Phosphoinositide 3-kinase (PI3K) may potentially influence intracellular [Ca 2ϩ ] 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 Ca 2ϩ signaling in rat airway smooth muscle (ASM) cells using fura-2 and imaging methodology. Wortmannin (1 M) and LY-294002 (1 and 10 M) had opposite effects: wortmannin caused a small increase, whereas LY-294002 caused a significant decrease of peak Ca 2ϩ responses to serotonin (5-HT). LY-294002 (10 M) 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 plateletderived 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--D-ribofuranosyl-benzimidazole (10 -40 M) reduced 5-HT-triggered responses to a similar extent as LY-294002. We conclude that LY-294002 modulates Ca 2ϩ signaling in rat ASM independently of its action on PI3K by acting on, or upstream of, PLC, possibly by inhibiting CK2.
We tested the hypothesis that, in airway smooth muscle cells, stimulation of G-protein-coupled receptors by contractile agonists activates Src kinase and that this kinase modulates cell contractility and Ca2+ signaling by affecting the levels of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PIP2). Stimulation of cultured rat tracheal smooth muscle cells with serotonin (5-HT) induced an increase in Src activity, Ca2+ mobilization, and contraction (decrease in cell area). 5-HT-evoked cell contraction was reduced by a specific inhibitor of Src family kinases, 4-amino-5(4-methylphenyl)-7-( t-butyl)pyrazolo[3,4 -d]pyrimidine (PP1). Peak Ca2+ responses to 5-HT were attenuated by PP1 and an anti-Src-blocking antibody and augmented by expression of constitutively activated Y529F Src. Sustained phases of Ca2+ responses to 5-HT and Ca2+ influx resulting from emptying of Ca2+ stores in the endoplasmic reticulum by thapsigargin were also decreased after PP1 treatment. PP1 significantly reduced the turnover of inositol phosphates produced on 5-HT stimulation and the amount of PIP2 in the Triton X-100-insoluble lipid fraction. Overall, these data demonstrate that, in rat tracheal smooth muscle cells, Src kinase modulates 5-HT-evoked cell contractility and Ca2+ signaling by regulating PIP2 levels and Ca2+ influx.
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