Investigating the role of phosphodiesterases (PDE) 3 and 4 in the activation of protein kinase A (PKA) in superior cervical ganglia using FRET

cAMP‐PKA signaling regulated by PDE activity induces release of transmitters from superior cervical ganglia (SCG) neurons. We used fluorescence resonance energy transfer (FRET) based imaging to investigate the role of PDE3 and PDE4 in cAMP/PKA activity in these neurons. Primary cultures of dissociated SCG neurons were obtained from SD rats at postnatal day 8. After 2 days of culture, SCG neurons were infected with an adenovirus containing A‐kinase activity reporter (AKAR3). SCG neurons (n=6) were imaged at 37°C and sequentially treated with rolipram (ROL, 1uM, selective PDE4 inhibitor), milrinone (MIL, 10uM, selective PDE3 inhibitor), IBMX (100uM, non‐selective PDE inhibitor), Forskolin (FSK, 50uM, transmembrane adenylate cyclase activator) and H89 (10uM, selective PKA inhibitor). Two different responses were observed: 1) high PDE4 activity (n=3) with 20.0±0.43% change in emission ratio in response to ROL with no further increase with MIL, IBMX and FSK, 2) high PDE3 activity (n=3) with 1.9±0.6 % change in response to ROL and 19.0±7.2 % change in response to MIL with no further increase with IBMX and FSK (figure shows representative curves). Addition of H89 reversed the response to baseline in cells with high PDE4 and 3 activities. CONCLUSIONS SCG neurons show basal activity of different PDE isoforms, which might be a mechanism that regulates neurotransmitter release in the sympathetic nervous system. ARN funded by SFRH/BD/39473/2007