Angiotensin type 1A receptors in C1 neurons of the rostral ventrolateral medulla modulate the pressor response to aversive stress

The rise in blood pressure during an acute aversive stress has been suggested to involve activation of angiotensin type 1A receptors (AT(1A)Rs) at various sites within the brain, including the rostral ventrolateral medulla. In this study we examine the involvement of AT(1A)Rs associated with a subclass of sympathetic premotor neurons of the rostral ventrolateral medulla, the C1 neurons. The distribution of putative AT(1A)R-expressing cells was mapped throughout the brains of three transgenic mice with a bacterial artificial chromosome-expressing green fluorescent protein under the control of the AT(1A)R promoter. The overall distribution correlated with that of the AT(1A)Rs mapped by other methods and demonstrated that the majority of C1 neurons express the AT(1A)R. Cre-recombinase expression in C1 neurons of AT(1A)R-floxed mice enabled demonstration that the pressor response to microinjection of angiotensin II into the rostral ventrolateral medulla is dependent upon expression of the AT(1A)R in these neurons. Lentiviral-induced expression of wild-type AT(1A)Rs in C1 neurons of global AT(1A)R knock-out mice, implanted with radiotelemeter devices for recording blood pressure, modulated the pressor response to aversive stress. During prolonged cage-switch stress, expression of AT(1A)Rs in C1 neurons induced a greater sustained pressor response when compared to the control viral-injected group (22 +/- 4 mmHg for AT(1A)R vs 10 +/- 1 mmHg for GFP; p < 0.001), which was restored toward that of the wild-type group (28 +/- 2 mmHg). This study demonstrates that AT(1A)R expression by C1 neurons is essential for the pressor response to angiotensin II and that this pathway plays an important role in the pressor response to aversive stress

The role of AT1A receptors in angiotensin II dependent hypertension

© 2014 Dr. Nikola JancovskiHypertension, or high blood pressure (BP), is a major contributing factor for the development of multiple cardiovascular and renal diseases. The pathogenesis of hypertension is multifactorial and despite decades of extensive research in this field, the exact mechanisms underlying the generation and maintenance of high BP remain largely unknown. However, the involvement of the renin-angiotensin system (RAS) and sympathetic nervous system is well recognized. Angiotensin II (Ang II), one of the effector peptides generated by the RAS, exerts modulatory actions on cardiovascular homeostasis via binding to its type 1A receptor (AT1AR) in several targets including the kidney, brain, vasculature and heart. Previous studies have demonstrated an important role of kidney AT1R in the pathogenesis of experimental hypertension. However, AT1Rs are expressed in several organ systems throughout the body including the nervous system, and the relative contribution of extra-renal AT1Rs to the pathogenesis of hypertension is not well understood. Therefore, the main focus of this PhD thesis is to examine the role of AT1R expression in the nervous system in the development and maintenance of Ang II hypertension. In Chapter 3, I examined whether lentiviral-mediated deletion of the AT1AR from C1 neurons in the rostral ventrolateral medulla (RVLM) would affect the pressor response to exogenous microinjection of Ang II in anesthetized mice. A Cre-lox approach was employed to delete AT1AR expression from C1 neurons in the RVLM by microinjection of a lentivirus expressing Cre-recombinase under the control of a phox2 (PRSx8) binding site promoter in mice with a conditional AT1AR allele (AT1ARfl/fl). I demonstrated that lentiviral-mediated Cre expression in the RVLM of AT1ARfl/fl mice had no effect either on the baseline cardiovascular parameters or the response to L-glutamate, but significantly attenuated the response to microinjection of Ang II. The response to Ang II was not altered in control mice injected with a lentivirus expressing the green fluorescent protein (GFP). Therefore, expression of AT1AR in C1 neurons is essential for the pressor response to exogenous Ang II in the RVLM. Chapter 4 describes a series of experiments to test whether expression of AT1ARs in catecholaminergic neurons contribute to hypertension induced by a low dose infusion of Ang II. A Cre-lox approach was used to selectively delete AT1ARs from all catecholaminergic cells (CAT-KO). In vitro autoradiography confirmed loss of AT1ARs from catecholaminergic cells in the adrenal medulla and sympathetic ganglia. Receptor deletion was also confirmed by loss of a functional response to exogenous microinjection of Ang II in the RVLM. Deletion of AT1ARs from catecholaminergic cells did not alter basal metabolism or arterial pressure, but delayed the onset of angiotensin-dependent hypertension, and reduced the maximal response. The reduced hypertensive response in the CAT-KO mice was accompanied by attenuated activation of the sympathetic nervous system, reduced cardiac hypertrophy and decreased reactive oxygen species production in the RVLM. Thus, activation of the AT1AR on catecholaminergic cells is required for the full development of Ang II-dependent hypertension and support an important role for the sympathetic nervous system in this model. In chapters 5 and 6, the role of AT1AR expression on catecholaminergic cells and activation of the sympathetic nervous system in Ang II-dependent hypertension was investigated further. I examined whether deletion of the AT1AR from catecholaminergic cells affects urinary catecholamine concentrations as well as fluid and electrolyte excretion during Ang II-dependent hypertension. The hypothesis that selective deletion of AT1ARs only from catecholaminergic C1 neuron in the RVLM would affect the development of hypertension in response to chronic infusion of Ang II was also tested. A Cre-lox approach was employed to delete AT1AR from all catecholaminergic cells (as described for Chapter 4) or from C1 neurons selectively (as described for Chapter 3). Experimental results showed that deletion of AT1ARs from all catecholaminergic cells is also associated with decreased renal fluid and electrolyte retention and urinary noradrenaline excretion. Mice with targeted deletion of AT1AR from the C1 neurons in the RVLM had normal baseline cardiovascular parameters, but altered BP responses to chronic infusion of Ang II. In particular, deletion of AT1ARs from C1 neurons decreases the hypertensive response during the second week of Ang II infusion, suggesting that AT1AR expression by C1 neurons plays a role in the later phase of the Ang II hypertension. The early divergence in the BP response in the CAT-KO mice during the first week of infusion suggests that AT1AR expression on cells, other than C1 neurons, is also playing a role in the development of Ang II-dependent hypertension. Together, the novel findings from this thesis highlight the importance of AT1AR expression in the nervous system for generation and maintenance of hypertension induced by Ang II

The role of AT1A receptors in angiotensin II dependent hypertension

© 2014 Dr. Nikola JancovskiHypertension, or high blood pressure (BP), is a major contributing factor for the development of multiple cardiovascular and renal diseases. The pathogenesis of hypertension is multifactorial and despite decades of extensive research in this field, the exact mechanisms underlying the generation and maintenance of high BP remain largely unknown. However, the involvement of the renin-angiotensin system (RAS) and sympathetic nervous system is well recognized. Angiotensin II (Ang II), one of the effector peptides generated by the RAS, exerts modulatory actions on cardiovascular homeostasis via binding to its type 1A receptor (AT1AR) in several targets including the kidney, brain, vasculature and heart. Previous studies have demonstrated an important role of kidney AT1R in the pathogenesis of experimental hypertension. However, AT1Rs are expressed in several organ systems throughout the body including the nervous system, and the relative contribution of extra-renal AT1Rs to the pathogenesis of hypertension is not well understood. Therefore, the main focus of this PhD thesis is to examine the role of AT1R expression in the nervous system in the development and maintenance of Ang II hypertension. In Chapter 3, I examined whether lentiviral-mediated deletion of the AT1AR from C1 neurons in the rostral ventrolateral medulla (RVLM) would affect the pressor response to exogenous microinjection of Ang II in anesthetized mice. A Cre-lox approach was employed to delete AT1AR expression from C1 neurons in the RVLM by microinjection of a lentivirus expressing Cre-recombinase under the control of a phox2 (PRSx8) binding site promoter in mice with a conditional AT1AR allele (AT1ARfl/fl). I demonstrated that lentiviral-mediated Cre expression in the RVLM of AT1ARfl/fl mice had no effect either on the baseline cardiovascular parameters or the response to L-glutamate, but significantly attenuated the response to microinjection of Ang II. The response to Ang II was not altered in control mice injected with a lentivirus expressing the green fluorescent protein (GFP). Therefore, expression of AT1AR in C1 neurons is essential for the pressor response to exogenous Ang II in the RVLM. Chapter 4 describes a series of experiments to test whether expression of AT1ARs in catecholaminergic neurons contribute to hypertension induced by a low dose infusion of Ang II. A Cre-lox approach was used to selectively delete AT1ARs from all catecholaminergic cells (CAT-KO). In vitro autoradiography confirmed loss of AT1ARs from catecholaminergic cells in the adrenal medulla and sympathetic ganglia. Receptor deletion was also confirmed by loss of a functional response to exogenous microinjection of Ang II in the RVLM. Deletion of AT1ARs from catecholaminergic cells did not alter basal metabolism or arterial pressure, but delayed the onset of angiotensin-dependent hypertension, and reduced the maximal response. The reduced hypertensive response in the CAT-KO mice was accompanied by attenuated activation of the sympathetic nervous system, reduced cardiac hypertrophy and decreased reactive oxygen species production in the RVLM. Thus, activation of the AT1AR on catecholaminergic cells is required for the full development of Ang II-dependent hypertension and support an important role for the sympathetic nervous system in this model. In chapters 5 and 6, the role of AT1AR expression on catecholaminergic cells and activation of the sympathetic nervous system in Ang II-dependent hypertension was investigated further. I examined whether deletion of the AT1AR from catecholaminergic cells affects urinary catecholamine concentrations as well as fluid and electrolyte excretion during Ang II-dependent hypertension. The hypothesis that selective deletion of AT1ARs only from catecholaminergic C1 neuron in the RVLM would affect the development of hypertension in response to chronic infusion of Ang II was also tested. A Cre-lox approach was employed to delete AT1AR from all catecholaminergic cells (as described for Chapter 4) or from C1 neurons selectively (as described for Chapter 3). Experimental results showed that deletion of AT1ARs from all catecholaminergic cells is also associated with decreased renal fluid and electrolyte retention and urinary noradrenaline excretion. Mice with targeted deletion of AT1AR from the C1 neurons in the RVLM had normal baseline cardiovascular parameters, but altered BP responses to chronic infusion of Ang II. In particular, deletion of AT1ARs from C1 neurons decreases the hypertensive response during the second week of Ang II infusion, suggesting that AT1AR expression by C1 neurons plays a role in the later phase of the Ang II hypertension. The early divergence in the BP response in the CAT-KO mice during the first week of infusion suggests that AT1AR expression on cells, other than C1 neurons, is also playing a role in the development of Ang II-dependent hypertension. Together, the novel findings from this thesis highlight the importance of AT1AR expression in the nervous system for generation and maintenance of hypertension induced by Ang II

Angiotensin type 1A receptor expression in C1 neurons of the rostral ventrolateral medulla contributes to the development of angiotensin-dependent hypertension

New Findings What is the central question of this study? This study addresses the mechanism by which deletion of angiotensinII type1A receptors from catecholaminergic neurons reduces angiotensin-dependent hypertension, as well as the identity of the cells involved.What is the main finding and its importance? Deletion of angiotensinII type1A receptors from catecholaminergic neurons results in reduced sympathetic nerve activation and fluid and electrolyte retention during angiotensin infusion. The C1 neurons of the rostral ventrolateral medulla are involved in the later phase of the hypertension. We demonstrate that at least two different populations of catecholaminergic neurons are involved in the sympathetic nerve activation required for the full development of angiotensin-dependent hypertension.Chronic low-dose systemic infusion of angiotensinII induces hypertension via activation of the angiotensinII type1A receptor (AT(1A)R). Previously, we have demonstrated that expression of the AT(1A)R on catecholaminergic neurons is necessary for the full development of angiotensin-dependent hypertension. In the present study, we examined the mechanism by which selective deletion of the AT(1A)R from these cells affects the development of hypertension. We also tested the hypothesis that AT(1A)Rs expressed by catecholaminergic C1 neurons in the rostral ventrolateral medulla play an important role in angiotensin-induced hypertension. A Cre-lox approach was used to delete the AT(1A)R from all catecholaminergic cells or from C1 neurons selectively. Subcutaneous administration of angiotensinII induced hypertension in all mice, with delayed onset and reduced maximal response in the global AT(1A)R catecholaminergic knockout mice. The AT(1A)R catecholaminergic knockout mice had decreased renal fluid and electrolyte retention and urinary noradrenaline excretion. The blood pressure response was reduced only during the second week of angiotensinII infusion in the mice with selective C1 AT(1A)R deletion, demonstrating that AT(1A)R expression by C1 neurons plays a moderate role in angiotensin-induced hypertension. The difference in the time course of development of hypertension between the mice with global AT(1A)R knockout from catecholaminergic cells and the mice with C1 AT(1A)R deletion suggests that other catecholaminergic neurons are important

Carbogen-Induced Respiratory Acidosis Blocks Experimental Seizures by a Direct and Specific Inhibition of NaV1.2 Channels in the Axon Initial Segment of Pyramidal Neurons

Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO2/95% O2) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demon-strate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experi-ments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant NaV1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neu-rons expressing the interneuron predominant NaV1.1 channel. Furthermore, knockdown of Scn2a mRNA in vivo using antisense ol-igonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in an Asic1a knock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of NaV1.2 channels, whereas inhibitory neuron firing is unaf-fected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification.Peer reviewe

Stimulation of angiotensin type 1A receptors on catecholaminergic cells contributes to angiotensin-dependent hypertension

Hypertension contributes to multiple forms of cardiovascular disease and thus morbidity and mortality. The mechanisms inducing hypertension remain unclear although the involvement of homeostatic systems, such as the renin-angiotensin and sympathetic nervous systems, is established. A pivotal role of the angiotensin type 1 receptor in the proximal tubule of the kidney for the development of experimental hypertension is established. Yet, other systems are involved. This study tests whether the expression of angiotensin type 1A receptors in catecholaminergic cells contributes to hypertension development. Using a Cre-lox approach, we deleted the angiotensin type 1A receptor from all catecholaminergic cells. This deletion did not alter basal metabolism or blood pressure but delayed the onset of angiotensin-dependent hypertension and reduced the maximal response. Cardiac hypertrophy was also reduced. The knockout mice showed attenuated activation of the sympathetic nervous system during angiotensin II infusion as measured by spectral analysis of the blood pressure. Increased reactive oxygen species production was observed in forebrain regions, including the subfornical organ, of the knockout mouse but was markedly reduced in the rostral ventrolateral medulla. These studies demonstrate that stimulation of the angiotensin type 1A receptor on catecholaminergic cells is required for the full development of angiotensin-dependent hypertension and support an important role for the sympathetic nervous system in this model

Excessive respiratory modulation of blood pressure triggers hypertension

The etiology of hypertension, the world’s biggest killer, remains poorly understood, with treatments targeting the established symptom, not the cause. The development of hypertension involves increased sympathetic nerve activity that, in experimental hypertension, may be driven by excessive respiratory modulation. Using selective viral and cell lesion techniques, we identify adrenergic C1 neurons in the medulla oblongata as critical for respiratory-sympathetic entrainment and the development of experimental hypertension. We also show that a cohort of young, normotensive humans, selected for an exaggerated blood pressure response to exercise and thus increased hypertension risk, has enhanced respiratory-related blood pressure fluctuations. These studies pinpoint a specific neuronal target for ameliorating excessive sympathetic activity during the developmental phase of hypertension and identify a group of pre-hypertensive subjects that would benefit from targeting these cells.10 page(s