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Compartmentalized cAMP Response to Prostaglandin EP2 Receptor Activation in Human Airway Smooth Muscle Cells
Author(s) -
Harvey Robert D,
Miyashiro Kathryn,
Agarwal Shailesh R,
Latt Htun,
Ostrom Rennolds S
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.710.13
Subject(s) - rolipram , prostaglandin e2 receptor , adenylyl cyclase , agonist , lipid raft , chemistry , receptor , microbiology and biotechnology , camp dependent pathway , phosphodiesterase , biology , biochemistry , enzyme
It has been previously demonstrated that prostaglandin EP2 receptors selectively couple to adenylyl cyclase type 2 in non‐lipid raft domains of human airway smooth muscle (HASM) cells, where they regulate specific cellular responses. In the present study, we used FRET‐based biosensors targeted to different subcellular locations to determine exactly where EP2 receptor production of cAMP occurs in these cells. The Epac2‐camps biosensor, which is expressed throughout the cell, was used to measure bulk cytoplasmic responses, while MyrPalm‐Epac2 and CAAX‐Epac2 were targeted to lipid raft and non‐lipid raft regions of the plasma membrane, and NLS‐Epac2 was targeted to the nucleus. All probes were able to detect cAMP responses produced by direct activation of adenylyl cyclase with forskolin or exposure to the beta‐adrenergic receptor agonist isoproterenol. On the other hand, butaprost, an EP2 receptor agonist, elicited a response that could be detected by CAAX‐Epac2, but not MyrPalm‐Epac2. The butaprost response could also be detected by Epac2‐camps, as well as NLS‐Epac2. Exposure to rolipram, a selective inhibitor of phosphodiesterase type 4 (PDE4), produced no effect on its own that could be detected by any of the probes. However, in the presence of rolipram, exposure to butaprost produced a response that could then be detected by the MyrPalm‐Epac2 probe. Rolipram also enhanced the butaprost responses detected by Epac2‐camps and CAAX‐Epac2. These results support the conclusion that EP2 receptor activation leads to cAMP production in subcellular compartments associated specifically with non‐lipid raft regions of the plasma membrane. Furthermore, the activity of PDE4 appears to play a role in maintaining the integrity of this compartmentalized response. Support or Funding Information Supported by NIH (GM107094)