help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Marx, R.
Right arrow Articles by Mains, R. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marx, R.
Right arrow Articles by Mains, R. E.
Endocrinology Vol. 138, No. 12 5108-5118
Copyright © 1997 by The Endocrine Society

ARTICLES

Adenovirally Encoded Prohormone Convertase-1 Functions in Atrial Myocyte Large Dense Core Vesicles1

Ruth Marx and Richard E. Mains

Departments of Neuroscience and Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185

Address all correspondence and requests for reprints to: Dr. Richard E. Mains, Departments of Neuroscience and Physiology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185. E-mail: dick.mains{at}jhu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bioactive peptides are usually synthesized as inactive precursor proteins that yield bioactive products only after specific biosynthetic processing events. Large dense core vesicles (LDCV) are usually the site of storage of mature peptides. Atrial myocyte LDCV are rather unique in their storage of intact prohormone, proatrial natriuretic factor (pro-ANF), with no storage of cleaved products. To investigate whether the lack of intracellular cleavage of pro-ANF is due to the absence of prohormone convertases (PCs) from the atrial granules or to other factors, we expressed PC1 in atrial myocyte cultures using a recombinant adenovirus vector. Pro-PC1 protein was processed to mature PC1 and to the COOH-terminally shortened neuroendocrine-specific form of PC1 and rapidly secreted. Integral membrane forms of peptidylglycine {alpha}-amidating monooxygenase (PAM) were processed by PC1, and two primary products were secreted: a monofunctional monooxygenase and a larger bifunctional form. The cleaved PAM products were stored in LDCV, as secretion of PAM-derived products was stimulatable. In addition, pro-ANF was processed to ANF within PC1-expressing cells. In primary atrial myocytes, virally encoded PC1 is active on three substrates; lack of cleavage of pro-ANF and PAM in atrial myocytes is not due to a fundamental inability of atrial LDCV to support endoproteolytic processing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PEPTIDE-CONTAINING secretory granules of neurons and endocrine cells are thought to be very similar and are often referred to as large dense core vesicles (LDCV) (1, 2). The secretory granules in atrial myocytes are morphologically very similar to neuroendocrine peptide-containing secretory granules. Atrial LDCV are the source of proatrial natriuretic factor (pro-ANF), precursor to a peptide hormone important for proper salt and water balance (3, 4, 5, 6, 7). Atrial secretory granules also contain many of the proteins characteristic of the endocrine and neuronal LDCV, including the peptide-amidating enzyme peptidylglycine {alpha}-amidating monooxygenase (PAM), chromogranin A and B, cytochrome b561, and carboxypeptidase E/H (8, 9, 10, 11, 12). Interestingly, the atrial LDCV form a ring around the nucleus in the region of the trans-Golgi network (TGN) (13), showing the same position as integral membrane PAM in endocrine cells (14).

Despite these similarities to LDCV, the atrial granules are unusual because the major form of ANF stored in the LDCV is pro-ANF-(1–126) (7). This is in contrast to the situation in endocrine cells and neurons, where prohormones are endoproteolytically processed and stored as bioactive product peptides. Pro-ANF is initially synthesized with two arginine residues at its COOH-terminal, but these residues are removed by the carboxypeptidase in the granules (8, 15, 16). In the atrium at the time of secretion, endoproteolytic cleavage of pro-ANF-(1–126) occurs at a single Arg residue (Arg98) to yield pro-ANF-(1–98) and the bioactive hormone ANF-(99–126) (5, 6, 7). The identification and localization of the atrial endoproteolytic cleavage enzyme that releases the bioactive hormone ANF-(99–126) are not yet clear.

Thus, the atrial myocyte LDCV may be very useful in understanding the biogenesis of mature peptide secretory granules, as they may be a case of arrested maturation. Atrial granules have many of the prerequisites for peptide processing (substrate, many of the enzymes, binding molecules, and pump proteins), but do not perform endoproteolytic cleavages of pro-ANF or PAM. Prohormone convertases 1 and 2 (PC1 and PC2; PC1 is also called PC3) are the subtilisin-like endoproteases believed to be crucial in prohormone cleavage in neurons and endocrine cells; PC1 and PC2 are expressed at very low levels in the atrium (17, 18). Thus, one possible explanation for the immature nature of the products stored in atrial LDCV is the lack of an active endoprotease. Other conceivable explanations include environmental factors, such as the Ca+2 concentration and pH in the LDCV, the possibility that endoproteases are not trafficked to atrial LDCV, as well as the potential presence of specific inhibitors of endoproteolytic activity, such as the peptide inhibitor packaged with the zymogens in exocrine pancreatic LDCV (19).

To explore the differences between the atrial LDCV and the neuroendocrine LDCV, we chose first to study PC1 expression, using an adenoviral system to introduce PC1 complementary DNA into the atrial myocytes. PC1 has the advantage that it is activated early in the endoplasmic reticulum, and active PC1 is produced by fibroblasts that do not contain LDCV (20, 21, 22, 23, 24). The fate of the virally encoded PC1 protein was examined as well as the effects of PC1 expression on endogenous PAM and pro-ANF metabolism. The conclusion is that the atrial myocytes are perfectly capable of trafficking PC1 to LDCV, where the PC1 is active as an endoprotease on itself, on PAM, and on pro-ANF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of primary atrial myocyte cultures and viral infections
Atrial cultures were prepared as previously described (7). Briefly, atria were taken from 2-day-old neonatal rat pups [Sprague-Dawley; purchased from Charles River (Lexington, MA) unless otherwise indicated] and dissociated in trypsin-EDTA solution. The dissociated cells were plated onto fibronectin-coated (12.5 µg/ml) 12-mm culture dishes at a density of 2 x 105 cells/well. Cells were maintained in DMEM-Ham’s F-12 medium (DMEM/F-12) supplemented with 10% FBS for the first 24 h and then maintained in DMEM/F-12, 2% FBS, 25 nM dexamethasone, and 10 µM cytosine arabinoside for another 48 h. Cytosine arabinoside was removed from the cultures, and the cells were maintained under these conditions for 7 days before they were either infected or not. At the time of viral infection, cultures were beating spontaneously at a rate of about 1 Hz. Infections were performed using a 1:5000 dilution of the viral stock (1 x 107 plaque-forming units/ml) of recombinant adenovirus encoding the PC1 enzyme [Ad5.PC1; Ad5.PC1 is the same as Ad5.PC1(S) (25)]. The next day, the medium was replaced; 48 h after the infection, cells were immunostained, extracted, or pulse labeled, and spent media were collected.

Biosynthetic labeling
Metabolic labeling of the cells was performed as described previously (26). Atrial cultures were incubated in complete serum-free medium (CSFM) lacking methionine for 5 min and then labeled for 30 min with 300 µCi L-[35S]methionine (Amersham, Arlington Heights, IL) in 300 µl Met- medium. Cells were either harvested immediately (pulse) or further incubated in CSFM (114 µM methionine) for different time periods. Secretion was stimulated by treating the cells with 1.6 µM phorbol 12-myristate 13-acetate (PMA) in the last 30 min of chase (the medium samples were collected from the last 30 min of chase). Spent media were centrifuged to remove debris and a mixture of protease inhibitors (50 µg/ml lima bean trypsin inhibitor, 50 µg/ml bacitracin, 2 µg/ml leupeptin, 16 µg/ml benzamidine, 2 µg/ml pepstatin, and 300 µg/ml phenylmethylsulfonylfluoride) was added. Cells were extracted in boiling 50 mM sodium phosphate containing 1% SDS, 20 mM ß-mercaptoethanol, and 2 mM EDTA, pH 7.4 (27). Samples were heated to 95 C for 5 min and centrifuged for 5 min, and the supernatants were stored frozen at -80 C before analysis.

Immunoprecipitations
Cell extracts were thawed by boiling for 5 min and centrifuged for 5 min. Aliquots (typically 10%) were diluted with a 7-fold excess of Nonidet P-40 and incubated with 10 µl rabbit polyclonal antisera to peptidylglycine {alpha}-hydroxylating monooxygenase (PHM; JH1761), PC1 (JH888), or ANF-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) (provided by Dr. Chris Glembotski, San Diego State University, San Diego, CA) for 4 h at 4 C. Spent media (typically 10%) were denatured by boiling for 5 min in 1% SDS before dilution with a 7-fold excess of Nonidet P-40 and immunoprecipitated. Immune complexes were collected after incubation with 20 µl protein A-Sepharose beads in 50 µM sodium phosphate-1% Triton X-100 for 1 h and analyzed by SDS-PAGE. Gels were fixed in 30% isopropanol-10% acetic acid and prepared for fluorography by incubation in Amplify (Amersham).

Western blot analysis
Primary atrial cells were extracted in boiling 50 mM sodium phosphate containing 1% SDS, 20 mM ß-mercaptoethanol, and 2 mM EDTA, pH 7.4 (as described above), and medium samples were collected for 16 h. Aliquots of cell extract (30 µl; 10% of the whole extract) and spent medium (40 µl; 10% of the spent medium) were subjected to 10% SDS-PAGE and Western blot analysis using a PHM antiserum (JH1761, 1:1000) or a PC1 antiserum (JH888, 1:1000), followed by ECL detection (Amersham). Oligosaccharide analyses were performed as previously described (28).

PHM and peptidyl-{alpha}-hydroxyglycine {alpha}-amidating lyase (PAL) enzyme activities
Primary atrial cells were extracted with 20 mM Na-N-Tris-[hydroxymethyl]methyl-2-aminoethanesulfonic acid and 10 mM mannitol, pH 7.4, to which protease inhibitors (50 µg/ml lima bean trypsin inhibitor, 50 µg/ml bacitracin, 2 µg/ml leupeptin, 16 µg/ml benzamidine, 2 µg/ml pepstatin, and 300 µg/ml phenylmethylsulfonylfluoride) were added, followed by 5-min centrifugation to remove debris (29). Supernatants were centrifuged at 350,000 x g for 15 min; the pellets were resuspended in 20 mM Na-N-Tris-[hydroxymethyl]methyl-2-aminoethanesulfonic acid, 10 mM mannitol, and 1% Triton X-100, pH 7.4; and protease inhibitors were added. Both supernatant and pellet were stored at -80 C. Spent media were collected for 16 h, and protease inhibitors were added. Enzyme assays were performed as described previously (28), and samples (5 µl cell extracts or 2.5 µl spent medium) were assayed in duplicate. PHM and PAL reactions were carried out in a final volume of 40 µl for 2 h.

Immunofluorescent staining
The localization of PAM, ANF, TGN38, and virally encoded PC1 in the atrial cells was detected using indirect immunofluorescence. Cells were fixed using 4% paraformaldehyde in PBS, followed by permeabilization with 0.075% Triton X-100 or cold methanol (for TGN38 staining); blocking was carried out with 2 mg/ml BSA for 1 h. Monoclonal antibodies against PAM COOH-terminal domain (mAb 6E6) and rabbit polyclonal antisera against TGN38 (JH1481) and PC1 (JH888) or ANF (Peninsula Laboratories, Belmont, CA) were used at a dilution of 1:1000 for 16 h at 4 C (14, 22). Antigen-antibody complexes were visualized using fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (Caltag, San Francisco, CA) and Cy3-conjugated donkey antimouse IgG (Jackson ImmunoResearch Laboratory, West Grove, PA). Samples were viewed with a Zeiss Axioskop microscope (Carl Zeiss, Thornwood, NY) and photographed using a Micromax CCD camera (Princeton Instruments, Princeton, NJ).

Electron microscopy
Cultures were fixed for 5 min in 3.75% acrolein and 2% paraformaldehyde in isotonic phosphate buffer, pH 7.4, followed by 30 min in 2% paraformaldehyde in the same buffer. Sections were prepared and analyzed as described by Fiore et al. (30).

RIA
Spent medium was diluted (1:1) with the column buffer and separated by carboxymethylcellulose-HPLC as described previously (31). RIA was performed using an antibody against ANF (Peninsula, 9103) and [125I]ANF (Peninsula, Y9103) as described by Eipper et al. (9). We verified that the antiserum detects ANF-(99–126) and ANF-(103–126) on an equimolar basis, as stated in the product brochure. Data were calculated with an Excel program using the logit-log transformation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Virally derived PC1 expression in primary atrial myocytes
Primary atrial cells were examined for the expression, processing, and secretion of PC1 after infection with Ad5.PC1 virus. After infection, high levels of the 87-kDa pro-PC1 protein and the 81-kDa PC1 protein and low levels of the 63-kDa C-terminally truncated form (PC1{Delta}C) protein (21, 24) were detected using Western blot analysis within the cells. PC1 and PC1{Delta}C were secreted into the medium (Fig. 1AGo, PC1). Specific immunostaining showed that PC1 expression was detected in the majority of myocytes after infection (Fig. 1BGo, PC1). By comparison, PC1 expression was not observed in noninfected cells (Fig. 1BGo, Con).



View larger version (47K):
[in this window]
[in a new window]
  		Figure 1. Virally derived PC1 expression in infected atrial myocytes. A, Equal aliquots of atrial cell extracts (Cell) and 16-h spent medium (Mdm) from noninfected cells (Con) and infected cells (PC1) were subjected to Western blot analysis using antibody to PC1. Samples were fractionated on a 10% SDS-PAGE gel; molecular masses of standards are given in kilodaltons at the left of the gel. Similar results were obtained in four independent experiments. Both pro-PC1 and PC1 are seen in the cell extracts; the increased apparent size of the PC1 in the medium is due to increased complexity of glycosylation, as demonstrated using N-glycanase digestion to remove the oligosaccharides. B, Noninfected atrial cultures (Con) and PC1-infected atrial cultures (PC1) were fixed and permeabilized. PC1 protein was visualized by incubation with affinity-purified rabbit polyclonal antibody to PC1 followed by FITC-conjugated goat antirabbit IgG antibody; both cultures were photographed under identical conditions. N, Nucleus. C, Schematic diagram of the three PC1 molecular forms; the position of the peptide used to raise the PC1 antiserum is indicated.

 
Localization of virally derived PC1 in myocytes
The localization of endogenous proteins that serve as subcellular markers was compared with the localization of virally encoded PC1 (Fig. 2Go). PC1 was found to adopt a perinuclear distribution overlapping that of PAM (Fig. 2AGo). Note the single myocyte in Fig. 2AGo that is not expressing high levels of PC1 (arrow); the distribution of PAM is not altered upon expression of PC1. PC1 staining was more prevalent than PAM staining in punctate structures distributed throughout the cytoplasm (Fig. 2AGo). The pattern for the TGN marker TGN38 was more restricted than that for PAM (Fig. 2BGo); note the nonmyocyte detected by the TGN38 antiserum, but not by the PAM antiserum. PAM staining was more evident in punctate structures removed from the perinuclear region. ANF staining was similar to the patterns for PAM (Fig. 2CGo) and PC1 (Fig. 2AGo), with granular staining visible at quite a distance from the TGN38-defined narrow ring around the nucleus.



View larger version (92K):
[in this window]
[in a new window]
  		Figure 2. Localization of PC1 in infected atrial myocytes. Primary atrial cultures were infected with Ad5.PC1, fixed, and permeabilized. Cells were simultaneously incubated with monoclonal antibody to PAM COOH-terminal domain and polyclonal antibody to PC1 (A), TGN38 (B), or ANF (C). Arrows indicate noninfected myocytes (A) or nonmyocyte cells (B). Antigen-antibody complexes were visualized using FITC-conjugated goat antirabbit IgG and Cy3-conjugated donkey antimouse IgG antibodies. FITC and Cy3 signals were distinguished using the appropriate filters, and photographs of the same cells are shown for comparison.

 
At the ultrastructural level (Fig. 3Go), the majority of the LDCV in cultured atrial myocytes were immunopositive for both ANF (gold particles; gp in inset) and PAM (diaminobenzidine reaction product; arrowheads in main image). Numerous dense core vesicles were seen surrounding the nucleus (nuc), consistent with the light microscopic images (Fig. 2Go). There was negligible staining of the stacks of the Golgi complex with either antibody. This localization of integral membrane PAM is quite unlike the situation for integral membrane PAM in endocrine cells, where there was little membrane PAM in LDCV, and the majority of integral membrane PAM was found in tubulo-vesicular structures closely associated with the TGN region (14).



View larger version (153K):
[in this window]
[in a new window]
  		Figure 3. Ultrastructural localization of PAM and ANF in cultured atrial myocytes. The low power image shows a cultured atrial myocyte exhibiting the diaminobenzidine product of avidin-biotin staining for PAL. Numerous dense core vesicles (DCV; arrowheads) surround the nucleus (nuc) and show the diaminobenzidine reaction product. In the inset at higher power, postembedding immunogold staining for ANF shows dense core vesicles containing numerous gold particles (gp). m, Mitochondrion. G, Golgi.

 
Processing, storage, and secretion of virally derived PC1 in myocytes
Western blot analyses (Fig. 1AGo) established that the atrial cells processed the virally encoded pro-PC1 to PC1 intracellularly, and that the cells secreted the PC1 rapidly, so that several times the PC1 content of the cells was present in the medium at the end of a 16-h collection period. To study the activation and cleavage of pro-PC1, metabolic labeling studies were carried out. Pulse-chase analyses (Fig. 4Go) established that the expression of PC1 in virally infected cells accounted for about 1% of the total protein synthesis, as defined by trichloroacetic acid-precipitable radioactivity; the level of expression of endogenous PAM was similar (32). The 87-kDa pro-PC1 protein was rapidly converted to the 81-kDa PC1 and 63-kDa PC1{Delta}C forms; these forms were detected intracellularly and were rapidly secreted into the medium. The majority of the newly synthesized PC1 molecules were secreted into the medium during the 90-min chase period, consistent with the high rate of secretion determined by Western analyses (Fig. 2Go). The 63-kDa forms of PC1 (PC1{Delta}C) was preferentially retained in the cells, and more of this form was found to be secreted at later chase times; conversion of the 81-kDa to the 63-kDa form of PC1 occurred in neurons and endocrine cells with LDCV, but not in fibroblasts (21, 22). To determine whether PC1 was secreted in a regulated or a constitutive fashion, cells were pulse labeled for 30 min, chased for 60 or 90 min, and then treated with PMA for the last 30 min of chase (Fig. 4Go, Mdm, -/+ PMA); no stimulation of PC1 release was observed. Despite using various paradigms of timing and stimulation, no significant stimulation of the release of any form of PC1 was seen. The 63-kDa PC1{Delta}C was released in a regulated manner from AtT-20 cells (21, 22). ANF secretion from atrial myocytes was stimulated 2.5-fold by PMA treatment (see below), as expected (33, 34).



View larger version (81K):
[in this window]
[in a new window]
  		Figure 4. PC1 is processed and secreted in atrial myocytes. Cells were infected with Ad5.PC1, labeled with 300 µCi [35S]methionine for 30 min (P), and chased (C) in CSFM for the times indicated (minutes). Spent medium was collected from cells treated with 1.6 µM PMA (+) or control medium (-) during the last 30 min of the chase (*). Immunoprecipitations of 10% of the cell extract (Cell) and spent medium (Mdm) were performed using antibody to PC1 and protease inhibitors as described. Samples were fractionated on a 10% SDS-PAGE gel; molecular masses of standards are given in kilodaltons at the left of the gel. The three major PC1 molecular forms are identified; similar results were obtained in two independent experiments.

 
PC1-infected atrial myocytes show intracellular cleavage and secretion of PAM
To determine whether virally derived PC1 enzyme was an active enzyme in the atrial myocytes, we studied the effects of its expression on the cleavage and secretion of PAM proteins, which are stored in the LDCV of atrial myocytes. Two integral membrane forms of PAM, PAM-1 (120 kDa) and PAM-2 (105 kDa), were expressed in the atrial myocytes, and both contained potential PC1 cleavage recognition sites (35), as indicated in Fig. 5Go. In addition, a low level of a soluble PAM isoform (PAM-3; 97 kDa) was expressed in the atrial myocytes (35). Using atrial cultures from rats that express more PAM-1 than PAM-2 (Fig. 5AGo), PC1 infection resulted in a dramatic decrease in the level of PAM-1 stored in the cells along with a dramatic increase in both the intracellular storage and secretion of 45-kDa PHM. Transfection of PAM-1 into AtT-20 cells, which express high levels of PC1, yielded PHM proteins of the same apparent molecular masses (29). Using atrial cultures from rats that express more PAM-2 than PAM-1 (Fig. 5BGo), the major released form of PAM was a bifunctional 69-kDa PAM protein; this protein was reactive with both PHM and PAL antisera (PAL not shown).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 5. Western blot analysis of PAM proteins in atrial myocytes expressing PC1. Extracts of atrial cells (Cell) and 16-h spent medium (Mdm) of noninfected cells (Con) and infected cells (PC1) were subjected to Western blot analysis using antibody to PHM (JH1761). Samples were fractionated on a 10% SDS-PAGE gel; molecular masses of standards are given in kilodaltons at the left side of the gel. A, Atrial cultures were prepared from 2-day-old Charles River Sprague-Dawley rat pups, which express more PAM-1 than PAM-2. For a positive control, a cell extract from AtT-20 cells expressing PAM-1 was also analyzed. B, Atrial cultures were prepared from 2-day-old Harlan Sprague-Dawley rat pups, which express more PAM-2 than PAM-1. A 69-kDa fragment (*) and a 45-kDa fragment (*) were detected in the spent medium of PC1-infected cells; similar results were obtained in four independent experiments. Schematic diagrams of the two membrane-bound PAM isoforms indicated the putative PC1 recognition sites in PAM-1 and PAM-2 (marked as ||) and their processing products.

 
To determine whether the dramatic changes in PHM protein shown in Fig. 5Go correlated with an increase in soluble and secreted enzyme activity, we measured PHM and PAL enzyme activities in cell extracts as well as secreted enzyme activity in the medium (Fig. 6Go). There was a doubling of the fraction of PHM activity that was soluble in PC1-expressing cultures and a smaller but clear increase in intracellular soluble PAL activity, with corresponding drops in membrane-bound enzyme activities. There was also a substantial increase in the PHM and PAL enzyme activities released into the medium (2.4-fold for both PAM and PAL activities). Thus, as a result of PC1 expression within the atrial myocytes, less PHM and PAL activities were retained in the membrane fraction, and higher levels of these activities were observed in the soluble fraction and secreted into the medium.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 6. PHM and PAL enzyme activities in PC1-expressing atrial myocytes. PHM activity (A) and PAL activity (B) were measured in triplicate samples of the supernatant (Sup) and solubilized crude particulate fraction (Pel) of noninfected cell extracts (Con) and PC1-infected cell extracts (PC1) and in the spent media that were collected for 16 h (Mdm); similar results were obtained in two independent experiments.

 
Next, we studied the kinetics of endoproteolytic processing of PAM and secretion of PHM using pulse-chase metabolic labeling (Fig. 7Go). Secretion of 45-kDa PHM was observed in medium from PC1-infected cells, but not in medium from noninfected cells. The secretion of 45-kDa PHM was detected after 60-min chase, but not after 30-min chase. To determine whether 45-kDa PHM were secreted in a regulated fashion, cells were pulse labeled for 30 min, chased for various times, and treated with PMA during the last 30 min of chase. The secretion of 45-kDa PHM was increased in the range of 2-fold by PMA stimulation. Thus, pulse-chase metabolic labeling showed that PC1 expression in atrial myocytes resulted in secretion of the PAM product (45-kDa PHM) in a regulated fashion.



View larger version (83K):
[in this window]
[in a new window]
  		Figure 7. Processing and secretion of PAM in atrial myocytes expressing PC1. PC1-infected cells (PC1) and noninfected cells (Con) were labeled with 300 µCi [35S]methionine for 30 min and chased in CSFM for 30, 60, or 90 min, and spent medium was collected. Cells were chased for 90 min and treated with 1.6 µM PMA (+) or control medium (-) during the last 30 min of chase (*). Immunoprecipitations of 10% of the spent medium (Mdm) were performed using antibody to PHM and protease inhibitors as described. Samples were fractionated on 10% SDS-PAGE gel; molecular masses of standards are given in kilodaltons at the left of the gel. The 97-kDa PAM protein secreted in the 60-min chase is intact PAM-3. Similar results were obtained in two independent experiments.

 
PC1-infected atrial myocytes show intracellular cleavage of ANF
ANF is stored in LDCV as a prohormone, and cleavage of pro-ANF to ANF occurs at the moment of secretion (7). Thus, we asked whether expression of PC1 would confer on atrial LDCV the ability to cleave pro-ANF and store the cleaved products or whether atrial LDCV had additional impediments to pro-ANF cleavage and storage. Endoproteolytic processing and secretion of ANF were examined with pulse-chase metabolic labeling and immunoprecipitation (Fig. 8Go). The 17-kDa pro-ANF protein was intracellularly converted to the 14-kDa ANF N-terminal product in PC1-infected cells, whereas the 14-kDa ANF N-terminal processing product was not detected in noninfected cells, even when the autoradiograms were deliberately overexposed. There was increased secretion of 14-kDa ANF N-terminal by PC1-infected cells compared with noninfected cells at all time points. Treatment with PMA resulted in an increase in the rate of secretion of ANF in the range of 2.5-fold in both noninfected and infected cells. When ANF production was examined by Western blot analysis, similar results were seen; only PC1-infected atrial cells had detectable ANF in cell extracts (not shown).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 8. Processing and secretion of ANF in atrial myocytes expressing PC1. A, PC1-infected cells (PC1) and noninfected cells (Con) were labeled with 300 µCi [35S]methionine for 30 min (P) and chased (C) in CSFM for the times indicated (minutes). Spent media were collected from cells treated with 1.6 µM PMA (+) or nontreated cells (-) during the last 30 min of chase (*). Immunoprecipitations of 10% of the cell extract (Cell) and the spent medium (Mdm) were performed using antibody to ANF-(1–16) and protease inhibitors as described. Samples were fractionated on a 15% SDS-PAGE gel; molecular masses of standards are given in kilodaltons at the right of the gel; similar results were obtained in two independent experiments. B, Schematic diagram of ANF molecular forms with the three Met (M), two Cys (C), and Phe (F) residues indicated. R, Arg; S, Ser; L, Leu.

 
Analysis of the pro-ANF cleavage site in PC1-infected atrial myocytes
To examine the site of PC1-induced cleavage of pro-ANF, spent media were analyzed by cation exchange HPLC followed by RIA (Fig. 9Go). We demonstrated that control cells produced almost exclusively ANF-(99–126), with the expected cleavage after the single Arg residue (R98). PC1-infected cells produced a significant peak of ANF-(103–126), demonstrating an additional cleavage after the pair of Arg residues (R101 and R102).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 9. Analyses of ANF cleavage site. Spent media from control and PC1-infected atrial myocyte cultures were analyzed by cation exchange HPLC and RIA. The top panel shows the separation of synthetic ANF-(103–126) and ANF-(99–126). The bottom panel shows the mean and SD of analyses from two independent sets of control and PC1-infected cultures.

 
Cleavages dependent on virally encoded PC1 occur primarily in LDCV
To analyze where in myocytes PC1-dependent cleavages were occurring, chase incubations were carried out at 20 C to block exit of newly synthesized proteins from the TGN (27, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). Cells were pulse labeled at 37 C for 30 min and then chased at 20 or 37 C for 2 h (Fig. 10Go). The chase at 20 C resulted in retention of the 81-kDa form of PC1 in the cells and largely prevented the cleavage that creates the neuroendocrine-specific 63-kDa PC1{Delta}C. The chase at 20 C also completely blocked cleavage of integral membrane PAM to liberate the soluble secreted 45-kDa PHM seen in the incubations performed at 37 C. These data indicate that COOH-terminal shortening of PC1 and cleavage of integral membrane PAM are primarily occurring after the site of the 20 C block and thus in the distal TGN or in LDCV.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 10. Effects of temperature blockade on PC1 and PAM processing in atrial myocytes. PC1-infected cells were labeled with 300 µCi [35S]methionine for 30 min (P) and chased (C) at 20 C (20) or 37 C (37) for 2 h in CSFM. A, Samples of cell extract (Cell) and spent medium (Mdm) were immunoprecipitated using antibody to PC1, and samples were analyzed by SDS-PAGE. B, Samples of cell extract (Cell) and spent medium (Mdm) were immunoprecipitated using antibody to PHM, and samples were analyzed by SDS-PAGE. The gel region within the rectangle is shown after a 7-fold longer exposure to film in the bigger rectangle. The molecular mass standards are shown on the left. Similar results were obtained in two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Atrial myocyte secretory granules exhibit many similarities to the LDCV of neurons and endocrine cells. However, their unique features suggest that a better understanding of their properties might offer insight into the biogenesis and function of these organelles.

LDCV from neurons, endocrine cells, and atrial myocytes share a number of critical features. All LDCV are larger than small synaptic vesicles, typically 100–300 nm in diameter instead of 50–100 nm for small synaptic vesicles; LDCV are denser than small synaptic vesicles during differential and density centrifugation; and LDCV share an electron-dense core (2, 13). The dense core includes chromogranins, carboxypeptidase E/H, PAM, ascorbate, and peptides and/or peptide precursors, whereas LDCV membranes contain cytochrome b561 for transporting electrons plus an adenosine triphosphatase to concentrate protons (8, 9, 10, 11, 12). By comparison, the lumen of small synaptic vesicles is widely believed to be devoid of protein.

The atrial LDCV have important unique properties as well. The localization of the endogenous integral membrane PAM in the atrial myocytes is quite unlike the pattern seen in endocrine cells (14). In endocrine cells, integral membrane PAM is largely restricted to the region of the TGN, both in the TGN itself and in vesicular structures closely associated with the TGN. The vesicular structures close to the TGN are thought to be the morphological correlate of immature LDCV, whereas smaller peptide products, such as those derived from POMC, are found in mature LDCV in the cellular processes (27). In the atrium, the endogenous integral membrane PAM and the endogenous pro-ANF are found together in LDCV clustered around the nucleus, but quite a distance from the TGN. This arrangement is consistent with the suggestion that atrial LDCV are granules that linger with many attributes similar to immature LDCV in other tissues.

In addition, atrial LDCV are immature LDCV from the point of view of peptide processing, and the cleavage of pro-ANF to the bioactive peptide occurs at the time of secretion (5, 6, 7); the precise mechanism of cosecretional cleavage remains unclear as do as the location and identity of the endoprotease that performs the processing. Similarly, atrial LDCV contain intact integral membrane forms of PAM, with negligible amounts of the soluble forms of PAM produced by cleavage from integral membrane PAM (32). Perhaps the inability of atrial LDCV to process pro-ANF and PAM endoproteolytically is a reflection of the observations that PC1 and PC2 messenger RNAs and proteins were undetectable in the atrium by Northern blot analysis (17, 18) and Western blot analysis. If the atrial myocyte LDCV simply lack the endoproteases prevalent in LDCV in neurons and endocrine cells, the expression of a PC in atrial myocytes should yield cleaved, but stored, peptide and protein products, as in neurons and endocrine cells.

In these studies we used the adenoviral system as a nontoxic approach to introduce complementary DNA for a subtilisin-like PC into primary atrial myocyte cultures. The exogenous endoprotease was highly expressed in most of the cultured cells. As a high percentage of the myocytes expressed the virally encoded PC, cleavage and secretion of endogenous substrates such as PAM and pro-ANF could be studied easily.

For these studies, PC1 had the advantage that it is activated early in the endoplasmic reticulum, and active PC1 is produced by fibroblasts that do not contain LDCV (20, 21, 22, 23, 24). Study of PC2 expression within the atrial myocytes would be more complicated to interpret, as PC2 enzyme activity is dependent on the coexpression of a neuroendocrine-specific protein, the 7B2 protein (46). Moreover, PC2 is activated late in the secretory pathway, in the TGN/LDCV compartments where the environment in the atrium may be inadequate. These data demonstrate that PC1 becomes activated in atrial myocytes, that some of the functional PC1 is trafficked to LDCV, and that three substrates are cleaved by the PC1: PC1 itself, PAM, and pro-ANF.

PC1 expression in atrial myocytes was largely colocalized with the endogenous PAM protein and ANF that are known to be present in the TGN and the LDCV compartment (3, 4, 5, 6, 7). Release of PC1-processing products (45-kDa PHM and pro-ANF-derived peptides) was stimulated by secretagogues, suggesting that they were stored in atrial LDCV. Furthermore, based on subcellular fractionation using sucrose gradients, some PC1 was found in the same granule fractions where PAM and ANF reside (not shown). However, the majority of the PC1 protein was secreted very rapidly, and little PC1 could remain in LDCV. A small portion of the 81-kDa PC1 protein was processed to the 63-kDa PC1 protein; it seems likely that this small portion of the PC1 proteins entered the LDCV and was responsible for PAM and pro-ANF cleavages. Similar phenomena were seen previously for sorting and secretion of C-terminal truncated PC1 expressed by transfection in AtT-20 pituitary cells, where only a small portion of exogenous PC1 entered LDCV (21, 22, 24).

PC1-mediated cleavage of pro-ANF occurs after the pair of basic residues (R101 and R102) to yield ANF-(103–126). Whether PC1 enhances the single Arg cleavage (R98) to produce ANF-(99–126) is unclear, as that cleavage already occurs cosecretionally in atrial myocytes (4). PC1 can perform cleavages at both dibasic amino acid residues and single Arg residues (47). In addition, it was shown that in pro-ANF-transfected AtT-20 cells, both ANF-(99–126) and ANF-(103–126) were secreted (48). It seems likely that endogenously expressed PC1 was responsible for both of these cleavages.

Thus, when atrial myocytes are made to express a PC that activates autocatalytically and is LDCV associated, then atrial LDCV adopt the ability to cleave and store pro-ANF and PAM, with PAM cleavage proceeding to a far greater extent than cleavage of pro-ANF. Therefore, it is clear that part of the immature nature of atrial LDCV can be attributed to a lack of expression of an appropriate PC, but there are also additional differences to note. Specifically, despite massive expression of PC1, pro-ANF is not cleaved very efficiently, and the PC1 is stored extremely poorly. Interestingly, at the level of the electron microscopy, PAM is localized to LDCV in the atrium, whereas in brain and pituitary the integral membrane PAM is expressed in the TGN and in vesicular structures primarily in the TGN region (14). This suggests that trafficking to LDCV is different within the atrium cells. One potential cause for the difference in trafficking may be the lack of kalirin, a protein with spectrin-like domains and guanine nucleotide exchange factor activity that was shown to interact with the PAM cytosolic domain (49).


   Acknowledgments
 
We thank Dr. Betty Eipper for innumerable suggestions and guidance throughout the project, Dr. Chris Glembotski for antisera to ANF, Dr. V. A. Bayer for performing the electron micrographs, Dr. Luc Paquet with help in the initial phases of the adenovirus work, many members of the Neuropeptide Laboratory for advice and counsel, and Marie Bell for general laboratory assistance.


   Footnotes
 
1 This work was supported by NIDA Grant DA-00266. Back

Received May 30, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rindler MJ 1992 Biogenesis of storage granules and vesicles. Curr Opin Cell Biol 4:616–622[CrossRef][Medline]
  2. Halban PA, Irminger J-C 1994 Sorting and processing of secretory proteins. Biochem J 299:1–18
  3. de Bold AJ, Kuroski-de Bold ML, Boer PH, Dube G, Mangat H, Johnson F 1991 A decade of atrial natriuretic factor research. Can J Physiol Pharmacol 69:1480–1485[Medline]
  4. Glembotski CC, Irons CE, Sprenkle AB, Sei CA 1990 Studies of ANF processing and secretion using a primary cardiocyte culture model. Can J Physiol Pharmacol 69:1525–1536
  5. Greenwald JE, Needleman P, Siegel N, Tetens E, Biel B, Ritter D 1992 Processing of atriopeptin prohormone by nonmyocytic atrial cells. Biochem Biophys Res Commun 188:644–654[CrossRef][Medline]
  6. Rosenzweig A, Seidman CE 1991 Atrial natriuretic factor and related peptide hormones. Annu Rev Biochem 60:229–255[CrossRef][Medline]
  7. Sei CA, Hand GL, Murray SF, Glembotski CC 1992 The cosecretional maturation of atrial natriuretic factor by primary atrial myocytes. Mol Endocrinol 6:309–319[Abstract]
  8. Lynch DR, Venable JC, Snyder SH 1988 Enkephalin convertase in the heart: similar disposition to atrial natriuretic factor. Endocrinology 122:2683–26891[Abstract]
  9. Eipper BA, May V, Braas KM 1988 Membrane-associated peptidylglycine alpha-amidating monooxygenase in the heart. J Biol Chem 263:8371–8379[Abstract/Free Full Text]
  10. Steiner HJ, Weiler R, Ludescher C, Schmid KW, Winkler H 1990 Chromogranins A and B are co-localized with atrial natriuretic peptides in secretory granules of rat heart. J Histochem Cytochem 38:845–850[Abstract]
  11. Pruss RM, Shepard EA 1987 Cytochrome b561 can be detected in many neuroendocrine tissues using a specific monoclonal antibody. Neuroscience 22:149–157[CrossRef][Medline]
  12. Aardal S, Helle KB 1991 Comparative aspects of the endocrine myocardium. Acta Physiol Scand 599:31–46
  13. Jamieson JD, Palade GE 1964 Specific granules in atrial muscle cells. J Cell Biol 23:151–172[Abstract/Free Full Text]
  14. Milgram SL, Kho ST, Martin GV, Mains RE, Eipper BA 1997 Localization of integral membrane peptidylglycine. J Cell Sci 110:659–706
  15. Fricker LD, Snyder SH 1983 Purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase. J Biol Chem 258:10950–10955[Abstract/Free Full Text]
  16. Thibault G, Lazure C, Chretien M, Cantin M 1989 Molecular heterogeneity of pro-atrial natriuretic factor. J Biol Chem 264:18796–18802[Abstract/Free Full Text]
  17. Bloomquist BT, Eipper BA, Mains RE 1991 Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA. Mol Endocrinol 5:2014–2024[Abstract]
  18. Beaubien G, Schafer MK, Weihe E, Dong W, Chretien M, Seidah NG, Day R 1995 The distinct gene expression of the pro-hormone convertases in the rat heart suggests potential substrates. Cell Tissue Res 279:539–549[Medline]
  19. Davenport HW 1978 Pancreatic secretion. In: A Digest of Digestion, ed 2. Year Book, Chicago, pp 69–74
  20. Thomas L, Leduc R, Thorne BA, Smeekens SP, Steiner DF, Thomas G 1991 Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acd Sci USA 88:5297–5301[Abstract/Free Full Text]
  21. Zhou Y, Lindberg I 1994 Enzymatic properties of carboxyl-terminally truncated prohormone convertase 1 (PC1/SPC3) and evidence for autocatalytic conversion. J Biol Chem 269:18408–18413[Abstract/Free Full Text]
  22. Zhou A, Paquet L, Mains RE 1995 Structural elements that direct specific processing of different mammalian subtilisin-like prohormone convertases. J Biol Chem 270:21509–21516[Abstract/Free Full Text]
  23. Benjannet S, Reudelhuber T, Mercure C, Rondeau N, Chretien M, Seidah NG 1992 Proprotein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity, and intracellular environment. Cell type-specific processing of human prorenin by the convertase PC1. J Biol Chem 267:11417–11423[Abstract/Free Full Text]
  24. Zhou A, Mains RE 1994 Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 or 2. J Biol Chem 269:17440–17447[Abstract/Free Full Text]
  25. Paquet L, Massie B, Mains RE 1996 Proneuropeptide Y processing in large dense-core vesicles: manipulation of prohormone convertase expression in sympathetic neurons using adenoviruses. J Neurosci 16:964–973[Abstract/Free Full Text]
  26. Zhou A, Bloomquist BT, Mains RE 1993 The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in a strict temporal order during proopiomelanocortin biosynthetic processing. J Biol Chem 268:1763–1769[Abstract/Free Full Text]
  27. Milgram SL, Mains RE 1994 Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins. J Cell Sci 107:737–745[Abstract]
  28. 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]
  29. 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]
  30. Fiore RS, Bayer VE, Pelech SL, Cooper JA, Baraban JM 1993 p42 mitogen-activated protein kinase in brain: prominent localization in neuronal cell bodies and dendrites. Neuroscience 55:463–472[CrossRef][Medline]
  31. Shields PP, Glembotski CC 1988 The post-translational processing of rat pro-atrial natriuretic factor by primary atrial myocyte cultures. J Biol Chem 263:8091–808[Abstract/Free Full Text]
  32. 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]
  33. Shields PP, Dixon JE, Glembotski CC 1988 The secretion of atrial natriuretic factor-(99–126) by cultured cardiac myocytes is regulated by glucocorticoids. J Biol Chem 263:12619–12628[Abstract/Free Full Text]
  34. Sei CA, Irons CE, Sprenkle AB, McDonough PM, Brown JH, Glembotski CC 1991 The {alpha}-adrenergic stimulation of atrial natriuretic factor expression in cardiac myocytes. J Biol Chem 266:15910–15916[Abstract/Free Full Text]
  35. Maltese JY, Eipper BA 1992 Developmental expression of peptidylglycine alpha-amidating monooxygenase (PAM) in primary cultures of neonatal rat cardiocytes: a model for studying regulation of PAM expression in the rat heart. Mol Endocrinol 6:1998–2008[Abstract]
  36. Saraste J, Kuismanen E 1984 Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface. Cell 38:535–549[CrossRef][Medline]
  37. Griffiths G, Pfeiffer S, Simons K, Matlin K 1985 Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane. J Cell Biol 101:949–964[Abstract/Free Full Text]
  38. Kuliawat R, Arvan P 1992 Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment. J Cell Biol 118:521–529[Abstract/Free Full Text]
  39. Arvan P, Castle D 1992 Protein sorting and secretion granule formation in regulated secretory cells. Trends Cell Biol 2:327–331[CrossRef][Medline]
  40. Griffiths G, Simons K 1986 The trans Golgi network: sorting at the exit site of the Golgi complex. Science 234:438–443[Abstract/Free Full Text]
  41. Baeuerle PA, Huttner WB 1987 Tyrosine sulfation is a trans-Golgi-specific protein modification. J Cell Biol 105:2655–2664[Abstract/Free Full Text]
  42. Hong W, Tang BL 1993 Protein trafficking along the exocytotic pathway. Bioessays 15:231–238[CrossRef][Medline]
  43. Mellman I, Simons K 1992 The Golgi complex: in vitro veritas? Cell 68:829–840[CrossRef][Medline]
  44. Chanat E, Huttner WB 1991 Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network. J Cell Biol 115:1505–1519[Abstract/Free Full Text]
  45. Milgram SL, Eipper BA, Mains RE 1994 Differential trafficking of soluble and integral membrane secretory granule-associated proteins. J Cell Biol 124:33–41[Abstract/Free Full Text]
  46. Zhu X, Lindberg I 1995 7B2 facilitates the maturation of pro-PC2 in neuroendocrine cells and is required for the expression of enzymatic activity. J Cell Biol 129:1641–1650[Abstract/Free Full Text]
  47. Nakayama K, Watanabe T, Nakagawa T, Kim WS, Nagahama M, Hosaka M, Hatsuzawa K, Kondoh-Hashiba K, Murakami K 1992 Consensus sequence for precursor processing at mono-arginyl sites. Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites. J Biol Chem 267:16335–16340[Abstract/Free Full Text]
  48. Shields PP, Sprenkle AB, Taylor EW, Glembotski CC 1990 Rat pro-atrial natriuretic factor expression and post-translational processing in mouse corticotropic pituitary tumor cells. J Biol Chem 265:10905–10911[Abstract/Free Full Text]
  49. Alam RM, Johnson RC, Darlington DN, Hand TA, Mains RE, Eipper BA 1997 Kalirin, a cytosolic protein with spectrin-like and GDP/GTP exchange factor-like domains that interacts with peptidylglycine {alpha}-amidating monooxygenase, an integral membrane peptide-processing enzyme. J Biol Chem 272:12667–12675[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Chem.Home page
I. Giuliani, F. Rieunier, C. Larue, J.-F. Delagneau, C. Granier, B. Pau, M. Ferriere, M. Saussine, J.-P. Cristol, A.-M. Dupuy, et al.
Assay for Measurement of Intact B-Type Natriuretic Peptide Prohormone in Blood
Clin. Chem., June 1, 2006; 52(6): 1054 - 1061.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
J. P. Goetze
Biochemistry of Pro-B-Type Natriuretic Peptide-Derived Peptides: The Endocrine Heart Revisited
Clin. Chem., September 1, 2004; 50(9): 1503 - 1510.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
R. El Meskini, R. E. Mains, and B. A. Eipper
Cell Type-Specific Metabolism of Peptidylglycine {alpha}-Amidating Monooxygenase in Anterior Pituitary
Endocrinology, August 1, 2000; 141(8): 3020 - 3034.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. Marx, R. El Meskini, D. C. Johns, and R. E. Mains
Differences in the Ways Sympathetic Neurons and Endocrine Cells Process, Store, and Secrete Exogenous Neuropeptides and Peptide-Processing Enzymes
J. Neurosci., October 1, 1999; 19(19): 8300 - 8311.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
D. C. Johns, R. Marx, R. E. Mains, B. O'Rourke, and E. Marban
Inducible Genetic Suppression of Neuronal Excitability
J. Neurosci., March 1, 1999; 19(5): 1691 - 1697.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
G. D. Ciccotosto, M. R. Schiller, B. A. Eipper, and R. E. Mains
Induction of Integral Membrane PAM Expression in AtT-20 Cells Alters the Storage and Trafficking of POMC and PC1
J. Cell Biol., February 8, 1999; 144(3): 459 - 471.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Marx, R.
Right arrow Articles by Mains, R. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marx, R.
Right arrow Articles by Mains, R. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals