This proposal requests funds for the purchase of optical and electronic equipment to perform dual-wavelength fluorescence measurements in isolated single cells, small multicellular preparations and crystalline enzymes. The equipment will be shared by six Project Leaders (all with active NIH grants) in three departments at the University of California, Riverside. The projects are studies involved in general areas of cardiovascular disease, cystic fibrosis, diabetes, endocrinology of Ca homeostasis (e.g. Vitamin D & PTH), toxicology, neuromuscular transmission and molecular aspects of enzyme catalysis. The following projects are described. Drs. Bers & Briggs will study the regulation of cytoplasmic Ca (and Nai & pHi) and Ca-currents in isolated cardiac myocytes, with respect to the sources of Ca which support contraction (SR & Ca influx) and also localization of arrhythmogenic sites. Dr. Luben will study the regulation of cytoplasmic Ca in suspensions of bone cells treated with parathyroid hormone (PTH) and in single bone cells (to study subcellular Ca distribution) and in cells transfected with the PTH receptor. Drs. Norman and Nemere will determine the subcellular localization of Cai (using fura-2) and beta-tubulin (using fluorescent antibodies) in isolated intestinal epithelial cells as functions of vitamin D status. Dr. Quinton will measure changes in [Ca]i, pHi and electrophysiological parameters in response to isotonic absorptive loads in isolated sweat gland ducts from both normal and cystic fibrosis subjects. Dr. Adams will use fluorescent Ca indicators to study the mechanism by which a novel spider toxin, omega-agatoxin inhibits Ca influx in presynaptic nerve terminals at neuromuscular junctions. He will also examine the means by which the neuropeptide proctolin mediates Ca entry in skeletal muscle. Dr. Dunn will study molecular interactions between enzymes and cofactors during different steps in the reaction sequence of four enzymes in structured (crystalline) states. The changes in polarization and spectral properties of intrinsically fluorescence cofactors will be measured.
