Airway smooth muscle is activated by neurotransmitters such as acetylcholine, and autocoids such as the leukotrienes and histamine. The resulting contractions are poorly inhibited by calcium channel blocking drugs, as is true for bronochoconstriction in asthma. Our proposed research responds to the need for a better understanding of the above agonist- induced contractions, so leading to the development of therapeutic control over bronchoconstriction. For this purpose we will study how the muscarinic agonist carbachol mobilizes Ca2+ from the extracellular space and the sarcoplasmic reticulum to raise the concentration of this final intracellular messenger in the myoplasm. Our postulate which is supported by the literature and our preliminary data, is that during agonist activation Ca2+ flows through receptor operated cation channels (ROCs), voltage dependent Ca2+ channels (VDCs) and the nonregulated """"""""leak"""""""", and exits from the SR through channels activated by inositol-1, 4, 5- trisphosphate, Ca2+ and another nonspecific leak. The Ca2+ and Na+ movements through the plasmalemma will be monitored as 45Ca and 22Na influx, and as whole cell and single channel currents measured by patch clamp techniques. Ion currents through ROCs and VDCs will be separated by the use of calcium channel blocking drugs. The extracellular and intracellular Ca2+ sources will be distinguished by extracellular Ca2+ depletion and the use of organic and inorganic Ca2+ entry blockers. The effects of the Ca2+ fluxes on myoplasmic Ca2+ activity will be measured by force development and Ca2+ - modulated Fura-2 fluorescence. The release of Ca2+ from the SR will be directly monitored in saponin skinned preparations of bronchial and tracheal smooth muscle and their tissue cultures. The sources for these preparations are both animal and human. Tissue culture procedures from these sources will be developed which will eventually yield sufficient airway smooth muscle for isolation of their ion channels. The basic properties and roles of the Ca2+ transporting channels elucidating during the proposed project combined with the culture of airway smooth muscle will provide a scientific basis for future molecular biological study of the ROCs and VDCs.
