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Articles in PresS, published online ahead of print January 4, 2002
Am J Physiol Lung Cell Mol Physiol, 10.1152/ajplung.00304.2001
Submitted on August 3, 2001
Accepted on December 28, 2001
1 Seymour Heisler Laboratory, MUHC, MCI Research Center, Montreal, Quebec, Canada
2 Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada
3 Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada; Seymour Heisler Laboratory, MUHC, MCI Research Center, Montreal, Quebec, Canada
* To whom correspondence should be addressed. E-mail: barbara.tolloczko{at}mcgill.ca.
We tested the hypothesis that in airway smooth muscle cells stimulation of G-protein-coupled receptors (GPCRs) by contractile agonists activates Src kinase and that this kinase modulates cell contractility and calcium (Ca2+) signalling by affecting the levels of phospholipase C (PLC) 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+ mobilisation 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 as well as an anti-Src blocking antibody and augmented by expression of constitutively activated, Y529F Src. Sustained phase of Ca2+ responses to 5-HT as well as 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 (IPs) produced upon 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+ signalling by regulating PIP2 levels and Ca2+ influx.
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