Effect of Δ 9 - and Δ 8 -Tetrahydrocannabinol on the Peripheral Autonomic Nervous System In Vitro

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<ul><li><p>Effect of A"- and Ax-Tetrahydrocannabinol on the Peripheral Autonomic Nervous System It2 Vi'ituol </p><p>'4. L. GASCON A N D M . 7'. Dkp:pm.temcjtrt r/c~ phar-,?lcrco/o,r(' </p><p>Received August 18. 1973 </p><p>GASCOX, .A. I,., and Ptnrks, hl. T. 1973. E f i c t of A'-'- and AX-tetrahydrocannabind on the peri- pheral autonomic nervous system in viruo. Can. J . Physiol. Pharmacol. 51, 12-21. </p><p>The purpose of this study was to evaluate the effect of A" -and Astetrahydrocannabinol (THC) on some isolated rnuscles. The results observed show that A',- and A T H C do not have agonistic activity on the vas deferens, the phrcnic nerve -- diaphragm. artd the ileum preparations. On the other hand, both compounds produce a transient potentiation followed by the inhibition of the acetylcholine- induced contraction as well as a potentiation of the rnusculotropic activity of noradrenaline on the isolated vas deferens. Furthermore, we have observed that AxTHC reverses the anti-cholinergic ac- tion of atropine, conteracts the anti-angiotensin activity of morphine, and finally, abolishes the potentiation of noradrenaline produced by cocaine. On the phrenic nerve - diaphragm preparation AWHC does not modify the contraction produced by nervc stimulation but alters the efkct of phy- sostigrnine, an anti-cholinesterase inhibitor. </p><p>GASCON, A. L., e l P ~ R ~ s , M. T. 1973. Effect of AO- and Ax-1etrahydroc:annabinol on the peri- pheral autonomic nervous system in vituo. Can. J . Physiol. Pharmacol. 51. 12-21. </p><p>ALL conrs de ce travail nous a w n s eterdie I'effet clu Lo- et du AXTHC silr diverses prkparations inuscuiaires isolees. I._es rksr~ltats qile nous avons obtenus montrent que ces deux derives du cannabi.; ne posskdent pas de propriete stimulante silr le canal dCferent, lVilCon et la preparation nerf phrCnique --- l~zuscle diaphragme. C'ependant, nous avons observd que le A"- e t le A V H C entrainent une poten- tialisation transitoire suivie de l'inhibition de 1'action stirnul:inte cie I'acdtylcholine sur lc canal clefd- rent et l'iieon du cobaye. De plus, ces cieux con~posCs possedent ilne inflnence potentialisatrice sur I'action de la noradrenaline ail niveai~ du canal deferent isole. Par la suite, nous avons observe que le AsTHC est capable de contrecarrer I'action anticholinergique dc I'atropine, de neutraliser I'aclion anti-angiotensine de In morphine et, de plus, d'annuler la potentialisation de la noradrenaline ii la suite de la cocaine. En dern'aer lieu, 13~x1s ;\VOIIS observi. que le LQTHC, quoique sans effet sur la contraction du diaphragme produite par la stinlulation nerveuse, 111ocIitie I'cfl'ek de la physostigminc selr la prkparntion taerf phrkniqne - - n~uscle diaphragmc de rat. </p><p>In the last Few years, it has been shown that cannabis and its chemical constituents are pharmaco~ogically active o n the cardiovascular system and isolated smooth muscle prepar- ations. However, variable effects were demon- strated on the dog heart rate, blood pressure. and respiratory rate (Loewe 1946; Sim and Tucker 1963; Bsse e f c r l . 1964; Garriot c,t nl. 1968: Dewey cr ui'. 1970; Hardman e f ui'. 1971). On the isolated smooth muscle prepar- ations, it is generally well-accepted that A')- tetrahydrocannabinol (THC) does not have agsnistic activity. Dewey rt ul. (1972) have shown that A" and A T T C are very weak inhibitors of acetylchoiline and histamine on the isolated guinea-pig ileum while Bose ct trl. (1963) reported that the resin of Ccrnnuhis </p><p>indic*ri antagonized to the same extent the spasmogenic actions of carbachol, histamine, barium chloride, and pitocin on the isolated guinea-pig ileum and rat uterus. There are many factors that might have contribnted to these conflicting findings. Among the most serious of these kactors has been the difficulty in controlling the dose level sf the active constituents of marihuana. </p><p>The purpose o f the present investigation was to determine the ;~ction of A')- and ATHC' on the isolated guinea-pig vas deferens, the isolated ileutn. and also the isolated rat phrenic nerve - diaphragm preparation. </p><p>Methods I n all our experiments. male gninea pigs weighing </p><p>between 250 and 300 1: and rats weighing 200 g mere lThis work was supported by a grant from the Medical used. In the first part o f our work we have slirdied the </p><p>Research Council of Canada (DA-101. influence of A')- and Ax-Tt-IC on the reactivity of the </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>UN</p><p>IV O</p><p>F N</p><p>OR</p><p>TH</p><p> CA</p><p>RO</p><p>LIN</p><p>A A</p><p>T o</p><p>n 11</p><p>/13/</p><p>14Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>(;-\SC(')V -kND I'ERES: TE'TR \IIY'IPROC.l\ </p><p>isolated \ a&gt; dcferens to acetylchoii~se, ~~oradrenalinc, barium cklloride. and histamine. In the second part, we havc rneabi~red the actaon of AVHC on the isolated r,at phren~c ncrvc - d~aphragrn preparataon in the third part, we have evaiuatccl the action of AsTHC on the spasmogersic activity of acetyl~holinc and angiotensin on the nsolated guinea-pig ller~rn and, finally, wz Iaavc rtudied the interactions between AxTHC :andios atrol-rlnc, morphine, and socairme. </p><p>1,solatcd h s .I~efi~r.cn.s Each isolated vas deferens was s ~ ~ s p e i ~ d e d in 10-ml </p><p>baths of Krebs-Henseleit solution maintained at 34 :: 0 . 5 "C and gasscd with 5'); carbon dioxide it1 oxygen. The co~?tractiol-ns indrlced by the various agonists weri: recorded on a smoked drum with a frontal-writing lever giving a fivefold magnification with a constant load of 0.5 g. Drugs were administered at 5-min intervals with a contact of 60 s. At tlae beginning of the assay. responses were obtained to a series of four doses of the agonist; each sequence was repeated two or three times. At this point, an alcoholic solution of A')- or AsTHC was added to the perfilsion mixt~1t.e to give a final concentration of either 10-8 or 10-7 M , and was kept in contin~lous con- tact with the preparation. The final cor~centration of ethanol was 0.044:,. After each addition of the niari- huana constituents, the isolated preparation was allowed to equilibrate for 30 rnin before the assay was repeated. </p><p>In order to study the interaction between THC and cocaine, the following protocol was followed. At the beginning of tlae assay, norjnal dose-response curves to noradrenaline were obtained after which cocaine (10 ,q$n~l) was added to the perfi~sion liquid and the reactivity of the vas deferens to noradrenalins: was reevaluated until a constant response was obtained. Then cocaine (18 pg/~nl) and AxTHC (10-6M) were added together to the Krebs-Henseleit solution and the dose-.response curves were repeated. </p><p>Isolated lie rttrm Segments. 2-3 cm long, were suspended a t 30 + 0 . 5 </p><p>'C in a 10-ml bath of Tyrode solution gassed with a rnixturc of 95C;;, oxygen ar.d 5(% carbon dioxide. Acetyl- choline was added at constant intervals of 5 n ~ i n and left in contact with the smooth muscle for 60 s. The contrac- tions induced by the agonist were recorded on a smoked drum with a frontal-writing lever. Responses were first obtained to a series of five doses of acetylcholine, each sequence being repeated twice. At this point, ABTHC was added to the perfusion medium in a final concentra- tion of 10-7 or 10-6 M and kept in contin~ious contact with the preparation. Between each addition of THC a 30-n~in period was allowed for equilibrium. </p><p>The interaction between AsTHC and morphine or atropine was studied on the agonistic activity of anglo- tensin and acetylcholine. For this purpose normal dose- response curces for ailgiotensin were recorded as well as the contraction produced by acetylcholine (5 *, 10-8 g/ml). At this point, the influence of atropine and mor- phine was studied either alone or in combination with AsTHC </p><p>IsolcPted Phrc~nic Nerve - Dia[~hl.cPgrn Pref~aration The phrenic nerve-diaphragm preparation de- </p><p>scribed by Bulbring (1946) was used. The isolated prepara- </p><p>tion &amp;as placed in a 108-ml bath containing Tyrodz solution, with double glucose concentration, maintained at r o o n ~ tenaperature, and gassed with 9sC: O2 and 5(,'/&gt; ('OL. The rmerve was stimulated at ,a frequency of 6 cycles! min with n duration of 0 . 2 ms at a supramaximal voltage (3-6 V). Thc contractions of the ciiaphragm lnuscle were recorded on a Pliysiograph 1V with an isometric trans- ducer (model R). After a 60-min recovery period, 1WHC and physostign~ine bere studied either alone or in com- bination. </p><p>Results /sobcited G'uin~6r-Pig Vn.s D~f1.re~l.s </p><p>Neither A"- nor ATT HC have agonistic activity on the isolated guinea-pig vas defer- ens. However, these two agcnts have the property to modify the action of acetylcho- line and of noradrenaline. The res~ilts in Fig. I show the action of ATTHC on the acetylclaoline-induced contraction (upper pa- nel). At a concentration of 10 + kf: ATHHC has a biphasic action characterized by a transient poterntiation of the acetylcl~oline effect Followed by rhe inhibition of the ~nusculotrophic activity of acetylcholine. At a higher concentration, 10- -' iW, A9TTHC has only an inhibitory ef'fecz on the acetylcholine contraction. </p><p>The middle panel of Fig. 1 repsesenmts the influence of A'JTHC on the noradrenaline- induced contraction of the isolated guinea- pig vas deferens. In this case, the eflect ob- served is a potentiation that does not seem to be related to the concentration of A T H C studied. </p><p>In the lower panel (Fig. 4 ) we can observe that ATHC at concentrations of 10-Qnd l O - - 7 M does not modify the response of tlme vas deferens to bariuna chloride. If the con- centration of A9THC is further increased (10-5 M ) , an inhibition of barium chloride induced contraction is obtained. </p><p>Figs. 2 and 3 illustrate the dose-response curves to acetylcholine before and after the addition sf either A9- or ASTHC. The inhibi- tion obtained is dose-dependent and the only difference between the two cannabinol deri- vatives is that A9THC is more active than A8TH@. On the noradrenaline effect, A9THC (Fig. 4) is much more active than A8THC (Fig* 5). Oil the other hand, the potentiation of the noradrenaline response by A9THC is not dose-dependent. The same degree of potentiation is also observed with a con- centration of 10-6 M. </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>UN</p><p>IV O</p><p>F N</p><p>OR</p><p>TH</p><p> CA</p><p>RO</p><p>LIN</p><p>A A</p><p>T o</p><p>n 11</p><p>/13/</p><p>14Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>'1 hi cF ;&gt;!&gt;I oh1 6v 1DdJJO;y 5 1 5 Z I Z P Z Z I S E S S S T 2 S S 2 r F E t E E E E E E E E E E E E E E E E </p><p>LC m L3 - - </p><p>3 i') - o m ! ? </p><p>ro m - ornu7 U-J rn - ,',"OLn </p><p>u w m m r c O P Y I ~ L O q q u-I u-1 ,a q - r u l m ~ q u o q o m o u 'Q 'Q ' Q ' Q ' ~ 0 Q Q 'Q 'Q '0 'Q '0 Q ' y ' ~ ' y ' L 2 ' ~ X X X </p><p>,-. 3 ' Q Q'Q Q'Q'S'Q Q m m m </p><p>X X T d X X X X X X X I X X X </p><p>V ) ,.? V&gt; r&gt; ,F&gt; r&gt; m lc, m 0 F&gt; V ) m m lc, </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>UN</p><p>IV O</p><p>F N</p><p>OR</p><p>TH</p><p> CA</p><p>RO</p><p>LIN</p><p>A A</p><p>T o</p><p>n 11</p><p>/13/</p><p>14Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>Effect m m </p><p>.4 </p><p>ieq dose log dose </p><p>Fie,. 2. Inhibitton of tisc acctylchol~ne contlactisn FK,. 3. Inhibition of the acetylcholine contrnction by L V H C on the isolated guinea-pig vas deferens. by AXTBIIC' an the isolatcd guinea-pig vas defercns. </p><p>If the potentiation of acetylcholine action by AOTHC and AXTH6' is the consequence of' an anti-cholinesterase activity. then these active pr inciplesof marihuana \houfd be active on the isolated rat phrenic nerve - diaphragm preparation. On this isolated preparation. A9THC at concentration5 o f 10 and 10- ,I/a does not n~odify the muscle conlractiorm produced by nerve stirtiaealation (Fig. 6 upper tracing). On the other hand, physostigminc (an anti-cholinesterase agelit) has a biphahrc action characteri~ed by a rise followed by a decrease in muscle contraction (middle tracing). When AOTHC ( 10-6 !%if) was studied on the physostigniine effect a complete block of the first phase wa\ observed and the second phase had a more rapid onsct (Fig. 61 middle tracing). </p><p>i.rolcrtec/ Guincri- Pig 8lc~a/11 This experiment was perfornaed in order to </p><p>confirm the potentiating ef'I'ect of T H C on the acetyicl1~7Blne-incluced contraction. 'The rebultc csbserved show that A T H C at l0 </p><p>potentiates the action of acety8choBine on the guinea-pig ileum. Furtlaeranore this poten- t ia~ion i b followed by an inhibition (Fig. 6 lower tracing). When the concentration ot' ATHH is increased to 10 Ad, a potent inhibition is obtained. </p><p>'I'lrc potentiation of acetylcholine action by 'T'HC may also be due t o a modification in the releace of the neuroml:diator. Ian order to verify such a hypothesis we have cornpared the action of AsTHC to that s f n-morphine o n the musculotropic action c,f am~giotensin in the guinea-pig ileum, since part of the action of dngiotensin is attributable to the release (sf acetylcholine. The results in Fig. 7 show the influence of morphine (aspper t rac~ng) </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>UN</p><p>IV O</p><p>F N</p><p>OR</p><p>TH</p><p> CA</p><p>RO</p><p>LIN</p><p>A A</p><p>T o</p><p>n 11</p><p>/13/</p><p>14Fo</p><p>r pe</p><p>rson</p><p>al u</p><p>se o</p><p>nly.</p></li><li><p>log dose </p><p>Frc,. 4. Potcntiatisn of the aloradlrenalil~c coaltrac- tion by 19THC o n tkc isoIated guinea-pig vas deferens. </p><p>and AsTHC (middie tracing) on the contrac- tion induced by angiotensin and acetylcholinc. At a concentration of 18 ~argil (upper tracing) morphine potentiates the action of acetyl- choline and slightly antagonizes the effect of angiotensin. On the other hand, ATTH-IC ( 141- .MI potentiates both t21c ~nusculotrcspic activity sf acetylcholine and angiotensin. When A q H C and morphine are studied toget her, t he con~rac t io i~ produced by angio- tensin tends to go back to normal while the effect of acetylcholine reanains potentiated (Fig. 7 middle tracing). </p><p>The lower tracing of Fig. 7 slloavs the inter- action between A V H C (lo--: M ) and atro- pine (10 --' g;'l). CVhen atropine is added to </p><p>log dose </p><p>Frc;. 5. I'otentiation of the noradrenaline contrac- tion by A V H C on the isolated guinea-pig vas deferens. </p><p>interiaction &amp;%t't,t'~en A V H C ~'criatf C-'ocuinc an the. VUS &amp; ) I &lt; ~ P Y P I ~ s </p><p>Since AqTHC and ATTHC potentiate the action of ~ioradrenalline on the isolated vas deferens, we wanted to compare this poten- tiation to the one produced by cocalne. i n the firct part of the experiment (Isft tracing) normal dose- response curves to noradren- aline were recorded. When cocaine (10 girnl) was added to the perf~asion mixture there was a marked potentiation of the noradrenaline response (Fig. 8 center tracing). tVB~en A T T C is added simultaneously with cocaine, the potentiation induccd by t%sc latter cornpound is abolished (Fig. 8 right araci~ig). </p><p>111 order t o assess the possibility that the antaeonism belween the alka8oids and A" </p><p>the Tyrodc solution thcre is a complete aboii- THC:' might be due to silnple cllemiwl inter- tion of acetylcholine contraction as well as actions, thin-layer chromatography (T.L.C.)...</p></li></ul>