Heart rate variability (HRV) has been used to evaluate cardiac autonomic function by measuring variations in electrocardiographic R-R intervals between cardiac cycles. HRV was first used to associate decreases in autonomic nervous system (ANS) control with an increased risk of mortality in coronary heart disease and in the diagnosis of diabetes (1). Current clinical research interest has extended to investigate uses of HRV to evaluate changes in the cardiovascular system due to disease, aging, physical activity, and cardiac rehabilitation treatment (2, 5). HRV scores are derivatives of R-R intervals and these may be represented as a function of either time or frequency domain parameters. Time domain analysis is the simplest and includes: the standard deviation of R-R intervals and the number of adjacent RR intervals that differ by >50ms (dRR50). Frequency domain measures involve more elaborate calculation and have been applied in studies to evaluate sympathetic and parasympathetic autonomic balance. The latter include: Low Frequency Power (LF), High Frequency Power (HF), and LF/HF ratio. HRV has been measured in a variety of ways, the most common being a continuous 24-hour collection of R-R data. In recent years, several investigators have sought to assess HRV by utilizing brief collection periods. Controversy exists about the potential of these short-term sampling intervals to yield reproducible and meaningful measurements of HRV. Many confounders such as respiration, stress, and body positioning can influence HRV, which is why a longer collection period has been accepted as the standard for providing a stable index of ANS function. However, short sampling periods would be useful to evaluate HRV when faced with time constraints. The purpose of the current study was to evaluate the reproducibility of HRV using 8-hour daytime measures with the Polar R-R RecorderTM (Polar Electro Oy, Kempele, Finland) and with short sampling duration of 512 cardiac cycles, using the Schiller AT-10TM device (Schiller AG, Baar, Switzerland). Methods: 10 apparently healthy adult volunteers participated in the study, which was conducted at the Sleep Disorders Clinic in Christiansburg, VA. Each subject performed two HRV trials with the Cardiovit AT-10TM device using recordings of 512 cardiac cycles. Within one or two days following the Schiller, the same subjects wore a Polar R-R RecorderTM device to obtain an 8-hour recording of HRV during waking hours; 24-hour urine samples were collected on the same day. Urine was analyzed for catecholamine levels, including norepinephrine and epinephrine in order to evaluate sympathetic nervous system globally. Each subject recorded their personal impressions of unavoidable physical activity and daytime stress demands on the day of the 8-hour recording and urine collection. This entire protocol was repeated one week later. On one of the days of the short sampling recording, VO2pk also was evaluated for each subject using a ramp protocol on the cycle ergometer and a metabolic cart. Results: The correlation analysis for the HRV response variables using the Schiller method indicated a high-to-very high correlation between trials within a day for the time domain measures (r = 0.75-0.99). The frequency domain measures, however, were low-to-moderately correlated (r = 0.24-0.66) between trials within a day for the Schiller method. Correlations between days for HRV response variables using the Schiller method were similarly low for both time (r < 0.5) and (r < 0.4) frequency domain measures. Correlation coefficients between days for the HRV response variables using the Polar method were moderate (r = 0.59-0.67) for the time domain and only low-moderate for the frequency domain measures (r = 0.37-0.69). However, an important finding was that Polar R-R data for two of the subjects contained excessive signal artifact, which affected the fidelity of the HRV scores. When these two cases were excluded from the group analyses, the resulting correlations were high-very high for all time and frequency domain measures (r = 0.70-0.93). The means for each response HRV time and frequency domain variable between the Polar method and Schiller method were significantly different (P < 0.05). Additional correlational analyses did not reveal any systematic associations between HRV measures and simple markers of sympathetic activity (urinary NE or E) and aerobic fitness (VO2pk) in this small sample of subjects. Conclusions: Due to this important change in reproducibility with the Polar method, the consequence of artifact-free recordings is unmistakable. Within the limitations of this small study sample it is concluded that, while HRV in apparently healthy adults may not be measured reliably with brief data collection periods, longer daytime sampling periods of 8 hours (e.g. Polar device) yields acceptable reliability for both time and frequency domain parameters of HRV. / Master of Science

Complications in pregnancy elevate fetal norepinephrine (NE) concentrations. Previous studies in NE-infused sheep fetuses revealed that sustained exposure to high NE resulted in lower expression of α2-adrenergic receptors in islets and increased insulin secretion responsiveness after acutely terminating the NE infusion. In this study, we determined if the compensatory increase in insulin secretion after chronic elevation of NE is independent of hyperglycemia in sheep fetuses and whether it is persistent in conjunction with islet desensitization to NE. After an initial assessment of glucose-stimulated insulin secretion (GSIS) at 129 ± 1 days of gestation, fetuses were continuously infused for seven days with NE and maintained at euglycemia with a maternal insulin infusion. Fetal GSIS studies were performed again on days 8 and 12. Adrenergic sensitivity was determined in pancreatic islets collected at day 12. NE infusion increased (P < 0.01) fetal plasma NE concentrations and lowered (P < 0.01) basal insulin concentrations compared to vehicle-infused controls. GSIS was 1.8-fold greater (P < 0.05) in NE-infused fetuses compared to controls at both one and five days after discontinuing the infusion. Glucose-potentiated arginine-induced insulin secretion was also enhanced (P < 0.01) in NE-infused fetuses. Maximum GSIS in islets isolated from NE-infused fetuses was 1.6-fold greater (P < 0.05) than controls, but islet insulin content and intracellular calcium signaling were not different between treatments. The half-maximal inhibitory concentration for NE was 2.6-fold greater (P < 0.05) in NE-infused islets compared to controls. These findings show that chronic NE exposure and not hyperglycemia produce persistent adaptations in pancreatic islets that augment β-cell responsiveness in part through decreased adrenergic sensitivity.

These studies provide evidence for novel properties of macrophages derived from bone marrow stem cells. In study 1, treatment of activated mouse bone marrow-derived macrophages (BMM) with either catecholamine synthesis inhibitors (α-methyl-para-tyrosine and fusaric acid) or the β2 adrenergic receptor antagonist ICI 118,551 demonstrated that BMM produce catecholamines. The catecholamines modulated macrophage cytokine production through autocrine actions on adrenergic receptors. In study II, undifferentiated human bone marrow cells were incubated in 30% mouse L929 fibroblast conditioned medium and generated adherent cells within three days. The cells were clearly identifiable as macrophages based on surface proteins and phagocytic activity but produced only low levels of the cytokines tumor necrosis factor-α and interleukin-lβ. Cytokine production did not increase in response to the bacterial endotoxin lipopolysaccharide (LPS). Generation of these macrophage-like cells was not repeatable with other samples of human bone marrow, but the cells continue to proliferate in cell culture and will be investigated further in future studies.

Phäochromozytome und Paragangliome (PPGL) sind seltene, katecholaminproduzierendeTumore des chromaffinen Gewebes. Die Erkrankung ist durch die Überproduktion von Katecholaminen gekennzeichnet und kann lebensbedrohliche Folgen haben. Die dieser Arbeit zugrunde liegende Studie untersuchte die interindividuellen Unterschiede im Metabolitenprofil bei Patient*innen mit PPGL im Vergleich zu Kontrollen mittels Flüssigchromatographie-Massenspektrometrie und einem Targeted Metabolomic Ansatz. Targeted Metabolomics beschreibt die Messung und Quantifizierung von im Voraus definierten Metaboliten in einer Probe. Von den 188 gemessenen Metaboliten zeigten vier Metabolite eine signifikanten Veränderung zwischen den Gruppen (Histidin, Threonin, LysoPC a C28:0 und Summe der Hexosen). Für alle vier Metabolite wurde ein Zusammenhang mit Katecholaminen im Urin beziehungsweise Metanephrinen im Plasma nachgewiesen. Subgruppenanalysen zeigten weitere Hinweise auf geschlechts- und phänotypspezifische Unterschiede im Metabolitenprofil zwischen Patient*innen mit PPGL und Kontrollen. / Pheochromocytomas and paragangliomas (PPGL) are rare, catecholamine-producing tumors arising from chromaffin cells. The disease is characterized by the overproduction of catecholamines and can have life-threatening consequences. The study on which this work is based investigated the interindividual differences in metabolite profiles in patients with PPGL compared to controls using liquid chromatography-mass spectrometry and a targeted metabolomics approach. Targeted metabolomics describes the measurement and quantification of predefined metabolites. Of the 188 metabolites measured, four metabolites showed a significant change between groups (histidine, threonine, LysoPC a C28:0 and sum of hexoses). A significant correlation with urinary catecholamines and/ or plasma metanephrines was identified for this metabolites. Subgroup analyses showed further evidence of sex- and phenotype-specific differences in metabolite profiles between patients with PPGL and controls.

In the isolated rat liver perfused in situ stimulation of the nerve bundles around the portal vein and the hepatic artery caused an increase of urate formation that was inhibited by the α1-blocker prazosine and the xanthine oxidase inhibitor allopurinol. Moreover, nerve stimulation increased glucose and lactate output and decreased perfusion flow. Infusion of noradrenaline had similar effects. Compared to nerve stimulation infusion of glucagon led to a less pronounced increase of urate formation and a twice as large increase in glucose output but a decrease in lactate release without affecting the flow rate. Insulin had no effect on any of the parameters studied.

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Tyrosine hydroxylase (TyrH) is the first and rate-limiting enzyme of catecholamine synthetic pathway, and its regulation is critical for controlling catecholamine synthesis. The well recognized regulatory mechanisms are inhibition by catecholamine binding and re-activation upon Ser40 phosphorylation. Catecholamines bind to TyrH tightly, while phosphorylation of TyrH at Ser40 decreases the binding affinity by several hundred-fold. Regulation of TyrH is accompanied by conformational changes of the protein. This study focuses on the identification of the conformational changes of TyrH upon dopamine binding and Ser40 phosphorylation, using hydrogen deuterium exchange mass spectrometry (HDMS) and fluorescence spectroscopy. HDMS identifies three peptides undergoing conformational changes upon dopamine binding, peptide 35-41, 42-71 and 295-299. Peptides 35-41 and 42-71 are on the regulatory domain, while peptide 295-299 is at the active site entrance. Upon dopamine binding, all three peptides are protected from exchange; phosphorylation of TyrH at Ser40 has opposite effects on the exchange kinetics of peptide 295-299, but peptides 35-41 and 42-71 could not be detected by MS after phosphorylation. This suggests that the structural effects of dopamine binding and Ser40 phosphorylation are opposite. The fluorescence spectroscopy of mutant enzymes containing a single tryptophan at position 14, 34 or 74 was performed before and after phosphorylation. F34W/F3W TyrH has a significant decrease in steady-state fluorescence anisotropy, an increase in the bimolecular quenching rate constant kq and dynamic anisotropy upon phosphorylation at Ser40, while F14W/F3W TyrH and F74W/F3W TyrH exhibit much smaller differences. This suggests that phosphorylation of TyrH at Ser40 increases the flexibility of the regulatory domain. The results are consistent with TyrH existing in two conformations, a closed conformation stabilized by dopamine in which the N-terminal regulator domain of TyrH covers the active site entrance and an open conformation stabilized by phosphorylation in which the regulatory domain has moved away from the active site entrance.

This thesis has examined the influence of the environment on aspects of the acute stress response in juvenile and pro-larval Acipenser fulvescens.The acute stress response was examined in grouped and isolated juveniles. Catecholamines significantly increased in both treatments one minute post-stress and plasma glucose was significantly higher in isolated as compared to grouped fish one minute post-stress. In the second series of experiments fertilized eggs of A. fulvescens were raised at 9, 12 & 15°C. Chromaffin-like cells were studied using light and electron microscopy techniques. Development of renal tissue was also examined in these treatment groups. Two populations of chromaffin-like cells were identified, one in close association with the proximal tubule of the kidney, and the other in close association with the neural tube. Results suggest this latter population were immature pheochromoblast like-cells. Development of renal tissue followed a predictable pattern that was most rapid in the 15°C treatment.

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.