©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Prohormone Thiol Protease (PTP) and MetEnkephalin by Forskolin
BLOCKADE OF ELEVATED [Met]ENKEPHALIN BY A CYSTEINE PROTEASE INHIBITOR OF PTP (*)

Nikolaos Tezapsidis (2)(§), Stephen Noctor (2), Rama Kannan (2), Timothy J. Krieger (2)(¶), Liane Mende-Mueller (3), Vivian Y. H. Hook (1)(**)

From the (1) From the Department of Medicine, University of California, San Diego, California 92103-8227, the (2) Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, and the (3) Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 [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.


INTRODUCTION

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)() (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).

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.


EXPERIMENTAL PROCEDURES

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 MeSO 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.

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.

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.

A 17-residue synthetic peptide corresponding to the NH-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).

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 [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.

For analysis of [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% MeSO) or control vehicle (0.1% MeSO) 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).

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 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).


RESULTS

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.

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.


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.

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.


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).




DISCUSSION

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 [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.

The forskolin-induced 10-fold stimulation of PTP biosynthesis, as indicated by [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).

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.

  
Table: Protease inhibitor profile of enkephalin precursor cleaving activity in chromaffin granules from forskolin-treated cells

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).


  
Table: Protease inhibitor profile of proteolytic activity remaining after immunodepletion of PTP

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.



FOOTNOTES

*
This work was supported by grants from the NINDS and NIDA of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present addresses: Dept. of Psychiatry and Fishberg Research Center for Neurobiology, Mt. Sinai School of Medicine, New York, NY 10029.

Cangene Corporation, Mississauga, Canada.

**
To whom correspondence should be addressed: Dept. of Medicine, University of California, UCSD Medical Center, 200 West Arbor Dr. #8227, San Diego, CA 92103-8227. Tel.: 619-543-7161; Fax: 619-543-7717.

The abbreviations used are: PTP, prohormone thiol protease; PPE, preproenkephalin; RIA, radioimmunoassy; PC, prohormone convertase; CG, chromaffin granule; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride.

V. Y. H. Hook and N. Tezapsidis, manuscript in preparation.


ACKNOWLEDGEMENTS

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.


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