Regulation of ACTH levels in anterior pituitary cells during stimulated secretion: evidence for aspartyl and cysteine proteases in the cellular metabolism of ACTH

Peptides 24 (2003) 717–725

Regulation of ACTH levels in anterior pituitary cells during stimulated secretion: evidence for aspartyl and cysteine proteases in the cellular metabolism of ACTH Catherine Sei a , Thomas Toneff a,b , Wade Aaron a , Vivian Y.H. Hook a,b,∗ a

Department of Neurosciences and Medicine, University of California, San Diego, La Jolla, CA, USA b Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945, USA Received 13 September 2002; accepted 26 February 2003

Abstract The regulation of cellular levels of adrenocorticotropin hormone (ACTH) in response to stimulated secretion was investigated to define the extent of cellular depletion of ACTH and subsequent increases to replenish ACTH levels in anterior pituitary cells (in primary culture). Treatment of cells with secretagogues for short-term incubation times (hours) resulted in extensive depletion of cellular ACTH. Corticotropin releasing factor (CRF) induced depletion of cellular levels of ACTH by 60–70% of control levels. The CRF-induced reduction of cellular ACTH was inhibited by the glucocorticoid dexamethasone. Phorbol myristate acetate (PMA), which stimulates protein kinase C (PKC), reduced ACTH levels by 50–60%. Forskolin, a stimulator of cAMP production, produced a moderate reduction in cellular ACTH. During prolonged incubation of cells (2 days) with these secretagogues, further reduction of ACTH levels by 70–80% was observed. However, increased cellular levels of ACTH occurred with continued treatment of cells with secretagogues, which provided nearly complete replenishment of cellular ACTH after 5 days treatment with secretagogues. Notably, the rising levels of cellular ACTH were inhibited by the aspartyl protease inhibitor acetyl-pepstatin A, and by the cysteine protease inhibitor E64d. These results demonstrate that depletion and recovery of ACTH levels are coordinately regulated, and that the increases in cellular levels of ACTH during the recovery phase involves participation of aspartyl and cysteine proteases. Thus, aspartyl and cysteine proteases may be involved in the cellular metabolism of ACTH. © 2003 Elsevier Inc. All rights reserved. Index terms: ACTH; anterior pitutiary; regulation of cellular ACTH; aspartyl protease; cysteine protease; ACTH biosynthesis Keywords: ACTH depletion; Secretion; Biosynthesis; Proteases

1. Introduction The peptide hormone adrenocorticotropin hormone (ACTH) is secreted from anterior pituitary for the regulation of glucocorticoid production in the adrenal cortex [31,32]. ACTH secretion is stimulated by corticotropin releasing factor (CRF) released from the hypothalamus [9,14,30], and is inhibited by glucocorticoids from the adrenal cortex by a feedback inhibitory mechanism. ACTH is produced by proteolytic processing of its proopiomelanocortin (POMC) precursor [17,27,39,40]. It is also known that the cellular metabolism of peptides and proteins involves production and clearance from cells. The regulation of cellular levels of ACTH is important for maintaining intracellular stores of ACTH, for its secretion, and physiological functions.

∗

Corresponding author. Tel.: +1-415-209-2069; fax: +1-415-209-2036. E-mail address: [email protected] (V.Y.H. Hook).

0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0196-9781(03)00126-8

Earlier studies have examined release of ACTH from pituitary cells [10,11,14,16,21,24,34,36,46], but have not defined the extent of depletion of cellular ACTH during its stimulated secretion. Coordinate regulation of cellular depletion and subsequent replenishment of ACTH is required to maintain stores of ACTH. The cellular metabolism of ACTH involves several processes consisting of the biosynthesis of the POMC precursor and proteolytic processing that converts POMC to ACTH, protein factors that coordinate the proper cellular trafficking of ACTH production in the regulated secretory pathway, endogenous positive or negative cofactors that influence ACTH production, and clearance of ACTH from cells by secretion or degradation. Thus, the overall cellular metabolism of ACTH is important for maintaining stores of ACTH within secretory vesicles to allow secretion of ACTH upon physiological stimuli. It is known that ACTH production by POMC processing is an important aspect of maintaining cellular levels of ACTH. POMC processing involves distinct proteases

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of different mechanistic classes [17,27,39,40]. The roles of PC1 and PC2 subtilisin-like proteases (PC: prohormone convertase) and related members of this protease family have been demonstrated [5,6,13,15,38,44,48]. Evidence also suggests involvement of aspartyl [8,12,28,29,33,37] and cysteine [28,37] protease activities for ACTH production and metabolism in pituitary secretory vesicles. To obtain an understanding of the cellular metabolism of ACTH with respect to recovery of cellular stores of ACTH after secretion, the goals of this study were to (a) determine the extent of ACTH depletion during stimulated secretion from anterior pituitary cells (in primary culture), (b) characterize the recovery of cellular stores of ACTH, and (c) gain further understanding of the proteolytic mechanisms involved in replenishment of depleted cellular stores of ACTH. Results demonstrated that the secretagogues CRF, phorbol myristate acetate (PMA), and forskolin induced the depletion of substantial amounts of cellular ACTH. Following depletion, increased ACTH production occurred to replenish cellular stores of the peptide hormone. Notably, the increases in cellular levels of ACTH were reduced by aspartyl and cysteine protease inhibitors. These findings demonstrate that a large portion of cellular ACTH is depleted during secretion, and that replenishment of cellular stores of ACTH involves aspartyl and cysteine proteases.

2. Methods and materials 2.1. Preparation of primary cultures of rat anterior pituitary cells For preparation of rat anterior pituitary cells in primary culture, 25–30 Sprague–Dawley rats (200–250 g, male, from Simonsen Laboratories, Gilroy, CA) were sacrificed and fresh anterior pituitary tissue was dissected. Tissue was placed in sterile phosphate-buffered saline (PBS), rinsed with PBS to remove red blood cells, and minced with a fine, sterile razor blade. Dissociation of cells was achieved by incubating minced tissue in digestion solution consisting of 0.2% trypsin (Calbiochem, San Diego,CA), 10 units/ml collagenase (Worthington Biochemicals, Freehold, NJ), 2% fetal calf serum (fcs; GIBCO/BRL), and 1% antibiotics (0.5% penicillin G and 0.5% streptomycin sulfate, in PBS, from GIBCO/BRL) in PBS at 37 ◦ C for 15 min with gentle shaking. DNase was added (50 ␮l of 1 mg/ml DNase I in PBS, from Sigma Biochemicals, St. Louis, MO) and the solution was incubated at 37 ◦ C for an additional 10–15 min. Remaining tissue fragments were allowed to settle to the bottom of the test tube, and the supernatant containing dispersed cells was removed and placed in 6–8 ml DMEM medium (GIBCO/BRL) containing 10% fcs. Cells were collected by centrifugation at 350 × g for 5 min, and the cell pellet was resuspended in 8 ml of DMEM containing 10% fcs.

Remaining tissue fragments were incubated again with digestion solution at 37 ◦ C for 20 min with addition of DNase. Dispersed cells were combined with the first group of dissociated cells in DMEM containing 10% fcs. Cell viability, assessed by trypan blue exclusion, showed that cells were greater than 95% viable. An additional 8–10 ml DMEM/10% fcs was added to the dispersed cells, and cells were centrifuged at 350 × g. The pelleted cells were resuspended in DMEM containing 10% fcs, glutamine, 0.1% antibiotics (0.05% penicillin and 0.05% streptomycin sulfate, GIBCO/BRL), and 1% non-essential amino acids. Cells were plated at 2–5 × 105 cells per well, in 24-well plates, and incubated at 37 ◦ C in a water-jacketed 5% CO2 /95% air, humidified incubator for 5–7 days before beginning the experiments. 2.2. Regulation of cellular ACTH in pituitary cells by secretagogues Experiments evaluated cellular levels of ACTH, as well as secreted ACTH, that were controlled by CRF, dexamethasone, and activators of PKA and PKC. Cells were washed with secretion medium consisting of DMEM with 0.1% bovine serum albumin (BSA), and 3 ␮g/ml bacitracin. Cells were then incubated at 37 ◦ C (up to 5 h) with secretion medium (1 ml per well) containing CRF, dexamethasone, forskolin, or PMA in an atmosphere of 5% CO2 /95% air. When cells were incubated with secretagogues for 2 or 5 days, the media was changed to fresh secretagogue and media each day (once every 24 h). CRF was from Peninsula Laboratories, San Carlos, CA; dexamethasone, forskolin, and PMA were from Calbiochem. When dexamethasone was tested, cells were preincubated with dexamethasone for 30 min at 37 ◦ C in a humidified incubator in 5% CO2 /95% air, and then incubated with inhibitor and stimulatory factors. At the end of the incubation period, media was collected and centrifuged (TJ-6 Beckman centrifuge; 3000 rpm for 5 min) to remove residual cells, and the supernatant was analyzed for ACTH content by radioimmunoassay (RIA). Cell extracts (from cells attached to culture plates) were obtained by rinsing cells with PBS (1 ml per well), resuspending cells in 0.1 N acetic acid (200 ␮l per well), heating cells at 95 ◦ C for 5 min, centrifugation at 15,000 × g for 20 min, and collection of the supernatant for measurement of ACTH by RIA. Secretagogues (CRF, dexamethasone, forskolin, and PMA) were tested in triplicate wells, and reproducibility of results was confirmed by at least 2–3 experiments. 2.3. Measurement of ACTH by radioimmunoassay ACTH in cell extracts and media was measured by RIA essentially as we have described previously [18]. The ACTH RIA utilized anti-ACTH serum from NIDDK/NIH, and 125 I-ACTH from Peninsula Laboratories. This RIA showed no crossreactivity with ␣-MSH, ␤-MSH, ␥-MSH, or ␤-endorphin. The RIA assay for ACTH does not detect

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recombinant POMC (prepared as we have previously described [20]).

3. Results 3.1. Concentration-dependence of CRF-induced depletion of cellular ACTH, and inhibition by dexamethasone Depletion of cellular ACTH by secretagogues that stimulate ACTH secretion was examined in primary cultures of rat anterior pituitary cells. The concentration-dependence and time course for CRF-induced depletion of cellular ACTH during stimulated secretion were characterized. CRF reduced cellular levels of ACTH in a concentration-dependent manner (Fig. 1a). Half-maximal depletion of cellular ACTH occurred at approximately 0.1 nM CRF, with maximal effects observed at 1–10 nM CRF (Fig. 1a(i)). At 1–10 nM CRF, cellular ACTH was reduced by more than 50% com-

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pared to unstimulated controls. In parallel, CRF was a potent stimulator of ACTH secretion, with half-maximal stimulation of ACTH secretion occurring at 0.1 nM CRF, and maximal ACTH secretion occurred at 1–10 nM CRF (Fig. 1a(ii)). These results demonstrate that a large proportion of cellular ACTH was depleted by low concentrations of CRF. The time course of CRF-mediated (10 nM CRF) reduction of cellular ACTH showed that cellular ACTH was reduced after 1–2 h, with maximal reduction of ACTH (75% reduction) occurring after 3–5 h treatment of cells with CRF (Fig. 1b(i)). CRF stimulation of ACTH secretion was detected after 1–2 h, and maximal secretion was observed at 3–5 h incubation with CRF (Fig. 1b(ii)). CRF-mediated depletion of cellular ACTH occurred in a time-dependent manner. The glucocorticoid dexamethasone inhibited CRF-mediated depletion of cellular ACTH, and inhibited CRF-stimulated ACTH secretion (Fig. 2). Concentration-dependence studies showed that nanomolar levels of dexamethasone (10–100 nM) inhibited CRF-induced depletion of cellular

Fig. 1. CRF-mediated depletion of ACTH in pituitary cells: dose-dependence and time-course studies. (a) Dose-dependence of CRF-mediated depletion and secretion of ACTH. The concentration-dependence of CRF-mediated regulation of cellular ACTH (panel i) and ACTH secretion (panel ii) during incubation of cells with different concentrations of CRF for 3 h are illustrated. Values are shown as the mean ± S.E.M. (from triplicate wells). All experiments of this study were performed at least 2–3 times. (b) Time-course of CRF-mediated depletion and secretion of ACTH. The time-course of CRF regulation of cellular ACTH (panel i) and CRF-induced secretion (panel ii) are shown. Cells were incubated for 1–5 h with 10 nM CRF. Values are calculated as the mean ± S.E.M. (from triplicate wells).

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Fig. 2. Dexamethasone inhibits CRF-mediated depletion of cellular ACTH. (a) Dose-dependence of dexamethasone inhibition of CRF-mediated depletion and secretion of ACTH. Concentration-dependent effects of dexamethasone on control cells (䊊) and CRF-treated (10 nM) cells (䊉) with respect to cellular levels of ACTH (panel i) and ACTH secretion (panel ii) are illustrated for cells incubated at 37 ◦ C for 3 h. Values are calculated as the mean ± S.E.M. (from triplicate wells). (b) Time-course of dexamethasone effects on CRF-mediated depletion and secretion of ACTH. The time course (0.5–5 h) of effects of dexamethasone on control cells and CRF-treated cells with respect to ACTH cell content (panel i) and ACTH secretion (panel ii) are shown. Cells were incubated alone (䊊, control), with CRF (䊉, 10 nM CRF), dexamethasone (, 1 ␮M dexamethasone), or with CRF and dexamethasone (䉱, 10 nM CRF/1 ␮M dexamethasone). Values are calculated as the mean ± S.E.M. (triplicate wells).

ACTH (Fig. 2a(i)). Similarly, dexamethasone reduced CRFstimulated ACTH secretion, with maximal inhibition occurring at approximately 100 nM dexamethasone (Fig. 2a(ii)). Time course studies showed that dexamethasone (1 ␮M) inhibited CRF-induced depletion of cellular ACTH after 3–5 h incubation (Fig. 2b(i)). Moreover, dexamethasone (1 ␮M) substantially reduced CRF-stimulated ACTH secretion, with the largest effects of dexamethasone on ACTH secretion evident after 3–5 h incubation of cells with dexamethasone and CRF (Fig. 2b(ii)). These results show that dexamethasone blocks CRF-induced depletion of cellular ACTH. 3.2. Involvement of protein kinase C (PKC) and cAMP in depleting cellular ACTH during secretion Phorbol myristate acetate stimulates PKC and is known to induce secretion in many cell types [42]. PMA induced a de-

crease in cell content of ACTH (Fig. 3a(i)), with concomitant stimulation of ACTH secretion (Fig. 3a(ii)). Half-maximal depletion of cellular ACTH and stimulation of ACTH secretion occurred at approximately 5 nM PMA, with maximal effects of PMA observed at 100 nM. At maximal concentrations of PMA, cellular ACTH was reduced by nearly 50%. The role of the second messenger cAMP in regulating ACTH levels was assessed by treating cells with forskolin that stimulates adenylate cyclase to synthesize increased cAMP, which activates cAMP-dependent protein kinase A [41]. Forskolin induced a modest reduction in cellular levels of ACTH (Fig. 3b(i)), and increased ACTH secretion by two- to three-fold (Fig. 3b(ii)), at concentrations of 1–10 ␮M forskolin. These findings implicate PKC and cAMP in regulating depletion of cellular ACTH. In PMA- and forskolin-treated cells, time course studies demonstrated more pronounced effects of PMA (at 100 nM)

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Fig. 3. PMA and forskolin-induced reduction of cellular ACTH. (a) PMA-induced reduction of ACTH cell content. The concentration-dependence of PMA regulation of ACTH cell content (panel i) and ACTH secretion (panel ii) are illustrated (2 h incubation of cells). Values are shown as the mean ± S.E.M. (b) Forskolin-induced reduction of ACTH cell content. The concentration-dependence of forskolin regulation of ACTH cell content (panel i) and ACTH secretion (panel ii) are illustrated (2 h incubation of cells at 37 ◦ C). Values are shown as the mean ± S.E.M.

compared to forskolin (50 ␮M) to deplete cellular ACTH (Fig. 4). PMA induced a large decrease in cellular ACTH that was reduced by nearly 60% after 4–5 h treatment of cells with PMA (Fig. 4a). Forskolin, on the other hand, resulted in a moderate reduction of cellular ACTH by approximately 30–40% after treating cells with forskolin for 4–5 h (Fig. 4a). Consistent with the greater loss of cellular ACTH by PMA compared to forskolin, PMA stimulated ACTH secretion to a greater extent than forskolin (Fig. 4b). Thus, PMA and forskolin demonstrate differential magnitudes of effects for reducing cellular levels of ACTH, during its stimulated secretion. 3.3. Depletion and recovery of ACTH after prolonged treatment with secretagogues: role of aspartyl and cysteine proteases The secretagogues examined in this study (CRF, PMA, and forskolin) reduced cellular levels of ACTH after 3–5 h incubation. Treatment of cells for an extended period of time with these secretagogues resulted in a larger reduction of cel-

lular ACTH occurring after 2 days incubation (Fig. 5), compared to 3–5 h treatment with secretagogues. After 2 days of treatment, cellular ACTH was depleted by 70–80% (Fig. 5). Importantly, the cells then show replenishment of cellular ACTH levels towards control levels after 5 days of treatment with these secretagogues (Fig. 5). Control studies showed that during treatment of cells with secretagogues, there was no change in the number of cells (monitored by determining protein content); therefore, ACTH measurements reflect changes in relative cellular content of ACTH after 2 or 5 days treatment. Thus, in the continuous presence of these secretagogues, increases in cellular ACTH occurred that replenished cellular stores of ACTH depleted by stimulated secretion. During the recovery phase, during which increases in cellular levels of ACTH occurred, the influence of aspartyl and cysteine protease inhibitors on ACTH content was examined. During treatment of cells with acetyl-pepstatin A (100 ␮M), an aspartyl protease inhibitor, this protease inhibitor significantly reduced the cell content of ACTH in control and CRF-treated cells (Fig. 6). Acetyl-pepstatin A

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4. Discussion

Fig. 4. Time-course of PMA- and forskolin-induced reduction of cellular ACTH. (a) Regulation of cellular ACTH by PMA and forskolin. The time-course of the effects of PMA (100 nM, 䊐), forskolin (FSK, 50 ␮M, ), or the absence of secretagogue (䊊, control) on cellular content of ACTH are shown. Values are illustrated as the mean ± S.E.M. (b) Regulation of ACTH secretion by PMA and forskolin. The time-course of the effects of PMA (100 nM, 䊐), forskolin (FSK, 50 ␮M, ), or the absence of secretagogue (䊊, control) on ACTH secretion are shown. Values are calculated as the mean ± S.E.M.

represents a cell permeable form of pepstatin A that selectively inhibits aspartyl proteases [45]. Acetyl-pepstatin A reduced ACTH levels by approximately 35 and 60% in untreated and CRF-treated cells, respectively. The cysteine protease inhibitor, E64d, also reduced cellular levels of ACTH during treatment of anterior pituitary cells with CRF (Fig. 7). E64d represents a cell permeable analog of the cysteine protease inhibitor E64c [7]. After 5 days treatment of cells with CRF, E64d significantly reduced ACTH content by approximately 70% in untreated cells. In CRF-treated cells, E64d reduced ACTH levels by about 40–50%. These results demonstrate a role for cysteine and aspartyl proteases during replenishment of cellular ACTH after its depletion by secretagogue-induced secretion.

Results of this study have examined the coordinate depletion and recovery of cellular ACTH upon secretion from anterior pituitary cells (in primary culture), with demonstration that aspartyl and cysteine proteases may participate in the cellular metabolism of ACTH. Specifically, rat anterior pituitary cells in primary culture were treated with the secretagogues CRF, forskolin, or PMA in concentration- and time-course studies. During short-term treatment (3–5 h) with secretagogues, CRF induced the greatest depletion of cellular ACTH, with reduction of ACTH levels by 60–70%. In addition, the CRF-induced reduction of cellular ACTH was inhibited by the glucocorticoid dexamethasone, an inhibitory regulator of CRF-stimulated ACTH production and secretion. PMA reduced cellular levels of ACTH by 50–60%. Forskolin produced a moderate reduction of cellular ACTH. Prolonged treatment of cells with CRF, PMA, or forskolin for 2 days resulted in further reduction of cellular ACTH levels by 70–80%. Notably, increased production of ACTH provided replenishment of cellular ACTH after 5 days of treatment with these secretagogues. Moreover, the rising levels of cellular ACTH during replenishment of cellular levels of ACTH were significantly inhibited by the aspartyl protease inhibitor acetyl-pepstatin A, and by the cysteine protease inhibitor E64d. These results demonstrate extensive reduction of cellular ACTH during stimulated secretion, which is followed by increases in cellular levels of ACTH that involves participation of aspartyl and cysteine proteases. With respect to production of ACTH, the reduced cellular levels of ACTH resulting from treatment with acetyl-pepstatin A and E64d may represent inhibition of POMC processing by aspartyl or cysteine proteases, respectively. Evidence for the role of an aspartyl protease in the production of ACTH has been provided by studies showing proteolytic processing of POMC by the aspartyl protease ‘POMC converting enzyme’ (PCE) [8,12,25,26,28,29,33] that is present in pituitary secretory vesicles. In addition, secretory vesicles from other neuroendocrine tissues including posterior pituitary [33] and adrenal medullary chromaffin granules [2,3] also contain ‘PCE’-like aspartyl protease activity. It has been proposed that the mammalian PCE resembles the yeast YAP3 aspartyl protease [1,4]. With respect to cysteine proteases, the reduction of cellular levels of ACTH by the cysteine protease inhibitor E64d may represent cysteine protease activity in intermediate pituitary secretory vesicles, which was shown to be inhibited by PHMB (which inhibits cysteine proteases) [28]. Also, cysteine protease activity in intermediate pituitary secretory vesicles has been observed with the enkephalin precursor as substrate [43]. Recently, the POMC-derived peptides ␣-MSH and ␤-endorphin in intermediate pituitary cells were shown to be reduced by E64d, implicating a role for cysteine protease activity in the metabolism of ␣-MSH [37]. Furthermore, secretory vesicles of adrenal medullary chromaffin

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Fig. 5. Replenishment of reduced cellular levels of ACTH during prolonged treatment with secretagogues. Cells were treated without agent (control), or with the secretagogues CRF (10 nM), forskolin (50 ␮M FSK), or PMA (100 nM) for 2 or 5 days. Culture media was replaced once every 24 h that provided renewed secretagogues. Cellular levels of ACTH were determined by RIA, calculated as the mean ± S.E.M. (from triplicate wells).

cells (also known as chromaffin granules), contain the cysteine protease ‘prohormone thiol protease’ (PTP) that represents proenkephalin cleaving activity in these vesicles [17,19,35,47]. Recent studies have demonstrated participation of the subtilisin-like PC1/3 protease in the production of ACTH, based on findings of reduced ACTH in pituitaries of PC1/3 knockout mice [48]. However, corticosterone levels in plasma of PC1/3 knockout mice were normal, suggesting that sufficient amounts of ACTH were available for the regulation of adrenal corticosterone production. These findings suggest that proteases other than PC1 may be involved in producing ACTH. In addition, a patient with normal PC1/3 gene sequences showed markedly reduced plasma ACTH and elevated POMC [23], suggesting defect(s) in processing proteases other than PC1/3 for the conversion of POMC to

ACTH. Other proteases such as the neuroendocrine PC2 or PC5 processing enzymes [5,13,38,39,44] may be involved in ACTH production in the absence of PC1. In addition, based on results of this study, aspartyl and cysteine proteases may participate in the cellular metabolism of ACTH. It is noted that species differences in the participation of PC1/3 in pro-neuropeptide processing has been observed [48]. PC1/3 knockout mice showed reduced growth hormone and GHRH that were consistent with dwarfism of the mice; however, a patient with compound heterozygote mutations in the PC1 gene showed normal stature and growth [22]. These findings suggest differences in participation of PC1/3 in pro-neuropeptide processing in mouse and human species. Studies of this report utilized anterior pituitary cells from the rat species. Thus, in rat pituitary cells, results from this study provide evidence for a role of aspartyl and cysteine

Fig. 6. Inhibition of ACTH production by an aspartyl protease inhibitor. Cells were treated in the absence or presence of CRF (10 nM) for 5 days, with or without the aspartyl protease inhibitor acetyl-pepstatin A. Culture media was replaced once every 24 h to provide renewed CRF and protease inhibitor. Cellular levels of ACTH were measured by RIA, calculated as the mean ± S.E.M. (from triplicate wells). (∗) Statistically significant (two-tailed t-test) with P < 0.005.

Fig. 7. Inhibition of ACTH production by a cysteine protease inhibitor. Cells were treated in the absence or presence or CRF (10 nM) for 5 days, with or without the cysteine protease inhibitor E64d. Culture media was replaced once every 24 h to provide renewed CRF and protease inhibitor. Cellular levels of ACTH were measured by RIA, calculated as the mean ± S.E.M. (from triplicate wells). (∗) Statistically significant (two-tailed t-test) with P < 0.005.

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proteases in the cellular metabolism of ACTH. Participation of aspartyl or cysteine proteases in the metabolism of ACTH may be occurring concurrently with roles for members of the PC enzyme family in ACTH production. Moreover, effects of aspartyl and cysteine protease inhibitors may involve a variety of proteolytic processes that participate in the cellular metabolism of ACTH. Such possibilities may include aspartyl or cysteine protease regulation of positive or negative cofactors involved in either the synthesis or clearance (turnover) of cellular ACTH. For example, reduction of a positive cofactor for ACTH production by aspartyl or cysteine proteases could result in decreased cellular levels of ACTH. Or, reduction of a negative cofactor involved in the clearance or turnover of ACTH could enhance clearance of ACTH that would result in decreased cellular levels of ACTH. In summary, results from this study demonstrate that aspartyl and cysteine proteolytic mechanisms participate in regulating cellular levels of ACTH during its cellular depletion induced by secretagogues.

Acknowledgments This work was supported by grants from NIDA and NINDS of the National Institutes of Health. Technical assistance by William Hahn and Joseph Thoits are appreciated.

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