PDE8 Activity Regulates cAMP Signaling by β2AR But Not Prostanoid EP2 Nor EP4 Receptors In Human Airway Smooth Muscle

Abstract

Human airway smooth muscle (HASM) cells exhibit two distinct cAMP signaling compartments centered around adenylyl cyclase (AC), one containing β2AR‐coupled to AC6 and another configured with E prostanoid receptors (EPR) coupled to AC2. Different cell types express a unique complement of PDE isoforms that create unique spatial and kinetic properties of cAMP signals so it may be possible to selectively modulate cAMP signaling at the PDE level based on localization to cAMP signalosomes. We previously reported that PDE8A is abundantly expressed in HASM and that shRNA knockdown of its expression enhanced forskolin stimulated cAMP responses. We sought to determine if PDE8 is specifically localized such that it regulates signaling by only certain GPCR. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin‐1 and AC5/6, indicating PDE8 is spatially associated with β2AR signaling complexes. We examined real‐time cAMP dynamics in live HASM cells using the downward cAMP difference detector in situ (cADDis) cAMP sensor (Montana Molecular, Bozeman MT). We observed large, rapid decreases in fluorescence of the downward cADDis sensor in response to various concentrations of forskolin without the addition of IBMX or other PDE inhibitors. PF‐04957325, a PDE8‐selective inhibitor, dose dependently decreased sensor fluorescence over 30 minutes, indicating some basal level of cAMP synthesis. We then measured cAMP responses to various concentrations of either isoproterenol or PGE2, with or without 1 μM PF‐04957325. PF‐04957325 increased 1nM isoproterenol‐stimulated cAMP responses in a concentration‐dependent manner, but PGE2‐stimulated cAMP production was not enhanced by the PDE8 inhibitor. HASM express both EP2 and EP4 receptors, so we used specific antagonists along with PGE2 stimuli to dissect the proportion of the PGE2 response could be attributed to each receptor subtype. PGE2 concentration response curves were significantly dampened by the inclusion of the EP2 receptor antagonist, PF‐04418948 (100 nM). Only small reductions in cAMP responses stimulated by PGE2 were observed with the inclusion of the EP4R antagonist, GW‐627368X (100 nM). These results are consistent with the idea that EP2 receptors predominate over EP4 receptors in HASM. Inclusion of 1 μM PF‐04957325 did not increase either EP2‐ or EP4‐mediated cAMP increases, confirming that PDE8 does not regulate signaling by either of these receptors. We conclude that β2AR signaling microdomains appear to contain functionally responsive PDE8 while EP2R microdomains do not. In airway diseases such as asthma and COPD, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.Support or Funding InformationThis work was supported by NIH grant GM107094 (RO)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.