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. Project 3: The long-term objectives of this proposal are to demonstrate that intracellular Ca2+ signaling in gastric antrum smooth muscle cells is regulated by phospholamban phosphorylation via CaM kinase II and that this novel pathway regulating myogenic excitability in the stomach is disrupted by diabetes. The overall hypothesis of the proposed studies is that phospholamban and CaM kinase II, through their modulation of sarcoplasmic reticulum Ca2+ uptake and release by SERCA, are critical determinants of antrum smooth muscle excitability, and that alterations in the expression or activities of phospholamban, CaM kinase II, and SERCA trigger the pathological remodeling that leads to the diabetic dystrophy of antrum smooth muscles and the loss of gastric muscle motility. The application's aims are to (i) test the hypothesis that regulation of intracellular Ca2+ by phospholamban and CaM kinase II modulates the excitability of gastric antrum smooth muscles. To evaluate this, phospholamban-/- mice will be utilized to show that phospholamban affects smooth muscle membrane potential by modulating sarcoplasmic reticulum Ca2+ uptake. (ii) Test the hypothesis that sarcoplasmic reticulum-targeted CaM kinase II phosphorylates phospholamban and modulates intracellular Ca2+ levels in gastric antrum smooth muscles. We propose that CaM kinase II anchoring to the sarcoplasmic reticulum membrane could be a mechanism to achieve spatio-temporal specificity of phospholamban phosphorylation. (iii) Test the hypothesis that altered phospholamban and SERCA expression, and phospholamban phosphorylation by CaM kinase II trigger the molecular remodeling that leads to the dystrophy of diabetic antrum smooth muscles. Several techniques will be employed, including mechanical measurements of antrum smooth muscles, SDS-PAGE and Western blotting, CaM kinase II assays, perforated-patch whole cell recordings of STOCs, sharp electrode recordings of membrane potential, and fluorescent Ca2+ indicator dyes to measure intracellular Ca2+ transients and [SR Ca2+ levels and release events] in antrum smooth muscle cells.
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