TOXICOLOGY

AND APPLIEDPHARMACOLOGY

37,517-524(1976)

Delta-9-Tetrahydrocannabinol : Effect on Adrenal Catecholamines G. MITRA, M. K. PODDAR, AND J. J.

GHOSH

University College of Science, Calcutta University, Department of Biochemistry, Calcutta-700 019, India Received January 20,1976; accepted April 27,1976

Delta-9-Tetrahydrocannabinol : Effect on Adrenal Catecholamines. G., PODDAR, M. K., AND GHOSH, J. J. (1976). Toxicol. Appl. Pharmacol. 37, 517-524. The single ip administration of A9-tetrahydrocannabinol (A9-THC) at dosesof 10 and 50 mg/kg produces a dosedependentincreasein the contentsof noradrenaline(NA) and adrenaline (AD) in the rat adrenalglandat the first phase(45-90 min), and thereafter, declinesignificantly (90-360 min). The content of adrenal dopamine(DA) continues to decreasesharply after the A9-THC administration and remains below normal even after 6 hr. Measurementsof turnover of adrenal catecholaminesmade with the use of FLA-63, an inhibitor of dopamine fi-hydroxylase and cc-methyl-p-tyrosine(a-M-pt), an inhibitor of tyrosine hydroxylase, show that A9-THC given acutely both at low and at high dose(a) decreases the rate of synthesisand increasesthe rate of depletionof DA, (b) decreases the rate of depletionof NA and (c) increases the rate of depletion of AD. Treatment with A9-THC (10 mg/kg/day) for 21 consecutivedayssignificantly elevatesonly the AD content with increase in its turnover rate. The cardiovascularresponses,reported to occur after A9-THC administration, appear to be consistent with the increased adrenalinereleaseobservedin the presentstudy. MITRA,

In recent years many studieshave beenmade on the psychotomimetic action of dg-THC in relation to the biogenic amine metabolism in the central nervous system (Holtzman er al., 1969; Schildkraut and Efron, 1971; Maitre et al., 1974). But relatively little attention has been paid to its effect on catecholamine metabolism in the peripheral sympathetic nervous system. dg-THC affects the cardiovascular system and its autonomic control mechanisms(Beaconsfield et al., 1972; Birmingham, 1973); depletes adrenal catecholamines (Welch et al., 1971); significantly decreasesserum dopamine fl-hydroxylase (Ng et al., 1973); and potentiates the sympathetic response in rats subjected to immobilization stress(Lamprecht et al., 1973).A9-THC and its metabolites accumulate significantly in the adrenal gland, following its acute and chronic administration (Ho et al., 1970; Klausner and Dignell, 1971) and it increasesadrenocortical activity markedly (Kubena and Barry, 1971; Dewey et al., 1970). Thus, it may affect adrenomedullary activity. The present article dealswith the effect of acute and subacute administration of d9-THC on catecholamine metabolism in the rat adrenal gland. Copyright 0 1976 by Academic Press, Inc. 517 All rights of reproduction in any form reserved. Printed

in Great

Britain

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METHODS

Male albino rats of the Charles Foster strain, weighing about 120-150 g and maintained on laboratory stock diet, were used. For acute studies, d9-THC (99% pure, suspended in saline containing 1% Tween-80) was administered ip at 10 and 50 mg/kg, and animals were decapitated (between 4 and 5 PM) at different time intervals (45-360 min) thereafter. In subacute studies, rats received d9-THC ip at 10 mg/kg/day for 21 consecutive days and were decapitated at different time intervals (45-360 min) after the last injection. Control rats in acute and subacute experiments received the salineTween-80 vehicle by the same route and were killed under similar conditions. Immediately after the rats were killed, adhering lipids of the glands were removed, the glands were weighed, and catecholamines were assayed. Contents of dopamine and noradrenaline were measured by the method of Welch and Welch (1969) and adrenaline was measured by the method of Laverty and Taylor (1968). The rates of turnover of adrenal catecholamines were estimated by the nonisotopic method of Brodie et al. (1966), based on the measurements of the rates of synthesis and the rates of disappearance of endogenous catecholamines after inhibition of the catecholamine biosynthesis by specific inhibitors. This method assumes that after the administration of the specific inhibitors, the amines are no longer formed and the amine concentrations decline at a rate that is proportional to the concentration of the remaining amine. This relationship is described by the equation dA

1.

dt =-kA

upon integration, 0.434 kt where,

A = A,edkf, and converting to common logarithms,

A,, = initial steady-state amine concentration A = concentration of amine at any time t after the administration

1ogA = logA, -

of the inhibitor.

A plot of 1ogA vs t yields a straight line, the slope of which is 0.434 times the rate constant of A efflux (k). The rate of depletion of amines was calculated by multiplying the rate constant (k) by the initial value A,. The rates of turnover of dopamine, noradrenaline, and adrenaline were estimated by the rate of synthesis of dopamine and rates of decline of noradrenaline and adrenaline following the ip administration of FLA-63, i.e., bis-(Cmethyl-1-homopiperazinyl thiocarbonyl)-disulfide (25 mg/kg), a potent inhibitor of dopamine P-hydroxylase (Corrodi et al., 1970). The rate of decline of dopamine was measured after administering a-methyl-p-tyrosine (200 mg/kg, iv), an inhibitor of tyrosine hydroxylase. Inhibitors were given 15 min after the dg-THC injection. In subacute experiments, animals received dg-THC for 21 days (10 mg/kg/day) and then were treated with FLA-63 or a-M-p-T, 15 min after the last injection of dg-THC. Rats in acute and subacute studies and their controls were decapitated at different time intervals (45-180 min) after administration of the inhibitor. RESULTS

Figures la and b show that a single dose of dg-THC affects noradrenaline and adrenaline concentrations in the rat adrenal gland biphasically. A dose-related increase in both concentrations occurs first (45-90 min) and, thereafter, they decline sharply

dg-THC

LOW

DOSE

AND

I lo

ADRENAL

mghg

519

CATECHOLAMINES

1

(a)

8 -60

0

’

’ 90

I 180 TIME

, 360

0

90

180

360

TIME(min.)

1 min.)

FIG. 1. The effect of acute administration of d9-THC at (a) low (10 mg/kg) and (b) high (50 mg/kg) doses on the changes in rat adrenal catecholamines. Vertical bars represent the SE (N = 6).

(90-360 min). On the other hand, the adrenal dopamine concentration continues to decrease sharply after the dg-THC administration and even after 6 hr remains below normal in rats receiving either dose (10 and 50 mg/kg). The adrenal gland seemed to be markedly depleted of its catecholamine store 6 hr after d9-THC administration. After subacute treatment with 10 mg/kg/day d9-THC, the noradrenaline content of the gland did not change markedly from that of the saline-Tween-80-treated controls, whereas the dopamine concentration was low during the first phase and the adrenaline content seemed to be elevated (Fig. 2). Results (Table 1) of measuring rates of turnover of adrenal catecholamines showed that d9-THC given acutely: (a) significantly decreased the rate of accumulation of dopamine and also increased its rate of decline o- DOPAMINE .-ADRENALINE aNORADRENALINE

I 0

I

I so

I 160

TIME

I

360

(min.)

2. The effect of administration of d9-THC (10 mg/kg/day) for 21 consecutive days on the changes in rat adrenal catecholamines. Vertical bars represent the SE (N = 6). FIG.

520

MITRA,

PODDAR

AND

TABLE EFFECT

OF

A9-THC

ON THE TURNOVER

GHOSH

1

OF ADRENAL THE RAT”

CATECHOLAMINES

Synthesis rate Treatment Control (Saline-Tween-80) A9-THC

Dose

10 50 10f

+ It k &

OF

Declination rate

DAb

18.82 14.21 9.26 16.14

IN THE ADRENAL

DA’

2.21 0.60d 1.01’ 2.03

18.00 f 1.30 21.44* l.OOd 23.80 + 1.84d 18.21 + 1.85

AD*

NA”

18.42 15.19 10.47 18.70

f + f f

1.01 2.70 1.40d 2.02

56.55 74.77 90.89 79.20

+ if rt

4.47 4.28d 6.89’ 5.2Sd

0Turnoverratesare expressed as micrograms of catecholamine/g of adrenal/hr. Results are given as mean + SE of six separate sets of experiments. b Dopamine (DA) synthesis and noradrenaline (NA) and adrenaline (AD) depletion rates were measured following FLA-63 administration. c Dopamine (DA) depletion rate was measured following a-Mpt administration. d Analysis of variance showed a statistically significant change from control animals (p < 0.05). N = 6 animals. e Analysis of variance showed a statistically significant change from control animals (p < 0.01). N = 6 animals. J Dosage per day for 21 consecutive days. (Figs. 3 and 4); (b) significantly decreasedthe rate of decline in noradrenaline (Fig. 5); and (c) significantly increasedthe rate of decline in adrenaline (Fig. 6). After subacute treatment with Ag-THC, no marked alteration in rates of either accumulation or depletion of dopamine (Figs. 3 and 4) or the rate of depletion of noradrenaline (Fig. 5) was observed. However, the rate of decline in adrenaline was increased (Fig. 6). 0 -CONTROL A -Ag-THC o9, loo)-

01 0

.-

1,

(FLA-63) ((0 mg/kg) (50 IlO

45

,, w

90

TIME

I

/day)

180

( min.)

FIG. 3. The effect of d9-THC on the synthesis rate of dopamine using FLA-63. Vertical bars represent the SE (N = 6).

521 0 -CONTROL

(4 MpTl

A-AS-THC(io BII

mg/kg) (SO

FIG. 4. The effect of P-THC on the depletion rate of dopamine using a-methyl-p-tyrosine. bars represent the SE (N = 6).

0

o-

CONTROL

B-

9.

Verticai

( FLA-63)

45

160

TIMESYmin.1

FIG. 5. The effect of k’-THC represent the SE (N = 6).

on the rate of decline in noradrenaline

O-CONTROL

0

(FLA-63)

45

so

TIME

FIG. 6. The effect of d9-THC represent the SE (N = 6).

using FLA-63. Vertical bars

160

(min.)

on the rate of decline in adrenaline

using FLA-63. Vertical bars

522

MITRA,

PODDAR

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GHOSH

DISCUSSION

The present results indicate that d9-THC exerts characteristic effects on the different catecholamines in the rat adrenal. The initial increase in its content of both noradrenaline and adrenaline with concomitant decrease in that of dopamine seems to be due to increased conversion of the latter to the former during the first phase. The observed more rapid depletion of dopamine (Table 1) is consistent also with the possibility of such conversion. The subsequent sharp decrease in content of the catecholamines in the gland (90-360 min) under acute treatment may be due to a number of factors : (a) Perhaps the initial increased concentrations of noradrenaline and adrenaline produced by d9-THC exert feedback inhibition on tyrosine hydroxylase, the ratelimiting enzyme of noradrenaline synthesis and, hence, decrease the catecholamine concentrations (Spector et al., 1967). (b) The marked decrease in the rate of synthesis of dopamine (Table I), possibly because d9-THC either inhibits tyrosine hydroxylase or impairs uptake of tyrosine from the plasma by the catecholamine-synthesizing cells (Molinoff and Axelrod, 1971), may be responsible for decreased catecholamine content. The normal maintenance of adrenal tyrosine hydroxylase requires ACTH (Mueller et al., 1970), and d9-THC stimulates secretion of ACTH (Dewey et al., 1970; Kubena and Barry, 1971); hence, inhibition of tyrosine hydroxylase is unlikely. The low blood tyrosine concentration caused by d9-THC (Leonard, 1971) may also cause decreased synthesis. (c) It also may be due to increased release of catecholamines, presumably adrenaline, as evident from its increased rate of decline. The diminished rate of decline of noradrenaline at the same time indicates that d9-THC may either inhibit its release or slow down its intracellular catabolism. The decreased activity of adrenal MAO caused by d9-THC under acute conditions (unpublished observations) also supports the latter possibility. Subacute treatment with d9-THC may increase the adrenaline concentration either by increasing its synthesis or decreasing its release. The rate of adrenaline synthesis appears to be controlled by phenylethanolamine-N-methyl transferase (PNMT), and adrenal glucocorticoid hormones help maintain PNMT activity (Wurtman and Axelrod, 1966). d9-THC elevates the plasma corticosterone content (Kubena and Barry, 1971), and no tolerance to this effect was observed even after 20 days of continuous drug treatment (Kokka and Garcia, 1974). Hence, continuously increased concentrations of corticosterone during subacute d9-THC administration probably induce the synthesis of PNMT and thus increase the adrenaline content of the gland. It has been postulated that increased glucocorticoids in the blood can cause noradrenaline cells to change to adrenaline cells (Coupland and MacDougall, 1966). The possibility of such interconversion taking place during repeated administration of d9-THC needs further elaboration. The observed increase in the rate of depletion of adrenaline in rats given d9-THC subacutely may be due either to increased breakdown of adrenaline within the gland or its increased release. Biswas et al. (1975) reported that chronic administration of A9THC increases the activities of acetylcholinesterase and adenosine-triphosphatase, as well as an increase in Ca2+ concentration in medullary cells. These phenomena are involved in increased release of catecholamines (Douglas and Rubin, 1963). Hence, the

dg-THC

AND

ADRENAL

CATECHOLAMINES

523

presently observed effect most likely results from the increased release of adrenaline. moderately increased concentrations of adrenaline in the adrenals under subacute condition of treatment indicate that rate of synthesis exceeds rate of release, and perhaps there may be a tendency of a portion of the adrenaline to remain stored within the gland, unlike the effect observed under the acute condition. The reported effects of dg-THC in increasing the heart rate, arterial blood pressure, and other cardioacceleratory responses, and in increasing adrenaline secretion in man (Weiss et al., 1972) are consistent with the increased adrenaline release as observed in the present study. However,

ACKNOWLEDGMENTS

Our thanks are due to Dr. Olav. J. Braendenand Dr. EsmeLumsden,Narcotics Division, United Nations, Geneva, for kindly supplyingd9-THC which has beenusedin the present study. We are alsothankful to Astra Chemicals,Sweden,for FLA-63 which wasobtainedasa gift. This work hasbeensupportedby the University Grants Commission,Calcutta University. REFERENCES P., GINSBERG, J., AND RAINSBURY, R. (1972). Marihuana smoking, cardiovasculareffectsin man and possible mechanisms. New Eng. J. Med. 287, 209-12. BIRMINGHAM, M. K. (1973).Reduction by d9-THC in the blood pressureof hypertensiverats bearingregeneratedadrenalglands.Brit. J. Pharmacol. 48, 169-171. BISWAS, B., DEB, C., ANDGHOSH, J. J. (1975). Effect of administration of d9-THC on rat adreno-medullaryactivity, histochemicalstudies.Acta Endocrinol. 80, 329-338. BRODIE, B. B., COSTA, E., DLABAC, A., NEFF,N. H., ANDSMOOKLER, H. H. (1966).Application of steady state kinetics to the estimation of synthesisrates and turnover time of tissue catecholamines.J. Pharmacol. Exp. Ther. 154,493-498. CORRODI, H., F~~xE,K., HAMBERGER, B., AND LJUNGDAHL, A. (1970). Studieson central and peripheral noradrenalineneuronsusing a new dopaminefi-hydroxylase inhibitor. Eur. J. BEACONSFIELD,

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