Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4,5-bisphosphate.
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 Ca(2+) signaling by affecting the levels of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PIP(2)). Stimulation of cultured rat tracheal smooth muscle cells with serotonin (5-HT) induced an increase in Src activity, Ca(2+) 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 Ca(2+) 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 Ca(2+) responses to 5-HT and Ca(2+) influx resulting from emptying of Ca(2+) 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 PIP(2) 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 Ca(2+) signaling by regulating PIP(2) levels and Ca(2+) influx.
Tolloczko
Barbara
B
Seymour Heisler Laboratory of Montreal Chest Institute Research Centre and Meakins Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada H2X 2P2.
Turkewitsch
Petra
P
Choudry
Sofia
S
Bisotto
Sandra
S
Fixman
Elizabeth D
ED
Martin
James G
JG
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases; D051381:Rats; D011916:Rats, Inbred F344; D012701:Serotonin / Q000494:pharmacology*; D013268:Stimulation, Chemical; D014132:Trachea; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000906:Antibodies; D004791:Enzyme Inhibitors; D019269:Phosphatidylinositol 4,5-Diphosphate; D012701:Serotonin; D019061:src-Family Kinases; D010749:Phosphoprotein Phosphatases