Compartmentalized cAMP responses to prostaglandin EP 2 receptor activation in human airway smooth muscle cells

Br J Pharmacol. 2017 Aug;174(16):2784-2796. doi: 10.1111/bph.13904. Epub 2017 Jul 12.

Abstract

Background and purpose: Previous studies indicate that prostaglandin EP2 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 EP2 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 β2 -adrenoceptors with isoprenaline increased cAMP in all subcellular locations. Activation of EP2 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 EP2 receptors to increase cAMP levels associated with lipid raft domains. Overexpression of AC2 selectively increased EP2 receptor-stimulated production of cAMP in non-raft membrane domains.

Conclusions and implications: EP2 receptor activation of AC2 leads to cAMP production in non-raft and nuclear compartments of human ASMs, while β2 adrenoceptor signalling is broadly detected across microdomains. The activity of PDE4 appears to play a role in maintaining the integrity of compartmentalized EP2 receptor responses in these cells.

MeSH terms

  • Biosensing Techniques
  • Bronchi / cytology
  • Cells, Cultured
  • Cyclic AMP / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Guanine Nucleotide Exchange Factors
  • Humans
  • Myocytes, Smooth Muscle / metabolism*
  • Receptors, Prostaglandin E, EP2 Subtype / metabolism*
  • Trachea / cytology

Substances

  • Guanine Nucleotide Exchange Factors
  • PTGER2 protein, human
  • RAPGEF4 protein, human
  • Receptors, Prostaglandin E, EP2 Subtype
  • Cyclic AMP