Bioactive Angiotensin Peptides

I Moeller, AM Allen, S-Y Chai, J Zhuo and FAO Mendelsohn The Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia

Angiotensin II is recognised as the principle active pep- involved in memory retention and neuronal develop- tide of the renin-angiotensin system, exerting effects on ment. Furthermore, our demonstration that a globin fluid and electrolyte homeostasis, and cardiovascular fragment, LVV-haemorphin-7, binds with high affinity to control including neural and long term trophic effects. the angiotensin IV binding site and is abundant in the However, recent studies indicate that other angiotensin brain, indicates that this may represent a novel brain peptides such as angiotensin III, angiotensin II (1–7) and neuropeptide system. It now appears, that the renin- angiotensin IV, may have specific actions. Interestingly, angiotensin system is more complex than previously recent work involving angiotensin IV demonstrates that thought and capable of generating multiple, active pep- this peptide binds to specific receptors and may be tides which elicit numerous diverse actions.

Keywords: angiotensin metabolites; angiotensin III; angiotensin II(1–7); angiotensin IV; globin

Bioactive angiotensin peptides tropic and chronotropic effects and facilitates sympathetic activity.13 The renin-angiotensin system (RAS) was initially Acting on the brain, Ang II induces fluid and salt identified as a circulating humoral system with the ingestion, modulates neuroendocrine systems, effector peptide, angiotensin II (Ang II), generated including vasopressin and corticotropin releasing by an enzymatic cascade. Angiotensinogen, which factor release, and interacts with the autonomic con- is synthesized in the liver, is cleaved by renin, a pro- trol of the cardiovascular system to influence blood duct of the juxtaglomerular cells of the kidney, to pressure.3,14,15 In many instances, these effects are form Ang I, which in turn is cleaved by angiotensin- complementary to those of the systemic peptide on converting enzyme (ACE) to form Ang II. ACE is peripheral target organs. Thus, systematic Ang II membrane bound and predominates on the endo- affects the brain through AT1 receptors located in thelial cells of all vascular beds. Apart from the pro- the circumventricular organs, regions with a duction of Ang II in plasma, Ang II, renin and ACE deficient blood brain barrier. In addition, endogen- have all been described in tissues such as the brain, ous neurally derived Ang II appears to act at many kidney, adrenal, vasculature, heart and ovaries. This central nervous system sites behind the blood brain suggests a separate and distinct RAS in these tissues barrier.16–18 and implies endocrine, paracrine and autocrine Most of the classical actions of Ang II are roles for Ang II.1 mediated via the AT1 receptor whereas AT2 receptor stimulation may cause opposing effects. This was Actions of Ang II observed in rat coronary endothelial cells, in which AT2 receptor stimulation blocks the proliferative Ang II regulates blood pressure, fluid volume 19 effect mediated by the AT1 receptor and in neu- homeostasis and pituitary hormone release via AT1 ronal cultures, where AT1 and AT2 receptors and AT2 receptors located in the kidney, adrenal mediate opposing actions on potassium currents.20 gland, and the cardiovascular and nervous systems.2–5 In the kidney, for instance, Ang II acts on renal vessels, glomeruli, tubules and renomedullary inter- Ang II metabolism stitial cells, to alter renal blood flow, glomerular fil- 21 6–9 Ang II is rapidly metabolised in the circulation tration rate and electrolyte reabsorption. Ang II and in the cerebral ventricles.22 The aminopepti- also acts on the adrenal cortex to stimulate dases A and N sequentially remove amino acids aldosterone secretion and hence renal sodium 10 from the N-terminus of Ang II to form the fragments reabsorption. Ang II(2–8), also known as Ang II, and Ang II(3–8), Ang II is a potent vasoconstrictor, acting directly also known as Ang IV.23 Ang II(1–7) is another on vascular smooth muscle and indirectly via facili- metabolite of Ang II24 but may be cleaved directly tation of noradrenaline release from sympathetic ter- 24–26 11,12 from Ang I by neutral endopeptidase 24.11 and minals. In the heart, Ang II exerts positive iono- prolyl endopeptidase.27 Of these metabolites, Ang III binds with the highest affinity to AT1 receptors (10- Correspondence: I Moeller, The Howard Florey Institute of fold lower affinity than Ang II) whilst both Ang IV Experimental Physiology and Medicine, University of Melbourne, and Ang II(1–7) bind weakly with affinities of a 100- Parkville, Victoria 3052, Australia to 1000-fold less.28 Bioactive angiotensin peptides I Moeller et al 290 Ang III cular smooth muscle cells.50,53,54 However, Ang II(1– 7) has been associated with increased phospholipase Ang III exhibits some of the classical actions of Ang A2 activity in renal proximal tubular cells.55 II such as stimulating aldosterone secretion, vaso- 29–31 From numerous studies, it appears that there may constriction, pressor and dipsogenic activity. In be a distinct Ang II(1–7) receptor, the physiological addition to these studies, recent findings suggest significance of which may be further characterised that Ang III may be an important agonist, if not the by the Ang II(1–7) specific antagonist A-799. final mediator of some actions of Ang II.23,32–34 This was clearly shown in the study by Zini et al,23 in which the use of a specific antagonist of aminopepti- Ang IV dase A, demonstrated that the central action of Ang Recent work has focused on Ang IV due to the wide II on vasopressin secretion in rats is dependent on distribution of a pharmacologically distinct binding its prior conversion to Ang III. site, specific for the peptide and designated the AT4 In contrast to the actions associated with Ang III, receptor.56,57 This binding site shows high selec- initial work involving other Ang II metabolites, such tivity for Ang IV followed by Ang III (10-fold lower as Ang II(1–7) and Ang IV, resulted in the belief that ӷ 1 8 ӷ 29,30,35 affinity) Ang II, [Sar Ile ] Ang II losartan, PD these peptides were functionally inactive. 58–62 123177 and CGP 42112A. The AT4 receptor also However, subsequently, both Ang II(1–7) and Ang displays a distinct distribution pattern, being found IV have been associated with a number of actions. in the cholinergic areas and motor and sensory regions in the brain.59,63 It is also found in the 60 57,64 Ang II(1–7) heart, adrenal cortex, vascular smooth muscle cells,61 and numerous other tissues.65 An Ang II(1–7) binding site that is distinct from Ang One of the first actions attributed to Ang IV was II receptors, has not yet been clearly described and its ability to increase memory recall in passive66,67 is complicated by Ang II(1–7) eliciting some actions and conditioned avoidance response studies.66 This similar to those of Ang II. For instance, Ang II(1–7) cognitive effect of Ang IV was thought to be via the and Ang II stimulated vasopressin secretion36 and hippocampus since intracerebroventricular admin- excited paraventricular hypothalamic neurons and istration of Ang IV induces c-fos expression in hip- 68 were inhibited by the partial antagonist of the AT2 pocampal pyramidal cells. In addition, central receptor, CGP 42112A.37 Also, Ang II(1–7) and Ang injection of Ang IV in the rat, enhances apomorph- II excited the same nucleus tractus solitarius cells ine-induced stereotypy.66 Also, Ang IV inhibited although several cells were only stimulated by one neurite outgrowth from cultured embryonic day 11 peptide.38 Furthermore, microinjection of Ang II(1– (E11) chicken paravertebral sympathetic neurons by 7) and Ang II into the rostral ventrolateral medulla 25%69 and therefore may have a potential role in caused a pressor effect with the effect of Ang II(1– neuronal development. 7), but not Ang II, being inhibited by the Ang II(1–7) Several studies have focused on the ability of Ang specific antagonist, A-799.39–41 These latter studies IV to dilate vessels. In the rabbit, Ang IV caused suggest that Ang II(1–7) might exert functions which vasodilatation of pial arterioles, only if given sub- are distinct from Ang II actions through a novel Ang sequent to L-arginine administration, suggesting an II(1–7) binding site. In support of this, lateral cere- involvement of nitric oxide.70 Infusion of Ang IV broventricular administration of an Ang II(1–7) anti- into the middle cerebral artery of the rat increased body, dose-dependently increased blood pressure in cerebral blood flow by 20%65 although in another the Ren-2 transgenic hypertensive rats, the opposite study, Ang IV had no effect on cerebral blood flow to that observed with an Ang II antibody.42 Also, in in control animals, but in rats with experimental contrast to Ang II, Ang II(1–7) is a vasodilator in por- subarachnoid haemorrhage and vasospasm, infusion cine,43 canine44 and feline45 vessels, possibly via of Ang IV returned blood flow to baseline levels.71 nitric oxide, although prostaglandins may be This effect was not inhibited by a nitric oxide syn- involved46–50 and Ang II(1–7) but not Ang II, thase inhibitor.71 Infusion of Ang IV into the rat inhibited [3H]thymidine incorporation in vascular renal artery increased renal cortical blood flow.57 smooth muscle cells.51 Although Ang II(1–7) stimu- Whilst these studies do lend support for a role of lated prostaglandin release in one study was blocked Ang IV in vasodilatation, others observed Ang IV 47 46 by CGP 42112A, in another study it was partially mediated vasoconstriction, probably via the AT1 blocked by Sar1, Ile8]Ang II, [Sar1, Thr8]Ang II and receptor.72–76 In addition to these vascular effects, Dup 753 but not by CGP 42112A. Also, Ang II(1– Ang IV increased the expression of plasminogen 7) induced vasodilatation and inhibition of vascular activator inhibitor-1 (PAI-1) mRNA in bovine aortic smooth muscle cell proliferation was inhibited by endothelial cells.77 1 8 [Sar , Thr ]Ang II but not by AT1 (L-158,809 and CV Ang IV also stimulated DNA synthesis in rat 78 11974) and AT2 (PD 123177/PD 123319) receptor anterior pituitary cells and stimulated DNA and antagonists.44,51 RNA synthesis in cultures of rabbit fibroblasts.79 The signalling mechanisms involved with the Ang Furthermore, the peptide synergistically enhanced II(1–7) actions are also different to those of Ang II basic fibroblast growth factor induced DNA syn- since the heptapeptide does not activate phospho- thesis in cultured bovine endothelial cells.62 Nle1- lipase C or release inositol phosphates in astro- Ang IV, an Ang IV agonist, decreased c-fos and egr cytes,47,52 or activate phospholipase D, produce 1 mRNA in the mechanically loaded, isolated rab- diacylglycerol or increase intracellular Ca2+ in vas- bit heart.80 Bioactive angiotensin peptides I Moeller et al 291 Only sparse evidence exists regarding second suggests that Ang II metabolites may also be of messengers associated with the AT4 receptor. In the physiological importance. Indeed, it appears that mouse neuroblastoma cell line, NG108-1581 and E18 Ang III may be important in the actions that have chicken myocytes,82 Ang IV did not alter intracellu- formerly been attributed to Ang II, especially in the lar Ca2+ levels although Ang IV did inhibit an Ang brain. Furthermore, Ang II(1–7) may play a role in II induced increase in protein synthesis and intra- modulating blood pressure via the central nervous cellular Ca2+ in E18 chicken myocytes.82 In contrast system and peripheral tissues, particularly in states to these studies, in rat vascular smooth muscle cells, where the RAS is markedly activated. In addition, Ang IV produced a small but sustained increase in receptors characterised by a high affinity for Ang IV, intracellular Ca2+ via extracellular influx, and an have a widespread distribution in central and peri- increase in inositol phosphates83 whilst in opossum pheral tissues, raising the possibility of important kidney cells, Ang IV stimulated a transient increase actions. Our finding of another peptide ligand, LVV- in intracellular levels of Ca2+ (via voltage-sensitive haemorphin-7, in the brain, suggests this receptor channels), independent of inositol-phosphates.84 may be part of a novel neuropeptide system, pos- Furthermore, in opossum kidney cells, Ang IV sibly involved in memory and neural development. neither produced an alteration in basal, or atrial As a result of these ongoing studies, the RAS is natriuretic factor stimulated guanylate cyclase emerging as a system with multiple effector peptides activity, nor basal or forskolin activated adenylate with diverse functions. cyclase levels.84 Whether Ang IV is the true ligand for the AT4 References receptor is doubtful since the localisation of the 1 Robertson JIS. Renin and angiotensin: a historical components of the RAS, in brain areas which review. In: Robertson JIS, Nicholls MG (eds). The include the circumventricular organs and the renin-angiotensin system. 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