Frederick A.O. Mendelsohn

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.

Remodeling and reparation of the cardiovascular system

Growth or altered metabolism of nonmyocyte cells (cardiac fibroblasts, vascular smooth muscle and endothelial cells) alters myocardial and vascular structure (remodeling) and function. However, the precise roles of circulating and locally generated factors such as angiotensin II, aldosterone and endothelin that regulate growth and metabolism of nonmyocyte cells have yet to be fully elucidated. Trials of pharmacologic therapy aimed at preventing structural remodeling and repairing altered myocardial structure to or toward normal in the setting of hypertension, heart failure and diabetes are reviewed. It is proposed that these are therapeutic goals that may reduce cardiovascular morbidity and mortality. Although this hypothesis remains unproved the primary goal of therapy should be to preserve or restore tissue structure and function.

Angiotensin receptors in the nervous system

In addition to its traditional role as a circulating hormone, angiotensin is also involved in local functions through the activity of tissue renin-angiotensin systems that occur in many organs, including the brain. In the brain, both systemic and presumptive neurally derived angiotensin and angiotensin metabolites act through specific receptors to modulate many functions. This review examines the distribution of these specific angiotensin receptors and discusses evidence regarding the function of angiotensin peptides in various brain regions. Angiotensin AT1 and AT2 receptors occur in characteristic distributions that are highly correlated with the distribution of angiotensin-like immunoreactivity in nerve terminals. Acting through the AT1 receptor in the brain, angiotensin has effects on fluid and electrolyte homeostasis, neuroendocrine systems, autonomic pathways regulating cardiovascular function and behavior. Angiotensin AT1 receptors are also found in many afferent and efferent components of the peripheral autonomic nervous system. The role of the AT2 receptor in the brain is less well understood, although recent knockout studies point to an involvement with behavioral and cardiovascular functions. In addition to the AT1 and AT2 receptors, receptors for other fragments of angiotensin have been proposed. The AT4 binding site, which binds angiotensin, has a widespread distribution in the brain quite distinct from that of the AT1 and AT2 receptors. It is associated with many cholinergic neuronal groups and also several sensory nuclei, but its function remains to be determined. Our discovery that another brain-derived peptide binds to the AT4 binding site in the brain and may represent the native ligand is discussed. Overall, the distribution of angiotensin receptors in the brain indicate that they play diverse and important physiological roles in the nervous system.

Localization and function of angiotensin AT1 receptors

The distributions of angiotensin AT1 and AT2 receptors have been mapped by in vitro autoradiography throughout most tissues of many mammals, including humans. In addition to confirming that AT1 receptors occur in sites known to be targets for the physiologic actions of angiotensin, such as the adrenal cortex and medulla, renal glomeruli and proximal tubules, vascular and cardiac muscle and brain circumventricular organs, many new sites of action have been demonstrated. In the kidney, AT1 receptors occur in high density in renal medullary interstitial cells. The function of these cells, which span the interstitial space between the tubules and the vasa rectae, remains to be determined. Renal medullary interstitial cells possess receptors for a number of vasoactive hormones in addition to AT1 receptors and this, in concert with their anatomic location, suggests they may be important for the regulation of fluid reabsorption or renal medullary blood flow. In the heart, the highest densities of AT1 receptors occur in association with the conduction system and vagal ganglia. In the central nervous system, high AT1 receptor densities occur in many regions behind the blood-brain barrier, supporting a role for neurally derived angiotensin as a neuromodulator. The physiologic role of angiotensin in many of these brain sites remains to be determined. The AT2 receptor also has a characteristic distribution in several tissues including the adrenal gland, heart, and brain. The role of this receptor in physiology is being elucidated, but it appears to inhibit proliferation and to participate in development. Thus, receptor-binding studies, localizing the distribution of AT1 and AT2 receptors, provide many insights into novel physiologic roles of angiotensin.

The angiotensin IV/AT4 receptor.

Abstract.The angiotensin AT4 receptor was originally defined as the specific, high-affinity binding site for the hexapeptide angiotensin IV (Ang IV). Subsequently, the peptide LVV-hemorphin 7 was also demonstrated to be a bioactive ligand of the AT4 receptor. Central administration of Ang IV, its analogues or LVV-hemorphin 7 markedly enhance learning and memory in normal rodents and reverse memory deficits observed in animal models of amnesia. The AT4 receptor has a broad distribution and is found in a range of tissues, including the adrenal gland, kidney, lung and heart. In the kidney Ang IV increases renal cortical blood flow and decreases Na+ transport in isolated renal proximal tubules. The AT4 receptor has recently been identified as the transmembrane enzyme, insulin-regulated membrane aminopeptidase (IRAP). IRAP is a type II integral membrane spanning protein belonging to the M1 family of aminopeptidases and is predominantly found in GLUT4 vesicles in insulin-responsive cells. Three hypotheses for the memory-potentiating effects of the AT4 receptor/IRAP ligands, Ang IV and LVV-hemorphin 7, are proposed: (i) acting as potent inhibitors of IRAP, they may prolong the action of endogenous promnestic peptides; (ii) they may modulate glucose uptake by modulating trafficking of GLUT4; (iii) IRAP may act as a receptor, transducing the signal initiated by ligand binding to its C-terminal domain to the intracellular domain that interacts with several cytoplasmic proteins.

Localization of vasopressin binding sites in rat brain by in vitro autoradiography using a radioiodinated V1 receptor antagonist

Vasopressin may act in the brain as a neurotransmitter or neuromodulator to influence blood pressure, memory, body temperature and brain development. In order to localize probable central nervous system sites for these actions, we have used 125I-labelled 1-d(CH2)5, 7-sarcosine-8-arginine vasopressin, a specific V1-receptor antagonist, and in vitro autoradiography to map brain vasopressin binding sites. High levels of binding were found in the choroid plexus, blood vessels, lateral septum, bed nucleus of stria terminalis, accumbens nucleus, central nucleus of amygdala, stigmoid hypothalamic nucleus, suprachiasmatic nucleus, arcuate nucleus, nucleus of the solitary tract, area postrema and parts of the hippocampus, thalamus, superior colliculus, and inferior olivary nuclei. Many of these regions are known to be vasopressin-sensitive and to contain vasopressin fibres. Significantly there was no binding to the paraventricular nor the supraoptic nuclei. Displacement of the radioligand from the lateral septum with unlabelled vasopressin analogues gave a rank order of potencies: d(CH2)5-D-Tyr2(Et)Val4-desGly9-arginine-vasopressin approximately equal to d(CH2)5-Tyr2-(Me)arginine-vasopressin approximately equal to arginine-vasopressin approximately equal to d(CH2)5-Sar7-arginine-vasopressin greater than [1-deamino, 8-D-arginine]-vasopressin approximately equal to oxytocin much greater than vasopressin4-9, consistent with binding to V1 receptor subtype. These studies confirm and extend previous findings of V1 receptors in the rat brain. In particular, several new regions of vasopressin receptor binding have been identified, possibly due to the advantages of a radioiodinated ligand with high receptor affinity without binding to neurophysins. Future study of these regions may prove fruitful in elucidating the central actions of vasopressin.

Mapping tissue angiotensin-converting enzyme and angiotensin AT1, AT2 and AT4 receptors.

Background The renin–angiotensin system (RAS) functions as both a circulating endocrine system and a tissue paracrine/autocrine system. As a circulating peptide, angiotensin II (Ang II) plays a prominent role in blood-pressure control and body fluid and electrolyte balance by acting on the AT1 receptor in the brain and peripheral tissues. As a paracrine/autocrine peptide, locally formed Ang II also plays additional roles in tissues involving the regulation of regional haemodynamics, cell growth and remodelling, and neurotransmitter release. Evidence is emerging that Ang II is not the only active peptide of the RAS, and other Ang II fragments may also have important biological activities. Objectives To provide a morphological basis for understanding novel actions of angiotensin-converting enzyme (ACE), Ang II and related peptides in tissues, this article will review the localization of ACE and AT1, AT2 and AT4 receptors in the central nervous system, blood vessels and kidney. Results and conclusion Autoradiographic mapping of the major components of the RAS has proved a valuable strategy to reveal, or suggest, cellular sites of novel actions for Ang II and related peptides in tissues. First, colocalization of ACE and AT1 receptors in the substantia nigra, the caudate nucleus and putamen of human and rat brain, which contain the dopamine-synthesizing neurons, suggests that the central RAS may be important in modulating central dopamine release. Secondly, the distribution of AT4 receptors with a striking association with cholinergic neurons, motor and sensory nuclei in the brain reveals that Ang IV may modulate central motor and sensory activities and memory. Thirdly, the occurrence of high levels of ACE and AT1 and/or AT2 receptors in the adventitia of blood vessels suggests important paracrine roles of the vascular RAS. Finally, the identification of abundant AT1 receptor and elucidation of its roles in the renomedullary interstitial cells of the kidney may provide a new impetus to study further the role of Ang II in the regulation of renal medullary function and blood pressure. Overall, circulating and locally produced Ang II and related peptides may exert a remarkable range of actions in the brain, kidney and cardiovascular system through multiple angiotensin receptors.

Localization and characterization of endothelin receptor binding sites in the rat brain visualized by in vitro autoradiography.

Endothelin binding sites in rat brain were mapped by quantitative in vitro autoradiography employing [125I]endothelin-1 as radioligand. [125I]Endothelin-1 bound with high affinity and specificity to rat cerebellar sections and was potently displaced by unlabelled endothelins (endothelin-1 greater than endothelin-2 = endothelin-3) and sarafotoxin 6B. The highest densities of endothelin binding sites were found in the cerebellum (especially Purkinje cell layer), choroid plexus and median eminence. High densities were found in the supraoptic and paraventricular hypothalamic nuclei, anterior hypothalamic area, ventromedial hypothalamic nucleus, mammillary nuclei and glomerular layer of olfactory bulb. Moderate densities were found in many thalamic nuclei, the pretectal region, interpeduncular nucleus, suprachiasmatic nucleus, raphe nuclei, tegmental nuclei, olfactory ventricle, red nucleus, subthalamic nucleus, central gray, reticular nuclei, vestibular nuclei, oculomotor and trochlear nuclei, hypoglossal nucleus, motor trigeminal nucleus, nucleus of the trapezoid body and lateral cerebellar nucleus. Low but detectable densities of endothelin binding sites were found in medial geniculate nucleus, fields of Ammons horn, caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra, anterior commissure, internal capsule, anterior pituitary, median preoptic nucleus, septohypothalamic nucleus, superior colliculus and area postrema. These patterns were completely abolished by 1 microM unlabelled endothelin-1, -2 and -3 and sarafotoxin S6B. Brain endothelin binding sites show high affinity for endothelin-1, -2 and -3 and sarafotoxin 6B with highest affinity for endothelin-1. Endothelin binding sites show a non-vascular pattern of distribution in the brain, suggesting that the peptide may have widespread functions as a modulator of neuronal function.

The globin fragment LVV-hemorphin-7 is an endogenous ligand for the AT4 receptor in the brain.

Abstract: Angiotensin IV (Val‐Tyr‐Ile‐His‐Pro‐Phe) has been reported to interact with specific high‐affinity receptors to increase memory retrieval, enhance dopamine‐induced stereotypy behavior, and induce c‐fos expression in several brain nuclei. We have isolated a decapeptide (Leu‐Val‐Val‐Tyr‐Pro‐Trp‐Thr‐Gln‐Arg‐Phe) from sheep brain that binds with high affinity to the angiotensin IV receptor. The peptide was isolated using 125I‐angiotensin IV binding to bovine adrenal membranes to assay receptor binding activity. This peptide is identical to the amino acid sequence 30–39 of sheep βA‐ and βB‐globins and has previously been named LVV‐hemorphin‐7. Pharmacological studies demonstrated that LVV‐hemorphin‐7 and angiotensin IV were equipotent in competing for 125I‐angiotensin IV binding to sheep cerebellar membranes and displayed full cross‐displacement. Using in vitro receptor autoradiography, 125I‐LVV‐hemorphin‐7 binding to sheep brain sections was identical to 125I‐angiotensin IV binding in its pattern of distribution and binding specificity. This study reveals the presence of a globin fragment in the sheep brain that exhibits a high affinity for, and displays an identical receptor distribution with, the angiotensin IV receptor. This globin fragment, LVV‐hemorphin‐7, may therefore represent an endogenous ligand for the angiotensin IV receptor in the CNS.

Localization of binding sites for calcitonin gene-related peptide in rat brain by in vitro autoradiography

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of 1.16 +/- 0.23 X 10(10) M-1 and a site concentration of 43.4 +/- 3.4 fmol/mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of 3.9 +/- 0.7 X 10(9) M-1 whereas salmon calcitonin was very weak with a binding affinity constant of only 6.8 +/- 4.0 X 10(5) M-1. CGRP binding localized by in vitro autoradiography, using [125I]rat calcitonin gene-related peptide, had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex, the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.