Angiotensin-(1−7) Augments Bradykinin-Induced Vasodilation by Competing With ACE
and Releasing Nitric Oxide
Ping Li, Mark C. Chappell, Carlos M. Ferrario and K. Bridget Brosnihan
Hypertension. 1997;29:394-398
doi: 10.1161/01.HYP.29.1.394
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1997 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
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394
Angiotensin-(l-7)
Augments Bradykinin-Induced
Vasodilation by Competing With ACE and
Releasing Nitric Oxide
Ping LJ, Mark C Chappell, Carlos M Ferrario, K Bridget Brosmhan
Abstract Recent studies have shown that angiotensm-( 1-7)
[Ang-( l-7)] interactswith kmmsandaugments
bradykmm(BK)induced vasochlator responses by an unknown mechanism In this
study, we evaluated whether the potentlatlon of the BK-induced
vasoddation by Ang-( l-7) may be attributable to mlubitlon of BK
metabolism, release of nitric oxide, or both Isometric tension was
measured m intact canine coronary artery rings suspended m organ chambers ‘251-[Tyro]-BK metabolism was determined m vascular rings by assessing the degradation of the peptide by hlghperformance hqmd chromatography Ang-( l-7) augmented the
vasodllatlonInducedby BK In a concentration-dependent
manner
m nngs preconstructed with the thromboxane analog U46619
The ECso of BK (2 45+-O 51 nmol/L versus 0 37+-O 08 nmol/L)
was shifted leftward by 6 6-fold m the presence of 2 PmollL
concentration of Ang-(l-7)
The response was specific for BK,
Since Ang-( l-7) did not augment the vasoddatlon induced by elther acetylchohne (0 05 pmol/L) or sodium mtroprusside (0 1
pmol/L) Moreover, neither angiotensm I nor anglotensm II (Ang
II) duplicated the augmented BK response of Ang-( l-7) Pretreatment of vascular rings with the mtnc oxide synthase mhlbitor,
NW-mtro+argmme
(L-NA, 100 pmol/L) completely abohshed
the effects of Ang-(l-7) on BK-induced vasoddatlon whereas
pretreatment with mdomethacm (10 pmol/L) was without effect
A
ngiotensm-(l-7) possesses
novel blologlcal functions that are chstmctfrom Ang II. 132
In contrast
to Ang II, Ang-(l-7) is not a dlpsogen or an
aldosteronesecretagogue,3,4but slrmlarto Ang II, it stlmulates the release of vasopressm,5prostaglanchns,6and
mtnc oxide.7 Ang-(l-7) counteracts several actions of
Ang II. In both camne and porcine coronary arteries,
Ang-(l-7) causesvaso&lation,*sg while Ang II dlvergently constricts the coronary arteries * Freeman et alI0
showedthat Ang-( l-7) mlnbits cultured vascular smooth
muscle cell growth, whereas equal molar concentration of Ang II stimulates cell growth In transgemc
(mRen-2)27 hypertensive rats, central admmlstration of
antibodIes to Ang-(l-7) or Ang II causes opposite
changesm blood pressure.11In addition, chronic mtravenous infusion of Ang-(l-7) reducesblood pressurem
SHR,‘z while acute infusion of Ang-( l-7) causesa longlasting depressorresponsem the pithed rat13and vasodilation of piglet pial arterioles.14
There 1sevidence that Ang-( l-7) may interact with lumns and augment BK-induced vasodllator responses
From The Hypertension Center, The Bowman Gray School of
Medicine of Wake Forest University, Wmston-Salem, NC
Correspondence to K Bridget Brosmhan, PhD, Hypertension Center, Bowman Gray School of Medicine, Medical Center Blvd, Wmston-Salem, NC 27157-1032 E-mall bbrosmh@bgsm edu
0 1997 Amencan Heart Association, Inc
The potent specific BK B1 receptor antagomst, Hoe 140, nearly
abolished the BK and the Ang-(1-7) potentlated responses at 2
pmol/L, whereas at a lower concentration (20 nmol/L) Hoe 140
shlftedthe response
curve to the right for both Ang-(l-7) and
vehicle; however, the augmented response to Ang-( l-7) persisted
Premcubation of vascular rings with 20 pmol/L of the AT,
(CV11974), AT? (PD123319), or nonselective (Sar’ Thr*-Ang II)
receptor antagonists had no significant effect on the Ang-( l-7)enhanced vasodllator response to BK Llsmopnl (2 pmol/L) slgmficantly enhanced the BK-induced vasoddator response while
at the same time It abohshed the synerglstlc action of Ang-(l-7)
on BK In addition, pretreatment with 2 pmol/L Ang-( l-7) slgmficantly mlntnted the degradation of ‘251-[Tyro]-BK and the appearance of the BK-( l-7) and BK-( l-5) metabohtes in coronary
vascularnngsAng-(l-7) mhlbltedpunfiedcanmeanglotensm
converting enzyme activity with an ICsOof 0 65 pmol/L In conclusion,
Ang-( 1-7) acts as a local synergstlc modulator of kmm-induced vasodilation by mhibltmg anglotensm converting enzyme and releasing
mtnc oxide (Hypertension. 1997;29[part 2]:394-400.)
Key Words l anglotensm peptldes l coronary artery l
angiotensin-convertmg enzyme 9 endothelmm-denved relaxing
factors l nitric oxide l kmms l dog
Porsti et al’ and Brosmhanet al* reported that the specific
BK B2 receptor antagonist Hoe 140 inhibited the Ang(1-7)-evoked vasodilator responsem coronary vesselsIn
rat ludney, Hoe 140 inhibited Ang-(1-7)-induced natriuresls and dmresis.15Paula et all6 found that Ang-( l-7)
infusion potentlated BK-induced hypotensive responses
m conszious rats The ACE1 qumapnlat enhancedAng(1-7)-induced vasodilatlon,g a finding that may be mterpreted as resulting from mhlbltlon of BK degradation17-19
Addmonally, ACE inhibition IS associatedwith sigmficant elevations of Ang-( 1-7) asblockade of ACE actlvlty
diverts the pathway of Ang II formation from Ang I mto
Ang-( 1-7) 20-z Thus, the vasodllator responseassociated
with ACE1 therapy may be due to local accumulation of
BK and Ang-(l-7) and amplification of the effects of BK
m the presenceof augmentedplasmaand tissueconcentratlons of Ang-( l-7) The presentstudieswere conducted
to explore further the mechanismsof action of Ang-( l-7)
m the BK-potentiatmg responsem isolated camnecoronary arteries.
Methods
Vascular Ring Reactivity
Following approval by the Institutional Animal Care and Use
Committee, 18 male dogs (body weight 15 to 25 kg) were anesthetized with a combmation of ketamme (15 mg/kg IM) and 2%
halothane Animals were euthanatized with a lethal dose of sodium pentobarbital(50 mg/kg IV) The hearts were removed Im-
Downloaded from http://hyper.ahajournals.org/ by guest on March 6, 2014
0 et al Angiotensin-(l-7) and Bradykinin
ACE1
Ach
Ang
BK
L-NA
=
=
=
=
=
Selected Abbreviations and Acronyms
anglotensm convertmg enzyme mhlbltor
acetylcholme
anglotensm
bradykuun
N”-tutro-L-argmme
mediately and kept m ice-cold modified Krebs-Henselelt buffer
while the left anterior descendmg coronary artery was carefully
dissected free of fat and adhering connective tissues The artery
was cut mto 3-mm-long rings and mounted with two stainless
steel wire triangles m glass organ chambers containing modified
Krebs-Henseleit solution (composition m mmol/L 1 I8 3 NaCl,
4 7 KCl, 2 5 CaCl*, 1 2 MgS04, 1 2 KH2P04, 25 NaHCO?, 0 026
CaNaEDTA, and 11 glucose) The Krebs’ solution was aerated
with 95% O2 and 5% CO* at 37’C (pH 7 4) and the rings were
allowed to equilibrate for 60 minutes at 1 g mltlal resting tensIon
Basal tenslon was Increased m a step-by-step fashion until the
optimal length-tension relatlonshlp was achieved by repeated exposure to 40 mmol/L KC1 (the optimal tension was 3 5 to 5 g)
Isometric tension of the vascular rings was measured contmuously using a polygraph (Grass No 7) The integrity of functional
endothehum was confirmed by the presence of Achmduced relaxation (more than 90% relaxation at 0 1 pmol/L of Ach) m rmgs
preconstructed with 10 nmol/L of the thromboxane A2 analogue
U46619 It was demonstrated m a previous study* that the stable
constriction induced by 10 nmol/L U46619 was 40% to 50% of
maxlmal contractlon produced by U46619
Measurements
of ACE Activity
ACE was purified =1200-fold from a membrane flactlon of
canme lung using a hsmopnl-coupled affimty column, described
elsewhere *?24 Tissue was homogenized m 25 mmol/L HEPES,
0 2 mol/L NaCl, pH 8 0 (Buffer A) m a blender followed by
centnfugatlon at 10 OOOgfor 30 minutes The pellet was washed
once with Buffer A and centrifuged, and then the pellet was solubihzed m Buffer A with 0 1% Triton X-100 and 0 05 mmol/L
ZnS04 (Buffer B) Followmg centrifugation at 30 OOOg for 30
minutes, the supernatant was applied (10 mL/h) to a Sepharose
6B affnuty column (Sigma Chemical Co) coupled to llsmopril via
a 2 8-nm spacer arm The column was washed with 100 mL of
Buffer B, 50 mL of Buffer B containing 0 5 mol/L NaCl, and 50
mL of Buffer B at 4”C, and the enzyme was eluted with 10 mL
of 50 mmol/L sodium borate, pH 9 5, 0 1% Trlton ACE was
further purified and concentrated on a DEAE cellulose ionexchange column (m 10 mmol/L HEPES, pH 7 0, 0 1% Trlton)
and eluted with Buffer B contammg 0 5 mol/L NaCl Protein
concentration was determmed by a BioRad Bradford assay kit
ACE actlvlty was determined using the synthetic substrate
Hip-His-Leu as described previously 25 Purified canine ACE (40
ng) was incubated m 0 48-mL Buffer A with 1 0 mol/L sodium
chloride with 1 mmol/L concentration of Hip-His-Leu for 15
minutes at 37°C The reactron was terminated by addmon of 1 2
mL 0 34N sochum hydroxide (NaOH) The product (His-Leu)
was reacted with 0 I mL o-phthalaldehyde (20 mg/mL methanol)
for 10 minutes followed by 0 2 mL 3N hydrochloric acid, and
the fluorescence was determined (365-nm excitation and 495-nm
emission) The specificity of the assay was assessed by addition
of 1 pmol/L hsmopril
1251-[Tyro]BK Metabolism
We employed a BK analog contammg tyrosme (Tyr’) at the
ammo terrnmus of the peptide for metabolism studies The [Tyr]‘BK analog (Bachem) was loclmated with I?-Na (Amersham)
usmg the chloramme-T method and purified by high-performance
hqmd chromatography (HPLC, see below) to a specific activity
of 2200 Cl/mmol 26 The peak of ‘251-[Tyr]o-BK was diluted m
1.5 mol/L Tns-HCl, pH 8 3, containing 0 05% BSA and stored
at 4°C The radiolabeled peptlde (final concentration of 1 nmol/L)
395
was added to the tubes contammg three rmgs premcubated for
60 mmutes with 1-mL Krebs’ buffer gassed with 95% O2 and 5%
CO* at 37°C Ahquots of the mcubation medium were obtained
at 5, 10, and 20 minutes and the metabolism was terminated by
ad&on of 1% heptafluorobutyric acid
The extent of ‘*‘I-[Tyr]‘-BK
metabolism was determmed by
HPLC using the HFBA solvent system 26 Ttus system consisted
of 0 1% HFBA (mobile phase A) and 80% acetomtnle/O 1%
HFBA (mobile phase B), and the analysis was performed on an
Applied Biosystems 400 HPLC equipped with a narrow-bore
Waters Nova-Pak Cl8 column (Waters, 2 1 X 150 mm), an Aquapore Cs guard column (Apphed Blosystems, 3 2X 15 mm), and a
Rheodyne 7125 injector (ABI) Samples (50 pL, 25 to 50 000
cpm) were analyzed under Isocratic conditions of 38% mobile
phase B at a flow rate of 0 35 mL/mm One-mmute fractions (0 35
mL) were collected and counted m a Packard gamma counter
(Packard Instruments, counting efficiency 75%) ‘*jI- [Tyr]‘-BK(l-7) and BK-(l-5) standards were prepared enzymatlcally by
incubating ‘251-[Tyr]o-BK with purified ACE, and the peptldes
were isolated by HPLC
Experimental
Protocol 1
BK-induced relaxation (1 nmol/L) was produced after I hour
eqmhbratlon m intact vascular rings preconstncted with 10
nmol/L U46619 Ang-(l-7)
at concentrations of 0.1 to 2
PmollL was used to pretreat quiescent coronary artery rmgs for
10 minutes, then 1 nmol/L BK-induced relaxation response was
repeated m the preconstructed nngs The specificity of the potentiation response for BK was assessed by measurement of the relaxation responses to either Ach (50 nmol/L) or sodmm mtroprusslde (0 1 hmol/L) before and after pretreatment with 2
pmol/L Ang-( l-7)
Experimental
Protocol 2
For experiments m protocol 2, It was determined that control
concentration-dependent, cumulative relaxation response curves
to BK (lo-” to 10e6 mol/L) were able to be generated m rmgs
preconstructed from 70% to 80% of maximal contraction with 50
nmol/L of U46619 The contnbutlons of endothehum-derived mtric oxide, vasoactive prostaglandms, ACE, and Ang and kmm
receptors to the Ang-(l-7) potentiation response for BK were
mvestlgated as follows (1) The contrlbutlon of vasodllator prostaglanchns was assessed by admmlstratlon of the cyclooxygenase
mhlbltor mdomethacm (10 ,umol/L) applied for 20 minutes before
mtroductlon of 2 pmol/L Ang-( l-7) for 10 minutes Then the BKinduced dose-dependent relaxation response curve was repeated
(2) In separate experiments, rings were smularly co-pretreated
wrth the nitric oxide synthase mhlbltor L-NA (100 pmol/L) for
20 mmutes followed by the addltlon of 2 pmol/L Ang-(l-7) for
10 minutes (3) To evaluate a potential mteractlon among Ang(l-7), kmms, and ACE, coronary artery rings were also exposed
for 20 minutes to either the specific BK B2 receptor blocker Hoe
140 (2 pmol/L and 20 nmol/L) or the ACE1 hsmoprd (2
pmol/L), and each was then followed with 2 pmol/L Ang-(l-7)
co-pretreatment for 10 minutes (4) The participation of Ang receptor subtypes was determined followmg admimstratlon of selective AT, (CV11974, 20 hmol/L [the active form of TCV116]*7), and AT2 (PD123319, 20 PmoliL) receptor antagomsts
A nonselectlve competltlve peptlde Ang II receptor antagonist,
Sar’ Thr8-Ang II (20 kmol/L), was also utilized to determine the
possible participation of other subtypes of Ang receptors
In addition, to evaluate whether Ang I or Ang II also potentlate
the BK-induced relaxation response m isolated coronary rmgs,
the BK-mduced relaxation response curves were produced m the
presence and absence of rings pretreated for 10 minutes with 2
pmol/L of Ang I or Ang II. All receptor antagonists and enzyme
mlubitors (hsmopnl and cyclooxygenase) chd not change basal
tension and the preconstrictlon induced by U46619 On the other
hand, Ang I and Ang II pretreatment caused typical phaslc constriction m intact qmescent rings, the mtnc oxide synthase mhlb-
Downloaded from http://hyper.ahajournals.org/ by guest on March 6, 2014
396
Hypertension
Vol29,
No 1, Part 2
January 1997
ltor L-NA resulted m either no effect or a shght rise m basal
tenslon
Experimental
Protocol 3
Competmon assays usmg punfied canine ACE were determined using a fixed concentration of the substrate Hip-His-Leu
(1 mmol/L) and varying the concentrations of the competmg
agents[hsmopnl(lo-” to lo-’ mom), Ang-(l-7) (lo-’ to lo-’
mol/L), or Sar’, Thr’-Ang
II (lo-*
to 10e5 mol/L)]
Inhlbltory
constants
(ICzo)weredetermined
fromtherespective
competmon
curves
To study the effect of Ang-(l-7) on BK metabohsm m mtact
coronary rings, ‘251-[Tyro]-BK (final concentration of 1 nmol/L)
was addedto the tubescontammg
threeringspremcubated
with
1 mL Krebs’ buffer and aerated with 95% O2 and 5% COP at
37°C Llsmoprd (2 PmollL), Ang-(l-7) (2 pmol/L), or Krebs’
buffer as control were added to the rmgs 10 minutes before addition of the radlolabeled BK Aliquots of the mcubatlon medium
were removed at 5, 10, and 20 mmutes and diluted with 1%
HFBA to mhlblt peptldase activity
Statistical Analysis
The concentration
of BK causing50%(ECSo)
of themaximal
relaxation and the ICso of ACE mhlbltlon were calculated usmg
a nonlinearregression
methodof aslgmoldcurve-fittingprogram
(PRISM. GranhnadInc) Resultsare renortedas mean+SEM
(standard errdr 6f meanj One-way ANO\A followed by Newman-Keuls multiple comparisons and Student’s t test for paired
observations
wereusedfor statlstlcalanalysisA valueof P< 05
wasconsidered
statisticallysignificant
Ang-( 1-7) at 2 pmol/L concentrationehclted a stgmficant
leftward shtft of the BK-induced relaxanonresponsecurve.
The ECSoof BK was reduced6.6-fold m the presenceof 2
pmol/L Ang-( l-7) (EGO, 2 4520 51 versus 0.37+0 08
nmol/L, P<.Ol) Premcubation with mdomethacm(10
pmol/L) had no significant effect on the BK-induced
relaxationresponseThe ECSoof BK wasnot changedm the
presenceof mdomethacmm both control (BK) and Ang-( l7)-treated groups [control. 2 4520 51 versus 3.06?0.86,
Ang-( 1-7) treated 0 3720 08 versus 0 4320 11 nmol/L,
without versus wtth mdomethacm] The potenttatton responseto BK producedby 2 pmol/L Ang-(l-7) was sttll
presentm the presenceof mdomethacm(EC,,. 3 06kO.86
versus0.4320 11 nmol/L, P< 01) (Fig 2, top) In contrast,
pretreatmentwith the nitric oxide synthaseinhibitor L-NA
(100 pmol/L) srgmficantlyshiftedthe BK-inducedrelaxatton
responsecurves to the nght of both control and Ang-( l-7)treated groups [control 2 4520.51 versus 28 8429 68
nmol/L, P< 01, Ang-(1-7)-treated. 0 37+-O08 versus
31.68k9.59 mnol/L,,P<.Ol, without versuswith L-NA] and
abolishedthe effect of Ang-(l-7) on the BK-induced relaxation response (EGO 28.8429 68 versus 31.68+9 59
nmol/L, P>.O5) (Fig 2, bottom)
Pretreatmentwith the potent specificBK B2 receptorantagonistHoe 140at a concentrationof 2 prnoI/I, nearly abolishedthe BK-inducedrelaxation response,resultmgm only
about 20% relaxation at the htghest concentrationof BK
tested(1 pmol/L) The Ang-( l-7) potenttatmgresponseto
Drugs and Solutions
Ang peptldes were purchased from Bachem Hoe 140 was
a gift of Hoechst-RoussellInc (Frankfurt, Germany)
PD1233 19 was generously supplied by Warner-Lambert
Parke-Davis Inc (Ann Arbor, Mlch), CV11974 by Takeda
Chemical Industries, Ltd (Osaka, Japan), and hsmoprll by
*
100-l
mBK
Ii nmoULI
DuPontMerck Co (Wilmington, Del) Other chemicalswere
purchased from Sigma Chemical Co Ang peptldes were pre-
pareddally in a Krebs-Henselelt
buffer solution Indomethacm
and CVl1974 were dissolvedin 0 2N Na2C03m stock solution and diluted with Krebs’ buffer upon use U46619was
prepared as stock solution m ethanol and diluted with Krebs’
buffer The concentrations of drugs reported are at a final con-
centrationm organchambers
BK
O.lpmol/L
IlrmoUL
PpmoUL
Results
Synergistic Action of Ang-(l-7) on BK-Induced
Vascular Relaxation
BK at a concentrationof 1 nmol/L causedendothelmmdependentvasorelaxatton of intact coronary artery rings
preconstructedwith 10 nmol/L of the thromboxane AZ analogue U46619 The BK-induced relaxatton responsewas
augmentedby Ang-(l-7) (0 1 to 2 pmol/L) m a dose-dependent manner (Fig 1, top) At a concentration of 2
pmol/L Ang-(l-7), relaxation to BK was increased92%
compared to BK alone (4124.4% versus 92+2.5%,
P< 01) The effect of Ang-(l-7) on the BK-mduced relaxation responsewasspecificfor BK, sinceAng-( l-7) dtd
not augmentrelaxation responsesinducedby the endothehum-dependentvasodtlator Ach (0 05 pmol/L) (6025 5%
versus6126.9%, P>.O5) or the endothelmm-independent
vasoddator sodmmmtroprusstde(0 1 pmol/L) (7028 2%
versus 6828.5%, P> 05) (Fig 1, bottom)
Contribution of Endothelium-Derived
Relaxing Factors
Autacoid
BK (lo-” to 10e6mol/L) elicitedconcentration-dependent
relaxation responsesm submaximallypreconstnctedrmgs
m control
-A&1-7)
BK
ACH
treated
SNP
FIG 1 Top, Average
dose-response
curves to 1 nmol/L BK before (hatched
bar) and after (black bars) lo-minute
pretreatment
with Ang-(l-7)
(0 1 to 2 pmol/L)
Bottom,
Effect of 2 pmol/L Ang(l-7) on BK (1 nmol/L),
Ach (ACH, 50 nmol/L),
or sodium nltroprusside
(SNP, 100 nmol/L)
Values
are meantSEM
BK responses
are the average
of 16 rings obtalned
from 6 dogs All
other groups contain 6 to 12 rings from 4 to 6 dogs *PC 05 vs
BK, **PC 01 vs control
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0 et al
. Ccflkd
-9
-9
Bradyklnln
.
lndo +Conkol
.
lndo+ An@(l-7)
-7
-6
Log mol/L
.
O-
2
If
1
CmkOl
. An@(l-7)
1;
397
by Ang-(l-7) [ECJo*0.2420 01 versus0.25t0.01 pmol/L,
P>.O5, without and with Ang-(l-7)] (Fig 3, bottom)
. An@(l-7)
-10
Angiotensin-(l-7) and Bradykinin
.
L-N4+C0Ilk0l
.
L-N4 +Ang-(l-7)
Effect of Ang Receptor Antagonists
Premcubation with either the AT, or AT2 receptor antagonists at a concentration 10 times higher (20
pmol/L CV11974 and 20 pmol/L PD123319) than Ang(l-7) did not stgmficantly inhibit the Ang-(l-7) potentrating response to BK [EC50: 0 37?0 08 versus
0.44-f-O 06 versus 0.5220.16 nmol/L, Ang-(l-7) versus
CV11974 versus PD1233191 (Fig 4) Similarly, pretreatment of precontracted rings with the nonselective
Ang receptor antagonist Sarr Thr’-Ang II (20 pmol/L)
had no significant effect on the enhanced responseto
BK produced by Ang-(l-7) [EcSO 0 37kO.08 versus
0 41~0.11 nmol/L, Ang-( l-7) versus Sar’ Thr’ Ang II]
(Fig 4). Sar’ Thr’-Ang II had no effect on the BK-mduced relaxatron response(data not shown)
Selectivity of Ang Peptides
In contrast to Ang-(l-7), pretreatment with 2 pmol/L
Ang I or Ang II for 10 minutes did not change the BK. Conkol
. An@+7)
. HcE14o+cOnkol
.
-10
-9
-8
Bradykinln
-7
I5
-6
Log mol/L
2 Top, Cumulatrve
dose-response
curves to BK (lo-”
to
1 OS6 mol/L) alone (control) and in the presence
of 2 pmol/L Ang(l-7)
Condrtrons
are the same as In Figs 2 to 5 Preincubatlon
wrth
the cyclooxygenase
rnhrbrtor
rndomethacrn
(Indo,
10
pmol/L)
had no signrflcant
effect on the BK-Induced
relaxatron
response.
Coincubatron
with indomethacin
(10 pmol/L)
and 2
pmol/L
Ang-(l-7)
showed
no difference
from the findings
obtained with 2 pmol/L Ang-(l-7)
alone Bottom, Cumulatrve
doseresponse
curves to BK (1 O-lo to 1 O-’ mol/L) alone (control)
and
in the presence
of 2 PmoRL Ang-(l-7)
Prerncubatron
with the
nitric oxide synthase
inhibitor
L-NA (100 pmol/L) shifted the BKinduced
relaxation
response
to the right Co-pretreatment
with
L-NA (100 pmol/L) and 2 pmol/L Ang-(l-7)
showed no difference
from the L-NA treated
alone L-NA abolished
the Ang-(l-7)
potentiating
effect on the BK-Induced
relaxation
response
Values
are mean?SEM
Control
group contains
33 rings from 12 dogs,
and Ang-(l -7)-treated
group has 44 rings from 10 dogs Other
treatments
include 6 to 10 rings from 3 to 4 dogs
IiCE 14O+Ang+7)
-20.
1
>
-4O-
i
-60.
2
0
s
a-
FIG
BK was no longer present(data not shown) Pretreatment
with Hoe 140 at a 20 nmol/L concentrattonshiftedthe BKInducedrelaxauonresponse
curvesto the nght of both control
and Ang-( 1-7)-treated group [control. 2 45?O 51 versus
547 65k-19 63 nmol/L, P<.Ol, Ang-(1-7)-treated 0 37&
0 08 versus115.14t23.96 nmol/L, P<.Ol, without versus
with Hoe 1401(Fig 3, top) However, m the presenceof
20 nmol/L Hoe 140, Ang-(l-7) still potentiated the BKInduced relaxation [EC&: 547 65219.63 versus 115 142
23 96 nmol/L, P<.Ol, without and with Ang-(l-7)]
Pretreatmentwith the ACE1 hsmopnl(2 PmollL) markedly shifted the BK-induced relaxation responsecurves to
the left of both control and Ang-( 1-7)-treated groups[control: 24502510 versus 0 2520.07 pmol/L, P<.Ol, Ang(1-7)-treated* 370280 versus0.24+0 05 pmol/L, P<.Ol,
without and with hsmoprtl]. Pretreatment with hsmopnl
also abolishedthe potentiation responseto BK produced
-lOO-10
-9
-9
Bradyklnln
-7
-6
Log mol/L
. ConkOl
A Ang(t-7)
ii Oi
do-
P
>
-40.
i
1;
0
s
.
Uslnopril + Cankol
. Uslnopril+An~(l-7)
-loo-
-i4
-i3
-i2
-ii
Bradyklnln
-io
-b
i
-i
4
Log mol/L
FIG 3
Top, Cumulative
dose-response
curves to BK (lOmqo to
1 Ov6 mol/L) alone (control) and in the presence
of 2 pmol/L Ang(l-7)
Prelncubatlon
with the BK Bz receptor
antagonist
Hoe 140
(20 nmol/L)
shifted the BK-induced
relaxation
response
to the
right. Preincubation
with Hoe 140 (20 nmol/L) and 2 pmol/LAng(l-7) reduced
BK-induced
response
Low concentration
of Hoe
140 did not abolish the Ang-(l-7)
potentrating
effect on the BKinduced relaxation
response
Bottom, Pretreatment
with the ACEI
llsinopnl
(2 pmol/L)
markedly
leftward
shifted the BK-induced
relaxation
response
curves and that co-pretreated
with Ang(l-7)
Pretreatment
with lisrnopnl abolished
the potentration
response to BK produced
by Ang-(l-7)
Values are mean?SEM
Each treated
group includes 6 to 10 rings from 3 to 4 dogs
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398
Hypertension
Vo129, No 1, Part 2
.
.
.
OE
;
-2o-
h
i
January 1997
Conllol
Ang-(l-7)
CV 11974 + An@(W)
.
PO 123319 + Ang-(l-7)
l
S&h?-Ang
II + Ang+7)
-i
-6
-4O-
:
84
6
5
E
-so-
-SO-
-1 oo-
-io
-‘s
-b
Braclyklnln
Log mot/L
FIG 4
Prelncubatlon
with either the AT, (CV11974,
20 pmol/L)
or AT? receotor
(PDI 23319, 20 umol/L)
antagonists
at a 1 O-fold
highe; co&entr&on
than Ang-(l-7)
did not-significantly
block
the Ang-(l-7)
(2 pmol/L)
potenbatlng
response
to BK (control)
Similarly,
pretreatment
with the nonselective
Ang receptor
antagonist
Sar’ Thra-Ang
II (20 pmol/L) had no significant
effect on
the potentlating
response
to BK produced
by Ang-(l-7)
Values
are meantSEM
Each treated group Includes 6 to 10 rings from
3 to 4 dogs
induced relaxation response.There was no difference m
the ECSO
of BK m Ang I- and Ang II-treated groupscompared with BK alone [Et&, 2.45?0 51 versus2.33?1 24
versus 2 09+074 nmol/L, control versus Ang I versus
Ang II] (Fig 5)
Ang-(l-7) Inhibits ACE Activity and Attenuates
Metabolism of ‘251-[Tyro]-BK
Ang-( l-7) inhibited ACE actlvlty purified from canine
lungs with an ICsOof 0.65 pmol/L (Fig 6, top). Lisinopnl,
as expected, was a more potent mhtbltor of canme ACE
than Ang-(l-7), having an ICsOof 1 5 nmol/L. At 10
PmollL, Sar’ Thr8-Ang II showedno effect on ACE actlvlty Pretreatment of coronary rmgs with 2 pmol/L Ang(l-7) or 2 pmol/L lismopnl for 10 minutes significantly
attenuatedor blocked the rapid degradationof ‘**I-[Tyr’]BK metabolism(Fig 6, bottom). Five minutes after the
addition of radiolabeledBK, both Ang-( l-7) and hsinoprll
were shownto be equally effective m blockmg its degradation Thereafter, Ang-( 1-7) and hsmopnl significantly
attenuatedthe degradationof BK, but at 20 minutesonly
hsmopnl was effective m blockmg the degradation of
1251-[Tyro]-BKin vascular rings
(l-7) may be producedby releaseof NO Furthermore,we
provide novel evidencethat mhlbmon of endothehalACE
activity may m part alsocontribute to the synerglstlcaction
of Ang-( l-7) on the vasodllator responseto BK
Ang-( l-7) actsasa vasochlatoropposmgthe pressorand
prohferatlve actionsof Ang II In wvo Ang-( l-7) produces
vasodllatlon m feline mesentencand hindquartervascular
beds,*8m piglet plal artenoles,14and m pithed rats 13In
porcme and canine coronary artenes,s.gAng-( 1-7) dilates
vesselsby eliciting the releaseof kmms and endothehalderived mtnc oxide. Our studiesprovide further evidence
that Ang-(l-7) may be acting as a local autocnne or paracrme hormone Since rerun, anglotensmogen,ACE, and
Ang receptors are found m the various cellular elements
of the vascular wall 29~0Local releaseof both Ang I and
Ang II has been demonstratedacrossthe hmdhmb and
mesentencbeds after bilateral nephrectomy 31Ang-( l-7)
hasbeen shownto be producedlocally in endothehaland
vascular smoothmusclecells.21J6
Kmmsare potent endogenousvasodllator peptidescausmg endothelium-dependentvasodllation by releaseof endothelmm-denved nitric oxide, hyperpolanzmg factor,
and prostacyclm (PG12)17~33The vascular endothelmm
contams a kmm-generatmgsystem,l7.3*+34
and kmms are
mainly degradedmto inactive peptidesby local ACE and
other endopeptldases.32,35
Recent studieshave shownthat
ACE inhibition increaseslocal kmm concentration. Kmm
levels and the half-life of exogenously added BK m cultured endothehal cells and artenal tissue were increased
fourfold and prolonged, respectively, by treatment with
ACE mlubltors 36-39 Other studiesshowedthat ACE mhlbltors potentlatedBK-induced vasodllatlon mediatedby mtric oxide and enhancedcGMP production m arteries x9-41
These studiesstrongly mdlcate that ACE inhibition 1sassociatedwith increasesm local kmm concentrations.
In agreementwith previous studies,38J9*42,43
we found
that lisinopnl Inhibited punfied canine ACE activity with
an ICsoof 1 5 nmol/L and also blocked the degradationof
BK in vascular rings. Importantly, Ang-(l-7) also mhlblted ACE activity with an ICsoof 0.65 ,umol/L, possibly
by competing with kmms asa substratefor ACE Matsufujl et aI4 showedthat m SHR the antihypertensiveeffects
m COrltrOl
A Ang-(l-7)
v Angl
l
Angll
Discussion
In isolated camne coronary arteries, Ang-(l-7) has a
synerglstlc, concentration-dependentaction on BK-mduced vasorelaxation The specificity of the responsefor
Ang-( l-7) was demonstratedby determmmgthe fslllureof
other vasodilator autocoids, namely Ach, sochummtroprusnde, and prostaglandms,to augmentthe BK-induced
relaxation Moreover, neither Ang I nor Ang II given m
concentrationseqmmolarto Ang-( l-7) causedpotentiatlon
of the BK responseThe synergistic effect of Ang-(l-7)
on the vasochlatorresponseproduced by BK is not medlatedby a known Ang receptor sincethe effect persistedm
the presenceof ATI, AT2, and Sar’ Thr’-Ang II receptor
antagonists.Our studiesm&ate that the effect of Ang-
-io
-b
-b
Bradyklnln
-i
Log mol/L
FIG 5 Pretreatment
with 2 pmol/L Ang I or Ang II for 10 minutes
had no effect on the BK-induced
relaxation
(control)
response
Two micromoles
per liter of Ang-(l-7)
potentlated
the BK-Induced
relaxation
Values are mean?SEM
Ang I or Ang II groups include
6 to 8 rings from 3 to 4 dogs
Downloaded from http://hyper.ahajournals.org/ by guest on March 6, 2014
Li et al Angiotensin-(107)
and Bradykinin
.
.
Llsinoprrl
&l&(1-7)
l
sar’l#-Ang
II
80b
a
F
g
40-
*
zo-
&I-
01
-11
-9
-6
-7
Peptide Log mot/L
-.-A-
Conlrol
Ang-(I-7)
. Lislnopril
*
0-l
0
5
10
15
20
FIG 6 Top, Competition
assays using purified canine ACE were
determined
using a fixed concentration
of the substrate
Hip-HisLeu (1 mmol/L) and varying the concentrations
of the competing
agents [Iisinopnl,
10-l” to lo-’
mol/L, Ang-(l-7),
10-O to iOF
mol/L,
and Sar’ Thr’-Ang
II, IO-* to 10m5 mol/L]
Values are
mean+-SEM
n=3 to 4 dogs
Ang-(l-7)
had an ICsO of 0 65
pmol/L
Llsinopnl
had an I&,, of 1 5 nmol/L
At 1 pmol/L,
Sar’
Thr’-Ang
II showed
no rnhlbltlon
of ACE. Bottom,
‘251-~yr0]BK
(final concentration
of 1 nmol/L) was added to tubes containing
three preincubated
coronary
rings with 1 mL Krebs’ buffer aerated with 95% O2 and 5% CO? at 37°C Lisinopnl
(2 pmol/L),
Ang-(l-7)
(2 PmollL), or Krebs’ buffer as control were added to
the rings 10 minutes before addition of the radiolabeled
BK Allquots of the incubation
medrum
were removed
at 5, 10, and 20
minutes
Results are reported
as mean?SEM
n=4 dogs *P < 05
vs control
of small Ang fragments, including Ang-( l-7), were due to
ACE mhibitlon Thus, our findings indicate that Ang(l-7) may mhlblt local endothehal ACE activity,
prevent
the degradation of kimns in local vessels, and as a consequence augment kmm levels. Furthermore, since It
would not be anticipated that ACE mlubition would have
an effect on Ach or sodium mtroprusslde, our findings
demonstrating a specific effect of Ang-(l-7) on BK, but
not these other vasodllators, are consistent with Ang-( l-7)
competitively binding with ACE and preventing BK degradation The specific interaction of Ang-( l-7) and BK
confirms previously reported results m conscious rats ‘6
There are some differences between our m vitro stuches
and those described m whole animal. Paula et alI6 showed
that mdomethacm but not treatment with enalapnlat
blocked the Ang-(l-7) potentlatmg effect to BK m rats m
vlvo In contrast, m canme coronary vessels mtnc oxide
rather than prostaglandins appears to account for the synerglstlc action of Ang-(l-7) on BK-induced relaxation
Similarly, nitric oxide was demonstrated to participate m
399
the potentiatron of relaxation to BK by ACE inhlbltlon m
canine coronary rings 40It has previously been shown that
Ang-( 1-7) stimulated prostaglandin release m pithed rats
and cerebral arterlolar vessels m piglets, ‘33’4whereas Ang(l-7) responses were medlated by nitric oxide release m
feline hmdhmb and mesentery preparations and m vitro
preparation of coronary vascular nngs.*,g The kmd of mediator contrlbutmg to the modulatory actions of Ang-( l-7)
on BK may be tissue or organ specific or less likely reflect
differences in species-specific mechanisms
There 1s another possible explanation for the reported
differences between m vivo and m vitro findings ACEIs
do not always produce an mcrease in circulating kmm concentration in v1v0.l~ Thus, the lack of an effect of ACE
inhibition m the conscious rat may suggest that kmms are
either not involved or exist at concentrations below those
that will be reqmred to demonstrate a synergistic action
between the two peptldes In our preparation, hsmoprll
pretreatment greatly enhanced BK-induced responses, mdlcatmg the effectiveness of lismopril to inhibit local ACE
Moreover, hsmopnl abolished the Ang-( l-7) potentlatmg
effect to BK Since hsinoprll has a much higher affinity to
ACE than Ang-(l-7) (=500-fold), at equal molar concentrations, hsinoprll may mask the ACE inhibitory effects of
Ang-(l-7) m vascular nngs.
Hoe 140 1s a potent competitive antagonist of kmm B2
receptors. ‘7.45346
Our results showed that high concentrations of Hoe 140 (2 pmol/L) nearly abolished BK-induced
responses and also eliminated the effect of Ang-(l-7)
At
lower concentrations (20 nmol/L), Hoe 140 caused a nghtward shift m the relaxation curve of BK. Under these conditions, however, the potentiatmg response produced by
Ang-(l-7)
was still present These findings may be
explained by a chrect action of Ang-(l-7) on ACE preventing bmdmg of BK to the enzyme Reduced BK metabolism in the presence of Ang-(l-7), as shown in our
experiments, would allow increased kimn concentration m
the presence of low concentrations of Hoe 140
How then 1s the synergistic effect of Ang-(l-7) on BK
different from the effect of Ang-(l-7) acting as a direct
vasodllator of coronary vesselsT8 Both the direct and the
synergistic effects of Ang-( 1-7) involve a B2 receptor and
are mediated by nitric oxide but not prostaglandms Furthermore, in our previous expenment,8 we found that the
direct effect of Ang-(l-7) is mediated by a non-AT1
or -AT, receptor While Sar’ Thr’-Ang II has been reported by us to block the vasodllator and antiproliferative
actions of Ang-( 1-7),8J0 this competltlve peptlde antagonist did not inhibit the amplification of the BK-induced
vasodllator response m the presence of Ang-(l-7). This
finding suggests that the interaction between BK and Ang(l-7) may be mediated by an Ang receptor subtype that 1s
not competed for by Sar’ Thr’-Ang II or the subtype-specific ATI and AT:! receptor antagonists Alternatively,
Ang-(l-7), m these circumstances, may act as an endogenous hgand for Bz receptors or as an ACEI, as discussed
above Further studies need to be conducted to evaluate
the nature of the receptor and the mechanism accounting
for this interaction between Ang-( l-7) and BK receptors.
In summary, we have demonstrated that Ang-(l-7)
augments BK-induced vasochlation m coronary arteries
by acting as a local modulator of ACE activity and by
enhancing the release of nitric oxide. These effects of
Ang-( l-7) may contribute to enhanced cardiovascular
protection
Downloaded from http://hyper.ahajournals.org/ by guest on March 6, 2014
400
Hypertension
Vol29,
No 1, Part 2
January 1997
Acknowledgments
This work was supported m part by grant l-POl-HL51952 and
ROl HL56973 from the National Heart, Lung, and Blood Inshtute
of the National Institutes of Health, Bethesda, Md The authors
wish to thank Nancy Pmo and Ahcla Jones for their contnbutlons
to this study
25
26
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