Browsing by Subject "Transport Phenomena"
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Item Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes(2008-05-01) Fuller, John Anthony; Wordinger, Robert J.; Clark, Abbot F.; Krishnamoorthy, Raghu R.Fuller, John Anthony Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes. Doctor of Philosophy (Biomedical Sciences), May, 2008, 140 pp., 2 tables, 25 illustrations, bibliography, 218 titles. Primary Open Angle Glaucoma (POAG) is a family of heterogeneous optic neuropathies characterized by progressive retinal ganglion cell (RGC) death that leads to peripheral vision loss and eventually blindness. Various risk factors are associated with glaucoma, however the molecular mechanisms leading to RGC cell death remain unknown. The optic nerve serves as the conduit for the transmission of retinal ganglion action potentials to the brain. The cells that compromise the optic nerve form a scaffold that forms a physical support for the RGC axons. One cell type found throughout the optic nerve and associated with the RGC axon is the optic nerve astrocyte (ONA). Astrocytes are a predominant cell throughout the CNS and are believed to play crucial roles in metabolic, growth factor, and structural support, and respond to protect neurons during injury. The neuronal-glial interface in the optic nerve is poorly understood and believed to plan an important role in POAG pathophysiology, as unmyelenated RGC axons have direct contact with astrocyte processes. IN this study, the subtilisin-like Proprotein Convertases, (SPC) a family of proteases responsible for cleaving a wide variety of protein substrates, were examined in the retina and optic nerve head. PACE4, an SPC found to be secreted and active in the extracellular matrix was found to be highly expressed in the optic nerve, and colocalized to Mϋller cells in the retina and astrocytes in the optic nerve. Exposure of primary optic nerve astrocytes to oxygen-glucose deprivation (OGD) induces an increase in PACE4 mRNA. Furthermore, protein levels of extracellular, processed PACE4 increase following transient ODG, whereas the pro form of the molecule is degraded, and is believed to be chaperoned by the cleaved cysteine rich domain, a product found at high levels in the optic nerve in situ and the ONA in vitro. Due to the extracellular activity of PACE4, we hypothesized that it may regulate the bioactivity of TGF-β2, a growth factor believed to be involved in glaucoma-associated ONH remodeling by inducing the production of extracellular matrix (ECM). When PACE4 is inhibited via siRNA-mediated knockdown, as well as extracellular inactivation, TGF-β2 levels decrease. In addition, fibronectin, a major component of the ECM, is decreased. Furthermore, there is an increase in latent TGF-β2 secreted from the cell. It is therefore possible that PACE4 plays an active role in extracellular growth factor maturation, and may be a central mediator for growth factor bioactivity in the glaucomatous ONA.Item Opioid and Nitric Oxide Interaction in the Control of Heart Rate(2002-12-01) Farias III, Martin; James Caffrey; Fred H. Downey; Patricia GwirtzFarias III, Martin, Opioid and Nitric Oxide Interaction in the Control of Heart Rate. Doctor of Philosophy (Biomedical Sciences), December 2002, 130 pp, 2 tables, 30 figures. Understanding of the role endogenous opioids play as modulators of parasympathetic function has increased. The endogenous opioid, methionine-enkephalin arginine phenylalanine (MEAP) attenuates vagal control of heart rate when delivered by microdialysis directly in the canine sinoatrial node. This effect was mimicked by the δ-2 agonist, deltorphin-II indicating involvement by a δ-opioid receptor. The nodal delivery of the δ-antagonist naltrindole abolished the effect of deltorphin-II, further supporting the delta character of the receptor. Although the findings suggested that the opioid receptor mediating vagolysis was delta in character, the exact subtype of δ-receptor remained in question. Selective agonist and antagonists for δ-1 and δ-2 opioid receptors were employed to determine which subtype of δ-receptor mediated MEAP vagolysis. In these experiments, vagolysis produced by the nodal delivery of MEAP was unaltered by the highly selective δ-1 antagonist BNTX but abolished by the δ-2 antagonist, naltriben. Nodal delivery of deltorphin-II attenuated vagal bradycardia similar to MEAP while δ-1 agonists, DPDPE and TAN-67 failed to interrupt vagal bradycardia. TAN-67 actually improved vagal transmission and this effect was reversed by BNTX. These data indicate that δ-2 opioid receptors in the sinoatrial node and vagolytic and support the presence of vagotonic δ-1-opioid receptors in the same location. Nitric Oxide/Opioid Interaction. The hypothesis that intranodal nitric oxide synthase (NOS) modulates vagal transmission and that MEAP attenuates vagal bradycardia via the interruption of the NOS-cGMP pathway was tested. The general (L-NAME) and neuronal (7-nitroindazole) NOS inhibitors each attenuated vagal bradycardia and both effects were reversed by adding excess of the NOS substrate, L-arginine. These findings suggested that nNOS was a necessary component of vagal bradycardia in the canine sinoatrial node. Various probes of the NOS-cGMP pathway (L-arginine, SNAP, cGMP, and IBMX) were employed to determine if MEAP interrupted this pathway to produce vagolysis. The delivery of MEAP into the sinoatrial node for sixty minutes exerted a consistent vagolytic effect during vagal simulations. When MEAP was combined with a NOS pathway components, the vagolytic effect was reversed after 15-45 minutes of treatment. These findings suggested that MEAP exerted its effect by interacting with the NOW-cGMP system. The site of convergence maybe cAMP since the phosphodiesterase inhibitor, IBMX (by allowing the accumulation of cAMP) reversed the vagolytic effect of MEAP. To rule out a postjunctional effect, MEAP and the NOS inhibitors were combined with the direct acting muscarinic agonist, methacholine. The bradycardia produced by methacholine was unaltered by MEAP or nNOS inhibitors. This suggested that the effect of NOS inhibitors and MEAP were prejunctional. In summary, the cumulative findings suggest that MEAP, by activating δ-2-opioid receptors, attenuated vagal bradycardia prejunctionally, through modulating the cAMP component of the NOS-cGMP pathway in the canine sinoatrial node.