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Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells
B Tolloczko1, F C Tao, M E Zacour
1Seymour Heisler Laboratory of the Montreal Chest Institute Research Centre and Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada.
Abstract:
Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.
Insights
Contractile agonists activate tyrosine kinases in airway smooth muscle, influencing calcium signaling. This study reveals tyrosine kinases modulate calcium responses, independent of mitogen-activated protein kinase pathways.
Area of Science:
- Pharmacology
- Cell Signaling
- Smooth Muscle Physiology
Background:
- Contractile agonists can trigger mitogenic responses in airway smooth muscle via tyrosine kinases.
- The precise role of tyrosine kinase activation in contractile signaling by agonists remains unclear.
Purpose of the Study:
- To investigate the role of contractile agonist-evoked tyrosine kinase activation in airway smooth muscle signaling.
- To elucidate the mechanisms by which serotonin (5-HT) affects calcium (Ca2+) signaling in rat airway smooth muscle cells.
Main Methods:
- Cultured rat airway smooth muscle cells were used to study responses to serotonin (5-HT).
- Inhibitors of tyrosine kinase (genistein, tyrphostin 23) and mitogen-activated protein kinase kinase (PD-98059) were employed.
- Calcium (Ca2+) levels, tyrosine phosphorylation, and inositol phosphate production were measured.
Main Results:
- Serotonin (5-HT) induced contraction and increased intracellular Ca2+, effects blocked by genistein.
- Genistein and tyrphostin 23 reduced 5-HT-evoked Ca2+ responses; genistein's effect was observed without extracellular Ca2+.
- 5-HT increased tyrosine phosphorylation of ~70 kDa proteins, inhibited by genistein, and attenuated 5-HT-induced inositol phosphate production.
Conclusions:
- Contractile agonists activate tyrosine kinases in airway smooth muscle, modulating Ca2+ signaling.
- Tyrosine kinases likely affect phospholipase C-beta (PLC-beta) activity, influencing Ca2+ release.
- The mitogen-activated protein kinase pathway and phospholipase C-gamma (PLC-gamma) are not significantly involved in 5-HT-induced Ca2+ signaling.