The long-term objectives have been to understand calcium regulation in airway smooth muscle. Intracellular calcium stores of the sarcoplasmic reticulum (SR) play a role in determining the cytosolic calcium concentrations ([Ca2+]i) that regulate smooth muscle functions, including contraction. Therefore, understanding the mechanisms by which stimuli cause release of calcium from the SR to generate a rapid increase in [Ca2+]i in the cell cytosol (calcium transient) and how the SR then refills with calcium is important. Work on SR refilling has led to the novel observation that interleukin (IL)-4 rapidly (<30 min) inhibits carbachol-stimulated calcium transients in bovine airway smooth muscle cells. This new finding is potentially important because it conceptually links calcium regulation in airway smooth muscle cells to the T helper 2-type cytokine profile characteristic of asthmatic airway inflammation. Notably, IL-4 is closely related to IL-l3, an important mediator of airway inflammation, and binds to a shared family of receptor complexes. In general, the aims are to determine how IL-4 rapidly inhibits calcium transients and to assess for effects on contraction. Specifically, the goals are to determine the effects of IL-4 on 1) SR calcium content; 2) calcium influx; and 3) coupling of receptors to inositol trisphosphate generation. Then, the goals are to 4) characterize signal transduction pathways underlying IL-4 effects on calcium and 5) characterize the effects of IL-4 on cell shortening. The principal methods are single cell, ratiometric imaging of [Ca2+]i, including three dimensional reconstruction of images; electrophysiology to assess calcium influx; and biochemistry to define signal transduction pathways. Studies will be done with freshly-dispersed bovine and cultured human airway smooth muscle cells.
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