Activation of non-excitable cells leads to an increase in intracellular calcium and the generation of other intracellular signals which together initiate the physiological response. Single-cell studies of stimulus-response coupling in non-excitable cells are proposed, using two immune effector cells, the mast cell and the eosinophil, as model systems. The mast cell has been a valuable model for many years. In contrast, remarkably little is known about signal transduction in eosinophils, in spite of their importance in a variety of immne responses. Studies on mast cells will continue to utilize the well-established rat basophilic leukemia cell line. Guinea pig eosinophils will be used for more preliminary studies of signal transduction in eosinophils. Quantitative image enhanced microscopy will be used to measure Ca2+, membrane potential, exocytosis and chemotaxis in single cells. In many cases more than one of these parameters can be monitored simultaneously in many individual cells. Electrophysiologcal measurements, utilizing the perforated patch configuration of the whole cell recording technique, will be used to identify calcium currents or any other conductances that are activated in response to various stimuli. Simultaneous measurements of intracellular Ca2+, ionic current, and exocytosis are also planned. The long term goal is to identify the mechanisms by which intracellular Ca2+ is increased and to determine what aspects of the Ca2+ signal are important in the exocytotic and/or chemotactic response of a cell. When mast cells or eosinophils interact with certain protein or peptide effectors (such as immunoglobulins), the effectors bind to receptors on the surfaces of these cells and result in a specific physiological response (such as secretion of the contents of specific storage granules). The question addressed in these studies is how the binding of a receptor at the outside of the cell results in the physiological response, a phenomenon termed stimulus-response coupling. It has already been established that intracellular calcium is increased as a result of stimulation, and that the increased calcium levels leads to the generation of other intracellular signals culminating in the response. This project will probe the role of calcium in stimulus-response coupling, using a combination of novel and established techniques (sometimes simultaneously) in order to extend our understanding of the mechanism of this form of transmembrane signal transduction.
