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Endocannabinoids at the skeletal neuromuscular junction: rat phrenic nerve and hemidiaphragm
Anandamide and oleamide are endocannabinoids thought to act via cannabinoid (CB) receptors of which there are, at present, two subtypes, CB1 and CB2. CB1 are thought to be the only CB receptor found in the CNS, CB2 receptors are found on immune cells. CB receptors are thought to have a role in analgesia, memory formation and inflammation (Pertwee, 1997; 2001). This investigation looked at the action of anandamide and oleamide at the neuromuscular junction (NMJ). Phrenic nerve hemidiaphragms isolated from female Wistar rats (250–300g) after the method of Bulbring (1946) were placed under a resting tension of 2g in McEwen’s solution and continually aerated (5% CO2 vv-1, 95% O2 vv-1). Supramaximal voltages (typically 10V) were applied to the phrenic nerve at frequencies of 0.5Hz, 2Hz and 50Hz (for 1s). Isometric contractions were measured using a Grass Force Displacement Transducer (model FT03C) and recorded on a Grass Polygraph (model 79D). Results were statistically tested using a student t-test comparing results to the relevant controls. Increasing anandamide and oleamide concentrations (0.1-10 µM) produced a significant decrease in twitch tension by 30% (10µM, P<0.01, n=8) and 25% (10µM, P<0.01, n=6) respectively, compared to vehicle controls, with no effect on train of four (TOF) or tetanus. This inhibition was reduced to control levels with both anandamide and oleamide in the presence of AM251 (100nM; Tocris, UK) a specific CB1 inverse receptor agonist, also in the presence of SOD (1Uml-1), a superoxide scavenger and L -NAME ( L -NG-nitroarginine methyl ester; 100µM) an inhibitor of nitric oxide (NO) synthesis. These findings suggest that the response is mediated at least to some extent by a CB1 receptor in the NMJ and further suggests that CB1 receptors are specifically present on the postsynaptic membrane as it has been shown that twitch is a post synaptic event whereas TOF and tetanus are mediated presynaptically (Gibb & Marshall, 1986) There is evidence suggesting that anandamide activation of CB receptors induces NO (Salzet et al., 2000), and the reactive relationship between NO and superoxide could be the reason for the similar findings in these experiments. The inhibition of the anandamide induced decrease of twitch in the presence of a CB1 receptor antagonist suggests a possible receptor mediated response.
Bulbring, E. (1946). Br. J. Pharmacol. Chem., 1(1), 38–61. |
