Local uterine Ang-(1–7) infusion augments the expression of cannabinoid receptors and differentially alters endocannabinoid metabolizing enzymes in the decidualized uterus of pseudopregnant rats

Reproductive Biology and Endocrinology Local uterine Ang-(1-7) infusion augments the expression of cannabinoid receptors and differentially alters endocannabinoid metabolizing enzymes in the decidualized uterus of pseudopregnant rats K Bridget Brosnihan 0 Victor M Pulgar 0 1 2 3 Patricia E Gallagher 0 Liomar AA Neves 0 Liliya M Yamaleyeva 0 0 Hypertension and Vascular Research Center, Wake Forest School of Medicine , Winston-Salem, NC , USA 1 Department of Obstetrics & Gynecology, Wake Forest School of Medicine , Winston-Salem, NC , USA 2 Physiology and Pharmacology, Wake Forest School of Medicine , Winston-Salem, NC , USA 3 Biomedical Research Infrastructure Center, Winston Salem State University , Winston-Salem, NC , USA Background: Endocannabinoids (ECs) are important contributors to implantation and decidualization and are suppressed in early pregnancy. Elevated levels of anandamide (AEA), the endogenous ligand for the CB1 and CB2 receptors (R), interfere with receptivity of the blastocyst. Ang-(1-7) is down-regulated in the implantation site (IS) in normal pregnancy at day 7 of gestation. We determined the effects of intra-uterine angiotensin-(1-7) [Ang-(1-7)] (24 microg/kg/h) or vehicle given into the left uterine horn on the ECs in the decidualized uterus. Methods: Ovariectomized rats were sensitized for the decidual cell reaction by steroid treatment and decidualization was induced by a bolus of oil injected into the left horn; the right horn served as a control. Results: Decidualization increased endometrial permeability (3.1+/0.2 vs. 7.1+/0.5 uterus/muscle of cpm of (125) I-BSA, p < 0.0001). VEGF mRNA was increased by the decidualization (1.4-fold, p < 0.05) and by Ang-(1-7) (2.0-fold, p < 0.001). CB1R mRNA was reduced by decidualization (2.7-fold, p < 0.001), but increased by Ang-(1-7) (1.9-fold, p < 0.05). CB2R mRNA was increased by decidualization (4-fold, p < 0.05) and by Ang-(1-7) (2.4-fold, p < 0.001). The enzyme metabolizing AEA, fatty acid amide hydrolase (FAAH), was reduced by decidualization (7.8 fold, p < 0.001) and unchanged by Ang-(1-7) (p > 0.05), whereas the enzyme metabolizing 2-arachidonoylglycerol, monoacyl glycerol lipase (MAGL), was unchanged by decidualization (p > 0.05) and increased by Ang-(1-7) (1.7 fold, p < 0.001). Conclusions: These findings report for the first time that Ang-(1-7) augments the expression of CB1R, CB2R and MAGL in the decidualized uterus and thus may interfere with the early events of decidualization. Pregnancy; Implantation; Decidualization; Anandamide; 2-arachidonoylglycerol; Angiotensin-(1-7); Pseudopregnancy - Background The outcome of pregnancy depends on the success of implantation and placentation. In previous studies, we discovered that in early normotensive pregnancy the renin-angiotensin system (RAS) [Ang(17) and Ang II] was down-regulated in the uterus as compared to virgin animals and in the implantation site (IS) as compared to the adjacent interimplantation site (IIS) at day 7 of gestation [1]. The observation of reduced Ang-(17) and Ang II in the decidualized horn of the pseudopregnant rat further confirmed that a down-regulation of RAS is important for the early stages of decidualization [2]. In human placenta at the first trimester of aborted pregnancy, we demonstrated that Ang-(17) was increased [3], suggesting that an increase in Ang-(17) in the early uteroplacental unit is detrimental. Similar to the down-regulation of Ang II and Ang-(17) at early pregnancy, low levels of anandamide (AEA), an endogenous endocannabinoid, are associated with the IS as compared to the IIS and are required for the receptivity of the blastocysts attachment to the uterus [4]. High levels of AEA cause embryotoxicity, reduced trophoblast proliferation, and implantation failure [4-7]. The striking similarity of the pattern of distribution of the two systems (endocannabinoid system (ECS) and RAS) in early events of pregnancy and their required down-regulation during normal pregnancy makes a compelling argument to compare their regulation in the early events of pregnancy when the balance between the two systems is disrupted. Our hypothesis is that Ang-(17) exerts an important regulatory role on the endocannabinoid system in the decidualization process of early gestation. In order to uncover this role, Ang-(17) was infused locally into one decidualized uterine horn of a pseudopregnant rat and its effects on the expression of the endocannabinoid receptors, CB1R and CB2R, and the enzyme metabolizing AEA, fatty acid amide hydrolase (FAAH), and the enzyme metabolizing another endogenous endocannabinoid, 2arachidonoylglycerol (2-AG), monoacyl glycerol lipase (MAGL), were evaluated, together with other markers of decidualization. Methods Surgical procedures All procedures were approved by the Wake Forest School of Medicine Animal Care and Use Committee. Female SpragueDawley rats (n = 9-10/group) were obtained from Harlan Laboratories at 10 weeks of age and were ovariectomized under 2% isofluorane anesthesia. Five days after surgery animals were treated with a hormone regime [17-beta estradiol (0.1. 0.2, or 0.3 g) and progesterone (1 or 4 mg)] as illustrated in Figure 1 and as described [8]. On day 5 as indicated on Figure 1, animals were anesthetized with 2% isoflurane and 0.1 ml of sesame oil was injected into the left uterine horn; an osmotic minipump (model 2ML2, pumping rate of 5 L/hr) was placed in the left uterine horn for delivery of either 24 g/kg/h of Ang-(17) in sterile phosphate sodium buffer (PBS, pH 7.4) or vehicle. PE60 tubing attached to the minipump was inserted into the uterus lumen until it reached a plastic cuff placed 1 mm from the tip of the catheter. Sutures secured the catheter (before and after the cuff ) and either Ang-(17) or PBS was infused. The right horn was not injected or infused and served as a control. After five days of treatment, animals were euthanized by decapitation and trunk blood was collected in a cocktail of inhibitors as previously described [9]. The non-infused and infused uterine horns were removed, weighed, snap-frozen on dry ice for mRNA analysis, or fixed in 10% neutral buffered formalin solution for immunostaining. Plasma angiotensins Angiotensin (Ang) I, Ang II, and Ang-(17) peptides were measured in plasma by three radioimmunoassays as previously characterized [10,11]. Uterine permeability A separate group of animals similarly treated was prepared for permeability studies. On the morning of day 10 the animals weight was recorded. Animals were anesthetized with 2% isoflurane, and 810(6) cpm/250 g of (125)I-BSA was administered by intracardiac injection of 0.2 mL of (125)I-BSA. The animals were allowed to recover for 15 minutes and then euthanized by decapitation. The infused and non-infused uterine horn and the ventral gastrocnemius skeletal muscle were dissected free of any fat and weighed and the amount of radioactivity was determined. The uterine permeability index (...truncated)