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

Department of Obstetrics and Gynecology, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center,Torrance, California, sheng{at}labiomed.org, Pacific Reproductive Center, Torrance, California

Fataneh Amidi, MD

Department of Obstetrics and Gynecology, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center,Torrance, California

Shengli Yin, MD

Division of Cardiac Surgery, Department of Surgery, First Affiliated Hospital, Sun Yet-sen University, Guangzhou, China

Marie Beall, MD

Department of Obstetrics and Gynecology, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center,Torrance, California

Michael G. Ross, MD, MPH

Department of Obstetrics and Gynecology, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center,Torrance, California

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.

Key Words: Aquaporin • amniotic fluid regulation • intramembranous pathway • amnion • cyclic adenosine monophosphate • cAMP

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