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Persistent cAMP Signaling via Vasopressin V2 Receptors Imaged in 3D Cultures Using Lightsheet Microscopy
Author(s) -
Jiang Jason,
Kuo Ivana,
AbiJaoude Joanne,
Lemos Fernanda,
Yang Yi,
Curci Silvana,
Ehrlich Barbara,
Hofer Aldebaran
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb632
Subject(s) - vasopressin , intracellular , vasopressin receptor , polycystic kidney disease , arginine vasopressin receptor 2 , receptor , endocrinology , creb , medicine , microbiology and biotechnology , biology , chemistry , pharmacology , kidney , biochemistry , antagonist , transcription factor , gene
Elevated intracellular cAMP is a key factor in cyst enlargement in the inherited disorder known as polycystic kidney disease (PKD). Therapeutic strategies are actively being sought to reduce cAMP production in cells lining the cyst. Antagonists of the cAMP‐linked V2R vasopressin receptor such as the competitive inhibitor tolvaptan have been assessed in clinical trials for the treatment of PKD, albeit with inconsistent results. Here we used a sensitive, new‐generation circularly permuted ratiometric cAMP sensor to monitor cAMP signals in response to arginine vasopressin (AVP) in LLC‐PK1 cells. This probe proved valuable both in epifluorescence measurements of cAMP in 2D cultures, as well as in Lightsheet microscopy of polarized 3D spheroids. We observed that brief (5 min.) treatment with AVP induced a long‐lasting cAMP increase (more than 1 hr), which was not reversed by washing or by acute addition of tolvaptan, as measured in both 2D and 3D cultures. In contrast, pretreatment with tolvaptan completely prevented the AVP response. The persistent intracellular cAMP increase triggered by AVP also led to primary cilia elongation and phosphorylation of downstream PKA targets such as CREB. Our results are consistent with a model in which endogenous V2R receptors continue to signal from endosomal compartments (as described previously for overexpressed V2R). Our results may have implications for optimizing dosing regimens for V2R antagonists, with increased efficacy predicted in hydrated (i.e. low circulating AVP) patients.