Agaridoxin: A fungal catecholamine which acts as an alpha 1 agonist of mammalian hypothalamic adenylate cyclase

Brain Research, 231 (1982) 387-398 Elsevier BiomedicalPress

387

AGARIDOXIN: A FUNGAL CATECHOLAMINE WHICH ACTS AS AN ALPHA 1 AGONIST OF MAMMALIAN HYPOTHALAMIC ADENYLATE CYCLASE

M A R C I A W H E E L E R , M A X T I S H L E R a n d M A R K W. B I T E N S K Y

Yale University School of Medicine, Department of Pathology, New Haven, CI 06510 and (M.T.) Wesleyan University, Department of Chemistry, Middletown, CI 06457 (U.S.A.) (Accepted June 4th, 1981)

Key words': agaridoxin - - fungal catecholamine - - adenylate cyclase - - h y p o t h a l a m u s

SUMMARY

Agaridoxin, a catecholamine isolated from mushrooms, and 4 synthetic analogues cause activation of adenylate cyclase in the presence of guanylyl imidodiphosphate (Gpp(NH)p) in membrane particles prepared from rat hypothalamus. These compounds also activate adenylate cyclase preparations from rat kidney, liver and cerebral cortex. In the presence of tyrosinase, these compounds are readily oxidized to quinones which lack agonist activity. Studies with selective adrenergic blockers suggest that agaridoxin acts at an al-type receptor. Agaridoxin-mediated adenylate cyclase stimulation is most effectively antagonized by WB-4101 and phenoxybenzamine, while proplanolol and yohimbine are without inhibitory effect. Agaridoxin and the al agonist methoxamine inhibited the binding of [aH]WB-4101 in rat hypothalamic and cerebral cortical membranes. The values of K~ for both compounds are lower than that of norepinephrine. The agaridoxin analogue, 4-aminocatechol hydrochloride, is a more effective and potent adenylate cyclase activator than agaridoxin or methoxamine.

INTRODUCTION

Agaridoxin (y-glutaminyl-3,4-dihydroxybenzene) is a catecholamine found in the edible mushroom Agaricus campestris. Agaridoxin (AGX) has been isolated, purified, structurally defined and synthesized28. In addition a number of analogues (Table I) of AGX have been synthesized. A prominent chemical proper ty of agaridoxin is the ease with which the hydroxybenzene moiety can be oxidized to a red quinone (2max 490 nM)aO,aS,a6 which is both cytotoxic32 and bacteriostatic33. AGX is formed in

388 the mushroom from its monohydroxy precursol 7,-glutaminyl-4 hydroxybenzene (PGH). Agaridoxin has been evaluated as a putative antitumor agent 1~,~:'-,'-'~ in part because its quinone inhibits DNA polymerase 3°, an effect which is prevented by dithiothreitol (DTT). We have examined the effects of synthetic AGX and its analogues on adenylate cyclase under conditions which inhibit quinone formation. We find that AGX and related compounds function as Gpp(NH)p dependent, activators of adenytate cyclase (at concentrations between 10 -7 and 10~-.~M) in particulate membrane preparations isolated from mammalian hypothalamus and other cell types (Table !). Studies of AGX in the presence of catecholamine antagonists indicate that this compound behaves as an al-type adrenergic agonistlS, 37 comparable to methoxamine, a potent a~ agonist 20 known to stimulate adenylate cyOase in several areas of the brain (refs. 1, 27). We have also observed that the quinoaes formed from the agaridoxin compounds not only lose agonist activity but actually inhibit adenylate cyclase at concentrations above 10 ~ M. For these studies we have chosen the rat hypothalamic adenylate cyclase system due to its reported responsiveness to a wide range of agonists which include dopamine, norepinephrine, methoxamine, NaF and Gpp(NH)p 1,2~. We have confirmed these observations in both homogenate and washed particle preparations. MATERIALS AND METHODS

Animals Male Sprague-Dawley rats (50-150 g) were used. Rats were reserpinized by ip injection of l m g serpasil/kg body weight, 24 and 48 h prior to sacrifice. Control animals received saline injections. Reserpinized rats exhibited a marked depression of spontaneous motor activity.

Membrane particle preparations Homogenates from hypothalamus were prepared by the method of Ahn et at. 2. Rats were sacrificed by cervical dislocation. Hypothalami were removed to ice-cold phosphate buffered saline (pH 7.4), and homogenized (ice temperature, teflon on glass) at 500 rpm at a ! :20 dilution (w/v) in ice-cold 2 mM Tris-maleate, containing 0.8 mM EGTA, pH 7.4 (homogenization buffer). The homogenate was then centrifuged (600 g, 5 rain, 4 °C) and the pellet discarded. The supernatant was recentrifuged (7500 g, 10 rain, 4 °C) and the resulting pellet was suspended (1 ml/hypothalamus) in 0.32 M sucrose, containing 50 mM Tris-maleate, pH 7.8, 5 mM MgSO4, t mM E G T A and 1 mM DTT (STM). This material was recentrifuged (7500 g, 10 rain, 4 °C) and the pellet resuspended and washed twice in STM (17). The washed pellet was resuspended in STM to a protein concentration of about 2 mg/ml and stored under liquid nitrogen for up to 30 days without loss of activity. Prior to adenylate cyclase assay, the hypothalamus preparation was thawed at 4 °C, STM was removed by centrifugation (7500 g, 5 rain, 4 °C) and the pellet resuspended in homogenization buffer to a protein concentration of about 2 mg]ml. Rat cerebellum membrane particles were also prepared by the same method.

389 A crude synaptosomal fraction was prepared from rat cerebral cortex by modification ~1 of the method of Gray and Whittaker 13. Rat renal medulla membrane particles were prepared by the method of Dousa 9. Rat liver membrane particles were prepared by the method of Bitensky et al. a.

Assay of adenylate cyclase and phosphodiesterase Membrane particles were suspended in the same buffer used for particle pIeparation unless otherwise noted. Adenylate cyclase was assayed for 5 rain at 30 °C in a reaction mixture containing 80 mM Tris-maleate, pH 7.4; l mM 3-methylisobutylxanthine (MIX), 2 mM MgSO4, 0.5 mM ATP with phosphoenol pyruvate (0.06 M) and pyruvate kinase (0.5 mg/ml), 0.5 mM DTT, 0.05 mg/ml BSA and 0.4 mM EGTA in a reaction volume of 50 #1. The leaction was stopped by boiling (2 rain). Rates were linear for more than 15 min and the ATP concentration did not measurably decline for 15 min as measured by the luciferase assay16. In order to permit maximal stimulation of adenylate cyclase by Gpp(NH)p or NaF, these agents were routinely preincubated with the membrane particles for 15 rain at 30 °C. DTT, 1 mM, and the tyrosinase inhibitor phenylthiocarbamide (PTC), 0.1 mM, were included in the adenylate cyclase reaction mixture to inhibit quinone formation. Cyclic AMP was measured by a modification of the method of Brown et al. 5 using a cyclic AM P binding protein prepared from rabbit skeletal muscle. Adenylate cyclase activity was expressed as pmol cAMP formed/rag protein/5 min and was corrected by substraction of a boiled enzyme blank. Cyclic nucleotide phosphodiesterase activity was assayed as previously described 19.

Synthes& of agaridoxin and its analogues Agaridoxin was synthesized as previously described 2s. Agaridoxin and analogues were synthesized by Dr. Max Tishler. Quinone analogues were prepared by treating AGX, 4-aminocatechol hydrochloride (CNC), PGH and 4-aminoacetyl catechol (AAC) with tyrosinase (Sigma Type 500 U) for 30 rain at 23 °C, in 5 mM Tris-maleate (pH 6.5).

Receptor binding studies Receptor binding studies with WB-4101 were done using the method of U'Prichard 14,31. Hypothalamic and cortical membrane particles were suspended in 50 mM Tris-HCI, pH 7.7 (0.31 and 0.27 mg/ml protein, respectively). Tubes containing 0.5 ml of the particles and 0.20 nM [ZH]-WB-4101 were incubated for 15 rain and 25 °C at which point antagonists were added and incubation continued for 15 rain. Contents were rapidly chilled to 4 °C, diluted and filtered through Whatman GF/B filters with 10 ml of the above buffer. Filters were counted by liquid scintillation spectrometry in 10 ml 5 j°/oProtosol, 95 ~ Econofluor. Specific [aH]-WB-4101 binding was defined and the difference in binding observed in the presence and absence of 0.1 ~M of unlabeled WB-4101 or 100/~M (--)-norepinephrine.

390 TABLE 1 Activation of adenylate cyclase by agaridoxin and its analogues'

H0 N - C - (CH2)2 - CH-CO 2H H 0

HO

R3

NH2

STRUCTURE A

STRUCTURE B

Rat hypothalamic membranes were added to a reaction mixture containing Gpp(NH)p (10- ~; M) with or without the above agonists and adenylate cyclase assayed. Structure R1

(1) (2) (3) (4)

R2

R3

Chemical name

A A A B

OH OH - H OH-OH H -N-C-CH~

(5) B

H0 NHa+CI-

Abbreviation

~,,~Cvclose activation* ..................

10 i M

I 0 t, M

Agonist

Agonist

Agaridoxin ),-glutaminyl- 3-hydroxy benzene 7-glutaminyl-4-hydroxybenzene 4-aminoacetylcatecbol

AGX MGH PGH AAC

35 0 24 74

55 86 118 104

4-aminocatechol hydrochloride

CNC

100

138

II

* Results are expressed as the per cent stimulation over GPP(NH)p treated membranes (which gave a value of 212 pmol cAMP/5 min/mg prot.) and are the averages of three experiments each done in triplicate. All observed activations were statistically significant.

Statistical analysis

All averages are presented as the mean ~ S.E.M. Student's t-test was used to evaluate the difference between the means o f two samples. Differences at the P <~ 0.05 level were considered statistically significant. RESULTS Comparison o f agaridoxin and its analogues

Table I gives the structure and abbreviation for agaridoxin and each of its analogues used in this study. Activation of adenylate cyclase for each agonist is given at two concentrations. A A C and C N C were better activators o f adenylate cyclase than A G X at all concentrations examined, while M G H and P G H were more effective agonists that A G X at lower concentrations. Dependence on guanyl nueleotides

Activation o f adenylate cyclase by A G X and its analogues showed a requirement for guanyl nucleotides similar to that f o u n d for other receptor-mediated adenylate cyclase activators 15,23. In the rat hypothalamic preparation, G T P and to a greater extent its hydrolysis-resistant analogue G p p ( N H ) p produced a time-dependent cyctase

391 TABLE II

Effect of Gpp( NH)p on agaridoxin activation of adenylate cyelase Membrane particles from hypothalamus were preincubated with or without Gpp(NH)p for 10 rain at 32 'C. Reaction mixture with or without agaridoxin was added and adenylate cyclase activity measured. Values given ( ± S.E.) are the results of two separate experiments with each determination done in triplicate.

Guanine nucleotide

Adenylate cyclase activity without AGX Adenylate cyclase activity with AGX pmol/5 min/mg ~ Stimulation pmol/5 min/mg % Stimulation prot. prot.

None 236 Gpp(NH)p (10 .6 M)§ 381 Gpp(NH)p (5 × 10-6M)§587 Gpp(NH)p (10 6 M)§§ 247 * ** § §8

± :]_ ± ±

9 35 13 3

-61 148 5**

263 712 819 439

~ ± ± ~z

9 40 25 3

11"* 170 211 69

AGX concentration was 2 × l0 -'~ M. Not statistically significant. Gpp(NH)p preincubated with membranes, 10 min at 32 °C. Gpp(NH)p added in reaction mixture.

TABLE III

Effect of reserpine treatment on the response of adenylate cyelase to different agonists Hypothalamic membrane particles were used throughout. Reaction mixture contained 10 -G M Gpp(NH)p. Values ( ± S.E.) given are the averages of two separate experiments with each determination done in triplicate.

Additions

None Agaridoxin(2 × 10 ~M) Methoxamine (10-4 M) Norepinephrine(10 4M) Clonidine(10-SM)

Untreated*

269 494 365 272 220

± ~ ± ± ~z

5 16 3 11 16

Adenylate cyelase activity % Stimulation

Reserpine treated*

% Stimulation

-83 36 1 --

270 520 379 390 475

-93 46 44 76

~ ± _± ~ ±

7 16 16 11 22

* Values expressed as pmol cAMP/5 min/mg protein.

a c t i v a t i o n , w h i c h was e n h a n c e d as m u c h as 1 0 0 K by A G X ( T a b l e II). C o n v e r s e l y , a c t i v a t i o n o f a d e n y l a t e cyclase b y A G X a p p e a r s a b s o l u t e l y d e p e n d e n t o n the p r e s e n c e o f G T P o r its a n a l o g u e s w h e n extensively purified A T P (free o f G T P c o n t a m i n a t i o n iv) is used as s u b s t r a t e ( T a b l e I[).

Differential effects o f reserpinization on the al and a2 receptor function R e s e r p i n e is k n o w n to e n h a n c e n o r e p i n e p h r i n e r e s p o n s i v e a d e n y l a t e cyclase a c t i v i t y by d e p l e t i n g c a t e c h o l a m i n e s in h y p o t h a l a m i c p r e p a r a t i o n s 1. W e e x a m i n e d the effects o f r e s e r p i n i z a t i o n o n b o t h al- a n d a2-type a g o n i s t s ( T a b l e IlI). A d e n y l a t e

392 cyclase response to clonidine (an a2 agonist) was dramatically increased by reserpinization, whereas the adenylate cyclase response to methoxamine (an a~ agonist) and A G X showed no change due to reserpinization. Reserpinization also enhanced the response to norepinephrine. Characterization o f the adenylate cyclase-linked agaridoxin receptor Blocking agents for the al, a2 and fl pre-adrenergic receptors were examined for their ability to prevent adenylate cyclase activation by A G X and CNC (Table IV). WB-41016 and phenoxybenzamine is are known to be effective a~ antagonists. Phenoxybenzamine did not distinguish between clonidine and AGX, since it inhibited the effects of both to about the same extent (data not shown). WB-4101, in contrast, was a more effective antagonist against A G X than against clonidine. Yohimbine (at lower concentrations) functions as an a2 blocker 4 and propranolol was used as a t%adrenergic blocking agent 22. The data indicate that A G X interacted primarily at an al-type receptor since its effects were clearly antagonized by phenoxybenzamine and WB-4101 and virtually unaffected by yohimbine and propranolol. Propranolol actually enhanced the activation of adenylate cylase by agaridoxin (Table IV). C N C activation was partially blocked by both propranolol and WB-4101. Characterization o f the agaridoxin receptor --- ?'3H /-WB-4101 binding studies The al antagonist [3H]-WB-41014,~4,31 was used to measure the dissociation constants of methoxamine, A G X and CNC. At concentrations ranging from 10-5 to 10-9 M, these agonists competed with 2 .:< 10-l° M [3H]-WB-4101. The values of ki observed for methoxamine and agaridoxin are 0.44 # M and 0.59 #M, respectively, as compared to 1.5/zM for norepinephrine 31. Similar results were obtained using a rat cerebral cortex synaptosomal fraction. The K¢ for C N C in hypothalamic preparations was 59. However, C N C was rapidly oxidized under the conditions of the receptor binding study and the true Ki may be lower.

TABLE IV Effect of specific antagonists on hypothalamic adenylate cyclase activity*

Hypothalamic membrane particles were prepared from reserpinized rats and were preincubated with the indicated antagonists (10-5 M) (3 min, 30 °C). Reaction mixture plus 10-6 M Gpp(NH)p with or without the above agonists (10-.5 M) was added. Values are the averages of two separate experiments, with each determination done in triplicate. Agonists (10 -~ M )

Antagonists ................................................................. None Propranolol WB-4101 Yohimbine

None CNC AGX Clonidine Methoxamine

167 580 261 258 340

3 2 _]- 15 & it :f: 9 _~: 9

160 340 372 240 320

i:': ~_ ~ -i

* Values expressed as pmol cAMP/5 rain/rag protein.

5 2 22 5 2

160 148 160 252 152

~ 2 ~:: 8 ~ 8 .:~ 8 .~z 6

184 707 475 100 180

~: 6 : 25 ~: 16 ~:: 3 -! 4

393

Effects of agaridoxin and its analogues in other cell types In addition to the hypothalamus, we have also examined the effects of agaridoxin in cerebellum and cerebral cortex (Table V). The most striking effects in neural tissue are seen with the hypothalamus while cerebral cortical and cerebellar particulate preparations exhibit less adenylate cyclase response to AGX or other known at or/3 agonists. AGX and CNC also increase hepatic adenylate cyclase activity 40 ~ above a control value of 80 pmol/5 min/ml protein. These agonists also increase renal medullary adenylate cyclase activity 40 ~o above a control value of 46 pmol/5 min/mg protein, only in the presence of the PTC (data not shown).

Compar&on of agaridoxin and analogues with other known al agon&ts CNC can activate adenylate cyclase at concentrations as low as 10-~ and I0 -v M with much greater efficacy than methoxamine, AGX, clonidine or phenylephrine (Fig. 1). Maximal activation of adenylate cyclase by AGX and methoxamine requires about 10-fold greater concentrations than needed for maximal activation by CNC (Fig. 1). Even at optimal concentrations the adenylate cyclase activations produced by AGX and methoxamine are about 30~ less than the adenylate cyclase activation produced by CNC (Fig. I.).

Eff'ect of quinoneformation AGX and its analogues (except AAC) are rapidly oxidized to quinones in the presence of tyrosinase. The conversion to the quinone form results in loss of agonist activity (Table VI). At higher concentrations the quinones inhibit adenylate cyclase activity. This inhibition appears unrelated to the al receptor site since inhibition can also occur when adenylate cyclase was activated by fluoride, Gpp(NH)p or any of the agaridoxin compounds. The inhibition by the quinones requires 10-100-fold higher concentrations than does activation by the parent compounds. Furthermore, this TABLE V

Effect of A DX and CNC on brain adenylate cyclase activity Membrane particles from cerebellum, cerebral cortex or hypothalamus were added to reaction mixtures which contained l0 -6 Gpp(NH)p plus or minus the agonists. Values are the averages of two separate experiments, with each determination done in triplicate.

Adenylate cyclase activity (pmol cAMP/mg/5 m&)

Gpp(NH)p (10 6 M) A G X ( 2 :,( 10-s M) C N C ( 1 0 'SM) A A C (10 -~ M) * ** § ~§

Cerebellum

CerebralcJrtex

Hypothalamus

184 171 208 165

193 234 211 207

218 316 426 396

Significantly different from Significantly different from Significantly different from Significantly different from

± J: ~ :L

9 7 10" 8

Gpp(NH)p Gpp(Nl-l)p Gpp(NH)p Gpp(NH)p

control control control control

4~ ± -L at at at at

3 11§ 5§ 5**

P P P P

<: 0.05. -~ 0.02. ~ 0.01. ( 0.001.

t: ± ~ 5:

11 16§~ 11§§ 20§§

394

500

/ / /

I-

/ /

¥ I

k'}

O")c

II

Z W

, / ~ "N / "~

~-"\

/,

0 ~ 300

"~

/

--

/

200

0 10-8

- ~

I 10.7

-----~-. . . . . . . . . . . . . . . . . . . .

_

i 10.6

•

CNC

[]

Methoxom,ne

•

Phenylephr ine

0

AGX

Z}, Clonidine1

CONCENTRATION

10-5

BASAL

I 10-4

(M)

Fig. 1. Concentration dependence of adenylate cyclase activation by selected adrenergic agonists. Hypothalamic membrane particles were from reserpinized animals and added to reaction mixtures containing 10 -6 Gpp(NI-I)p plus or minus the indicated agonists. Results are from replicate experiments done in triplicate.

TABLE VI

Inhibition of adenylate eyclase by the qu&ones of AGX and CNC: reversal by DTT The quinones of CNC and AGX were incubated with membranes ± 2.mM DTT (8 min, 32 ~C). Reaction mixture with or without 2 mM DTT, and in the presence of agonists as shown, was added to the membranes and adenylate cyclase assayed for 15 min (32 °C). Values are the result for three experiments with each determination done in triplicate. Basal adenylate cyclase activity (without agonist or quinones) was 138 3_ 5 pmol cAMP]mg prot./5 rain.

Agonist

Gpp(NH')p (5 x 10 -6 M) Gpp(NH)p (5 × 10 -6 M) Gpp(NH)p (5 × 10 -6 M) NaF (10 mM) NaF (10 raM) NaF (10 mM) NaF (10 mM) NaF (10 mM) NaF (10 mM)

Quinone

None CNC-Q(5 × 10 `4 M) AGX-Q (10 -4 M) None CNC-Q (5 :~ 10 -a M) CNC-Q (5 "< 10 -5 M) CNC-Q (5:4 10 -6 M) AGX-Q (10 -4 M) AGX-Q (5 x 10 -~ M)

Adenylate eyelase activity (pmol cAMP/ mg protein]5 min) No addition

DTT (2 mM)

414 90 104 296 72 195 30l 73 295

444 268 222 226 ..... ...... 211 --

:L 7 ~: 2 :? 3 ~ 6 i 5 :J-- 1 ± 9 + 2 _A:. 8

:L: 9 j 2 i 7 L= 12

.{: 4

395 it~hibition is prevented by the presence of sulfhydryl reducing agents such as DTT (Table VI). The agaridoxin compounds and their quinones were also tested for effects on phosphodiesterase (PDE) activity. While the parent compounds had no effect on PDE activity, the quinone of AGX (at 10-4 M) inhibited PDE by 20 ~. We note, however, that neither the activation of adenylate cyclase by AGX and its hydroxyanalogues, nor the inhibition of adenylate cyclase by the quinone derivatives are attributable to the small inhibition that the quinones cause of PDE activity. DISCUSSION The Gpp(NH)p dependent activation of hypothalamic adenylate cyclase requires the preservation of the reduced (hydroxy) form of the molecule. Oxidation of these compounds to the quinone form results not only in a loss of activation of adenylate cyclase but in the generation of an inhibitor which appears to oxidize enzyme sulfhydryl groups. While agaridoxin and its analogues activate adenylate cyclase in other mammalian tissues, hypothalamic particles isolated from the rat have been extensively studied because they (1) have a relatively large array of receptor types including an almediated cyclase1,25; (2) show cyclase activation by AGX and methoxamine 7 which is observed in the presence and absence of EGTA and therefore is Ca 2 ~ and calmodulin independent; (3) stimulation of adenylate cyclase by AGX is more effective in the rat hypothalamus than the cerebral cortex or cerebellum. The data obtained with al inhibitors indicate that AGX may function predominantly at an (21 receptor. CNC also interacts with the al receptor. Binding studies to further characterize agaridoxin and CNC were done with [ZH]-WB-4101, a known antagonist 14,31. Data using yohimbine and its sterioisomeric alkaloids indicate [ZH]WB-4101 binds to at adrenergic receptor, while [3H]clonidine binds to an (2z receptor 29. Therefore [3H]WB-410I is a good candidate for receptor binding studies since it binds with high affÉnity to the (21receptor, blocks AGX activation of adenylate cyclase and is available at high specific activities. Studies with [3H]-WB-4101 indicate agaridoxin has a Ki (0.59 /zM) that is similar to that of methoxamine (0.44/~M) and smaller than that of norepinephrine (1.5 #M) 14. CNC had no effect on [3H]-WB-4101 binding at concentrations between 10-7 and 10-5 M under the conditions of the assay. Since CNC activation of adenylate cyclase is partially blocked by propranolol and WB-4101, it appears to function as a mixed agonist. CNC is a considerably more potent agonist than norepinephrine, dopamine, clonidine, methoxamine agaridoxin or L-phenylephrine. This greater efficacy is observed as 2-fold higher adenylate cyclase activation than that found with conventional (2l agonists. CNC also displays efficacy at concentrations an order of magnitude lower than naturally occurring or pharmacological agonists. Its interaction at two classes of cyclase linked receptors may explain its enhanced efficacy. Reserpinization studies give further support for the al receptor interaction of

396 agaridoxin. Neither agaridoxin nor methoxamine effects were influenced by reserpinization, whereas norepinephrine and clonidine stimulation were (Table I I I). Our findings confirm previous studies which emphasize the abundance of a~ (methoxamine) stimulated adenylate cyclase in rat hypothalamus 1. These observations call attention to a new class of potent al agonists which are isolated from fungi. Agaridoxin and its analogues may have intriguing potential as neuropharmacological and neurochemical agents. The function of agaridoxin in plant physiology is unknown. The molecule could influence the development of sporulation where its function could depend on its availability as an oxidizable substrate rather than a hormonal role 34. The abundance of the A G X precursor P G H (100 mg/100 g Agaricus bisporus gill tissue 3~;)and its readily oxidizable nature support the latter possibility. The striking Gpp(N H)p dependent agonist effects of these fun gal compounds on synaptic membrane adenylate cyclase are intriguing. These effects exemplify once again the fascinating circumstance of a plant substance interacting with a specific class of neuronal receptors 10,26. ACKNOWLEDGEMENTS We gratefully acknowledge Drs. George Aghajanian, George Wheeler, Mark Rasenick and David Menkes for the critical reading of this manuscript. We also acknowledge Dr. Mark Rasenick for assistance with receptor binding studies. This work was supported in full by the National Cancer Institute, Grant No. CA 22301.

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