Title: New Physiological Concepts of the Renin-Angiotensin System From the Investigation of Precursors and Products of Angiotensin I Metabolism
Abstract: HomeHypertensionVol. 55, No. 2New Physiological Concepts of the Renin-Angiotensin System From the Investigation of Precursors and Products of Angiotensin I Metabolism Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBNew Physiological Concepts of the Renin-Angiotensin System From the Investigation of Precursors and Products of Angiotensin I Metabolism Carlos M. Ferrario Carlos M. FerrarioCarlos M. Ferrario From the Hypertension and Vascular Disease Research Center, Wake Forest University School of Medicine, Winston Salem, N.C. Search for more papers by this author Originally published21 Dec 2009https://doi.org/10.1161/HYPERTENSIONAHA.109.145839Hypertension. 2010;55:445–452Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: December 21, 2009: Previous Version 1 Research on the regulatory actions of the renin-angiotensin system (RAS) continues to provide a wealth of information on how cells maintain their internal homeostatic environment, regulate metabolic processes, and adapt or contribute to disease. Not that long ago, the active product of the system, angiotensin II (Ang II), was considered the single critical hormone product of an endocrine system involved in regulating blood volume and vascular tone. A revised concept emerged after the demonstration that renin and angiotensinogen (Aogen) are present in tissues. These findings suggested that the RAS is composed of dual, independently regulated, blood-borne and tissue systems. Today, a broader and more complex system is being revealed by advanced genetic and molecular tools, as well as by the outcome of clinical studies using medications selective for ≥1 of the proteins contributing to the generation of angiotensin peptides.Recognition that the RAS contains both a pressor and depressor arm in exerting regulatory functions on vascular tone and cellular signaling paved the way for the generation of an alternate hypothesis as to how an imbalance of their function contributes to cardiovascular disease.1 This review summarizes the data supporting the hypothesis of a counterregulatory arm that, within the RAS, opposes the actions of Ang II. We build on these earlier discoveries to provide a new insight into an additional pathway in which an extended form of angiotensin I (Ang I), proangiotensin-12 (Ang-[1-12]), may be an alternate substrate for the production of the biological active angiotensins. A comprehensive evaluation of this topic cannot be achieved within the assigned space; therefore, only the key elements of the topic will be addressed, asking for indulgence in not providing a detailed listing of all of the published studies.Angiotensin-(1-7): The Paradigm ShiftThe period from approximately 1970 to 1980 represented the beginning of a shift in the concept of how the RAS was involved in cardiovascular pathology. Renewed enthusiasm for its study was stimulated by the demonstration that the administration of the angiotensin-converting enzyme (ACE) teprotide had a dramatic effect in reducing the blood pressure of hypertensive subjects.2 These results prompted many laboratories to undertake newer approaches to the investigation of the physiology of Ang II, isolate its receptor, and undertake the eventual synthesis of orally active Ang II receptor antagonists.3The progress made throughout these exciting discoveries nevertheless continued to posit Ang II as the biologically relevant product of the biochemical cascade that, initiated by renin, culminated in the production of Ang II. Alternate processes were assumed to have no major relevance in terms of biological function. The publication of the first description that the N-terminal derived heptapeptide, angiotensin-(1-7) (Ang-[1-7]), stimulated the release of vasopressin from rat hypothalamic explants4 would, over time, decisively alter the former view.Although initial studies found that Ang-(1-7) acted as a vasodilator,5 further research showed that this effect could be best demonstrated in isolated vessels,6,7 in animals in which the baroreceptors are eliminated,5 or in conditions in which endogenous levels of Ang II are increased by maneuvers such as sodium depletion8,9 or renovascular hypertension.10 These findings underscored the concept that Ang-(1-7) acts as a paracrine hormone when formed in close proximity to the vascular smooth muscle or that its actions depend on a change in the signaling effector mechanisms associated with increased expression or activity of Ang II type 1 (AT1) receptors. This is not an unreasonable possibility, because it has been documented that the antihypertensive action of ACE inhibitors and Ang II receptor antagonists is amplified by concomitant use of thiazide diuretics.Over the following decades, research would demonstrate that Ang-(1-7) contributes to the cardiorenal control of blood pressure via actions that, within the heart, kidney, and the blood vessels, opposed the activity of Ang II.11–13 Ang-(1-7) was shown to reverse the hypertrophic and profibrotic effects of Ang II in the heart and the vasculature,14–17 oppose Ang II–mediated cardiac arrhythmogenic activity,18 possess antiatherogenic and antithrombotic actions,19–24 inhibit oxidative stress and the generation of radical oxygen species,25,26 and modulate hematopoietic function.27,28Identification of the mas receptor as the conveyor for the cellular signaling responsible for Ang-(1-7) actions29 and the demonstration that genetic deletion of this receptor abrogates the protective actions of the heptapeptide30–36 have affirmed its participation in the regulation of cardiovascular function. Second messenger mechanisms responsible for the cellular response mediated by the binding of Ang-(1-7) to the mas receptor include inhibition of the mitogen activated protein kinase kinase pathway, stimulation of cellular phosphatases, inhibition of cyclooxygenase 2, and facilitation of NO release.28,37–44ACE2 and Ang-(1-7)The pace of research on the counterlever role of Ang-(1-7) on Ang II expanded with the identification of an ACE homologue, ACE2, that cleaved Ang II into Ang-(1-7).45,46 As reviewed elsewhere,47–50 ACE2 differs from ACE in acting as a monocarboxypeptidase to cleave the Pro7-Phe8 bond of Ang II to form Ang-(1-7) and has not been inhibitable by exposure to ACE inhibitors. A stepping stone in the evolving understanding of the role of ACE2 in cardiac function followed the demonstration that deletion of the ACE2 gene is accompanied by severe cardiac contractility defects and that ACE2 mRNA protein and expression were reduced in 3 different models of experimental hypertension.51 Selective overexpression of cardiac ACE2 in rats is associated with reversal of cardiac hypertrophy52–54 and progression of atherosclerosis,55 whereas chemical inhibition of ACE2 worsened the progression of renovascular hypertension.56 Although ACE2 has been shown to convert Ang II into Ang-(1-7) in both animals57 and humans,58,59 further work is necessary to affirm whether the predominant effects of ACE2 inhibition are primarily attributed to prevention of Ang II metabolism. Nevertheless, this hypothesis is in keeping with the findings of reduced ACE2 expression or activity in experimental models of hypertension,51,56,60–64 human prehypertension,65 heart failure,65–68 renal disease, and type 2 diabetes mellitus.69–72 After the first demonstration that blockade of AT1 receptors in rats with myocardial infarction was associated with upregulation of cardiac ACE2 mRNA,73 newer studies suggest that ACE2 gene transcription is negatively regulated by Ang II, whereas Ang-(1-7) counteracts the inhibitory effect of Ang II on ACE2 gene expression.74–76Altogether, the proposal that Ang-(1-7) opposes the actions of Ang II became the underpinning for the recognition that the RAS is biochemically constituted by alternate enzymatic pathways leading to the generation of separate peptides acting at receptors, of which the AT1 receptor subtype is only a part of the system. In accepting a more complex view of the system and its function in the regulation of blood pressure and vascular structure, the concept that the arm of the RAS composed of the ACE2/Ang-(1-7)/mas receptor axis counterbalances the activity of the other arm (ACE/Ang II/AT1 receptor axis) has gained acceptance. The new knowledge is stimulating further research into its possible role as, at the very least, a permissive contributor of the cardiorenal remodeling accompanying the pathogenesis of hypertensive vascular disease and target-organ damage.Ang-(1-12)As the science dissecting the contribution of the RAS to cellular function continues to evolve, a study from Japan in 2006 challenged the idea that renin-dependent hydrolysis of Aogen constitutes the rate-limiting step for the formation of angiotensin peptides.77 Their observations opened a new window toward exploring how angiotensin peptides may be formed within the interior milieu of cells or their immediate surrounding environment. The isolation of a new Aogen-derived peptide by Nagata et al77 from the rat’s small intestine contains a shorter form of the synthetic tetradecapeptide synthesized by Skeggs et al78 in 1958. The Aogen-derived substrate was termed “proangiotensin-12” by them on the basis of its role as an Ang II precursor. In following the terminology approved by the Nomenclature Committee of the Council for High Blood Pressure Research in 1991,79 we use the term “angiotensin-(1-12)” (Ang-[1-12]) throughout this review, because it best follows the appropriate convention in defining the amino acid sequence of the polypeptide within the family of angiotensins (Table). Critically important, their study showed the endogenous presence of the peptide in Wistar rats and its ability to serve as a substrate for the in vitro and in vivo generation of Ang II, a finding that strongly differentiates Ang-(1-12) from the tetradecapeptide isolated previously. As illustrated in Figure 1, Ang-(1-12) levels in the small intestine, liver, lungs, adrenal gland, heart, brain, and pancreas are higher than corresponding concentrations of Ang I. The ability of Ang-(1-12) to act as an endogenous substrate for Ang II production followed the observation that Ang-(1-12) vasoconstrictor effects were prevented by previous blockade with either captopril or the AT1 receptor antagonist candesartan in both the isolated aorta and the systemic circulation.77 Their findings attracted the interest of our laboratory because this was the first time that an extended form of Ang I had been shown to exist endogenously. Table. Amino Acid Sequences of Main Angiotensin PeptidesPeptides1234567891011121314Ang indicates angiotensin.Ang-(1-14) NH2AspArgValTyrIleHisProPheHisLeuLeuValTyrSerCOOHHuman Ang-(1-12) NH2AspArgValTyrIleHisProPheHisLeuLeuValIleCOOHRat Ang-(1-12) NH2AspArgValTyrIleHisProPheHisLeuLeuTyrTyrCOOHOvine Ang-(1-12) NH2AspArgValTyrIleHisProPheHisLeuLeuValHisCOOHCanine Ang-(1-12) NH2AspArgValTyrIleHisProPheHisLeuLeuValTyrCOOHEquine Ang-(1-12) NH2AspArgValTyrIleHisProPheHisLeuLeuValTyrCOOHAng-(1-10) NH2AspArgValTyrIleHisProPheHisLeuCOOHAng-(1-8) NH2AspArgValTyrIleHisProPheCOOHAng-(1-7) NH2AspArgValTyrIleHisProCOOHAng-(2-8) NH2ArgValTyrIleHisProPheCOOHAng-(3-8) NH2ValTyrIleHisProPheCOOHDownload figureDownload PowerPointFigure 1. Data (mean±SEM), redrawn from Nagata et al,77 illustrate the relative concentrations of Ang-(1-12), Ang I, and Ang II in tissues and plasma from the Japanese-derived strain of normotensive Wistar rats.Intrigued by the potential importance of Ang-(1-12) as an alternate substrate for the formation of angiotensin peptides, we explored the location of Ang-(1-12) in cardiac and renal tissues of both Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). Polyclonal antibodies targeting the specific sequence of rat Ang-(1-12) revealed intense and selective staining of cardiac myocytes and renal tubular cells of both strains (Figure 2). The patchy staining observed in cardiac myocytes of the left ventricle of WKY rats was markedly altered in the SHR, because almost all of the cardiac tissue displayed intense staining.80 To more precisely determine whether the visual display of increased Ang-(1-12) immunoreactive staining reflected a greater endogenous expression of the peptide, we measured Ang-(1-12) tissue concentrations by radioimmunoassay. These experiments showed that the endogenous content of Ang-(1-12) in the cardiac tissue of SHRs was 47% higher than those found in WKY rats.80 Rat kidney also displayed selective expression of Ang-(1-12) in the proximal, distal, and collecting renal tubules within the deep cortical and outer medullary zones in both strains (Figure 2); however, Ang-(1-12) concentrations in renal tissue were slightly reduced in SHRs.80Download figureDownload PowerPointFigure 2. Representative micrographs of Ang-(1-12) immunostaining from cardiac (A) and renal (B) tissue of WKY rats and SHRs (top images in each panel). In A, faint immunostaining for Ang-(1-12) is observed in the myocardium of WKY rats, whereas intense punctuate staining is expressed in left ventricular myocytes of SHRs. B illustrates the expression of Ang-(1-12) immunostaining in sections of kidney cortex from WKY rats and SHRs. Fluorescent staining for Ang-(1-12) found in renal tubules is increased in sections obtained from SHRs. Nuclei are indicated by 4′,6-diamidino-2-phenylindole blue fluorescence staining. Bottom images in each panel illustrate merged confocal and phase-contrast immunostaining for each organ. Data are from studies reported in our reference.80Differential tissue expression of Ang-(1-12) led to a second study focusing on whether renin accounted for the cleavage of Ang-(1-12) into Ang II, Ang-(1-7), or both in isolated perfused hearts from 3 normotensive and 2 hypertensive rat strains.81 As illustrated in Figure 3, Ang-(1-12) caused the rapid appearance of both Ang I and Ang II in the perfusate of WKY rats and SHRs, with highest levels occurring between 30 and 60 minutes of recirculation (Figure 3). Although renin was present both in the cardiac tissue and in the effluent of isolated perfused WKY and SHR hearts (Figure 3), the addition of a selective rat renin inhibitor (WFML-1) did not prevent conversion of Ang-(1-12) into angiotensin peptides.81 These data, thus, excluded renin from acting on Ang-(1-12). Recent studies in anephric rats are in agreement with this interpretation.82 In the anephric state, the loss of renal-derived renin resulted in the expected fall in the circulating concentrations of Ang I and Ang II to barely detectable levels, whereas it had only a small reducing effect on plasma Ang-(1-12) levels (Figure 4). In contrast, parallel measures of the cardiac content of angiotensin peptides documented a 91% increase in cardiac Ang-(1-12) tissue concentrations 48 hours after bilateral nephrectomy.82 Furthermore, absence of renin transcripts in cardiac tissue of both sham and nephrectomized rats suggests that both the processing of Aogen into Ang-(1-12) and its secondary conversion to Ang I occurs via a nonrenin pathway. Therefore, the biochemical processes associated with the expression and cleavage of Ang-(1-12) through a nonrenin pathway suggest an additional level of complexity within the enzymatic pathways accounting for the expression of biologically active peptides. Download figureDownload PowerPointFigure 3. A to C are peak concentrations of angiotensin peptides resulting from the addition of 10 nmol of Ang-(1-12) to the perfusate of isolated hearts from WKY and SHR adult rats. D illustrated renin concentration in the effluent of isolated perfused hearts from WKY rats and SHRs. Before shows the peak angiotensin values obtained 60 minutes after administration of Ang-(1-12); after shows the absence of a change in the peak values of Ang-(1-12) after administration of 1 μmol/L of the rat-specific renin inhibitor WFML-1. Data (mean±SEM) are redrawn from our reference.81Download figureDownload PowerPointFigure 4. Plasma and cardiac tissue concentrations of angiotensin peptides in sham and 48-hour bilateral nephrectomized (BNX) rats reveal differential expression of Ang-(1-12), Ang I, and Ang II in adult WKY rats. Data are redrawn from our reference.82Insights into the enzymatic pathway(s) responsible for the conversion of Ang-(1-12) into Ang II have been expanded recently by studies from Prosser et al83 in the isolated heart of Sprague-Dawley rats. In this preparation, ex vivo and in vivo conversions of Ang-(1-12) into Ang II were prevented by the administration of chymostatin using combined high-performance liquid chromatography and tandem mass spectrometry analysis.83 Urata et al84 first implicated a functional role for chymase, a member of the serine protease family, as a tissue enzyme involved in the conversion of Ang I into Ang II. The location of chymase in secretory granules and its ubiquitous existence in mast, vascular endothelial, and mesenchymal cells provide a mechanism for the intracellular formation of angiotensin peptides and their functioning as intracrine and paracrine regulators.85 A potential limitation in terms of ascribing a primary role for chymase in the metabolism of Ang-(1-12) is the previous report by Nagata et al,77 who found that captopril abolished the constrictor response of aortic strips to Ang-(1-12) exposure and prevented the pressor effect of intravenous Ang-(1-12). Differences in the findings between both studies may have been influenced by methodology used and the use by Prosser et al83 of human recombinant chymase. In this connection, rat, but not human, chymase cleaves the Tyr4-Ile5 bond of Ang II.86 In addition, tissue damage and edema associated with saline perfusion of the preparation may have increased cell permeability, exposing the peptide to intracellular chymase.83The tantalizing evidence for the existence of an alternate renin-independent substrate for extracellular or intracellular processing of angiotensin peptides awaits further studies as to whether its biological activity is expressed through the formation of Ang II or may act independent of its processing into the known biological peptides of the RAS. Additional studies should be undertaken to unravel the potential importance of Ang-(1-12) as an alternate substrate for the formation of angiotensin peptides, as well as to determine the enzyme(s) accounting for the cleavage of Ang-(1-12) from Aogen and its subsequent conversion into Ang II, Ang-(1-7), or both. As research delves further into the potential role of Ang-(1-12) as a source of tissues angiotensin formation, the question of whether increased formation of Ang-(1-12) may contribute to pathology comes to the forefront. A preliminary answer to this question may be derived from the demonstration of increased expression and cardiac content of Ang-(1-12) in the myocytes of SHRs80 and increased expression of cardiac Ang-(1-12) and Ang II in both anephric rats82 and in those in which hypertension was abated by administration of a mineralocorticoid receptor antagonist.87 In addition, the recent demonstration that endogenous neutralization of Ang-(1-12) via infusion of a selective Ang-(1-12) antibody into the cerebrospinal fluid of transgenic hypertensive rats lowers blood pressure provides further evidence for its role as a precursor for the formation of Ang II.88SummaryEven from this limited overview of the intricate internal mechanisms regulating the pathways determining the production of angiotensin peptides, it is obvious that the RAS is embodied with a great capacity to use alternate mechanisms in bypassing the blockade of primary pathways. In unraveling the complexity of the biochemical physiology of the system, it is also apparent that formation of angiotensin peptides within the cellular environment may not follow what has been characterized in the circulation or even the extracellular compartment.A new level of regulatory complexity is added with the demonstration of Ang-(1-12) as an alternate substrate contributing to forming angiotensin peptides by a nonrenin-dependent mechanism. The tissue formation and processing of the biologically active products of the RAS may follow those outlined in Figure 5, whereby synthesis of Ang II and Ang-(1-7) may be determined by the availability of Ang-(1-12) formed through intracellular processing of Aogen, uptake from the extracellular compartment, or both. Although there are still many fundamental gaps in uncovering the conditions and processes that determine the enzyme(s) accounting for either the cleavage of Ang-(1-12) from Aogen or the processing of Ang-(1-12) to Ang II and Ang-(1-7), the fact remains that this substrate occurs endogenously, is altered in a genetic model of hypertension, and can clearly produce angiotensin peptides by a nonrenin-dependent mechanism. Because the ubiquitous functions of tissue RAS extends outside of the cardiovascular system, the detection of high concentrations of Ang-(1-12) in the rat gut needs to be explored, because Ang II contributes to jejunal motility and fluid transport.89,90 Indeed, the finding that high doses of the direct renin inhibitor aliskiren are associated in humans with diarrhea91,92 posits the question as to whether this adverse effect of the drug might be related to increases in the gut content of Ang-(1-12). Download figureDownload PowerPointFigure 5. Diagram of the biochemical pathways leading to the formation of angiotensin peptides in tissues. NEP indicates neutral endopeptidase 24.11 and related endopeptidases contributing to the formation of Ang-(1-7) from Ang I.12PerspectivesUnderstanding the structure and the dynamics of the complex intercellular web of interactions that contribute to the function of a living cell requires acceptance that a discrete biological action can rarely be attributed to an individual molecule. The intertwined relationship between the ACE/Ang II/AT1 receptor axis and its counterlever ACE2/Ang-(1-7)/mas receptor axis is now buttressed on firm literature. A further investigation of the potential clinical impact of pursuing the hypothesis of whether genetic or acquired deficiency in ≥1 of the components of the Ang-(1-7)/ACE2/mas-R axis may explain the progression of hypertensive vascular disease ought to be pursued with greater vigor, because research in this field is extending knowledge of disease processes both within and outside the cardiovascular system.Sources of FundingThis work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health (grant PO1 HL051952).DisclosuresNone.FootnotesCorrespondence to Carlos M. Ferrario, Hypertension and Vascular Disease Research Center, Wake Forest University School of Medicine, Winston Salem, NC 27157. E-mail [email protected] References 1 Ferrario CM, Chappell MC, Tallant EA, Brosnihan KB, Diz DI. Counterregulatory actions of angiotensin-(1-7). 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