Chapter 32 Isolation and Reactivation of Newt Lung Respiratory Cilia

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Publisher Summary Cilia are the moving parts of the respiratory mucociliary epithelium, assisting in defense against respiratory tract infections. The constant and coordinated beating of cilia minimizes the adverse effects of inhaled material by exporting out of the respiratory tract any foreign material trapped in the mucous blanket. Dysfunction of mucociliary clearance may result either from impaired cilia or from abnormalities in the fluid component. A series of models or simplifications of the respiratory epithelium from newt lungs have been developed, that possess several advantages for studying mucociliary transport and the regulation of respiratory cilia. These models include isolated lungs, isolated epithelial sheets, and epithelia in primary tissue culture, isolated ciliated cells, isolated and reactivated ciliary tufts, and populations of demembranated ciliary axonemes. Such models allow study of the mucociliary transport process at different levels of organization without the regulatory influences of higher levels of organization. In this chapter, the methods used to isolate and reactivate demembranated ciliary axonemes from lungs of the newt, Taricha grunulosa , are summarized. The solutions, the isolation and reactivation of normal ciliary axonemes and of outer-arm-depleted axonemes have been described. With populations of demembranated cilia, study of ciliary function independent of cellular controls and investigate regulatory mechanisms that are inherent to the axoneme itself. Such demembranated axonemes can be isolated in a simple, one step process from newt lungs. Statistically, beat frequency shows a gaussian distribution about the mean for healthy axonemes. These characteristics are so consistent that reactivating solution can be used as a quality control solution at the start of the day's reactivation experiments and then every several hours to be sure that reactivated cilia have the same motility potential. These reactivation characteristics are not dependent on the particular animal, do not vary between males and females, and are the same throughout the year.