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(Received for publication, August 14,
1995; and in revised form, November 10, 1995) From the
The rat pituitary cell line GH Endocrine cells exhibit a constitutive secretory pathway, common
to all eukaryotic cells, as well as a regulated secretory pathway
(Kelly, 1985). The latter is characterized by intracellular storage of
secretory proteins, slow basal release, and stimulated secretion in
response to secretogogues. Granins (chromogranin A, chromogranin B,
secretogranin II) are a family of sulfated, calcium-binding proteins
that are co-stored with peptide and amine hormones in secretory
granules of endocrine cells. Thus, granins can serve as general markers
for the regulated secretory pathway in endocrine cells (Scammell,
1993). It has been suggested that granins play a direct role in the
sorting and packaging of peptide hormones in secretory granules (Rosa et al., 1985; Gorr et al., 1987a; Huttner et
al., 1991; Scammell, 1993). In support of this hypothesis, granins
exhibit calcium-induced aggregation at low pH, i.e. the
conditions found in the trans-Golgi network and secretory granules of
endocrine cells (Gorr et al., 1987b, 1988, 1989; Gerdes et
al., 1989; Chanat and Huttner, 1991; Thompson et al.,
1992). These aggregates can include other regulated secretory proteins
but exclude constitutive secretory proteins (Gorr et al.,
1989; Huttner et al., 1991). Specific sorting receptors have
also been proposed to act in the segregation of regulated secretory
proteins (Kelly, 1985). However, such sorting receptors have not been
conclusively identified (Gorr et al., 1992). The rat
pituitary cell line GH
In
some experiments, 4-methylumbelliferyl
Fluorographs and
dot-blots were quantitated by densitometric scanning using a Bio Image
Visage 60 image scanner (Bio Image, Ann Arbor, MI). The film exposure
times were adjusted to ensure that the bands were in the linear range
of the film. In general, the samples from chase incubations were
exposed longer than the cell extracts and labeling medium samples and
are, therefore, not directly comparable. Due to some interexperimental
variation, the data on stimulated secretion of prolactin and SEAP were
normalized by dividing all values in an experimental group (±
stimulation) by the lowest value from the unstimulated samples. The
data were analyzed by two-tailed Student's t test or
analysis of variance with Student-Newman-Keuls multiple comparison post
test. p < 0.05 was considered statistically significant. Rat pituitary GH To determine if the storage of
secretogranin II was similar to that of prolactin, the relative
secretion of both proteins in 4 h was quantitated (Fig. 1). The
relative prolactin secretion was 68% from untreated cells and 36% from
hormone-treated cells, consistent with increased prolactin storage in
hormone-treated cells. In contrast, secretogranin II exhibited
15-20% secretion under both conditions, suggesting that the
protein is predominantly stored in both untreated and hormone-treated
cells.
Figure 1:
Relative secretion of prolactin (PRL) and secretogranin II (SgII) from
GH
Since an endogenous marker protein for the constitutive
secretory pathway has not been identified in GH
Figure 2:
Relative secretion of SEAP. The secreted
and cellular amounts of SEAP from untreated (-EIE) and
hormone-treated cells (+EIE) were quantitated enzymatically after
4 h of incubation at 37 or 16 °C. % Secreted =
secreted amount/total (secreted + cellular) amount
The rapid basal secretion of SEAP suggested that the
enzyme was secreted constitutively from both untreated and
hormone-treated GH
Figure 3:
Stimulated secretion of SEAP (A)
and prolactin (B) from untreated (-EIE) and
hormone-treated cells. The cells were incubated with 50 mM NaCl + 100 nM 4
Sulfate-labeled GH
Figure 4:
Metabolic labeling of
GH
Figure 5:
Relative secretion of granins and
glycosaminoglycan from [
To test if the 30% secretion rate
of sulfated granins represented the regulated (slow) or constitutive
(fast) secretory pathway, the cells were labeled in the presence of
methylumbelliferyl xyloside to stimulate synthesis of sulfated
glycosaminoglycans, a marker for the constitutive secretory pathway (Fig. 4; lanes 2 and 4). The xyloside did not
affect the proportion of sulfated granins detected in the secretion
medium (Fig. 5). Glycosaminoglycan synthesis was induced
approximately 11-fold by xyloside treatment. In contrast to the
granins, the glycosaminoglycans were predominantly (67% of total) found
in the secretion medium ( Fig. 4and Fig. 5). These
results are consistent with sorting and storage of granins in the
regulated secretory pathway in both hormone-treated and untreated
cells, while glycosaminoglycans are rapidly secreted by the
constitutive secretory pathway. Stimulated secretion of sulfated
granins was tested by incubating the cells for 15 min in the presence
of 50 mM KCl. In two experiments, secretion of chromogranin B
was stimulated about 2.3-fold from both hormone-treated and untreated
cells (Fig. 6A), while secretion of secretogranin II
was stimulated 2-fold from hormone-treated cells and 1.6-fold from
untreated cells (Fig. 6B). Due to the small amounts of
the diffuse sulfated glycosaminoglycan band present in the chase media,
the constitutive marker could not be reliably quantitated in the
stimulation experiments. However, in a control experiment, SEAP
secretion was stimulated 1.2-fold (p < 0.06) in 1 h by 50
mM KCl (not shown).
Figure 6:
Stimulated secretion of granins from
hormone-treated (+EIE) and untreated
(-EIE) cells. [
GH
Figure 7:
Stimulated secretion of granins in the
presence of methylumbelliferyl xyloside. GH
Chromogranin B and secretogranin II, which serve as markers
for the regulated secretory pathway in most endocrine cell types
(Scammell, 1993), are expressed in GH
Granins have been
suggested to play a role in the sorting and packaging of peptide
hormones in the regulated secretory pathway (e.g. Gorr et
al. (1987a) and Huttner et al.(1991)). This is supported
by the co-aggregation in vitro of parathyroid hormone and
chromogranin A and B under the conditions of low pH and millimolar
concentrations of calcium that are found in the trans-Golgi network and
secretory granules (Gorr et al., 1989). Similarly, prolactin
exhibits low pH-induced aggregation (Thompson et al., 1992).
Recent evidence, however, suggests that calcium-induced aggregation at
low pH is neither necessary (Schmidt and Moore, 1994) nor sufficient
(Chanat et al., 1994) for sorting of regulated secretory
proteins. The present findings that granins, but not prolactin, are
efficiently stored in untreated GH One possible explanation for the differential storage of prolactin
and granins is that the proteins are stored in separate granule
populations. This is directly supported by the finding that prolactin
and secretogranin II are stored in distinct secretory granule
populations in bovine pituitary somatomammotrophs (Hashimoto et
al., 1987). Similarly, peptide hormones derived from a common
prohormone are sorted to, and stored in, separate granule populations
in Aplysia neuronal cells (Jung and Scheller, 1991). Thus,
individual cell types can contain separate regulated secretory pathways
including separate granule populations. Differential storage in
separate granule populations may be mediated by distinct sorting
signals (Gorr and Darling, 1995), presumably including Asn A
truncated, soluble form of vesicular stomatitis virus G-protein has
been used as a marker for constitutive secretion from endocrine cells (e.g. Moore and Kelly(1986)). Here it is shown that a
truncated form of human placental alkaline phosphatase (SEAP) is
secreted constitutively from GH Stimulated secretion of prolactin
has been detected in both untreated and hormone-treated cells (this
study and Reaves et al. (1990)). The stronger stimulation of
prolactin secretion from hormone-treated cells is presumably accounted
for by the increased intracellular storage of the hormone. In untreated
cells, the stimulation of prolactin secretion is somewhat stronger than
that of SEAP. However, taken together with the Golgi localization of
prolactin and the relative secretion data, it appears that prolactin is
mainly secreted by the constitutive secretory pathway in untreated
cells. Since untreated and hormone-treated cells exhibit both regulated
and constitutive secretory pathways, as evidenced by granin and SEAP
secretion, respectively, these results suggest that a regulated pathway
for prolactin secretion is up-regulated by hormone treatment. Chromogranin B and secretogranin II are tyrosine-sulfated in the
trans-Golgi network (Rosa et al., 1985; Huttner, 1988); thus,
analysis of granins in untreated and hormone-treated cells allowed the
use of sulfation as a marker for the late stages of the secretory
pathway, thereby eliminating differences due to transit through the
endoplasmic reticulum and Golgi apparatus. Stimulation of sulfated
chromogranin B secretion was similar in both untreated and
hormone-treated cells, while a consistent but small difference was
detected for secretogranin II secretion. These results suggest that the
two granins are sorted to separate granule populations that are
exocytosed at different rates upon stimulation. These granules may
exist in the same cells or in different subpopulations of
GH Methylumbelliferyl xyloside
inhibits proteoglycan synthesis but stimulates synthesis of free
glycosaminoglycan chains (Schwartz, 1977; Burgess and Kelly, 1984).
GH
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3575-3580
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
C
Cells (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C
secretes
granins (chromogranin B and secretogranin II) and prolactin by the
regulated secretory pathway. The intracellular storage of prolactin is
preferentially induced by hormone treatment with estradiol, insulin,
and epidermal growth factor. The goal of this study was to determine
the effect of hormone treatment on storage of granins and constitutive
secretory markers. The granins were efficiently stored in both
hormone-treated and -untreated cells (17% of total secreted in 4 h).
Secreted alkaline phosphatase (SEAP), a truncated membrane protein that
would not be expected to enter secretory granules, and
glycosaminoglycan, a marker for the constitutive secretory pathway,
exhibited 70-80% secretion under both conditions. In comparison,
the relative prolactin secretion was 31 and 68% from hormone-treated
and -untreated cells, respectively. Phorbol ester and KCl stimulated
prolactin secretion 2.3-fold from untreated cells and 5.5-fold from
hormone-treated cells. In contrast, SEAP secretion was stimulated
1.5-fold from both treated and untreated cells, consistent with
secretion by the constitutive secretory pathway. Stimulated secretion
of granins was detected from both hormone-treated and -untreated cells.
These results suggest that granin and prolactin storage are
differentially regulated and that the constitutive secretory pathway is
not affected by hormone treatment.
C, a subclone of the GH3
cell line, stores prolactin, chromogranin B, and secretogranin II in
secretory granules (e.g. Scammell et al. (1990a)) and
secretes all three proteins in response to extracellular stimulation
(Hinkle et al., 1992). The expression of prolactin and granins
is induced by treating the cells with a combination of estradiol,
insulin, and epidermal growth factor (EGF) ()(Scammell et al., 1986, 1990a; Thompson et al., 1992). In
addition, this hormone treatment preferentially induces prolactin
granulogenesis and the intracellular storage of prolactin (Scammell et al., 1986; Reaves et al., 1990). However, the
expression or storage of growth hormone, which is present in small
amounts in these cells, is not induced by hormone treatment (Scammell et al., 1986). Also, when insulin is expressed in transfected
GHC cells, the peptide is sorted to the
regulated secretory pathway, but insulin does not exhibit a
preferential increase in storage in hormone-treated cells (Reaves et al., 1990). To determine if induction of storage and
granulogenesis is specific for prolactin, the storage of the
endogenous-regulated secretory proteins secretogranin II and
chromogranin B, as well as constitutive secretory markers were
compared. Hormone treatment induced granin synthesis, but it did not
preferentially stimulate granin storage. The secretion and storage of
secreted placental alkaline phosphatase (SEAP) and glycosaminoglycans
was not affected by hormone-treatment.
Materials
Gelding horse serum was from Hyclone
Laboratories, Logan, UT; Lipofectamine, geneticin (G418), and mouse EGF
were obtained from Life Technologies, Inc.;
Na
SO
(carrier-free) was purchased
from ICN Radiochemicals, Irvine, CA. Phorbol 12-myristate 13-acetate,
4-methylumbelliferyl 
-D-xyloside, -estradiol, and
bovine insulin were from Sigma; 4
-phorbol 12,13-didecanoate was
purchased from Calbiochem, La Jolla, CA, and bovine serum albumin,
fraction V (protease free), was from Boehringer Mannheim. The plasmid
pcDNA3 was obtained from Invitrogen Corp., San Diego, CA, while
pSEAP-control and the Great Escape SEAP detection kit were purchased
from Clontech, Palo Alto, CA. Horseradish peroxidase-conjugated sheep
anti-mouse immunoglobulin was from Amersham Corp.;
horseradish-conjugated goat anti-rabbit immunoglobulin was from
Bio-Rad. Nitrocellulose membrane (Protran BA83) was obtained from
Schleicher and Schuell, Keene, NH. Other reagents were analytical grade
and were obtained from various suppliers.Cell Culture
GHC cells
were cultured in Gelding medium (Dulbecco's modified
Eagle's medium (42.5%), Ham's F-10 medium (42.5%), and
gelding horse serum (15%) supplemented with penicillin (100 µg/ml)
and streptomycin (100 units/ml)) at 37 °C, in a humidified
atmosphere with 5% CO. For each experiment, the cells were
detached with phosphate-buffered saline, pH 7.2, containing 0.2 g/liter
of EDTA and then plated in gelding medium at 10
cells/well
in 6-well cell culture plates (10 cells/cm).
Hormone-treated cells were cultured for 4 days in gelding medium
supplemented with 1 nM estradiol, 300 nM insulin, and
10 nM EGF (+EIE). Untreated cells were cultured in
parallel in gelding medium (-EIE).Transfection and SEAP Assays
The plasmids
pSEAP-control and pcDNA3 were co-transfected at a ratio of 10:1 into
GHC cells using Lipofectamine, as recommended
by the manufacturer. Control cells were only transfected with pcDNA3,
which contains the neomycin resistance gene. Transfected cells were
selected with Geneticin (320 µg/ml). Colonies of resistant cells
were directly collected with a pipette, expanded, and tested for SEAP
secretion. Two transfected GHC cell lines, S17
(SEAP positive) and S2 (negative control) were used for the experiments
reported. Both transfected cell lines expressed and secreted prolactin.
SEAP activity in secretion media and cell extracts were quantitated
with the Great Escape chemiluminescent assay in a Berthold LB 9501
luminometer (Wallac, Inc., Gaithersburg, MD).Metabolic Labeling
This was conducted at 37 °C
in a humidified atmosphere containing 5% CO. Cells were
preincubated for 30-45 min in Labeling Buffer (15 mM Hepes, pH 7.4, 0.3 mM CaCl, 3.82 mM KCl, 0.61 mM KHPO, 0.62 mM MgCl, 127 mM NaCl, 1.08 mM NaHPO, 6.1 mMD-glucose,
1.0 mM sodium pyruvate, 2.05 mM glutamine, 1 mg/ml
bovine serum albumin) and then incubated for 4 h in Labeling Buffer
containing 200 µCi/ml of NaSO
.
The medium was collected and centrifuged for 2 min at 16,000 
g, and the supernatant fraction (Labeling Medium) was stored
at -20 °C until use. The cells were rinsed with Labeling
Buffer containing 5 mM NaSO and then
chase incubated for 15 min in the same buffer supplemented with 50
mM NaCl or 50 mM KCl. At the end of the chase period,
the medium was centrifuged and stored frozen (Chase Medium). The cells
were stored frozen until extraction. The cells were either directly
solubilized in SDS-sample buffer or extracted in 1% Triton X-100. In
both protocols, the cell extracts were centrifuged for 2 min at 16,000
g and the supernatant fractions were analyzed by
SDS-PAGE. The two extraction procedures produced similar results.-D-xyloside was
added from a 100 mM stock solution in MeSO to a final concentration of 1 mM. The xyloside was added
to the preincubation and labeling buffers. Control incubations
contained 1% MeSO.Secretion Experiments with Unlabeled
Proteins
Cells cultured in 6-well plates were rinsed with
Labeling Buffer or, for experiments with SEAP containing samples,
Krebs-Ringer-Hepes buffer (129 mM NaCl, 5 mM NaHCO
, 4.8 mM KCl, 1.2 mM KHPO, 1 mM CaCl, 1.2
mM MgCl, 2.8 mM glucose, and 10 mM HEPES, pH 7.4) and preincubated in this buffer for 1 h. Fresh
buffer was added to the wells, and incubation continued at 37 or 16
°C for 4 h. Stimulated secretion was measured in buffer containing
50 mM KCl and 100 nM phorbol 12-myristate 13-acetate.
Control samples contained 50 mM NaCl and 100 nM phorbol didecanoate. Medium samples were centrifuged for 2 min at
16,000 g to remove cell debris before use. Cells were
resuspended in 50 mM HEPES, 12.5 mM EDTA, pH 7.2,
frozen and thawed, and homogenized through a 26-gauge needle. SEAP
containing samples were homogenized in Krebs-Ringer-Hepes buffer.Immunoblotting
Total cellular and secreted
prolactin and secretogranin II were analyzed by dot-immunoblotting
using antibodies to ovine prolactin and rat secretogranin II,
respectively. The antibodies were a kind gift from Dr. Jonathan G.
Scammell, University of South Alabama. The specificity of the
antibodies has previously been established in GHC cells (Scammell et al., 1990b; Hinkle et al.,
1992). Medium and cell extracts were applied to a nitrocellulose
membranes in a 96-well dot-blotting apparatus (Bio-Rad). The wells were
washed with 100 µl of TTBS (20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5), and then the membranes were blocked
for 30 min with 5% nonfat dry milk in TTBS. The membranes were
incubated overnight with the monoclonal anti-prolactin antibody diluted
1:2000 or the polyclonal anti-secretogranin II antiserum diluted
1:100-1:500 in TTBS containing 1 mg/ml of bovine serum albumin.
The blots were incubated with horseradish peroxidase-conjugated sheep
anti-mouse or goat anti-rabbit immunoglobulin (1:2000 in TTBS with 1
mg/ml of bovine serum albumin), respectively, for 2 h and developed
with chemiluminescent substrate and recorded on x-ray film. The films
were quantitated by densitometric scanning, as described below.
Incubation with the second antibody alone produced a negligible signal. SDS-PAGE
Aliquots of media samples and cell
extracts were analyzed by SDS-PAGE on 10 or 15% gels (Laemmli, 1970).
The latter were used for analysis of glycosaminoglycans. The gels were
fixed and stained in 10% methanol, 15% acetic acid containing Coomassie
Blue R-250 followed by destaining in methanol/acetic acid. The gels
were rinsed for 30 min in distilled water followed by 30 min in 1 M salicylate (Chamberlain, 1979). The gels were then dried and
exposed to Kodak X-Omat film at -80 °C.
C cells were cultured
for 4 days with or without hormone treatment (EIE: 1 nM estradiol, 300 nM insulin, 10 nM EGF).
Immunofluorescence microscopy of untreated cells indicated that
prolactin was predominantly found in a perinuclear region consistent
with localization in the Golgi complex. Only few cytoplasmic granules
were detected in these cells. Hormone-treated cells, on the other hand,
exhibited a bright punctuate pattern throughout the cytoplasm,
consistent with storage of prolactin in secretory granules (not shown).
Prolactin was quantitated by dot-immunoblotting of cell extracts and
media samples. Densitometric scanning of the immunoblots indicated that
hormone treatment induced the cellular and extracellular prolactin
levels by 20- and 3-fold, respectively, in agreement with earlier
reports (Scammell et al., 1986; Reaves et al., 1990;
Hinkle et al., 1992).
C cells. The secreted and cellular amounts of
PRL and SgII from untreated (-EIE) and hormone-treated cells
(+EIE) were quantitated by dot-immunoblotting after 4 h of
incubation. % Secreted = secreted amount/total
(secreted + cellular) amount 100%. Results from seven (PRL) and four (SgII; n = 14)
independent experiments were analyzed and presented as mean ±
S.E. Asterisk, different from untreated PRL samples (p < 0.0001; n =
21-23).
C cells, a truncated form of human placental alkaline phosphatase
(SEAP) was expressed in these cells and evaluated as a potential marker
for constitutive secretion. SEAP was released linearly from transfected
cells, and about 80% of total SEAP activity was found in the medium of
untreated cells after 4 h (Fig. 2). Hormone treatment had no
significant effect on the relative secretion of SEAP. When the cells
were incubated at 16 °C, to block protein export, only about 30% of
total SEAP activity was secreted in 4 h (Fig. 2). Incubation at
16 °C did not lead to a build-up in intracellular SEAP, suggesting
that synthesis or degradation of SEAP was affected in addition to
secretion.
100%.
Results from two independent experiments were analyzed and presented as
mean ± S.E. asterisk, different from 37 °C samples (p < 0.001; n =
6-11).
C cells. To further test
this, secretion was stimulated with phorbol ester and KCl and compared
with unstimulated secretion. SEAP secretion was stimulated 1.5-fold
from both untreated and hormone-treated cells (Fig. 3A), consistent with secretion by the
constitutive secretory pathway. Basal SEAP secretion resumed when cells
that had been incubated at 16 °C were warmed to 37 °C, but
stimulated secretion did not increase (not shown). Phorbol ester and
KCl stimulated prolactin secretion 2.3-fold from untreated cells and
5.5-fold from hormone-treated cell, respectively (Fig. 3B). The stronger stimulation of hormone-treated
cells was in agreement with an earlier report (Reaves et al.,
1990).
-phorbol 12,13-didecanoate (CTRL) or 50 mM KCl + 100 nM phorbol
12-myristate 13-acetate (STIM). Data from two to six
independent experiments, each performed in triplicate, were normalized
as described under ``Experimental Procedures,'' and expressed
as mean ± S.E. Asterisk, different from control (SEAP,
-EIE, p < 0.03, n = 6; SEAP,
+EIE, p < 0.02, n = 6; prolactin,
-EIE, p < 0.02, n = 9; prolactin,
+EIE: p < 0.0001, n =
17-18).
C cells were used
to further analyze the secretion of granins from untreated and
hormone-treated cells. Since tyrosine-sulfation of secretogranin II and
chromogranin B occurs in the trans-Golgi network, this modification is
a specific marker for the late stages of the secretory pathways
(Huttner, 1988). GHC cells were radiolabeled
with [S]sulfate for 4 h to allow sufficient time
for the sulfated granins to reach the storage compartment. Medium and
extracts of hormone-treated cells contained a high molecular weight
sulfated molecule, presumably representing a heparansulfate
proteoglycan (Hinkle et al., 1992), chromogranin B, and
secretogranin II (Fig. 4, lanes 1 and 3).
Similar results have been reported for untreated cells (Hinkle et
al., 1992). The cellular and secreted amounts of secretogranin II
each increased about 6-fold upon hormone treatment, while chromogranin
B synthesis was increased about 2-fold (Table 1). The higher
stimulation of secretogranin II synthesis compared with chromogranin B
synthesis reflects similar differences in mRNA levels after
hormone-treatment (Thompson et al., 1992). Hormone treatment
did not appear to selectively increase the cellular amounts of sulfated
granins (Table 1). To further determine if hormone-treatment
affected granin storage, the relative secretion of sulfated granins was
calculated for hormone-treated and untreated cells. Approximately 33%
of each granin was secreted from both hormone-treated and untreated
cells, confirming that hormone-treatment did not increase the fraction
of stored granins (Fig. 5).
C cells with
NaSO
. Hormone-treated cells were
labeled with [S]sulfate in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of methylumbelliferyl xyloside. Aliquots of labeling medium (MED) and cell extracts (CELL) were analyzed by
SDS-PAGE and fluorography. The positions of sulfated proteoglycan (p), chromogranin B (c), secretogranin II (s), and glycosaminoglycans (g) are indicated. Lane 5 is a shorter exposure of lane 3 to better
visualize the chromogranin B band.
S]sulfate-labeled
GH
C cells. Hormone-treated (EIE) or
untreated (blank) cells were radiolabeled in the presence (MUX) or absence (blank) of methylumbelliferyl xyloside.
Aliquots of labeling medium and cell extracts were analyzed by SDS-PAGE
and fluorography. Radioactive chromogranin B (CgB),
secretogranin II (SgII), and glycosaminoglycans (GAG)
were quantitated by densitometric scanning of the radiographic film. % Secreted = secreted amount/total (secreted +
cellular) amount 100%. Results from three independent
experiments were analyzed and presented as mean ± S.E. Asterisk, different from granin samples (p <
0.001, n = 3-11).
S]sulfate-labeled
GH
C cells were chase incubated for 15 min in
the presence of 50 mM NaCl (control) or 50 mM KCl
(stimulated). Secreted chromogranin B (top) and secretogranin
II (bottom) were quantitated by SDS-PAGE and fluorography and
presented as mean ± S.D. Asterisk, different from
control; CgB, -EIE, p < 0.0002, n =
2-3; CgB, +EIE, p < 0.002; n =
3; SgII, -EIE, p < 0.004, n =
2-3; SgII, +EIE, p < 0.005; n =
3). IOD, integrated optical
density.
C cells
express a cell-associated heparansulfate proteoglycan (Hinkle et
al., 1992). Methylumbelliferyl xyloside reduced the amount of
sulfated proteoglycan in the cells about 30% (Fig. 4), but the
xyloside did not prevent stimulated secretion of sulfated granins (Fig. 7). Thus, neither the reduction of proteoglycan synthesis
nor the increased constitutive secretion of glycosaminoglycans
prevented sorting of granins to the regulated secretory pathway.
C cells were labeled with [S]sulfate in the
presence of 1 mM methylumbelliferyl xyloside and then chase
incubated for 15 min in the presence of 50 mM NaCl (control)
or 50 mM KCl (stimulated). Secreted granins were quantitated
by SDS-PAGE and fluorography and presented as mean ± S.D. Asterisk, different from control (CgB, p < 0.0007; SgII, p < 0.007; n = 3). IOD, integrated optical density.
C cells,
and their expression is induced by hormone treatment with estradiol,
insulin, and EGF (Scammell et al., 1990a; Thompson et
al., 1992). In contrast to prolactin (Scammell et al.,
1986; Reaves et al., 1990), the storage of granins is not
preferentially induced by hormone treatment, although the relative
secretion remained constant, indicating that granin storage was
increased in parallel with granin expression in hormone-treated cells.
Thus, both secretogranin II and chromogranin B are efficiently stored
in untreated cells, while prolactin is rapidly secreted from these
cells, as measured by the relative secretion in the absence of
stimulation. In hormone-treated cells, on the other hand, both granins
and prolactin are efficiently stored, i.e. they exhibit low
relative secretion (Table 2). The relative secretion of prolactin
in 4 h reported here is similar to that previously reported (Scammell et al., 1986; Reaves et al., 1990; Hinkle et
al., 1992) (Table 2), assuming constant cellular amounts and
linear secretion in 24 h (see Reaves et al. (1990)). These
results indicate that prolactin and granin storage are differentially
regulated in GHC cells.
C cells
indicates that granin storage is not sufficient for the efficient
storage of prolactin. This finding suggests that the granins do not
directly interact with prolactin in the formation of a storage complex.
Indeed, it appears that the prolactin storage complex is highly
specific since it is disrupted by the expression of small amounts of
human prolactin in the rat cell line (Arrandale and Dannies, 1994). and Ser
in human prolactin (Arrandale and Dannies,
1994) or differential retention during granule maturation (Kuliawat and
Arvan, 1994). A second, but not mutually exclusive, explanation for
differential storage is that prolactin and granin storage involves
different subpopulations of GH
C cells. Thus,
this cell line is heterogeneous and prolactin and granin expression are
not uniformly induced in hormone-treated cells, although most prolactin
expressing cells also express at least one of the two granins (Scammell et al., 1990a). Scammell and co-workers (Scammell et
al., 1990a; Thompson et al., 1992) also noted that granin
expression is less strongly induced than that of prolactin upon hormone
treatment, although granins exhibited a redistribution to secretory
granules, similar to that of prolactin, as judged by immunofluorescence
(Scammell et al., 1990a). It is possible that the preferential
storage of prolactin correlated with the large increase in expression
levels in hormone-treated cells. However, prolactin storage is not
directly induced by high prolactin levels, since cells treated with EGF
alone exhibit increased prolactin expression with no preferential
increase in prolactin storage (Reaves et al., 1990).C cells. Since
alkaline phosphatase activity is readily quantitated, SEAP is a
convenient marker for the constitutive secretory pathway. SEAP and the
constitutive secretory marker glycosaminoglycan (Burgess and Kelly,
1984) were rapidly secreted from both untreated and hormone-treated
cells. Thus, it appears that the constitutive secretory pathway is not
affected by hormone treatment. Furthermore, the rapid secretion of SEAP
and glycosaminoglycans from hormone-treated cells, demonstrates that
the slow release of prolactin and granins is not due to a general block
of the secretory pathways due to hormone-treatment. The relative
secretion of SEAP was blocked at 16 °C, but without an increase in
cellular storage of SEAP, suggesting that SEAP is not shifted to a
storage compartment at the lower temperature. Similarly, the absence of
increased stimulated SEAP secretion after return to 37 °C suggests
that reducing its rate of secretion is not sufficient to target SEAP to
the regulated secretory pathway.C cells, in agreement with fluorescent
localization of granins in different cells (Scammell et al.,
1990a), as discussed above. If chromogranin B and secretogranin II are
differentially sorted in individual cells, this would imply the
presence of functionally distinct sorting signals on chromogranin B and
secretogranin II. Recent reports indicate that this is indeed the case.
Thus, chromogranin B, but not secretogranin II, is rerouted to the
constitutive pathway in dithiothreitol-treated PC12 cells (Chanat et al., 1993). In addition, our recent proposal that an
amino-terminal hydrophobic peak serves as the sorting signal for
chromogranin B, but not secretogranin II (Gorr and Darling, 1995), is
consistent with this possibility.C cells express a heparan sulfate
proteoglycan that is cellularly located (Hinkle et al., 1992).
Xyloside treatment reduced sulfate incorporation into this proteoglycan
about 30% and reduced sulfation of granins while strongly stimulating
glycosaminoglycan synthesis. The decrease in granin sulfation does not
prevent their sorting to the regulated secretory pathway in
hormone-treated cells. Similarly, inhibition of sulfation with sodium
chlorate does not prevent sorting of granins in untreated cells (Hinkle et al., 1992). Based on these and earlier data (Burgess and
Kelly, 1984; Gorr and Cohn, 1990; Gorr et al., 1991), it is
concluded that tyrosine-sulfation, oligosaccharide-sulfation, or
proteoglycan synthesis are not required for sorting or storage of
secretory proteins in the regulated secretory pathway in endocrine
cells.
)
I thank Dr. David V. Cohn for support and advice, Dr.
Douglas S. Darling for advice on cloning techniques, and Yancy R. Moore
for expert technical assistance. I also thank Dr. Jonathan G. Scammell,
University of South Alabama for supplying the prolactin and
secretogranin II antibodies and advice on the use of
GHC cells.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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