CRH及内源性H_2S对临产前后子宫肌收缩性的不同调节作用
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摘要
早产发生率可达9-11%,不仅严重危害母婴健康,更是围产期新生儿死亡的首要原因,早产儿死亡率15%。然而,虽然全世界的医学家和生物学家几十年来不懈地努力,早产的发生率却并未得到有效的控制,其根本原因在于人类分娩启动的机理尚未明晰。
在正常情况下,妊娠足月之前,子宫都是处于一种相对静息的状态,其兴奋性低,不发生大的收缩。只有在临近分娩时才发生高频率、高强度的规律宫缩。因此,子宫由静息态向收缩态的转化无疑是启动分娩的关键。但迄今为止对于子宫静息状态终止进而转为收缩激活的机制,尚未阐明。目前的研究表明,妊娠子宫肌静息状态维持和收缩的发生受到了一系列内源性活性因子的调控,而且妊娠期间,这些因子的产生量及子宫对它们的反应性是随着妊娠的进行而发生变化的。目前认为,正是由于上述这种动态变化,使子宫在足月前处于静息状态,在妊娠末期出现收缩。因此全面地了解究竟有哪些内源性子宫舒缩性调控因子,深入地研究这些因子在妊娠不同阶段对子宫的作用以及作用机制,是发现妊娠子宫静息终止转为收缩激活分子的关键。
妊娠期间人类胎盘组织合成和分泌的促肾上腺皮质激素释放激素(corticotropin-releasinghormone,CRH)在人类分娩启动中发挥重要作用,被称为控制分娩的“生物钟”。人类子宫肌可以表达CRH受体,提示人类子宫肌是CRH作用的重要靶器官。因此在本课题的第一部分进行了以下四个方面的研究:1.观察CRH对临产和未临产子宫肌收缩性的不同调节作用;2.介导CRH对临产和未临产子宫肌不同调节作用的受体亚型;3.CRHR1介导CRH对临产和未临产子宫肌收缩性不同调节作用的细胞内信号转导通路;4.引起临产和未临产子宫肌中CRHR1偶联的G蛋白亚型发生改变的可能机制。
硫化氢是继NO和CO之后发现的第三种具有生物活性的气体信号分子,可舒张多种平滑肌组织,但是H_2S对于子宫平滑肌作用的研究还相对较少,仅有两篇报道表明外源性的H_2S能抑制子宫平滑肌自发性收缩,而在人体中存在内源性H_2S,并且本课题前期研究表明人类妊娠子宫肌产生内源性H_2S。那么内源性H_2S是否能够舒张人类妊娠子宫肌?本课题的第二部分我们用产生内源性H_2S的供体L-半胱氨酸等处理人类离体子宫肌,观察内源性H_2S对人类子宫肌收缩性的调节,并对其作用机制进行系统性研究,进一步比较内源性H_2S对临产前后子宫肌收缩性的调节是否不同。
在子宫肌由静息态向收缩态转变的过程中,子宫平滑肌上多种收缩相关蛋白的表达发生变化。本课题组前期的研究已经发现在临产后ATP激活的K~+通道(K_(ATP)),大电导Ca~(2+)激活的K~+通道(BK_(Ca))的表达较临产前明显降低,但是这种表达的变化对子宫肌收缩性有何影响尚不清楚。因此在本课题的第三部分我们用K_(ATP)和BK_(Ca)激动剂以及孕酮处理子宫肌组织,观察它们对临产前后子宫肌收缩性的调节是否不同。
主要实验结果如下:
一、CRH对临产前后子宫肌收缩性不同调节作用的分子机制
1.我们课题组前期的研究已发现CRH能抑制未临产子宫肌的自发性收缩,但不影响临产子宫肌自发性收缩。本实验进一步观察CRH对催产素诱发的子宫肌收缩的影响,发现10~(-9) -10~(-7)mol/L CRH能剂量依赖性的增强临产子宫肌的收缩幅度。
2.细胞内游离钙离子水平([Ca~(2+)]_i)是平滑肌收缩的关键因子,因此我们观察[Ca~(2+)]_i作为检测子宫肌收缩性的另一指标。在临产子宫肌中,我们发现CRH升高子宫肌细胞[Ca~(2+)]_i,而且此效应可以被CRHR1特异性受体拮抗剂阻断,但不能被CRHR2特异性受体拮抗剂阻断,提示CRH升高临产子宫肌细胞[Ca~(2+)]_i是由CRHR1介导的。siRNA干扰CRHR1的表达后,CRH升高临产子宫肌[Ca~(2+)]_i的作用消失,进一步证实了CRH升高临产子宫肌[Ca~(2+)]_i是通过CRHR1介导的。
在未临产子宫肌中,我们发现CRH不影响未临产子宫肌[Ca~(2+)]_i。但我们前期的研究表明培养的子宫肌细胞能够合成和分泌CRH,为确认子宫肌自身合成和分泌(即内源性)的CRH是否调节子宫肌[Ca~(2+)]_i,我们分别应用CRHR1特异性拮抗剂和CRHR2特异性拮抗剂处理细胞,结果发现CRHR1特异性拮抗剂可剂量依赖性的升高未临产子宫肌[Ca~(2+)]_i,提示CRHR1特异性拮抗剂通过阻断内源性CRH与CRHR1的作用,升高[Ca~(2+)]_i;用siRNA干扰CRHR1的表达后,CRHR1特异性拮抗剂升高细胞[Ca~(2+)]_i的作用消失,进一步证实了CRHR1特异性拮抗剂升高细胞[Ca~(2+)]_i的作用是阻断内源性CRH与CRHR1的作用产生的。CRHR2特异性拮抗剂和激动剂都不影响未临产子宫肌细胞[Ca~(2+)]_i,进一步证实CRHR2不参与CRH对未临产子宫肌细胞[Ca~(2+)]_i的调节。
这些结果提示CRH促进临产子宫肌的收缩,抑制未临产子宫肌的收缩,且CRH的作用都是由CRHR1介导的。
3. CRHR1介导CRH调节临产前后子宫肌收缩性不同作用的细胞内信号转导通路。
CRHR1是G蛋白偶联受体。G蛋白偶联受体若与Gαq偶联可激活磷脂酶Cβ(PLCβ),PLCβ催化PIP2水解产生IP_3和DAG,IP_3能引起内质网内钙离子释放,升高细胞[Ca~(2+)]_i;与Gαs偶联能激活AC,升高细胞内cAMP水平,cAMP激活PKA,PKA磷酸化PLCβ上的相应位点,PLCβ上这些位点的磷酸化使其丧失活性,降低细胞内IP_3水平,从而抑制[Ca~(2+)]_i升高;而与Gαi偶联则抑制AC-cAMP通路,减弱cAMP抑制[Ca~(2+)]_i升高的作用。CRHR1在多种组织中(如胎盘、垂体、心脏,神经等)能与包括Gαs、Gαi、Gαq等在内的多种Gα蛋白亚型偶联,而在子宫肌上的研究也表明,CRHR1能与多种Gα蛋白亚型偶联。因此,我们进一步研究在临产和未临产子宫肌细胞中CRH通过CRHR1激活的细胞内信号转导通路。
(1)CRHR1介导CRH促进临产子宫肌收缩的细胞内信号转导通路。我们发现CRH能够剂量依赖性升高临产子宫肌细胞内PLCβ3磷酸化水平和IP_3水平,此效应可被CRHR1特异性拮抗剂阻断,提示CRH通过CRHR1激活PLCβ3-IP_3通路。将特异性抑制Gαq的小肽GP-2A转染进入临产子宫肌细胞后,CRH升高细胞内IP_3的作用消失,提示在临产子宫肌中CRHR1与Gαq偶联升高细胞内IP_3。
如前所述,除了Gaq激活可升高细胞内Ca外,Gi蛋白的激活也可能升高[Ca~(2+)]_i。我们发现CRH能激活临产子宫肌Gαi蛋白,还能剂量依赖性的降低临产子宫肌细胞内cAMP水平,而且此效应能够被CRHR1的特异性拮抗剂阻断,提示CRH通过CRHR1降低临产子宫肌细胞内cAMP。而转染Gαi抗体进入临产子宫肌细胞,阻断Gαi的作用,能部分阻断CRH升高细胞内IP_3水平的作用,提示临产子宫肌中CRH通过CRHR1激活Gαi也能升高细胞内IP_3。PLC抑制剂和IP_3受体拮抗剂能阻断CRH升高临产子宫肌细胞内[Ca~(2+)]_i的作用,提示PLCβ-IP_3通路激活的确参与CRH升高临产子宫肌[Ca~(2+)]_i。
由上述结果提示在临产子宫肌中CRHR1偶联于Gaq和Gai,CRH通过CRHR1激活PLC-IP_3和抑制AC-cAMP细胞内信号通路,升高[Ca~(2+)]_i。
(2)CRHR1介导CRH抑制未临产子宫肌收缩性的细胞内信号转导通路。
在未临产子宫肌中,我们发现CRH能够激活Gαs。外源性给予CRH不影响未临产子宫肌细胞内cAMP水平,但是CRHR1的特异性拮抗剂能降低细胞内cAMP水平,这与CRHR1拮抗剂升高未临产子宫肌细胞[Ca~(2+)]_i的结果相符。腺苷酸环化酶(AC)激动剂能阻断CRHR1拮抗剂升高未临产子宫肌[Ca~(2+)]_i的作用,而AC抑制剂可以模拟CRHR1拮抗剂升高未临产子宫肌[Ca~(2+)]_i的作用,提示CRHR1拮抗剂的确是通过抑制AC升高未临产子宫肌[Ca~(2+)]_i。如前所述,cAMP升高可抑制PLC-IP_3通路,抑制[Ca~(2+)]_i升高。我们的研究表明外源性给予CRH不影响未临产子宫肌细胞内IP_3水平,但CRHR1的特异性拮抗剂能剂量依赖性的升高细胞内IP_3水平,提示内源性CRH通过CRHR1降低未临产子宫肌细胞内IP_3水平。
由上述结果提示在未临产子宫肌中CRHR1偶联于Gas,CRH通过CRHR1激活AC-cAMP细胞内信号通路,抑制[Ca~(2+)]_i升高。
比较CRH对临产和未临产子宫肌细胞[Ca~(2+)]_i的调节机制,我们发现CRH对临产和未临产子宫肌的作用不同是由于CRHR1偶联于不同的G蛋白亚型,激活不同的细胞内信号通路所致。
4.对未临产和临产子宫肌组织上各Gα亚型的蛋白表达量进行比较,发现临产后子宫肌上Gαs蛋白表达下降,Gαq蛋白表达上升,这可能是导致临产前后子宫肌上CRHR1偶联的Gα亚型发生改变的原因之一。
二、内源性H_2S对子宫肌收缩性的调节及其机制
1.在人类妊娠子宫肌中L-cysteine、SPRC都能产生内源性的H_2S;应用H_2S合成酶CBS和CSE的抑制剂可以明显降低子宫肌以L-cysteine为底物的H_2S生成速率,提示L-cysteine是在H_2S合成酶CBS和CSE的催化下产生内源性的H_2S的。
2.内源性H_2S对于人类妊娠子宫平滑肌收缩性的调节具有双重效应:能降低自发性收缩的幅度,而高浓度时能升高收缩的频率,引起紧张性收缩。K_(ATP)通道抑制剂能阻断内源性H_2S抑制子宫肌收缩的效应,提示内源性H_2S通过激活K_(ATP)通道产生抑制子宫肌收缩的效应。
3.比较内源性H_2S供体L-cysteine对临产前后子宫肌收缩性的调节,发现L-cysteine对临产子宫肌自发性收缩的抑制作用明显弱于对未临产子宫肌的作用。
三、多种药物对临产前后子宫肌收缩性的影响
1.用K_(ATP)和BK_(Ca)激动剂处理离体子宫肌组织,结果发现K_(ATP)和BK_(Ca)激动剂能明显抑制子宫肌的自发性收缩,而且它们对临产子宫肌收缩的抑制作用明显弱于对未临产子宫肌的作用。
2.孕酮能剂量依赖性的抑制未临产子宫肌的自发性收缩,但不能抑制临产子宫肌自发性收缩。
结论:1. CRH促进临产子宫肌的收缩,抑制未临产子宫肌的收缩,且CRH的作用都是由CRHR1介导的。2.在临产子宫肌中,CRHR1与Gαq和Gαi蛋白偶联,CRH通过激活PLC-IP_3和抑制AC-cAMP这两条细胞内信号通路,升高[Ca~(2+)]_i;在未临产子宫肌中, CRHR1与Gαs蛋白偶联,内源性CRH可能是通过激活Gαs-AC-cAMP信号通路,抑制细胞内IP_3水平升高,抑制细胞[Ca~(2+)]_i升高;3.与未临产子宫肌相比,临产子宫肌上Gαs蛋白表达下降,Gαq蛋白表达上升,这可能是导致CRHR1偶联的Gα亚基发生改变的原因之一。4.内源性H_2S可调节妊娠子宫平滑肌收缩,对临产子宫肌收缩性的抑制作用明显弱于对未临产子宫肌的作用。5. K_(ATP)通道激动剂、BK_(Ca)通道激动剂和孕酮对临产子宫肌收缩性的抑制明显弱于对未临产子宫肌的作用。
Premature birth remains a major cause of infant morbidity and is a serious health and financial burden to society. The pre-excitement of uterine smooth muscle will lead to preterm labor. And the etiology for premature labor is not clear. While in human the events that trigger parturition are not well defined, prior to the onset of parturition, a series of coordinate events prepare the myometrium to respond to contractant signals and to be less responsive to relaxant signals.
Placental CRH production increases exponentially as pregnancy progresses toward labor, and it has been proposed that CRH might act as a placental clock regulating the length of human gestation. The precise biological role of placental CRH during pregnancy and labor remains unknown, but the human myometrium appears to be a major target of their actions. In vitro studies in our laboratory suggest that, CRH acts on CRH-R1 to inhibit spontaneous contractions in term myometrium from women who were not undergoing labour, but not those who were undergoing labour. But the mechanisms of CRH different modulation on human myometrial contractility before and during labour is unknown.
Hydrogen sulfide (H_2S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H_2S produced locally in the control of uterine contractility during labour is unknown.
The aims of the present study were to explore effects of CRH and endogenous H_2S on contractility of human term myometrium, and compare these effects in labouring and nonlabouring myometrium. Further, study the mechanisms of CRH and endogenous H_2S different modulation in human myometrium contractility before and during labor.
Results:
1. CRH (10-10-10-7 mol/L) enhance oxytocin-induced contractions and produced dose-dependent increase in [Ca~(2+)]_i in myometrium from women who were undergoing labour, but not those who were not undergoing labour.
2. The effects, CRH increase in [Ca~(2+)]_i in myometrium cells from women underwent labour, were reversed by CRH receptor type 1 (CRHR1) antagonist antalarmin, but not by CRH receptor type 2 (CRHR2) antagonist astressin 2b. And CRHR1 small interfering RNA (siRNA) abrogate the effect of [Ca~(2+)]_i increase in laboring myometrium cell stimulated by CRH.
For the nonlabouring myometrium, CRH did not induce [Ca~(2+)]_i transient in myometium cells. Antalarmin(10-9-10-6 mol/L) dose-dependently increased [Ca~(2+)]_i in the cells, whereas astressin 2b did not. The effects of antalarmin could be blocked by exogenous CRH, suggesting that endogenous CRH has tonic inhibitory effects on [Ca~(2+)]_i . And CRHR1 siRNA abrogate the effect of [Ca~(2+)]_i increase in nonlaboring myometrium cell stimulated by antalarmin. CRHR1 exert the contrasting roles of maintaining myometrial relaxation before labor onset and stimulating contractility during labor.
3. The possible signaling pathways involved in the CRH regulation of [Ca~(2+)]_i were also investigated.
For the labouring myometrium, CRH (10-9-10-6 mol/L) induced active Gαi expression and dose-dependently decreased cAMP production in the cells. Blocking phospholipase C (PLC) activity with U73122 and application of IP_3 receptor antagonist blocked CRH-evoked [Ca~(2+)]_i transient in laboring myometrium cells. CRH (10-10-10-7 mol/L) dose-dependently induced phosphorylated PLC-?3 expression and increased in level of IP_3 in labouring myometrium cells. These effects were partly block by Gαi antibody and totally block by GP-2A, the inhibitor of Gαq.
For nonlabouring myometrium, CRH (10-10-10-7 mol/L) induced active Gαs expression and antalarmin (10-9-10-6 mol/L) dose-dependently decreased cAMP production in the cells. Forskolin, the adenylate cyclase activator, reversed antalarmin-induced [Ca~(2+)]_i transient. SQ 22536, an adenylate cyclase inhibitor, induced [Ca~(2+)]_i transient. Our results suggest that CRH act on CRHR1 to induce [Ca~(2+)]_i transient in labouring myometrium, but inhibit [Ca~(2+)]_i in nonlabouring myometrium. Before labour onset, CRHR1 may coupled with Gs protein and CRH enhances AC-PKA signaling pathway to decrease [Ca~(2+)]_i in myometrium cells. During labour, CRHR1 may couple with Gq protein and CRH activates PLC/IP_3 signaling pathway to increase [Ca~(2+)]_i in myometrium cells.
4. Cumulative administration of L-cysteine (10-7-10-2mol/L), a precursor of H_2S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10-3mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (K_(ATP)) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.
Conclusion:
Our results suggest that CRH act on CRHR1 to induce [Ca~(2+)]_i transient in labouring myometrium, but inhibit [Ca~(2+)]_i transient in nonlabouring myometrium. Before labour onset, CRHR1 may coupled with Gαs protein and CRH stimulates AC-PKA signaling pathway to decrease [Ca~(2+)]_i in myometrium cells. During labour, CRHR1 may couple with Gαq protein and Gαi protein, and CRH activates PLC/IP_3 signaling pathway to induced [Ca~(2+)]_i transient in myometrium cells, thereby facilitating contraction.
H_2S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H_2S synthetic enzymes is down-regulated during labour, suggesting that H_2S is one of factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.
在正常情况下,妊娠足月之前,子宫都是处于一种相对静息的状态,其兴奋性低,不发生大的收缩。只有在临近分娩时才发生高频率、高强度的规律宫缩。因此,子宫由静息态向收缩态的转化无疑是启动分娩的关键。但迄今为止对于子宫静息状态终止进而转为收缩激活的机制,尚未阐明。目前的研究表明,妊娠子宫肌静息状态维持和收缩的发生受到了一系列内源性活性因子的调控,而且妊娠期间,这些因子的产生量及子宫对它们的反应性是随着妊娠的进行而发生变化的。目前认为,正是由于上述这种动态变化,使子宫在足月前处于静息状态,在妊娠末期出现收缩。因此全面地了解究竟有哪些内源性子宫舒缩性调控因子,深入地研究这些因子在妊娠不同阶段对子宫的作用以及作用机制,是发现妊娠子宫静息终止转为收缩激活分子的关键。
妊娠期间人类胎盘组织合成和分泌的促肾上腺皮质激素释放激素(corticotropin-releasinghormone,CRH)在人类分娩启动中发挥重要作用,被称为控制分娩的“生物钟”。人类子宫肌可以表达CRH受体,提示人类子宫肌是CRH作用的重要靶器官。因此在本课题的第一部分进行了以下四个方面的研究:1.观察CRH对临产和未临产子宫肌收缩性的不同调节作用;2.介导CRH对临产和未临产子宫肌不同调节作用的受体亚型;3.CRHR1介导CRH对临产和未临产子宫肌收缩性不同调节作用的细胞内信号转导通路;4.引起临产和未临产子宫肌中CRHR1偶联的G蛋白亚型发生改变的可能机制。
硫化氢是继NO和CO之后发现的第三种具有生物活性的气体信号分子,可舒张多种平滑肌组织,但是H_2S对于子宫平滑肌作用的研究还相对较少,仅有两篇报道表明外源性的H_2S能抑制子宫平滑肌自发性收缩,而在人体中存在内源性H_2S,并且本课题前期研究表明人类妊娠子宫肌产生内源性H_2S。那么内源性H_2S是否能够舒张人类妊娠子宫肌?本课题的第二部分我们用产生内源性H_2S的供体L-半胱氨酸等处理人类离体子宫肌,观察内源性H_2S对人类子宫肌收缩性的调节,并对其作用机制进行系统性研究,进一步比较内源性H_2S对临产前后子宫肌收缩性的调节是否不同。
在子宫肌由静息态向收缩态转变的过程中,子宫平滑肌上多种收缩相关蛋白的表达发生变化。本课题组前期的研究已经发现在临产后ATP激活的K~+通道(K_(ATP)),大电导Ca~(2+)激活的K~+通道(BK_(Ca))的表达较临产前明显降低,但是这种表达的变化对子宫肌收缩性有何影响尚不清楚。因此在本课题的第三部分我们用K_(ATP)和BK_(Ca)激动剂以及孕酮处理子宫肌组织,观察它们对临产前后子宫肌收缩性的调节是否不同。
主要实验结果如下:
一、CRH对临产前后子宫肌收缩性不同调节作用的分子机制
1.我们课题组前期的研究已发现CRH能抑制未临产子宫肌的自发性收缩,但不影响临产子宫肌自发性收缩。本实验进一步观察CRH对催产素诱发的子宫肌收缩的影响,发现10~(-9) -10~(-7)mol/L CRH能剂量依赖性的增强临产子宫肌的收缩幅度。
2.细胞内游离钙离子水平([Ca~(2+)]_i)是平滑肌收缩的关键因子,因此我们观察[Ca~(2+)]_i作为检测子宫肌收缩性的另一指标。在临产子宫肌中,我们发现CRH升高子宫肌细胞[Ca~(2+)]_i,而且此效应可以被CRHR1特异性受体拮抗剂阻断,但不能被CRHR2特异性受体拮抗剂阻断,提示CRH升高临产子宫肌细胞[Ca~(2+)]_i是由CRHR1介导的。siRNA干扰CRHR1的表达后,CRH升高临产子宫肌[Ca~(2+)]_i的作用消失,进一步证实了CRH升高临产子宫肌[Ca~(2+)]_i是通过CRHR1介导的。
在未临产子宫肌中,我们发现CRH不影响未临产子宫肌[Ca~(2+)]_i。但我们前期的研究表明培养的子宫肌细胞能够合成和分泌CRH,为确认子宫肌自身合成和分泌(即内源性)的CRH是否调节子宫肌[Ca~(2+)]_i,我们分别应用CRHR1特异性拮抗剂和CRHR2特异性拮抗剂处理细胞,结果发现CRHR1特异性拮抗剂可剂量依赖性的升高未临产子宫肌[Ca~(2+)]_i,提示CRHR1特异性拮抗剂通过阻断内源性CRH与CRHR1的作用,升高[Ca~(2+)]_i;用siRNA干扰CRHR1的表达后,CRHR1特异性拮抗剂升高细胞[Ca~(2+)]_i的作用消失,进一步证实了CRHR1特异性拮抗剂升高细胞[Ca~(2+)]_i的作用是阻断内源性CRH与CRHR1的作用产生的。CRHR2特异性拮抗剂和激动剂都不影响未临产子宫肌细胞[Ca~(2+)]_i,进一步证实CRHR2不参与CRH对未临产子宫肌细胞[Ca~(2+)]_i的调节。
这些结果提示CRH促进临产子宫肌的收缩,抑制未临产子宫肌的收缩,且CRH的作用都是由CRHR1介导的。
3. CRHR1介导CRH调节临产前后子宫肌收缩性不同作用的细胞内信号转导通路。
CRHR1是G蛋白偶联受体。G蛋白偶联受体若与Gαq偶联可激活磷脂酶Cβ(PLCβ),PLCβ催化PIP2水解产生IP_3和DAG,IP_3能引起内质网内钙离子释放,升高细胞[Ca~(2+)]_i;与Gαs偶联能激活AC,升高细胞内cAMP水平,cAMP激活PKA,PKA磷酸化PLCβ上的相应位点,PLCβ上这些位点的磷酸化使其丧失活性,降低细胞内IP_3水平,从而抑制[Ca~(2+)]_i升高;而与Gαi偶联则抑制AC-cAMP通路,减弱cAMP抑制[Ca~(2+)]_i升高的作用。CRHR1在多种组织中(如胎盘、垂体、心脏,神经等)能与包括Gαs、Gαi、Gαq等在内的多种Gα蛋白亚型偶联,而在子宫肌上的研究也表明,CRHR1能与多种Gα蛋白亚型偶联。因此,我们进一步研究在临产和未临产子宫肌细胞中CRH通过CRHR1激活的细胞内信号转导通路。
(1)CRHR1介导CRH促进临产子宫肌收缩的细胞内信号转导通路。我们发现CRH能够剂量依赖性升高临产子宫肌细胞内PLCβ3磷酸化水平和IP_3水平,此效应可被CRHR1特异性拮抗剂阻断,提示CRH通过CRHR1激活PLCβ3-IP_3通路。将特异性抑制Gαq的小肽GP-2A转染进入临产子宫肌细胞后,CRH升高细胞内IP_3的作用消失,提示在临产子宫肌中CRHR1与Gαq偶联升高细胞内IP_3。
如前所述,除了Gaq激活可升高细胞内Ca外,Gi蛋白的激活也可能升高[Ca~(2+)]_i。我们发现CRH能激活临产子宫肌Gαi蛋白,还能剂量依赖性的降低临产子宫肌细胞内cAMP水平,而且此效应能够被CRHR1的特异性拮抗剂阻断,提示CRH通过CRHR1降低临产子宫肌细胞内cAMP。而转染Gαi抗体进入临产子宫肌细胞,阻断Gαi的作用,能部分阻断CRH升高细胞内IP_3水平的作用,提示临产子宫肌中CRH通过CRHR1激活Gαi也能升高细胞内IP_3。PLC抑制剂和IP_3受体拮抗剂能阻断CRH升高临产子宫肌细胞内[Ca~(2+)]_i的作用,提示PLCβ-IP_3通路激活的确参与CRH升高临产子宫肌[Ca~(2+)]_i。
由上述结果提示在临产子宫肌中CRHR1偶联于Gaq和Gai,CRH通过CRHR1激活PLC-IP_3和抑制AC-cAMP细胞内信号通路,升高[Ca~(2+)]_i。
(2)CRHR1介导CRH抑制未临产子宫肌收缩性的细胞内信号转导通路。
在未临产子宫肌中,我们发现CRH能够激活Gαs。外源性给予CRH不影响未临产子宫肌细胞内cAMP水平,但是CRHR1的特异性拮抗剂能降低细胞内cAMP水平,这与CRHR1拮抗剂升高未临产子宫肌细胞[Ca~(2+)]_i的结果相符。腺苷酸环化酶(AC)激动剂能阻断CRHR1拮抗剂升高未临产子宫肌[Ca~(2+)]_i的作用,而AC抑制剂可以模拟CRHR1拮抗剂升高未临产子宫肌[Ca~(2+)]_i的作用,提示CRHR1拮抗剂的确是通过抑制AC升高未临产子宫肌[Ca~(2+)]_i。如前所述,cAMP升高可抑制PLC-IP_3通路,抑制[Ca~(2+)]_i升高。我们的研究表明外源性给予CRH不影响未临产子宫肌细胞内IP_3水平,但CRHR1的特异性拮抗剂能剂量依赖性的升高细胞内IP_3水平,提示内源性CRH通过CRHR1降低未临产子宫肌细胞内IP_3水平。
由上述结果提示在未临产子宫肌中CRHR1偶联于Gas,CRH通过CRHR1激活AC-cAMP细胞内信号通路,抑制[Ca~(2+)]_i升高。
比较CRH对临产和未临产子宫肌细胞[Ca~(2+)]_i的调节机制,我们发现CRH对临产和未临产子宫肌的作用不同是由于CRHR1偶联于不同的G蛋白亚型,激活不同的细胞内信号通路所致。
4.对未临产和临产子宫肌组织上各Gα亚型的蛋白表达量进行比较,发现临产后子宫肌上Gαs蛋白表达下降,Gαq蛋白表达上升,这可能是导致临产前后子宫肌上CRHR1偶联的Gα亚型发生改变的原因之一。
二、内源性H_2S对子宫肌收缩性的调节及其机制
1.在人类妊娠子宫肌中L-cysteine、SPRC都能产生内源性的H_2S;应用H_2S合成酶CBS和CSE的抑制剂可以明显降低子宫肌以L-cysteine为底物的H_2S生成速率,提示L-cysteine是在H_2S合成酶CBS和CSE的催化下产生内源性的H_2S的。
2.内源性H_2S对于人类妊娠子宫平滑肌收缩性的调节具有双重效应:能降低自发性收缩的幅度,而高浓度时能升高收缩的频率,引起紧张性收缩。K_(ATP)通道抑制剂能阻断内源性H_2S抑制子宫肌收缩的效应,提示内源性H_2S通过激活K_(ATP)通道产生抑制子宫肌收缩的效应。
3.比较内源性H_2S供体L-cysteine对临产前后子宫肌收缩性的调节,发现L-cysteine对临产子宫肌自发性收缩的抑制作用明显弱于对未临产子宫肌的作用。
三、多种药物对临产前后子宫肌收缩性的影响
1.用K_(ATP)和BK_(Ca)激动剂处理离体子宫肌组织,结果发现K_(ATP)和BK_(Ca)激动剂能明显抑制子宫肌的自发性收缩,而且它们对临产子宫肌收缩的抑制作用明显弱于对未临产子宫肌的作用。
2.孕酮能剂量依赖性的抑制未临产子宫肌的自发性收缩,但不能抑制临产子宫肌自发性收缩。
结论:1. CRH促进临产子宫肌的收缩,抑制未临产子宫肌的收缩,且CRH的作用都是由CRHR1介导的。2.在临产子宫肌中,CRHR1与Gαq和Gαi蛋白偶联,CRH通过激活PLC-IP_3和抑制AC-cAMP这两条细胞内信号通路,升高[Ca~(2+)]_i;在未临产子宫肌中, CRHR1与Gαs蛋白偶联,内源性CRH可能是通过激活Gαs-AC-cAMP信号通路,抑制细胞内IP_3水平升高,抑制细胞[Ca~(2+)]_i升高;3.与未临产子宫肌相比,临产子宫肌上Gαs蛋白表达下降,Gαq蛋白表达上升,这可能是导致CRHR1偶联的Gα亚基发生改变的原因之一。4.内源性H_2S可调节妊娠子宫平滑肌收缩,对临产子宫肌收缩性的抑制作用明显弱于对未临产子宫肌的作用。5. K_(ATP)通道激动剂、BK_(Ca)通道激动剂和孕酮对临产子宫肌收缩性的抑制明显弱于对未临产子宫肌的作用。
Premature birth remains a major cause of infant morbidity and is a serious health and financial burden to society. The pre-excitement of uterine smooth muscle will lead to preterm labor. And the etiology for premature labor is not clear. While in human the events that trigger parturition are not well defined, prior to the onset of parturition, a series of coordinate events prepare the myometrium to respond to contractant signals and to be less responsive to relaxant signals.
Placental CRH production increases exponentially as pregnancy progresses toward labor, and it has been proposed that CRH might act as a placental clock regulating the length of human gestation. The precise biological role of placental CRH during pregnancy and labor remains unknown, but the human myometrium appears to be a major target of their actions. In vitro studies in our laboratory suggest that, CRH acts on CRH-R1 to inhibit spontaneous contractions in term myometrium from women who were not undergoing labour, but not those who were undergoing labour. But the mechanisms of CRH different modulation on human myometrial contractility before and during labour is unknown.
Hydrogen sulfide (H_2S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H_2S produced locally in the control of uterine contractility during labour is unknown.
The aims of the present study were to explore effects of CRH and endogenous H_2S on contractility of human term myometrium, and compare these effects in labouring and nonlabouring myometrium. Further, study the mechanisms of CRH and endogenous H_2S different modulation in human myometrium contractility before and during labor.
Results:
1. CRH (10-10-10-7 mol/L) enhance oxytocin-induced contractions and produced dose-dependent increase in [Ca~(2+)]_i in myometrium from women who were undergoing labour, but not those who were not undergoing labour.
2. The effects, CRH increase in [Ca~(2+)]_i in myometrium cells from women underwent labour, were reversed by CRH receptor type 1 (CRHR1) antagonist antalarmin, but not by CRH receptor type 2 (CRHR2) antagonist astressin 2b. And CRHR1 small interfering RNA (siRNA) abrogate the effect of [Ca~(2+)]_i increase in laboring myometrium cell stimulated by CRH.
For the nonlabouring myometrium, CRH did not induce [Ca~(2+)]_i transient in myometium cells. Antalarmin(10-9-10-6 mol/L) dose-dependently increased [Ca~(2+)]_i in the cells, whereas astressin 2b did not. The effects of antalarmin could be blocked by exogenous CRH, suggesting that endogenous CRH has tonic inhibitory effects on [Ca~(2+)]_i . And CRHR1 siRNA abrogate the effect of [Ca~(2+)]_i increase in nonlaboring myometrium cell stimulated by antalarmin. CRHR1 exert the contrasting roles of maintaining myometrial relaxation before labor onset and stimulating contractility during labor.
3. The possible signaling pathways involved in the CRH regulation of [Ca~(2+)]_i were also investigated.
For the labouring myometrium, CRH (10-9-10-6 mol/L) induced active Gαi expression and dose-dependently decreased cAMP production in the cells. Blocking phospholipase C (PLC) activity with U73122 and application of IP_3 receptor antagonist blocked CRH-evoked [Ca~(2+)]_i transient in laboring myometrium cells. CRH (10-10-10-7 mol/L) dose-dependently induced phosphorylated PLC-?3 expression and increased in level of IP_3 in labouring myometrium cells. These effects were partly block by Gαi antibody and totally block by GP-2A, the inhibitor of Gαq.
For nonlabouring myometrium, CRH (10-10-10-7 mol/L) induced active Gαs expression and antalarmin (10-9-10-6 mol/L) dose-dependently decreased cAMP production in the cells. Forskolin, the adenylate cyclase activator, reversed antalarmin-induced [Ca~(2+)]_i transient. SQ 22536, an adenylate cyclase inhibitor, induced [Ca~(2+)]_i transient. Our results suggest that CRH act on CRHR1 to induce [Ca~(2+)]_i transient in labouring myometrium, but inhibit [Ca~(2+)]_i in nonlabouring myometrium. Before labour onset, CRHR1 may coupled with Gs protein and CRH enhances AC-PKA signaling pathway to decrease [Ca~(2+)]_i in myometrium cells. During labour, CRHR1 may couple with Gq protein and CRH activates PLC/IP_3 signaling pathway to increase [Ca~(2+)]_i in myometrium cells.
4. Cumulative administration of L-cysteine (10-7-10-2mol/L), a precursor of H_2S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10-3mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (K_(ATP)) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.
Conclusion:
Our results suggest that CRH act on CRHR1 to induce [Ca~(2+)]_i transient in labouring myometrium, but inhibit [Ca~(2+)]_i transient in nonlabouring myometrium. Before labour onset, CRHR1 may coupled with Gαs protein and CRH stimulates AC-PKA signaling pathway to decrease [Ca~(2+)]_i in myometrium cells. During labour, CRHR1 may couple with Gαq protein and Gαi protein, and CRH activates PLC/IP_3 signaling pathway to induced [Ca~(2+)]_i transient in myometrium cells, thereby facilitating contraction.
H_2S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H_2S synthetic enzymes is down-regulated during labour, suggesting that H_2S is one of factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.
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