Modulation of carbachol-induced Ca oscillations in airway smooth muscle cells by PGE.

PGE 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 repetitive Ca oscillations in airway smooth muscle cells (ASMC) and the force of contraction is positively correlated with the frequency of the underlying Ca oscillations. The purpose of the present study was to examine if carbachol-evoked Ca oscillations in isolated ASMC were inhibited by PGE. 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 Ca transient followed by a series of Ca oscillations. This activity was abolished by PGE (300 nM) and the EPR agonist (R)-butaprost (3 μM) and the inhibitory effects of PGE were reversed by application of the EPR antagonist PF-04418948 (100 nM). Activation of adenylate cyclase using forskolin (1 μM) mimicked the effects of PGE. The PKA activator, 6-MB-cAMP (300 μM) reduced the frequency of CCh-induced Ca oscillations by 33% and the PKA inhibitor Rp-8-CPT-cAMPs partially reversed the inhibitory effects of PGE. The EPAC activator 007-AM (10 μM) reduced the frequency of the oscillations by 60% and joint application of 007-AM and 6-MB-cAMP reduced oscillation frequency by ∼85%. CCh-induced Ca oscillations were inhibited by 2-APB and tetracaine, but caffeine-evoked Ca transients were resistant to PGE. These data suggest that PGE inhibits CCh-induced Ca oscillations in murine ASMC via stimulation of EPRs and a mechanism involving activation of PKA and EPAC.