Binding affinity and antimuscarinic activity of σ and phencyclidine receptor ligands

151

UP 51768

The characterization of the ff receptor has been hampered by the lack of a functional bioassay system. Drugs that bind to o receptors have been reported to inhibit carbachoi-induced phosphatidylinositol turnover in rat brain; however, these drugs might directly affect muscarbaic ac~tylcho~ne receptors. The purpose of the present study was to determine the affinity for muscarkic receptors and the antimuscarinic activity of Q and phe~y~~id~n~ receptor ligands. All of the drugs tested inhibited the binding of j3HPIJ-methylscopolamine to guinea pig cerebral cortical membranes with KI values in the micromolar range and also inhibited carbachol-induced contractions in the guinea pig ileum. These results demonstrate that these compounds have substantial antimuscarinic activity which might limit the use of the inhibition of carbachol-induced phosphatidylinositol turnover as a functional assay system for studying 0 ligands. Furthermore, this antimuscarinic activity must be considered when evaluating the effects of these compounds after in viva ad~~stration* Acetylcholine; Ileum (guinea-pig); Muscarinic receptors; N-Methylscopolamine;

Phencyclidine (PCP) has been shown to bind to several distinct binding sites, including a site associated with dopamine reuptake (Chaudieu et al., 1989), and two other sites that have been termed the PCP and u receptors (Q.&ion et al., X987). FCP and e receptors can be d~ffe~ntiated on the basis of ligand selectivity (Largent et al., 1986; Wong et al., 1988), regional localization (Las-gent et al., 1986) and differences in ontogeny (Majewska et al., 1989; Paleos et al., 1990). tier the last several years there have been major advances in the characte~zation of the PCP receptor. It is now known that the PCP binding site is associated with the Nmethyl-D-aspartate (NMDA) subtype of the glutamate receptor, snd PCP has been found to be a nan-competitive antagonist at the NMDA receptor-ionophore complex (fohnson and Jones, 1990). The behavioral effects of PCP and other compounds that produce PCF-like behavioral effects, such as benzomorphans, substituted dioxolanes, and other arylcycloalkylamines, correlate with their binding affinities for PCP receptors and not (I receptors (&kin and Z&in, 1988; McCann et al., 1989), sugg~ting that their behavioral effects are mediated by PCP receptors. Less is known about the o receptor, its

Correspondence to: R.N. Pechnick, Department of pharmacology, UCLA Sc&ool d Medicine, Los Angefes, CA 90024-1735, USA.

Phencycfidine; Fhosphatidylinositot; of Receptors

ch~acte~~tion being primarily based on in vitro binding studies. The inability to find any unique pharmacological effect that can be linked to interactions with Q receptors, as well as the lack of a correlation between affinity for the a binding site and behavioral activity, have led some investigators to hypothesize that the (I receptor represents a binding site and not a functiunal receptor (Z&in and &kin, 1988; McCann et al., 198% Thus, the characterization of the a receptor has been hampered by the lack of a functional bioassay system. it has been reported that drugs that bind to u receptors inhibit carbachol-induced phosphatidylinositol turnover in rat brain (Fowler and Thorell, 1987; Bowen et al., 1988; 1990; Candura et al., 1989) and cell lines (Adams and Weber, 1989). Moreover, the rank order of potency for inhibition of cizrbachol-induced phosphatidylinositol turnover is highly correlated with the affinity of the compounds for (I receptors, suggesting that d receptors are coupled to the phosph~osi~de second messenger system (Bowen et al., 1988; in press). However, an important experimental confound is that drugs that bind to u receptors might also have affinity for muscarinic acetylcholine receptors. Thus, the- inhibition of c~bachol-i~du~ phospha~dy~no~~l turnover by u ligands could be due to a direct antimuscarinic act%ity rather than specific interactions with cr receptors coupled to the phosphoinositide system. In order to ascertain whether u ligands possess the ability to interact with muscarinic receptors, we de-

t~~?~~~~a~~i~potency of several @ and o as.says of m~s~a~nie function. First, nding studies were carried out to definity of the compounds for muscarinic pig cerebra? cortical membranes. the ~ti~~~sca~~ic potency of the compounds was e~~I~ate~ by their abi?ity to inhibit carbachd-induced ~o~t~~~tions in the guinea pig ileum.

of carbacho? step-wise by a factor of 2.15. The compounds were allowed to equilibrate with the tissue for 20 min prior to the addition of carbachol, and dose ratios (ratio of the EC,, values for carbacho? in the absence and presence of the test compounds) were calculated at three to four different concentrations of the te’st drugs. 2.3. Dara atla!rsis

Frozen guinea pig brains (Pel-Freez. Rogers. AR) were thaw&. and the cerebra? cortices were dissected out and suspended in 40 volumes of 50 mM sodiumta~ium ~Ilosphat~ buffer (pH 7.4). The tissue was mogenized in a glass homogenizer tube with a Tef?on pestle, and centrifuged at 30000 X g for 10 min at 4OC. The pellet was resuspended in buffer to a concentration of 10-O mg origina? wet weight/m? buffer. Receptor binding studies were carried out using the methods described by mert and Jenden (1985). Saturation stud. were conducted by incubating the homogenate (0.1 ) with [“WIN-methylscopolamine ([‘HINMS; 80.0 Ci/mmo?; New England Nuclear. Wilmington. DE) over a concemration range of 0.01-1.0 nM in a rota? volume of 2.0 ml. Non-specific binding was determined by incubating in tie presence of 10.0 PM atropine. For competition experiments, the homogenate was incubated with [‘H]NMS (0.3 nM) and increasing concentrations of non-labeled test compounds in a total volume of 2.0 ml. A?? binding assays were equilibrated for 60 min at 37 0 C and were performed in triplicate. The incubation was terminated by rapid filtration through Whatman GF/B glass fiber filters using a cell harvester (Brande?. Gaithersburg, MD). Protein concentration was determined by the method of Lowry et a?. (1951) using bovine serum a?bu~n as the standard. 2.2. Guinea pig ileum preparation The antitmuscarinic activity of the compounds was determined by assessing their ability to inhibit c~bacho?-induced con~actions in the guinea pig i?eum preparation as previously described (Ringdah?, 1984). The ilea were bathed in 37 OC Tyrode solution (pH 7.4) with the following composition (mM): NaC? 137.0; NaHCO, 12.0; KC? 2.7; MgSO, 1.0; NaH,PO, 0.4; CaC?, 1.8; glucose 5.0; and hexamethonium 0.3. Ilea? con~actions were recorded isoto~ca??y at 1.0 g of tension with a electromechanical displacement transducer and a potentiometric recorder. Concentration-response curves to carbacho? were constructed by the cumulative dose-response technique by increasing the concentration

For the receptor binding experiments, the data were analyzed by iterative non-linear least-squares regression analysis (Ehlert and Jenden, 1985). For competition studies. the I& values for each test compound were obtained by fitiing the experimental competition binding data to a one-site competitive inhibition mode?. Estimates of the dissociation constant (K,) were determined from the IC,, values using the Cheng and Prusoff equation (1973). For the guinea pig ileum studies. the dissociation constants (Ka) for each drug were determined by the method of Besse and Furchgott (1976): K, = [test compound]/(dose ratio - 1). The competitive nature of the antagonism was assessed by Schild analysis (A~nlakshana and Schild, 1959). Experimental values are reported as the means f S.E.M. 2.4. Drugs The drugs were obtained from the following sources: phencyciidine hydrochloride (PCP), ( + )-N-a?ly?-Nnormetazocine hydrochloride (( + )-SKF 10,047) and ( +)-pentazocine succinate were obtained from the National institute on Drug Abuse (Rockville, MD); haloperido? base was purchased from Sigma Chemical Company (St. Louis, MO); 1,3-di-o-tolyl-guani~ne (DTG) was purchased from Aldrich Chemical Company (Milwaukee, WI); R( + )-3-(3_hydroxyphenyl)-N-( l-propy?)piperidine hydrochloride (( + )3-PPP) was purchased from Research Biochemicals Incorporated (Natick, MA): ( +)-5-methyl-lO,ll-dihydro-5H-dibenzo [a,d]cyc?ohepten-5,10-i~ne ma?eate (MK-801) was provided by Merck, Sharpe and Dohme Research Laboratories (West Point, PA); and dexoxadro?, levoxadro? and l-[(2-thieny?)-cyclohexyll-piperidine (TCP) were gifts from Dr. Kenner C. Rice.

3. ResuIts The muscarinic antagonist (‘H]NMS exhibited saturable, specific binding in the guinea pig cortical homogenate, with a Ko of 0.17 f 0.02 nM and a B,,,,, of 791.9 P 50.8 fmol/mg protein (n = 4). A?? of the test compounds inhibited the binding of [3H]NMS to muscarinic receptors (fig. l), with K, values ranging from 0.10 PM for dexoxadro? to 33.32 PM for MK-801

@I PCP P HotDperidst . (+)%w 10.017 0 f+B-PPP

7 0

-6

-7

-6

-5

-4

-3

-2

-7

LOG [LIGAND]

Fig. 1. Percent specific binding of ~3H~N-methyl~opola~ne to guinea pig cerebral cortical membranes in the presence of various coneentrations of a or PCP ligands. Each curve represents the best fit of the experimental binding data to a single muscarinic receptor site.

(table 1). The competition curves were parallel, indieating a direct competitive interaction with musca~nic receptor sites. The rank order of potency for inhibiting [3H]NMS binding was dexoxadrol = levoxadrol > ( + )pentazocine > TCP > DTG > PCP > haloperidol > ( + )SKF 10,047 > ( + )3-PPP > MK-801. In the guinea pig ileum, carbachol produced a 50% maximal contraction at 0.66 f 0.04 FM (N = 12). With the exception of MK-801 (vide infra), none of the test TABLE 1

KB

Haloperidol (+ f ~ent~~in~ (IO-20 FM) a Levoxadrol (5-50 CM) Dexoxadrol (2.5-20 PM) DTG (48-130 PM) TCP (14-200 PM) ( + ) SKF-10,047 (20-200 fiM) PCP (10-200 ~.tMf ( + f 3-PPP (40-200 PM) MK-801

-5

Fig. 2. The percent maximal contraction of the guinea pig ileum induced by carbachol in the absence and presence of PCP. Standard errors varied between 4 and 7% of the mean values.

compounds caused contractions when applied in the absence of carbachol. PCP antagonized muscarinic agonist-induced contractions, producing rightwards shifts in the carbachol contraction curve (fig. 2). All of the compounds tested exhibited antimuscarinic activity in the micromolar range. The slopes of the Schild regressions were all near unity (table l), indicating that the compounds behaved as competitive muscarinic antagonists in the guinea pig ileum. However, it was not possible to determine the K, value for haloperidal because concentrations of haloperidol greater than 1.0 PM produced noncompetitive blockade of the carba7

7

Cortex

(PM)

Slope b

- b

-

0.89 f 0.04 =

1.30*0.08

-4

LOG [CARBACHOL]

Antimu~a~~c effects of o and PCP ligands assessed by muscarinic contraction of the ileum or [“HINMS binding to cerebral cortex in the guinea pig. Ileum

-6

DEXOXADROL

S

LEYOXADROL

e

K, (CM) 3.55 Ito. e

1.05f

0.22

0.65 f 0.03

1.36k0.27

0.14+0.01

1.03-1 0.30

1.61 f 0.45

0.10 fO.02

2.15f

l/44&0.29

1.71ItO.28

0.80f0.17

1.38f0.24

2.37

1.25kO.15

4.63 f 0.29

24.34k 12.02

0.86 kO.57

2.67 f0.52

36.19i _b

0.63 f 0.24 _b

20.55 f0.62 33.32f3.00

0.30

PCP 700&l PCP 200 j&M

2

f+)PENTAZOCtNE

6

xc) s I

5

6.73& 1.39 12.87k

4

6

5 -

3.15

a Values represent the concentration range used in the ileum organ bath. b The Kn values for haloperidol and MK-801 could not be determined (see text). ’ Numbers are the means f S.E.M. (N = 3-4).

7

LOG KB (M)

Fig. 3. Relationships of tbe negative logarithms of the affinities of the compounds for muscarinic receptors in the guinea pig brain (Kt) with the antimuscarinic activity in the ileum (KB). The Pearson correlation coefficient correlation was statistically significant (r = 0.87, P < 0.0047). The equation for the regression line is y = 1.02~ +0.43.

-~~~d~~~ ~~~tm~ti~t~s. ( + ~Fent~~ine

showedthe ~~~t~st ~~~t~~~~~rini~ activity with a KB Of 0.89 FM. + b3-PPP had the weakest antimuscarinic activa K Bof 36.19 FM. The rank order of potency as amagonists was ( -C)-pentazocine > dexoxadrol = ievoxadrol > DTG > TCP > ( + )-SKF 10,047 > PCP ) ( + k3-PPP. MK-801 displayed unusual properties in the ileum ere examined only qualiratively. Over the range of .O,uM, MK-801 produced unsustained twitches of the ilea when administered in the absence of carbachoi; the twitches were 8.~~0.0~ of the maximal carbacholinduced contractions. At iow concentrations (5-15 FM), MK-801 potentiated carbachol-induced contractions, but at a higher concentration (30 PM), MK-801 produced noncompetitive inhibition of carbachol-induced contractions, ~4th the c~b~chol-induced contractions never exceeding 4570% of the control response, These effects of MK-801 preclrrded the determination of its antimuscarinic activity in the guinea pig ileum. The Peamon correlation coefficient comparing the affinity of the compounds (excluding MK-801 and h~o~~doi~ for muscarinic receptors in the guinea pig brain (K,) with the antimusctitic activity in the ileum (K,) was statistically significant (r = 0.87, P < 0.0047, N = 8: fig. 3). All of the drugs had K Bvalues that were 1.3 to 10.3-fold greater than their K, values.

E

4. c)jiion

The data demonstrate that all of the compounds tested bind to brain muscarinic receptors with K, values

in the micromolar range. Moreover, with the exception MK-801 whose activity when administered alone prohibited the determination of a KB value, all of the compounds possess significant muscarinic antagonist activity in the ileum, with K, values also in the micromolar range. Although the correlation between binding to rn~~c receptors in the brain and ~timu~nic activity in the ileum was statistically significant, all of the drugs were more potent in the brain muscarinic receptor binding assay (Kc,< Ka). This discrepancy could be due to the different distribution of muscarinic receptor subtypes in cerebral cortex and ileum (Eglen and Whiting? 19851,or related to differences in the two methods used to determine ligand affinity (e.g. ligand binding in homogenates versus functional antagonism in intact tissues). In addition, differences in the incubation conditions of the ileum and binding assays (e.g. buffer and/or ionic strength] may have contributed to the unequal ligand affiities estimated by the two assay systems. This latter factor is supported by the observation of Birdsall et al. (1979) that muscarinic ligand exhibit lower affinity in buffers of high ionic strength. There have been other reports that compounds that of

bind to CTand/or FCP receptors also can interact with muscarinic receptors. For example, it has been shown that PCP can inhibit the contractile responses produced by muscarinic agonists in the guinea pig ileum (Maayani et al., 1973; Kloog et al., 1977; Vincent et al., 1978; Aronstam et al.. 1980; Gintzler et al., 1982) as well as bind to muscarinic receptors in brain (Kloog et al., 1977; Vincent et al., 1978; Aronstam et al., 1980). Haloperidol (Snyder et al., 1974), (+)-SKF 10,047, (Johnson and Hillman, 1982), DTG and (+)-pentazocine (Candura et al., 1989)” all of which have high affinity for CTreceptors, also bind to brain muscarinic receptors. Walker et al. (1989) found that f + )-SKF 10,047 can inhibit oxotremorine-induced analgesia, suggesting that it has antimuscarinic activity, and dexoxadrol also has been reported to have significant antimuscarinic properties (Gonzales and Moerschbaecher, 1989). It is interesting to note that dexoxadrol and levoxadrol were equipotent in both assay systems, and these two compounds have approximately equal binding affinities for Q receptors, but dexoxadrol has a much higher affinity for PCP receptors than levoxadrol (Largent et al., 1986). Thus- the binding affinity and potency of these two enantiomers for muscarinic receptors shows the same lack of stereoselectivity as that observed for their binding to u receptors. Although the 0 and PCP ligands tested bind to and functionally antagonize muscarinic receptors, the agents differ in potency. One of the compounds, (+)-3-PPP, had the lowest affinity in the ileal assay and had an affinity for cortical muscarinic receptors similar to MK-801. This finding is important because ( + )-3-PPP has a high degree of selectivity for u receptors compared to PCP receptors (Largent et al., 1986). Thus, ( -I-)-3-PPP may serve as a prototype compound for the develupmezt of new highaffinity a ligaands which exhibit low or negligible interactions with muscarinic receptors. Given the importance af trying to establish a link between the neurochemical and functional effects mediated by d receptors_ our results indicate that caution should be used in correlating the pha~acolo~cal activity of the u/PCP ligands with specific and selective interactions with the u receptor. For example, it is plausible that a major component of the inhibition of carbachol-induced phosphatidylin~itol turnover produced by D ligands (Bowen et aI., 1988), if not the whole phenomenon, is related to the antimuscarinic properties of these compounds. Moreover, even if u receptors are coupled to the phosphoinositide system to some degree, the inherent antimuscarinic activity of the a ligands will preclude the use of this system as a f~c~onal assay for D receptor activity. Similarly, Fo;vler and Thorell(1987) concluded that haloperidot and ( + )3-PPP inhibited carbachol-induced phosphatidylinositol turnover via the direct antagonism by these agents at muscarinic receptors. In fact, the raatio of the IC,, values of ( + )3-PPP

155

relative to haloperidol (6.07) for inhibiting phosphatidylinositol turnover is very close to the ratio of their respective K, values (5.77) as determined by [3HJNMS binding (table l), further supporting a direct interaction of these ligands with muscarinic receptors in vitro. Interactions with muscarinic receptors might underlie other effects of PCP and (I receptor ligands. For example, it has been found that PCP and TCP (Gintzler et al., 1982), as well as (&)-SKF 10,047 (Su et al., 1981; Kramer et al., 1982), can inhibit electrically stimulated contractions in the guinea pig ileum, and ali of these compounds were found to have antimuscarinic activity in the present study. Campbell et al. (1989) evaluated a large number of u and PCP receptor-selective compounds for their ability to inhibit electrically stimulated contractions of the guinea pig ileal longitudinal muscle/myenteric plexus, and concluded that the inhibition was mediated by (I receptors. It is interesting to note that the Spearman rank order correlation coefficient comparing the potency for inhibiting the electrically stimulated contraction of the guinea pig ileal longitudinal muscle/myenteric plexus as determined by Campbell et al. (1989) and the potency of the compounds for inhibiting carbachol-induced contractions in the present study was highly significant (r = 1.0, p = 0.0253, N = 6), suggesting that the inhibition of electrically stimulated contractions in the ileum by these drugs might be due to their antimuscarinic activity. Both haloperidol and MK-801 had effects in the guinea pig ileum that were qualitatively different from those of the other compounds.’ For example, concentrations of haloperidol greater than 1.0 ,uM produced noncompetitive blockade. It is interesting to note that haloperidol also produced noncompetitive blockade of carbachol-stimulated phosphoinositide turnover in NCB-20 cells (Adams and Weber, 1989). MK-801 produced biphasic effects on the ileqm; low concentrations producing unsustained twitches when administered alone, and higher concentrations causing noncompetitive inhibition of carbachol-induced contractions. The low dose twitches could have been due to MK-801 interacting with ileal glutamate receptors (Sawyer and Shannon, 1988; Moroni et al., 1989); however, in control experiments NMDA (0.01-100 FM) did not affect baseline ileal tengion or inhibit the contractile effects of carbachol, indicating that this glutamate receptor agonist had no effect in this preparation. It is conceivable that the concentration of magnesium present in the buffer (1.0 mM), a cation that can antagonize responses to NMDA, blocked any NMDA receptor-mediated contractile effects. The results of the present study demonstrate that many u and PCP receptor ligands possess antimuscarinic activity. Moreover, these compounds might reach concentrations at which they have significant antimuscarinic activity after drug administration in vivo.

For example, PCP reaches a peak plasma concentration of approximately 1.0 FM after the i-p. administration of 8.6 mg/kg (Cho et al., 1989), and the estimated K, of PCP for muscarinic receptors determined in the present study was 2.67 PM. Although PCP and u receptor ligands might have affinities for PCP and u receptors in the nanomolar range, the plasma and brain levels that occur in vivo administration could allow interactions with other binding sites, such as with the muscarinic receptor, to take piace. The results of the present study suggest that antimuscarinic activity might underlie some the effects that have been hypothesized to be mediated by specific interactions with u receptors, and demonstrate that the antimuscarinic activity of PCP and u ligands must be taken into consideration when evaluating the pharmacological activity of these compounds both in vitro and in vivo.

Acknowledgements We would like to thank Dr. Donald J. Jenden for helpful comments on the manuscript and Dr. Kenner C. Rice for providing the dexoxadrol. levoxadrol and TCP. Supported by USPHS Grants GM37816. DA-04113 and DA-05448.

References Adams, J. and E. Weber, 1989, Sigma drugs can block carbacholstimulated phosphoinositide turnover in NCB-20 cells, Proc. !3w. Neurosci. 15. 543. Aronstam, R.S.. M.E. Eldefrawi. A.T. Eldefrawi. E.X. Albuquerque. K.F. Jim and D.J. Triggle. 1980. Sites of action of phencyclidine. IfI. Interactions with muscarinic receptors, Mol. Pharmacol. 18. 179. Arunlakshana, 0. and H.O. Schild. 1959. Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 438. Besse, J.C. and R.F. Furchgott, 1976. Dissociation constanls and relative efficacies of agonists acting on alpha adrenergic receptors in rabbit aorta. J. Pharmacol. Exp. Ther. 197, 66. Birdsall. N.J.M., A.S.V. Burgen. E.C. Hulme and J.W. Wells. 1979. The effects of ions on the binding of agonists and antagonists to muscarinic receptors, Br. J. Pharmacol. 67, 371. Bowen, W.D., B.N. Kirschner, A.H. Newman and KC Rice. 1988. c~ Receptors negatively modulate agonist-stimulated phosphoinositide metabolism in rat brain, European J. Pharmacol. 149, 399. Bowen. W.D., P.J. Tolentino. B.N. Kirschner, P. Varghese. B.R. De Costa and K.C. Rice, Sigma receptors and signal transduction: negative modulation of signaling through phosphoinositide-linked receptor systems, NIDA Res. Monogr. (in press). Campbell, B.G., M. Schetz, J.F.W. Keana and E. Weber, 1989, Sigma receptors regulate contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation elicited by both electrical stimulation and exogenous serotonin. J. Neurosci. 9. 3380. Candura, S.M., T. Coccini, L. Manzo and L.G. Costa, 1989. Effects of sigma compounds on agonist-stimulated phosphoinositide metabolism in rat brain. Proc. Sot. Neurosci. 15. 1006. Chaudieu, I., J. Vignon M. Chicheportiche. J.-M. Kamenka. G. Trouiller and R. Chicheportiche, 1989. Role of the aromatic group in the inhibition of phencyclidine binding and dopamine uptake by PCP analogs. Pharmacol. Biochem. Behav. 32. 699.

1973, Relati0nship between the inhibicon~~ntrntion of inhibitor which causes f an enzymatic reaction, Biochem. Pechnick ax& E. Di Stefano. 1989. k0Hnetic and ~~a~yn~c evaluation of phencyciidine & its &&eurero variant. J. Pharmacol. Exp. Ther. 250. and R.L. Whiting. 1986. Muscarinic receptor subtypes: a oi the current classification and a proposal for a working abate. J. Auton. Pharmacol. 5,323. Ehfert. F.J. and D.J. Jenden. 198% The biding of a t-chlorethylamuue derivative of oxotrem0rine (BM123) to muscarinic receptors in the rat cerebral cortex. Mol. Pharmacol. 28, 107. Fowkr. C.J. and 6. Thorell. 1987. Antagonist effects of the enantiCm of 3-PPP towards a,-adrenoceptors coupled to inositol in the rat cerebral cortex, Pharmacof. Gintzler. A.R.. R.S. Zukin and S.R. &kin. 1982, Effects of phencychdine and its derivatives on entenc neurones. Br. J. Phannacol. 75. 261. Got&es R,4. and J.M. Moerschbaecher, 1989. A phencyclidinc recqnition site is associated with N-methyl-D-aspartate inhibition of ~~hol-stim~at~ p~sph~nositide hydrolysis in rat cortical slim J. Pharmac0L Exp. Ther. 35, X7. Johnson KM. and G-R. Hillman. 1982. Comparisons between phencyehdine. its monohydtoxylated metabolizes. and the stereoisomers of N-allyl-N-normetazoeine (SKF 10047) as inhibitors of the muscarinic receptor and acetylcholiiesterase, J. Pharrn. Pharmac0L 34.462. Johnson. K.M. and SM. Jones, 1990. Neuroph armacology of phene_ychdine: basic mechanisms and therapeutic potential, Ann. Rev. Pharma&. Toxicol. 30.707. Kl0og. Y.. M. Rebavi, S. Maayani and M. Sokotovsky, 1977, Antichohnesterase and antiacetylcholine activity of l-phenylcyclohexykunine derivatives, European J. Pharmacol. 45,221. Kramer. W.. N. Steigemann and G.T. Shearman, 1982, Differential effects of SKF 10.047 (N-~lyl-no~et~~~e) on peristalsis and l~~tu~n~ muscle contractions Of the isolated guinea-pig ileum, NaunynS&rniedeb. Arch. Pharmacol. 321,218. Largent. B.L.. A.L. Gundlach and S.H. Snyder, 1986, Pharmacologic and autoradiographic discrimination of sigma and phencyclidine receptor binding sites in brain with ( +)-13H]SKF 10,047, (+ )~~H~~y~x~henyl]-Nil-propyl)pi~~ne and [3H]-1-[l-(2thieny~~lohe~yt~i~piperidine. J. PharmacoI. Exp. ther. 238.739. Lowry. G.H.. NJ. Rosebrough. AL. Farr and RJ. Randall, 1951,

Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193. 265. Maayani. S., H. Weinstein. S. Cohen and M. Sokolovsky, 1973, Acetyl~holin~like molecular arrangement in psychoto~meti~ antichoIine~c drugs, Proc. Natl. Acad. !%i. U.S.A. 70, 3’103. Majewska, M.D., S. Parameswaran. T. Vu and E.D. London, 1989, Divergent ontogeny of sigma and phencyclidine binding sites in rat brain, Dev. Brain Res. 47, 13. McCann. D.J., R.A. Rabin, S. Rens-Domiano and J.C. Winter, 1989. Phencyclidine/SKF-lO,O47 binding sites: evaluation of function, Pharma&. Bi0chem. Behav. 32.87. Moroni. E. D. Pelle~td-Giampietro, M. Ah&am. G. Cherici. F. Mori and A. Galli, 1989. Glycine and kynurenate modulate the glutamate receptors in the myentetic plexus and in cortical membranes. European J. Pharmacol. 163,123. Paleos. G.A., Z.W. Yang and J.C. Byrd, 1990, Ontogeny of PCP and sigma receptors in rat brain, Brain Res. 51. 147. Quirion, R.. R. Chjche~r~che, P.C. Contreras, K.M. Johns0n. D. Lodge, SW. Tam, J.H. Woods and S.R. Z&tin, 1987, Classification and nomenclature of phencyclidine and sigma receptor sites, Trends Neurosci. 10.444. Ringdahl, B., 191LQ,Determination of dissociation cOnstams and relative efficacies of oxotremorine analogs at muscarinic receptors in the guinea pig ileum by pharmacological procedures, J. Pharmacol. Exp. Ther. 229, 199. Sawyer, B.D. and H.E. Shannon, 1988, N-Methyl-D-aspartate receptors in the guinea pig iieum myenteric plexus, Pharmacologist 30, A184. Snyder, S.H., D. Greenberg and H.I. Yamamura. 1974, Antischizophrenic drugs and brain chohnergic receptors, Arch. Gen. Psychiat. 31.58. Su, T-P.. T.H. Clements and C.W. Gorodetsky, 1981, Multiple opiate receptors in guinea-pig ileum, Life Sei. 28.2519. Vincent, J.P., D. Cavey, J.M. Kamenka, P. Geneste and M. Lazdunski. 1978, Interaction of phencyclidines with the muscarinic and opiate receptors in the central nervous system, Brain Res. 152,176. Walker, J.M., S.L. Patrick, A. Thurkauf, KC. Rice and W.D. Bowen, 1989, (+)-Opiate in~bition of oxotrenmrine analgesia, Proc. Sot. Neurosci. IS. 664. Won& E.H. F., A.R. Knight and G.N. Woodruff. 1988, [sH]MK-801 labels 3 site on the N-methyl-D-aspartate receptor channel complex in rat brain membranes, 1. Neuroehem. 50, 274. Zukin, R.S. and S.R. Zukin. 1988, Phencyclidine, sigma and NMDA receptors: Emerging concepts, in: Sigma and Phencyclidine-like compounds as Molecular Probes in Biology, eds. EF. Dominio and J.-M. Kamenka (NPP Books, AM Arbor, MI) p. 407.