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Epac-mediated Ca²⁺ mobilization and exocytosis in inner medullary collecting duct

Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida

Submitted 18 October 2005 ; accepted in final form 7 May 2006

PKA has traditionally been thought as the binding protein of cAMP for mediating arginine vasopressin (AVP)-regulated osmotic water permeability in kidney collecting duct. It is now known that cAMP also exerts its effects via Epac (exchange protein directly activated by cAMP) and that intracellular Ca²⁺ mobilization is necessary for AVP-induced apical exocytosis in inner medullary collecting duct (IMCD). The role of Epac as an effector of cAMP action in addition to PKA was investigated using confocal fluorescence microscopy in perfused IMCD. PKA inhibitors (1 µM H-89 or 10 µM KT-5720) at concentrations known to inhibit aquaporin-2 (AQP2) phosphorylation did not prevent AVP-induced Ca²⁺ mobilization and oscillations. Epac-selective cAMP agonist (8-pCPT-2'-O-Me-cAMP) mimicked AVP in triggering Ca²⁺ mobilization and oscillations, which was blocked by ryanodine but not by Rp-cAMP (a competitive antagonist of cAMP binding to PKA). 8-pCPT-2'-O-Me-cAMP also triggered apical exocytosis in the presence of a PKA inhibitor. Immunolocalization of AQP2 in perfused IMCD demonstrated that 8-pCPT-2'-O-Me-cAMP induces apical targeting of AQP2 and that AQP2 is abundant in junctional regions of basolateral membrane. Immunofluorescence study also confirmed the presence of Epac (isoform I) in IMCD. These results indicate that activation of Epac by an exogenous cAMP analog triggers intracellular Ca²⁺ mobilization and apical exocytotic insertion of AQP2 in IMCD.

aquaporin-2; vasopressin; FM1–43; fluorescence confocal microscopy

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