Shengbiao Wang

Aquaporin 3 Expression in Human Fetal Membranes and its Up-regulation by Cyclic Adenosine Monophosphate in Amnion Epithelial Cell Culture:

Objective: 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). Methods: 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. Results:: 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. Conclusion: 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.

Cyclic Adenosine Monophosphate Regulation of Aquaporin Gene Expression in Human Amnion Epithelia

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 µM forskolin, an adenylate cyclase activator that stimulates cellular production of cAMP, or 100 µM 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.