~~r~~~, Natl. Acad. Sci. USA \,ol 76, No. 3, pp. 1135-1139, March 1979 Biochemistry Adenylate cyclase and acetylcholine release regulated by separate serotonin receptors of somatic cell hybrids (lysergic acid diethylamide/neurotransmitters/synapse/neuroblastoma) JOHN MACDERMOT *, H~RUHIRO HIGASHIDA?, STEVEN P. WILSON, HIROSHI MATSUZAWAS, JOHN MINNAI, AND MARSHALL NIRENBERG L&ratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Warren Nirenberg, December 13,1978 .iBSTRACT Serotonin activates adenylate cyclase [ATP p!rophosphate-lyase (cyclizing), EC 4.6.1.11 of NCB-20 neuro- biastoma-brain hybrid cells with an activation constant of 530 n&l, but has little or no effect on cellular cyclic AMP or cyclic GhlP content of NlE-115 neuroblastoma or NG108-15 hybrid cells. In homogenates of NCB-20 hybrid cells, lysergic acid di- ethylamide stimulates adenylate cyclase activity (K,,, = 12 nM) and partially inhibits (Ki = 10 nM) the stimulation of adenylate cyciase activity by serotonin. No desensitization was detected of serotonin receptors coupled to adenylate cyclase. Serotonin also depolarizes NCB-20, NGlOS-15, and NlE-115 cells and in- creases acetylcholine release. Serotonin receptors mediating depolarizing responses desensitize rapidly and reversibly, and ihe depolarizing effects of serotonin are neither mimicked nor inhibited by lysergic acid diethylamide. These results indicate that (i) NCB-20 cells possess at least two species of serotonin receptors, which independently regulate cellular functions, (ii) activation of adenylate cyclase does not directly affect mem- brane potential or acetylcholine release, and (iii) serotonin- dependent cell depolarization does not affect cyclic AMP or cyclic GMP synthesis in the cell lines tested. Serotonin activates adenylate cyclase (ATP pyrophosphate-lyase i,cyclizing), EC 4.6.1.11 in mammalian brain (13) and increases or decreases the frequency of spontaneous action potentials in cerebral neurons (4,5). Serotonin also induces contractions in ileal smooth muscle (6). However, central neuronal responses to serotonin in mammals are predominantly inhibitory and are mediated through either pre- or postsynaptic receptors, which can be distinguished by the differences in their responses to D-lysergic acid diethylamide (LSD) (5, 7). In molluscan neu- rons, as many as six kinds of response to serotonin have been detected (8). Serotonin-dependent activation of adenylate cyclase is pre- \rrved after electrolytic lesion of the raphe nuclei, which greatly reduces the number of serotonergic neurons innervating the colliculus. This suggests that collicular serotonin receptors that mediate activation of adenylate cyclase are postsynaptic re- ceptors (2). In this report, effects mediated by two species of serotonin receptor in NCB-20 neuroblastoma-brain hybrid cells are de- scribed. One receptor is coupled to the activation of adenylate Wlase, and the other mediates cell depolarization and ace- tYlcholine release. The receptors are distinguished on the basis of functional response, specificity, and desensitization. `rb prA)lication costs of this article were defrayed in part bv page charge payment. This article must therefore be hereby markid "ad- "~rtiscment" in accordance with 18 U. S. C. 91731 solely to indicate this fact. MATERIALS AND METHODS Cells. The NlE-115 adrenergic neuroblastoma cell line was cloned from C-1300 mouse neuroblastoma (9). The NG108-15 neuroblastoma-glioma hybrid cell line (unpublished results) was derived by Sendai virus-induced fusion of C-1300 mouse neuroblastoma clone N18TG2 resistant to 6-thioguanine (10) with rat glioma clone CGBU-1, resistant to 5-bromodeoxyuri- dine (11). The NCB-20 neuroblastoma-fetal Chinese hamster brain hybrid cell line (12) was obtained by Sendai virus-induced fusion of NlSTG2 with fetal Chinese hamster brain cells dis- sociated from 18-day embryos. The NBr-1OA and NBr-20A neuroblastoma-rat liver hybrid cell lines (unpublished results) were obtained by Sendai virus-induced fusion of N18TG2 with the BRL30E Buffalo rat liver cell line, resistant to 5-bromo- deoxyuridine (H. G. Coon, personal communication). Adenylate Cyclase Assay. Cells were cultured as described (13). Confluent cells were detached from flasks by gentle tapping and were washed three times, each with 10 ml of Dulbecco's phosphate-buffered saline (without Ca"+ or Mg"+ ions) adjusted to 340 mOsm with NaCl. Cells were recovered by centrifugation (150 X g for 5 min) after each wash, and after the third wash were suspended in 25 mM Tris-HCI, pH 7.4/290 mM sucrose (2 m1/75-cm' flask) and homogenized at 4oC with 50 strokes of a Dounce homogenizer. Homogenates were frozen and stored in a liquid Nz freezer. Each lOO-~1 reaction mixture contained 50 mM Tris-HCI (pH 7.4); 87 mM sucrose; 20 mM creatine phosphate, disodium salt (Sigma); 10 International Units of creatine kinase, 150 units/mg of protein (ATP:creatine N-phosphotransferase, EC 2.7.3.2) from Sigma; 1 mM cyclic AMP (CAMP), sodium salt (Sigma); 0.25 mM Ro20-1724 (a gift from Hoffmann-La Roche); 0.25% ethanol; 1 mM [~u-~~P]ATP (3 &i, New England Nuclear; 1 Ci = 3.7 X lOlo Bq); and 100-200 pg of homogenate protein. Homogenates were thawedand maintained at 4oC in an ice bath for no longer than 10 min prior to incubation. Reaction mixtures were incubated for 8 min at 37oC. Adenylate cyclase activity was determined by a modification (14) of method C Abbreviations: LSD, D-lysergic acid diethylamide; PGEL, prostaglandin El; cAMP, cyclic AMP; Rt-cAhlP, iv", @-dibutvrvl cvclic AhiP; cGhlP, cyclid GMP; Gpp(NH)p, guanylyl-5'-imidddiphosphate. + Present address: Deut. of Clinical Pharmacoloev. Rovai Postgraduate Medical School, London W12 OHS, England ' u + Present address: Dept. of Aerospace Physiology, Research Institute of Environmental Medicine. Nagoya University, Nagoya 161, Japan. + Present address: Dept. of Agricultural Chemistry. University of Tokyo, Tokyo, Japan. 4 Present address: Human Tumor Biology Laboratory. VA Hospital. Washington, DC 20422. 1135 1136 Biochemistry: MacDermot et al. of Salomon et al. (15). The production of [32P]cAMP was pro- portional to protein concentration within the range 50-250 pg of homogenate protein per reaction mixture; similarly [32P]- CAMP synthesis increased linearly for 30 min. Values are the means of two to four determinations, except where noted; replicates usually varied by <8%. CAMP and Cyclic GMP (cGMP) Assays. Methods for cell culture and incubation, and for CAMP and cGMP separation and assay have been described by Matsuzawa and Nirenberg (16). Protein was determined by a modification of the method of Lowry el al. (17), with bovine serum albumin, crystallized three times, as standard. Electrophysiological Measurements. Hybrid cells were usually cultured for 5-15 days in the presence of 1 mM N6, O*`-dibutyryl-CAMP (BtzcAMP). Myoblasts were dissociated from newborn Fisher rat hind limb and cultured for 3 days. Nondividing myotubes were selected for by incubating cells with 10 PM 5-fluorodeoxyuridine and 100 PM uridine for 48 hr; then hybrid cells that had been treated with 1 mM BtzcAMP for 7-10 days were added and cells were cocultured for an additional l-3 days in the presence of 1 mM BtzcAMP (18). The techniques for intracellular recording of membrane potentials of cultured cells have been described (18, 19). Serotonin and LSD were applied to'cells iontophoretically or by addition of a solution from a micropipette. The following generous gifts were received: LSD from Judith R. Walters; bufotenineqreatinine sulfate (Upjohn) and N,N'- dimethyl-5-methoxytryptamine (free base) (Aldrich) from J. C. Gillin; mianserin.hydrochloride (Organon Products, West Orange, NJ) from S. H. Snyder; methysergidemhydrogen mal- eate from C. R. Creveling; prostaglandin El (PGE,) from Upjohn; metergoline from Farmitalia (Milan, Italy); cypro- heptadine.hydrochloride from Merck, Sharp and Dohme; fluphenazine2 HCl from Squibb; phentolaminehydrochloride from Ciba Pharmaceuticals (Summit, NJ); and pimozide (free base) from McNeil Laboratories Inc. (Fort Washington, PA). Serotoninecreatinine sulfate, 5-methoxytryptamine.hydro- chloride, and propranolol.hydrochloride were obtained from Sigma; tryptamine.hydrochloride from Regis Chemical Co.; and guanylyl-5'-imidodiphosphate [GPP( NH)p] from P-L Biochemicals. RESULTS Serotonin stimulated adenylate cyclase activity 75-l 15% in homogenates of NCB-20 hybrid cells (Fig. 1). The serotonin concentration required for half-maximal activation (K& was consistently within the range 40&8CMl nM and was not affected by 50 PM ascorbate or 100 PM pargyline. Homogenates were prepared and frozen for subsequent use since freezing and thawing had little or no effect on the serotonin-dependent stimulation of adenylate cyclase. An Eadie-Scatchard plot (Fig. 1, upper inset) was linear and the Kact for serotonin was cal- culated to be 530 nM. From a Hi11 plot (Fig. 1, lower inset), the interaction coefficient (n) was found to be 1.0, revealing no cooperativity in the serotonin-dependent activation of ade- nylate cyclase. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA; l-10,000 PM) had no effect on the stimulation of adenylate cyclase activity by serotonin, which suggests the ef- fect of serotonin is not dependent on Ca*+ ions (data not shown). Adenylate cyclase also was stimulated by LSD (Kact = 12 nM) and the LSD analogs metergoline (K,,t = 250 nM) or methy- sergide (Kact = 620 nM) (Fig. 2A). The maximum stimulations of adenylate cyclase activity by LSD or LSD analogs were less than those obtained in the presence of serotonin (Fig. 1). The synthesis of [32P]cAMP in the presence or absence of 10 PM PTOC. Nat 1. Acad. Sci. USA 76 (1979) 1, IOU i5Hfl. C1M 4O I I I 10-7 106 10-s 10-4 [Serotonin], M FIG. 1. Serotonin-dependent increase in adenylate cyclase ac- tivity in a homogenate of NCB-20 hybrid cells. Results show the means (&SD) of triplicate determinations of adenylate cyclase activity as a function of serotonin concentration. (lfpper Inset) Eadie- Scatchard plot; 1V 5HT is the serotonin-dependent increase in ["`PIcAMP synthesis (pmol/min per mg of protein). (Lower Inset) Hill plot; 9" is the increase in adenylate cyclase activity at each se- rotonin concentration and "R" is the maximum increase. serotonin or 0.1 PM LSD was linear with respect to time and did not desensitize within 16 min (Fig. 2B). In another exper- iment, no desensitization to serotonin was detected in 30 min (not shown). Adenylate cyciase activity also was increased by the following analogs of serotonin: tryptamine, bufotenine, 5-methoxytryp- tamine, and N,N'-dimethyl-5-methoxytryptamine (Fig. 3A). The Lt values for these compounds were 400-570 nb1, and maximum stimulations of adenylate cyclase activity were similar to that of serotonin (Table 1). The serotonin receptor antagonists, mianserin or cyprohep- tadine, inhibited the stimulation of adenylate cyclase by 10 &l serotonin (Fig. 3B). The Ki values were 43 nM for mianserin and 95 nM for cyproheptadine. Mianserin and cyproheptadine also inhibited the stimulation of adenylate cyclase by LSD (K, = 100 nM and 64 nM, respectively). The dopamine receptor antagonists, fluphenazine or pimozide, inhibited serotonin- dependent stimulation of adenylate cyclase (Ki = 4i nM and [ActIvatorI, M Minutes FIG. 2. Activation of adenylate cyclase in homogenates of NCB-20 hybrid cells as a function of concentration of lysergic acid derivatives (A) or by LSD and serotoniri as a function of incubation time (B). (A) Values are the means (*SD) of triplicate determina- tions: 0, LSD; A, metergoline; and 0, methysergide. (B) Volume of each reaction mixture was 2 ml; concentrations of reagents are given in Materials and Methods. At each time indicated, a loo-p1 aliquot was removed and the reaction was terminated. Adenylate cyclase activity was determined in the presence of water (0); 0.1 PM LSD (b): 10 WM serotonin (0). Biochemistry: MacDermot et al. Proc. Natl. Acad. Sci. USA 76 (1979) 1137 FIG. 3. Activation of adenylate cyclase by tryptamine derivatives (A) and the inhibition of serotonin-stimulated adenylate cyclase ac- t.ivity in homogenates of NCB-20 hybrid cells by mianserin and cyproheptadine (B). (A) Values a.re the means of triplicate determi- nations of adenylate cyclase activity in the presence of increasing concentrations of tryptamine derivatives: o , serotonin (3.891; 0, lryptamine [X90]; A, bufotenine 12.75); 0, 5-methoxytryptamine 1:1.98]; or X, N,N'-dimethyl-5-methoxytryptamine (3.511. (Values shown in brackets are the pmol of cAMP/min per mg of protein that correspond to 100% on the ordinate.) (B) Results show the means (*SD) of triplicate determinations of adenylate cyclase activity in the presence of 10 PM serotonin and increasing concentrations of ei- ther mianserin (A) or cyproheptadine (0). Basal enzyme activity was 4.93 (f0.27) pmol of cAMP/min per mg of protein. 250 Al, respectively). These Ki values are greater than those reported for the inhibition of ["Hlhaloperidol binding to do- pamine receptors in calf brain membranes (0.88 nM and 0.81 nM, respectively) (20). The cr- or a-adrenergic receptor an- tagonists, phentolamine or propranolol, respectively, had little or no effect on the stimulation of adenylate cyclase by serotonin. Results are summarized in Table 2. The effect of serotonin concentration on adenylate cyclase activity in the presence or absence of a saturating concentration of LSD (10 MM) is shown in Fig. 4A. LSD stimulated adenylate cycluse and also partially inhibited the serotonin-dependent increase in enzyme activity. The effect of LSD concentration on adenylate cyclase activity was determined by using a dif- !erent homogenate in the presence or absence of 10 &l sero- tonin (Fig. 4B). LSD increased adenylate cyclase activity (Kact = 12 nM), as shown previously, and also partially inhibited the serotonin-dependent activation of adenylate cyclase (Ki = 10 nM). Serotonin stimulated adenylate cyclase activity only slightly Table 1. Specificity of serotonin receptors coupled to adenylate cyclase of NCB-20 hybrid cells Maximum activation, Additions K,,,, nM % Tryptamine derivatives Serotonin 530 80 5Methoxytryptamine 570 18 Tryptamine 400 77 NJ'-Dimethyl-5-methoxytryptamine 410 73 Bufotenine 520 60 1,ysergic acid derivatives LSD 12 41 Riletergoline 250 35 Methysergide 620 32 The activation constant (K,,,) for each compound is the concen- tration required for half-maximal stimulation of adenylate cyclase ;lctivity. The salts of the compounds tested are given in Xlaterials and .~fclhods. Table 2. Effects of receptor antagonists on serotonin- or LSD-activated adenylate cyclase of NCB-20 hybrid cells Adenylate cyclase, Ki (nM) Additions 5HT-activated LSD-activated Mianserin 43 loo Cyproheptadine `95 64 LSD (partial agonist) 10 Fluphenazine 47 Pimozide 250 Phentolamine >10,000 Propranolol > 10,000 The Ki values were calculated from the following equation: Ki = ICsa/l + ([activator]/K,,t of activator), where ICW is the concentration of antagonist producing half-maximal inhibition. 5HT, serotonin (5-hydroxytryptamine). in homogenates prepared from NG108-15, NBr-lOA, NBr-20A hybrid, or parental N18TG2 neuroblastoma cells (Table 3). Treatment of intact NG108-15 or NlE-115 cells with 10 PM serotonin for 0.5, 1.0, 1.5,2.0,2.5,5.0, and 10 min had littleor no effect on intracellular levels of CAMP or cGMP (not shown). As shown in Table 4, GTP increased basal adenylate cyclase activity by 31% and increased serotonin-dependent activation by 265%. Sodium fluoride stimulated adenylate cyclase activity 850% and completely blocked the effect of serotonin. Gpp(NH)p also stimulated adenylate cyclase activity 125%; however, the stimulatory effects of Gpp(NH)p and serotonin were additive. PGEl activated adenylate cyclase, and serotonin reduced by 13% the PGEl-dependent activation of the enzyme. Sodium fluoride or Gpp(NH)p inhibited PGEl-stimulated adenylate cyclase activity, and serotonin further reduced the activity in the presence of PGE, and Gpp(NH)p. These results suggest that activation of adenylate cyclase by serotonin re- quires GTP and reveals no unusual interaction with sodium fluoride or PGE1, with respect to the coupling of serotonin re- ceptors to the adenylate cyclase complex. However, the additive rather than synergistic effects of serotonin and Gpp(NH)z may indicate a difference in the mechanism of coupling of serotonin receptors to adenylate cyclase compared to other species of receptors. Responses of a NG108-15 hybrid cell to serotonin applied iontophoretically are presented in Fig. 5, which are represen- tative of the responses of NCB-20 and NlE-115 cells. Serotonin application resulted in two action potentials followed by cell depolarization for more than 1 set (Fig. 5A), confirming pre- vious findings (19). Repetitive application of serotonin, at a 1 10-6 10-5 ,otoninl, [Serotoninl, M M ILSDI, M ILSDI, M FIG. 4. Effect of LSD and serotonin on adenylate cyclase activity in homogenates of NCB-20 hybrid cells. (A) Increase in adenylate cyclase activity in response to increasing concentrations of serotonin in the absence (0) or presence (0) of 10 PM LSD. (B) Response to increasing concentrations of LSD in the absence (A) or presence (A) of 10 PM serotonin. 1138 Biochemistry: MacDermot et al. Proc. Matl. Acad. Sci. USA 76 (19791 Table 3. Effect of serotonin (5HT) on adenylate cyclase activity in homogenates of neuroblastoma N18T62 or hybrid cell lines Cell line pmol cAMP/min per mg protein Activation, Basal 10 UM 5HT % NCB-20 7.03 14.8 111 NBr-20A 7.08 8.36 18 NG108-15 14.2 16.5 16 N18TG2 4.09 4.24 4 NBr-1OA 48.7 50.0 3 frequency of 0.51 Hz, almost completely desensitized the re- sponse to serotonin (Fig. 5B). Responsiveness to serotonin re- covered within a few minutes in the absence of serotonin. LSD did not inhibit serotonin-dependent cell depolarization (Fig. 5C), and furthermore addition of LSD did not depolarize the cell (not shown). Application of PGEl, which markedly in- creases adenylate cyclase activity (Table 4), had little or no effect on cell membrane potential (unpublished results) or acetylcholine release (21) from NG108-15 cells. NG108-15 (18) and NCB-20 (unpublished results) hybrid cells form synapses with rat striated muscle cells in uitro. Se- rotonin-dependent release of acetylcholine from an NGlOB-15 hybrid cell at a synapse was measured by intracellular recording of synaptic potentials in a rat myotube (Fig. 6A). Application of serotonin to the hybrid cell resulted in an increase in ace- tylcholine secretion from the NG108-15 hybrid cell and a greater than 5O-fold increase in the frequency of muscle syn- aptic responses. LSD neither mimicked nor inhibited seroto- nin-dependent synaptic responses of the muscle cell (not shown). Serotonin had no effect on the membrane potential of muscle cells that were not innervated by NGIOS-15 or NCB-20 cells. The effect of serotonin on (3H]acetylcholine release from NG108-15 cells into the medium was measured. Muscle cells were not present. Cells were incubated with [3H]choline and washed and [3H]acetylcholine released into the medium was separated from [3H]choline and assayed (Fig. 6B) (21). Sero- tonin stimulated acetylcholine release from cells as shown (2I), and acetylcholine release was not inhibited by 50-500 nM LSD. The same concentrations of LSD had little or no stimulatory effect on acetylcholine release. Table 4. Effect of GTP, sodium fluoride, Gpp(NH)p, PGE1, and serotonin on NCB-20 adenylate cyclase activity Addition pmol cAMP/min per mg protein Basal 10 PM 5HT Exp. A Control 16.3 20.0 1pMGTP 21.4 31.2 Exp. B Control 5.06 8.88 30 mM NaF 43.5 42.6 10 &I Gpp(NH)p 11.4 16.6 10 rM PGE, 90.2 78.2 10 PM PGEi + 20 mM NaF 58.1 55.0 10 PM PGE, + 10 ALM GppUWp 86.4 75.7 In Exp. A, each reaction mixture contained 81.6 fig of protein of a 30,000 X g particulate fraction, washed three times with 25 mM Tris.HCi (pH 7.4), recovered by centrifugation (20 min). In Exp. B, adenylate cyclase activity of an unfractionated NCB-20 homogenate is shown. Each value is the mean of triplicate determinations. 5HT, serotonin. Seconds 0 I 20 50 100 150 5rT A km ' LSD' c' -lO- 1 -30 : 1::. I 4 i.-L;: > Seconds FIG. 5. Serotonin (5HT)-dependent regulation of ionophore activity in NG108-15 hybrid cells. (A) Iontophoretic application of serotonin toa hybrid cell. The iontophoretjc pipette contained a so- lution of 25 mM serotonin in water; a current of 100 n.4 was passed for 1 msec (shown in the trace at the bottom of each panel). (H) Re- petitive serotonin application at a frequency of0.51 Hz, which results in desensitization. (C) Effect of LSD on the depolarizing response was determined with repeated iontophoretic applications of serotonin in the absence of LSD and then after addition of 20 ~1 of 100 PM LSD in 150 mM NaCI. Prior to each pulse, a vo!tage calibration of 10 mV was passed for IO msec. DISCUSSION Activation of serotonin receptors of NClO%15 (19, 21) or NCB-20 hybrid cells results in cell depolarization, action po- tentials, and release of acetylcholine into the medium. These responses desensitize in less than 15 set and are not inhibited Minutes FIG. 6. Serotonin-dependent release of acetylcholine from NG108-15 hybrid cells. (A 1 Depolarizing responses of a rat striated muscle cell to acetylcholine released from a N6108-15 cell in response to serotonin (5HT) at a synapse; recording obtained with an intra- cellular microelectrode. Two microliters of a solution containing 10 gM serotonin and 150 mM NaCl was applied to the hybrid cell (arrow). (Top) Trace of an AC recording at high gain; (Middle) trace of a DC recording at low gain; (Louser), the number of spontaneous and serotonin-evoked muscle responses per sec. (R) [:%H]Acetylcholine ([:`H]ACh) release from hybrid cells into the medium. Cells were grown in the presence of 1 mM BtzcAMP for 17 days and then transferred to capillary pipettes (21) and grown for an additional 2 days. Cells then were incubated in 25 PM [methylJH]choline (4.2 Ci/mmol) for 45 min and washed by perfusion (0.4 ml/min) for 17.5 min before l-ml fractions were collected. ["HjAcetylcholine in the perfusate was separated from [methyl-"HIcholine and measured (21). Cells were exposed to 10 PMserotonin (5HT) with 50 nM (o), 500 nM (A), or no (0) LSD at the times shown. Biochemistry: MacDermot et al. or mimicked by LSD. Serotonin also stimulates adenylate cy- clase activity of NCB-20 hybrid cells, and in contrast these re- ceptors do not desensitize. In addition, LSD activates adenylate cyclase and partially inhibits activation of the enzyme by se- rotonin. The results suggest that cell depolarization and acti- vation of adenylate cyclase are mediated by different species of serotonin receptors. The serotonin receptors that mediate excitatory, depolarizing responses resemble M-receptors of neurons in the peripheral nervous system (6). These receptors differ from both pre- and postsynaptic serotonin receptors of mammalian brainstem (5, `i), which inhibit neuronal firing and are also activated by LSD. Inhibitory serotonin responses not affected by LSD have been found in brainstem neurons (22), the suprachiasmic nucleus (4), and half the serotonin-sensitive neurons of cat cerebral cortex (23). The specificity of serotonin receptors coupled to activation of adenylate cyclase in NCB-20 hybrid cells resembles that of serotonin receptors coupled to adenylate cyclase in mammalian brain (3). The demonstration that activation of adenylate cyclase by serotonin is stimulated by GTP, that sodium fluoride uncouples activation by serotonin, and that serotonin inhibits to a small extent the stimulation of adenylate cyclase by PGEl suggests a common mechanism for the coupling of receptors for sero- tonin and other neurotransmitters to the adenylate cyclase complex. However, the additive, rather than synergistic effects of serotonin and Gpp(NH)p suggest that coupling of the sero- tonin receptors to the adenylate cyclase complex may differ from that of other neurotransmitters. Serotonin stimulates .idenylate cyclase activity SO-100% in homogenates of colliculus of neonatal rat brain, which is similar to the extent of activation of adenylate cyclase by serotonin in NCB-20 homogenates. Eadie-Scatchard analysis of the activation of adenylate cy- clase by serotonin suggests a bimolecular interaction and reveals no evidence of receptor heterogeneity. The Hill interaction coefficient (n) is 1.0, indicating independent, noncooperative reactions. LSD activates adenylate cyclase (K,,, = 12 nM) and inhibits the activation of the enzyme by serotonin (Ki = 10 nM). In addition, mianserin and cyproheptadine inhibit serotonin Jctivation of adenylate cyclase (Ki = 43 nM and 95 nM, re- spectively) and LSD activation of adenylate cyclase (Ki = 100 nM and 64 nM, respectively). These results show that serotonin and LSD interact at a receptor site(s) that mediates activation of adenylate cyclase. Enjalbert et al. (3) have shown a complex interaction between serotonin and LSD at the level of adenylate cyclase in mammalian brain. Interactions between serotonin and LSD have also been demonstrated in binding studies (24, 25). Binding sites for [3H]LSD were detected in NCBZO homog- enates (unpublished results); the Ko,,, was 36 nM, the Hill roefficient was l..O, and the receptor concentration was 385 fmol/mg of protein. [3H]LSD was displaced by serotonin (Ki = 1 lo-180 nM). These results agree well with those presented for the stimulation of adenylate cyclase activity by a serotonin receptor that is also responsive to LSD. Two binding sites for ["Hjserotonin were detected in NCB-20 homogenates ~~~~~~ = 200 nM and 3750 nM] and serotonin and LSD interactions hoc. Natl. Acad. SC% USA 76 (1979) 1139 also were shown. LSD does not displace serotonin from the low-affinity serotonin site, which suggests that these molecules may function as receptors mediating cell depolarization and acetylcholine release. We conclude that NCB-20 hybrid cells possess two species of serotonin receptors, that activation of adenylate cyclase does not affect the rate of acetylcholine release, and, conversely, that serotonin-dependent cell depolarization does not affect intra- cellular levels of CAMP or cGMP in the hybrid cells tested. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25 Von Hungen, K., Roberts, S. & Hill, D. F. (1975) Brain Res. 84, 257-267. Enjalbert, A., Bourgoin, S., Hamon, M., Adrien, J. & Bockaert, J. (1978) Mol. Pharmucol. 14,2-10. Enjalbert, A., Hamon, M., Bourgoin, S. & Bockaert, J. (1978) Mol. Pharmucol. 14,11-23. Bloom, F. E., Hoffer, B. J., Siggins, G. R., Barker, J. L. & Nicoll, R. A. (1972) Fed. Proc. Fed. Am. Sot. Erp. Biol. 31,97-106. Haigler, H. J. & Aghajanian, G. K. (1977) Fed. Proc. Fed. Am. Sot. Erp. Biol. 36,2159-2164. Gaddum, J. H. & Picarelli, 2. P. (1957) Br. j. Phurmacol. 12, 323-328. Aghajanian, G. K. & Haigler, H. J. (1975) Psychopharmacol. Commun. 1,619-629. Gerschenfeld, H. M. & Paupardin-Tritsch, D. (1974) J. Physiol. 243,427-456. Amano, T., Richelson, E. & Nirenberg, M. (1972) Proc. N&l. Acad. Sci. USA 69,258-263. Minna, J., Glazer, D. & Nirenberg. M. (1972) Nature (London) New Biol. 235,225-231. Amano, T., Hamprecht, B. & Kemper, W. (1974) Exp. Cell Res. 85,399-408. Minna, J. D., Yavelow, J. & Coon, H. G. (1975) Genetics, Suppl., 79,373-383. Klee, W. A. & Nirenberg, M. (1974) Proc. Nat/. Acad. Sci. USA 71,3474-3477. Sharma, S. K., Nirenberg, M. & Klee. W. A (1975) Proc. Nat!. Acad. Sci. USA 72,590-594. Salomon, Y., Londos. C. & Rodbell, M. (1974) Ana/. Biochem. 58,541-548. Matsuzawa, H. & Nirenberg, M. (1975) Proc. Natl. Acad. Sci. USA 72,3472-3476. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J, Biol. Chem. 193,265-275. Nelson, P., Christian, C. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73,123-127. Christian, C. N., Nelson, P. G., Bullock, P., Mullinax, D. & Nirenberg, M. (1978) Brain Res. 147,261-276. Burt, D. R., Creese. I. & Snyder, S. H. (1976) Mol. Phormaco[. 12,800-812. McGee, R., Simpson, P., Christian, C., Mata, M., Nelson, P. & Nirenberg, 41. (1978) Proc. Natl. &kad. Sci. USA 75, 1314- 1318. Boakes, R. J., Bradley, P. B., Briggs, I. & Dray, A. (1969) Brain Res. 15,529-531. Roberts, M. H. T. & Straughan, D. W. (1967) J, Physiol. 193, 269-294. Bennett, J. P.. Jr. & Snyder, S. H. (1976) `Vol. Pharmacol. 12, 373-389. Fillion, G. M. B., Rousselle, J. C., Fillion, M. P., Beaudoin, D. M.. Goiny, M. R., Deniau, J. M. & Jacob, J J. (1978) Mol. Pharmacol. 14,50-59.