Sigma Receptors Regulate Contractions of the Guinea Pig

Home , Benoxathian, Benzomorphan, Rimcazole, Tiospirone

The Journal of Neuroscience, October 1989, 9(10): 33803391

Sigma Receptors Regulate Contractions of the Guinea Pig Ileum Longitudinal Muscle/Myenteric Plexus Preparation Elicited by Both Electrical Stimulation and Exogenous Serotonin

Bruce G. Campbell,’ Michael W. Scherz,2 John F.W. Keana,* and Eckard Weber’ ‘Vellum Institute, Oregon Health Sciences University, Portland, Oregon 97201, and *Department of Chemistry, University of Oregon, Eugene, Oregon 94305

Sigma receptors are specific, highly localized binding sites These results suggest that there are functional sigma re- in limbic and sensorimotor structures of the brain that in- ceptors on cholinergic nerve terminals or within the myen- teract with many psychotropic drugs. These agents include teric plexus and that these receptors can inhibit stimulated the psychotomimetic benzomorphan opiates, the psychoto- ACh release through an opioid receptor-independent mech- mimetic drug phencyclidine and its analogs, as well as nu- anism. However, sigma receptor activation in the ileum has merous typical and atypical antipsychotics such as halo- the same effect on ACh release as activation of naloxone- peridol, chlorpromazine, and the novel drugs BMY 14802 and sensitive opioid receptors. The LMMP may be an in vitro rimcazole. So far, no physiological function has been as- bioassay system for characterizing the mechanism of action signed to these binding sites. We have synthesized a num- of sigma receptors and for determining the biological effi- ber of novel sigma receptor-active drugs derived from the cacy of drugs known to bind to sigma receptors in radioli- selective sigma ligand NJ!!‘-di(o-tolyl)guanidine (DTG). DTG gand binding assays. and its congeners were found to inhibit contractions of the guinea pig ileal longitudinal musclelmyenteric plexus (LMMP) Phencyclidine (PCP, “angel dust”) and certain benzomorphan preparation evoked by electrical stimulation. In addition, the (sigma) opiates have similar behavioral effects in humansand sigma ligands noncompetitively antagonized contractions of in animals (Holtzman, 1985; Katz et al., 1985). In humans, the LMMP preparation evoked by serotonin (5HT). The thesedrugs are known to inducepsychotomimetic effectssimilar 5-HT-evoked contractions were found to be largely due to those seen in schizophrenic psychosis(Keats and Telford, to 5-HT’s activation of 5-HT, receptors to release ACh. The 1964; Haertzen, 1970). Psychotomimetic effects are best doc- activity of DTG congeners in inhibiting electrically or umented for PCP sinceit is a widely abusedstreet drug (Aniline 5-HT-evoked contractions of the LMMP highly correlated with and Pitts, 1982). Becauseof its strong psychotomimetic effects their potency to inhibit binding of both 3H-DTG and (+)3H-3- in humans,PCP has beenproposed as a drug model for schizo- PPP [3(3-OH-phenyl)-K( 1 -propyl)piperidine] to sigma re- phrenia (Snyder, 1980).Recent radioligand binding studieshave ceptors in guinea pig brain homogenates. Two DTG con- shown that in brain membrane homogenatesPCP and sigma geners that did not bind to sigma receptors also showed no opiates bind to 2 anatomically and biochemically distinct re- activity in the bioassay. Many other (but not all) sigma receptor binding sites:the PCP receptor and the sigma receptor ceptor ligands showed a high correlation between their po- (Quirion et al., 1987;Sonders et al., 1988).The 2 receptorsshare tency to inhibit electrically evoked contractions of the LMMP some pharmacological properties in that they both bind PCP and their sigma receptor binding affinity. The benzomor- and its derivatives as well as the sigma opiates. However, the phans (+)SKF 10,047 and (+)cyclazocine potentiated elec- PCP receptor has a higher affinity for PCP than for sigmaopi- trically evoked contractions of the LMMP. Sigma ligands also ates, while the sigma receptor has a higher affinity for sigma inhibited the contractions of the LMMP in the presence of opiatesthan for PCP (Largent et al., 1984, 1986; Downeset al., the opiate antagonist naloxone and in preparations in which 1986; Weber et al., 1986).The main pharmacologicaldifference opioid receptors had been inactivated by treatment with the between the 2 receptors is that sigma sitesbind many typical irreversible opiate antagonist beta-chlornaltrexamine. Con- and atypical antipsychotic drugs, while PCP receptors are in- trol experiments suggested that the sigma ligands act via a sensitiveto antipsychotics (Su, 1982;Largent et al., 1984, 1986, neuronal mechanism to inhibit ACh release evoked by elec- 1988; Tam and Cook, 1984; Weber et al., 1986). It has been trical stimulation or by stimulation with 5-HT. proposedthat PCP receptorsare solely responsiblefor mediating the psychotomimetic effects of PCP and sigma opiates (Sircar et al., 1986), while sigma receptors-because of their high af- Received Oct. 14, 1988; revised Mar. 6, 1989; accepted Mar. 20, 1989. finity for many typical and atypical antipsychotic drugs- have We thank Barbara Tester and Nam Hoang for help with the binding assays, Dr. been suggestedas a site involved in mediating some of the Annmarie Surprenant for providing guinea pig ilea, and Dr. John Williams for comments on the manuscript. This work was supported by grants from the Na- therapeutic properties of the antipsychotic drugs that bind to tional Institute of Mental Health (MH40303 and MH42068) and by a grant from this site (Deutch et al., 1988; Snyder and Largent, 1989). Cambridee Neuroscience Research. Inc.. Cambridge. MA. Becauseof the proposed involvement of the 2 receptorsin Correspondence should be addressed to Eckardieber, M.D., Department of Pharmacology, University of California, Irvine, Irvine, CA 927 17. psychotomimetic and antipsychotic drug action, it is very im- Copyright 0 1989 Society for Neuroscience 0270-6474/89/103380-12$02.00/O portant to establishthe role of the 2 receptorsin neuronalfunc- The Journal of Neuroscience, October 1989, 9(10) 3381 tion (Sonders et al., 1988).Recent work hasunequivocally shown sigmareceptor bioactivity. Second,a seriesof novel compounds that the PCP receptor is associatedwith the N-methyl-D-aspar- is describedthat includes compounds of high sigma receptor trte (NMDA)-type glutamate receptor. Drugs that bind to the binding affinity and structurally related control compounds of PCP receptorblock the NMDA receptor-associatedcation chan- very low sigma receptor affinity and bioactivity. These com- nel after it has been opened by NMDA receptor agonists(Anis pounds may prove useful in the characterization of sigma re- et al., 1983; Wong et al., 1986; Kemp et al., 1987; Huettner and ceptor-mediated responses in the CNS and prove valuable in Bean, 1988) and the CNS distributions of PCP and NMDA the development of sigma receptor-active pharmaceuticals. sitesare nearly identical (Maragos et al., 1988). Unlike for the PCP receptor, no physiological function has been established Materials and Methods for the sigma receptor, although there is some evidence that Sigma receptor binding assays. Sigma receptor binding assays using sigma receptor-selectivedrugs can modulate neurotransmitter guinea pig brain membrane homogenates and the radioligands ‘H-DTG release(Campbell et al., 1987) interfere with central control of and (+),H-3-PPP were done as previously described (Weber et al., motor activity (Bowen et al., 1988b; Walker et al., 1988), and 1986). Briefly, frozen whole guinea pig brains (Biotrol, Indianapolis, modulate phosphoinositide metabolism (Bowen et al., 1988a). IN) were homogenized in 10 volumes (wt/vol) of ice-cold 320 mM sucrose using a Brinkman polytron. The homogenate was centrifuged Systematic investigations into the function of sigma receptors at 1000 x g for 20 min at 4°C. The supernatant was centrifuged at have beenhampered by the lack of a wide spectrum of selective 20,000 x g for 20 min at 4°C. The resulting pellet was resuspended in sigma receptor ligandsand by the lack of’an in vitro bioassay 10 initial volumes of 50 mM Tris/HCl buffer at pH 7.4 and centrifuged system in which to study sigma receptor function. at 20,000 x g for 20 min at 4°C. The resulting pellet was resuspended in 5 initial volumes ice-cold 50 mM Tris/HCl (DH 7.4). and the final We have recently synthesizedand characterized what appears volume was adjusted to yield a protein concentration of 3 mg/ml, as to be the most selective radioligand for sigma receptorsiden- determined by dye-binding protein assay (Biorad) using BSA as the tified to date (Weber et al., 1986). This compound, 3H-DTG standard. Aliquots of 20 ml were stored at -70°C until used. with no (N,N’-di(2-[5-3H]tolyl)guanidine), is a symmetrically substitut- detectable loss of binding. ed guanidine derivative. Structure-activity studies on com- For 3H-DTG binding assays, 20 ml aliquots of the frozen membrane suspension were thawed and diluted 1:3 in 50 mM Tris/HCl (pH 7.4). pounds synthesized in our laboratories have shown that nu- To 12 x 75 mm polystyrene test tubes were added 0.8 ml of diluted merous analogsof DTG have a similar sigma receptor binding membrane suspension, 0.1 ml of )H-DTG (46 Ci/mmol; see Weber et activity as the parent compound (unpublished observations). al., 1986) or (+)3H-3-PPP (New England Nuclear, 98 Ci/mmol) to yield We report here that DTG and 9 analogs with potent sigma a final concentration of 1.4 nM, and 0.1 ml of unlabeled drugs or buffer. receptor affinity dose-dependently and by a neuronal mecha- The protein concentration in the 1 ml final incubation volume was 800 ,&ml, corresponding to 32 mg of brain tissue (original wet weight) and nism inhibit musclecontractions of the isolatedguinea pig ileum to a tissue concentration within the linear range for specific binding. longitudinal muscle/myenteric plexus (LMMP) preparation Nonspecific binding was defined as that remaining in the presence of evoked by electrical stimulation. Two additional disubstituted 10 PM haloperidol. Specific binding constituted >90% of total )H-DTG guanidineswith very low sigma receptor affinity do not inhibit binding. Incubations were terminated after 90 min at room temperature by addition of 4 ml of ice-cold 50 mM Tris/HCl (pH 7.4) and rapid the electrically stimulated contractions of the LMMP. Addi- filtration of the membrane suspension through Whatman GF/B glass- tionally, we report here that DTG and its congenerswith high fiber filters under vacuum, using a 48 well cell harvester (Brandel, Gaith- sigmareceptor affinity noncompetitively inhibit contractions of ersburg, MD). The filters were washed 2 times with 4 ml of 50 mM Tris/ the LMMP evoked by stimulation of neuronal 5-HT, receptors HCl (pH 7.4). Total filtration and washing time was less than 20 sec. Each filter was dissolved in 10 ml Cytoscint (Westchem, San Diego, by serotonin (5HT). The potency of the DTG analogsin the 2 CA), and radioactivity was measured by liquid scintillation spectrom- bioassayscorrelates highly with their potency to inhibit binding etry at a counting efficiency of approximately 50%. IC,, values were of 3H-DTG to sigmareceptors in guinea pig brain homogenates, determined by interpolation from displacement-curve plots on semi- suggestingthe presenceof functionally active sigma receptors logarithmic graph paper. in the LMMP and a modulatory role for sigma receptors in Preparation of guinea pig ileum longitudinal muscle strips. Male Hart- ley guinea pigs (225-300 gm; Charles River Breeders, MA) were killed neurotransmissionin the myenteric plexus. by a blow to the head and exsanguinated. A length of small intestine The guinea pig LMMP has served as a well-characterized between 10 cm and 25 cm proximal to the ileocecal junction was re- bioassayfor opioid receptorsin which agonistsat mu and kappa moved, and its lumen was gently flushed with a Krebs solution of the receptorsinhibit the releaseof ACh from myenteric plexus neu- following composition (mM): NaCl, 118; KCl, 4.75; CaCl,, 2.54; MgCl,, 1.17; NaHCO,, 25.0; KH,PO,, 0.93; and D-glucose, 11.0. Strips of 2 rons that have beenexcited by a brief electrical stimulus (Leslie, cm length were stretched over a Lucite rod, the mesentery carefully 1987). Activity at opioid receptors can be distinguished from removed, and the longitudinal muscle layer with adherent myenteric that at sigma receptorsby sensitivity to the opiate antagonist plexus separated from the underlying circular muscle by gently stroking naloxone and by higher potency of opioid responsesfor the tangentially away from the mesenteric attachment with a cotton-tipped (-)benzomorphans comparedwith the (+)benzomorphans; sig- applicator. These longitudinal muscle strips were mounted in 5 ml organ baths containing Krebs solution at 37°C and bubbled with 5% carbon ma receptorsare insensitive to naloxone and have the reverse dioxide in oxygen. The strips were placed under a resting tension of 500 stereoselectivity. We show here that the same functional re- mg. Contractions were recorded isometrically with Grass FT .03C trans- sponse(inhibition of electrically stimulated contractions of the ducers connected to Grass 79D polygraphs. LMMP) can result from activation of opioid and of sigma re- Electrically stimulated longitudinal muscle strips. The strips were stimulated by a Grass S88 stimulator at 0.5 Hz (supramaximal voltage, ceptorsand that bioactivity of the (-)benzomorphans at sigma 1 msec duration) via platinum ring electrodes 3.0 cm apart. An equil- receptors can be demonstrated in tissuesin which opioid re- ibration period of 90 min was allowed before testing, with frequent ceptors are blocked. exchange of the bathing solution by overflow. Compounds were ad- The results reported here may prove useful in the further ministered by microliter syringe injections of 5 10 ~1 and were left in understanding of sigma receptor function in 2 principal ways. contact with the tissues until the response stabilized. The effect of compounds on the electrically stimulated twitch was measured as the ratio First, a robust, easily performed bioassay is described that dem- of the twitch amplitude in the presence of the compound to that im- onstratesa functional responsemediated by sigmareceptors and mediately prior to its addition. Intervals ofat least 30 min (with frequent provides a means to determine if novel compounds possess bathing solution changes) were allowed between single doses, during 3382 Campbell et al. * Functional Sigma Receptors in the Myenteric Plexus which contractions returned to their previous amplitude. Antagonists ysergide, ketanserin, and ICS 205-930 (Sandoz); BMY 14802 and tios- were allowed to preequilibrate for at least 10 min prior to addition of pirone (Mead Johnson); DAGO (Bachem); rimcazole (Burroughs test compound. The half-maximal inhibitory concentration (IC,,) for a Wellcome); 5-CT (5-carboxyamidotryptamine, Glaxo); (+) and (-)SKF drug in a given tissue was estimated by interpolation from a log-con- 10,047 (Sterling Winthrop) and 8-OH-DPAT [8-(hydroxy-2-(n-dipro- centration curve which contained 3 or 4 concentrations within the range pylamino)tetralin] and (+) and (-)3-PPP HCL (RBI) were dissolved in of 20-80% of maximal effect. The geometric mean of 4 or more of these H,O. Control injections using only the solvent vehicles (methanol and values was obtained as an estimate of the IC,, of the compound in ethanol) had no effect on the electrically or 5-HT-evoked responses. inhibiting the electrically evoked twitch. Differences between 2 mean Eleven disubstituted guanidines (all structural analogs of DTG) were IC,, values were analyzed by Student’s t test. synthesized and purified by two of us (M.W.S., J.F.W.K.). The synthesis In some experiments, naloxone was included in the Krebs buffer and chemical characterization of the compounds will be described else- during the entire experiment. In other experiments, the strips were where. See Figure 1 for their structure and its legend for their chemical treated with beta-chlornaltrexamine (B-CNA, an irreversible opiate re- names and the abbreviations used in this paper. ceptor antagonist), as follows. After the equilibration period, a concentration response curve to 2-D-Ala-Glyol-Met-enkephalin (DAGO) or Results U50,488H was obtained. B-CNA was injected into the bathing solution of each strip to yield a concentration of 100 nM, and the strips were Binding of novel DTG analogs to sigma receptors incubated for 30 min. The strips were extensively washed by repeatedly 3H-DTG has beenshown to be a selectiveradioligand for sigma changing the bathing solution, and B-CNA was injected a second time receptorsin guinea pig brain (Weber et al., 1986).Eleven novel to yield a concentration of 100 nM. After 30 min incubation, the strips DTG analogswere synthesizedand purified (Fig. 1; unpublished were washed extensively for 45 min. Only tissues in which 1 /IM concentrations of DAGO and of U50.488H elicited no chanae in twitch observations).Nine of thesecompounds were found to be potent amplitude subsequent to B-CNA treatment were used. - ligands of sigma receptors as determined by their ability to Agonist-stimulated longitudinal muscle strips. In experiments em- displace3H-DTG from sigmareceptors in guinea pig brain ho- ploying 5-HT as agonist, 1 PM methysergide or 1 PM ketanserin was mogenates(Table 1); the most potent of thesewas AdIpG, with included in the Krebs buffer during the entire experiment. Following an IC,, of 6.2 + 0.7 nM (n = 3). Thesecompounds also displaced the 90 min equilibration period, 3 noncumulative concentration-response curves for 5-HT were obtained, the first and third in Krebs buffer the sigma receptor ligand (+),H-3-PPP with high affinity from and the second in Krebs buffer containing a concentration of a com- guinea pig brain homogenates(data not shown), and there was pound tested as an antagonist, as follows. 5-HT was added to each organ a high correlation of IC,, values of these compounds in dis- bath by microliter syringe and remained in contact with the strips for placing the 2 sigmaselective radioligands (r = 0.98, p < 0.000 1; 1 min. The maximum height of the resulting contraction was recorded for each concentration of 5-HT. An interval of 20 min between 5-HT Fig. 2). Thus, these DTG analogsare potent sigma receptor applications was allowed during which the strips were washed exten- ligands. Two of the compoundswere inactive (IC,, > 100 PM) sively; preliminary experiments determined that this procedure avoided in displacing 3H-DTG; these were the conformationally con- tachyphylaxis of 5-HT responses. Concentrations of 5-HT over the strainedcongener Bridge-DPG and DMG (Fig. 1, Table 1).The range 20 nM to 60 MM were tested at half-log intervals and were applied inactive DTG congenersserved as controls to evaluate nonspe- in a varying, random sequence. A second concentration-response curve to 5-HT was obtained in the presence of an antagonist. The antagonist cific effects of disubstituted guanidinesin the bioassays. had been added to the Krebs buffer and was present during a 30 min equilibration period prior to and during the second 5-HT concentration- response curve. Concentrations of 5-HT were tested until no further Activity of novel DTG analogs and other sigma ligands on increase in the evoked-twitch amplitude was observed. The strips were electrically stimulated longitudinal muscle strips washed extensivelv for 30 min in normal Krebs before the third 5-HT The sigmareceptor selectivecompound DTG inhibited the elec- concentration-response curve was obtained. Only strips for which the trically evoked contractions of the guinea pig ileum LMMP maximum twitch heights obtained during the first and third concentration-response curves differed by 5 10% were used. For each strip in- preparation in a concentration-dependentmanner with an IC,, dividually the maximum response to 5-HT obtained during the second of 3.1 + 0.3 PM (Fig. 3A, Table 1). The time courseof DTG’s 5-HT concentration-response curve was divided by the mean of that action in inhibiting contractions was slowerthan that of opioid obtained in the first and third concentration-response curves, and for receptor agonists,taking 3-l 0 min to reachmaximum effect and each concentration of antagonist, the percentage values thus obtained from several preparations were expressed as a mean value. A concen- 5-10 min for the tissue to recover completely to its prior con- tration-response curve for the antagonist was constructed from inde- traction amplitude. In addition to DTG, 9 of the disubstituted pendent measures for each antagonist concentration. In other experi- guanidine compounds inhibited the electrically evoked con- ments, bethanachol (a muscarinic agonist) or 2-methyl-5-HT (with 1 tractions in a concentration-dependent manner (Table 1, Fig. /LM methysergide present during the entire experiment) were used a 3A) in the presenceof 1.25 PM naloxone. The most potent con- agonists in the 3-concentration-response curve paradigm as detailed above for 5-HT. In other experiments, DTG, (+)3-PPP, and (+)SKF generwas AdIpG with an IC,, of 1.0 + 0.2 PM (Fig. 3A). The 10,047 were employed as agonists in a single concentration-response 2 DTG analogsthat had no sigmareceptor binding activity were curve paradigm. also inactive in inhibiting the electrically evoked contractions Drugs used. B-CNA (RBI) was dissolved in anhydrous ethanol acid- of the muscle strips up to 100 KM; these were the conforma- ified with glacial acetic acid and used immediately for each application. 5-HT HCl (Sigma) was dissolved in H,O on each day of use and kept tionally constrainedcongener Bridge-DPG and DMG (Table 1). on ice in light-tight test tubes during the experiment. The following Numerous other compoundswith high affinity for the sigma compounds were dissolved in dilute HCl, brought up in H,O, and ti- receptor were tested for the ability to inhibit the electrically trated to pH 7 with dilute NaOH: DTG and disubstituted guanidines evoked contractions of the longitudinal muscle strips in the except AdIpG and Bridge-DPG (see below), (+) and (-)pentazocine presenceof 1.25 PM naloxone. The sigma receptor selective (Sterling Winthrop), and MK-801 maleate (Merck). Haloperidol (McNeil), (-)sulpiride, and perphenazine (Sigma); (+) and (-)cyclaz- compound (+)3-PPP inhibited contractions in a concentration- ocine (Sterling Winthrop); spiperone (RBI); and AdIpG and Bridge- dependentmanner with an IC,, of 5 1.3 + 7.5 PM (Table 1, Fig. DPG were dissolved in methanol. 5-HTP-DP (N-acetyl-5-hydroxy- 3A); the IC,, of (-)3-PPP was 111 * 12 PM. The sigma-selective tryptophyl-5-hydroxytryptophan amide, gift of A. Surprenant) was antipsychotic compounds BMY 14802 and rimcazole and the prepared on the day of use by dissolving it in a minimum volume of ethanol and diluting in Krebs buffer to a final concentration of < 10% antipsychotic compoundswith high sigmareceptor affinity halo- ethanol. Bethanechol, atropine, hexamethonium, tetrodotoxin, chlor- pet-idol, perphenazine, tiospirone, and chlorpromazine all in- promazine, and naloxone (Sigma); PCP HCl and TCP (NIDA); meth- hibited the electrically evoked contractions in a concentration- The Journal of Neuroscience. October 1989, 9(10) 3383

Table 1. Sigma receptor binding and bioactivity of DTG, its congeners and other sigma receptor ligands

Inhibition of Inhibition of electrically evoked twitch IC,, (PM) 5-HT- Inhibition of In presence of naloxone at: evoked ‘H-DTG binding In B-CNA- twitch IC,, Compound Go bf) 0 1.25 /.LM 20 PM treated tissues CPM) 1. DTG 28.0 k 1 3.1 f 0.3 3.0 + 0.2 2.9 + 0.3 2.9 -t 0.2 9.0 2. AdIpG 6.2 + 0.7 1.0 + 0.2 1.6 3. AdTG 7.6 f 0.3 1.6 + 0.2 5.0 4. DAG 11.8 f 3.4 1.6 -t 0.2 5. AdChG 12.5 f 2.2 2.0 k 0.2 6. ChTG 13.0 & 2.0 2.5 k 0.2 7. DChG 86.0 f 17 4.1 * 0.3 8. DXG 90.0 + 18 4.5 f 0.6 9. NHZ-DTG 280 + 20 7.5 f 1.2 16.0 10. DPG 397 + 21 10.1 f 0.8 34.0 11. Bridge-DPG > 100,000 >lOO > 50.0 12. DMG > 100,000 >lOO > 50.0 13. (+)3-PPP 76 f 4 54.3 * 5.0 51.3 + 7.5 50.8 f 8.0 49.0 f 4.5 14. (-)3-PPP 280 f 21 111 + 12 15. (+)Pentazocine 43 f 2 4.2 k 1.0 3.0 + 0.3 3.0 + 0.2 3.2 f 0.5 16. (-)Pentazocine 135 + 3 0.190 f 0.03 3.5 ? 0.3 4.7 + 1.0 5.7 + 0.8 17. (-)Cyclazocine 2600 + 210 0.031 f 0.004 0.28 k 0.03 14.4 + 2.4 17.8 f 3.3 18. (-)sKF10,047 4000 IL 566 0.033 f 0.003 0.93 + 0.08 25.0 f 4.6 n.a. 19. Haloperidol 5.0 Ifi 0.3 1.7 * 0.2 1.5 f 0.3 1.8 in 0.3 1.9 f 0.5 0.9 20. BMY 14802 120 +Z 15 5.8 f 0.7 4.3 f 0.4 4.0 k 0.7 5.5 f 0.5 9.5 2 1. Rimcazole 1400 -t 100 12.1 f 1.7 12.7 + 2.7 22. Tiospirone 233 f 52 2.5 + 0.2 23. Perphenazine 42 + 10 3.8 + 0.4 24. Chlorpromazine 1475 t 265 1.4 + 0.2 25. Sulpiride > 100,000 > 500 26. TCP 1100 + 110 14.0 & 1.3 27. PCP 1050 + 106 22.8 k 1.4 28. MK-801 > 10,000 263 k 24 Sigma receptor binding was assessed by the displacement of ‘H-DTG from homogenates prepared from guinea pig brain; IC,, values represent the mean + SEM from 2-t separate experiments (in triplicate; see Materials and Methods). Potency in inhibiting electrically evoked contractions of the guinea pig ileum longitudinal muscle/ myenteric plexus (LMMP) preparation was assessed in individual LMMP strips as described in Materials and Methods. These determinations were made in untreated strips, in strips bathed in Krebs buffer containing 1.25 or 20 NM naloxone during the entire experiment, and in strips treated with beta-chlomaltrexamine (B-CNA, see Materials and Methods). Potency in inhibiting 5-HT-evoked contractions was determined as described in Materials and Methods.

dependent manner (Table 1). The D,-selective antagonist (-)sulpiride did not affect the electrically evoked contractions Actions of mixed opioidkgma receptor active isomers of up to 500 PM. In addition, PCP and its analog l-[ I-(2-thi- benzomorphans on the electrically stimulated muscle strips enyl)cyclohexyl]piperidine (TCP), which have moderate affini- (-)Isomers ofbenzomorphan opiates(pentazocine, cyclazocine, ties for sigma receptors but high affinities for PCP receptors, and SKF 10,047) active at both opioid and sigma receptors inhibited the electrically evoked contractions with moderate potently inhibited electrically evoked contractions of the lon- potency (IQ, = 2 1.8 + 1.4 and 14.0 ? 1.3 PM, respectively). gitudinal muscle strips in the absenceof naloxone (Table 1). On the other hand, the high-affinity PCP receptor ligand MK- The concentration-responsecurves were shifted to the right in 80 1, which is very weak in displacing 3H-DTG at sigmarecep- presenceof 1.25 ~CLM naloxone and were shifted further to the tors, wasalso very weak in inhibiting the twitch (IC,, = 263 ? right by 20 PM naloxone (Table 1). Thus, the (-)benzomorphans 12 GM), indicating that the inhibition in the ileum is not me- seemedto inhibit the twitch primarily by interacting with nal- diated by PCP receptors. oxone-sensitive opioid receptors. The mu-opioid receptor-se- The inhibition of contractions by DTG and (+)3-PPP were lective peptide DAGO inhibited contractions with an IC,, value not affected by preincubation of the longitudinal musclestrips of 11 f 1.0 nM (n = 4; Fig. 3B), in closeagreement with pub- with 10 PM phentolamine, 1PM benoxathian, 1.25 I.LM idazoxan, lished values (Traynor et al., 1987). DAGO’s effectswere com- 40 PM (-)sulpiride, or 1.25 MM naloxone, suggestingthe inhi- petitively blocked by naloxone. The kappa-opioid receptor ag- bition of the electrically evoked contractions wasnot mediated onist U50,488H inhibited contractions with an IC,, value of by alpha, or alpha, adrenergic, D, dopaminergic, or opioid re- 10.0 f 0.2 nM (n = 4). ceptors. In order to determine if (-)benzomorphans can also inhibit 3384 Campbell et al. - Functional Sigma Receptors in the Myenteric Plexus

1 1 10 100 1000 NH [SHIDTG displacement,IC50 in r&I

Figure 2. Correlation of sigma receptor binding affinities of DTG and 9 congeners in radioligand binding assays using ;H-DTG and (+)3JH- 3-PPP to label siama receptors. 3H-DTG assavs were conducted as described in Materials and Methods, and (+)-;H-3-PPP assays were conducted as described in Weber et al. (1986). The affinities of DTG (I) and 9 congeners (2-10) as assessedin the 2 sigma receptor radioligand binding assays was highly correlated (r = 0.98, p < 0.0001). All numbers as in Table 1 and Figure 1.

obtained in the presenceof 20 PM naloxone (Table 1). (-)SKF 10,047 in B-CNA-treated strips inhibited contractions with a

NH, (-‘yHN:Ho flat concentration-responsecurve that did not reach 50% inhibition (data not shown). DTG, (+)pentazocine, (+)3-PPP, BMY 14802, and haloperidol inhibited contractions of the longitudinal muscle at IC,, values not statistically different in the

HN’NH absenceof naloxone and in B-CNA-treated strips (Table 1, Fig. 3B). These results suggestthat (-)benzomorphans can inhibit HN’NH the twitch by an opioid receptor-independentmechanism (pre- ‘CH, w sumably through action at sigmareceptors), and they confirmed 11 12 that the high-affinity sigmaligands DTG, (+)pentazocine, (+)3- PPP, BMY 14802, and haloperidol do not act through opioid Figure 1. Structures of the sigma receptor-selective ligand DTG and receptorsto inhibit the twitch. the 11 congeners characterized in this study. Compounds l-l 0 are active in sigma receptor binding and bioassays; compounds 11 and 12 are Actions of sigma receptor active isomersof benzomorphanson sigma receptor-inactive control substances that are chemically related the electrically evoked twitch responseof the musclestrips to the others. For each compounds pictured, the chemical name is given, followed by its abbreviation in parentheses. I, NJ’-di(o-tolyl)guanidine The sigma receptor active benzomorphan (+)pentazocine in- (DTG);2,N-(2-iodophenyl)-N’-(adamant-l-yl)guanidine (AdIpG); 3, hibited the electrically evoked contractions of the longitudinal N-(o-tolyl)-N-(adamant- 1-yl)guanidine (AdTG); 4, NJ-di(adamant- l- musclestrips with equalpotency in the absenceof naloxone and yl)guanidine (DAG); 5,N-(cyclohexyl)-N’-(adamant- 1-yl)guanidine (AdChG); 6, N-(o-tolyl)-N’-(cyclohexyl)guanidine (ChTG); 7, NJV-di- in the presenceof 1.25 and 20 PM naloxone (Table 1). Unlike cyclohexyl)guanidine (DChG); 8, N,N-di(2,6-methylphenyl)guanidine all other sigma ligands, the benzomorphans (+)SKF 10,047 (DXG); 9, N-(o-tolyl)-N’-(4-amino-2-methylphenyl)guanidine (NH,- [( +)N-allyl-normetazocine] and (+)cyclazocine potentiated DTG); IO, N,N’-di(phenyl)guanidine (DPG); 11, 2-amino- 1,3H-diben- contractions of the electrically stimulated longitudinal muscle zo[d,jl-[ 1,3]-diazepine (Bridge-DPG); 12, N,N’-di(methyl)guanidine strips in a concentration-dependent manner. (+)SKF 10,047 (DMG). Note that these numbers are used to identify DTG congeners in Table 1 and Figures 2 and 6 A, B, the abbreviations are used in the causeda maximal increasein the twitch of 93% and the con- text, table, and figure legends. centration that evoked 50% of this maximum (EC,,) was 3.2 PM (n = 16, data not shown). Additionally, (+)SKF 10,047 in- creasedthe basal tension of the electrically stimulated strips such that the baselinewas elevated. The threshold detectable increase(defined as 50 mg) was 1.0 PM, and the increasewas the twitch by interacting with sigma receptors, the longitudinal 570 mg at 50 PM (data not shown). Neither the twitch augmen- muscle strips were treated with the irreversible opioid receptor tation nor the baselineelevation was altered in the presenceof antagonist B-CNA twice for 30 min at 100 nM in order to in- 1.25 PM naloxone or 500 nM DTG, nor were they different in activate opioid receptors. In B-CNA-treated strips, DAGO was B-CNA-treated longitudinal muscle strips. (+)Cyclazocine in- inactive at 1 and 5 I.LM and inhibited contractions at 10 KM by creasedthe electrically stimulated contractions with a maximal only 13% (Fig. 3B) and U50,488H was inactive at 1 and 5 PM, increaseof 41% with an EC,, of 1.4 PM (n = 8) and increased suggestinga virtual inactivation of opioid receptors. In B-CNA- the resting tension with a threshold of 1 PM and an increaseof treated strips(-)cyclazocine and (-)pentazocine inhibited con- 145 mg at 50 FM (n = 8). These effectsof (+)cyclazocine were tractions at IC,, values not statistically different from those not significantly different in B-CNA-treated strips. The Journal of Neuroscience. October 1989, 9(10) 3385

.l 1 10 100 IO00 ,001 -01 .l I 10 I Concentration, p&l Concentration, @I Figure 3. A, Inhibition of electrically evoked contractions of the guinea-pig longitudinal muscle/myenteric plexus (LMMP) preparation. AdIpG (opensquares), DTG (filledcircles), DPG (opentriangles), and (+)3-PPP (jilled triangles.).B, Inhibition of electrically evoked contractions of the LMMP prior to and following beta-chlornaltrexamine (B-CNA) treatment. Compounds were evaluated in the same tissues prior to (solidlines) and followinp. B-CNA treatment (dashedlines). as described in Materials and Methods. DAGO before (filledsauares) and after (ooen sauares)B-CNA treatmeit, (-)pentazocine be‘fore (filledtr&gles) and after (open triangles)B-CNA treatment and DTG befbre (fiiled circles)&d aft& (opt& circles) B-CNA treatment. In both A and B, for each concentration tested, the effect is expressed as a percentage and plotted for the value (mean k SEM, n = 4-16) one minus the ratio of the twitch amplitude in the presence of the compound to that immediately prior to its addition to the bathing solution.

the ability of sigma ligandsto inhibit the releaseof ACh stim- Activity of sigma receptor ligands on unstimulated longitudinal ulated by 5-HT. 5-HT has been shown to have multiple effects musclestrips on myenteric neurons(see Fumess and Costa, 1987; Takaki et DTG and (+)3-PPP did not affect the resting tension of lon- al., 1985; Mawe et al., 1986; Galligan et al., 1988),to be capable gitudinal muscle strips at concentrations up to 100 PM, sug- of releasingACh from the myenteric plexus (Adam-Vizi and gestingthat the sigmaligands did not interact directly with the Vizi, 1978; Kilbinger and Pfeuffer-Friederich, 1985), and to muscleto reduce resting tension. However, (+)SKF 10,047 in- cause the LMMP to contract (Gaddum and Picarelli, 1957; creasedthe resting tension of the longitudinal muscle strips in Buchheit et al., 1985; Richardson et al., 1985). In the presence a concentration-dependentmanner with a detectable threshold of 1 PM methysergide,5-HT evoked contractions of the longi- (defined as 50 mg) of 1 FM and an increaseof 500 mg at 50 PM tudinal muscle strips over a broad concentration range of 20 (data not shown). This increasein resting tension was blocked nM-60 PM (Fig. 4A). Concentration-responsecurves were shal- by 1 PM TTX and 1 PM atropine. The increasein restingtension low and often notably biphasic with an inflection point near under unstimulatedconditions wasnot statistically different from 500 nM. 5-HT-evoked contractions below 500 nM constituted that observed during electrical stimulation. approximately 20% of the maximum response,were characterized by slow onset, and were maintained in the presenceof Activity of sigma ligands on musclestrip contractions evoked agonist. 5-HT-evoked responsesabove 500 nM constituted the by a muscarinic agonist remaining 80% of the maximum responseand were character- In order to determine whether sigma ligands acted directly on ized by rapid climb to peak amplitude followed by a return to the muscle or on myenteric cholinergic neurons (or nerve ter- baseline,although the agonist continued to be present. The bi- minals) to inhibit the electrically evoked contractions of the phasic nature of the 5-HT concentration-responsecurve has muscle strip, we tested the effects of sigma drugs on muscle previously been noted (Buchheit et al., 1985). Contractions contractions evoked by the muscarinic agonistbethanechol. Be- evoked by 5-HT were completely blocked by 1 WM TTX, con- thanecholevoked contractions of the longitudinal musclestrips firming their neuronal mediation. with an EC,, of 2.2 PM (n = 12). These contractions were pro- The pharmacologic specificity of 5-HT’s ability to contract duced rapidly and maintained in the presenceof agonist. The the LMMP was assessedby examining the contractile effectsof contractions were abolished in the presenceof 1 KM atropine selective 5-HT receptor agonistsand the contraction-inhibiting but were not affected by 100 PM hexamethonium or 1 PM TTX. effectsof antagonists.The selective5-HT,, agonists8-OH-DPAT DTG at 50 PM, (+)3-PPP at 100 PM, and (+)SKF 10,047 at 10 and 5-CT at 1 PM failed to alter the restingtension of the LMMP. PM did not affect the bethanecholconcentration-response curve, 2-Methyl-5-HT, an full agonistat enteric S-HT, receptors(Mawe suggestingthat the sigma ligands did not act on the muscle or et al., 1986; Surprenant and Crist, 1988), evoked contractions on muscarinicreceptors to inhibit (or potentiate for SKF 10,047) of the LMMP that plateaued at a maximal amplitude not sta- the electrically evoked contractions of the muscle strips. tistically different than that which 5-HT evoked in the same preparation (Fig. 4B). The concentration-response curve for Activity of sigma receptor ligands on S-HT-stimulated 2-methyl-5-HT was monophasicover a range of l-100 I.LM with longitudinal musclestrips an EC,, of 8.6 MM. Contractions evoked by 2-methyl-5-HT were The observations above support the hypothesis that sigma re- completely blocked by 1 PM TTX (data not shown). Both the ceptor ligandsact by a neuronal mechanismto inhibit the elec- 5-HT and the 2-methyl-5-HT responseswere obtained in the trically stimulated releaseof ACh that leads to LMMP con- presenceof 1 FM methysergideto block 5-HT, receptorslocated traction. To further characterizethis mechanism,we investigated on smooth muscle cells; similar results were obtained in the Campbell et al. * Functional Sigma Receptors in the Myenteric Plexus

.Ol I 10 100 -1 I IO 100 1000 5-HT concentration, pM Concentration, FM Figure 4. S-HT-evoked contractions of the LMMP and the noncompetitive antagonism of this effect by NH,-DTG and the augmentation of this effect by (+)SKF 10,047. 5-HT evoked contractions of the LMMP over the range 20 nM-60 FM with a biphasic concentration-response curve (filled circles; see Results). Each of the following 5 curves represent independent experiments (n = 4 each) employing the 3-concentration-response curve paradigm described in Materials and Methods. Four curves depict 5-HT-evoked contractions assayed in the presence of 4 PM NH,-DTG (open triangles), 10 PM NH,-DTG (open squares), 20 PM NH,-DTG (open diamonds), and 40 PM NH,-DTG (open circles). One curve depicts 5-HT- evoked contractions assayed in the presence of 10 PM (+)SKF 10,047 (crosses). B, 2-Methyl-5-HT-evoked contractions of the LMMP and the noncompetitive inhibition of this effect by DTG. Amplitude of contractions evoked by 2-methyl-5-HT (filled squares) increased in monophasic concentration-dependent manner to reach a plateau not statistically different from that of 5-HT (filled circles) obtained in the same preparation. DTG at 500 nM (open triangles), 1 PM (open circles), 3 PM (open diamonds), and 8 PM (open squares) noncompetitively antagonized the 2-methyl- 5-HT-evoked contractions.

presence of the specific 5-HT, antagonist ketanserin at 1 PM. In (n = 8, data not shown; see Richardson et al., 1985, figure 2~); the presence of 1 PM ketanserin, the following compounds did these values are in close agreement with those previously pub- not affect the 5-HT concentration-response curve: the nonse- lished: pA, = 8.1 t- 0.2 (Buchheit et al., 1985) and 7.85 + 0.1 lective 5-HT, and 5-HT, antagonist methiothepin at 1 PM, the (Richardson et al., 1985). ICS 205-930 also competitively an- 5-HT,, and 5-HT, antagonist spiperone at 1 PM and the selective tagonized 5-HT contractions in the presence of 1 PM ketanserin 5-HT,, antagonist 5-HTP-DP at 10 PM. In the presence of 1 I.LM in place of methysergide. These observations indicate that methysergide, the 5-HT response was competitively shifted to 5-HT’s contractile response in the LMMP is mediated primarily the right by increasing concentrations of the selective 5-HT, by 5-HT, receptors (see Bradley et al., 1986). receptor-selective antagonist ICS 205-930, and analysis of the Sigma receptor ligands were found to affect the 5-HT-evoked data in the method of Arnnlakshana and Schild (1959) gave a contractions. DTG inhibited the 5-HT-evoked contractions in straight line with slope 1.1 & 0.2 and pA, value of 8.1 f 0.2 a concentration-dependent manner with an IC,, of 9.0 PM (Table 1, Fig. 5); the inhibition of 5-HT-evoked contractions by DTG was noncompetitive in that it could not be overcome by in-

100 creasing 5-HT concentrations. The disubstituted guanidines AdIpG, AdTG, NH,-DTG, and DPG and haloperidoland BMY s l 14802 similarly inhibited the 5-HT-evoked contractions in a noncompetitive manner (Figs. 4A, 5; Table 1). The sigma receptor inactive congeners Bridge-DPG and DMG did not affect the 5-HT-evoked contractions. (+)SKF 10,047at 10 PM shifted the 5-HT concentration-responsecurve 3.5-fold to the left (Fig. 4A). DTG noncompetitively blockedthe 2-methyl-5-HT-evoked contractions with an IC,, of 2.8 PM (interpolated from Fig. 4B). The inhibition of contractions evoked by 5-HT and by 2-methyl-5-HT could be completely abolishedby sigma receptor ligands(e.g., Fig. 4B, open squares).

Concentration, ph4 Correlation of binding ajinity with bioactivity of sigma receptor active drugs Figure 5. Inhibition of the 5-HT-evoked twitch by sigma receptor ligands. The ability of each sigma receptor ligand at a given concentra- The IC,, values for most, but not all, of the sigma ligandsin tion to suppress the maximal amplitude of the 5-HT-evoked twitch was inhibiting the electrically evoked contractions of the guinea pig determined with a 3-concentration-response curve paradigm as de- ileum LMMP preparation were highly correlated with their abil- scribed in Materials and Methods. For example, as can be seen in Figure 4A, the maximum twitch evoked by S-HT in the presence of 4 PM NH,- ity to displace3H-DTG from sigmareceptors in guinea pig brain DTG was 80% of the maximum obtained in its absence; therefore, 4 homogenates.The highestcorrelation was found for the 10 sig- PM NH,-DTG inhibited the 5-HT-evoked twitch by 20%, as is plotted ma receptor-active DTG congeners(r = 0.98, p -C 0.0001; Fig. here. Curves for each sigma ligand were constructed in which each data 6A, filled circles). Note that the 2 sigmareceptor-inactive con- point represents a single 3-concentration-response paradigm experiment. AdIpG (open squares), DTG cfilled circles), NH,-DTG (open tri- genersdid not inhibit the electrically evoked contractions (Table angles), and DPG (filledsquares); similar curves were obtained for halo- 1). A high correlation was also found when numerous other peridol, BMY 14802, and AdTG (data not shown). chemically diverse sigma ligands were taken into account, in- The Journal of Neuroscience, October 1989, 9(10) 3387

,I 0 .1 I lb I 0 Inhibitiod of electricall~evoked Inhibition of 5-I-IT-evoked twitch, p,M contractions, IC.50 in PM Figure 6. A, Correlationof sigmareceptor ligand binding affinity and potency in inhibiting the electrically evoked twitch in the LMMP. Sigma receptor binding and potency in inhibiting the electrically evoked twitch were assessed as described in Table 1 and Materials and Methods. Correlation line is derived from regression analysis for compounds l-10, namely, DTG and its sigma receptor-active congeners (filled circles; r = 0.98, p < 0.0001). Eight other compounds with high sigma receptor affinity (open circles; 15, 19-23, 26, 27) also correlated well independently (r = 0.89,p < 0.01) and in conjunctionwith the DTG congeners(r = 0.93,p < 0.0001,n = 18).(-)Benzomorphans (open squares, 16-18) also correlatedwith aboveaggregate (r = 0.95,p < 0.0001, n = 2 1). Two sigmareceptor ligands, chlorpromazine (24, open triangle) and(+)3-PPP (13, open diamond) weredistant from the regressionline generatedby the DTG congeners.All numbersas in Table 1. B, Correlationof sigmareceptor ligandbinding affinity and potencyin antagonizingthe 5-HT-evokedcontractions in the LMMP. Sigmareceptor binding affinity wasassessed as describedin Table 1 and Materialsand Methods.Antagonism of the 5-HT-evokedcontractions was assessed as shownin Table 1 and Figures4A and 5 and as described in Materials and Methods. The inhibition of 5-HT-evoked contractions by 5 DTG congeners (DTG,l; AdIpG,Z; AdTG,3; NH,-DTG,9; DPG,10) and haloperidol(19) and BMY 14802(20) correlatedwell with their sigmareceptor affinity (r = 0.91, p < 0.0 1).

eludingthe typical antipsychoticshaloperidol and perphenazine, nerson S-HT- or electrically evoked contractions of the LMMP the novel antipsychoticsBMY 14802,rimcazole, and tiospirone, are mediated by sigma receptors. the arylcyclohexylamines PCP and TCP, and the benzomorphan The hypothesis that the DTG congenersact in the LMMP at (+)pentazocine; these 8 compounds alone also correlated well sigmareceptors is supportedby severalobservations. First, sim- (r = 0.89, p < 0.01; Fig. 6A, open circles), as did the aggregate ilar results were obtained using 2 different stimulation para- when they were included with the DTG congeners(r = 0.93, p digms-by electrical pulsesand by 5-HT-and high correlations < 0.0001, n = 18). Additionally, under conditions in which were found for eachparadigm. Second, 2 DTG congeners,Bridge- opioid receptorswere blocked, the bioactivity of the (-)ben- DPG and DMG, that lack sigma receptor binding affinity also zomorphanspentazocine, cyclazocine, and SKF 10,047also cor- lacked sigmareceptor bioactivity; becauseof their similar struc- related well with their sigma receptor binding (Fig. 6A, open tures, pK,‘s, and molecular weights to active congeners,these squares;aggregate r = 0.95, p < 0.000 1, n = 2 1). The compounds control compoundspresumably also sharetheir nonspecificcon- that correlated poorly were chlorpromazine, having a higher founding effects. Third, antagoniststo adrenergic, dopaminer- potency in the bioassaythat would be expected from its binding gic, and opioid receptors were inactive in altering the sigma affinity, and (+) and (-)3-PPP, both displaying lower potency receptor bioactivity of DTG. Fourth, the bioactivity of a broad in the bioassaycompared with their binding affinities. The ben- range of sigmareceptor ligandswith diverse chemical structures zomorphans(+)cyclazocine and (+)SKF 10,047potentiated the also correlated well with sigma receptor binding affinity, both contractions and therefore were not included in the correlation. independently of the DTG congenersand when grouped with There was also a high correlation for 5 DTG congenersand for them. Fifth, compounds with binding affinities over a range of haloperidol and BMY 14802 to inhibit 5-HT-evoked contrac- 3 orders of magnitudecorrelated well with respectto their sigma tions compared with their sigma receptor affinity (r = 0.9 1, receptor bioactivity. It is important to note, however, that the p < 0.01; Fig. 6B). slope of this correlation line is significantly greater than unity; i.e., a IO-fold increasein binding affinity yields a lessthan lo- Discussion fold increasein bioactivity. The basisof this discrepancyis not Sigma receptorbioactivity known, in part becausethe molecular and cellular correlatesof The cardinal finding of this study is that DTG and its sigma sigma receptor activation are not known. It is of interest that receptor-active congenersinhibit electrically or S-HT-evoked preliminary reports demonstrate that sigma receptor ligands contractions of the LMMP preparation by a neuronal mecha- negatively modulate phosphoinositide metabolism in rat brain nism and that the potency of these drugs in the bioassaysis (Bowenet al., 1988a;Kirschneret al., 1988);this effect resembles highly correlated with their sigma receptor binding affinity in the inhibition of LMMP contractions in that it occurred only guinea pig brain membranehomogenates. Sigma receptor bind- under agonist-stimulated conditions, the effective concentra- ing siteshave been shown to be present in the myenteric plexus tions for ligandscommon to both studieswere similar, and the of the guinea pig ileum (Roman et al., 1988). Thus, our results ability to inhibit phosphoinositide metabolism was highly cor- strongly suggestthat the inhibitory actions of the DTG conge- related with binding affinity with a slope significantly greater 3388 Campbell et al. - Functional Sigma Receptors in the Myenteric Plexus than one (greater difference in affinity than bioactivity). Negative however, competitively antagonized by ICS 205-930, and Schild modulation of agonist-stimulated phosphoinositide turnover has analysis of this effect yielded linear slope and a pA, value that previously been shown for D2 receptors on lactotrophs (Enjal- closely matched previously published results using this prepa- bet-t et al., 1986) and NMDA receptors in hippocampus (Baudry ration. These results suggest that 5-HT acts primarily at 5-HT, et al., 1987). receptors to contract the LMMP. The lack of contractile effects DTG inhibited electrically evoked contractions of the LMMP of selective 5-HT,, agonists and the ability of the 5-HT, agonist at concentrations approximately 100 times its affinity for CNS 2-methyl-5-HT to contract the LMMP to the same maximum sigma receptors; this difference in absolute potency is shared by tension as 5-HT in the same strips further supports this hy- the other sigma receptor ligands. The disparity is particularly pothesis. These data fully meet the criteria proposed by Bradley surprising in that agonists at opioid receptors are active in the et al. (1986) for characterization of 5-HT,-mediated responses, LMMP at potencies near their binding affinity for mu and kappa as do the results obtained by Buchheit et al. (1985) and Rich- sites. It is possible that the bioavailability of DTG and its con- ardson et al. (1985) using the same preparation. Kilbinger and geners is impaired in intact LMMP strips. Alternately, the myen- Pfeuffer-Friederich (1985) reached a similar conclusion by ex- teric sigma receptors may have lower affinities for these com- amining the efflux of tritiated ACh from preloaded LMMP in pounds relative to central sites; preliminary data by Roman et response to 5-HT. al. (1988) suggest that sigma receptors in. the LMMP possess Thus, the LMMP contractile response evoked by 5-HT is similar properties as those in the CNS, and the binding of the mediated primarily by 5-HT, receptors. Sigma receptor ligands selective sigma receptor radioligand 3H-DTG in the LMMP is were found to inhibit this response in a noncompetitive manner. currently being investigated. Numerous possible mechanisms can be proposed whereby sig- A further consideration raised by these data is whether sigma ma receptor ligands interfere with the chain of events initiated receptor-active compounds can be identified as agonists or an- by 5-HT’s binding to 5-HT,-recognition sites and completed by tagonists. All disubstituted guanidines tested and most other the release of ACh from cholinergic myenteric neurons. One compounds inhibited the LMMP contractions in accordance possible mechanism is that sigma receptor ligands block the with their sigma receptor binding affinity. The sigma receptor channel opened by 5-HT’s activation of 5-HT, receptors, anal- active benzomorphans (+)SKF 10,047 and (+)cyclazocine po- ogous to MK-801’s blockade of the channel opened by gluta- tentiated the contractions of the LMMP preparation, acting op- mate’s activation of NMDA receptors. That this does not occur posite to all other sigma ligands tested. It is not known whether was demonstrated by intracellular recordings performed in these 2 drugs acted through sigma receptors to potentiate the myenteric plexus neurons in which depolarization following contractions or through an unidentified mechanism. Their ef- 5-HT, receptor activation was not affected in the presence of fects were blocked by TTX, thus apparently neuronal, but were DTG (Galligan et al., 1989). A second possible mechanism is not affected by DTG. The mixed dopamine/sigma receptor li- that sigma receptors act to uncouple excitation from neurotrans- gands (+) and (-)3-PPP were notably less potent in the bioassay mitter release directly on cells that express both sigma and 5-HT, compared with DTG. No compound yet tested has demonstrat- receptors. The majority of myenteric S/type I neurons, a subset ed the ability to specifically antagonize the responses of the of which are the cholinergic excitatory neurons that release ACh sigma receptor ligands. to contract the longitudinal smooth muscle (Fumess and Costa, Sigma receptor ligands were found to inhibit LMMP con- 1987), demonstrate 5-HT,-mediated depolarizations to 5-HT tractions evoked by the bath application of 5-HT. The con- (Galligan et al., 1989); thus, sigma receptor activation may in- tractile effect of 5-HT in this preparation could be ascribed to terfere with excitation-neurotransmitter release coupling di- an effect on the neurons of the myenteric plexus because it was rectly in these cells. The negative modulation of phosphoino- blocked by TTX and was unaffected by the presence of antag- sitide metabolism by sigma receptor ligands could provide a onists of 5-HT, receptors located on the smooth muscle cells. mechanism for the observed inhibition of ACh release (Bowen The myenteric plexus, however, contains a diverse population et al., 1988a). It is not known, however, whether the cholinergic of neurons, and the experimentally measured parameter (tension motor neurons are among those S/type I cells that possess 5-HT, produced by smooth muscle contraction) reflects only the final receptors, and 5-HT,-mediated responses to 5-HT have also common output via excitatory cholinergic neurons of the com- been observed in most AH/type II cells (Takaki et al., 1985; plex myenteric circuitry. Interpretation is further complicated Mawe et al., 1986). If myenteric neurons with 5-HT, receptors in that 5-HT has multiple effects on myenteric neurons and acts synapse with cells with sigma receptors that either are, or even- on more than one type of neuronal receptor. Three distinct tually activate, the cholinergic final motor neurons, then 5-HT- effects of 5-HT on myenteric neurons have been characterized induced contractions would be antagonized without sigma re- in terms of their inhibition by specific receptor antagonists: a ceptors having either to affect 5-HT, receptor activation or 5-HT,,-mediated hyperpolarization that is blocked by spipe- transmitter release by the myenteric S/type I neurons them- rone (Galligan et al., 1988); a 5-HT,,-mediated slow depolar- selves. The mechanism that underlies the inhibition of ACh ization that is blocked by 5-HTP-DP (Takaki et al., 1985; Mawe release by DTG and other sigma receptor ligands in the myen- et al., 1986); and a 5-HT,-mediated fast depolarization that is teric plexus can only be established by careful electrophysiologic blocked by ICS 205-930 (Mawe et al., 1986). The contribution and biochemical studies; the series of disubstituted guanidine of activation of these 5-HT receptor types to the contractile compounds described here may prove useful in this pursuit. response was assessed by these specific antagonists in the LMMP 5-HT, receptors have recently been identified in the brain (Kil- preparation. Spiperone and 5-HTP-DP at concentrations that patrick et al., 1987) and shown to mediate rapid synaptic CNS blocked the effect of 5-HT on myenteric neurons did not affect neurotransmission (Yakel and Jackson, 1988). It will be of in- the 5-HT contractile effect on the LMMP, suggesting that ac- terest to see if sigma receptor activation in the brain-as in the tivation of 5-HT,, and 5-HT,, receptors does not significantly ileum-results in the inhibition of 5-HT,-mediated or other contribute to the contractile response. LMMP contractions were, rapid excitatory responses. The Journal of Neuroscience, October 1989, 9(10) 3389

Opioid receptor agonist drugs also inhibit electrically and The site of action for these effects is uncertain in the absence of 5-HT-stimulated ACh release in the ileum (Gaddum and Pi- any clear correlation between bioactivity and binding affinity at carelli, 1957; Leslie, 1987). However, it is clear that sigma sigma receptors and radioligand binding studies to demonstrate receptor-mediated inhibition is separate from opioid receptor- the presence of sigma sites. It remains to be determined if there mediated inhibition since the sigma ligands were not antago- are functional sigma receptors in the vas deferens, as has been nized by naloxone and sigma receptor-mediated inhibition was demonstrated here for the guinea pig ileum LMMP preparation. not altered in ilea that had been treated with the irreversible opioid receptor antagonist B-CNA. On the other hand, sigma receptor activation clearly has the same functional result as opioid receptor activation. This is most clearly illustrated by Sigma receptors and centrally mediated effects the effects of the opioid active (-)isomer of pentazocine on the Until now, sigma receptors were defined as specific and ana- electrically stimulated ileum. In the absence of naloxone, tomically highly localized binding sites in limbic and sensori- (-)pentazocine potently inhibited the electrically induced twitch motor structures of the brain. No function was assigned to these response. Naloxone shifted the dose-response curve of (-)pen- sites. However, it has been suggested that sigma sites may play tazocine to the right, suggesting that the low-dose effects of an important role in the action of psychotropic drugs (Deutch (-)pentazocine were mediated by opioid receptors. In ilea in et al., 1988; Snyder and Largent, 1989). Some novel nondo- which opioid receptors had been inactivated by the irreversible paminergic antipsychotic drug candidates such as BMY 14802, opiate antagonist B-CNA, (-)pentazocine still caused a dose- rimcazole and remoxipride have been shown to bind selectively dependent inhibition of the twitch response, presumably by to sigma receptors (Ferris et al., 1986; Largent et al., 1988; interaction with sigma receptors. Given these opioid receptor- Taylor and Dekleva, 1988). The common mode of action of all like properties of sigma receptors, it is important to note recent typical and novel antipsychotics tested in the ileum suggests results from drug discrimination studies performed by Holtz- that sigma receptor-mediated inhibition of neurotransmitter re- man (1989) showing that mu and kappa opioid receptor agonists lease could be responsible for the antipsychotic properties that were able to substitute for DTG in rats trained to discriminate these drugs have in common. This mechanism if active in mod- DTG from saline. It was shown in that study that the DTG- ulating dopamine release from mesolimbocortically projecting like discriminative stimulus control of behavior by the mu and ventral tegmental neurons would provide a conceptual basis for kappa agonists was mediated by a naloxone-sensitive opioid the antipsychotic properties of selective sigma ligands such as receptor, while the discriminative stimulus control of behavior BMY 14802, rimcazole, and remoxipride. Three recent obser- by DTG was naloxone insensitive. Our results show a naloxone- vations are of particular interest in this light. First, sigma re- insensitive effect of DTG on stimulated ACh release in the ileum ceptors and D, receptors have been shown to share dense con- that mimics that of opioid receptor activation. This opioid-like centrations in an anatomically discrete subdivision of the cat’s effect of sigma receptor activation by DTG may well explain substantia nigra pars compacta, the densocellular zone (Graybiel why mu and kappa opioid receptor agonists fully substitute for et al., 1989). Second, BMY 14802 in electrophysiological studies DTG in drug-discrimination studies. has been shown to modulate dopaminergic neurotransmission The actions of PCP and TCP in inhibiting the electrically in ventral tegmental neurons by a nondopaminergic (presum- evoked contractions of the LMMP have previously been re- ably sigma-mediated) mechanism (Wachtel and White, 1988). ported (Gintzler et al., 1982) and were ascribed to specific PCP Third, the sigma receptor-selective antipsychotic rimcazole has receptors in the LMMP. However, several lines of evidence been shown to block both behavioral hyperactivity and exci- suggest that PCP and TCP act in the LMMP at sigma rather tation of ventral tegmental dopamine neurons induced by than PCP receptors. First, the highly selective PCP receptor (+)SKF 10,047 (Ceci et al., 1988). ligand MK-801 was virtually inactive at inhibiting the electri- However, it remains to be seen whether inhibitory action in cally evoked contractions, although it has higher affinity than the ileum combined with high sigma receptor binding activity PCP and TCP for the PCP receptor and is more potent that is predictive of a drug’s antipsychotic activity in vivo. This ques- PCP and TCP in numerous CNS effects of the PCP receptor tion can be answered by testing a wide variety of selective sigma (Wong et al., 1986; Kemp et al., 1987). Second, the potency of receptor ligands (such as our DTG congeners) in the ileum bioas- PCP and TCP to inhibit electrically evoked contractions cor- says followed by evaluation of the drugs in animal tests that are related well with their sigma receptor binding affinity. Third, predictive of antipsychotic activity. Although the receptor se- activity at NMDA sites in the LMMP as demonstrated by ap- lectivity of the DTG congeners has not been firmly established, plication of exogenous glutamate are blocked in the presence of they have poor affinity (IC,, values > 1 FM) for the PCP receptor the Krebs buffer Mg2+ concentration used in these studies (1.17 and for opioid (unpublished observations), serotinergic (S. Pe- mM; see Luzzi et al., 1988). Thus, DTG, PCP, and TCP may routka, personal communication), dopaminergic, and gluta- act via a common mechanism in the LMMP, namely, the sigma matergic receptors (J. C. Fisher, personal communication). receptor. This finding may be useful in interpreting drug-dis- In summary, we have demonstrated that there are functional crimination studies recently performed by Holtzman (1989) in sigma receptors in the LMMP preparation and that sigma re- which rats trained to distinguish PCP from saline generalized ceptor ligands act to inhibit electrically or 5-HT-stimulated ACh dose dependently and completely to DTG as did rats trained to release by an opioid receptor-independent mechanism. Fur- DTG did for PCP. thermore, we have synthesized and characterized 9 novel DTG Recent studies have provided evidence that certain sigma congeners with potent sigma receptor binding and bioactivity, receptor-active drugs such as (+)3-PPP, (+)pentazocine, TCP, as well as 2 chemically related but inactive control compounds. and (+)SKF 10,047 can augment electrically stimulated con- The bioassay system and the novel drugs described here should tractions of the mouse and guinea pig vasa deferentia by a neu- be useful tools to clarify the role of sigma receptors in the mod- ronal mechanism (Campbell et al., 1987; Vaupel and Su, 1987). ulation of neurotransmitter release. 3390 Campbell et al. l Functional Sigma Receptors in the Myenteric Plexus

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