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Stereospecific presynaptic inhibitory effect of A'-tetrahydrocannabinol on cholinergic transmission in the myenteric plexus of the guinea pig'

Division of Pharmacology and Therapeutics, Faculty of Medicine, Tlle University of Calgary, Calgary, Alta., Canada T2N IN4

Received March 20, 1978

ROTH, S. H. 1978. Stereospecific presynaptic inhibitory effect of AD-tetrahydrocannabinol on cholinergic transmission in the myenteric plexus of the guinea pig. Can. J. Physiol. Pharmacol. 56, 968-975.

AD-Tetrahydrocannabinol (THC) is very lipid soluble, as are many anesthetic agents. The action of anesthetics is nonspecific; isomers are equieffective. T H C is optically active; therefore, the effects of its stereoisomers were studied on the electrically and chemically stimulated longitudinal muscle strip of guinea pig ileum. The results demonstrate that both isomers depress the response to electrical stimulation in a dose-related manner. The maximum effect is gradually reached in approximately 20 min. The (-) isomer is 24.6-fold more active than the (+) isomer (ED5,, for (-) THC is 1.25 x 10-'M, for (+) THC, 3.08 x lo-" M ) and the site of action appears to be presynaptic because responses to ACh are not significantly depressed. The depressant effects are relatively nonreversible. Membrane concentrations calculated at the EDjo values for the (-) isomer are of the order of 0.5 mM/kg dry membrane, well within the range for anesthesia. Thus THC may be regarded as a partial anesthetic since some of its actions are similar to those .

of the classical anesthetics, yet it possesses selective action at the neuronal membrane or tissue level.

ROTH, S. H. 1978. Stereospecific presynaptic inhibitory effect of AO-tetrahydrocannabinol on cholinergic transmission in the myenteric plexus of the guinea pig. Can. J. Physiol. Pharmacol. 56,968-975.

Tout comme plusieurs anesthdsiques, le A"-tetrahydrocannabinol (THC) est trks lipo- soluble. L'action des anesthdsiques n'est pas spCcifique; les isomkres QCmontrent la meme efficacitk. Le THC est optiquement actif; aussi a-t-on CtudiC les effets de ses stCrCoisomkres sur des bandes longitudinales musculaires d7ilCon de cochon d'inde, stimulkes Clectrique- ment et chimiquement. Les rCsultats dtmontrent que les isomkres diminuent tous deux la rCponse ii la stin~ulation Clectrique, et ce dkpendamment de la dose administrie. L'effet maximum est progressivement atteint en 20 min environ. L'isomkre (-) est 24.6 fois plus actif que l'isomkre (+), (EDjo pour THC (-) vaut 1.25 x 10 M, et pour THC (+), 3.08 x 1 0 - W ) ; le site d'action semble Stre prCsynaptique car les rCponses ii 1'ACh ne sont pas diminuCes de f a ~ o n significative. Les effets dCpressifs sont relativement irrCver- sibles. Les contractions de la membrane, calculCes aux valeurs ED,,, p u r l'isomkre (-), sont de l'ordre de 0.5 mM/kg de membrane skche, ce qui correspond bien aux valeurs ren- contrCes en anesthksie. Ainsi le T H C peut-il @tre considCrC comme un anesthksique partiel puisque certaines de ses actions sont semblables B celles des anesthksiques classiques; il a toutefois une action sClective au niveau de la membrane neuronale ou B celui du tissu.

[Traduit par le journal]

Introdl~ction coefficient of the order of 12 000 (Roth and Wil-

The pharmacological effects of cannabis are diffi- cult to classify; depending on the dose administered, the effects observed are very similar to those of hyp- notic sedatives, minor tranquilizers, and stimulants (Davis et al. 1972; Thomas and Chesher 1973; Domino 1976). The major active constituent of cannabis is believed to be A9-tetrahydrocannabinol (THC) (Hollister 1974; Isbell et al. 1967; Mechou- lam 1970). THC is very lipid soluble (Gill and Jones 1972a), having a membrane-buff er partition

liams 1977). This high partition coefficient suggests that THC may interact with hydrophobic regions in the membrane (Mahoney and Harris 1972; Roth and Williams, in preparation), as many anesthetic compounds do (Roth and Seeman 197 1 ) . THC has been described as a partial anesthetic (Paton et al. 1972; Lawrence and Gill 1975). Its many other actions, e.g., analgesic effects (Noyes et al. 1975), sedative effects (Frederickson et al. 1976)' pro- longation of barbiturate sleeping time (Paton et al. 1972; Takahashi and Karniol 1975), anticonvulsant

'Supported by the Medical Research Council of Canada. activity (Dwivedi and Harbison 1975; Karler et al.

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B974), membrane stabilizing property (Chari- Bitron 1971 ; Raz et crl. 1972; Bach er al. 1976), and ability to decrease the conce~itration of halothane required for anesthesia (Stoelting et crb. 1973), all support the hypothesis that THC is ancsthetic-like in action.

The effect of THC has been studied on several i r ~ vitro preparations, but the results are variable and often contradictory. THC depresses the resporsse of guinea pig ileum to electrical stimulation (Gill et a!. 1970; Layman and h4ilton 2974 ; Rose11 and Agu- sell 1975; Rssell rt a!. 1976). The response to exogenous acetylc%aoline has been reported to be either reduced (Dewey e f a / . 1970; Layman and lllilton 1971 ), not affected (Rosell et ak. 1976)- or poten- tiated (Gill et 01. 1970; Gascon and P6r;s 1973). THC inhibits the bissynthesis of acetylchoIine in brain (Friedman et al. 1976) and Iysolecitliin acyl transferase of lymphocytes (Greenberg et al. 2977) and may inhibit release of aselylclio8ine from presynaptic terminals (Paton et (al. 1972: Askew czt al. 1974; Fredericksorl et 01. 1 976).

The effects of THC on isolated neural preparations are not well established. Wyck and Witchie (1973) demonstrated that THC! decreased the action potential amplitude of imammalian ~ac~mimyelin- ated nerve fibers. However, Brady and Carbone (1973) were unable to demon~trate an effcct on the impulse conduction of the squid giant axon. Similar discrepancies have been repc~rtcd using the phrenic nerve diaphragm preparation. Although Layman and Milton (1 97 1 ) and Gascon and Pirks (1 973) ok- served no effect on electrically induced twitch responses, Kayaalp et 01. (8974) reported that THC decreased the twitch amplitude and slowly blocked conduction in the phrenic nerve trunk at concentrations of 3 X 18-"/ml.

Since it is assumed that a lack of a structure- activity relationship exists for most anesthetics, the action of anesthetics has been commonly referred to as nonspecific. Should THC be considered an anesthetic or anesthetic-like, then a study of the eflects of its isomers wouId be helpful in evaluating whether its mode of action is nonspecific as well. Tetrahydrocannabinol is optically active. Thc (+ ) isomer, being nonbiological, must be synthesized. Optical isomers are isolipophilic in organic solvents: therefore, there should he no apparcnt difference in their activities provided the interaction is purely nonspecific. The binding of the (+) and ( - ) isomers to proteins and blood cells is almost identical (Widman et al. 1974). However, differences in activities have been shown to exist in mice (Jones

et al. 1974). Recently Lawrencc and Gill (1975) have demonstrated a significarrt difference between the two enantiomers on the mcslccular mobility of the hydrocarbon phase of Iiposomes.

Since many of the effects in man and animals ap- pear to be anticholinergic (Friedman ct nl. 1976) and the isolated guinea pig ileurn preparation is very sensitive to cannabinoids (Wosell ut nl. 1976) the effects of tlme isomers of THC ota the responses of the electrically and chemically stimulated lot~gitudinal muscle strip of ilcuna were studied.

The results presented here detnonstratc that the effects of THC are dose related, that the site of action is presyr~aptic, that the ( - ) isomer is about 25-fold more active than the (+) isomer, and that the effect is relatively nonreversible in rjitro.

The method described for the longitr~dinal smooth nluscle strip of the guinea pig ileum was adapted from baton and Vizi (1969) and Paton and Z:tr (1968). Male guinea pigs weighing between 500 and 700 g were sacrificed by a blow to the head and bled from the carotid arteries. A suitable length of iIeum free of mesentery was carefully dissected and immediately placed into a dish containing oxygenated Kreb's solution. The Kreb's solution had the following composition (millimolar) : NaCl 95; KC1 4.7: MgSO,, 2.3: CaCI, 2.5; KHnPOt 1.2; NaHCO.% 25; and dextrose 12. Lengths (5-8 cm) were cut and the contents of the lulnen were washed out with fresh Kreb's solution. Each piece of ileum was then threaded over a glass pipette with 6 mrn outside diameter. The longitudinal muscIe Irmyer was gently teased away from the underlying circular muscle with cot- ton wool soaked in Kreb's solution, using a tangential mo- tion. A thin skeet of tissue thus removed consists of lnngi- tudinal smooth muscle, a portion of the Auerbach9s plexus, and a small proportion of circular smooth muscle (Paton and Vizi 1969; Henderson 1975). A length of silk suture was tied to each end of the strip and the strip was suspended in a 5- car 20-ml water-jacketed organ bath filled with Kreb's solution continuously bubbled with 95% oxygen and 4% COO-. The temperature was maintained at 37 a 1°C. One end of the strip was tied to a fixed rod at the bottom of the organ bath and the other end was attached to a force transducer (Grass FTO3G). Each strip was allowed to equilibrate for at least 1 h during which time tension was adjrlsted to 500 mg. The muscle strip was stimulated elec- tricalIy by 'field' stimulation using two platinum sIectrodes lying against the inside wall of the organ bath on either side of the tissue. Rectangular pulses a t l .O-ms duration, 0.2 Hz at supramaxima1 voltage, were delivered from a modified Grass S-88 stimulator. Contractions were recorded on a pen recorder (Becknian dynograph R42 1 ). The transducers were calibrated with brass weights before and after each experiment.

Drugs The (-1 THC (dissolved in dehydrated alcohol) was gen-

erously supplied by the Department of National HeaIth and Welfare, Health Protection Branch, Ottawa, Canada,

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970 CAN. J. PHYSIOL. PHARMACOL. VOL. 56, 1978

through the offices of Mr. R. G. Graham. The (+) THC isomer was synthesized by the method of Gill and Jones (19726). Cremophor E.L. was purchased from BASF Canada Limited, Montreal. Acetylcholine Br was purchased from Eastman Kodak Co., Rochester, NY, and absolute ethanol from Canadian Industrial Alcohols and Chemicals Limited, Corbyville, Ontario. All chemicals required for chromatographic analysis were purchased from Chroma- tographic Specialties, Brockville, Ontario.

Gas Chromatographic Analysis of THC Determinations of concentrations in ethanol stock and

aqueous solutions of both isomers of THC were made using a Hewlett-Packard gas chromatograph model 7620A with integrator. The column was packed with 3% S.E. 30 on Gas Chrom Q 80/100, and nitrogen was used as carrier gas. The flow rates for nitrogen and hydrogen were 20 ml/min. The oven temperature was programmed from 150 to 260°C, detector temperature set at 270°C, and injection port at 260°C. 5-a-Cholestane was used as the internal standard. The THC isomers were converted to their trimethylsilyl ether derivatives using a mixture of 1 part BSTFA, 0.5 parts trimethyl chlorosilane (TMCS), and 1 part acetonitrile. Aliquots of THC and internal standard were placed in small vials and evaporated to dryness under a stream of nitrogen. An aliquot of 200 p1 of silation mixture was added to the vial and the mixture was allowed to stand at room temperature for 10 min. Samples were dried and taken up in ethyl acetate and 1-2 pl were injected directly into the column. Under these conditions retention temperature of T H C was 219°C. Calculations of concentrations were made from peak integral ratios from the integrator printout of standard mixtures prepared from ( + ) THC.

Preparation o f Drug Solutions The (-) T H C (approximately 100 mg) was dissolved

in absolute ethanol to a final volume of 100 ml. The accu- rate concentration was determined by gas-liquid chromatography (GLC) analysis. An appropriate aliquot was transferred to a 20-ml glass vial, and the ethanol was evaporated under nitrogen. Cremophor E.L. dissolved in Kreb's solution (0.5 mg/ml) was added to the vial and the solution was sonicated for 5 min (Heat Systems, Ultra- sonics Ltd. sonifer model W140). The vial was contained in an ice bath during sonication. The sonicated solution was transferred to a volumetric flask with repeated washings of the vial with Kreb's solution containing Cremophor E.L. The final concentration of this solution was lo-" g/ml. Lower concentrations were prepared by diluting aliqtlots of this solution with Kreb's solution containing Cremophor E.L. (0.5 mg/ml). All solutions were prepared each day of the experiment from the stock ethanolic solution. The ethanolic stock solutions were stored under nitrogen in the dark at 4°C. The (+) T H C solutions were prepared in ex- actly the same manner, except that evaporation of the benzene solvent left a residual yellowish gum which was re- dissolved in ethanol and reevaporated to produce a solid residue.

Experimental Procedure All solutions were injected into the fluid in the organ

bath in 0.1- to 0.5-ml aliquots by syringe and needle. Cremophor E.L. controls were tested at the beginning of each experiment on each tissue. The T H C solutions were allowed to remain in the bath for at least 30 min. It was observed that even with repeated washings (overflow

method using fresh oxygenated and prewarmed Kreb's solution) the tissue would not recover following exposure to THC; therefore, only one concentration was tested on each tissue. The T H C isomers were tested on the responses to electrical stimulation and to chemical stimulation produced by acetylcholine ( ACh), barium chloride (BaCl,), and potassium chloride (KC1).

Results Since THC is relatively water insoluble, solu-

bilizers are often used to increase aqueous concentrations. The suitability of Cremophor E.L., chosen for this study, was established by preliminary experiments. A standard concentration of ( - ) THC was tested in the presence of varying concentrations of Cremophor E.L. and the effect of the combina- tion (THC and Cremophor E.L.) was compared with the effect of Cremophor E.L. alone. Minimal effects were observed in the presence of Cremophor E.L. (0.5 mg/ml) alone; therefore, this concentration was selected for all experiments. At this concentration, it was also found (Roth and Williams, in preparation) that the glass binding or loss of THC was decreased to less than l o % , where in the ab- sence of solubilizer, glass binding can be as great as 40% (Garrett and Hunt 1977). The final bath concentration of Cremophor E.L. was 0.01 mg/ml.

Both of the isomers of THC were effective in depressing the force af contraction of the electrically stimulated longitudinal muscle strip. The effective concentrations for the isomers fell in the range of lod9 to 1 0 - W . The maximum effect was reached very gradually, although the effect began within 4 min after addition of the drug (see Fig. 1 ) . The effect would begin to plateau at approximately 20 to 30 min after the administration of the drug (see Fig. 2 ) . However, it was observed in a few experiments with ( - ) THC, that the depression would continue to increase with time, eventually abolish- ing all response. It can be seen in Fig. 2 that equimolar solutions of the ( + ) and (- ) isomer were not equieffective in depressing the response. The activity of both isomers appeared to be irreversible, for repeated washing with fresh Kreb's or Kreb's- Cremophor solution would not return the response to normal. Occasionally it was observed that the depressant effect was enhanced after a wash. The effect of the solubilizing agent employed, 0.01 mg/ ml final concentration of Cremophor E.L., is also plotted. The response in the presence of the solvent alone was constant over a long period of time.

Dose response curves for each isomer were calculated by determining the effect of different concentrations of (+ ) and (- ) THC at a specific time

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ROTH

1 I I I I I 0 5 10 15 20 25

TlME (rnin)

FIG. 1. Example of a pen recording trace of the response (force of contraction expressed as grams of tension) of eIec- trically stimulated longitudinal muscle strip as a function of tin~e. Addition of (-1 THC solution to the organ bath is indicated by the arrow ( ) and designated time zero. The final bath concentration of THC was 6.9 X 10-7 rW. The response decreased gradually and plateaued a t 20/ ; of control, the initial efTect of the drug occurring at approximately 4 min, the maximunl effect at about 20 min.

*-------& ----- "'-* -------- *---------+-------- ;C--------

1 5 10 15 20 25 30 TIME (min)

FIG. 2. Time - dose response curves of electrically stimulated longitudinal muscle strip in the presence of equimolar concentrations of (+) and (-)THC isonlers (6.36 X lo-' M ) and control solution containing Crenlophor E.1,. (0.01 mg/ml). The (-) isomer produces a depression about 40; ( greater than the (+) isomer at nlaximunl eHect. The control solution depresses force of contraction by about 9;;) of control and its effect reniains constant for at least 60 min. Each data point is the mean 4 SEM of at least six tissues.

(20 min after injection of the drug into the bath fluid). The results are plotted in Fig. 3. The concentrations that reduced the maximum response of the longitudinal strip by 50% were calculated by linear regression analysis. For the (+ ) isomer the calculated EDB0 was 3.08 X 1 0 - W M 1.98 X 1 0 - W (SEE) and for the (- ) isomer it was 1.25 X lo-? M t 1.02 X lo-; M (SEE). The dose ratio of the two isomers was calculated to be 24.4. The sfopes of the linear portions of the dose response curves were determined by regression analysis and these were identical.

The response to stimulation by acetylcholine in the presence of the two isomers was also studied and the results are shown in TabIe 1. The response is expressed as a percentage of the control force of

contraction produced by a standard dose of acetylcholine after approximately a 30-min equilibration time with the drug. It can be seen that the response to acetylcholine is not depressed significarltly by either isomer.

The effects of the ( - ) isomer of THC on the response to stimulation by KC1 and BaC12 were also studied. Contractions produced by adding 2, 4.5, and 20 m:M KC1 or 0.1, 1, and 4 mM BaCl,, were depressed by at most 10% in the presence of 1.09 X 1 0 - W (-) THC, and these differences were not significant.

Discussion The maximum solubility of THC in a solution of

0.01 mg/ml Cremophor E.L. is of the order of

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CAN. J. PHYSIOL. PHARMACOL. VOL. 56, 1978

I I 1 1 1 1 1 1 1 I 1, 1 1 1 1 1

lo-' lo-' 1 o - ~ THC M

FIG. 3. The force of contraction as percent of control (response) of electrically stimulated tissues in the presence of varying concentrations of THC isomers 20 min after addition of drug. The dotted lines indicate the coilcentration of each isomer which reduced response by 50(,; of control (EDs0). Each point is the mean + SEM response of at least six tissues.

6 X 10-6 M, and the glass binding is reduced to TABLE 1. Response of the longitudinal muscle strip of

less than 10% (Roth and Williams, unpublished ileum to a standard dose of acetylcholine in the presence

data). The longitudinal strip in each organ bath was of A 9 (+)THC and A 9 ( - ) THC

approximately 30-50 mg of tissue and did not de- A C ~ response*, 5; of control crease the final concentration appreciably. Thus the concentrations reported in this study as final bath concentrations are correct within a 10% error, which may differ from those studies where no solubilizer is utilized and glass binding can be as high as 40% (Garrett and Hunt 1977). Maximum aqueous solubility is of the order of 2 X 1W6 M in Kreb's solution (Roth and Williams, unpublished data).

The slow onset of maximum or near maximum effect of the isomers of THC has been reported previously (Layman and Milton 1971 ; Kayaalp et al. 1974). This phenomenon has been suggested to be a result of physicochemical artifacts, such as the interaction of THC with the solubilizer (Clarke and Jandhyala 1977). The dispersion of THC in Cremo- phor E.L. may result in the formation of micelles. Attempts have been made to determine the critical micelle concentration (CMC) for Cremophor E.L.; however, these have not been successful and accu- rate CMC values were not obtained. It can be assumed, however, based on the knowledge of CMC values for other surfactants, and on the data obtained from the trials, that the CMC of Cremophor E.L. may well be in the range where optimum activity was observed, i.e., 0.01 mg/ml (Dr. R. Roche, personal communication).

A9 ( - )THC concn., M 6 . 4 ~ 6.4X10-8 3 . 2 ~ 6.4X10-7

A 9 (+) THC concn., M 6.4X 3.2X10-' 6.4X lo-' 6.4X - -

' V a l u e s are m e a n s and standard errors. N u m b e r s i n parentheses indicate n u m b e r o f de terminat ions .

The formation of micelles, therefore, may result in a rate-limiting step in the translocation of THC from the aqueous phase to the site of action and allow for the long duration required for maximal activity. It is interesting, however, that symptoms generally reach a peak in 30min in man (Manno et al. 197 1 ) and in dog (Martin et al. 1977). Ex- periments in this laboratory using THC at concentrations less than 1 0 - W without solubilizer also demonstrate a maximum effect attained in 20 to 30 min, which suggests the rate-limiting step for peak effect is a characteristic of the drug per se. The initial onset of activity occurs within 4 min after addition of the drug as noted previously for the 7-

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RBTH 97 3

OH-THC derivative (Wosell and Agurell 6975). The apparent irreversibility or persistence of effect in vitro has been reported by others (Gill et al. 1970; Layman and Milton 1971 ; Kayaalp et al. 1974), although others have stated the effect is reversible (Dewey et al. 1970).

It has been demonstrated that differences in activities do exist for the isomers of THC on mice (Jones et al. 1974) and the meail potency ratio was calculated to be 13 and estimated to be at least 20 for the pure compounds. The ( + ) isomer has been shown to be 20-fold less active than the (- ) isomer in msnkcys (Edery et al. 1971 ) . Recently Lawrence and Gill ( 1975) have shown a significant difference between the two enantiomers on the molecular mobility of the hydrocarbon phase of liposomes. suggesting the bilayer was suffciently chiral to differen- tiate between the isomers. Concentrations that depressed the electrically induced contraction by 50%) (i.e., ED,,,) were interpolated from the graph plotted in Fig. 3 . In the present study the ED,,, of the two enantiomers differed by 24.6, which agrees with previous values.

Optical isomers may be isolipophilic in membrane; therefore, we can assume that the rnembrane- buffer partition coefficients ( P ) are identical. The membrane concentrations for the THC isomers can be calculated using the equation

where Cf,,. is the EDso expressed as moIes per litre and C ,,,,, , ,,,,, ,,, is expressed as moles per kilogram dry membrane. In the presence of 0.01 mgjml Cremophor E.L., the P for ( - ) THC has been reported to be 4570 (Roth and Williams 1977) ; therefore, the C,n,,,l,,,,, for the ( - ) isomer would be of the order of 0.57 mM/kg dry me~nbrane which is well within the range required for general anesthesia (Woth and Seematl 1972; Seeman i972). The Clnchnl,,ral t. for the (+ ) isomer would be approximately 25-fold greater, i.e., 14.1 mM/kg dry membrane suggesting the intrinsic activities of the two isomers may not be similar.

The response to exogenous ACh was not reduced by either isomer. This suggests that the site of action is presynaptic in origin, with the depressant activity resulting from inhibition of release of neurotrans- mitter (Askew et nl. 1974; Hoekman et al. 1976; Frederickson et rrl. 1976). At the highest concentr-a- tion of the (- ) isomer a slight depression is evident. This agrees with the observations of previous invcsti- gations (Gill pt al. 1970; Gascon and P6ri.s 1973). Although there have been a few reports indicating

that THC inhibits release of acetylcholille from longitudinal strips (Layman and Milton 1971 ; Paton et nl. 1972), conclusive evidence has not been provided as yet that demonstrates a dose-dependent inhibition of release of ACh by THC. It has been reported recently that aX and a" THC both inhibit the biosynthesis of ACh in brain (Friedman et nl. 1976) and THC inhibits lysolecithin acyl transferase (Greenberg et al. 1977). However. choline acetyl- transferase activity is not affected (Askew ct al. 1974). The response to 5-liydroxytryptamine which stimulates cholinergic neurons in the ileum preparation is depressed by 7-OW-THC (Rose11 and Agurell 1975). In addition. Hattori and h4cCeer (1 977) have shown that tritium-labelled &THC preferentially binds on terminal boutons. Thus the biochemical, histochemical, and the pharmacological results pro- vide evidence to support a presynaptic site for action.

Inhibition of release of ACh may be a result of a blockade of propagation of action potentials along the fine terminal fibers or an inhibition of depolariza- tion-release process at terminals (Friedman et nl. 1976). Presynaptic inhibitory effect of THC is stereospecific yet it occurs at a membrane concentration similar to that of anesthetics (Hoekman et al. 1976). Thus THC may be regarded as a partial anesthetic since many of its properties and actions are similar to those of the classical anesthetics, yet it sIiows stereospecific and selective action on presynaptic events.

Acknowledgments

The author acknowledges the technical assistance of Mrs. T. Hanna and B. Gunn, and Dr. E. W'. Gill for initial synthesis of the (+) isomer and intro- ductioil to the problcm.

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