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Effects of acute cannabis use on urinary neurotransmitter metabolites and cyclic nucleotides in man
Drug and Alcohol
Dependence,
Elsevier
Publishers
Scientific
14 (1984) Ireland
175
175-178
Ltd.
EFFECTS OF ACUTE CANNABIS USE ON URINARY NEUROTRANSMITTER METABOLITES AND CYCLIC NUCLEOTIDES IN MAN
MANOLIS
MARKIANOS
and ANTONIS
Athens University Medical School, Sophias 74, Athens (Greece) (Received
VAKIS
Department
of Psychiatry,
Eginition
Hospital,
Vass.
June Sth, 1981)
SUMMARY
The noradrenaline, dopamine and serotonin metabolites methoxyhydroxyphenylglycol (MHPG), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), as well as the cyclic nucleotides c-AMP and c-GMP were estimated in urine samples of five normal volunteers. Ten control samples and two samples after cannabis use were analyzed for each volunteer. Cannabis use caused significant decreases in MHPG and c-AMP, and increases in HVA, while 5-HIAA and c-GMP excretion remained unchanged. The results indicate that cannabis use interferes with catecholaminergic mechanisms in man, decreasing the noradrenaline and increasing dopamine turnover, probably through action on presynaptic receptors.
Key
&lords:
Cannabis
- Noradrenaline
- Dopamine
- Serotonin
- c-AMP
- c-GMP
INTRODUCTION
In a previous paper we reported the effects of acute cannabis use and short-term deprivation on plasma dopamine-fl-hydroxylase (DBH) and plasma prolactin (PRL) of six long-term cannabis users [l] . The results suggested that ‘cannabis use in man reduces noradrenergic and enhances dopaminergic activity’. Further data are necessary to support this. Studies on neurochemical correlates in relation to tetrahydrocannabinol (THC) in man are rare. The available information on the mode of action of THC on Abbreviations: DA, dopamine; DBH, dopamine-p-hydroxylase; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; MHPG, methoxyhydroxyphenylglycol; NA, adrenaline; PRL, plasma prolactin; THC, tetrahydrocannabinol. 0376-8716/84/$03.00 01983 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
176
neurotransmission is derived mostly from animal experiments. In the latter dose-dependent biphasic effects have been found regarding the synthesis and utilization of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) [ 2-41, and a depletion or no significant change of acetylcholine [ 5,6]. Biphasic effects were also reported on the concentrations of c-AMP in mouse brain [ 71 . Generally, low doses stimulate and high doses suppress noradrenergic, dopaminergic, serotoninergic and acetylcholinergic activities. Taking into account that a certain dose can be ‘low’ for a particular neuronal formation and ‘high’ for another, we could ascribe the biphasic action as an action on more sensitive presynaptic receptors (low doses), or on post-synaptic less sensitive receptors (high doses). In this study, we investigated the overall effects of cannabis use on the turnover of neurotransmitters, as reflected on the concentration of their metabolites in urine. To our knowledge, they have not yet been studied in humans in relation to cannabis use. A large part of them derives from the amine metabolism in the central nervous system [8,9] . The understanding of the mechanism of action of THC is important in relation to its psychic and behavioural effects in man, but also with respect to its potential therapeutic uses [lo]. PROCEDURES
AND METHODS
Five normal volunteers (staff and students of the Athens University Medical School), four men and one women, aged 21.--44 years, participated in the study. Night urine was collected for the period 11 p.m. to 8 a.m. In order to establish control values, we collected urine samples from each volunteer on ten normal drug-free nights. At 11 p.m. cannabis oil was then administered which was smoked mixed with tobacco. This was repeated after 3 weeks. On both occasions, all experienced the subjective high. Twenty millilitres from each urine sample were kept at -30°C without preservative until measurements, which were completed within 3 months. MHPG and HVA were estimated by gas chromatography in 0.5-ml urine aliquots. MHPG was extracted in ethyl acetate after hydrolysis with glusulase and derivatized with trifluoroacetic anhydride. HVA was extracted in ethyl ether at acidic pH, transferred to buffer pH 7.0: re-extracted in ether at pH 2.0 and derivatized with trifluoroacetic anhydride and hexafluoroisopropanol. The derivatives, dissolved in ethyl acetate were injected into a 3% SE-30 column at 13O”C, with the electron capture detector at 180°C. The concentrations were calculated from the differences in peak area of known amounts of MHPG and HVA, added to samples at the beginning of the procedure. Each sample had its own internal standard to compensate possible differences in extraction. 5-HIAA was estimated photometrically according to the method of Goldenberg [ 111, with modifications so that the estimation could be made in 2 ml of urine. c-AMP and c-GMP were estimated in diluted urine samples using protein binding assay kits (Amersham Radiochemical Centre), and
creatinine using the Boehringer kit. The concentrations were expressed per mg creatinine. For the statistical evaluation of the differences the paired t-test was used. RESULTS
AND
DISCUSSION
The results are summarized in Table I. Cannabis use caused a significant reduction in MHPG excretion (40% in the mean), which implies a decrease in NA turnover. This, together with our previous finding of a reduction in plasma DBH [l],can be understood as a slow down of the exocytotic process in NA nerve terminals, and resembles the action of clonidine. This could also explain the reduction in c-AMP, which is also significant. Changes in plasma c-GMP levels may reflect cholinergic function in humans [ 121. If such changes occur during cannabis use, they are not reflected in changes in urinary c-GMP excretion. The same is valid for 5-HIAA, the concentrations of which did not change after cannabis use. If cannabis interferes with serotonin turnover, this is not represented in the urinary 5-HIAA output. Of special interest is our finding that cannabis use causes a significant increase in HVA excretion; this finding supports the idea of hyperdopaminergic activity in psychotic states. In connection with our previous finding of a reduction in plasma prolactin after cannabis use [l] , and the fact that by the suppression of prolactin secretion in the rat, THC acts on the central nervous system and not directly on the pituitary [ 131, we can exclude the TABLE
I
MHPG, HVA, 5HIAA, VOLUNTEERS
c-AMP AND c-GMP IN URINE
SAMPLES
FROM
Control values are the mean values from 10 urine samples from cannabis values the means of 2 samples after cannabis smoking. expressed in nmol/mg creatinine.
MHPG
HVA
5-HIAA
c-AMP
c-GMP
each volunteer, Concentrations
and are
r
P
f 1.2 +1.4
+3.04
0.025
9.5 14.2
f5.6 f 7.1
-6.40
0.001
11.3 12.3
+1.6 +6.1
-0.41
N.S.
1
2
3
4
5
Control Cannabis
5.6 4.6
5.6 3.8
5.1 3.1
7.6 1.9
7.6 5.6
6.3 3.8
Control Cannabis
9.8 15.1
7.3 10.2
4.9 9.7
19.0 26.2
6.3 9.9
Control Cannabis
11.1 9.8
11.7 9.3
8.8 11.2
13.2 23.0
11.5 8.0
Subject
FIVE NORMAL
f f S.D.
Control Cannabis
3.26 3.40
3.10 2.40
4.70 3.90
3.72 2.52
3.86 2.89
3.73 3.02
+ 0.63 + 0.63
+3.10
0.025
Control Cannibis
0.43 0.49
0.71 0.76
0.28 0.28
0.57 0.63
0.92 0.86
0.58 0.60
k 0.25 +0.23
-0.94
N.S.
178
possibility that the increase in DA turnover is caused by an action of THC on postsynaptic DA receptors. More probable is an antagonistic action on presynaptic autoreceptors that regulate DA release. The hypotensive action of cannabis could be explained by the clonidine-like action mentioned, and by the increase in DA turnover, i.e. the stimulation of peripheral dopamine receptors that cause vasodilatation, inhibition of ganglionic transmission and inhibition of transmitter release on the sympathetic nerve terminals [ 141 . REFERENCES 1 2 3 1 5 6 7 8 9 10 11 12 13
M. Markianos and C. Stefanis, Drug Alcohol Depend., 9 (1982) 251. A.S. Bloom and C.J. Kiernan, Psychopharmacology, 67 (1980) 215. B.T. Ho et al., Brain Res., 38 (1972) 163. D.A. Taylor and M.R. Fennessy, Eur. J. Pharmacol., 46 (1977) 93. W.E. Askew, A.P. Kimball and B.T. Ho, Brain Res., 69 (1974) 375. A.K. Bhattacharyya et al., Neuropharmacology, 19 (1980) 87. T.W. Dolby and L.J. Kleinsmith, Biochem. Pharmacol., 23 (1974) 1817. J.W. Maas et al., Science, 205 (1979) 1025. J.W. Maas et al., J. Neurochem., 3-l (1980) 1517. L. Lemberger, Annu. Rev. Pharmacol. Toxicol., 20 (1980) 151. H. Goldenberg, Clin. Chem., 19 (1973) 38. F. Okada, M. Honma and M. Ui, J. Clin. Endocrinol. Metab., 54 (1982) 645. C.L. Hughes, J.W. Everett and L. Tyrey, Endocrinology, 109 (1981) 876.
Dependence,
Elsevier
Publishers
Scientific
14 (1984) Ireland
175
175-178
Ltd.
EFFECTS OF ACUTE CANNABIS USE ON URINARY NEUROTRANSMITTER METABOLITES AND CYCLIC NUCLEOTIDES IN MAN
MANOLIS
MARKIANOS
and ANTONIS
Athens University Medical School, Sophias 74, Athens (Greece) (Received
VAKIS
Department
of Psychiatry,
Eginition
Hospital,
Vass.
June Sth, 1981)
SUMMARY
The noradrenaline, dopamine and serotonin metabolites methoxyhydroxyphenylglycol (MHPG), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), as well as the cyclic nucleotides c-AMP and c-GMP were estimated in urine samples of five normal volunteers. Ten control samples and two samples after cannabis use were analyzed for each volunteer. Cannabis use caused significant decreases in MHPG and c-AMP, and increases in HVA, while 5-HIAA and c-GMP excretion remained unchanged. The results indicate that cannabis use interferes with catecholaminergic mechanisms in man, decreasing the noradrenaline and increasing dopamine turnover, probably through action on presynaptic receptors.
Key
&lords:
Cannabis
- Noradrenaline
- Dopamine
- Serotonin
- c-AMP
- c-GMP
INTRODUCTION
In a previous paper we reported the effects of acute cannabis use and short-term deprivation on plasma dopamine-fl-hydroxylase (DBH) and plasma prolactin (PRL) of six long-term cannabis users [l] . The results suggested that ‘cannabis use in man reduces noradrenergic and enhances dopaminergic activity’. Further data are necessary to support this. Studies on neurochemical correlates in relation to tetrahydrocannabinol (THC) in man are rare. The available information on the mode of action of THC on Abbreviations: DA, dopamine; DBH, dopamine-p-hydroxylase; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; MHPG, methoxyhydroxyphenylglycol; NA, adrenaline; PRL, plasma prolactin; THC, tetrahydrocannabinol. 0376-8716/84/$03.00 01983 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
176
neurotransmission is derived mostly from animal experiments. In the latter dose-dependent biphasic effects have been found regarding the synthesis and utilization of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) [ 2-41, and a depletion or no significant change of acetylcholine [ 5,6]. Biphasic effects were also reported on the concentrations of c-AMP in mouse brain [ 71 . Generally, low doses stimulate and high doses suppress noradrenergic, dopaminergic, serotoninergic and acetylcholinergic activities. Taking into account that a certain dose can be ‘low’ for a particular neuronal formation and ‘high’ for another, we could ascribe the biphasic action as an action on more sensitive presynaptic receptors (low doses), or on post-synaptic less sensitive receptors (high doses). In this study, we investigated the overall effects of cannabis use on the turnover of neurotransmitters, as reflected on the concentration of their metabolites in urine. To our knowledge, they have not yet been studied in humans in relation to cannabis use. A large part of them derives from the amine metabolism in the central nervous system [8,9] . The understanding of the mechanism of action of THC is important in relation to its psychic and behavioural effects in man, but also with respect to its potential therapeutic uses [lo]. PROCEDURES
AND METHODS
Five normal volunteers (staff and students of the Athens University Medical School), four men and one women, aged 21.--44 years, participated in the study. Night urine was collected for the period 11 p.m. to 8 a.m. In order to establish control values, we collected urine samples from each volunteer on ten normal drug-free nights. At 11 p.m. cannabis oil was then administered which was smoked mixed with tobacco. This was repeated after 3 weeks. On both occasions, all experienced the subjective high. Twenty millilitres from each urine sample were kept at -30°C without preservative until measurements, which were completed within 3 months. MHPG and HVA were estimated by gas chromatography in 0.5-ml urine aliquots. MHPG was extracted in ethyl acetate after hydrolysis with glusulase and derivatized with trifluoroacetic anhydride. HVA was extracted in ethyl ether at acidic pH, transferred to buffer pH 7.0: re-extracted in ether at pH 2.0 and derivatized with trifluoroacetic anhydride and hexafluoroisopropanol. The derivatives, dissolved in ethyl acetate were injected into a 3% SE-30 column at 13O”C, with the electron capture detector at 180°C. The concentrations were calculated from the differences in peak area of known amounts of MHPG and HVA, added to samples at the beginning of the procedure. Each sample had its own internal standard to compensate possible differences in extraction. 5-HIAA was estimated photometrically according to the method of Goldenberg [ 111, with modifications so that the estimation could be made in 2 ml of urine. c-AMP and c-GMP were estimated in diluted urine samples using protein binding assay kits (Amersham Radiochemical Centre), and
creatinine using the Boehringer kit. The concentrations were expressed per mg creatinine. For the statistical evaluation of the differences the paired t-test was used. RESULTS
AND
DISCUSSION
The results are summarized in Table I. Cannabis use caused a significant reduction in MHPG excretion (40% in the mean), which implies a decrease in NA turnover. This, together with our previous finding of a reduction in plasma DBH [l],can be understood as a slow down of the exocytotic process in NA nerve terminals, and resembles the action of clonidine. This could also explain the reduction in c-AMP, which is also significant. Changes in plasma c-GMP levels may reflect cholinergic function in humans [ 121. If such changes occur during cannabis use, they are not reflected in changes in urinary c-GMP excretion. The same is valid for 5-HIAA, the concentrations of which did not change after cannabis use. If cannabis interferes with serotonin turnover, this is not represented in the urinary 5-HIAA output. Of special interest is our finding that cannabis use causes a significant increase in HVA excretion; this finding supports the idea of hyperdopaminergic activity in psychotic states. In connection with our previous finding of a reduction in plasma prolactin after cannabis use [l] , and the fact that by the suppression of prolactin secretion in the rat, THC acts on the central nervous system and not directly on the pituitary [ 131, we can exclude the TABLE
I
MHPG, HVA, 5HIAA, VOLUNTEERS
c-AMP AND c-GMP IN URINE
SAMPLES
FROM
Control values are the mean values from 10 urine samples from cannabis values the means of 2 samples after cannabis smoking. expressed in nmol/mg creatinine.
MHPG
HVA
5-HIAA
c-AMP
c-GMP
each volunteer, Concentrations
and are
r
P
f 1.2 +1.4
+3.04
0.025
9.5 14.2
f5.6 f 7.1
-6.40
0.001
11.3 12.3
+1.6 +6.1
-0.41
N.S.
1
2
3
4
5
Control Cannabis
5.6 4.6
5.6 3.8
5.1 3.1
7.6 1.9
7.6 5.6
6.3 3.8
Control Cannabis
9.8 15.1
7.3 10.2
4.9 9.7
19.0 26.2
6.3 9.9
Control Cannabis
11.1 9.8
11.7 9.3
8.8 11.2
13.2 23.0
11.5 8.0
Subject
FIVE NORMAL
f f S.D.
Control Cannabis
3.26 3.40
3.10 2.40
4.70 3.90
3.72 2.52
3.86 2.89
3.73 3.02
+ 0.63 + 0.63
+3.10
0.025
Control Cannibis
0.43 0.49
0.71 0.76
0.28 0.28
0.57 0.63
0.92 0.86
0.58 0.60
k 0.25 +0.23
-0.94
N.S.
178
possibility that the increase in DA turnover is caused by an action of THC on postsynaptic DA receptors. More probable is an antagonistic action on presynaptic autoreceptors that regulate DA release. The hypotensive action of cannabis could be explained by the clonidine-like action mentioned, and by the increase in DA turnover, i.e. the stimulation of peripheral dopamine receptors that cause vasodilatation, inhibition of ganglionic transmission and inhibition of transmitter release on the sympathetic nerve terminals [ 141 . REFERENCES 1 2 3 1 5 6 7 8 9 10 11 12 13
M. Markianos and C. Stefanis, Drug Alcohol Depend., 9 (1982) 251. A.S. Bloom and C.J. Kiernan, Psychopharmacology, 67 (1980) 215. B.T. Ho et al., Brain Res., 38 (1972) 163. D.A. Taylor and M.R. Fennessy, Eur. J. Pharmacol., 46 (1977) 93. W.E. Askew, A.P. Kimball and B.T. Ho, Brain Res., 69 (1974) 375. A.K. Bhattacharyya et al., Neuropharmacology, 19 (1980) 87. T.W. Dolby and L.J. Kleinsmith, Biochem. Pharmacol., 23 (1974) 1817. J.W. Maas et al., Science, 205 (1979) 1025. J.W. Maas et al., J. Neurochem., 3-l (1980) 1517. L. Lemberger, Annu. Rev. Pharmacol. Toxicol., 20 (1980) 151. H. Goldenberg, Clin. Chem., 19 (1973) 38. F. Okada, M. Honma and M. Ui, J. Clin. Endocrinol. Metab., 54 (1982) 645. C.L. Hughes, J.W. Everett and L. Tyrey, Endocrinology, 109 (1981) 876.