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Am J Physiol Heart Circ Physiol 293: H3550-H3557, 2007. First published September 28, 2007; doi:10.1152/ajpheart.00784.2007
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Carboxypeptidase B and other kininases of the rat coronary and mesenteric arterial bed perfusates

Eduardo B. Oliveira,1 Laura L. Souza,1 Disney O. Sivieri, Jr,2 Luiz B. Bispo-da-Silva,2 Hugo J. V. Pereira,1 Claudio M. Costa-Neto,1 Marcelo V. Sousa,3 and Maria Cristina O. Salgado2

Departamentos de 1Bioquímica e Imunologia e de 2Farmacologia da Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; and 3Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, Brazil

Submitted 6 July 2007 ; accepted in final form 20 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We describe the enzymes that constitute the major bradykinin (BK)-processing pathways in the perfusates of mesenteric arterial bed (MAB) and coronary vessels isolated from Wistar normotensive rats (WNR) and spontaneously hypertensive rats. The contribution of particular proteases to BK degradation was revealed by the combined analysis of fragments generated during incubation of BK with representative perfusate samples and the effect of selective inhibitors on the respective reactions. Marked differences were seen among the perfusates studied; MAB secretes, per minute of perfusion, kininase activity capable of hydrolyzing ~300 pmol of BK/min, which is ~250-fold larger amount on a per unit time basis than that of its coronary counterpart. BK degradation in the coronary perfusate seems to be mediated by ANG I-converting enzyme, neutral endopeptidase 24.11-like enzyme, and a DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid-sensitive basic carboxypeptidase; coronary perfusate of WNR contains an additional BK-degrading enzyme whose specificity resembles that of neurolysin or thimet oligopeptidase. Diversely, a des-Arg9-BK-forming enzyme, responsible for nearly all of the kininase activity of MAB perfusates of WNR and spontaneously hypertensive rats, could be purified by a procedure that involved affinity chromatography over potato carboxypeptidase inhibitor-Sepharose column and shown to be structurally identical to rat pancreatic carboxypeptidase B (CPB). Comparable levels of CPB mRNA expression were observed in pancreas, liver, mesentery, and kidney, but very low levels were detected in lung, heart, aorta, and carotid artery. In conclusion, distinct BK-processing pathways operate in the perfusates of rat MAB and coronary bed, with a substantial participation of a des-Arg9-BK-forming enzyme identical to pancreatic CPB.

bradykinin; angiotensin-converting enzyme; neutral endopeptidase; carboxypeptidase N; basic carboxypeptidase

THE NONAPEPTIDE BRADYKININ (BK) has important pharmacological effects on blood vessels, heart, and kidney; among these, the most conspicuous is the transient hypotensive effect elicited by the administration of BK into the systemic circulation of all species studied (3). BK circulates in the low nanomolar range, a concentration two orders of magnitude lower than that needed to decrease blood pressure (27), indicating that BK must act as an autocrine or paracrine hormone as a modulator of the cardiovascular function (2). Consequently, the concentration of BK at its site of action is a critical determinant for BK-mediated effects, which may be markedly influenced by the presence of kininases in the surrounding tissue and blood. Physiologically relevant kininases in most tissues or blood are ANG I-converting enzyme (kininase II; ACE), carboxypeptidase N (kininase I; CPN), carboxypeptidase M (CPM), and neutral endopeptidase 24.11 (NEP). ACE is a metallopeptidase that removes the COOH-terminal dipeptide from BK and eventually cleaves its primary metabolite BK(1-7) further into the shorter fragment BK(1-5); soluble plasma CPN and membrane-bound CPM are basic carboxypeptidases capable of generating des-Arg9-BK from BK, a reaction that may correspond to either inactivation of BK or alteration of its receptor specificity depending on the responsiveness of the surrounding tissues; and NEP inactivates BK by cleaving the Gly4-Phe5 or the Pro7-Phe8 bond of the nonapeptide (10, 11, 35, 36). The contribution of individual kininases to the metabolism of BK in different tissues or blood has been assessed by the effects of selective protease inhibitors on the pharmacological properties of the peptide or on its cleavage profile. Accordingly, in human and rat plasma, BK is degraded mainly by the actions of ACE and CPN (13, 15, 31). In human heart membranes, ACE alone was found to play a major role in BK metabolism (1), whereas in rat heart membranes both ACE and NEP participate in BK degradation (25), the same two enzymes that inactivate BK during a single passage of the peptide through the isolated rat coronary bed (7). On the other hand, we have recently shown that a basic carboxypeptidase is the most important kininase involved in the metabolism of BK that was allowed to recirculate through the rat isolated mesenteric arterial bed (MAB) (33).

We have previously described that isolated and perfused rat MAB secretes endo- and exopeptidases capable of metabolizing vasoactive peptides, among which a carboxypeptidase, referred to as CPN-like enzyme for its ability to generate des-Arg9-BK from BK, which accounted for nearly all of the kininase activity of the perfusate (23). Because it is well recognized that local kinins have beneficial effects in renal and cardiovascular diseases besides their role in lowering blood pressure (18), it is possible that locally secreted kininases influence BK activity in the vascular wall or in specific compartments of a particular tissue, including the heart and kidneys, and might have a role, together with membrane kininases, in pathophysiological conditions. In the present work, we describe the major soluble enzymes responsible for BK degradation in the perfusates obtained from MAB and hearts of normotensive and spontaneously hypertensive rats (SHR). Additionally, because a soluble kininase I-type carboxypeptidase constitutes the major BK-processing pathway in the MAB perfusates and because of the potential importance of product of this peptidase, the active agonist des-Arg9-BK that specifically binds to the B1 kinin receptor (17), we also present some essential biochemical and structural features of this basic carboxypeptidase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. In the experiments, we used 11- to 12-wk-old male Wistar normotensive rats (WNR; n = 16) and SHR (n = 12) that were maintained in a controlled environment with a 12:12-h light-dark cycle and provided food and water ad libitum. All experimental protocols used in this study were reviewed and approved by the Animal Care and Use Committee of the Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo.

Arterial pressure measurement. The day before the experiments, rats were anesthetized with tribromoethanol (250 mg/kg ip), and a polyethylene catheter was inserted into the abdominal aorta through the right femoral artery. The distal end was exteriorized through the animal's back. On the day of the experiment, after arterial pressures from conscious rats were individually recording (Hewlett Packard 7754 A recorder, Palo Alto, CA), each animal was anesthetized and the mesentery or the heart was rapidly removed and handled as described below.

Perfused, isolated mesenteric bed preparation. The mesentery was removed with a polyethylene cannula inserted into the superior mesenteric artery and placed ready for perfusion in a water-jacketed organ bath maintained at 37°C, as previously described (28, 33). Briefly, the MAB perfusion was carried out by infusing a modified Krebs solution (in mmol/l: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.64 MgSO4, 1.18 KH2PO4, 24.9 NaHCO3, 11.1 glucose) equilibrated with a 95% O2-5% CO2 mixture (pH 7.4) through the mesenteric artery at a constant flow rate of 4 ml/min. After a 20-min period of perfusion with input of fresh Krebs solution to ensure thorough removal of blood substances from the preparation, the piping connections of the perfusion setup were altered so as to keep 10 ml of the perfusing solution recirculating through the MAB for 2 h. The perfusate was then recovered, and its protein content was 10-fold concentrated using an Amicon apparatus fitted with a YM-10 membrane and kept at 4°C until use.

Perfused isolated heart preparation. The heart was rapidly removed and mounted on a modified Langendorff apparatus and perfused through a cannula inserted into the aorta with 100 ml of the above-mentioned Krebs solution containing 2 mmol/l sodium pyruvate. The perfusion was carried out at a flow rate of 10 or 8 ml/min in hearts isolated from WNR or SHR, respectively. Under these conditions, the isolated hearts showed stable heart rate and ventricular contractility for at least 2 h. After a 10-min period of stabilization with input of fresh Krebs solution to ensure thorough removal of blood substances from the preparation, the piping connections of the perfusion setup were altered so as to keep ~125 ml of perfusing solution recirculating through the coronary bed during 2 h. The perfusate was then recovered and its volume reduced to 0.5 ml, corresponding to a 250-fold concentration of its protein content, by using an Amicon apparatus fitted with a YM-10 membrane and kept at 4°C until use.

Determination of proteolytic activities in the cardiac and mesenteric perfusates and fractions thereof. The proteolytic activities of the rat mesenteric and cardiac perfusates toward BK were investigated by determining the HPLC profiles of BK fragments generated in the presence or absence of protease inhibitors. Assays were carried out in 150 µl of 30 mmol/l Tris-buffered saline (30 mmol/l Tris·HCl, pH 7.4, containing 150 mmol/l NaCl), by incubating 30 nmol of BK with 10 µl of concentrated MAB or cardiac perfusate at 37°C for 3-min or 6-h periods, respectively. The inhibitors used were 10 µmol/l captopril, an ACE inhibitor; 10 µmol/l phosphoramidon, a NEP inhibitor; 0.3–300 µmol/l DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA), a basic carboxypeptidase inhibitor; 20 µmol/l potato carboxypeptidase inhibitor (PCI), a 4.3-kDa polypeptide that inhibits pancreatic carboxypeptidases; and 1 mmol/l 1,10-phenanthroline, an inhibitor of zinc metalloenzymes. After reactions were terminated by the addition of 10 µl of 5% trifluoroacetic acid (TFA), the resulting BK fragments were separated by reverse-phase HPLC on Shimadzu 6B equipment fitted with a Shim-Pack CLC-ODS column (4.6 x 150 mm) and an ultraviolet detector set at 215 nm. Separations were performed at a flow rate of 1.0 ml/min with a 10–32% linear gradient of acetonitrile concentration in 0.1% TFA. The material corresponding to each peak of absorption at 215 nm was identified either by comparing its retention time with those of synthetic peptide standards or by its chemical composition determined by amino acid analysis after acid hydrolysis. One unit of kininase activity is defined as the amount of enzyme capable of forming 1 µmol of des-Arg9-BK per minute using BK as the substrate, under the described conditions.

Basic carboxypeptidase purification. The enzyme was purified by a combination of affinity chromatographies on PCI-Sepharose (40) and Arg-Sepharose (38) columns. Briefly, a solution of concentrated high-molecular-weight substances of eight pooled perfusates in 5 ml of equilibrating buffer (10 mmol/l Tris·HCl buffer, pH 7.5, containing 0.5 mol/l NaCl) was loaded on a PCI-Sepharose (8 x 20 mm) at room temperature at a flow rate of 0.3 ml/min. After removal of unbound material and washing of the column with 10 ml of equilibrating solution, the absorbed enzymes were recovered by percolating a 100 mmol/l Na2CO3 solution, pH 11.4, through the column. Samples of 0.7 ml were collected throughout the experiment, with pHs lowered to ~7.5 by addition of 1.0 mol/l Tris·HCl buffer, pH 7.0, as required. The fractions that had des-Arg9-BK-forming activity using BK as substrate were pooled and applied on an Arg-Sepharose column (8 x 100 mm) equilibrated and developed with 20 mmol/l Tris·HCl buffer, pH 8.1, containing 1.0 mol/l NaCl, conditions under which some of the known basic carboxypeptidases are retarded relative to other proteins (38). The active fractions were pooled, concentrated by ultrafiltration under N2 pressure, and stored at 4°C until use.

Peptide mass fingerprint. A sample of the affinity-purified basic carboxypeptidase from the MAB perfusate, containing 1.6 units of kininase activity, was subjected to SDS-PAGE on a 12% gel under reducing conditions and stained with amido black. The gel portion containing the single clearly stained protein band was excised from the gel slab and treated with trypsin (32). The tryptic peptides formed were extracted twice with 40 µl of acetonitrile-water-TFA (66:33:0.1) solution for 20 min with the aid of a sonicator apparatus, the extract dried in a vacuum centrifuge, and the product stored at –20°C before use. For mass spectrometric analysis, the product was solubilized in 4 µl of 0.1% TFA, followed by microscale concentration and desalting with C18 Zip-Tips (Millipore, Bedford, MA). Peptides were eluted directly onto a MALDI-TOF probe using 1 µl of 50% acetonitrile in 0.1% TFA solution containing matrix (20 µg/µl {alpha}-cyano-4-hydroxycinnamic acid). Mass spectra were determined with a Reflex IV (Bruker Daltonics, Karlsruhe, Germany) mass spectrometer in positive reflector mode and processed with XMASS and Biotools software (Bruker Daltonics). Spectra were internally calibrated by trypsin autolysis products (m/z 842.509 and m/z 2211.104). Protein identification was performed with MASCOT (24) at 50 ppm mass tolerance, which screened the fingerprint of the protein provided by the peptide mass information against National Center for Biotechnology Information (nonredundant) and Swiss-Prot databases.

PCR amplification of reverse-transcribed mRNA (RT-PCR). Total RNA was extracted from rat mesentery, pancreas, kidney, liver, lung, heart, aorta, and carotid with Trizol reagent, following the manufacturer's instructions (Invitrogen, Carlsbad, CA). RNA integrity was confirmed by agarose gel electrophoresis. Four micrograms of total RNA were used to perform reverse transcription of mRNAs into cDNAs using oligo-d(T) and SuperScript II protocols (Invitrogen). cDNAs for carboxypeptidase B (CPB) and β-actin were amplified by PCR using oligonucleotide primers [for CPB, 5'-GGGAATCCATGTTGCTGCTACTGGCC-3' (sense) and 5'-GGCTGCAGTCAATATAGATGTTCTCGGAC-3' (antisense); for β-actin, 5'-CTAAGGCAAACCGTGAAAAGA-3' (sense) and 5'-ATTGCCGATAGTGATGACCTG-3' (antisense)]. The sequences for the CPB primers were based on the full-length nucleotide sequence of the rat pancreatic pre-proCPB, available through the National Center for Biotechnology Information GenBank CoreNucleotide database under identification 6978696 (http://www.ncbi.nlm.nih.gov/entrez/). PCR amplification was performed with Taq DNA polymerase (Invitrogen). The process of thermal cycling consisted of initial denaturation for 2 min at 94°C followed by 43 cycles of amplification of the cDNA, each comprising 1 min of denaturation at 94°C, 1 min of annealing carried out at 60°C, and 1.5 min of extension at 72°C. Samples were incubated for an additional 30-min period at 72°C (terminal elongation) after completion of the 43 cycle process. A similar amplification protocol was used for β-actin, except for the annealing temperature at 45°C. For each set of primers, RT-PCR was performed on sterile water to check for contamination. Aliquots of 10 µl of each PCR product were run on a 1% agarose gel, stained with ethidium bromide, and subjected to densitometric scanning by ImageJ software (http://rsb.info.nih.gov/ij/); the intensity of each particular cDNA was normalized to the respective β-actin PCR product.

Statistical analysis. The results are expressed as means ± SE. Blood pressure values were compared by Student's t-test, and the amount of fragment generated by the perfusates was compared by one-way ANOVA followed by Newman-Keuls test. The statistical analyses were performed with GraphPad Prism Software, and differences were considered significant when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
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Blood pressure. Mean arterial pressures in SHR were significantly higher than those in WNR (156 ± 3 vs. 104 ± 2 mmHg; P < 0.0001).

Degradation of BK by MAB and cardiac perfusates. When BK was incubated with MAB perfusate from WNR, only two fragments were generated, corresponding to the major degradation product des-Arg9-BK (>94%) and the fragment BK(1-7), as shown in Fig. 1A. These data also indicate that, on average, an individual MAB released into the perfusate, per minute, an amount of kininase activity capable of hydrolyzing ~300 pmol of BK/min, under the described in vitro assay conditions. The results shown in Fig. 1B indicate that perfusates of MAB from WNR and SHR are nearly identical with regard to their proteolytic specificities and potencies toward BK. On the other hand, an individual isolated and perfused coronary bed from a WNR, on average, released an amount of kininase activity into the perfusate, per minute, that was ~250-fold less potent, and of a strikingly diverse proteolytic specificity, compared with that of its MAB counterpart as judged by the HPLC analysis of the fragments formed during the cardiac perfusate-catalyzed BK cleavage reaction (Fig. 2A). Moreover, although BK(1-5), BK(1-7), and des-Arg9-BK were the major degradation products formed during incubation of BK with both coronary perfusates, the generation of BK(1-5) was significantly greater in the reaction catalyzed by the perfusate from WNR than in that obtained with perfusate from SHR (Fig. 2B).


Figure 1
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  		Fig. 1. Proteolytic cleavage of bradykinin (BK) catalyzed by the perfusate of isolated mesenteric arterial bed (MAB) of Wistar normotensive rats (WNR) and spontaneously hypertensive rats (SHR). A: reverse-phase HPLC analyses of BK and its fragments generated by the incubation of 30 nmol of BK for 3 min at 37°C with a sample of perfusate containing the amount of proteolytic activity secreted by a single isolated MAB of a WNR during ~1.2 min of perfusion. B: comparison of the amounts of the 2 major BK degradation products released by incubating 30 nmol of BK with samples of perfusates of individual WNR (n = 8) or SHR (n = 6). C: inhibition of des-Arg9-BK generation on incubation of BK with perfusates of WNR (n = 3) or SHR (n = 3) by DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA), under the conditions described above. Values are means ± SE.

 

Figure 2
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  		Fig. 2. Proteolytic cleavage of BK catalyzed by the perfusate of isolated coronary beds of WNR and SHR. A: reverse-phase HPLC analyses of BK and its fragments generated by incubation of 30 nmol of BK for 6 h at 37°C with a sample of perfusate containing the amount of proteolytic activity secreted by a single isolated coronary bed of a WNR during 2.5 min of perfusion. Peaks eluting with retention times of 3 and 9 min correspond to BK(8-9) and BK(6-9), respectively. B: comparison of the amounts of the major BK degradation products released by incubating 30 nmol of BK with samples of perfusates of individual WNR (n = 8) or SHR (n = 6), under the conditions described above. Values are means ± SE. *P < 0.001 compared with peptides in the same group; §P < 0.001 compared with WNR.

 
Effects of protease inhibitors on the kininase activities. To investigate the contribution of enzymes potentially involved in the BK cleavage by the MAB and coronary perfusates, as suggested by the fragmentation profiles depicted in Figs. 1A and 2A, the effects of some particular enzyme inhibitors were monitored on the corresponding reactions. Thus the cleavage at the Phe8-Arg9 bond of BK, which accounts for nearly all of the kininase activity in samples of MAB perfusates and generates des-Arg9-BK, was almost fully inhibited by 30 µM MGTA (Fig. 1C) when BK was incubated with perfusates from both WNR (n = 3) and SHR (n = 3), under the conditions described in Fig. 1; similarly, the formation of des-Arg9-BK on incubation of BK with WNR perfusate was blocked by the presence of either 1 mmol/l 1,10-phenanthroline or 20 µmol/l PCI. Together, the inhibitory effects of these compounds on the formation of des-Arg9-BK indicate that a basic metallocarboxypeptidase is the most conspicuous kininase of the MAB perfusate. At least three proteases contribute to the kininase activity of the rat coronary perfusates, as judged by the effects of captopril, MGTA, and phosphoramidon on the degradation of BK by samples of perfusates (n = 5 each) from WNR and SHR (Fig. 3). The formation of des-Arg9-BK by these perfusates was inhibited by MGTA, suggesting the participation of a basic carboxypeptidase. The generation of BK(1-7) by incubation of BK with samples of perfusates from WNR and SHR was apparently carried out by two distinct enzymes, assuming nonoverlapping inhibitory effects of captopril and phosphoramidon on the kininases present in the perfusates; under the conditions described in Fig. 2, either of these compounds decreased the formation of BK(1-7) by 30–60%. Captopril also inhibited the formation of BK(1-5) catalyzed by coronary perfusate from SHR but not from WNR, revealing a distinction between the two perfusates concerning BK degradation.


Figure 3
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  		Fig. 3. Comparison of the amounts of the major BK degradation products released by incubating 30 nmol of BK with samples of coronary perfusates of individual WNR (n = 5) or SHR (n = 5), under the conditions described in Fig. 2, in the presence of captopril (10 µmol/l), MGTA (10 µmol/l), or phosphoramidon (10 µmol/l). Data are percent means ± SE of the control in the absence of inhibitors. *P < 0.001 compared with control without inhibitors.

 
Isolation and identification of the rat MAB perfusate basic carboxypeptidase. Affinity chromatography over PCI-Sepharose column proved an efficient means to isolate the BK-degrading enzyme from rat MAB perfusate (Fig. 4). It should be noted that the PCI-Sepharose resin absorbed all of the basic carboxypeptidase activity loaded on the column, wherefrom the corresponding enzyme was recovered (70% yield) in a single peak that coeluted with a carboxypeptidase A-like activity revealed with Cbz-Val-Phe as the substrate (not shown). The basic carboxypeptidase could be separated from contaminating proteins by percolating the pooled active fractions from the PCI-Sepharose chromatography through an Arg-Sepharose column, from which the enzyme was recovered (39% yield) in a broad peak that was retarded by the resin, in a fashion reminiscent of pancreatic CPB but not CPN (21). This purified basic carboxypeptidase released solely des-Arg9-BK on incubation with BK (Fig. 5A), even after prolonged incubation times, and was inhibited by MGTA and PCI (Fig. 5, B and C). Also, this protein migrated essentially as a single band on SDS-PAGE under reducing conditions, from which protein fragments were prepared by tryptic digestion and analyzed by mass spectrometry. Seven peptides of precisely determined masses were screened against a tryptic fragment database derived from over 50,000 proteins by MASCOT software and recognized as the following fragments of rat pancreatic CPB (pre-proenzyme numbering; GenBank Protein Database accession number P19223; http://www.ncbi.nlm.nih.gov/entrez/): Pro162-Arg176, Glu177-Arg189, Glu190-Lys202, Ala283-Arg289, Tyr371-Arg378, Asp379-Arg393, and Tyr405-Arg411. Thus these results strongly suggest that the basic carboxypeptidase isolated from the rat MAB perfusate is identical to the pancreatic CPB. Moreover, none of these fragments was found in the rat thrombin-activatable fibrinolysis inhibitor (TAFI), also known as rat plasma CPB, sequence (GenBank Protein Database accession number NP_446069).


Figure 4
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  		Fig. 4. Isolation of basic carboxypeptidase from rat MAB perfusate by affinity chromatography over potato carboxypeptidase inhibitor (PCI)-Sepharose column. Sample of 5 ml containing high-molecular-weight substances from pooled perfusates of 8 MAB preparations was loaded on the column (8 x 30 mm) at room temperature at a flow rate of 0.3 ml/min. After removal of unbound material by washing the column with 10 ml of 10 mmol/l Tris·HCl buffer, pH 7.5, containing 0.5 mol/l NaCl, bound enzymes were eluted with a 100 mmol/l Na2CO3 solution, pH 11.4. Absorbance at 280 nm and kininase activity were determined in each fraction and plotted against effluent volume, as indicated.

 

Figure 5
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  		Fig. 5. Kininase activity and inhibition of the affinity-purified carboxypeptidase B (CPB) from rat MAB perfusate. BK (30 nmol) was individually incubated with samples of purified CPB, equivalent to 50 µl of perfusate, in the absence (A) or presence of 30 µmol/l MGTA (B) or 20 µmol/l PCI (C) for 40 min at 37°C in 150 µl of Tris-buffered saline, pH 7.4. BK and its fragments were determined by reverse-phase HPLC analyses on a Shim-Pack CLC-ODS column (4.6 x 150 mm) using a linear gradient of acetonitrile concentration (10–32%) in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. A small peak eluting with retention time of 6–7 min (B) is also seen in a control run of MGTA alone.

 
Tissue distribution of mRNA encoding rat CPB. The expression of CPB mRNA was investigated in some of the rat tissues using RT-PCR (Fig. 6). This procedure, using the specific CPB oligonucleotides described in MATERIALS AND METHODS, amplified DNA fragments from total RNA extracts from the indicated tissues whose sizes matched the predicted value of 1,248 bp previously described for rat pancreas pre-proCPB (16). No PCR products were detected when sterile water was a substitute for the respective cDNA in the reaction (not shown). CPB mRNA was highly expressed in mesentery, pancreas, liver, and kidney, but its expression was below detection levels in lung, heart, aorta, and carotid artery.


Figure 6
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  		Fig. 6. Detection of mRNAs for rat CPB in different tissues using RT-PCR. Each lane of the ethidium bromide-stained agarose gels shows the RT-PCR products derived from total RNA for the indicated rat tissues using specific primers for CPB (1,248 bp) and β-actin (351 bp), the latter a control for matching the RT-PCR processes for the different total mRNA preparations (top). The expression level of mRNAs for CPB in the various tissues was estimated by densitometric scanning of the gels, normalized to the corresponding β-actin product, and expressed as carboxypeptidase A (CPA)-to-β-actin ratio in the column chart (bottom). Ki, kidney; Pa, pancreas; Li, liver; Lu, lung; Me, mesentery; He, heart; Ao, aorta; Ca, carotid artery.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Comparison between the kininase activities detected in the rat MAB and coronary perfusates revealed two prominent differences concerning their proteolytic capacities and specificities. First, it was shown that an individual rat MAB secretes, on average, 250-fold more kininase activity per minute than its coronary counterpart. Second, BK was cleaved almost exclusively at the Phe8-Arg9 bond on incubation with MAB perfusates from both normotensive and hypertensive rats, releasing des-Arg9-BK, whereas incubation of BK with coronary vessel perfusates generated des-Arg9-BK, BK(1-7), and, particularly, BK(1-5). Also, it was noted that SHR had a relatively low kininase activity in cardiac but not MAB perfusates of these rats, compared with their WNR analogs. Despite these conspicuous differences in the overall kinin processing between the coronary and MAB perfusates, one can only surmise the physiological significance of the contributing enzymes besides that they are likely to play a role in the local tissue control mechanisms of kinin action.

The analyses of the fragmentation profiles that resulted from treatment of BK with rat MAB (Fig. 1) and coronary (Fig. 2) perfusates and the corresponding alterations affected by some selective protease inhibitors allowed the identification of some of the soluble kininases in each perfusate. ACE and phospohoramidon-sensitive endopeptidase, such as NEP or endothelin-converting enzyme (11, 12), are present in small quantities in the coronary perfusates of WNR and SHR, the former having also a thus far unidentified BK(1-5)-forming enzyme whose specificity resembles that of neurolysin or thimet oligopeptidase (21). The specificities of these BK-destroying proteases present in the rat coronary perfusate are consistent with the products formed in previously described pathways of BK degradation studied in different isolated rat heart preparations (1, 7, 25); the pentapeptide BK(1-5), a major fragment formed by the action of coronary perfusate on BK, has also been shown to be a stable end product of BK degradation in human plasma (19). Although we did not characterize the kininases responsible for the release of BK(1-5), our results indicate that this activity had a major role in BK hydrolysis in coronary perfusate from normotensive rats because the ACE inhibitor did not affect the generation of the most abundant product BK(1-5); in contrast, this endopeptidase activity was greatly reduced in perfusates from SHR in view of the fact that BK(1-5) released from BK was greatly reduced by ACE inhibition. In effect, a soluble form of thimet oligopeptidase was reported by Chappell et al. (4) as the predominant protease present in hindlimb perfusates of normotensive rats responsible for the conversion of ANG I to angiotensin(1–7). The significantly lower kininase activity observed in cardiac perfusate of SHR compared with that of WNR (Fig. 2) may represent a compensatory mechanism associated with hypertension that endows the heart with protection against ischemic damage, owing to an enhanced preservation of the cardioprotective actions of BK (18).

Both coronary and MAB perfusates contain a MGTA-sensitive basic metallocarboxypeptidase reminiscent of CPN, particularly for being a soluble enzyme capable of generating the active kinin B1-receptor agonist des-Arg9-BK (17). The MAB perfusates of both WNR and SHR are undistinguishable by their basic carboxypeptidase contents (Fig. 1B), just as the plasma of these animals with regard to CPN (5), indicating that these BK-cleaving enzymes do not significantly contribute to the SHR pathophysiology. Interestingly, the average concentration of immunoreactive des-Arg9-BK was found to be much higher than that of BK in venous blood from normal humans as well as from patients with essential hypertension (22), indicating the effectiveness of human plasma basic carboxypeptidases in converting BK into des-Arg9-BK. A typical isolated and perfused rat MAB releases into the perfusate, per minute, an amount of basic carboxypeptidase capable of hydrolyzing ~300 pmol of BK/min, which is over two orders of magnitude larger than that produced by an individual coronary bed. Thus any attempt to ascribe function to this enzymatic activity must take into consideration the large difference of its production between different tissues, which may reflect the contribution of this basic carboxypeptidase to the overall BK metabolism in a particular tissue. It is worth mentioning that a full dose-vasodilation response curve was obtained with bolus injection of BK in the range of 1–160 pmol in the rat isolated MAB (33). However, a role for basic carboxypeptidase in modulating the vasodilator effect of BK induced by a single passage through the isolated and Krebs-perfused MAB could not be demonstrated in a previous study (33), indicating that membrane-bound carboxypeptidases, like CPM, did not have an important role in processing BK in this vascular bed.

The des-Arg9-BK-forming enzyme of the MAB perfusate was first described as CPN-like after two of its readily observable features: its occurrence as a soluble protease in the perfusate and its proteolytic activity toward the substrates hippuryl-Lys and BK (23). In the present work, we extended the enzymological characterization of the des-Arg9-BK-forming enzyme of the MAB perfusate by using some inhibitors for basic carboxypeptidases, including MGTA and PCI. Whereas CPN is known to be inhibited by MGTA (30) but refractory to CPI treatment (34), the basic carboxypeptidase of the rat MAB was inhibited by both compounds, setting a clear distinction between these two enzymes. Indeed, the latter enzyme could be isolated in good yield and highly purified form by a procedure that involved affinity chromatography over PCI-Sepharose column (Fig. 4). The amino acid sequence of the purified protein was shown to be identical to that of the rat pancreatic CPB by screening a tryptic peptide mass fingerprint of the enzyme against a tryptic fragment database derived from information of National Center for Biotechnology Information and Swiss-Prot databases (24). Thus, contrary to the general belief that CPB is a digestive protease whose production is restricted to the pancreas and that does not participate in regulatory processes (34, 37, 39), we found that it is the major BK-processing enzyme of the rat MAB perfusate. The rat MAB perfusate CPB shares some similarities with human- and rat-activated TAFI regarding its solubility, structure, and specificity toward synthetic substrates and inhibitors (8, 14, 20). Despite these biochemical similarities, any functional overlap between rat MAB perfusate CPB and activated TAFI remains to be established; activated TAFI is also known as plasma CPB, carboxypeptidase U, or carboxypeptidase R. During our study, we also observed that the expression of CPB mRNA was of the same order of magnitude in rat liver, mesentery, kidney, and pancreas, whose corresponding amplicons were of identical sizes, but very low in lung, heart, aorta, and carotid artery (Fig. 6). A relatively high level of expression of CPB mRNA in mesentery compared with that in heart correlates well with the CPB activities found in rat MAB and coronary perfusates, respectively (Figs. 1A and 2A). Another example of a prototypical digestive enzyme that seems to participate in regulatory processes is rat elastase-2, produced by endothelial cells of the rat MAB as a highly specific ANG II-forming enzyme (29, 30). Although there has been no direct evidence that CPB functions as a vasopeptidase in vivo, two reports have raised this possibility. It has been proposed that human serum would contain either small quantities of a CPB-like enzyme or a cofactor capable of augmenting about five times the activity of plasma CPN to account for the actual serum des-Arg9-BK-forming activity (31); indeed, CPB is known to remove the COOH-terminal Arg residue of BK far more readily than CPN (9). A second report that suggests that CPB might also function as a regulatory enzyme is the one that describes a significant linkage between the locus of CPB on chromosome 2 in the Lyon hypertensive rat strain and the pulse pressure component of blood pressure regulation (6). Despite this genetic linkage, experimental evidences demonstrating the involvement of CPB in this process remains to be established. Although the precise physiological roles for the soluble basic carboxypeptidases described here are not certain at present, a reasonable hypothesis is that they play a role in the processing of bioactive peptides to meet the metabolic requirements of different tissues, as suggested by the wide difference in the distribution of these enzymes in rat MAB and coronary perfusates.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo.


   ACKNOWLEDGMENTS
 
We thank Osmar Vettore and Orlando Mesquita, Jr., for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. O. Salgado, Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto-USP, 14049-900 Ribeirão Preto, SP, Brazil (e-mail: mcdosalg{at}fmrp.usp.br)

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
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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