The Effect of Anandamide on Uterine Nitric Oxide Synthase Activity Depends on the Presence of the Blastocyst

Abstract

Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot−1 h−1) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies.

Novel role for lysophosphatidic acid in vascular remodeling at the maternal–fetal interface

“…In this sense, we observed that LPA increases the production of nitric oxide in the rat uterus as augments iNOS activity through the activation of LPA3 receptor (Beltrame et al 2013). Also, NOS activity is increased at the sites of embryo implantation and depends on the presence of the blastocyst (Sordelli et al 2011). Other authors reported that the regulation of nitric oxide tone and NOS expression is associated to vascularization defects leading to reduced implantation rates in the rat uterus (Ota et al 1999, Purcell et al 1999.…”

Lysophosphatidic acid‐triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro

“…Previous results from our own support these observations. We reported that NOS activity is higher in rat implantation sites and is differentially regulated by the blastocyst . In addition, we found that LPA stimulates specifically iNOS isoform in the rat uterus, and this pathway is involved in the increase of interleukin‐10, a vascular marker .…”

“…We observed that NOS activity is increased at the sites of embryo implantation, and this regulation depends on the presence of the blastocyst. 13 Deregulation of nitric oxide tone and NOS expression is related to reduced implantation rates due to vascularization defects in the rat uterus. 14,15 Previously, we found that LPA augments COX-2 and iNOS during implantation in the rat uterus by activating LPA 3 receptor, and these pathways are involved in LPA-stimulated interleukin-10 expression.…”

Anandamide Levels Fluctuate in the Bovine Oviduct during the Oestrous Cycle

“…N -arachidonoylethanolamide or anandamide (AEA) is an EC that activates the specific cannabinoid receptors 1 (CB1) and 2 (CB2) and also activates the transient receptor potential vanilloid type I (TRPV1) ion channel. Several studies have shown that AEA is widely distributed in the mammalian reproductive tissues and fluids (oviduct, oviductal fluid, uterus, testis, seminal plasma, spermatozoa) and is involved in many reproductive functions such as sperm motility and capacitation, ovulation, embryo implantation, and oviductal embryo transport [9–15]. …”