Objective: To study the effect of cyclic adenosine monophosphate on aquaporin 8 (AQP8) expression and distribution in human amnion-derived WISH cells.
Methods: Human amnion-derived WISH cells were cultured. The cells were divided into control group and study group at random. The study group was established by exposure to various concentrations of 8-Br-cAMP. Western analysis was used to quantify AQP8 expression levels. RT-PCR was used to quantify AQP8 mRNA expression levels. Immunofluorescence was used to determine the localization of AQP8 in WISH cells.
Results: With increase of cAMP, AQP8 mRNA and protein expressions significantly increased in WISH cells in vitro. When the concentration of cAMP reached 200 micromol/L, AQP8 mRNA and protein expressions were highest. Incubation with cAMP (200 micromol/L) for 2 hours resulted in a 2-fold increase in AQP8 mRNA level, and incubation for 8 hours resulted in a peak. After incubation for 16 hours AQP8 mRNA level began to descend, and after 24 hours it decreased to baseline. Incubation with cAMP for 8 hours AQP8 protein level began to increase. Incubation for 24 hours resulted in a peak in AQP8 protein level, and after incubation for 48 hours it began to decline. By immunofluorescence microscopy after incubation with cAMP (200 micromol/L) cells, AQP8 labeling in plasma membrane was enhanced and intracellular AQP8 labeling was decreased.
Conclusion: The cAMP triggers translocation of AQP8 from cytosol to the plasma membrane via vesicle-transporting related protein instead of AQP8 itself. cAMP may upregulate the transcription of target gene protein kinase A. The cAMP may be the critical regulatory medium of AQP8 in WISH cells.