The major goal of this research project is to elucidate intracellular signal transduction pathways by which metabolic changes modulate and hormones regulate the Na+-Pi symport across brush border membranes (BBM). The studies are based on the following hypothesis. While hormones such as PTH or dopamine regulate Na+-Pi symport via cAMP-PKA and/or pathway(s), the change in metabolic states namely gluconeogenesis (GNG) of proximal tubular (PT) modulate the Na+ Pi symport via a novel pathway involving NAD+, cyclic adenosine diphosphoribose (CADPR), and Ca2+ release. The final steps of the cADPR signalling pathway is regulation of microtubule (MT) disassembly and assembly by microtubule associated proteins (MAP2), which are phosphorylated by Ca2+ dependent and cAMP- dependent protein kinases. Specific objective include: 1. To determine the characteristics of cADPR system in PT cells and OK cells. This includes subcellular localization and enzymatic properties of ADP-cyclase, cADRP-glucohydrolase (cADPR-GH), and dynamics of cellular cADPR levels. 2. To examine binding of cADPR onto specific cADPR-receptor and its characteristic (KD, Bmax, specificity, pH optimum, etc) and relationship between cADPR binding and release of Ca2+ from the intracellular Ca2+ stores. To determine whether multifunctional Ca2+/CaM protein kinases-II (Ca/CaM-PK-II) is activated by Ca2+ released in response to cADPR, and whether it phosphorylates microtubule associated proteins (MAP2), and whether phosphorylated MAP2 promote disassembly of MT in PT cells. Further, to test whether PKA also phosphorylates MAP2 and whether the effects of both protein kinases are additive. 3. To determine whether the cADPR signalling system operates in intact cells as integral pathway and modulates Na+-Pi symport across BBM in response to change in PT metabolism, namely GNG. 4. To delineate intracellular mechanisms by mutual """"""""crosstalk"""""""" between the hormonal signals acting via cAMP-PKA pathway and metabolic signals acting via cADPR-Ca2+ pathway in PT cells. We will also test the hypothesis which states that the degree of MT assembly in cytoplasm of PT cells determines the extent of internalization and reinsertion of Na+- Pi symporters into BBM, and thereby regulates the Na+-Pi reabsorption. We propose that MAP2 phosphorylation by the two kinases is the focal point of additivity or synergism.
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