Objective: 

In our previous studies, the prostaglandin receptor EP2 agonist butaprost (Sigma-Aldrich, MO, USA) increased aquaporin-2 (AQP2) plasma membrane targeting (PMT) and phosphorylation at ser-269 (pS269), but EP4 agonist CAY10580 (CA) (Cayman Chemicals, MI, USA) increased only PMT in MDCK cells. Butaprost increased AQP2 PMT in rat kidney slices ex vivo and relieved polyuria in a rat model. EP2 ^vs. EP4 mediated AQP2 trafficking events and the role of cAMP and pS269 were examined in the present studies.

Methods: 

Agonist/antagonist treatment was performed in MDCK cells, which natively express EP2 and EP4, and were stably transfected with wild type AQP2 or AQP2 constitutively non- phosphorylated at S269 (S269A-AQP2) where indicated. AQP2 PMT was quantified by cell surface biotinylation.

Results: 

Stimulation with 3mM CA (2 to 80min) increased PMT from 5–40min, but did not increase pS269 or cAMP at any time point. In contrast, butaprost (0.1–1mM, 2–80min) increased cAMP and AQP2 PMT after 10min. Whereas AQP2 PMT continued to increase from 10–80min, cAMP displayed no further increase over time. 20min agonist washout abolished CA stimulated AQP2 PMT whereas butaprost induced MT was upheld for at least 80min of washout. cAMP returned to control levels after butaprost removal. In 269A-AQP2 cells, AQP2 PMT increased in response to butaprost or CAY10580. After 40min butaprost washout, AQP2 PMT was still significantly increased.

Conclusion: 

1) EP4 increases AQP2 PMT without increasing cAMP and pS269. This is not due to receptor desensitization over time 2) EP4 mediated membrane targeted AQP2 is rapidly internalized after washout, whereas the effect of EP2 on AQP2 MT is upheld 3) the latter is not due to continued cAMP increase after washout and does not depend on pS269.