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The intracellular renin–angiotensin system: a new paradigm Rajesh Kumar1,2,3, Vivek P. Singh1,2,3 and Kenneth M. Baker1,2,3 1
Division of Molecular Cardiology, Cardiovascular Research Institute, Texas A&M Health Science Center, College of Medicine, Temple, TX 76508, USA 2 Scott & White, 2401 South 31st Street, Temple, TX 76508, USA 3 Central Texas Veterans Health Care System, 1901 South 1st Street, Temple, TX 76504, USA
More than a century after its discovery, the physiological implications of the renin–angiotensin system (RAS) continue to expand, with the identification of new components, functions and subsystems. These advancements have led to better management and understanding of a broad range of cardiovascular and metabolic disorders. The RAS has traditionally been viewed as a circulatory system, involved in the short-term regulation of volume and blood pressure homeostasis. Recently, local RASs have been described as regulators of chronic tissue effects. Most recently, studies have provided evidence of a complete, functional RAS within cells, described as an ‘intracrine’ or intracellular system. A more comprehensive understanding of the intracellular RAS provides for new strategies in system regulation and a more efficacious approach to the management of RAS-related diseases. Introduction: the renin–angiotensin system There has been significant advancement in knowledge regarding the renin–angiotensin system (RAS) over the past several decades. Originally, the RAS was viewed as consisting of a single, biologically active hormone angiotensin II (Ang II), generated by the sequential action of renin on angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) on the cleaved product from AGT, angiotensin I (Ang I) [1]. Ang II interacts with two wellcharacterized plasma membrane receptors, AT1 and AT2, and probably additional intracellular receptor(s) [2,3]. Recent advancements in the RAS field have included the discovery of: (i) novel bioactive peptides, (ii) additional specific receptors, (iii) alternative pathways of Ang II generation and (iv) additional roles for precursor components, other than Ang II synthesis. Some of these novel aspects of the RAS were the subject of recent reviews [4–6]. Here, we focus on the intracellular RAS, the existence of which has been suggested for a long time [7] but has gained support only recently. Classical RAS The classical RAS is systemic in nature, whereby Ang II is synthesized in the circulation and contributes to maintaining electrolyte balance, body fluid volume and arterial pressure primarily through vasoconstriction and aldosterone Corresponding author: Baker, K.M. (
[email protected]). Available online 16 May 2007. www.sciencedirect.com
production. One of the most significant advancements in the past two decades has been the discovery of local or tissue RASs [8,9]. A local system is characterized by the presence of RAS components, AGT and the conversion enzymes, local synthesis of Ang II, and binding to specific receptors, resulting in a physiological response. Local systems are regulated independently of the circulatory RAS but can also interact with the latter. Local and circulatory system interactions have attracted significant controversy, particularly with regard to the heart [10]. The identification of a functional cardiac RAS has been established through a variety of pharmacological and genetic approaches [11,12]. In addition to the heart, other sites in which local systems have been demonstrated include the kidneys [13], brain [14], pancreas [15], and reproductive [8], lymphatic [8], and adipose tissues [16]. Local system functions of the RAS include cell growth and remodeling in the heart; regulation of blood pressure; stimulation of food and water intake through the brain; and hormone secretion in the pancreas. Intracellular RAS Similar to the classification of the RAS into systemic or local systems, which were defined by circulatory or tissue synthesis of Ang II, the intracellular RAS is characterized by the presence of its components inside the cell and synthesis of Ang II at an intracellular site. The primary nature of RAS components, such as the presence of signal peptides in AGT and renin, and the transmembrane nature of ACE, is generally thought not to be supportive of an intracellular system. However, the existence of differentially glycosylated isoforms of AGT [17,18], alternatively spliced forms of renin [19,20], intracellular and secreted forms of ACE [21], alternative Ang II-generating enzymes (such as cathepsins and chymase) [22,23], and detection of these components intracellularly, under certain cellular conditions [21,24], support the hypothesis of an intracellular RAS. In addition to its intracellular presence or synthesis, Ang II should be able to mediate biological effects from an intracellular location, to be functionally relevant. Although Ang II is the main bioactive peptide of the intracellular RAS, other components, such as renin and prorenin, might also have intracellular effects [25]; a view strengthened by the recent demonstration of an intracellular renin receptor that couples to signal transduction cascades and cellular effects [26]. We here focus on the evidence for the intracellular presence of RAS components,
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Ang II generation, the functionality of intracellular Ang II, and putative mechanisms of action. Components of the intracellular RAS The intracellular RAS includes the following components: AGT, renin, ACE, chymase and various receptors. AGT AGT is synthesized locally and is believed to be secreted extracellularly, owing to the presence of a signal sequence and glycosylation. However, the signal peptide, required for endoplasmic reticulum (ER) translocation, does not necessitate extracellular secretion. The destination of proteins in the ER is further determined by glycosylation and the role of other targeting sequences. Based on structural similarity, AGT belongs to a family of serine protease inhibitors (serpins). Some serpins, such as ovalbumin, exhibit complex cellular distribution, being nuclear, cytoplasmic or both [27]. Another serpin, plasminogen activator inhibitor type 2 (PAI-2), is synthesized as a non-glycosylated, intracellular form and a glycosylated, extracellular form [28]. In addition, the secretion efficiency of PAI-2 differs depending on the cell type, differentiation state and culture conditions [28]. AGT seems to exhibit similar properties because brain astrocytes produce a non-glycosylated form of AGT, which is not secreted and is targeted to the nucleus [18]. Mutagenesis of human AGT at four putative N-glycosylation sites produced a non-glycosylated AGT that demonstrated higher intracellular retention and catalytic efficiency than the wildtype protein [17]. Certain natural variants of AGT have been reported to be abnormally glycosylated, resulting in altered plasma levels of AGT [29,30]. Thus, glycosylation of AGT seems to be a requirement for efficient secretion. The existence of multiple glycosylated forms of AGT suggests complex cellular distribution, including intracellular localization, depending on the cellular state. Consistent with this hypothesis, it has been observed that high glucose conditions decrease secretion and increase intracellular retention of AGT in neonatal rat ventricular myocytes (NRVMs) [31]. Renin Intracellular renin can be derived from local synthesis and/ or uptake from the circulation. Cardiac myocytes internalize both prorenin and renin, and activate prorenin after internalization [32]. Similar to AGT, (pro)renin exists with varying degrees of glycosylation, which can affect secretion as well as the functional consequence of internalization [33]. Glycosylated forms of pro(renin) are internalized through the mannose-6-phosphate receptor, which is considered to be a clearance mechanism [34]. By contrast, internalization of non-glycosylated prorenin, through unknown receptors, is accompanied by increased intracellular levels of Ang I and Ang II in cardiac myocytes [32]. This observation suggests the existence of an intracellular mechanism for prorenin activation in cardiac myocytes. A similar mechanism of prorenin activation might apply to locally synthesized prorenin that is retained intracellularly owing to the lack of a secretory signal or proper glycosylation. Additionally, alternative renin transcripts coding for prefragment-deficient prorenin have been reported and shown to be upregulated in the left www.sciencedirect.com
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ventricle following myocardial infarction [20]. These non-secretory, intracellular renin isoforms might participate in intracellular Ang I generation from AGT [19]. Importantly, high levels of glucose have been shown to decrease prorenin secretion in rat mesangial cells, resulting in increased intracellular renin activity and Ang II generation [24]. Similarly, increased intracellular levels of renin were observed in NRVMs when exposed to high glucose [31]. ACE ACE is a membrane-bound ectoenzyme, expressed in many tissues on endothelial, epithelial and neuroepithelial cells. ACE contains two catalytic domains, termed N- and C-terminal domains, which convert Ang I to Ang II. In addition to membrane-bound forms, secreted and intracellular forms of ACE have been identified in mesangial cells [21]. Confocal immunofluorescence microscopy revealed that N-domain ACE, which is catalytically active, was present inside the nuclei of mesangial cells of Wistar and spontaneously hypertensive rats [21]. ACE was also detected immunocytochemically inside cardiac myocytes and fibroblasts [35]. Chymase Chymase-mediated Ang II synthesis has emerged as an alternative pathway to ACE in renal, vascular and cardiac tissue, particularly in pathological conditions [22,36]. Chymase is an intracellular enzyme, shown by electron microscopic immunocytochemistry to reside in cytosolic granules of mast cells, endothelial cells and mesenchymal interstitial cells of the heart [37]. In kidney, chymase has been shown to be expressed by mesangial and vascular smooth muscle cells [38]. Interestingly, increased intracellular chymase levels have been observed in NRVMs grown in a high-glucose medium [31]. Receptors Intracellular Ang II binding sites have been demonstrated to be present on renal and hepatocyte nuclei and on chromatin [39–42]. Whereas nuclear Ang II binding sites seem to be AT1-like, the nature of chromatin binding is not known. These nuclear and chromatin binding sites are functional, as determined by modulation of gene expression by Ang II in isolated nuclei [43,44]. The translocation of plasma membrane AT1 to a nuclear site, after Ang II binding, has been described [45–47]; however, whether the nuclear AT1-like receptor represents the internalized plasma membrane receptor remains unknown. Recently, intracellular Ang II was shown to relocate AT1 receptor to the nucleus and reduce receptor density on the plasma membrane [48]. High-affinity Ang II binding sites; a soluble cytosolic binding protein; [49,50], atypical Ang II binding in cardiac fibroblasts [51]; and a non-AT1, non-AT2 Ang II binding site in brain membranes [52] have all been reported. The latter Ang II binding sites have not been characterized regarding functional coupling. Intracellular synthesis of Ang II Several studies have provided evidence for intracellular generation of Ang II in cultured kidney and heart cells and in the intact heart.
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Evidence based on intracellular colocalization of RAS components Intracellular staining for Ang II has been detected in several tissues, although the source of Ang II (i.e. intracellular synthesis or internalization) was not determined. Cellular uptake of Ang II is believed to contribute to intracellular Ang II levels in kidney and heart [10,53]; however, there is also evidence to suggest that intracellular synthesis occurs in both tissues (Figure 1). The intracellular colocalization of precursor components and enzymes of the RAS in the same cell (rat renal cortical, adrenal medullary chromaffin and pituitary glandular cells), and even in the same secretory granule, strongly suggests that Ang II is capable of being synthesized intracellularly [54–56]. Similarly, colocalization of N-domain ACE with Ang II in the cytoplasm and nuclei of renal mesangial cells from Wistar and spontaneously hypertensive rats suggests intracellular synthesis of Ang II [21]. In renal juxtaglomerular cells, which do not express AGT, colocalization of Ang II with renin in storage granules was suggested to be a result of intracellular synthesis after exogenous AGT internalization [57]. High glucose-induced intracellular synthesis of Ang II Elevated levels of glucose have been shown to result in the activation of the tissue RAS [58]. In a study on rat mesangial cells, high glucose resulted in a time-dependent increase in the expression of the genes encoding prorenin, AGT, ACE and cathepsin B [24]. Interestingly, prorenin secretion was reduced by high glucose, and intracellular renin activity was increased by 30-fold. Cathepsin B probably accounted for the intracellular activation of prorenin to renin. The
increase in renin activity was accompanied by a several-fold increase in intracellular Ang II concentrations, which localized predominantly in the nucleus [24]. In human renal mesangial cells, Ang II levels increased only in cell lysates, and not in the medium, following high-glucose exposure [59]. Although strongly suggestive of intracellular Ang II synthesis, these studies did not exclude the possible contribution of receptor-mediated internalization to the intracellular pool of Ang II. In another study, intracellular Ang II synthesis was studied in NRVMs, in response to elevated glucose levels and isoprenaline [31]. Receptor-mediated internalization of Ang II was prevented by an AT1 receptor blocker (candesartan) in the cellular medium. Isoprenaline stimulated Ang II synthesis, both extracellularly and intracellularly (probably through the secretory pathway). Conversely, high glucose increased Ang II levels only intracellularly in the perinuclear and nuclear regions. High glucose-induced intracellular Ang II synthesis was accompanied by increased intracellular retention and decreased extracellular levels of AGT and renin, consistent with intracellular Ang II generation. Localization of Ang II to the nucleus suggested a functional role of intracellularly synthesized Ang II. Additional experiments, using specific enzyme inhibitors, determined that renin and chymase, but not ACE, were responsible for high glucose-induced intracellular Ang II synthesis, whereas renin and ACE were involved in extracellular, in addition to intracellular, Ang II synthesis induced by isoprenaline. These observations corroborate those reported in mesangial cells, and suggest that the increase in intracellular Ang II was largely a result of ‘on site’ synthesis, and not uptake.
Figure 1. Putative pathways for intracellular Ang II synthesis. In the prevailing paradigm, Ang II synthesis occurs in the interstitial space (shown at the bottom right of the figure), from components either sequestered from the circulation or secreted from the tissue. Two possible pathways could be envisioned for synthesis of intracellular Ang II. In the first scenario (1), RAS components are synthesized in the rough endoplasmic reticulum and segregated together in the secretory vesicles [54–56], where Ang II generation would occur. Depending on the cellular state, Ang II would either be released extracellularly or relocated intracellularly. In a second scenario (2), RAS components would be synthesized outside of the secretory pathway or targeted intracellularly, instead of being secreted, resulting in intracellular synthesis. This latter mechanism could involve alternative Ang II-generating enzymes, such as chymase. Cytosolic Ang II is likely to be capable of freely entering the nucleus, owing to its small size. The recently demonstrated intracellular synthesis of Ang II in NRVMs, following stimulation with isoprenaline or high glucose [31], would be compatible with either of pathways 1 and 2. Isoprenaline stimulation increased Ang II levels, both in cell lysates and the medium. Intracellular Ang II staining was largely observed along actin filaments, suggestive of Ang II synthesis in secretory vesicles (pathway 1). By contrast, high glucose increased Ang II and RAS component (AGT, renin and chymase) levels only intracellularly. Intracellular Ang II staining was localized in the perinuclear and nuclear regions, suggestive of pathway 2. www.sciencedirect.com
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Additional evidence of intracellular Ang II synthesis in cultured cardiac myocytes In adult rat cardiac myocytes, generation of intracellular Ang II synthesis was demonstrated following incubation of the cells with non-glycosylated mouse ren-2d prorenin [32]. The latter study also demonstrated that uptake of glycosylated prorenin, which occurs through the mannose-6-phosphate receptor, probably represents a clearance mechanism; whereas uptake of non-glycosylated prorenin, through an unidentified mechanism, has functional consequences through Ang II generation. Because prorenin exists with different degrees of glycosylation and is upregulated in diabetes [60], its internalization and endogenous expression might significantly result in increased intracellular Ang II levels in diabetes. Thus, the increase in Ang II staining observed in cardiac myocytes from diabetic patients might be caused by intracellular synthesis [61]. Increased intracellular synthesis of Ang II has been shown indirectly in cardiac myocytes from cardiomyopathic hamsters, when renin and ACE levels are elevated [62]. At two months of age, when no signs of heart failure are detected in the hamsters, intracellular administration of enalaprilat into isolated cardiac myocytes had no effect on junctional conductance [62]. However, cells obtained from six-month-old cardiomyopathic hamster hearts showed increased cell coupling by intracellular enalaprilat, an ACE inhibitor (ACEI), suggesting the synthesis of Ang II through intracellular ACE, as previously noted [35]. Ang II synthesis in isolated hearts In isolated, perfused rat hearts, the infusion of renin resulted in Ang II formation, even after renin in the perfusate had been depleted, probably caused by the uptake of renin by cardiac tissue and continued Ang II formation intracellularly [63]. Additionally, Ang II-forming activity in heart homogenates has been shown to be only partially inhibited by cilazaprilat, another ACEI, but more so by chymostatin, a chymase inhibitor, suggesting the involvement of a chymase-like activity [63]. In another study, combined perfusion of isolated hearts with renin and AGT showed that cardiac Ang II generation was not restricted to the extracellular environment, but also occurred intracellularly [64]. Ang II accumulation in cells was observed even in the presence of losartan, an angiotensin II receptor antagonist, suggesting that local Ang II production in the heart, following perfusion of a combination of renin and AGT, was occurring intracellularly. Functional coupling of intracellular Ang II Conceptually, it needs to be determined whether intracellularly generated Ang II is secreted extracellularly or is directed to intracellular sites that are responsible for mediating biological effects. One study found that the site of Ang II localization seems to be stimulus dependent [31]. Whereas b-adrenoceptor stimulation causes Ang II synthesis in the secretory pathway, most is secreted extracellularly, and thus is unlikely be of functional relevance intracellularly. By contrast, high glucose induces synthesis of Ang II, in addition to its relocation to the cytoplasm and nucleus, strongly suggesting functional relevance of intracellular Ang II. Similar observations have been reported in www.sciencedirect.com
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mesangial cells, suggesting that the activation of the intracellular RAS by high glucose might be a widely occurring phenomenon [21]. However, more research will be necessary to validate this hypothesis. Functions of the intracellular RAS The earliest study suggesting functional effects of intracellular Ang II date back to 1971, when Robertson and Khairallah [7] demonstrated localization of injected Ang II in the nuclei of smooth and cardiac muscle cells, accompanied by ultrastructural cellular changes. Later, Re et al. [40,44] demonstrated Ang II binding sites in chromatin fragments that resulted in increased RNA synthesis by Ang II. The coupling of nuclear membrane Ang II receptors to increased transcription of the genes encoding AGT, renin and platelet-derived growth factor, has also been demonstrated in isolated nuclei [43]. Microinjection of Ang II into rat vascular smooth muscle cells (VSMCs) has been shown to increase cytosolic and nuclear Ca2+ ([Ca2+]i), which was secondary to an influx of extracellular Ca2+ [65]. Adjacent cells, through cell-to-cell contact, also showed increased [Ca2+]i, which was due to release from intracellular stores. In cultured cardiac myocytes from rats and hamsters, intracellular dialysis of Ang II had opposite effects on the inward Ca2+ current: inhibition in rats and enhancement in hamsters [66]. Consistent with the effect on Ca2+ influx, intracellular Ang II induced contractions in rat aortic rings [67]. Intracellular Ang II was also observed to stimulate growth in A7r5 VSMCs (derived from fetal rat aorta), as determined by DNA synthesis and cell count [68]. Transfection of rat hepatoma cells with AGT, lacking the secretory signal, resulted in intracellular generation of Ang II, which had a mitotic effect in these cells [69]. Similarly, enhanced proliferation of Chinese hamster ovary cells has been observed after transfection with a plasmid that coded for the non-secretory, mature Ang II peptide [70]. Using adenovirus expression vectormediated synthesis of Ang II, cell growth in cultured NRVMs was demonstrated, as determined by cell size and [3H]leucine incorporation [71]. Significantly, the in vivo effects of intracellular Ang II on the development of cardiac hypertrophy have been demonstrated in the adult mouse [71]. A plasmid construct that resulted in the expression of the Ang II peptide, under the control of an a-myosin heavy chain promoter, resulted in an increased concentration of Ang II in cardiac ventricles and a 68% increase in the heart weight-to-body weight ratio, within four days [71]. Intracellular Ang II increased the cardiac mRNA levels of several genes, including those encoding cjun, insulin-like growth factor-1, and transforming growth factor-b. Circulating Ang II levels and blood pressure were not affected by intracellular Ang II expression in the heart [71]. The possibility that some of the above-described effects are mediated by the breakdown fragments of intracellular Ang II has not been investigated. Mechanism of action of intracellular Ang II The effects of intracellular Ang II are varied, ranging from Ca2+ modulation, to cell growth and gene expression. Whereas extracellular Ang II induces both Ca2+ influx and release from intracellular stores, the effects
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of intracellular Ang II seem to vary among cell types. In rat aortic VSMCs, the increase in [Ca2+]i resulted from an influx of extracellular Ca2+ [65]. Likewise, the intracellular Ang IIinduced contraction of rat aorta rings was dependent on Ca2+ influx, not on inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 (IP3)]-mediated release from intracellular stores [67]. However, in A7r5 VSMCs, intracellular Ang II stimulated Ins(1,4,5)P3, the latter being required for the release of Ca2+ from intracellular stores [72]. In renal proximal tubule cells, chelation of extracellular Ca2+ with ethylene glycolbis(2-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA) did not affect intracellular Ang II-induced [Ca2+]i responses; however, the latter were attenuated by pretreatment with thapsigargin (to deplete intracellular Ca2+ stores) and by inhibition of phospholipase C [73]. Intracellular Ang IImediated growth effects in A7r5 VSMCs were blocked by inhibition of phosphatidylinositol 3 kinase and extracellular-signal-regulated kinase (ERK)-1 and ERK-2 [68]. In A10 VSMCs, intracellular Ang II activated cAMP-response element-binding protein (CREB) through phosphorylation of p38 mitogen-activated protein kinase (not ERK-1 or ERK2); however, extracellular Ang II resulted in the activation of both pathways [48]. Other seemingly contradictory findings regarding the mechanism of action of intracellular Ang II are related to the nature of intracellular Ang II receptors. AT1 receptor blockers (ARBs) have been shown to differ in their efficacy to block intracellular Ang II-mediated effects [74]. This observation seems to depend on the cell type and biological effects being investigated [74]. The methods of intracellular delivery of Ang II (microinjection, liposomes or expression vector), which result in the localization of Ang II to different intracellular sites (nucleus or cytoplasm), might also have contributed to the observed differences with ARBs [74]. A7r5 VSMCs, which are devoid of plasma membrane Ang II receptors, do not respond to extracellular Ang II. Importantly, intracellular Ang II-mediated growth effects have been shown in these cells, and were partially inhibited by both AT1 and AT2 receptor antagonists,
suggesting the involvement of atypical, intracellular Ang II receptors [68,72,75]. In CHO cells that do not express functionally significant levels of AT1 receptor, increased cell proliferation by intracellular Ang II was demonstrated, an effect that was not blocked by ARBs [70]. However, the effects on the inward Ca2+ current in VSMCs, and cell-tocell communication in cardiac myocytes, were blocked by intracellular application of ARBs [62,65]. The profile that emerges from these studies suggests that intracellular Ang II interacts with multiple intracellular proteins, both with and without AT1-like characteristics [74]. In addition, the observation of the binding of Ang II to chromatin, and the modulation of gene expression, suggests direct effects of Ang II, without involvement of putative signaling cascades [40,44]. In view of these diverse effects, the mechanism of intracellular Ang II-mediated responses could be classified as cytosolic, such as the observed effect on Ca2+, or as nuclear, such as gene expression, possibly mediated through a direct effect on chromatin remodeling and involving different Ang II-interacting partners. Significance of the intracellular RAS Several studies have demonstrated the existence of a functional intracellular RAS [76,77]; however, the physiological and pathophysiological role of this system remains to be determined. Is the intracellular RAS a general phenomenon or is it restricted to specific tissues, cells or pathophysiological conditions? The effects of intracellular Ang II in a variety of cells, namely cardiac [71], renal [73], hepatic [69] and vascular [45,78] cells, suggests a broad relevance of the system. Intracellular Ang II generation in cardiac myocytes stimulated by hyperglycemia demonstrates selective activation of the intracellular RAS [31]. Additional pathophysiological states are also likely to regulate the intracellular RAS. Increased intracellular Ang II staining of cardiac myocytes from diabetic patients and animals suggests a clinically relevant pathological role of the intracellular system [61,79]. The observed increase in intracellular Ang II in diabetes [80,81] is probably due to
Figure 2. Clinical significance of the intracellular RAS. Under hyperglycemic conditions in the heart, cardiac myocytes (a) can synthesize Ang II intracellularly, with redistribution to the nucleus, thereby mediating intracrine effects. Ang II can also be present in the interstitial space, synthesized locally from RAS components of circulatory origin and/or by other cell types in the heart, such as fibroblasts (b). Extracellular Ang II exhibits autocrine and paracrine effects, which can be blocked by ACEIs or ARBs. However, the intracrine effects of Ang II would not be blocked by these agents because plasma membrane AT1 receptors are not required for the intracrine effects, and ACE is not involved in intracellular Ang II synthesis by cardiac myocytes in high glucose conditions. By contrast, a renin-I, which can be internalized by cardiac myocytes, is able to block both extra- and intracellular generation of Ang II. Thus, ARBs and ACEIs might not completely block Ang II effects in diabetic conditions, whereas renin-Is might prove more efficacious. www.sciencedirect.com
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intracellular Ang II synthesis and receptor-mediated uptake. Reviews by Re and co-workers [77,82] have suggested that intracrine pathways have physiological and pathophysiological relevance. The intracellular RAS has potentially major therapeutic implications (Figure 2). The intracellular system is generally not inhibited by ARBs (because AT1 receptors might not be involved in mediating intracellular effects) or by ACEIs (because intracellular Ang II synthesis probably involves alternative enzymes, such as chymase, in pathological conditions such as hyperglycemia) [31,70]. The observed beneficial effects of ARBs and ACEIs in these conditions might be related to the inhibition of the extracellular RAS and RAS-independent mechanisms, such as effects on peroxisome proliferator-activated receptor-g and the kallikrein–kinin system [83–85]. The latter observations suggest that inhibition of the intracellular RAS might provide significant additional clinical benefits. This is consistent with recent reports that ‘standard’ therapeutic regimens of RAS inhibition (ACEIs and ARBs) might not have as much cardiovascular efficacy as anticipated [86,87]. Because renin is involved in the synthesis of both intra- and extracellular Ang II, a renin inhibitor (renin-I) might provide a particularly attractive therapeutic modality in situations in which the intracellular system is activated. Conclusion In summary, there is increasing evidence, both direct and indirect, in support of the existence of a complete and functional intracellular RAS in multiple tissues. The intracellular RAS probably does not represent an independent entity but an extension or alternative form of a local RAS, which might be manifested only under select pathophysiological conditions, such as hyperglycemia. This suggests a unique evolutionary role of the intracellular RAS. What remain to be determined are the mechanisms of regulation and actions, and a more precise understanding of the role of the intracellular RAS in (patho)physiology. Concerted efforts will be required to achieve these objectives, which are likely to have major clinical implications for the complete blockade of the RAS, in pathological conditions in which the intracellular RAS is activated. References 1 Hall, J.E. (2003) Historical perspective of the renin-angiotensin system. Mol. Biotechnol. 24, 27–39 2 de Gasparo, M. et al. (2000) International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol. Rev. 52, 415–472 3 Hunyady, L. and Catt, K.J. (2006) Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. Mol. Endocrinol. 20, 953–970 4 Kurdi, M. et al. (2005) Working outside the system: an update on the unconventional behavior of the renin-angiotensin system components. Int. J. Biochem. Cell Biol. 37, 1357–1367 5 Carey, R.M. and Siragy, H.M. (2003) Newly recognized components of the renin-angiotensin system: potential roles in cardiovascular and renal regulation. Endocr. Rev. 24, 261–271 6 Chappell, M.C. et al. (2004) Novel aspects of the renal reninangiotensin system: angiotensin-(1-7), ACE2 and blood pressure regulation. Contrib. Nephrol. 143, 77–89 7 Robertson, A.L., Jr and Khairallah, P.A. (1971) Angiotensin II: rapid localization in nuclei of smooth and cardiac muscle. Science 172, 1138– 1139 www.sciencedirect.com
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8 Paul, M. et al. (2006) Physiology of local renin-angiotensin systems. Physiol. Rev. 86, 747–803 9 Re, R.N. (2004) Mechanisms of disease: local renin-angiotensinaldosterone systems and the pathogenesis and treatment of cardiovascular disease. Nat. Clin. Pract. Cardiovasc. Med. 1, 42–47 10 Danser, A.H. (2003) Local renin-angiotensin systems: the unanswered questions. Int. J. Biochem. Cell Biol. 35, 759–768 11 Bader, M. (2002) Role of the local renin-angiotensin system in cardiac damage: a minireview focussing on transgenic animal models. J. Mol. Cell. Cardiol. 34, 1455–1462 12 Varagic, J. and Frohlich, E.D. (2002) Local cardiac renin-angiotensin system: hypertension and cardiac failure. J. Mol. Cell. Cardiol. 34, 1435–1442 13 Haller, H. et al. (2006) Spotlight on renin: intrarenal renin-angiotensin system – important player of the local milieu. J. Renin Angiotensin Aldosterone Syst. 7, 122–125 14 Sakai, K. and Sigmund, C.D. (2005) Molecular evidence of tissue reninangiotensin systems: a focus on the brain. Curr. Hypertens. Rep. 7, 135– 140 15 Leung, P.S. and Carlsson, P.O. (2005) Pancreatic islet renin angiotensin system: its novel roles in islet function and in diabetes mellitus. Pancreas 30, 293–298 16 Goossens, G.H. et al. (2007) Endocrine role of the renin-angiotensin system in human adipose tissue and muscle: effect of beta-adrenergic stimulation. Hypertension 49, 542–547 17 Gimenez-Roqueplo, A.P. et al. (1998) Role of N-glycosylation in human angiotensinogen. J. Biol. Chem. 273, 21232–21238 18 Sherrod, M. et al. (2004) Nuclear localization of angiotensinogen in astrocytes. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288, R539–R546 19 Lavoie, J.L. et al. (2006) Evidence supporting a functional role for intracellular renin in the brain. Hypertension 47, 461–466 20 Clausmeyer, S. et al. (2000) Tissue-specific expression of a rat renin transcript lacking the coding sequence for the prefragment and its stimulation by myocardial infarction. Endocrinology 141, 2963–2970 21 Camargo de Andrade, M.C. et al. (2006) Expression and localization of N-domain ANG I-converting enzymes in mesangial cells in culture from spontaneously hypertensive rats. Am. J. Physiol. Renal Physiol. 290, F364–F375 22 Miyazaki, M. and Takai, S. (2006) Tissue angiotensin II generating system by angiotensin-converting enzyme and chymase. J. Pharmacol. Sci. 100, 391–397 23 Belova, L.A. (2000) Angiotensin II-generating enzymes. Biochemistry (Mosc.) 65, 1337–1345 24 Vidotti, D.B. et al. (2004) High glucose concentration stimulates intracellular renin activity and angiotensin II generation in rat mesangial cells. Am. J. Physiol. Renal Physiol. 286, F1039–F1045 25 Re, R.N. (2003) Intracellular renin and the nature of intracrine enzymes. Hypertension 42, 117–122 26 Schefe, J.H. et al. (2006) A novel signal transduction cascade involving direct physical interaction of the renin/prorenin receptor with the transcription factor promyelocytic zinc finger protein. Circ. Res. 99, 1355–1366 27 Bird, C.H. et al. (2001) Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1. Mol. Cell. Biol. 21, 5396–5407 28 von Heijne, G. et al. (1991) The efficiency of the uncleaved secretion signal in the plasminogen activator inhibitor type 2 protein can be enhanced by point mutations that increase its hydrophobicity. J. Biol. Chem. 266, 15240–15243 29 Gimenez-Roqueplo, A.P. et al. (1996) The natural mutation Y248C of human angiotensinogen leads to abnormal glycosylation and altered immunological recognition of the protein. J. Biol. Chem. 271, 9838–9844 30 Nakajima, T. et al. (1999) Functional analysis of a mutation occurring between the two in-frame AUG codons of human angiotensinogen. J. Biol. Chem. 274, 35749–35755 31 Singh, V.P. et al. (2007) High glucose induced regulation of intracellular angiotensin II synthesis and nuclear redistribution in cardiac myocytes. Am. J. Physiol. Heart Circ. Physiol. (in press) 32 Peters, J. et al. (2002) Functional significance of prorenin internalization in the rat heart. Circ. Res. 90, 1135–1141 33 Paul, M. et al. (1988) Glycosylation influences intracellular transit time and secretion rate of human prorenin in transfected cells. J. Hypertens. Suppl. 6, S487–S489
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