Endogenous cannabinoid, anandamide, acts as a noncompetitive inhibitor on 5-HT₃ receptor-mediated responses in Xenopus oocytes

The cloned 5-HT₃ receptor from NCB-20 neuroblastoma cells was expressed in Xenopus oocytes and the effect of the endogenous cannabinoid ligand, anandamide, was investigated on the function of this receptor. The oocytes expressing the cloned 5-HT₃ receptors were voltage-clamped at - 70 mV. Anandamide, at the concentration range of 0.1-100 μM, reversibly inhibited 1 μM 5-HT induced currents. The inhibition of 5-HT induced currents by anandamide was concentration-dependent with an EC₅₀ of 3.7 μM and slope value of 0.94. This inhibitory effect was not dependent on the membrane potential and anandamide did not have an effect on the reversal potential of 5-HT-induced currents. In the presence of 10 μM anandamide, the maximum 5-HT-induced response was also inhibited and the respective EC₅₀ values were 3.4 μM and 3.1 μM in the absence and presence of anandamide, indicating that anandamide acts as a noncompetitive antagonist on 5-HT₃ receptors. CB₁ receptor antagonist SR-141716A (1 μM) and pertussis toxin (5 μg/ml) did not cause a significant change on the inhibition of 5-HT responses by anandamide. The effect of anandamide was not changed by preincubating the oocytes with 0.2 mM 8-Br-cAMP, a membrane-permeable analog of cAMP, or Sp-cAMPS (0.1 mM), a membrane-permeable protein kinase A activator. These results suggest that the effect of anandamide is independent of the activation of cAMP pathway and not mediated by the activation of PTX sensitive G-proteins. In conclusion, we demonstrated that the endogenous cannabinioid anandamide inhibits the function of 5-HT₃ receptors expressed in Xenopus oocytes in a cannabinoid-receptor independent and noncompetitive manner.