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Endocrinology Vol. 141, No. 8 3020-3034
Copyright © 2000 by The Endocrine Society

ARTICLES

Cell Type-Specific Metabolism of Peptidylglycine {alpha}-Amidating Monooxygenase in Anterior Pituitary1

Rajaa El Meskini, Richard E. Mains and Betty A. Eipper

Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Dr. Betty A. Eipper, Department of Neuroscience, WBSB 907, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205. E-mail: beipper{at}jhmi.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptidylglycine {alpha}-amidating monooxygenase (PAM) is a bifunctional enzyme expressed in each major anterior pituitary cell type. We used primary cultures of adult male rat anterior pituitary to examine PAM expression, processing, and secretion in the different pituitary cell types and to compare these patterns to those observed in transfected AtT-20 corticotrope tumor cells. Immunostaining and subcellular fractionation identified PAM in pituitary secretory granules and additional vesicular compartments; in contrast, in AtT-20 cells, transfected PAM was primarily localized to the trans-Golgi network. PAM expression was highest in gonadotropes, with moderate levels in somatotropes and thyrotropes and lower levels in corticotropes and lactotropes. Under basal conditions, less than 1% of the cell content of monooxygenase activity was secreted per h, a rate comparable to the basal rate of release of individual pituitary hormones. General secretagogues stimulated PAM secretion 3- to 5-fold. Stimulation with specific hypothalamic releasing hormones demonstrated that different pituitary cell types secrete characteristic sets of PAM proteins. Gonadotropes and thyrotropes release primarily monofunctional monooxygenase. Somatotropes secrete primarily bifunctional PAM, whereas corticotropes secrete a mixture of mono- and bifunctional proteins. As observed in transfected AtT-20 cells, pituitary cells rapidly internalize the PAM/PAM-antibody complex from the cell surface. The distinctly different steady-state localizations of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines may simply reflect the increased storage capacity of primary pituitary cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IMPORTANCE OF peptidylglycine {alpha}-amidating monooxygenase (PAM; EC 1.14.17.3) in the production of {alpha}-amidated peptides is well established, and the mechanisms through which copper, ascorbate, and molecular oxygen are used to catalyze the conversion of peptidylglycine substrates into {alpha}-amidated products are yielding to mechanistic and crystallographic studies (1). Our knowledge of how this enzyme functions in vivo, however, is largely based on studies of transfected endocrine cell lines (2). The fact that immortalized pituitary cell lines express much lower levels of PAM than those found in the pituitary and have relatively few secretory granules prompted us to study endogenous PAM in primary cultures of anterior pituitary cells. Although none of the major anterior pituitary hormones is {alpha}-amidated, PAM expression in the rat anterior pituitary is higher than that in any tissue other than the heart atrium (3). Joining peptide, a product of POMC processing (4), is amidated. In addition, small amounts of several other {alpha}-amidated peptides (substance P, neuropeptide Y, vasoactive intestinal peptide, galanin, GnRH, TRH, and pyroglutamyl-glutamyl-prolineamide) have been identified in the rat anterior pituitary (5, 6, 7, 8, 9, 10). These amidated peptides play important paracrine and autocrine roles in the pituitary (11).

The anterior pituitary is a complex tissue composed of several different major endocrine cell types. Early immunocytochemical studies indicated that the highest levels of PAM protein were found in gonadotropes, with detectable levels of PAM in each of the major pituitary cell types (12). Levels of thyroid hormone, sex steroids and glucocorticoids regulate PAM messenger RNA levels and activity in the anterior pituitary and affect the expression of selected {alpha}-amidated peptides in a cell type-specific manner (13, 14, 15). For example, during the estrous cycle, PAM expression in the female rat anterior pituitary is inversely related to circulating estrogen levels (14).

Peptide {alpha}-amidation is a two-step reaction that requires the sequential actions of the two catalytic domains of PAM. The first enzyme, peptidylglycine {alpha}-hydroxylating monooxygenase (PHM), is contained within the NH2-terminal third of the PAM precursor and catalyzes the copper-, molecular oxygen-, and ascorbate-dependent formation of peptidyl {alpha}-hydroxyglycine intermediates (1, 4). The second enzyme, peptidyl {alpha}-hydroxyglycine {alpha}-amidating lyase (PAL), catalyzes the conversion of this intermediate into an {alpha}-amidated product (4). Tissue-specific alternative splicing of the noncatalytic regions of the PAM gene generates messenger RNAs encoding integral membrane and soluble forms of PAM (4). Only when the noncatalytic exon A region is included between PHM and PAL can the two catalytic domains be separated by endoproteolysis (16). Although a similar collection of PAM transcripts is expressed in the anterior and neurointermediate lobes of the pituitary, tissue-specific endoproteolytic processing yields different products in these two regions (17). In the current study we employed cultured anterior pituitary cells to establish that expression and processing of PAM differ in the major endocrine cell types.

Subcellular fractionation studies showed that pituitary secretory granules contained integral membrane PAM along with a significant amount of soluble bifunctional PAM. In contrast to AtT-20 corticotrope tumor cells, little of the PAM in the anterior pituitary was recovered in the trans-Golgi network (TGN) fraction. However, a significant amount of the membrane PAM was located in either post-TGN organelles or an intracellular recycling compartment (18). Although pituitary cell lines have proven incredibly useful in elucidating pituitary cell function, major quantitative differences between cell lines and primary cells cannot be ignored. AtT-20 cells express far lower levels of POMC than do corticotropes; although AtT-20 cells have close to equimolar levels of POMC and prohormone convertase 1, pituitary cells (melanotropes) have 3000 times more POMC than prohormone convertase 1 (19).

We used primary cultures of anterior pituitary cells and immunohistochemical methods to compare the levels of PAM in somatotropes, lactotropes, gonadotropes, thyrotropes, and corticotropes. In each cell type, unlike AtT-20 corticotrope tumor cells, the majority of the PAM protein was localized to secretory granules. We employed general secretagogues to study regulated exocytosis of the products of PAM processing. In addition, we used individual hypothalamic releasing hormones to identify cell type-specific differences in the processing and exocytosis of PAM. As in transfected AtT-20 cells, significant internalization of PAM from the plasma membrane occurred under steady-state conditions. Although PAM trafficking in AtT-20 corticotrope tumor cells and primary pituitary cells appears to involve the same compartments, quantitative differences in their storage capacities lead to distinct differences in PAM metabolism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary anterior pituitary cell cultures
Anterior pituitary primary cultures were prepared as previously described (20). Anterior pituitaries from 5–20 adult male Sprague Dawley rats (175–200 g; Charles River Laboratories, Inc., Wilmington, MA) were separated from the neurointermediate lobes under a dissecting microscope. Cells were dissociated for 30 min at 37 C in DMEM-air medium containing 4 mg/ml collagenase (Worthington Biochemical Corp., Lakewood, NJ), 1 mg/ml hyaluronidase (Sigma, St. Louis, MO), 0.1 U/ml benzonase (EM Science, Germany), and 10 mg/ml fatty acid-free BSA, followed by a 15-min dissociation in medium containing 3 mg/ml trypsin (TO646; Sigma). This procedure consistently produced 1.5 x 106 cells/anterior pituitary. The dissociated cells were plated on protamine-coated culture wells in DMEM/Ham’s F-12 supplemented with 10% clone III FBS (HyClone Laboratories, Inc., Logan, UT) and 10% Nu-Serum IV (Collaborative Research, Bedford, MA) for the first 24 h. For all experiments, cells were used after growth for 4 days in the same medium containing 10 µM cytosine arabinoside (Sigma).

Secretion experiments and analysis
Cultured pituitary cells (750,000 cells/2-cm2 well) were initially rinsed for three 30-min periods in complete serum-free medium (CSFM)/BSA/DMEM/Ham’s F-12 with 1 µg/ml insulin, 0.1 µg/ml transferrin, and 0.1 mg/ml fatty acid-free BSA. Following this equilibration period, medium was collected after two 1-h periods of basal secretion (first basal and second basal) followed by a 1-h challenge period of stimulated secretion using one of the following secretagogues: 1 mM BaCl2, 1 µM phorbol 12-myristate 13-acetate (PMA; Sigma), 1 µM GnRH, 1 µM GH-releasing hormone (GHRH; Peninsula Laboratories, Inc., Belmont CA), 1 µM CRH (Sigma), 1 µM TRH (Peninsula Laboratories, Inc.), or 1 µM bromocriptine (Sigma). Each secretagogue was diluted in CSFM/BSA to the final concentration indicated above (21). The collected media were centrifuged to remove nonadherent cells and were stored until assay at -80 C after the addition of protease inhibitors (2 µg/ml leupeptin, 16 µg/ml benzamidine, and 300 µg/ml phenylmethylsulfonylfluoride). In one experiment, four wells (2 cm2 each) were used to test each secretagogue; quadruplicate samples were assayed in duplicate, and the mean ± SD are reported.

Cell extracts
Cells were scraped into 20 mM sodium N-Tris[hydroxymethyl]methyl-2-aminoethansulfonic acid (NaTES)/10 mM mannitol/1% Triton X-100 (TMT; pH 7.4) containing protease inhibitors. Cell extracts were frozen and thawed three times and centrifuged for 5 min to remove cell debris. For ACTH RIA, the cells were extracted in 5 N acetic acid with protease inhibitors, lyophilized, dissolved in immunoassay buffer, and stored frozen.

Protein analysis
For Western blot analysis, TMT extracts and medium samples were fractionated by 10% or 12% SDS-PAGE, transferred to Immobilon-P membranes (Millipore Corp., Bedfore, MA), and visualized using the indicated antibodies and the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL) (16). The antibodies used included rabbit polyclonal antisera to PHM (JH1761; 1:1000), exon A (JH629; 1:1000) (21, 22), and GH (JH89) (23) and a mouse monoclonal antibody to the cytosolic domain (CD) of PAM (6E6; 1:20) (2). PHM activity was measured in both cell extracts and media as previously described using {alpha}-N-acetyl-Tyr-Val-Gly as a substrate (24). Samples were assayed in duplicate, and reactions were carried out for 1.5 h. PHM specific activity is expressed as picomoles of product formed per h (units)/µg protein or as a percentage of the corresponding total enzyme activity in the cell extract. ACTH RIAs were performed on media and cell extracts using antibody Kathy (1:20,000) and [125I]ACTH-(1–39) (NEN Life Science Products, Boston, MA). Antiserum Kathy only recognizes POMC products in which the COOH-terminal end of ACTH-(1–39) is exposed (25).

Immunofluorescent staining
Pituitary cells (200,000 cells/well of a 4-well slide) were fixed with 4% paraformaldehyde in PBS (50 mM sodium phosphate and 150 mM sodium chloride, pH 7.4) for 30 min, permeabilized with 0.075% Triton X-100, and blocked with 2 mg/ml BSA in PBS for 1 h at room temperature. The antibodies used included rabbit polyclonal antibodies against exon A (JH629), TGN38 (JH1481) (2), chromogranin A (26), GH (23) and ACTH (Kathy antibody) and monoclonal antibodies to PAM CD (6E6) and ACTH (Novocastra Laboratories) (22). Antisera to rat LHß (IC-3, AFP571292393), FSHß (IC-2, AFPHFSH6), PRL (IC-5, AFP4251091), and TSH ß (IC-1, AFP1274789) were obtained through the generosity of the National Hormone and Pituitary Program, the NIDDK, and Dr. A. F. Parlow. Cells were incubated with primary antibodies overnight at 4 C, and the antigen-antibody complexes were visualized using fluorescein isothiocyanante (FITC)-conjugated goat antirabbit IgG (Caltag Laboratories, Inc., San Francisco, CA), Cy3-conjugated donkey antirabbit IgG, Cy3-conjugated goat antirabbit Fab, or Cy3-conjugated donkey antimouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). For quantification of cell types, all (n = 20–30) of the cells in 3 or 4 microscopic fields were counted; the data from 5 independent immunofluorescence staining experiments were then averaged (>500 cells total).

Sequential immunofluorescence staining using two rabbit polyclonal antibodies was performed as described by McCormick et al. (27). Immunofluorescent staining of PAM in pituitary cells was performed using PAM exon A antiserum followed by antirabbit Cy3-labeled Fab. In a sequential step, the cells were incubated with polyclonal antisera to FSH, LH, GH, PRL, ACTH, or TSH followed by FITC-tagged goat antirabbit IgG. Control experiments demonstrated that there was no Cy3 signal when the PAM primary antibody was deleted and no FITC signal when the pituitary hormone primary antiserum was omitted. Cells were viewed with a Carl Zeiss Axioskop microscope (Carl Zeiss, Thornwood, NY) and were photographed with a Micromax CCD camera (Princeton Instruments, Princeton, NJ) or a Spot RT camera (Diagnostic Instruments, Sterling Heights, MI).

Subcellular fractionation
Cultures prepared from 10 anterior pituitaries were harvested at 4 C in 10 vol homogenization buffer containing 0.32 M sucrose, 10 mM Tris-HCl (pH 7.4), and protease inhibitors and passed 6 times through a 26-gauge needle and then 12 times through a ball-bearing homogenizer (H&Y Enterprises, Redwood City, CA) (28). Cell debris was removed by centrifugation at 800 x g for 5 min. The supernatant was then centrifuged at 4,000 x g for 15 min to obtain a P1 pellet. The resulting supernatant was separated into a P2 pellet and soluble fraction by centrifugation at 10,000x g for 30 min (18). The P1 pellet (TGN enriched) and the P2 pellet (largely depleted of TGN38 and secretory granule enriched) were resuspended in homogenization buffer and fractionated further on 2 different types of discontinuous sucrose gradient. Resuspended P1 was layered onto a density gradient consisting of 200 µl 0.4 M sucrose; 250 µl 0.6 M sucrose; 350 µl each of 0.8, 1.0, 1.2, and 1.4 M sucrose; and 200 µl each of 1.6 M sucrose; this gradient was designed to separate TGN-enriched fractions from lighter membranes and from secretory granules, which collect at the bottom of the gradient. Resuspended P2 was layered onto a density gradient of 200 µl each of 0.4, 0.6, 0.8, and 1.0 M sucrose; 350 µl each of 1.2, 1.4, and 1.6 M sucrose; and 200 µl 2.0 M sucrose; this gradient was designed to keep even the most dense secretory granules from pelleting. Both gradients were centrifuged for 2 h at 120,000 x g; 150-µl fractions were collected from the top of the gradient, and proteins in an equal fraction of each sample were analyzed (18).

Antibody internalization experiments
Primary pituitary cells were incubated in rabbit polyclonal antiserum to PAL (JH471) diluted 1:50 in CSFM for 30 min at 37 C and either prepared immediately for immunofluorescence staining or chased in antibody-free medium for 1 or 2 h at 37 C (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of PAM in anterior pituitary cell culture
We first used indirect immunofluorescence staining to evaluate PAM expression in primary cultures of adult male rat anterior pituitary. Under the growth conditions used, the cultures consisted almost entirely of endocrine cells (Fig. 1Go). Visualization of filamentous actin with FITC-phalloidin revealed only a small number of fibroblast-like cells (not shown). Most of the anterior pituitary cells exhibited PAM staining, with some cells more intensely stained than others (Fig. 1AGo, white arrows). Punctate staining for PAM was observed throughout the cytosol of most of the endocrine cells (Fig. 1CGo). The images shown in Fig. 1Go were obtained using antiserum specific for the exon A region of PAM, but similar localization and intensity patterns were observed using antibodies to PHM or to the CD of PAM (Fig. 2AGo). In contrast to transfected AtT-20 cells, the staining pattern observed in anterior pituitary endocrine cells suggests that most of the PAM is localized in vesicular structures.



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  		Figure 1. PAM immunostaining in pituitary cell cultures. After 4 days in culture, primary anterior pituitary cells were fixed, permeabilized, and incubated overnight with polyclonal antibody to PAM exon A. A, Primary antibody was visualized with FITC-tagged goat antirabbit IgG. Pituitary cells typical of those expressing the highest levels of PAM are indicated with white arrows. B, The corresponding phase contrast image is shown. C, At higher magnification, the vesicular nature of the PAM staining (Ves) is apparent, as is the unstained cell nucleus (N).

 


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  		Figure 2. The steady-state subcellular localization of PAM differs in primary pituitary cells and transfected AtT-20 cells. A, Cells were simultaneously incubated with mouse monoclonal antibody to PAM-CD and rabbit polyclonal antibody to TGN38. The two primary antibodies were then visualized with fluorescently tagged secondary antisera (PAM, red; TGN38, green). B and C, PAM exon A and chromogranin A were visualized by the sequential application of two rabbit antisera; PAM staining was visualized with Cy3-tagged goat antirabbit IgG Fab (red), and chromogranin A with FITC-tagged goat antirabbit IgG Fab (green). Images were superimposed, and areas visualized by both antibodies appear yellow. In the higher magnification image (C), areas of PAM and chromogranin A coimmunostaining are indicated by arrows, vesicles stained only by the chromogranin A antibody by arrowheads, vesicles stained only by the PAM antibody by lines, and the nucleus by N. D, AtT-20 corticotrope tumor cells expressing rat PAM-1 were visualized as described in A. PAM-TGN38 colocalization is shown in yellow by arrows; three nontransfected cells showing intense green fluorescence for TGN38 are indicated by V shapes. E, AtT-20 PAM-1 cells were incubated with a rabbit polyclonal antibody to ACTH and a mouse monoclonal antibody to PAM-CD. Both antibodies were visualized with fluorescently tagged secondary antisera (PAM, red; ACTH, green). PAM staining colocalized with ACTH in the TGN region (shown in yellow with arrows). Arrowheads indicate ACTH staining in the tips of the cell. The magnification was the same for A, B, D, and E.

 
To identify the major subcellular compartments containing PAM, primary cultures were fixed and simultaneously visualized with an antibody for PAM and an antibody to TGN38, a marker for the TGN (30) (Fig. 2AGo). PAM (stained in red) and TGN38 (in green) images were superimposed. TGN38 was highly localized to the perinuclear region of each cell. In contrast, the PAM CD antibody immunostained vesicular structures distributed throughout the cell, with very little PAM concentrated in the TGN region. Simultaneous staining for PAM and chromogranin A, a soluble secretory granule marker (31, 32), was carried out to determine whether the punctate PAM staining corresponded to secretory granules (Fig. 2Go, B and C). In the pituitary endocrine cells expressing high levels of chromogranin A, PAM and chromogranin A were often localized to the same vesicular structures (Fig. 2Go, B and C, arrows). However, the distributions of chromogranin A and PAM were not identical, with some vesicles staining more intensely for chromogranin A (arrowheads), and others staining more intensely for PAM (lines).

These results are strikingly different from the steady-state localization of PAM in AtT-20 corticotrope tumor cells stably expressing exogenous membrane PAM (PAM-1; Fig. 2Go, D and E). The shape and organization of the TGN are distinctly different in primary pituitary cells and AtT-20 corticotrope tumor cells, but the TGN forms a compact, perinuclear structure in both systems. As reported previously (2), most of the PAM protein in stably transfected AtT-20 cells is colocalized with TGN38 in a complex of tubulovesicular structures localized to one side of the nucleus (Fig. 2DGo, arrows). As evidenced by the nontransfected cells included in Fig. 2DGo (V shapes), TGN38 staining was not affected by expression of PAM. As shown by ACTH staining (Fig. 2EGo), some secretory granules in PAM-1-expressing AtT-20 corticotrope tumor cells are present at the tips of the cellular processes (arrowheads), but most of the PAM protein colocalizes with ACTH in the TGN region (Fig. 2EGo, arrows).

PAM proteins in anterior pituitary cell culture
To evaluate the secretion of PAM proteins from the different anterior pituitary cell types, we first identified the major PAM proteins present in the cultures. PAM-1/{Delta}Bb, PAM-2/{Delta}Bb, and PAM-3 are the major PAM proteins expressed in the anterior pituitary of Sprague Dawley rats from Charles River Laboratories, Inc. (Fig. 3Go) (33). Intact PAM-1/{Delta}Bb (125 kDa), PAM-2/{Delta}Bb (105 kDa), and PAM-3 (93 kDa) were clearly identified using antisera to PHM, exon A, or PAM-CD (Fig. 3Go). Based on the patterns observed with the PHM and CD antibodies, PAM-2/{Delta}Bb is the most prevalent PAM protein, with lesser amounts of PAM-1/{Delta}Bb and PAM-3 expressed. The 73-kDa protein visualized by the PHM antibody (sPAM-2) is the major protein generated by the endoproteolytic cleavage of PAM-2 and PAM-3. In addition, pituitary cells cleaved PAM-1/{Delta}Bb to yield a 110-kDa soluble protein (sPAM-1) or a 70-kDa membrane PAL and a 45-kDa soluble PHM. Production of 50-kDa soluble PAL requires cleavage of PAM-1/{Delta}Bb both within exon A and between PAL and the transmembrane domain; only small amounts of soluble PAL were detected.



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  		Figure 3. Major PAM proteins in anterior pituitary cultures. Cultures extracted with TES-mannitol/TX-100 were subjected to Western blot analysis and visualized with antiserum to PHM, exon A, or PAM CD. Scale drawings of PAM-1/{Delta}Bb (right) and PAM-2/{Delta}Bb or PAM-3 (left) are shown. The major cleavage products (indicated in italics) of these bifunctional proteins are diagrammed. Apparent mol wts for the various proteins are indicated. TMD, Transmembrane domain; CD, cytosolic domain; lollipop, N-linked oligosaccharide.

 
Stimulated secretion of PHM enzyme activity and PAM protein from anterior pituitary cells
Most of our understanding of PAM protein trafficking and secretion has come from studies of AtT-20 corticotrope tumor cells expressing exogenous PAM (21, 34). However, secretory granules are far more abundant in anterior pituitary endocrine cells than in AtT-20 cells, and as shown in Fig. 2Go, the steady-state localization of PAM is quite different in primary pituitary cells and transfected AtT-20 cells. Before examining the effects of cell-type specific secretagogues, we analyzed the secretion of PHM enzymatic activity and PAM protein under basal conditions and in response to stimulation by two general secretagogues, BaCl2 and PMA (21) (Fig. 4Go). Ba2+ mimics the effect of Ca2+ in the regulation of exocytosis (35). Ba2+ blocks K+ channels, causing cell depolarization followed by voltage-gated Ca2+ channel opening and Ba2+ entry into the cytosol. Furthermore, Ba2+ triggers exocytosis by releasing Ca2+ from intracellular stores. PMA is known to stimulate protein kinase C, inducing a Ca2+-independent secretion of secretory granule constituents via phospholipase D activation (36). It has been shown in male rat pituitary gonadotropes that exocytosis of secretory vesicles can be stimulated independently by either Ca2+ elevation or PMA activation of protein kinase C (37). Thus, Ba2+ stimulates secretion from mature granules, whereas PMA stimulates Ca2+-independent exocytosis at earlier steps of the secretory pathway, affecting secretion from mature and immature pools of vesicles.



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  		Figure 4. Stimulated secretion of PHM activity and PAM protein from pituitary cells. Cultures were incubated in basal medium for two 1-h periods, then exposed to either 1 mM BaCl2 or 1 µM PMA for 1 h. Cultures were extracted in TMT. A, PHM enzymatic activity measured in duplicate samples of medium was expressed as units of product formed per microgram of protein (left) and as a percentage of the cell content of enzyme activity (right). Data are the mean ± SD for four cultures for each secretagogue; where error estimates are small, error bars are not visible. Similar results were obtained in two additional independent experiments. B, Equal amounts of basal and stimulated media were analyzed by Western blot and probed with antisera to PHM or exon A. The identity of PAM-3 was confirmed using the PAM-CD antibody (not shown).

 
Secretion experiments involved collecting medium for two sequential 1-h basal periods followed by the addition of secretagogue (BaCl2 or PMA) for a final 1-h stimulation period. The PHM activity in spent medium was assayed and compared with PHM activity in whole cell extracts (Fig. 4AGo). The basal rate of secretion of PHM activity (0.05 U/µg protein) corresponded to 1% of the cell content/h. The addition of PMA stimulated secretion of PHM activity 4.5 ± 0.5-fold over basal levels; 5.3 ± 0.4% of the cell content of PHM activity was released in a 1-h period. The addition of BaCl2 stimulated secretion of PHM activity 3.5 ± 0.5-fold over basal levels, with 3.3 ± 0.5% of the cell content of PHM activity released. The ability of BaCl2 to stimulate secretion is consistent with the presence of a significant amount of PAM in secretory granules. The fact that PMA consistently stimulated the release of PHM activity more effectively than BaCl2 could be due to differences in the responses of different pituitary cell types to the two secretagogues (38, 39) or to differences in the responsiveness of various compartments in the secretory pathway (37, 40), but is similar to the effects of the two secretagogues on transfected AtT-20 cells (41, 42).

To characterize the PAM proteins secreted upon stimulation with PMA or BaCl2, equal volumes of spent medium from basal and challenge periods were subjected to Western blot analysis and visualized using antisera to PHM or exon A (Fig. 4BGo). Under basal conditions, the major PAM proteins recovered from the medium were intact PAM-3 and sPAM-2. The production of sPAM-2 requires an endoproteolytic cleavage, but its precursor could be either PAM-3 or PAM-2/{Delta}Bb. The basal secretion of proteins derived from PAM-1/{Delta}Bb was barely detectable. Intact PAM-3 and sPAM-2 were more prevalent in medium from the stimulation period. Secretion of proteins derived from PAM-1/{Delta}Bb was also stimulated, with a robust increase in secretion of sPAM-1 and PHM. As expected from the enzyme assays, PMA was a more effective stimulant of PAM secretion than BaCl2. The endocrine cells of the anterior pituitary store a significant amount of PAM in compartments from which release can be stimulated.

Sucrose density gradient fractionation of anterior pituitary cells
We used sucrose density gradient fractionation to identify the PAM proteins enriched in secretory granules. These PAM proteins were then compared with the PAM proteins secreted in response to stimulation with secretagogue. Anterior pituitary cultures were subjected to differential centrifugation followed by sucrose gradient fractionation (18). Based on staining for TGN38, this marker was recovered entirely in the 4,000 x g pellet; no TGN38 was recovered in the 10,000 x g pellet (data not shown). Staining for GH indicated that secretory granules were recovered in both the 4,000 x g pellet and the 10,000 x g pellet (Fig. 5Go, A and B). Most of the pituitary hormone granules cofractionate in gradients of this type (43). The distribution of PAM proteins throughout both gradients was evaluated using an antibody to PHM. As evidenced by the presence of GH, secretory granules appeared in fractions 12–14 of the 4,000 x g pellet-gradient; this gradient resolved TGN from granules and lighter membranes (Fig. 5AGo). Fractions 12–14 were enriched in PAM-3, sPAM-2, and 45 kDa PHM. Intact, bifunctional PAM-2/{Delta}Bb and PAM-3 were located in the lighter fractions of the gradient. Fractions enriched in TGN markers were not enriched in PAM (Fig. 5AGo) (18).



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  		Figure 5. Sucrose gradient fractionation. Anterior pituitary cultures were subjected to differential centrifugation. The TGN-enriched 4,000 x g pellet (A) and the secretory granule-enriched 10,000 x g pellet (B and C) were further fractionated on discontinuous sucrose density gradients of different compositions (Oyarce 1995). Fractions were collected from the top of the gradient (fraction 1), and PAM proteins were identified by Western blot analysis using PHM antibody (A and B) or exon A antibody (C). Secretory granules were identified using a GH antiserum. The gradient in A was designed to pellet secretory granules and yield TGN in fractions 9–12, whereas the gradient in B and C was designed to prevent secretory granules from reaching the bottom of the tube.

 
Secretory granules appeared in fractions 8–14 of the 10,000 x g pellet gradient, which was designed to prevent pelleting of the densest secretory granules (Fig. 5BGo). These fractions contained primarily 45-kDa PHM, with sPAM-2 barely detectable. GH granules, which are very dense, were recovered further down the gradient than PHM-enriched granules (Fig. 5BGo). The exon A antibody was used to localize products of PAM-1 cleavage. The granule-enriched fractions from the 10,000 x g gradient (Fig. 5CGo) contained 110-kDa sPAM-1, membrane PAL (70 kDa), and 45-kDa PHM. The lighter density region (fractions 4–7) of this gradient contained primarily intact bifunctional PAM-1/{Delta}Bb, PAM-2/{Delta}B, and PAM-3, as confirmed by visualization with both PHM and exon A antisera (Fig. 5Go, B and C). Soluble PAM proteins were enriched in secretory granules, whereas membrane PAM proteins were prevalent in the lighter fractions.

PAM staining in individual anterior pituitary cell types
Anterior pituitary endocrine cells clearly fall into at least three different groups based upon their levels of expression of PAM (Fig. 1Go); a similar pattern of staining is observed with antisera to several different regions of PAM. To determine whether high, moderate, and low levels of PAM expression are consistently observed in individual pituitary cell types, sequential immunofluorescence was used to colocalize PAM and each major pituitary hormone: FSH, LH, GH, PRL, ACTH, and TSH (Fig. 6Go). Under the growth conditions used, approximately 40% of the primary pituitary cells were somatotropes (GH), 30% were lactotropes (PRL), 15% were corticotropes (ACTH), 15% were gonadotropes (LH and FSH), and less than 10% were thyrotropes (TSH).



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  		Figure 6. Colocalization of major pituitary hormones and PAM. PAM was localized in pituitary cells using PAM exon A antiserum followed by antirabbit Cy3-labeled Fab (red). The cells were then incubated with rabbit polyclonal antiserum to FSHß (A), LHß (B), GH (C), PRL (D), ACTH (E, right), or TSHß (F), followed by FITC-tagged antirabbit IgG (green). Control experiments demonstrated that there was no Cy3 signal when the PAM antibody was deleted and no FITC signal when the pituitary hormone antiserum was omitted. In addition, simultaneous visualization using the PAM exon A polyclonal antibody and a monoclonal antibody to ACTH followed by Cy3-tagged antirabbit and FITC-tagged antimouse secondary antisera (E, left) yielded the same result as the sequential staining. In each of the major pituitary cell types (arrows), PAM staining (in red) and pituitary hormone staining (in green) were superimposed. The combined images showing areas of PAM/hormone overlap are yellow.

 
Immunofluorescent images of PAM (in red) and each pituitary hormone (in green) were superimposed. The colocalization studies revealed a correlation between levels of PAM expression and pituitary cell type. Cells containing the highest levels of PAM protein were always visualized with antisera to LH and FSH (Fig. 6Go, A and B). PAM staining in the remaining pituitary cells types was above background, but was of moderate to low intensity. Somatotropes and thyrotropes expressed moderate levels of PAM (Fig. 6Go, C and F). By comparison, PRL cells and corticotropes contained the lowest levels of PAM protein (Fig. 6Go, D and E).

Stimulated secretion from individual anterior pituitary cell types
To evaluate the responsiveness of PAM stored in the different pituitary cell types to secretagogues, we challenged anterior pituitary cultures with individual hypothalamic releasing hormones and measured secretion of PHM activity (Fig. 7AGo). Secretion was examined during two sequential basal collections, as described above. The cells were then stimulated with GnRH, GHRH, CRH, or TRH during the final 1-h period. Overall, the PHM secreted during the basal collection period (1% of pituitary cell content of PHM activity/h) reflected constitutive-like secretion from somatotropes, gonadotropes, corticotropes, and thyrotropes plus secretion from lactotropes removed from the tonic inhibitory influence of dopamine.



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  		Figure 7. Stimulated secretion of PHM activity from individual pituitary cell types. Medium was collected after two consecutive 1-h periods of basal secretion and then 1 h of exposure to individual hypothalamic releasing factors: GnRH, GHRH, CRH, and TRH. The cultures were also treated with bromocriptine to inhibit secretion from lactotropes. A, Medium samples were assayed for PHM activity. The data are expressed as the mean ± SD for four independent wells of cells receiving each secretagogue; where error estimates are small, error bars are not visible. PHM specific activity was expressed as units of product formed per µg protein and as a percentage of the cell content after measuring enzyme activity in cell extracts. B, GH secretion was evaluated by Western blot analysis (using a GH antibody) of equal aliquots of the GHRH medium samples. C, ACTH secretion was determined by RIA of medium samples after CRH stimulation. Similar results were obtained in two additional complete experiments.

 
Application of GnRH or CRH stimulated secretion of approximately 1% of the total cell culture content of PHM activity. Application of GHRH stimulated secretion of 3% of the total pituitary content of PHM (Fig. 7AGo). As somatotropes are approximately 3-fold more prevalent than gonadotropes or corticotropes, the effects of the different secretagogues on a per cell basis were similar. Application of TRH also stimulated the release of PHM activity, with the relatively modest effect reflecting the scarcity of thyrotropes in these cultures. The application of bromocriptine, a dopaminergic agonist, decreased the secretion of PHM activity 2-fold, suggesting that half of the basal secretion represented unchecked secretion from lactotropes.

For corticotropes and somatotropes, we compared the ability of secretagogue to stimulate release of PHM activity and the pituitary hormone specific for each cell type (Fig. 7Go, B and C). Using the GH antibody, Western blot analysis of equal volumes of medium collected from the two basal collection periods and during application of GHRH showed a greater than 10-fold increase in GH secretion (Fig. 7BGo). Secretion of immunoreactive ACTH was determined under both basal and CRH-stimulated conditions and increased over 10-fold as well (Fig. 7CGo). Over 20% of the total cell culture content of ACTH was released during the 1-h incubation with CRH. The percentage of the total culture content of PHM that was released in response to all of the secretagogues was significantly lower (~7%). This was consistent with the localization of PAM to both secretory granule-enriched fractions and lighter fractions after density gradient centrifugation (Fig. 5Go) and with the staining of PAM in vesicles containing chromogranin A (presumably mature secretory granules) and also other vesicles (Fig. 2CGo).

Stimulated secretion of PAM from individual pituitary cell types
Along with mature hormone, secretory granules contain the final protein products produced by endoproteolytic cleavage of PAM; the enzymes responsible for cleaving PAM have not been established. We exposed cultures to the individual hypothalamic releasing hormones so that we could investigate PAM processing in individual anterior pituitary cell types. Equal volumes of spent medium from basal and challenge periods were visualized on Western blots using antisera to PHM and exon A (Fig. 8Go); exposure times for each pair of basal and stimulated medium were selected to provide an accurate (nonsaturated) image of the stimulated samples, which vary depending on the prevalence and stimulation of each cell type. Overall, basal medium was enriched in PAM-3 and sPAM-2. As the PHM antibody visualizes proteins derived from PAM-1/{Delta}Bb, -2/{Delta}Bb, and -3, Western blot analysis with the PHM antiserum is the most accurate way to compare the effects of the different secretagogues.



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  		Figure 8. Forms of PAM protein secreted in response to hypothalamic releasing hormones. Equal volumes of basal and stimulated medium were analyzed by Western blot using antisera to PHM and exon A. Exposure times for each pair of samples (basal and stimulated for each antibody) were selected so that no bands were saturated in the stimulated sample; as a result, some of the bands in the basal samples are faint to undetectable. The basal images in different panels cannot be compared directly, because their intensities are regulated by the number of cells of a given pituitary cell type and the relative stimulation of that cell type. Proteins derived from PAM-1 are shown on the right, and proteins derived from PAM-2 or PAM-3 are shown on the left. Additional Western blots using PAM-CD antibody (not shown) verified the identity of the PAM proteins. Similar results were obtained in two additional independent experiments.

 
In response to GnRH (Fig. 8AGo), gonadotropes showed little increase over basal levels of secretion of sPAM-2. However, the release of 45-kDa PHM, a product of PAM-1/{Delta}Bb processing, was very responsive to GnRH stimulation. In contrast, treatment with the somatotrope-specific secretagogue GHRH strongly stimulated the secretion of PAM-3 and sPAM-2 (Fig. 8BGo); 45-kDa PHM was not a major secretory product from somatotropes. Use of the more sensitive exon A antibody demonstrated that GHRH did stimulate the secretion of 45-kDa PHM and sPAM-1. In response to CRH (Fig. 8CGo), secretion of 45-kDa PHM was stimulated to a greater extent than secretion of PAM-3 or sPAM-2. Using the exon A antibody, CRH could be seen to stimulate the secretion of both sPAM-1 and PHM. PAM-1 may thus be the major form of PAM expressed in corticotropes (Fig. 8CGo). In response to TRH (Fig. 8DGo), thyrotropes showed increased secretion of 45-kDa PHM and PAM-3, with little effect on secretion of sPAM-2. This suggests that the major forms of PAM protein in thyrotropes are PAM-1 and PAM-3.

Anterior pituitary cells internalize PAM/PAM antibody complexes
The steady-state localizations of PAM in primary pituitary cells and in transfected AtT-20 cells are distinctly different despite the fact that cleavage, storage, and secretion of PAM are quite similar in the two systems. The steady-state pattern reflects the balance of PAM traffic into and out of a series of subcellular compartments. The dynamic nature of PAM trafficking is especially apparent when one examines the trafficking of PAM proteins that reach the plasma membrane. To examine the routing of membrane PAM in the endocytic pathway of pituitary cells, live anterior pituitary primary cultures were incubated under basal conditions with PAL antiserum for 30 min (pulse), washed, and either harvested or further incubated (chase) for 1 h. The lumenal epitope of PAM recognized by this antibody allows it to bind to any PAM present on the cell surface and to undergo endocytosis. A subpopulation of the cultured pituitary cells internalized easily detectable amounts of the PAM/PAM-antibody complex from the cell surface (Fig. 9Go). Immediately after the pulse with the PAL antibody, the PAM/PAM-antibody complex was internalized into small, uniform vesicles distributed all over the cell and extending to the edges of the cell (Fig. 9AGo). After the 1-h chase in antibody-free medium, the PAM/PAM antibody complex was present in a more heterogeneous collection of vesicular structures; some were much larger than the vesicular structures observed initially. There was no change in intensity after 1 h (Fig. 9BGo) or 2 h (not shown) of chase, suggesting that the PAM/PAM antibody complex was not degraded. In contrast, the PAM/PAM-antibody complex internalized by transfected AtT-20 cells is largely localized to the TGN region of the cell within 30 min of chase (34).



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  		Figure 9. Internalization of PAM ectodomain antibody under basal conditions. Anterior pituitary cells were incubated with PAL antibody for 30 min and either harvested (A) or chased in antibody-free medium for 1 h at 37 C (B). The cells were fixed and permeabilized, and the internalized PAM/PAM-antibody complex was visualized with fluorescently tagged antiserum. The narrow arrows indicate small uniformly sized vesicles present throughout the cell, even near the edges; the wide arrows indicate staining of the larger vesicular structures that become apparent during the chase.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
After the purification of PHM and PAL from tissue, most studies of PAM have used transfected cell lines and recombinant protein (1, 21, 29, 44). Although a great deal has been learned about the mechanism of the PAM reaction and differences in the trafficking of soluble and membrane forms of PAM, the behavior of PAM in cultured neuroendocrine cell lines cannot be fully representative of its behavior in the many different cell types in which it is present in vivo. For example, AtT-20 corticotrope tumor cells have fewer secretory granules than primary corticotropes and produce roughly 5% as much POMC as primary corticotropes (21, 45). The metabolism of endogenous PAM in primary atrial cardiomyocytes differed dramatically from its metabolism in transfected AtT-20 or hEK-293 cells (46). A brief summary of the metabolism of integral membrane PAM in AtT-20 cells is presented in Fig. 10AGo.



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  		Figure 10. Trafficking of PAM in primary pituitary cells and transfected AtT-20 cells. A, The major steps in PAM trafficking have been elucidated by studying soluble and membrane forms of PAM stably expressed in AtT-20 cells (reviewed in Refs. 2 and 67). The system is dynamic, with a major component of basal secretion (CLV, constitutive-like vesicle) and relatively little storage in secretory granules. About 1 h after synthesis, PAM exits the TGN; soluble PHM is generated only after exit from the TGN, and a great deal of it undergoes constitutive-like secretion. Very little PAM resides on the plasma membrane at steady-state, undergoing rapid endocytosis. The pool of mature granules (large dense core vesicles) is small, with the cell content of ACTH secreted roughly every 5 h, even in the absence of secretagogue. The half-life of integral membrane PAM is roughly 3 h, consistent with degradation after endocytosis. The width of the arrows gives a qualitative indication of the flux of PAM through each pathway. B, Assuming that all of the same compartments are accessible to PAM in primary pituitary cells, the dramatically different steady-state distributions of PAM may be explained by a single difference. PAM proteins exiting immature secretory granules have unlimited access to mature secretory granules, which are stored for very long times; as a result, very little PAM returns to late endosomes for recycling to the TGN or basal secretion. Basal rates of hormone secretion from primary pituitary cells are less than 1% of the cell content/h, and the pool of mature granules is large. Very little PAM is on the cell surface at steady-state, and PAM appearing on the plasma membrane is rapidly endocytosed.

 
We used our knowledge of PAM trafficking in AtT-20 cells to study PAM processing, storage, and secretion in primary pituitary cultures. We first evaluated PAM expression in the different endocrine cell types. The anterior pituitary cell cultures contained very few nonendocrine cells, with the major cell type under our growth conditions being the somatotrope. In vivo, the highest levels of PAM were detected in gonadotropes with moderate levels in corticotropes; PAM levels in somatotropes and lactotropes were distinctly lower (12). In cell culture, PAM expression was also highest in gonadotropes, but significant levels of PAM were present in somatotropes and thyrotropes, and the lowest levels were found in corticotropes and lactotropes. These differences may be explained by the substantial hormonal regulation of PAM expression known to occur in vivo.

As shown by subcellular fractionation experiments, soluble PAM proteins cleaved from membrane precursors are enriched in secretory granules, whereas the membrane PAM proteins are prevalent in post-TGN, light membrane fractions (18). The results of our immunohistochemical localization of PAM in anterior pituitary endocrine cells are consistent with these biochemical data. PAM staining is localized to a variety of vesicular structures, only some of which are recognized by antisera specific to secretory granule components such as chromogranin A. Vesicular structures recognized only by the PAM antibody or only by the chromogranin A antibody are also common.

The single most striking difference between the behavior of endogenous PAM in primary pituitary cells and that of exogenous PAM in AtT-20 cells is the steady-state localization of the protein. In pituitary cells, as in hypothalamic neurons and cultured atrial myocytes, PAM is not concentrated in the trans-Golgi network (46, 47). In sharp contrast, membrane PAM proteins expressed in AtT-20 corticotrope tumor cells are primarily localized to the same structures visualized by antisera to TGN38 (2). Even at the immunoelectron microscopic level, a significant amount of overlap was observed between transfected PAM and endogenous TGN38, with PAM expression enriched in more distal compartments of the TGN (2).

We compared the trafficking of integral membrane PAM in AtT-20 cells and anterior pituitary cells. Sorting of components out of immature secretory granules is mediated by adaptor protein-1 (AP1) and clathrin-coated vesicles (48). Immature secretory granules contain several membrane proteins not found in mature granules; these included furin, the cation-independent mannose-6-phosphate receptor, and carboxypeptidase D (CPD) (49, 50). PAM is also found in immature secretory granules (2). The localization of endogenous CPD in AtT-20 cells is like that of exogenous membrane PAM (50). Soluble PC1 is also localized to the TGN (51), whereas soluble CPE is localized to secretory vesicles in cell processes, as are soluble PHM and PAL (52). Like PAM, CPD and furin cycle to the plasma membrane, undergoing rapid endocytosis (50, 53, 54). Unlike PAM, the lumenal domains of CPD and furin are secreted by the constitutive pathway.

As proposed in Fig. 10BGo, alterations at a single step in the complex itinerary traversed by membrane PAM, i.e. exit from immature secretory granules, would produce the observed difference in steady-state localization. AtT-20 cells contain far fewer mature secretory granules than pituitary cells (25). In primary pituitary cells, PAM proteins leaving immature secretory granules largely go to mature granules; in AtT-20 cells, PAM proteins leaving immature secretory granules are shuttled to late endosomes, with constitutive-like secretion of soluble PHM and intracellular retention of membrane PAM (2). There is no simple correlation between the level of expression of processing enzymes and the ability of cells to make secretory granules and exhibit regulated secretion, as there are AtT-20 variant lines with normal processing enzyme levels but no secretory granules (55).

We first examined the secretion of PAM by quantifying the secretion of enzyme activity under basal and secretagogue-stimulated conditions. Making the assumption that the secretion observed in response to PMA or BaCl2 should come primarily from mature secretory granules (35, 36, 40), we compared the forms of PAM protein in the medium to the forms of PAM protein in the secretory granule fraction after gradient centrifugation. Lactotropes, released from tonic dopaminergic inhibition, account for about half of the basal rate of monooxygenase secretion (1% of the cell content/h with 0.5% from lactotropes). This rate is typical of basal rates of secretion of hormone from a variety of endocrine tissues (56, 57, 58, 59, 60, 61). Basal secretion from the anterior pituitary cultures (clearly from the whole mixture of cell types) is enriched in PAM-3 (93 kDa), a product requiring no endoproteolytic cleavage, and sPAM-2 (73 kDa), a bifunctional enzyme that could be created by a single endoproteolytic cleavage of PAM-3 or PAM-2. After the addition of PMA, approximately 5% of the total cell culture content of PHM activity is secreted in 1 h. The fact that secretagogues such as CRH can release a larger percentage of the culture content of hormone (>20%) is consistent with the biochemical and immunocytochemical observations that PAM is localized to secretory granules as well as other organelles.

The use of specific hypothalamic secretagogues made it possible to demonstrate that different pituitary cell types secrete distinct patterns of PAM proteins under stimulated conditions. For example, 45-kDa PHM, a product of PAM-1/{Delta}Bb processing, is the major form of PAM protein secreted by stimulated gonadotropes. Somatotropes secrete PAM-3 and sPAM-2 in response to stimulation. The major form of PAM expressed in corticotropes is PAM-1/{Delta}Bb, as evidenced by their secretion of primarily monofunctional PHM. Thyrotropes secrete predominantly PAM-3 and products derived from PAM-1/{Delta}Bb. Although the proteases that cleave PAM have not been identified, it is clear from transfection studies that both PC1 and PC2 can cleave within exon A; PC2 can also release PAL from the membrane anchor (22, 41, 62). Corticotropes, gonadotropes, and thyrotropes contain PC1 (63, 64), consistent with secretion of 45-kDa PHM by these three cell types. There is far less PC2 in the adult anterior pituitary than PC1 (63, 65). Somatotropes are not known to have high levels of PC1 or PC2; consistent with this, our data show secretion of primarily bifunctional PAM secreted from GH cells. Corticotropes, which produce amidated joining peptide as part of the metabolism of POMC, contain lower levels of PAM than most other anterior pituitary cell types and still {alpha}-amidate essentially all of the joining peptide produced as long as copper and ascorbate are present in adequate amounts (4).

As observed with transfected AtT-20 cells, robust internalization of PAM/PAM-antibody complexes from the surface of anterior pituitary cells occurred under basal conditions. In both systems, internalized PAM/PAM-antibody complexes are initially present in small uniform vesicles distributed all over the cell. Later, a more heterogeneous collection of intracellular vesicular structures is observed. The intensity of the signal obtained from the internalized PAM/PAM-antibody complexes remained constant for at least 2 h, suggesting that the internalized complexes are not degraded or rapidly resecreted. Unlike transfected AtT-20 cells, massive accumulation of PAM/PAM-antibody complexes in the TGN region is not observed in primary pituitary cells. This difference may simply reflect the prevalence of mature secretory granules in pituitary cells and differences in trafficking from immature secretory granules. Instead of remaining in endosomes or the TGN, PAM/PAM-antibody complexes retrieved from the surface of primary pituitary cells may have access to mature secretory granules (Fig. 10BGo).

By stably expressing wild-type and mutant PAM proteins in AtT-20 cell lines, we have identified domains of the protein that include important trafficking information (2, 21, 28, 29). Expressed independently, both soluble catalytic domains are efficiently stored in the secretory granules of AtT-20 cells, as occurs in primary pituitary cells (4). The cytosolic domain of membrane PAM contains essential trafficking information, and it is the handling of membrane PAM that differs most dramatically in AtT-20 cells and primary pituitary cells. Membrane PAM proteins lacking most of the cytosolic domain were less extensively cleaved by secretory granule endoproteases, were localized on the plasma membrane, and failed to undergo internalization (2). A protein lacking both PAM catalytic (lumenal) domains and consisting only of the PAM signal sequence followed by the transmembrane/cytosolic domain was highly localized to the TGN region of AtT-20 cells (66), and appending the cytosolic domain of PAM to Tac, a plasma membrane protein, rerouted Tac to the TGN (67). With our current knowledge that membrane PAM is not localized to the TGN in primary pituitary cells, it is clear that proper appreciation of the diversity of trafficking information encoded by the cytosolic domain of PAM and an understanding of the roles of interactor proteins will require the use of primary pituitary cells as well as stable cell lines.


   Acknowledgments
 
We thank the National Hormone and Pituitary Program, NIDDK, and Dr. A. F. Parlow for providing the antisera to pituitary hormones. We also thank Dr. Ruthann Nichols for sharing her sequential immunostaining protocol. We gratefully acknowledge Lixian Jin and Kate Deanehan for help with tissue culture and Marie Bell for general laboratory assistance. We thank Drs. Tami C. Steveson and Chenie Bell-Parikh for critically reading this manuscript.


   Footnotes
 
1 This work was supported by NIH Grant DK-32949. Back

Received February 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Prigge ST, Kolhekar AS, Eipper BA, Mains RE, Amzel LM 1999 Substrate-mediated electron transfer in peptidylglycine alpha- hydroxylating monooxygenase. Nat Struct Biol 6:976–983[CrossRef][Medline]
  2. Milgram SL, Kho ST, Martin GV, Mains RE, Eipper BA 1997 Localization of integral membrane peptidylglycine {alpha}-amidating monooxygenase in neuroendocrine cells. J Cell Sci 110:695–706[Abstract]
  3. Braas KM, Stoffers DA, Eipper BA, May V 1989 Tissue specific expression of rat peptidylglycine {alpha}-amidating monooxygenase activity and mRNA. Mol Endocrinol 3:1387–1398[Abstract]
  4. Eipper BA, Milgram SL, Husten EJ, Yun HY, Mains RE 1993 Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing and routing domains. Protein Sci 2:489–497[Abstract]
  5. DePalatis LR, Khorram O, Ho RH, Negro-Vilar A, McCann SM 1984 Partial characterization of immunoreactive substance P in the rat pituitary gland. Life Sci 34:225–238[CrossRef][Medline]
  6. Jones PM, Ghatei MA, Steel J, O’Halloran D, Gon G, Legon S, Burrin JM, Leonhardt U, Polak JM, Bloom SR 1989 Evidence for neuropeptide Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin. Endocrinology 125:334–341[Abstract]
  7. Arnaout MA, Garthwaite TL, Martinson DR, Hagen TC 1986 Vasoactive intestinal polypeptide is synthesized in anterior pituitary tissue. Endocrinology 119:2052–2057[Abstract]
  8. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, Chin WW 1988 Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 85:7408–7412[Abstract/Free Full Text]
  9. May V, Wilber JF, U’Prichard DC, Childs GV 1987 Persistence of immunoreactive TRH and GnRH in long-term primary anterior pituitary cultures. Peptides 8:543–558[CrossRef][Medline]
  10. Ashworth RJ, Morrell JM, Aitken A, Patel Y, Cockle SM 1991 Pyroglutamylglutamylprolineamide is present in rat anterior and posterior pituitary gland. J Endocrinol 129:R1–R4
  11. Schwartz J, Cherny R 1992 Intercellular communication within the anterior pituitary influencing the secretion of hypophysial hormones. Endocr Rev 13:453–475[CrossRef][Medline]
  12. May V, Ouafik L, Eipper BA, Braas KM 1990 Immunocytochemical and in situ hybridization studies of peptidylglycine {alpha}-amidating monooxygenase in pituitary gland. Endocrinology 127:358–364[Abstract]
  13. Ouafik L, May V, Saffen DW, Eipper BA 1990 Thyroid hormone regulation of peptidylglycine {alpha}-amidating monooxygenase expression in anterior pituitary gland. Mol Endocrinol 4:1497–1505[Abstract]
  14. El Meskini R, Delfino C, Boudouresque F, Hery M, Oliver C, Ouafik L 1997 Estrogen regulation of peptidylglycine alpha-amidating monooxygenase expression in anterior pituitary gland. Endocrinology 138:379–388[Abstract/Free Full Text]
  15. Thiele EA, Marek KL, Eipper BA 1989 Tissue-specific regulation of peptidyl-glycine {alpha}-amidating monooxygenase expression. Endocrinology 125:2279–2288[Abstract]
  16. Husten EJ, Eipper BA 1991 The membrane-bound bifunctional peptidylglycine {alpha}-amidating monooxygenase protein. Exploration of its domain structure through limited proteolysis. J Biol Chem 266:17004–17010[Abstract/Free Full Text]
  17. May V, Cullen EI, Braas KM, Eipper BA 1988 Membrane-associated forms of peptidylglycine {alpha}-amidating monooxygenase activity in rat pituitary. Tissue specificity. J Biol Chem 263:7550–7554[Abstract/Free Full Text]
  18. Oyarce AM, Eipper BA 1995 Identification of subcellular compartments containing peptidylglycine {alpha}-amidating monooxygenase in rat anterior pituitary. J Cell Sci 108:287–297[Abstract]
  19. Oyarce AM, Hand TA, Mains RE, Eipper BA 1996 Dopaminergic regulation of secretory granule-associated proteins in rat intermediate pituitary. J Neurochem 67:229–241[Medline]
  20. May V, Eipper BA 1986 Long term culture of primary rat pituitary adrenocorticotropin/endorphin-producing cells in serum-free medium. Endocrinology 118:1284–1295[Abstract]
  21. Ciccotosto GD, Schiller MR, Eipper BA, Mains RE 1999 Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1. J Cell Biol 144:459–471[Abstract/Free Full Text]
  22. Marx R, El Meskini R, Johns DC, Mains RE 1999 Differences in the ways sympathetic neurons and endocrine cells process, store and secrete exogenous neuropeptides and peptide processing enzymes. J Neurosci 19:8300–8311[Abstract/Free Full Text]
  23. Dickerson IM, Mains RE 1990 Cell-type specific posttranslational processing of peptides by different pituitary cell lines. Endocrinology 127:133–140[Abstract]
  24. Kolhekar AS, Keutmann HT, Mains RE, Quon AW, Eipper BA 1997 Peptidylglycine {alpha}-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core. Biochemistry 36:10901–10909[CrossRef][Medline]
  25. Schnabel E, Mains RE, Farquhar MG 1989 Proteolytic processing of pro-ACTH/endorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells. Mol Endocrinol 3:1223–1235[Abstract]
  26. Eskeland NL, Zhou A, Dinh TQ, Wu H, Parmer RJ, Mains RE, O’Connor DT 1996 Chromogranin A processing and secretion: specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway. J Clin Invest 98:148–156[Medline]
  27. McCormick J, Lim I, Nichols R 1999 Neuropeptide precursor processing detected by triple immunolabeling. Cell Tissue Res 297:197–202[CrossRef][Medline]
  28. Yun HY, Milgram SL, Keutmann HT, Eipper BA 1995 Phosphorylation of the cytosolic domain of peptidylglycine alpha-amidating monooxygenase. J Biol Chem 270:30075–30083[Abstract/Free Full Text]
  29. Steveson TC, Keutmann HT, Mains RE, Eipper BA 1999 Phosphorylation and dephosphorylation of Ser937 affect a discrete step in PAM-1 trafficking in AtT-20 cells. J Biol Chem 274:21128–21138[Abstract/Free Full Text]
  30. Traub LM, Kornfeld S 1997 The trans-Golgi network: a late secretory sorting station. Curr Opin Cell Biol 9:527–533[CrossRef][Medline]
  31. Winkler H, Fischer-Colbrie R 1992 The chromogranins A and B: the first 25 years and future perspectives. Neuroscience 49:497–528[CrossRef][Medline]
  32. LIoyd RV, Cano M, Rosa P, Hille A, Huttner WB 1988 Distribution of chromogranin A and secretogranin I (chromogranin B) in neuroendocrine cells and tumors. Am J Pathol 130:296–304[Abstract]
  33. Ciccotosto GD, Mains RE, Hand TA, Eipper BA 2000 Breeding stock-specific variation in peptidylglycine {alpha}-amidating monooxygenase mRNA splicing in rat pituitary. Endocrinology 141:476–486[Abstract/Free Full Text]
  34. Milgram SL, Mains RE, Eipper BA 1993 COOH-terminal signals mediate the trafficking of a peptide processing enzyme in endocrine cells. J Cell Biol 121:23–36[Abstract/Free Full Text]
  35. Von Ruden L, Garcia AG, Lopez MG 1993 The mechanism of Ba(2+)-induced exocytosis from single chromaffin cells. FEBS Lett 336:48–52[CrossRef][Medline]
  36. Sloan DC, Haslam RJ 1997 Protein kinase C-dependent and Ca2+-dependent mechanisms of secretion from streptolysin O-permeabilized platelets: effects of leakage of cytosolic proteins. Biochem J 328:13–21
  37. Billiard J, Koh DS, Babcock DF, Hille B 1997 Protein kinase C as a signal for exocytosis. Proc Natl Acad Sci USA 94:12192–12197[Abstract/Free Full Text]
  38. Herrington J, Hille B 1994 Growth hormone-releasing hexapeptide elevates intracellular calcium in rat somatotropes by two mechanisms. Endocrinology 135:1100–1108[Abstract]
  39. Tse FW, Tse A, Hille B, Horstmann H, Almers W 1997 Local Ca2+ release from internal stores controls exocytosis in pituitary gonadotrophs. Neuron 18:121–132[CrossRef][Medline]
  40. Von Ruden L, Neher E 1993 A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells. Science 262:1061–1065[Abstract/Free Full Text]
  41. Milgram SL, Johnson RC, Mains RE 1992 Expression of individual forms of peptidylglycine {alpha}-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules. J Cell Biol 117:717–728[Abstract/Free Full Text]
  42. Mains RE, Alam MR, Johnson RC, Darlington DN, Back N, Hand TA, Eipper BA 1999 Kalirin, a multifunctional PAM COOH-terminal domain interactor protein, affects cytoskeletal organization and ACTH secretion from AtT-20 cells. J Biol Chem 274:2929–2937[Abstract/Free Full Text]
  43. Nansel DD, Gudelsky GA, Porter JC 1979 Subcellular localization of dopamine in the anterior pituitary gland of the rat: apparent association of dopamine with prolactin secretory granules. Endocrinology 105:1073–1077[Abstract]
  44. Kulathila R, Merkler KA, Merkler DJ 1999 Enzymatic formation of C-terminal amides. Nat Prod Rep 16:145–154[CrossRef][Medline]
  45. Tooze J, Hollinshead M, Fuller SD, Tooze SA, Huttner WB 1989 Morphological and biochemical evidence showing neuronal properties in AtT-20 cells and their growth cones. Eur J Cell Biol 49:259–273[Medline]
  46. Maltese JY, Eipper BA 1993 Maturation, internalization, and turnover of soluble and membrane proteins associated with atrial myocyte secretory granules. Endocrinology 133:2579–2587[Abstract]
  47. Oyarce AM, Eipper BA 1993 Neurosecretory vesicles contain soluble and membrane-associated monofunctional and bifunctional PAM proteins. J Neurochem 60:1105–1114[CrossRef][Medline]
  48. Dittie AS, Thomas L, Thomas G, Tooze SA 1997 Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation. EMBO J 16:4859–4870[CrossRef][Medline]
  49. Dittie AS, Klumperman J, Tooze SA 1999 Differential distribution of mannose-6-phosphate receptors and furin in immature secretory granules. J Cell Sci 112:3955–3966[Abstract]
  50. Varlamov O, Eng FJ, Novikova EG, Fricker LD, Wu F, Shields D 1999 Localization of metallocarboxypeptidase D in AtT-20 cells. Potential ro