Download Modification of the Enzymatic Activity of Renin by

Modification of the Enzymatic Activity of Renin by
Acidification of Plasma and by Exposure of Plasma
to Cold Temperatures
THEODORE A.
KOTCHEN, M.D.,
WILLIAM J. WELCH,
M.S.,
A N D RAMESH T. TALWALKAR, P H . D .
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
SUMMARY Plasma renin activity (PRA) increases after acidification of plasma or exposure of plasma to
cold temperatures. The purpose of the present study is to evaluate the hypothesis that these increases of PRA
reflect inactivation of circulating renin inhibiting factors. In each of eight normotensive subjects, mean PRA
increased (p < 0.01) after acidification of plasma by acid dialysis (98%), after acidification of plasma by addition of HCI (47%), or after exposure of plasma to —4°C for 5 days (155%). Renin substrate-free plasma was
obtained by passing plasma over Sephadex G-50-40. Both untreated plasma and the substrate-free extract of
plasma inhibited the enzymatic activity of renin (p < 0.01); little or no inhibition occurred after addition of
acidified plasma or the protein-free extract of acidified plasma to renin-renin substrate. After addition of exogenous renin to cold-exposed plasma, the rate of angiotensin I generation was greater than that in control
plasma (p < 0.05), although renin substrate did not differ. These observations suggest that denaturation of a
circulating renin inhibitor may contribute to increased PRA after acidification or cold exposure of plasma. In
a separate experiment, the addition of polyunsaturated free fatty acids (linoleic and arachidonic) to renin-renin
substrate inhibited angiotensin production, although no inhibition occurred after dialysis of these fatty acids
against an acid buffer (pH 3.3). Prolonged exposure of linoleic acid to cold temperatures did not affect its
capacity to inhibit renin. Thus, loss of long chain, unSaturated free fatty acids may contribute to increased
PRA after acid dialysis of plasma but not to increased PRA after exposure of plasma to cold temperatures.
(Hypertension 1: 190-196, 1979)
KEY WORDS
acid activation
•
renin • renin inhibitor
• fatty acids
plasma renin activity
R
•
cyroactivation
plasma or to a preparation of renin substrate.10 The
measurement of plasma renin reactivity (PRR)
provides the opportunity to evaluate the effect of
plasma on the enzymatic activity of added renin.
The enzymatic activity of endogenous renin increases either after acidification of plasma1214 or after
prolonged incubation of plasma at -4°C. 16 ' lf> Acid
activation and cryoactivation have generally been attributed to the production of an enzymatically more
active renin from a prorenin. The present series of experiments was designed to evaluate the hypothesis that
increased PRA* after acidification or cold exposure is
related to denaturation of a normally occurring renin
inhibitor. In these experiments, PRR was compared
ENIN is a proteolytic enzyme that cleaves a
circulating alpha-2-globulin substrate to
form the decapeptide angiotensin I. Several
reports suggest that plasma contains inhibitors of the
reaction between renin and its substrate.19 Acetonesoluble neutral lipids extracted from plasma inhibit
renin,10 and we have recently demonstrated that
physiologic concentrations of long chain, unsaturated
fatty acids inhibit both the capacity of renin to.
generate angiotensin in vitro and the pressor response
to renin in vivo.11 Thus, in addition to renin and renin
substrate concentrations, the measurement of plasma
renin activity (PRA) may be affected by circulating
renin inhibitors. We have used the term renin reactivity to refer to the capacity of added renin to
generate angiotensin I either after its addition to crude
*By convention, PRA refers to the enzymatic activity of endogenous renin with endogenous renin substrate. The term plasma
renin concentration has been used to describe the rate of angiotensin
production after addition of excess renin substrate to plasma. In this
manuscript, we have retained the term PRA to refer to the enzymatic activity of endogenous renin after acidification of plasma
(which denatures endogenous renin substrate) and addition of exogenous substrate; the enzymatic activity of renin, not necessarily
renin concentration, is increased by acidification of plasma.
From the Department of Medicine, University of Kentucky
College of Medicine, Lexington, Kentucky.
Supported in part by Grant HL 15528 from the National
Institutes of Health.
Address for reprints: Theodore A. Kotchen, M.D., University of
Kentucky, College of Medicine, Department of Medicine, 800 Rose
Street, Lexington, Kentucky 40536.
190
ACID ACTIVATION AND CRYOACTIVATION OF PRA/Kotchen et al.
before and after exposure of plasma either to pH 3.3
or to - 4 ° C for 5 days.
Methods
Acid "Activation" of Plasma Renin
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
Peripheral venous plasma was obtained from each
of eight normotensive control subjects. One aliquot
was acidified by the Skinner plasma renin concentration (PRC) procedure.17 According to this procedure,
plasma is dialyzed against a glycine buffer to pH 3.3
over 18 hours at 4°C, incubated at 32°C for 45
minutes and then dialyzed back to pH 7.4 against a
phosphate buffer over 18 hours at 4°C. To control for
an effect of dialysis itself, a second plasma aliquot was
dialyzed against phosphate buffer at pH 7.4, incubated
at 32°C for 45 minutes and again dialyzed against the
same phosphate buffer. As another control for an
effect of dialysis, an additional aliquot of plasma was
acidified to pH 3.3 by dropwise addition of 1 N HC1,
without dialysis. Plasma was incubated at this pH for
18 hours at 4°C and then at 32°C for 45 minutes.
These samples were then adjusted to pH 7.4 by addition of 1 N NaOH and were again incubated for 18
hours at 4°C.
To determine if acidification denatures a renin inhibitor in plasma, untreated plasma and plasma
acidified both by dialysis and by addition of HC1 were
added to renin-renin substrate. A preparation of exogenous human substrate was diluted with 150 mM
phosphate buffer (pH 7.4) to a final concentration of
1000 ng/ml; 200 //I of untreated plasma and 1.1 X
10' Goldblatt units (GU) of renin were added to 1 ml
of this substrate solution, and the rate of angiotensin
production was measured after 30- and 60-minute incubations at 37°C. Control incubations containing
phosphate buffer instead of plasma were added to the
same concentrations of substrate and enzyme, and the
rate of angiotensin production was measured. Higher
concentrations of untreated plasma were not added to
renin-renin substrate because endogenous substrate
was present in plasma and higher total substrate concentrations (endogenous substrate plus added substrate) affect the rate of angiotensin production.
Acidification of plasma to pH 3.3 denatures endogenous renin substrate17 thus providing the opportunity to add higher concentrations of "substrate free"
plasma to renin-renin substrate. A constant concentration of exogenous human substrate was added to
acid dialyzed plasma (1000 ng/ml) and to HCl-treated
plasma, after the pH was readjusted to 7.4. Exogenous
substrate was also added to plasma dialyzed only at
pH 7.4. After addition of substrate, to measure the activity of endogenous plasma renin (endogenous
" P R A " ) , the concentrations of angiotensin I
generated were measured after 30- and 60-minute incubations at 37°C and pH 7.4. In addition, the
capacity of added renin to generate angiotensin I was
also measured (plasma renin reactivity); after addition
of substrate, a constant concentration of exogenous
renin was added (1.1 X 10^' GU/ml), and the concentrations of angiotensin I produced were measured
191
after 30- and 60-minute incubations at 37°C and pH
7.4. The activity of added enzyme was determined by
subtracting the relatively small concentrations of
angiotensin generated without added enzyme (endogenous "PRA") from the total concentrations of
angiotensin produced in the presence of exogenous
renin.
Sephadex Fractionation of Plasma
Conceivably, acidification of plasma may affect factors in plasma other than renin and renin substrate
that may also influence the velocity of the renin reaction. To extract substrate from plasma without
acidification, an additional aliquot of plasma was
passed over Sephadex G-50-40 (superfine) columns.
Pharmacia K15 (900 mm X 15 mm) columns were
used. Between 3.5 to 4.0 g of Sephadex was swollen
overnight and the columns were packed by gravity.
Sodium chloride (0.9%) was used to equilibrate and
elute the column. After the column bed settled to a
constant height, 5 ml of plasma sample was placed on
the column, taking care not to displace the top of the
gel bed. Fractions were collected from the time of
application of the sample. The protein separation was
monitored by reading the absorbancc of the eluate at
280 m^, and the protein peak was separated from the
rest of the collected eluate. More than 97% of plasma
protein (measured by the Lowry procedure) was
removed by this fractionation procedure. The pooled
eluate without protein was dialyzed (pore size 13,000)
against water, lyophilized and taken up in 0.15 M
phosphate buffer (pH 7.4) to the original sample
volume. This eluate contained no measurable renin or
renin substrate. The PRR was measured in these aliquots after addition of homologous substrate (1000
ng/ml).
"Cryoactivation" of Plasma Renin
To measure the reactivity of exogenous renin in
plasma exposed to cold temperatures, peripheral
venous blood was obtained from eight normotensive
subjects. A control aliquot was frozen at —20°C for 5
days. Another aliquot was incubated in a shaker bath
at - 4 ° C and pH 7.4 for 5 days. Both aliquots were
then incubated at 37°C and pH 7.4 for 60 minutes,
and the concentrations of angiotensin I generated
were measured (PRA). The reactivity of exogenous
renin was compared in both aliquots by measuring the
capacity of added renin (2.8 X 10 s GU/ml) to
generate angiotensin I during a 60-minute incubation
period. Again, the relatively low concentrations of
angiotensin produced without added enzyme were subtracted from the total to determine the reactivity of
exogenous renin. Renin substrate concentrations were
also measured in both aliquots of plasma.
Effect of Acidification or Cold Exposure on Renin Inhibition
by Fatty Acids
We have previously demonstrated that long chain,
unsaturated fatty acids inhibit the renin reaction.11 In
192
HYPERTENSION
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
a separate series of experiments, we evaluated the
capacity of linoleic (18:2) acid and arachidonic (20:4)
acid to inhibit renin after acidification. As we have
previously described, fatty acids were taken up in hexane (10 mg/ml), and butylated hydroxyanisol was
added to prevent oxidation (0.01 mg%). Each fatty
acid (0.5 mg) was aliquoted to each of 16 tubes and
was dried under vacuum. Phosphate buffer (0.75 ml)
with lipid free human albumin (30 mg/ml) was added
to each tube. Lipid free albumin alone does not affect
the in vitro renin reaction.1' Eight tubes with each fatty acid were then acidified according to the Skinner
PRC procedure, and to control for an effect of dialysis
itself the remaining eight tubes were dialyzed only
against phosphate buffer at pH 7.4. Dialyzed fatty
acids were added to renin substrate (1200 ng), and the
fatty acid-renin substrate mixture was sonicated at
room temperature for 90 seconds. Renin was then
added (1.1 X 10~" GU/ml), and the rate of angiotensin
production was compared in incubations containing
fatty acid that had been dialyzed only at pH 7.4, fatty
acid that had been exposed to pH 3.3, and control incubations containing only enzyme and substrate
without fatty acid. We have previously reported that
linoleic and arachidonic acids do not affect the
recoveries of added angiotensin I, indicating that these
fatty acids do not act as angiotensinases and do not interfere with the radioimmunoassay for angiotensin.11
To determine if cold exposure affects the capacity of
unsaturated fatty acids to inhibit renin, linoleic acid
(aliquoted with albumin as described above) was incubated at - 4 ° C for 5 days (n = 8). Control aliquots
of linoleic acid were incubated at — 20°C for 5 days
(n = 8). Both cold-exposed (-4°C) and control aliquots of linoleic acid were added to renin (2.8 X 10 s
GU/ml)-renin substrate (1000 ng/ml) as described
above, and the final fatty acid concentration was 1
mg/ml. The concentrations of angiotensin I generated
without fatty acid (n = 8), or with addition of coldexposed or control fatty acid, were measured after 30and 60-minute incubations.
Analytical Methods
For all experiments, angiotensinase-free renin was
prepared from human kidneys by the method of
Haas. 1 ' Based on comparing the concentrations of
angiotensin I generated in the presence of excess
human substrate (5000 ng/ml) with a human renin
standard obtained from the Medical Research Council
(London, England), our concentrated enzyme
preparation contained approximately 1.6 GU/ml.
Homologous renin substrate was prepared from
pooled plasma, obtained from normotensive women
taking oral contraceptives, by ammonium sulfate
precipitation between the molarity of 1.51 and 2.65 at
pH 7.4.20 The mean concentration of angiotensin I
produced after incubation of substrate alone without
enzyme (n = 10) was 1.2 ng/ml/hr ± 0.1 SE, and we
attribute this to a renin contaminant. This "contaminant" was constant for all incubations and
represents less than 2% of the concentrations of
VOL 1, No
3, MAY-JUNE
1979
angiotensin generated with added renin. Chromatographic grade free fatty acids were obtained (Sigma
Chemical, St. Louis, MO) and their purity was
verified by gas liquid chromatography (GLC). For
GLC, fatty acids were transmethylated with
borontrifiuride-3-methanol (14% w/v) and benzene,"
and GLC of the methylated fatty acids was carried out
using a Shimadzu GC-Mini 1 with an integrator.
Angiotensin I concentrations were measured in
quadruplicate by the radioimmunoassay procedure of
Haber et al.M Dimercaprol (10 fi\ of a 2% solution/ml)
and 8-hydroxyquinoline (10 ^1 of a 0.34 M
solution/ml) were added to all incubations to inhibit
converting enzyme activity. We have previously
demonstrated that the recovery of angiotensin I added
to plasma of normal subjects is essentially quantitative
using this assay system,10 indicating that the measurement of generated angiotensin I is not affected by
differences of converting enzyme activity or nonspecific angiotensinase activity. To measure renin substrate, high concentrations of renin were added to
plasma (0.2 GU/ml), and angiotensin I concentrations were measured after the reaction between
added enzyme and endogenous substrate had
proceeded to completion; angiotensin I concentrations
after 3 and 6 hours incubation did not differ and were
averaged.
For statistical analyses, either a paired or unpaired
Student's t test was used. In instances where multiple
comparisons were made, according to the Bonferroni
modification of the t test, to accept an overall 0.05
significance level, a 0.01 significance level was required for each individual t test.23 Consequently, for
multiple comparisons, the designation of statistically
significant differences will indicate a significance level
of 0.05 or less.
Results
Acid "Activation" of Plasma Renin
Mean renin substrate concentration in control
plasma dialyzed only at pH 7.4 was 966 ng/ml ± 74
SE. Substrate was almost totally denatured in plasma
that had been acidified by dialysis (55 ng/ml ± 318 SE)
and by addition of HC1 (33 ng/ml ± 3 SE), p > 0.02.
After addition of exogenous substrate, compared to
that in control plasma dialyzed only at pH 7.4, PRA
in each of eight subjects was higher after dialysis
against the buffer at pH 3.3 (fig. 1). The mean PRA of
9.1 ng/ml/hr ± 0.8 SE after acid dialysis was
significantly higher than the mean of 4.5 ng/ml/hr ±
0.3 SE in control plasma. Similarly, PRA increased
significantly after acidification of plasma with
dropwise addition of HC1 (6.6 ng/ml/hr ± 0.5 SE).
However, PRA after acidification with HC1 was
significantly lower than PRA measured after acidification of plasma by dialysis.
After addition of untreated plasma to renin-renin
substrate, significantly less angiotensin was generated
(p < 0.01) during both a 30- and a 60-minute incubation, than in control incubations containing only enzyme and substrate but no plasma (fig. 2). Recoveries
ACID ACTIVATION AND CRYOACTIVATION OF PRA/Kotchen et al.
193
AOD'N OF HCI
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
ACIDIF
CON
CON
ACIMF
FIGURE 1. Mean plasma renin activity (PRA) before and after acidification of plasma by either acid
dialysis or dropwise addition of HCI.
of standard angiotensin I in plasma (75 ng/ml) were
virtually quantitative, indicating that lower concentrations of angiotensin measured with plasma cannot
be attributed to angiotensinase activity, or to nonspecific interference with the radioimmunoassay.
Unlike crude plasma, compared to the concentration
of angiotensin generated in control incubations (72.5
ng/ml/hr ± 2.5 SE), no inhibition occurred after addition of plasma that had been acidified either by
dialysis or by HCI (fig. 3). However, after addition to
renin-renin substrate, significantly less (p < 0.01)
angiotensin was generated with plasma acidified by
HCI (65.8 ng/ml/hr ± 2.7 SE) than with plasma
acidified by dialysis (82.5 ng/ml/hr ± 2.3 SE).
Sephadex Fracrionation of Plasma
Addition of the substrate-free plasma fraction to
renin-renin substrate significantly inhibited angiotensin production (fig. 3). The capacity of this substratefree extract to inhibit renin after acid dialysis of whole
3U
80 -
70
Control- No plaimo
•h
60
50
40
°o 30
20
10
I
*
I
(p<0.0l)
60 -
Plaima
i
*
50
o
CD
40 30 20 10 -
8
CON
8
8
DIAL HCI
f-ACIDIF-|
8
SEPH
6U.
FIGURE 2. Effect of untreated plasma on the rate of
angiotensin I production after addition to renin-renin substrate.
FIGURE 3. Mean rates of angiotensin I production after
addition of acidified plasma and Sephadex fractionated
plasma to renin-renin substrate. Asterisk = p < 0.01 compared to control.
194
HYPERTENSION
VOL 1, No
3, MAY-JUNE
1979
TAHLK 1. Mean (± SK) Concentrations of Angiotensin I (ng/ml/CO nun) Generated After Addition of Acidified Fatty Acids to
Rcnin-lienin Substrate
Control
(no fatty acid)
74.S ± 2 .0
Arachidonic (20:4)
Dialysis to
Dialysis at
pH 3.3
pH 7.4
(glycine buffer)
51.7 ± 4.7*
Lmoleic. (18:2)
Dialysis to
pH 3.3
(glycine buffer)
Dialysis at
pi-r 7.4
49.2 ± 2..">*
67.3 ± 2.4
68.9 ± 5.1
Dialysis to
pH 3.3
(citrate buffer)
72.1 ± 3.7
*p < 0.05 (or less) compared to control incubations without fatty acid.
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
plasma was then evaluated. Two aliquots of plasma
from each of five subjects were passed over Sephadex:
intact plasma and plasma exposed to acid dialysis.
Again, during a 30-minute incubation, less angiotensin was generated with addition of renin-substrate-free
plasma than in control incubations containing only enzyme and substrate (fig. 4). However, no inhibition occurred after addition of substrate-free plasma that had
previously been acidified. During 60 minutes, compared to control incubations, significantly less
angiotensin was produced with addition of the proteinfree fraction of both intact plasma and acidified
plasma. However, significantly less inhibition occurred with the protein-free extract of acidified
plasma. Thus, both crude plasma and a substrate-free
extract of plasma inhibit the in vitro renin reaction.
After addition to renin-renin substrate, relatively little
or no inhibition occurred with acidified plasma or with
a protein-free extract of plasma that had previously
been exposed to pH 3.3. Taken together, these observations suggest that acidification of plasma denatures
a non-protein renin inhibiting factor.
"Cryoactivation" of Plasma Renin
Mean plasma renin substrate in control plasma
(1198 ng/ml ± 59 SE) and cold-exposed plasma (1105
ng/ml ± 66 SE) did not differ. The PRA increased in
plasma from each of the subjects after cold exposure,
and compared to that in control plasma (2.2 ng/ml/hr
± 0.4 SE) the mean was significantly elevated (p <
0.001) after cold exposure (5.6 ng/ml/hr ± 0.6 SE).
After adding exogenous renin and adjusting for endogenous PRA, the rate of angiotensin I production
was also significantly greater (p < 0.05) in coldexposed plasma (173 ng/ml/hr ± 16 SE) than in control plasma (134 ng/ml/hr ± 17 SE).
Effect of Acidification or Cold Exposure on Renin Inhibition
by Fatty Acids
Compared to control incubations containing no fatty acid, after'dialysis at pH 7.4 both arachidonic and
linoleic acids inhibited the concentrations of angiotensin I generated during a 60-minute incubation when
added to renin-renin substrate (table 1). When these
60
•
CONTROL - NO PLASMA
0
SUBSTRATE-FREE PLASMA
• p < 0 . 0 2 I COMPARED
* * p< 0.001 ( TO CONTROL
•}
50
40
H
§ 30
20
10
ACIDIF
- 3 0 MM
ACWF
- 6 0 MIN.
FIGURE 4. Mean rate of angiotensin I production after addition of Sephadex fractionated crude plasma
and Sephadex fractionated acidified plasma to renin-renin substrate.
ACID ACTIVATION AND CRYOACTIVATION OF PRA/Kotchen et al.
two fatty acids were initially dialyzed to pH 3.3
against a glycine buffer and then dialyzed back to pH
7.4, no significant inhibition occurred. However, the
concentrations of linoleic acid measured by GLC after
dialysis at pH 7.4 only (0.38 mg/ml ± 0.02 SE) or after
initial dialysis to pH 3.3 (0.41 mg/ml ± 0.03 SE) did
not differ. To determine if the loss of inhibitory activity after dialysis against the glycine buffer was
related to acidification or to glycine itself, linoleic acid
was also dialyzed to pH 3.3 against a citrate buffer
and then back to pH 7.4. Again, after acidification by
dialysis against the citrate buffer, linoleic acid did not
inhibit angiotensin production.
In a separate experiment incubation of linoleic acid
at - 4 ° C for 5 days did not impair its capacity to inhibit renin.
Discussion
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
Plasma renin activity increased both after acidification of plasma (either by dialysis against an acidic
buffer or by dropwise addition of HC1) and after incubation of plasma at —4°C for 5 days. As anticipated, acidification of plasma denatured endogenous renin substrate, and the measurement of
PRA required addition of exogenous substrate. After
addition to renin-renin substrate, both untreated
plasma and a renin-substrate-free extract of plasma
inhibited the rate of angiotensin production. However,
little or no inhibition occurred after addition of
acidified plasma or the substrate-free extract of
acidified plasma. These observations are consistent
with the hypothesis that increased PRA after
acidification of plasma is related to a loss of a nonprotein renin inhibiting factor. Compared to acidification by dialysis, PRA was lower and the enzymatic activity of exogenous renin was less in plasma acidified
with addition of HC1, suggesting that acid dialysis
more effectively impairs the capacity of plasma to inhibit renin. In separate experiments, the reactivity of
exogenous renin increased in "cryoactivated" plasma,
raising the possibility that loss of a renin inhibiting
factor may also contribute to increased PRA after exposure of plasma to cold temperatures.
Activation of renin by acid dialysis has been
reported to occur in kidney extracts, amniotic fluid,
and plasma.11"14- "~" Although the molecular weight
of renin is approximately 40,000 daltons, acid activation of renal renin has been attributed to conversion of
a larger molecular weight renin to the smaller more
active enzyme. 14 " However, Shulkes et al.14 reported
that the molecular weights of active and inactive renin
did not differ in plasma or amniotic fluid. Day et al."
found a high molecular weight renin (60,000) in amniotic fluid, in renal tumor extracts, in plasma from
diabetic patients with nephropathy, and to a lesser extent in plasma of a relatively small percentage of patients with hypertension and proteinuria. The enzymatic activity of this "big renin" increased after
acidification, although the molecular weight determined by sephadex gel filtration, did not change.88
We have recently demonstrated that long chain, unsaturated fatty acids inhibit renin.11 The current ex-
195
periments with linoleic and arachidonic acids suggest
that increased enzymatic activity of rcnin after
acidification of plasma by dialysis may at least in part
be related to loss of inhibition by fatty acids. In
plasma, free fatty acids are bound to albumin, and
consequently, in the present experiment, physiologic
concentrations of lipid free albumin were added to fatty acids before dialysis. Although the binding of fatty
acids to albumin is complex, when long chain, free fatty acids are incubated with albumin, the unbound concentrations of fatty acids increase as pH of the incubation medium is lowered." However, the recovery of
linoleic acid was not decreased after acid dialysis,
suggesting that acid dialysis may irreversibly affect
fatty acid-protein binding rather than result in denaturation of fatty acid per se.
Other investigators have suggested that denaturation of a renin inhibitor also accounts for acid activation of renal renin. The enzymatic activity of purified
hog and human renal renin does not increase after
acidification,50'S1 although acidification results in the
production of 40,000 molecular weight hog renin from
a partially purified 60,000 molecular weight enzyme.
Levine et al.31 reported that the enzymatic activity of
partially purified hog renin is inhibited by addition of
a crude kidney extract, and concluded that the purported activation of renin in crude unacidified kidney
extract is due to the presence of an inhibitor that is
destroyed by acidification. Leckie and McConnelP
reported that after acidification, a larger proenzyme
extracted from rabbit kidney dissociated into an active
enzyme plus a renin inhibitor of molecular weight
13,000 daltons. Similar to our results in plasma, this
inhibitor was inactivated after acidification either
by dialysis or addition of HC1.
In apparent contrast to the present results, Sealey
and Laragh16 reported that the rate of angiotensin
generation after addition of exogenous renin was
not increased after storage of plasma at — 20°C
for 12 months. However, cryoactivation at —4°C
for 5 days is considerably more effective than storage
at —20°C,16 and it is possible that small differences of
renin reactivity could not be detected in measurements
that had been obtained 1 year apart and in plasma that
had been stored at - 2 0 ° C . In the present experiment,
we did observe greater reactivity of exogenous renin in
cryoactivated plasma. Whether the relatively small increased reactivity of high concentrations of added
renin is sufficient to totally account for increased PRA
after cold exposure remains to be determined. Exposure of linoleic acid to cold temperatures did not
alter its capacity to inhibit renin. This finding suggests
that there may be additional inhibiting factors in
plasma that are modified by these incubation conditions.
Trypsin," 1 " " pepsin,14 and urinary kallikrein" activate plasma renin, and several neutral protease inhibitors have recently been reported to inhibit cryoactivation and partially inhibit acid activation." Plasma
itself also contains proteinase inhibitors that limit the
effect of proteolytic enzymes on renin." It has been
suggested that cryoactivation and acid activation may
HYPERTENSION
196
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
reduce the Content of a neutral serine protease inhibitor normally present in plasma and that the
liberated protease converts inactive prorenin to active
renin.3* However, trypsin activation of plasma renin
may not be entirely comparable to acid activation or
cryoactivation. After cryoactivation of plasma, Noth
et al.M reported that renin activity increased further
following subsequent incubation with trypsin. Trypsin
activation occurs with both plasma substrate and
tetradecapeptide substrate, whereas acid activation
occurs only with plasma substrate." Furthermore,
Slater et al." reported that trypsin activation also occurred with a 40,000 molecular weight renin separated
by gel filtration, whereas acid activation did not occur
with this relatively pure enzyme preparation. Thus,
two mechanisms may contribute to renin activation,
i.e., denaturation of a renin inhibitor and increased
protease activity.
In summary, compared to that in buffer, the reactivity of added renin was decreased in untreated
plasma and in a renin-substrate-free extract of
plasma. However, the enzymatic activity of renin was
not inhibited by acidified plasma. Renin reactivity was
greater in plasma incubated at -4°C for 5 days than
in plasma stored at -20°C. These observations are
consistent with the hypothesis that plasma contains a
renin inhibiting factor and that denaturation of circulating renin inhibitors contributes to acid activation
and possibly to cryoactivation of PRA.
References
1. Carretero O, Gross F: Evidence for humoral factors participating in the renin-substrate reaction. Circ Res 20,21 (suppl
II): 11-115, 1967
2. Craig M, Sullivan JM, Saravis CA, Hickler RB: Studies of the
renin-renin substrate reaction in man; kinetic evidence for inhibition by serum. Am J Mcd Sci 274: 45, 1977
3. Favrc L, Vallotton MB: Kinetics of the reaction of human renin
with natural substrates and tetradecapeptide substrate. Biochim
Biophys Acta 327: 471, 1973
4. Kotchen TA, Talwalkar RT, Welch WJ: Inhibition of the in
vitro renin reaction by circulating neutral lipids. Circ Res 41
(suppl II): 11-46, 1977
5. Lazar J, Hoobler SW: Studies on the role of the adrenal in
rcnin kinetics. Proc Soc Exp Biol Med 138: 614, 1971
6. Lucas CP, Waldnausl WK, Cohen EL, Berlinger FG,
McDonald WG, Sider RS: A plasma inhibitor of the renihantirenin reaction and the in vitro generation of angiotensin I.
Metabolism 24: 127, 1975
7. Regoli D, Brunner H, Gross F: Undersuchungen uber den
mechanisms der wirkungsverstaerking von renin nach
nephrektomic. Helv Physiol Acta 19: C101, 1961
8. Schaechtelin G, Baechtold N, Haefeli L, Regoli D, GaudryParedes A, Peters G: A renin-inactivating system in rat plasma.
Am J Physiol 215: 632, 1968
9. Smeby RR, Sen S, Bumpus FM: A naturally occurring renin
inhibitor. Circ Res 20, 21 (suppl II): 11-129, 1967
10. Kotchen TA, Talwalkar RT, Kotchen JM, Miller MC:
Evidence for the existence of an acetone soluble renin inhibiting
factor in normal human plasma. Circ Res 36,37 (suppl I): 1-17,
1975
11. Kotchen TA, Welch WJ, Talwalkar RT: In vitro and in vivo inhibition of renin by fatty acids. Am J Physiol 234: E593, 1978
12. Day RP, Luetscher JA, Gonzales CM: Occurrences of big renin
in human plasma, amniotic fluid, and kidney extracts. J Clin
Endocrinol Metab 40: 1078, 1975
13. Weinberger M, Aoi W, Grim C: Dynamic responses of active
and inactive renin in normal and hypertensive humans. Circ Res
VOL 1, No
3, MAY-JUNE
1979
41 (suppl II): 11-21, 1977
14. Shulkes AA, Gibson RR, Skinner SL: The nature of inactive
renin in human plasma and amniotic fluid. Clin Sci Mol Med
55: 41, 1978
15. Sealey JE, Laragh JH: "Prorenin" in human plasma?
Methodologic and physiological implications. Circ Res 36
(suppl I): I-10, 1975
16. Sealey JE, Moon C, Laragh JH, Alderman M: Plasma
prorenin: cryoactivation and relationship to renin substrate in
normal subjects. Am J Mcd 61: 731, 1976
17. Skinner SL: Improved assay methods for renin "concentration" and "activity" in human plasma. Circ Res 20: 391,
1967
18. Kotchen TA, Talwalker RT, Miller MC, Welch WJ: Modification of renin reactivity by lipids extracted from normal,
hypertensive, and uremic plasma. J Clin Endocrinol Metab 43:
971, 1976
19. Haas E, Goldblatt H, Gipson EC, Lewis L: Extraction,
purification, and assay of human renin free of angiotensinase.
Circ Res 19: 739, 1966
20. Rosenthal JH, Wolff HP, Webber P, Dahlheim H: Enzyme
kinetic studies on human renin and its purified homologous substrate. Am J Physiol 22: 1292, 1971
21. Morrison WR, Smith LM: Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron fluoride
methanol. J Lipid Res 5: 600, 1964
22. Haber E, Koerner T, Page LB, Kliman B, Purnode A: Application of a radioimmunoassay for angiotensin I to the physiologic
measurements of plasma renin activity in normal human subjects. J Clin Endocrinol Metab 29: 1349, 1969
23. Miller RG: Simultaneous Statistical Inference. New York,
McGraw-Hill, 1966, p 67
24. Skinner SL, Cran EJ, Gibson R, Taylor R, Walters WAW,
Catt KJ: Angiotensins I and II, active and inactive renin, renin
substrate, rcnin activity, and angiotensinase in human liquor
amnii and plasma. Am J Obstet Gynecol 121: 626, 1975
25. Boyd GW: A pfotein-bound form of porcine renal renin. Circ
Res 35: 426, 1974
26. Morris BJ, Johnston CI: Isolation of renin granules from rat
kidney cortex and evidence for an inactive form of renin
(prorenin) in granules and plasma. Endocrinology 98: 1466,
1976
27. Leckie BJ, McConnell A: A renin inhibitor from rabbit kidney:
conversion of a large inactive renin to a smaller active enzyme.
Circ Res 36: 513, 1975
28. Day RP, Luctschcr JA: Biochemical properties of big renin extracted from human plasma. J Clin Endocrinol Metab 40: 1085,
1975
29. Spector AA, John K, Fletcher JF: Binding of long-chain fatty
acids to bovine serum albumin. J Lipid Res 10: 56, 1969
30. Slater EE, Haber E: A large form of renin from normal human
kidney. Circulation 54 (suppl II): 11-143, 1976
31. Levine M, Lentz KE, Kahn JR, Dorer FE, Skeggs LT: Studies
on high molecular weight renin from hog kidney. Circ Res 42:
368, 1978
32. Cooper RM, Gordon EM, Osmond DH: Trypsin induced activation of renin precursor in plasma of normal and anephric
man. Circ Res 40 (suppl I): 1-171, 1977
33. Slater EE, Cohn RC, Haber E: Differences between trypsinand acid-activated plasma renin activity in normal human
plasma. Clin Res 26: 368A, 1978
34. Noth RH, Cariski AT, Havelick J: Trypsin activation of
plasma renin activity. Clin Res 26: 367A, 1978
35. Sealey JE, Atlas SA, Laragh JH, Oza NB, Ryan JW: Human
urinary kallikrein converts inactive to active renin and is a
possible physiological activator of renin. Nature 275: 144, 1978
36. Sealey JE, Atlas SA, Laragh JH: Linking the kallikrein and
renin systems via activation of inactive renin: new data and a
hypothesis. Am J Med 65: 994, 1978
37. Tewksbury DA, Premeau MR: The effect of proteolytic activity
on plasma renin activity assay. Clin Chim Acta 73: 67, 1976
38. Atlas SA, Sealey JE, Laragh JH: "Acid"- and "cryo"activated inactive plasma renin: similarity of their changes during /5-blockade. Evidence that neutral protcase(s) participate in
both activation procedures. Circ Res 43 (suppl I): 1-128, 1978
Modification of the enzymatic activity of renin by acidification of plasma and by
exposure of plasma to cold temperatures.
T A Kotchen, W J Welch and R T Talwalkar
Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017
Hypertension. 1979;1:190-201
doi: 10.1161/01.HYP.1.3.190
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1979 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on
the World Wide Web at:
http://hyper.ahajournals.org/content/1/3/190.citation
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally
published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center,
not the Editorial Office. Once the online version of the published article for which permission is being
requested is located, click Request Permissions in the middle column of the Web page under Services.
Further information about this process is available in the Permissions and Rights Question and Answer
document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/