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The roles of protein-tyrosine kinases in neutrophil and airway smooth muscle cell activation

University of British Columbia

Asthma is an inflammatory lung disease involving reversible airway obstruction. The characteristic features of asthma include eosinophilia, nonspecific bronchial hyperresponsiveness to inhaled spasmogens, airway epithelial damage, mucosal edema, and increased mucosal gland secretion. Two cellular components involved in this complex respiratory disease are neutrophils which are the major contributer to pulmonary inflammation and airway smooth muscle cells that are the ultimate responding cells to the released inflammatory mediators. In this study, the mechanisms of cellular activation of these two cell types are examined. Cellular stimulation involves receptor-induced activation of intracellular signalling pathways that are potential sites of intervention and targets for future drug design. Protein-tyrosine kinase activity has been demonstrated in neutrophils, although the role of these enzymes in neutrophil activation is not clearly defined. The effect of protein-tyrosine kinase inhibitors were used to determine the site of action of this class of compounds that are capable of inhibiting neutrophil activation. The rise in intracellular calcium is an important link between receptor activation and cellular stimulation in neutrophils. Changes in Fura-2 fluorescence has been used to determine the efficacy of several protein-tyrosine kinase inhibitors to determine the most potent inhibitor of neutrophil activation. α-Cyano-3,4- dihydroxythiocinnamamide (DHC) was found to be the most active inhibitor and was selected to be used for further investigations. Other assays used to assess the activity of this drug as an inhibitor of neutrophil activation include measurement of superoxide production and elastase release. Both these agonist-stimulated pathophysiological activities were diminished by DHC, suggesting that proteintyrosine kinase inhibitors have pharmacologically beneficial effects in the control of neutrophil activation and possibly inflammatory reactions. Protein-tyrosine kinase activation in neutrophils was detected by immunoblotting using an antiphosphotyrosine specific antibody and decreases in immunoreactivity were observed in tyrphostin-treated cells. DHC was found to have no effect on binding affinity or receptor number although decreased polyphosphatidylinositol hydrolysis and protein kinase C activation suggesting that the site of action was preceding PLC activation. Airway smooth muscle contraction leads to decreased airflow due to airway narrowing. A variety of external factors control signal transduction pathways leading to smooth muscle contraction. Guinea pig airway smooth muscle contraction in response to contractile agonists was diminished by a protein-tyrosine kinase inhibitor, suggesting that this class of enzyme has a role in smooth muscle contraction. On antiphosphotyrosine immunoblots, tyrosine phosphorylated substrates were identified in cultured canine airway smooth muscle cells. Also, phosphorylation of a copolymer containing poly(Glu, Tyr) 4:1 by smooth muscle extracts implies that these cells contain protein-tyrosine kinase activity that is dependent on magnesium and manganese. Using anion exchange chromatography, a major peak of protein-tyrosine kinase activity as well as lower activity peaks were obtained from both cytosolic and NP-40 solubilized particulate smooth muscle extracts. Gel filtration chromatography resolved an ~60 kDa cytosolic peak of protein-tyrosine kinase activity and a high molecular weight (~400-500 kDa) peak was fractionated from NP-40 solubilized particulate extracts. Therefore, detection of protein-tyrosine kinase activity in neutrophils and smooth muscle cells provides a potential site to inhibit the activation of these cells and control asthmatic symptoms.

Thesis/Dissertation

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2008-12-15

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http://hdl.handle.net/2429/2908

Experimental Medicine

University of British Columbia

UBCV

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