Effects of Cannabinoids on Synaptic Transmission in the FrogNeuromuscular Junction

Enrique Sanchez-Pastor, Xochitl Trujillo, Miguel Huerta, and Felipa AndradeUnidad de Investigacion Enrico Stefani del Centro Universitario de Investigaciones Biomedicas, Universidad de Colima,Colima, Mexico

Received October 28, 2006; accepted January 29, 2007

ABSTRACTThis study aimed to investigate the function of the cannabinoidreceptor in the neuromuscular junction of the frog (Rana pipi-ens). Miniature end-plate potentials were recorded using theintracellular electrode recording technique in the cutaneouspectoris muscle in the presence of the cannabinoid agonistsWIN55212-2 (WIN; R-(�)-[2,3-dihydro-5-methyl-3-[(morpholinyl)]-pyrolol[1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone)and arachidonylcyclopropylamide [ACPA; N-(2-cyclopropyl)-5Z,8Z,11Z,147-eicosatetraenamide] and the cannabinoidantagonists 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) and6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630). Adding WIN to the exter-nal medium decreased the frequency and amplitude of theminiature end-plate potentials (MEPPs); the WIN EC50 valuewas 5.8 � 1.0 �M. Application of ACPA, a selective agonist ofcannabinoid receptor CB1, also decreased the frequency of the

MEPPs; the ACPA EC50 value was 115.5 � 6.5 nM. The CB2antagonist AM630 did not inhibit the effects of WIN, indicatingthat its action is not mediated through the CB2 receptor. How-ever, the CB1 antagonist AM281 inhibited the effects of WINand ACPA, suggesting that their actions are mediated throughthe CB1 receptor. Pretreatment with the pertussis toxin inhib-ited the effects of WIN and ACPA, suggesting that their effectsare mediated through Gi/o protein activation. The N-type Ca2�

channel blocker �-conotoxin GVIA (�-CgTX) diminished thefrequency of the MEPPs, with an �-CgTX EC50 value of 2.5 �0.40 �M. Blocking the N-type Ca2� channels with 5 �M�-CgTX before addition of ACPA to the bath had no additionalinhibitory effect on the MEPPs, whereas in the presence of 1�M �-CgTX, ACPA had an additional inhibition effect. Theseresults suggest that cannabinoids modulate transmitter releasein the end-plate of the frog neuromuscular junction by activat-ing CB1 cannabinoid receptors in the nerve ending.

Cannabinoids, the active components of marijuana (Can-nabis sativa) cause psychoactive and motor effects when theplant is consumed. These effects are produced by the inter-action of these compounds with the cannabinoid membranereceptors named CB1 and CB2 (Howlett et al., 2002). Bothsubtypes of cannabinoid receptors alter cellular activitythrough activation of G proteins of the Gi/o subtype, whichare sensitive to pertussis toxin (Soderstrom et al., 2000),although some effects are thought to occur through Gs

(Bayewitch et al., 1995; Glass and Felder, 1997). Activationof Gi/o proteins by CB1 and CB2 receptors decreases adenyl-ate cyclase activity, leading to decreased levels of intracellu-

lar cyclic AMP and protein phosphorylation. G protein acti-vation by both cannabinoid receptors has been implicated inthe modulation of the mitogen-activated protein kinase sig-naling pathway and Krox-24 (zenk) induction (Bouaboula etal., 1995). However, activation of CB1, but not CB2, is nega-tively coupled through Gi/o proteins to N-type and P/Q-typecalcium channels and is positively coupled to the inward-rectifying potassium channels (Mackie et al., 1995; Twitchelet al., 1997; Vasquez et al., 2003).

The endogenous cannabinoid system, represented by can-nabinoid receptors, emerged early in evolution, and conser-vation of the CB1 receptor seems to be restricted to verte-brate species. Therefore, in amphibians, only the CB1

receptor ortholog has been reported to date (Soderstrom etal., 2000; McPartland et al., 2006). The endocannabinoids(endogenous cannabis-like substances) are fatty acids de-rived from arachidonic acid, and they notably include anan-damide and 2-arachidonylglycerol. Once anandamide and

This work was supported, in part, by Grant Consejo Nacional de Ciencia yTecnologıa (CONACyT)-Mexico 43446-M (to M.H.) and by Grant FRABA-03(Ramon Alvarez Buylla-Mexico) (to M.H. and X.T.) E.S.-P. is in receipt of aCONACyT-Mexico fellowship.

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.106.116319.

ABBREVIATIONS: CB, cannabinoid; MEPP, miniature end-plate potential; �-CgTX, �-conotoxin GVIA; WIN, WIN55,212-2, R-(�)-[2,3-dihydro-5-methyl-3-[(morpholinyl)]pyrolol[1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone; ACPA, arachidonylcyclopropylamide [N-(2-cyclopro-pyl)-5Z,8Z,11Z,147-eicosatetraenamide]; AM281, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxam-ide; AM630, 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone; PTX, pertussis toxin; ACh, acetylcholine.

0022-3565/07/3212-439–445$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 321, No. 2Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 116319/3195792JPET 321:439–445, 2007 Printed in U.S.A.

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2-arachidonoylglycerol are formed, in some cases they targetthe CB1 receptors in the same cell where they are formed, viadiffusion within the plasmalemma, or they can be releasedfrom the extracellular fluid, where they reach presynapticterminals (Piomelli, 2003; Rodriguez de Fonseca et al., 2005).

In the past three decades, the actions of cannabinoids havebeen shown to be related to the inhibition of transmitterrelease in different synapses of the central nervous systemand in smooth muscle (Ishac et al., 1996; Engler et al., 2006).The mechanism by which cannabinoids affect transmitterrelease is thought to work through the activation of potas-sium channels (IA) or by blocking voltage-dependent Ca2�

channels or both (Elphick and Egertova, 2001). The inhibi-tion of N-type calcium channels decreases neurotransmitterrelease in several tissues (Liang et al., 2004; Szabo et al.,2004). With �9-tetrahydrocannabinol present in the neuro-muscular junction, Kumbaraci and Nastuk (1980) reported adiminution in the frequency and an increase in the amplitudeof the MEPPs, whereas Turkanis and Karler (1986) reportedcontradictory data—an increase in the frequency of theMEPPs and a reduction in their amplitude. These studieswere performed before the cannabinoid receptors were iden-tified and before the development of specific agonists andantagonists for each type of cannabinoid receptor.

In contrast, although cannabinoids reduce motor activityat the central level in humans and other mammals, fewstudies exist about the effects of cannabinoids on skeletalmuscle. Therefore, we explored whether part of the effects ofcannabinoids (e.g., reduced motor activity; Dewey 1986; Sa-nudo-Pena et al., 2000) occurs directly at the level of theskeletal muscle. The aim of this study was to investigate thefunction of cannabinoid receptors in the neuromuscular junc-tion of the frog (Rana pipiens) and the effects of cannabinoidson synaptic transmission.

Materials and MethodsThe experiments were performed on the neuromuscular junction

of the cutaneous pectoris muscle of R. pipiens. Frogs were used inaccordance with the Institute for Laboratory Animal Research Guidefor the Care and Use of Laboratory Animals. Frogs were sacrificed bydecapitation and demedulated. The nerve and muscle were dis-sected, pinned on a Sylgard-coated dish (WPI, Sarasota, FL), andbathed in normal frog Ringer’s solution consisting of 117 mM NaCl,2.5 mM KCl, 1.8 mM CaCl2, and 2 mM imidazole-Cl, pH 7.4.

Electrophysiology. We used conventional methods for single-electrode intracellular recording in skeletal muscle. Glass microelec-trodes (A-M Systems, Inc., Sequim, WA) filled with 3 M KCl gave 10-to 20-M� resistance. Stock solutions for each drug (see “Drug Prep-arations”) were added to the bath solution by a three-way changer,and the solution was allowed to achieve the specific concentration bydiffusion. The resting membrane potential was monitored continu-ously, and an experiment was rejected when more than a 10-mVchange in membrane potential remained after the drug administra-tion. The frog cutaneous pectoris muscles were always bathed by thenormal solution. All recordings of MEPPs were performed at roomtemperature (22–24°C). Data were acquired through an Axoclamp2B (Axon Instruments, Foster City, CA) and digitized with a Digi-data 1322A (Axon Instruments) using Clampex, a subroutine ofpClamp 9.0 software (Axon Instruments), in 70-s periods. MEPPswere identified and analyzed by the Clampfit subroutine of pClamp9.0. This program summarizes the amplitudes of MEPPs and a meanis obtained. In addition, we obtained normalized cumulative ampli-tude histograms, which show the differences more clearly, to com-

pare the event amplitude distributions (Clements and Bekkers,1997).

To quantify the effect of each drug, MEPPs were recorded contin-uously from the same neuromuscular junctions every 5 min duringthe 15 min before the application of each drug (control conditions)and for 30 min in the presence of each drug, similar to protocolsestablished in previous investigations (Turkanis and Karler, 1986).After 30 min, drugs were removed from the bathing solution, exceptfor nifedipine and �-CgTX, unless otherwise specified.

Drug Preparations. Each of the cannabinoids—WIN, ACPA,AM630, and AM281 (Tocris Cookson Inc., Ellisville, MO)—was pre-pared as a 10 mM stock solution in dimethyl sulfoxide or ethanol(diluent concentration was �0.01%; Sigma-Aldrich, St. Louis, MO).Stock solutions were stored at �20°C. The L-type Ca2� channelblocker nifedipine (Sigma-Aldrich) was dissolved in ethanol to makea 100 mM stock solution. The N-type Ca2� channel blocker �-CgTX(Sigma-Aldrich) was prepared as a 0.5-mg/ml stock solution in water.Experiments using ACPA and nifedipine were performed in thedark.

Pretreatment with PTX. PTX (Sigma-Aldrich) was reconsti-tuted as a 50 �g/ml stock solution with 5 mg/ml bovine serumalbumin (Sigma-Aldrich) in water. The muscle was incubated with 2�g/ml PTX in normal frog Ringer’s for 22 to 24 h at 4°C (Sugiura andKo, 1997).

Statistical Methods. Differences between the means of the fre-quencies were evaluated using a t test; P � 0.05 was consideredsignificant. To evaluate the differences in the amplitude of theMEPPs, the Kolmogorov-Smirnov test for cumulative probabilitycurves was used; P � 0.05 was considered significant (Liang et al.,2004; Szabo et al., 2004). The dose-effect graphs were elaboratedusing Origin 6.0 (OriginLab Corp., Northampton, MA), and the datawere adjusted to a Hill equation of the type I � Imax/(1 � (EC50/x)h).

ResultsEffects of WIN on Spontaneous Transmitter Release

in the Frog Neuromuscular Junction. To examinewhether cannabinoids modulate transmitter release in theneuromuscular junction of the frog cutaneous pectoris mus-cle, we first used the cannabinoid agonist WIN, which bindsto both types of cannabinoid receptors. Adding this agonist(0.1–50 �M) to the external media decreased both the fre-quency and amplitude of the MEPPs compared with controlconditions. Figure 1 shows that adding 10 �M WIN to thebath reduced the amplitude of the MEPPs by 10.6 � 2.9%and reduced their frequency by 25.3 � 3.2% in all five cellstested (Fig. 1, A and B). Maximal inhibition by WIN wasreached 25 to 30 min after its application (Fig. 1), and thiseffect was sustained for at least 15 min after washout (datanot shown).

To test whether the effects of WIN involve G protein-mediated inhibition of transmitter release at the neuromus-cular junction, we used PTX, which inhibits certain types ofG proteins (Gi and Go) (Reisine and Law, 1992). We incu-bated cells overnight in PTX to allow internalization of PTXand inactivation of G proteins (Reisine and Law, 1992). Fig-ure 1 (A and B, compare the first versus the second columns)shows that 2 �g/ml PTX pretreatment for 22 to 24 h pre-vented inhibition of the MEPPs by WIN. However, the aver-aged amplitude and frequency of the MEPPs did not differsignificantly before and after WIN treatment. These resultssuggest that inactivation of PTX-sensitive G proteins pre-vents WIN from inhibiting MEPPs.

We next tested whether the receptor antagonists of CB1 orCB2 could reverse the effect of WIN. Adding the CB1 receptor

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antagonist AM281 at 1 �M reversed the inhibitory effect ofWIN on the MEPPs (Fig. 1, A and B, fourth column), sug-gesting that the effect of WIN occurs through the activationof CB1 receptors. When superfused alone for 30 min, 1 �MAM281 did not change the frequency or amplitude of theMEPPs (data not shown). Adding the CB2 receptor antago-nist AM630 at 1 �M to the bath 5 min before the addition ofWIN produced a similar effect to that observed in the absenceof AM630 (Fig. 1, A and B, fifth column), suggesting that theeffect of WIN is not mediated by CB2 receptors.

Figure 2 shows the dose-response curve for the inhibitoryeffect of WIN over the concentration range from 0.1 to 50 �M.The threshold concentration for the effect of WIN on the

frequency of the MEPPs was 1 �M, and the maximal effectwas reached with 50 �M WIN. Each point in the curve (Fig.2) corresponds to the frequency averaged from five experi-ments. Data were adjusted to a Hill equation, and the EC50

value for the inhibition of the MEPPs frequency by WIN was5.8 � 1.0 �M. The Imax value calculated by the Hill equationwas 52.68 � 3.26%, and the Hill coefficient was 0.96 � 0.11.

Inhibitory Effects of ACPA on Spontaneous Trans-mitter Release. We next examined whether the syntheticCB1-selective agonist ACPA, which is analogous to the endo-cannabinoid anandamide (Hillard et al., 1999), has similareffects to WIN on the frequency and amplitude of the MEPPs.Figure 3 shows the dose-response curve obtained usingACPA over a concentration range of 0.01 to 50 �M. ACPAproduced a pattern similar to that induced by WIN; eachpoint in the curve corresponds to the frequency averagedfrom five experiments. Data were adjusted by the Hill equa-

Fig. 1. A, effect of 10 �M WIN on the amplitude of the MEPPs. WINattenuated MEPP amplitude by 10.6 � 2.9% compared with controlconditions (compare first versus. second columns). Pretreatment with 2�g/ml PTX for 22 to 24 h and 1 �M CB1 antagonist AM281 reversed theeffect of WIN (third column). The CB2 antagonist AM630 at 1 �M had noadditional effect on the inhibition by WIN on MEPPs (see fifth column).B, effect of 10 �M WIN on the frequency of MEPPs. WIN decreased MEPPfrequency compared with control conditions (first versus second col-umns). Pretreatment with PTX and the CB1 antagonist AM281 at 1 �Mreversed the effect of WIN (third and fourth columns). The CB2 antago-nist AM630 at 1 �M had no effect on the inhibition of MEPPs by WIN(fifth column). Data are presented as mean � S.E.M. Asterisks indicateP � 0.05 compared with the control condition.

Fig. 2. Dose-response curve for the synthetic cannabinoid WIN. The EC50for the inhibition of MEPP frequency by WIN was 5.8 � 1.0 �M.

Fig. 3. Dose-response curve for the selective agonist of CB1 receptorACPA. The EC50 for inhibition of MEPP frequency by ACPA was 115.5 �6.5 nM.

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tion, and the EC50 value for the inhibition by ACPA of thefrequency of the MEPPs was 115.5 � 6.5 nM. The Imax valuecalculated by the Hill equation was 67.94 � 0.78%, and theHill coefficient was 1.40 � 0.10.

Adding 1 �M ACPA to the bath diminished both the meanfrequency and amplitude of the MEPPs compared with con-trol conditions (Fig. 4, A and B). Maximal inhibition wasreached 30 min after its application, when the mean inhibi-tion of MEPP frequency was 63.0 � 2.9%. ACPA decreasedmean MEPP amplitude 35.2% � 10.1%. Figure 4 shows thatACPA significantly decreased the cumulative amplitude dis-tribution compared with the control condition (Fig. 4B).

To test whether the effects of ACPA are mediated throughGi/o proteins (i.e., similar to the action of WIN), we incubatedthe cutaneous pectoris muscles with 2 �g/ml PTX for 22 to24 h. ACPA at 1 �M had no effect on the frequency oramplitude of the MEPPs. The mean frequency of MEPPsafter 30-min exposure to the presence of ACPA was 100.8 �3.6% compared with the control (n � 4). The cumulativeprobability plots indicated no significant difference in MEPPamplitude (P � 0.92, Kolmogorov-Smirnov test; data notshown). These data suggest that ACPA acts through a mech-anism that involves Gi/o proteins.

Adding the CB1 receptor antagonist AM281 at 1 �M 5 min

before adding ACPA did not reduce MEPP frequency or am-plitude. Mean MEPP frequency was 95.9 � 4.1%, and meanMEPP amplitude was 95.2 � 3.3%; these results were notstatistically significant. Likewise, the cumulative amplitudedid not differ significantly from control after 30 min in thepresence of ACPA. These results suggest that ACPA acti-vates pre- and postsynaptic CB1 receptors and that this ac-tivation decreases the transmitter release and/or probablyacetylcholine receptor sensitivity.

The N-Type Ca2� Channel Blocker �-CgTX Inhibitsthe Miniature End-Plate Potentials. To explore whetherCa2� channels are involved in spontaneous transmitter re-lease in the frog neuromuscular junction, we blocked the L-and N-type Ca2� channels, both of which are found in theneuromuscular junction of the frog (Meir et al., 1999). After30 min in the presence of 10 �M nifedipine, mean MEPPfrequency was 103.7 � 5.8%, and mean MEPP amplitude was98.6 � 5.1%. These data indicate that L-type Ca2� channelsdo not participate in spontaneous transmitter release andthat they do not affect acetylcholine receptors in the postsyn-aptic membrane. In contrast, Fig. 5A, shows the dose-re-sponse curve for the inhibitory effect of �-CgTX, which blocksN-type Ca2� channels, on the frequencies of the MEPPs, overthe concentration range from 0.1 to 10 �M. Each point in thecurve, corresponds to the frequencies averaged from fourexperiments. The threshold concentration for the effect of

Fig. 4. A, effects of the CB1 agonist ACPA at 1 �M on MEPP frequency.ACPA decreased MEPP frequency. Data are presented as means �S.E.M. Asterisks indicate P � 0.05 compared with the control condition(before ACPA administration) (paired Student’s t test). B, cumulativeprobability plots of the MEPPs before (continuous line) and during(dashed line) application of 1 �M ACPA is shown. ACPA attenuatedMEPP amplitude by 35.2 � 10.1% 30 min after application (n � 5; P �0.001, Kolmogorov-Smirnov test). The inset shows original MEPPs re-corded at time 0 and 30 min after the addition of ACPA.

Fig. 5. A, dose-response curve for the N-type Ca2� channel blocker�-CgTX. The EC50 value for the inhibition of MEPPs frequency by�-CgTX was 2.5 � 0.48 �M. B, cumulative probability plots of the MEPPsbefore and during application of 5 �M �-CgTX. �-CgTX caused no signif-icant changes in MEPP amplitude (dashed line) (Kolmogorov-Smirnovtest). The inset shows original MEPPs recorded at time 0 and 30 min afterthe addition of �-CgTX.

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�-CgTX on the frequencies of the MEPPs was 0.1 �M, andthe mean of the maximum effect was reached with 10 �M�-CgTX. Data were adjusted to the Hill equation, and theEC50 value for the inhibition of the MEPPs frequency with�-CgTX was 2.5 � 0.48 �M. The Imax value calculated by theHill equation was 102 � 4.72%, and the Hill coefficient was1.02 � 0.16. These results indicate that the Ca2� currentthrough the N-type Ca2� channels is involved in spontaneoustransmitter release in our preparation. Alternatively,�-CgTX had no significant effect on the cumulative ampli-tude, Fig. 5B shows an example when we used 5 �M �-CgTX.

N-Type Ca2� Channels Are Involved in the ACPA-Induced Synaptic Inhibition. To determine whetherACPA decreases transmitter release by a mechanism involv-ing inhibition of the presynaptic N-type Ca2� channels, weexplored the effects of adding 1 �M ACPA, which reducedMEPPs frequency by 63.1 � 2.9%, after blocking the N-typeCa2� channels with �-CgTX with maximal subthreshold con-centrations of 1 and 5 �M. Thirty minutes after the additionof �-CgTX, we recorded MEPPs for 15 min, and then weadded the ACPA to the bath and recorded MEPPs for anadditional 30 min. With the N-type Ca2� channels blocked (5�M �-CgTX), which reduced MEPPs frequency by 67.9 �5.2%, ACPA did not additionally decrease MEPP frequency(99.6 � 2.05%). However, MEPP amplitude was still dimin-ished (25.4 � 3.5%) by ACPA (Fig. 6, B and C), suggesting apostsynaptic site of action for this cannabinoid agonist. Whenwe used 1 �M �-CgTX, which reduced MEPPs frequency by29.6 � 1.0%, an additional decrease in the MEPPs frequencyby 60% in the presence of 1 �M ACPA was observed (Fig. 6A).These results suggest that ACPA decreased spontaneoustransmitter release by blocking the presynaptic N-type Ca2�

channels. However, we do not discard that ACPA has apresynaptic inhibition of the amount of acetylcholine (ACh)released.

DiscussionThe present results show that the cannabinoid WIN over a

concentration range of 1 to 50 �M (the WIN EC50 value was5.8 � 1.0 �M), decreased neurotransmitter release in a con-centration-dependent manner at the neuromuscular junctionof the frog cutaneous pectoris muscle by decreasing the fre-quency of the MEPPs. Activated cannabinoid receptors inter-act with Gi/o proteins, which can be blocked by PTX (Bokochet al., 1983). Pretreating the muscles with 2 �g/ml PTXbefore adding WIN caused WIN to have no significant effecton the MEPPs, indicating that WIN acts through Gi/o protein-coupled receptors. In contrast, because WIN has similar af-finity for CB1 and CB2 receptors (Howlett et al., 2002), todetermine which of these receptors are activated by WIN, weused selective antagonists for each type of cannabinoid re-ceptor. In the presence of the CB2 antagonist 1 �M AM630(Ross et al., 1999), WIN had a similar effect to that observedin the absence of AM630, indicating that the CB2 receptorsare not involved in the effects observed after addition of WIN.However, the presence of the CB1-selective antagonistAM281 at 1 �M (Howlett et al., 2002) blocked the effects ofWIN on the frequency and amplitude of the MEPPs, indicat-ing that WIN acts through activation of the CB1 receptors.These results are accord with only the CB1 receptor ortholog

having reported in amphibians (Soderstrom et al., 2000; Mc-Partland et al., 2006).

To confirm that cannabinoids inhibit neurotransmitter re-lease in the frog neuromuscular junction by their interactionwith CB1 receptors, we also used the synthetic CB1-selectiveagonist ACPA (the ACPA EC50 value was 115.5 � 6.5 nM),analogous to the endocannabinoid anandamide (Hillard etal., 1999), which diminished both the amplitude and fre-quency of the MEPPs in a concentration-dependent manner.WIN and ACPA reached the peak effect within 15 min. Sim-ilar results have been reported (Turkanis and Karler, 1986).However, more time-effects studies are necessary to clarifythis mechanism. These effects were more pronounced than

Fig. 6. A, blocking the N-type Ca2� channels with 1 �M �-CgTX for 45min before adding 1 �M ACPA did not inhibit the additional effect ofACPA on MEPPs frequency. B, blocking the N-type Ca2� channels with 5�M �-CgTX for 45 min before adding 1 �M ACPA did not inhibit the effectof ACPA on MEPP amplitude. In the presence of 1 �M ACPA, MEPPamplitude was reduced by 25.4 � 3.5% after 30 min (P � 0.002; n � 4).C, cumulative probability plots of the MEPPs before and during applica-tion of 5 �M �-CgTX. �-CgTX caused not significant change in MEPPamplitude (dashed line) (P � 0.05, Kolmogorov-Smirnov test). The insetshows original MEPPs recorded at time 0 and 30 min after the additionof ACPA, with the N-type Ca2� channels blocked by �-CgTX.

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those observed in the presence of WIN, confirming thatACPA is a more potent and efficient cannabinoid receptoragonist than WIN. A possible cause of the lower potency ofWIN versus ACPA could be that the amphibian CB1 receptorlacks some amino acid residues necessary for the binding ofWIN and for its efficacious functional coupling of the receptorto the G protein. Furthermore, it has been suggested thatWIN binds to an uncharacterized non-CB1 cannabinoid re-ceptor (Breivogel et al., 2001).

Moreover, it has been reported that the different efficaciesand potencies for anandamide (ACPA is analogous to anan-damide) and WIN, depend on sensitivity to differences inreceptor density, cell background, and/or receptor/G proteinstoichiometry (Huang et al., 2005). The ACPA binding site isat residue 192 and the WIN binding site will be next to thissite, it would be in the third transmembrane domain of CB1

(Song and Bonner, 1996). With respect to the WIN concen-tration used in this study, similar concentrations (10 �M)have been used in studies of synaptic transmission in thecerebellar cortex of rats (Szabo et al., 2004), and an IC50

value of 4.6 �M has been used in studies of glutamatergictransmission in rat brain stems (Liang et al., 2004).

The same results were obtained in the experiments withWIN; the treatment of cutaneous pectoris muscles with PTXto block Gi/o proteins inhibited the effects of ACPA on MEPPfrequency and amplitude, indicating that ACPA acts by ac-tivating G protein-coupled receptors. However, addingAM281 before adding ACPA blocked the agonist effects ofACPA, suggesting the functional presence of CB1 cannabi-noid receptors in the neuromuscular junction of the frog.These data indicate that ACPA diminish spontaneous trans-mitter release in the frog skeletal muscle neuromuscularjunction by activating presynaptic CB1 cannabinoid recep-tors. A previous study has reported the presynaptic locationof cannabinoid receptors (Schlicker and Kathmann, 2001).This diminution in transmitter release could generate, as aconsequence, a subliminal plate potential as was reported byKumbaraci and Nastuk (1980) using 0.3 to 30 �M �9-tetra-hydrocannabinol. In this way, action potential cannot begenerated.

Furthermore, it is known that the Ca2� entry throughvoltage-dependent Ca2� channels is essential for transmitterrelease at the presynaptic end. To address this issue, we haveused blockers for L- and N-type Ca2� channels, which arepresent in the frog motor end nerve (Kerr and Yoshikami,1984; Robitaille et al., 1993). The L-type Ca2� channelblocker nifedipine at 10 �M had no effect on the frequency oramplitude of the MEPPs, indicating that this type of Ca2�

channel does not participate in the entry of Ca2� into thepresynaptic cell, which is needed for spontaneous transmit-ter release. However, the N-type Ca2� channel blocker�-CgTX caused a rapid diminution of MEPP frequency in aconcentration-dependent manner, suggesting that Ca2� en-try occurs through this type of Ca2� channel and that thesechannels are involved in spontaneous transmitter release inthe frog neuromuscular junction. These results are in agree-ment with previous data reported by Grinnell and Pawson(1989). The amplitude of MEPPs was not affected by �-CgTXat all concentrations, and although the frequency decreasedby blocking the N-type of Ca2� channels with �-CgTX at 5�M before addition of ACPA, it had no additional inhibitioneffect on the frequency of the MEPPs. However, MEPPs

amplitude was diminished, whereas, in the presence of 1 �M�-CgTX, ACPA had an additional inhibition effect.

These data suggest that ACPA and WIN decreased trans-mitter release by blocking the presynaptic N-type Ca2� chan-nels. The effect of ACPA on MEPP amplitude is a postsyn-aptic effect, suggesting the presence of postsynaptic CB1

receptors. We have reported the presence of mRNA for CB1 infrog skeletal muscle fibers (Sanchez-Pastor et al., 2004). Can-nabinoid receptors are frequently located presynaptically,but they also occur postsynaptically, e.g., in the hippocampus(Schweitzer, 2000). Another possibility is that ACPA actspresynaptically, inhibiting the ACh release by diminishingthe quantal content of vesicles; therefore, the amplitude ofMEPPs should be diminished. However, reduction in theamplitude occurs within a few minutes, and motor end nervewas not electrically stimulated. Therefore, the turnover ofthe neurotransmitter is minimal; thus, the presynaptic effectof the drug on the neurotransmitter synthesis is unlikely(Hubbard et al., 1969).

To explain these postsynaptic effects, one possibility is thatcannabinoids might act in form similarly to how 5-hydroxy-tryptamine acts on the nicotinic ACh response diminution inthe currents (mediated by acetylcholine receptors) via amechanism involving one or more G proteins (Butt and Pit-man, 2002). Recently, the direct action of anandamide, butnot WIN, on neuron nicotinic ACh receptors was reported inXenopus oocytes expressing �7-ACh receptors (Oz, 2006).However, Nojima et al. (2000), using arachidonic acid andprostaglandin D2, cooperatively accelerated desensitizationof the nicotinic ACh receptor channel in mouse skeletal mus-cle. Those effects were inhibited by staurosporine, an inhib-itor of the protein kinase C. In this study, the experimentswere performed on intact fast muscle fibers, which permitmaintaining all cannabinoid signaling systems in the intra-cellular media.

Another possibility is that the cannabinoids modified themembrane input resistance and membrane time-constant atthe junctional region. However, more work is needed to elu-cidate the mechanism responsible for the effects of the can-nabinoid agonists on the amplitude of the MEPPs. Finally,the functional significance of the cannabinoid receptors inmuscle could be the modulation of tension.

Acknowledgments

We especially thank Dr. Clemente Vasquez for helpful suggestionand discussion of the data. In addition, we thank to Ezequiel Vierafor technical assistance.

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Address correspondence to: Dr. Miguel Huerta, Universidad de Colima,Centro Universitario de Investigaciones Biomedicas, Av. 25 de Julio #965,Colonia Villa San Sebastian, Apartado Postal 11, C.P. 28000-Colima, Colima,Mexico. E-mail: huertam@cgic.ucol.mx

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