Wang Shengbiao, Amidi Fataneh, Beall Marie, Gui Lizhen, Ross Michael G
Department of Obstetrics and Gynecology, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, California 90502, USA.
J Soc Gynecol Investig. 2006 Apr;13(3):181-5. doi: 10.1016/j.jsgi.2006.02.002.
The cell membrane water channel protein aquaporins (AQPs) may be important in regulating the intramembranous (IM) pathway of amniotic fluid (AF) resorption. The objective of the present study was to determine whether aquaporin 3 (AQP3) is expressed in human fetal membranes and to further determine if AQP3 expression in primary human amnion cell culture is regulated by second-messenger cyclic adenosine monophosphate (cAMP).
AQP3 expression in human fetal membranes of normal term pregnancy was studied by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). To determine the effect of cAMP on AQP3 expression, primary human amnion cell cultures were treated in either heat-inactivated medium alone (control), or heat-inactivated medium containing: (1) SP-cAMP, a membrane-permeable and phosphodiesterase resistant cAMP agonist, or (2) forskolin, an adenylate cyclase stimulator. Total RNA was isolated and multiplex real-time RT-PCR employed for relative quantitation of AQP3 expression.
We detected AQP3 expression in placenta, chorion, and amnion using RT-PCR. Using IHC, we identified AQP3 protein expression in placenta syncytiotrophoblasts and cytotrophoblasts, chorion cytotrophoblasts, and amnion epithelia. In primary amnion epithelial cell culture, AQP3 mRNA significantly increased at 2 hours following forskolin or SP-cAMP, remained elevated at 10 hours following forskolin, and returned to baseline levels by 20 hours following treatment.
This study provides evidence of AQP3 expression in human fetal membranes and demonstrates that AQP3 expression in primary human amnion cell culture is up-regulated by second-messenger cAMP. As AQP3 is permeable to water, urea, and glycerol, modulation of its expression in fetal membranes may contribute to AF homeostasis.
细胞膜水通道蛋白水通道蛋白（AQPs）可能在调节羊水（AF）重吸收的膜内（IM）途径中起重要作用。本研究的目的是确定水通道蛋白3（AQP3）是否在人胎膜中表达，并进一步确定原代人羊膜细胞培养中AQP3的表达是否受第二信使环磷酸腺苷（cAMP）的调节。
采用逆转录聚合酶链反应（RT-PCR）和免疫组织化学（IHC）研究足月妊娠人胎膜中AQP3的表达。为了确定cAMP对AQP3表达的影响，原代人羊膜细胞培养物分别用单独的热灭活培养基（对照）或含有以下物质的热灭活培养基处理：（1）SP-cAMP，一种膜通透性且抗磷酸二酯酶的cAMP激动剂，或（2）福斯可林，一种腺苷酸环化酶刺激剂。分离总RNA，采用多重实时RT-PCR对AQP3表达进行相对定量。
我们使用RT-PCR在胎盘、绒毛膜和羊膜中检测到AQP3表达。使用IHC，我们在胎盘合体滋养层细胞和细胞滋养层细胞、绒毛膜细胞滋养层细胞和羊膜上皮细胞中鉴定出AQP3蛋白表达。在原代羊膜上皮细胞培养中，福斯可林或SP-cAMP处理后2小时AQP3 mRNA显著增加，福斯可林处理后10小时仍保持升高，处理后20小时恢复到基线水平。
本研究提供了AQP3在人胎膜中表达的证据，并表明原代人羊膜细胞培养中AQP3的表达受第二信使cAMP上调。由于AQP3对水、尿素和甘油具有通透性，调节其在胎膜中的表达可能有助于羊水内环境稳定。
