Browsing by Subject "cAMP"
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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 Signaling crosstalk in cancers(2008-04-03) Zhenyu Ji; Xiaodong Cheng; Scott R. Gilbertson; Miriam Falzon; Mark B. Evers; Jingwu Xie; Jingson LiuDeregulation of cellular signaling contributes to the malignant phenotype of cancer. In this dissertation, cross-talk among oncogenic signaling pathways was investigated in two common human cancers. \r\n Pancreatic cancer has a well known spectrum of genetic alterations, among which KRAS mutation is the most prominent component with a detection frequency as high as 90%. Hedgehog signaling pathway is involved in developmental process and it’s reactivated in the early stage of carcinogenesis of pancreatic cancer. The coincidence of KRAS mutation and abnormal hedgehog activation indicates that there’s a potential link between them. We found that oncogenic KRAS activated hedgehog signaling in pancreatic cancer cells through RAF/MEK/MAPK subpathway, but not PI3K/AKT subpathway. Inactivation of KRAS/MEK pathway activity by a KRAS specific siRNA or MEK inhibitors inhibits GLI transcriptional activity and GLI1 expression, and promotes GLI1 protein degradation in pancreatic cancer cell lines with activating KRAS mutation. Furthermore, Suppressing Gli activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing pancreatic cancer cells. These results indicate that hedgehog pathway activation play an important role in oncogenic KRAS mediated transformation during the pancreatic cancer formation. \r\n In addition to its role in pancreatic cancer, hedgehog signaling was also activated in acute lymphoblastic leukemia (ALL). We found that hedgehog pathway activity was correlated to glucocorticoid (GC) sensitivity status in ALL cells. GC resistant ALL cells had higher hedgehog activity when compared to their sensitive counterpart. Increasing intracellular cAMP levels by forskolin overcomes GC resistance in ALL cells. Our studies demonstrate that PKA is responsible for the observed synergism with GC while Epac isn’t. We find that endogenous PKA activity is higher in the GC-sensitive clone than in the GC-resistant clones. Inhibiting hedgehog pathways activity by specific inhibitors alone leads to cell cycle arrest and apopotosis in ALL cells. These results suggest that Hh activity is critical for leukemia cell growth and survival and that the level of Hh activity is in part responsible for the synergism between cAMP and GC. \r\n To identify the mechanism contributing to the PKA activity difference, we measured PKA subunits expression levels and found that regulatory subunit RIIâ is preferentially expressed in the GC sensitive clone C7-14 cells compared to the GC resistant clone C1-15 cells. Down-regulation of RIIbeta by siRNA transfection leads to enhanced GC resistance in CEM cells, which indicates RIIbeta expression is required for GC sensitivity. We also found that activation of PKA II by selective agonists recapitulated forskolin’s effects of promoting apoptosis and antagonizing Akt activity in both GC sensitive and resistant cells. However, PKA I agonists did not have the same effect. These results demonstrate that PKA II is critical for cAMP-promoted apoptosis in ALL cells and its activation or with GC combination could be a novel approach against lymphoid malignancy. \r\nItem 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.