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Departments of 1 Pharmacology, 2 Internal Medicine, and 3 Biochemistry, Cardiovascular Research Institute COEUR, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (Kd) and maximum number of binding sites (Bmax) for prorenin binding to man-6-P/IGF-II receptors were 0.6 ± 0.1 nM and 3,840 ± 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times Bmax. Levels of internalized intact prorenin decreased rapidly (half-life = 5 ± 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.
local renin-angiotensin system; cardiomyocytes; fibroblasts; heart; kidney
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE BENEFICIAL CARDIAC
EFFECTS of angiotensin-converting enzyme (ACE) inhibitors in
subjects with heart failure are usually attributed to interference of
these drugs with ANG II generation at cardiac tissue sites. Although
initially it was thought that this generation depends on the de novo
synthesis of renin in the heart, it is now well established that such
synthesis does not occur either under normal circumstances (13,
24, 34, 35, 40, 56) or under pathological conditions (14,
31, 50). Therefore, the heart must sequester renin from the
circulation to synthesize ANG II locally. Renin may diffuse into the
interstitial space (18, 28) or bind to renin receptors
and/or renin-binding proteins (8, 46, 53). Because renin
in blood is predominantly present in the form of its inactive precursor
prorenin (11), it is also conceivable that the heart
sequesters prorenin instead of renin. This prorenin must then be
activated locally. In support of this concept we recently demonstrated
that endothelial cells and cardiac myocytes and fibroblasts, which do
not synthesize (pro)renin (1, 59), are capable of binding
recombinant human prorenin to cell-surface mannose-6-phosphate
(man-6-P)/insulin-like growth factor-II (man-6-P/IGF-II) receptors
(1, 58). Binding precedes rapid internalization and
appearance of renin-specific enzymatic activity. Furthermore, the
receptors also bound and internalized recombinant human renin. It is
well established that recombinant human prorenin produced in Chinese
hamster ovary (CHO) cells contains the man-6-P signal that is required
to bind to man-6-P/IGF-II receptors (2, 25). This is not a
unique property because many other prohormones [e.g., latent
transforming growth factor-
(19), procathepsin D
(29), and proliferin (39)] also carry the
man-6-P recognition marker, and these prohormones (like prorenin) are
activated after binding and internalization via man-6-P/IGF-II receptors.
At present it is unlikely that the man-6-P/IGF-II receptor is the only renin- and prorenin-binding receptor, because excess man-6-P did not block prorenin binding to microsomal membrane fractions prepared from various rat tissues (53). In addition, it is not known whether intracellular prorenin activation occurs proteolytically or nonproteolytically. Proteolytic activation involves the actual removal of the prosegment by any of the known prorenin-renin convertases [e.g., cathepsin B (45), kallikrein (37), and prohormone convertases (51)]. Nonproteolytic activation implies transient unfolding of the prosegment so that it no longer folds over the enzymatic cleft, thereby allowing prorenin to cleave angiotensinogen. In vitro, acid pH or cold storage favor the latter type of activation (21). Moreover, a recent study in mice has shown that nonproteolytically activated prorenin is capable of generating ANG I at tissue sites in vivo (43).
The aim of the present study was to investigate in neonatal rat cardiomyocytes and fibroblasts 1) the kinetics of man-6-P/IGF-II receptor-dependent prorenin binding [dissociation constant (Kd), maximum number of binding sites (Bmax)] and activation; 2) the possibility of prorenin binding independent of the man-6-P/IGF-II receptor; 3) the nature of the prorenin activation (proteolytic or nonproteolytic) and, in case of proteolytic activation, the nature of the prorenin-activating enzyme; and 4) possible differences between binding of recombinant human prorenin and binding of native human prorenin from renal and extrarenal sources.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell culture. All experiments were performed according to the regulations of the Animal Care Committee of Erasmus University Rotterdam, Rotterdam, The Netherlands, and in accordance with the "Guiding Principles in the Care and Use of Laboratory Animals" as approved by the Council of the American Physiological Society.
Primary cultures of rat neonatal cardiac cells were prepared as previously described (57). Briefly, ventricles of Wistar strain rat pups (age 1-3 days) were minced and cells were dispersed by eight subsequent trypsinization steps. Nonmyocytes were separated from myocytes by differential preplating. Myocytes were seeded in noncoated 12-well plates (Corning Costar Europe; Badhoevedorp, The Netherlands) yielding a confluent monolayer of spontaneously beating cells at 1.5 × 105 cells/cm2 after 24 h. The preplated cells (fibroblast fraction) were passaged after 4 days to noncoated 12-well plates yielding a confluent monolayer of 0.75 × 105 cells/cm2 after 2 days. The cells were maintained for 72 h in a humidified incubator at 37°C with 5% CO2 in air and 1.5 ml of growth medium consisting of a 4:1 (vol/vol) ratio of DMEM (GIBCO Life Technologies; Breda, The Netherlands) and medium 199 (GIBCO) supplemented with 5% FCS (Roche Diagnostics; Almere, The Netherlands), 5% horse serum (Sigma-Aldrich; Zwijndrecht, The Netherlands), 100 U/ml of penicillin (Roche), and 100 mg/ml of streptomycin (Roche). The incubations with prorenin (see Incubation of cells with prorenin at 4 or 37°C) were carried out under serum-free conditions. Before the start of each experiment, cells were washed with 1 ml of warm (37°C) PBS consisting of (in mM) 140 NaCl, 2.6 KCl, 1.4 KH2PO4, and 8.1 Na2HPO4 (pH 7.4). The cells were then preincubated either at 4 or 37°C for 30 min with 0.4 ml of incubation medium consisting of a 4:1 (vol/vol) ratio of DMEM and medium 199 supplemented with 1% (wt/vol) BSA (Sigma-Aldrich).Prorenin preparation. Recombinant human prorenin was a kind gift of Dr. S. Mathews (Hoffmann-LaRoche; Basel, Switzerland). It was secreted by CHO cells transfected with a vector containing human prorenin cDNA. To remove traces of renin, the prorenin was partially purified by Cibacron blue Sepharose affinity chromatography (Amersham Pharmacia Biotech; Roosendaal, The Netherlands). The intrinsic renin activity of the prorenin preparation before proteolytic activation was <2% of the activity after complete proteolytic activation when the prorenin preparation contained ~2 × 105 U/l (4 µM) renin.
Man-6-P receptor affinity chromatography.
To separate prorenin into fractions that do or do not contain the
man-6-P signal, recombinant human prorenin was applied to a 0.5-ml
bovine liver man-6-P/IGF-II receptor column (kindly provided by Dr. S. Kornfeld, St. Louis, MO). The column was equilibrated with column
buffer containing 50 mM imidazole at pH 6.5, 150 mM NaCl, 5 mM
Na--glycerophosphate, 0.1% (wt/vol) BSA, 6 µM antipain, 8 µM
leupeptin, 6 µM pepstatin A, 7 µM chymostatin (all from
Sigma-Aldrich), and 10 kallikrein inhibitory U/ml of aprotinin (Bayer;
Mijdrecht, The Netherlands) (26). All manipulations were
performed at 4°C. After the application of recombinant human prorenin
(500 units in 0.1 ml of column buffer), the column was washed with
column buffer ("column runthrough"). Subsequently, 10 mM man-6-P
was added to the column buffer and man-6-P-containing prorenin was eluted. Prorenin was measured in 0.5-ml fractions. Fractions
corresponding to column run-through (i.e., man-6-P-free prorenin) and
man-6-P-containing prorenin were separately pooled, concentrated, and
adjusted to contain PBS (pH 7.4) by Centricon C-30
ultrafiltration (Amicon Bioseparations; Bedford, MA).
Incubation of cells with prorenin at 4 or 37°C. After preincubation at 4 or 37°C for 30 min under serum-free conditions (see Cell culture), experiments were started by replacing the incubation medium by 4 or 37°C incubation medium containing either recombinant human prorenin (final concentration 3-1,000 U/l), man-6-P-free recombinant human prorenin (10 U/l), or man-6-P-containing recombinant human prorenin (10 U/l). Incubations at 37°C were also performed with pools of human plasma and human amniotic fluid diluted to a 1:3 ratio with incubation medium. Plasma was obtained from subjects with renal artery stenosis (one man and two women, age 41-66 yr). Amniotic fluid was obtained from three women (age 19-38 yr) after natural delivery. All incubations at 4 or 37°C lasted 4 h and were performed in both the presence and absence of 10 mM man-6-P to determine man-6-P/IGF-II receptor-specific prorenin binding (58). To investigate the intracellular presence of man-6-P/IGF-II receptors in myocytes, incubations with recombinant human prorenin (1,000 U/l) at 4°C were also performed after prior permeabilization of the cells with PBS containing 0.2% saponin (Merck; Amsterdam, The Netherlands) (52). Finally, to determine what proteases are responsible for prorenin activation, incubations at 37°C were performed in the presence of the following five protease inhibitors: 0.04 mM 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF, Calbiochem; LaJolla, CA); 0.1 mM leupeptin; 0.14 mM L-trans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64, Sigma-Aldrich); 1.0 mM 1,10-phenanthroline (Merck); or 0.1 mM pepstatin A.
At the end of the incubation period the culture medium was removed. Each well was washed three times with 1 ml of ice-cold PBS. Prorenin was not detectable in the last PBS wash. Cells were then lysed in 0.2 ml of ice-cold PBS containing 0.2% Triton X-100 (Merck), and the cell lysates were quickly frozen on dry ice. Cell lysates were stored at
70°C until assays for total and cell-activated prorenin were performed.
To determine whether prorenin had been internalized, the acid-wash
method was used (58). At low pH surface-bound prorenin dissociates from the cells; internalized prorenin, however, is acid
resistant. Briefly, after the cells had been washed three times with
ice-cold PBS, cells were incubated at 4°C with 0.4 ml of an acid
solution containing 50 mM glycine and 150 mM NaCl at pH 3.0. After 10 min the acid solution was removed, the cells were washed and lysed as
described above, and the cell lysates were stored at 70°C until
being assayed.
Incubation of cells at 4°C followed by incubation at 37°C.
To study the kinetics of prorenin activation in more detail, cells
cultured in six-well plates (Corning Costar) were loaded with 1 ml of
recombinant human prorenin-containing incubation medium (final
concentration 100 U/l) for 2 h at 4°C. After this period, free
prorenin was removed by washing the cells three times with 3 ml of
ice-cold PBS. After the last wash, 1 ml of fresh incubation medium
without prorenin at 37°C was added, and the cells were incubated at
37°C. The incubation was terminated after 15, 30, 60, 120, 180, or
240 min by removing the culture medium and subsequently washing the
cells three times with 3 ml of ice-cold PBS. The cells were then lysed
in 0.5 ml of ice-cold PBS containing 0.2% Triton X-100 as described
above. Culture medium and cell lysate were stored at 70°C until
assays for total prorenin, cell-activated prorenin, and intact
propeptide-containing prorenin were performed.
Prorenin measurement. In the experiments with recombinant human prorenin in myocytes, cell-activated prorenin and total prorenin (i.e., cell-activated plus nonactivated prorenin) were measured by immunoradiometric assay (IRMA). The proteolytic activation of recombinant human prorenin by myocytes was monitored with an IRMA specific for intact prorenin, i.e., prorenin in which the propeptide was still bound to the renin part of the molecule. The IRMAs are not sensitive enough to measure the low levels of cell-activated and total recombinant human prorenin in fibroblasts. The prorenin measurements in these cells were therefore performed by enzyme-kinetic assay. The renin and prorenin measurements in the experiments with human plasma and human amniotic fluid were also performed by enzyme-kinetic assay.
Enzyme-kinetic assay. Cell-activated recombinant prorenin and native renin were measured by incubating a 100-µl sample for 3 h with a saturating amount of sheep renin substrate at 37°C and pH 7.4 in the presence of serine protease and angiotensinase inhibitors (58). The generated ANG I was quantified by RIA. Results were expressed as microunits per 1,000,000 cells or microunits per milliliter medium using plasmin-activated recombinant human prorenin as a reference. The lower limit of detection was 1 µU/106 cells or 1 µU/ml of medium. To measure total recombinant prorenin and native prorenin, the samples were first incubated for 48 h at 4°C with plasmin (0.5 caseinolytic U/ml, obtained from Chromogenix; Mölndal, Sweden). This preincubation with plasmin causes complete proteolytic activation of prorenin. The serine protease inhibitor aprotinin (final concentration 100 kallikrein-inhibiting U/ml) was added to the incubation medium of the ANG I-generating step to inactivate plasmin.
Immunoradiometric assays.
The monoclonal antibodies (MAb) used in these assays were MAb
R3-36-16 (Nichols Institute; Wychen, The Netherlands), which reacts equally well with activated and nonactivated prorenin
(Kd = 0.6 pM) (30), MAb
R1-20-5 (Nichols Institute), which recognizes the active site
of renin (Kd = 250 nM) (60),
and MAb F258-37-B1 (a kind gift of Dr. S. Mathews,
Hoffmann-LaRoche), which is directed against the COOH-terminal part
(P20-P43) of the propeptide (Fig. 1) and
does not react (<0.1%) with renin (F. H. M. Derkx,
unpublished observation). MAbs R1-20-5 and F258-37-B1 do
not react (<0.1%) with intact inactive prorenin. However, they do
react with prorenin after the treatment of prorenin with the renin
inhibitor remikiren (0.1 mM; a kind gift of Dr. W. Fischli,
Hoffmann-LaRoche; Basel, Switzerland) for 48 h at 4°C
(16). The renin inhibitor enters the enzymatic cleft in
which the active site is located, thereby inducing a slow
conformational change of the inactive ("closed") form of the
prorenin molecule into the active ("open") form. This nonproteolytic conformational change not only allows subsequent recognition of the active site by MAb R1-20-5, it also causes the propeptide to move to the surface of the molecule so that it can
react with MAb F258-37-B1. In the IRMA for cell-activated and
total prorenin, 200 µl of untreated and remikiren-treated sample
(diluted to a 1:9 ratio in heat-inactivated sheep serum, obtained from
Biotrading; Mijdrecht, The Netherlands), respectively, were incubated
for 6 h at 37°C with biotinylated MAb R3-36-16, 125I-labeled R1-20-5 (250,000 counts/min), and an
avidin-coated bead as described (15). In the IRMA for
intact propeptide-containing prorenin, 250 µl of remikiren-treated
sample (diluted to a 1:4 ratio in heat-inactivated sheep serum) were
incubated for 6 h at 37°C with an avidin-coated bead to which
1.6 µg of biotinylated MAb F258-37-B1 had been bound
(1). The bead was then washed three times with 2 ml of PBS
containing 0.1% (wt/vol) BSA and subsequently incubated with 100 µl
(250,000 counts/min) of 125I-labeled MAb R1-20-5
and 200 µl of heat-inactivated sheep serum containing 0.1 mM
remikiren for 24 h at room temperature. After the 6-h and 24-h
incubation periods, the beads in both IRMAs were washed three times
with 2 ml of PBS containing 0.01% (vol/vol) Triton X-100, and bound
radioactivity was measured in a gamma-counter. The results of these
assays were expressed as microunits per 1,000,000 cells using intact
recombinant human prorenin as a reference. The lower limit of detection
was 5 µU/106 cells.
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Statistical analysis. Results are expressed as means ± SE. Data were compared using Student's t-test for paired observations or ANOVA. A value of P < 0.05 was considered to be significant. Binding data were analyzed by nonlinear regression analysis using the GraphPad Prism (version 3) computer program (GraphPad; San Diego, CA).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Incubation of myocytes and fibroblasts with recombinant human
prorenin at 4 or 37°C.
Myocytes (Fig. 2) and fibroblasts (Fig.
3) bound recombinant human prorenin in a
concentration-dependent manner at both 4 and 37°C.
Cell-associated prorenin at 37°C but not at 4°C was acid resistant
(data not shown), indicating that prorenin internalization occurred at
37°C only. For a given prorenin concentration in the medium, the
level of cell-associated total prorenin after 4 h of incubation
was 10-15 times higher at 37°C than at 4°C.
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Binding of recombinant human prorenin to man-6-P/IGF-II receptors: Kd and Bmax. Man-6-P significantly reduced recombinant human prorenin binding in both myocytes (Fig. 2A) and fibroblasts (Fig. 3A). The reduction was much smaller in fibroblasts than in myocytes, suggesting that fibroblasts may contain a second prorenin binding site that cannot be blocked by man-6-P. However, because the levels of cell-associated prorenin after 4 h of incubation in the presence of man-6-P were similar in myocytes and fibroblasts, a more likely explanation is that non-man-6-P/IGF-II receptor-mediated prorenin binding represents nonspecific binding and that the man-6-P-induced reduction in prorenin binding is smaller in fibroblasts because these cells contain less cell-surface man-6-P/IGF-II receptors. Indeed, Scatchard analysis revealed that binding of prorenin to man-6-P/IGF-II receptors occurred with similar affinity in both myocytes (Kd = 0.6 ± 0.1 nM; n = 8) and fibroblasts (Kd = 0.8 ± 0.2 nM; n = 4) and that Bmax was 3,840 ± 510 sites/myocyte and 650 ± 150 sites/fibroblast. Prior permeabilization of myocytes with saponin increased man-6-P/IGF-II receptor-dependent prorenin binding 7.7 ± 0.4-fold (n = 4), indicating that >85% of the man-6-P/IGF-II receptors is located intracellularly. Recycling of these intracellular receptors to the cell membrane most likely explains why the levels of cell-associated prorenin at 37°C are much higher than at 4°C.
Does prorenin binding occur independently of man-6-P/IGF-II
receptors?
To investigate whether prorenin binding occurs independently of
man-6-P/IGF-II receptors, recombinant human prorenin was separated into
man-6-P-free and man-6-P-containing fractions with the help of a bovine
man-6-P/IGF-II receptor-affinity column (Fig.
4). The amount of recombinant human
prorenin (38 ± 1%; n = 3) that was not bound by
this column (which did not contain the man-6-P signal) resembled the
amount of prorenin (38 ± 2%) that eluted only after the addition
of man-6-P to the elution buffer (which did contain the man-6-P
signal). The remaining prorenin eluted shortly after the first
runthrough peak (before the addition of man-6-P) and was not
investigated further.
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1.5-2.5 times
higher than observed after incubation with nonfractionated prorenin
(Fig. 5). Assuming that only
man-6-P/IGF-II receptors are involved in prorenin binding, this is
exactly what one would predict when exposing cardiac cells to a
prorenin solution in which either all or only 40% of the prorenin
molecules carry the man-6-P signal.
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Activation of recombinant human prorenin: effect of protease
inhibitors.
Activation of recombinant human prorenin was detectable at 37°C only
(Figs. 2B and 3B). Saturation of the activation
process did not occur because the percentage of cell-associated
prorenin that was activated was similar at all concentrations of
prorenin to which the cells were exposed [ranging from 88 ± 15%
at 3 U/l to 78 ± 8% at 1,000 U/l in myocytes (n = 7) and from 83 ± 9% at 3 U/l to 75 ± 9% at 1,000 U/l in
fibroblasts (n = 4)]. The serine protease inhibitor
AEBSF partially blocked the activation of prorenin in myocytes but had
no effect in fibroblasts (Table 1). None of the other protease inhibitors that were tested blocked prorenin activation in either myocytes or fibroblasts. The cysteine protease inhibitor E64 and the mixed serine-cysteine protease inhibitor leupeptin increased the level of cell-associated total prorenin by
40-50% in myocytes and by 40-80% in fibroblasts, thereby
indicating that cysteine proteases contribute to (pro)renin degradation
in cardiac cells.
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Proteolytic or nonproteolytic activation of prorenin in myocytes.
After 2 h of incubation at 4°C with 100 U/l prorenin and
repeated washing with ice-cold PBS, the level of cell-associated total
prorenin was 350 ± 45 µU/106 cells
(n = 7). Acid wash confirmed that at that time, all
cell-associated prorenin was located on the cell surface. Immediately
after the temperature was raised to 37°C, the level of
cell-associated intact (i.e., prosegment-containing) prorenin started
to decrease. The decrease followed a biphasic pattern (Fig.
6). The first phase [half-life
(t1/2) = 5 ± 3 min]
corresponds with the release of cell-surface-bound intact prorenin into
the medium and did not differ from the first phase that was observed
for total prorenin (t1/2 = 4 ± 1 min). This
phase is determined by the rapidity of the internalization process. The
second phase represents the proteolytic removal of the prosegment from
internalized prorenin (t1/2 = 21 ± 4 min),
because a rise in the cellular levels of activated prorenin was
simultaneously observed. These levels reached a maximum after 60 min
and then started to decrease, with a t1/2 (67 ± 8 min) similar to that of the second phase of total prorenin
(t1/2 = 74 ± 5 min). Release of
activated prorenin into the medium could not be demonstrated during the 6-h observation period (data not shown). Taken together, these findings
suggest that prorenin, after its internalization, is rapidly activated
by proteolytic cleavage of the prosegment and that activated prorenin
is subsequently metabolized by degrading enzymes without being released
into the medium.
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Incubation of myocytes with native human (pro)renin at
37°C.
To verify man-6-P/IGF-II receptor-dependent binding,
internalization, and activation of native human prorenin of renal and nonrenal origin, myocytes were incubated at 37°C during 4 h with human plasma or human amniotic fluid (diluted to a 1:3 ratio with incubation medium) in the presence or absence of man-6-P. Expressed as
a percentage of the sum of renin and prorenin, plasma and amniotic fluid contained 79 ± 10% and 94 ± 3% prorenin,
respectively (Fig. 7). Incubation with
plasma resulted in man-6-P/IGF-II receptor-mediated (pro)renin uptake
by myocytes. After incubation with plasma, myocytes contained
predominantly (>75%) renin (Fig. 7). Because man-6-P/IGF-II receptors
bind and internalize man-6-P-containing renin and prorenin equally well
(58), this is not due to selective uptake of plasma renin.
The increased renin-to-prorenin ratio in cell lysates therefore suggests that internalized plasma prorenin, like internalized recombinant human prorenin, is activated to renin by myocytes. The
cellular (pro)renin levels after incubation with amniotic fluid were
close to the detection limit and did not differ with or without man-6-P
(Fig. 7). Thus amniotic fluid does not contain prorenin that carries
the man-6-P signal.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The data of the present study show that man-6-P-containing prorenin binds with high affinity to man-6-P/IGF-II receptors on neonatal rat myocytes and fibroblasts. Binding is followed by internalization and subsequent proteolytic activation to renin, possibly by a serine protease. Internalization is greatly enhanced by receptor recycling. Obtained with recombinant human prorenin, these results could be fully reproduced with native human prorenin of renal origin (i.e., prorenin in human plasma) but not with native human prorenin of extrarenal origin (i.e., prorenin in human amniotic fluid), suggesting that local angiotensin production by nonrenin-producing cells such as myocytes and fibroblasts (59) depends on renin of renal origin.
Evidence for prorenin binding to receptors other than the
man-6-P/IGF-II receptor was not obtained. First, man-6-P significantly reduced prorenin binding in myocytes and fibroblasts. Although the
reduction was more modest in fibroblasts, prorenin binding in the
presence of man-6-P (i.e., "nonspecific" prorenin binding) was
similar in myocytes and fibroblasts and did not differ from prorenin
binding to human umbilical vein endothelial cells in the presence of
man-6-P (data not shown). These findings may point to the existence of
a second unidentified prorenin receptor. However, in view of the
similarity of the non-man-6-P/IGF-II receptor-mediated prorenin binding
in cardiac and endothelial cells, a more likely explanation is that
binding in the presence of man-6-P represents nonspecific binding.
Second, when exposing cardiac cells to a recombinant human prorenin
fraction in which all molecules contain the man-6-P signal (as opposed
to only 40% of the molecules in the nonfractionated recombinant human
prorenin preparation), binding and internalization increased 1.5- to
2.5-fold. This is within the range expected when increasing the level
of man-6-P-containing prorenin 2.5-fold and also indicates that the
true Kd is 2.5-fold lower than the values
reported here. The increases were somewhat smaller in fibroblasts,
which is most likely due to the fact that man-6-P/IGF-II
receptor-specific prorenin binding in these cells was comparable to
prorenin binding not mediated via man-6-P/IGF-II receptors (i.e.,
nonspecific prorenin binding). A low number of cell-surface
man-6-P/IGF-II receptors also explains why the decrease in prorenin
binding in the presence of excess man-6-P was more modest in
fibroblasts than in myocytes.
Binding of prorenin to man-6-P/IGF-II receptors occurred with high affinity (Kd < 1 nM), which suggests that prorenin occupies both man-6-P binding sites of this receptor (22, 38). In both fibroblasts and myocytes, the levels of cell-associated prorenin increased 10-15-fold when the cells were incubated with prorenin at 37°C instead of 4°C. This can be explained on the basis of the internalization and continuous recycling of man-6-P/IGF-II receptors among the cell surface and intracellular compartments (e.g., Golgi and endosomes) that are known to occur at 37°C but not at 4°C (10). In myocytes, >85% of the man-6-P/IGF-II receptors were found to be located in the cells. Internalization of the receptor-prorenin complex appeared to occur rapidly with a half-life of <5 min and was followed by activation of prorenin. The rapid decline in the levels of prorenin still containing the carboxy terminal part of its prosegment [as measured with monoclonal antibody F258-37-B1 (Fig. 6)] confirms that prorenin activation was a proteolytic process and that cleavage occurred in the correct manner. The cells did not release proteolytically activated prorenin (i.e., renin) into the medium. Instead, renin was degraded intracellularly with a half-life of ~1 h. This raises the possibility that the man-6-P/IGF-II receptor is involved in (pro)renin clearance. Alternatively and perhaps more likely in view of the rapid activation process, cell-activated prorenin may contribute to intracellular angiotensin generation before its destruction. Several studies have provided evidence for intracellular angiotensin generation (17, 32, 42), and ANG II is known to activate intracellular AT1 receptors in the cytosol and nucleus (23, 27). In the absence of local renin synthesis, such intracellular angiotensin generation will depend on the uptake of circulating renin or prorenin. Moreover, because in previous studies we were unable to demonstrate angiotensinogen synthesis in neonatal rat cardiac myocytes and fibroblasts (59), it seems that renin substrate also has to be internalized to allow intracellular angiotensin generation in the neonatal heart. This may be different in the adult heart or under pathological conditions (40). In addition, age as well as pathological conditions may affect the density of cardiac man-6-P/IGF-II receptors and/or give rise to the appearance of alternative (pro)renin receptors or uptake mechanisms (4, 8, 46, 48, 53).
At present it cannot be concluded what enzymes are responsible for the intracellular prorenin activation. With the exception of the serine protease inhibitor AEBSF, none of the protease inhibitors used in this study exerted an inhibitory effect on prorenin activation. This is not due to the inability of these blockers to reach the proper intracellular compartment because others have demonstrated efficacy in the same setup (7, 44). Moreover, the mixed serine-cysteine protease inhibitor leupeptin and the cysteine protease inhibitor E64 increased the levels of cell-associated prorenin by >40%, indicating that cysteine proteases contribute to (pro)renin degradation and that exogenous inhibitors apparently are capable of reaching the intracellular sites where degradation occurs. Possible candidates for the prorenin-activating enzyme are kallikrein (37), prohormone convertases (51), and cathepsin B (45), although the latter seems unlikely in view of the absence of an inhibitory effect of E64. These enzymes have been demonstrated in the heart (6, 41, 49, 55). Furthermore, Baba and colleagues (3) described a serine protease (mol mass 26 kDa) capable of activating prorenin in rat adrenal explant cultures. The pH optimum for prorenin activation by this enzyme was 6.5. The rapid activation of prorenin in the present study, which is indicative for early endosomal (i.e., at pH 6.5) removal of the prosegment, as well as the inhibitory effects of AEBSF on prorenin activation in myocytes, is in agreement with the existence of a similar serine protease in the heart.
Finally, our results are not limited to recombinant human prorenin but also apply to native human plasma prorenin. Plasma prorenin is predominantly of renal origin (11), although extrarenal prorenin sources such as the eye (12), ovary (20), placenta (33), and testis (54) are also known to contribute to circulating levels of prorenin. When incubated at 37°C for 4 h with plasma containing prorenin (and low levels of renin), myocytes were found to contain predominantly renin. It is unlikely that this is due to selective uptake of plasma renin because man-6-P/IGF-II receptors do not make a distinction between man-6-P-containing renin and prorenin (58). Therefore, these findings suggest that plasma prorenin, like recombinant prorenin, is activated intracellularly to renin. In absolute terms, the levels of cell-associated renin and prorenin after incubation with plasma were two to three times lower than after incubation with equal amounts of recombinant prorenin (Fig. 7). This may have at least two explanations. First, the percentage of plasma prorenin carrying the man-6-P signal may be lower than the percentage of recombinant human prorenin carrying this signal. Second, the high (in the nanomolar range) levels of soluble man-6-P/IGF-II receptors that have been reported in plasma (9) may affect prorenin binding to cellular man-6-P/IGF-II receptors, especially because the density of the latter (expressed per million cells) is in the femtomole range. Studies reporting on the presence of high-molecular-weight forms of prorenin in plasma (5, 47) are in agreement with the contention that plasma prorenin is in part bound to soluble man-6-P/IGF-II receptors. Further experiments are required to resolve this issue. Interestingly, prorenin present in human amniotic fluid did not bind to myocytes, which suggests that this prorenin does not contain the man-6-P signal. Amniotic fluid prorenin is derived primarily from the placental chorion laeve (33) (i.e., is synthesized extrarenally), and its isoelectric focusing pattern differs from that of plasma prorenin (36). The levels of soluble man-6-P/IGF-II receptors in amniotic fluid are ~100 times lower than in plasma (9). Whether prorenin from other extrarenal sources also lacks the man-6-P signal is currently unknown, but if so, this would imply that only prorenin of renal origin is meant to be taken up by the heart and thus that cardiac angiotensin generation is regulated by the kidney and not by other (pro)renin-producing tissues. Consequently, the release of large amounts of prorenin from extrarenal sources [e.g., during pregnancy (33)] would not necessarily result in increased cardiac angiotensin generation.
In conclusion, our data support the concept of cardiac angiotensin generation by renal (pro)renin. Prorenin is sequestered from the circulation by cardiac cells through binding to man-6-P/IGF-II receptors. Binding occurs with high affinity and is limited to prorenin containing the man-6-P signal. After binding, the prorenin-man-6-P/IGF-II receptor complex is internalized and prorenin is activated to renin, possibly by a serine protease in an early endosomal compartment. The receptor then returns to the cell surface to repeat the binding and internalization process. Activated prorenin may participate in intracellular ANG I production before its destruction by cysteine proteases in lysosomes.
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ACKNOWLEDGEMENTS |
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This study was supported by The Netherlands Heart Foundation Research Grant NHS96-019.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. H. J. Danser, Dept. of Pharmacology, Rm. EE1418b, Erasmus Univ. Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands (E-mail: danser{at}farma.fgg.eur.nl).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 June 2000; accepted in final form 7 November 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Admiraal, PJJ,
van Kesteren CAM,
Danser AHJ,
Derkx FHM,
Sluiter W,
and
Schalekamp MADH
Uptake and proteolytic activation of prorenin by cultured human endothelial cells.
J Hypertens
17:
621-629,
1999[Web of Science][Medline].
2.
Aeed, PA,
Guido DM,
Mathews WR,
and
Elhammer AP.
Characterization of the oligosaccharide structures on recombinant human prorenin expressed in Chinese hamster ovary cells.
Biochemistry
31:
6951-6961,
1992[Medline].
3.
Baba, K,
Doi Y,
Yamaguchi T,
Yano K,
and
Hashiba K.
Production of active and inactive renin by adrenal explant cultures and the existence of a prorenin activating enzyme in the adrenal gland.
Jpn Heart J
33:
465-476,
1992[Medline].
4.
Ballesteros, M,
Scott CD,
and
Baxter RC.
Developmental regulation of insulin-like growth factor-II/mannose 6-phosphate receptor mRNA in the rat.
Biochem Biophys Res Commun
172:
775-779,
1990[Web of Science][Medline].
5.
Barrett, JD,
Eggena P,
and
Sambhi MP.
Activation of rat plasma renin.
Endocrinology
108:
778-785,
1981
6.
Beaubien, G,
Schafer MK,
Weihe E,
Dong W,
Chretien M,
Seidah NG,
and
Day R.
The distinct gene expression of the pro-hormone convertases in the rat heart suggests potential substrates.
Cell Tissue Res
279:
539-549,
1995[Web of Science][Medline].
7.
Beynon, RJ,
and
Bond JS.
Proteolytic enzymes: a practical approach.
In: The Practical Approach Series (1st ed.), edited by Rickwood D,
and Hames BD.. Oxford: IRL, 1989, p. 259.
8.
Campbell, DJ,
and
Valentijn AJ.
Identification of vascular renin-binding proteins by chemical cross-linking: inhibition of binding of renin by renin inhibitors.
J Hypertens
12:
879-890,
1994[Web of Science][Medline].
9.
Costello, M,
Baxter RC,
and
Scott CD.
Regulation of soluble insulin-like growth factor II/mannose 6-phosphate receptor in human serum: measurement by enzyme-linked immunosorbent assay.
J Clin Endocrinol Metab
84:
611-617,
1999
10.
Dahms, NM,
Lobel P,
and
Kornfeld S.
Mannose 6-phosphate receptors and lysosomal enzyme targeting.
J Biol Chem
264:
12115-12118,
1989
11.
Danser, AHJ,
Derkx FHM,
Schalekamp MADH,
Hense HW,
Riegger GAJ,
and
Schunkert H.
Determinants of interindividual variation of renin and prorenin concentrations: evidence for a sexual dimorphism of (pro)renin levels in humans.
J Hypertens
16:
853-862,
1998[Web of Science][Medline].
12.
Danser, AHJ,
van den Dorpel MA,
Deinum J,
Derkx FHM,
Franken AAM,
Peperkamp E,
de Jong PTVM,
and
Schalekamp MADH
Renin, prorenin, and immunoreactive renin in vitreous fluid from eyes with and without diabetic retinopathy.
J Clin Endocrinol Metab
68:
160-167,
1989
13.
Danser, AHJ,
van Kats JP,
Admiraal PJJ,
Derkx FHM,
Lamers JMJ,
Verdouw PD,
Saxena PR,
and
Schalekamp MADH
Cardiac renin and angiotensins. Uptake from plasma versus in situ synthesis.
Hypertension
24:
37-48,
1994
14.
Danser, AHJ,
van Kesteren CAM,
Bax WA,
Tavenier M,
Derkx FHM,
Saxena PR,
and
Schalekamp MADH
Prorenin, renin, angiotensinogen, and angiotensin-converting enzyme in normal and failing human hearts: evidence for renin binding.
Circulation
96:
220-226,
1997
15.
Deinum, J,
Derkx FHM,
and
Schalekamp MADH
Improved immunoradiometric assay for plasma renin.
Clin Chem
45:
847-854,
1999
16.
Deinum, J,
Derkx FHM,
and
Schalekamp MADH
Probing epitopes on human prorenin during its proteolytic and non-proteolytic activation.
Biochim Biophys Acta
1388:
386-396,
1998[Medline].
17.
De Lannoy, LM,
Danser AHJ,
Bouhuizen AMB,
Saxena PR,
and
Schalekamp MADH
Localization and production of angiotensin II in the isolated perfused rat heart.
Hypertension
31:
1111-1117,
1998
18.
De Lannoy, LM,
Danser AHJ,
van Kats JP,
Schoemaker RG,
Saxena PR,
and
Schalekamp MADH
Renin-angiotensin system components in the interstitial fluid of the isolated perfused rat heart: local production of angiotensin I.
Hypertension
29:
1240-1251,
1997
19.
Dennis, PA,
and
Rifkin DB.
Cellular activation of latent transforming growth factor beta requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor.
Proc Natl Acad Sci USA
88:
580-584,
1991
20.
Derkx, FHM,
Alberda AT,
Zeilmaker GH,
and
Schalekamp MADH
High concentrations of immunoreactive renin, prorenin and enzymatically active renin in human ovarian follicular fluid.
Br J Obstet Gynaecol
94:
4-9,
1987[Web of Science][Medline].
21.
Derkx, FHM,
Schalekamp MP,
and
Schalekamp MADH
Two-step prorenin-renin conversion isolation of an intermediary form of activated prorenin.
J Biol Chem
262:
2472-2477,
1987
22.
Dong, JM,
and
Sahagian GG.
Basis for low affinity binding of a lysosomal cysteine protease to the cation-independent mannose 6-phosphate receptor.
J Biol Chem
265:
4210-4217,
1990
23.
Eggena, P,
Zhu JH,
Sereevinyayut S,
Giordani M,
Clegg K,
Andersen PC,
Hyun P,
and
Barrett JD.
Hepatic angiotensin II nuclear receptors and transcription of growth-related factors.
J Hypertens
14:
961-968,
1996[Web of Science][Medline].
24.
Ekker, M,
Tronik D,
and
Rougeon F.
Extra-renal transcription of the renin genes in multiple tissues of mice and rats.
Proc Natl Acad Sci USA
86:
5155-5158,
1989
25.
Faust, PL,
Chirgwin JM,
and
Kornfeld S.
Renin, a secretory glycoprotein, acquires phosphomannosyl residues.
J Cell Biol
105:
1947-1955,
1987
26.
Faust, PL,
Wall DA,
Perara E,
Lingappa VR,
and
Kornfeld S.
Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting.
J Cell Biol
105:
1937-1945,
1987
27.
Haller, H,
Lindschau C,
Erdmann B,
Quass P,
and
Luft FC.
Effects of intracellular angiotensin II in vascular smooth muscle cells.
Circ Res
79:
765-772,
1996
28.
Heller, LJ,
Opsahl JA,
Wernsing SE,
Saxena R,
and
Katz SA.
Myocardial and plasma renin-angiotensinogen dynamics during pressure-induced cardiac hypertrophy.
Am J Physiol Regulatory Integrative Comp Physiol
274:
R849-R856,
1998
29.
Helseth, DL, Jr,
and
Veis A.
Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen.
Proc Natl Acad Sci USA
81:
3302-3306,
1984
30.
Heusser, CH,
Bews JPA,
Alkan SS,
Dietrich FM,
Wood JM,
de Gasparo M,
and
Hofbauer KG.
Monoclonal antibodies to human renin: properties and applications.
Clin Exp Hypertens
9:
1259-1275,
1987.
31.
Hirsch, AT,
Opsahl JA,
Lunzer MM,
and
Katz SA.
Active renin and angiotensinogen in cardiac interstitial fluid after myocardial infarction.
Am J Physiol Heart Circ Physiol
276:
H1818-H1826,
1999
32.
Imig, JD,
Navar GL,
Zou LX,
O'Reilly KC,
Allen PL,
Kaysen JH,
Hammond TG,
and
Navar LG.
Renal endosomes contain angiotensin peptides, converting enzyme, and AT1A receptors.
Am J Physiol Renal Physiol
277:
F303-F311,
1999
33.
Itskovitz, J,
Rubattu S,
Levron J,
and
Sealey JE.
Highest concentrations of prorenin and human chorionic gonadotropin in gestational sacs during early human pregnancy.
J Clin Endocrinol Metab
75:
906-910,
1992[Abstract].
34.
Iwai, N,
and
Inagami T.
Quantitative analysis of renin gene expression in extrarenal tissues by polymerase chain reaction method.
J Hypertens
10:
717-724,
1992[Web of Science][Medline].
35.
Katz, SA,
Opsahl JA,
Lunzer MM,
Forbis LM,
and
Hirsch AT.
Effect of bilateral nephrectomy on active renin, angiotensinogen, and renin glycoforms in plasma and myocardium.
Hypertension
30:
259-266,
1997
36.
Khalidi, N,
McKenzie I,
and
McKenzie JK.
Isoelectric heterogeneity of human prorenin (inactive renin) in body fluids.
Am J Hypertens
4:
56-59,
1991[Web of Science][Medline].
37.
Kim, WS,
Nakayama K,
Nakagawa T,
Kawamura Y,
Haraguchi K,
and
Murakami K.
Mouse submandibular gland prorenin-converting enzyme is a member of glandular kallikrein family.
J Biol Chem
266:
19283-19287,
1991
38.
Kornfeld, S.
Structure and function of the mannose 6-phosphate/insulinlike growth factor II receptors.
Annu Rev Biochem
61:
307-330,
1992[Web of Science][Medline].
39.
Lee, SJ,
and
Nathans D.
Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors.
J Biol Chem
263:
3521-3527,
1988
40.
Lindpaintner, K,
Jin MW,
Niedermaier N,
Wilhelm MJ,
and
Ganten D.
Cardiac angiotensinogen and its local activation in the isolated perfused beating heart.
Circ Res
67:
564-573,
1990
41.
Lockwood, TD.
Inactivation of intracellular proteolysis and cathepsin B enzyme activity by dehydroascorbic acid and reactivation by dithiothreitol in perfused rat heart.
Biochem Pharmacol
54:
669-675,
1997[Web of Science][Medline].
42.
Mercure, C,
Ramla D,
Garcia R,
Thibault G,
Deschepper CF,
and
Reudelhuber TL.
Evidence for intracellular generation of angiotensin II in rat juxtaglomerular cells.
FEBS Lett
422:
395-399,
1998[Web of Science][Medline].
43.
Methot, D,
Silversides DW,
and
Reudelhuber TL.
In vivo enzymatic assay reveals catalytic activity of the human renin precursor in tissues.
Circ Res
84:
1067-1072,
1999
44.
Moore, RH,
Tuffaha A,
Millman EE,
Dai W,
Hall HS,
Dickey BF,
and
Knoll BJ.
Agonist-induced sorting of human 2-adrenergic receptors to lysosomes during downregulation.
J Cell Sci
112:
329-338,
1999[Abstract].
45.
Neves, FA,
Duncan KG,
and
Baxter JD.
Cathepsin B is a prorenin processing enzyme.
Hypertension
27:
514-517,
1996
46.
Nguyen, G,
Delarue F,
Berrou J,
Rondeau E,
and
Sraer JD.
Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen.
Kidney Int
50:
1897-1903,
1996[Web of Science][Medline].
47.
Nielsen, AH,
Malling C,
and
Poulsen K.
Characteristics and conversion of high molecular weight forms of renin in plasma and their incomplete activation by the current acid treatment.
Biochim Biophys Acta
534:
246-257,
1978[Medline].
48.
Nissley, P,
Kiess W,
and
Sklar M.
Developmental expression of the IGF-II/mannose 6-phosphate receptor.
Mol Reprod Dev
35:
408-413,
1993[Web of Science][Medline].
49.
Nolly, H,
Carbini LA,
Scicli G,
Carretero OA,
and
Scicli AG.
A local kallikrein-kinin system is present in rat hearts.
Hypertension
23:
919-923,
1994
50.
Passier, RCJJ,
Smits JFM,
Verluyten MJA,
and
Daemen MJAP
Expression and localization of renin and angiotensinogen in rat heart after myocardial infarction.
Am J Physiol Heart Circ Physiol
271:
H1040-H1048,
1996
51.
Reudelhuber, TL,
Ramla D,
Chiu L,
Mercure C,
and
Seidah NG.
Proteolytic processing of human prorenin in renal and non-renal tissues.
Kidney Int
46:
1522-1524,
1994[Web of Science][Medline].
52.
Rijnboutt, S,
Aerts HMFG,
Geuze HJ,
Tager JM,
and
Strous GJ.
Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells.
J Biol Chem
266:
4862-4868,
1991
53.
Sealey, JE,
Catanzaro DF,
Lavin TN,
Gahnem F,
Pitarresi T,
Hu LF,
and
Laragh JH.
Specific prorenin/renin binding (ProBP) identification and characterization of a novel membrane site.
Am J Hypertens
9:
491-502,
1996[Web of Science][Medline].
54.
Sealey, JE,
Goldstein M,
Pitarresi T,
Kudlak TT,
Glorioso N,
Fiamengo SA,
and
Laragh JH.
Prorenin secretion from human testis: no evidence for secretion of active renin or angiotensinogen.
J Clin Endocrinol Metab
66:
974-978,
1988
55.
Seidah, NG,
Hamelin J,
Gaspar AM,
Day R,
and
Chretien M.
The cDNA sequence of the human pro-hormone and pro-protein convertase PC1.
DNA Cell Biol
11:
283-289,
1992[Web of Science][Medline].
56.
Sinn, PL,
and
Sigmund CD.
Transgenic models as tools for studying the regulation of human renin expression.
Regul Pept
86:
77-82,
2000[Web of Science][Medline].
57.
Van Heugten, HAA,
Bezstarosti K,
Dekkers DHW,
and
Lamers JMJ
Homologous desensitization of the endothelin-1 receptor mediated phosphoinositide response in cultured neonatal rat cardiomyocytes.
J Mol Cell Cardiol
25:
41-52,
1993[Web of Science][Medline].
58.
van Kesteren, CAM,
Danser AHJ,
Derkx FHM,
Dekkers DHW,
Lamers JMJ,
Saxena PR,
and
Schalekamp MADH
Mannose 6-phosphate receptor-mediated internalization and activation of prorenin by cardiac cells.
Hypertension
30:
1389-1396,
1997
59.
van Kesteren, CAM,
Saris JJ,
Dekkers DHW,
Lamers JMJ,
Saxena PR,
Schalekamp MADH,
and
Danser AHJ
Cultured neonatal rat cardiac myocytes and fibroblasts do not synthesize renin or angiotensinogen: evidence for stretch-induced cardiomyocyte hypertrophy independent of angiotensin II.
Cardiovasc Res
43:
148-156,
1999
60.
Zuo, WM,
Pratt RE,
Heusser CH,
Bews JPA,
de Gasparo MM,
and
Dzau VJ.
Characterization of a monoclonal antibody specific for human active renin.
Hypertension
19:
249-254,
1992
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) or presence
(
) of 10 mM mannose-6-phosphate (man-6-P).
Man-6-P/insulin-like growth factor-II (IGF-II) receptor-specific
binding (
) was taken as the difference between levels
of cell-associated prorenin with and without man-6-P. Data are
means ± SE; n = 8. Dissociation constant
(Kd) and maximum number of binding sites
(Bmax) were calculated from a plot according to Scatchard
(inset; B/F represents the ratio of bound to free prorenin).
B: levels of cell-associated total (
) prorenin after incubation of myocytes
with recombinant human prorenin at 37°C for 4 h. Data are
means ± SE; n = 7.
) are shown.
P < 0.01 vs. plasma.