Characterization of the endocannabinoid system, CB(1) receptor signalling and desensitization in human myometrium

BACKGROUND AND PURPOSE

The endocannabinoid plays vital roles in several aspects of reproduction, including gametogenesis, fertilization and parturition. However, little is known regarding the presence or role of the endocannabinoid system in myometrial function. Here the presence of the endocannabinoid system and signalling properties of cannabinoid receptors were characterized.

EXPERIMENTAL APPROACH

Components of the endocannabinoid system were identified using qRT-PCR, immunohistochemical, immunoblotting and radioligand binding experiments. Cannabinoid receptor signalling pathways were characterized using standard MAPK and second messenger assays.

KEY RESULTS

Primary myometrium expresses the endocannabinoid synthesizing enzyme N-acyl-phosphatidyl ethanolamine-specific phospholipase D, endocannabinoid degrading enzyme fatty acid amide hydrolase and cannabinoid CB₁, but not CB₂ receptors or transient receptor potential vanilloid-type-1 channels. The CB₁ receptor ligand anandamide caused a Gα_i/o-dependent inhibition of adenylate cyclase reducing intracellular cAMP levels, and Gα_i/o, phosphoinositide-3-kinase, Src-kinase-dependent ERK activation. CB₁ receptor-generated signals declined following continual anandamide stimulation, possibly due to ligand metabolism since free anandamide concentrations declined during the experiment from 2.5 µM initially, to 500 nM after >30 min. However, identical loss of CB₁ receptor responsiveness occurred in the presence of the metabolically stable derivative methanandamide. Moreover, RNAi-mediated depletion of arrestin3 (a negative regulator of receptor signalling) prevented loss of CB₁ receptor activity, enhancing and prolonging ERK signals.

CONCLUSIONS AND IMPLICATIONS

The myometrium has the capacity to synthesize, respond to and degrade endocannabinoids. Furthermore, reduced CB₁ receptor responsiveness occurs as a consequence of receptor desensitization, not agonist depletion and we identify a key role for arrestin3 in this process.