Despite the importance of airway smooth muscle (ASM) excitability in reactive airway disease, very little information is currently available with respect to the precise ionic mechanisms which mediate the neurotransmitter induced excitation of this tissue. Further, the cellular mechanism or mechanisms by which important mediators of bronchoconstriction such as leukotrienes and histamine trigger contraction in airway smooth muscle is not known. In order to understand the processes by which neurohumoral agents trigger contraction and alter excitability in ASM, it is necessary to have a complete knowledge of the membrane ion channels, and to determine the effect of these agents on channel function. We hypothesize that modulation of ion channel function by neurohumoral agents released by inflammatory cells in the airways plays an important role in the development of ASM hyperreactivity. This proposal seeks to apply recently developed advances in cell disaggregation, smooth muscle cell culture, and patch clamp recording techniques to the understanding of these processes in ASM. Single-cell patch clamp experiments will provide a detailed view of ion channel function at the cellular level. Additionally, specific ion fluxes mediated by ion channels will be investigated on a larger scale through the use of radiolabeled ions.
The specific aims of these studies are thus to: (1) further characterize electrophysiological and pharmacological properties of calcium and potassium channels in ASM cells using cell dissociation, and whole-cell patch clamp methods; (2) identify the compounds of 45Ca++ and 42K++ flux in primary cultures of ASM cells monolayers that are attributable to voltage-dependent ion channel activity; (3) determine the influence of neurotransmitters, inflammatory autacoids, and other bronchoactive substances on specific ion channel currents and ion channel fluxes in acutely dissociated and cultured ASM cells; and (4) determine the mechanisms by which these agents alter ion channel function by examining the effect of cellular second messengers on specific ion channel currents and ion channel fluxes. These experiments will provide important information about the extent to which alteration of ion channel function is important in agonist-induced excitation, antagonist-mediated relaxation, and mediator-induced hyperreactivity of ASM.
