Browsing by Subject "Adenosine"
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Item Adenosine and blood flow regulatory mechanisms in hippocampal ischemia(Texas Tech University, 2002-05) Gervitz, Leon MAccording to the National Institutes of Health (NIH), stroke affects more than 700,000 people annually, making it the third leading cause of death and the most common cause of adult disability in the United States. The success of medical intervention after a stroke depends on its being started soon after the insult. Defined as an acute neurological disorder caused by disturbances of the cerebral blood supply, stroke can rapidly lead to the development of ischemic brain tissue that is comprised of a nonviable, necrotic core surrounded by a penumbral region. Although functionally depressed, the penumbral region remains metabolically intact making it potentially salvageable during the post-ischemic therapeutic window. As such, it is considered a promising target for acute therapeutic intervention. The limited success of current early interventions, however, argues for a greater understanding of the regulatory mechanisms governing the physiology of the ischemic brain. Of particular interest are the regulatory mechanisms governing neuronal function and blood flow within the ischemic hippocampus. An integral part of the limbic system that is involved in the processing of short-term memory, the hippocampus is a bilateral structure that is exquisitely sensitive to hypoxia and/or ischemia. It is well established that an early response to cerebral hypoxic and/or ischemic conditions is a reversible inhibition of evoked synaptic potentials. The suppression of synaptic function is thought to serve as a neuroprotective mechanism to reduce energy expenditure during metabolic stress, i.e. hypoxia/ischemia. There is substantial evidence in in vitro preparations that the initial reversible loss of synaptic activity during exposure to hypoxia or ischemic-like conditions in the hippocampus is due to the release of endogenous adenosine acting at neuronal Ai receptors. Such roles for adenosine in in vivo preparations, however, have not been as convincingly demonstrated. Using a rat model of unilateral common carotid artery occlusion coupled with hypoxia, this dissertation examines the regulatory mechanisms of hippocampal blood flow and the contribution of adenosine to the early hypoxic/ischemic inhibition of synaptic transmission in an in vivo model of ischemic penumbra, and additionally examines the role of adenosine in the initiation of a post-ischemic, anti-apoptotic signal transduction pathway, Akt/Protein Kinase B. Animals were placed in a stereotaxic apparatus and evoked excitatory postsynaptic potentials (fEPSPs) were recorded from CAl of the rat hippocampus. Additionally, body temperature, systemic blood pressure, arterial blood gases, and hippocampal blood flow using laser Doppler flowmetry were monitored during experiments. Akt/PKB activation was examined using Western blot analysis. We demonstrate for the first time in an in vivo preparation, that A1 receptor activation plays a central role in the early hypoxic-ischemic depression of the evoked hippocampal synaptic potentials. Moreover, we demonstrate that while hypoxia is a potent stimulus for the adenosine-mediated depression of the synaptic potentials in vitro, reduced hippocampal tissue p02 alone does not appear to be sufficient to induce an adenosine-mediated depression of synaptic transmission in vivo. There must be, it seems, an accompanying reduction in local hippocampal blood flow. Moreover, the adenosine A1- mediated depression of synaptic depression occurs in proportion to reductions in local cerebral blood flow over a wide range of flows typical of penumbra. We also demonstrated that A] receptor activation leads to the activation of the neurotrophic/anti-apoptotic protein kinase Akt/Protein Kinase B (PKB). This result suggest that Akt/PKB activation may play a heretofore unappreciated role in adenosine A1-mediated signal transduction and, therefore, in adenosine A 1-mediated neuroprotection. We conclude from this work that adenosine acts as both an endogenous mediator and a sensitive indicator of penumbral conditions throughout the range of penumbral blood flows, and is an important mediator of the cellular response to survivable levels of ischemia.Item Akt: it's role in neuronal viability and protection against ischemia in the rat hippocampus(Texas Tech University, 2006-05) Omidvar, Kamran; Fowler, John C.; Roghani, Ali; Strahlendorf, Jean C.According to the American Stroke Association, about 700,000 people suffer a new or recurrent stroke each year in the United States. Of these people, approximately 163,000 die, making stroke the third leading cause of death in the U.S., only behind heart disease and cancer. Depending on the area of the brain affected by the stroke, functions such as motor activity, speech, behavior, and/or memory can be hampered. The hippocampus is a bilateral structure that is highly susceptible to hypoxic and/or ischemic insult. One of the early responses to ischemia is the transient and reversible inhibition of synaptic activity mediated by endogenous adenosine acting on neuronal A1 receptors. Increase in adenosine during ischemia is thought to play a key prosurvival role by attenuating excitotoxic damage through inhibiting glutamate release and activating Akt. Akt is activated by PI3K-dependent and PI3K-independent mechanisms. Akt, also known as PKB, has been shown to be both necessary and sufficient to promote cell survival by growth factors in vitro. Akt directly phosphorylates multiple proteins resulting in the inhibition of apoptotic and/or necrotic cell death. Bcl-2 and Bcl-xL are two proteins that are disinhibited by the direct Akt phosphorylation of Bad. These two proteins function to maintain mitochondrial integrity during ischemia, thus inhibiting the release of cytochrome c which is a strong inducer of the apoptotic pathway. This thesis explores the activation mediated by PI3K and the significance of this activation in neuronal survival mechanisms.Item Akt: It's role in neuronal viability and protection against ischemia in the rat hippocampus(2006-05) Omidvar, Kamran; Fowler, John C.; Roghani, Ali; Strahlendorf, Jean C.According to the American Stroke Association, about 700,000 people suffer a new or recurrent stroke each year in the United States. Of these people, approximately 163,000 die, making stroke the third leading cause of death in the U.S., only behind heart disease and cancer. Depending on the area of the brain affected by the stroke, functions such as motor activity, speech, behavior, and/or memory can be hampered. The hippocampus is a bilateral structure that is highly susceptible to hypoxic and/or ischemic insult. One of the early responses to ischemia is the transient and reversible inhibition of synaptic activity mediated by endogenous adenosine acting on neuronal A1 receptors. Increase in adenosine during ischemia is thought to play a key prosurvival role by attenuating excitotoxic damage through inhibiting glutamate release and activating Akt. Akt is activated by PI3K-dependent and PI3K-independent mechanisms. Akt, also known as PKB, has been shown to be both necessary and sufficient to promote cell survival by growth factors in vitro. Akt directly phosphorylates multiple proteins resulting in the inhibition of apoptotic and/or necrotic cell death. Bcl-2 and Bcl-xL are two proteins that are disinhibited by the direct Akt phosphorylation of Bad. These two proteins function to maintain mitochondrial integrity during ischemia, thus inhibiting the release of cytochrome c which is a strong inducer of the apoptotic pathway. This thesis explores the activation mediated by PI3K and the significance of this activation in neuronal survival mechanisms.Item Evaluation and Characterization of Novel Signal Transduction Pathways in Striatum(2008-05-13) Sahin, Bogachan; Bibb, James A.In the mammalian central nervous system, protein kinases and protein phosphatases control the function of myriad target proteins in the pre- and postsynaptic compartments, including other protein kinases and phosphatases, neurotransmitter receptors, ion channels, transporters, metabolic enzymes, transcription factors, cytoskeletal elements, and vesicle-docking proteins. Using biochemical and pharmacological approaches, a number of novel striatal signal transduction pathways were evaluated and characterized in the following studies, with emphasis on protein kinase C-mediated signaling. 1) A known and novel form of mouse Adk encoding splice variants of adenosine kinase, the principal enzyme of adenosine metabolism, were cloned from a mouse brain cDNA library and expressed and purified as recombinant proteins with high enzymatic activity. The tissue distribution of adenosine kinase isoform expression was defined. A polyclonal anti adenosine kinase antibody was generated for further characterization of the enzyme. In vitro protein phosphorylation studies using purified protein kinases and in vivo radioimmunoprecipitation assays using the novel antibody for adenosine kinase indicated, however, that this metabolic enzyme is unlikely to be regulated by phosphorylation. 2) Further studies using a candidate approach demonstrated the regulation of several postsynaptic phosphoproteins by striatal adenosine A2A receptor signaling, including ionotropic glutamate receptor subunits, mitogen-activated protein kinase isoforms, a striatal inhibitor of protein phosphatase 1, a protein phosphatase 1- and actin-binding protein, and the cAMP-response element-binding protein. 3) In parallel studies, inhibitor-1, a protein phosphatase 1 inhibitor activated by cAMPdependent protein kinase, was characterized as a novel protein kinase C substrate in vitro and in vivo. Phosphorylation state-specific antibodies raised against this novel phosphorylation site showed that it is dephosphorylated by protein phosphatase 1 and positively regulated by group I metabotropic glutamate receptors in the striatum. Furthermore, protein kinase C-dependent phosphorylation was shown to reduce the efficiency with which inhibitor-1 serves as a substrate for cAMP-dependent protein kinase in vitro and in vivo. 4) Finally, protein kinase C activation was shown to decrease the level of phosphorylation of cyclin-dependent kinase 5 substrates in the striatum, suggesting a possible role for protein kinase C in regulating cyclin-dependent kinase 5 activity.Item Modulation as an Acidosis-Evoked Current by A1 Adenosine Receptors in the CA1 Region of the Mouse Hippocampus(2006-05-15) Galanis, Victor Chris; Greene, Robert W.Acidosis, along with hypoxia and hypoglycemia are immediate metabolic consequences of reduced blood flow to the brain. Acidosis exacerbates ischemic brain injury by activating non-selective cation currents that induce neuronal damage in a calcium-dependent manner, independent of glutamate receptor activation. Adenosine is released during periods of metabolic stress and exerts a neuroprotective role mediated by adenosine A1 receptor stimulation. The purpose of this project was to study the effect of adenosine A1 receptor stimulation in an in vitro model of acidosis. The findings suggest that acidosis activates a non-selective sustained cation current which is directly inhibited by adenosine, consistent with the neuroprotective role of adenosine.