Modulation of carbachol-induced Ca2+ oscillations in airway smooth muscle cells by PGE2

PGE2 is a potent bronchodilator, but the mechanisms underlying this effect have not been fully elucidated. Acetylcholine-induced contractions of airway smooth muscle (ASM) are associated with the generation of re- petitive Ca2+ oscillations in airway smooth muscle cells (ASMC) and the force of contraction is positively correlated with the frequency of the underlying Ca2+ oscillations. The purpose of the present study was to examine if carbachol-evoked Ca2+ oscillations in isolated ASMC were inhibited by PGE2.
Isolated murine ASMC loaded with fluo4-AM were imaged with a Nipkow spinning disk confocal microscope. Cells responded to application of CCh (1 μM) by generating an initial Ca2+ transient followed by a series of Ca2+ oscillations. This activity was abolished by PGE2 (300 nM) and the EP2R agonist (R)-butaprost (3 μM) and the inhibitory effects of PGE2 were reversed by application of the EP2R antagonist PF-04418948 (100 nM). Acti- vation of adenylate cyclase using forskolin (1 μM) mimicked the effects of PGE2. The PKA activator, 6-MB-cAMP (300 μM) reduced the frequency of CCh-induced Ca2+ oscillations by 33% and the PKA inhibitor Rp-8-CPT- cAMPs partially reversed the inhibitory effects of PGE2. The EPAC activator 007-AM (10 μM) reduced the fre- quency of the oscillations by 60% and joint application of 007-AM and 6-MB-cAMP reduced oscillation frequency by ~85%. CCh-induced Ca2+ oscillations were inhibited by 2-APB and tetracaine, but caffeine-evoked Ca2+ transients were resistant to PGE2.
These data suggest that PGE2 inhibits CCh-induced Ca and a mechanism involving activation of PKA and EPAC.
oscillations in murine ASMC via stimulation of EP2Rs