Retrieval and Reuse of Pituitary Secretory GranuleProteins
2005; Elsevier BV; Volume: 280; Issue: 27 Linguagem: Inglês
10.1074/jbc.m414156200
ISSN1083-351X
AutoresFrancesco Ferraro, Betty Eipper, Richard E. Mains,
Tópico(s)Receptor Mechanisms and Signaling
ResumoThe pituitary contains professional secretory cells, devoting a largefraction of their energy to the synthesis of hormones that are stored forsecretion in response to a complex mixture of inputs. Ba2+, asubstitute for Ca2+, and phorbol ester, a mimic for diacylglycerol,have a synergistic effect on exocytosis. By using these secretagogues, wedeveloped a paradigm in which phorbol ester potentiation ofBa2+-evoked exocytosis produces a robust secretory response inmultiple pituitary cell types. Because cells subjected to this stimulatoryparadigm remain healthy despite their greatly reduced hormone content, we usedthis paradigm to study the fate of granule membrane proteins. We examined theturnover of peptidylglycine α-amidating monooxygenase (PAM), a membraneenzyme involved in the final maturation of many peptides, and VAMP2, a vesiclesoluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE). The stability of recently synthesized PAM was increased by sustainedexocytosis. Biotinylation studies established that the appearance of integralmembrane PAM at the plasma membrane was stimulated along with hormonesecretion. PAM biotinylated on the cell surface undergoes cleavage to yieldsoluble peptidylglycine-α-hydroxylating monooxygenase that can then besecreted in a regulated fashion. Consistent with a kiss-and-run or cavicapturemode of secretion (Taraska, J. W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu,S., and Almers, W. (2003) Proc. Natl. Acad. Sci. U. S. A. 100,2070–2075), biotinylated prolactin was also retained by the cells andlater released in response to secretagogues. Thus, pituitary cells canretrieve and reuse components of the machinery involved in the final stages ofexocytosis (the SNAREs) as well as soluble and membrane granule proteins. The pituitary contains professional secretory cells, devoting a largefraction of their energy to the synthesis of hormones that are stored forsecretion in response to a complex mixture of inputs. Ba2+, asubstitute for Ca2+, and phorbol ester, a mimic for diacylglycerol,have a synergistic effect on exocytosis. By using these secretagogues, wedeveloped a paradigm in which phorbol ester potentiation ofBa2+-evoked exocytosis produces a robust secretory response inmultiple pituitary cell types. Because cells subjected to this stimulatoryparadigm remain healthy despite their greatly reduced hormone content, we usedthis paradigm to study the fate of granule membrane proteins. We examined theturnover of peptidylglycine α-amidating monooxygenase (PAM), a membraneenzyme involved in the final maturation of many peptides, and VAMP2, a vesiclesoluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE). The stability of recently synthesized PAM was increased by sustainedexocytosis. Biotinylation studies established that the appearance of integralmembrane PAM at the plasma membrane was stimulated along with hormonesecretion. PAM biotinylated on the cell surface undergoes cleavage to yieldsoluble peptidylglycine-α-hydroxylating monooxygenase that can then besecreted in a regulated fashion. Consistent with a kiss-and-run or cavicapturemode of secretion (Taraska, J. W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu,S., and Almers, W. (2003) Proc. Natl. Acad. Sci. U. S. A. 100,2070–2075), biotinylated prolactin was also retained by the cells andlater released in response to secretagogues. Thus, pituitary cells canretrieve and reuse components of the machinery involved in the final stages ofexocytosis (the SNAREs) as well as soluble and membrane granule proteins. In neurons, synaptic vesicle membrane components are rapidly retrieved atthe nerve terminal after secretion. The vesicles thus formed are replenishedwith locally synthesized neurotransmitter and are again able to undergostimulated release (1Sudhof T.C. Annu. Rev.Neurosci. 2004; 27: 509-547Crossref PubMed Scopus (1888) Google Scholar). Incontrast, neuroendocrine secretory granule membrane components must have adifferent fate following exocytosis. The peptides stored in secretory granulesare often generated from propeptides as they enter immature secretorygranules, which form in the trans-Golgi network region of the cell.If the membrane proteins of secretory granules are retrieved and reused, aconnection between granule biogenesis and granule membrane recycling mustoccur. Sporadic evidence of functional recycling (retrieval and reuse) ofsecretory granule membrane proteins has been collected over the last 2decades. In chromaffin cells, stimulation of secretion causes internalizationof glycoprotein III/clusterin and dopamine β-hydroxylase from the plasmamembrane and their subsequent re-entry into secretory granules(2Patzak A. Winkler H. J. CellBiol. 1986; 102: 510-515Crossref PubMed Scopus (123) Google Scholar, 3Hunter A. Phillips J.H. Exp.Cell Res. 1989; 182: 445-460Crossref PubMed Scopus (13) Google Scholar, 4Hurtley S.M. J. CellSci. 1993; 106: 649-656Google Scholar).In insulinoma cells, stimulated secretion resulted in the endocytosis of twosecretory granule membrane proteins, phogrin and ICA512, and their re-entryinto newly forming insulin granules(5Vo Y.P. Hutton J.C. Angleson J.K. Biochem. Biophys. Res. Commun. 2004; 324: 1004-1010Crossref PubMed Scopus (45) Google Scholar,6Solimena M. Dirkx Jr., R. Hermel J.-M. Pleasic-Williams S. Shapiro J.A. Caron L. Rabin D.U. EMBO J. 1996; 15: 2102-2114Crossref PubMed Scopus (231) Google Scholar). Both endogenous andheterologously overexpressed P-selectin, a protein involved in the first phaseof the inflammatory response, can be retrieved from the plasma membrane andre-inserted in newly formed granules(7Subramaniam M. Koedam J.A. Wagner D.D. Mol. Biol. Cell. 1993; 4: 791-801Crossref PubMed Scopus (155) Google Scholar). Peptidylglycine α-amidating monooxygenase(PAM), 1The abbreviations used are: PAM, peptidylglycine α-amidatingmonooxygenase; VAMP2, vesicle-associated membrane protein 2/(synaptobrevin 2);SNARE, soluble N-ethylmaleimide-sensitive factor attachment proteinreceptor; PHM, peptidylglycine-α-hydroxylating monooxygenase; PALm,membrane-bound peptidyl-α-hydroxyglycine-α-amidating lyase; PRL,prolactin; TGN, trans-Golgi network; PMA, phorbol 12-myristate13-acetate; sPHM, soluble PHM; ACTH, adreno-corticotropic hormone; TES,N-Tris(hydroxymethyl)methyl-2-aminoethansulfonic acid; GH, growthhormone; BSA, bovine serum albumin. a type I membraneprotein, is a processing enzyme present in secretory granules ofneuroendocrine cells and neurons that is responsible for one of the finalsteps in the biosynthesis of many peptide hormones and neuropeptides(8Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell. Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (381) Google Scholar). Studies on thebiosynthesis and trafficking of exogenous membrane PAM in a mouse corticotropetumor cell line, AtT-20, identified residues in the cytosolic domain of PAMthat were essential for granule entry, internalization from the plasmamembrane, and transit through the endosomal pathway(9Milgram S.L. Kho S.T. Martin G.V. Mains R.E. Eipper B.A. J. Cell Sci. 1997; 110: 695-706Crossref PubMed Google Scholar). These studies revealed arole for the phosphorylation and dephosphorylation of specific residues in thecytosolic domain of PAM in determining whether PAM internalized from theplasma membrane entered lysosomes(10Steveson T.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 1999; 274: 21128-21138Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Because overexpressionof membrane PAM in AtT-20 cells causes striking differences in the cellularlocalization of secretory granules, the cytoskeleton, and the basal and evokedrelease of mature hormone compared with cells in which only endogenous PAM ispresent (11Alam M.R. Steveson T.C. Johnson R.C. Back N. Abraham B. Mains R.E. Eipper B.A. Mol. Biol.Cell. 2001; 12: 629-644Crossref PubMed Scopus (29) Google Scholar), we wanted toexamine this process in primary pituitary cells, where secretory granules aremuch more prevalent. We used cultured rat anterior pituitary cells to establish a stimulatoryparadigm based on the synergistic actions of Ba2+, a substitute forCa2+, and phorbol myristate acetate (PMA) to mimic diacylglycerol,which caused massive secretion of hormone from multiple cell types. Massivesecretion in turn creates the necessity for sustained retrieval of secretorygranule membrane proteins, including PAM, from the plasma membrane. Theresponse to the stimulation paradigm of granule membrane proteins in primarypituitary cells was monitored using subcellular fractionation,immunocytochemistry, surface biotinylation and enzyme assays. The sustainedexocytosis yielded healthy cells with less than 20% of their normal content ofhormone, causing depletion or redistribution of secretory granule membraneproteins. Combining biosynthetic labeling with the stimulation paradigm, wefound that the rate of turnover of recently synthesized PAM in primarypituitary cells was decreased by sustained exocytotic activity. By usingbiotinylation to label PAM on the plasma membrane, we found that a fraction ofthe PAM tagged on the cell surface was subsequently endoproteolyticallyprocessed to yield soluble PHM that was secreted in a regulated fashion. Theability of secretory granule components to be reused, if not releasedfollowing exocytosis, is not limited to membrane proteins. Prolactin, asoluble granule protein, was also internalized following stimulation and wasdelivered to a cellular compartment from which it could be released again intothe medium. Together, these data suggest that secretory granule proteins, ifnot consumed during one round of exocytosis, can be functionally recycled forsubsequent use. Reagents—Antisera to growth hormone (JH89) and ACTH (Kathy)were described previously(12Dickerson I.M. Mains R.E. Endocrinology. 1990; 127: 133-140Crossref PubMed Scopus (41) Google Scholar), as was antiserum to exonA of PAM (JH629) (13Bell-Parikh L.C. Eipper B.A. Mains R.E. J. Biol. Chem. 2001; 276: 29854-29863Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar).Polyclonal antiserum to prolactin (IC-5) was from the National Hormone andPeptide Program (NIDDK, National Institutes of Health), and monoclonalantibody to actin (JLA20) was from the Developmental Studies Hybridoma Bank(University of Iowa). Monoclonal antibody to VAMP2 (clone 69.1) was obtainedcommercially from Synaptic Systems GmbH (Germany). Sulfo-NHS-LC-biotin andsulfo-NHS-SS-biotin were from Pierce (catalog numbers 21335 and 21331,respectively); all the other chemicals were from Sigma unless otherwisestated. Anterior Pituitary Primary Cell Cultures—Anterior pituitaryprimary cultures were prepared as described previously(14May V. Eipper B.A. Endocrinology. 1986; 118: 1284-1295Crossref PubMed Scopus (45) Google Scholar) with only minormodifications. Briefly, anterior pituitaries from adult male and femaleSprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) wereseparated from the neurointermediate lobes under a dissecting microscope,minced, and subjected to sequential collagenase and trypsin digestion at32–37 °C. Cells to be used for microscopy were subjected to oneadditional round of trypsin digestion in order to obtain a single cellsuspension. Chamber slides or plastic dishes were coated with protamine andNu-Serum I or IV (Collaborative Research, Bedford, MA). Dissociated cells wereplated at high density (1–4 pituitary/well) for biochemical analyses andat low density (1/25 pituitary/well) for microscopy. Cells were plated inDulbecco's modified Eagle's medium/F-12 supplemented with 10% Fetal Clone IIIfetal bovine serum (HyClone Laboratories, Inc., Logan, UT), 10% Nu-Serum, 10mm HEPES, 100 units/ml penicillin, 100 μg/ml streptomycin(Invitrogen); 10 μm cytosine arabinoside was included for thefirst 24 h. The next day, cells were fed with Complete Serum-free Medium(CSFM, Dulbecco's modified Eagle's medium/F-12, 10 mm HEPES, 100units/ml penicillin, 100 μg/ml streptomycin, insulin/transferrin/seleniumfrom Invitrogen or Mediatech, and 1 mg/ml fatty acid-free bovine serumalbumin). Cells were fed daily with the same medium until used. Secretion Stimulation Paradigm—Primary cells that had beenmaintained in culture for 3–7 days were pre-rinsed 2–3 times(15–30 min each) with secretion medium (CSFM containing 0.2 mg/ml BSA)devoid of secretagogues. Cells were then fed with secretion medium alone(control) or secretion medium containing secretagogue (stimulation): 2mm BaCl2 and/or 1 μm phorbol myristateacetate (PMA, 1 mm stock in Me2SO). For each experiment,the modality and the length of the stimulation periods are indicated. Thespent media were centrifuged to remove non-adherent cells and stored at–80 °C after addition of protease inhibitors (0.3 mg/mlphenylmethylsulfonyl fluoride, 50 μg/ml lima bean trypsin inhibitor, 2μg/ml leupeptin, 16 μg/ml benzamidine, and 2 μg/ml pepstatin). At theend of the last stimulation period, cells were extracted with 20 mmNaTES, 10 mm mannitol, 1% Triton X-100 (TMT; pH 7.4) at 4 °Ccontaining protease inhibitors. Fluorescence Microscopy—After secretagogue treatment, cellswere rinsed with warm HEPES saline buffer (HS: 120 mm NaCl, 4mm KCl, 5mm CaCl2, 15mm HEPES-KOH,pH 7.5) and fixed for 30 min with 4% formaldehyde in the same pre-warmedbuffer. After extensive rinsing, cells were permeabilized with 0.075% TritonX-100 in blocking buffer (2 mg/ml BSA in phosphate-buffered saline) for 20 minat room temperature and then further incubated in blocking buffer for 20 minat room temperature. Fixed cells were incubated with primary antibodiesdiluted in blocking buffer for 2 h at room temperature or overnight at 4°C. Following extensive rinsing with phosphate-buffered saline, cells werethen incubated in the appropriate Cy3-conjugated secondary antibody for 1 h atroom temperature in the dark. Slides were mounted with glass coverslips usingPermafluor mounting medium (Immunotech, France). Image stacks(Δz = 0.2 μm) were created using Openlab 3.4 software(Improvision, UK) and a Nikon TE300 fluorescence microscope with a Hamamatsudigital CCD camera C4742-95-12ERG connected to an Orca-ER camera controller.Images were acquired using an oil immersion 60× objective (NA 1.4) andwere deconvolved (iterative mode) using Volocity 3.0 software (Improvision,UK) and an appropriate point spread function. The pictures shown represent amid-stack region of the cells. Radioimmunoassays—ACTH radioimmunoassays were performed onmedia from cultured anterior pituitary(15Ciccotosto G.D. Schiller M.R. Eipper B.A. Mains R.E. J. Cell Biol. 1999; 144: 459-471Crossref PubMed Scopus (54) Google Scholar). Antiserum Kathy isspecific for the COOH terminus of CLIP, ACTH, and larger precursors like ACTHbiosynthetic intermediate but does not detect intact proopiomelanocortin(16Schnabel E. Mains R.E. Farquhar M.G. Mol. Endocrinol. 1989; 3: 1223-1235Crossref PubMed Scopus (148) Google Scholar). 125I-LabeledACTH was from Peninsula Laboratories or Amersham Biosciences, and syntheticrat ACTH-(1–39) was used as the standard (Bachem). Subcellular Fractionation—Control and stimulated cells werescraped from the culture dish into ice-cold homogenization buffer (150mm sucrose, 60 mm KCl, 2.5 mmMgCl2, 20 mm HEPES-KOH, pH 7.5) containing proteaseinhibitors and then passed 5 times through a 26-gauge needle and 12 timesthrough a ball-bearing homogenizer (H&Y Enterprises, Redwood City, CA).The resulting homogenates were centrifuged at 1,100 ×gmax for 5 min, removing cell debris and nuclei (P1). Theresulting supernatants were separated by centrifugation at 17,400 ×gmax for 15 min to yield a P2 pellet (enriched insecretory granules) and supernatants. These supernatants were then centrifugedat 435,000 × gmax for 15 min to separate a lightmembrane P3 pellet from the soluble, cytosolic supernatant fraction (SN).Pellets were resuspended in equal volumes of homogenization buffer, and thesoluble fractions were adjusted to the same volume of resuspended pellets withhomogenization buffer. Western Blot—After fractionation by SDS-PAGE andelectroblotting to polyvinylidene difluoride membranes (Schleicher &Schuell), membranes were probed with the indicated primary antibodies. Theantigen-antibody complexes were visualized using the appropriate horseradishperoxidase-conjugated secondary antibody and Super Signal West Picochemiluminescence substrate (Pierce). When quantification was performed,nonsaturated signals were desensitized using Scion Image software (NationalInstitutes of Health) or acquired with a GeneGnome work station through theGeneSnap software (Syngene) and analyzed with the GeneTools software(Syngene). Biosynthetic Labeling—Cultured rat anterior pituitary cellswere incubated with Met(–) CSFM for 5 min, then pulsed with[35S]Met containing CSFM for 30 min, and rinsed with nonradioactiveCSFM for 2 min. The labeling was performed either before or after challengingthe cells with the Ba2+/PMA paradigm. In the former case, the cellswere chased for 2 h and extracted or further chased in medium devoid orcontaining secretagogues (in the presence or absence of 20 μmchloroquine to dissipate the transmembrane pH gradients) and then extracted inTMT buffer. In the latter case, the control and Ba2+/PMA-challengedcells were labeled and extracted with TMT buffer. Aliquots of the extractswere precipitated with trichloroacetic acid, solubilized, and either countedor loaded on gel, fractionated by SDS-PAGE, and exposed for fluorography.Other aliquots of the extracts were immunoprecipitated with the indicatedantibodies, and the immunoprecipitates were fractionated by SDS-PAGE andexposed for fluorography. PAM Enzymatic Assays—Peptidylglycine α-hydroxylatingmonooxygenase assays were performed as described(15Ciccotosto G.D. Schiller M.R. Eipper B.A. Mains R.E. J. Cell Biol. 1999; 144: 459-471Crossref PubMed Scopus (54) Google Scholar) usingAc-125I-Tyr-Val-Gly as substrate. Surface Biotinylation—Cultured rat primary anteriorpituitary cells or AtT-20 cells stably expressing PAM-1 were fed with CSFM airsupplemented with 10 mm HEPES, 100 units/ml penicillin, 100μg/ml streptomycin, insulin/transferrin/selenium (from Invitrogen orMediatech), and 0.1 mg/ml fatty acid-free bovine serum albumin for 30 min at37 °C in order for the cells to adjust to the air conditions. Before theaddition of the biotin derivative, cells were rinsed with pre-warmed (37°C) or pre-chilled (wet ice) HSG buffer (15 mm HEPES-KOH, pH7.5, 120 mm NaCl, 2 mm CaCl2, 4mmKCl, 25 mm glucose) three times (5 min per rinse) in order to washout amino acids and proteins present in the medium that could quench thebiotinylation reaction. The reactions were performed with 5 mmsulfo-NHS-LC-biotin in HSG, dissolving the biotin derivative just before beingused in order to minimize its hydrolysis. The reaction was stopped, and theresidual unreacted biotin was quenched by rinsing the cells with CSFM airsupplemented as described above with the exception that the BSA concentrationwas 2 mg/ml. For the biotinylation reactions performed at 0 °C, afterbeing fed with CSFM air, cells were transferred to a bath containing meltingice. All the steps were as for the biotinylations at 37 °C except that allthe media and buffers used were pre-chilled at 0 °C. In some experiments,surface biotinylation was performed with 5 mm sulfo-NHS-SS-biotin,a derivative whose biotin moiety can be removed by incubation of cells withbuffer (50 mm Tris-HCl, pH 7.6, 100 mm NaCl) containingreduced glutathione (50 mm; GSH), a membrane-impermeable reducingagent (as described in Ref. 17Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholarwith modifications). Cells were extracted in TMT supplemented with proteaseinhibitors. The protein concentration of the lysates was determined by thebicinchoninic acid assay (Pierce), and equal amounts of lysates (by protein)were tumbled with avidin stripped of the carbohydrate moieties(NeutrAvidin™, Pierce) and immobilized on agarose beads for 1 h at roomtemperature. The beads were rinsed twice with TMT buffer and once with thesame buffer devoid of Triton X-100 (TM), both containing protease inhibitors.Finally, avidin-bound proteins were eluted by boiling in Laemmli sample bufferfor 5 min. Biotinylated proteins from the media were purified by the sameprocedure. Repetitive Stimulation Causes Massive Exocytosis and Depletion ofHormone Content in Pituitary Endocrine Cells—Our laboratory haslong been interested in the synthesis, storage, and secretion of proteinstargeted to the secretory pathway(18Eipper B.A. Mains R.E. Endocr. Rev. 1980; 1: 1-27Crossref PubMed Scopus (818) Google Scholar). Exocytosis is coupled tocompensatory endocytosis (19Gundelfinger E.D. Kessels M.M. Qualman B. Nat. Rev. Mol. Cell. Biol. 2003; 4: 127-139Crossref PubMed Scopus (203) Google Scholar);therefore, to study secretory granule membrane protein recycling, we developeda stimulation paradigm that causes massive secretion of hormone. Anteriorpituitary endocrine cells typically respond to multiple secretagogues, and weused BaCl2 and a phorbol ester (phorbol 12-myristate 13-acetate(PMA)) to mimic the physiological second messengers Ca2+ anddiacylglycerol, respectively(20TerBush D.R. Holz R.W. J.Neurochem. 1992; 58: 680-687Crossref PubMed Scopus (32) Google Scholar, 21von Ruden L. Garcia A.G. Lopez M.G. FEBS Lett. 1993; 336: 48-52Crossref PubMed Scopus (51) Google Scholar, 22Exton J.H. Annu. Rev. Pharmacol.Toxicol. 1996; 36: 481-509Crossref PubMed Scopus (300) Google Scholar).Phorbol ester and barium are known to have a synergistic effect on the evokedexocytosis of luteinizing hormone in chicken pituitary cells(23Davidson J.S. Wakefield I. King J.A. Millar R.P. J. Endocrinol. 1987; 114: 11-16Crossref PubMed Scopus (5) Google Scholar). We observed a similarresponse in cultured rat primary pituitary cells(Fig. 1A). Basalsecretion of growth hormone was compared with that elicited by PMA alone,barium alone, or their combination (Fig.1A). Barium and PMA alone elicited a similar exocytoticresponse, with a 2–3-fold increase of GH secretion over basal levels.However, when the two secretagogues were administered together, the evokedsecretion of GH was 8–9 times higher than basal levels(Fig. 1A,bottom). Next, we tested a paradigm involving sequential stimulation with thesesecretagogues (Fig.1B). The PMA exposure time was selected to maximize thesecretory response and minimize protein kinase C down-regulation. Whencultured anterior pituitary cells were subjected to this paradigm, a largesecretory response was produced by somatotropes, lactotropes(Fig. 1C), andcorticotropes (not shown); we refer to cells exposed to this stimulationparadigm as Ba2+/PMA-challenged. At the end of the stimulationparadigm, the GH and PRL cells had undergone a massive reduction of hormonecontent (Fig. 1D);Ba2+/PMA-challenged cells contained less than 20% as much hormoneas control cells. These biochemical data were qualitatively confirmed byimmunocytochemistry. Ba2+/PMA-challenged and control cells werefixed and probed for ACTH, GH, and PRL(Fig. 1E).Corticotropes and somatotropes under control conditions showed strongimmunoreactivity, mainly in the plasma membrane region(Fig. 1E,arrows). In stimulated cells, staining in the cortical region wasless pronounced, and punctate staining likely corresponding to single granuleswas prevalent throughout the cytoplasm(Fig. 1E,asterisks). Control lactotropes showed a characteristic strongstaining in the Golgi/TGN region along with punctate staining locatedeccentrically to the nucleus (Fig.1E, arrows). In contrast, in the stimulatedlactotropes immunoreactivity was present only in the perinuclear region(Fig. 1E,asterisks), with the cytoplasmic punctate staining almost completelylost. The 5-fold drop in hormone content measured by biochemical analysis(Fig. 1D) was moredramatic than the decrease observed by immunocytochemistry. This discrepancysuggested that some endocrine cells might be so depleted that they becamedifficult to detect based on their remaining hormone content. Each type ofanterior pituitary endocrine cell contains VAMP2, an integral membrane granuleprotein that participates in exocytosis through formation of the SNARE complex(24Jahn R. Sudhof T.C. Annu.Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1025) Google Scholar). VAMP2 is not found athigh levels in non-endocrine cells(25Jacobsson G. Meister B. Endocrinology. 1996; 137: 5344-5356Crossref PubMed Scopus (79) Google Scholar). When endocrine cellswere identified using VAMP2 and co-stained with hormone-specific antibodies,it became clear that responses ranged from almost complete depletion of thehormone content (very few granules stained) to little discernible effect (notshown). The stimulated cells shown in Fig.1E retained enough hormone following theBa2+/PMA challenge to allow easy identification based onvisualization of hormone. The Ba2+/PMA Stimulation Paradigm Is Not Toxic and PrimarilyAffects Secretory Granule Proteins—The Ba2+/PMA paradigminvolves long stimulation times, raising the possibility that cells could bedamaged. By phase contrast microscopy, the Ba2+/PMA-challengedcells did not show any evident signs of stress (not shown). To obtain aphysiological parameter of cellular health, protein synthesis was analyzed. Atthe end of the Ba2+/PMA stimulation paradigm, control andBa2+/PMA-challenged cells were pulse-labeled using[35S]methionine, chased briefly in nonradioactive medium, andextracted. Aliquots of the extracts were subjected to trichloroacetic acidprecipitation to measure total protein synthesis or to GH immunoprecipitationto look specifically at synthesis of a secretory product. The totalradioactivity incorporated was not significantly different (not shown), andfluorography of equivalent samples from control andBa2+/PMA-challenged cells revealed indistinguishable patterns oftotal protein synthesis and similar rates of GH synthesis(Fig. 2A). Therefore,this stimulation paradigm, while causing massive depletion of hormone, doesnot compromise the viability of the cells. We next analyzed the effect of the Ba2+/PMA challenge on avariety of cellular markers. Stimulated and control cells were homogenized inisotonic buffer and subjected to differential centrifugation. A crude nuclearpellet (P1), a secretory granule-enriched fraction (P2), a fractioncorresponding to lighter membrane compartments (P3), and soluble cytosolicproteins (SN) were subjected to Western blot analysis. The subcellularfractions were probed for a general marker, actin, and for three secretorygranule proteins, GH, PAM, and VAMP2 (Fig.2B). In control cells, actin localizes to the particulatefractions containing nuclei and secretory granules and to cytosol, whereas themicrosomal fraction (P3) is largely devoid of actin. In the stimulated cells,the actin distribution was unchanged. Analyzed in this way, theBa2+/PMA paradigm affects neither the actin distribution nor itstotal amount. As expected, the GH signal was reduced in all fractions from thestimulated cells when compared with control cells. The greatest reduction wasseen in the P2 fraction, where most of the secretory granules sediment. The GHsignal detected in the cytosolic fraction presumably reflects rupture of somegranules during homogenization (Fig.2B). We next analyzed the response of two secretory granule membrane proteins tothe Ba2+/PMA challenge. PAM)is a bifunctional integral membraneenzyme (8Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell. Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (381) Google Scholar). PAM-1, a majorsplice variant, undergoes an endoproteolytic cleavage that produces a solublePHM domain (sPHM) and a membrane-bound PAL domain (PALm). As expected, thestrongest signal for both sPHM and PALm was present in the secretorygranule-enriched P2 fraction of control cells(Fig. 2B). Thecorresponding fraction from the stimulated cells showed a marked reduction inthe signal for both full-length PAM-1 and its cleavage products, sPHM andPALm. The reduction was proportionally higher for sPHM, as expected for asoluble protein that undergoes exocytosis, compared with integral membraneproteins that can be recycled. As in the case of GH, the cytosolic fractioncontained a small amount of sPHM. VAMP2 is a resident granule membrane protein that is enriched in matureversus immature granules(24Jahn R. Sudhof T.C. Annu.Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1025) Google Scholar). After fusion of thesecretory granule membrane with the plasma membrane, VAMP2 is recycled. Incontrol cells, VAMP2 was largely associated with the P2 fraction, with smalleramounts recovered in the P3 fraction. In stimulated cells, the amount of VAMP2in the P2 fraction decreased, whereas the amount in the P3 fraction increased(Fig. 2B). This shiftis consistent with movement of VAMP2 into recycling compartments. The overalldecrease in the level of VAMP2 observed followingBa2+/PMA-challenge (an ∼17% decrease,Fig. 2B) indicatesthat this membrane protein is also subject to limited degradation. In order to confirm this observation, primary anterior pituitary cells wereeither challenged with the stimulation paradigm or not, fixed, andimmunostained for VAMP2 (Fig.2C). Control cells showed a localization of VAMP2resembling that of hormones such as GH and ACTH(Fig. 1E), withcharacteristic sub-plasmalemmal staining(Fig. 2C, top,arrows); perinuclear staining and some punctate cytoplasmic staining wasalso visible. When visualized for VAMP2, the Ba2+/PMA-challengedcells looked remarkably different, with the immunoreactivity mainly localizedto internal sites (Fig.2C, bottom, asterisks). As would be predictedfrom the changes in hormone staining, the cell periphery was essentiallydevoid of VAMP2 staining, except for a low intensity punctate staining(Fig. 2C, bottom,arrowheads). Tak
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