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Cardiovascular Research 1999 44(2):252-265; doi:10.1016/S0008-6363(99)00202-3
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Is there a local renin—angiotensin system in the heart?

A.H.Jan Dansera,*, Jasper J Sarisa,b, Martin P Schuijta,b and Jorge P van Katsb

aCardiovascular Research Institute COEUR, Department of Pharmacology, Erasmus University Rotterdam, 3015 GE Rotterdam, The Netherlands
bCardiovascular Research Institute COEUR, Department of Internal Medicine I, Erasmus University Rotterdam, 3015 GE Rotterdam, The Netherlands

* Corresponding author. Tel.: +31-10-408-7540; fax: +31-10-408-9458 danser{at}farma.fgg.eur.nl

Received 29 March 1999; accepted 10 June 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Studies in cardiac...
 3 Studies in isolated...
 4 Studies in intact...
 5 Cardiac renin-,...
 6 Site of tissue...
 7 Mannose 6-phosphate receptors...
 8 Role of alternative...
 9 Regulation of cardiac...
 References
 
The existence of a local renin—angiotensin system in the heart is still a controversial issue. This review discusses the evidence, obtained from studies in cardiac cells, in isolated perfused hearts and in intact animals and humans, both under normal and pathological conditions, for local production of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin I and angiotensin II at cardiac tissue sites. In addition, the role of alternative angiotensin-generating enzymes (cathepsin, chymase) and the possibility of (pro)renin uptake from the circulation is evaluated.

KEYWORDS Angiotensin; ACE inhibitors; Interstitial space; Myocytes; Renin angiotensin system


   1 Introduction
Top
 Abstract
 1 Introduction
2 Studies in cardiac...
 3 Studies in isolated...
 4 Studies in intact...
 5 Cardiac renin-,...
 6 Site of tissue...
 7 Mannose 6-phosphate receptors...
 8 Role of alternative...
 9 Regulation of cardiac...
 References
 
The renin—angiotensin system (RAS) has traditionally been viewed as a circulating system. Kidney-derived renin cleaves liver-derived angiotensinogen to form angiotensin (Ang) I in circulating blood. Subsequently, Ang I is converted into Ang II, the main effector peptide of the RAS, by angiotensin-converting enzyme (ACE) located at the luminal side of the endothelium. Ang II exerts its effects via stimulation of Ang II receptors, of which at least two types have been described, AT1 and AT2. Based upon discrepancies, observed more than 15 years ago, between RAS blocker-induced changes in the circulating levels of RAS components and the blood pressure-lowering effects of these drugs, it was proposed that so-called local renin—angiotensin systems exist in addition to the circulating RAS. Subsequent studies showing the presence of RAS components at tissue sites appeared to confirm this theory. However, one has to keep in mind that the mere presence of RAS components in tissue cannot be taken as direct evidence for their local production. With regard to the heart, more convincing evidence for local Ang II production comes from the recently discovered beneficial effects of the ACE inhibitors in heart failure, which are independent, at least partly, of their effect on blood pressure [1–4]. Local Ang II production may depend on (1) in-situ synthesis of all RAS components required for Ang II production (i.e., renin, angiotensinogen and ACE), (2) uptake of these components from the circulation, or (3) a combination in-situ synthesis and uptake of RAS components (e.g., uptake of circulating renin and angiotensinogen in combination with local synthesis of ACE). The latter two definitions would no longer require in-situ synthesis of renin, so that one should better speak of a system generating Ang II locally rather than a local RAS. This review addresses the question whether a local RAS exists in the heart.


   2 Studies in cardiac cells
Top
 Abstract
 1 Introduction
 2 Studies in cardiac...
3 Studies in isolated...
 4 Studies in intact...
 5 Cardiac renin-,...
 6 Site of tissue...
 7 Mannose 6-phosphate receptors...
 8 Role of alternative...
 9 Regulation of cardiac...
 References
 
The uncertainties concerning local synthesis arising from tissue measurements (i.e., local synthesis vs. uptake from circulating blood) can be avoided largely when measurements are made in cells cultured in the absence of serum. The use of serum-free medium is necessary to exclude the uptake of RAS components present in serum. However, serum-free conditions are always preceded by cell-culturing conditions in the presence of serum, so that even under ‘serum-free’ conditions RAS components may be detected in cells that have been sequestered from the serum to which the cells were exposed earlier. Table 1 summarizes the findings in cardiac cells that are discussed below.


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  		Table 1 Presence of renin, angiotensinogen and ACE (mRNA and/or protein) in serum-deprived cardiac myocytes and fibroblasts vs. their release into or uptake from the mediuma

 
2.1 Renin
Several attempts have been made to measure renin, both at the mRNA and protein level, in myocytes and fibroblasts obtained from hearts of neonatal and adult rats. Some [5–7], but not all [8] authors were able to detect renin mRNA, using sensitive PCRs. Renin immunoreactivity was present in the perinuclear region in neonatal rat cardiomyocytes and fibroblasts [5], or throughout the cytoplasm in adult rat cardiomyocytes and non-myocytes [6]. In these latter two studies, no distinction between renin and its inactive precursor, prorenin, was made, nor was (pro)renin release into the culture medium investigated. This is remarkable, since most so-called renin-expressing extrarenal cells produce prorenin rather than renin [9–12]. These cells do not store prorenin, and secrete it in a constitutive manner. Studies with cardiac cells where renin and prorenin (the latter after in-vitro activation to renin) were measured enzyme-kinetically, were unable to support either the release of (pro)renin into the medium [13] or the intracellular presence of (pro)renin [13,14]. The lack of (pro)renin release in combination with the low to undetectable renin mRNA levels in cardiac cells does not support the concept of (pro)renin synthesis by these cells. The discrepancy between the absence of renin-dependent Ang I-generating activity in cardiac cells on the one hand, and the presence of immunoreactive renin in these cells on the other hand can be explained as follows: 1) the polyclonal antibodies used to demonstrate intracellular renin immunohistochemically [5,6] may have crossreacted with renin-like enzymes such as cathepsin D [15], or 2) the cells may indeed have contained renin, not due to local synthesis but due to uptake from the serum to which the cells were exposed prior to the serum-free period. In support of the latter explanation we have recently demonstrated that neonatal rat cardiac cells are capable of internalizing both renin and prorenin [16]. Internalized prorenin is rapidly activated to renin. The amount of serum to which the cells were exposed, the serum source, the duration of exposure to serum, and the intracellular half life of internalized (pro)renin may have differed between studies, and this might explain why under certain conditions renin still is present within cells, whereas in others it is not.

2.2 Angiotensinogen
Studies on angiotensinogen synthesis by cardiac cells are scarce. Angiotensinogen mRNA has been demonstrated in both neonatal and adult rat cardiac cells by several investigators [5–8,17–20]. However, the actual presence of the angiotensinogen protein has been investigated in two studies only. Dostal et al. described positive immunoreactive staining in the perinuclear region for angiotensinogen in both neonatal rat cardiomyocytes and fibroblasts, without investigating its release into the medium [5]. Van Kesteren et al., who measured angiotensinogen by radioimmunoassay after its conversion to Ang I by renin, was unable to detect angiotensinogen in neonatal rat cardiac cells or in the conditioned medium of these cells [13]. All other cells described to synthesize angiotensinogen secrete this substrate constitutively, without storing it intracellularly [21–23]. In vivo, angiotensinogen is also limited to the extracellular fluid compartment, and not located in cells [14,24,25]. Thus, despite the reports demonstrating angiotensinogen mRNA in cardiac cells with the help of sensitive PCRs, evidence for the release of angiotensinogen from these cells is lacking.

2.3 ACE
ACE, a cell membrane-bound enzyme, has been demonstrated in cardiac cells by enzyme-kinetic and immunohistochemical methods [13,26–29]. Both neonatal rat cardiomyocytes and fibroblasts generate Ang II when incubated with Ang I under serum-free conditions, and ACE inhibitors fully inhibit this Ang II generation [13,29]. The presence of ACE protein or activity in cardiac cells correlates well with the demonstration of ACE mRNA in these cells [5–8].

2.4 Angiotensin I and II
According to several studies, serum-deprived cardiac cells release angiotensins into the culture medium. The Ang I and II levels in the medium, however, showed huge variations, from <10 fmol/ml to >1000 fmol/ml [13,17,26,28,30]. Part of these discrepancies may be due to the fact that angiotensins were sometimes measured by direct radioimmunoassays (i.e., without prior purification and/or separation from material crossreacting with the Ang I and II antibodies applied in these assays). This approach will lead to an overestimation of the "true" angiotensin levels, or even to the detection of angiotensins in medium that does not contain angiotensins [31]. It should also be kept in mind that, in view of the cardiac angiotensin levels measured in vivo (Ang I, {approx}5 fmol/g wet weight; Ang II, {approx}20 fmol/g wet weight) [24,32–34], even levels of 5–10 fmol/ml are very high, since in most studies medium was collected from only 1–10 million cells, with an estimated wet weight of less than 10 mg [35]. Sadoshima et al. [17] found the Ang II concentration in the medium of serum-deprived cardiomyocytes to increase nearly 100-fold upon stretch (Table 2). This Ang II, which is assumed to be responsible for the hypertrophic [8,17–19,28,30] or apoptotic [20] response of cardiomyocytes after stretch, appeared to originate from intracellular storage sites, since its release was not affected by captopril and not accompanied by Ang I release [17]. Immunoelectron microscopy confirmed the existence of secretory granule-like structures containing Ang II in ventricular cardiomyocytes [17]. Dostal et al. [26] did not observe these granule-like structures and localized intracellular Ang II in the perinuclear region of neonatal rat cardiomyocytes and fibroblasts. Stretch is assumed to cause an upregulation of RAS components in cardiomyocytes, and this would explain why the Ang II levels in the medium are also elevated 20–24 h after the initiation of stretch [20]. However, the reports on elevated renin – and ACE mRNA levels were not supported by protein measurements [17,28], suggesting that increases in expression may not be translated to the protein level. In addition, not all authors were able to observe a rise in Ang II following stretch [13,30]. Taken together therefore, the initial report by Sadoshima et al. on Ang II release after stretch has not been unequivocally confirmed by others. It is possible that differences in experimental conditions have played a role (Table 2). Furthermore, the Ang II in intracellular storage sites may have been derived, via AT1 receptor-mediated endocytosis [36], from the serum-containing medium used to culture the cells prior to stretch.


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  		Table 2 Effect of stretch on renin—angiotensin system components in cardiomyocytesa

 

   3 Studies in isolated perfused hearts
Top
 Abstract
 1 Introduction
 2 Studies in cardiac...
 3 Studies in isolated...
4 Studies in intact...
 5 Cardiac renin-,...
 6 Site of tissue...
 7 Mannose 6-phosphate receptors...
 8 Role of alternative...
 9 Regulation of cardiac...
 References
 
The isolated perfused heart has been used widely to study the effects of RAS blockers on coronary flow and cardiac function. In these studies, it is generally assumed that all RAS components are present in cardiac tissue and that Ang II is generated continuously. However, evidence to proof this notion is currently lacking. Studies investigating the presence of RAS components in the isolated perfused heart are scarce, despite the fact that an isolated perfused preparation is ideal to study local synthesis. The buffers used to perfuse the heart are free of renin and angiotensinogen, thereby eliminating the problems arising from in-vivo measurements, when the heart is perfused with blood containing these components.

3.1 Renin
Renin or prorenin release by the heart has never been demonstrated. Angiotensin release from isolated perfused hearts occurred only after the addition of renin to the perfusion fluid [37–40], thereby demonstrating that (1) renin is not present in the isolated buffer-perfused heart, and (2) renin is the only enzyme involved in cardiac Ang I generation. The kinetics of renin uptake into the heart have been studied extensively by de Lannoy et al., using a modified version of the rat Langendorff heart, allowing separate collection of both coronary effluent and interstitial fluid [38]. Renin could not be demonstrated in either the coronary effluent or the interstitial fluid of buffer-perfused hearts. When renin was added to the perfusion buffer, it slowly entered the interstitial space, reaching steady-state levels in interstitial fluid comparable to those in coronary effluent after approximately 30 min. Calculations on the basis of the steady-state renin levels in coronary effluent, interstitial fluid and cardiac tissue, revealed that the majority of cardiac renin was present in extracellular fluid. After stopping the renin infusion, the washout of renin from the heart followed a biphasic pattern, suggesting that renin may also be present in an additional compartment. In support of this finding, Müller et al. observed Ang II release from isolated hearts of rats overexpressing the human angiotensinogen gene even after discontinuation of renin infusion, at a time when renin had already disappeared from the coronary perfusate [40]. It is therefore possible that some renin is located outside the extracellular fluid compartment, for instance bound to the membrane of vascular or cardiac cells. This notion is in agreement with the recent observation that both endothelial cells [41] and cardiac cells [16] are capable of binding renin and prorenin. Prorenin kinetics in the isolated perfused heart were comparable to those of renin; release of activated prorenin into either the coronary effluent or interstitial fluid could not be demonstrated [42].

3.2 Angiotensinogen
Angiotensinogen release from isolated buffer-perfused rat Langendorff hearts has been investigated in two studies. Lindpaintner et al. [37] observed a rapid decline of the angiotensinogen levels in coronary effluent to levels that were <1% of the levels in blood plasma. De Lannoy et al. [38] were unable to demonstrate angiotensinogen in coronary effluent, but found low angiotensinogen levels (corresponding to <0.1% of the plasma levels of angiotensinogen) in interstitial fluid, which decreased even further (to levels below the detection limit) during prolonged buffer perfusion of the heart. The most likely explanation for these findings is that some blood-derived angiotensinogen is still present in the isolated heart preparation (for instance in the interstitial space), which is slowly washed away during perfusion with buffer. In support of this assumption, angiotensinogen, when added to the perfusion buffer of the isolated rat heart, entered the interstitial space [38]. Steady-state levels comparable to those in coronary effluent were reached 30–40 min after the start of the angiotensinogen perfusion. The steady-state tissue levels of angiotensinogen were also compatible with its presence in extracellular fluid. Following discontinuation of the angiotensinogen perfusion, angiotensinogen disappeared monophasically from cardiac extracellular fluid. This outcome contrasts with the findings on renin, which disappeared in a biphasic manner, and suggests that angiotensinogen is limited to one compartment (the extracellular fluid) only. Indeed, binding of angiotensinogen to cardiac or vascular membranes could not be demonstrated [24,43].

3.3 ACE
Many investigators have shown that Ang I is converted to Ang II in the isolated perfused rat heart [39,40,44–46]. ACE inhibitors prevented the Ang I–II conversion completely. Thus, there is no doubt that ACE is present and functionally active in the isolated perfused heart. The conversion of arterially delivered Ang I was usually low (<10%) and appeared to depend on both endothelial and extra-endothelial ACE [46].

3.4 Angiotensin I and II
Buffer-perfused rat hearts do not release Ang I or Ang II, unless renin is added to the perfusion buffer [37–39]. Renin-induced angiotensin release diminishes rapidly, suggesting that it depends on a limited amount of trapped plasma-derived angiotensinogen [37,38]. Cardiac angiotensin release during renin infusion can only be maintained over longer time periods by adding angiotensinogen simultaneously to the perfusion buffer [38,39], or by using hearts of rats overexpressing the angiotensinogen gene [40]. Interestingly, angiotensin release via coronary effluent reached a steady state after 30–40 min of combined renin and angiotensinogen perfusion, whereas renin and angiotensinogen in coronary effluent had reached a steady-state level within 5 min after the start of their infusion into the perfusion buffer. This finding, in combination with the fact that angiotensin release continues after discontinuation of the renin perfusion, at a time when renin is no longer present in coronary effluent [40], strongly suggests that cardiac angiotensin release depends largely on tissue-bound renin. The most likely binding site for renin involved in the release of Ang I into the intravascular compartment is the vascular wall. Angiotensin production also occurred in cardiac interstitial fluid, and under steady-state conditions, the interstitial fluid Ang I and II levels were 2–3 times higher than those in coronary effluent [38,39]. In view of the low interstitial fluid flow (50–100 times lower than the coronary flow) and the extensive metabolism of angiotensin in the vascular wall, it is unlikely that interstitial angiotensin production contributed importantly to the angiotensin levels in the coronary effluent. Finally, Ang II was present in cardiac tissue during combined renin and angiotensinogen perfusion, at levels higher than those expected on the basis of the Ang II levels in extracellular fluid [39]. This was not the case for Ang I; per gram tissue the amount of Ang I was as high as expected on the basis of the assumption that Ang I is present only in extracellular fluid. Thus, cardiac Ang I is largely confined to the coronary vascular bed and the interstitial fluid compartment, whereas cardiac Ang II is also located outside these compartments. Tissue Ang II may therefore be present in cells, either because it is synthesized intracellularly, or because, following its synthesis outside the cell, it is rapidly internalized via AT1 receptors [36].


   4 Studies in intact animals and humans
Top
 Abstract
 1 Introduction
 2 Studies in cardiac...
 3 Studies in isolated...
 4 Studies in intact...
5 Cardiac renin-,...
 6 Site of tissue...
 7 Mannose 6-phosphate receptors...
 8 Role of alternative...
 9 Regulation of cardiac...
 References
 
Results from in-vivo studies on the tissue levels of RAS components are difficult to interpret because these levels might be partly or wholly contributed to the presence of blood in tissues. Even when the levels are considerably higher per gram of tissue than per millilitre of plasma, one should keep in mind that an active uptake process (e.g., receptor binding) rather than local synthesis may underlie these high levels.

4.1 Renin
Renin mRNA concentrations in normal hearts are close to or below the detection limit [47–50], suggesting that under normal circumstances cardiac renin synthesis may not occur. We compared the renin levels in the heart with its level in blood plasma in normal and nephrectomized pigs [24]. Ang I-generating activity of cardiac tissue was identified as renin by its inhibition with a specific active site-directed renin inhibitor. The levels of renin in cardiac tissue (expressed per gram wet weight) were similar to those in blood plasma (expressed per ml plasma) and could therefore not be attributed to trapped blood plasma. However, both in cardiac tissue and in plasma renin fell to undetectable levels after nephrectomy. These data, which were confirmed in the rat heart by Katz et al. [25], suggest that most, if not all, renin present in the normal heart originates from the kidney. Apparently, the heart is capable of sequestrating renin from the circulation. Renin may either diffuse into the interstitial space [38] or bind to the recently described renin receptor(s) and/or renin binding proteins [16,41,43,51–53]. In support of the latter, we [24] and others [43] found renin to be enriched in a purified membrane fraction prepared from either left ventricular tissue or mesenteric arteries. Such enrichment in cardiac and vascular tissue is in agreement with the existence of a renin receptor. It is currently not known what cardiac cells are responsible for the binding of renin. Based on studies in isolated cells [16,41], endothelial cells as well as cardiomyocytes and fibroblasts might be involved in the uptake process. Interestingly, these cells were not only capable of binding renin, but bound prorenin as well. Moreover, following binding, renin and prorenin were internalized, and prorenin was activated to renin. These findings may explain why in the normal heart virtually no prorenin can be detected [24,53]: prorenin taken up by the heart from the circulation may have been activated locally to renin. The receptor involved in the binding and internalization process of (pro)renin and prorenin activation appeared to be the mannose 6-phosphate receptor [16,41]. Renin acquires phosphomannosyl residues during its biosynthesis that enable it to bind to this receptor [54]. Katz et al. [25] found that, following bilateral nephrectomy, high-mannose renin glycoforms disappeared from the heart at a much slower rate than from plasma, thereby indirectly confirming that (phosphorylated) oligosaccharide attachments to renin determine its binding to cardiac tissue.

4.2 Angiotensinogen
Angiotensinogen mRNA can be detected in the heart [49,55–57], at levels that are <0.1% of the angiotensinogen mRNA levels in the liver [57]. The angiotensinogen concentrations in porcine cardiac tissue are 10–25% of the levels in plasma, a figure compatible with the diffusion of angiotensinogen from plasma into the interstitium [24]. In hearts of humans and rats, the angiotensinogen levels, measured by enzyme-kinetic assay, were lower than expected on the basis of diffusional equilibrium between plasma and tissue [25,53], either because these hearts were washed with buffer after their removal from the body [25], or because significant consumption of angiotensinogen had occurred at cardiac tissue sites [53]. In support of the latter possibility, a negative correlation was found between angiotensinogen and renin in human hearts [53]. Despite the low levels of angiotensinogen that could be demonstrated in the human heart by enzyme-kinetic assay [53], Sawa et al. [58] found intense immunoreactivity for angiotensinogen in the atrial muscles, the muscles of the conduction system and those of the subendocardial layers of human autopsy hearts. Remarkably however, despite the intense immunoreactivity in myocardial cells, Sawa and colleagues were unable to show positive immunostaining of angiotensinogen in the liver. Taken together therefore, although the data for angiotensinogen are less clear than those for renin, it appears that the majority of cardiac angiotensinogen is derived from the circulation. Most likely angiotensinogen diffuses freely from plasma into the interstitial space, where cleavage by interstitial fluid renin and/or membrane-bound renin may occur.

4.3 ACE
ACE mRNA is readily detectable in cardiac tissue [45,59,60]. ACE has been demonstrated in the heart by autoradiography [61], using a radiolabelled ACE inhibitor, as well as by measurement of its activity in cardiac homogenates [45,62]. Most likely, cardiac ACE is normally limited to the coronary vascular endothelial cells and the endocardium [63].

4.4 Angiotensin I and II
Many groups have reported on the presence of Ang I and II in cardiac tissue [24,32–34]. In most cases the tissue levels (expressed per g wet weight) were similar to or higher than the concomitant plasma levels (expressed per ml plasma). This, however, cannot be taken as definite evidence for angiotensin production at cardiac tissue sites, since both Ang I and Ang II may have been actively sequestered from the circulating blood. According to two studies [24,32], cardiac angiotensin levels decreased to levels close to or below the detection limit following a bilateral nephrectomy, whereas a third study recently reported no change in cardiac angiotensin levels after nephrectomy [33]. The most likely explanation for these differences is that in the latter study cardiac angiotensin levels were measured in rats 24 h after nephrectomy, while the effects of nephrectomy on cardiac Ang II, at least in the rat, become apparent only at 48 h after nephrectomy [32]. Moreover, a bilateral nephrectomy is known to be accompanied by the release of large amounts of renin from the kidney, and this may have resulted in correspondingly high myocardial renin levels immediately during and after surgery [14,25].

What are the possible sources of Ang I and Ang II in cardiac tissue? Ang I in cardiac tissue might be derived from Ang I in the coronary artery, from Ang I generated in the coronary circulation by the reaction of circulating renin with circulating angiotensinogen ('plasma renin activity', PRA), or from Ang I synthesized in situ in cardiac tissue. Cardiac Ang II might be derived from Ang II in the coronary artery and, via conversion, from the above three sources of Ang I.

In order to quantify the contribution of each of these sources, we have measured the steady-state tissue and plasma levels of endogenous and radiolabelled Ang I and Ang II, as well as PRA, during infusions of 125I-labelled Ang I and Ang II [34]. Great care was taken to measure intact 125I-labelled and endogenous Ang I and Ang II rather than tissue radioactivity or immunoreactive angiotensin levels. The body does not distinguish between radiolabelled and endogenous angiotensins [64], and thus the steady-state levels of 125I-labelled Ang I and Ang II present in cardiac tissue during the infusion of these radiolabelled peptides are a measure for the uptake of angiotensins from the circulation. The contribution of angiotensins generated in the coronary circulation by PRA can be calculated by assuming that PRA-derived angiotensins are taken up by the heart in the same way as radiolabelled angiotensins.

The results indicated that, under steady-state conditions, the cardiac 125I-Ang I concentrations are less than 5% of its levels in plasma, whereas the concentrations of cardiac 125I-Ang II are approximately 90% of plasma 125I-Ang II. At the same time, the cardiac tissue concentration of endogenous Ang I was similar to the plasma concentration of endogenous Ang I, while the cardiac tissue concentration of endogenous Ang II was 4–5 times higher than the plasma concentration of endogenous Ang II (Fig. 1). Taking into consideration the small amounts of angiotensins generated by PRA in the coronary circulation, it can be calculated that over 90% of the Ang I in tissue is synthesized in the tissue itself and not derived from the circulation. Moreover, more than 75% of the Ang II in tissue is also synthesized in the tissue and its source is in-situ synthesized Ang I rather than Ang I from the circulation. Interestingly, locally synthesized Ang I, but not locally synthesized Ang II, was found to be released into the coronary circulation [65,66]. This suggests either that Ang I produced at tissue sites enters the blood at a level distal to the site where Ang I-to-II conversion occurs, or that Ang II produced in the tissue cannot leave the tissue, for instance because, following its formation, it rapidly binds to angiotensin receptors.


Figure 1
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  		Fig. 1 Plasma and cardiac tissue levels of angiotensin (Ang) I and II in the pig. The majority of cardiac Ang I and II is synthesized locally at cardiac tissue sites. The contribution of plasma renin activity in the coronary vascular bed to the cardiac tissue levels of Ang I and II is too small to be shown. Data are taken from Ref. [34].

 
Summarizing, the majority of cardiac Ang I and Ang II is synthesized at tissue sites by kidney-derived renin. Locally synthesized Ang II is kept in the tissue, whereas locally synthesized Ang I is capable of reaching the coronary circulation.


   5 Cardiac renin-, angiotensinogen- and ACE synthesis under pathological conditions
Top
 Abstract
 1 Introduction
 2 Studies in cardiac...
 3 Studies in isolated...
 4 Studies in intact...
 5 Cardiac renin-,...
6 Site of tissue...
 7 Mannose 6-phosphate receptors...
 8 Role of alternative...
 9 Regulation of cardiac...
 References
 
Although synthesis of renin and angiotensinogen at cardiac tissue sites does not appear to occur under normal circumstances, it is possible that the renin and angiotensinogen gene are switched on in response to pathological conditions. In addition, changes in ACE gene expression may occur. Most studies investigating the cardiac RAS under pathological conditions have determined changes at the mRNA level only. In view of the low to undetectable levels of renin and angiotensinogen mRNA in control hearts, as well as the uncertainties with regard to transcriptional regulation, it is difficult to establish the value of increased mRNA levels in the diseased heart. Boer et al. [50] and Pieruzzi et al. [67] described increases in renin mRNA in the rat heart in response to volume overload. These increases could only be demonstrated after 37 and 40 PCR cycles, and were not confirmed by Iwai et al. [57]. No change in cardiac angiotensinogen mRNA was found in the volume overload model [50,57]. Passier et al. found increased renin mRNA levels in the infarct zone following coronary artery ligation, and no change in angiotensinogen mRNA levels in either the infarcted and noninfarcted zone [49]. In contrast, Lindpaintner et al. reported a transient activation of angiotensinogen mRNA in the noninfarcted left ventricle of rats after a coronary artery ligation [56]. Heller et al. studied myocardial renin—angiotensinogen dynamics during pressure-induced cardiac hypertrophy, and found cardiac renin to vary directly with plasma renin [14]. Similarly, the increases in plasma renin occurring in subjects with end-stage heart failure were found to be accompanied by parallel increases in cardiac renin [53]. Thus, on the basis of renin protein measurements in cardiac tissue no evidence was obtained for significant cardiac renin production under pathological conditions. As far as angiotensinogen is concerned, decreased rather than increased levels were found in failing hearts, suggesting local consumption by cardiac renin [53]. Thus, demonstration of significant angiotensinogen production in the heart under pathological conditions is difficult, since increased consumption may mask local production. Finally, with regard to ACE, changes in its mRNA levels have been observed in diseased hearts that are supported by protein measurements. Both ACE protein and ACE mRNA increase following myocardial infarction, as well as during pressure – and volume overload-induced left ventricular hypertrophy [45,57,59,60,67]. Under these conditions, the localization of ACE may no longer be limited to the endothelium. In humans, following myocardial infarction, ACE can be detected in the remaining viable cardiomyocytes near the infarct scar of the aneurysmal left ventricle, as well as in fibroblasts, vascular smooth muscle cells, and macrophages in the scar area itself [68]. In rats, following coronary occlusion, ACE was demonstrated in fibroblasts in the healthy hypertrophying part of the heart [69].


   6 Site of tissue angiotensin generation
Top
 Abstract
 1 Introduction
 2 Studies in cardiac...
 3 Studies in isolated...
 4 Studies in intact...
 5 Cardiac renin-,...
 6 Site of tissue...
7 Mannose 6-phosphate receptors...
 8 Role of alternative...
 9 Regulation of cardiac...
 References
 
Tissue angiotensin generation may occur in interstitial fluid, on the cell membrane, or within cells (Fig. 2).


Figure 2
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  		Fig. 2 Proposed scheme of angiotensin (Ang) I and II production in the heart [39]. Intravascular and interstitial compartments as well as blood vessel wall and cardiac cells (endothelial cells, myocytes, fibroblasts, macrophages) are depicted. Circulating renin and angiotensinogen (Aog) both enter the interstitial fluid compartment. Renin may also bind to the vascular wall and cardiac cells. ACE is present on endothelial cells and cardiac cells. Ang I and II are metabolized by peptidases while passing through the vascular wall. Ang I and II in the interstitial fluid are mainly generated outside the vascular fluid compartment. Tissue Ang I and II generation may occur not only in the interstitial fluid or on the cell surface but also within cells, for instance after renin uptake by the cells. Binding of Ang II to the AT1 receptor is followed by internalization of the AT1 receptor—Ang II complex.

 
6.1 Interstitial fluid
According to our studies in the isolated rat heart, circulating renin and angiotensinogen are able to reach the interstitial space [38]. This offers the possibility of angiotensin generation within this fluid. Indeed, during combined renin/angiotensinogen perfusion of the Langendorff heart, we found the levels of Ang I and Ang II in interstitial fluid to be two—three times higher than the levels measured simultaneously in the intravascular compartment [38,39]. Data from in-vivo studies in the dog also demonstrated that the cardiac interstitial angiotensin levels are higher than the plasma levels of these peptides [70]. Since diffusion of intact Ang I and II from the intravascular compartment to the interstitial compartment is marginal (most likely because of rapid metabolism of angiotensins in the vascular wall [38,39]), the high interstitial levels can be taken as evidence for interstitial angiotensin generation.

6.2 Cell membrane
In support of a role for membrane-bound renin in local angiotensin generation, we [38,65] and others [66] observed that, both in vivo and in vitro, the amount of Ang I released by the heart via coronary effluent was too high to be explained by the renin—angiotensinogen reaction occurring in intravascular fluid during coronary passage. Moreover, in the isolated Langendorff heart preparation perfused with renin and angiotensinogen, Ang I release via coronary effluent reached a steady-state level long after renin and angiotensinogen had reached a steady state in this fluid [38], and angiotensin release continued after discontinuation of the renin perfusion [40]. These data suggest that tissue-bound renin rather than extracellular fluid renin is responsible for the high Ang I levels in coronary effluent. Endothelial cells, vascular smooth muscle cells, cardiomyocytes and cardiac fibroblasts may all be involved in the binding process.

6.3 Intracellular compartment
Direct evidence for intracellular angiotensin generation is not available. Renin dialysis into cultured cardiomyocytes leads to a decrease in the conductance of the adjacent myocytes [71]. The reduction of conductance was amplified when renin was infused together with angiotensinogen and attenuated when a renin inhibitor was co-administered, suggesting that these effects are mediated by renin-dependent angiotensin II formation within the cell. Our data on renin and prorenin internalization [16,41] might explain how renin normally enters the cell. When, concurrently with (pro)renin, angiotensinogen is taken up from the interstitial fluid via bulk fluid endocytosis, a scenario for intracellular angiotensin generation is provided (Fig. 3). Additional proof for intracellular angiotensin generation comes from studies where the extracellular and tissue levels of Ang II were measured during Ang II perfusion and during renin/angiotensinogen perfusion of the isolated rat Langendorff heart in the absence or presence of the AT1 receptor antagonist losartan [39]. During these infusions the heart is exposed to arterially delivered and locally generated Ang II, respectively. Losartan did not affect the extracellular Ang II levels during both infusions. It did however reduce the tissue Ang II levels during Ang II perfusion to almost undetectable levels, whereas tissue Ang II during renin/angiotensinogen perfusion was not affected. It appears therefore that arterially delivered Ang II binds to AT1 receptors at cardiac tissue sites and that losartan interferes with this process, thereby reducing the tissue Ang II levels. Locally synthesized Ang II present at tissue sites is not affected by losartan, and thus may have been generated at a site that cannot be reached by losartan, i.e. the intracellular compartment.


Figure 3
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  		Fig. 3 Binding and activation of prorenin by cardiomyocytes. After binding to the cell surface, the receptor—prorenin complex is internalized in a clathrin-coated pit, that pinches off to become a coated vesicle. The clathrin coat then depolymerizes, thereby leading to the formation of an endosome. Prorenin is activated to renin and dissociates from the receptor due to the acid environment in the endosome. The receptor returns to the cell surface, and the activated prorenin might participate in either intracellular or extracellular angiotensin generation.

 

   7 Mannose 6-phosphate receptors and cardiac (pro)renin binding
Top
 Abstract
 1 Introduction
 2 Studies in cardiac...
 3 Studies in isolated...
 4 Studies in intact...
 5 Cardiac renin-,...
 6 Site of tissue...
 7 Mannose 6-phosphate receptors...
8 Role of alternative...
 9 Regulation of cardiac...
 References
 
In view of the absence of significant renin synthesis at cardiac tissue sites, one may speculate that the heart possesses specific mechanisms to sequester (pro)renin from the circulation. Several groups have reported on the existence of (pro)renin binding proteins and/or receptors [16,41,43,51–53,72–74]. An intracellular renin-binding protein (RnBP) was discovered in the early eighties in humans, rats and pigs [72–74]. Binding to this RnBP reduces the Ang I-generating activity of renin by >80%. Recently, this RnBP was found to be equal to the enzyme N-acyl-D-glucosamine 2-epimerase, indicating that it might be involved in the intracellular processing of renin rather than renin uptake [75]. Subsequently, using chemical cross-linking, two vascular RnBPs were identified by Campbell and colleagues in membranes isolated from rat mesenteric arteries or cultured rat aortic smooth muscle cells [43]. Interestingly, binding to these RnBPs was inhibited by a specific, active site-directed renin inhibitor, suggesting that the active site of the renin molecule might be involved in the binding process. Nguyen et al. and Sealey et al., with the use of radiolabelled (pro)renin, demonstrated high-affinity renin binding sites/receptors (Kd{approx}1 nM) in human mesangial cells and in membranes prepared from rat tissues [51,52]. In the rat, these binding sites bound prorenin and renin equally well, which suggests that neither the prosegment nor the active site is involved in the binding process [51]. This contrasts with Campbell's findings.

If binding does not involve the prosegment or the active site, a further possibility would be binding to the carbohydrate portions which both proteins contain. Renin and prorenin display isoelectric heterogeneity; up to five or six forms with different isoelectric points have been described in rats and humans [76–79]. This heterogeneity most likely results from differential glycosylation (glycoforms). The carbohydrate portion appears to be involved in the clearance of renin by the liver, since deglycosylation greatly (>90%) reduced the hepatic uptake of renin [80]. We recently observed that the mannose 6-phosphate signal, present on both renin and prorenin, determines (pro)renin binding and internalization by cardiac and endothelial cells [16,41]. Most likely therefore, it is the mannose 6-phosphate receptor (MPR) which is involved in this process.

MPRs function in the process of intracellular lysosomal enzyme sorting [81,82]. At present, two different MPRs have been identified: a large MPR (mol. wt. 300 kDa), which binds ligand independent of divalent cations (cation-independent or CI-MPR), and a small MPR (mol. wt. 46 kDa), which requires divalent cations for optimal binding (cation-dependent or CD-MPR) [83,84]. In 1987, it was discovered that the CI-MPR and the insulin-like growth factor II receptor are the same protein [85]. Thus, the CI-MPR is now also known as the M6P/IGFII receptor. This receptor binds IGFII, which is non-glycosylated, and phosphomannosylated proteins at distinct sites (Fig. 4) [86,87]. The M6P/IGFII receptor is involved in the activation processes of several precursor proteins, such as procathepsin D and the latent form of transforming growth factor beta [88,89]. In view of the fact that we observed not only MPR-dependent binding and internalization of renin and prorenin, but also activation of prorenin to renin, it seems logical to assume that the MPR involved in these processes is the M6P/IGFII receptor.


Figure 4
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  		Fig. 4 Schematic representation of the mannose 6-phosphate receptors. The two ligands of the cation-independent mannose 6-phosphate are depicted. Arginine residues at positions 435 and 1334 in domains 3 and 9, respectively, are essential for high-affinity binding of mannose 6-phosphate. Sequences in domain 11 are involved in binding insulin-like growth factor (IGF) II. Modified from Refs. [81,82].

 
The M6P/IGFII receptor consists of a large extracellular domain, containing 15 repeat regions, and a small cytoplasmic domain (Fig. 4). The extracytoplasmic domain of the CD-MPR is similar to each of the repeating units of the extracellular domain of the M6P/IGFII receptor, suggesting that the two receptors may be derived from a common ancestor [90]. The M6P/IGFII receptor exists as a monomer, whereas the CD-MPR exists as a monomer, dimer or tetramer [84,91]. MPRs cycle constitutively among the Golgi, endosomes, and the plasma membrane. The majority (90%) of the M6P/IGFII receptors is located in a late endosomal/prelysosomal compartment, with the rest being distributed over the plasma membrane, early endosomes, and the Golgi [92]. Extracellular lysosomal enzymes which bind to the cell surface M6P/IGFII receptor are internalized via clathrin-coated pits. They dissociate from the receptor in acidified endosomal compartments and are subsequently delivered to lysosomes. The receptor is then reutilized; it can undergo many rounds of ligand delivery [92–95]. Binding and internalization of IGFII to the M6P/IGFII receptor results in the lysosomal degradation of this ligand [96]. In addition, IGFII mediates growth-stimulatory responses via this receptor [97]. At present it is not clear what the function of the M6P/IGFII receptor with regard to (pro)renin is: clearance, coupling to second messengers [52], or facilitation of local angiotensin production (Fig. 3)? The idea of prorenin contributing to local angiotensin production is attractive, in view of the fact that the prorenin concentrations in the circulation are tenfold higher than those of renin.


   8 Role of alternative angiotensin generating pathways
Top
 Abstract
 1 Introduction
 2 Studies in cardiac...
 3 Studies in isolated...
 4 Studies in intact...
 5 Cardiac renin-,...
 6 Site of tissue...
 7 Mannose 6-phosphate receptors...
 8 Role of alternative...
9 Regulation of cardiac...
 References
 
It has been suggested, on the basis of in-vitro experiments, that Ang II synthesis may occur independently of renin and ACE. The candidates that are generally assumed to replace renin and ACE are cathepsin D and chymase, respectively. Cathepsin D is a lysosomal enzyme that cleaves angiotensinogen, unlike renin, at low pH [98,99]. Consequently, measurement of Ang I-generating activity at acidic pH will yield results that are representative for lysosomal cathepsin D rather than renin. Studies in which Ang I-generating activity is quantitated as a measure for renin activity should therefore always be performed in the absence and presence of specific renin inhibitors to correct for non-renin-dependent Ang I generation. Evidence that cathepsin D is of importance in vivo is currently lacking. Under circumstances where cardiac angiotensinogen levels are high and cardiac renin levels are low or undetectable (e.g., after nephrectomy), cardiac Ang I and II levels are close to or below the detection limit [24,32]. Moreover, in human heart homogenates renin levels correlated negatively with angiotensinogen levels, thereby suggesting angiotensinogen consumption by renin at cardiac tissue sites [53]. Studies in the isolated perfused heart also do not support a role for cathepsin D, since angiotensin generation in this preparation only occurred after the addition of renin to the perfusion buffer [37–40].

Chymase is a serine protease present in the cardiac interstitium, and cardiac mast cells and endothelial cells are sites of chymase biosynthesis and storage [100]. Remarkably, chymase is the main enzyme in human heart homogenates responsible for Ang I–II conversion [101]. In contrast, Ang I–II conversion in the coronary vascular bed of intact humans and pigs depends on ACE only [65,102,103]. This raises the question whether chymase is of importance in vivo. Kokkonen et al. [104] have suggested that interstitial fluid contains an endogenous inhibitor of chymase, {alpha}1-antitrypsin, which would normally suppress any chymase-dependent Ang I–II conversion. However, the inhibitory effect of {alpha}1-antitrypsin may be limited to tissue homogenates only, since it could not be demonstrated in an intact preparation [105]. Cardiac chymase mRNA levels are unaltered in subjects with heart failure [60]. More detailed knowledge on the in-vivo role of chymase will be obtained once specific chymase inhibitors are available.


   9 Regulation of cardiac angiotensin generation
Top
 Abstract
 1 Introduction
 2 Studies in cardiac...
 3 Studies in isolated...
 4 Studies in intact...
 5 Cardiac renin-,...
 6 Site of tissue...
 7 Mannose 6-phosphate receptors...
 8 Role of alternative...
 9 Regulation of cardiac...
References
 
All RAS components are present in cardiac tissue, and both Ang I and II are generated in the heart. However, the renin responsible for this local angiotensin production originates from the circulation and is therefore kidney-derived. Thus, a local RAS in the sense that all RAS components are synthesized in situ does not appear to exist in the normal heart. This does not mean that cardiac angiotensin synthesis occurs in parallel with angiotensin generation in the circulation. There still are many ways by which the heart may regulate its Ang I and II concentrations independent of the circulating levels of these RAS components. Membrane binding could be a mechanism by which renal renin is sequestered in the heart. The density of the binding sites involved in the uptake process may vary, and this could modify the cardiac production of Ang I and II. Interestingly, the inactive precursor of renin, prorenin, also binds to these binding sites and becomes activated following internalization [16,41]. It is not yet known what enzyme(s) is/are responsible for the prorenin-to-renin conversion step. Their concentration may decrease or increase under different circumstances. In addition, one has to keep in mind that normally the circulating levels of prorenin are approximately 10-fold higher than those of renin.

Cardiac ACE levels will also influence local Ang II production. These are determined, at least in part, by the so-called insertion/deletion polymorphism [62]. Furthermore, enzymatic degradation of Ang II and AT1 receptor-mediated endocytosis could influence the Ang II concentrations at the cellular and subcellular level. Finally, under certain pathological conditions renin and/or angiotensinogen may be produced in the heart itself and this would create the possibility for the heart to regulate its own Ang II production, independent of kidney and liver.

More detailed knowledge on the actual sites of Ang II production in the heart and on its regulation under pathological conditions will further illuminate the role of the RAS in cardiac function, growth, and remodelling, and will help us to understand the beneficial cardiac effects of ACE inhibitors, AT1 receptor antagonists and renin inhibitors.

Time for primary review 28 days.


   References
Top
 Abstract
 1 Introduction
 2 Studies in cardiac...
 3 Studies in isolated...
 4 Studies in intact...
 5 Cardiac renin-,...
 6 Site of tissue...
 7 Mannose 6-phosphate receptors...
 8 Role of alternative...
 9 Regulation of cardiac...
 References
 

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