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Introduction: An increased gluteofemoral adipose tissue (AT) mass is associated with a protective cardiovascular and metabolic risk profile, and effective fatty acid retention in femoral AT has been proposed as a possible mechanism. Catecholamines are important regulators of AT lipolysis and blood flow (ATBF). The aim of the thesis was to investigate regional differences in the adrenergic regulation of fatty acid release and ATBF between abdominal and femoral AT in vivo. Furthermore, in vivo regional fatty acid trafficking was studied in a physiological setting over 24 h. Methods: Regional fatty acid trafficking, along with the measurement of ATBF, was studied with the arterio-venous difference technique and stable isotope tracers in healthy volunteers. Adrenergic agonists (isoprenaline, adrenaline) were infused either locally by microinfusion, or systemically. Local microinfusion of adrenoreceptor antagonists (propranolol, phentolamine) was used to characterize specific adrenoreceptor subtype effects. The trafficking of dietary fatty acids was studied over a 24 h period involving three meals containing stable isotope-labelled fatty acids along with intravenous infusions of another labelled fatty acid. Results: Femoral ATBF and lipolysis was less responsive to adrenergic stimulation with adrenaline compared to abdominal AT. This was due to increased femoral α-adrenoreceptor responsiveness. When studied over 24 h, femoral AT showed a lower lipolysis rate compared to abdominal AT, while dietary fatty acids were extracted more avidly by abdominal AT. Uptake of non-dietary fatty acids (derived from very-low-density lipoproteins or unbound non-esterified fatty acids) was comparable between abdominal and femoral AT. Conclusion: There are fundamental differences in response to adrenergic stimuli between abdominal and gluteofemoral tissues and the ability of femoral AT to trap non-dietary fatty acids may provide protection of other tissues from ectopic fatty acid deposition.

Capacity to perform prolonged exercise is reduced in high ambient temperatures, but this premature fatigue is not adequately explained by peripheral mechanisms. The aim of this thesis was to examine some possible underlying mechanisms of central fatigue operating during prolonged exercise in a warm environment. The first series of experiments investigated the effect of nutritional manipulation of central serotonergic activity through alterations to the plasma concentration ratio of free-tryptophan to branched-chain amino acids (f-TRP:BCAA). In contrast to previous reports, acute BCAA supplementation failed to alter perceived exertion and delay the onset of fatigue (Chapter 3). This response was similar when exercise was preceded by an exercise and diet regimen designed to reduce glycogen availability (Chapter 4). The ingestion of meals containing added carbohydrate and fat did not alter f-TRP:BCAA at rest (Chapter 5). Acute dopaminergic / noradrenergic reuptake inhibition with bupropion increased exercise perfonnance by 9 % in warm conditions (30C), but this effect was not apparent at 18C (Chapter 6). This response was accompanied by attainment of a higher core temperature and heart rate towards the end of the bupropion trial in the heat despite no detectable difference in perceived exertion and thermal stress. These data suggested that maintenance of catecholaminergic activity may dampen inhibitory signals from the CNS due to the attainment of a high core temperature, allowing power output to be maintained. The blood-brain barrier (BBB) regulates the exchange of substances between the cerebral interstitial fluid and the blood to maintain a stable environment for the CNS. If the BBB is compromised this may adversely influence nonnal brain function. Serum S1OOb, a proposed peripheral marker of BBB penneability, was increased following exercise in a warm environment (Chapter 7). These data indicate that exposure to combined exercise and heat stress may result in a loss of BBB integrity.

To improve animal performance and modify growth by increasing lean tissue accretion, beef cattle production has relied on use of growth promoting technologies such as beta-adrenergic agonists. These synthetic catecholamines, combined with the variable inclusion of rumen degradable (RDP) and undegradable protein (RUP), may improve feed efficiency and rate of gain in finishing beef cattle. However, research about the impact of beta-adrenergic agonists and protein level and source on the ruminal microbiome is limited. The objective of this study was to determine the effect of different protein concentrations and beta-adrenergic agonist (ractopamine hydrochloride; RAC usage) on ruminal bacterial communities in finishing beef heifers. Heifers (n=140) were ranked according to body weight and assigned to pens in a randomized complete design to 6 treatments, containing 3 protein treatments (Control: 13.9% CP, 8.8% RDP, and 5.0% RUP; High RDP: 20.9% CP, 13.4% RDP, 6.1% RUP; or High RUP: 20.9% CP, 9.1% RDP, 10.4% RUP) and 2 RAC treatments (0 and 400 mg/day). Rumen fluid samples were collected from heifers by oral lavage 7 days before harvest. The DNA from the samples were sequenced to identify bacteria based on the V1-V3 hypervariable regions of the 16S rRNA gene using the Illumina MiSeq. Sequences and data from the treatments were analyzed using the R environment and PROC MIXED in SAS 9.4 (SAS Inst.; Cary, NC). Beta diversity was analyzed using PERMANOVA based on PCoA Bray-Curtis distances and were significant among the protein and RAC treatments (P < 0.05). Alpha diversity metrics such as Chao1 and Shannon diversity indices were not significantly different (P > 0.05). Differences among treatments at variable taxonomic levels after analyses through DESeq2 were significantly different for the main effects of protein concentration, rather than the interaction of protein and RAC treatments (P < 0.05). These results suggest possible effects on the microbial communities with different concentration of protein, but limited positive impact with RAC. However, both may potentially act synergistically to improve performance in finishing beef cattle.

Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative facultative anaerobe and an opportunistic oral pathogen, strongly associated with localized periodontitis and other inflammatory diseases. Periodontitis is a chronic inflammation of the periodontium resulting from the inflammatory response of the host towards the dysbiotic microbial community present at the gingival crevice. The host immune response creates a hostile environment for microorganisms; therefore, it is important for Aa to be able to regulate the necessary genes to survive and thrive in such an environment. Aaexpresses several virulence factors such as a cytolethal distending toxin (Cdt), lipopolysaccharide (LPS) and leukotoxin A (LtxA), that allows it to evade the immune response. For example, LtxA, considered a major virulence factor, targets leukocytes by creating pores on their membranes leading to cell lysis. LtxA has also been shown to provide Aa resistance against internalization and killing by neutrophils, however the effect of the toxin on neutrophils at a non-lethal level is often overlooked. At the subgingival pocket Aa is in a tug-of-war scenario where immune cells will secrete molecules such as catecholamines (i.e., epinephrine) to sequester residual metals, so these are not available as nutrients. To compete for such mineral bacteria have evolved different mechanisms, such as two-component systems (TCS) that allow them to assess their environment and adjust accordingly. Aa expresses the QseBC TCS composed of the sensor protein QseC and the response regulator QseB. Previously, catecholamines and iron were identified as the signals that activate the QseBC TCS in Aa, necessary for the organism to acquire iron as a nutrient to survive in the anaerobic environment. However, the source of catecholamines had not been identified at the time. The main objectives presented in this dissertation are a) the characterization of epinephrine interaction with different components of QseBC TCS, b) to characterize the synthesis, storage, and release of catecholamines by neutrophils stimulated with Aa, and c) determination of the effect sublytic levels of LtxA has on neutrophils. Previously, our group showed that QseC, primarily the periplasmic domain of QseC, is required for biofilm formation and virulence. A third gene was found to be co-expressed in the qseBC operon, YgiW, a protein of unknown function, but essential for biofilm growth and virulence. Additionally, it was previously shown that in the presence of Cat-Fe the expression of the enterobactin operon remained unchanged. Among the genes in this operon there is the fepA gene encoding for the enterobactin receptor FepA. In this work we investigated the interaction of different components of the QseBC TCS and epinephrine. Using Aa mutants for the different QseBC component we determined that FepA, QseC and the QseC periplasmic domain are necessary for Aa to interact with epinephrine (Chapter 3). In contrast, YgiW was not required for epinephrine to interact with Aa. Due to the high infiltration of neutrophils that is characteristic of periodontitis we proposed neutrophils as the epinephrine source for Aa. We present evidence of the ability of human neutrophils to release epinephrine upon Aa challenge (Chapter 4). In addition, we demonstrated that epinephrine is stored in azurophilic granules and proposed that Aa gains access to it by inducing degranulation. To connect our findings, we demonstrated that chemically define media (CDM) supplemented with iron and epinephrine from human neutrophils promoted Aa growth and the induction of the qseBC operon. LtxA, a major virulence factor expressed by Aa, targets neutrophils and induces cell lysis. However, lysis only occurs when cells are exposed to high levels of the toxin, the sublytic effects of the toxin on neutrophils remains understudied. We show that the minimally leukotoxic Aa strain 652 caused minimal cytotoxicity in human neutrophils, even at MOI 50 (Chapter 5). Assessment of membrane permeability confirmed that human neutrophils suffered loss of membrane integrity. However, neutrophils were functionally active and were able to internalize Aa at MOI 10. In contrast, Aa internalization did not occur at MOI 50 for both the minimally leukotoxic strain or a LtxA mutant (JP2 DltxA). Aachallenge induced the formation of cytotoxic vacuoles, which have previously been associated with pore-forming toxins, but we observed cytoplasmic vacuolation regardless of LtxA expression of Aa strains. Siglec-9, a sialic acid-binding immunoglobulin-type lectin, has been associated with cytoplasmic vacuolation. Therefore, we measured Siglec-9 as an alternative inducer of cytoplasmic vacuolation. We were able to show that Aa 652 enhances Siglec-9 expression in human neutrophils and that IL-8 priming before Aa challenge further increases expression. The findings presented in this dissertation contribute to the current knowledge of nutrient acquisition mechanisms and provides the foundation for future studies of how Aa can modulates the inflammatory response of neutrophils through Siglec-9 activation.

Secondary metabolites are a broad class of specialized compounds that mediate plant-environment interactions and mitigate stress. It is increasingly clear that many phenylalanine-derived secondary metabolites are nearly indispensable for plant survival and that plants adjust their growth according to their secondary metabolic outputs. Consequently, many phenylalanine-derived secondary metabolites have influence over hormone activity. For instance, multiple phenylpropanoid intermediates and catecholamines alter the sensitivity of plants to the central hormone auxin, which in concert with cytokinin directs most aspects of plant growth and development. This dissertation reviews previous research on the influence of phenylpropanoid intermediates and catecholamines on plants, with a focus on their impact on auxin and cytokinin signaling. In addition, original data is presented on the impact of exogenous dopamine on auxin activity and plant physiology, illustrating that dopamine appears to affect plant growth by altering auxin transport, plant redox status and iron homeostasis. Expanding on the influence of phenylalanine-derived compounds on auxin signaling, data indicating that trans-cinnamic acid or its derivates influence auxin and cytokinin driven developmental processes. Collectively, this dissertation aims to highlight the profound impact of phenylalanine-derived secondary metabolites on plant growth and development, which presumably allows plants to simultaneously adapt their growth and metabolic outputs in response to their environment.

Cigarette smoking is a leading cause of preventable disease and premature death worldwide. The adverse effects of cigarette smoking, including proarrhythmia, are related to the mixture of chemicals, including nicotine (which sustains tobacco addiction). However, it remains unclear which individual tobacco smoke constituents and biological pathways mediate this increased risk. The purpose of this research was to explore the chronic effects of cigarette smoking, as well as compare the acute effects of nicotine and cigarette smoking, and the possible role of β-adrenoreceptors, on human cardiac electrophysiology. Chapter 1 is a comprehensive literature review of (a) the ex vivo and in vivo effects of nicotine and non-nicotine constituents of cigarette smoking on cardiac ion channels, (b) the direct and indirect effects of the autonomic nervous system on cardiac electrophysiology, and (c) studies of acute and chronic effects of cigarette smoking in humans. Chapter 2 consists of two studies in which we used cotinine levels to investigate the differences in baseline cardiac electrocardiogram between chronic smokers and non-smokers, and to define smoking status and its burden. We also explored the relationship between urinary catecholamines, cotinine, and electrocardiographic changes. Chapter 3 features the 2 x 2 factorial experimental study designed to compare the acute effects of cigarette smoking and nicotine, with and without a β-blocker (propranolol). We found that chronic cigarette smoking was associated with a shortened PR segment at baseline, and that dopamine possibly mediates this effect. There was also (corrected) QT interval shortening with increased cotinine levels. This experimental study revealed that the non-nicotine constituents in cigarette smoking were mainly responsible for PR segment shortening, through β-adrenoreceptors. Other evidence revealed that, although nicotine in cigarette smoke is primarily responsible for sympathetic activation and (corrected) QT interval shortening, it is the non-nicotine constituents that depress the ST segment. Collectively, acute and chronic exposure studies indicate that smoking may promote cardiac arrhythmia, primarily via β-adrenoreceptors, causing acceleration of dromotropy and ischemia (non-nicotine mediated), and ventricular repolarization (nicotine-mediated). This research elucidated a major physiological mechanism driving the effect of cigarette smoking and nicotine on cardiac electrophysiology. Consequently, these findings will inform U.S. Food and Drug Administration of tobacco and nicotine-containing products’ impact on the human cardiac electrical system, and potentially help regulate alternative forms of nicotine delivery and protect public health.

The dorsal striatum and the medial prefrontal cortex are part of a neurocircuitry that is affected by acute and chronic drug use. In the present studies, we sought to characterize the pharmacological effects of ethanol on extracellular catecholamine concentrations in the dorsal striatum and medial prefrontal cortex. To this end, we utilized two different routes of administration to quantify ethanol’s actions. We performed in vivo microdialysis in adult, male Long Evans rats as they received single or repeated intravenous infusions of ethanol. Following infusion of a 1-g/kg dose of ethanol, we observed no significant effects on extracellular dopamine in either the dorsomedial or dorsolateral striatum, but in a separate group of animals, we observed significant stimulation of extracellular norepinephrine in the medial prefrontal cortex. However, following a cumulative intravenous dosing protocol, we observed a gradual ramping up of tonic dopamine activity in the dorsal striatal subregions, which was more robust in the dorsomedial striatum. Subsequently, we performed in vivo microdialysis in separate groups of rats during an operant self-administration session to quantify the time course of extracellular dopamine and norepinephrine in the medial prefrontal cortex. In the seven operant sessions prior to the microdialysis test session, each group of rats had been assigned to a separate treatment group: one that received a sweetened ethanol solution, one that received a sucrose solution, and a handling control group that did not receive any drinking solutions. In the ethanol-experienced animals, we report a reduction in basal dopamine and norepinephrine in the medial prefrontal cortex, relative to control groups. However, there were no significant differences in the temporal profile of extracellular norepinephrine across the three treatment groups. These studies demonstrate that limited voluntary ethanol consumption appears to be sufficient to alter tonic catecholamine signaling in the medial prefrontal cortex. Additionally, we conclude that central catecholamine signaling pathways are a target for ethanol.

Sleep loss alters both the concentration and activity of various aspects of the immune system. These alterations lead to increased susceptibility to infection and the progression of pathologies such as insulin resistance and atherosclerosis. Two proposed mechanisms of this alteration in immune function are the changes in both cortisol and sympathetic nervous system activity that accompany sleep deprivation. This work reviewed literature that measured the effects of periods of sleep restriction upon both cortisol and catecholamine concentrations within human subjects. Furthermore, studies which measured the effects of sleep loss upon these hormone levels and the associated changes in immune parameters were included. This thesis asserts that there is no defined pattern in reference to alterations of cortisol levels as a result of sleep deprivation. Furthermore, more evidence must be collected before implementing cortisol as a main effector of sleep loss upon immune system function. This dissertation, although repeatedly noting increased levels of norepinephrine following periods of sleep restriction, similarly argues that more research must be completed in order to declare that altered catecholamine concentrations as a result of sleep loss is a mechanism for altered immune function.

In order for a pathogen to successfully colonize the host, it must be able to acquire essential nutrients and regulate gene expression to respond to environmental fluctuations. One mechanism bacteria have evolved in order to detect these fluctuations and respond is the two component signal transduction systems (TCS). Aggregatibacter actinomycetemcomitans, a dental commensal associated with localized aggressive periodontitis, contains a TCS designated QseBC that was previously shown to regulate expression of several iron acquisition genes, biofilm formation and virulence, but the activating signal was unknown []. QseBC is a conserved TCS in the Enterobacteriaceae and Pasteurellaceae, but the activating signal for QseC varies for each species [125]. The QseC in Escherichia coli responds to catecholamines whereas the QseC homolog (FirS) in Haemophilus influenzae responds to ferrous iron. Here we show that the activating signal for the A. actinomycetemcomitans QseC is a combination of iron and catecholamines (CAT-Fe). The sensing of the CAT-Fe complex leads to a significant stimulation of biofilm and planktonic growth, as well as induction of genes involved in anaerobic metabolism, electron transport and oxidative stress while down-regulating expression of genes involved in iron acquisition and fatty acid and LPS biosynthesis. Using a combination of RNA analysis and metabolomics, we identified an increased flux through the TCA cycle to pyruvate and then to anaerobic respiration and metabolism with exposure to CAT-Fe. Cells exposed to Cat-Fe also expressed higher levels of catalase and were more resistant than untreated cells to oxidative stress. Finally, QseB also appears to play a role in regulating the general stress response in A. actinomycetemcomitans since a ΔqseB mutant exhibits increased transcription of several stress response genes, including the Lon protease. Ectopic expression of lon resulted in an increase in transcription of ygiW-qseBC. Lon is involved in activating type II toxin/anti-toxin (TA) systems, one of which (D11S_2133-2134) was shown to be highly up-regulated during iron starvation. Further, it was found that ectopic expression of the putative anti-toxin D11S_2134 resulted in a significant increase in transcription of qseBC. Based on this data, it is likely that D11S_2133-2134 regulates expression of qseBC allowing QseBC and D11S_2133-2134 to tightly control iron acquisition and regulate adaptation to the host.

Phenylethanolamine-N-methyltransferase (Pnmt) catalyzes the conversion of noradrenaline to adrenaline and is the last enzyme in the catecholamine biosynthetic pathway. Pnmt serves as a marker for adrenergic cells, and lineage-tracing experiments have identified the embryonic heart and hindbrain region as the first sites of Pnmt expression in the mouse. Pnmt expression in the heart occurs before the adrenal glands have formed and prior to sympathetic innervation, suggesting that the heart is the first site of catecholamine production in the mouse. The function of these Pnmt+ cells in heart development remains unclear. In the present study, we test the hypothesis that (i) a genetic ablation technique utilizing a suicide reporter gene selectively destroys Pnmt cells in the mouse, and (ii) Pnmt cells are required for normal cardiovascular and neurological function. To genetically ablate adrenergic cells, we mated Pnmt-Cre mice, in which Cre-recombinase is under the transcriptional regulation of the Pnmt promoter, and a Cre -activated diphtheria toxin A (DTA) mouse strain (ROSA26-eGFP-DTA), thereby causing activation of the toxic allele (DTA) in Pnmt-expressing (adrenergic) cells resulting in selective "suicide" of these cells in approximately half of the offspring. The other half serve as controls because they do not have the ROSA26-eGFP-DTA construct. In the Pnmt+/Cre; R26+/DTA offspring, we achieve a dramatic reduction in Pnmt transcript and Pnmt immunoreactive area in the adrenal glands. Furthermore, we show that loss of Pnmt cells results in severe left ventricular dysfunction that progressively worsens with age. These mice exhibit severely reduced cardiac output and ejection fraction due to decreased LV contractility and bradycardia at rest. Surprisingly, these mice appear to have a normal stress response, as heart rate and ejection fraction increased to a similar extent compared to controls. In addition to baseline cardiac dysfunction, these mice fail to gain body weight in a normal manner and display gross neurological dysfunction, including muscular weakness, abnormal gaiting, and altered tail suspension reflex, an indicator of neurological function. This work demonstrates that selective Pnmt cell destruction leads to severe left ventricular dysfunction, lack of weight gain, and neurological dysfunction. This novel mouse is expected to shed insight into the role of Pnmt cells in the heart, and suggests a role for Pnmt cells in neurological regulation of feeding behavior, metabolism, and motor control.

Background: Patients with obstructive sleep apnea (OSA) suffer from increased mortality because of complications from metabolic and cardiovascular disease. Excess sympathetic nervous activity and catecholamine exposure contribute to the disease associations of OSA, but the underlying mechanism remains enigmatic. Because catecholamines cause overgrowth of bacteria in the class Enterobacteriaceae in the laboratory, this translational study proposed a role for altered gut microbiota in the complications of OSA. Objectives: We tested the hypothesis that catecholamine excess in disordered sleep alters intestinal microbiota by comparing urinary catecholamines and the fecal microbiome of 24 patients with obstructive sleep apnea and 23 controls. Results: Next-generation sequencing of the gut microbiome using the Illumina platform provided evidence for a trend toward altered community structure of gut microbiota in patients with sleep apnea. A positive linear relationship was seen in norepinephrine exposure and Enterobacteriaceae in patients with sleep apnea, but no such relationship occurred in controls. Conclusions: These findings provide preliminary support for a central role of gut microbiota in the complications of sleep.

Abstract: Caffeine is used by some athletes to improve endurance performance, however, the mechanism(s) by which caffeine elicits performance improvements have been unclear. The purpose of this study was to investigate the effects of caffeine on pain perception, fatigue perception, plasma catecholamine concentrations and plasma potassium concentrations to determine whether altered perception related to the central nervous system and potassium ion handling are associated with enhanced performance during a 1 km cycling time trial. A cross-over, double blind design of 13 well trained men (age: 27 ± 6 yrs, height: 180 ± 7 cm, body mass: 76.4 ± 6.4 kg, and VO2max: 57.5 ± 4.6 ml•kg-1•min-1) were randomized to a caffeine (5 mg•kg-1) or a placebo condition. Caffeine had no significant effects on the 1 km time-trial performance indicators; time, peak power, or average power. In addition, caffeine had no significant effect on the perception of pain or overall fatigue. There was a significantly greater increase in post-exercise blood lactate, post-exercise catecholamines and lower pre-exercise blood potassium concentrations when caffeine was consumed. The results suggest that although there were no differences in performance time, caffeine caused changes in metabolic markers. In conclusion, caffeine consumption prior to a 1 km simulated cycling time-trial did not improve performance and its use is not warranted.

Background: Urinary catecholamines and metanephrines have been proposed as a diagnostic tool for identifying canine pheochromocytomas, but the effects of critical illness on urine concentrations of catecholamines and metanephrines is currently unknown. Objectives: To examine the effects of illness on urine concentrations of catecholamines and metanephrines in dogs. Animals: Twenty-five critically ill dogs and twenty-five healthy age- and gender-matched control dogs. Methods: Prospective observational study. Urine was collected from healthy and critically ill dogs and urine concentrations of epinephrine, norepinephrine, metanephrine, and normetanephrine were measured by high-performance liquid chromatography (HPLC) with electrochemical detection. Urinary catecholamine and metanephrine:creatinine ratios were calculated and compared between groups. Results: Urinary epinephrine, norepinephrine, metanephrine, and normetanephrine:creatinine ratios were higher in critically ill dogs when compared to a healthy control population (P = 0.0009, P < 0.0001, P < 0.0001, and P < 0.0001 respectively). Conclusions and Clinical Relevance: Illness has a significant impact on urinary catecholamines and their metabolites in dogs. Further investigation of catecholamine and metanephrine concentrations in dogs with pheochromocytomas is warranted to fully evaluate this test as a diagnostic tool, however the findings of this study suggest that the results may be difficult to interpret in dogs with concurrent illness.

Polychlorinated biphenyls (PCBs) are environmental pollutants that bioaccumulate through the food web. Lipophilic endocrine disruptors, PCBs accumulate in adipocytes, liver, muscle and breast tissues. The adrenal medulla synthesizes the catecholamines, dopamine, norepinephrine and epinephrine, using several enzymes, including tyrosine hydroxylase (TH) and dopamine β-monooxygenase (DBM). Since catecholamines are important neurotransmitters, this study investigates the effect of PCB 47/77 on DBM and TH expression in adrenal glands of 3, 21 and 60 day old rats. PCB 47/77 was administered orally from conception until the rats were euthanized in doses of 12.5 and 25 ppm. Western blot analyses showed decreased DBM expression in 3 and 60 day old rats treated with both PCB doses, while decreased DBM expression only was observed in 21 day old rats after 25 ppm PCB treatment. TH expression was lower in 3 day old rats treated with 12.5 and 25 ppm PCB as compared to the controls. However, TH expression was elevated in 21 day old rats treated with 12.5 ppm PCB, while in 60 day old rats TH expression was elevated only following 25 ppm PCB treatment. Studies to determine the effect of PCB 47/77 on the developmental expression of DBM and TH showed that in control rats, the greatest DBM and TH levels were observed in 3 day old rats followed by a decrease in expression levels with the increasing age of the rats. Following treatment with 12.5 ppm PCB, DBM expression decreased only in 60 day old rats, while a decrease in 21 and 60 day old rats was observed with 25 ppm PCB treatment as compared to 3 day old rats. In contrast, TH levels were the lowest in 3 day old rats, while a steady increase in TH levels was observed with increasing age of the animals following 25 ppm PCB treatment. Overall, exposure of rat adrenal glands to PCB 47/77 results in alterations in DBM and TH levels that could adversely affect the catecholamine synthesis needed to regulate stress-induced homeostatic mechanisms involved in the flight or fight response.

Attentional dysfunction is associated with numerous genetic and environmental factors that disrupt brain development. Evidence implicates catecholaminergic systems in attentional processing, suggesting that alterations in catecholamine neurochemistry may underlie these attentional dysfunctions. The goal of the present experiments was to use a pharmacological challenge approach to examine underlying neural changes that relate to attentional dysfunctions induced by two developmental insults, lead (Pb) exposure and prenatal cocaine exposure, and to test the hypothesis that exposure-induced alterations in catecholaminergic systems contribute to these attentional deficits. In each experiment, adult rats were administered a visual attention task which assessed subjects' ability to monitor an unpredictable light cue and maintain performance when presented with olfactory distractors. In the first study, Pb-exposed subjects demonstrated significant impairments in accuracy relative to controls, providing evidence for Pb-induced attentional dysfunction. The alpha-2 adrenergic antagonist idazoxan improved accuracy, specifically in the most attentionally demanding conditions. However, this effect of idazoxan did not differ between Pb-exposed and control subjects, failing to support the hypothesis that the attentional dysfunction was due to alterations in noradrenergic systems. In the second study, prenatal cocaine-exposed subjects did not differ from controls in their response to idazoxan's effect on errors of commission, but were more sensitive to idazoxan's effects on errors of omission and nontrials. The pattern of effects suggested that the differential treatment response to idazoxan resulted from prenatal cocaine-induced alterations in norepinephrine-modulated dopamine release, reflecting lasting changes in dopaminergic and/or noradrenergic systems underlying attention. The final study was designed to assess whether cocaine exposed and control subjects differ in sensitivity to attentional effects of the D1 dopamine agonist SKF81297. Prenatal cocaine-exposed subjects were more sensitive to the impairing effects of SKF81297 on errors of omission, providing specific evidence that prenatal cocaine exposure produces lasting changes in dopaminergic systems mediating attention. In conclusion, environmental Pb exposure impairs attention; however, these impairments do not appear to reflect alterations in noradrenergic function, suggesting the involvement of other neurochemical systems. In contrast, prenatal cocaine-induced attentional impairments appear to be related to lasting changes in dopaminergic systems underlying attention, suggesting possible targets for pharmacotherapeutic intervention in affected children.

Heart Rate Variability (HRV) currently is utilized when assessing the risk of mortality in individuals suffering from coronary heart disease or diabetic neuropathy. Research has shown that patients with Obstructive Sleep Apnea (OSA) also show a decrease in HRV, as well as an increase in sympathetic drive characterized by an increase in the low-frequency component of HRV. HRV, in conjunction with other indicators, may represent a non-invasive, low cost method for the confirmation of severity of OSA in some patients and therefore may represent an additional tool for the assessment of risk in these individuals. This becomes especially true when urinary catecholamines, fitness level, and quality of life (QOL) assessment are included. The purpose of this study was to determine if a correlation exists between severity of OSA as assessed by respiratory distress index (RDI) and the selected measures HRV, fitness, QOL, and catecholamine output. Subjects were 6 men and 5 women who were recently diagnosed with OSA by polysomnographic (PSG) study. HRV and blood pressure was measured during two consecutive trials consisting of 512 heartbeats. Catecholamine levels were determined by HPLC following 24-hour urine collection. Fitness levels were established following cycle ergometer testing and QOL following questionnaire completion. Subjects with lower weight, BMI, and neck circumference had significantly higher parasympathetic influence as analyzed through the amount of high frequency component of HRV (r =.738, .726, .789, respectively; p

One important aspect of neural development is the differentiation of neurotransmitter phenotypes in developing neurons. The studies described in this dissertation explore mechanisms of transmitter differentiation using catecholaminergic (CA) phenotypic expression in sensory neurons as a model system. The results show that both environmental factors as well as intrinsic neuronal properties play key roles in regulating sensory CA phenotypic expression. Specifically, ganglionic non-neuronal cells (NNC) inhibit expression of the CA-biosynthetic enzyme tyrosine hydroxylase (TH) in cultured sensory neurons. The effect of NNC is partially mediated by a NNC-derived cytokine, Leukemia Inhibitory Factor (LIF). The detection of LIF and LIF receptor (LIFR) mRNAs in vivo suggests that LIF may be one factor that modulates CA potential in sensory neurons in vivo. In contrast to the inhibitory effect of LIF, depolarizing conditions up-regulate TH expression in cultured embryonic and newborn sensory neurons. These results raise the possibility that membrane depolarization during this critical period of development plays a role in stimulating CA differentiation in subsets of sensory neurons. Moreover, the effects of depolarization and LIF are antagonistic, suggesting that mature levels of CA expre ssion are regulated by a balance of positive and negative regulatory signals. Regulation of intrinsic neuronal properties, such as responsiveness to environmental influences, could be another general mechanism of neurotransmitter regulation. For example, nodose ganglion (NG) neurons lose their LIF responsiveness between embryonic and newborn stages, coincident with the development of a CA phenotype in a subset of NG neurons in vivo. This result raises the possibility that modulation of LIF responsiveness may contribute to stable, phenotypic TH expression in a subset of nodose neurons during late fetal development. To examine whether LIF inhibits sensory TH expression in vivo, I compared sensory TH expression in LIFR and LIF knockout mice with wildtype mice. TH cell numbers appeared unchanged in LIFR and LIF knockout mice compared to controls, indicating that the LIF system is dispensable for the normal patterning of TH expression in sensory neurons in vivo. These data suggest either (1) the loss of LIF function can be compensated for by other TH inhibitory factor(s), or (2) LIF may only be involved under culture conditions. In conclusion, my data indicate that spatial restriction of CA phenotypic expression in subsets of adult sensory neurons arises through modulation, by positive and negative extrinsic regulatory factors, of a widespread CA potential, as well as from the regulation of intrinsic neuronal responses to these environmental factors during development

Surface enhanced resonance Raman scattering (SERRS) of free flavins and flavoproteins on colloidal silver has been studied with 488 nm excitation. The fluorescence arising from the flavin 480 nm (pi) (--->) (pi)* electronic transition is completely quenched. At concentrations as low as 1 x 10('-9) M, free flavins give good Raman spectra, with evidence for strong adsorption at the silver surface. The active species on the surface has been identified. The several strong SERRS bands known to be ring III modes are perturbed from their frequencies in resonance Raman spectra. N(3) deuterated species give SERRS at the same or slightly shifted frequencies. N(3) substituted flavins do not give any SERRS. The pH dependence of SERRS intensity confirms the N(3) anion as the active species. The active species on the silver sol is the flavin N(3) anion, and it is coordinated to the silver surface through N(3) and the oxygen of C(4)=O. We have attempted to study flavoproteins by SERRS. No frequency differences are observed in the spectra of riboflavin binding protein (RBP), glucose oxidase (GO), flavodoxin (FD), lactate oxidase (LO), p-hydroxybenzoate hydroxylase (PHBH), and Old yellow enzyme (OYE). The resonance Raman spectra of the same flavoproteins are quite different. The SERRS intensities are 5% or less than those of equi-molar free flavins. The SERRS of any flavoprotein disappears on addition of excess apo-RBP. Control experiments demonstrate that SERRS intensities increase with laser exposure. The observed spectra are from dissociated free flavin, not from the intact flavoproteins. In exploratory experiments, the SERS of the neurotransmitters dopamine, epinephrine and norepinephrine have been obtained at a silver electrode. Dopamine gives intense SERS. Similar but weaker SERS is obtained for norepinephrine. Epinephrine gives very weak SERS. Good dopamine SERS is observed at 1(mu)M, and the strong bands are detectable at 0.1(mu)M. There is no interference from ascorbic acid, acetylcholine or glutamic acid. SERS is proposed as a useful technique in neurochemistry. Inverse Raman spectroscopy has been used to obtain the triplet state Raman spectra of 8-methoxypsoralen (8-MOP) and 3-carbethoxypsoralen (3-CP). The furan C=C stretch of 3-CP is perturbed in the triplet state, whereas the pyrone C=C stretch of 8-MOP is perturbed in the triplet state. Spontaneous Raman scattering has been applied to low temperature psoralen systems. The results confirm the inverse Raman findings.

The nucleus tractus solitarius (nTS) is a brainstem structure with an important role in the elicitation of the baroreceptor reflex. This reflex is triggered by increases in blood pressure, and serves to bring about decreases in heart rate and vascular resistance. Studies have revealed the presence of catecholaminergic nerve terminals within the nTS. While such findings suggest the involvement of catecholamines in autonomic regulation, little is known of catecholamine effects on the activity of individual nTS neurons receiving baroreceptive input. This issue was addressed by examining the responses to catecholamines of baroreceptive neurons in the rat nTS. Neuronal responses to microiontophoretically applied catecholamines were recorded extracellularly and correlated with responses to pharmacologically induced elevations of blood pressure. Responses to norepinephrine and epinephrine were further examined in the absence and presence of microiontophoretically applied antagonists selective for subclasses of alpha- and beta-adrenergic receptors. Of the 357 neurons studied, 193 responded to blood pressure elevation. One-hundred twenty-eight neurons responded in an inhibitory manner, while the remaining 65 were excited. Both classes of neurons were inhibited by application of catecholamines. No differences were found between the responses of the two groups of neurons. Responses to norepinephrine and epinephrine were antagonized in both groups of cells by the alpha-adrenergic antagonist tolazoline but not by the beta antagonist sotalol. Responses to norepinephrine were also blocked by the alpha-2 antagonist idazoxan, but the responses of only a few cells were blocked by the alpha-1 antagonist prazosin. In contrast, the effects of epinephrine were equally blocked by prazosin and idazoxan. These data indicate the presence of at least two groups of baroresponsive neurons in rat nTS. Neuronal activity in both groups was suppressed by catecholamine iontophoresis. The actions of epinephrine appeared to be mediated in either group by both alpha-1 and alpha-2 adrenergic receptors, whereas the actions of norepinephrine appeared to be primarily mediated by an alpha-2 receptor. However, this latter finding may reflect a possible methodological artifact. The results are discussed in relation to current knowledge regarding baroreceptor information processing in the nTS.

These studies tested the interrelated hypotheses that the ovarian hormones produce their positive feedback effects on luteinizing hormone (LH) secretion through activation of noradrenergic and adrenergic systems in specific hypothalamic regions. Furthermore, the ovarian hormones may alter the activity of opioid neuropeptide and Gamma-Aminobutyric Acid (GABA) systems to produce these alterations in catecholamine transmission and gonadotropin secretion. Radioimmunoassays were utilized to determine plasma LH and median eminence LHRH, and hypothalamic catecholamine concentrations were measured by radioenzymatic assay. The first two studies tested whether epinephrine (EPI) synthesis inhibition blocks the accumulation of median eminence LHRH that precedes the ovarian hormone-induced LH surge and also to test whether the stimulatory ovarian hormone regimen enhances the activity of hypothalamic EPI systems. Ovariectomized rats were primed with estradiol (EB), followed 2 days later by progesterone (Prog.). Animals were treated before Prog, administration with saline, one of the EPI synthesis inhibitors SKF 64139 or LY 78335, or the norepinephrine (NE) synthesis inhibitor, FLA-63. The catecholamine synthesis inhibitors blocked or delayed the LH surge. FLA-63 completely prevented the accumulation of LHRH in the median eminence that preceded the rise in LH release. However, selective reduction in EPI levels with SKF 64139 only partially prevented this increase in LHRH. A second EPI synthesis inhibitor, LY 78335, delayed both the LH surge and the rise in LHRH. In a second experiment, the administration of EB plus Prog, to ovariectomized rats increased the alpha-methyltyrosine (aMT) induced depletion of EPI in the medial basal hypothalamus (MBH). The depletion of NE after synthesis inhibition was enhanced in both the MBH and preoptic-anterior hypothalamus (POA). Experiments 3 and 4 examined a possible mechanism underlying these ovarian hormone effects on LH release and catecholamine activity. These studies tested whether the opiate antagonist, naloxone, which increases LH release, enhances the activity of NE and EPI neurons in the hypothalamus, and also tested whether morphine, an opiate agonist which decreases LH release, depresses the activity of hypothalamic NE and EPI activity. Administration of naloxone to EB-primed rats increased LH release and potentiated the depletion of NE in the POA and MBH, and enhanced the decline of EPI and dopamine (DA) in the MBH, suggesting increased catecholamine activity in these regions. Administration of the opiate agonist, morphine, to rats pretreated with EB and Prog., decreased LH and decreased the depletion of the catecholamines in the POA and MBH, suggesting reduced activity. In most cases, naloxone antagonized the inhibitory effect of morphine. Experiments 3, 6, and 7 examined the involvement of (GABA) systems in the positive feedback effects of EB and Prog, on LHRH and LH release. These studies tested 1) the effects of GABAergic drugs on the LH surge induced by EB and Prog., 2) whether GABA agonists reduce NE and EPI activity in the hypothalamus, and 3) whether a GABA agonist prevents the accumulation of median eminence LHRH induced by EB and Prog. Ovariectomized rats received the stimulatory EB plus Prog, treatment. Simultaneously with Prog., rats received either saline, the barbiturate, phenobarbital, the GABAg agonist, baclofen, the GABA^ agonist, muscimol, or either the GABA^ antagonist, bicuculline, or the putative GABAg antagonist, 5-aminovalerate. Additional experiments tested the effects of the GABA drugs on LH release in ovariectomized, hormonally untreated rats and in response to exogenous LHRH. The LH surge induced by EB+Prog. was blocked by treatment with either baclofen, muscimol, or phenobarbital. Bicuculline was ineffective in preventing the effect of baclofen and phonobarbital but partially prevented the effect of muscimol. Neither baclofen nor muscimol significantly affected LH release in hormonally untreated, ovariectomized rats or in rats receiving LHRH administration. In the results of Experiment 6, in EB plus Prog.-treated rats, baclofen and muscimol significantly reduced the concentrations of EPI and NE in the POA and MBH and prevented their decline after administration of otMT, suggesting decreased catecholamine transmission. In Experiment 7, rats were primed with the ovarian hormones and received, concurrently with Prog., either saline, or baclofen. The GABAg agonist, baclofen, blocked the LH surge and selectively increased LHRH concentrations. Experiment 8 tested 1) whether baclofen reverses the enhancement of LH release and catecholamine activity produced by naloxone, and 2) whether the opiate antagonist, nalmefene, prevents the blockade of the LH surge produced by baclofen. In the first study of Experiment 8, naloxone increased LH release and enhanced catecholamine activity in EB-primed rats. Baclofen was unable to reverse these effects. In the second study, baclofen administration to EB plus P treated rats blocked the LH surge and concomitant administration of nalmefene was unable to prevent this effect of baclofen. These results suggest that: 1) the ovarian hormones activate both NE and EPI systems to stimulate the early afternoon rise of LHRH in the median eminence and to induce the subsequent LH surge, 2) the ovarian hormones may produce their positive feedback effects on LH secretion by removing an inhibitory GABA or opioid neuropeptide influence on catecholamine transmission, allowing NE and EPI to stimulate LHRH, and subsequently, LH release, and 3) these modulatory actions of GABA and opiates may represent effects of two parallel, yet independent hypothalamic systems which regulate catecholamine neurotransmission and subsequently LH secretion.

Cocaine, the principal biologically active alkaloid of Erythroxylon coca, acts as both a local anesthetic agent and an indirect-acting sympathomimetic. These two very different activities make research on and subsequent interpretation of cocaine's multiplicity of varied effects difficult. Not surprisingly, investigations into the central effects of cocaine have yielded mixed results. However, there is a certain body of evidence which points to the euphoric and self-administrating properties of cocaine as appearing to involve the acute activation of central dopamine neuronal systems . With chronic cocaine use, central neurotransmitter alterations have been observed to occur. Dopamine depletion has been hypothesized to result from the overstimulation of these central neurons and the excessive synaptic metabolism of the neurotransmitter. This depletion may underlie the dysphoric aspects of cocaine abstinence and cravings. The potential cardiotoxicity of chronic cocaine abuse has been welldocumented. The possible mechanisms for this remain unclear. The present study was designed to evaluate the effects of repeated daily administration of two different doses (15 and 30 mg/kg/day) of cocaine on male Sprague-Dawely rat cardiac norepinephrine levels. Seven time intervals of sustained dosing were acute (1 dose), 1, 2, 4, 8, 12 and 20 weeks. The catecholamine levels of the whole heart were measured using an HPLC with electrochemical detection. Cardiac norepinephrine levels were unaffected by an acute injection of either cocaine dose. The lower dose elicited a slight increase of 5.8% while the higher dose evoked a small decrease at the 1 week administration period. At 2, 4, and 8 weeks cardiac neurotransmitter levels decreased an average of 13% compared to control values for both doses. At 20 weeks, the catecholamine content was similar to that of the controls. It was observed that cocaine in regard to cardiac norepinephrine content failed to elicit a dose-dependent response. The lower dose of 15 mg/kg/day evoked a greater reduction than that of the higher dose, especially at the 12 week time increment ( -23% and -11%, respectively) which was statistically significant. This is one of the first studies to investigate the effects that repeated daily cocaine dosing has on cardiovascular biochemical pharmacology. It is difficult to postulate the possible etiology and functionality of this decrease. Previous studies have indicated that cocaine might cause alterations in tyrosine hydroxylase activity, the rate-limiting enzyme in the biosynthesis of catecholamines which could be responsible for the reduction observed.

The purpose of this study is to investigate the change in catecholamine levels over the course of a training season in collegiate swimmers. Swimmers were chosen because there are no reports in the literature that look at the response to stress in actively training swimmers. Additionally, there are very few studies which use athletes trained at the intensity of collegiate swimmers (27, 64, 156). Thus, one of the first requirements of the study was to obtain data on collegiate swimmers training at elite levels. The study was designed to determine the long term changes in hormone secretion patterns when the intensity of training was continually increased. There are no reports of studies on athletes training for more than four months duration (287). Since most elite athletes train for much longer periods of time than four months, the first objective was to develop a research protocol that would result in information on catecholamine production over an extended period of time. A second objective was to test for a relationship between a simple psychological measure and the results obtained from physiological measurements. Since there are no reports about the psychological changes in elite athletes during a training season, a third objective was to determine if there was any correlation between psychological and physiological changes. A fourth objective was to evaluate whether a testing day could be broken into time periods and then assess if there was a particular time period of the day when there was more or less change due to stress. Although a few reports have described the variation in secretion in sedentary subjects with time of day (86, 104,183), none have looked at this variation in elite athletes. A fifth objective was to determine what variable or variables correlated with hormone secretion. Was it distance trained, psychological stress, time of day, or some other factor? Again, no one has reported attempting to determine the relationship of catecholamines to other variables in elite athletes. The last objective of this study was to determine whether any observed changes in catecholamine output were due to changes in production or to changes in metabolism. By measuring the metabolites of norepinephrine and epinephrine, it was anticipated that the factors responsible for changes in hormone excretion could be determined. Thus. this research sought to test three main hypotheses. First: there is a change in catecholamine production in swimmers throughout the training season. Second: this change could be correlated to changes in psychological stress evaluations and to the amount of distance trained. Finally: the change in catecholamine production could be directly correlated to a change in catecholamine metabolite production.