Inhibition of cAMP stimulated VAMP2 trafficking in renal epithelial cells by small molecule inhibitors (1181.13)

cAMP stimulates exocytosis in many cell types in the kidney. In juxtaglomerular cells, cAMP stimulates renin exocytosis. In thick ascending limbs and collecting ducts, cAMP stimulates exocytic delivery of NKCC2 and aquaporin 2, respectively. The mechanism by which this occurs includes the vesicle associated membrane protein VAMP2. New compounds aimed at inhibiting VAMP2‐mediated exocytosis could be useful to target these renal processes. We hypothesized that delphinidin, a small molecule that binds VAMP2, and 4‐AD, an inhibitor of aquaporin 2 trafficking, block cAMP‐stimulated VAMP2 trafficking to the surface in renal epithelial cells. We expressed C‐terminus GFP tagged VAMP2 (GFP faces vesicle lumen or surface) in LLC‐PK 1 cells. Confocal imaging showed that in polarized cells, VAMP2‐GFP localized to the apical surface, primarily in small clusters. We then measured cAMP‐stimulated surface VAMP2 delivery by surface biotinylation. We used forskolin/IBMX to maximally stimulate cAMP. In vehicle treated cells cAMP enhanced surface VAMP2‐GFP by 173±57% in 8 min (p<0.02). In cells pre‐treated with delphinidin chloride (50 µM) cAMP did not increase surface VAMP2‐GFP (baseline= 100, cAMP= 95±8%). Similarly, when cells were pretreated with 4‐AD (40 µM), cAMP failed to increase surface VAMP2‐GFP (baseline= 100, cAMP=108±14%). We concluded that cAMP stimulates VAMP2‐GFP trafficking to the surface in renal epithelial cells, most likely as part of the exocytic process. The small molecule delphinidin, and the V‐ATPase inhibitor 4‐AD block cAMP‐stimulated VAMP2 trafficking. Grant Funding Source : Supported by NIH, AHA