Role of the endocannabinoid system in the control of mouse myometrium contractility during the menstrual cycle

Abstract

Cannabis and cannabinoids are known to affect female reproduction. However, the role of the endocannabinoid system in mouse uterine contractility in the dioestrus and oestrus phases has not been previously investigated. The present study aimed at filling this gap. Endocannabinoid (anandamide and 2-arachidonoylglycerol) levels were measured in mouse uterus at dioestrus and oestrus phases by liquid chromatography-mass spectrometry; quantitative reverse transcription-PCR and western blot were used to measured the expression of cannabinoid receptors and enzymes involved in the metabolism of endocannabinoids. Contractility was evaluated in vitro either on the spontaneous contractions or by stimulating the isolated uterus with exogenous spasmogens. The tissue concentrations of anandamide and 2-AG were reduced in the oestrus phase, compared to dioestrus. Uteri obtained in the dioestrus, but not oestrus, phase showed spontaneous phasic prostaglandin-mediated contractions that were reduced by ACEA (CB₁ receptor agonist) and to a lower extent by JWH133 (CB₂ receptor agonist). These inhibitory effects were counteracted by the corresponding selective antagonists. Neither ACEA nor JWH133 did affect the contractions induced by exogenous PGE₂ in the uterus from the oestrus phase. The FAAH inhibitor JNJ1661010 and, to a lower extent, the MAGL inhibitor JZL184 also reduced spontaneous contractions. It is concluded that the endocannabinoid system undergoes to adaptive changes between the oestrus and dioestrus phases. CB₁ and, to a lower extent, CB₂ receptor activation results in selective inhibition of myometrial contractility, without un-specific relaxing effects on the smooth muscle. These results might be of interest for female marijuana smokers as well as for the design of novel tocolytic agents.

Introduction

Marijuana, a preparation from the Cannabis sativa plant, continues to be the most frequently used illicit drug among women of childbearing age [1] and its use has been demonstrated to affect adversely reproduction [2]. Women smoking marijuana show impaired fertility, aberrant hormonal regulation, or impaired embryo implantation and development [3]. Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the main psychotropic component of marijuana, binds two G_i/o coupled membrane receptors, named CB₁ and CB₂ receptors, which are also activated by endogenous ligands (endocannabinoids). The main endocannabinoids are anandamide and 2-arachydonoylglycerol (2-AG), and are generated on demand rather than stored in cells. Endocannabinoids are biosynthesized from membrane phospholipids by the action of a number of enzymes including N-acyl-phosphatidylethanolamine-selective phospholipase D (NAPE-PLD, involved in anandamide biosynthesis) and diacylglycerol lipases α and β (DAGL α and β, involved in 2-AG biosynthesis), and are inactivated through a reuptake process (facilitated by a putative endocannabinoid membrane transporter), followed by enzymatic degradation. The latter process occurs predominantly through fatty acid amide hydrolase (FAAH, in the case of anandamide) and through the serine hydrolases monoacylglycerol lipase (MAGL), and α,β-hydrolase 6 (ABHD6) and 12 (in the case of 2-AG) [4].

The endocannabinoid system has been found in the female reproductive system of different species, from sea urchins to humans, thus suggesting its possible role in female reproduction [5]. Components of the endocannabinoid system have been identified in the rodent and human uterus and changes in anandamide synthesis and/or expression of cannabinoid receptors in this organ have been suggested to be responsible for early pregnancy failure or female infertility [6]. In addition, endogenous and exogenous cannabinoid receptor agonists, including THC and anandamide, exert a direct, CB₁-mediated relaxant effect on myometrial contractility in vitro [7]. Despite these reports, no conclusive data exist on a possible tonic action of the endocannabinoid system on the contractility of the myometrium. Specifically, the possible involvement of the endocannabinoid system in the oestrus phase (i.e. when the uterus is ready for implantation) and dioestrus phase (i.e. when ovulation normally occurs) is not known to date. In the present study, first we have measured endocannabinoid levels as well the mRNA and protein expression of cannabinoid receptors and enzymes involved in endocannabinoids biosynthesis and degradation in the uterus from mice in dioestrus and oestrus phases. Then, we have investigated the role of the endocannabinoid system in uterine smooth muscle contractility in both phases. For this latter purpose, we used the selective cannabinoid CB₁ receptor agonist, ACEA; the selective CB₂ receptor agonist, JWH133; the selective CB₁ receptor and CB₂ receptor antagonists, rimonabant and SR144528; the selective FAAH inhibitor, JNJ16610, the selective MAGL inhibitor, JZL184 and the ABDH6 inhibitor, WWWL70 [8].