What controls calcium homeostasis in human airway smooth muscle?

Asthma is a major cause of lost working days in the UK: currently, according to Asthma UK 5.2 million are taking asthma treatment. The majority of symptoms are caused by narrowing of the airways. A major component of airway narrowing is due to contraction of the muscle (called airway smooth muscle) in the airway wall. This contraction is due to calcium release within the muscle cell or entry of calcium into the muscle cell. The mechanisms underlying these responses are poorly understood. This project will systematically define the important pathways and signalling molecules responsible for these processes. By defining these mechanisms, it should be feasible to design new treatments for asthma. We will also gain a fuller understanding of the mechanisms underlying the development of asthma.

Short term changes in the calibre of the human airway are primarily influenced by the tone of the airway smooth muscle (ASM) bundles found in the airway wall. Contraction of ASM produces bronchoconstriction. In contrast relaxation produces bronchodilation, a response which underlies the mechanism of action of beta2 adrenoceptor agonists, the most widely used agents for symptom relief in asthma and COPD. Thus understanding the mechanisms underlying contraction and relaxation of the ASM cell is critical to the study of the pathophysiology of airway disease. Recent studies including those by the applicant using siRNA and other approaches has led to the recognition of Ca2+ homeostasis being fundamental to these processes. The hypotheses to be examined in this programme of work are therefore as follow:

(i) contraction of airway smooth muscle (ASM) is dependent upon Ca2+ entry via both receptor and store dependent pathways
(ii) signalling of Ca2+ entry into ASM following store depletion is mediated by STIM1 and Orai1 induced activation of as yet undefined Ca2+ entry channels
(iii) agonist induced Ca2+ entry is dependent in part on store dependent pathways but also involves separate, receptor activated pathways, involving trp channels and related homologues
(iv) using a systematic siRNA based approach in a range of physiologically relevant model systems including human primary cultured cell and lung slice models these pathways can be defined
To address the role of these pathways in ASM contraction, we will (i) delineate Ca2+ entry pathways in primary cultured human ASM cells using both measurement of intracellular Ca2+ and electrophysiological (patch clamp) approaches, and (ii) delineate the contribution of these pathways to ASM contraction using lung slice preparations from murine and human lung to simultaneously monitor ASM Ca2+ oscillations and contractile responses. Inhibition of components potentially important in contractile signalling will be achieved by use of selective antagonists (where available), siRNA approaches using a panel of characterised siRNA species we are developing and with which we have undertaken preliminary proof of principle work, and exploratory screening using two siRNA libraries targeted at ion channels and Ca2+ signalling pathways.