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(Hypertension. 2005;46:1368.)
© 2005 American Heart Association, Inc.

Original Articles

Hydrolysis of Angiotensin Peptides by Human Angiotensin I–Converting Enzyme and the Resensitization of B₂ Kinin Receptors

Zhenlong Chen; Fulong Tan; Ervin G. Erdös; Peter A. Deddish

From the Departments of Pharmacology (Z.C., F.T., E.G.E., P.A.D.), Anesthesiology (F.T., E.G.E.), and Anatomy and Cell Biology (P.A.D.), University of Illinois College of Medicine, Chicago.

Correspondence to Ervin G. Erdös, MD, Professor, Department of Pharmacology, 835 S Wolcott (MC 868), Chicago, IL 60612. E-mail EGErdos{at}uic.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We measured the cleavage of angiotensin I (Ang I) metabolites by angiotensin I–converting enzyme (ACE) in cultured cells and examined how they augment actions of bradykinin B₂ receptor agonists. Monolayers of Chinese hamster ovary cells transfected to stably express human ACE and bradykinin B₂ receptors coupled to green fluorescent protein (B₂GFP) or to express only coupled B₂GFP receptors. We used 2 ACE-resistant bradykinin analogues to activate the B₂ receptors. We used high-performance liquid chromatography to analyze the peptides cleaved by ACE on cell monolayers and found that Ang 1-9 was hydrolyzed 18x slower than Ang I and &30% slower than Ang 1-7. Ang 1-7 was cleaved to Ang 1-5. Although µmol/L concentrations of slowly cleaved substrates Ang 1-7 and Ang 1-9 inhibit ACE, they resensitize the desensitized B₂GFP receptors in nmol/L concentration, independent of ACE inhibition. This is reflected by release of arachidonic acid through a mechanism involving cross-talk between ACE and B₂ receptors. When ACE was not expressed, the Ang 1-9, Ang 1-7 peptides were inactive. Inhibitors of protein kinase C-, phosphatases and Tyr-kinase blocked this resensitization activity, but not basal B₂ activation by bradykinin. Ang 1-9 and Ang 1-7 enhance bradykinin activity, probably by acting as endogenous allosteric modifiers of the ACE and B₂ receptor complex. Consequently, when ACE inhibitors block conversion of Ang I, other enzymes can still release Ang I metabolites to enhance the efficacy of ACE inhibitors.

Key Words: angiotensin • angiotensin-converting enzyme • bradykinin • enalapril • protein kinases

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiotensin I–converting enzyme (ACE) inhibitors are used by tens of millions of patients to block conversion of angiotensin I (Ang I) to Ang II and inactivation of bradykinin; the latter contributes significantly to the beneficial effects.^1–4 After ACE inhibitors increase Ang I concentration, it can be converted to derivatives such as Ang 1-9 and Ang 1-7.^5,6 Ang 1-7 actively antagonizes the vasoconstrictor and proliferative effects of Ang II.^7,8 Ang 1-9, believed to be inactive until converted by ACE to the active Ang 1-7, which is subsequently inactivated by release of the C-terminal dipeptide to form Ang 1-5. The heptapeptide has its own receptor, its specific activities differ from those of Ang II,^9,10 and it also enhances the activity of bradykinin on its B₂ receptors.^11,12

The major enzymes that cleave Ang I to derivatives other than Ang II are ACE2^13–15 and deamidase or cathepsin A.¹² ACE2 is a carboxypeptidase instead of a peptidyl dipeptidase like ACE.¹⁶ ACE2 hydrolyzes Ang II orders of magnitude faster than Ang I.¹⁵ ACE2 generates Ang 1-7 by releasing the C-terminal Phe⁸ of Ang II much more efficiently than cleaving the C-terminal Leu¹⁰ of Ang I (Figure 1). ACE2 commanded much attention because a genetically determined lack of the enzyme can block normal heart development^13,14 and because of its identity with the corona virus receptor.¹³ Ang I is rapidly converted to Ang 1-9 by deamidase or cathepsin A, found in the heart and throughout the body.¹² A carboxypeptidase A-type enzyme¹⁷ also releases Ang 1-9, which inhibits ACE at relatively high concentrations.¹¹

View larger version (30K): [in this window] [in a new window]   Figure 1. Peptides and enzymes that hydrolyze them. Arrows indicate the bonds cleaved by enzymes and the relative rate of hydrolysis. () indicates rapid hydrolysis; (--), slow hydrolysis.

To determine whether Ang 1-9 is active per se or it becomes active only after conversion to Ang 1-7 under our conditions, we examined the metabolism of Ang I, Ang 1-9, and Ang 1-7 in stably transfected Chinese hamster ovary (CHO) cells that express human ACE and human bradykinin B₂ receptors coupled to green fluorescent protein (B₂GFP). We then used the membranes of living cells to establish that Ang I cleavage products, in addition to potentiating bradykinin activity,^12,18,19 also resensitized the B₂GFP receptors desensitized by agonist. We found that they were active at much lower concentrations than suggested by their IC₅₀ values for ACE. Because ACE, or kininase II, is a major bradykinin inactivator,³ we used an ACE-resistant bradykinin analogue (BKan)²⁰ and synthesized another, didansyl-lysyl-bradykinin (DidnsKBK), as B₂GFP receptor agonists.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Materials section is available online at http://www.hypertensionaha.org.

Enzyme Purification
Human renal ACE was purified from human cadaver kidneys.²¹ Approval for use of human tissue was granted by the internal review board of our university.

Measurement of [³H]Arachidonic Acid
The release of [³H]arachidonic acid ([³H]AA) from monolayers of cells loaded for 18 to 24 hours in culture medium containing 1 mCi/L [³H]AA was measured as before.²²

ACE Assay
ACE activity was assessed fluorometrically with the dipeptide cleaved from hippuryl-histidyl-leucine coupled to o-phthalic-dicarboxaldehyde.¹¹

Binding of [³H]Bradykinin to B₂ Receptors
Cell monolayers expressing ACE and B₂ receptors or only B₂ receptors were incubated with [³H]bradykinin to achieve saturation binding.²⁰

B₂GFP Expression Construct
Production and subcloning of the B₂GFP fusion protein was done in 2 steps. First, the coding sequence of GFP was altered by polymerase chain reaction (PCR) purification to introduce an amino terminal XhoI restriction site and a carboxyl-terminal XbaI restriction site following the stop codon.

The amplified fragment of GFP digested with XhoI and XbaI was ligated to the pcDNA3 expression vector digested with XhoI and XbaI. To obtain the B₂GFP fusion protein, the sequence of B₂ receptor was amplified by PCR. HindIII restriction site and a partial Kozak site were introduced in front of the initiator Met of the B₂ receptor, and XbaI restriction site was introduced in front of the stop codon of B₂ receptor. Finally, the GFP construct in pcDNA3 was digested with XhoI and HindIII and was ligated together with the PCR product of the B₂ receptor amplification, which was also digested with XhoI and HindIII. The open reading frame representing the coding sequence of B₂GFP was fully sequenced before its expression and analysis.

Construction of CHO/ACE–B₂GFP Cells
The cDNA of B₂GFP was stably transfected into CHO-K1 cells, and transfectants were selected by fluorescence-activated cell sorting. Selected clones were tested for B₂GFP function by measuring [Ca²⁺]_i response to bradykinin stimulation. Cells with high expression of B₂GFP (CHO/BG) were selected and transfected with cDNA of human ACE and the transfectants selected with geneticin. Then a clone of cells with high ACE activity, CHO/ACE-BG (CHO/ABG), was chosen.

Synthesis of DidnsKBK
The molecular mass of samples was determined with electron spray mass spectroscopy, corresponding closely to the calculated values for monodansyl-KBK (1421.8) and DidnsKBK (1655.1). The NH₂ group in the monodansyl compound and both -amino and -amino groups of Lys¹ in DidnsKBK were coupled (please see online supplement, available at http://www.hypertensionaha.org).

Hydrolysis of Lys¹-Bradykinin, Monodansyl-KBK and DidnsKBK by ACE
Hydrolysis was assayed by incubating each of the peptides (100 µmol/L) with 2 nmol/L purified human ACE in 50 mmol/L Tris–maleate buffer with 150 mmol/L NaCl, pH 7.8, at 37°C for 30 minutes. Ice-cold 5% trifluoroacetic acid (TFA) precipitated the enzyme. Percent hydrolysis was determined by changes in substrate peak area in high-performance liquid chromatography (HPLC).

Hydrolysis of Peptides by Cell Monolayers
CHO/ABG cells were grown in DMEM containing 10% FBS in 12-well plates. Cell monolayers were washed with serum-free DMEM, and 400 µL serum-free DMEM containing 50 to 800 µmol/L angiotensin peptide was added to each well. Plates were incubated at 37°C with shaking for appropriate times. Then 140-µL aliquots were drawn and added to 70 µL of 5% ice-cold TFA. Fifty-microliter aliquots were analyzed by reversed-phase HPLC. Less than 1% ACE activity was released spontaneously into the medium.

Lisinopril Titration of ACE
ACE activity on cell monolayers was titrated with the tight binding inhibitor lisinopril. Cells were grown to confluence in 12-well plates (&10⁶ cells per well). After washing, 0 to 375 nmol/L lisinopril was added, and the plates were incubated with shaking at 37°C for 30 minutes. Then 50 µmol/L Ang I was added and incubation was continued for 5 minutes. Hydrolysis products were analyzed.

Hydrolysis of Peptides by ACE In Vitro
For kinetics, Ang I and Ang 1-9 (50 to 800 µmol/L) were incubated with 2 or 10 nmol/L of purified human ACE in 50 mmol/L Tris–maleate buffer with 150 mmol/L NaCl, pH 7.8, at 37°C. The hydrolysis products were analyzed by HPLC.

Analysis of Hydrolysis Products
Peptides and their hydrolysis products were separated on a Bondclone 10µ C18 reversed-phase column (Phenomenex) with a 10% to 28% linear gradient of acetonitrile/0.05% TFA in H₂O/0.05% TFA over 30 minutes. Products were detected at 214-nm wavelength. The cleavage of peptides followed zero-order kinetics during the assay, and amounts of peptide fragments were determined with standard peptides.

Resensitization of Bradykinin Receptors in CHO/ABG Cells
After desensitization of B₂GFP receptors by initial exposure of cells to bradykinin or BKan to induce tachyphylaxis,¹¹ we assessed the extent of resensitization, or the return of sensitivity to kinin in the medium, by measuring [³H]AA release from cells. Cells were stimulated and desensitized with 1 µmol/L bradykinin, BKan, or DidnsKBK (10 µmol/L) for 30 minutes, then exposed again to agonist or Ang 1-7, Ang 1-9, or enalaprilat for an additional 5 minutes. [³H]AA release was determined by sampling AA release during the first 30 minutes and corrected for the spontaneous release. B₂ receptor antagonist HOE 140 (1 µmol/L) was added as a control 10 minutes before Ang 1-7 or Ang 1-9.

Effect of Inhibitors on Resensitization
CHO/ABG cells were incubated with 1 of the following inhibitors: 1.0 µmol/L staurosporine, 0.5 µmol/L calphostin C, 1.0 µmol/L okadaic acid, 0.1 µmol/L calyculin A, 0.1 µmol/L Gö 6976, or 20 µmol/L genistein for 30 minutes at 37°C. The cell monolayers were then washed with fresh medium before testing for resensitization.

Statistics
Data are expressed as mean±SEM (n3). Statistical evaluation was performed by 1-way ANOVA for matched values.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of B₂GFP Receptor and ACE
CHO cells transfected with B₂GFP cDNA were selected for high expression; then we transfected them with ACE cDNA and isolated CHO/ABG clones with high ACE activity. We determined the number of B₂ receptors expressed per cell by saturation binding of [³H]bradykinin and the number of ACE molecules per cell by active site titration of ACE in cell monolayers with lisinopril.¹⁵ CHO/BG and CHO/ABG cells had &1x10⁶ B₂ receptors per cell, and the CHO/ABG cells had &5x10⁶ ACE molecules per cell.

Hydrolysis of N-Terminally Protected BKan Analogue
To study resensitization, we synthesized a derivative of bradykinin with a bulky N terminus that would protect it from inactivation by ACE but would allow it to act as an agonist of the B₂ receptor. KBK was dansylated at its NH₂ or at the NH₂ and NH₂ groups of lysine (Table 1). Whereas human ACE cleaved monodansyl-KBK at about one third the rate of KBK, DidnsKBK was only 0.4% hydrolyzed in 30 minutes.

View this table: [in this window] [in a new window]   TABLE 1. Hydrolysis of DidnsKBK Peptides*

Kinetics of Hydrolysis of Peptides by Cell Monolayers
Table 2 shows that Ang I had the highest Michaelis constant (K_m; 119 µmol/L) when CHO/ABG cell monolayers hydrolyzed Ang peptides, followed by Ang 1-9 (61 µmol/L) and Ang 1-7 (35 µmol/L). The catalytic constant (k_cat) of Ang I was also the highest (2890/min^–1) and was 18x higher than that of Ang 1-9 and 12x higher than Ang 1-7. Ang 1-7 was 4x less efficiently hydrolyzed (k_cat/K_m=6.5 [µmol/L]^–1 min^–1) and Ang 1-9 had the lowest specificity constant (2.6 [µmol/L]^–1 min^–1), &10x less than that of Ang I.

View this table: [in this window] [in a new window]   TABLE 2. Hydrolysis of Angiotensin Peptides by Human ACE on Intact CHO Cell Surface* and Purified Human ACE

Comparison of Kinetics of Hydrolysis by Purified Human ACE and Cell Monolayers
Table 2 also compares the hydrolysis of Ang I and Ang 1-9 by purified human renal soluble ACE with the plasma membrane–bound ACE of CHO/ABG cells. The apparent K_m for the cell surface enzyme was approximately twice that of the purified enzyme (119 versus 67 µmol/L for Ang I; 61 versus 28 µmol/L for Ang 1-9). The k_cat of ACE on the plasma membrane of intact cells was much higher than with purified enzyme, which suggests some loss of activity of during purification or a lack of negative cooperativity between the 2 active sites of ACE on the cell membrane. Also, the specificity constant (k_cat/K_m) for each peptide was lower for the purified enzyme than for the cell surface enzyme (1.3 versus 2.6 µmol/L/min) for Ang 1-9. The lower K_m with the purified enzyme did not offset the lower k_cat.

Resensitization of B₂GFP Receptors by Ang 1-7 and Ang 1-9
The bradykinin B₂ receptor was desensitized by pretreating CHO/ABG cells with 1 µmol/L BKan, and the agonist was tested again to establish that desensitization had occurred. Ang 1-7 or Ang 1-9 was then added in concentrations varying from 1 to 10 nmol/L. Figure 2A shows [³H]AA release in cells expressing B₂GFP and ACE. The results indicate that exposure to the peptides resensitized the B₂GFP receptor to BKan in a concentration-dependent manner. Ang 1-9 (10 nmol/L) was significantly more active than Ang 1-7 (P<0.01). The bradykinin B₂ receptor antagonist HOE 140 completely blocked resensitization. The expression of ACE on the cell surface was required because in CHO/BG cells lacking ACE, the B₂ receptors could not be resensitized by either Ang 1-7 or Ang 1-9 or by enalaprilat (please see online supplement). In the absence of ACE expression, the desensitized B₂ receptor remains inactive. In Figure 2B, the B₂ receptor was desensitized with 10 µmol/L DidnsKBK, which is unaffected by ACE, then 1 µmol/L Ang 1-9 or enalaprilat was added without adding more B₂ agonist. This treatment resensitized the receptor to release [³H]AA, and HOE 140 blocked it. DidnsKBK, even at 50-µmol/L concentration, did not inhibit ACE using hippuryl-his-leucine as substrate (data not shown).

View larger version (36K): [in this window] [in a new window]   Figure 2. Resensitization of bradykinin B2GFP receptors to ACE-resistant BKans. A, CHO/ABG cells were stimulated with 1 µmol/L BKan for 30 minutes to desensitize, then treated with medium alone, 1 µmol/L BKan, or 1 to 10 nmol/L Ang 1-7 or Ang 1-9 for 5 minutes; 1 µmol/L HOE 140 blocked resensitization. Ordinate: [3H]AA released by the peptides (5 minutes) relative to amount released by medium alone. Values are mean±SEM (n=8). *P<0.001; P<0.01; experiments done in triplicate. B, CHO/ABG were desensitized with 10 µmol/L DidnsKBK, then resensitized to DidnsKBK in medium with 1 µmol/L Ang 1-9 or EPT (n=3). P<0.001. HOE 140 blocked the effect (data not shown).

Effect of Protein Kinase C and Protein Phosphatase Inhibitors on Resensitization
Figure 3 shows the effects of the protein kinase C (PKC), phosphatase, and PKC inhibitors on the resensitization of B₂ receptors in CHO/ABG cells. Several diverse signal transduction inhibitors abolished the resensitization of the B₂ receptor by Ang 1-9: stauroporine, calphostin C; okadaic acid, calyculin A; and Gö6976. Uninhibited cells were resensitized by treatment with 10 nmol/L Ang 1-9. The results with Ang 1-7 or enalaprilat were identical (data not shown). None of the inhibitors altered the basal response to a first dose of BKan.

View larger version (24K): [in this window] [in a new window]   Figure 3. Effect of signal transduction inhibitors. CHO/ABG cells were incubated with 1 of the following inhibitors for 30 minutes: 1.0 µmol/L staurosporine, 0.5 µmol/L calphostin C, 1.0 µmol/L okadaic acid, 0.1 µmol/L calyculin A, or 0.1 µmol/L Gö976, washed, desensitized with BKan (1 µmol/L), then exposed to medium alone or 1 µmol/L BKan or 10 nmol/L Ang 1-9. Values are mean±SEM (n=3). *P<0.01 vs medium control. Kinase or phosphatase inhibitors blocked resensitization also by Ang 1-7 and enalaprilat (data not shown).

Effect of Tyrosine Kinase Inhibitor on Resensitization
The tyrosine kinase inhibitor genistein also completely blocked resensitization of CHO/ABG cells by Ang 1-7, Ang 1-9, or enalaprilat (EPT) (Figure 4). As with PKC inhibitors, preincubation of the cells with genistein did not block the initial release of [³H]AA to 1 µmol/L BKan before resensitization.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effect of tyrosine kinase inhibitor. CHO/ABG cells were incubated with or without 20 µmol/L genistein, desensitized with BKan (1 µmol/L) followed by medium, 1 µmol/L BKan, 10 nmol/L Ang 1-7 or Ang 1-9, or enalaprilat for 5 minutes. Values are mean±SEM (n=4); *P<0.005 vs medium control. Tyrosine kinase inhibitor abolished resensitization by peptide or enalaprilat. EPT indicates enalaprilat.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although the role of angiotensin in hypertension was well established during the past century,²³ this peptide has become increasingly relevant to various pathological conditions other than hypertension. Ang II, released by ACE, acting on its angiotensin type 1 (AT₁) receptor is usually noxious, whereas activation of the Angiotensin type 2 (AT₂) receptor can counteract some of these effects, possibly through the bradykinin B₂ receptor.²⁴ ACE is also kininase II;^3,25 its inhibitors block the inactivation of kinins. Because of the proven and probable pathological functions of Ang II and the widely reported counter-regulatory actions of Ang 1-7,^7–9 we wished to establish how Ang II metabolites influence bradykinin activity. We included Ang 1-9 because it has not been well studied and is thought to be active only after conversion to Ang 1-7 (Figure 1). Both peptides inhibited ACE^11,19 but only in µmol/L concentrations. We reported that Ang 1-7 was cleaved only by one of the active sites of ACE, by the so-called N domain and not by native testicular C domain.¹⁹ Rice et al described that Ang 1-9 was converted more readily to Ang 1-7 by the separate recombinant C domain of ACE than by the N domain, but as shown above, they also found that Ang 1-9 was hydrolyzed much slower than Ang I.¹⁵ Cleavage of peptides by ACE can be influenced by its high carbohydrate content; for example, the degrees of glycosylation of ACE from human lung and kidney differ.²⁶ Carbohydrate structures of enzymes can depend on their tissue and cellular origin.

To learn more about Ang I derivatives, we transfected CHO cells to express human ACE and bradykinin B₂ receptors. Lisinopril titration of active ACE sites on the cell surface indicated &5x10⁶ ACE molecules on the apical surface. Using saturation binding with labeled bradykinin, we found &1x10⁶ B₂ receptors per cell. This estimate assumes 2 active sites per molecule of ACE rather than an apparent single active site attributable to negative cooperativity between the 2 sites.^27,28

We compared the rates of hydrolysis of Ang I and its derivatives by ACE in CHO cells. Ang 1-9 was converted to Ang 1-7 at a rate <6% of Ang II release with a 90% lower specificity constant. Ang 1-7 was inactivated to Ang 1-5 by ACE at a relatively low turnover rate and specificity constant (Table 1). Thus, Ang 1-7 and Ang 1-9 are more resistant to hydrolysis by ACE than Ang I. Although Ang 1-7 and Ang 1-9 have a slow turnover rate, their K_m was lower than that of Ang I but still much higher than the K_m for bradykinin: 1 to 0.1 µmol/L.^25,26 Consequently, neprilysin (neutral endopeptidase 24.11) is the major enzyme that liberates Ang 1-7 from Ang I^5,6 and ACE2,¹⁵ and prolylcarboxypeptidase releases it from Ang II.²⁹ Human chymase liberates only Ang II from Ang I³⁰ (Table 1).

Ang I is hydrolyzed only very slowly by ACE 2,¹⁵ whereas deamidase (cathepsin A) converts it effectively to Ang 1-9.^12,31 Ang 1-9 is more than a poor precursor of Ang 1-7 attributable to low conversion rate by ACE; it is also an active derivative of Ang I because enhances bradykinin actions in cultured cells and resensitizes its receptor. Ang 1-9 may antagonize Ang II, just as Ang 1-7 does in laboratory animals.^7,8 Ang 1-7 and Ang 1-9 inhibited ACE only at relatively high µmol/L concentrations, but in human pulmonary endothelial cells, they increased NO release by bradykinin at a much lower concentration than the IC₅₀ values.¹²

ACE inhibitors acted beyond ACE inhibition when they potentiated kinins in cultured cells or surviving organs.³² ACE and B₂ can colocalize and form heterodimers.²⁰ These findings suggest that ACE inhibitors can function as indirect allosteric enhancers³³ of B₂ bradykinin receptor agonists by inducing conformational changes via ACE.

Indeed, because Ang 1-7 and Ang 1-9 resensitize the receptors to bradykinin, they may be endogenous allosteric enhancers for B₂ receptor agonists if ACE and B₂ are expressed in the same cells. The peptides resensitized receptors at concentrations nearer to physiological levels than their IC₅₀ values.

To show that resensitization of the receptors cannot be explained by blocking bradykinin inactivation,^11,20,32 we used ACE-resistant BKans. One ACE-resistant kinin analogue has a modified Phe⁸–Arg⁹ bond;²⁰ in the other, we dansylated both amino groups of lysine in Lys¹-bradykinin. The size of the peptide substrate restricts ACE activity, which drops beyond undecapeptide.³⁴ Because B₂ receptors are more permissive and accept N-terminally enlarged agonists, such as dextran-coupled bradykinin with intact C terminus,³⁵ we synthesized the DidnsKBK analogue. This modified kinin was hydrolyzed by ACE at <3% of the rate for Lys¹-bradykinin; it was not a competitive inhibitor and was blocked by HOE 140 on the B₂ receptor. As with BKan, CHO/ABG cells desensitized to DidnsKBK were resensitized by Ang 1-9 or enalaprilat. We believe that resensitization depends on the induction of conformational changes in ACE–B₂ complex and certainly not on blocking a potential inactivation of the ACE-resistant BKans.

Inhibition of protein kinases abolished potentiation³² and resensitization without affecting basal bradykinin activities. This process unlikely involves phosphorylation of the cytosolic portion of ACE³⁶ because truncating the enzyme there did not affect resensitization by enalaprilat.²⁰ Resensitization was blocked by inhibitors of PKC, PKC, tyrosine kinase, and serine phosphatases, and the findings indicate that resensitization depends on rapid phosphorylation and dephosphorylation of the receptor with PKC as a key component.³⁷

B₂ receptor activation liberates prostaglandins and other arachidonic acid metabolites^38,39 and involves first G_i protein leading to downstream activation of phospholipase A₂. Because inhibition of PKC abolished resensitization, that suggests a complex cascading signal transduction pathway, which could result in the downstream opening of a transient receptor potential channel-1 Ca²⁺ channel.⁴⁰

Perspectives
ACE inhibitors block Ang I conversion, enhancing its concentration as substrate for enzymes such as neprilysin or deamidase. ACE2 and prolylcarboxypeptidase convert Ang II to Ang 1-7. The well-documented effects of Ang 1-7 underscore the potential importance of Ang 1-9 as an active metabolite of Ang I. Ang 1-9 and Ang 1-7 are quite resistant to ACE on the plasma membrane of cultured cells, indicating that these peptides are relatively stable derivatives. Ang 1-7 and 1–9 potentiate bradykinin B₂ receptor agonists and resensitize the desensitized receptors to ACE-resistant kinins independent of ACE inhibition, provided human ACE and B₂ receptors are expressed. Peptides resensitized B₂ receptor similarly to enalaprilat, although Ang 1-7 and Ang 1-9 are poor ACE inhibitors. Resensitization of receptors restored their ability to react to the agonist in the medium and initiate signal transduction. The fact that serine/threonine kinase and even tyrosine kinase inhibitors can block the process without affecting basal bradykinin activity suggests different pathways of transduction leading to phospholipase A₂ activation and arachidonic acid release. ACE appears to be an "acceptor" of Ang 1-7 and Ang 1-9, which, in low concentrations, may modify the conformation of an enzyme–receptor complex. By counteracting the actions of Ang II, the angiotensin-derived peptides can enhance the therapeutic usefulness of ACE inhibitors.

	Acknowledgments

 
These studies were supported by National Institutes of Health, National Heart, Lung, and Blood Institute grants HL36473 and HL68580.

Received August 5, 2005; first decision August 26, 2005; accepted September 26, 2005.

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