Ligand-induced downregulation of receptor-mediated clearance
of hepatocyte growth factor in rats
Ke-Xin
Liu1,
Yukio
Kato1,
Ichiro
Kino1,
Toshikazu
Nakamura2, and
Yuichi
Sugiyama1
1 Graduate School of
Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033; and
2 Biomedical Research Center,
Osaka University School of Medicine, Osaka 565-0871, Japan
 |
ABSTRACT |
The change in tissue uptake clearance of
125I-labeled hepatocyte growth
factor (HGF) after an intravenous injection of an excess (120 µg/kg)
of unlabeled HGF was examined in rats. The heparin-washable component
of the hepatic uptake clearance of
125I-HGF was only slightly
changed, whereas the heparin-resistant component was significantly
reduced 30 min after injection of excess HGF, followed by gradual
recovery with a half-life of 3.2 h. Because the former clearance mainly
represents 125I-HGF association
with heparan sulfate proteoglycan on the cell surface and/or
extracellular matrix, whereas the latter includes relatively specific
clearance, such as receptor-mediated endocytosis, this result suggests
that injection of excess HGF selectively causes downregulation of
receptor-mediated HGF clearance in the liver. Downregulation could also
be observed for HGF receptor density in isolated liver plasma membrane,
assessed by Western blot analysis by means of anti-receptor antibody,
30 min after injection of excess unlabeled HGF, supporting the
hypothesis that the overall elimination of HGF from the systemic
circulation can be affected by a change in HGF receptor density on the
liver cell surface.
receptor-mediated endocytosis; internalization; heparin; c-Met; pharmacokinetics
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INTRODUCTION |
MANY TYPES OF BIOLOGICALLY ACTIVE POLYPEPTIDES exert
their activity through binding to their own receptors on the cell
surface. The polypeptide receptor is internalized with its ligand after binding to the ligand. This process is termed receptor-mediated endocytosis (RME) (8, 27). RME is responsible for the systemic clearance of many types of polypeptides (27). Thus the polypeptide receptor acts as a clearance receptor as well as a pharmacological receptor (27). Actually, the silent receptor that acts as an elimination mechanism for polypeptides has been identified for atrial
natriuretic factor (16).
In RME, excess ligand in the extracellular space causes the following
two phenomena, which affect the efficiency of receptor-mediated clearance of the ligand. One is the saturation of receptor binding on
the cell surface, and the other is the reduction in the density of the
cell-surface receptors themselves (8). The latter phenomenon is called
downregulation, one of the unique characteristics in RME (2, 8, 18).
Downregulation of cell-surface receptors has been demonstrated for many
types of biologically active polypeptides (2, 4, 17, 18, 21, 23). To
give a detailed description of the pharmacokinetics of polypeptides, it
is necessary to examine the intracellular kinetics of the receptor as
well as the ligand (8).
Changes in growth hormone (GH) receptor density induced by excess GH
have been investigated (17, 18). A single injection of GH induces
downregulation of the GH receptor in the liver, whereas repeated
injections cause very little reduction in receptor density on the cell
surface (18). On the other hand, a continuous infusion of an equal
amount of GH induces upregulation of GH receptors (17). Thus the
receptor density of polypeptides can be affected by the dose of
polypeptides used and also by how the polypeptides are exposed to the
cells. However, little information has been reported that gives a
quantitative analysis of the mechanism for the change in receptor
density induced by exposure to ligand.
Hepatocyte growth factor (HGF) was first discovered as a potent mitogen
for mature hepatocytes and now is recognized as a mitogen, morphogen,
and motogen for a variety of types of epithelial cells (20, 22).
Because HGF exhibits marked pharmacological effects in vivo in animal
models of liver disease (5, 6, 26) and renal failure (9), it is
expected that HGF will be developed as a treatment for certain types of
diseases. We previously suggested, using both in vivo and liver
perfusion systems, that receptor-mediated endocytosis in the liver
contributes to the elimination of HGF from the circulation in rats
(12-15). In addition, we found that there is another mechanism
with a lower affinity for HGF than RME that is also involved in HGF
uptake by the liver (7, 12-15). In an analysis of its nonlinear
pharmacokinetics, the low-affinity component for the plasma clearance
of HGF was identified, and it was shown that it cannot be completely
saturated even when there is a sufficient plasma concentration to
occupy the cell-surface receptor (30). We believe that the heparan sulfate proteoglycan (HSPG) on the cell surface and/or
extracellular matrix is involved in such nonspecific elimination of
HGF, because this substance can bind to HGF (7, 19, 33), and a large amount of cell-surface binding of
125I-labeled HGF can be
dissociated by heparin in perfused rat liver (13).
In the present study, we examined the downregulation induced by
injection of excess HGF on these two types of clearance mechanisms in
the liver. To separately determine the kinetics of the hepatic clearance mediated by such HSPG in vivo, we determined both
heparin-washable and heparin-resistant hepatic uptake clearances
(12-15). In addition, we directly determined the density of the
HGF receptor (protooncogene c-met
product c-Met) on the liver cell surface by use of Western blot analysis to show that HGF receptor density governs the hepatic elimination of HGF.
 |
MATERIALS AND METHODS |
Animals.
Male Wistar rats weighing 250 g (Nisseizai, Tokyo, Japan) were used.
All animals were treated humanely. The studies described in this
article have been carried out in accordance with the Declaration of
Helsinki and with the Guide for the Care and Use of Laboratory Animals
as adopted and promulgated by the National Institutes of Health.
Materials.
Porcine intestinal mucosa heparin was from Sigma (St. Louis, MO);
rabbit polyclonal antibody recognizing the COOH-terminal 21-amino acid
sequence of mouse c-Met, which also cross-reacts with rat c-Met, was
from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit antiserum
recognizing the NH2-terminal
16-amino acid sequence of rat
Na+-dependent
taurocholate-cotransporting polypeptide (Ntcp) (3) was from BioLink
(Tokyo, Japan); human recombinant HGF purified from a culture medium of
C-127 cells transfected with plasmid containing human HGF cDNA (22) was
radiolabeled with 125I-Na by the
chloramine-T method, as described previously (13). The specific
activity of 125I-HGF prepared in
this way was 70-160 Ci/g.
Induction of receptor downregulation.
With rats under light ether anesthesia, excess unlabeled HGF (120 µg/kg body wt) dissolved in saline was administered through the tail
vein. Plasma HGF concentrations were determined by an enzyme
immunoassay (EIA) kit (Institute of Immunology, Tochigi, Japan).
Measurement of tissue uptake clearance of
125I-HGF.
At 10 and 30 min and 1, 2, 3, 6, and 24 h after the induction of
downregulation, a tracer amount of
125I-HGF (5 µCi, 0.6 pmol/kg
body wt) was administered through the femoral vein under light ether
anesthesia. The TCA-precipitable radioactivity in plasma was determined
as described previously (13, 14). At 10 min after intravenous
administration of 125I-HGF, a
piece (100 mg tissue) of the liver was removed by biopsy (14). Eleven
minutes after 125I-HGF
administration, heparin (100 mg/kg body wt) dissolved in saline (1 ml/kg body wt) was injected through the opposite side of the femoral
vein, and plasma samples were collected (14). Four minutes after the
injection of heparin, rats were killed, and the liver, adrenal, spleen,
kidney, lung, duodenum, and muscle were excised (14). Both the liver
biopsy specimen and an aliquot of each tissue were weighed and counted
(14).
The plasma concentration-time profiles of TCA-precipitable
125I-HGF before the injection of
heparin were fitted to the following biexponential equation by use of a
nonlinear iterative least squares method (14)
|
(1)
|
where
Cp is the plasma concentration of
TCA-precipitable radioactivity, A and
B are the corresponding
time 0 intercepts,
and
are the
apparent rate constants, and t is
time.. The plasma clearance
(CLplasma) was calculated as
follows (13)
|
(2)
|
The heparin-resistant component of tissue uptake clearance
(CLuptake,HR) was obtained by
|
(3)
|
where
Xt is the amount of
heparin-resistant radioactivity in the tissue determined after the
heparin injection, and
AUC(0-10) is the area under
the plasma concentration-time curve from time
0 to 10 min, calculated from
|
(4)
|
For the liver, the heparin-washable component of the hepatic
uptake clearance (CLuptake,HW),
which probably represents HGF binding to HSPG on the cell surface
and/or extracellular matrix (14), was calculated from
|
(5)
|
where
CLuptake,HW+HR was determined from
Eq. 3, where
Xt is the radioactivity in the
liver biopsy sample removed before heparin injection.
Calculation of the recovery half-life of tissue uptake clearance.
For the data in the recovery phase, from 30 min to 24 h after induction
of downregulation, the values of
ln[CLuptake,HR(control)
CLuptake,HR(t)]
were plotted vs. time (Sigma-minus plot), where CLuptake,HR(control) represents
the values for CLuptake,HR
determined in the control rats, and
CLuptake,HR(t)
represents the values for
CLuptake,HR determined at
time t after intravenous
administration of an excess of unlabeled HGF. The recovery half-life
[t1/2 (recovery)] was calculated from the slope of the linear regression line of the
Sigma-minus plot as follows (32)
|
(6)
|
Western blot analysis of the HGF receptor in isolated
plasma membrane.
Rat liver plasma membrane was separated as reported (25) and stored as
a solution, 1 mg protein/ml, at
100°C. Each membrane preparation originates from one rat liver, and three independent series
of experiments were performed, each experiment series using one
untreated rat and a rat each at 30 min and 24 h after induction of
downregulation. Marker enzyme activities such as
Na+-K+-ATPase,
acid phosphatase, and glucose-6-phosphatase were determined by the
method of Yachi et al. (31), an assay kit for acid phosphatase (Wako
Pure Chemical Industries, Osaka, Japan), and Yachi et al. (31),
respectively, to check the purity of the prepared plasma membranes. An
aliquot, 16-30 µl, of the stored membrane was centrifuged at
20,000 g (Optima TLX, Beckman
Instruments, Fullerton, CA) at 4°C for 10 min. The supernatant was
discarded, and the precipitate was solubilized with 30 µl of Laemmli
buffer (11). The solubilized sample (10 µl) was subjected to SDS-PAGE
under reducing conditions with a separating gel containing 5%
acrylamide and then was transferred onto Immobilon-P (Millipore,
Tokyo), which was subsequently treated with anti-c-Met antibody or
antiserum recognizing Ntcp. The Immobilon-P was then incubated with an
125I-anti-rabbit immunoglobulin
F(ab')2 fragment (Amersham,
Arlington Heights, IL), and blots were quantified using a Bio-Image
Analyzer (BAS-2000, Fuji Photo Film, Tokyo, Japan). The background
values were subtracted from the amounts measured on the blots. All
values were expressed as a percentage of the immunoreactive amount that came from 10 µg of membranous protein prepared from untreated (control) rats.
 |
RESULTS |
Plasma concentration-time profile of HGF after an intravenous
administration of excess unlabeled HGF as a bolus.
After intravenous administration of unlabeled HGF (120 µg/kg), its
plasma concentration-time profile was determined using EIA (Fig.
1). Plasma HGF rapidly disappeared after
being given intravenously and exhibited concentrations of 145 ± 37, 35.1 ± 5.3, 14.5 ± 1.9, and 4.39 ± 1.01 pM at 30 min and 1, 2, and 3 h after administration (Fig. 1).

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Fig. 1.
Time profiles of plasma hepatocyte growth factor (HGF) concentrations
after iv administration of excess unlabeled HGF (120 µg/kg body wt).
Plasma concentrations were determined using an enzyme immunoassay (EIA)
kit. Each point represents the mean of 3 different rats; SE values were
smaller than symbols for all data points.
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Change in tissue uptake clearance and plasma clearance of
125I-HGF after intravenous administration of
excess unlabeled HGF.
After an intravenous administration of excess unlabeled HGF (120 µg/kg), a tracer amount of
125I-HGF was subsequently given to
determine its tissue uptake clearance and plasma clearance (Figs.
2-4).
At 10 min after the injection of excess HGF, only a slight change in
the CLuptake,HR of
125I-HGF could be observed for the
liver, adrenal, spleen, kidney, lung and duodenum compared with control
levels (the values in untreated rats) (Fig. 2), whereas at 30 min after
injection of excess HGF, the
CLuptake,HR for these organs was
significantly reduced (Fig. 2). The
CLuptake,HR for the liver at 30 min after injection of excess unlabeled HGF was 44% that of the
control level (Fig. 2). The
CLplasma was determined from
TCA-precipitable radioactivity in plasma after injection of a tracer
amount of 125I-HGF and was also
reduced 30 min after injection of excess HGF (Fig. 2). When a tracer
amount of 125I-HGF was
coadministered with excess HGF (120 µg/kg), both
CLplasma and
CLuptake,HR in the liver exhibited
very little reduction and were not significantly different from the
control values (Fig. 2). The
CLuptake,HW in the liver did not
show any significant change either after injection of excess HGF or
after coinjection of excess HGF and the tracer
125I-HGF (Fig. 2).

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Fig. 2.
Time-dependent reduction in tissue uptake clearance of
125I-labeled HGF after iv
administration of excess unlabeled HGF. At 10 (2nd bar) or 30 min (3rd
bar) after iv administration of excess unlabeled HGF (120 µg/kg body
wt), a tracer amount of 125I-HGF
was also administered, and heparin-resistant tissue uptake clearance
(CLuptake,HR) for the indicated
organs, heparin-washable clearance
(CLuptake,HW) for the liver, and
plasma clearance (CLplasma) of
125I-HGF were determined. First
bar, those values obtained without previous injection of unlabeled HGF
(control values); 4th bar, those values obtained after coadministration
of excess HGF with a tracer amount of
125I-HGF, respectively. Values are
means ± SE of 3 rats. * Significantly different from control
values (P < 0.05).
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Fig. 3.
Change in hepatic uptake clearance and plasma clearance of
125I-HGF after iv administration
of excess unlabeled HGF. At specified times after iv administration of
excess unlabeled HGF (120 µg/kg body wt), a tracer amount of
125I-HGF was also administered.
Both CLuptake,HW
(A) and
CLuptake,HR
(B) in liver, and
CLplasma of
125I-HGF
(C) were determined. Each point and
vertical bar represent mean ± SE of 3 different rats.
* Significantly different from control values
(P < 0.05).
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Fig. 4.
Change in tissue uptake clearance of
125I-HGF after iv administration
of excess unlabeled HGF (120 µg/kg body wt). At specified times, a
tracer amount of 125I-HGF was also
administered, and CLuptake,HR of
125I-HGF in extrahepatic organs
was determined. Each point and vertical bar represent mean ± SE of
3 different rats. * Significantly different from control values
(P < 0.05).
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In the liver, the CLuptake,HR
gradually recovered, starting 30 min after injection of excess
unlabeled HGF (Fig. 3). When both the
CLuptake,HW and
CLuptake,HR were calculated per
kilogram body weight values, the sum of these clearances was 85, 89, and 86% of the CLplasma at
baseline and 30 min and 24 h after injection of excess HGF (Fig. 3).
The CLuptake,HR for extrahepatic
organs such as the adrenal, spleen, kidney, lung, and duodenum
exhibited minimum values around 30 min after injection of excess
unlabeled HGF, followed by a gradual recovery (Fig. 4). On the other
hand, the CLuptake,HR for muscle
did not show any clear reduction or subsequent recovery (Fig. 4). The
CLuptake,HR
t1/2 (recovery)
was calculated from Sigma-minus plots shown in Fig.
5. The
t1/2 (recovery)
in the liver was 3.15 h, comparable with the
t1/2 (recovery)
values for the adrenal, spleen, lung and duodenum (3.28, 2.97, 1.85, and 2.65 h, respectively; Fig. 5).

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Fig. 5.
Sigma-minus plots for determination of the recovery half-life
[t1/2 (recovery)]
values of CLuptake,HR. Data shown
in Figs. 3 and 4 were used to calculate the
t1/2 (recovery)
values of CLuptake,HR of
125I-HGF by use of
Eq. 6. Each point and vertical bar
represent mean ± SE of 3 different rats. Half-lives obtained are
shown in parentheses.
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Change in HGF receptor density in isolated plasma membrane from
liver after injection of excess unlabeled HGF.
At 30 min and 24 h after injection of excess unlabeled HGF (120 µg/kg), plasma membranes were isolated from the livers and solubilized to determine their HGF receptor content by Western blot
analysis (Fig. 6). The immunoreactive band
was observed at 140 kDa (Fig. 6A).
This band was reduced 30 min after injection of unlabeled HGF compared
with that in untreated rats [62.1 ± 4.5% (SE) that in
untreated rats of three independent series of experiments; Fig.
6A], followed by its recovery to
the control level at 24 h after the HGF injection (110 ± 19% that
in untreated rats). As a control experiment, Ntcp content in the liver
plasma membranes was also determined (Fig.
6A). The 49-kDa immunoreactive band
was observed, and the change in the intensity of this band was minimal
after the injection of unlabeled HGF (98.5 ± 8.5 and 83.8 ± 12.3% of that in normal liver at 30 min and 24 h after injection of
unlabeled HGF; Fig. 6A). To confirm
the validity of this quantification of receptor density by Western
blotting, the linearity of the intensity of the 140-kDa band was
investigated by varying the amount of membranous protein prepared from
untreated liver and subjected to electrophoresis (Fig.
6B). A significant (P < 0.05) correlation could be
observed between the intensity of the 140-kDa band and the membranous
protein subjected to electrophoresis (Fig.
6B).

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Fig. 6.
Western blot analysis of HGF receptor density in isolated plasma
membranes from liver after ligand-induced downregulation.
A: in an untreated rat
(lanes A and
D) and rats 30 min
(lanes B and
E) and 24 h (lanes
C and F) after iv
administration of excess unlabeled HGF (120 µg/kg body wt), plasma
membrane was isolated from liver and solubilized. Solubilized samples
(10 µg protein) were subjected to SDS-PAGE and then transferred onto
Immobilon-P, which was subsequently treated with anti-c-Met antibody
(lanes A, B, and
C) or
anti-Na+-dependent
taurocholate-cotransporting polypeptide (Ntcp) antiserum
(lanes D, E, and
F), and then with
125I-labeled anti-rabbit
immunoglobulin F(ab')2
fragment. Values shown are typical examples of results, and
reproducible results could also be obtained in another two series of
experiments. B: plasma membrane was
isolated from an untreated rat ( ) and a rat 30 min ( ) after iv
administration of excess unlabeled HGF (120 µg/kg body wt).
Solubilized membranes (0, 5.2, 7.8, and 10 µg protein) were then
subjected to SDS-PAGE and Western blot analysis using anti-HGF receptor
antibody. Intensity of the 140-kDa band was quantified using a
Bio-Image Analyzer and plotted against the amount of membranous protein
applied to SDS-PAGE. Values are means ± SE of 3 different rats
shown as a % of the immunoreactive amount from 10 µg of membranous
protein prepared from an untreated rat. Straight line, linear
regression line (P < 0.05).
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Purity of isolated plasma membranes.
To check the purity of the plasma membranes, we examined the relative
enrichment (ratio of specific activity in membranes to the specific
activity in homogenate) of the marker enzymes for plasma membrane
(Na+-K+-ATPase),
lysosomes (acid phosphatase), and microsomes (glucose-6-phosphatase). The enrichment for
Na+-K+-ATPase
was 71.6 ± 17.0, 89.9 ± 3.1, and 72.6 ± 14.5 (means ± SE of three independent series of experiments) in the control condition
and at 30 min and 24 h after HGF injection, respectively. The
enrichment for acid phosphatase was 1.32 ± 0.07, 1.23 ± 0.17, and 1.34 ± 0.19, whereas that for glucose-6-phosphatase was 1.35 ± 0.16, 1.48 ± 0.14, and 1.11 ± 0.10 in the control
condition and at 30 min and 24 h after HGF injection, respectively.
Thus enrichment of the plasma membrane fraction was much higher than that of the other intracellular organella.
 |
DISCUSSION |
We have previously suggested that the elimination mechanism of HGF from
circulating plasma consists of two components in the liver; one is RME,
and the other is a nonspecific uptake mechanism probably mediated by a
cell-surface HSPG (12-15). If RME is involved in hepatic
clearance, decrease in this clearance should be observed, resulting
from downregulation of HGF receptors on the liver cell surface. In the
present study, we confirmed that excess unlabeled HGF given
intravenously to rats induces receptor downregulation, which affects
both hepatic uptake clearance and plasma clearance of a tracer amount
of 125I-HGF.
HGF receptors on the liver cell surface were downregulated by injection
of excess unlabeled HGF. Such receptor downregulation was confirmed by
Western blot analysis (Fig. 6). The
CLuptake,HR for the liver, a major
clearance organ for HGF (13), was also significantly reduced by an
injection of excess HGF, followed by a gradual recovery, whereas the
CLuptake,HW did not show any clear
change (Fig. 3). We have previously identified two types of cell
surface-associated 125I-HGF in
perfused rat liver after perfusion of tracer concentrations: heparin-washable, and heparin-resistant acid-washable
125I-HGF (13). The former has a
lower affinity for HGF than the latter and may represent
125I-HGF bound to HSPG on the cell
surface and/or extracellular matrix (13). The latter mainly
represents receptor binding of
125I-HGF, because this binding
could be reduced by coperfusion with excess unlabeled HGF (13). The
radioactivity was still present in liver after both heparin and acid
washing and could be partially reduced either by coperfusion with
excess HGF or an inhibitor of RME, phenylarsine oxide; however,
approximately one-half of this radioactivity could not be inhibited
(13). Therefore, HGF is taken up by the liver through both RME and
another nonspecific uptake mechanism, which has a lower affinity for
HGF and cannot be inhibited by either excess HGF or inhibitor (13). On
the basis of these findings, the
CLuptake,HW should reflect
125I-HGF binding to HSPG, whereas
CLuptake,HR should reflect its receptor binding and subsequent internalization in the liver. Therefore, the CLuptake,HR
includes receptor-mediated clearance of
125I-HGF. This is
suggested by the following findings.
1) Only the CLuptake,HR was reduced, whereas
the CLuptake,HW was not
(Fig. 3). 2) HGF receptor density in
the liver plasma membrane, assessed by Western blot analysis, was also
reduced (Fig. 6), and the degree of such reduction was almost
comparable for both CLuptake,HR
and receptor density (44 and 62% of the control level, respectively) 30 min after injection of excess unlabeled HGF. These results suggest
that receptor-mediated clearance in the liver is decreased because of
the receptor downregulation induced by injection of excess unlabeled HGF.
Tajima et al. (30) showed that plasma membranes from liver, adrenal,
spleen, kidney, and lung exhibited specific
125I-HGF binding in rats. Prat et
al. (24) reported that immunoreactive protein against the HGF receptor
antibody was detected in liver and jejunum of humans, although such
protein was undetectable in spleen, kidney, and lung. These reports
imply that the HGF receptor is expressed in these organs. For these
organs in the present study, the
CLuptake,HR showed reduction and
subsequent recovery (Fig. 4). On the other hand, no specific binding of
125I-HGF could be detected for
plasma membrane of muscle (30), and no immunoreactive protein was
detected for skeletal and smooth muscle (24). Therefore, the HGF
receptor density in muscle is undetectable; hence, no clear reduction
was observed for the CLuptake,HR in muscle (Figs. 4 and 5).
After injection of excess unlabeled HGF, both
CLuptake,HW and
CLuptake,HR in the liver were
calculated in terms of kilogram body weight (Fig. 3). The sum of these
values was close to the CLplasma
any time after injection of unlabeled HGF (Fig. 3), suggesting that the
liver is a major clearance organ for HGF after the induction of
receptor downregulation. As indicated in Fig. 3, the reduction and
subsequent recovery of CLplasma
can be attributed to the reduction and recovery of
CLuptake,HR in the liver. Such a
reduction and subsequent recovery of the
CLuptake,HR result from the
downregulation and subsequent recovery of HGF receptors on the liver
cell surface, as we have discussed. Therefore, these results
demonstrate that receptor downregulation in the liver has a critical
effect on the overall elimination of HGF from the circulating plasma.
In the present study, the
CLuptake,HR in the liver was not
significantly reduced at 10 min, whereas a significant reduction occurred 30 min after injection of excess unlabeled HGF (Fig. 2). We
previously reported that the half-life of HGF internalization is
7-11 min in perfused rat liver (14, 15, 29). When excess unlabeled
HGF was injected intravenously as a bolus, most of the cell-surface
receptors can be occupied by HGF and subsequently become internalized,
resulting in a transient reduction in HGF receptor density on the cell
surface (Fig. 6). If the binding and subsequent internalization occur
in such a gradual manner, a gradual reduction in
CLuptake,HR for the liver (Fig. 2)
can be explained.
The CLuptake,HR in the liver,
adrenal, spleen, kidney, lung, and duodenum showed minimum values
around 30 min after injection of excess unlabeled HGF (Figs. 3 and 4).
At this time the plasma concentration of unlabeled HGF was still 145 pM
(Fig. 1). Therefore, the reduction in
CLuptake,HR could be explained if
we assume that any unlabeled HGF remaining in the circulating plasma
competitively inhibits the tissue uptake of
125I-HGF. However, this hypothesis
can be refuted, because the reduction in
CLuptake,HR was minimal when a
tracer amount of 125I-HGF was
simultaneously injected with excess unlabeled HGF (Fig. 2). Although a
slight amount of saturation was observed for the CLuptake,HR in the liver
and CLplasma, the reduction after
coadministration of excess unlabeled HGF was very small and not
significant, compared with the reduction 30 min after injection of
excess unlabeled HGF (Fig. 2). If we assume that the unlabeled HGF
remaining in the circulating plasma competitively inhibits tissue
uptake of a tracer amount of
125I-HGF, the reduction after
coadministration of excess unlabeled HGF should be more marked than
that 30 min after injection of excess HGF, because the remaining HGF
level in circulating plasma was higher after coadministration than at
30 min. In addition, the
CLuptake,HR for the liver and
other extrahepatic organs 10 min after injection of excess unlabeled
HGF was still higher than for those organs at 30 min (Fig. 2). For the
same reason, as we have mentioned, this result also suggests that the
decrease in the CLuptake,HR found
at 30 min cannot be attributed only to competitive inhibition of
125I-HGF uptake by the unlabeled
HGF remaining in the circulation.
We have previously investigated the change in tissue uptake clearance
of a tracer amount of 125I-labeled
epidermal growth factor (EGF) after intravenous administration of
excess unlabeled EGF (32). We here would like to compare the
downregulation of EGF and HGF receptors. According to our analysis, the
t1/2 (recovery)
of the hepatic uptake clearance of
125I-EGF is 22 min, much higher
than that for other extrahepatic organs such as spleen, kidney,
duodenum, and stomach (2-5 h) (32). The major clearance organ for
EGF is also the liver (10). Hepatocytes highly express EGF receptors
(100,000-150,000 sites/cell) (28), and RME via the receptors is
responsible for the hepatic elimination of EGF (10). Also, in
extrahepatic organs such as kidney, spleen, stomach, duodenum, and
jejunum, EGF is taken up by a saturable uptake mechanism representing
RME (10). Therefore, such a rapid recovery of hepatic EGF uptake,
compared with extrahepatic organs, suggests the importance of RME in
the liver as a homeostatic regulator that maintains the level of
circulating EGF. The recruitment of cell-surface receptors is rapid in
the liver so that RME in the liver can quickly recover to maintain the
EGF concentration in plasma at an appropriate level. The liver is also
a major clearance organ for HGF (13). Nevertheless, the
t1/2 (recovery)
for CLuptake,HR in the liver is
3.2 h, comparable with that for extrahepatic organs (1-3 h) (Fig.
5). As we have mentioned, the hepatic clearance mechanism for HGF
involves not only RME but also another nonspecific uptake mechanism,
probably mediated by cell-surface HSPG (12-15). The
receptor-mediated clearance in the liver is decreased not only by
injection of excess unlabeled HGF (Figs. 2 and 3) but also by the
administration of a hepatotoxin such as
CCl4 (15) and by partial
hepatectomy (14). The reduction in receptor-mediated clearance in such
cases also results from receptor downregulation (3, 14, 15). However,
the nonspecific uptake mechanism is not downregulated and is still
functional both after injection of excess HGF (Fig. 3) and after
hepatic damage (14, 15). Because there is an uptake mechanism other
than RME, the HGF level in circulating plasma may be under control even
when the receptor-mediated clearance has not recovered very rapidly
from its downregulation.
The CLuptake,HR at 30 min after
HGF injection was 44% of control (Fig.
3B), whereas the receptor density in
plasma membrane at that time was 62% of control (Fig. 6). One of the
possible explanations for such a slight difference is that, at 30 min
after HGF injection, a fraction of cell-surface receptors was still bound to unlabeled HGF and could not bind to tracer
125I-HGF. This could result in a
reduction in CLuptake,HR; however, such receptors on the cell surface should be detected by Western blot
analysis. Another possible explanation is contamination of the plasma
membrane preparations by intracellular vesicles. Although the
enrichment of
Na+-K+-ATPase
is much higher than that of other marker enzymes, a small degree of
contamination by intracellular vesicles may affect the receptor density
in plasma membranes assessed by Western blot analysis. After the
injection of excess HGF, a fraction of the cell-surface receptors is
internalized, possibly resulting in the increase in receptor density in
intracellular vesicles. Therefore, the impact of such contamination on
the assessment of receptor density in plasma membrane is more marked at
30 min after HGF injection than in control rats. It could also be that
some of the receptors on the cell surface at 30 min after HGF injection were functionally inactive and could not bind to HGF. However, such
"inactive" receptors can still be detected by Western blot analysis and have a molecular mass similar to the active receptor (140 kDa). To examine the validity of a such possibility, equilibrium binding studies and Scatchard analysis with
125I-HGF are necessary. In fact,
we have tried to perform such analysis but could not get a clear
result, probably because of the high degree of nonspecific binding of
125I-HGF to the membranes. Further
studies are needed to clarify whether all of the HGF receptors
remaining in the plasma membrane after the induction of downregulation
are functionally active.
In conclusion, 1) injection of
excess unlabeled HGF induces receptor downregulation in the liver,
resulting in a transient reduction in receptor-mediated clearance,
concomitantly with a transient reduction in plasma HGF clearance, and
2) the recovery half-life of
receptor-mediated uptake is 1-3 h, independent of the organs. The
present study demonstrates that HGF receptors are directly involved in
the systemic clearance of HGF.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: Y. Kato, Dept. of Pharmaceutics,
Graduate School of Pharmaceutical Sciences, Univ. of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Received 16 March 1998; accepted in final form 15 July 1998.
 |
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