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 repetitive 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). Activation 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 frequency 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 Ca²⁺ oscillations in murine ASMC via stimulation of EP₂Rs and a mechanism involving activation of PKA and EPAC.