Physiological genomic analysis of the brain renin-angiotensin system

The brain renin-angiotensin system (RAS) has long been considered pivotal in cardiovascular regulation and important in the pathogenesis of hypertension and heart failure. However, despite more than 30 years of study, the brain RAS continues to defy explanation. Our lack of understanding of how the brain RAS is organized at the cellular and regional levels has made it difficult to resolve long-sought questions of how ANG II is produced in the brain and the precise mechanisms by which it exerts its actions. A major reason for this is the difficulty in experimentally dissecting the brain RAS at the regional, cellular, and whole organism levels. Recently, we and others developed a series of molecular tools for selective manipulation of the murine brain RAS, in parallel with technologies for integrative analysis of cardiovascular and volume homeostasis in the conscious mouse. This review, based in part on a lecture given in conjunction with the American Physiological Society Young Investigator Award in Regulatory and Integrative Physiology (Water and Electrolyte Homeostasis Section), outlines the physiological genomics strategy that we have taken in an effort to unravel some of the complexities of this system. It also summarizes the principles, progress, and prospects for a better understanding of the brain RAS in health and disease.     

The brain renin-angiotensin system: location and physiological roles

Angiotensinogen, the precursor molecule for angiotensins I, II and III, and the enzymes renin, angiotensin-converting enzyme (ACE), and aminopeptidases A and N may all be synthesised within the brain. Angiotensin (Ang) AT(1), AT(2) and AT(4) receptors are also plentiful in the brain. AT(1) receptors are found in several brain regions, such as the hypothalamic paraventricular and supraoptic nuclei, the lamina terminalis, lateral parabrachial nucleus, ventrolateral medulla and nucleus of the solitary tract (NTS), which are known to have roles in the regulation of the cardiovascular system and/or body fluid and electrolyte balance. Immunohistochemical and neuropharmacological studies suggest that angiotensinergic neural pathways utilise Ang II and/or Ang III as a neurotransmitter or neuromodulator in the aforementioned brain regions. Angiotensinogen is synthesised predominantly in astrocytes, but the processes by which Ang II is generated or incorporated in neurons for utilisation as a neurotransmitter is unknown. Centrally administered AT(1) receptor antagonists or angiotensinogen antisense oligonucleotides inhibit sympathetic activity and reduce arterial blood pressure in certain physiological or pathophysiological conditions, as well as disrupting water drinking and sodium appetite, vasopressin secretion, sodium excretion, renin release and thermoregulation. The AT(4) receptor is identical to insulin-regulated aminopeptidase (IRAP) and plays a role in memory mechanisms. In conclusion, angiotensinergic neural pathways and angiotensin peptides are important in neural function and may have important homeostatic roles, particularly related to cardiovascular function, osmoregulation and thermoregulation.     

The renin-angiotensin system in the rat brain

Summary The distribution of angiotensinogen containing cells was determined in the brain of rats using immunocytochemistry. Specific angiotensinogen immunoreactivity is demonstrated both in glial cells and neurons throughout the brain, except the neocortical and cerebellar territories. Positive neurons are easily and invariably detected in female brains, and haphazardly in male brain (sex hormone dependent). Angiotensinogen immunoreactivity in male brain neurons can be induced by water deprivation or binephrectomy in some areas and particularly in paraventricular nuclei. Finally, the highest concentrations of positive neurons are found in the anterior and lateral hypothalamus, preoptic area, amygdala and some well known nuclei of the mesencephalon and the brainstem.Our results confirm the wide distribution of angiotensinogen mRNA in the brain reported recently by Lynch et al. (1987). Thus the demonstration of angiotensinogen in neurons and glial cells allows a greater understanding of the biochemical and physiological data in accordance with multiple brain renin angiotensin systems.     

Cellular organization of the brain renin-angiotensin system

A model of intracellular Ang II formation (Figure 1) implies that angiotensinogen neurons exist and that CNS Ang II acts both as a neurotransmitter as well as a neurohormone. Such a mechanism is consistent with the immunocytochemical localization of a fraction of brain Ang II in neurosecretory vesicles. To date, several dozen peptide neurotransmitters and neurohormones have been studied. Those assigned to peptidergic systems follow the generalized pathway of biosynthesis shown in Figure 1. In peptidergic systems, a prohormone and all of its processing enzymes are synthesized in the rough endoplasmic reticulum of a cell and move into the Golgi apparatus (Figure 1: #1-3). In the Golgi the prohormone and processing enzymes are packaged into the same vesicle (#3). These secretory vesicles then migrate toward the plasma membrane, frequently via axonal or dendritic projections to terminals. Within these cytoplasmic vesicles and prior to release, the processing enzymes are activated (#4) and the prohormone enzymatically processed, yielding the active peptide (#5-6). Only then do the vesicles fuse with the plasma membrane (in a calcium-dependent process), releasing their contents (#7-8). Once released, the active peptide migrates across the extracellular space and interacts with specific cell surface receptors to initiate a response (#9). Finally, receptor-bound peptide degradation is initiated by receptor-mediated endocytosis (#10-11). For angiotensin peptides to be produced intracellularly, the cell must present only one secretory pathway for Golgi packaging of renin and angiotensinogen; otherwise current theories of protein sorting would predict that these two proteins would be segregated even if synthesized within the same cell. Small quantities of co-packaged renin and angiotensinogen occurring via "spill-over" between compartments seems an unsatisfactory process for a regulated hormone system. Figure 2, depicting an extracellular mechanism for producing Ang II in the brain, has also been proposed. The mechanism of extracellular angiotensin formation is consistent with the molecular information encoded within the component proteins, known mechanisms of protein secretio, well-defined systemic renin-angiotensin enzymatic cascades, and demonstration of all the components of the renin-angiotensin system in the extracellular compartments of the brain. This model (Figure 2) allows independently coordinated gene expression and synthesis of renin (#1R), angiotensinogen (#1A), and angiotensin-converting enzyme (# 1C) in the same or different cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The renin-angiotensin system and the central nervous system.

One of several factors affecting the secretion of renin by the kidneys is the sympathetic nervous system. The sympathetic input is excitatory and is mediated by beta-adrenergic receptors, which are probably located on the membranes of the juxtaglomerular cells. Stimulation of sympathetic areas in the medulla, midbrain and hypothalamus raises blood pressure and increases renin secretion, whereas stimulation of other parts of the hypothalamus decreases blood pressure and renin output. The centrally active alpha-adrenergic agonist clonidine decreases renin secretion, lowers blood pressure, inhibits ACTH and vasopressin secretion, and increases growth hormone secretion in dogs. The effects on ACTH and growth hormone are abolished by administration of phenoxybenzamine into the third ventricle, whereas the effect on blood pressure is abolished by administration of phenoxybenzamine in the fourth ventricle without any effect on the ACTH and growth hormone responses. Fourth ventricular phenoxybenzamine decreases but does not abolish the inhibitory effect of clonidine on renin secretion. Circulating angiotensin II acts on the brain via the area postrema to raise blood pressure and via the subfornical organ to increase water intake. Its effect on vasopressin secretion is debated. The brain contains a renin-like enzyme, converting enzyme, renin substrate, and angiotensin. There is debate about the nature and physiological significance of the angiotensin II-generating enzyme in the brain, and about the nature of the angiotensin I and angiotensin II that have been reported to be present in the central nervous system. However, injection of angiotensin II into the cerebral ventricles produces drinking, increased secretion of vasopressin and ACTH, and increased blood pressure. The same responses are produced by intraventricular renin. Angiotensin II also facilitates sympathetic discharge in the periphery, and the possibility that it exerts a similar action on the adrenergic neurons in the brain merits investigation.     

The CNS renin-angiotensin system

The renin-angiotensin system (RAS) is one of the best-studied enzyme-neuropeptide systems in the brain and can serve as a model for the action of peptides on neuronal function in general. It is now well established that the brain has its own intrinsic RAS with all its components present in the central nervous system. The RAS generates a family of bioactive angiotensin peptides with variable biological and neurobiological activities. These include angiotensin-(1–8) [Ang II], angiotensin-(3–8) [Ang IV], and angiotensin-(1–7) [Ang-(1–7)]. These neuroactive forms of angiotensin act through specific receptors. Only Ang II acts through two different high-specific receptors, termed AT1 and AT2. Neuronal AT1 receptors mediate the stimulatory actions of Ang II on blood pressure, water and salt intake, and the secretion of vasopressin. In contrast, neuronal AT2 receptors have been implicated in the stimulation of apoptosis and as being antagonistic to AT1 receptors. Among the many potential effects mediated by stimulation of AT2 are neuronal regeneration after injury and the inhibition of pathological growth. Ang-(1–7) mediates its antihypertensive effects by stimulating the synthesis and release of vasodilator prostaglandins and nitric oxide and by potentiating the hypotensive effects of bradykinin. New data concerning the roles of Ang IV and Ang-(1–7) in cognition also support the existence of complex site-specific interactions between multiple angiotensins and multiple receptors in the mediation of important central functions of the RAS. Thus, the RAS of the brain is involved not only in the regulation of blood pressure, but also in the modulation of multiple additional functions in the brain, including processes of sensory information, learning, and memory, and the regulation of emotional responses.     

The renin-angiotensin system in the rat brain. Immunocytochemical localization of angiotensinogen in glial cells and neurons

The distribution of angiotensinogen containing cells was determined in the brain of rats using immunocytochemistry. Specific angiotensinogen immunoreactivity is demonstrated both in glial cells and neurons throughout the brain, except the neocortical and cerebellar territories. Positive neurons are easily and invariably detected in female brains, and haphazardly in male brain (sex hormone dependent). Angiotensinogen immunoreactivity in male brain neurons can be induced by water deprivation or binephrectomy in some areas and particularly in paraventricular nuclei. Finally, the highest concentrations of positive neurons are found in the anterior and lateral hypothalamus, preoptic area, amygdala and some well known nuclei of the mesencephalon and the brainstem. Our results confirm the wide distribution of angiotensinogen mRNA in the brain reported recently by Lynch et al. (1987). Thus the demonstration of angiotensinogen in neurons and glial cells allows a greater understanding of the biochemical and physiological data in accordance with multiple brain renin angiotensin systems.     

The pyruvate kinase-coupled assay for ATPases: a critical analysis.

The pyruvate kinase assay for ATPases suffers from major systematic errors due to the variable amounts of the inhibitory product, ADP, present as the rates change. A simple method is presented to estimate the true uninhibited rates and the ADP inhibition constant for the ATPase.     

Side chain-oxidized oxysterols regulate the brain renin-angiotensin system through a liver X receptor-dependent mechanism

Disturbances in cholesterol metabolism have been associated with hypertension and neurodegenerative disorders. Because cholesterol metabolism in the brain is efficiently separated from plasma cholesterol by the blood-brain barrier (BBB), it is an unsolved paradox how high blood cholesterol can cause an effect in the brain. Here, we discuss the possibility that cholesterol metabolites permeable to the BBB might account for these effects. We show that 27-hydroxycholesterol (27-OH) and 24S-hydroxycholesterol (24S-OH) up-regulate the renin-angiotensin system (RAS) in the brain. Brains of mice on a cholesterol-enriched diet showed up-regulated angiotensin converting enzyme (ACE), angiotensinogen (AGT), and increased JAK/STAT activity. These effects were confirmed in in vitro studies with primary neurons and astrocytes exposed to 27-OH or 24S-OH, and were partially mediated by liver X receptors. In contrast, brain RAS activity was decreased in Cyp27a1-deficient mice, a model exhibiting reduced 27-OH production from cholesterol. Moreover, in humans, normocholesterolemic patients with elevated 27-OH levels, due to a CYP7B1 mutation, had markers of activated RAS in their cerebrospinal fluid. Our results demonstrate that side chain-oxidized oxysterols are modulators of brain RAS. Considering that levels of cholesterol and 27-OH correlate in the circulation and 27-OH can pass the BBB into the brain, we suggest that this cholesterol metabolite could be a link between high plasma cholesterol levels, hypertension, and neurodegeneration.     

The renin-angiotensin system and sodium appetite

Intracranial renin is a potent stimulus to sodium appetite and thirst, the effects being mediated by local generation of angiotensin II. Intakes are persistent and lead to fluid retention during the first 24 h (Avrith and Fitzsimons, 1983). Increased circulating renin after captopril treatment in adrenalectomized rats (Elfont and Fitzsimons, 1981), or in renal hypertension following partial inter-renal aortic ligation (Costales et al., 1982), also leads to increased intakes of 2.7% NaCl and water. Fluid intakes after aortic ligation were independent of the severity of hypertension produced by this procedure. In both the examples given, additional stimulation resulting from the hypovolaemia itself is required for the full expression of increased sodium appetite, but in both cases angiotensin makes a significant contribution to sodium appetite as well as thirst. Therefore, as has been shown for thirst, angiotensin is one of a number of factors that act together to cause increased sodium appetite in hypovolaemia.     

Receptors for angiotensin: a critical analysis.

Angiotensin exerts numerous contractile and secretory effects by activating specific receptors. Recent pharmacological findings obtained with this peptide in various laboratories are analyzed, using the order of potency of agonists and the affinity of competitive antagonists as criteria for the classification of receptors for angiotensin in several systems. The analysis is restricted to experiments in which biological effects have been measured. Desensitization (the third criterion for classification of receptors) is discussed and a new protocol is proposed for its utilization. The analysis reveals that receptors for angiotensin in intestinal and vascular smooth muscles, in the heart, and in the vas deferens are all of the same type, while the receptors mediating the release of catecholamines from the adrenal medulla and those subserving the steroidogenic action on the adrenal cortex remain still unidentified. The recently proposed role of ATI as mediator of renin in the adrenal medulla is not substantiated by pharmacological findings with decapeptide antagonists. Moreover, the utilization of ATI as an agonist to determine the order of potency of angiotensins and the use of SQ 20881 as an inhibitor of the converting enzyme have shown serious limitations and should be reconsidered. The hypothetical role of ATIII as mediator of the renin-angiotensin system in the adrenal cortex, at least in other species than the rat, appears to be supported by the high affinity of heptapeptide antagonists for the adrenocortical receptor. However, these antagonists have generally been compared with [Sar1,Ala8]-ATII, A compound which is definitely inadequate for evaluating the affinities of octapeptides in the adrenal cortex. Therefore most of the data supporting the role of ATIII in this system have to be carefully reconsidered. Analogues of ATII are proposed for using as agonists and as antagonists instead of the natural angiotensins (for determining the order of potency of agonists) and instead of [Sar1,Ala8]-ATII (for measuring the affinities of competitive antagonists).     

Diabetes-induced stimulation of the renin-angiotensin system in the rat brain cortex

Cerebrovascular disease is a threat to people with diabetes and hypertension. Diabetes can damage the brain by stimulating the renin-angiotensin system (RAS), leading to neurological deficits and brain strokes. Diabetes-induced components of the RAS, including angiotensin-converting enzyme (ACE), angiotensin-II (Ang-II), and angiotensin type 1 receptor (AT1R), have been linked to various neurological disorders in the brain. In this study, we investigated how diabetes and high blood pressure affected the regulation of these major RAS components in the frontal cortex of the rat brain. We dissected, homogenized, and processed the brain cortex tissues of control, streptozotocin-induced diabetic, spontaneously hypertensive (SHR), and streptozotocin-induced SHR rats for biochemical and Western blot analyses. We found that systolic blood pressure was elevated in SHR rats, but there was no significant difference between SHR and diabetic-SHR rats. In contrast to SHR rats, the heartbeat of diabetic SHR rats was low. Western blot analysis showed that the frontal cortexes of the brain expressed angiotensinogen, AT1R, and MAS receptor. There were no significant differences in angiotensinogen levels across the rat groups. However, the AT1R level was increased in diabetic and hypertensive rats compared to controls, whereas the MAS receptor was downregulated (p < 0.05). These findings suggest that RAS overactivation caused by diabetes may have negative consequences for the brain's cortex, leading to neurodegeneration and cognitive impairment.     

Antisense inhibition of the renin-angiotensin system in brain and peripheral organs

Antisense inhibition is a method of attenuating the target at the gene expression level. There are two main groups of molecular tools for this goal. The first includes the use of short synthetic stretches of DNA-antisense oligodeoxynucleotides. The second tool is the use of vectors (plasmids or viruses) containing the gene of interest subcloned in the antisense orientation, which in the cells produces the antisense RNA. Both antisense DNA and RNA can bind to the complementary sense mRNA and interfere with its translation. Effects are usually short lasting (days) for oligodeoxynucleotides and longer lasting (weeks or months) for vectors. In this article we briefly describe techniques of antisense inhibition in the context of the renin-angiotensin system.