Browsing by Subject "PKA"
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Item cAMP and oxidative mechanisms of plasmalemmal sealing and the effects on rapid and long lasting repair of severed axons in vivo by polyethylene Glycol(2011-05) Spaeth, Christopher Scott; Bittner, George D.; Zakon, Harold; Ben-Yakar, Adela; Morgan, Jennifer; Dalby, KevinTraumatic neuronal injury inevitably causes plasmalemmal damage, and sometimes leads to axonal severance. For any eukaryotic cell to survive following traumatic injury, the plasmalemma must be repaired (sealed). Plasmalemmal sealing occurs via a Ca²⁺-dependent accumulation of vesicles or other membranous structures that form a plug at the damage site. Using uniquely identified and damaged rat hippocampal B104 cells that extend neurites with axonal properties, or rat sciatic nerves, plasmalemmal sealing is assessed by exclusion of an extracellular dye from each damaged B104 cell, or sciatic nerves ex vivo. B104 cells with neurites transected nearer (<50 [micrometres]) to the soma seal at a lower frequency and slower rate compared to cells with neurites transected farther (>50 [micrometres]) from the soma. Sealing in B104 cells is enhanced by 1) increased [cAMP], 2) increased PKA activity, 3) increased Epac activity, 4) H₂O₂ and 5) Poly-ethylene glycol (PEG). Sealing is decreased by 1) PKA inhibition, 2), Botulinum toxins A, B, E, 3) Tetanus toxin 4), NEM, 5) Brefeldin A, 6) nPKC inhibition, 7) DTT, 8) Melatonin and 9) Methylene Blue. Substances (NEM, Bref A, PKI, db-cAMP, PEG) that affect plasmalemmal sealing in B104 cells in vitro have similar effects on plasmalemmal sealing in rat sciatic nerves ex vivo. Based on data from co-application of enhancers and inhibitors of sealing, I propose a plasmalemmal sealing model having four partly redundant, parallel pathways mediated by 1) PKA, 2) Epac, 3) cytosolic oxidation and 4) nPKCs. The identification and confirmation of these pathways may provide novel clinical targets for repairing and/or recovery from traumatic injury. The fusogenic compound PEG rapidly repairs axonal continuity of severed axons, potentially by rejoining severed proximal and distal axons. PEG-fusion is influenced by plasmalemmal sealing, since unsealed axons are easier to PEG fuse. I demonstrate that PEG restores morphological continuity, and improves behavioral recovery following crush-severance to sciatic nerves in rats in vivo. Co-application of Mel or MB prior to PEG application further improves PEG fusion (as measured by electrophysiology) and behavioral recovery following crush-severance in vivo. These PEG data may provide novel clinical techniques for rapidly repairing axonal severance.Item Investigating the role of Bruno interactions with oskar regulatory proteins(2014-08) Kim, Goheun; Macdonald, Paul M.; Browning, Karen; Fischer, Janice; Stein, David; Stevens, ScottOskar (Osk) is a posterior body-patterning determinant in Drosophila melanogaster and is highly concentrated at the posterior pole of the oocyte. osk mRNA is translationally repressed until it reaches the posterior of the oocyte where Osk protein is made. Bruno (Bru) represses translation during osk mRNA localization by direct binding, but how Bru-mediated repression is relieved at the posterior of the oocyte is unknown. Two types of Bru protein interactions are implicated in repression of osk: Bru-Cup interaction and Bru dimerization. By mapping the Bru domains that are important for these interactions, I found that the amino-terminal domain of Bru contributes to both interactions, and deletion of this domain caused a defect in translational repression. However point mutations, within the amino-terminal domain, that disrupt both types of interaction in vitro did not interfere with translational repression in vivo. The difference may be due to other factors stabilizing the Bru-Cup interaction in vivo, as the mutant Bru still associates with Cup in vivo. My work supports the model of repression that relies on Bru interaction with Cup. I also build a new model in which Bru dimerization promotes translational activation of osk, based on my unexpected results: dimerization-defective Bru only weakly accumulated Osk::GFP fusion protein encoded by an osk::GFP reporter RNA bearing a Bru-binding region, while dimerization-competent Bru showed the opposite effect. This suggests that dimerization may contribute to switching Bru from a repressor to an activator, with dimerization controlled via a post-translational modification. Consistent with this, I found that a small fraction of Bru in ovaries is phosphorylated. PKA is a positive regulator of osk expression and phosphorylates Bru in vitro. To test if PKA regulation of osk is mediated through Bru, I examined the effect of altering PKA activity on Bru phosphorylation and Bru-mediated repression. Modulating PKA activity caused small, yet detectable changes in Bru phosphorylation and Bru-dependent translational repression using an osk::GFP reporter. However, while the studies with Bru mutants suggest that phosphorylation promotes repression by Bru, these studies argue for a role in promoting activation. Further work will be required to explain these phenomena.Item The Non-genomic Signaling Pathways Mediated By G-protein Coupled Estrogen Receptor 1 (GPER) In Coronary Arteries(2014-12-04) Yu, XuanCoronary heart disease (CHD) remains the leading cause of death throughout the world, and postmenopausal women are at particularly high risk for CHD. A promising new avenue of study is the novel G protein-coupled estrogen receptor (GPER) which mediates estrogen action. The major purpose of my studies in this dissertation is to investigate the role of GPER in porcine coronary artery tone regulation. In a series of studies, we tested four hypotheses: 1) activation of GPER regulates coronary artery tone by paradoxically inducing relaxation and potentiating contraction; 2) activation of GPER induces coronary artery relaxation by Gs/cAMP-dependent pathway(s); 3) activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets: the exchange proteins directly activated by cAMP (Epac) as well as PKA; and 4) activation of GPER potentiates coronary artery contraction by a G??/EGFR-dependent pathway. Isometric tension studies were performed on endothelium-denuded porcine coronary arteries to test the function role of GPER and its signaling pathways. RT-PCR, Western blots, patch-clamp experiments and kinase activity assays also were employed in these studies to confirm the expression and phosphorylation of subjective proteins, channel activities and kinase activities in porcine and human coronary artery smooth muscle cells (SMCs) and coronary artery tissues. Results from these studies suggest: 1) GPER is expressed in porcine and human coronary artery SMCs; 2) GPER mediated coronary artery relaxation is NO-independent and involves BKCa channel activity; 3) activation of GPER stimulates the production of cAMP, thus activates its downstream targets PKA and Epac; 4) GPER mediates coronary relaxation through activation of MLCP via inhibition of RhoA activity by both PKA and Epac; 5) the interaction between AKAP and PKA is involved in the cAMP/PKA signaling mediated by GPER in coronary artery; and 6) GPER potentiates coronary artery contraction via G?? signaling to stimulate transactivation of EGFR and activation of ERK1/2. These findings provided evidence of the dual effects of GPER in coronary regulation, which may help reveal the controversial actions of estrogen and provide a molecular basis for developing new compounds that better target estrogen signaling for a variety of clinical applications.