The biogenesis of adrenal chromaffin granules

Neurmcience.

1977. Vol. 2, pp. 657-683.

Pergsmon

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Britain.

COMMENTARY THE BIOGENESIS

OF ADRENAL GRANULES H.

Department

of Pharmacology,

~HRO~AFFIN

%WLER

University of Innsbruck, A-6020 Innsbruck, Austria

Introduction Biogenesis of macromolecular components Studies with radioactively labelled precursors E3H]leucine C3H]fucose [35S]sulphate [32P]phosphate Characterization of newly formed chromaffin granules after cateoholamine depletion Morphological studies Origin and final fate of membranes of chromaffin granules Is there a concomitant synthesis of the membranes of chromatlin gram&s and of their exportable proteins? Are membranes of chromaffin granules retrieved after exocytosis? What is the fate of the granule membranes after retrieval? How are membranes of chromafiin granules m-used? Relationship between stimulation of adrenal medulla and biogenesis of chromaffin granules Biogenesis and uptake of small molecules Calcium Nucleotides Catecholamines Bios~~esis of ~t~holam~es Uptake of catecholamines by chromaffin granules Biogenesis of chromaffin granules and catecholamine uptake Conclusions THE CATECHOLAMINE-STORING organelles

of adrenal medulla, the chromaflin granules, have been analysed in great detail (SMITH, 1%8; STIARNE, 1972; K~~HNER, 1974; WINIUEX & SMITH,1975;HELLE& SERCK-HANSSEN, 1975; WINKLER,1976). It is a characteristic feature of these membrane-limited vesicles that their content, which is destined for secretion, consists of both macromolecular components, i.e. glycoproteins and rnu~o~ly~~ha~~s, and small molecules including catecholamines, nucleotides and calcium. It is therefore not surprising that the biogenesis of these multicomponent organelles has proved to be rather complex in that it occurs in more than one step. In fact it is only just now that the available data enable us to present a fairly coherent concept. It is hoped that this commentary, dealing with the formation of chromaflin granules, will also provide a conceptual basis (see also HOLTZMAN,1977) for studies on the biogenesis of other organelles storing small molecules. This hope may be fulfilled since many si~l~~es have already been revealed between the biochemical composition of the ‘standard’

Abbreviations: AMP, ADP and ATP, adenosine 5’-mono-, di- and triphosphate, respectively; GERL, Golgiassociated endoplasmic reticulum with attached lysosomes. 657

organelle, the chromafhn granule, and the composition of noradrenergic nerve vesicles (see Sbfrrq 1972; LAGERCRANTZ,1976), of cholinergic vesicles (see WHITI-AKER, 1974)and of 5-hydroxytryptamine-storing particles in thrombocytes (PLEWXIX, DA PRADA, BERNQS,STEFFEN,LUIIILD & WEDER,1974). By analogy with the actual sequence of events in the thrown cell in uim, I will first discuss the synthesis of the ~cromol~u~r components and their assembly into newly formed chromaffin granules. This will be followed by a description of the various uptake mechanisms which endow these particles with the ability to accumulate a high concentration of small molecules. BIOGENESIS OF MACROMOLECULAR COMPONENTS Evidence for the biogenesis of these components has been obtained mainly by three methods which will be discussed in the following three sections. The important topic of the origin and final fate of the granule membranes will be dealt with in a separate discussion.

Finally,

of chromafIin stimulation

the way in which

the biogenesis

granules can be modified by prolonged of the adrenal

will be considered

H. WINKLER

658

Studies with radioactively

labelled precursors

ponent is the enzyme dopamine /3-hydroxylase (see WINKLER, 1976). In these studies bovine adrenal glands, which were [3H]leucine. This amino acid was used (WINKLER, HBRTNAGL, SCH~~PF, H~~RTNAGL & ZUR NEDDEN, perfused in vitro, were ‘pulse’ labelled with C3H]leu1971; WINKLER, SCHGPF, HGRTNAGL & H~~RTNAGL, tine. The subcellular distribution of the newly synthesized proteins within the gland depended on the time 1972) in order to study the biosynthesis of the secreinterval which elapsed between the pulse and the subtory proteins of chromaffin granules, which comprise cellular fractionation. After 1.5 min the labelled sola family of acidic proteins (chromogranins) and, as uble proteins gave a distribution after density graa minor component, the enzyme dopamine /I-hydroxdient centrifugation consistent with a microsomal ylase. It was also intended to study the synthesis rate localization. After 45 min they were present in a parof the granule membrane whose main protein com-

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FIG. 1. Synthesis of macromolecular components of chromaffin granules: analysis by gradient centrifugation. Results included in this figure are taken from several papers (WINKLER et al., 1972; BAUMGARTNERet al., 1974; GEISSLERet al., 1977). In these studies perfused bovine adrenal glands were ‘pulse’ labelled with either C3H]leucine, [35S]sulphate, C3H]fucose or [32P]phosphate. Four hours after the radioactive pulse a large granule fraction was isolated from the adrenal medulla and then subjected to sucrose (1.3-2.0 M) density gradient centrifugation. The cell particles in the gradient fraction were

subjected to hypotonic lysis and the soluble material and the membranes were separated by high-speed centrifugation. The fractions were then analysed and the results expressed in histograms as shown above. In each histogram the columns from left to right correspond to the fractions in the gradient from top to bottom. The abscissae are divided according to the volumes of the gradient fraction. The ordinates give the percentage of the total activity per ml of gradient fraction. ‘Soluble’ refers to the radioactivity found in the soluble macromolecules, whereas ‘insoluble’ refers to membrane-bound activity. Glucose-6-phosphatase (G-6-ph. E.C.3.1.3.9) indicates the distribution of elements of the endoplasmic reticulum in the gradient. Mitochondria have a similar distribution (not shown). Rn-ase (acid ribonuclease) shows the distribution of lysosomes. Catecholamines pinpoint the localization of chromaffin granules in the gradient. Newly synthesized soluble macromolecules which have become radioactively labelled are present in particles which equilibrate in less dense sucrose, i.e. slightly above mature granules (compare catecholamines). The membranes of these particles are not significantly labelled since there is no peak of radioactively labelled material in the fractions where the new granules equilibrate (see also values for counts per min [c.p.m.] in these fractions).

Biogenesis of the chromaffin granule

659

title denser than microsomes, and after 4 h (see Fig. granules, which do not yet contain their full comple1) they were found in a particle equilibrating at a ment of catecholamines (see below) and which are more fragile. This latter property is not Surprising if density consistently less than that of mature chromaffin granules. The labelled soluble proteins of these we consider that in their early stages granules are particles were identified by electrophoresis as chro- likely to go through a process of fusion and fission mogranins (WINKLERet al., 1972) which led to the with coated vesicles (see below) which may render conclusion that these particles were newly formed them more vulnerable to the shearing forces during chrornathn granules. The relatively low density of homogenization. It seems therefore justified that we these particles is a significant feature which will be consider the enzyme pool in the supematant fraction as being mainly derived from the soluble pool in vesidiscussed in subsequent sections. These results obtained with C3H]leucine are consis- cles. In this case the total soluble pool incorporated tent with a synthesis of the chromogranins in the 23OOc.p.m. (GAGNONet al., 1976) 3 h after the end rough endoplasmic reticulum but they do not reveal of the C3H]leucine pulse, when probably most of the newly synthesized enzyme was already transferred to anything about the transport from this cell compartment to newly formed granules. This gap in our new chromaffin granules. The total membrane-bound pool contained 2OOOc.p.m. Since in rat chromaffin knowledge has been filled by a recent autoradiographic study on mouse adrenal (COUPLAND& KOBAYASHI, granules only about 15-20x of the total enzyme is soluble (CLUANELLO,WOOTEN& AXELROD,1975), we 1976). The Golgi region showed the highest labelling can calculate that the specific radioactivity, and there1.5min after the injection of C3H]leucine, whereas 30 min after the radioactive pulse the granules adja- fore the synthesis rate of the soluble enzyme is about cent to this region were most heavily labelled. Ap- five times greater than that of the membrane-bound parently the newly synthesized secretory proteins pass enzyme (see Fig. 2). These results are therefore in through the Golgi region as has been so well estab- good agreement with the studies on bovine glands lished for other secretory tissues (see PALADE,1975). where the membrane of chromaffin granules did not A significant finding which emerged from the bio- incorporate anything like as much C3H]leucine as did the soluble proteins. The functional significance of chemical studies on bovine.glands was that although this finding will be discussed in the section on the the soluble proteins of newly formed chromaffin origin and final fate of the granule membranes. granules were signiftcantly labelled, the membrane In the experiments with bovine glands (WINKLER proteins had not incorporated comparable amounts et a!., 1971) it was observed that as early as 30min of [3H]leucine. As can be seen in Fig. 1 the labelled membrane proteins in the gradient exhibit a micro- after the injection of [3H]leucine, stimulation of the somal distribution. In the region of the newly formed gland elicited the release of radioactively labelled chromogranins. This seems to indicate that the transgranules there is no peak of radioactively labelled membrane proteins. Furthermore, electrophoresis of port time of the secretory proteins from their site of the labelled proteins in these gradient fractions gave synthesis to the plasma membranes for exocytosis is no indication that the main components of the rather short, comparable to the results in exocrine pancreas (JAMIESON & PALADE,1967b). For this tissue granule membranes, chromomembrin A (dopamine fi-hydroxylase) and chromomembrin B, were signiti- it has been suggested that the discharge of newly synthesized secretory proteins is random, implying an cantly labelled (WINKLER et al., 1972). These results were recently extended by an elegant immediate mixing of old and new granules. On the study with rat adrenals (GAGNON,SCHATZ,Om & other hand, in the parotid gland there appears to be THomm, 1976). Cultured adrenals were pulse a preferential release of ‘old’ secretory products labelled with [3H]leucine. Various times after .the (SHARONI,EIMERL& SCHRAMM,1976), which corre‘pulse’ dopamine /I-hydroxylase was isolated from the lates with the autoradiographic finding that new secparticle-free supematant as well as from the soluble retory granules remain near the Golgi region for a and the membrane-bound pools in the sedimentable long time (CASTLE,JAMIESON& PALADE,1972). For particles. All three pools of the enzyme were signifi- the adrenal medulla the early secretion of some of cantly labelled; but with’the method used (immunothe newly synthesized chromogranins does not necessprecipitation) it was not possible to measure the arily indicate random release of the secretory specific radioactivities of these enzyme forms. How- products since this secretion may only represent a ever, these figures can be calculated from the results, minute fraction of the total new pool. In fact, the albeit only in relative terms, but with reasonable relia- autoradiographic results of CCXJPLAND & KOBAYASHI bility. In order to do so we have first to discuss the (1976)are consistent with such a non-random release, relationship between the enzyme found in the since most of the new granules remain near the Golgi particle-free supematant and the soluble enzyme region for several hours, as indicated by the time present in the cell particles. As concluded by GAGNON course of the labelling over this region. From an et al. (1976) and previously suggested by VIVEROS, economical point this would be very sensible since ARQUEROS,CONNET~ & KIRSHNER (1968) it seems the new granules have still to acquire their catechollikely that the enzyme in the particle-free supematant amine content (see below), therefore random disis mainly derived from newly formed chromaffin charge would appear a wasteful procedure.

H. WINKLER

660 Soluble 5

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FIG. 2. Synthesis rate of dopamine /?-hydroxylase in cultured rat adrenal. This figure is based on results given in the paper by GAGNON et al. (1976). In this study cultured rat adrenals were ‘pulse’ labelled with C3H]leucine. Various times after the pulse the soluble dopamine /I-hydroxylase, which is present in the particle free supernatant and in the cell particles, and the membrane-bound enzyme were precipitated by antibodies and purified by polyacrylamide-gel electrophoresis. The amount of radioactivity which was incorporated into these pools of enzyme was then determined. This method does not yield specific radioactivities (per mg protein of enzyme); however, relative values for soluble and membrane-bound enzyme can be obtained, since we know that about 80% of the enzyme in chromaffin granules is membrane-bound (CIARANELLO et al., 1975). The data in Fig. 1 of the paper by GAGNON et al. (1976) were therefore recalculated using this value. For this purpose the counts found in the soluble enzyme present in cytosol and in cell particles were combined (see text) and divided by 20, whereas the membrane-bound counts were divided by 80. The above figure gives these calculated specific radioactivities (synthesis rates) of the two enzyme pools in arbitrary but comparable units.

[3Hlfucose. This sugar was chosen as a precursor for the glycoproteins since it offered the chance to obtain data on the biosynthesis of soluble dopamine fi-hydroxylase, which contains 1.32 pmol fucosef 1OOmg protein (WALLACE, KRANTZ & LOVENBERG, 1973; LJONES, SKOTLAND & FLATMARK, 1976; GEISSLER, MARTINEK, MARGOLIS, MARCOLIS, SKRIVANEK, LEDEEN, KOENIG& WINKLER, 1977), whereas the acidic chromogranins contain only 0.2 pmol/ 1OOmg protein (GEISSLER et al., 1977). Furthermore, the membranes of chrornallin granules, with their main protein component dopamine B-hydroxylase, are also rich in fucose (2.1 pmol/lOO mg protein; GEISSLERet al., 1977). In contrast to C3H]leucine which is incorporated into secretory proteins already in the rough endoplasmic reticulum, [3H]fucose is added at a later stage, i.e. in the Golgi region (see SCHACHTER,1974). The results (GEISSLERet al., 1977) with [3H]fucose were in line with those obtained with C3H]leucine (see Fig. 1). Newly formed chromaffin granules which contain C3H]fucose labelled proteins were less dense than mature granules. About 80% of the labelled soluble proteins were adsorbed by Concanavalin A, which indicates that most of the [3H]fucose had been incorporated into dopamine /?-hydroxylase, since the en&ne binds specifically to this lectin (see RUSH, THOMAS, KINDLER & UDENFRIEND, 1974; AUNIS, MIRAS-PORTUGAL & MANDEL, 1974; WALLACE & LOVENBERG, 1974). The membranes of these newly assembled granules were not significantly labelled. These results are therefore in agreement with those of GAGNON et al. (1976) as discussed above and indicate a different synthesis rate of soluble and mem-

brane-bound dopamine /I-hydroxylase (see section on origin and final fate of membranes for further discussion). [35S]sulphate. Chromaffin granules contain mucopolysaccharides, which can be labelled with [35S]su1phate (FILLION, Noti~ & Uvr&& 1971; DA PRADA, VON BERLEPSCH & PLETSCHER, 1972; MARGOLIS, JAANUS & MARGOLIS, 1973; MARGOLIS & MARGOLIS, 1973). [35S]sulphate like [3H]fucose is incorporated into secretory proteins of other tissues in the Golgi region (see Yomc, 1973 ; BERG & AUSTIN, 1976). When bovine adrenal glands were labelled with [3sS]sulphate, newly formed chromaffin granules which had incorporated this isotope equilibrated in their typical position in the gradient (see Fig. 1; BAUMGARTNER, GIBB, H~~RTNAGL, SNIDER & WINKLER, 1974). Again the membranes of these particles were not significantly labelled. This was not due to the absence of mucopolysaccharides from the granule membranes since they contain nearly as much mucopolysaccharide (2.9 pmol hexosamine/lOO mg protein) as the soluble proteins (3.6pmol hexosamine/100 mg protein: GEISSLERet al., 1977). By using this isotope we were able to study the biosynthesis of chromaffin granules in the living rat over longer time periods than were possible in the perfusion studies on bovine glands (BAUMGARTNERet al., 1974). Significantly, under in oiuo conditions the newly formed chromaffin granules are also less dense than mature ones and the ‘maturation’ process leading to a density identical with that of old granules takes from 24 to 48 h. [32P]phosphate. This isotope was used in order to study the synthesis rate of the phospholipids which

661

Biogenesis of the chromaffingranule are mainly or even exclusively localized in the granule membranes (see WINKLER,1976). For bovine adrenal medulla no evidence could be obtained (WINKLERet al., 1972) that the lipids of chromal%n granules incorporated significant amounts of this isotope, when compared with other subcellular organelles. The labelled phospholipids (see Fig. 1) gave a microsomal distribution in the gradient. Most of the label was present in phosphatidylinositol and this was true for all fractions of the gradient. In contrast, TRIFAR~ & DWORKIND(1975) reported for similar experiments on bovine glands--which, however, were stimulated by acetylcholine-that the lipids of chromafhn granules became significantly labelled, since a high peak of lipid-bound radioactivity was found in the relevant gradient fractions. Most of the label was again confined to phosphatidylinositol. TRIFAR~ & DWORKIND (1975) suggested that the discrepancy between these two studies may have been caused by differences in the lipid-extraction procedure (chloroform/methanol: WINKLERet al., 1972; acidified chloroform/methanol: TRIFAR~ & DWORKIND, 1975). However, this explanation seems unlikely since the gradient distribution of total membrane-bound [32P]phosphate was found to be similar (WINKLER& AEZRER,unpublished observations) to that obtained with the lipid-extracts of the gradient fractions as shown in Fig. 1. The only other explanation which might be offered is that stimulation of the gland induces a much’ higher tumover of phosphatidylinositol in chromalhn granules than it does in the other cell fractions. Studies in another secretory tissue, i.e. in the exocrine pancreas do not support a high turnover of the lipids in secretory organelles. GERBER,DAVIES 8z HOKM (1973) concluded that newly synthesized phosphatidylinositol whose synthesis from C3H]myoinositol was stimulated by secretagogues was mainly confined to microsomal membranes and that it was not transferred to zymogen granules. In similar experiments with and without stimulation DE CAMILLI& MELDOLES(1974) also found relatively little labelling of the zymogen granules; however, they considered the possibility that some labelled phosphatidylinositol was taken up by zymogen granules by a phospholipid exchange reaction.

For the adrenal medulla it seems advisable to perform further experiments also with different phospholipid precursors in order to obtain a fhral answer on the turnover of phosphatidylinositol which is a minor component (about So?, WINKLER,1976) of the granule phospholipids. On the other hand, it is already obvious that one of the specific major phospholipids of the granule membranes, i.e. lysolecithin, has a low turnover when adrenal glands are labelled with [3zP]phosphate. Under these conditions neither the lysolecithin in total adrenal medulla (TRIFAR~,1969) nor the lysolecithm in subsequently isolated granules (WINKLERet al., 1972) became significantly labelled. Thus it seems likely that the turnover of the major lipids of granule membranes is relatively low, which would be in agreement with the results on the glycoproteins and mucopolysaccharides of these membranes as discussed above. Characterization of newly formed chromafin granules after catecholam’ne depletion

In an unstimulated adrenal gland newly formed chromaffin granules represent a minority of the total granules making their isolation and subsequent biochemical analysis difficult, if not impossible. Kirshner and collaborators overcame this problem by first depleting the adrenal of mature granules, which enabled them to characterize the subsequently produced population of new chromalhn granules. In an early paper (VIVEROS, ARQUEROS & KIRSCHNER, 1971b; see also KIRSHNER & VIVEROS,1972) they studied the recovery of granule stores in rabbit glands after insulin stimulation. Twenty-four hours after insulin, which depleted more than 90% of the catecholamines, newly formed chromal?in granules, identified by the marker enzyme dopamine /I-hydroxylase, first appeared in the less denser fractions after sucrose density gradient centrifugation (see Table 1, fraction B at 24 and 48 h). It took about another 24 h before these new vesicles became again identical with mature granules equilibrating in the dense fractions of the gradient (see Table 1, fraction A). In addition VI~EROSet al. (1971b) observed that these new vesicles had a low content of.catecholamines (see Table 1). This finding offered an explanation for their reduced density, since it was

TABLET. DSTRIBUTIONMSUCROSE DENSI~GRADIENTSOFCATECHOWMINESAND DOPAr+u~~~-HYDROXYLASE FROM RABBIT.~DRENALS AFTER ~N~LJLIN ADMINIS~~ON A DBH 0 3h 24 h 48 h 96-144 h

412 43 40 244 434

A Catecholmnines 32 5.4 3.7 7.6 29.0

B DBH 441 73 128 501 462

B Catecholatnines 25.9 4.5 6.7 11.0 19.6

These results are taken from a paper by VI~EROSet al. (1971b). Large granule fractions of rabbit adrenals were subjected to density gradient centrifugation. The dense fractions of the gradient containing the chromafhn granules were divided into two parts, where A represents the densest fractions. The content of these fractions in dopamine b-hydroxylase (DBH) and catecholamines is presented above for various times after treatment of the rabbits with insulin.

662

H. WINKLER

FIG. 3. Ultrastru~ture of adrenal medulla. These electron barographs were kindly provided by I. Benedeczky and A. D. Smith (see BENEDECZKY & SMITH,1972). (a) Golgi region of a chromaffin cell from hamster adrenal. Rough endoplasmic reticulum is seen on one side of the Golgi stacks with a transitional zone interposed. Arrows indicate the prosecretory granules. Several coated vesicles can be seen. One such vesicle (double arrow) is in the process of fusion with a Golgi cisterna. In the lateral part of this cisterna electron-dense material is accumulating. (b) Golgi region of a chromaffin cell from hamster adrenal. In the lateral part of a Golgi cisterna electron dense material is present (arrowhead). Two prosecretory granules (arrows) and several coated vesicles are seen adjacent to it. (c) Golgi region of a chromaffin cell from hamster adrenal. The arrow indicates a prosecretory granule which is in the process of fusion with two coated vesicles. A coated vesicle (double arrow) with a more electron dense content can also be seen. (d) Golgi cisterna with a coated region (arrow). (e) Exocytosis in hamster adrenal medulla. Numerous. coated pits are seen along the membrane of a chromaffin granule which is in the process of exocytosis.

Biogenesis of the chromaffin granule also shown that mature vesicles which were depleted of catecholamines by the action of reserpine had a similar low density (VIV-EROS, ARQUEROS & KIRSHNER, 1971a). A low catecholamine content of new vesicles is in agreement with a study using both morphometrical and bidchemical methods (BR~CKE, GRAF & PICHLER,1971). After reserpine-induced depletion the number of chromaffin granules in rabbit adrenals recovered several days before the catecholamine content returned to normal. Furthermore, SERCK-HANSSEN & HELLE(1972) reported for perfused bovine glands that after prolonged stimulation the remaining population of granules, which is probably enriched in newly formed ones, was poor in catecholamines and nucleotides. All these results were confirmed and extended in thorough experiments with r’ats after insulin stimulation (SLOTKIN& KIRSHNER,1973a,b). The properties of new vesicles were studied various times after insulin administration. New vesicles were not only poor in catecholamines but also in ATP. However,. the nucleotide stores recovered before the concentration of amines became normal (see section on nucleotides). Furthermore, new vesicles could already take up metaraminol before they could accumulate adrenaline, which was attributed to the fact that metaraminol uptake is relatively independent of the presence of ATP stores (see SLOTKIN& KIRSHNER,1971). The studies just discussed are in good agreement with the results obtained with radioactively labelled precursors. Based on both approaches we can conclude (see Fig. 7): (i) newly formed chromaflin granules are assembled in or near the Golgi region from their macromolecular constituents; however, they do not yet contain significant amounts of small molecules, at least not catecholamines and ATP (for calcium see below). They are therefore less dense than mature chromaffin granules when characterized by sucrose density centrifugation; (ii) new vesicles require more than 24 h to become as dense as old vesicles, implying a rather slow accumulation of catecholamines and ATP. These studies have not yet told us any details of the cellular mechanism involved in granule formation and we have to turn to morphological studies to obtain an answer. Morphological studies

In electron micrographs of glutaraldehyde-fixed tissue chromal%n granules appear as membrane-limited vesicles with a dense core. In the case of adrenaline granules this dense core represents the secretory’proteins whereas in noradrenaline granules a highly dense reaction product derived from the amine is superimposed on the proteinaceous content (see C~JPLAND & HOPWO~D, 1966). Thus morphological studies can tell us something about the origin of the membranes and the secretory proteins and in addition (see section on catecholamines) provide information on the timing of the entry of noradrenaline into newly formed chromatlin granules.

665

When chromaffin cells are severely depleted of their chromaffin granules, e.g. following the administration of reserpine or insulin to an animal, then in the recovery phase the rough endoplasmic reticulum appears swollen and contains electrondense material (CLEMENTI8~ Z~CCHE, 1963; ABRAHAMS& HOLTZMAN, 1973, see also P~HORECKY& RUST, 1968). These signs of increased activity are consistent with the synthesis of secretory proteins in the rough endoplasmic reticulum. It is agreed by most morphologists that the packaging of the secretory proteins occurs in the Golgi region (DE ROBERTIS& SABATINI,1960; BARGMANN & LINDNER,1964; COUPLAND, 1965; MOPPERT,1966; ELFVIN, 1967; AL-LAMI, 1969; HOLTZMAN & DOMINITZ,1968; COUPLAND & WEAKLEY,1970; BENEDECZKY & SMITH, 1972; HOLTZMAN, TEICHBERG, ABRAHAMS, CITKOWITZ,GRAIN, KAWAI & PETERSON, 1973). However, it is rather difficult to give a detailed account of the morphological sequence of events in this region, and only by taking observations from several papers and by adding admittedly speculative points can a somewhat coherent proposal be made. In the chromaffin cell ‘the Golgi complex consists of a collection of smooth-surfaced membranes of tubular or vesicular profile which may be loosely arranged or aggregated’ (COUPLAND,1965; see also references cited above). Numerous coated vesicles (see Fig. 3) are present in the Golgi region, some of them translucent (BARGMANN & LINDNER,1964; HOLTZMAN & D~MINITZ, 1968) but some apparently containing electron-dense material (BENEDECZKY & SMIITH,1972). There is also some indication of a transitional zone (see Fig. 3) between the rough endoplasmic reticulum and the Golgi region (Fig. 14: BARGMANN & LINDNER, 1964; HOLTZMAN& DOMINITZ,1968; Figs. 4, 5: ALLAMI,1969; Fig. 10: BENEDECZKY & SMITH,1972; Fig. 27: HOLTZMANet al., 1973), where vesicles and membranous sacs sometimes studded on one side with ribosomes are interposed between these two cell compartments. What is the functional integration of these structures in the process of granule formation? Several reports have stated (BARGMANN& LINDNER, 1964; &UPLAND, 1965; ELFVIN,1967; POHORECKY & RUST, 1968; HOLTZMAN& DOMINITZ, 1968; AEZRAHAMS & HOLTZMAN,1973; Fig. 13: BENEDECZKY& SMITH, 1972) that relatively dense material is seen to accumulate in terminal parts of Golgi cistemae (see Fig. 3). Similarly dense material is observed in vesicles adjacent to the Golgi region which was taken as an indication that these vesicles were formed by a buddingoff process, which of course is analogous to the events occurring in other secretory tissues (see BEAM & I@~EL, 1968; FARQUHAR,1971; PALADE,1975). These newly formed vesicles with their special morphological features have been termed ‘prosecretory granules’ (BENEDECZKY & SMITH,1972). Their content (see Fig. 3) when compared with mature granules appears uneven, more flocculent (Fig. 5: AL-LAMI, 1969 ; Fig. 8 :

666

H. WINKLER

HOLTZMAN& DOMINITZ,1968; BENEDECZKY & SMITH, 1972) and there is no indication that these granules contain significant amounts of noradrenaline since they do not exhibit the highly dense core typical for the presence of this amine (COUPLAND,1965; BFNEDECZKY& SMUX, 1972; Fig. 2: MA~CORRO& YATJZS, 1973; see section on catecholamines for discussion). The membranes of these prosecretory granules are often of irregular shape (see BENEDECZKY & SMI~, 1972; AL-LAMI, 1969; MASCORRO & YATES,1973 ; Fig. 8: HOLTZMAN& DOM~NZTZ, 196Q which may be due to fusion with coated vesicles (see below). As far as their size is concerned it has been stated that they are smaller than the majority of mature granules. Some papers quoted in support for this were based on studies after stimulation of the adrenal, e.g. by reserpine. However, in this case the chromaffin cell apparently switches to the production of smaller granules (see below). In most papers the prosecretory granules near the Golgi region, when clearly separated from the cisternae, appear roughly of the same size as mature granules (see e.g. Fig. 9: HOLTZMAN & DOMINITZ, 1968; Fig. 11: BENEI)ECZKY & SMITH, 1972; Figs. 4, 5: AL-LAMI, 1969; Fig. 2: MA~C~RRO & YATES,1973). What is the further fate of these prosecretory granules? In the adenohypophysis it has been observed (FARQUHAR,1971) that several prosecretory granules fuse to form larger granules. No evidence for this can be seen in the published electron micrographs of the adrenal medulla, which may be related to the fact that chromaffin granules are much smaller (mean diameter: 25OOw; COUPLAND,1968) than the granules of the mammotroph cells (600&9OOOA; FARQUHAR,1971), in which the aggregation process is best documented. On the other hand, in adrenal medulla (see Fig. 3) fusion of small coated vesicles with prosecretory granules has been observed, as pointed out by BENEDECZKY & SMMITH (1972), but also seen in Fig. 5 of the paper by AL-LAMI (1969). This might indicate that prosecretory granules becomi enlarged by vesicles containing additional secretory proteins. However, it seems unlikely that the formation of a large granule containing a high concentration of exportable proteins can occur by this process. Incorporation of small vesicles wiih their high ratio of membrane (surface) to content (volume) would add excess of membrane to the new granules and would never enable these granules to increase the concentration of their content by a condensation process. Another more likely explanation for this fusion of coated vesicles is (compare discussion by BENEDECZKY & SMITH, 1972) that they represent a shuttle service frbm the endoplasmic reticulum or from the Golgi region to the new granules, a suggestion which is, of course, based on the work by JAMIESON& PALADE(1967a,b, see also PALADE, 1975) on the exocrine pancreas. Vesicles containing newly synthesized chromogranins would carry these proteins to prosecretory granules, fuse with them discharge their

content and return to the cytoplasm. The question which can not be answered at present is, whether these vesicles can transport secretory material directly from the endoplasmic reticulum to the prosecretory granules by bypassing the Golgi cisternae. If this is the case then we have to postulate that these vesicles can carry out biochemical reactions typical for the Golgi region, i.e. introduction of certain sugars like fucose and sulphation of mucopolysaccharides. On the other hand, there is no evidence to exclude the possibility that the Golgi region is interposed between two shuttle services of vesicles. One ~pulation of vesicles would then be involved in the transport from the endoplasmic reticulum to the Golgi region. A second population would act between this compartment and the prosecretory granules. The coating, which has been observed (HOLTBMAN & DO~EU’ITZ; 1968; BENEDECZKY & SMITH,1972) in the lateral portion of Golgi cistemae (see Fig. 3), indicates that at least one shuttle service exists, but it would also be consistent with a second one to the new granules. In other secretory tissues two main pathways seem to exist for the production of new secretory organelies. In the exocrine pancreas of the guinea-pig a shuttle service of vesicles is considered to carry secretory proteins from the rough endoplasmic reticulum directly to condensing vacuoles bypassing the Golgi cisternae (JAMIESON & PALADE,1967a,b). On the other hand in the stimulated guinea-pig pancreas (JAMIE~ON & PALADE,1971b) and in many endocrine tissues secretory material is seen to accumulate first in the lateral part of the Golgi cisternae (see BEAMS& KESSEL, 1968; FARQUHAR,1971). In the scheme for the adrenal medulla depicted in Fig. 7 both processes seen in other secretory tissues have been provisionally included. However, as just discussed above, we do not yet know whether coated vesicles can actually bypass the Golgi cistemae. Figure 7 presents a simplified and partly speculative proposal, and several points have been omitted since their significance is at present difficult to eyaluate. Thus, the possible for~tion ;of chro~ffin granules directly from elements of the endoplasmic reticulum (see HOLTZMANet al., 1972; see HOLTZMAN, 1977) was not discussed. Furthermore, we have included nothing on the formation of lysosomes in the Golgi region. At least some Golgi sacs (GERL region? see HOLTZMAN& DOMINITZ, 1968; see review by NOMKOFF,1976) and small vesicles, but in rat adrenal also newly formed chromaffin granules, give a histochemical reaction for acid phosphatase (HOLTZMAN 8~ DOMINITZ, 1968). Golgi cisternae (on opposite ends?) may therefore be involved both in the formation of secretory granules and primary lysosomes (see SMITH& FARQUHAR,1966, for adenoh~ophysis). The set of proposals outlined in Fig. 7 is of course intimately connected with the question of the origin and the specificity of the granule membrane which will be discussed in the next section and should add some further weight to this scheme.

Biogenesis of the chromagm granule Origin and final fate of membranes of chromqfin granules In discussing this rather complicated subject I will

667

rough endoplasmic reticulum to the Golgi region were carried out not by vesicles but within continuous channels (for liver see CLAUDE, 1970) where secretory concentrate on two proposals: (i) According to the proteins could ‘overtake’ the membranes. Therefore, membrane-flow hypothesis (see e.g. BENNE-IT,1956; in the pulse labelling experiments which were deCLAUDE, 1970; Mona&, MOLLENHAUER & BRACKW, scribed in the section on [3H]leucine (see WINKLFZR 1971; FRANKE,MORR&,DEUMLING,CHEIXHAM,KAR- et al., 1972) one might miss the appearance of the TENBECK, JARA~CH& ZENTGRAF,1971) secretory pro- labelled membranes in the chromafBn granules when they are isolated 4 h after the radioactive pulse. Simiteins and their surrounding membranes pass through larly one might suggest that there is a dissociation the cell via the same undirectional route, perhaps concomitantly, from the rough endoplasmic reticulum to (see WALLACHet al., 1975) of the synthesis of the membrane and the secretory proteins in the rough the plasma membranes, where the secretory proteins endoplasmic reticulum. Membrane synthesis might are discharged and the membranes of the secretory vesicles become incorporated into the plasma mem- occur only in cycles at certain periods whereas secrebrane. (ii) Each cell compartment participating in the tory proteins are synthesized continuously. All these possibilities seem unlikely if we consider the experitransport of secretory proteins has its own membrane, therefore only the exportable proteins, but not the ments with [3sS]sulphate and [3H]fucose discussed membranes are transferred from one ~orn~~rn~t to in previous sections. These precursors of gly~proteins the other. Membranes of chromaffin granules only and mucopolysaccharides are incorporated in the temporarily fuse with the plasma membrane, but then Golgi region and there is no evidence to suggest that this is only true for exportable proteins and not for they are retrieved and possibly reused. (compare AUTUORI, S~ENSSON & Is there a concomitant synthesis of the membranes membranes of chromafin granules and of their exportable proteins? DALLNER,1975). Thus at least with these precursors, The membrane flow hypothesis requires that the syn- which will be incorporated just before the new vesicle thesis rate of the secretory proteins and of the mem- buds off from the Golgi region, one would expect branes of chromal% granules is identical since the a parallel labelling of secretory and membrane proformer are discharged by exocytosis whereas the latter teins of chromaflin granules if a similar synthesis rate are continuou~y ‘lost’ by ~co~ration into the exists at all. However, as discussed above only the secretory proteins became signi&antly labelled. plasma membrane. The simplest mechanism consistent with this model is a concomitant synthesis and Further convincing evidence against any form of contransport of the secretory proteins and their sur- comitant synthesis comes from experiments in which rounding membranes. Evidence for such a mechanism the synthesis rate of membrane-bound dopamine seemed to be forthcoming for the parotid gland fi-hydroxylase in rat adrenals was found to be much (AM~TIZRDAM, SCHIZAMM, GHAD, SALOMON & !&LINGER, lower than that of the soluble enzyme (see GAGNON 1971) where after a pulse of [i4C]amino acids the etab,1976; see Fig. 2). The available data are theremembranes and the soluble proteins of the secretory fore not consistent with the membrane flow concept vesicles appeared to be similarly labelled However, requiring similar synthesis rates of membranes and in the adrenal medulla, as discussed in the section exportable proteins of chro~ffin granules. We will on [3H]leucine, a different result, i.e. no ~~ific~t therefore turn to the second model and discuss first labelling of the granule membranes was observed one of its crucial points, namely the question of (W~KLER etal., 1972). Similarly, in exocrine pancreas specific membrane retrieval, which clearly disthe membranes of zymogen granules had a much tinguishes this concept from that of unidirectional lower synthesis rate than that of the proteins in the membrane flow. content (MELDOLESI,1974). Subsequently, the results Are membranes of chroma#in granules retrieved after for the parotid gland were accounted for by the findexocytosis? Secretionfrom the adrenal medulla occurs ing of a highly labelled, proline-rich protein which by exocytosis (see SMI~, 1968; DOUGLAS, 1968; adhered to the membranes giving the false impression KIRsHNER& vnmaos, 1972; SMXTH & WINKLER,1972; of a high synthesis rate of these structures (WALLACH, DINER, 1967, Sm, S~-IH, WINKLER& RYAN, 1973). Therefore some m~hani~ of membrane retrieval KIR~HN~ & ~~, 1975). GHAD & These results provide strong arguments against a must exist (see PALADE,1959; AM.QERDAM, %HRAMM,1969), since otherwise the continuous addimodel requiring concomitant synthesis and transport; tion of membranes of secretion organelles to the however, one might still try to uphold it in modified versions, assuming either dissociation of synthesis or plasma membrane would lead to an enlargement of of transport. Thus, the possibility was considered (see the cell. The first morphological evidence of such a process in the adrenal medulla was provided by WALLUX et al., 1975) that membranes and secretory 1971). proteins are synthesized concomitantly but that the DINER (1967; see also GRYNSZPAN-WINOGRAD, Coated vesicles indicative of membrane retrieval were membranes become ‘diluted’ within the Golgi region and therefore no longer reach the storage vesicles at seen along the exocytotic profiles at the plasma membrane in hamster adrenals. This was confirmed and the same time as the secretory proteins. A similar dissociation would occur if the transport from the extended by the use of exogenous tracers like horse-

66X

H. WINKLER

radish peroxidase and thorium dioxide. Their uptake into chromaffin cells could be traced from exocytotic figures into coated and smooth vesicles in the cytoplasm (ABRAHAMS& HOLTZMAN, 1973; NAGASAWA & DOUGLAS, 1972). In a quantitative morphological study (KOERKER, HAHN & SCHNEIDER,1974) the chromaffin cell after in uiuo stimulation by insulin was found to contain numerous translucent microvesicles. There was a good correlation in time between the disappearance of chromaffin granules and the appearance of small vesicles. In all these studies the coated vesicles representing the retrieved membranes appeared considerably smaller (1000 A: BEZNEDECZKY & SMITH, 1972) than chromaffin granules (25OOA: COUPLAND, 1968). This seems to be, at least partly, at variance with subcellular fractionation studies in stimulated cat adrenal (MALAMED, POISNER, TRIFAR~ & DOUGLAS, 1967). After severe catecholamine depletion many electrontranslucent vesicles overlapping in size (30&37OOA) with chromaffin granules were present in the isolated fractions instead of the usual dense-core granules. However, in this case the larger vesicles may have been formed artifactually during homogenization from granule membranes still fused with the plasma membrane since in an acute experiment with short stimulation (see Fig. 4: MALAMED et al., 1968) the larger vesicles appeared to be more frequent than in an experiment with prolonged stimulation (Fig. 3: MALAMED et al., 1968). In any case, there is convincing evidence that membrane retrieval does occur in adrenal medulla during exocytosis. It is less certain to what extent the specific membranes of chromaffin granules are regained; either all of the membranes could be retrieved or some of them may become permanently incorporated in the plasma membrane. Coating apparently occurs on most exocytotic profiles since in one plane of section it was already seen in half of the exocytotic figures (NAGASAWA& DOUGLAS, 1972) and in some profiles two or more coated vesicles seemed to bud off (see Fig. 3), which all indicates rather efficient retrieval (NAGASAWA & DOUGLAS, 1972; BENEDECZKY& SMITH, 1972). These results do not, however, prove complete removal. From a functional point of view one might of course argue that incomplete removal would ‘dilute’ the plasma membranes with granule membranes and subsequently change their functional properties. The chromaffin cell would, for example, start to pump catecholamines from the cytoplasm to the extracellular space, if granule membranes, which possess such a pump, are permanently incorporated. Thus complete retrieval, at least of the proteins, seems the only sensibie mechanism. In fact, some evidence consistent with this view is available. Immunohistochemistry at the ultrastructural level (see WINKLER, SCHNEIDER,RUFENER, NAKANE & H~~RTNAGL,1974) indicated that chromomembrin B, one of the major proteins of the granule membranes, was practically absent from the plasma membrane. Furthermore, a recent biochemical study (WIL-

SON & KIRSHNER, 1976) revealed profound differences in the protein composition of these two membrane pools. For the parotid gland a different technique also provided support for complete retrieval (DE CAMILLI,PELUCHEITI & MELDOLESI, 1976). Freezefracturing revealed differences in particle density between plasma membranes and membranes of secretory granules, which made it possible to investigate the fate of the membranes during and after exocytosis. Specific and complete removal of the membranes of secretory granules from the plasma membranes seemed to occur. What is the fate of the granule membranes after retrieoalr Two possibilities seem to be supported by experimental data: (i) The membranes end up in lysosomal structures for digestion. (ii) Membranes are reused for packaging new secretory material. The first proposal is consistent with several studies. VIVEROS et al. (1971 b; see also KIRSHNER & VIVEROS, 1972) isolated subcellular fractions after severe depletion of catecholamines by insulin in order to investigate the fate of the granule membranes. In the recovery phase a considerable proportion of the membranes of empty chromaffin granules, as measured by the marker dopamine fi-hydroxylase, disappeared (or was degraded) before an equivalent amount of new granules had been formed. In a study with an exogenous tracer, i.e. thorium dioxide (NAGASAWA & DOUGLAS, 1972) vesicles which had taken up this compound were finally observed in lysosomal structures. Experiments with horseradish peroxidase (ABRAHAMS& HOLTZMAN, 1973) yielded similar findings for chromaffin cells which were stimulated following administration of insulin. In addition no evidence was obtained that any of the exogenous tracer that had been taken up into the cell was transported back to the Golgi cisternae or finally ended up in new vesicles. In other secretory tissues results obtained with such tracers appear controversial. In one study on the adenohypophysis the exogenous tracer taken up by the cell did not appear in the Golgi sacs (TIXIER-VIDAL, PICART & MOREAU, 1976). On the other hand in similar experiments PELLETIER (1973) discovered the presence of the exogenous tracer (horseradish peroxidase) in Golgi cisternae and even in immature secretion granules, which is consistent with a re-use of secretory granule membranes. Similar findings were reported for seminal vesicle cells (MATA & DAVID-FERREIRA, 1973). In the endocrine pancreas vesicles in the Golgi area also became positive when horseradish peroxidase was applied extracellularly (ORCI, MALAISSE-LAGAE, RAVAZZOLA, AMHERDT & RENOLD, 1973): However, at least some of these vesicles may be related to lysosomes as suggested by a similar study on the parotid gland in which ferritin was used as a tracer and acid phosphatase reaction was employed to characterize lysosomal involvement (KALINA & ROBINOVITCH,1975). For the adrenal medulla we can conclude that especially after intensive stimulation a considerable

Biogenesis of the chro~n

granule

669

On the other hand in the hedgehog adrenal the size proportion of the membranes of empty chromalhn of these vesicles was found to vary from 500 to 1000 A granules is degraded by lysosomes. This, however, & LINDNER,1964). In any case a more does not exclude re-use since, during the type of (BARGMANN stimulation used in most of the studies just discussed, direct analysis of these vesicles is now possible since an isolation method for two populations of vesicles excessive amounts of empty membranes become available, only part of which can be m-used. As will differing in size has been reported for the adrenal be pointed out in the next section, the strongly stimumedulla (PEARSI?,1976). Surprisingly, both these vesicle populations contained only one major protein lated chromaffin cell will start to produce smaller chromaffin granules in order to use up more ‘wrap- which was named clathrin. There was no evidence ping material’. However, any surplus membrane for additional major proteins in these vesicles, which one would expect from a membmne-lilted particle. material is likely to become degraded. What is then the evidence that at least some of the membranes are The explanation may be that the pu~fication proreused? As previously pointed out (WINKLER, 19’71; cedure preferentially yielded the coating of these vesiWINKLER et al., 1972; SMITH & WINKLER, 1972; cles (see PEAME, 1976). It is to be hoped that future WINKCERet al,, 1974) studies on the synthesis rate studies based on this method will make it possible of the membranes and of the secretory proteins can to isolate the whole vesicles, i.e. coating plus memprovide an answer. If membranes are re-used their brane, which should then be compared with other synthesis rate shauld be lower than that of the secre- membrane systems of the chromafhn cell. According tory proteins, which are continuously lost by export. to the scheme given in Fig. 7 one has to postulate As already discussed in previous sections, convincing that coated vesicles representing retrieved membranes evidence is available that the membranes and their should contain the membrane proteins typical of main protein dopamine /I-hydroxylase have a lower chromaffin granules, whereas coated vesicles involved synthesis rate than the secretory proteins. The only in a shuttle service from the endoplasmic reticulum feasible expl~ation for this f%ing seems to be that to the Golgi region should have a different composimembranes or their constituents are re-used (for exo- tion. crine pancreas, see JAMIESON& PALADE, 1971~; MELObviously, the detailed events occurring in the DOLEX, 1974). In fact, from the data on the synthesis Golgi region have to remain a matter of speculation. rate of dopamine &hydroxylase, one might even sug- However, the available evidence at least favours the gest that the .membranes are reused for about five basic concept that the membranes of chromaffin secretory cycles. This would imply that in cells which granules provide a shuttle service from the Golgi are not excessively stimulated about 20% of the region to the plasma membrane and back and simiretrieved membranes are directed towards digestion larly that such a shuttle service (see JAMIE~~N& by lysosomes. PALADE,1971a) also exists between the rough endoHow are membranes of chromafin granules re-used? plasmic reticulum and the Golgi region. This model, when compared with that of the uniRetrieved membranes of chromafhn granules might either be reused as intact pieces or first disintegrate dir~tional membr~e flow already discussed, offers into proteins and lipids which are then used again several functional advantages. In the rough endoplasfor assembling a new membrane. The morphological mic reticulum membrane-attached ribosomes synthestudies discussed above favour the first possibility, size the secretory proteins. Therefore many attachsince they have shown that in secretory tissues ment sites with relatively little inner membrane space retrieved coated vesicles reach the Golgi region and are required. The chromafhn granules, on the other it seems therefore quite likely that intact pieces of hand, are specialized for storage, therefore relatively membranes are fed into this system for reuse, or even little membrane and more content is needed which directly into newly formed chromahin granules. Thus, is provided by a larger vesicle. Concomitant memcoated vesicles in the Golgi region again represent brane flow could not fulfill these functional requirea crucial element in the formation of granules. We ments, since the change from small to large vesicles have already pointed out in the section on morphowould lead to a surplus of membranes. Secondly, logy that some coated vesicles may be involved in shuttle services enable the cell to main~in the specithe transport of secretory material from the endoplasficity of its membranes without a constant need for mic reticulum and the Golgi region to the new transformation as required during flow of membranes granules. A further population of these vesicles may from one cell compartment to the other. In fact, the now be involved in membrane reuse., FRIEND& FAR- membranes of the endoplasmic reticulum (H~RTNAGL, QUHAR (1967) have pointed out for the vas deferens 1976), of the Golgi region (TRIFAR~& DUERR, 1976; that coated vesicles derived from the plasma mem- TRIFA@ Dunna & PINTD, 1976) and the membranes brane are usually larger (> loo0 A) than those ori- of chrome granules are profoundly different (see ginating from the Golgi region (> 750 A). In the ham- Table 2), which is consistent with the model given ster, adrenal medulla coated vesicles near the Golgi in Fig. 7. For example, dopamine /?-hydroxylase, region have a mean diameter of 150 A (BENEDIXXKY which is the major protein of the granule membranes, & SMIITH,1972), which would indicate that the majoris practically absent from the other membranes, inity is not derived from retrieved granule membranes. eluding the plasma membrane (see Table 2 and WIL-

H.

670

WINKLER

TABLE 2. COMWSITION OF MEMBRANES FROMENWPLASMICRETICULUM,GOLGI REGIONAND CHROMAFFIE GRANULESIN BOVINE ADRENAL MEDULLA Membranes ol:

Endophsmlc reticulum Golgi Chromafin granules

DBH - (3) 4.91-l 60(100)

Chrom.B 2 lOa

Transph. G-Cph. 2.2 345

61 7 0

GaL 11. 28 145 0.6

Y-llucl. Chol/Phl. _ 2080 0.6

0.29 0.42 0.58

LL 1.5 3.1 13.4-16.8

The values given in this table are taken from three papers (TRIFAR~& DUERR,1976; TRIFAR~et QL,1976; NBRTNAGL, 1976). They are expressed either in enzyme units or in relative terms (DBH = dopamine ~-hydroxylase: values in parentheses from H~~RTNAGL,1976; Chr.B = Chromomembrin B; Transph. = transphosphorylation from ATP; G-6ph. = glucose 6-phosphatase; Gal. tr. = galactosyltransferase; 5’-nucl. = S-nucleotidase). Chol./Phl. refers to the molar ratio of cholesterol to phospholipids. LL: % lysolecithin of total lipid-phosphorus. -, not measured.

by a morphometric study on rat adrenals treated for 7 days with reserpine (GAGNON, PFALLER, FISCHER, SCHWAB, WINKLES & TH~JZNEN, 1977). In another quanti~tive morphologi~l study a preponderance of small chromaffin granules (<950A) was observed in rabbit adrenals in the recovery phase after insulin stimulation (KOERKER et al., 1974). Reduction in granule size has also been reported after stimulation course, exclude the possibility that some form of of the exocrine pancreas (JAMIESON& PALADE, 19716). membrane flow takes place in this tissue. It seems What are the functional implications of these observalikely that plasma membranes are replenished by vesi- tions? Since this change in granule size is also seen cles arising from the Golgi region (see e.g. MICHAELS in exocrine tissues it is probably related to a basic principle in the formation of secretory particles. Dur& LEBLOND,1976; for colonic epithelium). However, in adrenal medulla this transfer by membrane flow ing continuous exocytotic release more recycled membrane material (see previous section) becomes availmust occur by special vesicles and not via chromaffin able for packaging the secretory proteins. The progranules. Even if membranes of chromafi granules are re- duction of these proteins will then become rate-limiting and in order to conserve the membranes their used there is still the need for a constant replenishment of ‘aged’ membranes. We have fro direct evi- continuous re-use will require the formation of smaller-sized particles, which have a greater surface to dence to suggest where these membranes originate volume ratio. Only during further severe stimulation from. The most likely mechanism is a synthesis and formation in the rough endoplasmic reticulum where would too many membranes become available and proteins and lipids for membranes are known to be would lysosomal degradation prevail (see previous produced (compare DALLNER, SIEKEVITZ & PALADE, section). 1966). A further adaptive measure seen after stimulation Finally, it seems worthwhile to note that a return of the adrenal medulla is an increased production of of granule membranes to the Golgi region and their the enzyme tyrosine hydroxylase (see THOENEN,1975) re-use for several secretory cycles may not only occur and dopamine ~-hydroxylase (VIVEROS et al., 1969; in adrenal medulla but may also take place in sym- MOLINOFF‘,BRIMIJOIN,WEINSHILROUM & AXELROD, pathetic nerve since at least one essential feature has 1970). Since the latter enzyme is a typical constituent recently been established, i.e. retrograde axonal flow of chromaffin granules, the question arose what kind of membranes containing dopamine ~-hy~oxylase of relationship exists between this increased enzyme from the terminal to the cell body (ZIEGLER, THOMAS production and the biogenesis of chromaffin granules & JACOBOWITZ, 1976; BRIMIJOIN& HELLAND, 1976; or more specifically the biogenesis of the granule’s FILLENZ, GAGNON, STOECKEL & THOENEN,1976). membrane sinceJhis enzyme is the major protein constituent of the membrane (see WINIUER, 1976)? Two possibilities were considered for adrenals stimulated Relationship between stimulation ofadrenal medulla by reserpine treatment (see GAGNON et al., 1977): (i) and biogenesis of chromafin granules The chromaffin cells contain a larger pool of granule There is considerable evidence that during pro- membranes which have the same concentration of longed stimulation the adrenal gland reacts with what enzyme per unit of membrane. (ii) The ~hroma~n cell are probably adaptive measures. One such reaction contains the same amount of granule membranes, but as seen in the electron microscope is a reduction in with an increased enzyme concentration. The comgranule size. Thus, after reserpine treatment leading bined morphometric and biochemical approach to severe cat~holamine depi’etion, the ~pula~on of (GAGNONet al., 1977) favoured the second possibility. newly formed granules is smaller than normal (CLE- Chromaffin granules became smaller and more MENTI & ZOCCHE, 1963). This was recently confirmed numerous, whereas the total pool of granule memKIRSHNER, 1976). Similar findings have been reported for the membranes of the various cell compartments in the exocrine pancreas (MELDOLESI& COVA, 1972). The experimental evidence and the points just raised argue against the occurrence of membrane flow from the endoplasmic reticulum via chromaffin granules to the plasma membrane. This does not, of SON &

671

Biogenesis of the chromaffin granule branes remained the same. On the other hand, the specific activity of dopamine &hydroxylase in isolated membranes was signi6cantly elevated. Apparently during prolonged stimulation the chromafhn cell switches to the production of membranes having a higher concentration of this enzyme. It is premature to regard this as a final answer which applies to all situations. It is quite possible that under different conditions the chromaffin cell uses both principles, i.e. an increase in the amount of granule membranes and an insertion of more enzyme molecules into them. This may, for example, be the case when the dopamine /I-hydroxylase activity after stimulation rises as high as five times that of the control as observed by DIXON, GARCIA & KIRPEKAR (1975). Tyrosine hydroxylase is usually the rate limiting step in catecholamine synthesis (see UNDENFRIEND, 1966). However, since the chromafhn ‘cell during stimulation inserts more dopamine /I-hydroxylase into chromat%n granules, an elevated level of this enzyme may also be essential for an increased rate of biosynthesis. One wonders whether future studies will discover that after prolonged stimulation chromatIln granules also have an increased capacity for dopamine uptake. BIOGENESIS AND UPTAKE OF SMALL MOLECULES The assembly of the macromolecular components as discussed above leads to the formation of a particle which has still to acquire the small molecules so typical for these organelles. The following sections will discuss the mechanisms involved in this process. Calcium In this section two questions will be discussed: (i)

---

30

r

-

How do chromatIin granules accumulate their significant concentration of calcium? (ii) What role does calcium play in the general biogenesis of chromafhn granules? When bovine adrenal glands are perfused with 45Caz+, this isotope is taken up by chromaf-hn granules (Bo~owrrz, 1969; WINKLERet al., 1972; see also SERCK-HANSEN & CHRISIIANSEN, 1973). The uptake of 45Ca2+ (see Fig. 4) apparently occurs into mature granules. In these experiments 45CaZ+ may also have entered newly formed granules: however, an uptake into the small population of these granules could only have been demonstrated if they had taken up calcium exclusively or at least preferentially, which apparently was not the case. In a recent study (KOSTRON,WINKLER,GEI~~LFR& KOENIG,1977) the capacity of isolated chromagin granules to take up Ca ‘+ from an incubation medium was investigated. From the start it seemed unlikely that calcium could enter these organelles by a process of simple diffusion since the membranes of chromaffin granules are likely to be impermeable to cations (JOHNSON& SCARPA, 1976a,b; DOLAISKITAGBI & PERLMAN,1975). In fact, a highly temperature-dependent process for calcium uptake by chromafhn granules was discovered, which, however, did not require energy in the form of ATP. This latter feature distinguishes this process from the uptake mechanisms described for adrenal mitochondria and micros0me.s (POISNER& &VA, 1970; LESLIE& BOROWITZ, 1975). The rate of uptake into chromafhn granules levelled off with high calcium concentrations and was inhibited by Sr’+. These results are so far consistent with a process of facilitated diffusion through calcium-impermeable membranes. For the parotid gland WALLACH& SCHRAMM (1971) have proposed that ‘calcium serves in the aggregation

Cot.

3H-Cat.

l----l

If--d -

I

OL

II

IIIIIII

Mic.

45,-o2+

Lys. NG.

I MG.

1

I

Mic.

I

1

I

Lys. NG.

1

I

I

MG.

RG. 4. Uptake of newly synthesized catecholamines and 45Ca2+ by chromaffin granules. Perfused bovine adrenal glands were ‘pulse’ labelled either with [‘I-Ijtyrosine or 45Ca2+ (see WINKLERet al., 1972). In the experiment shown on the left a large granule fraction was isolated 3 min after a pulse of C3H]tyrosine and then subjected to density gradient centrifugation as described in Fig. 1. The distribution of endogenous catecholamines and 3H-labelled catecholamines (Cat.; mainly noradrenaline; see text) in the gradient is given. Newly synthesized catecholamines are present in mature chromaffin granules (MG). The position in the gradient of microsomes (MIC), lysosomes (LYS) and newly formed chromafTIn granules (NG) is indicated (compare Fig. 1). The right histogram gives the distribution of 45Ca2+ in the gradient, In this experiment the perfusion was stopped 4 h after the ‘pulse’. Radioactive calcium was taken up and retained by mature granules.

granules. When perfused bovine glands were ‘pulse’ labelled with [32P]phosphate, a specific labelling of the ATP pool in chromailin granules was obtained since these cell particles retained the labelled ATP much longer than the other cell organelles like mitochondria CINDER et al., 1972). This was recently confirmed by TRIFAR~ & DWORKIND(1975). In independent experiments (STEVENS, ROBINSON,VAN DVKE & ST~TZEL,1972; 1975) it was shown that the ATP pool in chromaffin granules could also be labelled with a more convenient precursor, i.e. [‘Hladenosine. In subsequent studies with this compound (PEER, WINKLER,SNIDER,GIBB & BAUMGARTNER, 1976) the origin of the newly synthesized ATP, which was the major labelled nucleotide, was investigated. The time course of the labelling and the effect of cyanide, atractyloside (see Fig. 5) and dinitrophenol, which block ~t~hondrial ATP production, indicated that this nucleotide was synthesized in mitochondria and subsequently transferred to chromaffin granules. Thus it became obvious that these organelles must possess an uptake mechanism for nucleotides and therefore we tried to discover it in experiments with isolated granules. Previous studies with such a preparation (KIRSHNER,1962; CARLSSON,HILLARP & WALDECK, 1963; BAUMGARTNER, WINKLER& HGRTNAGL,1973) had failed to provide clear evidence for any nucleotide uptake. However, after many methodological pitfalls we (KOSTRON,WINKLER,PEER & K~NIG, 1977) were able to establish such a process under in nitro conditions (see Fig. 5). The properties of this uptake are compiled in Table 3 in comparison with the uptake for catecholamines. Since there is a close relationship between two processes we will discuss their underlying mechanism together in the next section, Here it suffices to say that isolated chroma~n granules can take up ATP, ADP and AMP by an energy-dependent, carrier-mediated process. It is most likely that this uptake process represents the mechanism by which chromaffin granules in viuo accumulate their high concentration of nucleotides. There is good evidence that new chroma~n granules start to accumulate ATP before they begin to acquire catecholamines. This was indicated in earlier studies (BURACK, WEINER & HAGEN, 1960; SCH~HMANN, 1958) when a low catecholamine/ATP ratio in adrenal glands was observed during the recovery phase after Nucleootides severe catecholamine depletion caused by administraHow do newly formed chrom~n granules ac- tion of reserpine or insulin (see, however, KESWANI, cumulate their high concentration (see WINKLER, D’IORO & MAVRIDES,1971). This was confirmed in 1976) of nucleotides, mainly ATP? This question had a detailed subcellular fractionation study on rat remained a puzzling one for a long time and only adrenals (SILOTKIN & KIRSHNER,19736). Similarly durrecently an answer was found. The first discovery ing the ontogenesis of the adrenal of various species which helped to solve this problem was the observathe ~techolamine/ATP ratios are very low, indicating tion that the nucleotide pool in chroma~n granules that nucleotides are accumulated before the catecholcould be radioactively labelled. STJKRNE,HEDQVIST& amines when chromaffin granules appear in the LAGERCRANTZ (1970) injected cats with several doses adrenals for the first time (STUDNITZ,1968; O’BRIEN, of [3zP]phosphate which indiscriminately labelled all DA PRADA& PLETSCHER,1972). Thus newly formed the nucleotides of adrenal medulla, and therefore only granules first acquire the nucleotides, before catecholabout lo”/, of the total label was present in chromaffin amines are stored (see next section). This may be due of the exportable material’ in the early stages of granule formation. This was also discussed by AMSTERDAM & JAM~EWN(1974) for the exocrine pancreas, when they observed that the content of condensing vacuoles in isolated cells appeared looser and more flocculant than in intact tissue, which might have been caused by the lack of calcium in the media used for cell separation. Along a similar line CECCARELLI, CLEMENTE & MELD~LESI(1975) suggested that calcium is preferentially entering immature zymogen granules. Similar mechanisms may operate in the adrenal medulla. Calcium may be involved in the early aggregation process, which is likely to occur in the prosecretory granule step, where secretory proteins are added to the newly ‘formed granules (see section on morphology). In this context it is interesting to note that in vitro calcium can precipitate the acidic proteins of the granule content, i.e. the chromogranins (H. WINKLER, unpublished observations), which is reminiscent of the fact that calcium precipitates casein, an acidic protein of the milk secretion granules (FARELL,1973). Furthermore, the condensation process, at least in exocrine pancreas, is apparently not energ~dependent (JAMIE~N & PALADE, 1971~). This would be consistent with the involvement of a calcium uptake which does not require energy as found for chromaffin granules. It seems therefore justified to speculate that during ‘the condensation process’ calcium enters newly formed chromaffin granules before the other small molecules, i.e. ATP and catecholamines are accumulated. However, there is no direct evidence to support such a sequence of events. In addition to this role of calcium in an early stage of granule formation, which may be a general function for protein storing organelles, its uptake into chromaffin granules certainly serves a second purpose. Calcium is an important participant in the catecholamine/ATP storage complex (see PLETXHER et al., 1974). New granules have, therefore, to accumulate this metal ion in order to form the calcium/nucleotide core of the complex (see next section). Furthermore mature granules from which there is always some leakage of stored molecules, i.e. catecholamines, ATP and calcium, have to replenish these losses by a continuous calcium uptake.

673

Biogenesis of the chromaffin granule Perfused .-.---

/I

isolated

~iond

---

Control Atmct. Cyanide

11 Mic.

1

IJ

11

I

NG.

I

granules

Control Atroct.

I

I

I

I

I

I

I

I

I

MG.

FIG. 5. Uptake of nucleotides by chromaffin granules. The histograms on the left were derived from experiments with perfused bovine adrenals fabelled with [3H]adenosine (see PEERet al., 1976). Four hours after the radioactive pulse the subcellular fractions were isolated (compare Fig. 1). The ordinate gives the amount of C3H]ATP (d.p.m) found per ml of each gradient fraction. The distribution of [“H]ATP is consistent with its presence in mature chro~ffin granules (MG). NG indicates the position of newly formed granules. When the labehing of the gland was performed in the presence of cyanide (PEER et al., 1976) or atractyloside (atract., ~publi~ed experiments), chromatfin granules did not accumulate C3H]ATP (see dashed lines). The histograms on the right were derived from experiments with isolated chromathn granules which were incubated at 37°C with c3H]ATP, washed and then subjected to density gradient centrifugation. The distribution of radioactivity (more than 60% 13H]ATP) indicates that the isolated chromaffin granules have taken up [“HIATP. The ordinate indicates the amount of C3H]ATP taken up per ml of gradient fraction per g of adrenal medulla. In the presence

of atractyloside the uptake of [“H]ATP by isolated chromafCn granules was blocked (see et al., 1977).

to an inability of the granules to retain catecholamines before a ‘core’ (BERNEIS,DA PRADA & F%~CHER, 1970) of the storage complex consisting of nucleotides and calcium is present. On the other hand, ATP uptake is not only confined to newly formed chromati granules, since mature granules also take up recently synthesized nucleotides (see WINKLERet al., 1972). As seen in Fig. 5, these granules hecame lahelled when C3H]adenosine was injected into perfused glands. Under the& conditions a lahelling of the small popnlation of newly formed granules could not possibly have been observed. Uptake of ATP by mature granules in uivo is likely to replenish any loss of ATP occurring during the lifespan of these granules. In this context it is interesting to compare the uptake rates for catecholamines and ATP (see Table 3). The lower rate for nucleotide uptake may indicate that the losses of ATP from chromafBn granules in vioo are rather small. This is supported by in vitro experiments (BAUMGARTNER et al., 1973). Isolated granules do not lose sign&ant amounts of ATP when incubated under conditions whereby the catecholamine level is maintamed by a continuous m-uptake. The proposed model for the catecholamine/ATP storage complex (see PLErXXEJl et al, 1974) is consistent with these experiments ATP is supposed to form a rather stable core to which catecholamines are loosely bound.

~OSTRON

Biosynthesisof catecholamines.The synthetic pathways for the biosynthesis of the catecholamines are well established (see BLAXHKO, 1959; KIRCHNER, 1975) and will be discussed here only in connection with the function of chromafhn granules. The generally accepted view is that dopamine is formed from dopa in the cytoplasm. Dopamine is then taken up by chromafiin granules where it is converted to noralkaline. This latter amine has to leave the granules in order to become converted into adrenaline in the cytoplasm which is then again taken up by granules. This shuttling in the final biosynthesis step seemed to some a rather unlikely mechanism and therefore two modifications were proposed: (i) One group suggested (THOMAS,VAN ORDEN, bDICK ,& KOPM, 1974; RJZDICK,THOMAS, VAN OF~DEN,~'ANORDEN& KOPIN, 1974) that at least the activecenter of dopamine /I-hydroxylase may be localized on the outside of chromafhn granules allowing synthesis of nom~enal~e from dopamine without its previous uptake into granules. In this case only the final product, adrenaline, would enter the granules. Evidence for such a localization of the enzyme was seen in immunological experiments in which antisera against dopamine fl-hydroxylase partly inhibited the enzyme in a preparation of intact granules. This suggestion was in contrast with earlier studies of

674

H. WINKLER

KIRSHNER(1962), who found an inhibition of noradrenaline synthesis when the uptake of dopamine into chromaffin granules was blocked by reserpine. Similarly, LADURON(1975) reported more recently that the enzyme in intact granules was completely latent, i.e. it was not available to substrate outside of the granules. Finally, experiments in which intact and lysed granules were treated with a non-penetrating radioactive label for protein (diazobenzene [35S]su1phonate) gave no evidence that dopamine /3-hydroxylase or part of this enzyme was present on the outer surface of chromaffin granules, whereas chromomembrin B, the second major membrane protein, was found in such a position (K~NIG, HBRTNAGL,KOSTRON, SAPINSKY & WINKLER,1976). (ii) As another modification for the synthesis of adrenaline (LADURON,1972; LADURON,VAN GOMPEL, LEYSEN& CLAEYS,1974) it was suggested that dopamine becomes converted to epinine by the enzyme phenethanolamine N-methyltransferase in the cytoplasm. Epinine, after uptake into chromaffin granules, would then become hydroxylated to adrenaline by dopamine P-hydroxylase. The presence and formation of epinine in adrenal medulla was reported in support of this concept. However, SCH~~MANN & BRODDE (1976), in contrast to the results of LADURONet al. (1974) on pig and bovine adrenals, did not detect any epinine in rat adrenals. PENDLET~N& GESSNER(1975) found no evidence for a conversion of dopamine to epinine by purified phenethanolamine N-methyltransferase. An extremely low affinity of this enzyme for dopamine was also reported by another group (HOFFMAN, CIARANELLO& AXELROD,1976). Furthermore, after injection of C3H]dopamine into rats no C3H]epinine appeared in the adrenal medulla (PENDLETON & GESSNER,1975). Experiments with radioactively labelled precursors argue strongly against both proposals for a modification of the synthetic pathway. In one study (WINKLER et al., 1972) perfused bovine adrenals were pulselabelled with [3H]tyrosine. Three minutes after the pulse most of the labelled catecholamines were present in chromaffin granules and consisted of 15% dopamine, 81% noradrenaline and 4% adrenaline, whereas 3 h after the pulse the distribution was 0.4% dopamine, 74% noradrenaline and 23% adrenaline. Apparently, dopamine which had entered the granules was subsequently converted to noradrenaline and this latter amine to adrenaline. In another study (PENDLETON & GESSNER,1975) C3H]dopamine was administered to rats. The time-course of the conversion from C3H]dopamine to C3H]adrenaline was consistent with a formation of the latter from C3H]noradrenaline as a direct precursor. Similar results were obtained by COUPLAND,KOBAYASHI & KENT (1976) when C3H]dopamine injections were made into mice. In both these studies it took about 12 h before all the newly synthesized noradrenaline was converted to adrenaline. After severe catecholamine depletion caused by a high dose of acetylcholine (BUTTERWORTH & MANN, 1957)

the conversion of the newly synthesized noradrenaline pool to adrenaline seems to be even slower, lasting about a week. All these results are consistent with the view that dopamine has to enter chromaffin granules before it can be converted to noradrenaline. Since the binding of noradrenaline in the storage complex is by no means completely stable (see PLET~CHERet al., 1974), there will always be some leakage of noradrenaline to the cytosol. Noradrenaline lost from the granules in this way can be converted to adrenaline before this amine is taken up again. If one takes this view then one can not argue that this shuttle process consumes additional energy or that it is too ‘laborious’ (see e.g. LADURON,1972). From this discussion it has become obvious that a critical step in the biosynthesis of catecholamines is their uptake into chromaffin granules. Uptake of catecholamines by chromajjin granules.

The properties of this uptake process, which was discovered some time ago (KIRSHNER,1962; CARLSWN et al., 1963), are by now well established (see Table 3 ; see also TAUGNER& HASSELBACH, 1966; SLOTKIN, 1973 ; for further references see WINKLER & SMITH, 1975). The uptake is localized in the membranes of these organelles since it can also be demonstrated with isolated membrane ghosts (TAUGNER, 1971a; 1972; PHILLIPS, 1974; DA PRADA, OBRIST & PLETSCHER,1975). Already in the early studies it was recognized (see Table 3) that the catecholamine uptake was activated by ATP and Mg”, but inhibited by N-ethylmaleimide, which later led to the proposal that this uptake was driven by the SH-dependent Mg-activated ATPase (BANKS,1965). This concept has recently been significantly extended by the discovery that the uptake of catecholamines in chromaffin granules is inhibited by uncouplers of oxidative phosphorylation (BASHFORD,CASEY,RADDA & RITCHIE, 1975; 1976; CASEY, NJUS, RADDA & SEHR, 1977; see EULER& LISHAJKO,1969, for sympathetic nerve vesicles). The effect of these compounds was kxplained by their ability to abolish proton gradients across a membrane. Furthermore, dicyclohexylcarbodiimide which blocks ‘proton channels’ also interferes with catecholamine uptake (BASHFORDet al., 1976). The proton gradient across the granule membrane was considered to be generated by an inward directed electrogenic proton pump driven by the ATPase. Thus, the addition of ATP and Mg’+ to chromaffin granules enhances the fluorescence of the probe I -anilinonaphtalene-8-sulphonate as in other proton accumulating systems (BASHFORDet el., 1976). Furthermore, in the presence of ATP and permeant anions isolated chromaffin granules are lysed due to the coupled uptake of anions and protons (CASEY et al., 1976). When such a lysis is prevented by hypertonic media, the pH inside the granules which is already low, being about 5.5 (BASHFORD et al., 1976; JOHNSON & SCARPA,1976b), drops further due to significant proton uptake under these conditions (CASEY et al.,

Biogenesis of the chro~ffin

1977).In these experiments the internal pH of chromaflk granules incubated at WC in hypertonic media was estimated from the distribution of the weak base methylamine and from phosphorus-31 nuclear magnetic resonance spectra of the ATP contained in granules. In contrast, POLLARD, ZINDER, HOFFMAN& NIKODEJEVIC (1976) suggested that addition of ATP leads to an increase of internal pH in granules. However, their interpretation of the data was considered to be incorrect by CASEY et al. (1977). In any case, since POLLARD et al. (1976) incubated the granules at 2”C, their results can hardly be considered representative of physiological processes. In conclusion, the results just discussed have provided good evidence that an ATPase dependent proton translocation occurs in chromaffin granules and that this process is inti~tely linked to ~t~holamine uptake. In this context it is extremely interesting to note that the newly discovered uptake of nucleotides (see previous section) is apparently related to that of catecholamines (see Table 3 and Fig. 6). Both processes seem to depend on the Mg-activated, SH-dependent ATPase in the granule membranes, since they are both activated by MgZf, but inhibited by EDTA, N-ethylmaleimide and ATP-analogues. Furthermore, uncouplers abolish both uptakes, Thus we can conclude that the two uptake mechanisms apparently depend on the same driving force, namely the protontranslocating ATPase. On the other hand, two different carriers seem to be involved. Atractyloside, a wellknown blocker of the nucleotide carrier in mitochondria (see KLINGJZNBERG, 1970; VIGNAIS,1976) specifically interferes with the transport of nucleotide into

675

granule

chromailin granules. Reserpine inhibits only the uptake of catecholamines, which is in agreement with the previous suggestion that this alkaloid interacts with a specific carrier for catecholamines (Summ, 1973). The action of oligomycin on catecholamine uptake (see Table 3) is at present not well understood; however, it may react with the carrier for catecholamines like reserpine (CASEY, 1976). Antimycin, on the other hand, may interfere with the cytochrome b-561 which is a com~nent of the electron transfer system of chromatKn granules (TERGND & FLATMARK,1973). It has been proposed that this system is necessary to prevent oxidation of catecholamines during transport (BASHFORD et al., 1976). I have tried to present the results just discussed in a comprehensive diagram in Fig. 6. The weakest point in this scheme is the exact re~tionship between the driving force (proton pump) and the actual transport of the catecholamines and nucleotides. The pH inside the chromaffin granules is rather low (around 5.5). Therefore, one might argue (JOHIWN & SC..YR~A, 1976b) that catecholamines as weak bases might be taken up by chro~ffin granules, like me~yl~ne is, as uncharged molecules followed by reprotonation inside these organelles. Maintained catecholamine uptake would then require a proton pump which counteracts the pH increase inside the granules caused by the accumulated catecholamines (see JOHNSON & SCARPA, 1976b). However, uncouplers block active catecholamine uptake without changing the pH inside the granules (CASEY et al., 1977). Apparently the pH gradient existing in granules in the absence of ATP is not sufficient to drive catecholamine

TABLE3. A COMPARISOP~ OFHIE UPTAKE MECHANISMS FORNUCLEOTIDES ANDFORCATECHOLAMINES IN BOVINE CHROMAFFIN GRANULES Properties

Nucleotides

Substrates

ATP= ADP> AMP(2)

Rate

0.27stttol,‘mg protein/ mitt(ATP;1)

I.4mu(ATP:it

ElIactof Resetpine Oligomycin Antimycin Abactyloside ~~x~~actylosi& Unc0uplers ~~~~OP~Oi, compound 513, carbottylcyakle chlorophenylhydmzonc) N-ethylmaleimide Dicyclohexylcarbodiimide M$ + EDTA Nip&& ( + KCl) ATPahalo~ues: Ad~ylyl-~~~~yl~ ~phoaphoMt~ Adenylyl-imidodiphosphate

Catecholamines Noradrendine, adrenaline, dopamine. tyramine, J-OH-tryptamine (3-7) (-) noradrenaline > (+ ) noradrenaline (6 8) 30jt&t (rat:9) S-18PM&winemembrane ghost:6) 3.4nmol/mg prot/min (10)

0 (1) 0 (2) 0 (2) Inhibits Ii) Inhibits (2) InhibitII. 21

Inhibits (3) Inhibits(11,2) Inhibits (11,2) 0 (1) 0 (2) Inhibit(11.1)

Inhibits (1) Activates (1) Inhibits (2) 0 (2)

Inhibits (4) Inhibits (11) Activate8 (3) Inhibits (3,4) Inhibits (2)

Inhibits (2) Inhibits (2)

Inhibits (6,2) Inhibits (2)

The numbers in the parentheses refer to the following references: (1) KOSTRON et al. (1977); (2) WINRLER,KOSTRON, ABERER L K~NIG, unpublished observations; (3) Krasn~ax (1962); (4) CARLSWN et al. (1963); (5) SLOTKIN & KIRSHNER (1971); (6) PHILLIPS (1974); (7) DA PRADA et al. (1975); (8) TAUGNER(1972); (9) SLOTKIN,ANDERSON, SEIDLW& LAU

(1975); (10) TAWNER (1971b); (11) BASKFORD et al.(1976).

H. WINKLER

676

Co2+ (1 nmol/mg

/mm)

I

ATP ADP>AMP .27 nmol / mg / min q

I

Corboxyatr.

H+ Mg2+

ATP-analogues EDTA NEM

FIG. 6. Uptake of small molecules by chromaffin granules. The three uptake mechanisms in chromaffin granules are schematically presented. Uptake rates are given for Ca*+ (substrate concentration: S.)OpM; KOSTRONet al., 1977), nucleotides (2mM ATP; KOSTRON ef al., 1977) and catechoiatnines (c. 1Oph1; TAUGNER, 1971b). The common driving force for the uptake of catechofamines (see BASHFORD et al., 1976) and of nucleotides (KOSTRON et al., 1977) is considered to be a proton-translocating ATPase, whereas specific carriers are thought to be responsible for the translocation of these molecules through the membrane. Inhibitors (-4) and activators (---+) of the uptake mechanisms for ATP and catecholamine are indicated in the appropriate places. The exact coupling between the driving force (proton translocation) and the carrier-mediated transport of nucleotides and catecholamines is still a matter of speculation (see text). Atract., atractyioside; Catech., catecholamines; EDTA, ethyienedia~ne tetraacetate; NEM, N-ethylmaleimide.

uptake Additional protons accumulated by the ATPase apl ar necessary. These protons might be used for chart ‘g neutral catecholamines transported by the Carrie On the other hand the protons may directly drive the catecholamine carrier. The uptake of catecholamines depends on the chemical part of the proton gradient, whereas that of nucleotides is driven by the electrical component as shown by experiments with nigericin. This ionophore, which exchanges the H+ inside the granules against K’ in the incubation medium, abolishes the chemical component of the proton gradient but does not alter the electrical part. It inhibits catecholamine transport but does not block that of nucleotides. Thus we are beginning to understand the relationship between the electrochemi~l proton gradient and the uptake of these small molecules but many further studies are required. In any case it is already obvious that chro~~n granules have an elegant solution for their uptake problems. A common driving force provided by the granule ATPase is able to transport two quite different molecules. This mechanism enables these organelles to accumulate the two main partners of their storage complex. It remains now to discuss how this catecholamine uptake is integrated in the biogenesis of chromaffin granules. Biogenesis uptake.

of Carolyn

granules

and catechola~ine

Newly formed chromaffin granules have to

take up catecholamines in order to become fully mature storage organelles. However, they have to compete in this process with the total population of chromaffin granules since considerable evidence indicates that there is no preferential or at least no exclusive uptake into new granules. After a pulse of C3H]tyrosine the newly synthesized catecholamines are found (see Fig. 4) in the total population of granules (WINKLERet al., 1972) without any preference for the new granules which are found in less dense regions of the gradient. This is in agreement with radioautographic studies in mice and hamsters in which, at all times after the injection of the precursor, newly synthesized catecholamines were found to be distributed over the total pool of chromaffin granules in the cytoplasm (ELFVIN,APPELGREN & ULLBERG, 1966; C~UPLANDet al., 1976). These studies did not confirm MOPPERT(1966), who stated that 1 h after the injection of E3H]dopa there was a transient concentration of the labelled material over the Golgi region of adrenal medullary cells. As pointed out in previous sections the maturation process, and therefore the filling of chromaffin granules with catecholamines, is slow, requiring more than 24 h. This may be caused by two factors. (i) Firstly, before the granules can maintain a stable store of catecholamines, they have to acquire at least some core of calcium/nu~leotide complexes. As already pointed out, nucleotides appear in the

‘H-Leucine

FIG. 7. Biogenesis of chromaffin granules. In this diagram the solid arrows indicate movement of cell particles and the broken arrows indicate movement of small molecules. The secretory proteins of adrenal medulla are synthesized in the rough endoplasmic reticulum. Their transport to the Golgi region and to the prosecretory granules is supposed to be carried out by a shuttle service of coated vesicles. It is not yet established whether coated vesicles can carry secretory proteins from the endoplasmic reticulum directly to prosecretory granules or whether the Golgi asternae are interposed between two shuttle services of such vesicles. The membrane of the chromaffin granule is not provided from the endoplasmic reticulum by concomitant membrane-flow together with the secretory proteins. It is suggested that the chromaffin granule membrane is retrieved after exocytosis and re-used for several secretory cycles. Part of the membrane becomes degraded by lysosomal digestion, whereas new membrane for the chromaffin granules may be supplied from special synthesis sites in the rough endoplasmic reticulum. The special origin and use of this membrane is indicated by the red colour, which suggests that the membrane is specific for this cell compartment. Newly formed chromaffin granules start to accumulate calcium, nucleotides and catecholamines. Calcium may be involved in the condensation process whereas all three components are partners in the storage complex. Chromaffin granules have specific uptake systems for these small molecules (see Fig. 6).

Biogenesis of the chro~~n

granule

677

granules before the catecholamines. Furthermore, nu- These ‘prosecretory granules’ have a special morphocleotides can form aggregates with calcium in the logical appearance and also differ from mature granules (see below) in their composition. absence of the amines (BERNEIS et al., 1970). UnfortuThe membranes of chromaffin granules are not synnately, we have no direct information whether this thesized and transported concomitantly with the secprocess is rate-biting in the maturati~ of granules. However, it may be significant that the uptake rate retory protein. The synthesis rate of the membrane proteins (including dopamine /I-hydroxylase) is sigfor nucleotides is lower than that for catecholamines. (ii) On the other hand the synthesis rate for cate- nificantly lower than that of the exportable proteins. cholamines may be the rate-limiting process. Some This observation makes it very likely that these membranes are re-used several times in a shuttle service indication for this can be seen in the fact that after from the Golgi region to the cell membrane and back. prolonged stimulation of the adrenal there is a concell to preserve siderable increase in the synthesizing capacity as evi- Such a process enables the chrom~n the specificity of these membranes which is necessary denced by the increased production of dopamine /I-hydroxylase and tyrosine hydroxylase (VIVEROSet for their special functions, e.g. in the uptake of small al., 1969; MOLINOFFet al., 1970; see THOENEN,1975). molecules and fusion with the plasma membrane during exocytosis. Only those membranes which after It seems rather surprising that the synthesis of catecholamines might represent the rate-limiting step in several cycles become degraded by lysosomes have to be replenished by new membranes which may be the formation of mature granules when we consider that the main function of these organelles is the dis- synthesized in the rough endoplasmic reticulum. The specific handling of these membranes is illustrated in charge of catecholamines. However, the possibility Fig. 7 by colouring them red. that the new granules are kept near the Golgi region Newly formed chromaffin granules do not yet confor a prolonged time was already discussed in the tain their complement of small molecules, i.e. calcinm, section on [3H]leucine. This would reduce secretion nucleotides and ~t~hol~nes and are therefore less from granules still poor in cat~holamines. Furthermore, the long maturation process should be seen in dense than mature granules in density gradient centriconnection with the slow turnover of the total cate- fugation. Chromaffin granules possess a temperaturedependent uptake system for calcium, which, howcholamine pool in chromaffin granules which amounts to a half life of more than a week (UDEZNFRIEND,ever, does not require energy. Nucleotides enter chroCOOPER,CLARK & BAER, 1953; PENDLETON,SNOW, mat& granules before the catecholamines become GESSNER& Gm, 1973; HEMEL & K&, 1968). stored in them. This is probably related to the fact This turnover is in reasonable agreement with the that nucleotides can form large complexes with calsynthesis rates (2.2-9.3 nmol/h/kg of body weight) cium, which may provide the storage core to which found in rat adrenals which contain 575 nmol cate- catecholamines are subsequently bound. Chromaffin cholamines/kg of body weight; WALDECK, SNIDER, granules can take up both nucleotides and catecholBROWN& CAR-N, 1975). In this context the longer amines by a carrier-mediated transport. The carriers ~turation time appears sensible and only during are different, as shown by the possibi~ty of ~fferential prolonged stimulation has the gland to switch to a blockade with reserpine and atractyloside, but the more speedy procedure of catechofamine synthesis driving force seems to be provided for both uptake and, therefore, of granule maturation. processes by a proton gradient which is generated by an electrogenic proton pump driven by the ATPase (see Fig. 6). This commen~ry has shown that most of the many CONCLUSIONS steps involved in the biogenesis of chromafhn An attempt has been made in Fig. 7 to combine granules have already been studied experimentally. I all the available data discussed above into a coherent hope that the sometimes speculative interpretations picture of the biogenesis of chromaffin granules. The will induce further studies that may finally establish, macromolecular components of these granules des- but could also disprove, certain aspects of the contined for secretion are glycoproteins and mucopolycepts I have presented. saccharides. In accordance with other secretory tissues these components are synthesized in the rough Acknowledgements-Studies performed by the author endoplasmic reticulum with further additions on their quoted in this commentary were supported by the Fonds way to the Golgi region where the secretory products zur FGrderung der wissenschaftlichen Forschung, Austria, become packaged into new chromafhn granules. and by Dr. LEGWLOTZ~TIFFUNG.

REFERENCES

ABRAH~M~ S. J. & HOLTZMAN E. (1973) Secretion and endocytosis in insulin-stimulated rat adrenal medulla cells. J. Cell Bid. 56, 540-558. AL-LAMIF. (1969) Light and electron microscopy of the adrenal medulla of Macacu mulata monkey. Anat. Rec. 164, 317-332.

678

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(Accepted 24 April 1977)