Stimulus-induced alteration in the concentration of intracellular second messengers is a key mechanism by which cell function can be regulated. With the development of fluorescent probes, it became possible to directly measure changes in the concentration of a number of second messengers such as calcium and hydrogen ions in both resting and stimulated cells. Until recently, fluorometric measurements of intracellular ions monitored generalized changes, but were not sufficiently sensitive nor had adequate temporal resolution to measure very brief, localized ion release from internal stores. However, with laser scanning confocal microscopy, it now is possible to measure elevations in intracellular ions or pH changes within micron-thin optical sections through living cells. For example, the brief, localized elevation of intracellular calcium, that results from the opening of calcium channels in the sarcoplasmic reticulum/endoplasmic reticulum can be measured as "calcium sparks" (3,8). These local calcium sparks have a profound influence on muscle function by summing to cause a global elevation of intracellular calcium leading to contraction in heart muscle (3) or by activating surface membrane K+ channels leading to vasodilitation in smooth muscles (8). Calcium spark activity, in turn, is intimately controlled by local calcium entry through surface membrane channels. These extraordinary insights into fundamental mechanisms of cardiac muscle and smooth muscle control were permitted by the unparalleled resolution of the laser-scanning confocal microscope (3,8). This approach permits one to follow the fate of key ions, such as calcium, which after crossing the cell membrane are then sequestered into internal stores. Although calcium sparks occur as random events, their frequency and kinetic properties commonly are affected by cell voltage. Cons equently, the local changes in intracellular calcium, monitored as calcium sparks, must be measured in cells with membrane voltage controlled. It is proposed that these fluorescence measurements will be made on cells voltage clamped with whole cell patch clamp techniques. Also, these measurements will be made under conditions which allow for rapid exchange of bathing solutions. A Major objective of five of the six projects is to establish the relationships between calcium entry through surface membrane calcium channels and activation of calcium sparks and then subsequent effects on membrane ionic conductances in neurons and in different smooth muscle cells. The last project investigates the role that intracellular pH plays, in the regulation of chemotaxis in Paramecium, by measuring local intracellular changes in proton concentration. Each project asks a very specific question concerning the role of brief, highly localized changes in calcium or pH which may be critical to a specific cell function. Further, the requested equipment will permit the investigation of other key issues such as local homeostatic regulation of intracellular ionic concentrations by extrusion mechanisms. The critical requirement of all of the proposed studies is the ability to measure very rapid, highly localized changes in the concentration of calcium or pH in voltage-clamped cells. Funds are requested to create a multiuser facility comprised of three closely integrated components: a Noran Odyssey XL digital confocal laser scanning imaging system, a Nikon Diaphot 200 inverted microscope and an Axon Instrument based voltage clamp system. The presence of this highly sophisticated instrumentation will greatly enhance the research capabilities of the project directors and also will provide significant new training opportunities for our current and future gradua te students and postdoctoral fellows.
