京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12876号 / 論農博第2803号 / 新制||農||1028(附属図書館) / 学位論文||H26||N4875(農学部図書室) / 31594 / (主査)教授 伏木 亨, 教授 保川 清, 教授 入江 一浩 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

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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. / Master of Science

Circadian rhythm is a biological rhythm with period of about 24 hours. Circadian rhythm is universal in phyla from bacteria to mammals and exists in different level from gene expression to behavior. Circadian system consists of three components: 1) a self-sustained oscillator; 2) an input pathway which can alter the phase of the oscillator; and 3) an output such as gene expression, enzyme activity, hormone production, heart rate, body temperature or locomotor activities. The way the oscillator regulates its outputs is complicated, in that on one hand usually the oscillator is not the only factor affecting the outputs, and on the other, the oscillator itself is incorporated in intricate pathways. Chicken pineal cell culture is a well-established in vitro model to study circadian rhythm. It contains a self-sustained oscillator which can be phase-shifted by light as input and rhythmically releases melatonin as an output. Here I have characterized the role of norepinephrine (NE), the sympathetic regulatory input of pineal gland, and the microenvironment of pineal cells in melatonin rhythmicity of cultured chicken pineal cells. Chapter 1 of this dissertation provides a review of circadian rhythm with a focus on melatonin regulation in pineal gland. Chapter 2 describes the methods to build up a fraction collector which offers high time resolution of sampling for a superfusion system. Chapter 3 is a technical report of a melatonin enzyme-linked immunosorbent assay suitable for high throughput measurement of melatonin. Chapter 4 presents data demonstrating that daily administration of NE recovers damped melatonin rhythm in constant darkness. In addition, NE does not change the expression of clock genes but the recovery effect of NE depends on the internal clock. Furthermore, the data indicates that NE administration stimulates the gene expression of phosphodiesterase 4D (PDE4D) and adenylate cyclase 1 (AC1) in a time order, potentially corresponding to the trough and peak of recovered melatonin rhythm. Chapter 5 presents data showing that the amplitude of melatonin rhythm in cultured pineal cells is affected by microenvironments of the cell culture and connexin plays a role in this effect. Finally, in Chapter 6 I discuss how the results of each chapter demonstrate multiple regulatory mechanism of the melatonin rhythm of chicken pineal cells. Furthermore, I discuss the implications of this work in the field of developmental biology and how the current data will shape future investigations. My dissertation incorporates engineering, immunocytochemistry, chicken genetics, and biochemical analyses, and will help in better understanding the regulation mechanism of output in a circadian system.

More than 30% of the population suffers from obesity, which increases the risk of death and secondary health problems. Body fat [white adipose tissue (WAT) and brown adipose tissue (BAT)] are innervated and regulated by the sympathetic nervous system (SNS). WAT stores energy, while BAT generates heat for thermoregulation. Fat also has sensory innervations, but the roles of sensory nerves are still being elucidated. Hence, understanding the neuroanatomy of the SNS innervations of fat and the neural regulation of fat metabolism will be valuable for advancing obesity treatment. Using trans-synaptic tract tracers with unique fluorescent proteins, we defined and compared the SNS innervations of visceral fat [mesenteric WAT (MWAT)] and subcutaneous fat [inguinal WAT (IWAT)] and of IWAT and interscapular BAT (IBAT) in Siberian hamsters. MWAT and IWAT have moderately shared SNS innervations within the hindbrain, but separate SNS innervations in rostral regions. In contrast, IWAT and IBAT have relatively separate SNS circuitries throughout the brain yet some overlap in SNS nuclei known to regulate thermogenesis. We tested for the presence of functional coordination between IWAT and IBAT defined by overlap in IWAT SNS and IBAT SNS innervations. When IBAT function was impaired by SNS denervation, IWAT SNS drive, thermogenic activity, and beige adipocyte recruitment increased in cold exposed hamsters likely through coordination with IWAT SNS pathways. Conversely, we found that only SNS drive to IWAT increased during acute food deprivation suggesting that populations of SNS neurons singly innervating each fat depot may contribute to differential SNS drive to fat. Lastly, we demonstrated that IWAT sensory nerves mediate the functional coordination between IWAT and IBAT and the regulation of SNS drive to fat. The absence of IWAT sensory feedback via sensory denervation differentially decreased SNS drive to IBAT and IWAT itself, but not to MWAT, retroperitoneal WAT, and epididymal WAT in cold exposed hamsters. Collectively, the studies in this dissertation provide neuroanatomical evidence of separate and shared SNS brain sites likely receiving sensory signaling and regulating SNS drive to fat, and direct evidence of the roles of SNS and sensory nerves innervating fat to energetic homeostasis and thermoregulation.

Opioid/adrenergic interactions were studied to evaluate two hypotheses: (1) naloxone potentiates the effect of epinephrine on cardiac contractility by increasing circulating epinephrine concentrations; and (2) endogenous and exogenous opioids alter left cardiac nerve stimulationinduced norepinephrine release and cardiac function. A canine isolated heart-lung preparation was used for the first study. Plasma epinephrine was determined and myocardial epinephrine uptake was calculated during intravenous epinephrine infusion. Naloxone (4 mg) was given and the epinephrine infusion was repeated. Naloxone increased cardiac contractility, coronary blood flow, and the coronary sinus epinephrine concentration. When coronary blood flow was subsequently held constant (100% above resting), naloxone increased only contractility. This result indicated that the previously observed increase in coronary sinus epinephrine was flow dependent. Corticosterone (an uptake II blocker) was employed as a positive control. Corticosterone increased the contractile response to epinephrine, but unlike naloxone, corticosterone was accompanied by a clear decrease in myocardial epinephrine uptake. The stereospecificity of the response to naloxone was investigated and (+) naloxone equaled or exceeded (-) naloxone in potentiating the inotropic effect of epinephrine. In the second study, the left cardiac nerve was isolated and electrically stimulated in intact dogs. Norepinephrine overflow gradually declined during successive control stimulations. Pretreatment with naloxone (100 Mg/kg) prevented or delayed the decline. An intracoronary dynorphin 1-9 infusion (2 nmol/min/kg for 20 minutes) reduced both norepinephrine overflow and cardiac performance, and both effects were prevented by pretreatment with naloxone (100 /xg/kg) . To summarize, naloxone potentiated the inotropic effect of infused epinephrine without altering circulating epinephrine concentrations or myocardial epinephrine uptake. This effect of naloxone was not stereospecific and probably not mediated through a traditional opiate receptor. Endogenous and exogenous opioids inhibited the left cardiac nerve stimulation-induced norepinephrine overflow, suggesting that opiate receptors may regulate cardiac excitability by modulating norepinephrine release.

A series of rigid azetidenyl-based methamphetamine analogs were synthesized from commercially available N-Boc-azetidinone. The benzylideneazetidine analogs were prepared via a Wittig olefination via the ylides generated from the corresponding triphenylphosphonium benzylhalide salts. The substituted benzylazetidine analogs were synthesized from the corresponding benzylideneazetidienes via hydrogention over palladium and platinum catalysts. The benzylideneazetidine and benzyliazetidine analogs were evaluated at monoamine transporters as a part of preliminary structure-activity study for the development of novel monoamine transporter ligands. The binding affinities of the azetidine analogs were determined at dopamine (DAT) and serotonin (SERT) transporters in rat brain tissue preparations. The preliminary in vitro binding studies revealed that the rigid scaffold of the azetidine ring system was an effective substitution for the 2-aminopropyl group of methamphetamine and led to compounds with nanomolar binding affinity at dopamine and serotonin. In general, the benzylideneazetidine analogs were more potent than the corresponding benzylazetidine analogs. In addition, the azetidine analogs were more selective for the serotonin transporter than the dopamine transporter. The 3-(3,4-dichlorobenzylidene)azetidine (24m) was the most potent analog of the series with Ki values of 139 nM for SERT and 531 nM for DAT (DAT/SERT = 3.8).

Hypertension affects 1 in 3 adults and is the single greatest risk factor for premature death. Salt-sensitive hypertension occurs in approximately 50% of hypertensive patients and results in a 3-fold increase in the risk of adverse cardiovascular events. However, the pathophysiology of salt-sensitive hypertension remains to be fully elucidated. There has been increased interest in the interaction between the sympathetic nervous system and the kidney and how that interaction mediates sodium excretion to drive the development of salt-sensitivity. Previous studies show that sympathetic over-activity increases expression of the sodium chloride cotransporter (NCC) resulting in increased NCC-mediated sodium reabsorption, and the development of salt-sensitive hypertension. In this thesis, I show the effect of increased norepinephrine (NE) and high salt intake in salt-resistant vs. salt-sensitive rat phenotypes on blood pressure regulation, NCC activity, and the adrenoreceptor-mediated regulatory kinase network signal transduction pathway. A high salt diet 1) exacerbates NE-induced hypertension in salt-resistant Sprague-Dawley (SD) rats and 2) results in hypertension in Dahl salt-sensitive (DSS) rats. In contrast to salt-resistant phenotypes (SD & Dahl salt-resistant), dietary sodium-evoked suppression of NCC expression and activity is prevented in salt-sensitive rats (SD-NE infusion & DSS) - I show that this occurs through a failure of a high salt intake to suppress renal OxSR1, SPAK, and WNK1 (NCC regulatory proteins). I demonstrate that α1-adrenoreceptors are responsible for mediating the salt-sensitive component of hypertension and restore dietary sodium-evoked suppression of the NCC via a predominant OxSR1 pathway. Chronic β-adrenoreceptor antagonism significantly reduces blood pressure in NE-mediated hypertension. How the body senses salt remains unknown, but my data show that selective removal of the afferent renal nerves prevents dietary sodium-evoked suppression of NCC expression and activity resulting in salt-sensitive hypertension, suggesting that the afferent renal nerves play an important role as a sodium-sensing mechanism. Overall, these data demonstrate that attenuated afferent renal nerve feedback drives renal efferent nerve release of NE to prevent the downregulation of the NCC via an α1-adrenergic receptor-gated WNK1-OxSR1 signal transduction pathway to evoke the development of salt-sensitive hypertension.