From the
Proenkephalin and other prohormones require proteolytic
processing at paired basic and monobasic residues for the biosynthesis
of active neuropeptides. The novel ``prohormone thiol
protease'' (PTP) has been proposed as a candidate proenkephalin
processing enzyme for the production of [Met]enkephalin in
chromaffin granules (Krieger, T. J., and Hook, V. Y. H.(1991) J.
Biol. Chem. 266, 88376-8383). In this study, PTP was examined
during elevation of cellular [Met]enkephalin by forskolin, a
direct activator of adenylate cyclase that produces cAMP. Treatment of
chromaffin cells with forskolin for 72 h increased enkephalin precursor
cleaving activity (measured by following the conversion of the model
substrate [
Proteolytic processing of prohormones within neurosecretory
vesicles is required to generate the smaller biologically active
neuropeptides (Docherty and Steiner, 1982; Hook et al., 1994).
Chromaffin cells of adrenal medulla contain several neuropeptides
including enkephalin (Udenfriend and Kilpatrick 1983), neuropeptide Y
(Carmichael et al., 1990), somatostatin (Lundberg et
al., 1979), and others whose biosynthesis requires proteolytic
processing of respective precursors at paired basic and monobasic
sites. The presence of precursor forms and mature peptides in
chromaffin cell secretory vesicles, known as chromaffin granules,
indicates the presence of corresponding prohormone processing enzymes
in this organelle. Thus, chromaffin cells should serve as an excellent
model neuroendocrine cell for investigations of prohormone processing.
Investigations of chromaffin granule proteases involved in
converting proenkephalin to [Met]enkephalin have resolved
enkephalin precursor cleaving activity into three classes of processing
proteases. These candidate processing proteases in chromaffin granules
are represented by the novel cysteine protease ``prohormone thiol
protease'' (PTP)
In chromaffin granules, the cysteine protease PTP was found as a
major contributor of enkephalin precursor cleaving activity,
representing approximately 60% of total activity in chromaffin granules
(Krieger and Hook, 1991). The PC enzymes (PC1/3 and PC2) and the
chromaffin granule aspartic protease (Azaryan et al., 1995a,
1995b) contributed an estimated 20 and 10%, respectively, to total
chromaffin granule enkephalin precursor cleaving activity. In contrast,
secretory vesicles of insulinoma cells contain PC2 and PC1/3 as major
activities for proinsulin processing (Bennett et al., 1992;
Bailyes et al., 1992). Furthermore, pituitary secretory
vesicles contain primarily a 68-70-kDa aspartic protease for
processing proopiomelanocortin (Loh et al., 1985) and
provasopressin (Parish et al., 1986) in intermediate and
neural lobes, respectively. These findings suggest that prohormone
processing in several neuroendocrine cell types involves several
proteases of different mechanistic classes.
In chromaffin granules,
in vitro processing studies indicate PTP as the predominant
enkephalin precursor processing activity. These results lead to the
prediction that PTP could be involved in the regulation of
[Met]enkephalin biosynthesis. The production of
[Met]enkephalin and other active neuropeptides is highly
regulated by the second messenger cAMP (Hook et al., 1985a;
Eiden et al., 1984). cAMP is involved in receptor-mediated
stimulation of enkephalin synthesis and secretion. In this study, the
role of candidate prohormone processing proteases in mechanism(s) of
cAMP stimulation of [Met]enkephalin levels in chromaffin
cells was examined. Results demonstrate stimulation of PTP and
[Met]enkephalin by forskolin, a direct stimulator of
adenylate cyclase and cAMP (Seamon and Daly, 1981). Importantly, the
forskolin-mediated rise in [Met]enkephalin was completely
blocked when cells were pretreated with an effective cysteine protease
inhibitor of PTP. These findings suggest PTP as an important
proenkephalin processing enzyme, and that both PTP and
[Met]enkephalin are regulated by cAMP.
To examine whether the
lipophilic cysteine protease inhibitor Ep453 (Buttle et al.,
1992) (Ep453 was from Taisho Pharmaceutical Company, Japan) could block
the forskolin-induced rise in cellular [Met]enkephalin, cells
were preincubated with 100 µM Ep453 24 h prior to the
addition of forskolin. Cells were then further incubated with 50
µM forskolin in the presence of 100 µM Ep453,
for 72 h, and subsequently harvested for measurement of
[Met]enkephalin levels by radioimmunoassay.
A
17-residue synthetic peptide corresponding to the
NH
IgG
immunoglobulins were purified from anti-PTP serum and preimmune serum
by protein A-Sepharose affinity chromatography. This purification of
IgGs was necessary to remove serum proteases and protease inhibitors
that could interfere with immunochemical analyses of PTP activity.
Immunoglobulins were bound to protein A-Sepharose resin by rocking the
resin (5 ml bed volume) with 5 ml of 10% serum in 0.1 M sodium
phosphate, pH 7.0. The resin and serum mixture was poured into a column
and washed with 0.1 M sodium phosphate, pH 7.0, with elution
of immunoglobulins by 0.1 M sodium citrate, pH 4.0, and
immediate neutralization (by the addition of 1.0 M Tris-HCl,
pH 8.3) of eluted fractions to pH 6-7. Eluted fractions were
dialyzed against 10 mM sodium phosphate, pH 7.0, and
concentrated by lyophilization. Immunochemical Analyses of PTP: Immunodepletion of PTP Activity,
Immunoprecipitation of Cellular [
For analysis of
[
Western blots of PTP
in isolated chromaffin granules with anti-PTP serum (1:1000 dilution)
were performed as described previously (Hook et al., 1985b) [Met]enkephalin Radioimmunoassay (RIA)-After treatment
of chromaffin cells with Ep453 and forskolin (1.5
Identification of the
elevated granule membrane-associated cysteine protease activity was
achieved by immunoprecipitation with anti-PTP immunoglobulins
(Fig. 3). Control experiments showed that the anti-PTP
immunogloblulins were capable of complete immunoprecipitation of
purified PTP (data not shown). Immunoprecipitation by anti-PTP of
enkephalin precursor cleaving activity in solubilized membranes was
indicated by immunodepletion of 40% of activity. Immunodepletion of 40%
of the activity in the granule membrane fraction by anti-PTP antibodies
is consistent with the presence of a similar level, 48%, of
E-64c-sensitive cysteine protease activity. Evidence that the
E-64c-sensitive activity represented PTP was indicated by the removal
of E-64c-sensitive activity in the solubilized granule membranes by
immunodepletion of PTP (). Immunodepletion of
E-64c-sensitive activity by anti-PTP immunoglobulins provides evidence
for forskolin stimulation of PTP activity in the chromaffin granule
membrane fraction.
Chromaffin cells were pretreated with
Ep453 (100 µM) and then incubated with forskolin (72 h) in
the presence of Ep453. Ep453 at 100 µM was chosen since
this level of Ep453, combined with its intracellular conversion to
E-64c, would be predicted to fully inhibit PTP. Importantly, results
showed that the forskolin-induced increase in cellular
[Met]enkephalin was completely blocked by Ep453
(Fig. 6). These data indicate a role for a cysteine protease in
cAMP-mediated elevation of [Met]enkephalin in chromaffin
cells. It is proposed that stimulation of PTP may be responsible for
the forskolin-mediated increase in [Met]enkephalin
production.
Results from this study demonstrate that the candidate
proenkephalin processing enzyme PTP is involved in cAMP stimulation of
[Met]enkephalin production in chromaffin cells. Treatment of
chromaffin cells in primary culture with forskolin, a stimulator of
adenylate cyclase and cAMP (Seamon and Daly, 1981), increased
enkephalin precursor cleaving activity in chromaffin granules by 170%
over controls (100%). The elevated proteolytic activity was associated
with the membrane fraction of these granules. Complete inhibition of
the elevated activity by E-64c, and immunoprecipitation of the
increased activity by anti-PTP immunoglobulins, indicated that PTP
activity was stimulated. Forskolin treatment of chromaffin cells
resulted in a 10-fold increase in the production of
[
The
forskolin-induced 10-fold stimulation of PTP biosynthesis, as indicated
by [
One criteron
expected of a prohormone-processing enzyme (Docherty and Steiner, 1982)
is that inhibition of the candidate enzyme should reduce cellular
production of the peptide hormone. This criteron was addressed in this
study by treating chromaffin cells with Ep453, a lipophilic cysteine
protease inhibitor that is converted by intracellular esterases to the
more active E-64c inhibitor. E-64c and Ep453 both inhibit PTP (Azaryan
and Hook, 1994b); E-64c is effective against PTP at low concentrations
in the nanomolar range, and Ep453 is inhibitory at 0.1 µM.
Ep453 (100 µM) completely blocked the forskolin-mediated
rise in cellular [Met]enkephalin levels. These results
indicate the involvement of a cysteine protease in cAMP stimulation of
[Met]enkephalin. Because only PTP and no other cysteine
protease was detected in chromaffin granules (Krieger and Hook, 1991),
the use of E-64c and its modified form, Ep453, to demonstrate blockade
of forskolin-stimulated [Met]enkephalin implicates the
involvement of PTP in [Met]enkephalin production.
Thus
far, PTP fulfills most of the criteria expected of prohormone
processing enzymes: (a) localization to secretory vesicles,
the major site of prohormone processing (Krieger and Hook, 1991);
(b) conversion of authentic prohormone (proenkephalin) to
appropriate intermediates and peptide product,
[Met]enkephalin, known to be present in vivo (Krieger and Hook, 1991; Krieger et al., 1992; Schiller
et al., 1995); (c) cleavage at typical paired basic
and monobasic prohormone processing sites (Krieger and Hook, 1991;
Krieger et al., 1992; Azaryan and Hook, 1994a, 1994b; Schiller
et al., 1995); (d) optimal activity near the
intragranular pH of 5.5-5.8 (Krieger and Hook, 1991); and
(e) reduction of cellular neuropeptide levels by a protease
inhibitor of the enzyme (this study). These properties indicate PTP as
a strong candidate prohormone processing enzyme.
It is of interest
that results implicated PTP, and not the subtilisin-like PC1/3 and PC2
or aspartic protease processing enzymes in chromaffin granules (Azaryan
et al., 1995a, 1995b), in cAMP stimulation of
[Met]enkephalin. The elevated enkephalin precursor cleaving
activity was completely inhibited by the cysteine protease inhibitor
E-64c, but it was not inhibited by inhibitors of serine or aspartic
proteases. Apparently, PC1/3 and PC2 and the CG aspartic protease were
not highly regulated by cAMP in this study, at least not under the
conditions of these experiments; these enzymes may be controlled under
other conditions.
Cyclic AMP is an important regulator of enkephalin
and neuropeptide biosynthesis. In chromaffin cells, cAMP (induced by
forskolin) increases levels of high molecular weight
enkephalin-containing peptides and also raises
[Met]enkephalin levels (Hook et al., 1985a; Eiden
et al., 1984). In chromaffin cells, the increase in enkephalin
involves positive control by cAMP of PPE gene expression (Eiden et
al., 1984; Wan et al., 1991). Results from this study
demonstrate that forskolin increases PTP synthesis and activity. cAMP
is known to stimulate protein kinase A phosphorylation of regulatory
proteins, including cAMP response element-binding proteins that bind to
cAMP response elements of the PPE gene (Comb et al., 1986;
Nguyen et al., 1990; and Konradi et al., 1993). It is
not known whether enhanced PTP gene expression occurs concomitantly
with forskolin-stimulated PPE gene expression. It will be of interest
in future studies to understand how PPE and proenkephalin processing
enzyme genes may be coordinately regulated, since both precursor and
processing enzyme(s) are required for production of the active opioid
peptide [Met]enkephalin.
Chromaffin granule samples were preincubated
with inhibitors (10 µM E-64c, 10 µM pepstatin
A, and 1.0 mM PMSF) for 30 min at 4 °C, and enkephalin
precursor cleaving activity was measured. Enkephalin precursor cleaving
activities are expressed as X ± S.D. (n = 6).
Final inhibitor concentrations were 10
µM E-64c, 10 µM pepstatin A, and 1.0 mM PMSF. During the immunoprecipitation procedure, it is evident that
the E-64c-sensitive activity was stable. However, the aspartyl and
serine protease activities detected by inhibition by pepstatin A and
PMSF, respectively, were lost during the immunoprecipitation procedure.
We thank the Protein/Nucleic Acid Shared Facility of
the Medical College of Wisconsin for peptide microsequencing. We thank
Dr. Steven Sabol (National Institutes of Health, Bethesda, MD) for the
gift of RB-4 anti-[Met]enkephalin serum and the Taisho
Pharmaceutical Company, Japan, for the gift of Ep453. We also thank Dr.
A. V. Azaryan for helpful discussions concerning effective protease
inhibitors of PTP.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
S-Met]preproenkephalin to
trichloroacetic acid-soluble radioactivity) in isolated chromaffin
granules by 170-180% over controls (100%). The increased activity
was associated with the membrane fraction, rather than the soluble
fraction, of chromaffin granules. The elevated activity was inhibited
by E-64c, which is a potent inhibitor of PTP and cysteine proteases;
however, the activity was not inhibited by serine or aspartic protease
inhibitors. The elevated activity was identified as PTP based on
immunoprecipitation by anti-PTP immunoglobulins. Stimulation of PTP
synthesis was involved in the forskolin-induced increase in PTP
activity, as demonstrated by a 10-fold increase in
[
S]PTP pulse labeling in forskolin-treated
chromaffin cells. Forskolin elevation of PTP protein levels within
chromaffin granules was also detected in Western blots. Importantly,
the forskolin-mediated rise in cellular [Met]enkephalin
levels was completely blocked when cells were preincubated with the
cysteine protease inhibitor Ep453, which is known to be converted by
intracellular esterases to the more effective inhibitor E-64c (Buttle,
D. J., Saklatvala, J., Tamai, M., and Barrett, A. J.(1992) Biochem.
J. 281, 175-177). Both E-64c and Ep453 inhibit PTP, with
E-64c being more potent (Azaryan, A. V., and Hook, V. Y. H. (1994b)
Arch. Biochem. Biophys. 314, 171-177). These results
demonstrate a role for PTP in proenkephalin processing in chromaffin
cells and indicate that [Met]enkephalin formation and PTP are
both regulated by cAMP.
(
)
(Krieger and Hook, 1991;
Krieger et al., 1992; Azaryan and Hook, 1994a, 1994b; Schiller
et al., 1995), subtilisin-like PC1/3 and PC2 enzymes (Steiner
et al., 1992; Azaryan et al., 1995a), and the
chromaffin granule aspartic proteinase (Azaryan et al., 1995a,
1995b). These enzymes cleave at paired basic and single basic sites
that typically flank the peptide hormone within its precursor. They are
active at acidic pH optima of 5.0-6.5, which is compatible with
the intragranular pH of 5.5-5.8 (Pollard et al., 1978).
Treatment of Chromaffin Cells in Primary Culture with
Forskolin and Ep453
Primary cultures of bovine chromaffin cells
were prepared from fresh bovine adrenal glands as described previously
(Hook et al., 1985a). Cells in Dulbecco's modified
Eagle's medium (with 100 units/ml penicillin, 100 µg/ml
streptomycin, 10% fetal calf serum, and 10M cytosine arabinofuranoside) were plated at a density
of 5
10
cells/well in 24-well plates (Costar),
precoated with poly-L-lysine. The viability of the cells,
determined by trypan blue exclusion, was greater than 95%. After 3 days
in culture at 37 °C in a humidified atmosphere of 5%
CO
, 95% air, forskolin was added to culture medium to a
final concentration of 50 µM, and Me
SO was
added (final concentration of 0.1%) as drug vehicle to control cells.
After 72 h of treatment at 37 °C with forskolin or vehicle, cells
were harvested for isolation of chromaffin granules and assay of
enkephalin precursor cleaving activity.
Enkephalin Precursor Cleaving Activity in Isolated
Chromaffin Granules
Chromaffin granules (CG) were isolated from
chromaffin cells (12 10
cells from 24 wells) by
discontinuous sucrose gradient density centrifugation, as described
previously (Hook et al., 1985a). This method results in
purified chromaffin granules free from lysosomes as assessed by
lysosomal enzyme markers (Hook et al., 1985a). The isolated CG
from one sucrose gradient were resuspended in 200 µl of 15
mM KCl. Membrane and soluble fractions were obtained by
centrifuging the lysed CG (lysed by freeze-thawing) at 100,000
g for 20 min. The membranes, washed once in 15 mM
KCl, were resuspended in 200 µl of buffer consisting of 50
mM sodium acetate, pH 6.0, 150 mM NaCl. Enkephalin
precursor cleaving activities in CG (10 µl/assay), as well as
membrane and soluble fractions of CG (20 µl/assay), were determined
by incubation with the model precursor substrate
[
S-Met]preproenkephalin
([
S-Met]PPE) for 16 h at 37 °C, and
measuring production of trichloroacetic acid-soluble radioactivity, as
described previously (Krieger and Hook, 1991).
Preparation of Anti-PTP Immunoglobulins
The 33-kDa
PTP, purified from 4000 adrenal medullae, as described previously
(Krieger and Hook, 1991), was subjected to peptide microsequencing, as
described previously (Krieger and Hook, 1991; Krieger et al.,
1992). The sequence of 17 residues at the NH terminus of
PTP was obtained. Search of the NBRF protein sequence data bank
indicated that the 17-residue sequence of PTP was novel.
-terminal sequence of PTP was synthesized and conjugated
to thyroglobulin (synthesis and conjugation by Peninsula Laboratories)
for injection into rabbits (rabbit antisera produced by Hazelton,
Vienna, VA). Antisera were screened in enzyme-linked immunosorbent
assays to detect specific antibody binding to the 17-residue peptide,
as described previously (Hook et al., 1985b).
S]PTP, and
Anti-PTP Western Blots-For immunoprecipitation of PTP
activity from the granule membrane fraction of forskolin-treated cells,
chromaffin granule membranes were isolated from forskolin and control
cells. Membranes (in 200 µl, from 12
10
cells)
were then solubilized in 1.0 mM CHAPS, 50 mM sodium
acetate, pH 6.0, and 150 mM NaCl (buffer A), and incubated
with anti-PTP and preimmune immunoglobulins (purified by protein
A-Sepharose, 1:50 dilution) for 16 h at 4 °C. Protein A-Sepharose
(30 µl of slurry in buffer A) was added, and the mixtures were
rocked for 2 h. PTP immunoglobulin-protein A Sepharose complexes were
pelleted by centrifugation for 10 min at 10,000
g at 4
°C. Enkephalin precursor cleaving activity in the resultant
supernatants was then assayed (as described above) to assess
immunodepletion of proteolytic activity.
S]PTP pulse labeling in chromaffin cells
treated with or without forskolin, culture medium was replaced with
Dulbecco's modified Eagle's medium without methionine (Life
Technologies, Inc.), supplemented with
[
S]methionine (>800 Ci/mmol, DuPont NEN) to a
final concentration of 150 µCi of
[
S]methionine/ml of medium. After 1 h incubation
with [
S]methionine, forskolin (final
concentration, 50 µM in 0.1% Me
SO) or control
vehicle (0.1% Me
SO) were added to the culture medium. Cells
were incubated at 37 °C for an additional 24 h, and then cells were
collected and centrifuged at 1,000
g. The pelleted
cells (1.8
10
cells) were solubilized in 0.4 ml of
50 mM sodium acetate, pH 6.0, 1.0 mM CHAPS with
protease inhibitors consisting of 5 µM pepstatin A, 1.0
mM phenylmethylsulfonyl fluoride (PMSF), and 10
µM leupeptin (buffer A). To remove nonspecific
S-labeled protein binding to protein A-Sepharose,
solubilized cells (0.2 ml) were incubated with protein A-Sepharose (30
µl) at 4 °C for 2 h and centrifuged (10,000
g), and the supernatant was used for immunoprecipitation. The
supernatant was incubated with anti-PTP or preimmune immunoglobulins
(15 µl) for 16 h at 4 °C. Protein A-Sepharose was added (30
µl), and the mixture was rocked for 2 h; after centrifugation at
10,000
g, the pelleted protein A-Sepharose was washed
5 times with 1 ml of washing buffer consisting of 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.02%
Triton X-100. The washed resin was heated in 30 µl of SDS-PAGE
sample buffer (as described by Krieger and Hook(1991)) at 95 °C for
10 min and then subjected to SDS-PAGE gels, and autoradiography as
described previously (Krieger and Hook, 1991).
10
cells for each treatment group, with n = 6),
cells were lysed in 1.0 N acetic acid and centrifuged at
20,000
g for 30 min at 4 °C. The resultant
supernatant was lyophilized and resuspended in RIA buffer consisting of
50 mM Tris-HCl, pH 8.3, 0.3% bovine serum albumin, and 0.2%
-mercaptoethanol. [Met]Enkephalin levels in the extract
was assayed by RIA using anti-[Met]enkephalin serum (RB-4)
(kindly provided by Dr. Steven Sabol, NIH, Bethesda, MD) as described
previously (Hook et al., 1985a).
Enkephalin Precursor Cleaving Activity and PTP in
Chromaffin Granules from Forskolin-treated Cells
Forskolin
treatment of chromaffin cells results in a 2-fold increase in
[Met]enkephalin levels (Hook et al., 1985a; Eiden
et al., 1984). To determine if stimulation of cellular
[Met]enkephalin may involve regulation of candidate
proenkephalin processing enzyme activities, enkephalin precursor
cleaving activity was measured in chromaffin granules isolated from
control and forskolin-treated cells. Incubation of cells with forskolin
for 72 h resulted in a statistically significant increase in enkephalin
precursor cleaving activity of 170% over controls (100%)
(Fig. 1). Comparison of activities in membrane and soluble
fractions of chromaffin granules indicated that the elevation in
enkephalin precursor cleaving activity resided with the membrane
fraction of the chromaffin granule (Fig. 2).
Figure 1:
Increased enkephalin precursor cleaving
activity in chromaffin granules from forskolin-treated chromaffin
cells. Chromaffin cells in primary culture were incubated with 50
µM forskolin for 72 h, and enkephalin precursor cleaving
activity in chromaffin granules was measured by following the
conversion of [S-Met]preproenkephalin to
trichloroacetic acid-soluble
S-labeled peptides. Average
activities obtained from several determinations (n = 6)
are expressed as X ± S.D. *, statistically significant
p < 0.001 (Student's t test).
Figure 2:
Elevated enkephalin precursor cleaving
activity in the membrane and soluble fractions of chromaffin granules.
After 72 h of forskolin treatment of chromaffin cells, the membrane and
then soluble components of chromaffin granules were isolated as
described under ``Experimental Procedures.'' Enkephalin
precursor cleaving activities in membrane and soluble granule
components from control and forskolin treated cells are expressed as
X ± S.D. *, statistically significant, p <
0.005 (Student's t test).
Class-specific
protease inhibitors were used to assess whether cysteine, aspartyl, or
serine protease activities were responsible for the elevated enkephalin
precursor cleaving activity in forskolin-treated cells ().
The protease inhibitor profile of activity in the soluble fraction of
chromaffin granules was similar in control and forskolin-treated cells.
The soluble enkephalin precursor cleaving activity from both control
and forskolin-treated cells were inhibited 28-31% by the cysteine
protease inhibitor E-64c, inhibited 41-45% by the aspartic
protease inhibitor pepstatin A, and inhibited 13-14% by the
serine protease inhibitor PMSF. In contrast, the granule membrane
fraction from forskolin-treated cells showed significantly higher
levels of E-64c-sensitive activity, 48% inhibition by E-64c, compared
with control membranes that showed only 11% inhibition by E-64c.
Importantly, it is noted that the forskolin-stimulated activity
associated with granule membranes was nearly completely inhibited by
the cysteine protease inhibitor E-64c. On the other hand, granule
membranes from forskolin and control cells showed no differences in
pepstatin A- or PMSF-sensitive activities. These results indicate that
forskolin induced an increase in cysteine protease activity in the
membrane fraction of chromaffin granules.
Figure 3:
Immunoprecipitation of PTP from chromaffin
granule membranes. Solubilized chromaffin granule membranes from
forskolin-treated cells were incubated without immunoglobulins (NoAb), with preimmune immunoglobulins, and anti-PTP
immunoglobulins. IgG fractions from antisera were obtained by
purification on protein A-Sepharose affinity chromatography. After
anti-PTP immunoprecipitation, enkephalin precursor cleaving activity
remaining in the supernatant was measured, as described under
``Experimental Procedures.'' Average activities are expressed
as X ± S.D. *, statistically significant, p < 0.005 (Student's t test).
PTP Production during Forskolin
Treatment
[S]Methionine pulse labeling of
PTP in chromaffin cells was examined to determine whether forskolin
(24-h treatment) stimulates PTP synthesis. After 24-h incubation of
cells with forskolin and [
S]methionine,
[
S]PTP was isolated by immunoprecipitation with
anti-PTP immunoglobulins and analyzed by SDS-PAGE gels and
autoradiography (Fig. 4). [
S]PTP was
detected in control cells as a single 33 kDa band that corresponds in
molecular size to purified 33-kDa PTP (Krieger and Hook, 1991). Of
particular interest was the finding that forskolin stimulated the
incorporation of [
S]methionine into PTP by
10-fold compared with controls (according to cpm of
[
S]PTP obtained by immunoprecipitation for
SDS-PAGE analysis, Fig. 4). Furthermore, Western blots of
chromaffin granules indicated an increase by approximately
2-3-fold (determined by densitometry) in 33-kDa PTP from
forskolin-treated cells (48 and 72 h of treatment) compared with
control cells (Fig. 5). These data indicate that forskolin
stimulates the biosynthesis of PTP.
Figure 4:
Stimulation of
[S-Met]PTP pulse labeling in chromaffin cells.
Cells were incubated with [
S]methionine and
forskolin, and [
S]PTP was immunprecipitated as
described under ``Experimental Procedures.'' Anti-PTP
immunoprecipitation of control and forskolin-treated cells resulted in
6.0
10
and 6.4
10
cpm
[
S]PTP, respectively. Immunoprecipitated
[
S]PTP was analyzed by SDS-PAGE gels (12%
polyacrylamide) and autoradiography.
Figure 5:
PTP immunoblot of chromaffin granules from
forskolin-treated cells. Chromaffin granules (15-µl granule sample,
from 1 10
cells) isolated from control cells and
cells treated with forskolin for 48 h (lanes1 and
3) or 72 h (lanes2 and 4) were
analyzed in anti-PTP immunoblots (anti-PTP serum at 1:1000 dilution).
The arrow indicates the known molecular size of PTP (Krieger
and Hook, 1991).
It is noted that
immunoprecipitation of [S]PTP under native
buffer conditions resulted in identification of a single 33-kDa
PTP-related band (Fig. 4). However, Western blot analysis of
denatured chromaffin granule proteins detects two bands, 33 and 55 kDa,
of PTP immunoreactivity (Fig. 5). Differences in binding of the
antibody under native compared to denaturing conditions may be one
explanation for detection of the 55 kDa band under only denaturing
conditions. The 55 kDa band could represent a precursor form of PTP,
since proteases are synthesized as zymogens. Future studies will
resolve the identity of the 55 kDa band. Ep453 Blockade of Forskolin-mediated Rise in Cellular
[Met]enkephalin-If PTP is involved in the increased
production of [Met]enkephalin during forskolin treatment,
inhibition of PTP in chromaffin cells should block the
forskolin-mediated rise in [Met]enkephalin levels. PTP is
potently inhibited by nanomolar levels of the cysteine protease
inhibitor E-64c and by 0.1 µM of the related cysteine
protease inhibitor Ep453 (also known as E-64d) (Azaryan and Hook,
1994b). Ep453 is a nonpolar ethyl ester form of E-64c and is more
capable of entering cells where it is converted by cellular esterases
to E-64c cysteine protease inhibitor (Buttle et al., 1992).
Thus, Ep453 can be used to assess the role of cysteine protease
activity in mediating forskolin stimulation of cellular
[Met]enkephalin.
Figure 6:
Ep453 blocks the forskolin-stimulated rise
in [Met]enkephalin. Chromaffin cells were incubated with the
cysteine protease inhibitor Ep453 24 h prior to addition of forskolin.
After additional incubation with Ep453 and forskolin for 72 h, cells
were harvested as acid extracts, and [Met]enkephalin was
measured by RIA as described under ``Experimental
Procedures.'' [Met]enkephalin levels (picograms of
peptide/1 10
cells) are expressed as X ± S.D. (n = 6). *, statistically
significant compared with control cells without Ep453, p <
0.001 (Student's t test).**, statistically significant
compared with forskolin cells without Ep453; p < 0.001
(Student's t test).
S]PTP compared with controls; elevated PTP
synthesis was also indicated by Western blots. In addition, treatment
of cells with an effective protease inhibitor of PTP resulted in
complete blockade of the forskolin stimulation of
[Met]enkephalin levels. These data lead to the hypothesis
that cAMP regulation of [Met]enkephalin production involves
stimulation of the candidate processing enzyme PTP.
S]PTP pulse analysis in chromaffin cells,
may suggest a greater increase than the observed 1.7-fold increase in
enkephalin precursor cleaving activity in chromaffin granules. It must
be realized, however, that the presence in chromaffin granules of an
endogenous protease inhibitor of PTP,
-antichymotrypsin (Hook et al., 1993), will
limit the detectable rise in activity measured by in vitro assay of enkephalin precursor cleaving activity. Therefore, the
elevation in PTP activity may be greater than that actually measured in
this study. In addition, the presence of
-antichymotrypsin inhibitor in the soluble fraction
and the apparent lack of
-antichymotrypsin in the
membrane fraction of chromaffin granules
(
)
indicate that in vitro assay of enkephalin
precursor cleaving activity will detect changes in the membrane
fraction but may not be able to detect possible changes in the soluble
fraction, as observed in this study. Also, the presence in chromaffin
granules of the endogenous
-antichymotrypsin inhibitor
limits the detectable level of E-64c-sensitive PTP activity in granules
to 31% of total granule enkephalin precursor activity. However, after
purification of PTP from chromaffin granules, which removes endogenous
-antichymotrypsin, results indicate that PTP
represents 54-60% of total enkephalin precursor cleaving activity
purified from these granules (Krieger and Hook, 1991).
Table:
Protease inhibitor profile of enkephalin
precursor cleaving activity in chromaffin granules from
forskolin-treated cells
Table:
Protease
inhibitor profile of proteolytic activity remaining after
immunodepletion of PTP
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.