EPAC drives the anti-tussive effect of EP4 receptors on airway sensory nerves

Chronic cough is a burden of many respiratory diseases with no effective & safe treatment. The prostaglandin PGE2 acts on EP receptors (EP1-4) with beneficial & adverse effects in the lungs. We have shown EP4 agonism inhibits activation of airway sensory nerves, blocking cough1. We aimed to determine the signalling downstream of EP4 in airway sensory nerves, to better understand its anti-tussive effect. We isolated guinea pig (GP) vagal ganglia neurons, using retrograde tracer dye to identify airway neurons in vitro. We assessed intracellular calcium & cAMP in these neurons using chemical & virally transfected fluorophores. We also used a grease-gap recording technique to pharmacologically examine EP4 signalling in GP & human vagus nerves in vitro. We have shown that EP4 agonist inhibits capsaicin-induced activation of GP airway neurons. EP4 is known to be a Gαs-coupled GPCR, so we imaged cAMP, observing that EP4 agonism causes cAMP elevation. Dual-fluorophore imaging showed cAMP elevation in neurons where EP4 agonism had inhibitory effects. From previous work we know EP4 agonist inhibits capsaicin-induced GP & human vagus nerve activation. We looked whether the main cAMP effectors (PKA & EPAC) were involved. EPAC (but not PKA) blockers (ESI-09 & KT5720) abolished the inhibitory effect of EP4 agonism. EPAC activator (8-CPT-2Me-cAMP) mimicked the effect of EP4 agonism; this was blocked by EPAC inhibitor. These data reveal a role for cAMP & EPAC in the inhibitory & anti-tussive effects of EP4 agonism on airway sensory nerves. Understanding how EP4 exerts its beneficial effects on airway sensory nerves is important to better inform drug discovery efforts for anti-tussive therapies. 1.Wortley(2019)AJRCCM;199:A2862