Date of Award

8-1-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

J. Thomas Cunningham

Second Advisor

Steve W. Mifflin

Third Advisor

Ann M. Schreihofer

Abstract

The brain Renin Angiotensin System (RAS) is quickly becoming recognized as a critical mediator of blood pressure and body fluid homeostasis. In the forebrain, the median preoptic nucleus (MnPO) responds to Angiotensin II (Ang II) stimulation by increasing thirst and blood pressure. Understanding how this nuclei regulates blood pressure and body fluid homeostasis in response to Ang II has the potential to open new therapeutic avenues for treatment of hypertension. In the studies following series of studies I investigated the role of MnPO Angiotensin Type Ia receptors in the sustained hypertension induced by Chronic Intermittent Hypoxia (CIH) and thirst regulation.

The first project focuses on the role of the MnPO in the sustained hypertension of CIH. Sleep apnea leads to hypertension that persists throughout the waking period. The neural mechanisms that underlie this pathophysiological increase in blood pressure are not well known. CIH is a model of the hypoxemia experienced by sleep apnea sufferers. This model produces a sustained increase in blood pressure, and numerous studies have indicated that the central renin-angiotensin system is involved in this prolonged hypertension. The MnPO receives inputs from the subfornical organ (SFO) and the organum vasculosum lamina terminalis, both circumventricular organs (CVOs) outside the blood brain barrier allowing for it to receive input from nuclei exposed to peripherally circulating hormones. Downstream the MnPO projects to brain regions that control sympathetic activity, such as the paraventricular nucleus (PVN), making it a prime target for integrating peripheral signals with central sympathetic drive. Previous studies have shown that administration of Losartan, and AT1 receptor blocker, directly into the ventricles of the brain prevents the sustained component of CIH induced hypertension and decreaeses FosB in the MnPO. Inhibition of the transcription factor FosB in the median preoptic nucleus via a dominant negative construct prevents the sustained hypertension from CIH. CIH also increases Angiotensin Type 1a receptor mRNA in the MnPO via the FosB transcription factor. We hypothesize that knockdown of AT1a receptors locally in the MnPO will prevent the sustained hypertension of CIH. Utilizing a short hairpin RNA we knocked down the Angiotensin Type 1a receptor in the MnPO to determine its contribution to 7 days of CIH hypertension. AT1a receptor knockdown in the median preoptic nucleus blocked the sustained component of hypertension from CIH as compared to scramble injected animals. Immunohistochemistry revealed significantly less FosB positive cells in the MnPO of animals injected with the knockdown vector compared to animals given the control vector prior to 7 days of CIH. The rostral ventrolateral medulla, a key regulator of sympathetic outflow, also expressed significantly less FosB with the AT1a knockdown in the MnPO. Our results indicate that AT1a receptors in the MnPO are regulated by FosB and contribute to CIH hypertension.

Thirst is a critical function the MnPO regulates and the second project aimed to determine if Ang II is necessary for thirst generation at the MnPO. Ang II is known to produce a dipsogenic response when administered peripherally, or centrally. Peripheral Ang II acting as a hormone activates CVOs, brain regions lacking a blood brain barrier, such as the SFO. These circumventricular organs synapse upon the MnPO which leads to activation of higher cortical centers and thirst. Intracerebroventricular (ICV) administration of Ang II produces thirst without interacting with CVOs by directly activating the MnPO, and it is therefore hypothesized that Ang II is the neurotransmitter released at the CVO-MnPO junction. We sought to test this hypothesis by knocking down AT1a receptors in the MnPO using a short hairpin RNA complimentary for the receptor subtype. Knockdown of AT1a in the MnPO significantly reduced drinking in animals administered ICV Ang II. This reduction in water consumption was mirrored by a reduction in cFos, a transcription factor and marker of neuronal activity, in the MnPO, SON, and PVN. Thirst generated by subcutaneous administration of Ang II was unaffected by the knockdown of AT1a in the MnPO. cFos counts in the SFO, OVLT, MnPO, PVN, and SON also were not significantly influenced. These data indicates that AT1a receptors in the MnPO are not necessary for water intake stimulated by peripheral Ang II. While experimental administration of Ang II directly to the ventricles is sufficient to induce drinking it may not play a prominent role in stimulating drinking under physiological conditions.

Comments

Brent, Shell, The Role of Angiotensin II in Central Control of Blood Pressure and Body Fluid Homeostasis. Doctor of Philosophy (Biomedical Sciences), July 2016. Available August 2017.

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