The RAS (renin–angiotensin system) is one of the earliest and most extensively studied hormonal systems. The RAS is an atypical hormonal system in several ways. The major bioactive peptide of the system, AngII (angiotensin II), is neither synthesized in nor targets one specific organ. New research has identified additional peptides with important physiological and pathological roles. More peptides also mean newer enzymatic cascades that generate these peptides and more receptors that mediate their function. In addition, completely different roles of components that constitute the RAS have been uncovered, such as that for prorenin via the prorenin receptor. Complexity of the RAS is enhanced further by the presence of sub-systems in tissues, which act in an autocrine/paracrine manner independent of the endocrine system. The RAS seems relevant at the cellular level, wherein individual cells have a complete system, termed the intracellular RAS. Thus, from cells to tissues to the entire organism, the RAS exhibits continuity while maintaining independent control at different levels. The intracellular RAS is a relatively new concept for the RAS. The present review provides a synopsis of the literature on this system in different tissues.
- angiotensin II
- angiotensin type 1 receptor
- cardiovascular system
- renin–angiotensin system
The RAS (renin–angiotensin system) is one of the most significant and extensively studied hormonal systems in humans. Beginning with the discovery in 1898 by Tigerstedt and Bergman of a factor released from kidneys that controlled blood pressure, the RAS has expanded into an extensive system of enzymatic cascades, bioactive molecules and multiple receptors [1,2]. Consequently, this system has pleiotropic effects which control functions at multiple levels, from cells to tissues to the entire organism. Thus it is not surprising that this system is regulated at each level independently and in concert with others to fine tune the desired outcome. It is important to elucidate the RAS at every level to completely understand the interplay and mechanisms by which it regulates bodily functions.
In the simplest form, the RAS encompasses sequential conversion of AGT (angiotensinogen) by renin and ACE (angiotensin-converting enzyme) into AngI (angiotensin I) and AngII (angiotensin II) respectively. AngII binds to two specific and ubiquitous GPCRs (G-protein-coupled receptors), AT1 receptors (AngII type 1 receptors) and AT2 receptors (AngII type 2 receptors), to carry out biological functions. However, the RAS is significantly more complex. To deal with the complexity, the RAS will be divided into sub-systems based on two parameters: (i) new compared with old knowledge, and (ii) the site of synthesis and actions of AngII.
On the basis of traditional compared with new knowledge, the RAS can be categorized as the classical and the novel RAS (Figure 1). The former is the simplest form of the RAS described above. This consists of a single bioactive peptide AngII and two specific receptors AT1 and AT2 receptors. The physiological function of the classical RAS is to maintain vascular tone through direct effects on VSMCs (vascular smooth muscle cells) and salt and water homoeostasis through aldosterone secretion . The novel RAS includes several new bioactive peptide products, their receptors and newly identified activities of previously inert components. Among the new angiotensin peptides, Ang-(1–7) [angiotensin-(1–7)], AngIII (angiotensin III) and AngIV (angiotensin IV) are notable, with significant biological activity via binding to Mas, AT1 and AT2 receptors, and IRAP (insulin-regulated aminopeptidase) respectively. Interestingly, the actions of Ang-(1–7) are opposite to those of AngII . AngIV has an independent profile of biological actions, which include memory formation and control of water intake . In addition to these new angiotensin peptides, prorenin and renin have been assigned new biological roles that are independent of AngII production. Both prorenin and renin bind to a specific receptor, the PRR [(pro)renin receptor], and initiate intracellular signalling pathways that include MAPK (mitogen-activated protein kinase) and Wnt . The novel RAS represents a dramatic expansion of the classical RAS in terms of both the number of active molecules and biological functions.
Using the second parameter of the site of synthesis and actions, the RAS can be classified into systemic/circulating, local/tissue and intracellular systems (Figure 2). At the systemic level, AngII is formed in the circulation from AGT secreted from liver, renin from kidneys and ACE located on vascular endothelial cells. Actions of circulation-derived AngII on the vascular wall and elsewhere in tissues are termed endocrine functions. The local or tissue RAS is defined by the synthesis of AngII within tissues, from AGT and enzymes produced locally . The local RAS may utilize enzymes other than renin and ACE for the synthesis of AngII, such as cathepsins and chymase [8,9]. Since AGT is a secretory protein, the synthesis of AngII is thought to occur largely in the interstitial space. Newly synthesized AngII acts on neighbouring cells and these actions of AngII are termed autocrine/paracrine effects. The function and regulation of the tissue RAS is independent of the circulating system . The intracellular RAS is characterized by the synthesis and actions of AngII within a cell . The actions of AngII that are initiated inside the cell are termed intracrine effects. Intracrine effects may also be produced by AngII that is internalized from the interstitial space. The intracellular RAS is one of the newest concepts in the area of studies related to the RAS and thus is a subject of considerable discussion and debate . The nature of the intracellular RAS in different cell types and its pathophysiological significance has not been thoroughly investigated. However, recent studies have suggested that the intracellular RAS provides another dimension to the regulation and function of RAS in tissues. There is evidence that, in some cell types and pathological conditions, the intracellular RAS may have a predominant role [13,14]. In the present review, we focus on the presence and composition of the intracellular RAS and intracrine function of AngII in different tissues.
INTRACRINE ACTIONS OF ANGII: PRELUDE TO THE INTRACELLULAR RAS
Intracrine actions of AngII are those that are initiated from an intracellular location, rather than by binding to plasma-membrane AT1 or AT2 receptors. The concept of intracrine actions of AngII began with the observation that, when labelled AngII was injected into the left ventricle of adult rats, it localized to the nuclear zone of vascular and cardiac muscle cells . Later studies confirmed that AngII can bind to isolated nuclei and chromatin, resulting in a change in gene expression [16–18]. We and others have discovered that nuclear AngII-binding sites on hepatocytes were AT1-receptor-like, further strengthening the concept of intracrine functionality of AngII [19,20]. More systemic studies were subsequently performed in different cell types to establish the intracrine nature of AngII, as detailed below.
The intracrine RAS in cardiac myocytes and fibroblasts
Direct effects of intracellular AngII in intact cells were first reported on cellular communication in cardiac myocytes. Intracellular dialysis of AngII caused a decrease in junctional conductance in isolated cell pairs of rat cardiac myocytes . These effects of AngII were blocked by intracellular administration of losartan, suggesting the involvement of an intracellular AT1-receptor-like receptor. A similar decrease in junctional conductance was observed following the dialysis of renin or renin plus AGT in cardiac myocyte cell pairs . The observation that these effects were prevented by a renin or ACEi (ACE inhibitor) suggested that intracellular AngII was involved. To further confirm that endogenous intracellular AngII influences cell communication in the failing heart, isolated cell pairs from failing hearts of cardiomyopathic hamsters (at 2 and at 6 months of age) were studied . In this model, signs of heart failure were not evident and activity of the RAS was low at 2 months compared with 6 months. Both AngI and AngII decreased junctional conductance in cell pairs from 2-month-old hamsters, while causing cell uncoupling in cell pairs from 6-month-old animals. In separate experiments, ACEis and an ARB (angiotensin receptor blocker) alone did not produce any effect on cell coupling at 2 months of age, while increasing it at 6 months. These effects correlated with the activity of the RAS at these stages of heart failure, suggesting a role of endogenous AngII in cell communication in the failing heart . Additionally, intracellular administration of AngII increased the peak inward calcium current density in cardiac myocytes isolated from cardiomyopathic hamsters , which was inhibited by the mineralocorticoid receptor antagonist eplerenone . The latter reduced the expression of plasma-membrane and intracellular AT1 receptors, which might have contributed to the observed effects.
We studied the effect of intracellular AngII on cardiac myocyte cell growth and cardiac hypertrophy. Intracellular AngII was produced in cardiac myocytes using a recombinant adenovirus coding for the AngII peptide without a signal sequence. This approach resulted in increased AngII levels in cell lysates, but not in culture medium. NRVMs (neonatal rat ventricular myocytes), infected with the adenoviral vector, showed significant hypertrophic growth as determined by cell size, protein synthesis and enhanced cytoskeletal re-arrangement . Adult mice, injected with a similar intracellular AngII-coding plasmid vector, developed significant cardiac hypertrophy, without an associated increase in blood pressure or plasma AngII levels. Losartan did not block the growth effects, excluding the involvement of extracellular AngII and the plasma-membrane AT1 receptor . However, the latter observation suggested that AngII interacts with intracellular proteins other than the AT1 receptor. Our later studies identified high ambient glucose as a potent stimulus for endogenous intracellular AngII production in cultured cardiac myocytes and fibroblasts [14,27]. We observed that endogenous intracellular AngII stimulated the expression of AGT in cardiac myocytes and was involved in increased TGF-β (transforming growth factor-β) and collagen expression by cardiac fibroblasts when exposed to high glucose [14,27].
In a recent study, AngII microinjected into isolated adult rat cardiac myocytes was localized to the perinuclear and nuclear region . Exposure of isolated nuclei obtained from cardiac myocytes to AngII resulted in enhanced de novo RNA synthesis, specifically NF-κB (nuclear factor κB) mRNA expression. AngII applied to purified nuclei compared with intact cardiac myocytes showed a greater increase in NF-κB mRNA levels following nuclear application, suggesting preferential nuclear signalling.
Most effects of intracellular AngII are similar to that of extracellular AngII. However, it was reported that, although extracellular AngII increased the cell volume of cardiac myocytes isolated from cardiomyopathic hamsters, intracellular AngII reduced it . Interestingly, aldosterone enhanced the effects of both extracellular and intracellular AngII, partly due to the effect on AT1 receptor expression . It was proposed that intracellular AngII might have a protective role, in conditions such as myocardial ischaemia, by reducing cell volume [7,31]. Together, these studies demonstrate intracrine functionality of AngII in cardiac cells, both in vitro and in vivo, by artificially delivered, recombinant and endogenous intracellular AngII.
Intracrine AngII in VSMCs
In VSMCs, AngII was localized in endosomes and nuclei following microinjection. This was accompanied by a rapid increase in [Ca2+]i (intracellular Ca2+ concentration) in the cytosol and nucleus due to the influx of extracellular Ca2+. The increase in [Ca2+]i was also observed in adjacent cells; however, that increase was due to Ca2+ release from intracellular stores. These effects were blocked by intracellular, but not extracellular application of candesartan, suggesting the involvement of an intracellular AT1-receptor-like receptor . When delivered intracellularly in de-endothelized rat aorta rings using liposomes, AngII caused a dose-dependent contraction, which was dependent on Ca2+ influx from the medium and not on Ins(1,4,5)P3-mediated release from intracellular Ca2+ stores, similar to that observed in the previous study. These effects of intracellular AngII were sensitive to intracellular, but not extracellular, administration of candesartan . In A7r5 VSMCs, which are devoid of extracellular AngII effects, intracellular administration of AngII through liposomes or cell permeabilization resulted in enhanced Ca2+ influx, DNA synthesis and cell proliferation. Additionally, intracellular AngII modulated Ca2+ influx caused by serotonin receptor stimulation in these cells. Using various AT1 and AT2 receptor antagonists, the effects of intracellular AngII in A7r5 VSMCs were ascribed to two distinct low- and high-affinity-binding sites [34–37]. In the rat VSMC cell line A10, transfection with intracellular AngII and AT1 receptor expression vectors resulted in enhanced cell proliferation, which was mechanistically linked to p38 MAPK and CREB (cAMP-response-element-binding protein) activation .
Intracrine AngII in kidney cells
Renal PTCs (proximal tubule cells) grown in high-glucose medium showed elevated intracellular AngII levels that were associated with increased N-acetyl-β-glucosaminidase secretion and p22phox protein levels, which were normalized by treatment with different ARBs, suggesting the involvement of intracellular AngII in mediating the high-glucose effects . Microinjection of AngII into single PTCs elicited a marked increase in cytoplasmic [Ca2+]i, probably from intracellular Ca2+ stores . These effects were blocked by the intracellular, but not extracellular, application of losartan, an observation similar to that described for VSMCs.
Intracrine AngII in other cell types
Using a recombinant DNA approach, a mutated AGT lacking the secretory signal was expressed in rat hepatoma cells that also produced renin and ACE. These cells had an increased mitotic index and PDGF (platelet-derived growth factor) expression, which was blocked by a renin antisense oligonucleotide and losartan, but not by candesartan . The latter is less lipophilic than losartan, and thus probably did not enter cells in a sufficient amount to block the intracellular AT1 receptor . Co-expression of AngII and AT1 receptors in COS-7 and CHO (Chinese-hamster ovary)-K1 cells significantly enhanced cell proliferation, compared with the expression of any single protein . The increase in cell proliferation was prevented by losartan, but not an anti-AngII antibody added to the culture medium, excluding the possibility of extracellular AngII effects. Significantly, co-expression of intracellular AngII with AT1 receptors resulted in an increased localization of the latter to the nucleus, which was associated with CREB activation. Studies in our laboratory have demonstrated that, when AngII was overexpressed as a native peptide, without fusion to a fluorescent protein, a significant increase in cell proliferation was observed in CHO-K1 cells in the absence of functional AT1 receptors . These studies corroborated the lack of effect of ARBs on intracellular AngII-mediated cardiac myocyte cell growth and cardiac hypertrophy, suggesting novel AT1-receptor-independent effects of intracellular AngII in the absence of AT1 receptors. In rat luteal cells, AngII was shown to be localized to the cytoplasm and nucleus by confocal microscopy . Losartan inhibited progesterone production in permeabilized, but not intact, luteal cells, which was reversed by AngII, suggesting that AngII modulated steroidogenesis through intracellular AT1 receptors .
Intracrine AngII in vivo
A direct demonstration of the intracrine effects of AngII in an animal model had not been possible due to difficulty in separating intracellular AngII effects from those initiated at the plasma membrane. However, an association of intracellular AngII levels with the pathological state has been demonstrated in both human and animal studies. Increased intracellular AngII staining was observed in the myocardium from diabetic patients, which also showed increased apoptosis, necrosis and oxi-dative stress compared with non-diabetic controls . Intracellular AngII levels were shown to be increased further in hypertensive diabetic patients, suggesting an association with disease severity, as determined by cardiac hypertrophy, cavity dilation and depressed ventricular performance . Interestingly, the study patients were on ACEi therapy, which corroborated our in vitro studies in cardiac myocytes of chymase-mediated AngII generation following exposure to high glucose . Similarly, our in vivo animal studies demonstrated that increased intracellular AngII in the diabetic rat heart was associated with enhanced cardiac myocyte apoptosis, fibrosis and oxidative stress. The latter conditions were alleviated better by a renin inhibitor, which blocks the intracellular RAS compared with an ARB and ACEi .
Recently, a transgenic mouse line was generated that expressed AngII fused downstream of CFP (cyan fluorescent protein) without a secretory signal . The transgene expression was observed in multiple tissues, without an associated increase in circulating AngII levels, compared with controls. These animals were hypertensive and showed thrombotic microangiopathy in glomerular capillaries and small vessels. These studies were the first direct demonstration that an intracellular fluorescent fusion protein of AngII caused elevated blood pressure and kidney pathology in mice . In another study, a similar adenoviral construct of intracellular AngII was expressed in the superficial cortex of rat and mouse kidneys under an sglt2 (sodium and glucose co-transporter 2) promoter . These animals had an increase in systolic blood pressure (28 mmHg) and decrease in sodium and lithium excretion by 20–24%. The effects of intracellular AngII were blocked by losartan in wild-type animals and were prevented in AT1a-KO (AT1a-receptor-knockout) mice, suggesting a requirement of intracellular AT1 receptors in the kidneys.
THE INTRACELLULAR RAS: CONCEPT AND CONTROVERSIES
As alluded to above, the intracellular RAS is defined by the presence of RAS components and the synthesis and actions of AngII, and possibly other active angiotensin peptides, within a cell. Although conceptually simple, the presence of such a system was difficult to reconcile in view of the evolution of our knowledge regarding the RAS . In the 1930s, AngII was originally identified as hypertensin/angiotonin that was released in the plasma from ischaemic kidneys. Later studies revealed that the kidneys produced an enzyme that acted on a substrate in plasma, which was identified as renin and AGT respectively. ACE, which converts AngI into AngII, was discovered in 1956 and the concept of the classical RAS originated . AGT, the only known precursor of AngII, is a secretory protein. Renin is also a secretory protein expressed as the inactive precursor prorenin, and only renal juxtaglomerular cells appear to have the capacity to convert it into active renin. Another component of the RAS, ACE, is a membrane-bound enzyme with catalytic sites located on the extracellular domain. Furthermore, AngII receptors are plasma-membrane GPCRs. In view of the evolutionary history and the nature of individual components, the concept of the RAS as a circulating and extracellular system is pre-eminent in our thinking. Thus intracellular synthesis of AngII seemed less plausible and was not explored until the intracrine effects of AngII were identified.
To describe an intracellular RAS, two possible characteristics of the system were considered . One of these was the intracellular RAS as a transient system present in secretory vesicles. Being secretory proteins of a single system, it was hypothesized that renin, AGT and ACE were co-sorted in secretory vesicles, resulting in the generation of AngII due to the close proximity of the components. A portion of the newly generated AngII could move into the cytoplasm and the remaining secreted through constitutive or regulated pathways. There is evidence of co-localization of different RAS components in the same secretory vesicles of different cell types. Electron microscopic immunohistochemistry in neonatal and adult rat kidney sections has showed co-localization of renin, AngI and AngII in cytoplasmic granules of juxtaglomerular cells . AGT was distributed throughout the cytoplasm. Using a reverse haemolytic plaque assay, the release of AngI and renin from individual renocortical cells was demonstrated, suggesting the intracellular generation of AngI . It has been reported that the presence of large amounts of AngII in the storage granules (that contained renin) of renal juxtaglomerular cells was due to intracellular synthesis, probably following uptake of exogenous AGT [50,51]. The subcellular fractionation of renin granules from kidney homogenates further demonstrated the presence of AngI and AngII in renin granule fractions . In the rat anterior pituitary, AGT, prorenin and renin were co-localized in the same secretory vesicles in all glandular cell types, using immuno-electron microscopy, suggesting intragranular processing of these components. These studies further suggested sorting of RAS components into a regulated, in addition to a constitutive, secretory pathway . The observation of the release of AngII by cardiac myocytes cultured in serum-free medium, following mechanical stretch, also supported intracellular generation and regulated secretion [53,54].
The second characteristic of the intracellular RAS was an intracellular system present in non-secretory cellular compartments in the cytoplasm, nucleus, mitochondria and/or other cellular organelles. The precise cellular trafficking of RAS components in different cell types and pathological conditions is not known. Different isoforms of RAS components, formed as a result of post-translational modifications or alternative transcription or post-transcription processing of mRNA, might traffic to intracellular locations other than secretory vesicles. A human astrocyte cell line has been reported to express primarily the non-glycosylated form of AGT, which was localized to the nucleus . Similarly, primary cultures of glial cells, isolated from transgenic mice expressing human AGT, showed nuclear localization . Using fusion constructs of human AGT fragments with GFP (green fluorescent protein), a nuclear localization signal was demonstrated at the C-terminus of AGT . With regard to renin, three human and two rat renin mRNA isoforms, resulting from alternative transcription or splicing, have been described [56,57]. These different renin mRNA species translate into two renin protein isoforms. One is the classical secretory renin, whereas the second isoform lacks the pre-pro-fragment and is intracellular. The expression of renin isoforms is tissue-specific, with intracellular isoforms being expressed in the adrenal gland, brain and heart [58,59]. In transgenic animals, intracellular renin enhanced aldosterone production in rats and increased blood pressure and drinking volume when overexpressed in the mouse brain [60,61]. Cytosolic renin, when overexpressed in H9c2 cardiomyoblasts, was localized in mitochondria, resulting in enhanced apoptosis . Similar to AGT and renin, ACE and its fragments have been shown to be localized in intracellular compartments, including the nucleus, in mesangial, smooth muscle and endothelial cells [63,64]. Taken together, these studies support the concept of a functionally relevant intracellular RAS in several cell types. In the following section, we will review the literature demonstrating the intracellular synthesis of AngII.
The cardiac intracellular RAS
Cardiac cells constitutively express most components of the RAS, with the exception of renin, the expression of which is more often associated with pathological conditions [65,66]. That these endogenous components constitute an intracellular system has been suggested by functional studies or directly demonstrated by de novo intracellular AngII synthesis. As discussed above, intracellular AngII has effects on cellular communication in cardiac myocytes. The effect of dialysis of AngI on junctional conductance in cardiac myocyte cell pairs was abolished by an ACEi . Similarly, intracellular dialysis of renin reduced junctional conductance, which was reversed by a renin inhibitor and enalaprilat, suggesting the intracellular conversion of AngI into AngII . In rat aortic rings, intracellular administration of AngI caused contraction similar to AngII, which was abolished by candesartan but not by captopril, suggesting AngII-mediated effects through intracellular AngII generation independent of ACE . In a study on isolated perfused rat hearts overexpressing human AGT, perfusion with renin caused a sustained increase in AngII formation even after renin was no longer detectable in the perfusate, whereas AngI perfusion caused increased AngII levels only during the perfusion period . These studies suggest that renin is taken up by cardiac cells, resulting in sustained intracellular AngII generation.
In a more direct demonstration of intracellular AngII synthesis, NRVMs were incubated in serum-free medium and exposed to isoprenaline (isoproterenol) or high glucose in the presence of candesartan . The latter was used to prevent AT1-receptor-mediated internalization of AngII. AngII was measured in cell lysates and the culture medium, which represented intra- and extra-cellularly synthesized AngII respectively. Isoprenaline increased the AngII concentration in both the cell lysate and culture medium, whereas high glucose markedly increased AngII synthesis only in the cell lysate of NRVMs. Western blot analysis revealed increased intracellular levels of AGT, renin and chymase in high-glucose-exposed cells. Confocal immunofluorocytometry confirmed the presence of AngII in the cytoplasm and nucleus of high-glucose-exposed NRVMs and along the actin filaments in isoprenaline-exposed cells. The results of the immunostaining experiments suggested that AngII synthesis occurred in the secretory pathway in response to isoprenaline and in non-secretory locations in response to high glucose. Consistent with the latter conclusion is that AngII synthesis was dependent on renin and chymase in high-glucose-exposed cells and on renin and ACE in isoprenaline-exposed cells . Similarly, high-glucose-induced intracellular AngII synthesis was observed in adult mouse cardiac myocytes . However, in cardiac fibroblasts, intracellular AngII synthesis appeared to occur only in the secretory pathway mediated through the actions of renin and ACE, following both isoprenaline and high-glucose stimulation . In summary, the site of AngII synthesis, intracellular localization and synthetic pathways were dependent on the stimulus and cell type. Significantly, NRVMs synthesized and retained AngII intracellularly, which was redistributed to the nucleus under high-glucose conditions, suggesting a role for an intracrine mechanism in diabetic conditions.
Cardiac intracellular AngII synthesis was also suggested by animal studies. Diabetes for 1 week in rats resulted in significantly increased intracellular AngII levels in cardiac myocytes, which were not normalized by candesartan, suggesting that AngII was synthesized intracellularly and not internalized through AT1 receptors . Increased intracellular levels of AngII, AGT and renin were observed by confocal microscopy. Intracellular AngII synthesis was blocked by aliskiren, but not by benazepril, findings that were consistent with in vitro studies. Diabetes-induced superoxide production and cardiac fibrosis were partially inhibited by candesartan and benazepril, whereas aliskiren produced complete inhibition, suggesting a link with intracellular AngII .
In addition to intracellular AngII synthesis, both AT1 and AT2 receptors were detected in nucleus-enriched fractions from isolated adult rat cardiac myocytes by Western blot analysis . These receptors were localized on nuclear membranes by confocal microscopy and were demonstrated to functionally couple to signalling pathways, as was evident from increased nuclear [Ca2+] levels and NF-κB expression, following exposure of isolated nuclei to AngII . Recently, AT1 and AT2 receptors were also detected in mitochondria from human monocytes, mouse hepatocytes, kidney tubular cells, neurons and cardiac myocytes by immuno-electron microscopy . Significantly, AT2 receptors co-localized with endogenous AngII/AngIII and modulated NO production and mitochondrial respiration . The origin of intracellular AngII receptors is not clear and could be due to direct trafficking of newly formed receptors to different intracellular locations or internalization following stimulation with AngII. Although there is no direct evidence for the former, the latter has been extensively studied [42,70–72]. In summary, a complete intracellular system for AngII synthesis and actions, including signalling mechanisms and cellular effects, has been described in the heart .
The renal intracellular RAS
Human mesangial cells grown in high-glucose medium showed increased levels of AngII in the cell lysate, but not in the culture medium. The observed increase in AngII was blocked by chymostatin, but not captopril, and was associated with a 3-fold increase in chymase mRNA levels. Significantly, blocking AT1 receptors by losartan did not affect intracellular AngII levels, suggesting intracellular generation and not uptake of AngII in these cells . A significant amount of AngII induced by high glucose in a mouse mesangial cell line was localized to the nucleus . It was demonstrated that high glucose induced the increased expression of prorenin at early time points, reduced prorenin secretion and increased the rate of prorenin conversion into renin, resulting in increased intracellular AngII generation in mesangial cells . Similarly, other investigators have reported chymase-mediated intracellular AngII synthesis in rat mesangial cells in response to high glucose . Significantly, it was reported in patients that healthy kidneys weakly expressed chymase in mesangial and VSMCs; however, there was a 10–15-fold increase in chymase expression in the diabetic kidney . Chymase expression further increased 4–7-fold in hypertensive patients compared with normotensives, a finding which correlated with the increase in blood pressure and the severity of collagen matrix deposition, in contrast with ACE expression .
Immortalized human podocytes have been shown to contain intracellular AngII, the levels of which were significantly increased following exposure to prorenin . Prorenin-induced intracellular AngII levels were reduced by an ACEi and renin inhibitor, but not by an ARB. The latter observation suggested that AngII was formed intracellularly in human podocytes and was not internalized through AT1 receptors. How prorenin was internalized, activated and interacted with AGT in podocytes is not clear. However, siRNA (small interfering RNA)-induced down-regulation of the PRR attenuated prorenin-induced intracellular AngII generation, suggesting that the PRR is an important component of the intracellular AngII synthesis machinery in human podocytes . A significant role for the PRR in diabetic kidney diseases has been demonstrated previously [79–81]. Similarly, high glucose increased renin-mediated intracellular AngII levels in cultured podocytes [82,83].
Significant levels of AT1 receptors have been demonstrated on nuclei isolated from rat and sheep kidneys [84,85]. Importantly, in Lewis rats, renal cortical nuclei exhibited higher AT1 receptor sites, in comparison with the plasma membrane, whereas receptor density was similar in the medulla . In sheep, nuclei isolated from kidneys of young adult animals showed the AT2 receptor as the predominant receptor (80%), whereas the AT1 receptor accounted for the majority (approximately 85%) of AngII-binding sites in older sheep . The Mas protein, the Ang-(1–7) receptor, was also detected on sheep renal nuclei by immunoblotting . Thus, similar to the heart, intracellular synthesis of AngII and its receptors have been described in the kidney. The renal intracellular RAS and nuclear angiotensin receptor signalling mechanisms have been reviewed recently [86–88].
Other tissues or cell types
Rat VSMCs resemble cardiac myocytes in several aspects regarding the high-glucose-induced intracellular RAS. Although AngII was primarily localized in the culture medium and was blocked by an ACEI in normal-glucose conditions, high glucose significantly increased intracellular AngII levels that were primarily the result of vascular chymase-dependent synthesis . Corroborating these observations, high glucose significantly decreased ACE expression and knockdown of vascular chymase by siRNA prevented the increase in AngII levels. Significantly, cathepsin D, not renin, catalysed the conversion of AGT into AngI in VSMCs .
In Capan-1, a human pancreatic cancer-derived cell line, high levels of AngI and AngII were detected in cells, but not in the medium . Neither AT1 nor AT2 receptors were detected by RT (reverse transcription)–PCR or immunohistochemistry; however, specific binding to the cell membrane was identified for AngII. The authors speculated that intracellular AngII may have a role in pancreatic cancer through non-AT1-receptor-, non-AT2-receptor-binding sites .
In the rat sensory vagal complex, AT1 receptor immunoreactivity was observed in endosome-like granules, Golgi lamellae and outer nuclear membranes in addition to the plasma membrane . These sites also labelled for AngII, suggesting intracrine effects of AngII in the modulation of postsynaptic excitability.
AngII accumulation studies in serum-free culture medium of a bovine aortic endothelial cell line revealed significant levels in the second 24-h period compared with the first. This was accompanied by undetectable levels of AGT and renin in the medium, but significant levels intracellularly. The authors concluded that these cells probably synthesized AngII intracellularly and that it was constitutively secreted .
INTRACRINE MECHANISMS: CANONICAL COMPARED WITH NON-CANONICAL PATHWAYS
As discussed above, angiotensin receptors have been localized on several intracellular organelles, particularly nuclei, in multiple cell types that are primary targets of the actions of AngII. Although earlier studies had demonstrated the direct effects of AngII on isolated nuclei, recent studies have also begun to identify the signalling mechanisms involved. Essentially, nuclei are ‘cells within cells’ that contain major signalling pathways. Thus the signalling pathways activated by AngII through intracellular AT1 and AT2 receptors could be classified as canonical pathways. There is also evidence of actions of AngII through novel interactions with intracellular proteins. These non-AT1-receptor, nonAT2-receptor mechanisms have been categorized as non-canonical pathways. Our categorization of canonical and non-canonical pathways differs from that described in a recent review on a similar topic, in which the intracrine mechanism of several peptide hormones has been discussed . In that review, the authors considered signalling activated by the plasma-membrane receptors as canonical and by cognate intracellular receptors as non-canonical pathways. Nonetheless, the knowledge of intracellular mechanisms of intracrine peptides is essential to understand the full repertoire of their biological functions. An extensive review of GPCR signalling in the cardiac nucleus has been published recently .
In rat VSMCs, both intra- and extra-cellular AngII activated CREB though by different mechanisms. Extracellular AngII activated p38 MAPK and ERK (extracellular-signal-regulated kinase) 1/2, whereas intracellular AngII activated only p38 MAPK . In studies describing the effects of intracellular AngII on cytosolic Ca2+, the production of Ins(1,4,5,)P3 was identified, whereas cell growth effects required the activation of PI3K (phosphoinositide 3-kinase) and MAPK/ERK [28,34,35]. AngII, applied to renal nuclei isolated from Lewis rats, increased intranuclear ROS (reactive oxygen species) generation, which could be blocked by an ARB or NADPH oxidase (NOX) inhibitor . Furthermore, exposure of nuclei to a PKC (protein kinase C) agonist increased ROS, whereas a PKC or PI3K inhibitor abolished the AngII-induced ROS production, suggesting the involvement of an AngII/AT1 receptor/PKC axis at the nuclear level . A Mas receptor antagonist and ACE2 inhibitor exacerbated the AngII-dependent ROS formation in sheep renal nuclei . In isolated nuclei from rat renal cortical cells, AngII increased the transcription of TGF-β1, MCP-1 (macrophage chemoattractant protein-1) and NHE3 (Na+/H+ exchanger 3) in an AT1-receptor-dependent manner; however, the molecular signalling pathways were not investigated . AT2 receptors on sheep renal cortical nuclei coupled to NO production when stimulated with AngII . AngII application to nuclei isolated from cardiac myocytes increased NF-κB expression, which appeared to be mediated by both AT1 and AT2 receptors . Similar to nuclear receptors, AT2 receptors identified in mitochondria of several cell types modulated mitochondrial NO production and respiration . These studies suggested that intracellular AngII elicits similar signalling pathways and responses as extracellular AngII with few exceptions.
Non-canonical pathways encompass the interaction of AngII with intracellular proteins other than known angiotensin receptors, and binding to the nucleolus and chromatin. A possibility of the interaction with novel intracellular proteins has been suggested by the observed effects of intracellular AngII in cells that do not express sufficient levels of AT1 receptor for demonstrable extracellular AngII effects. These cells include CHO-K1 and A7r5 VSMCs, in which intracellular effects of AngII on cell proliferation and [Ca2+]i were not prevented by ARBs [35,43]. Direct binding of AngII to chromatin was demonstrated in some of the early studies of the intracrine actions of AngII [16,97]. A direct interaction with chromatin has been reported for other intracrine factors as well . Recently, a non-AT1-receptor, non-AT2-receptor AngII-binding site in rodent and human brain membranes was identified to be the membrane-bound variant of the metalloendopeptidase neurolysin . The functional significance of the high affinity and specific interaction between neurolysin and AngII remains to be determined. The non-canonical pathways of AngII deserve future investigation to more completely elucidate the intracellular RAS.
The actions of the intracellular RAS and intracrine AngII are slowly being accepted as a reality, rather than just a myth. There is a growing body of literature supporting the existence of this system and its role in various pathophysiological conditions . Significantly, the intracellular RAS is not confined to a particular tissue; instead, there are a number of cell types in different organs that have been shown to have a biologically relevant system. The composition of the intracellular RAS might differ between cell types . Furthermore, the activation of this system is probably affected by the pathophysiological state of an organism. There remain many unanswered questions, particularly those related to the precise site of intracellular AngII synthesis, source and mechanism of renin activation in some tissues, and the nature of intracellular receptors and other binding partners. The role of the intracellular system in association with, or independent of, the extracellular system in normal physiological or in pathological states needs to be determined. These studies might lead to new therapeutic avenues for RAS modulation in conditions such as diabetes.
Our own work was supported by the National Institutes of Health [grant number 5R01HL090817]. This material is the result of work supported with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, TX, U.S.A.
Abbreviations: ACE, angiotensin-converting enzyme; ACEi, ACE inhibitor; AGT, angiotensinogen; Ang-(1–7), angiotensin-(1–7); AngI etc., angiotensin I etc.; ARB, angiotensin receptor blocker; AT1 receptor, AngII type 1 receptor; AT2 receptor, AngII type 2 receptor; [Ca2+]i, intracellular Ca2+ concentration; CHO, Chinese-hamster ovary; CREB, cAMP-response-element-binding protein; ERK, extracellular-signal-regulated kinase; IRAP, insulin-regulated aminopeptidase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor κB; NRVM, neonatal rat ventricular myocyte; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PRR, (pro)renin receptor; PTC, proximal tubule cell; RAS, renin–angiotensin system; ROS, reactive oxygen species; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell
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