Immunochemical evidence for exocytosis in isolated chromaffin cells after stimulation with depolarizing agents

Journal of Neuroimmunclogy, I (I 981) 353-364

35.*

Elsevier/North-HollandBiomedicalPress

Immunochemical evidence for exocytosis in isolated chromaffin cells after stimulation with depolar~ng agents Johannes Wildmann, Mahraoud DewaJr * and Heinrich Matthaei Max.~'lanck.bt~titut f ~ experimentehe Medizin, D-3400 G6ttingen(ER.G.)

(Received6 April, 19~1) (Accepted I May, 19~',1)

After chemical stimuh~tic,n with depolarizing agents'(Ba 2+ or Ca2+/carbachol) isolated riving chromaffhl ceUs display a drastically increased binding capacity for anti-DBH, distributed spotw~se on or near the outer cell membrane. This effect is inhibited by noradrenaliae; s~ is not evoked by the non-exoc~otically releaftag agents tyraraine and rese~in¢. The effect of apparent externalization of DBlt is paralleled by the observation of a DBH-dependeat binding of ~251-1abeUedprotein A upon the same depol~uizing stimuli. These .~b~ervations are discussed as poraible evidence for exocytotic activities.

Introduatm Physiological secretio:a of catecholamines (CA) by chromaffin celL~of the adrenal medulla probably occurs via exocytosis (Douglas 1968). During this process, the membrane of the chromaffin granule and the outer cytoplasmic merabrane become fused, for a short pe:iod at least. These two types of membr,tnes differ in thtir constituent proteins, dopamine-p-hydroxylase (DBH), an enzyme of CA-biosynthesis, e.g. being characterL.~ic for granule membranes (Winkler 1976).

~t Correspondence should be tent to Prof. Dr. H. Matthaei, Max-Planck-Insfimtf~r experimentelk Medi,zln,Hermman-Rei~a-Str.3, D-3400GSttlngen,F.R.G. *PreKnt address: Abt. Pulmo~ologie, Medizlnlsche Klini~ ], Klinikum C,rtn~adem, Ludw~Maximiliam-UaiverJitk, D.g)00 Munich, F.R.G. 0165-~728/81/0000-f000/$02.50 ~' 1981Elsevier/North-HollandBiomedicalPress

354 As evidence for exocytosis in chromaffin ceils are so far considered (I) the "exocytotic pre,files" seen in freeze-fracture studies (Smith et al. 1972) an,", upon stimulation, (2) increased numbers of omega-shaped struet~ares (exocytotie ~'sieles) concomitant with decreased numb,.rs of filled vesicles, as observed by Uectrortmicroscopic investigation of thin sections (Grynszpan-Winograd 1971) :.nd (3) release of CA plus non-diffusible proteins in the same molar ratio as fou-.; inside the storage granules (~hneider et al. 1967). A sensitive light-mi:rroseopie recognitiol~ of exocytosis is still missing. Thi,; would facilitate the study of the: release mechanism o£ cultured cells, the eff~ts of different depolarizing and release-inducing agents, an~ the relationship between different cellular components involved in storage and secretion. Using chromaffin cells as a model, we have therefo~'e attempted to find a method by which a stimulated and exoc~tosis-dependent incorporation of granule membrane antigens~ like those of DBH into the plasma membrane, could be demonstrated with anti-DBH end f!uoreseein-labelled anti-a,atibodie.~. Antibodies have usually been produced in rabbits against readily available bovine DBH :rod used for immunological investigat:ons in other species. The strateg'¢ of using heterologous system,; however, has not a'ways been successful because of weak interspecific cross-reaction, s of DBH (Ebstein 1973; Grzanna and Curie 1976). Anti-bovine DBH has been successfully appli,-.d in the investigation of CA storing neurons and of chromaffi~l cells. Jacobowitz et ,~.1.(1975) have observed the uptake of intravenously administered heterologous anti.DBH into corresponding synaptic elements of rats: ~lective nerve terminal antiDBH uptake and a subsequent retrograde transport in vivo were observed in a peripheral sympathetic system (Fillenz et al. 1976). In recent years, several n~ethod,,; have been reported for the isolation of chromaffin cells from bovine adrena! medulla. A homologous system could thus be obtaincA. Anti-bovine amphiphilie E,BH was produced in rabbits, and the indirect immunoiluorescence assay w~t; applied for its detection. 12~l-labeiled protein A was also used instead c,f the fluorescent anti-1,-globulin in order to obtain a more quantitative assay for externalized DBH.

Methods isolation of chromaffi n cells

Cells were isolated by a modification of the method of Brook~ (1977). The whole adrenal medulEr tissue was u~ed--undissected ar.d well.washed For removal of blood cells--for the incubation wi,'.h enzyme buffer in order to obtain preferentially the A-cells located near the cortex (Palkama 1964). Incubation time was limited to 1 h. Only a single gland was t~,kt:n for every preparation (thus obl~.aining "monogenetic" cell suspensions). In orde: to exclude a possible influence of bovine serum albumin (BSA) on CA release, BSA was omitted in some experiments. In these cases, the pH was adjusted to 6.0 and lhe cells were purified by repeated ¢entrifugations at 50 × g rather than through BSA gradients. Yield: 5.5-6.5 ;< 10~ ceils/gland.

3~5

Isolation of DBH from chromaffin granules Chromaffin granules were pre!3ared by the method of Smi:h ~LndWinkler (1967). Their lysate was centrifuged at 37,000 rpm (60 Ti rotor of Bcc',l::rn~ ce~,trifuge) for 60 rain. T[,e supernatant w~s taken for the preparation of soktble DBH ~o=ording to Rush et al. {1974), using a 0.5 cm × 10 cm column of Con A-Se~haros~" (Pharma~ia, Uppsala, Sweden). The pellet was washed in 0.1 M acetate buffet" pH 6.5, containin$ 1 mM MgCI 2 and 25000 U / m l oi" bovine fiver catalase (B,celu'ing~, Mannheim, F.R.G.), the latter for protection against 13eroxides. After this v~.,:~a ~ne pellet was dissolved by homogenization in acetate buffer containing ?,~ Triton ~.-100 {Serva, Heidelberg, F.R.G.) and stirring for 2 h at 21°C, and centrifuge~a at 6~.~'I)3Cg for 20 min. The supernatant was charged onto a Con ~:, column, previol,,3iy was~'t¢.:i t~~ remove non-covalently bound lecfin. The column was then extensively wasbed to remove all the unbound m~terial. Elution of DBH was carried out with the acetate buffer containing 10~ of a-~ethylD-mannoside (Sigma St. Louis, U.S.A.). Under these conditions, howt,~er, a considerable part of applied DBH act:~vity remained bound to the column ~n,:l could be released only by including Triton-X 100 to a final concentration of 195 in the mannoside elution buffer. This detergem-mannoside eluted DBH (about 6095 of the DBH activity applied) is called amphiphilic DBH. The eluates were dialyzed at room temperature for 24 h against bi-di~:tilled water (3 changes), concen~.rated in an Amicon-Diaflo cell uslng XM 50 filters, lyophilized and dissolved ir~ C*.IM acetate buffer, pH ~.5 to a concentration of l m g protein/ml. SDS-disc-electrophoretic analysis revealed only a single protein baa¢~ when 0.6 x 10 em gels were charged with ~ 100/xg protein.

Preparation of antisera DBH-specifie antibodies were produced in rabbits (1.5-2.0 kg). For immuniza. tion, 1 mi eor, talning i mg antigens mixed with 1 ml complete Ffeund's adjuvant (Behring, Marburg, F.R.G.) was injecled subcutarteously into d~ffexeat places of:the n~ck fold. Producing a stable emulsion was easier with ampi~philic D~tH than with soluble DBH. Injections with 1 mg antigens were repeated after 2~ 4, 6, 8 and 10 weeks, using inconiplete adjuvant. 4 weeks after the last injection, 50 ml of test serum were taken from the vein of the ear. Harvesting was repeated after 6 weeks, when the animal w~ts totally bled. Sera were obtained from blood by ¢m,~xifegation ($00 × g, 30 rain) and stored in small portions at - 3 0 ° C . Single ra¢fi~ h n~munodiffusion was performed in 10% serum (stabilized with 1.2~ agar in 50 mI~4 PBS, 10 mM NaCi, 0,2 mM PMSF). Precipitation rings had been obtained after 7 days at 7°C. Pre-immune sera ,~ere obtained before immmtization.

Stimulation and labelling of chromaffin cells Monogenetic cell suspensions containing 0.7-0.8 × 106 cells pe~ final volume of 0.5 ml were incubated at 37°C in standard b'fffer ( = incomplete b~ffer of Brooks)

356 for 1, 5, 15 or 30 n,Jn, respectively, with the dq~olarizing agents Ba 2÷ (1-.]~ raM) or 0.2 mM earbacbol (Sienna St. Louis, U.S.A.) and Ca ~+ (1-10 raM). Shorter stimulation periods were stopped by chelating t~e divalent cations with an equimolar amount of EDTA-Na 2. CA, released into the incubation medium were determined. Incubations with anti-DBI'l were done either simultaneously with the stimulant or after its removal by 2 cenmfugations at 100 × g for 10 rain at 5°C. Anti-DBH was provided either 30 nun at 37°C or 45 rain at 21°C or 60 rain at 7°C in a final dilution of 1 : 80. 10% BSA ~;gasadded to minimize nonspe~fie binding. Stimulations with 5 mM tyramine or 0.2 mM reserpine were performed in the same way for 30 min at 37°C in the presence of anti.amphiphilic DBH serum. Control samples were incubated for 30 min with aa.~iserum also but without additional stimuli. Tbereafter, cells were washed 3 times with standard buffer at 10~ X g for 10 rain at 5°C to remove exc".'ss antiserum. They were then incubated at 37°C for 30 rain in the dark in 0.5 mJ fontaining a i :40 dihitioa of fluorescein-conjugated goat anti-rabbib7globulin (P.ehring, Marburg, F.R.G.) and 10~ BSA. Excess conjugate was removed by 3 washings in standard buffer at 5°C. Aliquots of the cells were dried onto slides and covered in nora-fluorescent glycerol-standard buffer (9:1). Preparatwn of thin sections Adrenal glands, ectomized appzpx. 30 rain post mortem, were dissected in~.o 4 m m × 4 mm cubes and frozen in i~pentane chilled with liquid nitrogen. 4-8-/zm sections were cut from such cubes a~'. - 2 0 ° C using a Cryocut II microtome. They ~'ere air-dried for 20 rain at room temperature and incubated with few drops of 1:40 diluted antise;~m for 30 min at 37°C in a moist chamber, either unfixed or after 10 nfin fixation in ethanol/ether (1 : 1 at 21°C) or glutaraldehyde '.0.5% at 7°C) and subsequent washings with standard buffer. After three 30 min-was:lings at 21 °C in standard buffer, sections were incubated with fluorescein-conjugate as above fc,r 30 rain at 37°C, washed 3 times again and covered in glycerol standard buffer (9:1). Fluorescence microscopy and microphotography Fiuorescein-labelled obj,)cts were inspected with a Zeiss photomicro~cope 11 equipped with a condenser for incident, fluorescen*, illumination, and the fi)llowing filters: Excitation filter 490 nm, emission filter 540 nm, diehroic mirror 510 am. The oil ir~rLmersionplanapochromate 63 × / 1 . 4 was used routinely. Exposure times for Kc~lak 100 ASA films were les.q than 30 s. Objects were normally photographed 2*) h after fluorescence" staining. Photographic prints were made on Agfa Brovira, grade 4. Labe, ging with [ t 2sI ] protein A In ~ome experiments fluorescein-conjugated goat anti-antiserum was replaced by [1251~protein A as a detec'~or. As described before, chromaffin cells were stimulat~ with 3 mM l~a2+ for 5, 15 or 30 rain, respectively. Alternatively, 3 mM Ca2+/0.2 mM carbachol was used. Incubation with antiserum occurred simultaneously with

357 the stimuli or after their removal by 2 washings. Basal labelling after 30 rain of incubation with antiserum however, w,.'.'.thoutadditional stimulus was measured as a control. After removal of .excessive antibodies, cells were incubated with 0.05 pg of labelled 50 ttCi/ml ( = 0.8 PS) protein A together with 0.4/tg of cold protein A for 30 min at 2! °C. They were washed 5 times by ¢~ntrifugation at 100 × g (5°(:9 and counted in a Prias y-counter (Packard) at a court;ring efficiency of 68~.

Determinalion of eatecholamines The method of Sailer and Zigmond (1978) was ~,pp~ied as modified by Witte and Matthaei (1980).

Determination of DBH The method of Pisano et al. (1968) was used as modified by Aunis et al. (1973). Tyrosin was used as substrate; 0.5 mM pargyllne, 25000 U / m ! of catalase and 0 . ~ Triton X-100 were always present. The octop~unine formed was determined spectrophotometrically after its oxidation ~o p-hydroxybenzaldehyde with periodate.

Protein determination Amounts of proteins were assayed after l)recipitation with TCA according to Lowry et al. (1951), using tryst. B3A as a staItdard.

Remits

Specificity of antisera Similar to anti-amphiphilic DBH, anti-sc]uble DBH precipitated amphiphilic DBH at a much higher efficiency compared to anti-membrane protein serum (Table 1). Ami-soluble as well as anti-amphiphilic DBH proved suitable for labelling of DBH located on the outer cell membrane as seen by fluorescence microscopy. In the experiments reported however, anti-amphiphific DBH was used routinely. 18 ~tg TABLE I INACTIVATIONOF AMPHIPHILICDBH BY VARIOUSANTISEKA Serum

Activityin supema~ant 0,mo~ octopamine) (30 rain.rag protein)

~$Inactivation

100~1pre.wmmn©serum 100pl attfi-amphiphilicDBH 100#1 anti-soinbi¢DBH 100~1 anti.membnmeprmein~

715___28 I I -+- 4 SO-+- 3 136± 10

0 98.5 93 81

35,";

amphichilic DBH were !.lcubated with the sera at 37°C for 30 min. After 2 h standi~'~g in ice and 15 rain centrifugation at 5000 rpm. activity was assayed in the

supernatant. 1)elect,on of DBtt with fluoresc,?nt y-globulin Whec thin sections made through the cortex/medulla boundary of the bovine ad enal gland were treatec with anti-amphiphilic DBH and subsequently with flu.;rescent y-globulin, the latter was found to bind specifically to cells of the medulla {Fig. 1). This immunofiuorescence could be suppressed by pre-incubation of the rabbit antiserum with amphiphilic DBH. If pre-immune serum was used instead of ,mtiserum or if the latter was omitted, only a very weak autofluorescence of the tissve could be seen. The antiserum did, however, not discriminate medullary from cortical cells of other species: in sections from rat and rabbit only some scattered structures showing a strong yellow autofluorescence were observed. Isolated bovine chromaffin cells, after such immunofluorescence treatment, appeared labelled within their whole cytoplasm {not in the nucleus), if their membranes bad been made permeable for macromolecules by either air-drying a n d / o r fixation ,vith ether/ethanol. This rather general cytoplasmic fluorescence distribution in dried a n d / o r fixed cells was not influenced by the presence or absence of Ba "~' (3 raM) during the incubation with anti-DBH. In intact living chromaffin cells, however, the interior of the cytoplasm was apparently not reached by the immunochemical reagents; t~u'; only the outer plasma membrane appeared fluorescence-labelled. Without special stimui, a low but rather uniform level of fluorescence was seen (Fig. 6), Thi, basic labelling of the cell membrane could not be lowered significantly by the omission of BSA from the isolation or incubation media: thus in order to obtain a higl'.er purity the ceils were isolated routinely iJccording to Brooks.

l-ffects of det~olarizing stimuli upon fluorescont antihody binding of viable cells However. if chromaffin ceils and anti-DfH were incubated in the presence of Ba:' (3 raM). considerably more antibody was bound near the outer plasma nlcmbrilnc (Figs. 2.5a and b). Correspondingly. the amount ol CA released illto l he Fig. I. I:luorescencc nficrophotograph of an unfixed 5 t i m freeze section through cortex (('l/riled.ilia t'onhlfl region of a bovine adrenal gl~tJd. )]~ar IO #I1. ]:ig~,. 2 and 5a tl. Fluorescence rain with i m m u n e serum and cquimolar E I ) T A - N a 2. 5a and n e a r the upper surface I a ) and

NIl

microphotographs of viable chromaffin cell sw, pcnsions incubate J for 30 with Wig, 2) 3 m M Ba 2' for the first 5 rain. thereafter cbelal,.'d with h: 3 m M Ba: ' for 3(i rain. a and h show the same group of cells. I.~,:u:.~.e,J near the equatorial plane { h h respectively. Bar = I0 # m

I:ig~.. 3..,1.6.7. I:luoresccnce microphotographs of viable chromaffin cell .,,uspensions ineabated for 30 rain i~th immut'.c sertml anti with {Fig. 61 m~ additional stmmli. (Fig. 3) 3 m M Ba: ' . 0.2 m M N A . (Fil;. 4) 5 m M lyraminc or (Fig. 71 3 m M Ca 2 ' . (1.2 m M carbachol. Bar : lO tim.

imm

mm

360 TABLE2 INCREASE OF :ATECHOLAMINESFOUND IN THE CELL CULTURE MEDIUM AWI'F_R30 MIN INCUBATIONWITH VARIOUSAGFaMTSABOVEBASALLEVEL 1N THE PRESENCEOF ANTI-DBH (1007o=5.6 t~molesCA per 73<105 coils,i.e. 5.0 t, molesA+0.5/tmole$ HA+0.08/,moles I)A) Agents added Pre-immtmeserumor Con.A 3 mM Ba2÷ 3 mM Ba2+ +0.2 mM NA 3 mM C a 2+ +0.2 mM carbacbol 0.2 mM reserpine 5 mM tyramine n=4,'n=2.

t$ increaseof CA above anti.DBH blanks(~SD) ± 0('4- 4)" +420 (±24) +215 (-¢-18) + 133 (-4-i I) +390 ('4-17) +220 (+21)

-:

medium was much iaoreased (TaMe 2). The apparent accumulation of antigen on the membrane appeared locate.~ in distinct green-fluorescent small spots or paKhes. Whereas the inner bounda U of these patches appeared rather sharp and coincident with the light-microscopic haage of the outer cell membrane, their outer margin(s) looked often rather fuzzy. "tlle overall fluorescence intensity located r.ear the ceii membrane increased strongly over a r~fiod of 30 mitt; but even after ~ l-min exposure to Ba2+ , its subs~quent complexlng with. EDTA and completio~ of the 30-min incubation period with anti-DBH, a distinct r~ot.wise accumulation of fluorescence near the outer cytoplasmic membrane could be observed. Upon substitution of Ca2+/carbachol (3 raM/0.2 raM) for Ba2+, a simi'~r pattern of fluorescence labelling, but of much lower intensity, was to be observed (Fig. 7). With both of these depolarizing stimuli the intemity of labelling was dependent upon con~ntration of the stimulus and the length of incubation time; a plateau of maximum iraensity labelling was apparently reached with ~ mM Ira':~ or 3 ink.,* Ca2+/0.2 mM carbachol within 30 mix. After stimulation for shorter periods, fluorescence labelling was spread over the cell surface in tiny spots, whereas after 30 rain it seems to be accumulated in strongly fluorescent patches. If 0.2 mM NA wa~: present in additfon to Ba2+, the intensity of membrane fluorescence was lower (Fig. 3). The .lmoum of CA found in the ~tedium was reduced ~y 50f6 under these conditions (Table 2). Prc-immbation of ~.he ceil:; for several hours with the MAO inhibitor pargyline similarly diminished both fluorescence ~r~tensity and CA release. Tyramine (.~ raM) or reserpine (0.2 raM), which non-exocytotically release CA into the laedium (Table 2), did twt significantly increase labelling of the p~sma membrane above the level observed in unstimulated cells (Fig. 4).

Labellin3 of viable chromaffin cells with [72al]protein A If and-DBH binding was monitored with ['~l]proteinA, known to bind to 7-globulins, the results obtained with fluorescent goat anti-y-globulin were ¢ ~ n -

"3~M Ba2+ 3ruMBa2+ 3ruMBa 2+ 3 mM Ba2+ + anti-DBH 3mMBa2+ 3ruMBa2+ 3 mMBa2+ 3 mM Ca2+ + 0.2 mM carbachol

Reagents

n=3;an=2.

5 15 30 30 5 15 30 30

37°C

(n~n)

Tune

II~tlblttiol~ with s~mu]allt

+ + + + + + + +

Wa~hin~ (2 × )

-

+ + + +

Postincubation (2 h, 37°C)

Incubation with ~t~-DBU (30 m/n, 37°C)

SUMMARY OF RESULTS OBTAINED WITH nSI-LABELLED PROTEIN A

TABLE 3

196 (+_. 5.1) 206 (_.+ 4.2) •

182 (_.+ 5.7)

318 (_+ II)* 180 ( ± 5.4)

77(_+ 2.9) 15o (___ 4,3) 261 (+__6) 281 (__+ 9.3) 3o8 (_ 9.8)

(±SD)

Protein A binding

cpmX 103/ 0.7-0.8 x 10~ cells

Incubation , - ~ p~o~clnA

(30 min, 21°C)

131 |58 168 29 32 46 56

0 III

cpm x t03/ 0.7-0.8× 106 cells, ra/n~ ur.stimulated anti-DBH blank

362

tinily confirme:! (Table 3). The high background radioactivity obtained in this assay is probably not reflecting a high level of plasma membrane-integrated DBH in the mtstimulated state but is rather due partly to I~onspe~ific binding of iodinat~ grole~n A and partly to entering of this tracer into a few protein-permeable cells and debri:; (cf. controls without serum, and without s_~:+,amand stimulant, respectively). With the tinge of stimulation, the amount of ~'adioactive labelling increased. Tim final level dep~nding again on the kind of stimulating agent. Thus after 30 mix of in,'.abation w~h Ba2+ protein A binding reached a 100~ increase above the nonstimulated controls exposed to anti-DBH, whereas CaZ+/carbaehol also raised the labelling significantly b~t only by 30q,. Anti-DBH binding, as indicated by protein A labelling, had drastically decreased, however, when the cell suspension, after a 30 rain stimulation period with Ba2+ , had been int~ubated for a further period of 2 h at 36°C before anti-DBH application. This decrease during a 2-h post-incubation period apparently did not occur, when anti-DBH was present simultaneously with the stimulant (Table 3). When comparing the fluorescence labelling results with those of the protein A binding experiments, it should be remembered that immunofluorescence has a linear sensitivity, amplified b), a cascade effect (several tracer molecules per antibody molecule bound), whereas sensitivity in the [125I]protein A binding assay decreases with increasing density of antigen bound to the membrane, because one protein A molecule may bind to 1-3 IgG molecules.

Di~u~sion In this study using isolated chromaffin cells w~th DBH-specif:c homologous antiserum and 2 alternative indicators, fluorescent anti-y-globulin or ~2Sl-I~:belled protein A, light-microscopic evidence was provided for the occurren,:¢ of DBH on the outer plasma membrane as a result of such stimuli which lead to release of catecholamines (CA) via exocy~o~is. When either Ba2"~ (3 mM) or Ca 2÷/carbachol (3 raM/0.2 raM) was used as the stimulating agent, incubation with ant,-DSH, either during exposure to the s~imulant or aft:r its removal, followed by incubation with fl~Jorescein-con.iugated antibody led to the observation of fluorescent spots or patches only on the external cell membrane, but not witifin the cytoplasm. With both stimulating agen,.s no re-uptake seems to take 91ace during the time period stud~cd. It may be concluded that at le;~st one chrom~ffin granule-specific proton (DBH), perhaps only temporari+y, is imegrated into the cellular l~!asn~a membrane via exocytosis, and thus becomes available lur binding of both fluorescence and radioimmune labelling agents. Several arguments s~tpport this conclusion: (a) Control experimcrts showed that neither fluorescent spots nor increa.~,ed protein A binding could be produced when anti-DBH was either omitted or substituted by pre-immune serum, indicating that tbe obser,/ed fluorescence and protein A binding were not due to a nonspecific precipitador, by Ba2+ ions, e.g. -Jf immune complexes formed by rabbit ~,-globulin and either goat anti-rabbit y-globubn or protein A. •" ,+

(b) The typical localization of fluorescent antibody binding in spots and pa~ches, located apparently only on the outer cell membrane, was limited to the living cells, whereas just a small number of injured cells and debris present showed im~tead, intensive diffuse fluorescence. (c) The stimuli-evoking exocytodc CA release (BA2+ , Ca2+/carbachol) led to a spot-wise accumulation of DBH in the outer membrane or to incre~',ed p:rot(,'in & binding, respectively. (d) The amounts of externalized DBH thus indicated appeared generally correlated with strength and duration of these stimuli. (e) Stimuli which deplete CA stores without exocytosis (tyramine, reserpine), however, led only to a weak background fluorescence with no increased intensity on the cell membrane. (f) Under inhibition of the Ba2+-eff¢ct upon CA release, either by NA or by pretreatment of the cells with pargyline (via a-receptors located in the ce,it m~nbr.'me probably controlling exoc~'tosis (Gutman and Boonyavir,oj 1974; Starkc et al. 197 3); a decreased DBH-sp~ific fluorescence was found. (g) The apparently lower efficienc7 of Ca~+/carbachol relative to Bae+ in CA release was correlated with a lower stimulation of both fluorescent anti-anti-DBH or protein A binding, respectively. These arguments altogether strongly support the conclusion that the an~i-DBHdependent binding of fluores~mt atlti-anti-y-globulin .to spots or patches on the outer membrane of chromaffin cells is a rather closely linked consef4uenc~ of the release of either A or NA via e~ocytosis. Light-microscopic assay for exocytesis may pot~entially be based on this inm,unochemical observation. A combined light- and electron-microscopic exan'6nation is required ~or the ultrastructural identification of t:ae phenomena seen by fluorescent n~icroscopy. The spots observed might represent reagent bindin$ into the omega-shaped vesicles opened towards outside and/or flattened membrane areas into which these may perhaps be converted. The patches seen may be accumulations of many such structures. When they appeared towards outside less sharply limited (fuzzy) than towards inside, particularly after Ba2+ stimulation, such patches may have been correlated to the areas with groups of many "microspikes" observed by Englert (1980) using Nomarsky ~hase optics t,t highest resolution. Thus, the assay ma} be useful for a qualitatiw: demonstration of exocytotic activities in isolated chromaffin and other A or HA secreting cells. y

~

t

s

We would like to thank Prof. K. Weber for critical examination of the immunofluorescence results and Prof. H. Wink]er for helpful discussions and advice on the manuscript. References Aunis. D., M.T. Miras-Portu8~l and P. Mandel, Inhibition of adrenal dopamine-B-hydro~:ylaseby 6-hydroxydopamine,Bicci~m. Pharmacol.,22 (1973)2581-2589. Brooks, J.C., The isolated i:ovineadre~omedullarychromaffincell~A model of neuronal ¢~xcitation,¢~¢refion,Enriocrinol., 101 0977) 1369-1378.

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