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

BACKGROUND AND PURPOSE

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

CONCLUSIONS AND IMPLICATIONS

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