© 2001 American Heart Association, Inc.
Vascular Biology |
Prorenin Accumulation and Activation in Human Endothelial Cells
Importance of Mannose 6-Phosphate Receptors
From the Cardiovascular Research Institute COEUR, Departments of Pharmacology (M.M.E.D.v.d.E., J.J.S., A.H.J.D.), Internal Medicine (M.M.E.D.v.d.E., J.J.S., R.J.A.d.B., M.A.D.H.S., F.H.M.D.), and Biochemistry (E.d.W., W.S.), Erasmus University Rotterdam, Rotterdam, the Netherlands, and the Laboratory of Molecular Biochemistry of Hypertension (T.L.R.), Clinical Research Institute of Montréal, Montréal, Canada.
Correspondence to Dr A.H.J. Danser, Department of Pharmacology, Room EE1418b, Erasmus University Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, Netherlands. E-mail danser{at}farma.fgg.eur.nl
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Abstract—ACE inhibitors improve endothelial dysfunction, possibly by blocking endothelial angiotensin production. Prorenin, through its binding and activation by endothelial mannose 6-phosphate (M6P) receptors, may contribute to this production. Here, we investigated this possibility as well as prorenin activation kinetics, the nature of the prorenin-activating enzyme, and M6P receptor–independent prorenin binding. Human umbilical vein endothelial cells (HUVECs) were incubated with wild-type prorenin, K/A-2 prorenin (in which Lys42 is mutated to Ala, thereby preventing cleavage by known proteases), M6P-free prorenin, and nonglycosylated prorenin, with or without M6P, protease inhibitors, or angiotensinogen. HUVECs bound only M6P-containing prorenin (Kd 0.9±0.1 nmol/L, maximum number of binding sites [Bmax] 1010±50 receptors/cell). At 37°C, because of M6P receptor recycling, the amount of prorenin internalized via M6P receptors was >25 times Bmax. Inside the cells, wild-type and K/A-2 prorenin were proteolytically activated to renin. Renin was subsequently degraded. Protease inhibitors interfered with the latter but not with prorenin activation, thereby indicating that the activating enzyme is different from any of the known prorenin-activating enzymes. Incubation with angiotensinogen did not lead to endothelial angiotensin generation, inasmuch as HUVECs were unable to internalize angiotensinogen. Most likely, therefore, in the absence of angiotensinogen synthesis or endocytosis, M6P receptor–mediated prorenin internalization by endothelial cells represents prorenin clearance.
Key Words: human umbilical vein endothelial cells • mannose 6-phosphate receptors • prorenin • renin • angiotensin
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Vascular angiotensin II (Ang II), through stimulation of Ang II type 1 (AT1) receptors, induces the generation of superoxide anions, thereby resulting in endothelial dysfunction.1 Blockade of this process, with ACE inhibitors or AT1 receptor antagonists, reverses endothelial dysfunction in human atherosclerosis.2 At present, it is still being debated whether vascular Ang II generation depends on locally synthesized or circulating kidney-derived renin. In support of the latter, angiotensins cannot be demonstrated in the perfusate of vascular preparations obtained from nephrectomized animals, unless renin is added to the perfusion buffer.3 Because renin in circulating blood plasma is predominantly present in the form of its inactive precursor, prorenin,4 it is also conceivable that kidney-derived prorenin, after its local activation, contributes to vascular angiotensin generation.
Renin and/or prorenin may enter the vascular wall through binding to (pro)renin receptors. Indeed, we have recently demonstrated that human umbilical vein endothelial cells (HUVECs) bind renin and prorenin to cell surface mannose 6-phosphate (M6P) receptors.5 Binding was followed by internalization and proteolytic activation of prorenin. The latter process is not unique, inasmuch as M6P receptors are known to be involved in the activation of several other prohormones carrying the M6P recognition marker, such as thyroglobulin.6 Moreover, M6P receptor–mediated prorenin activation also occurs in cardiac cells.7 The enzyme responsible for prorenin activation is currently not known. Possible candidates include cathepsin B, glandular kallikreins, and members of the prohormone convertase family.8 9 10 Furthermore, receptors other than the M6P receptor may also contribute to (pro)renin binding.11 12
It was the aim of the present study to investigate the kinetics of prorenin binding and activation in HUVECs, the nature of the prorenin-activating enzyme in these cells, endothelial prorenin binding independent of M6P receptors, and whether endothelial prorenin binding and activation result in angiotensin generation.
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Human Prorenin Preparations
Recombinant human prorenin (wild-type prorenin), produced in Chinese hamster ovary cells transfected with a vector containing human prorenin cDNA, was kindly provided by Dr S. Mathews (Hoffmann-LaRoche, Basel, Switzerland).5 To remove traces of renin, it was partially purified by Cibacron blue Sepharose affinity chromatography (Pharmacia).5 7 Wild-type prorenin was stored at -80°C in aliquots containing
2x105 U/L (4 µmol/L) in 0.1% BSA. It
was also separated into an M6P-containing and an M6P-free fraction by
use of a bovine M6P receptor affinity column, provided by Dr S.
Kornfeld (Washington University School of Medicine, St. Louis,
Mo).13 In short, 1 U
recombinant wild-type prorenin was applied to a 0.5-mL bovine M6P
receptor affinity column. The column was washed with column
buffer,13 and M6P-containing
prorenin was eluted by adding 10 mmol/L M6P to the column buffer.
Fractions corresponding to the column run-through material (ie,
M6P-free prorenin) and to M6P-eluted material (ie, M6P-containing
prorenin) were separately pooled and stored at -80°C in aliquots
containing 100 U/L.
K/A-2 prorenin, ie, prorenin that cannot be cleaved by known
proteases, was produced in GH4 cells transfected with a vector
containing human prorenin cDNA in which Lys42 is mutated to
Ala.14 Nonglycosylated
prorenin was produced in GH4 cells transfected with a vector containing
human prorenin cDNA in which Asn at positions 5 and 75 of renin is
mutated to Ser.15 The K/A-2
and nonglycosylated prorenin mutants were stored at -80°C in
aliquots containing 700 and 30 U/L, respectively, in DMEM with 5%
FCS.
Cell Culture
HUVECs were isolated from umbilical cords, cultured
to confluence, trypsinized, and stored in liquid nitrogen as described
earlier.5 For an experiment,
an aliquot of HUVECs (passages 2 to 5) was thawed. The cells were
cultured to confluence in a 75-cm2 tissue
culture flask coated with fibronectin (10
µg/cm2) in modified medium 199 containing
10% newborn calf serum, 10% human serum, 150 µg/mL
endothelial cell growth
factor,5 15 U/mL heparin, 50
U/mL penicillin, and 5 µg/mL streptomycin at 37°C in a humid 5%
CO2 atmosphere. Cells were trypsinized and
seeded in 12-well plates. Studies were carried out 1 day after
confluence (corresponding to a density of
4x104 cells/cm2)
had been reached. Before the start of each experiment, the cells were
washed 3 times with 2 mL warm (37°C) PBS (140 mmol/L NaCl,
2.6 mmol/L KCl, 1.4 mmol/L
KH2PO4, and 8.1
mmol/L Na2HPO4, pH 7.4).
The cells were then preincubated either at 37°C or 4°C for 30
minutes with 0.5 mL incubation medium consisting of modified medium 199
without newborn calf serum or human serum but supplemented with 1%
human serum albumin.
Incubation With Prorenin at 4°C or
37°C
After preincubation at 37°C or 4°C for 30
minutes, experiments were started by replacing the incubation medium by
incubation medium of 37°C or 4°C containing wild-type prorenin
(final concentration 3 to 300 U/L), M6P-containing wild-type prorenin
(10 U/L), M6P-free wild-type prorenin (10 U/L), K/A-2 prorenin (100
U/L), or nonglycosylated prorenin (10 U/L) with or without 10
mmol/L M6P. Cells were then incubated at 37°C or 4°C for maximally
4 hours. To investigate the nature of the prorenin-renin–converting
enzyme, incubations at 37°C with wild-type prorenin (100 U/L) were
also performed in the presence of the serine protease
inhibitors 4-(2-aminoethyl)benzenesulfonyl fluoride
hydrochloride (AEBSF, 0.5 mmol/L, Calbiochem), aprotinin
(0.001 mmol/L, Hoffmann-LaRoche), and chymostatin (0.1
mmol/L, Hoffmann-LaRoche), the cysteine protease inhibitor
L-trans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine
(E-64, 0.1 mmol/L, Sigma Chemical Co), the mixed serine/cysteine
protease inhibitor leupeptin (0.1 mmol/L, Sigma), the
metalloprotease inhibitors EDTA (1 mmol/L),
phenanthroline (1 mmol/L, Merck), and
phosphoramidon (0.5 mmol/L, Sigma), and the
aspartic protease inhibitor pepstatin A (0.1 mmol/L,
Sigma). At the end of the incubation period, the culture medium was
removed. The cells were washed 3 times with 2 mL ice-cold PBS and lysed
in 0.2 mL ice-cold PBS containing 0.2% Triton X-100. Subsequently, the
cell lysate was frozen on dry ice. Cell lysates were stored at
-80°C.
To distinguish cell surface–bound from internalized prorenin, the acid-wash method was used.7 Cells were then lysed as described above.
Incubation With Prorenin at 4°C Followed by
Incubation at 37°C
The kinetics of prorenin internalization and
activation were studied by incubating HUVECs, which were cultured in
6-well plates, with 1 mL wild-type prorenin-containing incubation
medium (final concentration 100 U/L) for 2 hours at 4°C. Thereafter,
the cells were washed 3 times with 3 mL ice-cold PBS and further
incubated at 37°C with prorenin-free incubation medium. The
incubation was terminated after various times (ranging from 10 to 360
minutes) by washing the cells 3 times with 3 mL ice-cold PBS. The cells
were then lysed in 0.5 mL ice-cold PBS containing 0.2% Triton X-100 as
described above. Cell lysates were stored at
-80°C.
Angiotensin Generation During
Incubation With Prorenin at 37°C
To study prorenin-induced endothelial
angiotensin generation, HUVECs, which were cultured in
6-well plates, were incubated at 37°C for 4 hours with 1 mL
incubation medium containing 10 U/L wild-type prorenin and/or 150
nmol/L human angiotensinogen (Sigma). HUVECs incubated
without prorenin or angiotensinogen served as the control.
After 4 hours of incubation, the medium was rapidly mixed with 50 µL
inhibitor solution (containing 0.1 mmol/L
remikiren, 200 mmol/L disodium EDTA, and 0.2 mmol/L
lisinopril) and frozen at -70°C. Cells used for the
measurement of angiotensinogen were washed 3 times with 3
mL ice-cold PBS and lysed in 0.2 mL ice-cold PBS containing 0.2%
Triton X-100 as described above. Cells used for the measurement of
angiotensin I (Ang I) and Ang II were scraped with a rubber
policeman in a volume of 0.5 mL ice-cold PBS. The cell-PBS mixture was
centrifuged at 1000g at
4°C for 1 minute, after which the pellet was homogenized
in 0.5 mL 0.1 mol/L HCl/80% ethanol by using a hand-operated Dounce
homogenizer. 125I-Ang I was
added to the samples before the homogenization
procedure to determine angiotensin recovery. Ethanol was
evaporated under vacuum rotation at 4°C by using a Speed Vac
Concentrator. The concentrated homogenates were dissolved
in 0.5 mL 1% orthophosphoric acid and applied to Sep-Pak columns (see
below).
In view of the partial catalytic activity of prorenin,16 Ang I was also measured in incubation medium containing 10 U/L wild-type prorenin and/or 150 nmol/L angiotensinogen that had been incubated without HUVECs for 4 hours at 37°C.
Finally, HUVEC-mediated Ang I–Ang II conversion and Ang I degradation was studied by incubating HUVECs for up to 4 hours at 37°C with 1 nmol/L Ang I in the presence or absence of 10 µmol/L quinaprilat. Samples (0.1 mL) for the measurement of Ang I and II were taken at 0, 0.5, 1, 2, and 4 hours, rapidly mixed with 10 µL inhibitor solution, and frozen at -70°C.
Biochemical Measurements
Total prorenin (ie, cell-activated plus
nonactivated) and cell-activated prorenin in the cell
lysates obtained from the 37°C experiments were measured by
immunoradiometric assay
(IRMA).17 Proteolytic
activation of prorenin in these experiments was verified with an IRMA
specific for intact prorenin, ie, prorenin in which the C-terminal part
of the prosegment is still attached to the renin part of the
molecule.5 The results of
these IRMAs are expressed as microunits per
106 cells with intact recombinant human
prorenin used as a reference. The lower limit of detection was 5
µU/106 cells. The IRMAs are not sensitive
enough to measure the low levels of prorenin that were present in
the cell lysates at 4°C. Therefore, the prorenin measurements in
these experiments were performed by enzyme-kinetic
assay.7 The results of this
assay are expressed as microunits per 106
cells with plasmin-activated recombinant human prorenin used as
a reference. The lower limit of detection was 1
µU/106 cells.
The concentration of angiotensinogen in the cell lysates was determined as the maximum quantity of Ang I that was generated during incubation at 37°C and pH 7.4 with excess recombinant human renin in the presence of a mixture of angiotensinase, ACE, and serine protease inhibitors.17 18 19 The lowest level that could be measured was 20 fmol/106 cells.
Ang I and II levels in medium and in cell homogenates were measured by radioimmunoassay after Sep-Pak extraction and reversed-phase high-performance liquid chromatography separation.18 19 Recovery was better than 65%, and results were corrected for incomplete recovery. The lower limits of detection for Ang I and II were 2 and 1 fmol per milliliter medium or per 106 cells, respectively.
Statistical Analysis
All data are expressed as mean±SEM. Differences
between the cellular prorenin levels at 37°C and 4°C and between
the cellular levels of total prorenin and cell-activated
prorenin in the presence or absence of protease inhibitors
were evaluated for statistical significance by ANOVA. Statistical
significance was accepted at
P<0.05.
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Results |
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Incubation With Prorenin at 4°C or 37°C
HUVECs bound wild-type prorenin in a concentration-dependent manner at 4°C and at 37°C (Figure 1
). 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 prorenin
after 4 hours of incubation was 10 times higher at 37°C than at
4°C. Prorenin activation was detectable at 37°C only. Saturation of
the activation process did not occur, inasmuch as the percentage of
cell-associated prorenin that was activated was similar at all
concentrations of prorenin to which the cells were exposed (ranging
from 82±5% at 3 U/L to 76±6% at 300 U/L, n=6;
P=NS). None of the protease
inhibitors that were tested (n=5 for each
inhibitor) blocked the activation of prorenin (ranging from
68±5% [with AEBSF] to 81±9% [with EDTA]), although some
inhibitors affected the amount of cell-associated total
prorenin. E64, leupeptin, and pepstatin A increased the levels of
cell-associated total prorenin to 135±9%, 138±11%, and 126±7% of
control (P<0.05 versus
control), respectively, thereby indicating that cysteine and aspartic
proteases contribute to (pro)renin degradation in
endothelial cells. AEBSF and phenanthroline reduced the
levels of cell-associated total prorenin to 49±7% and 39±7% of
control, respectively
(P<0.05). This was not due to
interference with the internalization process, because the percentage
of cell-associated prorenin that was acid resistant was similar
in the presence and absence of these inhibitors (data not
shown). Aprotinin (96±9% of control), chymostatin (119±6%), EDTA
(85±8%), and phosphoramidon (94±9%) did not affect
the levels of cell-associated total prorenin.
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K/A-2 prorenin was bound and internalized by HUVECs to the
same degree as wild-type prorenin
(Figure 2, left). Moreover, after 4 hours of incubation with
K/A-2 prorenin at 37°C, 80±2% of cell-associated total prorenin was
in the activated form. The activation was due to proteolytic
cleavage of the prosegment, inasmuch as <30% of cell-associated total
prorenin still contained the C-terminal part of the prosegment.
Furthermore, the rate of prorenin activation over the 4-hour incubation
period was identical for wild-type and K/A-2 prorenin
(Figure 2, right). Taken together, therefore, HUVECs
activated K/A-2 prorenin in a manner indistinguishable from the
activation of wild-type prorenin, thereby supporting the idea that
activation was not mediated by any of the known prorenin-renin
convertases.
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M6P significantly inhibited native prorenin binding at
37°C and at 4°C
(Figure 3). In the presence of M6P, prorenin binding was
observed only at prorenin concentrations >10 U/L, and the levels of
cell-associated prorenin were identical at 37°C and at 4°C.
Scatchard analysis of the results obtained at 4°C revealed
that M6P receptor–specific binding of prorenin occurred with high
affinity
(Kd
0.9±0.1 nmol/L). The number of prorenin-binding M6P receptors on the
cell surface (Bmax) was 1010±50 sites per
cell.
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Binding studies after the separation of wild-type prorenin
into an M6P-free and an M6P-containing fraction, with the help of a
bovine M6P receptor affinity column, revealed that only M6P-containing
prorenin and not M6P-free prorenin was bound by HUVECs
(Figure 4). In agreement with this finding, the cells did not
bind or internalize nonglycosylated prorenin (n=4, data not
shown).
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Incubation With Prorenin at 4°C Followed by
Incubation at 37°C
After 2 hours of incubation at 4°C with 100 U/L
wild-type prorenin, followed by repeated washing with ice-cold PBS, the
level of cell-associated total prorenin was 197±26
µU/106 cells (n=5). Acid wash confirmed
that all cell-associated prorenin at that time was located on the cell
surface. Immediately after elevating the temperature to 37°C, the
level of cell-associated total prorenin started to decrease. The
decrease followed a biphasic pattern
(Figure 5). The rapid phase (half-time [t1/2] 7±1 minutes)
corresponds with the release of cell-associated prorenin into the
medium, whereas the slow phase (t1/2 405±72 minutes)
represents intracellular degradation after internalization. As
soon as prorenin release into the medium no longer occurred (ie, at the
time all remaining cell surface–bound prorenin had been internalized,
after 40 minutes), the cellular levels of activated prorenin
started to rise rapidly, reaching a maximum after 60 minutes.
Thereafter, these levels decreased with a half-life (t1/2 305±33
minutes) similar to that of the degradation of cell-associated total
prorenin. Taken together, these findings suggest that activation
precedes degradation and that the half-lives of both processes differ
40- to 60-fold.
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Angiotensin Generation During
Incubation With Prorenin at 37°C
HUVECs that had been incubated under serum-free
conditions for 4 hours in the absence of prorenin and
angiotensinogen did not contain detectable levels of
angiotensinogen, Ang I, or Ang II (n=4), nor could these
renin-angiotensin system components be demonstrated in the
medium (n=4) of these cells. Cellular angiotensinogen and
angiotensin levels remained undetectable after a 4-hour
incubation with 10 U/L wild-type prorenin and/or 150 nmol/L
angiotensinogen (n=4 for each condition). Medium of HUVECs
that had been incubated for 4 hours with 10 U/L wild-type prorenin plus
150 nmol/L angiotensinogen contained 569±85 pmol/L Ang I
and 125±26 pmol/L Ang II (n=4). A 4-hour incubation of medium
containing the same amount of wild-type prorenin and
angiotensinogen in the absence of HUVECs also resulted in
the appearance of Ang I (2520±130 pmol/L, n=4) but not Ang II. Medium
containing either prorenin or angiotensinogen, after
incubation with or without HUVECs, did not contain
angiotensins (n=4 for each condition).
HUVECs metabolized Ang I (t1/2 1.0±0.1 hour, n=4), and this resulted in the appearance of Ang II in the medium, which reached a peak level (191±24 pmol/L) after 2 hours. Quinaprilat fully prevented the generation of Ang II. On the basis of the half-life of Ang I in the presence of the ACE inhibitor (t1/2 1.7±0.2 hours, n=4; P<0.01 versus control), calculations19 revealed that 42±7% of the Ang I metabolism by HUVECs was due to conversion by ACE.
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With the use of prorenin mutants as well as wild-type prorenin, the present study shows that endothelial internalization and activation of prorenin are mediated exclusively via M6P receptors. We have demonstrated earlier that these receptors also bind and internalize renin, in a manner indistinguishable from that of prorenin.5 Thus, despite evidence of the presence of other M6P-independent (pro)renin receptors in vascular preparations,11 our data do not support the idea that such receptors are localized on human endothelial cells. M6P receptor–dependent (pro)renin binding to endothelial cells also provides an explanation for the selective endothelial staining of renin in human arteries.20
At present, 2 different M6P receptors have been identified: the cation-independent type (also known as insulin-like growth factor II [IGFII] receptor) and the cation-dependent type. These receptors contain 2 M6P binding sites and 1 M6P binding site, respectively.21 The high-affinity binding of prorenin in the present study resembles the high-affinity binding of diphosphorylated oligosaccharides that occupy 2 M6P binding sites.22 Most likely, therefore, the endothelial prorenin-binding receptor is the cation-independent M6P receptor. In this respect, prorenin resembles other M6P-carrying prohormones that are internalized and activated after binding to cell surface cation-independent M6P receptors.6
Internalized prorenin was rapidly activated to renin, and the activation occurred proteolytically, as evidenced by the use of an antibody directed against the C-terminal part of the prosegment. Inhibitors of known proteases did not prevent cleavage, although some inhibitors interfered with the subsequent renin degradation by cysteine and aspartic proteases. The latter shows that protease inhibitors do enter endothelial cells and thus confirms that their lack of effect on prorenin activation is not due to their inability to get into the cells. Unexpectedly, K/A-2 prorenin, a prorenin mutant that is not cleaved in vitro in isolated cells or in vivo in rat pituitary glands,10 14 23 was also cleaved to renin. Remarkably, its activation occurred as rapidly as that of wild-type prorenin, although in vitro we found that plasmin activated K/A-2 prorenin 4 to 5 times as slowly as it did wild-type prorenin (authors’ unpublished data, 2000). Taken together, therefore, endothelial cells contain an as-yet-unidentified protease that is capable of cleaving prorenin at or near its natural cleavage site. The rapidity of this process after internalization suggests that this enzyme is located in endosomes.
Activated intracellular prorenin may contribute to endothelial angiotensin generation. This would require the simultaneous uptake or endothelial synthesis of angiotensinogen. The demonstration of Ang II in the cytoplasm of rat endothelial cells24 and of the release of intracellularly generated Ang II from bovine endothelial cells25 supports this concept. However, we were unable to demonstrate intracellular endothelial angiotensin generation during the incubation of HUVECs with prorenin and angiotensinogen. Although Ang I and II could be detected in the medium during these experiments, it is important to note that Ang I generation also occurred during the incubation of prorenin and angiotensinogen in the absence of HUVECs and that the addition of Ang I to HUVECs resulted in the immediate appearance of Ang II in the medium. Taken together, therefore, the presence of Ang I and II in the medium of cells incubated with prorenin and angiotensinogen most likely reflects the partial catalytic activity of prorenin that is due to the temporal unfolding of its prosegment16 rather than endothelial activation of prorenin. On the basis of the Ang I level measured after 4 hours of prorenin plus angiotensinogen incubation in the absence of HUVECs, it can be estimated that <2% of prorenin is catalytically active; ie, it exists in an "open" form.
The absence of intracellular angiotensin generation, despite the prorenin internalization (and subsequent activation) that occurred at 37°C, is most likely due to the fact that HUVECs did not sequester angiotensinogen. Although this does not necessarily apply to all endothelial cells in the human body, it suggests that intraendothelial angiotensin generation will occur only in endothelial cells that synthesize angiotensinogen. Evidence for the latter is currently not available.
In the absence of intracellular angiotensin generation, the high vascular levels of Ang II26 can be explained only on the basis of AT1 receptor–mediated internalization of Ang II after its extracellular generation.27 28 Such extracellular angiotensin generation most likely involves interstitial renin29 or renin bound to the surface of vascular cells via receptors other than the M6P receptor.11 12 30
Finally, in view of the intracellular degradation of activated prorenin, it is conceivable that M6P receptors function as clearance receptors of (pro)renin and that prosegment cleavage is a first step toward intracellular destruction. For instance, binding and internalization of IGFII to M6P/IGFII receptors results in the lysosomal degradation of this ligand.6 An argument against this concept is the large difference in half-life between prorenin activation and degradation. This difference would leave activated prorenin ample time to contribute to intracellular angiotensin generation. However, even if M6P receptors serve as clearance receptors for (pro)renin, this would still allow these receptors to affect vascular angiotensin generation.
In conclusion, prorenin internalization by HUVECs is mediated exclusively via high-affinity M6P receptors and is greatly enhanced by receptor recycling. Internalized prorenin is rapidly activated to renin by a protease that is different from any of the known prorenin-activating enzymes. Activation is followed by degradation and/or, if angiotensinogen is present, may result in intracellular angiotensin generation. Both possibilities support the regulation of vascular angiotensin generation by M6P receptors.
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Acknowledgments |
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This study was supported by the Netherlands Heart Foundation (grant No. NHS 97.186).
Received December 8, 2000; accepted February 16, 2001.
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