Willets Jonathon M, Brighton Paul J, Mistry Rajendra, Morris Gavin E, Konje Justin C, Challiss R A John
Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom.
Mol Endocrinol. 2009 Aug;23(8):1272-80. doi: 10.1210/me.2009-0047. Epub 2009 May 7.
Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration (Ca(2+)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.
催产素在分娩过程中子宫收缩的进展、时间和调节方面发挥着重要作用，并且被广泛用作宫缩剂。我们研究了在人原发性子宫肌层平滑肌细胞和ULTR细胞系中调节催产素受体（OTR）信号传导的机制。催产素使总[³H]肌醇磷酸积累和细胞内Ca²⁺浓度（[Ca²⁺]i）均呈浓度依赖性增加；然而，在ULTR细胞系中的反应更大且更具重复性。对单细胞中磷脂酶C活性的评估显示，在存在催产素（100 nM）的情况下，OTR迅速脱敏（在5分钟内）。为了进一步表征OTR脱敏，用最大有效浓度的催产素（100 nM，30秒）刺激细胞，随后进行可变的洗脱期，然后再次相同应用催产素。这种对催产素的短暂暴露导致OTR对再次刺激的反应性显著降低（>70%），并且通过增加激动剂刺激之间的时间间隔可完全逆转。为了评估G蛋白偶联受体激酶（GRKs）在OTR脱敏中的作用，用小干扰RNA转染细胞，导致GRKs 2、3、5或6特异性敲低≥75%。在原发性子宫肌层细胞和ULTR细胞中，GRK6的敲低在很大程度上阻止了催产素诱导的OTR脱敏；相反，GRKs 2、3或5的选择性缺失没有效果。这些数据表明GRK6的募集是OTR反应性的主要效应器，并为子宫平滑肌中OTR信号传导可能的体内调节提供了机制性见解。
