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Am J Physiol Cell Physiol 290: C420-C426, 2006. First published September 21, 2005; doi:10.1152/ajpcell.00409.2004
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RECEPTORS AND SIGNAL TRANSDUCTION

Distinct roles for ANG II and ANG-(1–7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes

Patricia E. Gallagher, Mark C. Chappell, Carlos M. Ferrario, and E. Ann Tallant

The Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Submitted 18 August 2004 ; accepted in final form 19 September 2005


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE that preferentially forms angiotensin-(1–7) [ANG-(1–7)] from angiotensin II (ANG II). Incubation of neonatal rat cerebellar or medullary astrocytes with ANG II reduced ACE2 mRNA by ~60%, suggesting transcriptional regulation of the enzyme. In contrast, ANG II had no effect on ACE mRNA in astrocytes isolated from either brain region, demonstrating a differential regulation of the two enzymes by ANG II. The ANG II-mediated reduction in ACE2 mRNA was blocked by the angiotensin type 1 (AT1) receptor antagonists losartan or valsartan; the angiotensin type 2 (AT2) antagonist PD123319 was ineffective. The reduction in ACE2 mRNA by ANG II also was associated with a 50% decrease in cerebellar and medullary ACE2 protein, which was blocked by losartan. Treatment of medullary astrocytes with ANG-(1–7), the product of ACE2 hydrolysis of ANG II, did not affect ACE2 mRNA; however, ANG-(1–7) prevented the ANG II-mediated reduction in ACE2 mRNA. The addition of [D-Ala7]-ANG-(1–7), a selective AT(1–7) receptor antagonist, blocked the inhibitory actions of ANG-(1–7). These data are the first to demonstrate transcriptional regulation of ACE2 by ANG II and ANG-(1–7). Because ACE2 preferentially converts ANG II to ANG-(1–7), downregulation of the enzyme by ANG II constitutes a novel positive feed-forward system within the brain that may favor ANG II-mediated neural responses. Furthermore, the modulatory role of ANG-(1–7) in the transcriptional regulation of ACE2 by ANG II suggests a complex interplay between these peptides that is mediated by distinct receptor systems.

central nervous system; glia; angiotensin receptor

THE RENIN-ANGIOTENSIN SYSTEM is a primary regulator of blood pressure, homeostasis, and cell growth. The major bioactive components of the renin-angiotensin system are produced from the conversion of the precursor angiotensinogen to the decapeptide angiotensin I (ANG I). The cascade diverges with the processing of ANG I to the peptide hormones, ANG II and ANG-(1–7), products with different carboxy termini and contrasting biological actions mediated by distinct high-affinity membrane receptors. ANG II acts through two pharmacological classes of G protein-coupled receptors, AT1 and AT2 receptors (4, 8), whereas the G protein-coupled receptor mas was recently identified as a functional ANG-(1–7) receptor (37, 44, 46). In addition to the classic endocrine system, the renin-angiotensin system is active within tissues, resulting in the local synthesis, release, and action of angiotensin peptides (1, 6, 11).

Angiotensin-converting enzyme 2 (ACE2) is a newly identified component of the renin-angiotensin system that catalyzes the conversion of ANG I to ANG-(1–9) (48, 50, 52). More important, ACE2 converts the vasoconstrictor and growth promoter ANG II to ANG-(1–7), a peptide with vasodilator and anti-proliferative properties (11, 36, 42). ACE2 exhibits a high catalytic efficiency for this reaction, almost 500-fold greater than that for the conversion of ANG I to ANG-(1–9) (52). From an array of >120 peptides, only dynorphin A and apelin 13 were hydrolyzed by ACE2 with comparable kinetics to the conversion of ANG II to ANG-(1–7). ACE2 thus provides an apparent mechanism to directly balance the levels of ANG II and ANG-(1–7) to modulate the pressor/mitogenic and depressor/growth inhibitory arms of the renin-angiotensin system.

ACE2 was originally characterized as a homolog of ACE, sharing ~42% nucleotide sequence homology with conservation of active site residues (10, 48). Similar to ACE, ACE2 is present in a wide variety of cells and tissues with high concentrations in cardiorenal and gastrointestinal tissues and limited expression in the central nervous system and lymphoid tissues (17, 19). Although ACE and ACE2 are type I glycoproteins, there are a notable differences. Both enzymes have two domains; however, ACE has two catalytic sites, whereas ACE2 has only one. ACE2 is a carboxymonopeptidase with a preference for hydrolysis between a proline and carboxy-terminal hydrophobic or basic residues, whereas ACE cleaves two amino acids from its substrate (53). ACE2 also shares a ~48% sequence homology of its COOH domain with the 35-kDa protein collectrin; however, collectrin lacks the carboxypeptidase domains found in ACE and ACE2 (54). Important clinically, ACE2 activity is not directly affected by ACE inhibitors (48); however, we showed that treatment of rats with the ACE inhibitor lisinopril caused a marked upregulation of cardiac ACE2 mRNA (21), indicating that ACE inhibitors indirectly affect the enzyme. In the present study, the transcriptional regulation of ACE2 by its substrate ANG II or product ANG-(1–7) was examined in neonatal rat cerebellar or medullary astrocytes as a first step in the evaluation of the mechanisms that regulate expression of this enzyme.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Preparation of astrocytes. Timed pregnant Sprague-Dawley rats were obtained from Charles River Laboratories and maintained in an Analytical Laboratory Accreditation Criteria Committee animal facility at our institution. Primary cultures of astrocytes were prepared from the cerebellum and medulla oblongata of neonatal pups by physical dissociation as previously described (47). Astrocytes were incubated routinely for 24 h in media depleted of serum before treatment with angiotensin peptides or receptor antagonists as indicated. The purity of the cultures was determined using antibodies to glial fibrillary acidic protein as well as for neurons, fibroblasts, or oligodendrites as described previously (47). Cells isolated by this procedure were routinely >95% positive for glial fibrillary acidic protein. All experimental procedures approved by the Institutional Animal Care and Use Committee.

ACE2 antibody. A polyclonal antibody to rat ACE2 was generated in rabbits against a 19-amino acid peptide from the NH2-terminal region of the enzyme. The peptide sequence had no homology with ACE, collectrin, or other proteins as assessed by the National Institutes of Health Basic Local Alignment Search Tool database. An NH2-terminal cysteine residue was added, and the peptide was conjugated to maleimide-activated keyhole limpet hemocyanin. Male New Zealand rabbits were immunized over an 8-wk period, and whole serum was used for Western blot hybridization. The specificity of the antibody was verified by the visualization of immunoreactive bands of the appropriate size compared with purified rat and human ACE2.

Preparation of cell lysates and Western blot hybridization. Cultured astrocytes were solubilized in a Triton lysis buffer containing 100 mmol/l NaCl, 50 mmol/l NaF, 5 mmol/l EDTA, 1% Triton X-100, 50 mmol/l Tris·HCl (pH 7.4), with 0.01 mmol/M NaVO4, 0.1 mmol/l phenylmethylsulfonylfluoride, and 0.6 µmol/l leupeptin. The supernatant was clarified by centrifugation (10,000 g for 10 min at 4°C), and protein concentration was quantified using the Lowry method (26). Solubilized proteins (20 µg/well) were separated on 10% polyacrylamide gel with the use of Laemmli buffer and transferred by electrophoresis to polyvinylidene difluoride membranes (Amersham Pharmacia, Piscataway, NJ). Nonspecific binding to the membranes was blocked by incubation in 5% Blotto (5% evaporated milk, 0.1% Tween 20 in Tris-buffered saline) and probed with an ACE2-specific polyclonal antibody. The reaction product was visualized by incubation with goat anti-rabbit antibody (1:1,000 dilution) coupled to horseradish peroxidase and its reaction with ECL, a luminescent substrate (Amersham, Piscataway, NJ). Protein loading was verified using an antibody to actin (Sigma, St. Louis, MO). The data were quantified by densitometry using the Microcomputer Imaging Device system and software.

RNA isolation and mRNA quantification. RNA was isolated from cultured astrocyte, using TRIzol reagent (GIBCO Invitrogen, Carlsbad, CA) as directed by the manufacturer. The RNA concentration and integrity were assessed using a bioanalyzer with an RNA 6000 nano LabChip (model 2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA). Approximately 1 µg of total RNA was reverse transcribed using avian myeloblastosis virus reverse transcriptase (RT) in a 20-µl mixture containing deoxyribonucleotides, random hexamers, and RNase inhibitor in RT buffer. When the RT reaction product was heated at 95°C, the reaction was terminated. For real-time PCR, 2 µl of the resultant cDNA was added to TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) with an ACE2 primer/probe set (forward primer, 5'-CCCAGAGAACAGTGGACCAAAA-3'; reverse primer, 5'-GCTCCACCACACCAACGAT-3'; and probe, 5'-FAM-CTCCCGCTTCATCTCC-3') or ACE primer/probe set (Applied Biosystems), and amplification was performed using an ABI 7000 Sequence Detection System. The mixtures were heated at 50°C for 2 min and at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. All reactions were performed in triplicate and 18S ribosomal RNA, amplified using the TaqMan Ribosomal RNA Control Kit (Applied Biosystems), served as an internal control. The results were quantified as Ct values, where Ct is defined as the threshold cycle of PCR at which amplified product is first detected, and expressed as the ratio of target to control (Relative Gene Expression).

Statistics. All data are presented as means ± SE. Statistical differences were evaluated using repeated-measures one-way ANOVA, followed by Dunnett's post hoc test or by Student's t-test. The criterion for statistical significance was set at P < 0.05.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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ACE2 regulation by ANG II. Cultured astrocytes, isolated from neonatal rat cerebellum or medulla oblongata, were incubated with 100 nmol/l ANG II to determine whether the peptide regulates ACE2 gene expression. EDTA (0.5 mmol/l) was added to the culture media to prevent the degradation of ANG II by blocking the activity of metalloproteases such as neprilysin or ACE2. HPLC analysis showed that ~50% of the added peptide remained after 12 h, indicating that EDTA effectively blocked the catabolism of ANG II. After 12 h, total RNA was isolated from the cells and ACE2 mRNA was quantified by RT real-time PCR with the use of gene-specific primers. The results were expressed as a ratio compared with the 18S rRNA, which served as an unregulated control. ACE2 mRNA was markedly reduced in astrocytes from the medulla oblongata and cerebellum after incubation with ANG II compared with untreated controls (Fig. 1), suggesting transcriptional regulation of the enzyme by ANG II. In contrast, no change in ACE mRNA was observed in astrocytes from either brain area treated with ANG II, demonstrating the differential regulation of the two enzymes.



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  		Fig. 1. Regulation of angiotensin-converting enzyme 2 (ACE2) mRNA by ANG II. Cultured astrocytes isolated from the medulla oblongata or cerebellum (n = cells isolated from 6 litters of neonatal rats) were incubated for 12 h in serum-free media containing 0.5 mmol/l EDTA (control; CTL) with or without the addition of 100 nM ANG II (AII). The relative gene expression of ACE2 or ACE was measured using real-time PCR as described in METHODS. *P < 0.05 compared with control.

 
Angiotensin receptor antagonists were used to identify the receptor subtype that mediated the downregulation of ACE2 mRNA by ANG II. The addition of the AT1 receptor antagonists losartan or valsartan at a concentration of 1.0 µmol/l effectively blocked the ANG II-mediated reduction of ACE2 mRNA in medullary astrocytes (Fig. 2), resulting in mRNA levels comparable to the control concentrations. Incubation of cultured astrocytes with an equivalent concentration of either AT1 receptor antagonist alone had no effect on ACE2 mRNA. As shown in Fig. 2, similar results were obtained after a 12-h treatment of cerebellar astrocytes with 100 nmol/l ANG II. The approximate 50% reduction in ACE2 mRNA by ANG II was prevented by the AT1 receptor antagonists, resulting in mRNA levels comparable to the control.



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  		Fig. 2. Inhibition of ANG II-mediated reduction of ACE2 by angiotensin type 1 (AT1) receptor antagonists. Cultured astrocytes isolated from the medulla oblongata or cerebellum (n = cells isolated from 6–8 litters of neonatal rats) were incubated for 12 h in serum-free media containing 0.5 mmol/l EDTA (CTL) with or without the addition of 100 nM ANG II (AII) and/or 1 µmol/l losartan (LOS), or valsartan (VAL) as indicated. The relative gene expression of ACE2 was measured using real-time PCR as described in METHODS. *P < 0.05 compared with control.

 
In contrast, the AT2 receptor antagonist PD123319 had no effect on the ANG II-mediated reduction in ACE2 mRNA in either cerebellar or medullary astrocytes (Fig. 3). This study demonstrates that the downregulation of ACE2 by ANG II is an AT1 receptor-mediated process.



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  		Fig. 3. Effect of AT2 receptor antagonist on ACE2 mRNA regulation. Cultured astrocytes isolated from the medulla oblongata or cerebellum (n = cells isolated from 6–8 litters of neonatal rats) were incubated for 12 h in serum-free media containing 0.5 mmol/l EDTA (CTL) with or without the addition of 100 nM ANG II (AII) and/or 1 µmol/l PD123319 (PD), as indicated. The relative gene expression of ACE2 was measured using real-time PCR as described in METHODS. *P < 0.05 compared with control.

 
ANG II regulation of ACE2 protein. The transcriptional downregulation of ACE2 mRNA by ANG II also reduced the concentration of ACE2 protein. Immunoreactive ACE2 was decreased to an extent similar to that of ACE2 mRNA, ~50% in cerebellar astrocytes and 60% in medullary astrocytes after incubation with ANG II for 12 h as determined using Western blot hybridization with an ACE2-specific antibody (Fig. 4). Again, EDTA (0.5 mmol/l) was added to the astrocyte cultures to prevent the degradation of ANG II by metalloproteases. No decrease in ACE2 immunoreactivity was observed after incubation of the astrocytes with ANG II and the AT1 receptor blocker losartan, confirming that the transcriptional downregulation of ACE2 is mediated by AT1 receptors.



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  		Fig. 4. Regulation of ACE2 protein by ANG II. Cultured astrocytes isolated form the medulla oblongata or cerebellum (n = cells isolated from 4 litters of neonatal rats) were incubated for 12 h in serum-free media containing 0.5 mmol/l EDTA (CTL) with or without the addition of 1 µmol/l ANG II (AII) and/or losartan (LOS) as indicated. ACE2 mRNA was determined using Western blot hybridization and is presented as the percentage of the control. *P < 0.05 compared with control.

 
Dose- and time-dependent regulation of ACE2 by ANG II. Cultured medullary astrocytes were used to further characterize the transcriptional regulation of ACE2 by ANG II. The reduction of ACE2 mRNA by the peptide was both dose and time dependent as shown in Fig. 5. At 1 nmol/l, ANG II caused a 25% reduction in ACE2 and the inhibition reached a maximal level of ~50% after treatment with 10 nmol/l ANG II for 12 h. Time course studies revealed a marked reduction of ACE2 mRNA with 100 nmol/l ANG II as early as 8 h after addition of the peptide, and expression remained attenuated for up to 24 h.



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  		Fig. 5. Time- and dose-dependent regulation of ACE2 by ANG II. A: astrocytes isolated from the medulla oblongata (n = cells isolated from 7 litters of neonatal rats) were incubated for 12 h in serum-free media containing 0.5 mmol/l EDTA with or without increasing concentrations of ANG II. B: medullary astrocytes (n = cells isolated from 4 litters of neonatal rats) were incubated for increasing time periods in serum-free media containing 0.5 mmol/l EDTA with or without 100 nmol/l ANG II ({bullet}) or ANG-(1–7) ({circ}). The control was harvested at time 0. The relative gene expression of ACE2 was measured using real-time PCR as described in METHODS. *P < 0.05 compared with control.

 
ANG-(1–7) modulation of ANG II-mediated ACE2 downregulation. Additional experiments were performed to assess whether ANG-(1–7), the product of the enzyme reaction, had any effect on the transcriptional regulation of ACE2. No reduction in ACE2 mRNA was observed after treatment of cultured astrocytes with 100 nmol/l ANG-(1–7) for up to 24 h, as shown in Fig. 5. Although ACE2 mRNA tended to increase in the cells incubated with ANG-(1–7) at the latter time points compared with control, the differences were not statistically significant. While no significant change in ACE2 mRNA was observed after incubation with 1.0 µmol/l ANG-(1–7) alone, the ANG II-mediated downregulation of ACE2 was blocked completely by the addition of equimolar concentrations of ANG-(1–7), as shown in Fig. 6. The heptapeptide modulation of the ANG II decrease in ACE2 mRNA was attenuated by coincubation with 1.0 µmol/l [D-Ala7]-ANG-(1–7), an ANG-(1–7) receptor antagonist. This study verified that the heptapeptide blockade of the ANG II-mediated downregulation of ACE2 required interaction at a specific ANG-(1–7) receptor.



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  		Fig. 6. ANG-(1–7) counterregulation of the ANG II-mediated reduction in ACE2 mRNA. Left: cultured astrocytes isolated from the medulla oblongata (n = cells isolated from 8 litters of neonatal rats) were incubated for 12 h in serum-free media containing 0.5 mmol/l EDTA (CTL) with or without the addition of 1.0 µmol/l ANG II (AII), 100 µmol/l ANG-(1–7) (A7), or the combination of both peptides (AII/A7). Right: cultured astrocytes isolated from the medulla oblongata (n = cells isolated from 4 litters of neonatal rats) were incubated for 12 h in serum-free media containing 0.5 mmol/l EDTA and 1.0 µmol/l [D-Ala7]-ANG-(1–7) (CTL) with or without the addition of 1.0 µmol/l ANG II, 1.0 µmol/l ANG-(1–7), or the combination of both peptides. The relative gene expression of ACE2 was measured using real-time PCR. *P < 0.05 compared with control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have demonstrated that treatment of cultured astrocytes with ANG II caused a marked reduction in neural ACE2 mRNA and protein, a response mediated by the AT1 receptor. These results suggest that the transcriptional downregulation of ACE2 by ANG II may serve as a mechanism within the brain to favor ANG II-mediated neural responses. The previous demonstration that inhibition of ANG II synthesis or AT1 receptor blockade is associated with a marked increase in cardiac ACE2 mRNA is consistent with these results in isolated cells, which provide a more specific explanation for the findings reported elsewhere (12, 21). Thus agents that attenuate ANG II actions or reduce ANG II production may cause upregulation of ACE2 and increased formation of the vasodilator antiproliferative peptide ANG-(1–7) and reduce the concentration of ANG II. The additional demonstration that ANG-(1–7) prevents the inhibitory effects of ANG II on ACE2 mRNA constitutes a novel observation shedding insight into the cellular mechanisms regulating ACE2 gene expression.

ANG-(1–7), produced from ANG II by ACE2, had no effect on ACE2 mRNA. However, ANG-(1–7) blocked the down-regulation of ACE2 expression by ANG II, an effect mediated by a selective AT(1–7) receptor. This suggests that ANG-(1–7), acting at its own receptor, prevents reduction of ACE2 transcription by ANG II to enhance the production of the heptapeptide (Fig. 7). Studies conducted by our group as well as others showed that ANG-(1–7) produces unique physiological responses, which are often opposite to those of the well-characterized angiotensin peptide ANG II (11). The ability of ANG II to increase blood pressure is well documented; it is a potent vasoconstrictor and it stimulates thirst and aldosterone release, and inhibition of its production or effect using ACE inhibitors or AT1 receptor antagonists reduces mean arterial pressure (43). In contrast, ANG-(1–7) reduced the blood pressure of hypertensive dogs and rats (2, 30), inhibited renal fluid absorption (9, 15, 18, 20), caused vasodilation (2, 3, 28, 31, 32, 49), and participated in the antihypertensive responses to ACE inhibition or AT1 receptor blockade in hypertensive rats (22, 23). While ANG II is a potent mitogen, stimulating vascular growth as well as hypertrophy in terminally differentiated cells (13, 35), we showed that ANG-(1–7) inhibits the growth of vascular smooth muscle cells, cardiomyocytes, cardiac fibroblasts, and human lung cancer cells (14, 38, 42, 46). Taken together, these studies suggest that ANG-(1–7) acts as a physiological modulator of ANG II, with opposing actions on body fluid volume, blood pressure, and cell growth that are particularly evident under conditions of an activated renin-angiotensin system. Thus ACE2 may be considered a key regulatory mechanism between ANG II and ANG-(1–7) to balance the pressor/mitogenic and depressor/growth inhibitory arms of the renin-angiotensin system. Importantly, the results of the present study also implicate distinct receptor systems that differentiate the actions of ANG II and ANG-(1–7) in the transcriptional regulation of ACE2.



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  		Fig. 7. Schematic representation of ACE2 regulation by angiotensin peptides. Our results suggest that ANG II activates the AT1 receptor on astrocytes to reduce ACE2 mRNA and protein. Reduced ACE2 activity would decrease the conversion of ANG II to ANG-(1–7) and favor ANG II-mediated neural responses. In contrast, ANG-(1–7) activation of an AT(1–7) receptor blocked the ANG II-mediated reduction in ACE2 to maintain the balance between ANG II and ANG-(1–7) in the brain.

 
All of the components of the renin-angiotensin system are present in the brain, including angiotensinogen, renin, ACE, and angiotensin peptide receptors, suggesting that the central nervous system contains an endogenous system for angiotensin peptide production (27). The identification of ACE2 in human brain areas (17, 19) and by us in cultured astrocytes as well as the high catalytic efficiency of ACE2 for ANG II provide evidence for a biosynthetic pathway in the brain, where ANG II can be converted into the bioactive peptide ANG-(1–7). ANG-(1–7) is present in brain tissues such as the medulla oblongata at concentrations equivalent to or greater than those of ANG II (5). Similar to the periphery, many of the responses to ANG-(1–7) in the central nervous system oppose those of ANG II. Moriguchi et al. (29) showed that intracerebroventricular administration of an ANG II antibody lowered blood pressure in [mRen-2]27 transgenic hypertensive rats; conversely, an antibody that selectively bound ANG-(1–7) raised pressure. In the dorsal medulla oblongata, ANG II attenuated baroreceptor reflex control of heart rate, whereas ANG-(1–7) augmented the reflex, suggesting that the balance of the two peptides sets the sensitivity of the baroreflex. In contrast, ANG II and ANG-(1–7) produce similar neural responses in some brain areas, such as the ventral lateral medulla. Both ANG-(1–7) and ANG II regulated arterial pressure and renal sympathetic nerve activity when injected into the ventral lateral medulla, responses blocked by AT1 receptor antagonists (33); however, higher doses of ANG-(1–7) were required because of a lower affinity for ANG-(1–7) at the AT1 receptor (34). These studies suggest that the balance of ANG II and ANG-(1–7) is critical for central control of blood pressure.

Although ACE2 has a high catalytic efficiency for the conversion of ANG II to ANG-(1–7), other peptides also serve as substrates for the enzyme. Vickers et al. (52) examined >120 potential peptide substrates and found that only apelin 13 and dynorphin A (1–13) were hydrolyzed by ACE2 with comparable kinetics to the conversion of ANG II to ANG-(1–7). Apelin, synthesized as a 77-amino acid preprohormone, is processed to a 36-amino acid peptide, apelin 36, with further proteolytic cleavage to yield apelin 13, composed of the COOH-terminal region (24). The biological effects of the apelin peptides are mediated by the APJ receptor, a G protein-coupled, seven-transmembrane receptor, which shares >50% sequence homology with the AT1 receptor in the transmembrane region. Intracerebroventricular administration of apelin 13 increased water intake and stimulated the release of several hypothalamic factors that regulate neuroendocrine control (41). The opioid peptide dynorphin A (1–13), also generated from a precursor molecule, activates G protein-coupled {kappa}- and {delta}-opioid receptors, which are important in pain perception (51). In addition, ACE2 cleaved another opioid peptide, casomorphin, and the neurotransmitters neurotensin and kinentensin (52). Thus, ACE2 likely plays multiple roles in the brain, maintaining the balance of peptides important in blood pressure regulation, water homeostasis, and neurotransmission, including pain perception.

In the current study, ANG II caused a significant reduction in ACE2 mRNA and protein, which was blocked by the AT1 receptor antagonist losartan or valsartan, but not by an AT2 receptor antagonist. In previous studies (47), we identified AT1 receptors on neonatal rat brain cerebellar and medullary astrocytes. Furthermore, activation of these receptors resulted in the stimulation of a phosphoinositide-specific phospholipase C (39, 40, 47). ANG II induced calcium release from astrocytes that was mediated by activation of AT1 receptors (16, 47), suggesting that ANG II activates glial AT1 receptors that are coupled to the activation of phospholipase C and the mobilization of intracellular calcium. ANG II also caused a dose-dependent release of prostaglandins from astrocytes, an effect mediated by the AT1 receptor (25, 45). These studies demonstrated that ANG II binds to AT1 subtype angiotensin receptors on astrocytes to activate multiple signaling pathways, suggesting the potential complexity of the AT1 receptor-mediated, transcriptional regulation of ACE2.

The transcriptional downregulation of ACE2 by ANG II agrees with our published studies in rat heart, which showed a twofold increase in ACE2 mRNA in rats with or without coronary artery ligation, which were treated with the AT1 receptor antagonists losartan or olmesartan (12, 21). Cardiac ACE2 mRNA correlated directly with plasma levels of ANG-(1–7) and inversely with plasma ANG II. In addition, plasma ANG-(1–7)/ANG II ratios were significantly greater in the losartan-treated group, a finding that suggests increased formation of ANG-(1–7) from ANG II. Similarly, we found that treatment of normotensive Lewis rats with the ACE inhibitor lisinopril or an AT1 receptor antagonist caused a significant upregulation in cardiac ACE2 mRNA (12). Furthermore, ACE2 is upregulated in spontaneously hypertensive rats after combined ACE-neprilysin blockade with omapatrilat, which causes reduced ANG II and increased ANG-(1–7) (7). Taken together, our previous studies and the results reported herein suggest that the protective effect of ACE inhibitors and AT1 receptor blockers may be due, at least in part, to increased ACE2, which shifts the angiotensin peptide balance favoring metabolism of ANG II to produce ANG-(1–7).

In summary, the present study is the first to demonstrate the transcriptional regulation of ACE2 by ANG II in brain astrocytes, a finding that further implicates a physiological role for this novel component of the renin-angiotensin system in the generation of ANG-(1–7) and ANG II degradation. Furthermore, the results reported herein suggest that the transcriptional regulation of ACE2 mRNA in astrocytes is dependent on the relative concentrations of both ANG II and ANG-(1–7) as well as on interaction with their respective receptors.


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 ABSTRACT
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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-51952 and HL-56973. We thank Unifi (Greensboro, NC) and Farley-Hudson Foundation (Jacksonville, NC) for financial support.


   ACKNOWLEDGMENTS
 
We acknowledge the excellent technical assistance of L. Tennille Howard, Robert Lanning, Brian Bernish, and Nancy Pirro. Losartan was a kind gift from Merck, and valsartan was kindly provided by Takeda Chemical.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. E. Gallagher, The Hypertension and Vascular Disease Center, Wake Forest Univ. School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1032 (e-mail: pgallagh{at}wfubmc.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
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