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Am J Physiol Heart Circ Physiol 290: H1244-H1250, 2006. First published November 4, 2005; doi:10.1152/ajpheart.00934.2005
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Kallikrein activates bradykinin B2 receptors in absence of kininogen

Dauren Biyashev,1 Fulong Tan,1,2 Zhenlong Chen,1 Kai Zhang,1 Peter A. Deddish,1 Ervin G. Erdös,1,2 and Claudie Hecquet1

Departments of 1Pharmacology and 2Anesthesiology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois

Submitted 1 September 2005 ; accepted in final form 31 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1–10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 µM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors.

arachidonic acid; angiotensin I-converting enzyme inhibitor; kallidin; immunoassay

THE FUNCTIONS of the kallikrein-kinin system have been widely studied since the discovery of its components (1, 14, 19, 46). For example, decrease in urinary excretion of kallikrein is a characteristic feature of clinical or experimentally induced hypertension (36). Kinins release potent vasodilators, such as PGs, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF) (3–5), which influence blood pressure and vessel tone, but they can cause pain and enhance capillary permeability in inflammation as well (3). The beneficial effects of angiotensin I-converting enzyme (ACE) (18, 40) or kininase II inhibitors are attributed at least in part to prolongation of the half-life of bradykinin (BK) (6–8, 20–22) and potentiation of its effects on the B2 receptor (15, 33–35). Although plasma kininogen is the primary substrate of kallikreins, these serine proteases can cleave other proteins as well, such as factor XII, the activator of prokallikrein (12, 41, 42). Generation of BK or kallidin (Lys-BK) by kallikrein results from a complex, multistep enzymatic cascade starting with prokallikrein activation and ending with kinin release from plasma kininogen (12, 29, 32, 41, 42). The peptides must then evade cleavage by the kininases we characterized, such as carboxypeptidase N or M as kininase I and ACE and neprilysin as kininase II, to activate the B2 receptors (3, 16, 26, 43, 47). When carboxypeptidases N or M cleave the COOH-terminal arginine from either BK or Lys-BK (14, 44), the resulting des-Arg kinins become ligands for the second kinin receptor B1 (3).

Because activation of B2 receptors has such important consequences in many diverse tissues, we considered that another pathway, a shunt, may activate B2 receptors independent of the complex enzymatic cascade required for kinin release (23–25). Similar dual backup systems occur in numerous other biological reactions. Indeed, we found that B2 receptors can be activated directly by kallikreins and certain other serine proteases. The BK B2 receptors, similar to the protease-activated receptors (PAR) 1–4 of thrombin or trypsin (38), belong to a G protein-linked, hepta-helical transmembrane receptor group that is activated by serine proteases. However, our experiments with cultured cells of various origins indicate that activation of the BK B2 receptor proceeds through a different mechanism than for PARs by thrombin or thrombin receptor activator peptide (TRAP) ligand (24).

Culture of specialized mammalian cells usually requires a rich medium that contains 5–10% fetal bovine serum. Previously, we used cells washed free of added proteins (23, 25) or washed and serum-starved. Nevertheless, we wanted to exclude a possibility that proteases could activate B2 receptors by cleaving traces of adherent kininogen. Consequently, we have grown and maintained cells transfected with human BK B2 receptors, or B2 and ACE, in media entirely free of serum and animal proteins.

We measured release of [3H]arachidonic acid (AA) from Chinese hamster ovary (CHO) cells and found that kallikreins activated transfected human BK B2 receptors in the absence of any cell-bound kininogen. We also investigated the effects of ACE inhibitors, because these agents appear to potentiate kinin effects through cross talk between ACE and B2 receptors (15, 33–35). The results from these experiments enabled us to exclude kinin release as a mechanism for kallikrein activation of B2 receptors. We showed that ACE inhibitors potentiated the actions of kallikrein by a mechanism independent of kinin inactivation also by employing an ACE-resistant BK analog in CHO cells expressing human ACE and B2 receptors.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Materials. CHO cells were purchased from American Type Culture Collection (Rockville, MD). The cDNA of human B2 receptor was obtained from Dr. K. Jarnigan (Syntex, Palo Alto, CA), and cDNA of human ACE was from Prof. P. Corvol (College de France, Paris, France). Superfect was from Qiagen (Valencia, CA), geneticin (G418) was from Invitrogen (Carlsbad, CA), and [3H]BK was from Amersham (Piscataway, NJ). CHO protein-free, animal component-free medium for attached cells (CHO PFAF) was purchased from Sigma-Aldrich (St. Louis, MO). [3H]AA was purchased from American Radiolabeled Chemicals (St. Louis, MO). BK, porcine pancreatic kallikrein, HOE-140, fatty acid-free BSA, protease inhibitor cocktail, and other reagents were from Sigma-Aldrich, and human plasma kallikrein was purchased from Enzyme Research (South Bend, IN). Crystalline BSA and crystalline aprotinin were from Calbiochem (San Diego, CA). ELISA high-sensitivity kit for BK was purchased from Bachem (King of Prussia, PA). RNA STAT-60 kit for total RNA isolation was from Tel-Test (Friendswood, TX). Hamster liver was from Pel-Freez (Rogers, AR), and the rat liver total mRNA sample was a gift from D. Riley (Dept. of Anesthesiology, University of Illinois at Chicago). SuperScript one-step RT-PCR with Platinum Taq kit was purchased from Invitrogen. We synthesized a BK analog by increasing its size at the NH2 terminus (11, 39), dansylating both the {alpha}- and {epsilon}-NH2 of Lys1 [didansyl-Lys-BK (didansyl-kallidin; DidnsKD)] that rendered it resistant to ACE (11).

Cell culture. Initially, CHO cells were grown in 100-mm petri dishes in Ham’s F-12 culture medium (Cellgro), supplemented with antibiotic and 10% fetal bovine serum under 5% CO2 and a water-saturated environment. Cells were routinely subcultured with the use of trypsin-EDTA for detachment and transfer. CHO cells stably transfected with either BK B2 receptor or both ACE and B2 receptor were adapted to culture in PFAF medium for attached cells (Sigma). The medium was supplemented with 200 mM L-glutamine, and the cultures were maintained at 37°C under 5% CO2 in a water-saturated environment. The cells were subcultured by rinsing with the same serum-free medium, and they were then scraped and transferred to 100-mm petri dishes in fresh medium. For measurements of AA release and other experiments, cells were cultured in CHO PFAF medium for at least 8–10 passages to exclude artifacts from prior exposure to fetal calf serum before transfer into 12-well plates for experiments.

Transfection with human B2 receptor cDNA and human ACE cDNA. pcDNA3 plasmid containing human BK B2 receptor was used to transfect CHO cells (24, 25). To obtain CHO cells that coexpress both human ACE and BK B2 receptor (AB), the cells were first transfected with human ACE pcDNA6 plasmid, and individual clones were screened for ACE activity (11, 34). Clones with high ACE activity were then transfected with human BK B2 receptor cDNA as described above (CHO AB cells; 33).

[3H]AA release. Experiments were performed essentially as previously described (11, 24). Briefly, the cells were grown to confluence in a 12-well dish. The medium was replaced with 1 ml of culture medium containing 0.5 mCi/ml of [3H]AA, and cells were loaded for 16 h at 37°C. After three washings with incubation medium (Ham’s F-12 medium containing 0.1% albumin), the cells were incubated for 10 min at 37°C in the presence of enalaprilat (1 µM). Kallikrein, DidnsKD, or other agonist was then added, and cells were incubated for an additional 20 min at 37°C. In parallel samples we added a B2 receptor blocker, HOE-140, before the addition of the test reagent. After completion of the incubation, the medium was removed and the [3H]AA content was measured in a scintillation counter. To trap released [3H]AA and block its reuptake, we added albumin that adsorbed the [3H]AA to be counted. We used defatted BSA in some experiments and crystallized albumin in others, but the results were the same. Neither preparation had contaminating kininogen.

ELISA. Enzyme immunoassays used a high-sensitivity kit for BK (Bachem) according to the manufacturer’s protocol. Briefly, 50 µl of standard solutions or test samples were added to immunoplate multiwells, 25 µl of each primary antisera and biotinylated peptide solution were then added, and the plates were incubated for 2 h at room temperature with mild agitation. The plates were then washed five times, and 100 µl of diluted streptavidin-conjugated horseradish peroxidase solution was added to each well. After a 60-min incubation at room temperature, the immunoplates were washed five times, and 100 µl of 3,3',5,5'-tetramethyl benzidine dihydrochloride (TMB) solution was added to each well. After a further 20-min incubation at room temperature, the reaction was stopped with 100 µl of 2 N HCl. Absorbance was read at 450 nm, using 100 µl of TMB solution and 100 µl 2 N HCl as a blank control.

Radioligand binding. Cells were grown in 24-well plates. In [3H]BK saturation-binding experiments, cells were washed with serum-free Ham’s F-12 medium, and increasing concentrations of [3H]BK were added to wells (24). After 1 h incubation at 4°C, cells were washed three times with serum-free Ham’s F-12 medium; solubilized in 0.5 ml of a solution containing 0.1 M NaOH, 0.1 M NaHCO3, and 1% SDS; and counted. Nonspecific binding was determined in the presence of 10 µM unlabeled BK, and specific binding was calculated as the difference between total and nonspecific binding. CHO AB/PFAF cells were preincubated with ACE inhibitor enalaprilat (1 µM) for 20 min before adding BK.

In radioligand displacement experiments, ~1 nM [3H]BK competed for the binding with increasing concentrations of DidnsKD (ranging from 10–12 to 10–4 M). As a control, homologous displacement using unlabeled BK was performed.

Total RNA/mRNA isolation. We used RNA STAT-60 isolation reagent according to the manufacturer’s protocol. The cells were lysed by using RNA STAT-60 and homogenized, chloroform was then added, and the samples were vortexed and held at room temperature for 2–3 min. All samples were centrifuged at 12,000 g for 15 min at 4°C, and the aqueous phase was transferred to a fresh tube and mixed with isopropanol. After 10-min incubation at room temperature, the samples were centrifuged at 12,000 g for 10 min at 4°C. The resulting RNA pellet was washed with 75% ethanol by vortexing and centrifugation at 7,500 g for 5 min at 4°C. The samples were then air-dried briefly and dissolved in RNase-free water.

RT-PCR. We used SuperScript one-step RT-PCR with Platinum Taq kit (Invitrogen) according to the manufacturer’s protocol. PCR primers for kininogen (KNG) were KNG3 (5'-GCCCAGAGCTGAAGGAGG) and KNG4 (5'-CATGTACACGTTAGCATTGCAG), and GAPDH1 (5'-CGACCCCTTCATTGACCTC) and GAPDH2 (5'-CTCCACGACATACTCAGCACC) for GAPDH.

Statistical analysis. Values as means ± SE were calculated for the experiments, and statistical significance of differences between means was tested by a paired t-test (Microsoft Excel).


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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CHO cells in serum-free medium. CHO cells stably transfected with human BK B2 (CHO B2) receptors were grown and maintained in medium entirely free of animal proteins for eight or more passages. Activation of the B2 receptor on these cells released [3H]AA, presumably by a mechanism involving a G{alpha}i protein coupled to the receptor, which in turn activates phospholipase A2 (4). With basal release as 1.0, (Fig. 1) tissue kallikrein (1 and 10 nM; Fig. 1A) increased the amount of released [3H]AA by 1.9 ± 0.3- or 2.9 ± 0.4-fold (n ≥ 4 experiments). All experiments were done in triplicate, and in a pilot experiment, human plasma kallikrein was as active as tissue kallikrein. BK (0.1–1 nM; Fig. 1B) also significantly enhanced the release of [3H]AA, increasing it 1.8 ± 0.2- and 3.3 ± 0.4-fold beyond the basal level. The B2 receptor blocker HOE-140 (0.5 µM) almost completely abolished the activation of the receptor by either BK or kallikrein.


Figure 1
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  		Fig. 1. Release of [3H]arachidonic acid (AA) from Chinese hamster ovary (CHO) cells by tissue kallikrein (KLK) or bradykinin (BK). Cells were stably transfected with human BK B2 receptors and cultured and maintained in entirely serum-free medium. Incubation with tissue KLK (1 and 10 nM; A) and BK (0.1 and 1 nM; B) for 30 min stimulated release of [3H]AA from previously labeled cells. Pretreatment of cells with HOE-140 (HOE, 0.5 µM) inhibited both KLK and BK-induced release of [3H]AA. Data are relative amount of [3H]AA released, with basal (spontaneous) release = 1. Data are means ± SE from 4 or more separate experiments done in triplicate. ***P < 0.001 indicates significant difference.

 
ELISA. To rule out any possible release of BK by kallikrein from kininogen expressed by or adhering to CHO B2 cells even in protein-free medium, we used an ELISA for BK. The linear portion of the assay standard curve ranged from 2 x 10–9 to 1 x 10–10 M BK in >4 experiments. Tissue kallikrein (10 nM) was tested first by adding it to a medium containing 10% fetal bovine serum. It released BK in excess of 10–8 M in 30 min. Serum-free culture medium and conditioned medium from kallikrein-treated cells were assayed along with appropriate reagent controls. We measured only a basal value 0.2 ng/ml from protein-free medium sampled both in the presence or absence of CHO B2 cells (data not shown); these findings confirm the absence of kininogen on cell membranes or in the culture medium. Because immunoreactivity of the medium collected from kallikrein-treated (10 nM) cells did not exceed the reagent baseline, we concluded that kallikrein failed to release kinin from either cell-free medium or CHO B2 cells and that the enzyme activated the BK B2 receptor by another mechanism.

Lack of kininogen expression. We considered the possibility that CHO cells constitutively express kininogen, even when grown in protein-free medium, so we used RT-PCR to test for kininogen expression. Because the exact sequence of hamster kininogen was not available, primers were synthesized from identical regions found after comparison of sequences from rat (Rattus norvegicus) kininogen 1 and mouse (Mus musculus) kininogen 2 mRNA (obtained from GenBank). We also synthesized primers based on identical regions of rat and mouse GAPDH mRNA sequences for RNA quality control. The total RNA isolated from CHO B2 cells cultured in serum-free medium was compared with the total RNA from rat and hamster liver. RNA was isolated with RNA STAT-60 and quantified by measuring UV absorbance at 260 nm. As control kininogen cDNA fragments of expected size (~247 bp) were amplified from 50 ng rat liver RNA after 35 cycles. Samples not subjected to RT-PCR had no detectable signal (not shown). Similarly, CHO B2 cell RNA mixtures, either with or without reverse transcription, yielded no detectable bands. In hamster liver RNA samples, strong bands of kininogen cDNA appeared with RT-PCR after 35 cycles with the use of 50 or 500 ng RNA (Fig. 2A ). The 200 bp cDNA fragments of GAPDH were amplified by RT-PCR from either the CHO B2 cells RNA preparation or the rat liver RNA preparation (Fig. 2B).


Figure 2
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  		Fig. 2. Kininogen mRNA is detected in hamster liver but not in CHO B2/PFAF cells. Total RNA was reverse-transcribed, and resulting cDNA was amplified by PCR and separated by agarose gel electrophoresis. A: PCR products of total RNA samples from hamster liver and CHO B2/PFAF cells amplified using kininogen primers. Lane 1, hamster liver (500 ng); lane 2, hamster liver (50 ng); lane 3, hamster liver (500 ng); lane 4, marker (M); lane 5, CHO B2/PFAF cells (500 ng); lane 6, CHO B2/PFAF cells (50 ng); and lane 7, CHO B2/PFAF cells (500 ng). B: PCR products of total RNA samples from rat liver, and CHO B2/PFAF cells amplified using GAPDH primers. Lanes 1 and 2, CHO B2/PFAF cells; lane 3, M; lanes 4 and 5, rat liver. Numbers at right are in bp.

 
Kininogens originate mainly from the liver in mammalian species (3). Thus the rat and hamster livers were strongly positive, showing appreciable amounts of mRNA, but cultured CHO cells lacked kininogen mRNA, confirming the results of our experiments (23, 25) that kallikrein indeed activates the BK B2 receptors without releasing kinin.

Effect of ACE inhibitor. We previously reported that inhibitors of ACE potentiated the effects of BK B2 receptor agonists in cultured cells and organs ex vivo (15, 33–35) by a mechanism independent of blocking BK inactivation. To determine how ACE inhibitors potentiate kinin receptor agonists, we stably transfected CHO cells bearing human BK B2 receptors with human ACE and adapted them to grow in serum-free medium (CHO AB/PFAF cells). These cotransfected CHO cells (CHO AB/PFAF) were less sensitive to B2 receptor agonists than CHO B2 cells. Despite this, both cell lines had very similar maximum binding capacity (Bmax) and Kd values [CHO B2/PFAF cells: Bmax, 214 ± 62 fmol/106 cells, Kd, 17.7 ± 5.3 nM; and CHO AB/PFAF cells: Bmax, 174 ± 70 fmol/106 cells, Kd, 11.3 ± 5.7 nM (n = 3 experiments)]. Figure 3 shows that kallikrein (100 nM) increased release of [3H]AA 1.73 ± 0.14-fold over the basal level, and the ACE inhibitor enalaprilat (1 µM) significantly enhanced it, augmenting release of [3H]AA 5.6 ± 0.47-fold over the basal level. This potentiation was blocked when B2 receptor antagonist HOE-140 (0.5 µM) was added to the reaction mixture (Fig. 3). We also used the ACE-resistant peptide DidnsKD as a positive control. This B2 receptor agonist resists degradation by ACE by 97% (11), because dansylation of both {alpha}- and {epsilon}-NH2 groups of Lys1 blocks hydrolysis by ACE, which cleaves peptides only of restricted size (13, 16, 43). In the displacement of labeled BK, this B2 agonist had a Ki of 1.96 ± 0.2 µM at 4°C. DidnsKD (1 µM) stimulated [3H]AA release 3.07 ± 0.2-fold over the basal level. Addition of enalaprilat enhanced [3H]AA release by DidnsKD even further to 6.3 ± 0.9-fold (Fig. 3), and it was inhibited by HOE-140 (0.5 µM). Enalaprilat potentiates only active kallikrein. When inhibited by crystalline aprotinin (1 µM; Fig. 4A), it was inactive and enalaprilat had no effect.


Figure 3
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  		Fig. 3. Effect of enalaprilat (EPT) on [3H]AA release by either kallikrein or ACE resistant BK analog in CHO with ACE and B2 receptors coexpression (AB)/PFAF cells. Cells expressing both BK B2 receptor and ACE were grown in serum-free medium as described in MATERIALS AND METHODS. Pretreatment of cells with EPT (1 µM) for 10 min significantly enhanced release of [3H]AA in response to either KLK (100 mM) or didansyl-kallidin (DidnsKD; 1 µM). HOE (0.5 µM) blocked activation of B2 receptor by either agonist. Data are relative release of [3H]AA above basal (spontaneous) release and are means ± SE of 3–6 separate experiments done in triplicate. ***P < 0.001 indicates significant difference.

 

Figure 4
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  		Fig. 4. Effects of aprotinin and lack of ACE. Conditions and ordinate as in Fig. 1. A: release of [3H]AA in CHO AB/PFAF cells after pretreatment of cells with EPT (1 µM), before addition KLK (100 nM) treated with crystalline aprotinin (1 µM) for 20 min. Data are means ± SE of 4–5 separate experiments. B: release of [3H]AA in CHO B2/PFAF cells that lack ACE but express BK B2 receptors. Cells were pretreated with EPT (1 µM) for 10 min before addition of either KLK (10 nM) or BK (1 nM) and incubated for 20 min. Data are means ± SE from 4 separate experiments done in triplicate. EPT did not potentiate [3H]AA release when KLK was inhibited or ACE was not expressed in cells.

 
Lack of ACE expression. To show that the ACE inhibitor is effective only when both B2 receptors and ACE are expressed, we tested CHO B2 cells that have no ACE. CHO B2 cells also lack B1 receptors that might be activated by an ACE inhibitor (28). Figure 4B shows that, as previously noted (15), enalaprilat was ineffective in the absence of ACE. It failed to enhance [3H]AA release by either BK or kallikrein (n ≥ 3 experiments). We concluded that kallikrein must be active to act as a B2 receptor agonist (25), and to be potentiated by an ACE inhibitor requires the expression of ACE.

Finally, we used an ELISA to see whether enalaprilat might increase the putative presence of BK locally by blocking its inactivation by CHO AB/PFAF cells. However, cells pretreated with enalaprilat (1 µM) before stimulation by kallikrein did not release BK (results not shown). BK was absent in the samples with or without added enalaprilat, allowing us to rule out BK release or protection from degradation as a cause of enhanced AA release.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 
We found that cells grown and maintained in protein-free medium can be activated by kallikrein, despite the fact that they were never exposed to plasma kininogen, the precursor of kinins. Human plasma has two kininogens. These proteins, high and low molecular weight kininogen, are products of the same gene; both are synthesized in the liver and released into circulation (3, 14). Plasma kallikrein cleaves the high molecular weight substrate, whereas tissue kallikrein hydrolyzes both proteins. When blood coagulates, much of the plasma kininogen is metabolized, which is likely what happens to the fetal bovine serum commonly used to supplement media for cultured cells.

Nevertheless, in our experiments we eliminated the possibilities that kallikrein activated the B2 receptors of cultured cells because of the presence of kininogen. Initially, we used serum-starved CHO cells to exclude the possibility of an uptake of kininogen from the medium. Next, although Zn2+ facilitate adsorption of kininogen from the medium to the cell membrane (29, 41, 42), we demonstrated previously that activation of B2 receptors by kallikrein was unaffected in the absence of zinc (25). Here we report that cells were cultured in entirely serum-free medium. When we applied an ELISA to measure putative kinin release from these cells, the results were negative. Finally, to eliminate the possibility that the CHO cells could synthesize kininogen, we showed that the cells we used lack kininogen mRNA. Taken together, these findings effectively eliminate participation of kinin release by kallikrein as a mechanism of BK B2 receptor activation in our studies.

We measured [3H]AA release from CHO cells by BK or kallikrein as an indication of receptor activation and showed that HOE-140, a B2 receptor blocker, inhibited release by either agonist.

ACE inhibitors are successfully used in therapy for a variety of cardiovascular diseases (18, 20, 22, 40). At least part of the effects of an ACE inhibitor may depend on actions beyond blocking the inactivation of BK by ACE, specifically the direct potentiation of BK at its B2 receptors (33–35). Although the first clinically tested ACE inhibitor was a peptide derived from the so-called BK potentiating factor in snake venom (22), BK potentiation and kininase ACE inhibition were not parallel (15). Indeed, they differed a great deal in a series of synthetic peptide congeners (37); peptides also potentiated in the absence of ACE expression. Synthetic ACE inhibitors can enhance the actions of BK on its B2 receptors by a cross talk between the receptor and ACE (15). For this reason, we wondered whether ACE inhibitors would potentiate kallikrein effects on the BK B2 receptors in a similar manner. To confirm that potentiation occurred in the absence of kinin generation and independent of BK inactivation, we synthesized a BK analog (DidnsKD) with a modified NH2 terminus. This structural change rendered the peptide almost entirely (97%) resistant to ACE (11). ACE inhibitor can enhance ACE and cyclooxygenase-2 expression in endothelial cells. This involves the phosphorylation of Ser1270 of the COOH-terminal cytosolic sequence of ACE (30, 31). Our acute experiments would unlikely involve protein synthesis, which may take 24–36 h (30, 31). ACE inhibitor potentiated BK and resensitized B2 receptors to the peptide; even when in a mutated, truncated ACE, the COOH-terminal 19 amino acids of the cytosolic portions of human ACE were deleted (33), thus very likely in the absence of phosphorylation.

Enalaprilat had no effect on the release of [3H]AA from labeled CHO B2 cells in serum-free medium. These cells were transfected to express B2 receptors but lacked ACE. In contrast, the addition of the ACE inhibitor to CHO cells that stably express both human ACE and B2 receptors potentiated the effect of kallikrein and an ACE-resistant BK analog. These findings suggest that an additional contribution to the multiple beneficial effects of ACE inhibitors could include the enhanced activation of B2 receptors by kallikrein.

We reported that kallikreins (tissue or plasma) activate B2 receptors differently than BK. For example, kallikreins and BK both desensitized B2 receptors, but there was no cross-desensitization (25). Experiments with a carboxypeptidase BK inactivator and site-directed mutagenesis underscored the differences between the protease and the peptide B2 agonists. Our investigations confirm that ACE inhibitors act beyond blocking kinin inactivation by ACE. Enalaprilat enhanced both the ACE-resistant BK analog and kallikrein activity in an apparently kinin-free system. Because ACE and B2 can form heterodimers (33) and can colocalize closely on cell membranes (Chen Z, Tan F, Erdös EG, and Deddish PA, unpublished data), our findings suggest that ACE inhibitors may induce conformational changes via ACE, acting as indirect allosteric enhancers (9) of BK B2 receptor agonists. Once the receptor is activated, the mechanism presumably follows a common path, namely, the coupling G{alpha}i protein and then phospholipase A2 activation.

Over the long history of kallikrein research, some experimental results could not be explained by kallikreins acting exclusively on plasma kininogen to release a kinin (45). For example, rat urinary kallikrein or trypsin applied at short intervals stimulated the isolated estrogen-primed rat uterus repeatedly (2, 10, 17). This repetitive action on the isolated organ cannot be due to a continuous replenishment of kininogen.

Furthermore, kallikreins release one molecule of kinin from each kininogen molecule, and the liberated kinins are rapidly cleaved by kininases. Kallidin (Lys-BK) is converted to BK by the removal of the NH2-terminal Lys (16). The half-life of injected BK is only 15–20 s, indicating that kinin release and action would necessarily occur within a discrete, local environment. In this regard, kinins function as paracrine agents (8). Kinin generation requires a very rapid reaction where possibly only a fraction of the kininogen substrate is cleaved. If only 10% of the plasma protein kininogen substrate was hydrolyzed, high or low molecular weight kininogen with a molecular mass of ~50 or 100 kDa would need to be in 500- to 1,000-fold excess to release a single molecule of BK (1 kDa) near to a receptor. This assumption then raises the question as to whether this weight ratio would always be obtained at the outer surface of cellular plasma membranes.

The direct activation of the human B2 peptide receptor (25, 27) offers an alternative pathway of activation for this multistep enzymatic process. We have identified such a pathway in cultured cells, and we believe that this mechanism may explain some of the phenomena associated with the therapeutic actions of ACE inhibitors in vivo.


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This work was supported by National Heart, Lung, and Blood Institute Grants HL-36473 and HL-68580.


   ACKNOWLEDGMENTS
 
The authors thank Cynthia Sanders of University of Illinois at Chicago College of Medicine for help in editing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. G. Erdös, Dept. of Pharmacology (MC 868), 835 S. Wolcott, Rm. E403, Chicago, IL 60612 (e-mail: egerdos{at}uic.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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 ABSTRACT
 MATERIALS AND METHODS
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  1. Beraldo WT and Andrade SP. Discovery of bradykinin and the kallikrein-kinin system. In: The Kinin System, edited by Farmer SG. San Diego, CA: Academic, 1997, p. 1–8.
  2. Beraldo WT, Araujo RL, and Mares-Guia M. Oxytocic esterase in rat urine. Am J Physiol 211: 975–980, 1966.[Free Full Text]
  3. Bhoola KD, Figueroa CD, and Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol Rev 44: 1–80, 1992.[Web of Science][Medline]
  4. Campbell WB and Halushka PV. Lipid-derived autacoids: eicosanoids and platelet-activating factor. In: Goodman and Gilman’s The Pharmacological Basis of Therapeutics (9th ed.), edited by Hardman JC, Limberd LE, Molinoff PB, Ruddon RW, and Gilman AG. New York: McGraw-Hill, 1996, p. 602–616.
  5. Campbell WB and Harder DR. Endothelium-derived hyperpolarizing factors and vascular cytochrome P450 metabolites of arachidonic acid in the regulation of tone. Circ Res 84: 484–488, 1999.[Free Full Text]
  6. Carretero OA, Miyazaki S, and Scicli AG. Role of kinins in the acute antihypertensive effect of the converting enzyme inhibitor, captopril. Hypertension 3: 18–22, 1981.[Abstract/Free Full Text]
  7. Carretero OA and Scicli AG. The kallikrein-kinin system as a regulator of cardiovascular and renal function. In: Hypertension: Pathophysiology, Diagnosis, and Management (2nd ed.), edited by Laragh JH and Brenner BM. New York: Raven, 1995, p. 983–999.
  8. Carretero OA, Scicli AG, and Maitra SR. Role of kinins in the pharmacological effects of converting enzyme inhibitors. In: Angiotensin Converting Enzyme Inhibitors. Mechanisms of Action and Clinical Implications, edited by Horovitz Z. Baltimore, MD: Urban & Schwarzenberg, 1981, p. 105–121.
  9. Changeux JP and Edelstein SJ. Allosteric mechanisms of signal transduction. Science 308: 1424–1428, 2005.[Abstract/Free Full Text]
  10. Chao J, Buse J, Shimamoto K, and Margolius HS. Kallikrein-induced uterine contraction independent of kinin formation. Proc Natl Acad Sci USA 78: 6154–6157, 1981.[Abstract/Free Full Text]
  11. Chen Z, Tan F, Erdös EG, and Deddish P. Hydrolysis of angiotensin peptides by human angiotensin I-converting enzyme and the resensitization of B2 kinin receptors. Hypertension 46: 1386–1373, 2005.
  12. Colman RW. Plasma prekallikrein and kallikrein. In: Handbook of Proteolytic Enzymes (2nd ed.), edited by Barrett AJ, Rawlings ND, and Woessner JF. San Diego, CA: Academic, 2004, p. 1644–1651.
  13. Corvol P, Eyries M, and Soubrier F. Peptidyl-dipeptidase A/angiotensin I-converting enzyme. In: Handbook of Proteolytic Enzymes (2nd ed.), edited by Barrett AJ, Rawlings ND, and Woessner JF. San Diego, CA: Academic, 2004, p. 337–346.
  14. Erdös EG. Bradykinin, Kallidin and Kallikrein. Handbook of Experimental Pharmacology (suppl. to XXV), edited by Erdös EG. Berlin, Germany: Springer-Verlag, 1979, p. 1–817.
  15. Erdös EG, Deddish PA, and Marcic BM. Potentiation of bradykinin actions by ACE inhibitors. Trends Endocrinol Metab 10: 223–229, 1999.[CrossRef][Web of Science][Medline]
  16. Erdös EG and Skidgel RA. Metabolism of bradykinin by peptidases in health and disease. In: The Kinin System, edited by Farmer SG. London: Academic, 1997, p. 111–141.
  17. Erdös EG, Tague LL, and Miwa I. Kallikrein in granules of the submaxillary gland. Biochem Pharmacol 17: 667–674, 1968.[CrossRef][Web of Science][Medline]
  18. Frohlich ED. Left ventricular enlargement in hypertension: it’s not only LVH. In: Angiotensin-Converting Enzyme (ACE): Clinical and Experimental Insights, edited by Giles TD. Fort Lee, NJ: Health Care Communications, 2001, p. 189–195.
  19. Gaddum JH. Polypeptides Which Stimulate Plain Muscle. London: Livingston, 1955, p. 1–140.
  20. Gainer JV, Morrow JD, Loveland A, King DJ, and Brown NJ. Effect of bradykinin-receptor blockade on the response to angiotensin-converting-enzyme inhibitor in normotensive and hypertensive subjects. N Engl J Med 339: 1285–1292, 1998.[Abstract/Free Full Text]
  21. Gavras I and Gavras H. Hypertension, vasoactive peptides and coagulation factors. J Hypertens 22: 1091–1092, 2004.[CrossRef][Web of Science][Medline]
  22. Gavras I and Gavras H. The use of ACE inhibitors in hypertension. In: Angiotensin Converting Enzyme Inhibitors, edited by Kostis JB and DeFelice EA. New York: Liss, 1987, p. 93–122.
  23. Hecquet C, Becker RP, Tan F, and Erdös EG. Kallikreins when activating bradykinin B2 receptor induce its redistribution on plasma membrane. Int Immunopharmacol 2: 1795–1806, 2002.[CrossRef][Web of Science][Medline]
  24. Hecquet C, Biyashev D, Tan F, and Erdös EG. Positive cooperativity between the thrombin and bradykinin B2 receptors enhances arachidonic acid release. Am J Physiol Heart Circ Physiol 290: H000–H000, 2006.
  25. Hecquet C, Tan F, Marcic BM, and Erdös EG. Human bradykinin B2 receptor is activated by kallikrein and other serine proteases. Mol Pharmacol 58: 828–836, 2000.[Abstract/Free Full Text]
  26. Hess JF. Molecular pharmacology of kinin receptors. In: The Kinin System, edited by Farmer SG. San Diego, CA: Academic, 1997, p. 45–55.
  27. Hess JF, Borkowski JA, Macneil T, Stonesifer GY, Fraher J, Strader CD, and Ransom RW. Differential pharmacology of cloned human and mouse B2 bradykinin receptors. Mol Pharmacol 45: 1–8, 1994.[Abstract]
  28. Ignjatovic T, Tan F, Brovkovych V, Skidgel RA, and Erdös EG. Novel mode of action of angiotensin I converting enzyme inhibitors. Direct activation of bradykinin B1 receptor. J Biol Chem 277: 16847–16852, 2002.[Abstract/Free Full Text]
  29. Kaplan AP, Joseph K, and Silverberg M. Pathways for bradykinin formation and inflammatory disease. J Allergy Clin Immunol 109: 195–209, 2002.[CrossRef][Web of Science][Medline]
  30. Kohlstedt K, Brandes RP, Muller-Esterl W, Busse R, and Fleming I. Angiotensin-converting enzyme is involved in outside-in signaling in endothelial cells. Circ Res 94: 60–67, 2004.[Abstract/Free Full Text]
  31. Kohlstedt K, Busse R, and Fleming I. Signaling via the angiotensin-converting enzyme enhances the expression of cyclooxygenase-2 in endothelial cells. Hypertension 45: 126–132, 2005.[Abstract/Free Full Text]
  32. Mahdi F, Shariat-Madar Z, and Schmaier AH. The relative priority of prekallikrein and factors XI/XIa assembly on cultured endothelial cells. J Biol Chem 278: 43983–43990, 2003.[Abstract/Free Full Text]
  33. Marcic B, Deddish PA, Skidgel RA, Erdös EG, Minshall RD, and Tan F. Replacement of the transmembrane anchor in angiotensin I-converting enzyme (ACE) with a glycosylphosphatidylinositol tail affects activation of the B2 bradykinin receptor by ACE inhibitors. J Biol Chem 275: 16110–16118, 2000.[Abstract/Free Full Text]
  34. Marcic BM, Deddish PA, Jackman HL, Erdös EG, and Tan F. Effects of the N-terminal sequence in the N-domain of ACE on the properties of its C-domain. Hypertension 36: 116–121, 2000.[Abstract/Free Full Text]
  35. Marcic BM and Erdös EG. Protein kinase C and phosphatase inhibitors block the ability of angiotensin I-converting enzyme inhibitors to resensitize the receptor to bradykinin without altering the primary effects of bradykinin. J Pharmacol Exp Ther 294: 605–612, 2000.[Abstract/Free Full Text]
  36. Margolius HS. The kallikrein-kinin system and the kidney. Annu Rev Physiol 46: 309–326, 1984.[CrossRef][Web of Science][Medline]
  37. Mueller S, Gothe R, Siems WD, Vietinghoff G, Paegelow I, and Reissmann S. Potentiation of bradykinin actions by analogues of the bradykinin potentiating nonapeptide BPP9alpha. Peptides 26: 1235–1247, 2005.[CrossRef][Web of Science][Medline]
  38. O’Brien PJ, Molino M, Kahn M, and Brass LF. Protease activated receptors: theme and variations. Oncogene 20: 1570–1581, 2001.[CrossRef][Web of Science][Medline]
  39. Odya CE, Levin Y, Erdös EG, and Robinson CJG. Soluble dextran complexes of kallikrein, bradykinin, and enzyme inhibitors. Biochem Pharmacol 27: 173–179, 1978.[CrossRef][Web of Science][Medline]
  40. Pfeffer M. Angiotensin-converting enzyme (ACE) inhibitors in the prevention of cardiovascular disease: clinical evidence. In: Angiotensin-Converting Enzyme (ACE): Clinical and Experimental Insights, edited by Giles TD. Fort Lee, NJ: Health Care Communications, 2001, p. 219–225.
  41. Schmaier AH. The kallikrein-kinin and the renin-angiotensin systems have a multilayered interaction. Am J Physiol Regul Integr Comp Physiol 285: R1–R13, 2003.[Abstract/Free Full Text]
  42. Schmaier AH, Rokjaer R, and Shariat-Madar Z. Activation of the plasma kallikrein/kinin system on cells: a revised hypothesis. Thromb Haemost 82: 226–233, 1999.[Web of Science][Medline]
  43. Skidgel RA and Erdös EG. Angiotensin converting enzyme (ACE) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies. Peptides 25: 521–525, 2004.[CrossRef][Web of Science][Medline]
  44. Skidgel RA and Erdös EG. Lysine carboxypeptidase. In: Handbook of Proteolytic Enzymes (2nd ed.), edited by Barret AJ, Rawlings ND, and Woessner JF. San Diego, CA: Academic, 2004, p. 837–839.
  45. Trabold F, Pons S, Hagege AA, Bloch-Faure M, Alhenc-Gelas F, Giudicelli JF, Richer-Giudicelli C, and Meneton P. Cardiovascular phenotypes of kinin B2 receptor- and tissue kallikrein-deficient mice. Hypertension 40: 90–95, 2002.[Abstract/Free Full Text]
  46. Werle E. Discovery of the most important kallikreins and kallikrein inhibitors. In: Bradykinin, Kallidin and Kallikrein. Handbook of Experimental Pharmacology, edited by Erdös EG. Berlin, Germany: Springer Verlag, 1970, vol. 25, p. 1–6.
  47. Yang HY, Erdös EG, and Levin Y. Characterization of a dipeptide hydrolase (kininase II: angiotensin I converting enzyme). J Pharmacol Exp Ther 177: 291–300, 1971.[Abstract/Free Full Text]



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