Browsing by Subject "Comparative and Laboratory Animal Medicine"
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Item Acetoacetate: A Cardioprotective Antioxidant(2002-05-01) Squires, Jeffrey E.; Mallet, Robert T.; Caffrey, James L.; Carroll, JoanSquires, Jeffrey E., Acetoacetate: A Cardioprotective Antioxidant. Master of Science, June 2002, 100 pp., 1 table, 18 illustrations, bibliography, 70 titles. The purpose of this study was to test the effectiveness of acetoacetate and β-hydroxybutyrate as myocardial protectants following peroxide injury and to determine acetoacetate’s ability to potentiate β-adrenergic responsiveness following ischemia-reperfusion injury. This study utilized antegradely perfused isolated working hearts exercised from male guinea pigs and sustained with glucose-fortified Krebs-Henseleit. Hearts were challenged by either 10 min perfusion with 100 μM H2O2 or 45 min of low flow ischemia exacerbated by ι-norepinephrine infusion. H2O2-challenged hearts were treated with 5 mM acetoacetate or β-hydroxybutyrate, whereas hearts injured by ischemia/reperfusion were treated with 5 mM acetoacetate. In the case of the ischemically injured hearts, acetoacetate treatment was combined with 2 nM isoproterenol to delineate acetoacetate’s ability to enhance β-andrenergic responsiveness to submaximal inotropic stimulation. Data were compared to non-injured time control hearts and injured untreated hearts to determine the impact of ketone body treatment. Acetoacetate increased citrate and glucose 6-phosphate content, nearly restored power, and increased the glutathione antioxidant redox potential (GSH/GSSG) by 140% in H2O2-injured myocardium. Although β-hydroxybutyrate increased citrate, an activator of NADPH-generating pathways, and glucose 6-phosphate, the substrate for the hexose monophosphate shunt to the same extent as acetoacetate, β-hydroxybutyrate raised GSH/GSSG by only 60% and did not enhance cardiac power. Therefore, acetoacetate enhances contractile function by augmenting the glutathione redox potential, and does so by additional mechanisms independent of the citrate and hexose monophosphate pathway. In hearts stunned by ischemia/reperfusion, acetoacetate and isoproterenol each increased power and glutathione redox potential three-to-fourfold, but phosphocreatine potential was 70% higher in acetoacetate hearts. Combined, acetoacetate + isoproterenol synergistically increased power and GSH/GSSG 16- and 17- fold respectively, doubled {NADPH/NADP+}, and increased cyclic AMP content 30%. These findings support the conclusion that acetoacetate enhances myocardial sensitivity to β-adrenergic stimulation possibly by enhancing GSH/GSSG.Item Adenosine Receptor Blockade Increases Lactate and Purine Release But Does Not Affect Functional Recovery in Isolated Rabbit Myocardium(1995-12-01) Wang, Sheng; Downey, H. Fred; He, Miao-Xiang; Mallet, Robert T.Wang, Sheng, Adenosine Receptor Blockade Increases Lactate and Purine Release but does not Affect Functional Recovery in Isolated Rabbit Myocardium Master of Science (Biomedical Sciences), December 1995; 67 pp; 3 tables; 8 figures; bibliography, 121 titles. This study tests the hypothesis that endogenous adenosine mediates recovery of cardiac function in ischemia/reperfused rabbit hearts. Isolated isovolumic rabbit hearts perfused at constant pressure was subjected to mild ischemia (perfusion pressure 50 cm H2) or moderate ischemia (perfusion pressure 30 cm H2O) for 90 min followed by 60 min of reperfusion. In treated hearts, infusion of 100 μM 8-p-sulfophenyl theophylline (SPT) was initiated 20 min before ischemia and maintained throughout the experiment. Adenosine receptor blockade did not affect left ventricular function assessed from pressure-heart rate product (PRP). Lactate release increased to 152 ± 24% of baseline during mild ischemia and 259 ± 26% of baseline during moderate ischemia in untreated hearts. Lactate release was markedly elevated at baseline, ischemia and reperfusion by SPT treatment (p [less than] 0.05 compared to untreated). Purine nucleoside release was 4.1 ±0.7 nmol · min-1 · g-1 in SPT treated group and 1.8 ± 0.24 nmol · min-1 · g-1 in untreated group during moderate ischemia (P [less than] 0.05). Myocardial efficiency was significantly lower in the SPT treated hearts (240 ± 11 mmHg · g=1 · μl-1 O2) compared to untreated hearts (300 ± 22 mmHg · g-1 · μl-1 O2) during reperfusion after moderate ischemia. In conclusion, adenosine receptor blockade stimulates glycolysis in normoxic and ischemic myocardium, but does not affect post-ischemic functional recovery.Item Age Related Changes in Rabbit Cornea: Permeability and Membrane Properties(1994-12-01) Tai-Lee, Ke; Clark, Abbot F.; Gracy, Robert W.; McConathy, Walter J.Ke, Tai-Lee, Age Related Changes in Rabbit Cornea: Permeability and Membrane Properties. Doctor of Philosophy (Biochemistry), December, 1994, 139 pp., 26 tables, 13 illustrations, bibliography, 117 titles. This investigation was designed to characterize age-related changes in corneal function and biochemical structure. The specific aims were to: 1) systematically assess changes in permeability to compounds of different molecular weights and lipophilicities, 2) examine differences in tissue binding by utilizing a theoretical transport model, and 3) evaluate the biochemical changes in lipid composition and distribution. Experiments to compare young (six weeks) versus old (three to four years) rabbit corneal permeability were carried out utilizing an in vitro diffusion model. Changes in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. Permeabilities of selected compounds with different physicochemical properties were evaluated in young and old intact and denuded (wounded) rabbit corneas. With age, the membrane permeability significantly decreased in parallel with an increase in transmembrane resistance. Age-related changes in activities of esterase and phosphatase were also found. For some compounds, the aged corneas exhibited longer lag times in penetration studies. This suggested that the binding constant in the cornea from older animals was higher than in young animals. Maximum binding capacity from theoretical model calculations correlated well with experimental results in the young corneal stroma but correlation was less rigorous for old corneal stroma. Age-related changes in lipid composition and distribution in corneas were observed and provide indirect evidence for a decrease in membrane fluidity (decrease in the ratio of phosphatidylcholine/sphingomyelin) in the aged cornea. Results indicate that the aging process in the cornea is associated with changes in biochemical structural matrix including membrane lipid composition and physical properties such as fluidity (microviscosity). Functional correlations include changes in: 1) transmembrane resistance, 2) membrane permeability, 3) enzymatic activities (esterase and phosphatase), and 4) binding properties of the cornea. A possible mechanism for understanding and developing an intervention for age-related changes in the cornea is postulated.Item Alterations in mRNA Levels of Selected Gene Products During Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells(2001-08-01) Vopat, Kelly S.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouVopat, K., Alterations in mRNA Levels of Selected Gene Products during Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells. Master of Science (Biomedical Science), August 2001. 54 pp., 2 tables, 10 illustrations, bibliography, 105 titles. In order to explore the mechanisms involved in the signal transduction pathways of ischemia-induced apoptosis of RGCs in glaucoma, an in vitro ischmia model of transformed rat retinal ganglion cells (RGC-5) was utilized. RGC-5 cells were exposed to hypoglycemia, hypoxia, and ischemia for six hours. Hypoxia and ischemia resulted in apoptosis of RGC-5 cells as determined by TUNEL assay. The bax mRNA levels increased significantly in cells exposed to hypoxia. The mRNA levels of hemoxygenase, c-fos HSP 70, and BDNF showed a trend of increase in both the hypoxic and ischemic conditions. These results demonstrate that retinal ganglion cells undergo apoptosis in hypoxic conditions likely via an increase in bax/bcl-2. The up-regulation of BDNF and some stress proteins may be part of a cellular rescue effort trying to overcome the damage created by hypoxic and ischemic stresses.Item Cardiac Parasympathetic Dysfunction in Morphine Addiction(1997-12-01) Napier, Leslie D.; Caffrey, James L.; Raven, Peter B.; Gwirtz, Patricia A.Napier, Leslie D., Cardiac Parasympathetic Dysfunction in Morphine Addiction. Doctor of Philosophy (Biomedical Sciences), December, 1997, 137 pp., 9 tables, 22 figures, references, 163 titles. The effects of chronic morphine treatment on parasympathetic control of the heart and associated cellular mechanisms were examined using a canine model. Vagal bradycardia was significantly blunted in dogs treated for one week with subcutaneous morphine pellets. In a separate group of dogs, heart rate and high frequency fluctuations in heart rate declined during the first three hours of subcutaneous morphine infusion consistent with the vagatonic action of acute morphine. Heart rate remained below baseline on Day 2 of the morphine infusion but had returned to normal by Day 10. Ambient sympathetic tone was increased on Days 2 and 10, and plasma catecholamines were elevated on Day 2. The intrinsic heart rates on Days 2 (160 bpm) and 10 (162 bpm) of morphine treatment were lower than the pre-treatment rate (182 bpm). Suggested mechanisms include a fundamental change in sinoatrial nodal cell function or attenuated tachycardia induced by vasoactive intestinal peptide co-released with acetylcholine from post-ganglionic parasympathetic neurons. The time to 50% maximal bradycardia during vagal nerve stimulation was increased with chronic and acute morphine suggesting an effect on the rate of acetylcholine synthesis, release or degradation. Muscarinic receptor density in left ventricular and right atrial sarcolemmal membranes from dogs treated chronically with morphine were 34% and 17% higher, respectively, than in control animals. Chronic morphine had no effect on basal or MnCl2-stimulated cyclase activity in either region. Similarly, maximal β-adrenergic and muscarinic receptor/G-protein coupling to adenylate cyclase were not altered by chronic morphine. Atrial norepinephrine content was higher than that in the ventricles and was unaltered by morphine. Ventricular norepinephrine was decreased with chronic but not acute morphine treatment. Epinephrine was evenly distributed throughout the myocardium and was reduced in both the atria and the ventricles by either acute or chronic morphine. This pattern suggests that morphine may reduce extraneuronal uptake of catecholamines. Collectively these studies show that chronic morphine treatment and the accompanying persistent vagal activity may reduce parasympathetic function. This attenuated function, however, is short-lived since sympathetic systems adapt with compensatory responses masking, or perhaps reversing, initial parasympathetic deficits.Item Characterization of a Novel Extracellular Superoxide Dismutase Allele Discovered in Mouse Models of Atherosclerosis(2004-07-01) Pierce, Anson; Dory, Lad; Easom, Robert; Basu, AlakanandaAnson Pierce, Characterization of a Novel Extracellular Superoxide Dismutase Allele Discovered in Mouse Models of Atherosclerosis. Doctor of Philosophy (Biochemistry and Molecular Biology), July 2004, 128 pp., 3 tables, 22 illustrations, references, 230 titles. Many diseases display some involvement with oxidative mechanisms and could potentially benefit from antioxidant therapy designed to restore the balance between reductive and oxidative factors. Data presented in this dissertation explore and establish the protective effect hyperbaric oxygen (HBO) has on the development of atherosclerosis, an oxidation-driven inflammatory disease mediated through low-density lipoproteins in the vasculature. Atherosclerosis in the apolipoprotein E-/- (apoE-/-) mouse is drastically reduced after 10 weeks of HBO treatment. Macrophages in HBO treated mice have an increased antioxidant capacity and reduced ability to generate oxidants. From this work, a new polymorphism of a key antioxidant enzyme, extracellular superoxide dismutase (ecSOD), is identified and characterized in mice. The new polymorphism is termed the “short” allele, and has the potential to alter the regulation of ecSOD mRNA and protein, as well as enzyme activity. Examination of its effect on the ecSOD phenotype in mice shows dramatic changes in enzyme levels and activity. In the plasma compartment ecSOD activity and mass are elevated, and indicate based on heparin injection studies that a change in ecSOD distribution results in tissues of mice expressing the short allele. Systematic examination of ecSOD in tissues of mice shows that its distribution is altered such that it is more accessible to heparin; this is most evident in the liver and kidney of mice expressing the short allele. The finding that HBO is protective against atherosclerosis highlights a potentially promising approach to treatment for this devastating disease, sheds light on the role oxidative processes play in atherosclerosis, and identifies potential targets for antioxidant therapy. This study also shows for the first time that two alleles for a major antioxidant enzyme exist in mice that display markedly different effects on the ecSOD phenotype, a finding that underlines the importance of genetic homogeneity in mouse models and adds to our knowledge concerning the role antioxidants play in human health and disease.Item Characterization of the Myo-inositol Efflux Pathway in Cultured Bovine Lens Epithelial Cells(1997-12-01) Reeves, Rustin E.; Victoria Rudick; Robert Easom; Thomas YorioReeves, Rustin E., Characterization of the Myo-inositol Efflux Pathway in Cultured Bovine Lens Epithelial Cells. Doctor of Philosophy (Biomedical Sciences), December, 1997, 173 pp., 1 table, 28 figures, bibliography, 94 titles. The basic cellular requirement of volume regulation utilizes many different channel and transport pathways working on concord to maintain a constant cell volume. Among these are distinct pathways spontaneously activated by changes in cell volume that modulate the gain or loss of certain organic osmolytes, such as myo-inositol (MI). The major goal of this dissertation is to characterize and identify the mechanism involved in the MI effux pathway and explore its relationship with intracellular polyol accumulation in cultured bovine lens epithelial cells (BLECs). Hypertonic exposure of BLECs causes an increase in MI uptake and aldose reductase enzymatic activity, two events which ultimately influence osmolyte efflux. A biphasic efflux pathway induced by rapid cell swelling (hypotonic-induction) was demonstrated in BLECS switched from hypertonice to physiologic medium. Also, intracellular polyol accumulation from galactose exposure resulted in enhanced activation of the MI efflux pathway (polyol-induction). Chloride channel inhibitors effectively blocked MI efflux suggesting a relationship between anion (chloride) movement and intracellular MI loss from cell to medium. Expression of a chloride channel regulatory protein, pICln, was demonstrated by Northern blot analysis in cultured BLECs. Hypertonic exposure upregulates the expression of pICln mRNA while hypotonicity downregulates expression. The volume-sensitivity for transcription of PICln mRNA in BLECs lends strong support for its role in both anion and osmolyte loss associated with the MI efflux pathway. The MI efflux pathway functions as a “relief value” in cell volume regulation by providing a conduit to alleviate intracellular osmotic stress. The mechanism which evolved to function under normal cellular circumstances in relief of excessive accumulation of intracellular osmolytes (i.e. polyols), may, by design, inadvertently promote the loss of essential intracellular volume and nonvolume regulatory organic solutes. Ironically, under certain pathological conditions, this mechanism, designed to protect the cell from intracellular osmotic stress, may instead be detrimental to the cell by promoting the excessive loss of osmolytes essential for normal cell function.Item Characterization of the Serotonin Receptors in the Long Posterior Ciliary Artery of the Bovine Eye(2000-08-01) Landry, Theresa A.; Quist, Eugene; Martin, Michael; Pang, Iok-HouLandry, Theresa A., Characterization of the Serotonin Receptors in the Long Posterior Ciliary Artery of the Bovine Eye. Doctor of Philosophy (Biomedical Science), August 2000, 14 pp., 5 tables, 29 illustrations, bibliography, 104 titles. Vascular disease and vasospasm are implicated in the etiology of glaucoma. The long posterior ciliary (LPCA) is the major blood supply for the ciliary body including the ciliary processes that produce aqueous humor. Information about the pharmacological control of this vessel would be helpful in understanding its normal and pathologic function. Serotonin (5-HT) is a neurotransmitter that effectively constricts the LPCA. The objective of this research is to identify the serotonin receptor subtype responsible for the 5-HT induced vasoconstriction of the LPCA and to characterize the cellular mechanisms that mediate that contraction. Ring segments of the LPCA were dissected from bovine eyes and mounted on tungsten triangles attached to a force transducer. Changes in vascular tension were measured and recorded using a physiography recorder. Dose response curves with 5-HT, 5-HT 1-like agonist, 5-CT, and 5-HT2 agonist, α-methyl-5-HT, indicate that the 5-HT 1-like receptor contributed about 15.13% to the contraction and the 5-HT2 receptor contributed to 61.61%. The EC50 for the three agonists were 283 nM (5-HT), 336 nM (5-CT), and 1.7 μM (α-methyl-5-HT). Inhibition curves with selective antagonists indicate that the IC50 is (5-HT 1-like antagonist) and ketanserin (5-HT2 antagonist). Following incubation of the rings with diltiazem 10 μM or nifedipine 10μM, the response to 5-HT was reduced 65.*% and 61.7% respectively. Incubation in calcium free PB produced similar results. Ryanodine inhibited the 5-HT contraction by 58.1% and caffeine inhibited the response 100%. PKC inhibitors bisindolymaleimide II 1 μM, disindolylamalemide II 10 μM, chelerythrine 25 μM and H-7 5 μM decreased the 5-HT response by19.8%, 55.7%, 31.1% and 61.5% respectively. Incubation of the ring segments with one of three PLC antagonists, 2-NCDC 70 μM, U73122 0.5μM, or neomycin 5 mM, prior to the addition of 1 μM serotonin, significantly reduced the contraction of each vessel, p [less than] 0.0001. The 5-HT-induced vasoconstriction of the LPCA of the bovine eye is mediated through activation of both 5-HT2 and 5-HT 1-like receptors. The contraction is dependent on the mobilization of calcium and is mediated in part through PLC activated intracellular calcium release from IP3 sensitive stores.Item Coronary Perfusion Pressure-Induced Changes in Coronary Vascular Volume in the Canine Right Ventricle(1997-12-01) Yu, Ying; H. Fred Downey; Robert T. Mallet; Michael L. SmithYu, Ying, Coronary Perfusion Pressure-induced Changes in Coronary Vascular Volume in the Canine Right Ventricle. Master of Science (Biomedical Sciences), December 1997; 33 pp; 4 tables; 5 figures; bibliography, 24 titles. Changes in coronary perfusion pressure cause changes in myocardial contractile function and oxygen consumption (MVO2), particularly in the right ventricle (RV). This study determined the effects of right coronary (RC) perfusion pressure (RCP) on RC vascular volume (RCB) and its relationship to MVO2 of in situ, working canine hearts, and also investigated whether changes in MVO2 are due primarily to altered RCP or RC blood flow (RCF). In 15 open chest, anesthetized dogs, the RC artery was cannulated and perfused with arterial blood diverted from a femoral artery. To blunt RCP-induced changes in RCF, vasopressin was infused into the RC perfusion line in seven dogs. RCV was measured by an indicator dilution method as RCP was varied without vasopressin (RCP=60, 100, 140, and 180 mmHg) and with vasopressin (RCP=60 and 100 mmHg). Without vasopressin, changes in RCP induced changes in MVO2 which were associated with changes in RCV and RCF. With vasopressin, increasing RCP from 60 mmHg to 100 mmHg produced no changes in RCF, RCV, or MVO2. These results indicate that RCP-induced changes in RV MVO2 are mediated by RCV and/or RCF, but not by RCP per se.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 Differential Gene Expression Profiling in a Small Animal Model of Progressively Pacing-Induced Heart Failure(2006-06-01) Selby, Donald Evan; Stephen R. Grant; Patricia A. Gwirtz; Dan DimitrijevichDonald Evan Selby, Differential Gene Expression Profiling in a Small Animal Model of Progressively Pacing-Induced Heart Failure. Doctor of Philosophy (Biomedical Sciences), July 2006, 235 pp, 4 tables, 35 illustrations, references, 328 titles. Pacing induced tachycardia (PIT), in mammals, is known to cause a change from normal heart function to early left ventricular dysfunction. Progression to heart failure in experimental animals, such as dogs, pigs, and sheep, takes place in a relatively short period of time compared to the disease development observed in humans. Due to the cost and nature of using such animals, there is a need for a small animal model of PIT, which would delineate the etiology of the disease state by impairing the systolic function. The mode of action of overpacing inducement of cardiomyopathy, as the data suggests, may be through a sarcomere stretch sensor and its length-dependent signaling mechanism. In this study, an internal electrical-overpacing of an isogenic rabbit strain over a 52-day period was used to initiate a pathology consistent with human CHF. The data presented demonstrated that PIT causes alterations in the systolic ability of the heart, observed as reduced fractional shortening of the heart. This is seen in changes of the message pool population for proteins of the contractile architecture. Initially the heart is being paced rapidly and therefore there is insufficient time to get blood into the chamber. Thus, the data suggests that a mechanical stretch sensor is the process by which overpacing the heart leads to changes in gene expression which ultimately cause a compounding cellular condition which exists during heart failure. The data shows that there are gene isoform ratio changes that occur as the disease develops these include changes in differential expression of cardiac titin alternative splicing isoforms. The data suggests that there is also isoform switching occurring with alternative splicing of the gene encoding for SERCA2a, the probe 1587641_at shows a moderate decrease in expression and using BLAST for this probe this sequence is homologous to an alternative splicing variant of SERCA2a of the rabbit accession number J04703. The data shows that ferritin heavy chain also has an alternative splicing variant that are differentially regulated, this dysregulation of the isoform ratio may be linked to ADAMSTS1, a disintegrin and metalloproteinase isoform 1, which is seen to be downregulated in the data, these play a role in negative regulation of cellular proliferation. In addition to these detected isoform changes in the ratios of alternative splice variants changes are seen in genes linked to sarcomere integrity such as dystrophin probe 1582958_at is significantly increased in its expression, also integrin beta-1 probe 1584175)at shows a marginal increase in expression. The protease calpain probe 1604384)at, which uses a substrate the aforementioned integrin, dystrophin, and titin is also significantly upregulated in the data. Interestingly calpastatin, probe 1591603_at the inhibitor of calpain is marginally increased in its expression. Only recently has titin become to be appreciated as the protein that is responsible for the Frank Starling law as it undergoes an isoform ratio change as heart failure develops. These changes are initially caused by changes in ion concentration and stress upon the contractile proteins but as seen in the study, leads to altered gene expression. In this model, these gene alterations lead to diastolic dysfunction and the compounded problems constitute heart failure. This work shows that heart failure induced by over-pacing creates physical demands upon the framework of the heart and these physical stresses are transmitted through mechanical sensors leading to differential expression of the message pools for proteins involved in the way the heart contracts, and fills upon relaxation which ultimately ends in a heart that can do neither, thus leading to death.Item Dobutamine Increases Mechanical Function and Cytosolic Phosphorylation Potential During Moderate Right Ventricular Hypoperfusion(2000-08-01) Yi, Kun Don; H. Fred Downey; Robert Mallet; Eugene E. QuistYi, Kun Don. Dobutamine increases mechanical function and cytosolic phosphorylation potential during moderate right ventricular hypoperfusion Master of Science (Biomedical Sciences), August, 2000, 101 pp, 4 tables, 18 figures, references, 108 titles. This study was conducted to investigate the functional and metabolic effects of regional inotropic stimulation with dobutamine during right ventricular (RV) hypoperfusion. Right coronary perfusion pressure was incrementally lowered to 40 mmHg from 100 mmHg, and two-doses of dobutamine (0.01 and 0.06 μg/kg/min) were continuously infused for 15 min into the right coronary artery in pentobarbital sodium-anesthetized mongrel dogs of either sex. Myocardial energy metabolites, glycolytic intermediates, glycogen, and phosphorylation potential were measured in freeze-clamped RV biopsies. RV hypoperfusion caused a 54% decrease in right coronary blood flow, a decrease in lactate uptake, and an increase in glucose uptake. Systolic segment shortening, isometric force, MVO2, and oxygen utilization efficiency (O2UE: power/MVO2) decreased significantly. Energy reserves were unaffected by the hypoperfusion. Low-dose dobutamine during hypoperfusion improved regional mechanical function without increasing MVO2, and thus, improved O2UE. Remarkably, low-dose dobutamine markedly increased phosphocreatine content and phosphorylation potential. In contrast, high-dose dobutamine produced only transient improvements in function and efficiency and sharp decreases in energy reserves. Analysis of glycolytic intermediates showed a sustained augmentation of glycolysis during low-dose dobutamine, but glycolysis was limited by high-dose dobutamine at the level of glyceraldehyde-3-phosphate dehydrogenase. Dobutamine is capable of improving both contractile function and cellular energetics in underperfused RV myocardium at low but not high dose dobutamine. Therefore, dosage should be carefully selected.Item Effects of Nitric Oxide on Right Ventricular Metabolism and Coronary Blood Flow(2000-01-09) Setty, Srinath; H. Fred Downey; Patricia A. Gwirtz; James L. CaffreySetty, Srinath Varadaraj. Effects of Nitric Oxide on Right Ventricular Metabolism and Coronary Blood Flow Doctor of Philosophy (Biomedical Sciences), January, 9, 2001, 123 pp, 3 tables, 16 figures, references, 211 titles. Nitric oxide (NO) formed from L-arginine and released from vascular endothelium causes relaxation of vascular smooth muscle via a cGMP mechanism. However, the of NO as a regulator of coronary blood flow control is unclear. NO has been shown also to reduce oxygen consumption in various in-vitro preparations, but its effect on myocardial oxygen consumption (MVO2) in the left ventricle of the working heart is controversial. The effect of NO on MVO2 in the right ventricle (RV) is unknown. This investigation delineated the effects of NO on RV MVO2 during controlled systemic and coronary hemodynamic conditions. In open chest dogs, NO synthesis was blocked by intracoronary infusion of NO synthesis with Nω-nitro-L-arginine methyl ester (L-NAME, 150 μg/min). To avoid effects of NO synthesis blockade on right coronary blood flow (RCBF), which might have altered RV MVO2, experiments were conducted during adenosine-induced maximal right coronary vasodilation (n=12). RCBF, RV MVO2, and other variables were measured at baseline and at elevated right coronary perfusion pressures (RCP). Under these conditions, L-NAME significantly increased RV MVO2 at baseline and at elevated RCP (P [less than] 0.05 vs. untreated control condition). These results indicate that NO acts to retard RV oxidative metabolism. We further characterized the role of NO on RV MVO2 during increases in RV workload, estimated as a product of heart rate X RV peak systolic pressure X RV dP/dt. RV workload, RCBF, and RV MVO2 were increased by intracoronary norepinephrine infusions at baseline RCP (n=5). L-NAME significantly reduced RCBF (P [less than] 0.05 vs. untreated control condition), and RV MVO2 was significantly higher at any measured RV workload during L-NAME (P [less than] 0.05 vs. untreated control condition). These findings indicate that NO is an important component of RCBF control and that NO blunts norepinephrine-induced increase in RV MVO2. If NO reduced RV MVO2 it may be cardioprotective during moderate right coronary hypoperfusion. Thus, we sought to determine if in fact the RV MVO2 was reduced by NO during moderate right coronary hypoperfusion (n=9). RCP was reduced to 60 (n=5) and 40 mmHg (n=4), and RCBF and RV MVO2 fell as RCP was reduced. L-NAME significantly increased RV MVO2 at RCP of 60 and 40 mmHg (P [less than] 0.05 vs. untreated control condition), although RV workload was not altered. Since NO reduced RV MVO2 without compromising RV mechanical performance, RV oxygen utilization efficiency was enhanced. Taken together, these findings demonstrate that NO has a significant dampening effect on RV MVO2.Item Effects of Testosterone on Obesity-Related Cardiac Hypertrophy and Fibrosis(2009-08-01) Wilson, Ana Kaye; Joan F. Carroll; James L. Caffrey; Robert T. MalletWilson, Ana Kaye. Effects of testosterone on obesity-related cardiac hypertrophy and fibrosis. Master of Science (Integrative Physiology), August 2009, 71 pp, 3 tables, 6 figures. Both testosterone and obesity are known to increase renin-angiotensin system activity, leading to cardiovascular dysfunction. This study determined the interactive effects of obesity and testosterone on left ventricular hypertrophy and cardiac fibrotic factors. Male New Zealand White rabbits were fed a lean or 10% added fat diet. After 12 weeks, fat-fed rabbits exhibited increased left ventricular weight (6.05±0.16 vs. 4.75±0.10 g, respectively, p≤0.05) and cardiomyocyte cross-sectional area compared to lean rabbits (372.3±19.0 vs. 305.0±13.4μm2, respectively; p≤0.01). These effects were attenuated by both castration and treatment with the angiotensin type 1 receptor blocker, losartan. Obese rabbits did not exhibit increased myocardial collagen as expected. However, castration and losartan treatment increased matrix metalloproteinase-2 (MMP-2) activity in obese rabbits. Despite the effects of castration hypertrophy and MMP-2 activity, castration did not attenuate plasma renin activity of aldosterone. These data suggest that testosterone contributes to obesity-related left ventricular hypertrophy and decreases collagen degradation, independent of renin activity.Item Endothelin-1 Mediated Regulation of Extracellular Matrix Collagens- A Role in Pathology of Primary Open Angle Glaucoma(2007-11-01) Rao, Vidhya Ramachandiran; Thomas Yoroi; Neeraj Agarwal; Raghu KrishnamoorthyEndothelin -1 Mediated Regulation of Extracellular Matrix Collagens –A role in Pathology of Primary Open Angle Glaucoma. Vidhya R. Rao, Doctor of Philosophy. (Pharmacology and Neuroscience), November, 2007, 157 pp., 3 tables, 18 figures. Summary. Primary Open Angle Glaucoma (POAG) is a progressive optic neuropathy characterized by loss of retinal ganglion cells, optic nerve degeneration and characteristic extracellular matrix (ECM) remodeling of the optic nerve head. An increase in collagen type I and VI is observed at the level of lamina cribosa (LC), a distinct connective tissue region of optic nerve in POAG subjects. Extensive ECM remodeling with enhanced collagen deposition observed in POAG is consistent with the pathology of fibrosis. Mechanisms contributing to ECM remodeling in POAG is not known. Endothelin-1(ET-1), a potent vaso-active peptide plays a key role in glaucoma pathology. Intra-vitreal administration of ET-1 in animal models results in optic neuropathy, RGC apoptosis, axonal transport block and ONA activation. An upregulation of ET-1 and ETB receptors is observed in glaucomatous LC and animal models of glaucoma and ET-1 mediated detrimental effects in POAG appears to be mediated by ETB receptors. ET-1 initiatives and maintains enhanced collagen synthesis and deposition in various tissues under pathological conditions and is recognized as a potent profibrotic factor. In the present study we hypothesized that ET-1 increases extracellular matrix collagen deposition in lamina cribrosa and this change in ECM contributes to optic nerve fibrosis. We have demonstrated that cells of lamina cribrose (LC) cells, express functional ETA and ETB receptors. ET-1 increases intracellular calcium mobilization via ETA receptors and increases NO release by mechanisms involving both ETA and ETB receptors. Consistent with POAG pathology we have observed an upregulation ETB receptors in LC cells in response to chronic treatment with ET-1. LC cells also express prepro-ET-1, the primary gene transcript of ET-1. We have demonstrated for the first time that ET-1 exerts its profibrotic effects by enhancing collagen type I and type VI mRNA, protein synthesis, deposition and secretion in LC cells. ET-1 enhanced collagen deposition in LC cells appears to involve both ETA and ETB receptors, as both of the receptor antagonist, individually inhibit ET-1 mediated collagen synthesis. We have demonstrated that ET-1 also exerts its profibrotic effects in vivo by enhancing collagen deposition in rat optic nerve head. We have also observed an apparent decrease in ET-1 mediated collagen VI deposition in optic nerve heads of ETB deficient transgenic rats suggesting that ET-1 mediated collagen VI synthesis involves ETB receptor activation. In conclusion, endothlein-1 stimulates collagen synthesis and deposition both in vitro in LC cells as well as in vivo at the level of rat optic nerve head. ET-1 mediated increase in collage synthesis at the level of optic nerve head could render a fibrotic mechanism that contributes to the progression of POAG.Item Exercise-Evoked Metabolic Adaptations in Canine Myocardium(1999-12-01) Stuewe, Steven Richard; Robert Mallet; Neeraj Agarwal; Patricia GwirtzStuewe, Steven Richard, Exercise-Evoked Metabolic Adaptations in Canine Myocardium. Doctor of Philosophy (Biomedical Sciences), November 1999; 128 pp; 4 tables; 17 figures; bibliography, 130 titles. Aerobic exercise training evokes adaptations in the myocardial contractile machinery that enhance cardiac functional capabilities, and the myocardium’s capacity to consume energy. Despite considerable investigative effort, the effects of exercise training on myocardial intermediary metabolism, the source of energy for cardiac function, have not been defined. The investigations described herein were undertaken to delineate the effects of aerobic exercise training on key rate-controlling enzymes of myocardial intermediary metabolism and energy transport, and to characterize the effects of acute exercise on cardiac messenger RNA transcripts encoding metabolic enzymes. To address these questions, dogs were conditioned by a 9 wk treadmill running program or cage rested for 4 wk. Exercise conditioning was documented by a significant decrease in heart rate at rest and during submaximal exercise. A panel of glycolytic and oxidative enzymes was measured in myocardial extracts. It was demonstrated that aerobic exercise training of dogs selectively increased capacities of key rate-controlling enzymes of each of the major pathways of intermediary metabolism in ventricular myocardium. In addition, it appeared that the training-evoked increases in enzyme activities were due to increased enzyme contents, not to changes in substrate affinity. The same training program was implemented to investigate the effects of aerobic exercise training on the myocardium’s energy shuttling system. Total creatine kinase (CK) activity and content of the CKMB isoenzyme were measured in canine myocardial extracts. It was demonstrated that aerobic exercise training increased total myocardial CK activity and CKMB content, although the CKMB isoenzyme remained minor component of the myocardial CK system. A third investigation was conducted to examine the effects of aerobic exercise on the abundance of messenger RNA (mRNA) encoding key enzymes involved in myocardial energy production and transport. Left ventricular myocardium was sampled 30 min after an exercise bout, and messenger RNA transcripts were analyzed by reverse transcriptase polymerase chain reaction. Exercise increased in the myocardial abundance of mRNA transcripts encoding glyceraldeheyde 3-phosphate dehydrogenase, citrate synthase, and the CK-M subunit. These mRNA enhancements could be responsible, at least in part, for exercise-evoked adaptations in myocardial metabolic enzymes demonstrated in the first two investigations.Item Function and Regulation of the Natural Killer Cell Receptor 2B4 (CD244)(2005-05-01) Vaidya, Swapnil V.; Porunelloor A. Mathew; Richard Easom; Hriday DasThe purpose of these studies was to investigate two issues related to the natural killer (NK) cell receptor, 2B4 (CD244) – its in vivo function and transcriptional regulation. In previous in vitro studies, ligation of 2B4 with a monoclonal antibody enhanced the cytotoxicity of NK and CD8 T cells against various tumor cell lines, indicating that 2B4 is an activating receptor. To study the role of 2B4 in vivo, 2B4 deficient (2B4-/-) mice were used. The initial characterization of the 2B4-/- mice indicate a thymic developmental defect with an increase in the immature CD4-/CD8- population in the thyme of female but not male mice. NK cells from the 2B4-/- mice were impaired in activation by IL-2 as compared to wild type NK cells. These results suggest a role of 2B4 in lymphoid development. The in vivo role of 2B4 in tumor rejection was studied in a mouse tumor model in which melanoma cells were injected intravenously and pulmonary metastases enumerated 14 days later. The murine melanoma cell line, B16, was stably transfected with CD48, the counter-receptor for 2B4. Using CD48+ and CD48- B16 cells in tumor experiments indicated that 2B4 functioned as an inhibitory receptor. In addition, a gender-specific role of 2B4 in the rejection of B16 melanoma cells was discovered. 2B4-/- male mice cleared B16 cells more efficiently than wild type male mice, while female 2B4-/- mice were impaired in controlling tumor growth as compared to wild type female mice. In vitro and in vivo studies indicate a complex role for NK cells in the mechanism of this gender effect. Several studies have shown that the expression of 2B4 is upregulated during viral infections and under certain cytokine stimulation. Previously, it has been shown activator protein-1 (AP-1) plays an important role in the transcription of the 2B4 gene. In this study an Ets transcription factor was shown to upregulate the transcription of the gene. This element functions in an AP-1 dependent manner. Stimulation of surface 2B4 down-regulates its own expression by decreasing the activity of the Ets element in the 2B4 promoter. These studies identify a role of 2B4 in lymphoid development and tumor rejection in vivo. The gender-specific defect in 2B4 knock-out mice implicates its role in lupus. The transcriptional studies provide insights into the regulation of 2B4 gene.Item Functional Heterogeneity in Canine Coronary Resistance Arteries(1994-06-01) Parker, James Bruce; Peter B. Raven; Patricia A. Gwirtz; James CaffreyParker, James B., Functional Heterogeneity in Canine Coronary Resistance Arteries. Doctor of Philosophy (Biomedical Sciences), June, 1994, 89 pages, 21 illustrations, bibliography, 82 titles. Two thirds of the coronary vascular resistance resides in the smallest arteries and investigators have hypothesized that they may respond differently to endogenous vasoactive substances. The arterial responses to norepinephrine, acetylcholine, and adenosine were evaluated in large ([greater than] 700 μm, n=24), intermediate (400 600 μm, n=24), and small arteries (μm, n=24). Maximal vessel lumen diameter (Dmax) was determined in CA++ free medium. A reference diameter (84 ± 4.3% of Dmax) was established by re-equilibration in medium containing 2.0 mM Ca++. Arterial maximal responses as a percentage of Dmax to norepinephrine, acetylcholine, and adenosine are given in table 1: Table 1; Large % of Dmax; Inter. % of Dmax; Small % of Dmax; Norepinephrine; 41 ± 2.3; 50 ± 4.2; 83 ± 2.4; Acetylcholine; 96 ± 2.7; 88 ± 3.9; 78 ± 1.9; Adenosine; 71 ± 1.8; 81 ± 4.2; 96 ± 1.4. The sensitivity of canine coronary arteries to norepinephrine, acetylcholine, and adenosine in terms of ED50’s are given in table 2: Table 2; Agonists; Large ED50 μM; Inter. ED50 μM; Small ED50 μM; Norepinephrine; 0.037 ± 0.002; 0.078 ± 0.004; no response; acetylcholine; 0.028 ± 0.003; 0.087 ± 0.005; 0.309 ± 0.03; Adenosine; 0.295 ± 0.002; 0.095 ± 0.004; 0.035 ± 0.03. These data indicate that canine arterial responses to the native agonists norepinephrine, acetylcholine, and adenosine are heterogeneous and that neural control predominates in the larger “transport” arteries while local control predominates in the smaller “distributive” arteries. Responses of small and intermediate isolated canine coronary arteries (lumen diameter 147±42μm, and 531±37μm respectively) to norepinephrine were evaluated after pharmacological or mechanical interruption of endothelial relaxing activity. Following with the nitric oxide synthase inhibitor N-Nitro-L-Arginine Methylester (L-NAME) 10^-5 M the small and intermediate vessels spontaneously constricted to 73±4.1% of Dmax indicating a significant basal release of nitric oxide. After L-NAME or endothelial disruption graded additions of norepinephrine now reduced the vessel diameter in previously unresponsive small arteries. These data suggest that the weak and equivocal response of coronary resistance arteries to norepinephrine results from the competitive dilatory influence of endothelial derived nitric oxide production and not to the absence of norepinephrine receptors.Item Hypoxic Conditioning Suppresses Cytotoxic Nitric Oxide Production Upon Myocardial Reperfusion(2007-05-01) Ryou, Myoung-Gwi; H. Fred Downey; Rong Ma; Raghu KrishnamoortyRyou, Myoung-gwi. Hypoxia conditioning suppresses nitric oxide production upon myocardial reperfusion. Master of Science (Integrative Physiology), May 2007, 61pp, 2 tables, 9 figures. This study was conducted in mongrel dogs to test the hypothesis that 20 d normobaric intermittent hypoxic conditioning (IHC) evokes cardioprotective adaptations of the myocardial nitric oxide synthase (NOS) system. Specifically, the proposal that IHC suppresses myocardial NOS activity sufficiently to dampen the cytotoxic burst of NO formation upon reperfusion of ischemic myocardium was tested. Mongrel dogs were conditioned by a 20 d program of IHC (FIO2 9.5-10%; 5-10 min hypoxia/cycle, 5-8 cycles/d with intervening 4 min normoxia). On day 21, ventricular myocardium was sampled for measuring NOS activity (colorimetric assay) and endothelial NOS (eNOS) content (immunoblot). In separate experiments, myocardial nitrite (NO2) release, an stable product of NO oxidation, was measured at baseline and during reperfusion following 1 h occlusion of the left anterior descending coronary artery (LAD). Values in IHC dogs were compared with respective values in non-conditioned, control dogs. IHC lowered left and right ventricular NOS activity by 60%, from 100-115 to 40-45 mU/g protein (P [less than] 0.01), and decreased eNOS content by 30%. IHC dampened cumulative NO2 release during the first 5 min reperfusion from 32 ± 7 to 14 ± 2 μmol/g (P [less than] 0.05), but did not alter hyperemic LAD flow (15 ± 2 vs. 13 ± 2 ml/g). Attenuation of the NOS/NO system may contribute to IHC-induced protection of myocardium from ischemia-reperfusion injury.
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