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Am J Physiol Heart Circ Physiol 293: H939-H948, 2007. First published May 4, 2007; doi:10.1152/ajpheart.00391.2007
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High-glucose-induced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes

Vivek P. Singh,1 Bao Le,2 Vadiraja B. Bhat,3 Kenneth M. Baker,1 and Rajesh Kumar1

1Division of Molecular Cardiology, Cardiovascular Research Institute, Texas A & M Health Science Center, College of Medicine, Scott & White and Central Texas Veterans Health Care System; and 2Department of Internal Medicine and 3Division of Investigative Pathology, Scott & White, Temple, Texas

Submitted 29 March 2007 ; accepted in final form 3 May 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The prevailing paradigm is that cardiac ANG II is synthesized in the extracellular space from components of the circulating and/or local renin-angiotensin system. The recent discovery of intracrine effects of ANG II led us to determine whether ANG II is synthesized intracellularly in neonatal rat ventricular myocytes (NRVM). NRVM, incubated in serum-free medium, were exposed to isoproterenol or high glucose in the absence or presence of candesartan, which was used to prevent angiotensin type 1 (AT1) receptor-mediated internalization of ANG II. ANG II was measured in cell lysates and the culture medium, which represented intra- and extracellularly synthesized ANG II, respectively. Isoproterenol increased ANG II concentration in cell lysates and medium of NRVM in the absence or presence of candesartan. High glucose markedly increased ANG II synthesis only in cell lysates in the absence and presence of candesartan. Western analysis showed increased intracellular levels of angiotensinogen, renin, and chymase in high-glucose-exposed cells. Confocal immunofluorocytometry confirmed the presence of ANG II in the cytoplasm and nucleus of high-glucose-exposed NRVM and along the actin filaments in isoproterenol-exposed cells. ANG II synthesis was dependent on renin and chymase in high-glucose-exposed cells and on renin and angiotensin-converting enzyme in isoproterenol-exposed cells. In summary, the site of ANG II synthesis, intracellular localization, and the synthetic pathway in NRVM are stimulus dependent. Significantly, NRVM synthesized and retained ANG II intracellularly, which redistributed to the nucleus under high-glucose conditions, suggesting a role for an intracrine mechanism in diabetic conditions.

renin-angiotensin system; hyperglycemia; renin; chymase; intracrine

THE RENIN-ANGIOTENSIN SYSTEM (RAS) is activated in several pathological conditions, e.g., hypertension, diabetes, obesity, and other cardiovascular conditions, contributing to a significant role in the etiology and morbidity associated with these diseases (11, 29, 33, 40). Classically, ANG II is synthesized in the circulation by the sequential actions of kidney-derived renin and endothelium-anchored angiotensin-converting enzyme (ACE) on liver-generated angiotensinogen (AGT). The demonstration of components of the RAS in several tissues has established the concept of a tissue or local RAS, the regulation of which is independent of the systemic RAS (16). In heart, blood pressure-independent cardioprotective effects of angiotensin receptor blockers (ARBs) and ACE inhibitors have demonstrated the significance of the cardiac RAS (13, 38). Locally synthesized ANG II probably acts in an autocrine/paracrine manner via binding to plasma membrane angiotensin type 1 (AT1) receptors, which are present on most cells. However, the site of ANG II synthesis (intracellular and/or extracellular) has been a subject of debate (7). The existing paradigm is that ANG II is synthesized in the interstitial space from components of local and/or circulatory origin. The recent demonstration by others and us (2, 3, 10) of intracrine effects of ANG II has again raised the question regarding the site of ANG II synthesis. As an intracrine peptide, ANG II has been shown to act from an intracellular location, resulting in cardiac myocyte growth, cardiac hypertrophy, and proliferation in several cell lines (2, 3, 10, 19). In these earlier studies, ANG II was introduced intracellularly by microinjection or recombinant means. In a physiological setting, ANG II is internalized through AT1 receptor-mediated endocytosis, resulting in lysosomal degradation and/or intracellular redistribution to locations such as the nucleus (24, 52, 53). Several studies have suggested that ANG II is synthesized intracellularly (12, 35, 41, 44, 54); however, a direct demonstration of this process is lacking. The question of intracellular ANG II synthesis is important not only to elucidate the intracrine system but, also, from the perspective of therapeutic inhibition of the local RAS by commonly used ACE inhibitors and the potential use of renin and chymase inhibitors. The involvement of renin and ACE in intracellular synthesis of ANG II would require further study in view of the secretory and membranous nature of these enzymes. Here, we demonstrate, for the first time, that cardiac myocytes synthesize significant amounts of ANG II intracellularly, the secretion and intracellular localization of which are stimulus specific. We also demonstrate an alternative ANG II synthesis pathway in cardiac myocytes in response to high-glucose conditions.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Neonatal rat ventricular myocytes (NRVM) were isolated from hearts of 0- to 2-day-old Sprague-Dawley rat pups and cultured in growth medium containing 5.5 mM glucose, as previously described (2). NRVM were exposed to 10 µM isoproterenol or high-glucose (25 mM) medium after 24 h in serum-free medium. To correct for hyperosmolarity, we added equivalent amounts of mannitol to control sets of cells. To exclude internalization of ANG II through AT1 receptor-mediated endocytosis, we exposed the NRVM to the agents in the absence or presence of 1 µM candesartan (AstraZeneca).

Enzyme inhibition. To identify the enzymes involved in ANG II synthesis, we added specific inhibitors [aliskiren (a renin inhibitor, 1–100 µM; Novartis), benazeprilat (an ACE inhibitor, 0.1–10 µM), and chymostatin (a chymase inhibitor, 1–100 µM)] to the culture medium at the time of isoproterenol or high-glucose exposure.

ANG II measurement. ANG II was measured in cell lysates and the culture medium by a quantitative, competitive ELISA using a specific anti-ANG II antibody (Peninsula Laboratories), as previously described (2).

HPLC-chip/mass spectrometric analysis. To validate the results of ELISA, ANG II content in cell lysates of high-glucose-exposed cells was analyzed by tandem mass spectrometry (MS). ANG II was immunoprecipitated using an agarose bead-coupled monoclonal anti-ANG II antibody (Biogenesis), and coeluting proteins were removed by passage through a Microcon-3K filter. Samples were dried in a Speedvac and redissolved in 3% acetonitrile + 0.1% trifluoroacetic acid. A 0.1-µl aliquot of each sample was injected into an ion trap mass spectrometer (1100 series HPLC-Chip-LC/MSD Trap XCT Ultra, Agilent Technologies). The liquid chromatography-MS/MS analysis was performed as reported previously, with minor modifications (26). Peptides were loaded onto the enrichment column with 97% solvent A (water with 0.1% formic acid) and 3% solvent B (acetonitrile with 0.1% formic acid) at 4 µl/min. Elution was performed with a gradient of solvent B (3–80% in 7 min) at a flow rate of 0.3 µl/min. Data-dependent MS/MS analysis was performed on the six most intense peaks in each full-scan spectrum, as reported previously (26). Spectrum Mill Server software (revision A.03.02) (5, 26) was used to search tandem MS spectra against a custom peptide database that contained angiotensin peptides. The relative abundance of ANG II in high- and low-glucose-exposed cells was calculated using total ion intensities of the extracted ion chromatogram.

Western analysis. NRVM were exposed for 24, 48, and 72 h to high glucose (25 mM) or isoproterenol (10 µM) as described above. Equal amounts of cell lysates or culture medium proteins (30 µg) were separated on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes as previously described (44, 45). Blots were probed with anti-angiotensinogen (Swant), anti-renin (Swant), anti-chymase (Abcam), or anti-ACE-1 (Abcam) antibody. Equal protein loading was confirmed by anti-GAPDH or anti-actin antibody for cell lysates and Coomassie staining for culture medium. Protein bands were detected using anti-mouse and anti-rabbit secondary antibodies labeled with infrared dye 680/800 and the enhanced Odyssey Infrared Imaging System (LI-COR, Biosciences).

Confocal immunofluorescence microscopy. NRVM, grown on two-well, collagen-coated chamber slides at a density of 0.25 x 106 cells/well, were exposed as described above and immunostained as described previously (2) using Alexa Fluor 594-conjugated phalloidin (Molecular Probes) and anti-ANG II antibody (Peninsula Laboratories) overnight at 4°C and then incubated with Alexa 488-labeled goat anti-rabbit IgG (Invitrogen). Specificity of the anti-ANG II antibody was confirmed by preadsorption with 10 molar excess of synthetic ANG II. Images were visualized with a x60 objective on a three-dimensional confocal fluorescence microscope (Olympus Fluoview 300). To study internalization of ANG II, NRVM were exposed to Alexa 488-labeled ANG II (Molecular Probes; see Flow cytometry) before visualization with the confocal microscope.

Flow cytometry. Alexa 488-labeled ANG II (50 nM) was added to NRVM culture medium at the time of exposure to isoproterenol and high glucose, as previously described. After 24 h, cells were washed with PBS, detached, and analyzed by BD FACScan Calibur for the presence of labeled ANG II. Chinese hamster ovary (CHO) cells stably expressing high levels of AT1 receptor (CHO-AT1 cells, 5,399 ± 1,229 fmol/mg protein) (3) were used as a positive control. CHO-AT1 cells were cultured as previously described (3), exposed to Alexa 488-labeled ANG II for 24 h, and analyzed by flow cytometry.

Statistical analysis. Values are means ± SE. ANOVA with Tukey's post hoc test was used for statistical analysis. P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The basal rate of ANG II synthesis in cardiac myocytes is very low. The ANG II detectable intracellularly in unstimulated cardiac myocytes could be due to AT1-mediated internalization or intracellular synthesis. To circumvent the former, we exposed NRVM to agents that stimulated de novo ANG II synthesis but added the AT1 antagonist candesartan to block any cellular uptake. Under these conditions, an increase in ANG II above the basal levels in cell lysates was considered to be due to intracellular synthesis. To confirm the absence of significant ANG II internalization, we detected the uptake of Alexa Fluor 488-labeled ANG II added to the cells at the time of exposure to isoproterenol and/or high glucose. The cells were analyzed after 24 h by flow cytometry and confocal microscopy. Additionally, 1–25 µM candesartan was used to study the effect on extra- and intracellular ANG II levels by ELISA. The results (supplemental data for this article are available online at the American Journal of Physiology-Heart and Circulatory Physiology website) showed that NRVM internalized very little ANG II compared with the amount of ANG II that is synthesized, probably as a result of low AT1 expression (50), which was significantly inhibited by candesartan. The results presented here are from NRVM that were separated from nonmyocytes by double-differential plating. Intracellular ANG II synthesis was confirmed on Percoll gradient-purified myocytes as well (data not shown).

Isoproterenol increases intracellular synthesis of ANG II. Isoproterenol increased ANG II levels in cell lysates, as well as in the culture medium of NRVM (61 ± 7 and 88 ± 3%, respectively; Fig. 1, A and B), compared with control. Intracellular ANG II levels were also significantly increased in the presence of candesartan (94 ± 10%), confirming that ANG II was synthesized intracellularly, rather than internalized through the AT1 receptor. Isoproterenol also increased ANG II concentration in culture medium in the presence of candesartan (133 ± 4%; Fig. 1B). The latter could be due to secretion of intracellularly synthesized ANG II or resultant extracellular synthesis from secreted AGT.


Figure 1
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  		Fig. 1. Isoproterenol and high glucose stimulate intracellular ANG II synthesis in neonatal rat ventricular myocytes (NRVM). NRVM were grown in serum-free normal glucose medium [5.5 mM glucose (Cont and NG)] for 24 h and exposed to 10 µM isoproterenol (Iso), 25 mM glucose (HG), or 5.5 mM glucose + 19.5 mM mannitol (NG + M) in the absence or presence of 1 µM candesartan. A quantitative competitive ELISA was used to measure ANG II in cell lysates (A and C) and culture medium (B and D). Values are means ± SE from 3 independent experiments performed in duplicate. *P < 0.05 vs. Cont (A and B) or NG (C).

 
High glucose increases intracellular synthesis of ANG II. High glucose significantly increased intracellular ANG II levels in the cell lysates of NRVM (605 ± 26%) compared with control cells (Fig. 1C). The high-glucose-induced increase in ANG II synthesis (492 ± 36%) was also observed in the presence of candesartan, confirming that the increase was due to intracellular synthesis, rather than uptake. Interestingly, extracellular ANG II levels were not significantly affected by high glucose (16 ± 5 and 22 ± 5% in the absence and presence of candesartan, respectively; Fig. 1D). Mannitol, which was used as a control for osmolarity, had no significant effect on ANG II concentrations (14 ± 3 and 5 ± 8% in cell lysates and medium, respectively). These observations indicated that, in the presence of high glucose, ANG II was synthesized, as well as retained intracellularly, by NRVM.

Quantification of ANG II in cell lysates by MS. To confirm the specificity of the ANG II measurement by ELISA, we performed tandem MS analysis of the cell lysates after anti-ANG II antibody immunoprecipitation. The 19.4 ± 7-fold increase in ANG II (Fig. 2 ) in high-glucose-exposed cells compared with control cells corroborated the ELISA values.


Figure 2
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  		Fig. 2. Mass spectrometric (MS) measurement of intracellular ANG II in NRVM exposed to high glucose. NRVM were grown in 5.5 or 25 mM glucose medium for 24 h. ANG II was immunoprecipitated using anti-ANG II antibody, and coeluting proteins were cleared from the sample. Samples were subjected to liquid chromatographic-MS/MS analysis. Samples 1, 2, and 3 are from 3 independent experiments. A: extracted ion chromatogram (EIC) of ANG II peptide, with normal medium stained blue and high-glucose medium stained red. B: MS spectrum at 3.2 min showing doubly charged ANG II peptide [mass-to-charge ratio (m/z) = 523.8, MH + 2]. C: collision-induced dissociation spectrum of ANG II peptide (m/z = 523.8, MH + 2).

 
Dose response and time course of stimulated ANG II synthesis by high glucose. To demonstrate that enhanced ANG II production is dependent on the concentration and duration of exposure to high glucose, we incubated NRVM in medium containing 5.5, 12.5, 17, or 25 mM glucose for 24 h. Mannitol was added to the control cell incubation medium to equalize osmolarity. Intracellular ANG II increased dose dependently in response to high glucose (Fig. 3A), but ANG II in the medium did not change significantly (Fig. 3B). We incubated cells in 5.5 or 25 mM glucose medium for 12, 24, 48, and 72 h. Intracellular ANG II levels in high-glucose-exposed cells increased as early as 12 h and continued to increase until 72 h (Fig. 3C), whereas extracellular ANG II showed a small, but significant, increase at 12 h, but not at 24, 48, and 72 h (Fig. 3D).


Figure 3
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  		Fig. 3. Time and dose dependency of high-glucose-stimulated intracellular ANG II synthesis. A and B: NRVM grown in culture medium containing 5.5–25 mM glucose for 24 h. Mannitol was used as a control for hyperosmolarity. C and D: NRVM grown in 5.5 or 25 mM glucose medium for 12–72 h. ANG II was measured in cell lysates (A and C) and culture medium (B and D) as described in Fig. 1 legend. Values are means ± SE from 3 separate experiments performed in duplicate. *P < 0.05 vs. mannitol (A and B) or NG (C and D).

 
Intracellular localization of ANG II in NRVM. To confirm the above-mentioned results and determine the localization of intracellularly synthesized ANG II, we stimulated NRVM with isoproterenol or high glucose, costained them with anti-ANG II antibody and phalloidin, and visualized them via confocal microscopy. In control cells, cytoplasmic ANG II staining was very faint (Fig. 4). Staining was more intense in isoproterenol-exposed cells, and a dramatically high level of staining was observed in high-glucose-exposed cells, which correlated with the intracellular ANG II levels detected by ELISA. In isoproterenol-stimulated cells, the majority of ANG II colocalized with actin filaments, possibly in secretory granules. Some diffuse cytoplasmic and nuclear staining was also observed. In high-glucose-exposed cells, some of the ANG II staining was visualized along the actin filaments, whereas the majority of staining was in the perinuclear and nuclear regions. A few cells showed mainly nuclear, punctate staining (see supplemental data). Peptides of up to ~40 kDa (~9 nm) can freely pass through the nuclear membrane of eukaryotic cells (30, 62). ANG II is only eight amino acids long and ~1 kDa and, thus, probably does not require a nuclear localization signal. From visualization along the complete z-axis of the cell, we confirmed that ANG II was localized inside the cell, rather than on the cell surface.


Figure 4
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  		Fig. 4. Immunofluorocytometric localization of intracellular ANG II by confocal microscopy. NRVM were grown in 5.5 mM glucose (a–d) or 25 mM glucose (i–l) medium or exposed to 10 µM isoproterenol (e–h) in the presence of 1 µM candesartan for 24 h. Cells were costained for nucleus (blue; a, e, and i), ANG II (green; b, f, and j), and cytoplasmic actin (red; c, g, and k). Cells were visualized using z-axis optical sectioning through a x60 objective in a confocal microscope. Images, shown in different channels, represent the same z-axis plane. Staining for ANG II and actin filaments is very faint in NRVM grown in 5.5 mM glucose and more intense in cells grown in 25 mM glucose and exposed to isoproterenol. ANG II colocalizes with actin filaments in isoproterenol-exposed cells, whereas a significant amount of ANG II is present in the cytoplasm and nucleus of cells exposed to 25 mM glucose. Identical ANG II staining was observed in the absence of candesartan. DAPI, 6-diamidino-2-phenylindole.

 
High glucose increases intracellular AGT, renin, and chymase in NRVM. De novo synthesis of ANG II intracellularly would require the presence of components of the RAS intracellularly. Additionally, the levels of these components would need to be regulated by the stimuli that modulate ANG II levels. To test this hypothesis, we analyzed AGT, renin, ACE, and chymase in cell lysates and the medium of NRVM exposed to isoproterenol or high glucose for 24, 48, and 72 h by Western blot. AGT increased significantly in cell lysates of high-glucose-exposed cells compared with control cells at all time points (Fig. 5A), whereas the levels in the medium decreased (Fig. 5B). Similarly, renin levels were significantly increased in cell lysates but did not change in the medium (Fig. 5, C and D). ACE levels were not altered by high glucose exposure (Fig. 5E). Interestingly, chymase levels increased significantly in cell lysates from high-glucose-exposed cells compared with control (Fig. 5F), although chymase levels in the medium were not detectable. The above-described observations confirm de novo intracellular ANG II synthesis and regulation by NRVM in response to high glucose.


Figure 5
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  		Fig. 5. Western analysis of renin-angiotensin system (RAS) components in NRVM after exposure to high glucose. NRVM were grown in 5.5 or 25 mM glucose medium for 24, 48, and 72 h. Cell lysates (A, C, E, and F) and culture medium (B and D) were analyzed by Western blotting for changes in the level of angiotensinogen (AGT, A and B), renin (C and D), angiotensin-converting enzyme (ACE, E), and chymase (F) using specific antibodies. ACE and chymase were not detectable in the medium (not shown). Equal protein loading was confirmed by GAPDH or actin detection in cell lysates and by Coomassie staining of medium (not shown). Densitometric data were obtained from 3 separate experiments. With renin, quantitative data represent ~35-kDa band. A representative blot of each analysis, with the position of molecular weight markers, is shown. Values are means ± SE. *P < 0.05 vs. NG.

 
In contrast to high glucose, isoproterenol exposure resulted in only small changes in AGT (nonsignificant) and renin (significant) intracellularly, whereas a significantly greater increase was observed for both in the medium (Fig. 6, A–D). There was no change in chymase (in contrast to our finding after glucose exposure) and ACE expression after isoproterenol exposure (Fig. 6, E and F).


Figure 6
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  		Fig. 6. Western blot analysis of RAS components in NRVM after exposure to isoproterenol. NRVM were exposed to 10 µM isoproterenol for 24, 48, and 72 h. Cell lysates (A, C, E, and F) and culture medium (B and D) were analyzed by Western blot for changes in levels of AGT (A and B), renin (C and D), ACE (E), and chymase (F) using specific antibodies. ACE and chymase were not detectable in the medium (not shown). Equal protein loading was confirmed by GAPDH or actin detection in cell lysates and by Coomassie staining of medium (not shown). Densitometric data were obtained from 3 separate experiments. With renin, quantitative data represent ~35-kDa band. A representative blot of each analysis, with the position of molecular weight markers, is shown. Values are means ± SE. *P < 0.05 vs. Cont.

 
Enzymes involved in intracellular ANG II synthesis by NRVM. Differential cellular distribution of ANG II in isoproterenol- and high-glucose-exposed cells and increased levels of chymase with high-glucose exposure suggested involvement of distinct ANG II synthesis mechanisms with these stimuli. To further elucidate the synthesis pathways, we measured ANG II levels in cells exposed to isoproterenol and/or high glucose in the presence of specific inhibitors of renin (aliskiren), ACE (benazeprilat), and chymase (chymostatin). Aliskiren, a very potent inhibitor of human renin, has also been shown to inhibit rat renin (57, 58). Aliskiren is internalized by NRVM, resulting in concentrations that are about threefold higher intracellularly than in the medium 24 and 48 h after addition (data not shown). Benazeprilat ranks at the top of the relative order of tissue potency of the ACE inhibitors, suggesting efficient membrane permeability (18). Chymostatin was solubilized in DMSO, which enhances cell penetration of the drug (61). Thus the above-described inhibitors were suitable to study the enzymes involved in intracellular ANG II synthesis. Isoproterenol-induced ANG II synthesis in cell lysates and medium was completely blocked by aliskiren and benazeprilat, indicating a conventional pathway (Fig. 7, A and B). In high-glucose-exposed cells, aliskiren and chymostatin inhibited intracellular ANG II synthesis, whereas benazeprilat had no effect (Fig. 7C). Although there was no significant increase in extracellular ANG II levels with exposure to high glucose, basal levels were reduced by renin and ACE inhibitors, suggesting that extracellular ANG II synthesis is ACE, not chymase, dependent (Fig. 7D). These results indicate different sites and pathways of ANG II synthesis in response to high glucose and isoproterenol.


Figure 7
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  		Fig. 7. Enzymes involved in ANG II synthesis. NRVM were exposed to isoproterenol (A and B) or high glucose (C and D) in the presence of 1, 10, 30, and 100 µM aliskiren (a renin inhibitor), 0.1, 1, and 10 µM benazeprilat (an ACE inhibitor), and 10 and 100 µM chymostatin (a chymase inhibitor). ANG II was measured in cell lysates (A and C) and culture medium (B and D), as described in Fig. 1 legend. Data are from a single inhibitor concentration that completely blocked the stimulated increase in ANG II synthesis. Values are means ± SE from 3 separate experiments performed in duplicate. *P < 0.05 vs. Iso (A and B) or HG (C and D) without inhibitor.

 
Intracellular ANG II is a positive regulator of AGT expression. Feedback regulation of the cardiac RAS by its components is an established mechanism. To determine whether intracellular ANG II functions similarly as a regulator, we studied the effect of inhibition of intracellular ANG II synthesis on AGT expression. NRVM were exposed to high glucose in the absence or presence of aliskiren (renin inhibitor), and AGT levels were determined by Western blot. Compared with control cells, the high-glucose-induced increase in AGT was blocked by aliskiren (Fig. 8A). Aliskiren did not have a significant effect on AGT expression in cell lysates of control cells but did reduce AGT significantly in the medium (Fig. 8B).


Figure 8
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  		Fig. 8. Intracellular ANG II regulates AGT protein expression in NRVM. NRVM were grown in 5.5 or 25 mM glucose medium for 24 h in the absence or presence of 10 µM aliskiren. AGT protein expression was determined in cell lysates (A) and culture medium (B) by Western blot analysis. Equal protein loading was confirmed by GAPDH or Coomassie staining in cell lysates and medium, respectively. A representative blot with the position of the molecular weight marker is shown. Values are means ± SE of 2 independent experiments. *P < 0.05 vs. NG. **P < 0.05 vs. HG.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have demonstrated that ANG II synthesis, intracellular localization, and the synthetic pathway in NRVM are dependent on the nature of the stimulus. Significantly, after exposure to high glucose, NRVM synthesize and retain ANG II intracellularly, with redistribution of the peptide to the nucleus. In contrast to the circulating RAS, intracellular ANG II synthesis in NRVM is renin and chymase, not ACE, dependent. This is the first study to demonstrate that endogenous renin, which is produced by NRVM, contributes significantly to local ANG II production. Additionally, we provide substantive evidence that chymase is the major ANG II-producing enzyme when NRVM are exposed to high glucose.

Earlier studies suggested that ANG II can be produced intracellularly in different cell types; however, this finding has not been directly demonstrated under pathophysiological conditions. Cultured NRVM have been shown to release ANG II after mechanical stretch; however, it was suggested that this was internalized and stored ANG II (7, 44). High glucose was reported to decrease renin secretion and increase intracellular renin activity, resulting in increased ANG II generation by rat mesangial cells; however, the site of ANG II production was not identified (54). A study of high-glucose-induced superoxide accumulation in human mesangial cells demonstrated an increase in ANG II in cell lysates, but not in the culture medium (35). Indirect evidence based on the effects of intracellular dialysis of renin and ACE inhibitors on gap junction conductance in isolated cardiac myocytes of cardiomyopathic hamsters was also presented (12). Increased levels of intracellular ANG II were reported in rat myocytes after incubation with exogenous prorenin of mouse ren-2d gene origin (41). Although the above evidence was certainly suggestive, intracellular ANG II generation was not directly demonstrated, largely because of difficulty in differentiating extracellularly generated and internalized ANG II from that synthesized intracellularly.

To demonstrate intracellular ANG II synthesis, we hypothesized that it was important to stimulate the RAS, inasmuch as the low basal activity of the system under nonstimulated conditions would preclude a reliable determination of changing ANG II levels. We used two physiologically relevant stimuli, sympathetic activation and hyperglycemia, to activate the RAS in NRVM. In vitro culture conditions provided the milieu to demonstrate that all components of the RAS were of endogenous origin. We excluded the possibility that extracellularly synthesized ANG II contributed to the intracellular pool by measuring uptake of Alexa 488-labeled ANG II in the absence or presence of candesartan in cells exposed to isoproterenol or high glucose. We observed very little (by confocal microscopy) or undetectable (by fluorescence-activated cell sorting) uptake of ANG II by NRVM, which was attenuated by 1 µM candesartan. This finding is in agreement with the previously reported very low level of AT1 receptor expression in these cells (50). The latter finding was further confirmed by our observation of significant uptake of labeled ANG II by AT1-transfected CHO cells. Additionally, increasing candesartan concentrations from 1 to 25 µM had no effect on intracellular ANG II in glucose-exposed NRVM (see supplemental data). The authenticity of ANG II, as determined by competitive ELISA, was confirmed by MS analysis. Confocal microscopy demonstrated that ANG II was not associated with the cell surface but, rather, was located intracellularly.

The expression of RAS components by cardiac myocytes has been demonstrated at the message and the protein level by several investigators, including us (4, 16, 17, 36, 48, 49). Past skepticism regarding intracellular synthesis of ANG II arose from the fact that AGT, which contains a signal peptide, is normally secreted into the interstitial space. A similar argument was advanced regarding renin, even though an intracellular form of active renin has been demonstrated in several tissues (8, 34). ACE is a membrane-bound enzyme with extracellular catalytic sites. Recently, however, active N-domain forms of ACE have been localized intracellularly in rat mesangial cells (6). In the present study, we observed an increase in the intracellular accumulation of AGT and renin in high-glucose conditions; however, the mechanism of this accumulation is not known. A signal peptide is required for endoplasmic reticulum targeting; however, intracellular sorting of the proteins largely depends on the glycosylation pattern (25). Interestingly, a human astrocytoma cell line produces a nonglycosylated form of AGT that is targeted to the nucleus (47). The glycosylation of the proteins is dependent on the metabolic state of the cells, which is significantly affected by hyperglycemia (60). In addition, intracellular calcium is an important mediator of protein secretion that has been well studied with regard to the enzyme renin. Increased intracellular calcium concentrations have been reported to stimulate renin secretion (22). Hyperglycemia has been shown to decrease calcium entry in NRVM and to depress intracellular calcium transients in adult cardiac myocytes of diabetic rats (14, 39). Thus the effect of high glucose on calcium homeostasis in these cells might be responsible for the intracellular retention of renin, as was observed in our studies. Significantly, in rat mesangial cells, exposure to high glucose has been shown to increase intracellular accumulation and activity of renin (54).

In cardiac myocytes, is the complete or truncated (exon 1A) (8) prorenin responsible for ANG II synthesis? In Western blots, using anti-renin antibody, we detected three bands (~52, 50, and 35 kDa) in cell lysates and the medium from NRVM. The intensity of the 35-kDa band (probably the active renin) was most clearly increased by exposure to high glucose. The 52- and 50-kDa bands possibly represent prorenin and the alternatively spliced form of prorenin, respectively, the latter of which has been shown to be expressed in cardiac myocytes (8). The band representing active renin was unlikely of exogenous origin, inasmuch as the intensity was increased by high glucose in NRVM grown in serum-free medium. However, it remains unclear how prorenin was activated to renin in cardiac myocytes. Recently, the expression of kallikrein-like prorenin-converting enzyme (PRECE) was shown to be increased in ischemic and diabetic myocardium and to contribute to the activation of the RAS in these conditions (28). Although the types of cardiac cells that express PRECE and the cellular localization were not determined in that study, the involvement of PRECE in our studies is possible. Our studies demonstrate that NRVM produce endogenous renin, which contributes to ANG II generation in high-glucose conditions.

The role of chymase in cardiac ANG II generation and the physiological significance of this conversion pathway have been subjects of discussion (1, 15, 32, 37). In this study, we did not observe a change in the levels of ACE in response to elevated glucose; however, chymase levels increased significantly in lysates from cells exposed to high glucose. The chymase inhibitor chymostatin completely prevented intracellular ANG II synthesis induced by exposure to high glucose. Interestingly, there was no effect of isoproterenol on chymase expression, suggesting that this enzyme participates in cardiac ANG II synthesis in selective pathophysiological conditions. It was also recently demonstrated that vascular and renal ANG II production in diabetic patients is chymase dependent (27, 31). This latter observation corroborates our observations and lends support to an intracrine mechanism of action of ANG II in diabetes.

From these studies using NRVM, it is apparent that the site of ANG II production and distribution (intracellular or extracellular) and the conversion enzymes involved (chymase or ACE) are dependent on the nature of the stimulus. Isoproterenol-stimulated expression and secretion of RAS components, which probably interacted in secretory granules, resulted in intra- and extracellular ANG II synthesis. The involvement of ACE, ANG II localization along actin filaments, and the increase in extracellular ANG II after isoproterenol exposure suggest that peptide was synthesized in the secretory pathway. An increase in AGT and renin in the medium was suggestive of additional ANG II production extracellularly. In contrast, in cells exposed to high glucose, the involvement of chymase, cytoplasmic and nuclear ANG II localization, and no detectable change in ANG II levels in the extracellular medium indicate that synthesis was solely cytoplasmic. These experiments also suggest that, in high-glucose conditions, AGT and renin did not enter or were removed from the secretory pathway before cleavage of AGT to smaller peptides.

The functional significance of intracellular ANG II has been demonstrated by us and others (2, 3, 9, 12, 20). Cardiac myocytes from diabetic patients showed a 3.4-fold increase in ANG II labeling compared with nondiabetic subjects and an additional 2-fold increase in diabetic patients with hypertension (23). Additionally, the above and other studies suggest (12, 21, 23) that our observation of intracellular ANG II synthesis in neonatal cells would likely be applicable to adult cells as well. In the present study, we observed that inhibition of intracellular ANG II synthesis by aliskiren prevented the increase in AGT levels induced by high glucose. This latter finding strongly suggests that intracellular ANG II positively regulates AGT gene expression, as was originally hypothesized by Re (42, 43).

Clinical relevance. The demonstration of intracellular ANG II synthesis in cardiac myocytes is of major significance. These findings provide strong support for our observations that ANG II can act as an intracrine peptide, resulting in myocyte growth and cardiac hypertrophy (2). Additionally, the retention and nuclear distribution of ANG II by high glucose suggest a significant contribution of the intracrine system in association with conditions such as diabetes. In diabetes, the systemic and local RAS are activated and contribute to diabetic cardiomyopathy (21, 23). ARBs and ACE inhibitors would block the extracrine (circulating and autocrine/paracrine), but not the intracrine, system. This is supported by our observation that the cardiac hypertrophy induced by intracellular ANG II is not inhibited by AT1 receptor antagonists (2). Additionally, as we report here, chymase, rather than ACE, is the enzyme responsible for intracellular ANG II synthesis in high-glucose conditions; thus ACE inhibitors would probably be ineffective in blocking the intracrine effects of ANG II. The beneficial effects of ACE inhibitors and ARBs may involve RAS-independent mechanisms, such as an effect on the kallikrein-kinin system and peroxisome proliferator-activated receptor-{gamma} (46, 55, 59). Finally, consideration should be given to the concept that ACE inhibitors and ARBs, observed in other studies, may not provide as much reduction in negative cardiovascular outcomes as anticipated (51, 56), suggesting that inhibition of the intracrine RAS may provide additional clinical benefits. This study suggests consideration of new therapeutic strategies that would result in blockade of the extra- and intracellular RAS. Renin inhibition appears likely to inhibit the intracrine RAS in cardiac myocytes and may provide a more efficacious approach to preventing or treating cardiovascular complications in diabetic patients. Since renin is involved in the synthesis of intra- and extracellular ANG II, a renin inhibitor would be a particularly attractive therapeutic modality in situations where the intracrine system is activated. In conclusion, we demonstrate, for the first time, the existence of an intracellular RAS that is activated and regulated at the cellular level, independent of the circulating or local RAS, and is differentially regulated by sympathetic activity and high-glucose conditions.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by an American Heart Association, Texas Affiliate, Beginning Grant-in-Aid (R. Kumar).


   ACKNOWLEDGMENTS
 
We thank the Proteomics Core Facility at Scott & White Memorial Hospital for performing mass spectrometry analysis of the samples.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Kumar, Cardiovascular Research Institute, Texas A & M HSC, College of Medicine, 1901 South 1st St., Bldg. 205, Temple, TX 76504 (e-mail: kumar{at}medicine.tamhsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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