Vol.
174,
No.
January
2, 1991
BIOCHEMICAL
AND
RESEARCH
BIOPHYSICAL
COMMUNICATIONS
Pages
31, 1991
EFFECT
928-933
OF CELL DENSITY ON INDUCTION OF GROWTH HORMONE RECEPTORS BY DEXAMETHASONE IN PRIMARY CULTURED RAT HEPATOCYTES' Shingo
Division
of
Institute
Takao
Niimi*. Biological
of
Hayakawa,
Chemistry
Hygienic
Received
December
22,
and
Akira
158,
Tanaka
Biologicals.
1-18-1
Sciences, Tokyo
and
National
Kamiyoga.
Setagaya-ku,
Japan
1990
Summary:
The effect of cell density on the regulation of growth (GH) receptors was studied by measuring specific binding ~ypi” IlhGH to primary cultured hepatocytes with or without dexamethasone, which induces GH receptors. In cell cultures without dexamethasone, the cell density did not affect the level of binding of labeled GH appreciably. On the other hand, in the presence of dexamethasone, which induced an increase in the level of GH receptors on the4cells. GH-binding by cultured cells at low cell density (3. 3 x 10 cells/cm2) was about one-third of that of cells at high cell density (lo5 cells/cm2). Scatchard plot analysis showed that the cell-density dependent change in induction of GH binding by dexamethasone was due to change in the number of binding sites without significant change in their affinity. The binding capacity of g,jucocorticoid receptors, measured as specific binding of 1 Hldexamethasone to the hepatocytes, was not significantly affected by cell density. These results suggest that cell density modulates GH receptor induction by dexamethasone via events after glucocorticoid receptor binding. 0 1991 Academic Press, Inc.
Growth liver
hormone
(l-6).
These
interaction are
of
several
change
in
the to
Recently, receptors
’ This Research *To
whom
clarify
work from
was the
presumably
cell
the its
the
found on
actions
factors
that
various
supported Ministry
in part of Health should
be
growth
rat
928
responses
addressed.
in
initiated GH are
the
by
receptors,
and
the there
controlled
Therefore,
by it
seems
GH receptors. regulate
the
hepatocytes
by a Grant and Welfare
hormone.
$1.50
Copyright 0 1991 by Academic Press. Inc. Ali right.s of reproduction it? arr~ form reserved.
regulate
hormones
cultured
human
of (7-9).
primary
: hGH.
surface
receptors
that
correspondence
Abbreviation 0006-291X/91
of
biological
are
specific that
level we
various
responses
GH with
reports
important GH
induces
of
level (10):
for Scientific Japan.
of
Vol.
174,
No.
2, 1991
BIOCHEMICAL
dexamethasone number
and
of
Many known
to
be rat
various
hormones
functions
whereas
several
only
at
changes have
of
cell
and
examined
whether
of
cells.
cell
hormones
functions regulated are
and
growth
cell
Our
(13,18).
results by
and
growth
factors.
and density
primary as well
low
that
the
23). level
highly
growthexpressed dependent
rat
hepatocytes cell-plasma
In
this
of
extent
increased
are
(11-151,
cell-cell, the
dexamethasone
some
cell-density
cultured
affects
:
density are
(13,
showed
hepatocytes
isolated by cell
m
factor
rat
(11-22)
of
the
glucagon.
These
mediated
density
induced
growth
at
interactions
cell
epidermal
functions
be
cell-matrix
in
only
functions
to
increase
factors
induced
density
suggested
membrane these
various
are
various
been
GH receptors
by
COMMUNICATIONS
glucagon plus
cultured
differentiated
higher
and
primary
hepatocytes
related
insulin
of cell
RESEARCH
plus
dexamethasone
regulated some
cultured by
by
functions
Interestingly, as
and
increase
cell
BIOPHYSICAL
dexamethasone
GH receptors, the
suppress
AND
work,
we
GH receptors of
with
increase
in
increase
in
density.
MATERIALS
AND METHODS
The materials used for cell i$olation and culture were as reported by Tanaka et al. (24). [ HlDexamethasone (49. 9 Ci/mmol) Other materials were as was obtained from New England Nuclear. reported previously (10). Parenchymal hepatocytes were isolated from adult male Wistar strain rats (180-2OOg) by perfusion of the liver in situ with ase (24). Isolated hepatocytes were cultured, and binding ; ; 1 ‘;1Y? IlhGH to the cells was measured as reported previously (10). Binding of [‘Hldexamethasone to the cells was measured by the methods of Griffin et al. (25) and Kondo et al. (26) with slight the cells in 30-mm plastic modification. After 18 h of culture, Modified Eagle’s medium dishes were washed with Dulbecco’s cgntaining 25 mM HEPES. pH 7. 4, and then incubated with 2 nM [ Hldexamethasone for 45 min at 37 ‘C in lml of the same medium. solution The cells were then washed first with cold Hanks’ albumin and then with phoshate containing 0. 2% bovine serum buffered saline and solubilized in 1 N NaOH. The samples were then neutralized and then radioactivity was determined in Nonspecific binding was Aquasol(New England Nuclear). determined in the presence of lOOO-fold excess of unlabeled Specific binding was defined dexamethasone in parallel cultures. total binding as the difference between and nonspecific binding. Protein was measured by the method of Bradford (27).
RESULTS Figure binding
1 shows of
[12511hGH.
the
effect In
this
AND DISCUSSION of
cell
experiment.
929
densities the
on cells
the specific were seeded
Vol.
174,
No.
2
01
BIOCHEMICAL
2, 1991
0
4
Cell
6
density
8
AND
BIOPHYSICAL
10
RESEARCH
0
(x 104 cells/c&
2
02
12
4
COMMUNICATIONS
6
Bound
hGH
8
10
(fmol/mg
72
14
protein)
a 1, Effects of cell density on specific binding of C12511hCH. Experimental conditions were as reported previously (10). The cells were cultured at various cell densities with 1 nM insulin and incubated with (0) for 18 h and without (0) dexamethasone nM) for 24 h and 8 h, respectively. Specific binding of ;t%S IIhCH to the cells was calculated as cpm/mg protein. Values are sho n as percentages of the specific binding at 10 cell /cm E in the presence of 100 nM dexamethasone, which was 4. 3 x 10 5 cpm/mg protein. Values are means for duplicate dishes in two experiments. Fia. 2_ Scatchard plot analysis of cell-deln24ty dependent change in increase in specific binding of C IlhCH induced by dexamethasone. The cells were cultured at low and high density with 1 nM insulin for 18 h and incubated h 100 nM dexamethasone for 1 day. Various concentrations of [w15k IlhGH were used in binding experiments. Other experimental conditions were as reported previously (10). Slopes of plots wer determined b 2 (0, the leas squares method. (0) 3. 3 x IO4 cells/cm 10 x 10 x 8 cells/cm .
at
various
and
8
densities. h
further
for
and
without
with
respectively.
In
almost
cultured
equal
at
cultures
cell
104
cells/cm2,
but
5.0
104
cells/cm2.
After
of
binding
was
because
of
the
cultured
for
induces
increase
the of
cell the
density cells:
dexamethasone
low
tended
24
h in had
induction increased
105
to
for
in
more
the
of
a marked of
low
effect
increase with
of
increase 930
on in
on
the
the in
densities, cells
the
GH binding
GH binding cell
below
measurement
cell the
to
were
dexamethasone,
GH receptors
h
was
of
8 h,
When
presence
binding
(subconfluent)
densities
than
at
24
dexamethasone,
2 at
binding.
level
for
the
less
especially
the
nM
cells/cm
be
culture
specific
incubated
dexamethasone,
of
difficult,
h and 100
without
densities
6. 6 x x
18
density,
which
cells
(lo), capacity
capacity
by being
Vol.
174,
No.
2, 1991
BIOCHEMICAL
lowest
at
a density
higher
at
subconfluent
Figure
2
binding
densities.
of
single
a
hepatocytes calculated
capacity
(3.3
x
cells/cm2)
at
or
low
109.
the
plots
were
of
binding
about
three
times
3.73
3 times
(lo5
The
dexamethasone-dependent
that
at
increase
of
these
low
cell
in
Ka
values same,
that
11. 94
of
the
of
fmol/mg
receptors
being
1. 5
density
the
plots. density
whereas
the
on cell
capacities
was
cell
the hGH
binding
cells
high
different
protein,
were
at for
two
cells/cm2)
higher. show
the and
fmol/mg
densities
results
sites
of
specific
indicating
intercepts
receptor
density cell
linear,
at
and
in
dexamethasone
(Ka)
slopes the
was
high
These
COMMUNICATIONS
changes
with
constants of
about
and
the
dexamethasone
subconfluent
protein, at
and
of
treated
Both
from
binding
cells
cells
affinity
The
104
plots
with
The
RESEARCH
cells/cm2,
class
treated
densities. were
to
cell
presence
x lo4
Scatchard
1125 IlhGH
low
3.3
BIOPHYSICAL
density.
shows
of
and
of
AND
MM1
x
regulates
number
of
GH
binding
sites. We next the
examined
level
change
of in
r3Hl
whether
GH
receptors to
after
x
lo4
receptors
by
to the
increase
of
GH
The at
various
is
not
Probably by
due
were
binding at
density
to of
densities cell
(average,
cell
due
bindings
Specific
cells/cm2
receptors
specific
significantly
of
was
in
densities
4. 0 x increase
to
modulation
density
dexamethasone
at
of
dpm/mg
103
on
cell
of
of
of
affects a
GH the
level
after
binding.
The
present
study
dexamethasone showed
was
that
sites
on
was
inhibited cell
showed regulated
primary by
on
dependent
binding
capacity
markedly
affected
density
on
GH is
by
by
known
to
(1, 2)
and
increase vitro
and
of
of that
by of
The
protein.
the seems
increase effect
dexamethasone
new
Therefore,
we binding
this
dexamethasone
receptors
by
hGH
D (10).
GH receptors synthesis
receptors Previously
number
actinomycin
density. GH
GH
density. the
without
of
action LQ
the
cultures cell
in
hepatocytes of
on
level the
cell
and
increase in
the
with
rat
cycloheximide
also
increase
increased
cultured
density
connected
that
dexamethasone
probably
viva
lo5
effect
receptors.
receptor
in
vary
change
dexamethasone
culture.
dexamethasone
glucocorticoid
by
seeded
h not
cells/cm2 Thus
of
cells
18 did
protein).
density-dependent
receptors.
dexamethasone
C3Hldexamethasone
cell
induced
glucocorticoid
measured
3. 3
this
is The
was effect to
be
of
GH not cell
closely
glucocorticoid. the
level
(28).
of
Interestingly,
931
IGF-I
mRNA IGF-I
in
rat liver decreases
Vol.
174,
in
No.
BIOCHEMICAL
2, 1991
liver
tissue reduces
liver
(30-32).
cells
hepatectomy to
may
reduction
several
of by
increases
are
caused
cell-cell
m
and
suggest such
-~in that
a way
higher The
that
intercellular
by also
(6,33,34).
of
These may
GH regulated
the
shown
regulated
the
be
cultured
and
02u
our
present
by
are
to their
rat
differentiated
and
functions
hand,
and
typical
facts
due
other
been
of
albumin
be
GH receptors
have
induces
of
partial
hepatocytes density
of
after
On
liver. liver
the
contact
IGF-I of
cultured
synthesis
GH receptors
mechanism by
of
regulate
the
was
in
glucocorticoid to
clarify
of is
not
increase
after
effect
dexamethasone
dexamethasone
required
GH the
of
Partial
globulin findings
cell
highly
in
density
in
expressed
at
density.
receptors
event
as
vitro
cell
of
in
affected
COMMUNICATIONS
(29).
reduction
contact
(13,181.
functions,
by
functions
also
such
and decrease
dexamethasone
hepatocytes
of
junctions
be
RESEARCH
hepatectomy
Therefore,
differentiated
increased
may
gap
well
BIOPHYSICAL
partial
after
hepatectomy
AND
cell
unknown.
affected
As
by
cell
GH receptors
by
receptor the
density
mechanism
binding.
on
we
increase
found
density, dexamethasone Further
of
that
GH
binding
cell
density via
studies
some are
involved.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Mathews, L.S., Norstedt, G. & Palmiter, R.D. (19861 Proc. Natl. Acad. Sci. USA 83, 9343-9347. Roberts Jr, C.T., Brown A.L.. Graham, D.E., Seelig. S., Berry K.H. & Rechler, M.M. (1986) J. Biol. Chew. 261, s. , Gabby, 10025-10028. P. & Gustafsson. J-i. Norstedt. G., Mode, A., Eneroth, (1981) Endocrinology 108, 1855-1861. Baxter, R.C., Zaltsman, Z. & Turtle. J.R. (1984) Endocrinology 114, 1893-1901. Norstedt, G., Andersson, G. & Gustafsson, J-i. (1984) Endocrinology 115, 672-680. Chatterjee, B., Demyan. W.F. & Roy, A.K. (1983) J. Biol. Chem. 258, 688-692. Maes, M., De Hertogh, R.. Watrin-Granger. P. & Ketelslegers J.M. (1983) Endocrinology 113, 1325-1332. Maes, M., Underwood, L.E. & Ketelslegers, J.-M. (1983) J. Endocrinol. 97 243-252. Su, T.. Liu, W.. Han, S., Jansen, M., Yang-Fen, T.L., P'eng, F. & Chou, C. (1989) Cancer Res. 49, 1773-1777. Niimi, S., Hayakawa, T. & Tanaka, A. (19901 Endocrinology 127, 688-694. Michalopoulos, G., Cianciulli, H.D., Novotny, A.R., Kligerman. A. D. , Strom, S. C. & Jirtle, R.L. (1982) Cancer Res. 42, 4673-4682. Nakamura, T.. Tomita, Y. & Ichihara, A. (1983) J. Biochem. (Tokyo) 94, 1029-1035. Nakamura, T., Yoshimoto. K., Nakayama, Y.. Tomita, Y. & Ichihara, A. (1983) Proc. Natl. Acad. Sci. USA 80, 7229-7233. 932
Vol. 174, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Voss. A., Reinhart. G., Hogan. S. & Roehrig, If. (198'71 Biochea. Blophys. Rea. Commun. 14g 118-124. R.R. & HcCune, S.A. (1985) J. Cell. Physiol. 123 15, Jurin, 442-448. C.D. 8 Baxter, R.C. (19871 J. Cell. Phyaiol. 133 53216. Scott, 14.
538.
17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
30. 31. 32. 33. 34.
Schleifer. L.S., Black. I.B. & Reid. L.M. (1989) J. Cell. Physiol. 140 53-58. Noda, C., Ohguri. M. 8 Ichihara. A. (1990) Biochee. Biophys. Res. Commun. 168, 335-342. Edwards, A, It.. Lucas, C. M. & BaddamE. H.H. (1987) Carcinogenesis 8 1837-1842. 0 Jensen, E., Fror&n. C.H. % Nilsson. A. (1985) Biochim. Biophys. Acta 834 279-283. Jensen, E., Fror&n, C.H. & Nilsson. !. (1987) Biochim. Biophys. Acta 917 74-80. Jensen, E., Hansson, P., Fror;?n, C.H., Nilsson, ii. & Nilsson-Ehle. P. (1989) Horm. Wetab. Res. 21 4-7. Ben-Ze'ev. A., Robinson, G.S., Bucher. N.L.R. & Farmer, S.R. (1988) PFOC. Natl. Acad. Sci. USA 85, 2161-2165. Tanaka, K., Sato. M., Tomita, Y. Ih Ichihara, A. (1978) J. Biochem. (Tokyo) 84. 937-946. Griffin, J.E.. Punyashtiti, K. & Wilson, J.D. (1976) J. Clin. Invest. 57 1342-1351. Kondo, H., Kasuga, H. L Nomura. T. (1985) Exp. Cell Res. 158 342-348. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Norstedt, G. & Moller, C. (1987) J. Endocrinol. 115, 135-139. Russel, W.E.. D'Ercole. A.J. & Underwood. L.E. (1985) Am. J. Physiol. 128, E618-E623. Yee. A. G. & Revel, J.-P. (1978) J. Cell Biol. 78 554-564. Meyer, D.J.. Yancey. S.B.. Revel, J.-P. & Peskoff. A. (1981) J. Cell Biol. 91 505-523. Traub, O., Driige, P.M. & Willecke, K. (1983) Proc. Natl. Acad. Sci. USA 80 755-759. Motwani. N.M., Unakar. N. J. & ROY, A. K. (1980) Endocrinology 107. 1606-1612. Keller, G.H. 8 Taylor, J.M. (1979) J. Biol. Chem. 254, 276278.
933
174,
No.
January
2, 1991
BIOCHEMICAL
AND
RESEARCH
BIOPHYSICAL
COMMUNICATIONS
Pages
31, 1991
EFFECT
928-933
OF CELL DENSITY ON INDUCTION OF GROWTH HORMONE RECEPTORS BY DEXAMETHASONE IN PRIMARY CULTURED RAT HEPATOCYTES' Shingo
Division
of
Institute
Takao
Niimi*. Biological
of
Hayakawa,
Chemistry
Hygienic
Received
December
22,
and
Akira
158,
Tanaka
Biologicals.
1-18-1
Sciences, Tokyo
and
National
Kamiyoga.
Setagaya-ku,
Japan
1990
Summary:
The effect of cell density on the regulation of growth (GH) receptors was studied by measuring specific binding ~ypi” IlhGH to primary cultured hepatocytes with or without dexamethasone, which induces GH receptors. In cell cultures without dexamethasone, the cell density did not affect the level of binding of labeled GH appreciably. On the other hand, in the presence of dexamethasone, which induced an increase in the level of GH receptors on the4cells. GH-binding by cultured cells at low cell density (3. 3 x 10 cells/cm2) was about one-third of that of cells at high cell density (lo5 cells/cm2). Scatchard plot analysis showed that the cell-density dependent change in induction of GH binding by dexamethasone was due to change in the number of binding sites without significant change in their affinity. The binding capacity of g,jucocorticoid receptors, measured as specific binding of 1 Hldexamethasone to the hepatocytes, was not significantly affected by cell density. These results suggest that cell density modulates GH receptor induction by dexamethasone via events after glucocorticoid receptor binding. 0 1991 Academic Press, Inc.
Growth liver
hormone
(l-6).
These
interaction are
of
several
change
in
the to
Recently, receptors
’ This Research *To
whom
clarify
work from
was the
presumably
cell
the its
the
found on
actions
factors
that
various
supported Ministry
in part of Health should
be
growth
rat
928
responses
addressed.
in
initiated GH are
the
by
receptors,
and
the there
controlled
Therefore,
by it
seems
GH receptors. regulate
the
hepatocytes
by a Grant and Welfare
hormone.
$1.50
Copyright 0 1991 by Academic Press. Inc. Ali right.s of reproduction it? arr~ form reserved.
regulate
hormones
cultured
human
of (7-9).
primary
: hGH.
surface
receptors
that
correspondence
Abbreviation 0006-291X/91
of
biological
are
specific that
level we
various
responses
GH with
reports
important GH
induces
of
level (10):
for Scientific Japan.
of
Vol.
174,
No.
2, 1991
BIOCHEMICAL
dexamethasone number
and
of
Many known
to
be rat
various
hormones
functions
whereas
several
only
at
changes have
of
cell
and
examined
whether
of
cells.
cell
hormones
functions regulated are
and
growth
cell
Our
(13,18).
results by
and
growth
factors.
and density
primary as well
low
that
the
23). level
highly
growthexpressed dependent
rat
hepatocytes cell-plasma
In
this
of
extent
increased
are
(11-151,
cell-cell, the
dexamethasone
some
cell-density
cultured
affects
:
density are
(13,
showed
hepatocytes
isolated by cell
m
factor
rat
(11-22)
of
the
glucagon.
These
mediated
density
induced
growth
at
interactions
cell
epidermal
functions
be
cell-matrix
in
only
functions
to
increase
factors
induced
density
suggested
membrane these
various
are
various
been
GH receptors
by
COMMUNICATIONS
glucagon plus
cultured
differentiated
higher
and
primary
hepatocytes
related
insulin
of cell
RESEARCH
plus
dexamethasone
regulated some
cultured by
by
functions
Interestingly, as
and
increase
cell
BIOPHYSICAL
dexamethasone
GH receptors, the
suppress
AND
work,
we
GH receptors of
with
increase
in
increase
in
density.
MATERIALS
AND METHODS
The materials used for cell i$olation and culture were as reported by Tanaka et al. (24). [ HlDexamethasone (49. 9 Ci/mmol) Other materials were as was obtained from New England Nuclear. reported previously (10). Parenchymal hepatocytes were isolated from adult male Wistar strain rats (180-2OOg) by perfusion of the liver in situ with ase (24). Isolated hepatocytes were cultured, and binding ; ; 1 ‘;1Y? IlhGH to the cells was measured as reported previously (10). Binding of [‘Hldexamethasone to the cells was measured by the methods of Griffin et al. (25) and Kondo et al. (26) with slight the cells in 30-mm plastic modification. After 18 h of culture, Modified Eagle’s medium dishes were washed with Dulbecco’s cgntaining 25 mM HEPES. pH 7. 4, and then incubated with 2 nM [ Hldexamethasone for 45 min at 37 ‘C in lml of the same medium. solution The cells were then washed first with cold Hanks’ albumin and then with phoshate containing 0. 2% bovine serum buffered saline and solubilized in 1 N NaOH. The samples were then neutralized and then radioactivity was determined in Nonspecific binding was Aquasol(New England Nuclear). determined in the presence of lOOO-fold excess of unlabeled Specific binding was defined dexamethasone in parallel cultures. total binding as the difference between and nonspecific binding. Protein was measured by the method of Bradford (27).
RESULTS Figure binding
1 shows of
[12511hGH.
the
effect In
this
AND DISCUSSION of
cell
experiment.
929
densities the
on cells
the specific were seeded
Vol.
174,
No.
2
01
BIOCHEMICAL
2, 1991
0
4
Cell
6
density
8
AND
BIOPHYSICAL
10
RESEARCH
0
(x 104 cells/c&
2
02
12
4
COMMUNICATIONS
6
Bound
hGH
8
10
(fmol/mg
72
14
protein)
a 1, Effects of cell density on specific binding of C12511hCH. Experimental conditions were as reported previously (10). The cells were cultured at various cell densities with 1 nM insulin and incubated with (0) for 18 h and without (0) dexamethasone nM) for 24 h and 8 h, respectively. Specific binding of ;t%S IIhCH to the cells was calculated as cpm/mg protein. Values are sho n as percentages of the specific binding at 10 cell /cm E in the presence of 100 nM dexamethasone, which was 4. 3 x 10 5 cpm/mg protein. Values are means for duplicate dishes in two experiments. Fia. 2_ Scatchard plot analysis of cell-deln24ty dependent change in increase in specific binding of C IlhCH induced by dexamethasone. The cells were cultured at low and high density with 1 nM insulin for 18 h and incubated h 100 nM dexamethasone for 1 day. Various concentrations of [w15k IlhGH were used in binding experiments. Other experimental conditions were as reported previously (10). Slopes of plots wer determined b 2 (0, the leas squares method. (0) 3. 3 x IO4 cells/cm 10 x 10 x 8 cells/cm .
at
various
and
8
densities. h
further
for
and
without
with
respectively.
In
almost
cultured
equal
at
cultures
cell
104
cells/cm2,
but
5.0
104
cells/cm2.
After
of
binding
was
because
of
the
cultured
for
induces
increase
the of
cell the
density cells:
dexamethasone
low
tended
24
h in had
induction increased
105
to
for
in
more
the
of
a marked of
low
effect
increase with
of
increase 930
on in
on
the
the in
densities, cells
the
GH binding
GH binding cell
below
measurement
cell the
to
were
dexamethasone,
GH receptors
h
was
of
8 h,
When
presence
binding
(subconfluent)
densities
than
at
24
dexamethasone,
2 at
binding.
level
for
the
less
especially
the
nM
cells/cm
be
culture
specific
incubated
dexamethasone,
of
difficult,
h and 100
without
densities
6. 6 x x
18
density,
which
cells
(lo), capacity
capacity
by being
Vol.
174,
No.
2, 1991
BIOCHEMICAL
lowest
at
a density
higher
at
subconfluent
Figure
2
binding
densities.
of
single
a
hepatocytes calculated
capacity
(3.3
x
cells/cm2)
at
or
low
109.
the
plots
were
of
binding
about
three
times
3.73
3 times
(lo5
The
dexamethasone-dependent
that
at
increase
of
these
low
cell
in
Ka
values same,
that
11. 94
of
the
of
fmol/mg
receptors
being
1. 5
density
the
plots. density
whereas
the
on cell
capacities
was
cell
the hGH
binding
cells
high
different
protein,
were
at for
two
cells/cm2)
higher. show
the and
fmol/mg
densities
results
sites
of
specific
indicating
intercepts
receptor
density cell
linear,
at
and
in
dexamethasone
(Ka)
slopes the
was
high
These
COMMUNICATIONS
changes
with
constants of
about
and
the
dexamethasone
subconfluent
protein, at
and
of
treated
Both
from
binding
cells
cells
affinity
The
104
plots
with
The
RESEARCH
cells/cm2,
class
treated
densities. were
to
cell
presence
x lo4
Scatchard
1125 IlhGH
low
3.3
BIOPHYSICAL
density.
shows
of
and
of
AND
MM1
x
regulates
number
of
GH
binding
sites. We next the
examined
level
change
of in
r3Hl
whether
GH
receptors to
after
x
lo4
receptors
by
to the
increase
of
GH
The at
various
is
not
Probably by
due
were
binding at
density
to of
densities cell
(average,
cell
due
bindings
Specific
cells/cm2
receptors
specific
significantly
of
was
in
densities
4. 0 x increase
to
modulation
density
dexamethasone
at
of
dpm/mg
103
on
cell
of
of
of
affects a
GH the
level
after
binding.
The
present
study
dexamethasone showed
was
that
sites
on
was
inhibited cell
showed regulated
primary by
on
dependent
binding
capacity
markedly
affected
density
on
GH is
by
by
known
to
(1, 2)
and
increase vitro
and
of
of that
by of
The
protein.
the seems
increase effect
dexamethasone
new
Therefore,
we binding
this
dexamethasone
receptors
by
hGH
D (10).
GH receptors synthesis
receptors Previously
number
actinomycin
density. GH
GH
density. the
without
of
action LQ
the
cultures cell
in
hepatocytes of
on
level the
cell
and
increase in
the
with
rat
cycloheximide
also
increase
increased
cultured
density
connected
that
dexamethasone
probably
viva
lo5
effect
receptors.
receptor
in
vary
change
dexamethasone
culture.
dexamethasone
glucocorticoid
by
seeded
h not
cells/cm2 Thus
of
cells
18 did
protein).
density-dependent
receptors.
dexamethasone
C3Hldexamethasone
cell
induced
glucocorticoid
measured
3. 3
this
is The
was effect to
be
of
GH not cell
closely
glucocorticoid. the
level
(28).
of
Interestingly,
931
IGF-I
mRNA IGF-I
in
rat liver decreases
Vol.
174,
in
No.
BIOCHEMICAL
2, 1991
liver
tissue reduces
liver
(30-32).
cells
hepatectomy to
may
reduction
several
of by
increases
are
caused
cell-cell
m
and
suggest such
-~in that
a way
higher The
that
intercellular
by also
(6,33,34).
of
These may
GH regulated
the
shown
regulated
the
be
cultured
and
02u
our
present
by
are
to their
rat
differentiated
and
functions
hand,
and
typical
facts
due
other
been
of
albumin
be
GH receptors
have
induces
of
partial
hepatocytes density
of
after
On
liver. liver
the
contact
IGF-I of
cultured
synthesis
GH receptors
mechanism by
of
regulate
the
was
in
glucocorticoid to
clarify
of is
not
increase
after
effect
dexamethasone
dexamethasone
required
GH the
of
Partial
globulin findings
cell
highly
in
density
in
expressed
at
density.
receptors
event
as
vitro
cell
of
in
affected
COMMUNICATIONS
(29).
reduction
contact
(13,181.
functions,
by
functions
also
such
and decrease
dexamethasone
hepatocytes
of
junctions
be
RESEARCH
hepatectomy
Therefore,
differentiated
increased
may
gap
well
BIOPHYSICAL
partial
after
hepatectomy
AND
cell
unknown.
affected
As
by
cell
GH receptors
by
receptor the
density
mechanism
binding.
on
we
increase
found
density, dexamethasone Further
of
that
GH
binding
cell
density via
studies
some are
involved.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Mathews, L.S., Norstedt, G. & Palmiter, R.D. (19861 Proc. Natl. Acad. Sci. USA 83, 9343-9347. Roberts Jr, C.T., Brown A.L.. Graham, D.E., Seelig. S., Berry K.H. & Rechler, M.M. (1986) J. Biol. Chew. 261, s. , Gabby, 10025-10028. P. & Gustafsson. J-i. Norstedt. G., Mode, A., Eneroth, (1981) Endocrinology 108, 1855-1861. Baxter, R.C., Zaltsman, Z. & Turtle. J.R. (1984) Endocrinology 114, 1893-1901. Norstedt, G., Andersson, G. & Gustafsson, J-i. (1984) Endocrinology 115, 672-680. Chatterjee, B., Demyan. W.F. & Roy, A.K. (1983) J. Biol. Chem. 258, 688-692. Maes, M., De Hertogh, R.. Watrin-Granger. P. & Ketelslegers J.M. (1983) Endocrinology 113, 1325-1332. Maes, M., Underwood, L.E. & Ketelslegers, J.-M. (1983) J. Endocrinol. 97 243-252. Su, T.. Liu, W.. Han, S., Jansen, M., Yang-Fen, T.L., P'eng, F. & Chou, C. (1989) Cancer Res. 49, 1773-1777. Niimi, S., Hayakawa, T. & Tanaka, A. (19901 Endocrinology 127, 688-694. Michalopoulos, G., Cianciulli, H.D., Novotny, A.R., Kligerman. A. D. , Strom, S. C. & Jirtle, R.L. (1982) Cancer Res. 42, 4673-4682. Nakamura, T.. Tomita, Y. & Ichihara, A. (1983) J. Biochem. (Tokyo) 94, 1029-1035. Nakamura, T., Yoshimoto. K., Nakayama, Y.. Tomita, Y. & Ichihara, A. (1983) Proc. Natl. Acad. Sci. USA 80, 7229-7233. 932
Vol. 174, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Voss. A., Reinhart. G., Hogan. S. & Roehrig, If. (198'71 Biochea. Blophys. Rea. Commun. 14g 118-124. R.R. & HcCune, S.A. (1985) J. Cell. Physiol. 123 15, Jurin, 442-448. C.D. 8 Baxter, R.C. (19871 J. Cell. Phyaiol. 133 53216. Scott, 14.
538.
17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
30. 31. 32. 33. 34.
Schleifer. L.S., Black. I.B. & Reid. L.M. (1989) J. Cell. Physiol. 140 53-58. Noda, C., Ohguri. M. 8 Ichihara. A. (1990) Biochee. Biophys. Res. Commun. 168, 335-342. Edwards, A, It.. Lucas, C. M. & BaddamE. H.H. (1987) Carcinogenesis 8 1837-1842. 0 Jensen, E., Fror&n. C.H. % Nilsson. A. (1985) Biochim. Biophys. Acta 834 279-283. Jensen, E., Fror&n, C.H. & Nilsson. !. (1987) Biochim. Biophys. Acta 917 74-80. Jensen, E., Hansson, P., Fror;?n, C.H., Nilsson, ii. & Nilsson-Ehle. P. (1989) Horm. Wetab. Res. 21 4-7. Ben-Ze'ev. A., Robinson, G.S., Bucher. N.L.R. & Farmer, S.R. (1988) PFOC. Natl. Acad. Sci. USA 85, 2161-2165. Tanaka, K., Sato. M., Tomita, Y. Ih Ichihara, A. (1978) J. Biochem. (Tokyo) 84. 937-946. Griffin, J.E.. Punyashtiti, K. & Wilson, J.D. (1976) J. Clin. Invest. 57 1342-1351. Kondo, H., Kasuga, H. L Nomura. T. (1985) Exp. Cell Res. 158 342-348. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Norstedt, G. & Moller, C. (1987) J. Endocrinol. 115, 135-139. Russel, W.E.. D'Ercole. A.J. & Underwood. L.E. (1985) Am. J. Physiol. 128, E618-E623. Yee. A. G. & Revel, J.-P. (1978) J. Cell Biol. 78 554-564. Meyer, D.J.. Yancey. S.B.. Revel, J.-P. & Peskoff. A. (1981) J. Cell Biol. 91 505-523. Traub, O., Driige, P.M. & Willecke, K. (1983) Proc. Natl. Acad. Sci. USA 80 755-759. Motwani. N.M., Unakar. N. J. & ROY, A. K. (1980) Endocrinology 107. 1606-1612. Keller, G.H. 8 Taylor, J.M. (1979) J. Biol. Chem. 254, 276278.
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