蛋白酶激活受体亚型在妊娠和未妊娠大鼠子宫肌层组织中的表达。
Protease-activated receptor isoform expression in pregnant and nonpregnant rat myometrial tissue.
作者信息
Chien Edward K, Sweet Leigh, Phillippe Mark, Marietti Sarah, Kim Terrence T, Wolff David A, Thomas Leandra, Bieber Eric
机构信息
Department of Obstetrics and Gynecology, University of Vermont, Given Building C258, 89 Beaumonmt Drive, Burlington, VT 05405, USA.
出版信息
J Soc Gynecol Investig. 2003 Dec;10(8):460-8. doi: 10.1016/s1071-5576(03)00148-5.
OBJECTIVE
Three protease-activated receptor (PAR1, 3, and 4) isoforms have been shown to be responsible for the cellular effects of thrombin; another PAR isoform (PAR2) is responsible for the cellular effects of trypsin. The present studies sought to test the hypothesis that one (or more) of these PAR isoforms is expressed in myometrial tissue, thereby accounting for the uterotonic effects of these novel agonists.
METHODS
The rat PAR3 and 4 isoforms were cloned from a rat spleen cDNA library. PAR isoform mRNA expression was determined by using reverse-transcriptase polymerase chain reactions (PCR) in Sprague-Dawley rats. Confirmation of the identity of the amplified mRNA was done by sequence analysis. Relative quantification of the PAR1 and PAR2 isoforms was performed using a real-time quantitative reverse transcriptase PCR (RT-PCR) technique. PAR protein expression was confirmed by Western blots using polyclonal antibodies.
RESULTS
The rat PAR3 and 4 homologues showed significant sequence homology to the mouse and human amino acid and nucleotide sequences. The RT-PCR studies confirmed PAR1-4 expression in myometrium from rats in estrus. PAR3 was expressed in uterus, spleen, kidney, liver, lung, brain, and heart. PAR4 was expressed in uterus, spleen, and lung. Messenger RNA for the PAR1 and 2 isoforms was expressed during the second half of gestation in myometrium from timed-pregnant rats. In contrast, mRNA for the PAR3 and 4 isoforms was not detected in gestational myometrium. PAR protein expression appeared to match tissue mRNA expression patterns.
CONCLUSION
These RT-PCR studies confirmed ubiquitous expression of the PAR1 and PAR2 isoforms in myometrium and other rat tissues; in contrast, the PAR3 and PAR4 isoforms are expressed in a tissue-specific and gestationally related pattern.
目的
三种蛋白酶激活受体（PAR1、3和4）亚型已被证明介导凝血酶的细胞效应；另一种PAR亚型（PAR2）介导胰蛋白酶的细胞效应。本研究旨在验证以下假设：这些PAR亚型中的一种（或多种）在子宫肌层组织中表达，从而解释这些新型激动剂的子宫收缩作用。
方法
从大鼠脾脏cDNA文库中克隆大鼠PAR3和4亚型。采用逆转录聚合酶链反应（PCR）测定Sprague-Dawley大鼠子宫肌层中PAR亚型mRNA的表达。通过序列分析确认扩增mRNA的身份。使用实时定量逆转录PCR（RT-PCR）技术对PAR1和PAR2亚型进行相对定量。使用多克隆抗体通过蛋白质免疫印迹法确认PAR蛋白的表达。
结果
大鼠PAR3和4同源物与小鼠和人类的氨基酸及核苷酸序列具有显著的序列同源性。RT-PCR研究证实动情期大鼠子宫肌层中存在PAR1-4的表达。PAR3在子宫、脾脏、肾脏、肝脏、肺、脑和心脏中表达。PAR4在子宫、脾脏和肺中表达。PAR1和2亚型的信使RNA在定时妊娠大鼠妊娠后半期的子宫肌层中表达。相比之下，在妊娠子宫肌层中未检测到PAR3和4亚型的mRNA。PAR蛋白表达似乎与组织mRNA表达模式相符。
结论
这些RT-PCR研究证实PAR1和PAR2亚型在子宫肌层和其他大鼠组织中普遍表达；相比之下，PAR3和PAR4亚型以组织特异性和与妊娠相关的模式表达。
Zotero 插件