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In vitro study on the effects of bradykinin potentiating factor and angiotensin II analogue on degradation and conversion of angiotensin I and II in plasma
287
Clinica Chimica Acta, 52 (1974) 287-292 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6258
IN VITRO STUDY ON THE EFFECTS OF BRADYKININ POTENTIATING FACTOR AND ANGIOTENSIN II ANALOGUE ON DEGRADATION AND CONVERSION OF ANGIOTENSIN I AND II IN PLASMA
TOSHIO OGIHARA’, TOSHIHIDE YAMAMOTO**, KEI DOI**, TERUTOSHI KIMURA*** and SHUMPEI SAKAKIBARA***
YUICHI
KUMAHARA*,
*Central Laboratory for Clinical Inuestigation, Osaka University Medical School, Fukushimaku, Osaka, **Center for Adult Diseases, Osaka and ***Peptide Institute, Protein Research Foundation, Mino, Osaka (Japan) (Received
November
5, 1973)
Summary Inhibition of converting enzyme by bradykinin potentiating factors and of angiotensin II by angiotensin antagonists have been shown by alteration of the pressor activity of angiotensin II by bioassays. By using specific radioimmunoassays of angiotensin I and II, the degradation of angiotensin I and the generation of angiotensin II were quantitated serially in plasma which was enriched with 20 to 40 ng angiotensin I per ml of plasma and incubated at 37” in the presence of synthetic bradykinin potentiating peptide B (BPPb) or l-sarcosine,&isoleucine-angiotensin II ( [ Sar’ ,Ile8 ] A II). Similarly, the degradation of angiotensin II added to plasma at a concentration of about 22 ng/ml was assessed in the presence of BPPb or [Sar’ ,Ile* ] A II. Inhibition of plasmaconverting enzyme was confirmed by the reduced generation of immunoreactive angiotensin II from angiotensin I in the presence of BPPb. Neither BPPb nor [Sari ,Ile* ] A II accelerated the degradation of angiotensin II.
Introduction Bradykinin potentiating factors are small peptide mixtures originally isolated from the venom of Bothrops jarurucu and characterized by Ferreira in 1965 [l]. Bakhle et al. demonstrated that bradykinin potentiating factors also prevent angiotensin I conversion into angiotensin II in vitro using rat colon preparation and ginea-pig ileum [2,3]. Many analogues were subsequently synthesized as bradykinin potentiating peptides (BPP), which were demonstrated to inhibit the converting enzyme in vivo and in vitro [4-71. On the other hand, l-sarcosine&isoleucine angiotensin II (Sar-ArgVal-Tyr-Ile-His-Pro-Be) ([Sar’ ,Ile* ] A II), one of angiotensin II anta-
288
gonists, was introduced by Khosla et al. [8] as a competitive inhibitor against the pressor action of angiotensin II. The competitive action of the compound against angiotensin II was shown in the ileum or aortic strip preparations and in whole animals made hypertensive by the infusion of angiotensin II [9]. Currently available radioimmunoassays of angiotensin I and II made it possible to estimate the action of bradykinin potentiating factors or angiotensin II antagonists on plasma converting enzyme and possibly on angiotensinases by direct quantitation of angiotensin I or II. In the following study, we measured the conversion of angiotensin I and the degradations of angiotensin I and II in plasma enriched with angiotensin I or II, in the presence of synthetic bradykinin potentiating peptide B (PyroGly-Leu-Pro-Arg-Pro-Lys-IlePro-Pro) (BPPb) [lo] or [ Sar’ ,Ile* ] A II in order to document (i) the inhibition of converting enzyme by BPPb by quantitation of the generation of angiotensin II from angiotensin I and (ii) possible acceleration of the degradation of angiotensin II in the presence of BPPb or [Sar’ ,Ile* ] A II. Materials and Methods Venous blood was drawn with a heparinized syringe and immediately transferred into chilled tubes. Plasma was separated by a refrigerated centrifuge. After the addition of approximately 20-40 ng angiotensin I to 1 ml of plasma, plasma was incubated in a water bath at 37” in the presence of varying concentrations of either BPPb or [Sar’ ,Ile* ] A II. The degradation of added angiotensin I and the generation of angiotensin II from angiotensin I were estimated by taking small portions from the incubated plasma at 15, 30 and 60 min using specific radioimmunoassays for angiotensin I and angiotensin II; Similarly the degradation of added angiotensin II at the concentration of approx. 22 ng/ml was assessed in the presence of BPPb or [Sar’ ,Ile* ] A II. Experiments were repeated with plasma from different individuals to confirm the reproducibility. Radioimmunoassay of angiotensin I based on the method described by Haber et al. [ll] was carried out using a commercial kit (Dainabot Laboratory, Tokyo, Japan). Radioimmunoassay of angiotensin II was done using a similar kit supplied by Cea-Cen-Sorin (Paris, France). Synthetic BPPb and [Sar’ ,Ile’ ] A II were synthesized at the Peptide Institute, Protein Research Foundation, Mino, Osaka, Japan. Results and Discussion Antibodies to angiotensin I did not show cross-reactivity with angiotensin II in the range of concentration used in the experiments nor did antibody to angiotensin II cross-react with antibodies to angiotensin I. BPPb did not crossreact with antibodies to angiotensin I and II significantly: [Sar’ ,Ile8 ] A II did cross-react with antibody to angiotensin I (0.005%). Therefore, the assays for angiotensin I and II can be considered highly specific even in the presence of BPPb or [Sar’ ,Ile* ] A II in the samples except when a high concentration of [Sar’ ,Ile8 ] A II is employed in the assay of angiotensin II degradation. The inhibition of converting enzyme by BPP has been shown by a reduced
289
I
A”gde”*m
%/mn 50-
Fig.
1. Effect
Fig. 2. Effect
of BPPb on angiotensin
of BPPb on angiotensin
I degradation.
I conversion
to angiotensin
11.
290
generation of angiotensin II from angiotensin I, which was demonstrated by reduced pressor response in a smooth muscle preparation by angiotensin II generated from angiotensin I in the presence of BPP ]4---71. BPPb is one of the BPP isolated from the venom of Agki~~ru~~~ haa’ys bbmhoffii and characterized by Kato and Suzuki [9]. Converting enzyme was inhibited by 0.1 gg/rnl BPPb in vitro [9]. In the present study, the degradation of angiotensin I in the plasma incubate was partially inhibited by BPPb and the magnitude of inhibition by BPPb was dose-related (Fig. 1). The generation of angiotensin II, however, in the same incubation mixture was almost compietely inhibited (Fig. 2). BPPb did not interfere with the degradation of added angiotensin II (Fig. 5). These findings indicate that BPPb does inhibit plasma converting enzyme but not angiotensinases. The inhibition of converting enzyme in plasma by BPPb was confirmed by the reduced production of immunoreactive angiotensin II. The pressor action of angiotensin II was counteracted by 1 @g/ml ]Sar’ ,Ile’ ] A II (lo). Addition of [ Sa.r* ,Ile’ f A II at a concentration of I pg/rnl in plasma did not inhibit the de~adation of angiotensin I or the generation of ~~otensin II in the same system (Figs 3 and 4), nor was the conversion of angiotensin I to angiotensin II affected by [Sar’ ,Ile8 ] A II, [Sar’ ,Ile* ] A II did not interfere with the de~adation of added an~otensin II in the plasma incubate (Fig. 5). Pressor action of ~~otensin II was shown to be blocked by [Sar’ ,Ile8 ] A II in
A II analog concentrati
Fig. 3. Effect
of [Sari .Ile* 1 A II on angiotensin
I degradation,
291
Angiotensin n nglmP
A n analog concentration 0:
0
A : O.O01/1g/mQ B : 0.01 ,ug/mQ C : 0.1 ,ug/mQ D : 1.0 ,ug/mQ
min Fig. 4. Effect
of [Sari .Ile8] A II on angiotensin
I conversion
to angiOtensin
If.
vivo in animals made hypertensive by infusion of angiotensin II [IO] . The other possibility that [Sar’ ,Ile* ] A II might antagonize angiotensin II by accelerating the degradation of angiotensin II by plasma angiotensinases was not supported by the present experiment.
A B C
15 Fig. 5. Effects
30
: : :
BPPb lOOrg/mQ ALlanalogO.lpg/mQ AIIanalog lpg/mQ
mm
60
of BPPb and [Sar’ .11e8] A II on angiotensin
II degradation.
292
References 1
S.H.
Ferreira,
2
Y.S.
Bakhle,
3
S.H.
Ferreira.
L.J.
Greene,
V.A.
4
J.M.
Stewart,
S.H.
Ferreira
and
5
G.R.
Keim.
EXP.
Biol.
Med., T.R.
6
S.L.
Engel.
7
J.G.
Collier.
8
M.C. 15
9
Brit. A.M.
R.K.
H. Kato
il
E. Haber,
140
R.A.
Leese,
(1965)
163
Vane,
Nature,
Alavaster, L.J.
Y.S.
Greene,
222
Bakhle
Biochem,
Peterson.
B.F.
Gold
B. Rubin.
(1969)
956
and J.R.
Vane,
Pharmacol.,
Murphy,
G.L.
Nature.
20 (1971)
Hassert,
225
(1970)
379
1557
Zr and J.W.
Poutsiaka,
Proc.
Sac.
149 B.1.
Robinson
24
and J.R.
A.E.
(1972)
Schaeffer.
B.F.
and J.R. W.L.
and Vane.
Malay,
Lancet,
Proc.
Sot.
i (1973)
A.T.
Ferreira,
C.S.
Sweet
R.R.
Exp.
Biol.
Med.,
140
(1972)
240
72 Smeby
and
F.M.
Bumpus.
J. Med.
Chem.,
792
Tiirker,
10
1349
Jr, J. Kirpan.
Khosla.
(1972)
J. Pharmacol.. Reynard
and T.
M.M.
Hall,
T. Suzuki, Koerner.
M. Yamamoto, Biochemistry, L.B.
Page.
B.
10
(1971)
Kliman
and
and
F.M.
Bumpus,
Science.
177
(1972)
1203
Metab..
29
972 A.
Purnode.
J. Clin.
Endocrinol.
(1969)
Clinica Chimica Acta, 52 (1974) 287-292 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6258
IN VITRO STUDY ON THE EFFECTS OF BRADYKININ POTENTIATING FACTOR AND ANGIOTENSIN II ANALOGUE ON DEGRADATION AND CONVERSION OF ANGIOTENSIN I AND II IN PLASMA
TOSHIO OGIHARA’, TOSHIHIDE YAMAMOTO**, KEI DOI**, TERUTOSHI KIMURA*** and SHUMPEI SAKAKIBARA***
YUICHI
KUMAHARA*,
*Central Laboratory for Clinical Inuestigation, Osaka University Medical School, Fukushimaku, Osaka, **Center for Adult Diseases, Osaka and ***Peptide Institute, Protein Research Foundation, Mino, Osaka (Japan) (Received
November
5, 1973)
Summary Inhibition of converting enzyme by bradykinin potentiating factors and of angiotensin II by angiotensin antagonists have been shown by alteration of the pressor activity of angiotensin II by bioassays. By using specific radioimmunoassays of angiotensin I and II, the degradation of angiotensin I and the generation of angiotensin II were quantitated serially in plasma which was enriched with 20 to 40 ng angiotensin I per ml of plasma and incubated at 37” in the presence of synthetic bradykinin potentiating peptide B (BPPb) or l-sarcosine,&isoleucine-angiotensin II ( [ Sar’ ,Ile8 ] A II). Similarly, the degradation of angiotensin II added to plasma at a concentration of about 22 ng/ml was assessed in the presence of BPPb or [Sar’ ,Ile* ] A II. Inhibition of plasmaconverting enzyme was confirmed by the reduced generation of immunoreactive angiotensin II from angiotensin I in the presence of BPPb. Neither BPPb nor [Sari ,Ile* ] A II accelerated the degradation of angiotensin II.
Introduction Bradykinin potentiating factors are small peptide mixtures originally isolated from the venom of Bothrops jarurucu and characterized by Ferreira in 1965 [l]. Bakhle et al. demonstrated that bradykinin potentiating factors also prevent angiotensin I conversion into angiotensin II in vitro using rat colon preparation and ginea-pig ileum [2,3]. Many analogues were subsequently synthesized as bradykinin potentiating peptides (BPP), which were demonstrated to inhibit the converting enzyme in vivo and in vitro [4-71. On the other hand, l-sarcosine&isoleucine angiotensin II (Sar-ArgVal-Tyr-Ile-His-Pro-Be) ([Sar’ ,Ile* ] A II), one of angiotensin II anta-
288
gonists, was introduced by Khosla et al. [8] as a competitive inhibitor against the pressor action of angiotensin II. The competitive action of the compound against angiotensin II was shown in the ileum or aortic strip preparations and in whole animals made hypertensive by the infusion of angiotensin II [9]. Currently available radioimmunoassays of angiotensin I and II made it possible to estimate the action of bradykinin potentiating factors or angiotensin II antagonists on plasma converting enzyme and possibly on angiotensinases by direct quantitation of angiotensin I or II. In the following study, we measured the conversion of angiotensin I and the degradations of angiotensin I and II in plasma enriched with angiotensin I or II, in the presence of synthetic bradykinin potentiating peptide B (PyroGly-Leu-Pro-Arg-Pro-Lys-IlePro-Pro) (BPPb) [lo] or [ Sar’ ,Ile* ] A II in order to document (i) the inhibition of converting enzyme by BPPb by quantitation of the generation of angiotensin II from angiotensin I and (ii) possible acceleration of the degradation of angiotensin II in the presence of BPPb or [Sar’ ,Ile* ] A II. Materials and Methods Venous blood was drawn with a heparinized syringe and immediately transferred into chilled tubes. Plasma was separated by a refrigerated centrifuge. After the addition of approximately 20-40 ng angiotensin I to 1 ml of plasma, plasma was incubated in a water bath at 37” in the presence of varying concentrations of either BPPb or [Sar’ ,Ile* ] A II. The degradation of added angiotensin I and the generation of angiotensin II from angiotensin I were estimated by taking small portions from the incubated plasma at 15, 30 and 60 min using specific radioimmunoassays for angiotensin I and angiotensin II; Similarly the degradation of added angiotensin II at the concentration of approx. 22 ng/ml was assessed in the presence of BPPb or [Sar’ ,Ile* ] A II. Experiments were repeated with plasma from different individuals to confirm the reproducibility. Radioimmunoassay of angiotensin I based on the method described by Haber et al. [ll] was carried out using a commercial kit (Dainabot Laboratory, Tokyo, Japan). Radioimmunoassay of angiotensin II was done using a similar kit supplied by Cea-Cen-Sorin (Paris, France). Synthetic BPPb and [Sar’ ,Ile’ ] A II were synthesized at the Peptide Institute, Protein Research Foundation, Mino, Osaka, Japan. Results and Discussion Antibodies to angiotensin I did not show cross-reactivity with angiotensin II in the range of concentration used in the experiments nor did antibody to angiotensin II cross-react with antibodies to angiotensin I. BPPb did not crossreact with antibodies to angiotensin I and II significantly: [Sar’ ,Ile8 ] A II did cross-react with antibody to angiotensin I (0.005%). Therefore, the assays for angiotensin I and II can be considered highly specific even in the presence of BPPb or [Sar’ ,Ile* ] A II in the samples except when a high concentration of [Sar’ ,Ile8 ] A II is employed in the assay of angiotensin II degradation. The inhibition of converting enzyme by BPP has been shown by a reduced
289
I
A”gde”*m
%/mn 50-
Fig.
1. Effect
Fig. 2. Effect
of BPPb on angiotensin
of BPPb on angiotensin
I degradation.
I conversion
to angiotensin
11.
290
generation of angiotensin II from angiotensin I, which was demonstrated by reduced pressor response in a smooth muscle preparation by angiotensin II generated from angiotensin I in the presence of BPP ]4---71. BPPb is one of the BPP isolated from the venom of Agki~~ru~~~ haa’ys bbmhoffii and characterized by Kato and Suzuki [9]. Converting enzyme was inhibited by 0.1 gg/rnl BPPb in vitro [9]. In the present study, the degradation of angiotensin I in the plasma incubate was partially inhibited by BPPb and the magnitude of inhibition by BPPb was dose-related (Fig. 1). The generation of angiotensin II, however, in the same incubation mixture was almost compietely inhibited (Fig. 2). BPPb did not interfere with the degradation of added angiotensin II (Fig. 5). These findings indicate that BPPb does inhibit plasma converting enzyme but not angiotensinases. The inhibition of converting enzyme in plasma by BPPb was confirmed by the reduced production of immunoreactive angiotensin II. The pressor action of angiotensin II was counteracted by 1 @g/ml ]Sar’ ,Ile’ ] A II (lo). Addition of [ Sa.r* ,Ile’ f A II at a concentration of I pg/rnl in plasma did not inhibit the de~adation of angiotensin I or the generation of ~~otensin II in the same system (Figs 3 and 4), nor was the conversion of angiotensin I to angiotensin II affected by [Sar’ ,Ile8 ] A II, [Sar’ ,Ile* ] A II did not interfere with the de~adation of added an~otensin II in the plasma incubate (Fig. 5). Pressor action of ~~otensin II was shown to be blocked by [Sar’ ,Ile8 ] A II in
A II analog concentrati
Fig. 3. Effect
of [Sari .Ile* 1 A II on angiotensin
I degradation,
291
Angiotensin n nglmP
A n analog concentration 0:
0
A : O.O01/1g/mQ B : 0.01 ,ug/mQ C : 0.1 ,ug/mQ D : 1.0 ,ug/mQ
min Fig. 4. Effect
of [Sari .Ile8] A II on angiotensin
I conversion
to angiOtensin
If.
vivo in animals made hypertensive by infusion of angiotensin II [IO] . The other possibility that [Sar’ ,Ile* ] A II might antagonize angiotensin II by accelerating the degradation of angiotensin II by plasma angiotensinases was not supported by the present experiment.
A B C
15 Fig. 5. Effects
30
: : :
BPPb lOOrg/mQ ALlanalogO.lpg/mQ AIIanalog lpg/mQ
mm
60
of BPPb and [Sar’ .11e8] A II on angiotensin
II degradation.
292
References 1
S.H.
Ferreira,
2
Y.S.
Bakhle,
3
S.H.
Ferreira.
L.J.
Greene,
V.A.
4
J.M.
Stewart,
S.H.
Ferreira
and
5
G.R.
Keim.
EXP.
Biol.
Med., T.R.
6
S.L.
Engel.
7
J.G.
Collier.
8
M.C. 15
9
Brit. A.M.
R.K.
H. Kato
il
E. Haber,
140
R.A.
Leese,
(1965)
163
Vane,
Nature,
Alavaster, L.J.
Y.S.
Greene,
222
Bakhle
Biochem,
Peterson.
B.F.
Gold
B. Rubin.
(1969)
956
and J.R.
Vane,
Pharmacol.,
Murphy,
G.L.
Nature.
20 (1971)
Hassert,
225
(1970)
379
1557
Zr and J.W.
Poutsiaka,
Proc.
Sac.
149 B.1.
Robinson
24
and J.R.
A.E.
(1972)
Schaeffer.
B.F.
and J.R. W.L.
and Vane.
Malay,
Lancet,
Proc.
Sot.
i (1973)
A.T.
Ferreira,
C.S.
Sweet
R.R.
Exp.
Biol.
Med.,
140
(1972)
240
72 Smeby
and
F.M.
Bumpus.
J. Med.
Chem.,
792
Tiirker,
10
1349
Jr, J. Kirpan.
Khosla.
(1972)
J. Pharmacol.. Reynard
and T.
M.M.
Hall,
T. Suzuki, Koerner.
M. Yamamoto, Biochemistry, L.B.
Page.
B.
10
(1971)
Kliman
and
and
F.M.
Bumpus,
Science.
177
(1972)
1203
Metab..
29
972 A.
Purnode.
J. Clin.
Endocrinol.
(1969)