Several previously approved NIH and new research proposals by the user group require temporal and spatial analysis of intracellular molecules in viable biological systems. Among these substances are calcium, sodium, potassium, pH, and other cellular constituents. Quantitative fluorescence imaging analysis has been found to be a powerful tool capable of dynamic measurement of these molecules with the use of appropriate fluoroprobes. The shared instrument request in this proposal is for a dual excitation macro/micro spectrofluorometer and image analysis system with accessories to provide needed flexibility for a variety of biological entities including small arterial vessels, renal tubules, cell suspension, monolayers and dispersed cells. The research protocols are focused on time/location quantitation of fluorometrically identifiable molecules. The first protocol (Dr. Conger) involves the assessment of smooth muscle cell calcium in the post-ischemic renal vasculature. Second, changes in calcium, pH, membrane potential and potassium in variable hypoxic carotid body cells will be examined (Dr. Weil). Third, Dr. Stelzner proposes to determine the roles of fluorescence detectable cytoskeletal constituents in the modulation of endothelial barrier properties. Fourth, the link between calcium, pH, membrane potential and endothelium-derived relaxing factor activity in the endothelial- response to hypoxia will be assessed (Drs. Rodman and McMurtry). Fifth, the relationship of calcium flux to differential vasoactive stimuli and the coupling of electrophysiologic events to calcium movement in small mesenteric arteries and veins will be determined by Drs. Hottenstein and Jacobson, and sixth, Dr. Dillingham will assess the role of cellular calcium in regulating hydraulic conductivity in the collecting duct. While the projects are distinct there is sufficient scientific overlap for effective utilization of the proposed instrument system.A monthly conference will be held by the members of the user group.Operational plans, including designation of a maintenance-operator individual and a technical consultant in fluorescence and fluoroprobe chemistry, have been formulated.
