Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells.
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.
Tolloczko
B
B
Seymour Heisler Laboratory of the Montreal Chest Institute Research Centre and Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada.
Tao
F C
FC
Zacour
M E
ME
Martin
J G
JG
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D007295:Inositol Phosphates / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009124:Muscle Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051966:Phospholipase C gamma; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D011916:Rats, Inbred F344; D012701:Serotonin / Q000494:pharmacology; D014132:Trachea / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D007527:Isoenzymes; D009124:Muscle Proteins; D020032:Tyrphostins; D012701:Serotonin; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium