Plasma membrane reregionalization in cultured mouse hepatocytes.

THE ANATOMICAL RECORD 214~1-711986)
Plasma Membrane Reregionalization in Cultured
Mouse Hepatocytes
JUN WATANABE, SHINSUKE KANAMURA, AND KAZUO KANA1
Department of Anatomy, Kansai Medical Universtty, Moriguchi, Osaka 570, Japan
ABSTRACT
The cytochemical localization of alkaline phophatase (ALPase) activity and the autoradiographic distribution of glucagon receptors were examined in
the plasma membrane of cultured mouse hepatocytes. After 24 hours of culture,
ALPase activity was exclusively localized on the plasma membrane in areas of cellcell contact, and glucagon receptors were more numerous in the plasma membrane
at the periphery of re-formed cell trabeculae. These results indicate that plasma
membrane regionalization of hepatocytes, lost by cell isolation, reappeared during
culture. The cells maintained this plasma membrane regionalization until 48 hours
of culture. By 72 hours of culture, however, ALPase activity was seen on the external
surface of all regions of plasma membrane, and the glucagon receptors decreased
markedly in number and became scattered in all regions of plasma membrane.
Thus, the re-formed plasma membrane regionalization disappeared in the cells by
72 hours of culture.
In the intact liver, alkaline phosphatase (ALPase) activity is exclusively localized on the bile canalicular
membrane (De Wolf-Peeters et al., 1972; Karasaki, 19751,
and glucagon receptors are more numerous in the sinusoidal membrane (Asada-Kubota et al., 1983). Isolated
hepatocytes, however, lose the polarity of the plasma
membrane, i.e., the plasma membrane regionalization;
ALPase activity (Groothuis et al., 1981) and glucagon
receptors (Watanabe et al., 1984) are present over the
entire cell surface.
During monolayer culture, hepatocytes reconstruct cell
trabeculae and bile canaliculi (Chapman et al., 1973;
Wanson et al., 1977; Miettinen et al., 1978). It is therefore of interest to examine whether hepatocytes also
regain the plasma membrane regionalization of ALPase
activity and glucagon receptors during culture. Little is
known about the localization of ALPase activity in cultured hepatocytes. Jung et al. (1982) reported that the
activity was seen on all regions of the plasma membrane
of rat hepatocytes until 72 hours of culture. In previous
study, we observed high ALPase activity on the plasma
membrane a t the periphery of re-formed cell trabeculae
after 72 or 96 hours of culture (Watanabe et al., 1983).
There have been no reports in the literature on the
distribution of glucagon receptors in cultured hepatocytes.
In the present study, therefore, we examined the distribution of ALPase activity and glucagon receptors in
cultured mouse hepatocytes. The purpose was to determine whether hepatocytes regain the plasma membrane
regionalization under culture conditions.
MATERIALS AND METHODS
Male ddY mice 2-3 months old were used. They had
free access to food and water prior to the experiments.
0 1986 ALAN R. LISS, INC.
Cell Culture
Cells were isolated from livers by perfusion of collagenase solution via the portal vein (Klauning et al.,
1981) under Nembutal anesthesia. The parenchymal
cells were separated from nonparenchymal cells by four
centrifugations at 50g for 1minute at 4°C (Watanabe et
al., 1983, 1984) and suspended in Williams’ medium E
containing 10% calf serum, 1 nM dexamethasone, and
100 nM insulin. One milliliter of the cell suspension (6
x lo5 cells/ml) was poured onto a 35-mm plastic dish
(Falcon, #3001). The cells were cultured as a monolayer
at 37°C under 5% C 0 2 and 95% air. The medium was
changed after 24 and 48 hours.
Cytochemical A LPase Activity
After 12, 24, 48, or 72 hours of culture, hepatocytes
were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) at 4°C for 5 minutes and washed in 0.1
M sodium cacodylate containing 5% sucrose (pH 7.4) a t
4°C for 1 hour. Then, the cells were incubated in the
reaction medium for cytochemical demonstration of ALPase activity (20 mM P-glycerophosphate; 3.9 mM magnesium sulfate; 2.0 mM lead citrate; 250 mM sucrose;
28 mM Tris-HC1 buffer, pH 9.2) (Mayahara et al., 1967)
at 20°C for 30 minutes. As controls, the cells were incubated in the reaction medium containing 10 mM levamisole as a n enzyme inhibitor (Van Belle, 1972)or lacking
(3-glycerophosphate.For light microscopy, the cells were
washed in distilled water, immersed in 1%ammonium
sulfide for 10 seconds, washed again in distilled water,
and mounted in glycerin-jelly. For electron microscopy,
the cells were postfixed in buffered 1%osmium tetroxide
Received February 15, 1955; accepted July 11, 1985
J. WATANABE, S. KANAMURA, AND K. KANA1
2
(pH 7.4) at 4°C for 1hour, dehydrated in graded ethanol
and embedded in Spurr's resin. Thin sections were
stained with uranyl acetate and lead citrate and examined in a Hitachi H-700H electron microscope.
Biochemical ALPase Activity
Cultured hepatocytes were removed from the dishes
by a rubber policeman. The cells were homogenized with
10 volumes of 0.25 M sucrose in a Potter-Elvehjem homogenizer at 2,000 rpm for 2 minutes at 4°C and centrifuged a t 3,OOOg for 10 minutes at 4°C. The supernatant
was used for measurement of ALPase activity. The activity was assayed by a modified Bodansky's method
(Shinowara et al., 1942). Protein was measured by the
method of Lowry et al. (1951). The activity was expressed as nanomoles of phosphorous liberated per milligram of protein per minute.
Glucagon Receptor
Incubation with '25Cglucagon
Hepatocytes (6 x lo5 cells/dish) cultured for 12,24,48,
or 72 hours were incubated in triplicates with 1 ml of
Krebs-Ringer bicarbonate buffer containing 0.2 nM 1251glucagon (specific activity of 94-114 pCi/,ug, NEN, Boston, MA), 0.6 mM bacitracin, and 3% bovine serum albumin a t 37°C for 10 minutes. Nonspecific binding was
estimated by adding 100 pg of unlabeled glucagon to the
medium before the incubation.
Autoradiography
After incubation with l"I-glucagon, hepatocytes were
fixed in 2%) glutaraldehyde in 0.1 M phosphate buffer
(pH 7.4) at 4°C for 1 hour, postfixed in buffered 1%~
osmium tetroxide (pH 7.4) at 4°C for 1hour, dehydrated,
and embedded in Spurr's resin. Semithin and ultrathin
sections were cut from the Spurr blocks. For light microscopy, semithin sections (1 pm in thickness) were
attached to slide glasses, stained with methylene blue,
coated with evaporated carbon (5 nm in thickness), and
coated with Sakura NR-HZ emulsion by a dipping
method. For electron microscopy, ultrathin sections were
placed on collodion-coated copper grids, stained with
Fig 1. Phase-contrast micrographs of hepatocytes cultured for a1 24
and b) 72 hours. Note polyhedral hepatocytes forming wide cell trabeculae. ~ 4 0 0 .
Fig. 2. Light micrographs of hepatocytes cultured and treated with
alkaline phosphataae (ALPase) reaction. a ) Cultured for 12 hours. The
staining reaction for ALPase activity is seen on the entire surface of
the cells. However, intense reaction is observed in the intercellular
space between aggregated cells (arrowhead). ~ 4 0 0 bl
. Cultured for 24
hours. The staining reaction is seen in the intercellular space between
adjoining cclls. ~ 4 0 0c)
. Cultured for 24 hours. Hepatocytes incubated
in the reaction medium lacking 8-glycerophosphate. No reaction is
seen in the cells except for slight staining in round cells. x400. d)
Cultured for 48 hours. The staining reaction is observed i n the intercellular space between adjoining cells. x400. e ) Cultured roo,. 7 2 hours.
Moderate to weak reaction for ALPase activity is seen in the intercellular space between adjoining cells and on the upper free surface of the
cells. However, intense reaction is occasionally seen a t the periphery
of cell traheculae (arrowheads). x400.
PLASMA MEMBRANE IN CULTURED HEPATOCYTES
Fig. 3. Electron micrographs of portions of hepatocytes cultured and
treated with ALPase reaction. a) Cultured for 24 hours. Horizontal
section. The reaction product is localized in the narrow intercellular
space between adjoining cells (arrowheads). There is very little or no
reaction product on the plasma membrane at the periphery of cell
trabeculae (P). x 16,600. b,c) Cultured for 24 hours. b) Horizontal and
c) vertical sections. The reaction product is observed in the narrow
intercellular space between adjoining cells (C), but very little or no
3
reaction product is present on the bile canalicular membrane (BC)and
the plasma membrane adjoining the dish (D). b, ~ 9 , 0 0 0c,; x 12,000.
d,e) Cultured for 48 hours. d) Horizontal and e) vertical sections. The
reaction product is localized in the narrow intercellular space between
adjoining cells (arrowheads) and bile canalicular membrane (BC,, hut
very few or no reaction product is present on upper free surface of the
cells (FSI. The reaction product is occasionally seen also on the plasma
membrane adjoining the dish (D). d, x 8,600; e, x 7,000.
4
J. WATANABE, S. KANAMURA, AND K. KANA1
uranyl acetate, coated with evaporated carbon, and
coated with the emulsion by a wire-loop method (Caro et
al., 1962). Then these specimens were exposed at 4°C for
6 weeks and developed with Konidol X.
Grain counting
Four independent cultures were used per each time
point. Eight Spurr blocks (2 blocks/culture) were chosen
at random. Sixteen horizontal sections cut from eight
blocks (2 sectionsiblock) were prepared for light microscopic autoradiography as described above. Four photographs taken at ~ 5 0 0from each autoradiographic
section were enlarged to a final magnification x3,400.
An area in each photograph covered about 3,360 pm2.
Then, two of four photographs were chosen at random.
The length of plasma membrane was measured by the
point-counting method (Weibel, 1979). The number of
grains associated with plasma membrane (within 0.5
pm from the plasma membrane) was counted in the
same photograph. The values were expressed as number
of grains per unit length (1pm) of plasma membrane in
each region.
Assay of '251-glucagonbound to the cells
Cultured hepatocytes were incubated with 0.2 nM '"1glucagon as described above. The cells were washed four
times with 0.25 M sucrose, removed from the dishes by
a rubber policeman, and suspended in 1 ml of 0.25 M
sucrose. Then radioactivity of the cells was assayed by
an auto gamma counter (Packard, 5330).
RESULTS
About 99%.of the cells used for culture were parenchymal cells as determined by phase-contrast microscopy.
On the basis of trypan blue exclusion, about 90% of the
cells were viable. Twelve hours after plating, hepatocytes adhered to the bottom of dish, and some of the cells
formed short cell trabeculae. After 24,48, or 72 hours of
culture, hepatocytes formed wide and flattened cell trabeculae (Fig. la,b). A few round cells were seen on the
upper free surface of the flattened polyhedral cells.
For the facility of describing the results, the plasma
membrane of the cultured cells was divided into four
regions; 1)upper free surface, 2) periphery of trabeculae,
3) portion in cell to cell contact and bile canaliculus, and
4)portion adjoining to the dish.
Cytochemical ALPase Activity
Light microscopy
After 12 hours of culture, the staining reaction for
ALPase activity was generally observed on the entire
surface of hepatocytes (Fig. 2a). However, the reaction
was not seen on the upper free surface, but was present
in the intercellular space between some of the cells
forming short cell trabeculae. After 24 or 48 hours of
culture, the reaction became exclusively observed in the
intercellular space between adjoining cells (Fig. 2b,d).
After 72 hours of culture, however, both the intercellular space and upper free surface showed moderate to
weak reaction (Fig. 2e). On the other hand, moderate to
intense reaction was sometimes seen at the periphery of
cell trabeculae, as reported in our previous paper (Watanabe et al, 1983).
In the cells incubated in the reaction medium containing levamisole or lacking 0-glycerophosphate, no stain-
ing reaction was observed in the cells except for round
cells lying on the upper free surface of the main sheet of
cells (Fig. 2c).
Electron microscopy
The reaction product for ALPase activity was mainly
localized on the external surface of all regions of the
plasma membrane except that adjoining the dish after
12 hours of culture. However, abundant reaction product
was occasionally seen in the narrow intercellular space
between aggregated cells, i.e., on the plasma membrane
in cell-cell contact. After 24 or 48 hours of culture, the
reaction product was almost exclusively localized on the
plasma membrane in cell-cell contact (Fig. 3a-e). Reformed bile canalicular membranes showed irregular
reaction; some displayed an intense reaction and others
showed weak or no reaction (Fig. 3b-e). After 72 hours
of culture, the reaction product was observed on the
external surface of all regions of the plasma membrane.
The plasma membrane adjoining the dish was devoid of
the reaction product after 12 or 24 hours of culture (Fig.
3c), and displayed only slight reaction product after 48
or 72 hours of culture (Fig. 3e).
Biochemical ALPase Activity
ALPase activity in hepatocytes cultured for 12, 24, or
48 hours was similar to that in freshly isolated hepatocytes (Fig. 4). The activity, however, increased significantly in the cells cultured for 72 hours.
Protein contents of the cells were constant until 72
hours of culture (mg per d i s h means k S.D. for five
experiments: 1.08 & 0.12, isolated cells; 1.01 k 0.10, 12
hours; 0.98 t 0.13,24 hours; 1.04 k 0.15, 48 hours; 0.94
0.18, 72 hours).
T
o
12
24
48
TIME IN CULTURE
72
(HOURS)
F g 4.Changes in ALPase activity duringculture Vnlucs a r e means
i S.U. for f i v e experiments. :'.P < .05: significantly different from
value of freshly isolated hepatocytes tStudent's t-test).Pi. phosphorous.
PLASMA MEMBRANE IN CULTURED HEPATOCYTES
5
Flg. 5 Light microscopic (a-di a n d electron microscopic ie.11 autoradiohvaphs of cultui-ed mouse hepatocytes incubated with 0.2 nM '"1~
glucagon a t 37°C for 10 minutes. a,bi Cultured tor ai 12 and hi 24
hours. Plasma-membrane-associated hvains are numerous 'IL t h c periphery of cell trabeculae. x 1,000. ci Cultured for 48 hours. M a n y
g a i n s are localized a t the peripherv of cell trabeculae, but some grains
a r c present in the intercellular space hetween adjoining cells. x 1,000.
di Cultured lor 72 hours. The grains decrease markedly In number.
~ 1 , 0 0 0ei
. Ciiltiired for 24 hours. The gyains are numerous in the
plasma membrane a t the periphery ofcell trabeculae (Pi. A fen p a i n s
are seen in the narrow interccllular space between aghyegated cells
iC). ~ 6 , 3 0 011
. Cultured for 48 hours. Some grains become seen also i n
t h e plasma membrane in cell-cell contact tCi. x 7,000.
Glucagon Receptor
Grain counting
The values are summarized in Table 1.The number of
plasma-membrane-associated grains was significantly
greater in region I (the plasma membrane a t the periphery of cell trabeculae and upper free surface) than in
region I1 (the plasma membrane in cell-cell contact and
bile canalicular membrane) in the cells cultured for 12,
24, or 48 hours. After 72 hours of culture, however, no
difference was seen between region I and region 11.
Autoradiography
About 30% of the cell-associated labels were internalized. However, we paid attention to the plasma-membrane-associated grains. After 12 or 24 hours of culture,
developed grains associated to the plasma membrane
were mainly seen at the periphery of cell trabeculae and
upper free surfaces (Fig. 5a,b,e). Very few grains were
seen a t the plasma membrane in cell-cell contact or at
bile canaliculi. After 48 hours of culture, although many
grains were still seen at the periphery of cell trabeculae,
more grains became present in the plasma membrane
in cell-cell contact (Fig. 5c,f). After 72 hours of culture,
the grains decreased markedly in number, and were
scattered a t the periphery of cell trabecuale and at regions of cell-cell contact (Fig. 5d). There were a few
grains after 12,24, and 48 hours of culture and no grains
after 72 hours of culture in the plasma membrane adjoining the dish.
Radioactivity Associated With the Cells
The amounts of l"I-glucagon bound t o the cells after
12, 24, or 48 hours of culture were similar to that of
freshly isolated hepatocytes (Fig. 6). After 72 hours of
culture, however, the binding decreased markedly.
DISCUSSION
In the intact liver, hepatocytes maintain plasma membrane regionalization (sinusoidal, lateral, and bile can-
6
J. WATANABE, S. KANAMURA, AND K. KANA1
TABLE 1. Distribution of cell-surface grains in cultured mouse hepatocytes'
Time in
culture
(hours)
12
E
C
No. of surface bp-ains per cell
circumference (1 um)
No. of surface
grains per photograph
(3,356 pm2)
Region I
Region I1
Total
Region I
Region I1
75.0
(4.1)
11.8
(7.1)
0.170
(0.023)
0.072
(0.031)
9.8
(2.2)
5.4
(1.3)
0.029
(0.006)
0.021
(0.004)
91.5
(11.0)
'3
0.195
(0.053)
(0.008)
Region I
Specific
Region I1
Significance
0.141
(0.019)
0.050
(0.028)
P < .05
0.159
(0.041)
0.047
(0.005)
P < .01
0.122
(0.032)
0.078
(0.014)
P < .05
0.038
(0.008)
0.031
(0.010)
NS
24
E
-)
.2
0.062
1.9)
0.033
(0.009)
0.015
(0.004)
88.0
(8.6)
32.3
(4.1)
0.147
(0.038)
0.085
(0.017)
9.5
(3.1)
6.9
(2.4)
0.015
(0.009)
0.008
(0.004)
E
29.3
(2.5)
16.7
(4.4)
0.049
(0.012)
0.036
(0.O 13)
C
9.2
(2.3)
4.5
(2.0)
0.011
(0.005)
0.006
(0.002)
C
10.4
(2.4)
48
E
n
CI
72
'Hepatocytes were incubated with 1251-glucagon(El or '"I-glucagon plus excess amounts of unlabeled glucagon (0.
No. of grains in region I
(the plasma membrane a t the periphery of cell trabeculae and a t the upper free surface) and region I1 (the plasma membrane in cell-cell contact
and bile canaiicular membrane) were counted as described in t h e text. Values a r e means (?S.D.) for four independent cultures. Student's ttest was used for comparison of values between region I and region 11. NS, not significant.
0
12
24
48
TIME I N CULTURE
72
(HOURS)
alicular sites) (De Wolf-Peeters et al., 1972; Karasaki,
1975; Wisher and Evans, 1977; Groothuis et al., 1981;
Asada-Kubota et al., 1983). However, the plasma membrane regionalization i s lost in freshly isolated hepatocytes (Groothuis et al., 1981; Wantanabe et al., 1984).
After 24 hours of culture, as revealed in the present
study, ALPase activity becomes exclusively localized in
the plasma membrane in cell-cell contact, and glucagon
receptors are more numerous in the plasma membrane
a t the periphery of cell trabeculae. Thus, hepatocytes
regain plasma membrane regionalization of ALPase and
glucagon receptor during 24 hours of culture, although
the plasma membrane in cell-cell contact may be not
equivalent to bile canalicular and lateral membrane in
hepatocytes of the intact liver.
The plasma membrane regionalization of ALPase and
glucagon receptor was maintained in the cells until 48
hours of culture, but disappeared after 72 hours of culture. Thus, the plasma membrane regionalization reestablished a t early culture time was lost after longer
culture. Ichihara et al. (1980) found that protein synthesis, insulin receptors, and 5'-nucleotidase activity in hepatocytes, damaged by cell isolation, recovered during
culture for a few davs but disawweared after longer culture. The recovery-and disappearance of the plasma
membrane regionalization during culture, observed in
the present
might be
to the results of
Ichihara et al. (1980).
I
Fig. 6. Changes of '""I-glucagon binding to hepatocytes during c u l ~
ture. The cells were incubated with 0.2 nM '"I-glucagon a t 37°C for
10 minutes. Nonspecific binding (7-16%) is subtracted. Values are
means
S.D. for five experiments. * P < .05: significantly different
from value of freshly isolated hepatocytes (Student's t-test).
I
U~~
~
~
PLASMA MEMBRANE IN CULTURED HEPATOCYTES
7
hepatocytes of intact mouse liver. An autoradiographlc study. Exp.
There have been few studies on cytochemical ALPase
Pathol.. 23:95-101.
activity in cultured rat hepatocytes. Jung et al. (1982)
M.N., and D.S. Friend (19691High-yield preparation of isolated
L activity
~ localized
~
~ in all
~ regions of the Berry,
reported A
ra t liver parenchymal cells. A biochemical and fine structural
plasma membrane of hepatocytes cultured for 24 or 72
study, J. cell Biol., 43,.506-52~,
hours. In the present results, ALPase activity was exclu- Caro, L.G., R.P. Van Tubberghen, and J.A. Roll (196'1 High resolution
autoradiogaphy. 1. Methods. J. Cell Biol., 15.173-188.
sively localized in the plasma membrane in cell-cell conG.S., A.L. Jones, U.A. Meyer, a nd D.M. Bisscl(1973l Parentact after 24 or 48 hours of culture. ~h~
for the Chapman,
chymal cells from adult r a t liver in nonproliferating monolayer
discrepancy between Our finding and that Of Jung et al.
culture. 11. Ultrastructural studies, J.Cell Biol., 59735-747.
is unknown.
.DeWolf-Peeters, C., R. De Vos, and V. Desmet (1972)Electron microscopy a nd histochemistry of canalicular differentiation in fetal and
Berry and Friend (1969), Drochmans et al, (1975) and
neonatal rat liver. Tissue Cell, 4r379-388.
Penasse et
(1979) reported that
hepatocytes Drochmans,
P., J.C. Wanson, and R. Mosselmans (19751Isolated and
lost the former polarity Of the plasma membrane; the
subfractionation on ficoll gradients of adult rat hepatocytes. Size,
cells were covered uniformly with short microvilh. Durmorphology, and biochemical characteristics of cell fractions. J.
Cell Blol., 66:1-22.
ing primary monolayer culture for a few days, the cells
Groothuis, G.M., C.E. Hulstaert, D. K a llc hxa n, and M.J. Hardonk
attached to each other and formed cell trabeculare
11981)Plasma membrane specialization and intracellular polarity
(Chapman et al., 1973; Wanson e t al., 1977; Miettinen et
offl-eshiy isolated rat hepatocytes. ELir. J. Cell B i d , 26.43-51.
al., 1978). Wanson et al. (19771, Miettinen et al. (19781, Ichihara, A., T. Nakamura, K. Takada, Y Tomita, K. Anoyama, S.
Kato, a nd H. Shinno (19801 Biochemical functions of adult r a t
and Jung e t ale (1982) suggested that cell to cell attachhepatocyles in primary culture. Ann. N.Y. Acad Sci., 34!1:77-84.
merit was important for the maintenance Of
Jung,W., R. Gebhardt, a nd D. Mecke (19821Alteration in activity and
functions Of the plasma membrane and Of the plasma
ultrastructural localization of several phosphatases on the surfacc
of adult r a t hepatocytes in primary monolayer culture. Eui- J . Cell
membrane regionahzation in hepatocytes. Also in the
Blol., 27t230-241.
present study, the plasma membrane regionalization of
s. 11975) Cell proliferation and subcellular localization of
A
L and ~glucagon
~ receptors
~
~ occurred in parallel with Karasaki,
alkaline phosphatase activity in rat liver parenchyma during azo
the reestablishment of cell to cell attachment during
dve carcinotrenesis, cancer
R ~ ~~ 5, : ,~ 8 3 - 4 9 1 ,
Klau"ning, J.E.:'P.J. Goldblatt, D.E. Hilton, M.M. Lipsky, J . Chacku,
culture.
and B.F. Trump (1981, Mouse liver cell culture. 1. Hepatocyte
Wanson et al. (1977), Miettinen et al. (1978,, and Robisolation. In Vitro, 17:913-925.
enek et al. (1982) reported reestablishment of focal tight
O H . N.J. Rosebrough, A.L. Far!., and R.J. Randall i 1951)
junctions a t the plasma membrane in cell-cell contact Lowry,
Protein measurement with the Folin phcnol reagent. J. B i d . Chem.,
during monolayer culture of rat hepatocytes. In the re193365-275.
sults of their experiments, hepatocytes cultured as a Mavahara, H., H. Hirano, T. Saito, a nd K. Ogawa (1967, 'The nvw lead
citrate method for the ultracytochemlcal dcmonstration if activity
monolayer could not reestablish complete sealing of bile
of non-specific alkaline phosphatase rortho-phosphoric monoester
canaliculi by tight junctions. We previously found that
phosphohydrolase,. Histochenne, 11:88-96.
hepatocytes began to be separated from neighboring Micttinen, A,, I. Virtanen, a nd E. Lindncr (19781Cellular actin and
junction formation during rcaggregatlon of adult rat hepatocytes
cells and culture substratum after 96 hours of culture
into epithelial cell sheets J Cell Sci., 33:341-353.
(Watanabe et al., 1983).Thus, tight junctions appear to
Penasse,
W., D. Bernaert, K. Mosselmans, J:C. Wanson, and P. Drochbreak down after longer culture. It may be therefore
mans i 19791 Scanning electron microscopy of adult rat hrpatocytes
possible that early in culture newly formed tight juncin situ, after isolation of pure fractions by elutriation and after
tions exclude glucagon receptors from the plasma memculture. Biol. Cell., 34:175-186.
brane in cell-cell contact, but later the barriers break Robenek, H.. W. J ung, and R. Gebhardt 11982)The topography of
filipin-cholesterol complexes in the plasma membrane of cultured
down and the receptors float into the plasma membrane
hepatocytes a nd their relation to cell junction (iirmation. J . Cltrain cell-cell contact. Similarly, i t may be that ALPase
struct. Re-:, 78:95-106.
activity is initially localized to the plasma membrane in Shinowara, C.Y., L.M. Jones. and H.L. Reinhart I 19421The estimation
of serum inorganic phosphate and "acid" and "alkalinc~" phosphacell-cell contact and becomes free to move to other memtase activity. J. Biol. Chem., 142921-933.
brane areas with time of culture.
K., M. Sato, Y. Toniitii. a nd A. Ichiliala 19781i'hochemical
Tanaka et al. (1978) reported that protein synthesis Tanaka,
studies on liver functions in primary culturcd hcpatocytes of' adult
increased in hepatocytes until 5 days of culture. In the
rats. 1 Hormonal effects on cell viabillty and protein syntheslh. J.
Biocheni., 84:937-946.
present results, however, protein content per dish did
not change significantly until 72 hours of culture. There- Van Belle, H. (1972) Kinetics and inhihition of alkaline pho3phatase
from canine tissues. Biochim Biophys. Acta, 289:158-168.
fore, the present results suggest that the increase in the Wanson.
J.-C., P. Drochmans, R. Mosselmans, and M.F. Konveaux
biochemical ALPase activity is not due to loss of protein
(1977)Adult rat hepatocytes in primary monolayer culture. Ultrastructural
characteristics oC intercellular contacts and cell memrelative to a n unchanged ALPase amount. However, it
brane differentiations. J . Cell Biol., 745358-877.
is unknown whether the increasing ALPase activity is
J.. S. Kanamura, M. Asada-Kubota, K. Kanai, and M. Oka
due to de novo synthesis of ALPase molecules. On the Watanabe,
(1983) Observation of cytochemical alkaline phosphatase activity
other hand, we previously found that very few or no
on the plasma membrane of culturcd rat hepatocytes by hackscattered electron imaging. Anat. Rec., 207r635-542.
glucagon receptors were synthesized in rat hepatocytes
cultured under the same conditions (Watanabe et al., Watanabe, J., S. Kanamura, M. Asada-Kubota, K. Kanai, a nd M. Oka
(1984)Receptor-mediated endocytosis of glucagon in isolated mouse
1985). This may be the reason why bound glucagon
hepatocptes. Anat. Rec., 210:557-567.
decreased in number in hepatocytes cultured for 72 Watanabe, J., S. K a na mura , K. Kanai, M. Asada-Kubota, and M. Oka
hours. Therefore, the changes in glucagon receptor dis11985) Inhibitory effect of colchicine a nd vinblastine on transport
of glucagon receptors to the plasma membrane in cultured rat
tribution during culture is probably due to the reshufhepatocytes. J. Endrocrinol., 106t125-131.
fling of the receptors that were present a t the time of Weibel,
E.R. 11979) Stereologicul Methods 1. Academic Prcss;,London,
culture initiation.
nn 76-27
rr.
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'I
LITERATURE CITED
Asada-Kubota, M., J. Watanabe, S. Kanamura, K. Kdnai, and M.
Yoshikawa (1983)Binding and internalization of '".-'I-glucagon in
Wisher, M.H., and W.H. Evans (1975)Functional polarity of rat hepatocyte surface membrane. Biochem. J., 146:375-388.