Clinical Science (1992) 83, 477-482 (Printed in Great Britain)
477
Generation of angiotensin II from human plasma by
tissue kallikrein
N. KRIVOY*, H. SCHLUTERt, M. KARASS and W. ZlDEKt
TMedizinische Poliklinik and SMedizinische Physik, Westfalische Wilhelms-Universiti, Miinster,
Federal Republic of Germany, and *Medicine A and Clinical Pharmacology Unit, Rambam
Medical Center and B. Rappaport Faculty of Medicine, Haifa, Israel
(Received 17 January/l9 May 1992; accepted 26 May 1992)
1. Human plasma was incubated with tissue kallikrein from porcine pancreas, dialysed to obtain a
fraction with a molecular mass < 10 kDa and further
purified by reverse-phase chromatography.
2. Vasopressor activity in the fractions obtained was
tested in the isolated perfused rat kidney.
3. In one fraction a strong vasopressor action was
found, which was blocked by saralasin and by an
angiotensin I1 antibody.
4. Aprotinin inhibited the formation of vasopressor
substances by tissue kallikrein.
5. U.v.-laser desorption/ionization mass spectrometry
revealed a molecular mass of 1046Da in the purified
active fraction.
6. It is concluded that tissue kallikrein forms not only
kinins, but also angiotensin 11, from human plasma
under physiological conditions.
_____
INTRODUCTION
A number of serine proteases in several tissues
such as the submandibular gland and prostate of
the rat, including glandular kallikrein, tonin and
another newly isolated protease, are capable of
releasing vasoactive peptides from larger proteins.
The physiological role of these proteases, which are
encoded for by the so-called kallikrein gene family,
in the local regulation of vascular tone is poorly
understood. On the other hand, the role of the local
renin-angiotensin system is increasingly recognized.
In earlier studies it has been suggested that glandular kallikrein may not only produce vasodilator
peptides such as bradykinin, but also vasopressor
compounds. The nature of the latter has been a
matter of debate. It was suggested that porcine
kallikrein may generate an angiotensin-like peptide
from Cohn’s fraction IV-4 of human plasma [l]. In
rainbow trout, kallikrein was found to generate a
peptide which was clearly distinct from angiotensin
I1 (ANG 11) [2, 31. Later it was also inferred that
kallikrein could produce ANG I1 from human
plasma [4, 51. However, in these studies the pep-
tides released by kallikrein were not identified directly, but indirectly by pharmacological inhibition
and chromatographical characteristics. Therefore in
the present study the peptides released by kallikrein
were identified directly by their molecular mass
using u.v.-laser-desorption/ionization (LDI) mass
spectrometry.
MATERIALS AND METHODS
Chemicals
Porcine pancreatic kallikrein, bradykinin, captopril, ANG 11, noradrenaline (NA) and saralasin
were obtained from Sigma (Deisenhofen, Germany),
aprotinin was from Boehringer (Mannheim, Germany), and methanol, acetonitrile, trifluoracetic acid
(TFA) and bonded-C18 silica columns (lg/column)
were from J. T. Baker (Phillipsburg, NJ, U.S.A.).
ANG I1 antibody was purchased from Amersham
(Amersham, Bucks, U.K.).
Isolated perfused rat kidney preparation
Normotensive Wistar-Kyoto rats, 4-6 weeks old,
were used. The isolated perfused kidney was
prepared as described previously [6]. Briefly, the
abdominal cavity was opened, and the infrarenal
aorta and left renal artery were isolated and cannulated. Immediately after the cannulation, 500 units
of heparin sodium were flushed into the aorta and
renal artery. Perfusion was started immediately after
the cannulation. The left kidney was excised by
blunt dissection excluding the adrenal gland and
was mounted in the perfusion system.
Perfusion system
A single-pass system at a constant flow rate of
9 mlmin-’ 8-l weight with Tyrode’s solution kept
at 37°C and equilibrated with 5% C0,/95% 02,
was used as described previously [7]. Vasoactive
hormones were injected in bolus doses of 1 5 0 ~ 1
each to test kidney responsiveness. Fig. 1 shows a
Key words: angiotensin 11, plasma, tissue kallikrein.
Abbreviations: ANG 11, angiotensin II; LDI, ux-laser desorptionlionization; NA, noradrenaline; SPE, solid-phase extraction; TFA, rrifluoroacetic acid.
Correspondence: Professor W. Zidek. Medizinische Poliklinik, Albert Schweitzer Strasse 33, Minster, Federal Republic of Germany.
N. Krivoy et al.
250
I
I
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The molecular mass was determined by matrixassisted LDI mass spectrometry (see below). As
shown in Fig. 2, isoelectric focusing of the fraction
from the MonoQ column containing kallikrein (row
9, as described by Johansen et al. [8], revealed two
bands in the pH range from 3.8 to 3.9. LDI mass
spectrometry of this fraction revealed two masses of
25 904 Da and 26 901 Da. These two bands represent
isoforms of kallikrein with different carbohydrate
residues in the glycoprotein [9].
50f
Plasma collection and incubation with kallikrein
0
14 13
12
II
10
9
8
7
6
-log [Dose (mol)]
Fig. I. Dose-response curve for A N G II ( 0 )and N A
isolated perfused kidney. Values are means fSD.
(0)
in the
dose-response curve for ANG I1 and NA in the
isolated perfused kidney.
Plasma fractions were injected in bolus doses of
1 5 0 ~ 1each. Drugs and plasma fractions were dissolved in Tyrode’s solution before injection. Before
testing these fractions for vasoactivity in the isolated
kidney preparation, the pH and osmolarity were
measured in order to avoid non-specific effects on
perfusion pressure due to deviations from the physiological range. Osmolarity was maintained
between 272 and 3l0mosmol/l; pH was kept
between 7.2 and 7.5. The experiments did not last
more than 3h. After perfusion, the mean weight
(+SD) of the perfused kidney was 108.4+7.2%
(n = 34) of that of the non-perfused kidney, indicating that no significant oedema formation occurred
during the perfusion period.
Purification of kallikrein
To exclude the action of other proteases such as
tonin, the commercial kallikrein preparation
obtained from Sigma was purified as described by
Johansen et al. [S]. The kallikrein was subjected to
chromatography on a MonoQ column. The starting
buffer (eluent A) consisted of 0.1 mol/l ammonium
acetate (pH 4.8) and the gradient was run from 0.1
to lmol/l ammonium acetate (pH 4.8) within
60min. The fraction between 33 and 40% lmol/l
ammonium acetate (pH 4.8) (eluent B) contained
kallikrein and was used for the incubation experiments. The purity of the final kallikrein preparation
was tested by isoelectric focusing using the Phastsystem (Pharmacia, Freiburg, Germany). The readyto-use gel (Phastgel IEF 3-9; Pharmacia, Freiburg,
Germany) had a pH gradient from 3 to 9. The
electrophoretic and the silver staining procedures
were performed as described in the Phast-Manual.
Pooled human plasma (20ml) from normotensive
subjects ( n = 10) was used for this purpose. Blood
was collected in heparinized syringes and plasma
was separated immediately. Plasma was immersed
in a 65°C water bath for 1h to inactivate the
proteolytic enzymes, as described elsewhere [3].
After the inactivation period, plasma was centrifuged at 4000 rev./min for lOmin and the supernatant incubated with purified porcine pancreatic
kallikrein (10units/ml of plasma) was dialysed overnight at 37°C in tubular dialysis membranes
(Spectra/POR; Spectrum Medical, Los Angeles, CA,
U.S.A.) with an exclusion diameter pore of 8000Da
against the physiological buffer [solution 1: 0.05 mol/l
4-(2-hydroxyethyl-1-piperazine-ethanesulphonicacid,
0.2 mol/l NaOH, 0.08 mol/l NaCl dissolved in 1 litre
of water; solution 2: 180mmol/l NaCl, 20 mmol/l
KCl, 3.6 mmol/l CaCl,, 2 mmol/l MgCl,; solutions 1
and 2 were mixed and titrated to pH 7.41. To the
dialysate (fraction < 8000 Da) TFA was added to a
concentration of 0.1%. The solution was frozen at
- 30°C until a solid-phase extraction (SPE) was
performed.
Separation of the proteolytic products
The SPE was preceded by conditioning the
columns on a SPE vacuum manifold (Burdick and
Jackson, Muskegon, MI, U.S.A.) with 5ml of acetonitrile and subsequently washing with a solution of
0.1% (v/v) TFA in water. To extract peptides out of
the ultrafiltrate, the thawed solution was aspirated
through the column by a gentle suction, allowing a
flow rate of lml/min. The elution fractions were
dried in a Speed Vac concentrator (Savant,
Framingdale, NY, U.S.A.).
The residue from the Speed Vac, reconstituted in
loop1 solution of 0.1% (v/v) TFA in water, was
further purified by reverse-phase h.p.1.c. (HPLC
gradient pump; Merck-Hitachi Darmstadt, Germany;
Nucleosil 300-RP 18 column; Macherey and Nagel,
Duren, Germany). Separation was performed using
0.1% (v/v) TFA in water (solution A) isocratically
for lOmin, followed by a linear gradient of 0-30%
of a solution containing 0.1% (v/v) TFA in acetonitrile (solution B) within 30 min (see Fig. 3) and
followed by a linear gradient of 30-60% solution B
within 15min. A flow rate of 0.5ml/min was main-
Angiotensin II formation by kallikrein
I
2
3
4
S
5
6
Fig. 2. Iwelectric focusing of the different fractions obtained from the purification of the commercially available
kallikrein preparation. Abbreviations: pl, isoelectric point; S, standard [glucose oxidase (fungal), pl=4.25; acetylated cytochrome c
(hone heart), PI=3.95; amyloglucosidase (Aspergillus), pl=3.65]. Lanes 1-6 show the fractions from the MonoQ column: I, 0% eluent
B (fraction with non-binding substances); 2, 0-7% eluent B 3, 7-20% eluent B 4, 20-33% eluent B 5, 3240% eluent B (kallikrein
fraction); 6, 6 5 0 % eluent 8.
tained. The U.V. absorption was monitored at
215nm (step I). Fractions (lml) were collected. As
control experiments, the same amount of human
plasma without kallikrein and the kallikrein preparation without plasma underwent the same procedure as described above up to step I.
The vasoactive fraction from step I was further
purified on the reverse-phase system described
above with an isocratical elution of 5min with 100%
solution A, a linear gradient of 0-20% solution B
within lOmin, a linear gradient of 2WO% solution
B within 40min and a linear gradient of 40-60%
solution B within 10min (step 11; see Fig. 4). The
flow rate was kept constant at 0.5 ml/min.
A re-fractionation (see Fig. 5 ) of the vasoactive
fraction from step I1 was performed using a
microbore-h.p.1.c. system (SMART; Pharmacia,
Freiburg, Germany) and a pRPC C2/C18 reversephase column (Pharmacia, Freiburg, Germany).
After an isocratical elution of 5min with 100%
solution A, a linear gradient of 0-20% solution B
within 5min, a linear gradient of 20-25% solution B
within 40min and a linear gradient of 25-30%
solution B within 5min followed (step 111).
Effects of proteinase inhibitors
For the inhibition experiments portions (2ml) of
pooled plasma were prepared as described in the
section Purification of kallikrein (above). Each 2 ml
of plasma, incubated with kallikrein, was mixed
with one of the following inhibitors and then dialysed as described in the section Purification of
kallikrein (above). The inhibitors were used with a
final concentration as indicated captopril (20 ,umol/
ml of plasma), aprotinin (1 pg/ml of plasma).
Pharmacological experiments
To observe the action of bradykinin on the
isolated perfused kidney preparation, different concentrations (1, 3 and long) were injected. The
isolated kidney was perfused with Tyrode’s solution
D
containing lOpmol/l saralasin to block the ANG I1
receptors.
To test whether the vasopressor substance
released by kallikrein is bound to antibodies against
A N G 11, 500pl of the vasoactive fraction after step I
was incubated for 30min with ANG I1 antibody,
equivalent to an ANG I1 binding capacity of 400ng
(n= 4). Control experiments were performed by
incubating 200ng of A N G I1 with the same amount
of antibody. In the isolated perfused kidney ANG I1
and the vasoactive fraction were tested with and
without the ANG I1 antibody. Furthermore, the
same amount of antibody alone was tested.
Analysis of the vasoactive fraction by
spectrometry after step II
LDI mass
The vasoactive material was examined by LDI
mass spectrometry. A reflector-type time-of-flight
mass spectrometer equipped with a nitrogen laser
(337nm, pulse length 411s) was used for ion generation and mass analysis. Details of the matrixassisted LDI mass spectrometry technique have
been reported elsewhere [lo]. Briefly, samples were
dissolved in water so as to obtain a concentration of
10-5-10-6 mol/l, if necessary. A portion (1 pl) of
this solution was mixed with lop1 of an aqueous
solution of 2,5-dihydroxybenzoic acid (10 g/l; U.V.
absorption maxima at 337 and 355 nm) representing
the u.v.-absorbing matrix. A portion (1 pl) of the
final solution was dripped and dried on to a
metallic substrate. Desorption of analyte ions was
achieved by laser shots of irradiances in the 106-107
W/cm2 region focused to spot sizes of typically
50-100 pm in diameter. The spectra were registered
by a LeCroy 9400 transient recorder and typically
accumulated from 10 single laser shots. The total
time of measurement including preparation was 1015min. The results were expressed as molecular
mass/electrical charge of the substance ( z ) (see Fig.
8). Since with this form of mass spectrometry substances with a single charge are produced, molecular
mass/z is identical with the molecular mass [lo].
N. Krivoy et al.
480
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10
20
30
40
10
50
30
40
50
60
Elution time (min)
Elution time (min)
Fig. 3. Reverse-phase chromatography (step I) of the elution fraction of the SPE step. Conditions for chromatography: column, C,,
reversephase (4mm x 250mm); flow rate, 0.5ml/min; N O m i n , solution A
I 0 4 m i n , 0-30% solution B &55min, 3040% solution B; sample, organic
fraction of the SPE equivalent t o lOml of plasma dissolved in I ml of
solution A. The vasopressor fraction is indicated by (D).
20
Fig. 4. Reverse-phase chromatography (step II) of the active fraction
of step I (Fig. 3). Conditions for chromatography: column, C,, reverse
-phase (4mm x250mm); flow rate, O.Sml/min; O-Smin, 100% solution A;
S-ISmin, 040% solution B; 15-55min. 2 0 4 % solution B; SMSmin,
4040% solution B; sample, active fraction (IOml of plasma equivalent) of
step I (Fig. 3) dissolved in I ml of solution A. The vasopressor fraction is
indicated by (D).
RESULTS
After dialysis and SPE the extract of the plasma
incubated with tissue kallikrein pursed as indicated
above was subjected to reverse-phase chromatography (step I: Fig. 3). The bioassay for vasopressor
activity revealed that in the fraction eluted at 43-45
min a vasopressor agent was present. The other
fractions and the physiological salt solution used
did not affect the perfusion pressure. Furthermore,
human plasma and kallikrein each processed alone
up to step I did not show significant vasoactivity
(data not shown). The second chromatographic
purification step (step 11) of the active fraction on
the same reverse-phase chromatography system but
with a flatter gradient is shown in Fig. 4. As
indicated the vasopressor activity was eluted at
47-49 min.
A re-fractionation of the active fraction of step I1
was chromatographed a third time (step 111) on a
microbore reverse-phase column (Fig. 5). The vasopressor activity was observed at a retention time of
50-52 min. Thus the retention times of the active
fractions in all three chromatography procedures
were identical with that of ANG I1 on these
columns.
Fig. 6 shows a representative recording out of five
identical experiments showing the response of the
renal vasculature to ANG 11, various doses of
bradykinin and the active fraction obtained by
reverse-phase chromatography (Fig. 3). Addition of
saralasin to the perfusion medium blocked the
responses to ANG I1 and the active fraction.
Fig. 7 demonstrates the effect of various concentrations of the active fraction on the perfusion
pressure (n = 5). The change in perfusion pressure
was established to be dose-dependent.
After co-incubation of plasma and kallikrein
r
I
10
20
30
Elution time (min)
40
50
Fig. 5. Reversephase chromatography (step 111) of the active fraction of step II (Fig. 4). Conditions for chromatography: column, C,/C,,
reversephase (3mm x 250mm); flow rate, O.Sml/min; O-Smin, 100% 10111tion A S-lOmin, 0-20% solution B; 10-50min, 20-25% solution 6
SMOmin, 2540% solution B; sample, active fraction (IOml of plasma
equivalent) of step II dissolved in I ml of solution A. The vasopressor
fraction is indicated by (D).
(n = 5 ) with captopril (20 pnollml), the formation of
vasopressor substance was not decreased compared
with in the absence of captopril. When aprotinin
(1 pg/ml) was co-incubated with the plasma/kallikrein preparation (n= 4), no vasoactive material was
recovered. Incubation of the vasoactive fraction and
of ANG I1 with an ANG I1 antibody abolished the
effect on perfusion pressure completely. Injection of
the ANG I1 antibody alone had no effect on
perfusion pressure.
Fig. 8 shows the results of LDI mass spectrometry of the active fraction obtained by rechromatography. The analysis shows that purifica-
Angiotensin I1 formation by kallikrein
481
c
.-0
Y
Y
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.-:
Y
Y
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lo:
O
1
I000
I200
Molecular mass/z (Da)
I
-10-
Fig. 8. LDI mass spectrum of the active fraction obtained after
reversephase chromatography (Fig. 4)
10 min
Fig.6. Typical pattern of changes in perfusion pressure in the
isolated perfused kidney produced by bradykinin (BK; I, 3 and long)
ANC II (Idng) and the active fraction (equivalent to 0.6~11of
plasma) from the chromatographic purification (Fig. I) before and
after adding IOpnol/l saralasin to the perfusion medium
0‘
200
I
1
I
600
800
Dose (PI of plasma equivalent)
400
lo00
Fig. 7. Dose-response curve for the vasopressor fraction obtained by
incubation of human plasma with tissue kallikrein in the isolated
perfused kidney. Values are means fSD (n = 5).
tion of this fraction was satisfactory. There was only
one significant mass peak at 1047Da, which is
identical with the mass of the protonated ion of
ANG 11.
DISCUSSION
In the earlier literature there were several hints
that kallikrein may produce a vasopressor compound. Arakawa & Maruta [l] found that tissue
kallikrein released an angiotensin-like substance
from human plasma at a pH of 4-6. Vasopressor
activity released from Cohn’s fraction IV-4 by kallikrein corresponded well with radioimmunologically
determined ANG I1 levels and was suppressible by
saralasin [S]. Similar results were reported by Lipke
et al. [2, 31, who studied the effects of incubation of
rat and trout plasma with kallikrein. They produced
evidence against the release of ANG I1 by kallikrein. The vasopressor released by kallikrein did not
replace ANG I1 at rat adrenal ANG I1 receptors
and was not bound to ANG I1 antibodies in a
radioimmunoassay. Furthermore, in an earlier study
[111 it was demonstrated that kallikrein releases
ANG I1 from Cohn’s fraction IV-4 at a pH of 4.
Thus a review of the pertinent literature shows that
kallikrein may release a vasopressor from plasma
proteins, but there is considerable uncertainty as to
the nature of this substance. Under special reaction
conditions, the release of ANG I1 was demonstrated, but it remained unclear as to whether ANG
I1 production by kallikrein was relevant under
physiological conditions.
The present results indicate that tissue kallikrein
from porcine pancreas is able to generate ANG I1
under physiological conditions, when incubated with
human plasma. The vasoactive substance generated
by tissue kallikrein was established to be ANG 11,
since: (1) the retention time of the vasoactive material was identical with that of ANG II; (2) saralasin,
a known ANG I1 receptor blocker, inhibited the
vasoactivity of the active fraction completely; (3) the
vasoactivity was abolished by incubation with an
ANG I1 antibody; and (4) in the purified vasoactive
fraction only the molecular mass of ANG I1 could
be detected by LDI mass spectrometry.
482
N. Krivoy et al.
The isolated perfused normotensive rat kidney [6,
71 was used as a model to examine the effect of the
generated material on vascular smooth muscle directly. This model offered the advantage that the
small peripheral arteries, which are mainly responsible for the vascular resistance, could be studied.
Furthermore, a counterregulatory activation of
vasodepressor systems in the intact animal was
avoided.
Since the commercially available tissue kallikrein
preparations may not be highly purified, the ANG
I1 produced may not stem from the action of tissue
kallikrein, but from other proteases. Therefore, the
commercially available preparation was further purified by anion-exchange chromatography as described in the literature 181, and only the fraction
containing tissue kallikrein was incubated with
plasma. Further evidence indicating that it is kallikrein which generates ANG I1 in these experiments
is the effect of the proteinase inhibitors applied.
Aprotinin, a kallikrein inhibitor, blocked the
vasoactivity. Furthermore, an ANG 11-converting
enzyme inhibitor did not affect the vasopressor
activity. Therefore it is unlikely that the ANG I1
production observed is due to the formation of
renin from plasma prorenin by kallikrein [lZ].
ACKNOWLEDGMENT
N.K. was a Visiting Investigator in the Medizi-
nische Poliklinik, Miinster, Federal Republic of
Germany.
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