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An adequate supply of oxygen (02) is essential to the survival of all higher organisms. The mammalian carotid body, located at the common carotid artery senses blood levels of 0 2, carbon dioxide (C02) and acidity. Glomus cells, or type I cells in the carotid body are the main 0 2-sensors which regulate blood p02 via reflex control of ventilation. The carotid body secretes multiple neurotransmitters including dopamine (DA), which is potentiated during low p02 levels and is thought to modulate sensory signaling by apposing afferent nerve fibers. Catecholamine (CA) release is also critical for the animal's ability to survive hypoxic stress associated with the birthing process and the transition to extrauterine life. However, the source for this CA release (primarily epinephrine; EPI) is from adrenal chromaffin cells. The primary 02-sensor in both adrenal chromaffin cells and carotid body type I cells is unknown. One potential candidate is the cytochrome b55s/NADPH oxidase complex that generates the respiratory burst in phagocytes. To test this hypothesis, cultured adrenal medulla chromaffin cells and intact carotid bodies from wild type (WT) and oxidase deficient (OD) mice (knockout gp91 phox, the glycoprotein subunits in the NADPH oxidase complex) were investigated. High performance liquid chromatography and immunocytochemistry were used to quantify amine release in these two chemoreceptors following exposure to hypoxia. Both WT and OD chromaffin cells and carotid bodies responded to the hypoxic challenge with increased monoamine secretion. Norepinephrine and epinephrine were the principal amines released from chromaffin cells, compared to dopamine and serotonin from carotid bodies. These findings suggest that NADPH oxidase is not the primary 02- sensor in either chemosensory system. Quantification of monoamine secretion in intact carotid body from mouse and rat was also compared under basal conditions and after exposure to hypoxia and acid/hypercapnia (pH 7.10). Significantly larger amounts of basal serotonin was secreted from mouse carotid body as compared to the rat. Interestingly, serotonin release was potentiated by hypoxia in mouse carotid body, but this was not observed in the rat. Additionally, ratio of basal level serotonin-to-dopamine secretion was significantly higher in mouse than rat CB. Surprisingly, acid/hypercapnic (pH 7.1 0) had no detectable effect on amine secretion from either mouse or rat carotid body. / Thesis / Master of Science (MSc)

For animals at high altitude, low oxygen (hypoxia) is an unremitting stressor that has the potential to impair metabolism and performance. The hypoxic chemoreflex senses reductions in the partial pressure of O2 in the arterial blood and thus elicits many of the physiological responses to hypoxia, including increases in breathing and activation of the sympathetic nervous system. The hypoxic chemoreflex is vital to surviving acute exposure to severe hypoxia, but the advantage of this reflex during chronic hypoxia is less clear. The goals of my thesis were to examine how control of breathing by the hypoxic chemoreflex has evolved in high-altitude natives to maintain O2 transport in chronic hypoxia, and to elucidate the potential genetic mechanisms that were involved. This was accomplished using deer mice (Peromyscus maniculatus) native to high- and low-altitudes, in addition to a strictly low-altitude species (P. leucopus). I found that high-altitude deer mice breathe with higher total ventilation using preferentially deeper breaths, contributing to higher O2 saturation of arterial blood, but in contrast to lowland mice highlanders do not exhibit ventilatory plasticity in response to chronic hypoxia. These phenotypes appeared to be uniquely evolved in the highland population and arise during the onset of endothermy in early post-natal development. I then used second-generation inter-population hybrids to evaluate the effects of genetic variation (specifically, in the hypoxia-inducible factor 2a gene Epas1 and in haemoglobin genes) on an admixed genomic background. The high-altitude variant of α-globin could completely explain the deep breathing pattern of highland mice, whereas the high-altitude variant of Epas1 and possibly β-globin contributed to their apparent lack of ventilatory plasticity in response to chronic hypoxia. Together, the physiological changes elicited by these mutations contribute to maintaining O2 uptake and metabolism in the cold and hypoxic environment at high altitude. / Thesis / Doctor of Philosophy (PhD) / High-altitude environments are amongst the harshest on earth, with extremely low levels of oxygen, but some animals not only survive but thrive in these conditions. How these animals do so was previously not well understood. My thesis has uncovered how the evolution of respiratory physiology contributes to high-altitude adaptation in the deer mouse, the species with the broadest altitudinal distribution of any North American mammal, and has elucidated the genetic mechanisms involved. My work contributes to understanding nature’s solutions to oxygen deprivation – an all too common problem in many human and animal diseases.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Siu-Yin Sylvia Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 123-140). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / 英中譯名對照 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.viii / Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Overview of Carotid Body --- p.1 / Chapter 1.1.1 --- Type I Cells --- p.3 / Chapter 1.1.2 --- Type II Cells --- p.4 / Chapter 1.1.3 --- Blood Vessels --- p.5 / Chapter 1.1.4 --- Innervation --- p.5 / Chapter 1.1.5 --- Biochemistry --- p.6 / Chapter 1.1.6 --- Physiology and Function --- p.7 / Chapter 1.2 --- The Renin-Angiotensin System (RAS) --- p.8 / Chapter 1.2.1 --- Circulating RAS --- p.8 / Chapter 1.2.1.1 --- Angiotensinogen --- p.10 / Chapter 1.2.1.2 --- Renin --- p.10 / Chapter 1.2.1.3 --- Angiotensin I --- p.11 / Chapter 1.2.1.4 --- Angiotensin Converting Enzyme --- p.12 / Chapter 1.2.1.5 --- Angiotensin II --- p.12 / Chapter 1.2.1.6 --- Angiotensin II Receptors --- p.13 / Chapter 1.2.1.7 --- Angiotensin IV and Angiotensin IV Receptor --- p.15 / Chapter 1.2.2 --- Tissue RAS --- p.16 / Chapter 1.3 --- Hypoxia and Carotid Body --- p.18 / Chapter 1.4 --- Hypoxia and RAS --- p.21 / Chapter 1.5 --- Hypoxia and RAS in Carotid Body --- p.23 / Chapter 1.6 --- Aims of Study --- p.24 / Chapter 1.6.1 --- Existence of Functional Angiotensin II Receptors --- p.24 / Chapter 1.6.2 --- Regulation and Function of Angiotensin II Receptors by Chronic Hypoxia --- p.24 / Chapter 1.6.3 --- Existence of an Intrinsic Angiotensin-generating System --- p.25 / Chapter 1.6.4 --- Regulation of Local RAS by Chronic Hypoxia --- p.25 / Chapter 1.6.5 --- Studies of AT4 Receptor --- p.26 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Experimental Animals and Rat Models --- p.27 / Chapter 2.1.1 --- Rat Model of Chronic Hypoxia --- p.27 / Chapter 2.1.2 --- Isolation of Carotid Body --- p.28 / Chapter 2.2 --- Semi-quantitative Reverse Transcriptase-polymerase Chain Reaction (RT-PCR) --- p.30 / Chapter 2.2.1 --- Total RNA Extraction and Quantification --- p.30 / Chapter 2.2.2 --- Reverse Transcription (RT) --- p.31 / Chapter 2.2.3 --- Polymerase Chain Reaction (PCR) --- p.31 / Chapter 2.2.4 --- Gel Electrophoresis --- p.34 / Chapter 2.2.5 --- Optimization of Semi-quantitative RT-PCR for RAS Gene Analysis --- p.34 / Chapter 2.3 --- Northern Blotting --- p.35 / Chapter 2.3.1 --- Transfer of Denatured RNA to Nitrocellulose Membrane By Capillary Elution --- p.35 / Chapter 2.3.2 --- Hybridization --- p.36 / Chapter 2.4 --- In-situ Hybridization --- p.38 / Chapter 2.4.1 --- Linearization of Angiotensinogen cDNA --- p.38 / Chapter 2.4.2 --- Riboprobe Preparation --- p.38 / Chapter 2.4.3 --- Quantification and Gel Electrophoresis of Riboprobes --- p.39 / Chapter 2.4.4 --- In-situ Hybridization Histochemistry --- p.39 / Chapter 2.5 --- Immunohistochemistry --- p.42 / Chapter 2.5.1 --- Preparation of Cryosection --- p.42 / Chapter 2.5.2 --- Indirect Immunoperoxidase Staining --- p.42 / Chapter 2.5.3 --- Immunofluorescent Double Staining --- p.43 / Chapter 2.6 --- Western Blot Analysis --- p.45 / Chapter 2.6.1 --- Preparation of Angiotensinogen Protein --- p.45 / Chapter 2.6.2 --- Quantification of Protein Concentration --- p.45 / Chapter 2.6.3 --- Sample Preparation --- p.45 / Chapter 2.6.4 --- Sodium Dodecyl-sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.6.5 --- Electroblotting and Immunodetection of Proteins --- p.46 / Chapter 2.7 --- Spectrofluorimetric Measurement and In-vitro Electrophysiology --- p.48 / Chapter 2.7.1 --- Dissociation of Carotid Body Type I Cells and Spectrofluorimetric Measurement --- p.48 / Chapter 2.7.2 --- In-vitro Electrophysiology --- p.49 / Chapter 2.8 --- Assay of ACE Activity --- p.51 / Chapter 2.8.1 --- Crude Membrane Preparation --- p.51 / Chapter 2.8.2 --- Basic Principle for ACE Activity Measurement --- p.51 / Chapter 2.8.3 --- Measurement of ACE Activity --- p.51 / Chapter 2.8.4 --- Fluorescence Measurement --- p.53 / Chapter 2.9 --- In-vitro Autoradiography and Fluorescence-labeled Binding Assay for Angiotensin IV --- p.54 / Chapter 2.9.1 --- Preparation of Frozen Tissue Sections --- p.54 / Chapter 2.9.2 --- Localization and Density of AT4 Receptor --- p.54 / Chapter 2.10 --- Statistics and Data Analysis --- p.57 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Functional Expression of Angiotensin II Receptors --- p.58 / Chapter 3.1.1 --- [Ca2+]i Response to Angiotensin II --- p.58 / Chapter 3.1.2 --- Antagonistic Blockade of Angiotensin II Receptor Subtypes --- p.58 / Chapter 3.1.3 --- Expression of AT1 Receptors mRNA --- p.61 / Chapter 3.1.4 --- Cellular Localization of AT1 Receptors Protein --- p.61 / Chapter 3.2 --- Effect of Chronic Hypoxia on the Expression and Function of Angiotensin II Receptors --- p.64 / Chapter 3.2.1 --- Effect of Chronic Hypoxia on the Expression of AT1 Receptors --- p.64 / Chapter 3.2.2 --- Effect of Chronic Hypoxia on the Expression of AT2 Receptors --- p.67 / Chapter 3.2.3 --- Cellular Localization of the AT1 Receptor by Chronic Hypoxia --- p.69 / Chapter 3.2.4 --- Increase of Afferent Nerve Activities of the Carotid Body In-vitro by Angiotensin II --- p.71 / Chapter 3.2.5 --- Inhibition of Angiotensin II-mediated Response in Chronically Hypoxic Carotid Body by Losartan --- p.73 / Chapter 3.3 --- Evidence for the Existence of an Intrinsic Angiotensin-generating System --- p.75 / Chapter 3.3.1 --- Expression and Localization of Angiotensinogen mRNA --- p.75 / Chapter 3.3.2 --- Expression and Localization of Angiotensinogen Protein --- p.78 / Chapter 3.3.3 --- Expression of Renin mRNA --- p.81 / Chapter 3.3.4 --- Expression of ACE mRNA --- p.81 / Chapter 3.4 --- Effect of Chronic Hypoxia on the Locally-generated Angiotensin System --- p.85 / Chapter 3.4.1 --- Effect of Chronic Hypoxia on the Expression of Angiotensinogen mRNA --- p.85 / Chapter 3.4.2 --- Effect of Chronic Hypoxia on the Localization of Angiotensinogen mRNA --- p.87 / Chapter 3.4.3 --- Effect of Chronic Hypoxia on the Expression of Angiotensinogen Protein --- p.89 / Chapter 3.4.4 --- Effect of Chronic Hypoxia on the Expression of ACE --- p.91 / Chapter 3.5 --- Time-course Effect of Chronic Hypoxia on ACE Activity --- p.93 / Chapter 3.6 --- Preliminary Studies of AT4 Receptor --- p.98 / Chapter 3.6.1 --- In-vitro Autoradiographic Study of AT4 Receptors --- p.98 / Chapter 3.6.2 --- Localization of AT4 Receptors --- p.100 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Functional Expression of Angiotensin II Receptors --- p.102 / Chapter 4.2 --- Upregulation and Function of Angiotensin II Receptors --- p.105 / Chapter 4.3 --- Existence of a Local RAS --- p.108 / Chapter 4.4 --- Regulation of the Local RAS --- p.112 / Chapter 4.5 --- Time-dependent Changes of ACE Activity --- p.155 / Chapter 4.6 --- Presence and Regulation of AT4 Receptor --- p.117 / Chapter 4.7 --- Conclusion --- p.120 / Chapter 4.8 --- Future Works --- p.121 / Chapter Chapter 5 --- References --- p.123

Ascent to altitude presents a significant challenge to the human body. Specifically, it is associated with an increased ventilation and pulmonary vasoconstriction. In healthy subjects these are related such that a high ventilatory drive is associated with blunted pulmonary vasoconstriction. Adults born prematurely and given supplemental oxygen at birth have a blunted ventilatory response to hypoxia. We hypothesized that the hypoxic ventilatory and pulmonary vasoconstrictor responses would be unrelated following perinatal supplemental oxygen exposure. To test our hypothesis, we used a well-established rat model of 80% O2 (80%) exposure for 14 days post-natally, with 21% O2 exposure as a control (21%). We assessed the ventilatory response to graded hypoxia using barometric plethysmography 6-9 months post hyperoxia exposure. The left and right ventricles were catheterized to evaluate the hemodynamic response to 10 minutes of 12% O2 (hypoxia). To our surprise we found that 80% animals did not demonstrate a depressed ventilatory response to hypoxia. However, these animals experienced increased right ventricular systolic pressure in response to 12% O2. An increase in cardiac output was the primary driving force behind the increase in right ventricular end systolic pressure, not an increase in vascular resistance. We found no relationship between the hypoxic ventilatory drive and right ventricular pressure. In 21% animals exposed to hypoxia, the increase in right ventricular pressure was driven primarily by vasoconstriction and, as previous studies have shown, there was a relationship between the ventilatory and pressure responses. These data suggest that neonatal supplemental oxygen alters the hemodynamic response to hypoxia, possibly through enhanced sympathetic drive. The relationship between ventilation and pulmonary pressure may not translate to individuals born prematurely.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-162). Also available in print.

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