From the
The sodium-dependent glucose transporter SGLT1 is expressed on
the apical plasma membrane of fully differentiated enterocytes.
Recently, we have found that the cotransport function appears gradually
during the process of differentiation of the human intestinal
epithelial cell clone HT-29-D4. However, the SGLT1 protein was detected
in both undifferentiated and differentiated HT-29-D4 cells suggesting
that sodium-glucose cotransport was dependent on post-translational
events controlling the efficient targeting of the protein in the plasma
membrane. In the present study, we have analyzed the molecular
mechanisms controlling the functional expression of the SGLT1 protein
during the course of HT-29-D4 differentiation. We show that the
appearance of the cotransport function in the apical membrane is
blocked by 1-5-isoquinolinesulfonyl)-2-methylpiperazine-HCl
(H-7), a potent inhibitor of protein kinase C activity. Moreover, H-7
treatment was associated with an unability of HT-29-D4 cells to
organize into a polarized monolayer of differentiated cells.
Reciprocally, short term treatment (15 min) of undifferentiated cells
by 0.1 µM phorbol myristyl acetate resulted in the
appearance of the cotransport function. In contrast, inhibition of cAMP
and cGMP-dependent protein kinases by
N-(2-guanidinoethyl)-5-isoquinolinesulfonamide-HCl did not
prevent the development of sodium-glucose cotransport during the
differentiation of HT-29-D4 cells. In addition, stimulation of
cAMP-dependent protein kinases by 8-Cl-cAMP did not induce the
cotransport function in undifferentiated HT-29-D4 cells. By using
immunogold labeling at the electron microscopy level, we demonstrated
that phorbol myristyl acetate induced the redistribution of SGLT1
protein from intracellular sites to the plasma membrane. In conclusion,
our data show that the appearance of a functional sodium-glucose
cotransporter in HT-29-D4 cells is controlled, at least in part, by
intracellular pathways regulated by the activity of protein kinase C.
The study of transport mechanisms across the intestinal
epithelium has been considerably facilitated by the availability of
several cultured epithelial cell lines with differentiation
characteristics mimicking enterocytic maturation
(1, 2) .
Among them, the HT-29-D4 clonal cell line possesses the advantage to
undergo differentiation following a simple alteration of the culture
medium, that is, the replacement of glucose by galactose
(3) .
This differentiation process occurs without cell loss nor progressive
adaptation, so that the differentiated cells can be considered as the
true counterpart of undifferentiated ones
(4) . HT-29-D4 cells
differentiated in galactose medium (HT-29-D4 gal) exhibit
morphological, biochemical, and electrophysiological properties of
fully differentiated intestinal absorptive
cells
(3, 4, 5, 6, 7) , including
the presence of a functional sodium-glucose cotransporter (SGLT1)
located in the apical membrane (8). Interestingly, undifferentiated
HT-29-D4 cells (HT-29-D4 glu) are unable to absorb glucose using this
pathway. This property is dependent on the efficient plasma membrane
targeting of the cotransporter, since the SGLT1 protein was expressed
in both HT-29-D4 gal and glu cells, but remained inside
undifferentiated cells.
In the present
study, we have analyzed the role of protein kinases in the mechanims
associated with the appearance of a functional sodium-glucose
cotransporter in the plasma membrane. Our results demonstrate that
protein kinase C is involved in the morphogenesis of the differentiated
epithelial phenotype associated with a functional sodium-glucose
cotransport. Moreover, we show that stimulation of this kinase by
phorbol myristyl acetate (PMA)
To investigate the role of protein kinases during the
functional differentiation of HT-29-D4 cells, we first tested the
influence of the protein kinase C inhibitor H-7 on the cotransport
function. In these experiments, HT-29-D4 glu cells were switched to the
galactose medium in either the absence or presence of 30
µM H-7 (i.e. 5-fold the value of the
k
These
data would suggest that the inhibition of protein kinase C activity
affect either the expression of the SGLT1 protein or its cellular
localization. Therefore, we have analyzed by Western blot the presence
of the protein in cells treated or not by the H-7 inhibitor. Using
affinity-purified antibodies directed against a nonadecapeptide
(Ser
In this study, we demonstrate the influence of protein kinase
C on the establishment of a functional state of differentiation in the
human epithelial intestinal cell clone HT-29-D4. One of the main
characteristic of this differentiation process is the organization of
the cells into a polarized epithelial monolayer which absorbs glucose
using the sodium-glucose cotransport pathway. This cotransport function
is specifically expressed by differentiated HT-29-D4 cells, and it
gradually appears when undifferentiated cells are switched in a
differentiating medium. The establishment of this
differentiation-associated function does not seem to be regulated at
the level of gene or protein level since the SGLT1 protein was detected
in both HT-29-D4 glu and HT-29-D4 gal cells.
The main result of the present study
is that an inhibitor of protein kinase C (H-7) is able to disturb the
organization of HT-29-D4 cells into a differentiated epithelial
monolayer and to prevent the appearance of the cotransport function.
Moreover, short term treatments of undifferentiated HT-29-D4 cells by
PMA, a potent protein kinase C activator, was sufficient to induce the
expression of the cotransport function. These data reinforce the
hypothesis of the involvement of protein kinase C in the establishment
of a functional state of differentiation in HT-29-D4 cells. This is
consistent with a previous report showing that the expression of SGLT1
cotransporter in the LLCPK-1 cell line was inhibited by
H-7
(19, 20) . In our study, the possible implication of
protein kinase A in the regulation of the cotransport function has been
evaluated by two ways. On the one hand, treatment of HT-29-D4 cells by
HA-1004 (an inhibitor of protein kinase A) did not prevent the
appearance of the function. The only effect of this inhibitor was a
delay in the detection of the sodium-dependent AMG uptake during the
course of HT-29-D4 differentiation. On the other hand, the cotransport
function could not be detected following short term treatments of
HT-29-D4 glu cells by 8-Cl-cAMP. These data suggest that protein kinase
A is not involved in the early events leading to the establishment of a
functional state of differentiation. In contrast, protein kinase C was
found to play a critical role throughout the process of epithelial
differentiation of HT-29-D4 cells. Therefore, the HT-29-D4 cell line
clearly differs from LLCPK-1 since in the later, an increase in cAMP
was associated with the development of the differentiated transport
mechanisms
(21) . Moreover, in Madin-Darby canine kidney cells,
the exocytosis of vacuolar apical compartment, which consists of large
vacuoles containing apical membrane proteins, was efficiently
stimulated by 8-Br-cAMP, while PMA induced only a modest exocytic
response
(22) .
At the morphological level, the inhibition of
protein kinase C activity was associated with an unability of the cells
to organize into a polarized epithelium. Obviously, the expression of
differentiated functions is highly dependent on the supracellular
organization of epithelial cells. For this reason, it is difficult to
separate the effect of protein kinase C on morphological versus functional parameters of HT-29-D4 differentiation. The fact that
the cotransporter protein was expressed in both untreated and
H-7-treated cells is consistent with the involvement of protein kinase
C in the post-translational events leading to the correct targeting of
the SGLT1 protein in the apical plasma membrane. According to these
results, the activation of protein kinase C by short term treatments
with PMA elicited the appearance of the cotransport function in
HT-29-D4 glu cells. This function was detectable after 15 min of PMA
treatment, which strongly suggests that the effect was not dependent on
de novo protein synthesis. By using immunocytochemical
techniques at the electron microscopy level (immunogold labeling), we
demonstrated that PMA induced the translocation of the SGLT1 protein
from intracellular sites to the plasma membrane in HT-29-D4
undifferentiated cells. Interestingly, similar data were recently
reported for the
In conclusion, our data show that
protein kinase C regulates protein plasma membrane targeting and thus
plays an important role in the morphogenesis and the functional
differentiation of intestinal epithelial cells in vitro.
(
)
(
)
induces the
occurrence of SGLT1 function in undifferentiated HT-29-D4 cells. In
contrast, stimulation of cAMP-dependent protein kinases had no effect
under the same experimental conditions. We conclude that the efficient
targeting of the sodium-glucose cotransporter is modulated by protein
kinase C.
Materials
1-(5-Isoquinolinesulfonyl)-2-methylpiperazine-HCl
(H-7) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide-HCl
(HA-1004) were purchased from France Biochem (Meudon, France).
-Methyl-D-glucopyranoside (AMG) was purchased from Sigma.
[U-
C-
-Methyl]D-glucopyranoside was
from Du Pont de Nemours (France). Phlorizin was obtained from Aldrich
Chimie (France) and prepared as a 100 mM stock solution in
Me
SO.
Cell Culture
HT-29-D4 cells were routinely grown
in 75-cm flasks (Falcon) in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) containing 25
mM glucose and supplemented with 10% fetal calf serum,
penicillin (40 units/ml) and streptomycin (40 µg/ml). To induce
differentiation, half-confluent HT-29-D4 cells were grown in
glucose-free Dulbecco's modified Eagle's medium
supplemented with 5 mM galactose and 10% dialyzed fetal calf
serum. Cells cultured in galactose medium will be referred to as
HT-29-D4 gal cells. Cells grown in glucose medium will be referred to
as HT-29-D4 glu cells.
AMG Uptake Measurements
Confluent monolayers of
HT-29-D4 cells were washed in modified Earle's solution (B
medium: 137 mM NaCl, 5.36 mM KCl, 0.4 mM
NaHPO
, 0.8 mM MgCl
, 1.8
mM CaCl
, 20 HEPES adjusted to pH 7.4 with NaOH).
Then, cells were washed twice in sodium-free B medium (137 mM
choline chloride instead of NaCl and 0.4 mM
K
HPO
instead of Na
HPO
adjusted to pH 7.4 with KOH). The cells were incubated at 37
°C, with 0.1 mM
C-labeled AMG (0.15
µCi/ml) in B medium or in sodium-free B medium. At the end of the
incubation, the medium was removed and the monolayer was washed three
times with 1 ml of B medium or sodium-free B medium at 4 °C. Cells
were disrupted in 0.5 ml of 0.1 N NaOH, 0.1% SDS, and the
radioactivity was determined using a Packard
counter. The results
were expressed as nanomoles of AMG/milligram of proteins. The protein
content was evaluated using a Pierce kit and bovine serum albumin as
standard.
Membrane Preparations
HT-29-D4 cells grown in
75-cm flasks were washed in B medium containing 5.5
mM glucose and scraped in the same medium using a rubber
policeman. Cells were pelleted for 7 min at 150
g and
were homogenized in a glass Teflon Potter homogenizer (10 strokes), in
a hypotonic solution (10 mM HEPES, 1 mM EDTA, 0.2
mM phenylmethylsulfonyl fluoride) at 4 °C. The homogenate
obtained was diluted in 1 M sucrose to obtain a final
concentration of 0.25 M sucrose and was spun at 150
g for 10 min. The supernatant was centrifuged at 15,000
g for 30 min, and the resulting membrane pellet was
resuspended in the centrifugation medium and stored at -80
°C.
Western Blot Analysis
A sequence-specific
(Ser-Lys
) antibody was prepared against a
synthetic peptide (19 amino acids) corresponding to the known amino
acid sequence of the sodium glucose transporter
(9) . A cysteine
was added to the NH
-terminal end to link the peptide to
keyhole limpet hemocyanin, and antibodies were raised in rabbit as
described previously
(8) . Specific IgG were isolated from serum
using affinity chromatography. Electrotransfer of 10 µg of membrane
proteins from 7.5% SDS-polyacrylamide gel electrophoresis to
nitrocellulose membrane (Hybond-ECL, Amersham) was carried out for 2 h
at 100 V according to Towbin et al.(10) . After
transfer, the membrane was preincubated overnight at 4 °C in
blocking buffer (5% non-fat milk and 0.05% Tween 20 in PBS, pH 7.4) to
reduce nonspecific binding. The membrane was then probed with
affinity-purified rabbit IgG in blocking buffer. After 1 h of
incubation, the nitrocellulose paper was washed with 0.05% Tween 20 in
PBS for 30 min, then incubated with anti-rabbit IgG peroxidase-coupled
antibodies (1/5000, Amersham) for 1 h in blocking buffer.
Peroxidase-loaded proteins were revealed using the ECL detection system
(Amersham).
Transmission Electron Microscopy
HT-29-D4 cells
were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer,
pH 7.3, for 1 h, washed for 10 min in the same buffer containing 6.84%
sucrose, post-fixed in 1% osmium tetroxide followed by uranyl acetate
treatment, then dehydrated in ethanol, and embedded in Epon. Ultrathin
sections were cut perpendicularly to the plane of the cell layer and
observed with a transmission electron microscope (Jeol 1200X).
Immunogold Labeling
Undifferentiated HT-29-D4
cells were grown in Transwell cell culture chambers
(catalog number 3426, Costar, Cambridge, MA) and treated or not with
0.1 µM PMA for 1 h at 37 °C. The cells were then fixed
in Sorensen-Phosphate 0.1 M, pH 7.4, containing 1%
glutaraldehyde and 0.2% picric acid for 3 h. After 70% ethanol
treatment, the cells were embedded in LR White (Agar Scientific Ltd,
Essex, United Kingdom). Ultrathin sections were deposited onto 200-mesh
nickel grids, quenched in PBS containing 50 mM
NH
Cl, saturated in PBS 10% normal goat serum, and
subsequently incubated with primary antibodies (anti-
-tubulin
monoclonal antibody was from Boehringer Mannhein and anti-CD26
monoclonal antibody was from Immunotech, Marseille, France). The
labeling was detected using protein A-gold 10 nm (Sigma). The cells
were contrasted with uranyl acetate and observed with a Jeol 1200X
transmission electron microscope.
Immunofluorescence Analysis
HT-29-D4 cells grown
on glass coverslips were washed with PBS containing 0.1 mM
CaCl and 1 mM MgCl
(PBS-CM). They were
fixed in 3.7% paraformaldehyde in PBS-CM at 4 °C. All subsequent
steps were performed at room temperature. Cells were incubated in
PBS-CM containing 10% normal goat serum to block nonspecific binding
sites. Monolayers were incubated for 45 min with the monoclonal
antibody MAC 601 raised against CEA (Biosys, Compiègne, France).
After several washings, the primary antibody was revealed with
fluorescein-conjugated goat anti-mouse antibodies (Immunotech,
Marseille, France). Immunofluorescence analysis was performed with a
Zeiss epifluorescence microscope.
for protein kinase C) and their
capacity to absorb AMG was tested. This non-metabolizable analog of
glucose is specific for the sodium-glucose pathway as already
described
(11) . AMG uptake experiments were performed with or
without sodium or in the presence of 100 µM phlorizin (a
specific inhibitor of the cotransporter) by time point analysis (10
min). As shown in Fig. 1a, a significant uptake of AMG
was measured as soon as 5 days after switching HT-29-D4 glu cells to
galactose medium. This uptake increased with time and was completely
abolished in the absence of sodium or in the presence of 100
µM phlorizin. In contrast, no significant
sodium-dependent, phlorizin-sensitive AMG uptake could be observed when
the cells were cultured in the presence of the protein kinase C
inhibitor H-7. The absence of the cotransport function in H-7-treated
cells was not due to an effect on cellular proliferation as assessed by
determination of cellular protein content (data not shown).
Figure 1:
Effect of H-7 and HA-1004 treatments on
AMG uptake during HT-29-D4 cell differentiation. HT-29-D4 glu cells
were switched in galactose medium at day 0 with (filled
symbols) or without (open symbols) 30 µM H-7
(a) or 11 µM HA-1004 (b). AMG uptake
measurements were analyzed for 10 min in the presence of 140
mM sodium chloride (squares), choline chloride
(circles), or sodium chloride and 100 µM
phlorizin (triangles). Values reported are means ± S.D.
of three replicate determinations.
These
results indicate that the functional differentiation of HT-29-D4 cells
is regulated by protein kinases. However, the type of the protein
kinase(s) involved is uncertain since H-7, although known as a potent
inhibitor of protein kinase C, may also alter the function of cGMP- and
cAMP-dependent protein kinases
(12) . To further document this
point, we have tested the influence of an inhibitor of both cAMP- and
cGMP-dependent protein kinases (HA-1004) on the appearance of
sodium-glucose cotransport function. The results indicate that HT-29-D4
cells treated with HA-1004 (at 11 µM, a concentration that
corresponds to 5-fold the K for protein
kinase A and 10-fold the value for protein kinase G) express the
cotransport function (Fig. 1b). However, one should note
that this function is not detectable after 6 days of culture in
galactose medium in contrast with untreated control cells. Therefore,
HA-1004 does not inhibit the appearance of the cotransport function but
moderately delays it in time. Indeed, the values of AMG uptake after 20
days are similar for both HA-1004-treated and control cells. Thus, it
seems that the functional appearance of the sodium-glucose cotransport
function is mainly dependent on protein kinase C activity.
-Lys
) issued from the sequence of
SGLT1
(9, 13) , we detected the specific 64-kDa protein
in both cell extracts, showing that SGLT1 expression was not altered by
H-7 treatment (Fig. 2). Thus, the absence of the cotransport
function could not be related to an effect at the level of protein
synthesis.
Figure 2:
Immunoblotting of SGLT1 protein in
membrane extracts form HT-29-D4 control and H-7-treated cells. 10
µg of membrane preparation from HT-29-D4 untreated cells (lane
A) or H-7 treated cells (lane B) were solubilized in
reduction buffer and resolved by 7.5% SDS-polyacrylamide gel
electrophoresis. Proteins were transferred to the nitrocellulose paper
and probed with anti-SGLT1 affinity-purified antibodies. The arrow indicates the specific band of 64 ± 2
kDa.
Next, we studied the effect of H-7 treatment on the
overall organization of HT-29-D4 cells by transmission electron
microscopy. When HT-29-D4 cells are cultured for more than 20 days in
galactose medium, the cells organize into a monolayer of highly
polarized columnar cells resembling normal enterocytes
(Fig. 3a). Treatment with the protein kinase C inhibitor
H-7 (30 µM) resulted in a marked perturbation of the
cellular organization (Fig. 3b). In this case, the cells
formed a multilayer of non-polarized cells in which tight junctions
were only occasionally observed. Several cells showed typical
intracellular lumina lined with regularly arranged microvilli
(Fig. 3d), structures which are representative of an
abortive targeting of the apical plasma
membrane
(14, 15, 16) . Large intercellular
lumina with rare microvilli were also noted (Fig. 3c).
The H-7-induced disorganization observed at the ultrastructural level
was further documented by an analysis of the localization of an apical
marker, CEA, by immunofluorescence techniques. As shown in
Fig. 4a, the anti-CEA mAb labeled the apical side of
differentiated HT-29-D4 cells. In contrast, a fundamentally different
pattern of CEA labeling was observed in H-7-treated cells
(Fig. 4b). In the latter case, the periphery of the
cells was strongly fluorescent, indicating that the antibody had access
to the entire plasma membrane. This result is consistent with the
electron microsopy study, confirming that H-7 inhibited the
organization of HT-29-D4 cells into a polarized monolayer.
Figure 3:
Ultrastructure of HT-29-D4 cells upon H-7
treatment. HT-29-D4 cells were cultured for 30 days in galactose medium
in either the absence (a) or the presence (b-d) of 30
µM H-7. The cells were fixed, and ultrathin sections cut
perpendicularly to the cell layer were analyzed by transmission
electron microscopy. In absence of H-7, the cells are fully
differentiated and formed a regular monolayer of columnar
enterocyte-like cells (a). In the presence of 30
µM H-7, the cells grew as unorganized multilayers
displaying large intercellular lumina (b and c). In
some cells, the presence of intracellular lumina lined with regularly
arranged microvilli was evidenced (b and d). In some
areas, an apical striated border was observed locally in cells
belonging the last cell layer (b). d, desmosomes;
g, Golgi apparatus; il, intracellular lumen;
is, intercellular space; itl, intercellular lumen;
ly, lysosomes; m, mithochondria; mv,
microvilli; n, nucleus; seb, striated border;
tj, tight junction. Bars: a-c, 5 µm;
d, 1 µm.
Figure 4:
Immunofluorescence localization of the CEA
antigen. HT-29-D4 grown in galactose medium in either the absence
(A) or presence (B) of 30 µM H-7 were fixed as
described under ``Experimental Procedures,'' labeled with
anti-CEA monoclonal antibody, then revealed with fluorescene
isothiocyanate-conjugated anti-mouse antibodies. Bar, 20
µm.
If the
inhibition of protein kinases is associated with a defect of the
appearance of differentiated parameters in HT-29-D4 gal cells, one
would expect that, in HT-29-D4 glu cells, the activation of these
kinases would entail the expression of such functions. To investigate
this possibility, we tested the influence of PMA and 8-Cl-cAMP on the
AMG uptake. As shown in Fig. 5a, HT-29-D4 glu cells
treated for 15 min by 0.1 µM PMA express a functional
sodium-glucose cotransporter. This expression increased as a function
of time to reach a plateau after 1 h of PMA treatment. Under these
conditions, the AMG uptake was phlorizin sensitive, which was not the
case for untreated control cells. In contrast, the activation of
cAMP-dependent protein kinase by 0.5 mM 8-Cl-cAMP did not
induce the appearance of the cotransport function even after 2 h of
treatment (Fig. 5b).
Figure 5:
Stimulation of protein kinases in HT-29-D4
undifferentiated cells. HT-29-D4 undifferentiated cells grown in
glucose medium were incubated with 0.1 µM PMA (a)
or 0.5 mM 8-Cl-cAMP (b) and tested for their capacity
to absorb AMG by time point analysis (10 min). AMG measurments were
performed in either the presence (filled squares) or absence
(open squares) of 100 µM phlorizin. Values
reported are means ± S.D. of three replicate
determinations.
These data would suggest that
protein kinase C is involved in the translocation of the SGLT1 protein
from intracellular sites to the plasma membrane. However, one cannot
rule out the alternative possibility that protein kinase C may directly
phosphorylate the cotransporter already localized to the plasma
membrane. Two experiments were thus conducted in order to further
clarify this particular point. First, we tested the influence of PMA
and H-7 treatments on the cotransport activity in fully differentiated
HT-29-D4 cells. As shown in Fig. 6, the sodium-dependent
phlorizin-sensitive AMG uptake was not altered in the presence of
either 0.1 µM PMA or 30 µM H-7, suggesting
that the cotransport activity is not directly influenced by protein
kinase C phosphorylation. Moreover, immunocytochemical studies were
conducted to confirm that the PMA-induced appearance of the cotransport
function in undifferentiated HT-29-D4 cells (Fig. 5a)
was associated with SGLT1 protein plasma membrane targeting. In these
experiments, anti--tubulin antibodies were used as a control for
intracellular labeling (Fig. 7b), while anti-CD26
(dipeptidylpeptidase IV) antibodies served as a reference for plasma
membrane proteins (Fig. 7c). When affinity-purified
antibodies directed against SGLT1 protein were used, the labeling was
exclusively detected in restricted areas corresponding to intracellular
membranes (Fig. 7d). Upon PMA treatment, the SGLT1
labeling was found in vesicles close to the plasma membrane
(Fig. 7e) and, in some cells, unambiguously at the level
of the plasma membrane (Fig. 7, f-h). This process
could not be evidenced in all observed cells, in agreement with the
level of cotransport activity induced by PMA in undifferentiated
HT-29-D4 cells (Fig. 5a). However, the redistribution of
the SGLT1 protein appeared to be highly specific since the localization
of
-tubulin and CD26 was not affected by PMA (not shown). Taken
together, these data strongly support the concept that protein kinase C
activation results in the SGLT1 protein translocation from
intracellular compartments to the plasma membrane.
Figure 6:
Effect of PMA and H-7 treatment on
sodium-glucose cotransport. Fully differentiated HT-29-D4 cells were
either treated or not treated with 0.1 µM PMA or 30
µM H-7 for 4 h. AMG uptake experiments were performed as
described under ``Experimental Procedures'' with (filled
bars) or without (open bars) 140 mM sodium or in
the presence of 100 µM phlorizin (hatched bars).
Values reported are means ± S.D. of three replicate
determinations.
Figure 7:
Immunolocalization of SGLT1 protein.
Undifferentiated HT-29-D4 cells were either treated (e-h) or
not treated (a-d) with 0.1 µM PMA for 1 h and
then embedded in LR White for immunogold labeling. a, negative
control with the primary antibody omitted; only two gold particles were
found in 100 observed cells. b, immunolocalization of
-tubulin in a cytoplasm area with numerous cytoskeletal fibrillar
elements. c, CD26 labeling exclusively localized on the plasma
membrane. d, typical SGLT1 labeling associated with
intracellular membranes in a HT-29-D4-untreated cell. e,
localization of SGLT1 in a vesicle close to the plasma membrane in a
PMA-treated cell. f-h, localization of SGLT1 protein on the
plasma membrane of PMA-treated HT-29-D4 cells. Abbreviations:as, apical space; is, intercellular space;
m, mitochondria; n, nucleus; in f, the gold
particles are indicated with arrowheads. Bars: 100 nm
(a-d, g, and h); 30 nm (e); 1
µm (f).
Thus, the
functional appearance of the cotransport function in HT-29-D4 cells
seems to be regulated by protein targeting in the apical plasma
membrane, which may also be the case for normal intestinal epithelial
cells
(17, 18) .
-aminobutyric acid transporter whose plasma
membrane targeting is dependent on protein kinase C
activation
(23) . In agreement with our results on the SGLT1
protein, the activity of this transporter is regulated by protein
kinase C-mediated subcellular redistribution rather than direct
phosphorylation of the protein.
-methyl-D-glucopyranoside; PBS, phosphate-buffered
saline.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.