Brain Research, 94 (1975) 57-66

57

© ElsevierScientific Publishing Company, Amsterdam - Printed in The Netherlands

INTERACTIONS BETWEEN LYSERGIC AClD DIETHYLAMIDE AND DOPAMINE-SENSITIVE ADENYLATE CYCLASE SYSTEMS IN RAT BRAIN

KERN VON H U N G E N , SIDNEY ROBERTS AND DIANE F. HILL

Department of Biological Chemistry, School q[ Medicine and the Brain Research Institute, University qf California Center for the Health Sciences, Los Angeles, Calif. 90024 (U.S.A.) (Accepted March 13th, 1975)

SUMMARY

Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-bromo-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation ofadenylate cyclase activity by equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10#M concentration, D-LSD not only completely eradicated the response to 10 E~M dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80/~M) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1/~M D-LSD. Activation of striatal adenylate cyclase by either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the fl-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and, in relatively high concentration (1130 ~M), partially blocked the activation by l0/~M dopamine, but was without effect on the stimulation by 10/~MD-LSD. The present results indicate that serotonin antagonists, in general, are potent inhibitors of

58 catecholamine-induced stimulation of adenylate cyclase systems in brain celt-lrce preparations. In addition, these results, coupled with earlier findings on the capacity of D-LSD to interact with serotonin-sensitive adenylate cyclase systems from rat brain'S3,24 and other neural systemsTM, strongly suggest that this hallucinogenic agent is capable of acting as an agonist at central dopamine and serotonin receptors, as well as functioning as an antagonist at dopamine, norepinephrine, and serotonin receptors in the brain. I NTRODUCTION

Considerable evidence supports the concept that the psychotomimetic drug, Dlysergic acid diethylamide (D-LSD), may exert certain of its effects on behavior by interactions with serotonin receptors in the central nervous system1,5,1s,ag. This hypothesis is based principally on the chemical similarity between D-LSD and serotonin and the effects of this hallucinogenic agent on central2,4,~° and peripheral 9,2s serotonergic mechanisms. Recent investigations suggest that adenylate cyclase systems may be involved in these central actions of D-LSD. Thus, o-LSD has been shown to stimulate adenylate cyclase activity, as well as antagonize the activation of this enzyme system by serotonin, in cell-free preparations from rat brain colliculi23,z4 and cockroach ganglion ~6. Although adenylate cyclase activity in collicutar preparations from newborn rats was highly sensitive to serotonin, stimulation by low concentrations of D-LSD was relatively small and the serotonin-btocking action of this drug was incomplete ~4. These observations suggested that the hallucinogenic effects of D-LSD might also involve interactions with central receptors for neurotransmitters other than serotonin. The present investigations provide support for this concept by revealing that D-LSD and other serotonin antagonists have profound effects on catecholamine-sensitive adenylate cyclase systems in rat brain. A preliminary report of some of this work has appeared 26. MATERIALS AND METHODS

The methods employed for preparation of brain subcellular fractions and measurement of adenylate cyctase activity have been described in detail elsewhere22,2.L Brains were obtained from young adult male rats of an inbred Sprague-Dawley strain. These rats were approximately 6 weeks old and weighed about 200 g. Most of the experiments were carried out on particulate fractions from various brain regions prepared by centrifugation of the homogenized tissue at 10,0C0 × g for 20 min. Adenylate cyclase activity was determined from the conversion of [14C]ATP to cyclic AMP by a procedure 22 which was based on the method of Krishna et al, 13. The incubation medium (0.1 ml) contained the following components in the final concentrations indicated: 3.6 mM (0.5 /tCi) [8-14C]ATP, 5.0 mM MgC12, 1.0 mM cyclic AMP, 40 mM Tris-HC1 (pH 7.3 at 37 °C), 10 mM phosphoenolpyruvate, 4 /~g pyruvate kinase, 15 mM (NH4)eSO4 (added with pyruvate kinase), 20 mM caffeine, 0.1 mg bovine serum albumin, 0.2 mM EGTA, 5 #g phosphatidylserine and 50-1130

59 /zg brain protein. Incubation was conducted in air for 5 rain at 37 °C. Protein was measured by the method of Lowry et al. 14. Biogenic amines, psychotropic drugs and all related compounds used in these investigations were of the highest purity obtainable. Sigma supplied L-norepinephrine hydrochloride, dopamine hydrochloride, serotonin creatinine sulfate, and propranolol hydrochloride. The following compounds were obtained from Sandoz: D-LSD, L-lysergic acid diethylamide (L-LSD), 2-bromo-D-lysergic acid diethylamide bitartrate (BOL), 1-methyl-D-lysergic acid diethylamide (MLD), 1-methyl-D-lysergic acid butanolamide (UML), thioridazine hydrochloride and N,N-dimethyl-4-hydroxytryptamine (psilocin). California Corp. for Biochemical Research was the source of N,Ndimethyltryptamine monooxalate and N,N-dimethyl-5-hydroxytryptamine monooxalate hydrate (bufotenine). Mescaline monooxalate was obtained from Aldrich Chemical Company, cyproheptadine hydrochloride from Merck, Sharp and Dohme, chlorpromazine and trifluoperazine hydrochlorides from Smith, Kline and French, haloperidol from McNeil Labs and promethazine hydrochloride from Wyeth Labs. RESULTS

Previous investigations from this laboratory revealed that adenylate cyclase systems in cell-free preparations from cerebral cortex of the adult rat were highly responsive to norepinephrine and dopamine, but were quite insensitive to serotonin 22, 2.5. Nevertheless, the serotonin-blocking agents, D-LSD and BOL, in 10/JM concentration, completely abolished the stimulation of cerebral adenylate cyclase activity by 10/zM norepinephrine plus I 0/zM dopamine 26. Comparable results were obtained in the present studies when serotonin antagonists were tested against equimolar amounts of either norepinephrine or dopamine at concentrations of these amines which produced maximal stimulation of adenylate cyclase activity in particulate preparations from adult rat cerebral cortex (see ref. 22). Norepinephrine (1130/zM) and dopamine (IC0/~M) resulted in increases in cerebral adenylate cyclase activity of approximately 60% and 50 ~o, respectively (Table I). The addition of D-LSD, BOL, or UML, each in 100/~M concentration, obliterated or markedly reduced these responses. Catecholamine-sensitive adenylate cyclase systems in particulate preparations from the hippocampus of adult rats were also strongly inhibited by D-LSD. The cerebral and hippocampal adenylate cyclase systems closely resembled one another in their responses to the catecholamines. Parallel studies from this laboratory demonstrated that D-I_SD was capable of producing a small stimulation of adenylate cyclase activity, as well as partially inhibiting the response to serotonin, in particulate preparations from newborn rat colliculi23, 24. These collicular preparations appeared to be unusually rich in serotonin receptors and poor in catecholamine receptors for adenylate cyclase24. However, o-LSD evoked a considerably larger increase in adenylate cyclase activity in particulate preparations from corpus striatum of adult rats 26. These striatal preparations contain adenylate cyclase systems which respond to norepinephrine or dopamine with large increases in activity but are relatively unresponsive to serotonin (see below). Adenylate

60 TABLE 1 INFLUENCE OF LYSERG1C ACID DERIVATIVES ON ACTIVATION OF ADENYLATE CYCLASE B' t CATECHOLAMINFS IN PARTICULATE PREPARATIONS FROM CEREBRAL CORTEX AND HIPPOCAMPUS OF A D U L T RATS

']7he particulate preparation from the cerebral cortex was a crude mitochondrial fraction obtained by centrifuging the 1000 i< g supernatant fraction from the cerebral homogenate at 10,000 ;;i g for 20 min 22. The preparation from the hippocampus was obtained by centrifuging directly the hippocampal homogenate at 10,000 ;~: g for 20 min. Values for cyclic AMP formed are means J_ S.E. for triplicate samples in representative experiments. Where indicated, basal values shown were obtained in the presence of the lysergic acid derivative. P values were calculated for the difference between the increase in cyclic AMP formed with only the catecholamine added and the increase with both the catecholamine and the lysergic acid derivative added. The catecholamines and lysergic acid derivatives were tested at 100 ltM concentration.

Lysergic acid derivative

Cyclic AMP Jbrmed (nm:~le~/mgprotein/h) Basal

bwrease with norepinephrine

Stimulation ( %)

Increase w i t h dopamine

Stimulation (%)

5.64 1.48 0.49 2.47

63 16 6 29

4.21 0.78 0.11 0.93

47 9** 1"* 11'*

49 1"**

2.85 ± 0.73 0.08 ~ 0.53

Cortex None D-LSD BOL UML

8.91 9.13 8.02 8.66

± ± ± ±

0.04 0.29 0.29 0.10

± 0.12 ± 0.30* :~::0.43* ± 0.11'

{_ 0.45 ± 0.44 ± 0.34 A: 0.40

Hippocampus None D-LSD

7.43 ± 0.67 8.53 ± 0.52

3.64 :L 0.69 0.08 ~_ 0.58

38 l***

* P < 0.001. ** P < 0.01. *** P g for 20 rain. Values for cyclic A M P formed are means i S.E. for 3-5 samples in representative experiments. Where indicated, basal values shown were obtained in the presence of the blocking agent. P values were calculated for the difference between the increase in cyclic A M P formed with only 10 ~tM dopamine or 10/~M D-LSD and the increase with the blocking agent added as well.

Agent

Cyclic' AMP Jbrmed Basal Cnmoles/mg protein~h)

Increase with dopamine

Increase with D-LSD

nmoles/mg protein/h

%

nmoles/mg protein/h %

25.19 4- 0.52 31.08 A: 0.73 24.53 4- 0.40

12.41 4- 0.69 1.59 4- 0.95* 12.61 4- 0.71

49 5 51

5.89 4- 0.90

23

6.45 4- 0.68

26

24,06 30.35 24.59 23.95 24.20 25.64

~: 0.37 4- 0.33 + 0.90 4- 0.91 4- 0.53 4- 0.63

10.66 2.26 4.63 0.05 12.75 10.58

~ 1.12 4- 0.42** ± 1.02"** ± 1.00"* 4- 0.74 4- 0.75

44 7 19 2 53 41

6.29 £ 0.49

26

5.30 2.05 7.98 5.10

22 9 33 20

18.93 23.16 24.43 18.23 17.78 19.54 18.95 20.09 20.33 21.37 19.12

:t: 0.23 4- 0.30 4- 0.31 ± 0.24 4- 0.27 4- 0.50 4- 0.34 i 0.63 4- 0.56 i 0.47 4- 0.39

9.62 4.22 0.72 5.95 1.45 3.60 1.59 1.87 -- 0.81 8.06 8.75

i 0.28 ± 0.86** i 0.76* :t: 0.42** 4- 1.00"* 4- 0.62* 4- 0.42* 4- 0.67* ± 0.61" i 0.89 4- 0.50

51 18 3 33 ;3 I8 8 9 -- 4 38 46

23.87 29.09 23.35 22;3t 22.58 21.06 23.88 23.09

i 0.46 ± 0.27 4- 0.36 + 0.55 4- 0.32 4- 0.29 :t: 0.23 4- 0.16

11.89 1.93 0.51 0.48 1.28 1.46 1.14 1.20

4- 0.65 4- 0.40* 4- 0.51" 4- 1.07"* 4- 0.71" 4- 0.45* _4- 0.23* 4- 0.25*

50 7 2 2 6 7 5 5

Experiment 1 None o-LSD, 10 # M L-LSD, 80/~M

Experiment 2 None D-LSD, 10 ArM Haloperidol, 1 # M Haloperidol, 10 # M Propranolol, 10/~M Propranolol, 100/~M

± 4± 4-

0.97 1.18~ 0.90 0.86

Experiment 3 None D-LSD, 1/~M D-LSD, 10 # M Chlorpromazine, 1/~M Chlorpromazine, 10 p M Thioridazine, I / ~ M Thioridazine, 10 p M Trifluoperazine, 1 / z M Trifluoperazine, 1 0 / t M Promethazine, 1 p M Promethazine, 1 0 / t M

5.50 =3:0.39

29

5.49 2.42 5.38 1.42 3.84 1.08 4.61 6.61

30 I4 28 7 19 5 22 35

4- 0.57 4- 0.31"* 4- 0.68 ± 0.70** ± 0.67 4- 0.57** 4- 0.71 _% 0.57

Experiment 4 None D-LSD, 10 # M BOL, 10 p M BOL, 100 p M Cyproheptadine, 10 p M Cyproheptadine, 100/zM MLD, 10 p M MLD, 100 p M *

P < 0.001.

** P < 0.01. *** P < 0.02. P < 0.05.

5.22 ~ 0.53 3.19 0.94 1.85 1.72 4.31 1.54

=~: 0,41 ± 0.92"* :L 0.38** 4- 0.40** ± 0.38 4- 0.50~

22 !4 .... 4 8 8 8 7

63 TABLE III INFLUENCE OF SEROTONINON ACTIVATIONOF ADENYLATE CYCLASEBY DOPAMINEIN PARTICULATE PREPARATIONS FROM CORPUS STRIATUMOF ADULT RATS

The particulate fractions were prepared by centrifuging striatal homogenates at I0,000 x g for 20 rain. Values for cyclic AMP formed are means ± S.E. for 3 4 samples in a representative experiment. Where indicated, basal values shown were obtained in the presence of se, otonin. P values were calculated for the difference between the increase in cyclic AMP formed with only 10 ttM dopamine or D-LSD added and the increase with both 10 I~M dopamine or D-LSD and serotonin added.

Serotonin (/~M)

Basal (nmoles/mg protein/h)

Cyclic AMP formed Increase with D-LSD

Increase with dopamine nmoles/mg protein/h

%

nmoles/mg protein/h

%

23.87 ~ 0.46 27.59 ± 0.83 27.23 -E 0.52

11.89 ::E 0.65 10.18 ± 0.88 8.67 ± 0.61"

50 37 32

5.22 -3- 0.53 4.37 ±. 0.97 6.31 ± 0.85

22 16 23

23.84 26.33 25.82 25.79

14.60 12.52 11.83 3.31

61 48 46 13

Experiment 1 0 10 100

Experiment 2 0 10 100 1000

~ 0.48 ± 0.29 ± 0.49 :k: 0.22

:~: 0.68 ± 0.67 ± 0.57** ± 0.26***

* P < 0.05. ** P

Interactions between lysergic acid diethylamide and dopamine-sensitive adenylate cyclase systems in rat brain.

Brain Research, 94 (1975) 57-66 57 © ElsevierScientific Publishing Company, Amsterdam - Printed in The Netherlands INTERACTIONS BETWEEN LYSERGIC AC...
595KB Sizes 0 Downloads 0 Views