Siew Yeen Chai

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 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.

Angiotensin AT4 ligands are potent, competitive inhibitors of insulin regulated aminopeptidase (IRAP)

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT4 receptor. However the AT4 receptor has recently been identified as the insulin‐regulated aminopeptidase (IRAP). In this study, we demonstrate that AT4 receptor ligands, including Ang IV, Nle1‐Ang IV, divalinal‐Ang IV, and the structurally unrelated LVV‐hemorphin‐7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leu‐β‐naphthylamide by recombinant human IRAP. Both Ang IV and divalinal–Ang IV display competitive kinetics, indicating that AT4 ligands mediate their effects by binding to the catalytic site of IRAP. The AT4 ligands also displaced [125I]‐Nle1‐Ang IV or [125I]‐divalinal1‐Ang IV from IRAP‐HEK293T membranes with high affinity, which was up to 200‐fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT4 ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met‐enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.

Different cross-presentation pathways in steady-state and inflammatory dendritic cells

Presentation of exogenous antigens on MHC class I molecules, termed cross-presentation, is essential for the induction of CD8 T-cell responses and is carried out by specialized dendritic cell (DC) subsets. The mechanisms involved remain unclear. It has been proposed that antigens could be transported by endocytic receptors, such as the mannose receptor (MR) in the case of soluble ovalbumin, into early endosomes in which the cross-presentation machinery would be recruited. In these endosomal compartments, peptides would be trimmed by the aminopeptidase IRAP before loading onto MHC class I molecules. Here, we have investigated the contribution of this pathway to cross-presentation by steady-state CD8+ DC and inflammatory monocyte-derived DC (moDC) generated in vivo. We demonstrate that IRAP and MR are dispensable for cross-presentation by CD8+ DC and for cross-priming. Moreover, we could not find any evidence for diversion of endocytosed antigen into IRAP-containing endosomes in these cells. However, cross-presentation was impaired in moDC deficient in IRAP or MR, confirming the role of these two molecules in inflammatory DC. These results demonstrate that the mechanisms responsible for cross-priming by steady-state and inflammatory DC are different, which has important implications for vaccine design.

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.

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.

Potentiation of cholinergic transmission in the rat hippocampus by angiotensin IV and LVV-hemorphin-7

Recent evidence demonstrates that the fragment of angiotensin II, angiotensin II (3-8) termed angiotensin IV, binds with high affinity to a specific binding site, the AT(4) receptor. Intracerebroventricular injection of AT(4) receptor agonists improves the performance of rats in passive avoidance and spatial learning paradigms. AT(4) receptors and cholinergic neurons are closely associated in regions involved in cognitive processing, such as the hippocampus and neocortex. We therefore postulated that AT(4) receptors affect cognitive processing by modulating cholinergic neurotransmission. To test this, we examined the effect of AT(4) receptor ligands, angiotensin IV and LVV-hemorphin-7, on potassium-evoked [(3)H]acetylcholine ([(3)H]ACh) release from rat hippocampal slices. Hippocampal slices from male Sprague--Dawley rats were incubated with [(3)H]choline chloride, perfused with Krebs--Henseleit solution and [(3)H]ACh release was determined. Angiotensin IV and LVV-hemorphin-7 both potentiated depolarisation-induced [(3)H]ACh release from the rat hippocampus in a concentration-dependent manner with the maximal dose (10(-7)M) of each inducing an increase of 45+/-7.5% (P<0.01) and 95.8+/-19% (P<0.01) above control, respectively. Potentiation of release by both agonists was attenuated by the AT(4) receptor antagonist, divalinal-Ang IV. Angiotensin IV-induced potentiation was not affected by AT(1) and AT(2) receptor antagonists. These results indicate that stimulation of AT(4) receptors can potentiate depolarisation-induced release of ACh from hippocampal slices and suggest that potentiation of cholinergic transmission may be a mechanism by which AT(4) receptor ligands enhance cognition.

Angiotensin‐Converting Enzyme Modulates Dopamine Turnover in the Striatum

Abstract: The effect of chronic inhibition of the angiotensin‐converting enzyme on dopamine content and release in the striatum was investigated using in vivo microdialysis in awake, freely moving rats. Rats were treated for 1 week with the angiotensin‐converting enzyme inhibitor perindopril (1 mg/kg) via the drinking water, whereas the controls were given water alone. One week after perindopril treatment, striatal dopamine dialysate levels in the treated group were markedly elevated compared with control values: control, 233 ± 43 pg/ml; perindopril, 635 ± 53 pg/ml (p < 0.001). These results were confirmed by a complementary study in which dopamine content was measured in striatal extracts (3.5 ± 0.4 µg of dopamine/g of tissue for controls compared with 9.2 ± 2.4 µg of dopamine/g of tissue for the treated group; p < 0.05). In the rats that were dialyzed, angiotensin‐converting enzyme levels in the striatum were decreased by 50% after perindopril treatment. Levels of dopamine D1 and D2 receptors and of preprotachykinin and tyrosine hydroxylase mRNAs were unchanged after angiotensin‐converting enzyme inhibition. A small, but significant, increase was detected in striatal preproenkephalin mRNA levels in the angiotensin‐converting enzyme inhibitor‐treated group. These results indicate that peripherally administered angiotensin‐converting enzyme inhibitors penetrate the blood‐brain barrier when given chronically and modulate extracellular dopamine and striatal neuropeptide levels.

Distribution of angiotensin IV binding sites (AT4 receptor) in the human forebrain, midbrain and pons as visualised by in vitro receptor autoradiography

Angiotensin IV and other AT4 receptor agonists, improve memory retention and retrieval in the passive avoidance and swim maze learning paradigms. Angiotensin IV binding sites (also known as the AT4 receptors) are widely distributed in guinea pig and monkey (Macaca fascicularis) brains where high densities of the binding sites have been detected in the hippocampus, neocortex and motor nuclei. However, the distribution of the binding sites in the human brain is not known. We have recently localised the angiotensin IV binding sites (AT4 receptors) in post-mortem human brain using iodinated Nle-angiotensin IV, a higher affinity and more stable analogue of angiotensin IV. This radioligand bound with relatively high affinity and specificity to angiotensin IV binding sites. In competition studies on consecutive sections through the prefrontal cortex and claustrum, angiotensin IV, Nle-angiotensin IV and LVV-hemorphin 7 competed for the binding of 125I[Nle]-angiotensin IV with nanomolar affinities. Angiotensin II and the AT1 and AT2 receptor antagonists were ineffective in competing for the binding at concentrations of up to 10 microM. We found high densities of 125I[Nle]-angiotensin IV binding sites throughout the cerebral cortex including the insular, entorhinal, prefrontal and cingulate cortices. Very high densities of the binding sites were observed in the claustrum, choroid plexus, hippocampus and pontine nucleus. Some thalamic nuclei displayed high densities of binding including the anteroprincipal, ventroanterior, anteromedial, medial dorsal and ventrolateral nuclei. The caudate nucleus, putamen, many amygdaloid nuclei and the red nucleus all displayed moderate densities of binding with a higher level detected in the substantia nigra pars compacta. In the hypothalamus, high densities binding sites were found in the ventromedial nucleus with lower levels in the dorsomedial and paraventricular nuclei. The distribution of 125I[Nle]-angiotensin IV binding sites in the human brain is similar to that found in other species and supports multiple roles for the binding sites in the central nervous system, including facilitation of memory retention and retrieval.

Distribution of angiotensin II receptor subtypes in the rabbit brain

We have determined the distribution of angiotensin II receptor subtypes in rabbit brain using in vitro autoradiography. AT1 receptors were found in very high concentrations in the forebrain circumventricular organs--the subfornical organ, organum vasculosum of the lamina terminalis, and the median eminence as observed in other mammals. However, there was very little labeling in the area postrema. In the paraventricular nucleus, median preoptic nucleus, supraoptic nucleus there were high levels of predominantly AT1 receptors. High densities of AT1 receptors were also found in the nucleus of the solitary tract and the rostral and caudal ventrolateral medulla. All of these regions have putative roles in the regulation of blood pressure and fluid and electrolyte balance. In the rabbit brain there is less AT2 receptor binding than the rat, with most AT2 binding found in the molecular layer of the cerebellum and in the septohypothalamic nucleus. In the subthalamic nucleus, the mediodorsal and ventroposterior nuclei of the thalamus, locus coeruleus and inferior olivary nuclei, areas containing mostly AT2 receptors in the rat, no binding was detected in the rabbit except in the locus coeruleus which contains moderate levels of AT1 receptors. Taken in conjunction with our previous results in the rat and human brains, these results reveal that AT1 receptors predominate in rostral forebrain, hypothalamus and autonomic control centers of the medulla oblongata in all three species. However, the distribution and density of AT2 bearing sites in regions such as the septum, thalamus subthalamic nuclei, locus coeruleus, cerebellum and inferior olivary nuclei show marked species differences.