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Background: Prostaglandin E 2 (PGE 2 ) increases pulmonary vascular permeability by activation of the PGE 2 receptor 3 (EP 3 ), which may explain adverse pulmonary effects of the EP 1 /EP 3 receptor agonist sulprostone in patients. In addition, PGE 2 contributes to pulmonary oedema in response to platelet-activating factor (PAF). PAF increases endothelial permeability by recruiting the cation channel transient receptor potential canonical 6 (TRPC6) to endothelial caveolae via acid sphingomyelinase (ASMase). Yet, the roles of PGE 2 and EP 3 in this pathway are unknown. We hypothesised that EP 3 receptor activation may increase pulmonary vascular permeability by activation of TRPC6, and thus, synergise with ASMase-mediated TRPC6 recruitment in PAF-induced lung oedema., Methods: In isolated lungs, we measured increases in endothelial calcium (ΔCa 2+ ) or lung weight (Δweight), and endothelial caveolar TRPC6 abundance as well as phosphorylation., Results: PAF-induced ΔCa 2+ and Δweight were attenuated in EP 3 -deficient mice. Sulprostone replicated PAF-induced ΔCa 2+ and Δweight which were blocked by pharmacological/genetic inhibition of TRPC6, ASMase or Src-family kinases (SrcFK). PAF, but not sulprostone, increased TRPC6 abundance in endothelial caveolae. Immunoprecipitation revealed PAF- and sulprostone-induced tyrosine-phosphorylation of TRPC6 that was prevented by inhibition of phospholipase C (PLC) or SrcFK. PLC inhibition also blocked sulprostone-induced ΔCa 2+ and Δweight, as did inhibition of SrcFK or inhibitory G-protein (G i ) signalling., Conclusions: EP 3 activation triggers pulmonary oedema via G i -dependent activation of PLC and subsequent SrcFK-dependent tyrosine phosphorylation of TRPC6. In PAF-induced lung oedema, this TRPC6 activation coincides with ASMase-dependent caveolar recruitment of TRPC6, resulting in rapid endothelial Ca 2+ influx and barrier failure., Competing Interests: Conflict of interest: T. Jiang has nothing to disclose. Conflict of interest: R. Samapati has nothing to disclose. Conflict of interest: S. Klassen has nothing to disclose. Conflict of interest: D. Lei has nothing to disclose. Conflict of interest: V. Jankowski has nothing to disclose. Conflict of interest: S. Simmons has nothing to disclose. Conflict of interest: J. Yin has nothing to disclose. Conflict of interest: C. Arenz has nothing to disclose. Conflict of interest: A. Dietrich has nothing to disclose. Conflict of interest: T. Gudermann has nothing to disclose. Conflict of interest: D. Adam has nothing to disclose. Conflict of interest: M. Schaefer has nothing to disclose. Conflict of interest: J. Jankowski has nothing to disclose. Conflict of interest: V. Flockerzi has nothing to disclose. Conflict of interest: R. Nüsing has nothing to disclose. Conflict of interest: S. Uhlig has nothing to disclose. Conflict of interest: W.M. Kuebler has nothing to disclose., (Copyright ©The authors 2022. For reproduction rights and permissions contact permissions@ersnet.org.)