Stimulation of the EP3 receptor causes lung oedema by activation of TRPC6 in pulmonary endothelial cells

Abstract

Background: Prostaglandin E₂ (PGE₂) increases pulmonary vascular permeability by activation of the PGE₂ receptor 3 (EP₃), which may explain adverse pulmonary effects of the EP₁/EP₃ receptor agonist sulprostone in patients. In addition, PGE₂ 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 PGE₂ and EP₃ in this pathway are unknown. We hypothesised that EP₃ 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 (ΔCa²⁺) or lung weight (Δweight), and endothelial caveolar TRPC6 abundance as well as phosphorylation.

Results: PAF-induced ΔCa²⁺ and Δweight were attenuated in EP₃-deficient mice. Sulprostone replicated PAF-induced ΔCa²⁺ 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 ΔCa²⁺ and Δweight, as did inhibition of SrcFK or inhibitory G-protein (G_i) signalling.

Conclusions: EP₃ 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 Ca²⁺ influx and barrier failure.