Phosphodiesterase 4D forms a cAMP diffusion barrier at the apical membrane of the airway epithelium

Anthony Barnes, Gabriel Livera, Pingbo Huang, Chuanwen Sun, Wanda K. O'Neal, Marco Conti, M. Jackson Stutts, Sharon L. Milgram

Research output: Contribution to journalArticle

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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. PBE4 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.

Original languageEnglish (US)
Pages (from-to)7997-8003
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number9
DOIs
StatePublished - Mar 4 2005
Externally publishedYes

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Type 4 Cyclic Nucleotide Phosphodiesterase
Cystic Fibrosis Transmembrane Conductance Regulator
Diffusion barriers
Phosphoric Diester Hydrolases
Epithelium
Cyclic AMP-Dependent Protein Kinases
Membranes
Chemical activation
Adenosine
Adenosine A2B Receptors
Phosphodiesterase 4 Inhibitors
Rolipram
Chloride Channels
Transcription
Adenylyl Cyclases
Isoenzymes
Reverse Transcription
Western Blotting
Epithelial Cells
Immunohistochemistry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphodiesterase 4D forms a cAMP diffusion barrier at the apical membrane of the airway epithelium. / Barnes, Anthony; Livera, Gabriel; Huang, Pingbo; Sun, Chuanwen; O'Neal, Wanda K.; Conti, Marco; Stutts, M. Jackson; Milgram, Sharon L.

In: Journal of Biological Chemistry, Vol. 280, No. 9, 04.03.2005, p. 7997-8003.

Research output: Contribution to journalArticle

Barnes, A, Livera, G, Huang, P, Sun, C, O'Neal, WK, Conti, M, Stutts, MJ & Milgram, SL 2005, 'Phosphodiesterase 4D forms a cAMP diffusion barrier at the apical membrane of the airway epithelium', Journal of Biological Chemistry, vol. 280, no. 9, pp. 7997-8003. https://doi.org/10.1074/jbc.M407521200
Barnes, Anthony ; Livera, Gabriel ; Huang, Pingbo ; Sun, Chuanwen ; O'Neal, Wanda K. ; Conti, Marco ; Stutts, M. Jackson ; Milgram, Sharon L. / Phosphodiesterase 4D forms a cAMP diffusion barrier at the apical membrane of the airway epithelium. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 9. pp. 7997-8003.
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