The uterus is a potential site for anandamide synthesis and hydrolysis: differential profiles of anandamide synthase and hydrolase activities in the mouse uterus during the periimplantation period.

Arachidonoylethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors. We demonstrated previously that ligand-receptor signaling with cannabinoids is operative in both the mouse embryo and uterus during the periimplantation period. In the present investigation, we provide evidence that mouse uterus has the enzymatic capacities to form (synthase) and hydrolyze (amidase) anandamide. These activities were primarily localized in uterine microsomes and were dependent upon pH, time, protein, and substrate concentrations. The rate of formation of anandamide was dependent on arachidonic acid (Km: 3.8 microM and Vmax: 2.5 nmol/h/mg protein) and ethanolamine (Km:1.2 mM and Vmax:4.1 nmol/h/mg protein) concentrations. The amidase activity showed an apparent Km of 67 microM and Vmax of 3.5 nmol/min/mg protein with anandamide as a substrate. While the synthase showed maximal activity at pH 9.0, the amidase activity was maximal at pH 8.5. As reported previously, phenylmethylsulfonyl fluoride (PMSF) or arachidonyl trifluoromethyl ketone (ATK) inhibited the amidase activity in a dose-dependent manner. In contrast, PMSF was not inhibitory to synthase activity, rather it stimulated synthase activity at lower concentrations. Further, inhibitory effects of ATK were only modest toward the synthase activity and the effects were not concentration-dependent. To determine whether uterine synthase and/or amidase activity have any physiological significance with respect to uterine receptivity and implantation during early pregnancy, profiles of synthase and amidase activities were analyzed in mouse uterine microsomes obtained during early pregnancy or pseudopregnancy. It should be noted that the synchronized development of the embryo to the blastocyst stage and differentiation of the uterus to the receptive state are critical to the embryo implantation process. In the mouse, the uterus becomes receptive for implantation only for a limited period during pregnancy or pseudopregnancy. The uterus becomes receptive on day 4 (the day of implantation) and by day 5, it becomes nonreceptive for blastocyst implantation (Paria et al., 1993: Proc Natl Acad Sci USA 90:10159-10162.). Both anandamide synthase and amidase activities remained virtually unaltered on days 1-4 of pregnancy. In contrast, while the synthase activity increased, the amidase activity decreased in the uterus on day 5 of pseudopregnancy (nonreceptive phase) as compared to those observed on day 4 of pregnancy or pseudopregnancy (receptive phase). The synthase and amidase activities in surgically separated implantation and interimplantation sites showed an interesting profile on days 5-7 of pregnancy; the synthase activity was lower in implantation sites as compared to that in interimplantation sites. In contrast, amidase activity was higher in implantation sites compared with that in interimplantation sites. Since we have shown previously that cannabinoids including anandamide interfere with preimplantation mouse embryo development, the local modulation of anandamide formation and hydrolysis by the implanting blastocysts could be critical for successful embryonic growth, implantation, and pregnancy establishment. The finding of increased synthase activity with concomitant decrease in amidase activity in the uterus on day 5 of pseudopregnancy, when the uterus in hostile to blastocyst survival and implantation, is consistent with this assumption. Further indomethacin, known to interfere with arachidonate metabolism and embryo implantation, stimulated the synthase activity, while inhibiting the amidase activity in the uterus in vivo and in vitro. Finally, considering the kinetics and profiles of these two enzymatic reactions during early pregnancy, the results suggest that synthase and amidase may be two separate enzymes in the mouse uterus. This investigation constitutes the first detailed studies on anandamide synthase and amidase activities in the female reproductive t