This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Core D will support Projects 1, 2 and 4 requiring expertise in fluorescent imaging. This core has purchased two fluorescent high speed imaging systems that can be used for examining activity in both intact tissues and in isolated cells. One imaging set up is based around an upright microscope (Nikon TS100) with water immersion lenses and has a wavelength switcher (DG-5) for ratio-metric (Fura 2-AM) imaging. The latter allows for calibrating calcium activity in pressurized blood vessels, cells in intact tissues and in isolated cells. The second imaging system is based around an inverted microscope and incorporates an electrophysiology set up. This system is used for determining the relationship between voltage and fluorescent calcium signals. Both systems use a highly sensitive back illuminated camera (Cascade 512). These imaging systems will support projects that need to examine the spread of intracellular and intercellular calcium waves that regulate the membrane potential and contractile activity respectively of the smooth muscle layers of the intact but isolated fundus and colon. Project 2 will examine whether these waves are modified PLB knockout mice compared to their wild type littermates. It will also be determined whether CaMKII inhibitors affect the propagation and duration of these waves. Project 4 will use this apparatus to examine the changes in the spread of intracellular and intercellular calcium waves in the smooth muscle layers from hypertrophied regions of bowel. In addition, Project 4 will use confocal calcium imaging to determine if changes occur to the spread of activity through the neurons of the enteric nervous system. This is possible because [Ca 2+]I is a reliable indicator of action potential dependent changes in the activity of enteric neurons. Project 4 will also use this technology in combination with patch clamp studies to determine if there are changes in the ionic conductances underlying spontaneous Ca2+ transients (Sparks and Puffs) in isolated smooth muscle cells taken from different regions of bowel with a partial obstruction. Project 1 will utilize the ratio-metric/pressurized vessel imaging system to determine whether Ca2+ signaling and vascular reactivity are altered in arteries from mdx mice lacking dystrophin compared to their wild type litter mates. In addition, single cell Ca2+ imaging experiments combined with patch clamp techniques will be utilized to provide a detailed understanding of how integrins affect Ca2+ signaling and currents in isolated vascular smooth muscle cells. Ca2+-permeable (L-type and nonselective) channels will be compared in isolated myocytes from mdx and wildtype mice.
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