Stereospecific presynaptic inhibitory effect of Δ 9 -tetrahydrocannabinol on cholinergic transmission in the myenteric plexus of the guinea pig

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<ul><li><p>Stereospecific presynaptic inhibitory effect of A'-tetrahydrocannabinol on cholinergic transmission in the myenteric plexus of the guinea pig' </p><p>Division of Pharmacology and Therapeutics, Faculty of Medicine, Tlle University of Calgary, Calgary, Alta., Canada T2N IN4 </p><p>Received March 20, 1978 </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>[Traduit par le journal] </p><p>Introdl~ction coefficient of the order of 12 000 (Roth and Wil- </p><p>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 </p><p>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 </p><p>'Supported by the Medical Research Council of Canada. activity (Dwivedi and Harbison 1975; Karler et al. </p><p>Can</p><p>. J. P</p><p>hysi</p><p>ol. P</p><p>harm</p><p>acol</p><p>. Dow</p><p>nloa</p><p>ded </p><p>from</p><p> ww</p><p>w.n</p><p>rcre</p><p>sear</p><p>chpr</p><p>ess.</p><p>com</p><p> by </p><p>WA</p><p> ST</p><p>AT</p><p>E U</p><p>NIV</p><p> LIB</p><p>RA</p><p>RIE</p><p>S on</p><p> 11/</p><p>20/1</p><p>4Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>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. </p><p>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 exo- genous 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 pre- synaptic terminals (Paton et (al. 1972: Askew czt al. 1974; Fredericksorl et 01. 1 976). </p><p>The effects of THC on isolated neural prepara- tions are not well established. Wyck and Witchie (1973) demonstrated that THC! decreased the ac- tion 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 re- sponses, Kayaalp et 01. (8974) reported that THC decreased the twitch amplitude and slowly blocked conduction in the phrenic nerve trunk at concentra- tions of 3 X 18-"/ml. </p><p>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 identi- cal (Widman et al. 1974). However, differences in activities have been shown to exist in mice (Jones </p><p>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. </p><p>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. </p><p>The results presented here detnonstratc that the effects of THC are dose related, that the site of ac- tion is presyr~aptic, that the ( - ) isomer is about 25-fold more active than the (+) isomer, and that the effect is relatively nonreversible in rjitro. </p><p>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 1C. 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 experi- ment. </p><p>Drugs The (-1 THC (dissolved in dehydrated alcohol) was gen- </p><p>erously supplied by the Department of National HeaIth and Welfare, Health Protection Branch, Ottawa, Canada, </p><p>Can</p><p>. J. P</p><p>hysi</p><p>ol. P</p><p>harm</p><p>acol</p><p>. Dow</p><p>nloa</p><p>ded </p><p>from</p><p> ww</p><p>w.n</p><p>rcre</p><p>sear</p><p>chpr</p><p>ess.</p><p>com</p><p> by </p><p>WA</p><p> ST</p><p>AT</p><p>E U</p><p>NIV</p><p> LIB</p><p>RA</p><p>RIE</p><p>S on</p><p> 11/</p><p>20/1</p><p>4Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>970 CAN. J. PHYSIOL. PHARMACOL. VOL. 56, 1978 </p><p>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. </p><p>Gas Chromatographic Analysis of THC Determinations of concentrations in ethanol stock and </p><p>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 260C, detector temperature set at 270C, and injection port at 260C. 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 tem- perature 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 219C. Calculations of concentrations were made from peak integral ratios from the integrator printout of standard mixtures prepared from ( + ) THC. Preparation o f Drug Solutions </p><p>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 chroma- tography (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 4C. 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. </p><p>Experimental Procedure All solutions were injected into the fluid in the organ </p><p>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 </p><p>method using fresh oxygenated and prewarmed Kreb's so- lution) 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). </p><p>Results Since THC is relatively water insoluble, solu- </p><p>bilizers are often used to increase aqueous concen- trations. The suitability of Cremophor E.L., chosen for this study, was established by preliminary ex- periments. 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 concentra- tion was selected for all experiments. At this con- centration, it was also found (Roth and Williams, in preparation) that the glass binding or loss of...</p></li></ul>

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