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Compartmentalized cAMP responses to prostaglandin EP 2 receptor activation in human airway smooth muscle cells
Author(s) -
Agarwal Shailesh R,
Miyashiro Kathryn,
Latt Htun,
Ostrom Rennolds S,
Harvey Robert D
Publication year - 2017
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.13904
Subject(s) - lipid raft , microbiology and biotechnology , raft , receptor , forskolin , phosphodiesterase 3 , chemistry , biology , signal transduction , biochemistry , protein kinase a , kinase , organic chemistry , copolymer , polymer
Background and Purpose Previous studies indicate that prostaglandin EP 2 receptors selectively couple to AC2 in non‐lipid raft domains of airway smooth muscle (ASM) cells, where they regulate specific cAMP‐dependent responses. The goal of the present study was to identify the cellular microdomains where EP 2 receptors stimulate cAMP production. Experimental Approach FRET‐based cAMP biosensors were targeted to different subcellular locations of primary human ASM cells. The Epac2‐camps biosensor, which expresses throughout the cell, was used to measure bulk cytoplasmic responses. Epac2‐MyrPalm and Epac2‐CAAX were used to measure responses associated with lipid raft and non‐raft regions of the plasma membrane respectively. Epac2‐NLS was used to monitor responses at the nucleus. Key Results Activation of AC with forskolin or β 2 ‐adrenoceptors with isoprenaline increased cAMP in all subcellular locations. Activation of EP 2 receptors with butaprost produced cAMP responses that were most readily detected by the non‐raft and nuclear sensors, but only weakly detected by the cytosolic sensor and not detected at all by the lipid raft sensor. Exposure to rolipram, a PDE4 inhibitor, unmasked the ability of EP 2 receptors to increase cAMP levels associated with lipid raft domains. Overexpression of AC2 selectively increased EP 2 receptor‐stimulated production of cAMP in non‐raft membrane domains. Conclusions and Implications EP 2 receptor activation of AC2 leads to cAMP production in non‐raft and nuclear compartments of human ASMs, while β 2 adrenoceptor signalling is broadly detected across microdomains. The activity of PDE4 appears to play a role in maintaining the integrity of compartmentalized EP 2 receptor responses in these cells.