Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium

Abstract

We demonstrated previously that Calu-3 airway epithelial cells sense adenosine on their luminal surface through adenosine A2B receptors coupled to adenylyl cyclase. Occupancy of these receptors leads to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel through protein kinase A (PKA) anchored at the apical membrane. Because luminal A2B receptor activation does not raise total cellular cAMP levels, we hypothesized that activation of phosphodiesterases (PDEs) confines cAMP generated by apical A2B receptors to a microdomain that includes the CFTR channel. Using reverse transcription-PCR, Western blotting, and activity measurements, PDE4D was identified as the major PDE species in airway epithelia. Consistent with these results, inhibitors of PDE4, but not PDE3, selectively abolished the lateral confinement of cAMP signaling in apical membrane patches during cell-attached recordings. Furthermore, stimulation of the CFTR in excised apical patches by rolipram and RS25344 indicated that PDE4 is localized in close proximity to the CFTR channel. Indeed, immunohistochemistry of human airway sections revealed that PDE4D is localized in the apical domain of the cell. PDE4 was activated after luminal adenosine exposure in a PKA-dependent manner. Because PDE4 activity is positively regulated by PKA, our results support a model whereby the PDE diffusion barrier is proportional to the degree of receptor stimulation. These findings underscore the concept that subcellular localization of individual PDE isozymes is an important mechanism for confining cAMP signaling to functional domains within cells.

Dynamics of Protein Kinase A Signaling at the Membrane, in the Cytosol, and in the Nucleus of Neurons in Mouse Brain Slices

“…Phosphodiesterases probably play a role in maintaining this low cAMP concentration in neurons because IBMX, a nonspecific blocker of phosphodiesterases, produces a strong AKAR2 ratio increase even in the absence of any agonist (our unpublished data). Phosphodiesterases have also been shown to play a major role in the confinement of the cAMP response in various other cell types (Jurevicius and Fischmeister, 1996;Rich et al, 2001;Rochais et al, 2004;Barnes et al, 2005). Furthermore, if cAMP concentration never reaches a concentration sufficient to activate PKA in the cytosol, the signal may be broadcasted by protein diffusion: either the free catalytic subunit of PKA diffuses from the membrane to the cytosol or AKAR2 diffuses from the cytosol to the cAMP signaling domains.…”

Characterizing the cancer genome in lung adenocarcinoma

“…Homogeneous mass extension (hME) genotyping for validation of sequencing candidates was performed in 96-well plates with up to 7-plex reactions. PCR was performed with final concentrations of 0.83 mM dNTPs, 1.563 of 103 buffer, 3.38 mM MgCl, 0.03 U ml 21 HotStar Taq (Qiagen), 0.10 mM PCR primers. Thermocycling was performed at 92 uC for 15 min, followed by 45 cycles of 92 uC for 20 s, 56 uC for 30 s and 72 uC for 1 min, with an additional extension at 72 uC for 3 min.…”

“…5). These deletions may be significant for lung biology because PDE4D encodes the major phosphodiesterase responsible for degrading cyclic AMP in airway epithelial cells 21 . Another single-gene deletion occurs within AUTS2, a gene of unknown function in chromosome 7q11.22 (Table 1 and Supplementary Table 8).…”

Mechanistic Insight into Control of CFTR by AMPK

“…For example, competitive interaction of EBP50-PKA and Shank2-PDE4D with CFTR has been demonstrated recently (19). In addition, Barnes and co-workers (20) demonstrated that phosphodiesterase 4D generates a cAMP diffusion barrier local to the apical membrane of the airway epithelium. It is therefore likely that activator pathways through cAMP and inhibitory AMP/AMPK signaling occur in a local CFTR-organized compartment.…”