LY-294002 [2-(4-Morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one] Affects Calcium Signaling in Airway Smooth Muscle Cells Independently of Phosphoinositide 3-Kinase Inhibition

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Abstract
Phosphoinositide 3-kinase (PI3K) may potentially influence intracellular [Ca2+]i concentration by several mechanisms. We have investigated the effects of phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] on Ca2+ signaling in rat airway smooth muscle (ASM) cells using fura-2 and imaging methodology. Wortmannin (1 μM) and LY-294002 (1 and 10 μM) had opposite effects: wortmannin caused a small increase, whereas LY-294002 caused a significant decrease of peak Ca2+ responses to serotonin (5-HT). LY-294002 (10 μM) diminished 5-HT-induced ASM cell contraction, measured as a change in cell surface area, and inositol phosphate formation, measured by anion exchange chromatography. Thin layer chromatography revealed that the levels of phospholipase C (PLC) substrate phosphatidylinositol 4,5-bisphosphate were not affected. SDS polyacrylamide gel electrophoresis and Western blotting have shown that both wortmannin and LY-294002 inhibited platelet-derived growth factor-induced PI3K activation. However, PI3K activation could not be detected after 5-HT stimulation. The specific casein kinase-2 (CK2) inhibitor 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (10-40 μM) reduced 5-HT-triggered responses to a similar extent as LY-294002. We conclude that LY-294002 modulates Ca2+ signaling in rat ASM independently of its action on PI3K by acting on, or upstream of, PLC, possibly by inhibiting CK2.