Paria B C, Deutsch D D, Dey S K
Department of Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City 66160-7338, USA.
Mol Reprod Dev. 1996 Oct;45(2):183-92. doi: 10.1002/(SICI)1098-2795(199610)45:2<183::AID-MRD11>3.0.CO;2-2.
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
花生四烯酸乙醇酰胺（阿南达胺）是大麻素受体的内源性配体。我们先前已证明，在植入前期，大麻素的配体 - 受体信号传导在小鼠胚胎和子宫中均起作用。在本研究中，我们提供证据表明小鼠子宫具有形成（合成酶）和水解（酰胺酶）阿南达胺的酶促能力。这些活性主要定位于子宫微粒体中，并且取决于pH、时间、蛋白质和底物浓度。阿南达胺的形成速率取决于花生四烯酸（Km：3.8微摩尔，Vmax：2.5纳摩尔/小时/毫克蛋白质）和乙醇胺（Km：1.2毫摩尔，Vmax：4.1纳摩尔/小时/毫克蛋白质）的浓度。以阿南达胺为底物时，酰胺酶活性显示出表观Km为67微摩尔，Vmax为3.5纳摩尔/分钟/毫克蛋白质。虽然合成酶在pH 9.0时显示出最大活性，但酰胺酶活性在pH 8.5时最大。如先前报道，苯甲基磺酰氟（PMSF）或花生四烯酰三氟甲基酮（ATK）以剂量依赖性方式抑制酰胺酶活性。相反，PMSF对合成酶活性没有抑制作用，反而在较低浓度下刺激合成酶活性。此外，ATK对合成酶活性的抑制作用仅适度，且其作用不依赖于浓度。为了确定子宫合成酶和/或酰胺酶活性在妊娠早期子宫接受性和植入方面是否具有任何生理意义，分析了在妊娠早期或假孕期间获得的小鼠子宫微粒体中的合成酶和酰胺酶活性谱。应当指出，胚胎同步发育到胚泡阶段以及子宫分化为接受状态对胚胎植入过程至关重要。在小鼠中，子宫仅在妊娠或假孕期间的有限时间段内对植入具有接受性。子宫在第4天（植入日）变得具有接受性，到第5天，它对胚泡植入变得不具有接受性（Paria等人，1993年：美国国家科学院院刊90：10159 - 10162）。在妊娠的第1 - 4天，阿南达胺合成酶和酰胺酶活性实际上均未改变。相比之下，与妊娠或假孕第4天（接受期）观察到的情况相比，在假孕第5天（非接受期）子宫中的合成酶活性增加，而酰胺酶活性降低。在妊娠第5 - 7天，手术分离的植入部位和植入间隙部位的合成酶和酰胺酶活性呈现出有趣的谱；与植入间隙部位相比，植入部位的合成酶活性较低。相反，与植入间隙部位相比，植入部位的酰胺酶活性较高。由于我们先前已表明包括阿南达胺在内的大麻素会干扰植入前小鼠胚胎发育，因此植入的胚泡对阿南达胺形成和水解的局部调节对于胚胎的成功生长、植入和妊娠建立可能至关重要。假孕第5天子宫中合成酶活性增加而酰胺酶活性降低的发现与这一假设一致，此时子宫对胚泡存活和植入具有敌意。此外，已知会干扰花生四烯酸代谢和胚胎植入的消炎痛在体内和体外均刺激子宫中的合成酶活性，同时抑制酰胺酶活性。最后，考虑到妊娠早期这两种酶促反应的动力学和谱，结果表明合成酶和酰胺酶可能是小鼠子宫中的两种不同酶。本研究构成了对雌性生殖系统中阿南达胺合成酶和酰胺酶活性的首次详细研究。
