absence of endogenous choline, was then converted by eel ACh esterase and [32P]ATP to [~2P]choline for quantitation by liquid scintillation spectrometry. For determination of choline acetyltransferase and acetylcholine esterase the animals were sacrificed by decapitation. Choline acetyltransferase activity was measur* To whom reprint requests should be addressed: Texas Research Institute of Mental Sciences, Houston, Tex. 77025, U.S.A.
376
SHORT COMMUNICATIONS
TABLE 1 RAT BRAIN
ACh
I.EVEI.S [ h AFTER INTRAVENOUS INJECTION O1- 5
mg/kg OFTHE TEl RAHYI)RO(
,\NNABINOI
Control animals received 4 !~,;Tween-80 in saline only. Values in parentheses represent the mean Assay method
d C h (nmoles/g) ('onttvl
1S-THC
13.79 19.71 15.8/ 19.63 16.94 16.08 15,36 13.90 20.72 22,61 (17.46 ! 3.02)
Reid et al."
Goldberg and McCaman :~ 28.51 29.86 25.84 26.89 28.62 (27.94 * P < 0.01,
S.E.
** P
1.58)
5.27 10.67 4.67 3.99 2.59 19.58 19.14
I',~-THC
] 1- HO- ] 8_TIt("
14.38 18.99 18.27 10.63 9.71 10.43 13.73
(8.30 :L 4.64)*
11.5() t 8.4O 13.7 i 12~62 10.41 16.29 2 l.S0 15,4 l /4,7t~ 20,83 (13.73 i: 3.77)** 115.57
19.54 22.75 22.03
21.45 25.07 21.86
27.32 19.04
(21.44 L 1.68)*
(22.79 ! 1,98)*
(2'3,7:t . 424)
3.82)
24,82
0.05 (significance of differences between drug and control valuesL
ed by the m e t h o d o f Schrier and Shuster 1° using [1-t4C]acetyl C o A and choline, and acetylcholine esterase activity by the colorimetric m e t h o d o f Ellman e t al. z using the substrate acetylthiocholine iodide. Protein determinations were performed by the m e t h o d o f Lowry5. The effect of 5 mg/kg of AS-THC, 1 t - H O - A S - T H C and Ag-THC on whole brain A C h content is shown in Table I. A8-THC and Ag-THC produced a marked depletion of A C h in the rat brain, while I 1-HO-AS-THC had no significant effect on the A C h content, which suggests that the metabolite o f AS-THC is not active in the depletion of A C h (Table I). These results are consistent regardless of the method used for assaying ACh. N o significant decrease in choline acetyltransferase activity was found in the brain o f A8-THC treated animals (Table II), suggesting that the decrease in brain A C h was not due to decreased synthesis. The finding of a 50~o depletion o f ACh in the synaptosomes by AS-THC (Table IIl) implies that the primary mode of action o f the tetrahydrocannabinol is on the release of A C h from the storage. Since only a 10%£ decrease in activity of A C h esterase was observed (Table ll), the enzyme is likely to be present in quantities sufficient to hydrolyze any A C h released: the activity o f A C h esterase in rat brains is much higher than that of choline acetyltransferase with the rate of hydrolysis o f A C h being reported as 80 to I00 mg/g/h compared to a reported synthesis rate o f 2.0 to 2.5 mg/g/h (see ref. 6). Our results, therefore, suggest that AS-THC exerted its anticholinergic effect by increasing the release o f A C h
377
SHORT COMMUNICATIONS TABLE lI EFFECT OF
A8-THC ON A C T I V I T I E S
OF C H O L I N E A C E T Y L T R A N S F E R A S E A N D A C E T Y L C H O L I N E
ESTERASE I N
THE RAT BRAIN
Each value represents the mean (~: S.E.) of 6 assays. Group
Choline acetyltranaJerase (nmole/h/mg)
A Ch esterase (mmole substrate hydrolyzed/min/g)
Control AS-THC
24.79 ~ 0.03 24.05 _~: 0.02
1.80 ~_ 0.18 1.61 L 0.15"
* P -< 0.05 (significance of difference between the drug and control values). TABLE 111 ACh CONTENTIN RAT BRAIN SYNAPTOSOMESISOLATED I tl AFTERINTRAVENOUSINJECTIONOF 5 mg/kg OF AS-THC 1N RATS Control animals received 4~o Tween-80 in saline only. Values in parentheses represent the mean :: : S.E. Assay method
ACh (nmoles/g) Control
al s_THC
Reid et al5
10.81 15.25 6.88 11.83 10.47 (11,05 :k 3.00)
4.05 6.56 6.66 2.30 8.04 (5.52 - 2.31)
Goldberg and McCaman 3
9.53 6.80 10.24 (8.85 -- 1.81)*
3.37 4.25 1.55 (3.07 :- 1.37)**
* P < 0.002,
** P < 0.01 (significance of differences between drug and control values).
which, u p o n s u b s e q u e n t d e g r a d a t i o n , decreases its a m o u n t f o r n e u r o t r a n s m i s s i o n . A C h levels e s t i m a t e d by b o t h e n z y m a t i c a n d b i o a s s a y t e c h n i q u e s usually v a r y f r o m l a b o r a t o r y to l a b o r a t o r y . Sacrifice by m i c r o w a v e r a d i a t i o n d e n a t u r a t e s b o t h c h o l i n e a c e t y l t r a n s f e r a s e a n d A C h esterase by h e a t while n o t affecting ACh9,11. B e c a u s e o f the i n a c t i v a t i o n o f A C h esterase r e s p o n s i b l e for d e g r a d a t i o n o f A C h , the m e t h o d gives h i g h e r A C h values t h a n the m o r e c o m m o n sacrifice t e c h n i q u e 11. T h e t r a u m a o f c o n v e n t i o n a l sacrifice p r e s u m a b l y
releases A C h
which is t h e n r a p i d l y
m e t a b o l i z e d by the still active A C h esterase resulting in l o w e r A C h c o n t e n t in tissues.
1 BROWN, I-l., Some anticholinergic-like behavioral effects of trans(--)-A~-tetrahydrocannabinol, Psychopharmacologia (Bed.), 21 (1971 ) 294-301. 2 ELLMAN, G,, COURTNY, D., ANDRES, V., AND FEATHERSTONE,R., A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. PharmacoL, 7 (1961) 88-95.
378
SHORT COMMU NICATIONS
3 GOLDBERG, A. M., AND MCCAMAN, R. E., The determination of picomole amount~ ot acetytcholine in mammalian brain, J. Neurochem., 20 (1973) 1-8. 4 GRAY, E. G., AND WHITTAKER, V. P., The isolation of nerve endings from brain. A~ electronmicroscopic study of cell fragments derived by homogenization and centrifugatiol:, J. Anat. (Lond.), 96 (1962) 79-87. 5 LOWRY, O. H., ROSEBROUGH, N. H., FARR, L., AND RANDALL, R. 3., Protein measurements with the folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 6 NACItMANSOI4N, D., Cholinesterases and anticholinesterase agents. In G. B. KOELLi-' (Ed.), Handbuch der experimentelh, n Pharmakolog&, XVI, Springer, Berlin, 1963, p. 44. 7 REID, W., HAUBRICH, D., AND KRISHNA, G., Enzymic radioassay for acetylcholine and choline in brain, Analyt. Biochem., 42 (1971) 390-397. 8 SAELENS,J. K., ALLEN, M. P., AND SIMKE, J. P., Determination of acetylcholine and choline by an enzymatic assay, Arch. int. Pharmacodyn., 186 (1970) 279-286. 9 SCHr~IDT, D. E., SPETH, R. C., WELSCH, F., AND SCHMIDT, M. J., The use of microwave radiation in the determination of acetylcholine in the rat brain, Brain Research, 38 (1972) 377 -389. 10 SCHRIER, B. K., aNo St~USTER, L., A simplified radiochemical assay for choline acetyl transferase, J. Neurochem., 14 (1967) 977-985. 11 STAVINOHA,W. B., WEINTRAUB, S. T., AND MODAK, A. T., The use of microwave heating to inactivate cholinesterase in the rat brain prior to analysis for acetylcholine, J. Neurochem., 20 (1973) 361-371
375
Effect of tetrahydrocannabinols on brain acetylcholine
WILLIAM E. ASKEW, A. P. KIMBALL AND BENG T. HO* Texas Research Institute of Mental Sciences, Houston, Tex. 77025 and Department of Biophy~'ical Sciences, University of Houston, Houston, Tex. 77004 (U.S.A.)
(Accepted December 14th, 1973)
A tetrahydrocannabinol (THC), trans(--)-AS-THC, was reported to have an effect similar to that of anticholinergic agents in abolishing the behavioral inhibition of habituating experience in mice 1. It was then suggested that the tetrahydrocannabinol perhaps exerts its effect via an anticholinergic mechanism1; however, little biochemical evidence supports this postulation. We have investigated the effects of Ag-THC, AS-THC and its major metabolite, 11-hydroxy-AS-THC (11-HO-AS-THC) on brain acetylcholine (ACh) in the rat. This report describes the result of these findings. Male Sprague-Dawley rats, 200-250 g, were injected intravenously with various doses of tetrahydrocannabinols in 4% Tween-80 and saline suspension. Animals were sacrificed 1 h after the injection by exposure to microwave radiation in a commercial microwave oven for 30 sec (see ref. 9). ACh was extracted from brain tissue into a mixture of 90% acetone and 1 0 ~ 1 N formic acid (1:3, w/w) by a modified method of Saelens et al. s. ACh content in brain was determined by the method of Reid et al. 7, which involves the separation of ACh electrophoretically on Schlecher and Schnell paper strips, hydrolysis to choline, and incubation with choline kinase in the presence of [32P]ATP. The radioactive phosphorylcholine was then assayed by liquid scintillation spectrometry against a standard curve. Choline kinase was prepared for Ehrlich's ascites tumor fluid as described by Reid et al. 7. Synaptosomes were isolated by the procedure of Gray and Whittaker 4. For confirmation ACh was also assayed by the procedure of Goldberg and McCaman 3' ACh was extracted into a mixture of 85 % acetone and 15 ~/o I N formic acid (1 : 6.6, w/w) and in the absence of endogenous choline, was then converted by eel ACh esterase and [32P]ATP to [~2P]choline for quantitation by liquid scintillation spectrometry. For determination of choline acetyltransferase and acetylcholine esterase the animals were sacrificed by decapitation. Choline acetyltransferase activity was measur* To whom reprint requests should be addressed: Texas Research Institute of Mental Sciences, Houston, Tex. 77025, U.S.A.
376
SHORT COMMUNICATIONS
TABLE 1 RAT BRAIN
ACh
I.EVEI.S [ h AFTER INTRAVENOUS INJECTION O1- 5
mg/kg OFTHE TEl RAHYI)RO(
,\NNABINOI
Control animals received 4 !~,;Tween-80 in saline only. Values in parentheses represent the mean Assay method
d C h (nmoles/g) ('onttvl
1S-THC
13.79 19.71 15.8/ 19.63 16.94 16.08 15,36 13.90 20.72 22,61 (17.46 ! 3.02)
Reid et al."
Goldberg and McCaman :~ 28.51 29.86 25.84 26.89 28.62 (27.94 * P < 0.01,
S.E.
** P
1.58)
5.27 10.67 4.67 3.99 2.59 19.58 19.14
I',~-THC
] 1- HO- ] 8_TIt("
14.38 18.99 18.27 10.63 9.71 10.43 13.73
(8.30 :L 4.64)*
11.5() t 8.4O 13.7 i 12~62 10.41 16.29 2 l.S0 15,4 l /4,7t~ 20,83 (13.73 i: 3.77)** 115.57
19.54 22.75 22.03
21.45 25.07 21.86
27.32 19.04
(21.44 L 1.68)*
(22.79 ! 1,98)*
(2'3,7:t . 424)
3.82)
24,82
0.05 (significance of differences between drug and control valuesL
ed by the m e t h o d o f Schrier and Shuster 1° using [1-t4C]acetyl C o A and choline, and acetylcholine esterase activity by the colorimetric m e t h o d o f Ellman e t al. z using the substrate acetylthiocholine iodide. Protein determinations were performed by the m e t h o d o f Lowry5. The effect of 5 mg/kg of AS-THC, 1 t - H O - A S - T H C and Ag-THC on whole brain A C h content is shown in Table I. A8-THC and Ag-THC produced a marked depletion of A C h in the rat brain, while I 1-HO-AS-THC had no significant effect on the A C h content, which suggests that the metabolite o f AS-THC is not active in the depletion of A C h (Table I). These results are consistent regardless of the method used for assaying ACh. N o significant decrease in choline acetyltransferase activity was found in the brain o f A8-THC treated animals (Table II), suggesting that the decrease in brain A C h was not due to decreased synthesis. The finding of a 50~o depletion o f ACh in the synaptosomes by AS-THC (Table IIl) implies that the primary mode of action o f the tetrahydrocannabinol is on the release of A C h from the storage. Since only a 10%£ decrease in activity of A C h esterase was observed (Table ll), the enzyme is likely to be present in quantities sufficient to hydrolyze any A C h released: the activity o f A C h esterase in rat brains is much higher than that of choline acetyltransferase with the rate of hydrolysis o f A C h being reported as 80 to I00 mg/g/h compared to a reported synthesis rate o f 2.0 to 2.5 mg/g/h (see ref. 6). Our results, therefore, suggest that AS-THC exerted its anticholinergic effect by increasing the release o f A C h
377
SHORT COMMUNICATIONS TABLE lI EFFECT OF
A8-THC ON A C T I V I T I E S
OF C H O L I N E A C E T Y L T R A N S F E R A S E A N D A C E T Y L C H O L I N E
ESTERASE I N
THE RAT BRAIN
Each value represents the mean (~: S.E.) of 6 assays. Group
Choline acetyltranaJerase (nmole/h/mg)
A Ch esterase (mmole substrate hydrolyzed/min/g)
Control AS-THC
24.79 ~ 0.03 24.05 _~: 0.02
1.80 ~_ 0.18 1.61 L 0.15"
* P -< 0.05 (significance of difference between the drug and control values). TABLE 111 ACh CONTENTIN RAT BRAIN SYNAPTOSOMESISOLATED I tl AFTERINTRAVENOUSINJECTIONOF 5 mg/kg OF AS-THC 1N RATS Control animals received 4~o Tween-80 in saline only. Values in parentheses represent the mean :: : S.E. Assay method
ACh (nmoles/g) Control
al s_THC
Reid et al5
10.81 15.25 6.88 11.83 10.47 (11,05 :k 3.00)
4.05 6.56 6.66 2.30 8.04 (5.52 - 2.31)
Goldberg and McCaman 3
9.53 6.80 10.24 (8.85 -- 1.81)*
3.37 4.25 1.55 (3.07 :- 1.37)**
* P < 0.002,
** P < 0.01 (significance of differences between drug and control values).
which, u p o n s u b s e q u e n t d e g r a d a t i o n , decreases its a m o u n t f o r n e u r o t r a n s m i s s i o n . A C h levels e s t i m a t e d by b o t h e n z y m a t i c a n d b i o a s s a y t e c h n i q u e s usually v a r y f r o m l a b o r a t o r y to l a b o r a t o r y . Sacrifice by m i c r o w a v e r a d i a t i o n d e n a t u r a t e s b o t h c h o l i n e a c e t y l t r a n s f e r a s e a n d A C h esterase by h e a t while n o t affecting ACh9,11. B e c a u s e o f the i n a c t i v a t i o n o f A C h esterase r e s p o n s i b l e for d e g r a d a t i o n o f A C h , the m e t h o d gives h i g h e r A C h values t h a n the m o r e c o m m o n sacrifice t e c h n i q u e 11. T h e t r a u m a o f c o n v e n t i o n a l sacrifice p r e s u m a b l y
releases A C h
which is t h e n r a p i d l y
m e t a b o l i z e d by the still active A C h esterase resulting in l o w e r A C h c o n t e n t in tissues.
1 BROWN, I-l., Some anticholinergic-like behavioral effects of trans(--)-A~-tetrahydrocannabinol, Psychopharmacologia (Bed.), 21 (1971 ) 294-301. 2 ELLMAN, G,, COURTNY, D., ANDRES, V., AND FEATHERSTONE,R., A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. PharmacoL, 7 (1961) 88-95.
378
SHORT COMMU NICATIONS
3 GOLDBERG, A. M., AND MCCAMAN, R. E., The determination of picomole amount~ ot acetytcholine in mammalian brain, J. Neurochem., 20 (1973) 1-8. 4 GRAY, E. G., AND WHITTAKER, V. P., The isolation of nerve endings from brain. A~ electronmicroscopic study of cell fragments derived by homogenization and centrifugatiol:, J. Anat. (Lond.), 96 (1962) 79-87. 5 LOWRY, O. H., ROSEBROUGH, N. H., FARR, L., AND RANDALL, R. 3., Protein measurements with the folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 6 NACItMANSOI4N, D., Cholinesterases and anticholinesterase agents. In G. B. KOELLi-' (Ed.), Handbuch der experimentelh, n Pharmakolog&, XVI, Springer, Berlin, 1963, p. 44. 7 REID, W., HAUBRICH, D., AND KRISHNA, G., Enzymic radioassay for acetylcholine and choline in brain, Analyt. Biochem., 42 (1971) 390-397. 8 SAELENS,J. K., ALLEN, M. P., AND SIMKE, J. P., Determination of acetylcholine and choline by an enzymatic assay, Arch. int. Pharmacodyn., 186 (1970) 279-286. 9 SCHr~IDT, D. E., SPETH, R. C., WELSCH, F., AND SCHMIDT, M. J., The use of microwave radiation in the determination of acetylcholine in the rat brain, Brain Research, 38 (1972) 377 -389. 10 SCHRIER, B. K., aNo St~USTER, L., A simplified radiochemical assay for choline acetyl transferase, J. Neurochem., 14 (1967) 977-985. 11 STAVINOHA,W. B., WEINTRAUB, S. T., AND MODAK, A. T., The use of microwave heating to inactivate cholinesterase in the rat brain prior to analysis for acetylcholine, J. Neurochem., 20 (1973) 361-371