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Date of Award

Spring 2012

Degree Type

Thesis

Degree Name

Master of Science (MSc)

Department

Medical Sciences (Division of Physiology/Pharmacology)

Supervisor

Luke Janssen

Language

English

Abstract

Introduction

Asthma is a chronic respiratory disease that is becoming more prevalent. Airway hyperresponsivness, a key feature of asthma, involves increased narrowing of the airways in response to bronchoconstricting agents. Airway smooth muscle (ASM) functioning is largely responsible for hyperresponsiveness yet the mechanisms behind excitation-contraction coupling are not fully understood. Src tyrosine kinase contributes to contraction in other smooth muscle types. Furthermore, STIM1, Orai1, IPLA2b and RyRs play a role in ASM excitation-contraction coupling.

Aim

We sought to determine whether Src activity is involved in serotonin (5-HT)- and acetylcholine (ACh)-induced ASM contraction. We also examined whether the gene expression of molecules involved in sarcoplasmic reticulum emptying and refilling is altered during airway hyperresponsiveness.

Methods

Bovine tracheal ASM strips were pre-treated with the non-specific tyrosine kinase inhibitor genistein (10-4 M), src kinase family inhibitors PP1 (10-5 M) and PP2 (10-5 M) or vehicle and challenged with either 5-HT or ACh to determine the involvment of Src in contraction. Western blotting was used to examine Src activity following 5-HT or ACh treatment. Female BALB/c mice were exposed to an intranasal injection of [1.7mg/ml] HDM extract or saline. Real time, reverse-transcriptase polymerase chain reaction was used to examine gene expression.

Results

Genistein, PP1 and PP2 significantly reduced 5-HT-induced ASM contractions and Src activity was significantly increased in response to 5-HT. ACh-induced contractions were significantly reduced by genistein, but not PP1 and PP2. However, Src activity was significantly increased by ACh. RyR3 mRNA expression was significantly increased, Orai1 was significantly decreased, and STIM1, IPLA2b, RyR1 and RyR2 were unchanged by the house dust mite treatment.

Conclusion

These data suggets 5-HT-induced ASM contraction involves Src activity. However, ACh-induced ASM contractions might not require Src. The changes in RyR3 and Orai1 expression might alter Ca2+-handling in such a way as to potentiate airway hyperresponsiveness but further investigation is required.

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