Carboxypeptidase B and other kininases of the rat coronary and mesenteric arterial bed perfusates

1

Carboxypeptidase B and other kininases of the rat coronary and

mesenteric arterial bed perfusates

Eduardo B. Oliveira1, Laura L. Souza1, Disney O. Sivieri Jr2, Luiz B. Bispo-da-Silva2,

Hugo J. V. Pereira1, Claudio M. Costa-Neto1, Marcelo V. Sousa3, Maria Cristina O.

Salgado2

Departamentos de Bioquímica e Imunologia1 e de Farmacologia2 da Faculdade de

Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil, and Departamento

de Biologia Celular3, ICB, Universidade de Brasília, Brazil

Running head: Kininase activity of rat carboxypeptidase B

Corresponding Author:

Dr. Maria Cristina O. Salgado

Departamento de Farmacologia Faculdade de Medicina de Ribeirão Preto-USP

14049-900 Ribeirão Preto, SP Brazil

Phone: 55-16-36023046 Fax: 55-16-3633-2301

e-mail: [email protected]

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Copyright Information

Articles in PresS. Am J Physiol Heart Circ Physiol (September 28, 2007). doi:10.1152/ajpheart.00784.2007

Copyright © 2007 by the American Physiological Society.

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Abstract

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 (WNR) and spontaneously hypertensive (SHR) 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 about 300 pmol of BK/min, which is

about 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 ACE,

NEP-like enzyme and a MGTA-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 SHR,

could be purified by a procedure that involved affinity chromatography over potato

carboxypeptidase inhibitor-Sepharose column and shown to be structurally identical with

rat pancreatic 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 with pancreatic CPB.

Key words: bradykinin, angiotensin converting enzyme, neutral endopeptidase,

carboxypeptidase N, basic carboxypeptidase.

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Introduction

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 angiotensin 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 C-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 either to 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), while

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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).

Since 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 heart of normotensive and spontaneously

hypertensive (SHR) rats. Additionally, since a soluble kininase I-type carboxypeptidase

constitutes the major BK-processing pathway in the MAB perfusates and considering

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.

Material and Methods

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Animals: The experiments were conducted employing 11-12 weeks old male Wistar

normotensive rats (WNR, n=16) and SHR (n=12) that were maintained in a controlled

environment with a 12-hour light: 12-hour dark lighting 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 individually recording the arterial

pressure of the conscious rats (Hewlett Packard 7754 A recorder, Palo Alto, CA, USA),

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 37ºC, as previously described

(28,33). Briefly, the mesenteric arterial bed perfusion was carried out by infusing a

modified Krebs' solution (in mmol/L: NaCl 118; KCl 4.7; CaCl2 2.5; MgSO4 1.64; KH2PO4

1.18; NaHCO3 24.9; glucose 11.1) 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 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

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altered so to keep 10 mL of the perfusing solution recirculating through the mesenteric

arterial bed for 2 h. The perfusate was then recovered and its protein content was10-fold

concentrated using an Amicon apparatus fitted with a YM-10 membrane, and kept at

4oC 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 to keep approximately 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 4oC 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 (TBS, 30 mmol/L Tris-HCl, pH 7.4, containing

150 mmol/L NaCl), by incubating 30 nmol of BK with 10 µL of concentrated mesenteric

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arterial bed or cardiac perfusate at 37oC for 3 min or 6 h periods, respectively. The

inhibitors used were: 10 µmol/L captopril, an ACE inhibitor; 10 µmol/L phosphoramidon,

a neutral endopeptidase 24.11 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 terminating the reactions by the addition of 10 µL of 5%

trifluoroacetic acid (TFA), the resulting BK fragments were separated by reversed phase

HPLC on a 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 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 min 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

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percolating a 100 mmol/L Na2CO3 solution, pH 11.4, through the column. Samples of 0.7

mL were collected throughout the experiment, whose pHs were lowered to about 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 40C until

use.

Peptide mass fingerprint: A sample of the affinity-purified basic carboxypeptidase from

the mesenteric arterial bed 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 -20oC prior to use. For mass spectrometric analysis the product was

solubilized in 4 µL 0.1% TFA, followed by microscale concentration and desalting using

C18 Zip-Tips. (Millipore, Bedford, MA, USA). Peptides were eluted directly onto a

MALDI-TOF probe using 1 µL of 50% ACN in 0.1% TFA solution containing matrix (α-

CHCA 20 µg/µL). Mass spectra were determined using a Reflex IV (Bruker Daltonics,

Karlsruhe, Germany) mass spectrometer in positive reflector mode, and processed

using XMASS and Biotools software (Bruker Daltonics). Spectra were internally

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calibrated using trypsin autolysis products (m/z 842.509 and m/z 2211.104). Protein

identification was performed using MASCOT (24) at 50 ppm mass tolerance, which

screened the fingerprint of the protein provided by the peptide mass information against

NCBI (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 using the

Trizol reagent, following the manufacturer instructions (Invitrogen, Carlsbad, CA, USA).

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 (sense 5’-

gggaatccatgttgctgctactggcc-3’ and antisense 5'-ggctgcagtcaatatagatgttctcggac-3' for

CPB, and sense 5’- ctaaggcaaaccgtgaaaaga-3’ and antisense 5’-

attgccgatagtgatgacctg-3’ for β-actin). The sequences for the CPB primers were based

on the full-lenght nucleotide sequence of the rat pancreatic pre-proCPB, available

through the NCBI's GenBank CoreNucleotide database under identification 6978696

(http://www.ncbi.nlm.nih.gov/entrez/). PCR amplification was performed using Taq DNA

polymerase (Invitrogen). The process of thermal cycling consisted of initial denaturation

for 2 min at 94 ºC followed by 43 cycles of amplification of the cDNA, each comprising 1

min of denaturation at 94ºC, 1 min of annealing carried out at 60 ºC, and 1.5 min of

extension at 72ºC. Samples were incubated for additional 30 min period at 72ºC

(terminal elongation) after completion of the 43 cycles process. Similar amplification

protocol was used for β-actin, except for the annealing temperature at 45ºC. For each

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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 were expressed as mean±SEM. Blood pressure values

were compared by Student 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 using GraphPad Prism Software, and differences

were considered significant when P<0.05.

Results

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 mesenteric arterial bed and cardiac perfusates: When BK was

incubated with mesenteric arterial bed 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 Figure 1A. These data also indicate that, on the average,

an individual mesenteric arterial bed released into the perfusate, per minute, an amount

of kininase activity capable of hydrolyzing about 300 pmol of BK/min, under the

described in vitro assay conditions. The results shown in Figure 1B indicate that

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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 the average, released an amount

of kininase activity into the perfusate, per min, that was about 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 (Figure 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 as compared with that obtained with

perfusate from SHR (Figure 2B).

Effects of protease inhibitors on the kininase activities: In order 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 Figures 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 (Figure 1C) when BK

was incubated with perfusates from both WNR (n=3) and SHR (n=3), under the

conditions described in Figure 1; similarly, the formation of des-Arg9-BK upon incubation

of BK with WNR perfusate was blocked by the presence of either 1 mmol/L 1,10-

phenanthroline or 20 µmol/L PCI. Taken together, the inhibitory effects of these

compounds on the formation of des-Arg9-BK indicate that a basic

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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 (Figure 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 non-overlapping inhibitory effects of captopril and phosphoramidon on the

kininases present in the perfusates; under the conditions described in Figure 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.

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 (Figure 4). It should be noted that the

PCI-Sepharose resin absorbed all the basic carboxypeptidase activity loaded on the

column, wherefrom the corresponding enzyme was recovered (70% yield) in a single

peak that co-eluted 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

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reminiscent of pancreatic carboxypeptidase B (CPB) but not CPN (21). This purified

basic carboxypeptidase released solely des-Arg9-BK upon incubation with BK (Figure 5-

A), even after prolonged incubation times, and was inhibited by MGTA and PCI (Figure

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 P19233;

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

with the pancreatic CPB. Moreover, none of these fragments were found in the rat TAFI,

also known as rat plasma CPB, sequence (GenBank Protein database accession

number NP_446069).

Tissue distribution of mRNA encoding rat CPB: The expression of CPB mRNA was

investigated in some of the rat tissues using RT-PCR (Figure 6). This procedure, using

the specific CPB oligonucleotides described in Methods, amplified DNA fragments from

total RNA extracts from the indicated tissues whose sizes matched the predicted value

of 1248 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,

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and kidney but its expression was below detection level in lung, heart, aorta and carotid

artery.

Discussion

Comparison between the kininase activities detected in the rat MAB and coronary

perfusates revealed two prominent differences concerning their proteolytic capacities

and specificities. Firstly, it was shown that an individual rat MAB secretes, on the

average, 250-fold more kininase activity per min than its coronary counterpart; secondly,

BK was cleaved almost exclusively at the Phe8-Arg9 bond upon incubation with MAB

perfusates from both normotensive and hypertensive rats, releasing des-Arg9-BK, while

incubation of BK with coronary vessels 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

their cardiac but not MAB perfusates, as compared with their WNR analogues. In spite

of 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 treating BK with rat

MAB (Figure 1) and coronary (Figure 2) perfusates, and the corresponding alterations

effected by some selective protease inhibitors, allowed the identification of some of the

soluble kininases in each perfusate. ACE and phospohoramidon-sensitive

endopeptidase, like NEP 24.11 or endothelin converting enzyme (11, 12), are present in

small quantities in the coronary perfusates of WNR and SHR, the former having also a

so far unidentified BK(1-5)-forming enzyme whose specificity resembles that of

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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 since 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 rat responsible for

the conversion of angiotensin I to angiotensin(1-7). The significantly lower kininase

activity observed in cardiac perfusate of SHR compared with that of WNR (Figure 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 (Figure 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

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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 about 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 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 a

CPN-like after two of its readily observable features, namely, 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, among which MGTA and PCI. Whereas CPN is known to be

inhibited by MGTA (30) but refractory to CPI treatment (34), the basic carboxypeptidase

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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 (Figure 4). The amino acid sequence of the purified protein was shown to be

identical with 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

NCBI 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,

CPU or CPR. During our studies 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 (Figure 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 (Figures 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 angiotensin

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

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proposed that human serum would contain either small quantities of a CPB-like enzyme

or a co-factor 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 C-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.

Acknowledgements: We thank Osmar Vettore and Orlando Mesquita Jr. for their

technical assistance.

Grants: This research was supported by the Fundação de Amparo à Pesquisa do

Estado de São Paulo (FAPESP).

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Figure Legends

Figure 1. Proteolytic cleavage of BK catalyzed by the perfusate of isolated MAB of WNR

and SHR. (A) Reversed phase HPLC analysis of BK and its fragments generated by the

incubation of 30 nmol of BK for 3 min at 37oC with a sample of perfusate containing the

amount of proteolytic activity secreted by a single isolated MAB of a WNR during about

1.2 min of perfusion. (B) Comparison of the amounts of the two 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 upon incubation of

BK with perfusates of WNR (N=3) or SHR (n=3) by MGTA, under the conditions

described above. Data are expressed as mean ±SEM.

Figure 2. Proteolytic cleavage of BK catalyzed by the perfusate of isolated coronary

beds of WNR and SHR. (A) Reversed phase HPLC analysis of BK and its fragments

generated by the incubation of 30 nmol of BK for 6 h at 37oC 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. The 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. Data are expressed as mean ±SEM. * P<0.001 compared with peptides in the

same group and §P<0.001 compared with WNR.

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25

Figure 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 Figure 2, in the presence of captopril

(10 µmol/L), MGTA (10 µmol/L), or phosphoramidon (10 µmol/L). Data are expressed as

the percentage mean ±SEM of the control in the absence of inhibitors. *P<0.001

compared to control without inhibitors.

Figure 4. Isolation of basic carboxypeptidase from rat MAB perfusate by affinity

chromatography over PCI-Sepharose column. Sample of 5 mL containing high

molecular weight substances from pooled perfusates of eight 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. Kininase activity and inhibition of the affinity-purified CPB from rat MAB

perfusate. Thirty nmol of BK were 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 37oC in 150 µL of Tris-buffered saline, pH 7.4. BK

and its fragments were determined by reversed phase HPLC analyses on a Shim-Pack

CLC-ODS column (4.6 x 150 mm) using a linear gradient of acetonitrile concentration

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26

(10-32%) in 0.1% TFA 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.

Figure 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 (1248 bp) and

β-actin (351 bp), the latter a control for matching the RT-PCR processes for the different

total mRNA preparations (top panel). 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 CPA/ β-actin ratio in the column chart

(bottom panel). Tissues are indicated as Ki (kidney), Pa (pancreas), Li (liver), Lu (lung),

Me (mesentery), He (heart), Ao (aorta) and Ca (carotid artery).

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BK

BK(1-7)

des-Arg9-BK

0.4

0.2

0

Abs

orba

nce

215

nm 0.6

Retention time (min)250 20105 15

0.8 A

Injection

BK

BK(1-7)

des-Arg9-BK

0.4

0.2

0

Abs

orba

nce

215

nm 0.6


0.8 A

Injection

-7 -6 -5 -4 -30

20

40

60

80

100

MGTA (mol/L)

Inhi

bitio

n(%

)

C

NWRSHR

0

3

6

9

12

des-Arg9-BK BK(1-7)

nmol

ofpr

oduc

t

B NWRSHR

-7 -6 -5 -4 -30

20

40

60

80

100

MGTA (mol/L)

Inhi

bitio

n(%

)

C

NWRSHR

-7 -6 -5 -4 -30

20

40

60

80

100

MGTA (mol/L)

Inhi

bitio

n(%

)

C

NWRSHR

0

3

6

9

12

des-Arg9-BK BK(1-7)

nmol

ofpr

oduc

t

B NWRSHR

0

3

6

9

12

des-Arg9-BK BK(1-7)

nmol

ofpr

oduc

t

B NWRSHR

Figure 1

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Figure 2

des-Arg9-BK

BK

BK(1-7)BK(1-5)

0.4

0.2

0

Ab

sorb

ance

215

nm

0.6


A

Injection

BK(8-9)BK(6-9)

des-Arg9-BK

BK

BK(1-7)BK(1-5)

0.4

0.2

0

Ab

sorb

ance

215

nm

0.6


A

Injection

BK(8-9)BK(6-9)

0

3

6

9

12

15

18

des-Arg9-BK BK(1-7) BK(1-5)

§

*

*

* *

nmol

of p

rodu

ct

B NWRSHR

0

3

6

9

12

15

18

des-Arg9-BK BK(1-7) BK(1-5)

§

*

*

* *

nmol

of p

rodu

ct

B NWRSHR

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Figure 3

0

25

50

75

100

125 NW RSHR

**

Des-A

rg9 -B

K

0

25

50

75

100

125

**

* *

BK

(1-7

)

0

25

50

75

100

125

*

BK

(1-5

)

Captopril MGTA Phosphoramidon

0

25

50

75

100

125 NW RSHR

**

Des-A

rg9 -B

K

0

25

50

75

100

125

**

* *

BK

(1-7

)

0

25

50

75

100

125

*

BK

(1-5

)

Captopril MGTA Phosphoramidon

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Figure 4

0 7 14 21

0

0.5

1.0

1.5

2.0

2.5

Ab

sorb

ance

280

nm

0

0.5

1.0

1.5

2.0

Effluent Volume (mL)

En

zym

e U

nit

s

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Figure 5

Ab

sorb

ance

215

nm

0

0.4

0.8

1.2

1.6B

Retention time (min)

0

0.4

0.8

1.2

1.6

0 5 10 15 20

C

des-Arg9-BK

BK0

0.4

0.8

1.2

1.6 A InjectionA

bso

rban

ce 2

15 n

m

0

0.4

0.8

1.2

1.6B

Ab

sorb

ance

215

nm

0

0.4

0.8

1.2

1.6B


0

0.4

0.8

1.2

1.6

0 5 10 15 20

C


0

0.4

0.8

1.2

1.6

0 5 10 15 20

C

des-Arg9-BK

BK0

0.4

0.8

1.2

1.6 A Injection

des-Arg9-BK

BK0

0.4

0.8

1.2

1.6 A Injection

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Figure 6

CPB

β-actin

Ki Pa Li Lu Me He Ao Ca

Ki Pa Li Lu Me He Ao Ca0.0

0.5

1.0

1.5

2.0

2.5

CPB

/ β-a

ctin

Rat

io

CPB

β-actin

Ki Pa Li Lu Me He Ao Ca

Ki Pa Li Lu Me He Ao Ca0.0

0.5

1.0

1.5

2.0

2.5

CPB

/ β-a

ctin

Rat

io

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Carboxypeptidase B and other kininases of the rat coronary and mesenteric arterial bed perfusates

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Transcript of Carboxypeptidase B and other kininases of the rat coronary and mesenteric arterial bed perfusates