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 cann abinoid receptors. We demonstrated previously that ligand-receptor sig naling with cannabinoids is operative in both the mouse embryo and ute rus 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 w ere 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 (K-m: 3.8 mu M and V- max: 2.5 nmol/h/mg protein) and ethanolamine (K-m: 1.2 mM and V-max: 4 .1 nmol/h/mg protein) concentrations. The amidase activity showed an a pparent K-m of 67 mu M and V-max of 3.5 nmol/min/mg protein with anand amide as a substrate. While the synthase showed maximal activity at pH 9.0, the amidase activity was maximal at pH 8.5. As reported previous ly, phenylmethylsulfonyl fluoride (PMSF) or arachidonyl trifluoromethy l ketone (ATK) inhibited the amidase activity in a dose-dependent mann er. In contrast, PMSF was not inhibitory to synthase activity, rather it stimulated synthase activity at lower concentrations. Further, inhi bitory effects of ATK were only modest toward the synthase activity an d the effects were not concentration-dependent. To determine whether u terine synthase and/or amidase activity have any physiological signifi cance with respect to uterine receptivity and implantation during earl y pregnancy, profiles of synthase and amidase activities were analyzed in mouse uterine microsomes obtained during early pregnancy or pseudo pregnancy. It should be noted that the synchronized development of the embryo to the blastocyst stage and differentiation of the uterus to t he receptive state are critical to the embryo implantation process. In the mouse, the uterus becomes receptive for implantation only for a l imited 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 Nat l Acad Sci USA 90:10159-10162.). Both anandamide synthase and amidase activities remained virtually unaltered on days 1-4 of pregnancy. In c ontrast, while the synthase activity increased, the amidase activity d ecreased in the uterus on day 5 of pseudopregnancy (nonreceptive phase ) as compared to those observed on day 4 of pregnancy or pseudopregnan cy (receptive phase). The synthase and amidase activities in surgicall y separated implantation and interimplantation sites showed an interes ting 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 compar ed with that in interimplantation sites. Since we have shown previousl y that cannabinoids including anandamide interfere with preimplantatio n mouse embryo development, the local modulation of anandamide formati on 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 t he uterus in hostile to blastocyst survival and implantation, is consi stent with this assumption. Further indomethacin, known to interfere w ith arachidonate metabolism and embryo implantation, stimulated the sy nthase activity, while inhibiting the amidase activity in the uterus i n vivo and in vitro. Finally, considering the kinetics and profiles of these two enzymatic reactions during early pregnancy, the re suits su ggest that synthase and amidase may be two separate enzymes in the mou se uterus. This investigation constitutes the first detailed studies o n anandamide synthase and amidase activities in the female reproductiv e tract. (C) 1996 Wiley-Liss, Inc.