Immunofluorescent evidence for exocytosis and internalization of secretory granule membrane in isolated chromaffin cells

Neuroscience Vol 10,No. 3, pp. 1025-1033, 1983 Printedin Great Britain

0306-4522/83 $3.00+ 0.00

Pergamon PressLtd IBRO

IMMUNOFLUORESCENT EVIDENCE FOR EXOCYTOSIS AND INTERNALIZATION OF SECRETORY GRANULE MEMBRANE IN ISOLATED CHROMAFFIN CELLS D. J. DOWD*, C. EDWARDS, D. ENGLERT~,J. E. MAZURKIEWICZ$ and H. Z. YE NeurobiologyResearchCenterand Departmentof BiologicalSciences,StateUniversityof New York at Albany, Albany, NY 12222;and SDepartmentof Anatomy, Albany MedicalCollege,Albany, NY 12208,U.S.A.

Abstract--Cultured bovine adrenalmedullary chromffi cells were stimulatedwith the secretogogues Ba2+or carbamylcholineplus Ca’+ m the presenceof a monospecificrabbit IgG fraction directedagainst bovine dopaminefi-hydroxylase.The anti-clopamineb-hydroxylasewas labeledeither with fluorescent proteinA or with a fluorescentsecondantibodyto rabbit IgG. Stimulationproduceda patchycell surface distribution of fluorescence. Therewasno noticeableinternalizationof the fluorescence for up to 2 h. In similar experimentsusing fluorescentmonovalentfragments(Fab) of the samemonospecificidopamine-fi-hydroxylase. IgG, a more uniform distribution of the fluorescence wasobserved.A few min after a 5min period of stimulationwith Ba2+,the fluorescence appearedto be on or nearthe cell surface; however,after 20min or moreit wasdistributedthroughoutthe cytoplasmexceptthat the cell nucleiwere not labeled.Thus, dopamine/?-hydroxylasewhich appearedon the cell surfaceas a consequenceof exocytosiswasinternalizedin the presenceof monovalentantibodyfragments,but not in the presenceof the divalent (polyclonal) antibody, presumablybecauseendocytosisof dopamine/?-hydroxylasewas inhibited by crosslinkingof the dopamineb-hydroxylasemolecules.The internalizedanti-dopaminejhydroxylaseFab fragmentswerefound to reappearon the cell surfaceduring a secondsecretoryresponse. It is concluded that the interior of the chromaffin granule membrane,for which dopamine /3hydroxylaseis a marker, becomesexposedon the surfaceof the cell during secretionand that the membraneis then retrievedbackinto the cell whereit can be re-usedin a further secretorycycle.

The adrenal medulla, which synthesizesand secretes catecholamines, has heen studied intensively as a model of the mechanisms involved in neurosecretion. The catecholamines are stored in membrane-limited vesicles or granules along with proteins, ATP, and other constituents30 and are released in discrete quanta (discussedby Kirshner & Viveros16). Acetylcholine or certain other stimuli cause the release of the granule contents by a Ca2+ dependent process.‘O*” Most biochemical and morphological evidence indicates that the secretory mechanism is exocytosis10*16*31a process during which the chromaffin granules fuse with the plasma membrane and empty their contents into the extracellular space. Direct evidence for exocytosis is the finding that the enzyme, dopamine j?-hydroxylase (DBH), which is normally found only on the inner surface of the granule membrane, appears on the outer surface of the plasma membrane following stimulation.12*29 The presence of DBH was demonstrated by using an antibody to DBH, which was localized by means of a

second fluorescently-lahelled antibody. The DBH was localized in patches on the cell surface and there was no evidence of internalization over a period of 2 h. However, the results of experiments using cationized ferritin or labeled 1”’ proteins indicate that components of the granule membrane are selectively retrieved from the plasma membrane and reused.z6 Thus, although this patchiness might he physiological, an alternative explanation is that, since both the anti-DBH and the second antibody are divalent and polyclonal, they can crosslink the enzyme and this crosslinking can somehow block its internalization. To determine whether the distribution of the DBH is affected by the divalent antibodies, the experiments were repeated with fluorescently labeled, univalent Fab fragments of the sameantibody. EXPERIMENTAL PROCEDURES Preparation of DBH

Chromatlin granules were isolated from fresh bovine adrenal medullas according to the method of Smith & Winklerzs as modified by Schneider.24 Dopamine j*Presentaddress:Section of Pharmacology,Division of hydroxylase was purified from the lysate of chromaffin Biology and Medicine, Brown University, Providence, granules according to the procedure of Rush et aLz3 RI 02912,U.S.A. Following centrifugation to remove chromaffin granule tPresentaddress:PharmaciaFine Chemicals,800 Centen- membranes, the supematant was applied to a concanavalin nial Avenue,Piscataway,NJ 08854,U.S.A. A Sepharose column and the DBH was eluted with amethyl+-mannoside. The eluate was then applied to a Abbreviations: ATP, adenosine S-triphosphate; DBH, dopamine b-hydroxylase; FITC, fluorescein iso- Sepharose4B column to separatethe DBH from the smaller molecular weight glycoproteins.8 Enzymatic activity was thiocyanate; RITC, rhodamine isothiocyanate. 1025

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D. J. Dowd et al.

washed with Ml99 plus 10% fetal calf serum or with ;I solution containing 3.4mM Mg2+ and no Ca2+. and then stimulated with a solution m which the Mg’ + was replaced with Ba’+. When carbamyl choline was used as the secretogogue the cells were first washed with solution containing 3.4 mM Ca’ +, and then carbamyl choline was added. Ba’ + (5 mM) has been reported to be about 2; times as effective as 0.1 mM carbachol m releasmp catecholamine.” The immunofluorescence experiments were done with Preparation qf antuera unfixed ceils, so that only antigens on the external surface of Dopamine /l-hydroxylase-specific antiserum was raised in the cells were labeled. The cells were exposed to the a male New Zealand white rabbit. Two hundred and fifty gg antibodies in the physiological solutions for 5-20mm and of DBH in Freund’s complete adjuvant was injected into the were washed for 2-5min before addition of secondary fat pads of the back followed by intramuscular boosters 20 labels or observation of fluorescence. Anti-dopamine /?and 44 days later with 50 pg of DBH in Freund’s incomplete hydroxylase IgG was used at a concentration of about adjuvant. Sera were collected and tested for the presenceof 0.4mg/ml in Ml99 plus 10% fetal calf serum, and the specific antibody using Ouchterlony double diffusion plates. fluorescein- or rhodamine-labeled sheep anti-rabbit IgG An IgG fraction was prepared from an ammonium second antibodies (FITC- or RITC-ant]-rabbit IgG) for sulfate precipitate of the serum using DEAE cellulose indirect immunofluorescence were used at l/50 or l/l00 chromatography.” This fraction was characterized by dilutions in the Ca*+, Mg*+-free buffered salt solution acrylamide agarose crossed immuno-e1ectrophoresiis.SAli(Cappel Laboratories). In some experiments fluoresceinquots of chromaffin granule lysate or purified DBH were labeled protein A (FITC-Protein A. Sigma Chemical Co ) electrophoresed in the first dimension using a native 7.5% was used to label the anti-DBH IgG; this was used at about polyacrylamide gel according to the technique of Aunis et O.O5mg/ml and was either mixed with the IgG or applied a1.j using a stock solution of 30% acrylamide, 0.8% bis- after wash-out of unbound IgG. Fluorescein-labeled deacrylamide. 1* The separated components were then electro- xtran with a molecular weight of 70,000 daltons (FITCphoresed at l-2 volts/cm for 19h into 1% agarose (low dextran, Sigma Chemical Co.) was used at 2.0mgjml in electroendosmosis grade, Seakeam Co.) In Tris-glycine some experiments as a marker for the extracellular solution. buffer, pH 8.7, containing anti-DBH at 0.13 mg/ml. Cells were observed using a Zeiss Universal mtcroscope Fab fragments were prepared from the anti-DBH IgG by equipped with either a 100x planachromat objective (N.A. a modification of the method of Porte? as presented in 1.25) or a 63 x planapochromat objective (N.A. 1.4), Garvey et al. I3 The Fab fragments from this preparation Nomarski differential interference contrast optics and were equilibrated with protein A-Sepharose CL-4B (Phar- selective epi-illumination systemsfor fluorescein and rhodamacia Fine Chemicals) in phosphate-buffered saline, pH mine fluorescence. Fluorescence was photographed using 7.4, to remove any remaining intact IgG. The Fab Tri-X film developed with Diafine or was observed with a fragments were labelled with fluorescein isothiocyanate Dage SIT camera and either photographed from the video (FITC) and the FITC-Fab was isolated by ion exchange monitor with Plus-X film or recorded on video tape. chromatography.’

assayed photometrically. 21 Protein was measured by a microvolumetric adaptation of the method of Lowry et ~1.” Analysis using SDS polyacrylamide gel electrophoresls’ * demonstrated a single prominent protein band with an M, x 75,000daltons. A few minor bands were present under these reduced and denatured conditions (Fig. Ic). However, analysis of the preparation under conditions which retain the nature conformation of proteins’,4 gave only a single band (Fig. la).

Preparation of cells

Bovine adrenal glands were obtained at a local slaughter house and transported to the laboratory on ice within about 1h of slaughter. Chromaffin c&s were prepared essentially by the method of Kilpatrick et a/.15 except that a 0.05% non-specific protease (Sigma type XIV) was used to perfuse the gland, and the minced tissue was then incubated with 0.1% collagenase (Sigma type A) and 0.1% hyaluronidase (Sigma). The buffered salt solution used to prepare the cells was: 140mM NaCl, 5.6mM KCI, 3.6mM NaHCO,, 15mM Na-HEPES, pH 7.4 and lOmM glucose. The cells were purified from red blood cells, dead cells and debris by centrifugation on a previously prepared Percoll gradient (Pharmacia Fine Chemicals). The cells were at least 95% viable. and the vield was about 5 x IO’ cells from one gland.‘The cells were suspended in tissue culture medium (Medium 199 with 10% fetal calf serum, 130units/ml penicillin, 160pg/ml streptomycin and 1 pg/ml fungazone. In some cultures, 50yg/ml gentamycin and 5yM 5fiuorodeoxyuridine were added. The cells were cultured for 2-3 weeks on collagen-coated cover slips at 37°C in a humid atmosphere of 3% CO*, 97% air. The medium was changed every 2 or 3 days. Experimental technique

Coverslips were mounted with wax to become the top of a uerfusion chamber, and the cells were perfused either with b 199 plus 10% fetal calf serum or with a buffered salt solution containing 1.2mM Mg2 + and 2.2mMCazC pnd ts approximately 1% bo~iN scEum a&W&n. were done at 22-30°C. A e p\tnrt, N@ ~&&to w solutions at rates from 0.19 ml/min to @.7ml/min. when Ba’+ was used as the secretogogue the cells were first

RESULTS

SpeciJicity of anti-DBH

Antibody generated against DBH was characterized by crossed immunoelectrophoresis with purified DBH or chromaffin granule lysate (Fig. 1). The purified anti-DBH IgG fraction gave a sin& precipitin peak at the position of the purified DBH when run against purified DBH (Fig la). A single precipitin peak was also seen when run against a complete lysate of the secretory granules (Fig. lb). These results indicate that the antibody was monospecific for DBH. ikbeliing

cells with anti-DBH IgG

Cells stimulated with Ba2 + in the presence of antiDBH IgG became fluorescent after addition of

FITC-protein A or FITC-anti-rabbit IgC (Figs 2c, d). The same results were observed when the antiDBH IgG and FITC-protein A were both present during the stimulation

of secretion or when 10min

washout was interposed between the Ba2* stimulation and the exposure to anti-QW IgC$. Unstimulated cells. &owed no &&e%%&# &ix ad@&&& dition of anti or FITC protein -A (Figs 2a, b). The sur%ces of stimulated cells showed a patchy distribution of

Fig. 1. Crossed immunoelectrophoresis of chromaffin granule lysate and dopamine-/I-hydroxylase. First dimension: (a) 10.5 pg DBH and (b) 956 ng lysate were run in native gels (non-reduced, non-denaturing) of 7.5% acrylamide. Second dimension: 1% agarose containing O.l3mg/ml anti-DBH. See Experimental Procedures for details. (c) SDS-polyacrylamide gradient gel electrophoresis of purified DBH used for immunization (17 pg).

UNSTIMULATED

STIMULATED

1Oum

Fig. 2. Indirect immunofluorescence of chromaffin cells treated with anti-dopamme-P-hydroxylase. Cells were Incubated for 20 min in the absence (a, b) and m the presence of BaCl, (c, d) both with anti-DBH, washed, and then incubated with FITC sheep anti-rabbit I&i. Note in the control that the fluorescence material is debris, and that the cell shows little, if any, fluorescence. In the stimulated cells, the Nomarski image on the left shows that the equatorial plane of the cell in the lower left was in focus, while the section of the cells in the upper left and right was somewhat higher and thus some of the surface is seen. 1027

FtTC

Dextran

RtTC

2nd

Antibody

Fig. 3. Fluorescent images of equatorial sections showing localization of FITC dextran (left) and rhodamine second antibody (right).

Fig. 4. Fluorescent images of equatorial sections showing localization of FITC Fab fragments of anti-DBH at 5min (a) and 30min (b) after stimulation. Paired Nomarski images are also shown (calibration 10pm).

1058

Rh

2nd

F-Fab

antibody

Surface

Fig. 5. Distribution of fluorescence on cells exposed to Ba *+ twice. Cells were exposed to Ba*+ in the presence of FITC-Fab fragments, washed, left for 60 min, and then perfused with Ba* + and FITC sheep antt-rabbit IgG. The photographs from the monitor show surface and equatorial views for both rhodamine and fluorescein. The patterns appear to be quite similar.

1029

1031

Exocytosisand intema~~tion of vesiclemembrane fluorescence as seen in photographs taken near or through an equatorial plane (Figs 2c, d). This distribution of the anti-DBH fluorescence was unchanged for up to 2 h; there was little or no internali~tion. Cells stimulate with Baz+ in the presence of anti-DBH IgG and FITC-dextran and then labeled with RITC-anti-rabbit IgG had patches of rhodamine fluorescence on their surfaces and fluorescein-labeled vacuoles in the cytoplasm (Fig. 3). These results indicate that although endocytosis occurred in the cells in the presenceof anti-DBH, the antibody was not internalized. Since DBH is released from the chromaffin granules during stimulation, it was necessary to exclude the possibility that it could bind to the cell surface after release and then combine with the antibody. Therefore, unstimulated chromaffin cells were preincubated with 13yglml of purified DBH prior to treatment with anti-DBH. There was little or no labelling of the chromaffin cells under these conditions and so binding of released DBH cannot account for the results. Cells activated with Ba*’ solution for 5 min were labelled with FITC Fab fragments of anti-DBH. In this case the fluorescence recorded 5min after washout of the Ba’+-FITC Fab solution appeared to be rather uniformly distributed and was mostly near or on the surface. This is shown in the pictures taken at an equatorial section (Fig. 4a). By 30min after washout, much of the surface fluorescence had disappeared, and the fluorescence was now distributed throughout the cell in a somewhat punctate pattern (Fig. 4b) except that the region of the cell nucleus was not labelled. In these experiments, the FITC-Fab fragments were usually added with the Ba’+. However, the response was the same if the Fab fragments were added only after the Ba*+ was washed out. This controls for the possibility that the Fab fragments reacted with the soluble DBH released by Ba*+, and that the immunocomplex was subsequently absorbed onto the surface membrane. Cells that had been exposed to the Ba*+ FITCFab solution, washed and left for 30-60min so that the FITC-Fab-DBH was internalized were exposed to Ba2’ a second time in the presenceof rhodaminelabelled sheep anti-rabbit IgG. This protein will bind with the Fab fragments, which were presumably still bound to the DBH which came to the surface during the initial period in Ba’+. The cells showed both rhodamine and fluorescein fluorescence on the surface (Fig. 5). In both cases the fluorescence was patchy. The dist~butions of the two ~uore~nt agents were similar to each other and to that in the case where anti-DBH IgG was labelled with FITC sheep anti-rabbit IgG. This was to be expected since the rhodamine-labelled sheep anti-rabbit IgG was divalent and polyclonal and so would produce a

similar crosslinking of the DBH on the surface. Cells treated with the rhodamine-labelled sheepanti-rabbit IgG after the fluorescein Fab had been internalized but before the second exposure to Ba2+ showed no rhodamine fluorescence. Therefore, the DBH apRearedon the surface and was labelled by FITC-Fab fragments; it was then internalized, and finally reappeared on the cell surface in responseto a second exposure to Ba*+. DlSCU~ION

Dopamine /I-hydroxylase is a biochemical marker for chromaffin granules’4v33and the antigenic determinants for DBH are located on the inner surface of the chromaffin granule membrane17*32. Further, DBH has been shown not to be present on the plasma membrane of unstimulated cells2 and we also found this to be so. Therefore, the labelling pattern observed on stimulated chromaffin cells suggeststhat the vesicles must have fused with the plasma membrane in an inside-out orientation to expose the DBH to the externally applied antibody. These results are in ag~ement with those of Wildmann et a1.29 and demonstrate that the chromaffin granule membrane is transferred to the plasma membrane of cells stimulated to release catecholamines. Stimulated chromaffin cells treated with antiDBH and FITC sheep anti-rabbit IgG (see Fig. 2) exhibited a patchy pattern of fluorescencearound the perimeter of equatorial sections of the cells and on the flattened surface where the cell adhered to the coverslip. Therefore the fluorescence was present only on the surface of the cells. Further, the DBH in the patches was not internalized over a period of 2 h. The punctate fluorescence reported here and by Wildmann et a1.29 suggests that the distribution of DBH on the cell surface was not homogeneous. If the vesticle membrane is retrieved selectivelyz6 (also Edwards and Ye, unpublished), then it is likely that the vesicle membrane remains segregatedafter fusion with the surface membrane. The rapid release of chromaffin material induced by Ba2” causesvacuoles to form, which are presumably a consequence of exocytosis. These vacuoles, which were demonstrated by the uptake of FITC-dextran (molecular weight 70,000) (Edwards, Englert and Ye, unpublished), have been observed previously in electron micrographs of chromaffin cells6 and are probably due to the endocytotic uptake of the excess membrane added to the cell surface by exocytosis. The formation of vacuoles could be induced by repeated localized exocytic events. This localized exocytosis and the segregation of vesicle membrane are probably the cause of the patchy distribution of fluorescence.The apparent absence of patchiness with FITC-Fab is likely due to the rapid internalization of DBH; the half time of internalization is about 1Omin” (also Edwards and Ye, unpublished), and since the cells were not examined until almost 20 min

1032

D. J. Dowd et ul

after Ba* + and FITC-Fab were first added (15 mm In this solution and then several minutes for washout), more than half of the DBH which reached the surface during the first 10 min would have become internalized. In other cells, patching of cell surface molecules exposed to multivalent ligands often leads to endocytosis, but this is not always the case. Cell systemssuch as H-2 antigens on mouse L-cells; blood group antigens on monkey kidney cells; immunoglobulin determinants on human lymphocytes do not exhibit endocytosis following patching and capping.‘* In the study of each of these systems, as with our study, the indirect immunofluorescent technique was used. On the other hand, monovalent antibody, which does not induce patching and capping, has been observed to be internalized by pinocytosis in lymphocytes.‘q9 The DBH did appear to be internalized in the presence of the monovalent Fab fragments of the anti-DBH, suggestingthat it was the crosslinking and aggregation of the antigen by the divalent antibodies that prevented its internalization. Other evidence indicates that DBH is internalized after exocytosis in the absence of antibodies. Wildmann et al.29 reported that the binding of anti-DBH was less if the cells were incubated for 2 h after washout of Ba2’

before addition of the antibody; this was presumably due to internalization. They found that this decreased binding did not occur if the anti-DBH was present during stimulation; this suggests that internalization was blocked by the antibody. Suchard cjt a1.26 found that cell surface DBH labeled with ‘251 during stimulation of exocytosis became incorporated into secretory granule membranes. With Fab fragments and 125I labelled second antibody, internalization of the DBH has been found to have a half time of about 10min (C. Edward & H. Z. Ye, unpublished). Similar measurements, using an antiserum against a membrane glycoprotein from granules and the cytotoxic effects of complement also showed that the antigen had largely dlsappeared within 30min”. In addition, the results of the experiments with two exposures to Ba*+ and two fluorescent labels show that the DBH is internalized in such a way that it can be made to reappear on the cell surface within 1h. Acknowledgements--Part of the work in this paper was

presentedby D. .I. Dowd in partial fulfillment of the requirementsof the Ph.D. degree.We are indebtedto C. Izzard for assistancewith the microscropy. to M. Hoyle for assistancewith tissue culture procedures and to D. Rice for help with the immunization. This researchwas supported by a grant from NIH (NS07681) to C. Edwards.

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