Proc. Nat. Acad. Sci. USAVol. 71, No. 3, pp. 797-801, March 1974

Serotonin-Sensitive Adenylate Cyclase in Neural Tissue and Its Similarityto the Serotonin Receptor: A Possible Site of Action of LysergicAcid Diethylamide

(dopamine/octopamine/norepinephrine)

JAMES A. NATHANSON AND PAUL GREENGARD

Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510

Communicated by Edward A. Adelberg, November 3, 1973

ABSTRACT An adenylate cyclase (EC 4.6.1.1) that isactivated specifically by' low concentrations of serotoninhas been identified in homogenates of the thoracic gangliaof an insect- nervous system. The activation of this enzymeby serotonin' was selectively inhibited by extremely lowconcentrations of D-lysergic acid diethylamide (LSD),2-bromo-LSD, and cyproheptadine, agents which areknown to block certain serotonin receptors in vivo. Theinhibition was competitive with respect to serotonin, andthe calculated inhibitory constant of LSD for this sero-tonin-sensitive adenylate cyclase was 5 nM. The data areconsistent with a model in which the serotonin receptor ofneural tissue is intimately associated with a serotonin-sensitive adenylate' cyclase which mediates serotonergicneurotransmission. The results are also compatible withthe'possibility that 'some of the physiological'effects ofLSD may be mediated through interaction with serotonin-sensitive adenylate'cyclase.

There is at present some evidence that the neuronal receptorsfor certain neurotransmitters may be intimately associatedwith the enzyme adenylate cyclase (EC 4.6.1.1), and thatthe action of these neurotransmitters may be mediatedthrough an increase in the intracellular level of adenosine3':5'-monophosphate (cyclic AMP) in postsynaptic neurons(1). In the mammalian caudate nucleus, for example, a dopa-mine-sensitive adenylate cyclase has been identified (2) whichhas properties quite similar to those of the caudate "dopaminereceptor," characterized previously by a variety of biochem-ical, pharmacological, and behavioral data in animals and inman. In the cerebellum, a norepinephrine-sensitive adenylatecyclase may mediate the inhibitory effect of norepinephrineon Purkinje cells (3).Recent work suggests that the indoleamine, serotonin (5-

hydroxytryptamine), may act as a neurotransmitter at cer-tain synapses in both vertebrate and invertebrate centralnervous systems. In the rat, for example, serotonin appearsto exert an inhibitory effect both on the serotonin-containingneurons of the raphe nuclei, as well as on neurons receivingserotonergic connections from the raphe nuclei (4). In mollus-can central ganglia, serotonin has an excitatory effect oncertain neurons, mediated through a selective increase in so-dium permeability (A receptors), and inhibitory effects onother neurons, mediated through an increase in permeability

to potassium (B receptors) or to chloride (C receptors) (5, 6);all three of these effects of serotonin in the mollusc areknown to be blocked by D-lysergic acid diethylamide (LSD).Serotonin can also decrease membrane ion permeability inthese same ganglia, an effect not blocked by LSD (7). In addi-tion, serotonin appears to have direct excitatory effects onvertebrate and invertebrate smooth muscle (8, 9) and to stim-ulate fluid secretion in the insect salivary gland (10).

In 1960, Mansour et al. (11) made the important observa-tion of the existence, in cell-free preparations of whole liverflukes, of an adenylate cyclase activated either by low (10-v-10-6 M) concentrations of serotonin or by somewhat higher(10-5-10-4 M) concentrations of LSD. (There was no reportin that study of an antagonistic action of LSD on serotonin-stimulated adenylate cyclase activity.) In view of the largeamount of evidence suggesting a role for serotonin in neuralfunction, it seemed of great importance to establish the existenceof serotonin-sensitive adenylate cyclase in neural tissue. Despitethe clear demonstration by Kakiuchi and Rall (12) that sero-tonin can increase cyclic AMP levels in slices of rabbit brain,efforts in several laboratories, including our own, to demon-strate serotonin-sensitive adenylate cyclase in cell-free prepa-rations of mammalian nervous tissue have not yet provensuccessful. However, recently we were able to demonstratethe existence, in homogenates of insect nervous tissue, of anadenylate cyclase activated specifically by low concentrationsof serotonin (13). This serotonin-sensitive adenylate cyclase,which was found in cockroach thoracic ganglia, was shown to bedistinct from two other adenylate cyclases (activated bydopamine and octopamine, respectively) found in the sametissue.We now report that this serotonin-sensitive adenylate cy-

clase present in neural tissue has pharmacological propertiessimilar to those of known serotonin receptors and suggest thatserotonin-sensitive adenylate cyclase may, in fact, mediatethe physiological action of serotonin at some serotonergicsynapses. Included among our evidence is the finding thatthe hallucinogen D-lysergic acid -diethylamide, which isthought to exert its behavioral effects through interactionwith serotonin receptors, is capable, at extremely low concen-trations, of completely inhibiting the activation-by serotoninof serotonin-sensitive adenylate cyclase.

METHODSThe effect of putative neurotransmitters on adenylate cyclaseactivity was measured as described previously (13). In our

797

Abbreviations: LSD, -lysergic acid diethylamide; BOL, 2-bromo-LSD; EGTA, ethyleneglycol bis(,6-aminoethyl ether)-N,N'-tetraacetate.

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798 Biochemistry: Nathanson and Greengard

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FIG. 1. Effect of various concentrations of LSD on the activityof adenylate cyclase, in insect ganglion homogenate, in the pres-ence (broken line) or absence (solid line) of 2.5 X 10-6 M sero-tonin. Control activity in the absence of serotonin and LSD was10.8 1 1.2 pmol/mg of protein per min (mean 4 mean deviationfor four samples). In the absence of LSD, 2.5 X 10- M serotoninstimulated enzyme activity to 163% of control. The values shownin Figs. 1-5 are the means and ranges for two to three identicalsamples, each assayed in duplicate.

earlier experiments we obtained evidence for distinct sero-tonin-, octopamine-, and dopamine-sensitive adenylate cy-clases in homogenates of cockroach ganglia. However, wewere not able to obtain evidence for-a distinct norepinephrine-sensitive adenylate cyclase, and it seems probable that theobserved stimulatory effect of norepinephrine on adenylatecyclase activity of this preparation is attributable to a partialactivation by norepinephrine of octopamine- and dopamine-sensitive adenylate cyclases.

Briefly, pro-, meso-, and metathoracic ganglia from adultmale cockroaches, Periplaneta americana, were homogenized(20 mg/ml) in 6 mM Tris- maleate buffer (pH 7.4) containing2 mM EGTA [ethyleneglycol bis(P-aminoethyl ether)-N,N'-tetraacetate]. Adenylate cyclase activity was measured bydetermining the rate of formation of cyclic AMP from ATPin an assay system containing (in mmol/liter): Tris * maleate(pH 7.4), 80; theophylline, 10; MgSO4, 2; EGTA, 0.5; ATP,0.5; and tissue homogenate (0.5 mg of wet weight of cockroachganglia), plus putative neurotransmitter or other test sub-stances as indicated, in a final volume of 200 Al. Incubationwas for 3 min at 30° in a shaken water bath. The reaction wasinitiated by the addition of ATP, and terminated by boilingfor 30 sec. The reaction mixture was then centrifuged at lowspeed to remove insoluble material. Cyclic AMP in the super-natant was measured by the method of Brown et al. (14).Under the experimental conditions used, enzyme activity waslinear with respect to time and enzyme concentration. In allexperiments the adenylate cyclase activity of homogenates,measured in the presence of added test substance(s), was ex-pressed as a percentage of control activity measured in theabsence of added test substance.LSD and 2-bromo-D-lysergic acid diethylamide (BOL) were

obtained from the Food and Drug Administration-NationalInstitute of Mental Health. Cyproheptadine [4-(5H-dibenzo-[a,d]cyclohepten-5-ylidene)-1-methylpiperidine] was sup-plied through the courtesy of Dr. Clement Stone, Merck andCo., Inc.

All experiments were replicated 2-4 times. The resultsshown in Figs. 1-5 represent typical experiments.

RESULTSIn invertebrate ganglia, LSD is known to block the effects ofserotonin on certain neurons which receive serotonergic input(5, 6). In the present study of insect ganglion homogenates, verylow concentrations of LSD inhibited the stimulation of gangli-onic adenylate cyclase due to serotonin. In the experimentshown in Fig. 1, in the absence of LSD, 2.5 X 106 M serotonincaused a stimulation of adenylate cyclase activity to 163% ofthe control value. Increasing concentrations of LSD pro-gressively inhibited the stimulation due to this concentrationof serotonin. The stimulation due to serotonin was reducedby 50% in the presence of 2 X 10-8 M LSD and was almostabolished in the presence of 1 X 10-7M LSD.

Concentrations of LSD greater than 1 X 106 M stimulatedenzyme activity, in the presence or absence of serotonin (Fig.1). For example, 1.25 X 10-5 M LSD stimulated enzymeactivity to about 210% of the control value in the presenceor absence of 2.5 X 106 M serotonin. Thus, depending uponthe concentration present, LSD had a dual effect on adenylatecyclase activity: at extremely low concentrations it inhibitedthe stimulation of activity due to serotonin, while at somewhathigher concentrations LSD, itself, stimulated the formation ofcyclic AMP.The inhibitory effect of low doses of LSD was found to be

specific for stimulation of adenylate cyclase activity by sero-tonin. In the experiment shown in Fig. 2, in the absence ofLSD, 2.5 X 106 M dopamine caused a 1.85-fold stimulation,2.5 X 10-6 M octopamine, a 3.8-fold stimulation, and 5 X10-5 M norepinephrine, a 3.7-fold stimulation of adenylate

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FIG. 2. Effect of various concentrations of LSD on the activityof adenylate cyclase, in insect ganglion homogenate, in thepresence of either 2.5 X 10- M dopamine, 2.5 X 10-6 M octo-pamine, or 5 X 10- M norepinephrine. For comparison, thedata showing the effect of LSD on enzyme activity in the pres-ence of 2.5 X 106 M serotonin are reproduced from Fig. 1.Stimulation due to octopamine or norepinephrine is shown on theright-hand ordinate; stimulation due to serotonin or dopamine isshown on the left-hand ordinate. Control activity in the absenceof added compound was 12.0 ± 1.6 pmol/mg of protein per min(mean ± mean deviation for four samples).

Proc. Nat. Acad. Sci. USA 71 (1974)

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Serotonin-Sensitive Adenylate Cyclase and LSD 799

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FIG. 3. Effect of various concentrations of the serotoninantagonists, BOL or cyproheptadine, on the activity of adenylatecyclase, in insect ganglion homogenate, in the presence (solidlines) or absence (broken lines) of 2.5 X 10-6 M serotonin.Control activity in the absence of serotonin, BOL, and cypro-heptadine, was 13.3 4 1.0 pmol/mg of protein per min (meanmean deviation for four samples).

cyclase activity, compared with that of control. Concentra-tions of LSD which inhibited the stimulation of adenylatecyclase activity by- serotonin had little or no effect on thestimulation of adenylate cyclase activity by octopamine,dopamine, or norepinephrine. For example, in the presenceof 1 X 10-7M LSD, a concentration which virtually abolishedthe stimulation of adenylate cyclase activity by serotonin,there was little or no inhibition of dopamine-, octopamine-,or norepinephrine-stimulated activity.The LSD derivative, 2-bromo-D-lysergic acid diethylamide

(BOL), and the structurally unrelated compound, cyprohep-tadine, are known to block serotonin receptors in both verte-brate and invertebrate tissues (8, 9, 15). Fig. 3 shows thatthese two serotonin antagonists, like LSD, inhibited the stim-ulation, by serotonin, of adenylate cyclase activity in insectganglion homogenates. A 50% inhibition of the stimulationdue to 2.5 X 10- M serotonin was obtained with either 1 X10-8 M BOL or 2 X 10-7 M cyproheptadine. In other experi-ments it was found that the inhibitory effects of both BOLand cyproheptadine on enzyme activity were relativelyspecific for serotonin stimulation: both BOL and cyprohepta-dine were considerably less effective in inhibiting stimulationof adenylate cyclase activity due to dopamine, octopamine,or norepinephrine (see below and Table 1).

Like LSD, BOL had a direct stimulatory effect on adenylatecyclase activity, when used alone at concentrations of 1 X10- M or more (Figs. 3 and 4). (Unexpectedly, the combina-tion of 2.5 X 106M serotonin plus 1 X 106 M BOL causedless stimulation of enzyme activity than did either substancealone.) Fig. 4 compares the activation of adenylate cyclaseactivity by either LSD (Ka > 4 X 10- M), BOL (K, = 1.5 X10- M), or serotonin (K. = 6 X 10-7 M). (Because of thesmall amounts available to us, it was not practical to use con-

centrations of LSD greater than 1 X 10-4 M.) It should beemphasized that the stimulatory effect of BOL, like that ofLSD, was obtained at concentrations much greater than theconcentrations required to inhibit the activation of the enzymeby serotonin. In contrast to the stimulation of adenylate cyclaseactivity by LSD, BOL, or serotonin, cyproheptadine, at similarconcentrations, caused a depression of basal adenylate cyclase

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BOL, or cyproheptadine on adenylate cyclase activity in insectganglion homogenate. Control activity in the absence of addedcompound was 10.0 4 1.5 pmol/mg of protein per min (mean 4

mean deviation for four samples).

activity. Even in the presence of serotonin, a depression ofenzyme activity below control levels was seen in the presenceof high concentrations of cyproheptadine (Fig. 3).The effect of a fixed concentration of LSD, BOL, or cy-

proheptadine was tested on adenylate cyclase activity in thepresence of various concentrations of serotonin. Fig. 5 showsthat LSD and BOL each caused a shift in the serotonin dose-response curve to the right, resulting in an increase in theactivation constant, Ka (the concentration required to causea half-maximal increase in enzyme activity), for serotonin,without causing any significant change in the maximal activa-tion of the enzyme (Vm.s). A similar shift in the dose responsecurve was seen in the presence of cyproheptadine (data notshown). This type of interaction between an agonist and an

TABLE 1. Inhibition by various antagonists of thestimulation of adenylate cyclase activity by various putative

neurotransmitters

Stimula- Approximate Ki for

tion of Cypro-enzyme LSD BOL heptadine Phentby (nM) (nM) (Mm) (MM)

5-HT 5 5 0.25 3DA >10,000 100 1.0 2OCT >10,000 >10,000 1.0 0.5NE >10,000 >10,000 1.0 0.6

The Ki values represent the calculated inhibitory constantsof the serotonin antagonists, LSD, BOL, and cyproheptadine,as well as of the alpha-adrenergic antagonist, phentolamine(Phent), for adenylate cyclase activity of cockroach ganglionhomogenates, stimulated by either serotonin (5-HT), dopamine(DA), octopamine (OCT), or norepinephrine (NE). The dataare derived from experiments like those shown in Figs. 1-3 andFig. 5. Further details are in the text.

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FIG. 5. Effect of various concentrations of serotonin on the activity of adenylate cyclase, in insect ganglion homogenate, in the absenceof inhibitor (control) and in the presence of LSD (1 X 10-7 M) or BOL (2.5 X 10-7 M). Basal enzyme activity in the absence of addedcompound was 15.8 4 2.0 pmol/mg of protein per min (mean 4 mean deviation for four samples). Inset: double reciprocal plot of thepercent increase in enzyme activity due to serotonin, as a function of the serotonin concentration from 2 X 106 M to 1.5 X 10-4 M. A:control;B:1 X 10-7MLSD;C:2.5 X 10-7MBOL.

inhibitor indicates inhibition of a competitive type. A doublereciprocal plot of the data (inset in Fig. 5) also illustrates thecompetitive nature of the inhibition, by LSD or BOL, of theserotonin-induced increase in enzyme activity.

In other experiments it was found that stimulation ofadenylate cyclase activity by dopamine, octopamine, ornorepinephrine, could be inhibited to some extent by suffi-ciently high concentrations of any of the serotonin antagonists,LSD, BOL, or cyproheptadine. The inhibition appeared in allinstances to be of a competitive nature. Using competitivekinetics, the inhibitory constants (Ki values) of LSD, BOL,and cyproheptadine for serotonin-sensitive adenylate cyclaseactivity, as well as for octopamine-, dopamine-, and norepi-nephrine-stimulated activity, were calculated from the ex-perimental data. The inhibitory constants shown in Table 1were calculated from data of the type presented in Fig. 5, inwhich the inhibitor concentration was held constant andthe concentration of putative neurotransmitter was varied, aswell as from data of the type shown in Figs. 1-3, in which theconcentration of putative neurotransmitter was held constant,and the inhibitor concentration was varied. The two typesof experiments gave similar results. The apparent Kj of LSDand of BOL for the serotonin-sensitive adenylate cyclase wasapproximately 5 X 10-9 M. (The apparent Ki of LSD for theserotonin-sensitive adenylate cyclase was independent of theconcentration of ATP present.) Both LSD and BOL werequite ineffective in inhibiting octopamine- and norepi-nephrine-stimulated adenylate cyclase activity (Ki > 1 X 10-5M). LSD was also quite ineffective in inhibiting dopamine-sensitive adenylate cyclase activity (Kj > 1 X 10-5 M),whereas BOL was somewhat more effective (K1, 1 X 10-v M).The inhibition of serotonin-sensitive adenylate cyclase bycyproheptadine was much less specific than that shown byLSD: the Ki of cyproheptadine for serotonin-sensitiveadenylate cyclase was 2.5 X 10-7 M, whereas the Ki of cypro-heptadine for dopamine-, octopamine-, and norepinephrine-sensitive adenylate cyclase was about 1 X 10- M, i.e., onlyabout 4 times greater.

Table 1 also includes the results of studies of the effect ofthe alpha-adrenergic antagonist, phentolamine, on the neuro-transmitter-sensitive adenylate cyclase activity of insectganglion homogenates. Phentolamine competitively inhibitedthe stimulation of adenylate cyclase activity caused by sero-tonin, dopamine, octopamine, or norepinephrine. In contrastto the serotonin antagonists, phentolamine was not moreeffective in inhibiting serotonin-sensitive adenylate cyclaseactivity (K1, 3 X 10- M) than in inhibiting dopamine-sensitive (Ki, 2 X 10- M), octopamine-sensitive (Ki, 5 X10-7 M), and norepinephrine-sensitive (Ki, 6 X 10-7 M)enzyme activity.

DISCUSSIONThe present results demonstrate the presence of a serotonin-sensitive adenylate cyclase in invertebrate neural tissue, andshow that activation of this enzyme is selectively inhibited byagents which are known to block certain serotonin receptorsin vivo. Thus, LSD and BOL (and cyproheptadine to a lesserextent) were far more effective in blocking stimulation due toserotonin than they were in blocking stimulation due todopamine, norepinephrine, or octopamine. Conversely,phentolamine, an alpha-adrenergic antagonist, was not moreeffective in inhibiting stimulation due to serotonin than ininhibiting stimulation due to the other putative neurotrans-mitters.Extremely low concentrations of both LSD and BOL were

effective in inhibiting serotonin-stimulated enzyme activity.The calculated inhibitory constants for these two serotoninantagonists were 5 X 10-9 M, and that for cyproheptadine was2.5 X 10-7 M. The fact that LSD inhibits serotonin-sensitiveadenylate cyclase activity at such low concentrations is ofparticular interest in view of the great potency of LSD as ahallucinogenic agent in man: the K1 for LSD (5 X 10-9 M)is similar to the concentration (2.5 X 10-9 M) estimated byAxelrod et al. (16) to be present in human brain when ahallucinogenic dose (1 Ag/kg) of LSD (17) is administered.

Although, at low concentrations, LSD and BOL inhibited

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Serotonin-Sensitive Adenylate Cyclase and LSD 801

serotonin-sensitive adenylate cyclase, at higher concentra-tions (1 X 10-lIM or greater) both LSD and BOL directlystimulated adenylate cyclase activity. This concentration-dependent dual effect of LSD and BOL is of interest, since, inphysiological studies, LSD has been variously reported to actas either a serotonin agonist or antagonist depending upon thetissue studied and the concentration of LSD used (4, 6, 8, 9,18-20). It has also been reported (21) that synaptic mem-brane fractions of rat cerebral cortex contain separate lowaffinity and high affinity binding sites for LSD. Interestingly,the reported dissociation constant for the high affinity bindingsites (9 X 10-9 M) and that for the- low affinity binding sites(1.2 X 10-6 M) are similar, respectively, to the inhibitoryconstant of LSD for serotonin-sensitive adenylate cyclase (5X 10- M) and the activation constant of LSD for adenylatecyclase (slightly greater than 4 X 10- M) found in the pres-ent experiments.

Current evidence suggests that serotonin may be a neuro-transmitter in invertebrate ganglia (5-7, 22). Using intactcell preparations, Cedar and Schwartz (22) demonstratedthat serotonin raises cyclic AMP levels in isolated Aplysiaganglia. However, because they could not demonstrate aneffect of serotonin in homogenates, these authors were not ableto show that serotonin directly affected adenylate cyclase ac-tivity. The results of the present study, which demonstratethe presence in insect ganglia of an adenylate cyclase which isselectively activated by serotonin and specifically inhibitedby low concentrations of agents which are known to blockinvertebrate serotonin receptors, support the possibility thatserotonin-sensitive adenylate cyclase may mediate serotoner-gic neurotransmission in invertebrate ganglia. It will be im-portant, in future studies, to determine the electrophysiologi-cal effects of cyclic AMP derivatives on vertebrate and in-vertebrate nerve cells which are known to contain serotoninreceptors.The pharmacological properties of the serotonin-sensitive

adenylate cyclase studied in the present investigation aresimilar to those of serotonin receptors in the rat uterus and A-receptors in molluscan ganglia (6, 8). In both of the lattercases, as well as in the present study, LSD and BOL antago-nized the actions of serotonin and were approximately equi-potent in doing so. In man, however, LSD is known to bemuch more potent than BOL as a hallucinogenic agent (17).Therefore, if LSD exerts its behavioral effects through inter-actions with serotonin receptors, then one must postulatethat the serotonin receptors in various tissues have differentcharacteristics. There is, in fact, some evidence which sug-gests that serotonin receptors in various tissues do differ intheir characteristics. In the rat, for example, LSD blocks theeffects of serotonin on uterine muscle (8) but mimics theeffects of serotonin on neurons of the raphe nucleus (4). Fur-thermore, BOL, which is as effective a serotonin blocker asLSD in the rat uterus (8), and gastropod mollusc (6), hadconsiderably less effect than LSD in directly inhibiting rapheneuron discharge (4) and has effects opposite to those of LSDin the liver fluke (9).

In the present experiments LSD had a dual effect onadenylate cyclase activity (inhibition of serotonin-stimulatedactivity at low concentrations but direct stimulation of en-zyme activity at higher concentrations). It is possible thatin the mammalian brain LSD might either stimulate adenyl-ate cyclase activity directly or inhibit the stimulation of sero-tonin-sensitive adenylate cyclase activity by serotonin. Inany case, because of the present evidence that LSD affectsserotonin-sensitive adenylate cyclase activity of invertebratenervous tissue at very low concentrations, it will be importantto examine extracts of the raphe nuclei, as well as of otherselected parts of the mammalian brain, for serotonin-sensitiveadenylate cyclase activity, and to determine whether theeffects of LSD, BOL, and other serotonin antagonists on thisenzyme can provide an explanation, at the molecular level,for the effects of LSD in man.

This work was supported by USPHS Grant NS-08440 andMH-17387.

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