Browsing by Subject "Pharmacology"
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Item 17 Beta-Estradiol, Integrins, and Synaptic Proteins(2009-05-01) Chandra, Manjari; Simpkins, James W.Item [3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors(2000-05-01) Yagle, Monica A.; Dillon, Glenn; Martin, Michael; de Fiebre, ChristopherYagle, Monica A., [3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors. Master of Science (Pharmacology), May 2000, 59 pp., 3 tables, 7 illustrations, bibliography, 75 titles. Modulation of the GABAA receptor has been studied with noncompetitive convulsant ligands such as tert-butylbicyclophosphorothionate (TBPS) and picrotoxin (PTX). EBOB is a more recently developed ligand that appears to bind in the same region of the channel at TBPS, but with a higher affinity. While only a few studies have examined the binding of EBOB to vertebrate brain tissue and insect preparations, none have examined potential subunit-dependent binding of EBOB. We have thus examined [3H] EBOB binding in rat cerebellum and HEK293 cells stably expressing human α1β2γ2, human α2β2γ2, and rat α6β2γ2 GABAA receptors. For comparison, [35S] TBPS binding was also examined in α1β2γ2 receptors. Saturation and Scatchard analyses revealed saturable [3H] EBOB binding at one site in all tissue preparations with Kd values ranging from 3 to 9nM. [3H] EBOB binding, like [35S] TBPS binding was inhibited by the CNS convulsants dieldrin, lindane, tert-butylbicyclophosphorothionate (TBOB), PTX, TBPS, and pentylenetetrazole (PTZ) at one site in a concentration dependent fashion. Affinities were in the high nM to low μM range for all compounds except PTZ (low mM range). GABA modulated [3H] EBOB binding in a biphasic manner in α1β2γ2 receptors with a 100-fold difference between stimulatory and inhibitory affinities. Inhibition of GABA-mediated current by TBOB in α1β2γ2 receptors resulted in a functional IC50 of 0.2 μM, in agreement with binding study results. Differences seen in binding between the different receptor subtypes examined suggest that some characteristics of EBOB binding are subunit dependent. In addition, we have shown that [3H] EBOB is a useful ligand in the study of recombinant GABAA receptors and that results obtained with [3H] EBOB are comparable to those obtained with [35S] TBPS.Item Androgens and Cardiovascular Disease(1998-05-01) Dickerman, Rob D.; Walter J. McConathy; Thomas Yorio; Robert GracyDickerman, Rob D., Androgens and Cardiovascular Disease Doctor of Philosophy (Biomedical Sciences), May 1998; 111 pp; 10 tables, bibliography, 197 titles. Anabolic steroids are commonly used by many muscle and strength dependent athletes due to their ability to enhance the hypertrophic effects of resistance training. The use of anabolic steroids by bodybuilders appears to carry significant health risks, most commonly reported are sudden death, myocardial infarction and cardiomyopathy. To investigate the effects of anabolic steroids on cardiovascular risks, a study was designed to analyze the effects of androgens on lipoprotein levels and structure/function of the heart. For the study on lipid-related risk, twelve competitive bodybuilders were recruited for a comprehensive analysis of serum apolipoprotein A-I, B, total cholesterol, HDL-cholesterol, LDL-cholesterol, and testosterone. Serum total cholesterol, HDL- and LDL-cholesterol, apolipoproteins A-I and Be were significantly lower in androgen-users. Consistent with previous reports, androgens were associated with decreases in HDL-cholesterol and apolipoprotein A-I. However, androgens were also associated with reduced serum total cholesterol, LDL-cholesterol and apolipoprotein B. Despite the significantly higher total cholesterol/HDL-cholesterol ratio, the low levels of serum total cholesterol levels (percentile) in the androgen-users raises questions as to whether there is increased risk for cardiovascular disease and the exact role of androgens in cardiovascular risk. To investigate the effects of anabolic steroids in pathologic concentric left ventricular hypertrophy, the effects of androgens on left ventricular size and function were analyzed. Previous investigations conducted on left ventricular size and function have yielded inconclusive results. Problems existing in each of the previous investigations were small body mass, short length of myocardial exposure time to resistance training (years of training), significantly different body mass between steroid-users and steroid-free subjects and monitoring/reporting of steroid use. These problems may have contributed to the discrepancies between studies. Therefore, we selectively recruited eight competitive heavy weight drug-free bodybuilders and eight matched competitive weight bodybuilders on self-directed regimens of anabolic steroids for examination of left ventricular size and function via echocardiography. Increases in left ventricular posterior wall (LVPW) and ventricular septal thickness (VST) were apparent in the steroid-user group (p [less than] 0.05). Ratio of echocardiographic findings to body mass index (BMI) revealed a significantly smaller left ventricular and diastolic dimension (LVDEd/BMI, p [less than] 0.05) in the steroid-user. The smaller LVDEd in steroid-users is coupled with a significantly disproportionate septal and posterior wall thickness in steroid-users. There was no direct evidence of diastolic dysfunction. Thus it appears from these studies that androgens alter lipoproteins leading to a questionable increased risk for cardiovascular disease and may potentiate concentric left ventricular hypertrophy without affecting cardiac function.Item Cellular and Molecular Mechanisms that Distinguish the Effects of Progestorone and Medroxyprogesterone Acetate on Neuroprotection(2006-07-28) Kaur, Paramjit; Goldfarb, Ronald; Singh, Meharvan; Agarwal, NeerajKaur, Paramjit. Cellular and Molecular Mechanisms That Distinguish the Effects of Progesterone and Medroxyprogesterone Acetate on Neuroprotection., Doctor of Philosophy, (Pharmacology and Neuroscience), July, 2006, 203 pp., 5 illustrations, 20 figures and bibliography. Women have a higher prevalence for Alzheimer’s disease (AD) than men, suggesting that the precipitous decline in gonadal hormone levels following the menopause may contribute to the risk of developing AD. However, principal results from the Women’s Health Initiative concluded that women taking conjugated equine estrogens combined with medroxyprogesterone acetate (MPA, tradename: Prempro) incurred more harmful than beneficial outcomes versus the placebo group (Rossouw et al., 2002). This dissertation was aimed at determining if the discrepancy between basic science reports and these clinical studies could have been due to the synthetic progestin, MPA. I hypothesized that P4 and MPA differed in their ability to protect against the excitotoxic/oxidative insult, glutamate. Further, I proposed that this difference in neuroprotective potential would be reflected in the difference in the ability of these hormones to elicit key effectors of two neuroprotection-associated signaling pathways, the ERK/MAPK and P13-Kinase pathways. Finally, studies were initiated to evaluate the potential importance of BDNF (brain-derived neurotrophic factor) in mediating the protective effects of P4. I used organotypic explants of the cerebral cortex, and found that both P4 and MPA elicit the phosphorylation of ERK and Akt, two signaling pathways implicated in neuroprotection, with maximal phosphorylation occurring at a concentration of 100 nM. Interestingly, P4 protected against glutamate- induced toxicity however, while an equimolar concentration of MPA (100nM) did not. Further, P4 resulted in an increase in BDNF, while MPA did not. Our data bring into question the relevance of using MPA as a component of hormone therapies in postmenopausal women, and instead, argue that the relevant progestin for use in treating brain-related disorders is progesterone. Collectively, the data presented here suggest that P4 is protective via multiple, and potentially related mechanism, and importantly, its neurobiology is different from the clinically used progestin, MPA.Item Characterization of Protein Kinase C in Cisplatin Sensitive and Resistant Human Cervical Cancer HeLa Cells(2000-12-01) Mohanty, Sanghamitra; Basu, Alakananda; Simecka, Jerry; Dimitrijevich, DanMohanty, S., Characterization of protein kinase C in cisplatin sensitive and resistant human cervical cancer HeLa cells. Master of Science (Microbiology and Immunology), December, 2000. 37 pp., 11 illustrations, bibliography, 27 titles. Signal transduction plays a crucial role in carcinogenesis. A defect in signaling, by evading cell death or promoting cell proliferation, may result in neoplastic transformation or protection of cells from the cytotoxicity of anticancer drugs. Therefore, in order to understand the complex mechanism of drug resistance, it is relevant to probe into the important signal transduction pathways. Protein kinase C, a key signal transducer, influences cisplatin sensitivity in many cell lines. We examined whether or not the PKC signal transduction pathway is affected during development of resistance to cisplatin by tumor cells. PKC activators increased cisplatin sensitivity in both parental and cisplatin-resistant cells. Western blot analysis showed a slight decrease in cPKCα and nPKCε, an evaluation in nPKCδ and no change in the abundance of PKCϚ in HeLa/CP cells compared to HeLa cells. Though TPA-induced translocation of PKC isoforms was identical in both cell lines, down regulation of PKCδ was defective in resistant cells. Therefore, a deregulation in PKCδ was associated with cisplatin resistance.Item Corticotropin-Releasing Factor and Corticosterone Modulate the Anxiogenic-Like Effects of mCPP(1998-06-01) Jenkins, Jennifer A.; Michael Forster; Robert Luedtke; Patricia GwirtzJenkins, Jennifer A., Corticotropin-Releasing Factor and Corticosterone Modulate the Anxiogenic-Like Effects of mCPP. Doctor of Philosophy (Pharmacology), June 1998, 119 pp., 2 tables, 29 figures, bibliography, 100 titles. The administration of PTZ or mCPP produces anxiety-like behavior as measured by an increase in the percentage of entries into the open arms and the time spent on the open arms of the elevated plus maze (Prunell et al., 1994). Reportedly, PTZ and mCPP substitute for each other in the drug discrimination paradigm (Wallis and Laz, 1998). It is therefore suggested that commonality exists among anxiogenic drugs as perceived by trained animals. Andrews and Stephen (1990) suggested that this overall parallelism is an indication that anxiogenic agents may possess similar properties. Therefore, the question posed is as follows: Is there a common denominator anxiety? The global hypothesis is that the core component of anxiety produced by anxiogenic agents or processes involves stimulation of the HPA axis to release CRF, ACTH and/or CORT. Long Evans rats were trained to discriminate either mCPP (1.4 mg/kg) or PTZ (16mg/kg) from saline in a two-lever choice procedure (FR10) which is food reinforced. Animals were pretreated with CRF, α-helical CRF (a CRF antagonist), two steroid synthesis inhibitors (ketoconazole, KETZ and aminoglutethimide, AMG), CORT or underwent an adrenalectomy prior to behavioral testing in order to test the hypothesis that the release of CRF and/or CORT are components of the discriminate stimulus of the mCPP and/or PTZ. Pretreatment with CRF, KETZ, AMG and an adrenalectomy facilitated mCPP level selection. However in the absence of mCPP neither drug nor adrenalectomy produced drug lever selection. In addition CORT did not alter the mCPP dose response curve. However, CORT replacement therapy returned the does response curve to baseline in adrenalectomized animals. Alpha-helical CRF did not block mCPP discrimination. Unlike mCPP-trained animals, KETZ and AMG decreased PTZ-lever selection in PTZ-trained animals. In addition, CORT enhanced and partially substituted for the discriminative stimulus of PTZ. However, adrenalectomy completely abolished drug lever selection in PTZ animals. To compare the discriminative stimulus effects of mCPP and PTZ, PTZ-trained animals were injected with cumulative doses of mCPP. mCPP-trained animals were injected with cumulative doses of PTZ. mCPP and PTZ minimally substituted for each other. The results suggested that neither CRF nor CORT are components of the discriminative stimulus of mCCP and that the role of the HPA axis in mCPP discrimination maybe be a modulator of the stress response. However, CORT is a component of the discriminative stimulus of PTZ such that CORT is necessary for drug lever selection in PTZ trained animals.Item Cross-Tolerance Between the Discriminative Stimulus Properties of Ethanol, Diazepam and Pentobarbital(1995-12-01) Lytle, Douglas A.; Michael Forster; Glenn Dillon; Thomas YorioLytle, Douglas A., Cross-Tolerance Between the Discriminative Stimulus Properties of Ethanol, Diazepam and Pentobarbital. Doctor of Philosophy (Biomedical Sciences), December, 1995, 132 pp., 8 tables, 19 figures, bibliography, 176 titles. Ethanol, benzodiazepine agonists and barbiturates all facilitate GABA-mediated CT flux. The present experiments tested the hypothesis that, because these agents share this common action, tolerance to discriminative stimulus properties of one of these drugs would result in cross-tolerance to the others. Rats were trained to detect either ethanol (EtOH; 1.0 g/kg), the benzodiazepine diazepam, (DZP; 5.6 mg/kg), or the barbiturate pentobarbital (PB; 10.0 mg/kg) from vehicle using a two-lever choice procedure where food was available under a fixed-ration ten schedule of reinforcement. Subsequently, dose-effect curves for EtOH (0.1-1.78 g/kg), DZP (0.56-17.8 mg/kg), or PB (1.0-17.8 mg/kg) were tested before and after chronic administration of EtOH 96.0 g/kg/12hrs for seven days), DZP (20.0 mg/kg/8hrs for seven days), or PB (32.0 mg/kg/8hrs for seven days). The chronic administration of EtOH conferred tolerance to itself in all cases and cross-tolerance to DZP and PB in subjects trained to detect EtOH, but did not confer cross-tolerance to these agents in their respective discriminations. The chronic administration of DZP conferred tolerance to itself substituting for DZP. Although tolerance developed to DZP substituting for PB after treating animals with chronic DZP, this regimen on DZP did not confer tolerance to itself substituting for EtOH. This regimen of DZP failed to confer significant cross-tolerance to either EtOH or PB under any conditions. The chronic administration of PB conferred tolerance to itself substituting for PB. Although tolerance developed to PB substituting for DZP after treating animals with chronic PB, this regimen of PB did not confer tolerance to itself substituting for EtOH. This regimen of PB failed to confer significant cross-tolerance to either EtOH or DZP under any conditions. In summary, EtOH was found to confer cross-tolerance to DZP and PB only in animals trained to detect EtOH. The chronic administrations of DZP and PB failed to confer tolerance to themselves substituting for EtOH. These results are parsimonious with the heterogeneous nature of the GABA receptor. Finally, tolerance to either DZP or PB does not result in cross-tolerance to the discriminative stimulus properties of the other drug. These results suggest that the mechanisms mediating tolerance to BZs and barbiturates are not linked.Item Elucidation of the Mechanism of Action of Carisoprodol at GABAA Receptors(2009-05-01) Gonzalez, Lorie A.; Dillon, Glenn H.Carisoprodol is an increasingly abused, centrally-acting muscle relaxant. Its sedative effects, which contribute to its therapeutic and recreational use, are attributed to its metabolite, meprobamate, a controlled substance with barbiturate-like activity at GABAA receptors (GABAARs). GABAARs are ion channel-coupled protein complexes underlying the majority of fast synaptic inhibition in the central nervous system. Recent evidence suggests carisoprodol may act independently of meprobamate. Thus, we used behavioral and pharmacological approaches to investigate carisoprodol’s effects on GABAAR function with the ultimate goal of elucidating its mechanism of action at these receptors. In mice, the time course of locomotor depression was comparable for carisoprodol (intraperitoneal or oral) versus meprobamate (intraperitoneal). GABAergic ligands substituted for carisoprodol in drug discrimination studies using carisoprodol trained rats. As observed in vitro, carisoprodol’s effects were antagonized by bemegride, a barbiturate antagonist, but not by the benzodiazepine site antagonist flumazenil, suggesting carisoprodol produces barbiturate-like effects in vivo. Moreover, whole-cell patch clamp recordings were obtained from HEK293 cells expressing human α1β2 and αxβzγ2 (where x = 1-4 and z = 1-2) GABAARs. Each receptor configuration was directly activated and allosterically modulated by carisoprodol in a barbiturate-like manner. Carisoprodol efficacy, but not potency, was subunit-dependent with α and β isoforms contributing to carisoprodol site(s) of action. Notably, carisoprodol was more efficacious at α1-containing receptors, consistent with its sedative effects and abuse potential. Homomeric glycine α1 and GABA ρ1 receptors were carisoprodol-insensitive. Despite similarities between carisoprodol and barbiturates, their sites of action are likely not equivalent as barbiturate-sensitive ρ1W328M subunits were carisoprodol-insensitive. However, chimeric ρ1/α1 receptors gained sensitivity to modulation, but not direct activation by carisoprodol. Our findings indicate carisoprodol modulates GABAARs in a subunit- and receptor-dependent manner, contributing to its pharmacological profile and possibly its abuse potential. Furthermore, partial restoration of modulation, but not direct gating by carisoprodol suggests this drug may mediate its effects via multiple sites on GABAARs.Item Evaluation of Reconstituted High-Density Lipoprotein as a Drug Delivery System in Drug-Sensitive and Drug-Resistant Ovarian Cancer Cell Lines(2004-05-01) Buttreddy, Sabitha; Borejdo, Julian; McConathy, Walter J.; Nair, Maya P.The purpose of this study was to perform uptake and efflux studies of ORB in drug-sensitive (OV1063) and drug-resistant (SK-OV-3 and OVCAR-3) cells. Experiments were carried out by delivering ORB to the cells with and without rHDL to compare the amount of ORB taken up and expelled by the respective cells. Because serum contains lipoproteins, the experiments were also done in the absence of serum to show the specific effect of the rHDL vehicle. Because the efflux pumps responsible for multi-drug resistance are overexpressed and amplified in resistant cells, the efflux of ORB should be higher in these cells compared to the drug-sensitive cell line, OV1063. But, since our rHDL system is designed to reduce drug resistance by releasing the drug directly inside the cell bypassing the efflux pumps, the efflux of ORB should be less when it is delivered with rHDL.Item Genetic Modulation of β-Amyloid Neurotoxicity and Protection by Nicotinic Agents(2007-05-01) Martin, Shelley E.; Basu, Alakananda; Forster, Michael; Singh, MeharvanMartin, Shelley E., Genetic Modulation of β-Amyloid Neurotoxicity and Protection by Nicotinic Agents. Master of Science (Pharmacology and Neuroscience), May, 2007, 53 pp., 7 figures, 2 tables, bibliography, 95 titles. Β-amyloid1-42 (Aβ42) has been implicated in the pathogenesis of Alzheimer’s disease (AD); however, the amount of this peptide in the brain does not correlate well with the presence or severity of AD. This project tested the hypothesis that individual differences exist in susceptibility to Aβ42 neurotoxicity arising from the differences in the expression of α7 nicotinic acetylcholine receptors α7 nACHRs). This hypothesis was tested in primary neuronal cultures derived from inbred mouse strains which differ in expression of α7 nAChRs. Also, the ability of nicotinic agents to modulate Aβ42 toxicity was examined. Significant strain differences in susceptibility to Aβ42 toxicity were found; however, these were not related to levels of α7 nAChRs. Additionally, strain differences were found in the ability of α7-selective partial agonist, an α7-selective antagonist and a α4β2 nAChR-selective antagonist to protect against this toxicity. Inbred strains of mice may be useful in uncovering the pathophysiology of AD.Item Horse Serum High Density Lipoproteins as Drug Transporters(2004-05-01) Johnson, Shemedia; Walter McConathyJohnson, Shemedia J., Horse Serum High Density Lipoproteins (HDL) as Drug Transporters. High-density lipoproteins (HDL) are complex particles composed of specific proteins and lipids that facilitate blood and tissue cholesterol homeostasis by transporting excess peripheral cholesterol to the liver. In association with cholesterol ester transfer protein (CETP) and the enzyme, lecithin: cholesterol acyltransferase (LCAT), HDL contributes to the transport of hydrophobic lipids, including cholesterol ester and triglycerides through the blood. The studies presented here involve the evaluation of horse serum HDL as a carrier of water insoluble drugs and an improved process to isolate and purify horse serum HDL utilizing hydrophobic affinity chromatography. Dilauryl fluorescein (DLF) has been chosen as a model compound for the study of horse HDL as a drug carrier. The prepared HDL/DLF particles have similar flotation densities and size properties to native horse serum HDL. The amount of DLF incorporated into HDL is 30μg/mg protein. Various cancer cell lines internalized DLF from horse HDL/DLF particles successfully. While human plasma contains cholesterol ester transfer protein (CETP), horse plasma does not. Horse plasma/serum can be supplemented by human plasma to study the role of CETP in drug transport and the stability of the horse HDL/drug complex.Item Intravenous pyruvate to protect heart and brain during closed-chest resuscitation and recovery from cardiac arrest(2014-08-01) Cherry, Brandon H.; Mallet, Robert T.; Olivencia-Yurvati, Albert H.; Raven, Peter B.Cardiac arrest is a leading cause of death in the United States and Western Europe. Cardiopulmonary resuscitation (CPR) is the only means of sustaining the victim until application of defibrillatory countershocks. Although it has been over 50 years since its advent, CPR remains a work in progress. Many initially resuscitated victims later die from the damage sustained from ischemia-reperfusion, and treatments to combat the extensive ischemia-reperfusion injury sustained during cardiac arrest-resuscitation remain elusive. The major mechanism of injury underlying ischemia-reperfusion is the intense overproduction of reactive oxygen and nitrogen species (RONS) that accumulate during reperfusion and compromise normal cell function. RONS formed during resuscitation trigger lipid peroxidation, disable enzymes vital for cell metabolism and survival and, ultimately, induce cell death within affected organs. In order to prevent extensive damage to the central nervous system culminating in permanent neurocognitive disability and death, prospective treatments must possess robust antioxidant properties, traverse the blood-brain barrier between the cerebral circulation and brain parenchyma, and be non-toxic at effective doses. Pyruvate is a natural intermediary metabolite, energy-yielding substrate and antioxidant. Pyruvate neutralizes RONS, thereby dampening oxidative stress and preventing covalent oxidative modification of enzymes and lipid membranes, and generates ATP to support brain function. Pyruvate readily traverses the blood-brain barrier and is non-toxic over a wide range of doses, including those previously demonstrated to protect the heart during cardiopulmonary bypass and the brain during stroke, thereby supporting oxygen and fuel delivery to the recovering brain. Moreover, pyruvate has been shown to promote cardiac electromechanical and metabolic recovery following cardiac arrest and open-chest CPR. This study tested whether infusion of pyruvate during, CPR and early recovery can decrease the biomarkers of oxidative stress after cardiac arrest. Isoflurane-anesthetized pigs were subjected to 6 min electrically-induced, untreated ventricular fibrillation, followed by 4 min closed-chest CPR, defibrillation and either 1 or 4 h recovery. Beginning at 5.5 min arrest, either sodium pyruvate or NaCl control were infused iv for the duration of CPR and for the first 60 min after recovery of spontaneous circulation (ROSC). Arterial blood was sampled pre-arrest and at 5, 15, 30, 60, 120, 180, and 240 min ROSC for analyses of blood gases and plasma constituents. At either 1 h (i.e. end of treatment infusion) or 4 h ROSC, a craniotomy was performed, the pig was euthanized, the brain was removed, and biopsies from hippocampus and cerebellum were snap-frozen in liquid nitrogen for biochemical analysis. The first phase of this project tested the hypothesis that intravenous administration of sodium pyruvate during precordial compressions and the first 60 min ROSC restores hemodynamic, metabolic, and electrolyte homeostasis in a closed chest porcine model of cardiac arrest. Resuscitation with pyruvate sharply decreased the incidence of lethal pulseless electrical activity (PEA) following defibrillatory countershocks, and lowered the dosage of vasoconstrictor phenylephrine required to maintain systemic arterial pressure. Pyruvate also enhanced glucose clearance, elevated arterial bicarbonate, and raised arterial pH. The second phase of this project tested the hypothesis that pyruvate prevents the decrease in activity of the brain’s antioxidant enzymes following cardiac arrest and hyperoxic (100% O2). Activities of glutathione peroxidase and glutathione reductase were decreased at 60 min ROSC vs. sham in both the hippocampus and cerebellum. Pyruvate partially preserved glutathione peroxidase activity at 1 h ROSC, but by 4 h, after 3 h of pyruvate clearance from the circulation, the enzyme’s activity fell to the same extent as in NaCl-infused pigs. Interestingly, the glutathione peroxidase/reductase activity fell sharply in non-arrested sham pigs between the time points corresponding to 1 and 4 h ROSC, suggesting that hyperoxia resulting from ventilation with 100% produced sufficient oxidative stress to inactivate the enzymes. Similarly, lactate dehydrogenase activity fell between 1 and 4 h ROSC in hippocampus and especially cerebellum. In sham pigs, lactate dehydrogenase activity decreased from the time points corresponding to 1 and 4 h ROSC, and pyruvate had no effect on lactate dehydrogenase in either region of the brain. Thus, cardiac arrest and hyperoxic ventilation disabled a critical antioxidant system in two ischemia-sensitive brain regions. Pyruvate afforded partial protection of these enzymes which waned after pyruvate cleared from the circulation. We conclude that 1) Pyruvate infusion during cardiac arrest, CPR and early recovery promotes conversion from ventricular fibrillation to a productive sinus rhythm instead of lethal PEA; 2) Pyruvate hastened glucose clearance, a prognostic measure used clinically; 3) Pyruvate elevated the arterial bicarbonate concentration and raised arterial pH, which combats the acidemia normally observed following ROSC; 4) Cardiac arrest-resuscitation and hyperoxic ventilation disabled the glutathione peroxidase-reductase system, a critical component of the brain’s antioxidant defenses, in hippocampus and cerebellum; and 5) Pyruvate delayed oxidative inactivation of glutathione peroxidase in the cerebellum, but this effect subsided as pyruvate elevated. These investigations demonstrate the therapeutic effects and limitations of pyruvate as a resuscitative treatment to hasten electrocardiographic and metabolic recovery post cardiac arrest.Item Opioid Receptors in Aging and Oxidative Stress(2007-01-01) Raut, Atul M.; Ratka, Anna; Simpkins, James W.; Dillon, GlennRaut, Atul M., Opioid Receptors in Aging and Oxidative Stress. Doctor of Philosophy (Pharmacology and Neuroscience), January 2007, 181 pp, 4 illustrations, 21 figures, 159 titles. Oxidative stress has been implicated in aging and neurodegenerative disorders. Pain sensitivity and responses to opioids change with aging. The effect of aging and oxidative stress on opioid receptor system is not yet well understood. To study the effects of aging on pain sensitivity and opioid-induced antinociception, and to determine the possible association of oxidative stress with these pain parameters, in vivo studies were conducted. To further elucidate the effects of oxidative stress on opioid receptor proteins and their function, in vitro studies were carried out. The effects of aging on pain sensitivity and opioid-induced antinociception were studied in male Fischer 344 rats. Oxidative stress markers in cerebral cortex, hippocampus, striatum and midbrain of these rats were estimated. It was concluded that sensitivity to nociceptive stimulus increases and responses to opioids decrease with aging and age-related oxidative damage is negatively correlated with opioid-induced antinociception. To characterize the effects of oxidative stress on function of opioid receptors, changes in intracellular cyclic adenosine monophosphate (cAMP) was measured in human SK-N-SH neuronal cells under oxidative stress conditions. It was found that oxidative stress decreased the function of mu opioid receptor (MOR) but not that of delta or kappa opioid receptors (DOR and KOR respectively). Antioxidant intervention preserved the function of MOR. Western immunoassays revealed that MOR but not DOR and KOR proteins were decreased under oxidative stress conditions. Thus, these findings show a selective impairment of the MOR function and reduction in MOR protein under conditions of oxidative stress. The results from the in vivo and in vitro projects demonstrate the involvement of aging and oxidative stress in modulation of pass sensitivity, opioid-induced antinociception and opioid receptor function and expression.Item Pharmacological Evaluation of the D2 and D3 Dopamine Receptor Selective Compounds on L-Dopa Dependent Abnormal Involuntary Movements in Rats(2008-05-01) Kumar, Rakesh; Robert Luedtke; Hriday K. Das; Nathalie SumienKumar Rakesh, Pharmacological Evaluation of the D2 and D3 Dopamine Receptor Selective Compounds on L-dopa Dependent Abnormal Involuntary Movements in Rats. Master of Science (Pharmacology & Neuroscience), May 2008, 106 pp, 21 illustrations, references, 31 titles. Parkinson’s Disease (PD) is a progressive, neurodegenerative disease of the dopamine neurons that innervate the striatum and is characterized by resting tremor, rigidity, bardykinesia and postural instability. L-dopa treatment is the most common and effective therapy for PD. However, both motor (wear-off phenomena, rigidity and dyskinesia) and non-motor (sweating, tachycardia, restless leg syndrome, anxiety, depression, confusion, reduced alertness, psychosis and/or dementia) side effects are associated with long term L-dopa therapy. Motor complications depend on the duration of L-dopa treatment and the abnormal involuntary movements are known as L-dopa-induced dyskinesia (LID). Several studies have suggested a possible role of the dopamine D3 receptor subtype in LID. Here I evaluated the effects of various D2 and D3 dopamine receptor selective compounds on LID in 6-hydroxydopamine-induced complete lesioned hemi-parkinsonian model of rat. D3 dopamine receptor selective compounds (agonists, partial agonists or antagonists) have antidyskinetic effects on LID. Co-administration of D3 dopamine receptor agonist and D3 dopamine receptor antagonist has additive effects in attenuating the LID instead of antagonizing the effects of each other in vivo. D3 dopamine receptor selective compounds attenuated apomorphine-induced dyskinesia.Item Psalmotoxin-1 and nonproton ligand interactions with acid-sensing ion channels(2015-05-01) Smith, Rachel N.; Gonzales, Eric B.; Dillon, Glenn H.; Sumien, NathalieAcid-sensing ion channels (ASICs) are trimeric, sodium-selective channels activated by extracellular protons. Although ASICs are intriguing molecular targets for pharmacological agents, there remains a lack of selective compounds that differentiate ASIC subtypes. The peripherally located ASIC3 activates with the simple removal of calcium. Additionally, nonproton ligands, like 2-guanidine-4-methylquinazoline (GMQ), have been identified to selectively activate ASIC3 via the nonproton ligand sensor domain (NPLSD). A pair of glutamates in rat ASIC3 (E79 and E423) responsible for GMQ activation is present in ASIC1, despite having no direct modulation effect on the channel. We proposed that nonproton ligand activation of ASIC1 may be state dependent, and relies on expansion of the NPLSD in order for GMQ to reach the binding site and exert its effects. We utilized two features of ASICs in order to test our hypothesis with whole cell and outside out patch-clamp electrophysiology. First, we induced a persistent current in chicken ASIC1 (cASIC1) via a natural venom toxin, Psalmotoxin-1 (PcTx1). We determined that GMQ acts as a molecular wedge by prying apart the transmembrane domains of the cASIC1-PcTx1 protein complex and potentiating ASIC-current. This led us to better understand that the NPLSD is intact in cASIC1 and is sensitive to GMQ additions, albeit in a state-dependent manner. We then theorized that direct activation of rASIC3 by GMQ is possible due the channel’s interaction with extracellular calcium, and were interested in introducing feature into the cASIC1 channel. We generated a chimeric ASIC combining the extracellular, transmembrane, and intracellular domains of rASIC3 and cASIC1 in order to individually isolate the calcium and nonproton ligand effects on channel activation. This chimera, termed cASIC1 (rASIC3-TM/ITC), is comprised of the extracellular domain of cASIC1 and the transmembrane/intracellular domains of rASIC3, and can be activated by GMQ in the absence of calcium similarly to wild-type rASIC3. Thus, GMQ activation was introduced in cASIC1 by replacing the transmembrane domains with those of ASIC3 suggesting that the ASIC3 TM domains dictate NPLSD influence on channel activity. Taken together, we identified that channel state influences nonproton ligand interaction with ASICs, and the transmembrane domains are critical for this interaction.Item Review of the History of Coronary Stenting, Role and Evolution of Adjunctive Pharmacotherapy and Experience with an Ongoing Drug-Eluting Stent Trial(2005-12-01) Kang, Mi Jung; Bens, Annita V.; Ratka, Anna; Oglesby, MichaelKang, Mi Jung. Review of the History of Coronary Stenting, Role and Evolution of Adjunctive Pharmacotherapy and Experience with an Ongoing Drug-Eluting Stent Trial. Master of Science (Clinical Research Management), December 2005, 116 pp., 6 tables, 12 figures, bibliography, 61 titles. The history of stent development with regard to the use of coating materials and eluting drugs, with special emphasis on mechanism of release and duration of action of drugs used in drug-eluting stents, was summarized. The general safety profile of currently used adjunctive pharmacotherapy to coronary stenting, with special emphasis on the clinical trials providing the scientific assessments of the effectiveness and safety of the regimens was reviewed. The enrollment process and the critical role of the Clinical Research Coordinator (CDC) in the process of the implementation of a clinical study with a new drug-eluting stent were described.Item Safety and Efficacy of Alefacept, Efalizumab, Etanercept, and Infliximab in Treating Moderate to Severe Psoriasis: A Meta-Analysis of Randomized Controlled Trials(2007-05-03) Brimhall, Angela K.; John Licciardone; Roberto Cardarelli; Richard VirgilioBrimhall, Angela K., Master of Science, Clinical Research and Academic Medicine, May 19, 2007, Safety and Efficacy of Alefacept, Efalizumab, Etanercept, and Infliximab in Treating Moderate to Severe Plaque Psoriasis: A Meta-Analysis of Randomized Controlled Trials. Pages=41, Tables=3, Figures=9, Bibliography=61 titles. To evaluate and compare efficacy and safety of biological agents in the treatment of plaque psoriasis, data was identified through four parallel systematic reviews. Randomized, controlled, double-blind, monotherapy trials of alefacept, efalizumab, etanercept and infliximab were selected. Sixteen studies comprising 7,931 patients met inclusion criteria. Efficacy was measured by pooled relative risk (RR) and number needed to treat (NNT) of PASI 75 achievement as compared to placebo. Each biological agent was efficacious (P [less than] .001); however, there was a graded response for achievement of PASI 75. The risk of one or more adverse event was increased in the alefacept, efalizumab, and infliximab groups compared to placebo.Item The Effects of Chronic Intermittent Hypoxia on Oxidative Stress and Inflammation(2015-08-01) Snyder, Brina D.; Rebecca L. Cunningham; Robert C. Barber; Weiming MaoObstructive sleep apnea (OSA) is increasingly implicated in neurodegenerative diseases. Evidence points to OSA as contributing to inflammation similar to inflammation observed in neurodegeneration. The chronic intermittent hypoxia (CIH) experienced by OSA patients may be an early contributing factor to chronic inflammation that leads to neurodegeneration. Experiments in this study identify circulating biomarkers affected by OSA and their early impact on neurodegeneration. In the first experiment, oxidative stress and inflammation were observed to increase in male rats exposed to CIH. In the second set of experiments, inflammation within brain nuclei implicated in the onset of Parkinson’s disease and Alzheimer’s disease were correlated to circulating OS elevated by CIH.Item The Role of Dopamine, Nicotine Acetylcholine, Opioid and Sigma Receptors in Ketamine Self-Administration and Reward(2000-05-01) Stoffel, Stephen A.; Michael Forster; Glenn Dillon; Robert LuedtkeStoffel, Stephen A., The Role of Dopamine, Nicotinic Acetylcholine, Opioid and Sigma Receptors in Ketamine Self-Administration and Reward. Doctor of Philosophy in Pharmacology, May 2000, 114 pp 15 figures, bibliography. The rewarding effects of ketamine were postulated to involve dopaminergic neural tracts modulated by nicotinic, sigma, or opioid receptor mechanisms. In support of the hypothesized involvement of dopamine, an increase in extracellular dopamine was detected in the nucleus accumbens using electrochemical chronoamperometry following intravenous ketamine self-administration. When rats were permitted unlimited access to ketamine via self-administration, a greater concentration of dopamine was detected in the nucleus accumbens than was detected in the nucleus accumbens than was detected when self-administration was limited. In a subsequent set of experiments, the effects of agonists or antagonists of dopaminergic, nicotinic, sigma, or opioid receptors were examined for their effect on ketamine self-administration. Decreases in the rate of self-administration following treatment were interpreted to represent an increase in rewarding effect, whereas increases in self-administered were interpreted as a decrease in rewarding effect. The rate of self-administered intraperitoneally prior to ketamine self-administration sessions, but intravenous BMS181-100 would not substitute for ketamine in the self-administration occurred following intraperitoneal (i.p.) administration of: ketamine, SCH23390 (a D1 receptor antagonist), naloxonazine (a mu opioid receptor antagonist), and mecamylamine, a central nicotinic acetylcholine receptor antagonist. An increase in the rate of ketamine self-administration followed nicotine and dihydrexidine (a D1 receptor agonist) intraperitoneal injection. In previous studies, published in the literature, SCH23390 increased the rate of self-administration of amphetamines and cocaine, indicating a competitive effect on drug reward. However, the current studies indicate that the rewarding effects of ketamine were facilitated by SCH23390. The results are consistent with the hypothesis that the rewarding effects of ketamine are mediated through dopaminergic neural pathways. The rewarding effects of ketamine were facilitated by SCH23390. The results are consistent with the hypothesis that the rewarding effects of ketamine are mediated through dopaminergic neural pathways. The rewarding effects of ketamine may be modulated, in an inhibitory fashion, via sigma receptors, presynaptic D1 receptors, nicotinic acetylcholine receptors, and/or μ opioid receptors. Ligands at nicotinic acetylcholine and dopamine receptors yielded effects opposite to that hypothesized based on their ability to modulate the rewarding effects of other abused chemicals.Item The Role of Glycogen Synthase Kinase-3β in the Regulation of Mitochondrial Membrane Permeability(2014-12-01) Brooks, Morgan M.; Patrick R. CammarataLens epithelial cells in a fully mature lens thrive in a hypoxic environment by developing several pro-survival mechanisms that can prevent cellular dysfunction. Many of these mechanisms focus on maintaining mitochondrial membrane integrity. Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mitochondrial electrochemical gradient in a process termed mitochondrial membrane permeability transition (mMPT). The project herein focuses primarily on understanding the role of glycogen synthase kinase-3β (GSK-3β) in preventing mMPT in human lens epithelial (HLE-B3) cells; and, understanding that role in relation to extracellular signal-regulated kinase 1/2 (ERK1/2), a known regulator of GSK-3β activity. These studies further define mitoprotective mechanisms of lens cells by identifying how ERK1/2 and GSK-3β can directly (through the mitochondrial transition pore) or indirectly (through the induction of apoptosis) effect mitochondrial membrane potential). Additionally, we extended the GSK-3β studies into the field of epithelial to mesenchymal transition (EMT) research. Specifically we focused on understanding how GSK-3β in conjunction with the hypoxia inducible factor (HIF) proteins can influence the persistence of EMT and the production of vascular endothelial growth factor (VEGF). Collectively, these studies demonstrate important roles in lens epithelial cell mitoprotection for GSK-3β and ERK1/2; and, demonstrate a pivotal role for HIF-1α in the persistence of EMT under hypoxic conditions. Overall, the work described herein has provided invaluable information and understanding in the field of mitoprotection research as well as EMT research.