Amidi Fataneh, Beall Marie, Ross Michael G
Department of Obstetrics and Gynecology, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California90502, USA.
Reprod Sci. 2007 Apr;14(3):234-40. doi: 10.1177/1933719107300970.
Cell membrane aquaporins (AQPs) may con t r i b u t e importantly to the regulation of intramembranous absorption of amniotic fluid. Recently, the authors demonstrated that human amnion AQP3 expression is upregulated by second-messenger cyclic adenosine monophosphate (cAMP). The present study was undertaken to determine the cAMP regulation of other AQP types, specifically AQP1, 8, and 9, in human amnion epithelia in vitro. Human amnion epithelial cell cultures were prepared from amnion of normal-term pregnancy. To investigate the effect of cAMP on AQP expression, primary human amnion cell cultures were incubated for 2, 10, and 20 hours with culture medium containing either 50 microM forskolin, an adenylate cyclase activator that stimulates cellular production of cAMP, or 100 microM SP-cAMP, a cAMP agonist that stimulates protein kinase A. Total RNA was isolated from the cultured cells, and semiquantitative real-time reverse transcription polymerase chain reaction was carried out to determine the relative level of AQPs mRNA expression. In primary amnion epithelial cell culture, AQP1 mRNA expression increased significantly at 10 hours (0.219 +/- 0.006 to 0.314 +/- 0.008, P < .05) and remained elevated for 20 hours (0.223 +/- 0.004 to 0.323 +/- 0.012, P < .05) following forskolin treatment. AQP8 mRNA expression increased significantly at 2 hours (0.069 +/- 0.003 to 0.086 +/- 0.012, P < .05) and remained upregulated for 20 hours following forskolin treatment. Forskolin stimulation of AQP9 mRNA expression was evidenced by 10 hours (0.098 +/- 0.005 to 0.115 +/- 0.006, P < .05) and maintained for 20 hours. In contrast to forskolin, SP-cAMP incubation resulted in no change in AQP1, 8, or 9 mRNA expression. Human amnion epithelial cell AQP1, 8, and 9 mRNA expression is upregulated by cAMP as their expression is simulated by forskolin. Lack of effect of SP-cAMP, the protein kinase A activator, on AQP1, 8, and 9 mRNA expression suggests that cAMP upregulates human amnion AQP1, 8, and 9 mRNA expression via the protein kinase A independent pathway.
细胞膜水通道蛋白（AQPs）可能对羊水膜内吸收的调节起着重要作用。最近，作者证明人羊膜AQP3的表达受第二信使环磷酸腺苷（cAMP）上调。本研究旨在确定体外培养的人羊膜上皮细胞中cAMP对其他类型AQP，特别是AQP1、8和9的调节作用。从足月妊娠的羊膜制备人羊膜上皮细胞培养物。为了研究cAMP对AQP表达的影响，将原代人羊膜细胞培养物与含有50微摩尔毛喉素（一种刺激细胞产生cAMP的腺苷酸环化酶激活剂）或100微摩尔SP-cAMP（一种刺激蛋白激酶A的cAMP激动剂）的培养基孵育2、10和20小时。从培养的细胞中分离总RNA，并进行半定量实时逆转录聚合酶链反应以确定AQPs mRNA表达的相对水平。在原代羊膜上皮细胞培养中，毛喉素处理后10小时AQP1 mRNA表达显著增加（从0.219±0.006增至0.314±0.008，P<.05），并在20小时内保持升高（从0.223±0.004增至0.323±0.012，P<.05）。毛喉素处理后2小时AQP8 mRNA表达显著增加（从0.069±0.003增至0.086±0.012，P<.05），并在20小时内保持上调。毛喉素刺激AQP9 mRNA表达在10小时时得到证实（从0.098±0.005增至0.115±0.006，P<.05），并维持20小时。与毛喉素相反，SP-cAMP孵育导致AQP1、8或9 mRNA表达无变化。人羊膜上皮细胞AQP1、8和9 mRNA表达受cAMP上调，因为它们的表达受毛喉素模拟。蛋白激酶A激活剂SP-cAMP对AQP1、8和9 mRNA表达无影响，提示cAMP通过蛋白激酶A非依赖途径上调人羊膜AQP1、8和9 mRNA表达。
