Commentaries in the Neurosciences

LLINÁS

CHEMISTRY AND DYNAMICS OF NEUROTRANSMITTER STORAGE PARTICLES

Outline

Chapter 1: SYNAPTOSOMES AND CELL SEPARATION

Chapter 2: THE COMPOSITION OF ADRENAL CHROMAFFIN GRANULES: AN ASSESSMENT OF CONTROVERSIAL RESULTS

Chapter 3: THE BIOGENESIS OF ADRENAL CHROMAFFIN GRANULES

Chapter 4: ON THE COMPOSITION AND FUNCTION OF LARGE DENSE CORED VESICLES IN SYMPATHETIC NERVES

Chapter 5: THE ORIGIN AND FATE OF SECRETORY PACKAGES, ESPECIALLY SYNAPTIC VESICLES

Chapter 6: VESICLE RECYCLING AND TRANSMITTER RELEASE

Chapter 7: CONTRACTILE PROTEINS IN TISSUES ORIGINATING IN THE NEURAL CREST

SYNAPTOSOMES AND CELL SEPARATION

IAN G. MORGAN,     Department of Behavioural Biology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City, A.C.T. 2601, Australia

Publisher Summary

When fractionating a complex tissue like brain, experiments should begin by separating it into its constituent cell classes. Any subcellular fraction prepared from brain is intrinsically heterogeneous as it is derived from several cell types. But even if it were possible to separate cell types satisfactorily, the fractions obtained would still be heterogeneous because of the fact that it is possible to subdivide the classes of neurons and glial cells into a series of subclasses. This chapter discusses the preparation of synaptosomes and synaptosomal subfractions and the related problem of distinguishing glial from neuronal material. Methods for preparing synaptosomal subfractions often begin with a crude mitochondrial fraction. However, it is preferable to begin with a synaptosomal fraction—best prepared on a Ficoll-sucrose gradient because of the greater ease of osmotic shock. As the synaptosomal fraction has lower levels of mitochondrial, myelin, and membranous contamination, a crude mitochondrial fraction should be used only if time is an absolutely crucial factor.

Preparation of synaptosomes

Preparation of subfractions from synaptosomes

Separation of neurons from glia

Conclusions

Logically, when fractionating a complex tissue like brain, experiments should begin by separating it into its constituent cell classes. Unfortunately, at the present time there are practical limitations on doing this, as will become clear later. As a result, any subcellular fraction prepared from brain is intrinsically heterogeneous since it is derived from several cell types. But even if it were possible to separate cell types satisfactorily, the fractions obtained would still be heterogeneous due to the fact that it is possible to subdivide the classes of neurons and glial cells into a series of subclasses. Moreover, even within a given cell there are different regions: an important problem for the neuron where dendritic, cell body, axonal and synaptic regions can be distinguished.

Any brain subcellular fraction suffers from some of these types of heterogeneity, although certain fractions, such as myelin, and nerve-endings and their subfractions, escape them to some extent. Very often, this heterogeneity is not important for the interpretation of results, but the finer the analysis, the more likely it is that it will be necessary to take it into account.

A crucial point in subcellular fractionation experiments, that of deciding on the aim of the experiment, applies to any tissue—not specifically to the nervous system. Basically there are two types of fractionation experiment: analytical experiments aiming to draw a parallel between the distribution of markers, and the constituent under study, and preparative experiments aiming to obtain as pure a fraction as possible for detailed studies of its composition or metabolism. While, ideally, the criteria for the techniques used in both types of experiment should be the same, in practice increases in the purity of a fraction are generally accompanied by decreases in yield, and vice versa. In practice, therefore, the optimum fractionation procedures for the two types of experiment are different.

In the classical analytical experiment—that of determining if a given tissue constituent is associated with a given subcellular fraction—a parallel is drawn between the distribution of the constituent and that of a known marker. In these experiments a standard procedure of high resolution is required, yet complete recovery of material is also necessary so that balance-sheets can be drawn up. The latter point is generally accepted, but the former is often neglected. Many experiments have reported localizations’ of enzymes in synaptosomal plasma membrane using procedures which do not effectively resolve them from glial, or even microsomal elements. This problem is compounded by the difficulty of choosing markers. Many are chosen by analogy with other tissues, particularly liver, which, given that few reference fractions can be isolated in pure form from brain, is unfortunately the best that can be done.

This sort of experiment generally gives some idea of whether the constituent is associated with the marker. But conclusions should always be confirmed by isolating the particular subcellular fraction absolutely pure. Whether the localization reported is exclusive can be answered by looking at the percentage distribution results, and the preparative enrichment of the constituent compared to the marker. The ultimate test would be to isolate all other subcellular fractions in pure form and test them for the presence of the constituent. In practice, our technology falls somewhat short of this, since all the fractions obtained are contaminated to some extent. Care needs to be exercised in adapting these criteria to reality.

In analytical experiments a method need only give a fraction in which the appropriate marker is highly enriched, and in which other markers are less enriched. This does not mean that the fraction is suitable for preparative work, since, to take the case of lysosomes, the specific activities of relevant enzymes in pure lysosomal fractions from liver may be 100 times those of the homogenate (Stahn, Maier & Hannig, 1970). Fractions enriched 10–20 times can be obtained from brain (Koenig, Gaines, Mcdonald, Gray & Scott, 1964), yet the lysosomes may only make up 10–20% of these fractions. It is obviously ludicrous to study the composition of lysosomes using such fractions, yet they are useful ‘reference fractions’ in the analytical approach.

For preparative work, in order to be able to judge the significance of the results, as complete an inventory as possible of the components of the fraction is necessary. It is not possible to put an arbitrary limit on the degree of purity necessary before results become meaningful. Generally, several minor components are less important than one major contaminant, even if the total percentage contamination is the same. Moreover, if the contaminant has itself been purified, it may be possible to allow for its presence. Particular care over contamination must be taken in experiments involving immunology, since biochemical contaminants may be highly antigenic and so give rise to major immunological components.

After these preliminary remarks, the main focus of this article will be upon the preparation of synaptosomes, and synaptosomal subfractions, and upon the related problem of distinguishing glial from neuronal material.

PREPARATION OF SYNAPTOSOMES

Preparation of synaptosomes has been in the past, and remains today, an empirical affair which has recently been reviewed by COTMAN (1974) in an admirably succinct article. Within certain limits, the precise conditions of homogenization do not appear to be crucial for isolating synaptosomes. Nor do the lower, and higher, centrifugal conditions of the first step, the preparation of the ‘crude mitochondrial’ fraction, appear to be absolutely crucial, although the levels of contamination with endoplasmic reticulum can be reduced by choosing the appropriate conditions (Cotman, Brown, Harrell & Anderson, 1970), and by washing the fraction exhaustively (Morgan, Wolfe, Mandel & Gombos, 1971; Gurd, Jones, Mahler & Moore, 1974).

Various gradients have been used for isolating synaptosomes, primarily made with sucrose or Ficoll. A synaptosomal peak can be easily defined by measuring occluded lactate dehydrogenase, but for more knowledge of the composition of the fractions, morphological studies are necessary. These have been performed on sucrose (Whittaker, 1968) and Ficoll (Joó & Karnushina, 1975) gradients. As a generalization it appears that synaptosomal fractions are never pure, but fractions containing up to 40–50% synaptosomes can be obtained. More importantly, by selecting dense fractions the contamination with membranous particles and fragments can be reduced, but at the expense of a corresponding increase in mitochondrial contamination. Conversely, less dense fractions are much less contaminated with mitochondria, but are more contaminated with membrane fragments. In practice, this means that the gradient used to isolate synaptosomes can only be chosen empirically once the aim of the experiments has been decided.

If the synaptosomes are to be used for subsequent fractionation, then, in general, denser fractions are preferable. As mentioned above, the denser fractions tend to be less contaminated with membranous particles, which therefore reduces the potential contamination with glial membranes and endoplasmic reticulum. If synaptosomal soluble components are required then denser fractions are also to be preferred since there is some evidence that there are less contaminating glial structures (Joó & Karnushina, 1975). When synaptosomal mitochondria are to be prepared, it is obviously preferable to start off from the lighter synaptosomal fractions where mitochondrial contamination is less marked.

Preparation of subfractions from synaptosomes

Methods for preparing synaptosomal subfractions often begin with a crude mitochondrial fraction. (There is a tendency to refer to this sort of fraction as a ‘synaptosome-mitochondria fraction’, an acceptable designation, or a ‘crude synaptosomal fraction’. Given that even the best synaptosomal fractions are crude, the latter is just a verbal trick for increasing the purity of the fraction studied.) But it is preferable to begin with a synaptosomal fraction—best prepared on a Ficoll-sucrose gradient because of the greater ease of osmotic shock. Since the synaptosomal fraction has lower levels of mitochondrial, myelin and membranous contamination, a crude mitochondrial fraction should be used only if time is an absolutely crucial factor.

Whether a crude mitochondrial, or a crude synaptosomal fraction is used as the starting point, the next step in the process is the disruption of the synaptosomal particles, normally by osmotic shock. Control of the pH and ionic strength at this step seems to be of some importance, since in part higher pH and ionic strengths seem to prevent absorption of soluble enzymes to membrane fragments (FONNUM, 1967, 1968). Moreover, Cotman & Matthews (1971) have shown that brief incubation of the disrupted material at higher pH gives better separation of membrane fragments from mitochondria. These variables have not been studied in detail, but it seems likely that osmotic shock in a relatively large volume of buffer, coupled with control of the pH and ionic strength, is desirable.

The shocked material can be directly layered onto a final gradient (usually sucrose (Whittaker, Michaelson & Kirkland, 1964)) to resolve the various synaptosomal components. Often, particularly when working with large volumes, it is more convenient to initially sediment the particulate material which is then suspended and layered onto a gradient. This does not interfere with subsequent resolution in most cases. However, the composition of the gradient appears to have some influence on the resolution of material: GURD et al. (1974) have noted that substituting phosphate-ethylenediamine tetra-acetate buffer for N-2-hydroxyethylpiperazine-N?-2-ethane-sulphonate buffer gave synaptosomal plasma membrane fragments with higher Na + K-activated ATPase activities.

In the most commonly used gradient (Whittaker et al., 1964), the material which remains at the top of the gradient and is not sedimented by centrifugation at 100,0000 g for 1 h (or thereabouts) is primarily the soluble component of the synaptosomes. It has been estimated that 70–80% of it is neuronal soluble material on the basis of the level of the S-100 protein fraction detected in it (Morgan, Reith, Marinari, Breckenridge & Gombos, 1972). However this point needs to be further investigated, particularly in view of the conflicting report by Donato & Michetti (1974), and the presence of particulate S-100 protein (Rusca, Calissano & Alema, 1973). In addition to the glial contamination, the upper fractions of the gradient are likely to be contaminated with mitochondrial soluble material, since this is released from osmotically shocked mitochondria.

The soluble fraction tends to spread into lower fractions in the gradient, and it is always preferable to sediment other fractions, particularly the synaptic vesicles, in order to separate them from contaminating soluble proteins. This can be done at higher ionic strength to remove possible adsorbed material.

Fraction D is usually taken as the synaptic vesicle fraction, and under the appropriate conditions can be quite pure. However, we have found that when working with large quantities of material, the synaptic vesicles produced in this way can be quite contaminated (Morgan, Vincendon & Gombos, 1973a), and further tricks are necessary to purify them. The most satisfactory we have used involves sequential Millipore filtration to remove contaminating membranes. We have also observed that addition of 50 μM CaCl2to the material layered onto the gradient gives purer vesicle fractions.

Using the method we have described (Morgan et al., 1973a) synaptic vesicles can be routinely prepared around 90% pure. Analogous methods give synaptic vesicles in which acetylcholine (De Robertis, Rodriguez De Lores Arnaiz, Salganicoff, Pellegrino De Iraldi & Zieher, 1963: Whittaker et al., 1964) and the catecholamines (Michaelson, Whittaker, Laverty & Sharman, 1963; Maynert, Levi & De Lorenzo, 1964; De Robertis, Pellegrino De Iraldi, Rodriguez De Lores Arnaiz & Zieher, 1965) are concentrated, but there is little evidence for the association of other transmitters with these vesicles (RASSIN, 1972). This could be due to loss during preparation, but in any case it seems likely that the synaptic vesicles are intrinsically heterogeneous, since they are possibly derived from the whole synaptic population of the brain.

Fragments of the synaptosomal plasma membrane tend to be distributed all over the gradient, but with most of the membrane in the E, F, G and H fractions. Superimposed on this distribution are the synaptic vesicles in fractions D and E, myelin in fractions F and G and mitochondria in fractions G, H and I. We have found that centrifugation of the crude fractions using around the same centrifugation conditions as were used for preparing the crude mitochondrial fraction removes most, if not all, of the contaminating myelin—but with a marked loss of synaptosomal plasma membrane as well (Morgan et al., 1971). Other workers do not appear to have had the same problems with myelin contamination (Cotman, Blank, Moehl & Snyder, 1969; Cotman & Matthews, 1971).

Similar centrifugations appear to eliminate most contaminating mitochondria from fractions F and G. An alternative procedure has been used by Davis & Bloom (1970; 1973) and Cotman & Taylor (1972) which exploits a histochemical reaction for succinate dehydrogenase. The reaction product weighs down the mitochondria, thus improving the resolution of the gradient. This technique has been particularly used for the preparation of synaptosomal plasma membranes from which synaptic junctions are to be prepared, since there is some evidence (as yet not quantitative) that denser fractions of synaptosomal plasma membranes contain more synaptic junctions.

Using our technique (Morgan et al., 1971) the purest synaptosomal plasma membrane fractions have been in fractions F and G—as measured by the levels of ganglioside sialic acid and (Na + K)ATPase. The improvement of this technique, and those of others (Cotman & Matthews, 1971; GURD et al., 1974; Levitan, Mushynski & Ramirez, 1972) over the more classical procedures (Whittakeret al., 1964, De Robertis, Alberici, Rodriguez De Lores Arnaiz & Azcurra, 1966) can be appreciated from the markedly greater enrichments of these markers, which for the moment we will assume to be overwhelmingly neuronal. Exhaustive testing in several laboratories for markers of possible contaminants suggests that there is some contamination with external mitochondrial membrane, Golgi apparatus and smooth endoplasmic reticulum, but other contaminants can largely be eliminated (Morgan et al., 1971; Morgan, Breckenridge, Vincendon & Gombos, 1973b; Cotman & Matthews, 1971; Jones, Mahler & Moore, 1975; GURD et al., 1974). Synaptosomal plasma membranes can be prepared 70–90% pure as plasma membrane but it must be borne in mind that there are several uncertainties in this calculation. The estimation of glial plasma membrane contamination is difficult but several estimates suggest that it may be somewhat between 5 and 20% (Morgan et al., 1972; 1973b). There is then the unsolved problem of distinguishing between synaptosomal, axonal and cell body plasma membranes. The preparations could therefore be anywhere from 50 to 80% pure. There is some support for the higher figure from morphological studies (Jones & Matus, 1974).

Finally, it must be remembered, as for synaptic vesicles, that the synaptosomal plasma membranes are intrinsically heterogeneous in that they are derived from the total neuronal population. Thus, even more than in the case of the synaptic vesicles, only the common major constituents of synaptosomal plasma membranes can be studied.

Mitochondria can also be isolated from synaptosomal fractions. Given that these fractions are heavily contaminated with extra-synaptosomal mitochondria, it is perhaps surprising that synaptosomal mitochondria can be prepared. However two populations of mitochondria can be isolated from brain, one of which resembles, in its high docosahexaenoic acid content, synaptic vesicles and synaptosomal plasma membranes, and differs significantly from the total brain mitochondrial population in this respect (Breckenridge, Gombos & Morgan, 1972; Brenckenridge, unpublished results). To what extent high levels of docosahexaenoic acid are characteristic of synaptosomal, or more generally of neuronal mitochondria remains to be established.

Despite the critical nature of my comments on the classical techniques for preparing synaptosomes and synaptosomal subfractions, the techniques of Whittaker and De Robertis both give synaptosomal preparations which are as good as any available. For many purposes, the preparations of synaptic vesicles and synaptosomal plasma membranes obtained by the method of Whittaker et al. (1964) are quite satisfactory, but more care is needed when using the methods of De Robertis and colleagues (for review, see De Robertis & Rodriguez De Lores Arnaiz, 1969). Whittaker et al. (1964) discard material sedimentable at low speeds before applying the shocked synaptosomes to a gradient, and our approach has been modelled on this. However De Robertis and his colleagues use the discarded material to prepare synaptosomal plasma membranes, and in our experience this leads to massive contamination with myelin (Morgan et al., 1971). That this may in fact be the case is borne out by the lipid analyses of synaptosomal plasma membranes reported by the Argentinian group (Lapetina, Soto & De Robertis, 1967, 1968). In addition, their simple differential separation of synaptic vesicles and synaptosomal plasma membranes is not as satisfactory as gradient separation, as has been implicitly recognised by the later introduction of modifications to their methodology (Lapetina et al., 1967).

The most recent development in this field, the dissection of the synaptosomal plasma membrane to obtain synaptic junctions and post-synaptic densities was pioneered by De Robertis, Azcurra & Fiszer (1967). Progress was hampered by the failure of other workers to appreciate the importance for the preservation of synaptic morphology of the small amounts of Ca²+ used by the De Robertis group. However, in 1970, Davis and Bloom reported a method which has become the basis for most methods for preparing synaptic junctions (Davis & Bloom, 1973; Cotman & Taylor, 1972). There are some unanswered questions about these preparations: primarily, the degree of purity and the degree of preservation of synaptic morphology or in other words what is there that should not be there, and what is not there that should be. Some progress has been made towards answering these questions—the degree of purity seems only moderate and the preservation of ultrastructure, and more importantly, of composition is also only moderate (Morgan, unpublished results). Walters & Matus (1975) have introduced a method for preparing ‘synaptic junctions’ using sodium deoxycholate instead of the classical Triton X-100, but the resulting particles, while undoubtedly of synaptic origin, appear to be highly disrupted. They may in fact be fundamental units of the post-synaptic density. Cotman, Banker, Churchill & Taylor (1974) have recently reported a method for preparing post-synaptic densities by treating synaptosomal plasma membranes or synaptic junctions with sodium lauroyl sarcosinate. The purity, based on morphological data, of these fractions is quite high. Much more work needs to be done to clarify the nature of these preparations, but there is probably a range of purities obtainable, depending upon the conditions used, and higher purities are probably obtained at the expense of synaptic morphology and composition.

From all these studies on synaptic plasma membranes, vesicles and mitochondria several points are clear. The lipid composition of these fractions shows several important features; notably a high concentration of polyunsaturated fatty acids (particularly docosahexaenoic acid) in the serine and ethanolamine phosphoglycerides, and the virtual absence of long chain fatty acids from sphingomyelins. In addition the synaptosomal plasma membrane contains high concentrations of gangliosides, (Na + K)ATPase and neuraminidase (Morgan et al., 1973b; Morgan, Zanetta, Breckenridge, Vincendon & Gombos, 1973c).

The unsolved question is to what extent these characteristics are specific to synaptic structures, more generally specific to neuronal structures, or even more generally distributed throughout the brain. The evidence, albeit far from conclusive, favours a neuronal localization of these constituents (which in no way excludes a more specifically synaptosomal localization) and should be taken into account when assessing the validity of methods for separating neurons and glial cells from brain.

SEPARATION OF NEURONS FROM GLIA

Numerous methods have been published for these separations (for reviews see Rose, 1969; Poduslo & Norton, 1972; Sellinger & Azcurra, 1974), some of which have been extensively characterized morphologically—particularly at a light microscope level. However, while this is adequate to demonstrate that the two types have been resolved satisfactorily, it is not sufficient to determine the degree of purity of the fractions obtained since many types of subcellular contamination would not be seen by this method. I will now proceed to argue that there is a strong case for believing that glial cell fractions, as presently isolated, are highly contaminated with synaptosomes (or similar particles), and that until this problem is resolved little reliance can be placed on many of the results obtained using these fractions.

If (Na + K)ATPase is primarily neuronal, then it would be expected that neuronal cell bodies would contain more of the enzyme than glial cells. However, the results obtained on separated cells show that the glial cells contain higher ATPase activities (Medzihradsky, Nandhasri, Idoyaga-Vargas & Sellinger, 1971). Glial cells in tissue culture do not appear to have high (Na + K)ATPase activities (Embree, Hess & Shein, 1971; Cotman, Herschman & Taylor, 1971) but the strength of this argument is weakened by the fact that neuroblasts, or neuroblastoma cells have even lower (Na + K)ATPase activities (Kimelberg, 1974, M. Sensenbrenner, personal communication). Thus high ATPase could be a sign of a differentiated cell, although attempts to induce ATPase activity under differentiating conditions in these tissue culture systems have been unsuccessful. Alternatively, (Na + K)ATPase might be relatively specifically synaptic in its localization.

Similarly, isolated glial cells have higher ganglioside levels than do neuronal cell bodies (Norton & Poduslo, 1971; Hamberger & Svennerholm, 1971). The two ganglioside patterns are qualitatively essentially the same. Glial cells in tissue culture contain much lower levels of simpler gangliosides (Shein, Britva, Hess & Selkoe, 1971; Dawson et al., 1971 Robert et al., 1975), as do glial cells isolated by micro-dissection (Derry & Wolfe, 1967). Unfortunately neuroblasts or neuroblastoma cells in culture do not contain markedly different gangliosides (Rebel, Treska-Ciesielski & Mandel, 1973; Yogeeswaran et al., 1973; Dawson et al., 1971), so that the gangliosides of total brain could be related to differentiated cells rather than to neurons. However differentiating conditions do not provoke the ‘appropriate’ changes (Yogeeswaran et al., 1973).

There is moreover one strange observation: isolated glial cells, despite their high ganglioside levels, do not appear to be able to synthesize gangliosides whereas isolated neuronal cell bodies can (Jones, Ramsey, Aexel & Nicholas, 1972; Radin, Brenkert, Arora & Sellinger, 1972). This apparent contradiction could be explained if the gangliosides detected in the isolated glial cells were primarily associated with contaminating synaptosomes, since there are indications that synaptosomes are supplied with gangliosides by anoxal flow (Forman & Ledeen, 1972). However it has also been claimed that synaptosomes can synthesize gangliosides (Di Cesare & Dain, 1971, 1972; Den, Kaufman & Roseman, 1970) so this argument is far from unequivocal. The other characteristics of the lipid compositions of the various cell types have not yet been studied in sufficient detail to enable even tentative conclusions to be drawn.

A better way of testing the purity of isolated neuronal and glial cells would be to look for less equivocal markers. Probably the brain-specific proteins like S-100 and 14-3-2 (for review see MOORE, 1972) and the glial fibrillary acidic protein (Dahl & Bignami, 1973a, b) could be used for this. However the studies so far performed are limited (Packman, Blomstrand & Hamberger, 1971), and are complicated by problems of diffusion and the uncertainty of the localization of the proteins.

One point on which there may be general agreement is the probable localization of enzymes involved in the synthesis of transmitters within neurons. It is therefore disturbing that several transmitter-synthesizing enzymes, choline acetyltransferase, glutamic acid decarboxylase, tyrosine hydroxylase, plus other enzymes such as acetylcholinesterase (Hemminki, Hemminki & Giacobini, 1973; Arbogast & Arsenis, 1974; Nagata, Mikoshiba & Tsukada, 1974) are found in isolated glial cells in high specific activities. From the levels detected, up to 30–50% of the protein of the isolated glial cell fractions could be derived from synaptosomes, or other neuronal material.

These studies are also not unequivocal since enzymes can diffuse, or be absorbed to membranes, However they seem to provide the strongest evidence that the isolated glial cells are quite heavily contaminated. Thus, for the moment, it would seem to be unwise to use these isolated glial cells for preparing glial cell plasma membrane, or any other glial structure. Preparations of neuronal cell bodies seem to be purer and can probably be used with caution for such studies.

CONCLUSIONS

This commentary on the present state of analysis of the isolation of synaptosomes and cell fractions may be somewhat pessimistic—but it is a measure of our progress that we can now start to raise in a precise form the questions which have to be answered when using these techniques.

Studies using synaptosomal fractions and subfractions have been clarified by a more precise knowledge of the likely contaminants of these fractions, since some of the results in the literature can be explained by problems of contamination. Moreover, the knowledge of the degree of purity of the fractions enables limits to be set to the experiments which can be meaningfully done with these fractions (for a more detailed discussion, see Morgan & Gombos, 1975). Some workers have recognised the problems of contamination which arise when using neuronal and glial (or neuropil, ROSE, 1969) fractions, and when the limitations involved are fully recognised, isolated cells will undoubtedly be as useful as synaptosomes have proven to be.

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THE COMPOSITION OF ADRENAL CHROMAFFIN GRANULES: AN ASSESSMENT OF CONTROVERSIAL RESULTS¹

HANS WINKLER,     Dept of Pharmacology, University of Innsbruck, A– 6020 Innsbruck, Austria

Publisher Summary

Considerable work has been done on the biochemical composition of the catecholamine-storing organelles of adrenal medulla, the so-called chromaffin granules. Chromaffin granules, especially those of bovine adrenal medulla, are, therefore, well characterized and provide a basis of comparison for results obtained with other storage organelles. Studies on the biochemical composition of chromaffin granules require methods of isolation that combine both good yield and high purity. These two criteria are met by a simplified density gradient procedure in which the cell particles of adrenal medulla are centrifuged on a layer of 1.6 M sucrose solution. Chromaffin granules are obtained as sediment that is only slightly contaminated by other cell particles. A minor modification of the method reduces the mitochondrial contamination.

Introduction

Isolation of chromaffin granules

Methods for isolation

Enzyme markers for chromaffin granules

Composition of isolated chromaffin granules

Overall composition

Composition of the soluble content and of the membrane of chromaffin granules

Separation of the membrane from the content

Soluble and membrane-bound constituents

Lipids’ of chromaffin granules: constituents of the membrane and/or of content?

The proteins of chromaffin granules

Soluble proteins:

Chromogranin A and the other acidic chromogranins

Dopamine β-hydroxylase

The relationship between dopamine β-hydroxylase and chromogranin

A Membrane proteins of chromaffin granules:

Characterisation and isolation

The presence of chromogranin A in membranes

The lipids of chromaffin granules

Conclusions

Considerable work has been done on the biochemical composition of the catecholamine-storing organelles of adrenal medulla, the so-called chromaffin granules. This is not surprising since these cell particles are less difficult to isolate than other hormone or transmitter storing organelles. Chromaffin granules, especially those of bovine adrenal medulla, are therefore well characterised and provide a basis of comparison for results obtained with other storage organelles. Thus, for example, the composition of the noradrenaline-storing vesicles of sympathetic nerve has been found to be similar to that of chromaffin granules (Lagercrantz, 1971; Lagercrantz, 1976) and even cholinergic vesicles exhibit some related properties (Whittaker, 1974). Unfortunately quite a few of the published results on chromaffin granules are contradictory, which reduces the usefulness of this preparation as a ‘standard’ organelle. It is the main purpose of this review to assess these controversial results and if possible to establish the genuine properties of these organelles.

Since the writer himself is engaged in this field there is of course the danger of his being biased in drawing conclusions. In order to avoid this as much as possible a great effort has been made to compile all data available on a controversial point and to reach a conclusion only in case of overwhelming results. On the other hand if there was general agreement on one particular point no attempt has been made to give a complete list of references. Further references can be found in recent reviews which also discuss the functions of the various constituents of chromaffin granules (Smith, 1968; Stjärne, 1972; Smith & Winkler, 1972; Kirshner, 1974; Helle & Serck-Hanssen, 1975; Winkler & Smith, 1975).

ISOLATION OF CHROMAFFIN GRANULES

Methods for isolation

Studies on the biochemical composition of chromaffin granules require methods of isolation which combine both good yield and high purity. These two criteria are met by a simplified density gradient procedure in which the cell particles of adrenal medulla are centrifuged on a layer of 1.6 M sucrose solution (Smith & Winkler, 1967a, see Bartlett & Smith, 1974). Chromaffin granules are obtained as a sediment, which is only slightly contaminated by other cell particles. A minor modification of the method (larger volume of the 1.6 M sucrose solution) reduces the mitochondrial contamination (Helle, Flatmark, Serck-Hanssen & Lönning, 1971). By increasing the molarity of the sucrose solutions (e.g. 1.8 M: Schneider, 1972) one can enhance the purity of the granule preparation; however one has to be satisfied with a lower yield. The chromaffin granules purified by these procedures are a mixture of granules containing either noradrenaline or adrenaline. Pure noradrenaline granules can be obtained when the particles are centrifuged on 2.2 M sucrose solution (Winkler, 1969). An isolation of chromaffin granules under isoosmotic conditions is achieved by the replacement of the 1.6 M sucrose with Ficoll/D2O (Trifaró & Dworkind, 1970), whereas the Millipore filter method recommended for this purpose (Oka, Ohuchi, Yoshida & Imaizumi, 1966) was subsequently shown to give granules of low purity (Trifaró & Dworkind, 1970).

Enzyme markers for chromaffin granules

Even highly purified granules are not completely free from contamination with other cell particles. Therefore, if one attempts to establish the presence of a certain constituent in chromaffin granules it is not sufficient to demonstrate the occurrence of this particular component in a preparation of ‘pure’ granules. The only reliable approach is to compare the distribution of the component in question with that of markers for the various cell particles in fractions obtained by density gradient centrifugation. In fact, such an application of sucrose gradient centrifugation has already proved very useful for characterizing the various cell particles from adrenal medulla (Blaschko, Hagen & Hagen, 1957; Hillarp, 1958c; Banks, 1965). For a gradient ranging from 1.3 to 2.0 M sucrose solution the distribution of various markers for mitochondria, lysosomes, elements of the endoplasmic reticulum and chromaffin granules has been established (Smith & Winkler, 1966; Smith & Winkler, 1968). Results from several other laboratories have confirmed these findings (Laduron & Belpaire, 1968; Kirshner, 1969; Schneider, 1970).

Table 1 compiles some references to papers which deal with the occurrence of enzymes in chromaffin granules. There appears to be overwhelming evidence that chromaffin granules contain dopamine β-hydroxylase, Mg++-activated ATP-ase (also Activated by Ca++), cytochrome b-561, a NADH: oxidoreductase and phosphatidylinositol kinase. For most of these enzymes convincing evidence for their presence in chromaffin granules was obtained by sucrose density gradient centrifugation in which the distribution of the enzyme was compared with that of chromaffin granules. In the case of adenylate kinase the data of Lagercrantz, Kuylenstierna & Stjärne (1970) strongly indicate that this enzyme is absent from chromaffin granules.

TABLE 1

ENZYMES IN BOVINE CHROMAFFIN GRANULFS

References supporting the presence of a particular enzyme in chromaffin granules are given on the left, whereas those indicating the absence of an enzyme are given on the right.

Apart from dopamine β-hydroxylase, chromaffin granules do not contain enzymes involved in the synthesis or degradation of catecholamines (see Table 1). There is overwhelming agreement that these organelles are also free of enzymes typical for other cell organelles like succinic dehydrogenase (mitochondria), acid phosphatase and acid ribonuclease (lysosomes), glucose-6-phosphatase (endoplasmic reticulum), galactosyltransferase (Golgi complex) and 5′-nucleotidase (plasma membrane).

It would be useful to present quantitative data for the activity of the various enzymes in chromaffin granules. However for dopamine β-hydroxylase this is rather difficult since this enzyme has been measured at various, and not saturating, substrate concentrations; thus the values published are not easily comparable. With a substrate (tyramine) concentration of 10 mM Helle (1971a) finds an activity of 670 nmol/min/mg protein (calculated from her Table III), whereas Aunis, Miras-Portugal & Mandel (1973) report a value of 280 (calculated from their Table I). With reduced substrate concentration much lower values have been reported (Winkler, HÖRtnagl, HÖRtnagl & Smith, 1970; De Potter, Smith & De Schaepdryver, 1970; Schneider, 1972).

The ATP-ase activity of chromaffin granules according to Banks (1965) amounts to 28.5 nmoles/mg protein/min (37°C), Taugner (1971b) reports values ranging from 16.7 to 26.8 (at 31°C) depending on the incubation medium, whereas Trifaró & Dworkind (1971) arrive at a value of 15.9 (at 31°C). For the ATP-ase activity of the granule membrane, which contains all of the enzyme but only about 20% of the total protein (see below), Winkler et al. (1970) found a value of 30 nmol/mg protein/min and Schneider (1972) one of 47 which are obviously rather low activities. For this preparation Banks (1965) gives a figure of 105, Taugner (1971a) one of 155, Taugner & Wähler (1974b) one of 120, whereas Pollard et al. (1973) published the rather high figure of 250, which agrees with a value of 282 given by Muller & Kirshner (1975). Thus the values reported do not agree well, which suggests that the enzyme may be rather unstable.

COMPOSITION OF ISOLATED CHROMAFFIN GRANULES

Overall composition

A first complete analysis of bovine chromaffin granules was performed by Hillarp (1959) and his results are given in Table 2. This composition has been generally accepted and apparently no further complete analyses have been performed. However, during the last few years numerous data on the various components of chromaffin granules have been published which can be used for recalculating the composition of total granules. Table 2 presents such a composition computed from the data discussed in the following paragraphs.

TABLE 2

COMPOSITION OF ISOLATED BOVINE CHROMAFFIN GRANULES

The values in the left column (a) are from Hillarp (1959), those in the two columns on the right (b,c) were computed from the results quoted in the text. Molecular weights (MW) used for these calculations are 180 for catecholamines, 750 for phospholipids and 507 for nucleotides.

Most of the reported data on the catecholamine concentration of bovine chromaffin granules isolated by density gradient centrifugation fall into a range of 2.0–2.9 μmol catecholamines/mg protein (Banks, 1965; Smith & Winkler, 1967a; Trifaró & Dworkind, 1970; Serck-Hanssen & Christiansen, 1973; Da Prada, Berlepsch & Pletscher, 1972a). The mean value obtained from these figures from 5 laboratories is 2.5 μmol catecholamine/mg protein. In contrast, Hillarp (1958c) reports a value of 5.5 μmol catecholamines/mg protein (34.7 μmol/mg protein-N) which is completely outside the range given above. This is rather peculiar since from the figures given by Hillarp (1959) on the dry weight (see Table 2) one arrives at a value of 3.3 μmol catecholamines/mg protein, which is high when compared with the other figures, but considerably lower than the value of 5.5. Since there is good agreement between many laboratories the value of 2.5 can be considered to be the most representative one and this value was used for Table 2. Catecholamine concentrations for chromaffin granules of other species including those of a human phaeochromocytoma have already been reported (Smith & Winkler, 1967a; Blaschko, Jerrome, Robb-Smith, Smith & Winkler, 1968). In addition to noradrenaline (27% of total catecholamines) and adrenaline (72%: Eade, 1958), adrenal chromaffin granules contain also trace amounts of dopamine. Eade (1958) showed that this compound was present (0.8% of total catecholamines) in a large granule fraction from bovine adrenal medulla (see Lishajko, 1970, for sheep adrenal). After density gradient centrifugation of such a fraction the distribution of dopamine matched that of the other catecholamines (Snider & Winkler, unpublished observation) indicating that this dopamine was present in chromaffin granules.

Nucleotides are found in chromaffin granules in a high concentration. ATP is the major component (see Table 3) but significant amounts of ADP and AMP were also discovered (Hillarp & Thieme, 1959). Table 3 presents the values for bovine granules. In other species the relative amounts of these nucleotides vary, for example in granules from fowl adrenal AMP and ADP represent up to 40% of the total adenine nucleotides (Hillarp & Thieme, 1959). In addition (see Table 3) bovine chromaffin granules contain guanine and uridine nucleotides (Goetz, Da Prada & Pletscher, 1971). If one attempts to relate the nucleotide content to the other components of bovine granules it is best to use the frequently published molar catecholamine/ATP ratios. There is general agreement that this ratio lies between 4.2–4.85 with a mean value of 4.5 calculated from results of four groups (Hillarp, 1958b; Banks, 1965; Smith & Winkler, 1967a; Trifaró & Dworkind, 1970). Similar ratios were obtained for other species (Hillarp & Thieme, 1959) and for noradrenaline granules from pig adrenal (Winkler, 1969); however an extremely wide range of values (4.7–35) has been reported for human phaeochromocytoma granules (see Winkler & Smith, 1972). For Table 2 the total nucleotide content was calculated from the catecholamine/ATP ratio (4.5) and from the relative amount of ATP (70%) of the total nucleotides.

TABLE 3

NUCLEOTIDES IN BOVINE CHROMAFFIN GRANULES

The main lipids in bovine chromaffin granules are cholesterol and phospholipids (Blaschko, Firemark, Smith & Winkler, 1967; Winkler, Strieder & Ziegler, 1967), only traces of fatty acids, triglycerides and cholesterolesters are found (Winkler et al., 1967). Three research groups agree on a phospholipid content of total granules amounting to 0.45–0.51 μmol lipid phosphorus/mg protein (mean value: 0.48; Blaschko et al., 1967b; Winkler et al., 1967; Mylroie & Koenig, 1971; calculated from their Table 1; Helle & Serck-Hanssen, 1975), whereas a value of 0.29 is reported from another laboratory (calculated from Trifaró, Poisner & Douglas, 1967; Trifaró & Dworkind, 1970). A better agreement exists on the molar cholesterol/phospholipid ratios of total granules: values ranging from 0.53 to 0.68 (mean value: 0.60) have been published (Blaschko et al., 1967b; Winkler et al., 1967; Trifaró & Dworkind, 1970; Mylroie & Koenig, 1971).

Recent studies have established that chromaffin granules contain mucopolysaccharides. The first evidence was provided by Fillion, Nosal & Uvnäs (1971), who demonstrated that chromaffin granules can be labelled by ³⁵S-sulfate (see also Margolis, Jaanus & Margolis, 1973; Baumgartner, Gibb, HöRtnagl, Snider & Winkler, 1974). The main components of these mucupolysaccharides are chondroitin-4-sulfate and chondroitin-6-sulfate (Margolis & Margolis, 1973). Quantitative data are still scarce and not in good agreement. Margolis & Margolis (1973) find 15 μg mucopolysaccharides/g protein, whereas a value of 53 μg/g protein can be calculated (based on molecular weight for the disaccharide repeating unit of 456) from the data for uronic acid given by Da Prada et al. (1972a).

Calcium is a minor constituent of chromaffin granules, however the concentration is significantly higher than for example in mitochondria (Borowitz, Fuwa & Weiner, 1965; Borowitz, 1969). In a large series of determinations (Borowitz, 1967) a calcium content of 76 nmol calcium/mg protein was found and this value (n=25) was used for Table 2. However, significantly higher values (150 nmol/mg protein) were reported by Serck-Hanssen & Christiansen (1973: calculated from their Table II and IV, n = 7). Ascorbic acid is the most recently discovered component of chromaffin granules (Terland & Flatmark, 1975). A relatively high concentration of this component is present in these organelles (see Table 2).

Ribonucleic acid was considered to be a minor constituent of chromaffin granules by Hillarp (1958c) and by Philippu & Schümann (1964). However, the distribution of ribonucleic acids in density gradients after centrifugation of medullary cell particles indicates that this compound is confined to elements of the rough endoplasmic reticulum (Kirshner, 1969).

In conclusion, there is a surprisingly good agreement between the composition of chromaffin granules as given by Hillarp (1959) and that calculated from results which have been published subsequently by the numerous groups engaged in research on chromaffin granules. For most constituents, the values that are presented are supported by ample data. The available results for calcium and mucopolysaccharides are conflicting.

Composition of the soluble content and of the membrane of chromaffin granules

In this section we will assign the various granule constituents either to the content or to the membrane of these organelles. In order to achieve this we first have to discuss methods for separating these two components.

Separation of the membrane from the content.

When chromaffin granules are subjected to hypotonic media they are lysed and their content goes into solution. High speed centrifugation of such a suspension yields a sediment which as judged from electron micrographs consists of membranes without the osmiophilic contents (Winkler et al., 1972a; Helle, 1971a). Membranes can be further purified by several washes (Winkler, et al., 1970) or by centrifugation through sucrose gradients (Schneider, 1972; Phillips, 1973; Helle, 1973a; Muller & Kirshner, 1975). This latter method was introduced by Schneider (1972). He used a gradient ranging from 0.4 to 1.6 M sucrose solution and recovered the granule membranes, which were identified by their high activity of dopamine β-hydroxylase, from a layer just above 1.0 M sucrose. A similar density of the granule membranes was observed by Phillips (1973) and Muller & Kirshner (1975). The membranes of chromaffin granules are purified by this procedure since membranes of contaminating cell particles (mitochondria, microsomes) equilibrate at a higher sucrose molarity. Helle (1973a), on the other hand, obtained different results when the membranes were subjected to a cycle of freezing and thawing before centrifugation on the gradient. In this case a bimodal distribution of the membranes was obtained whereas membranes not pretreated in this way exhibited only one peak (at 1.1M sucrose). The second peak in the bimodal distribution was present in a region of the gradient corresponding to 0.4 M sucrose. It contained most of the phospholipids, some dopamine β-hydroxylase, but little protein. It cannot be stated whether there is a discrepancy between the results obtained by Helle (1973a) and those by the other authors, since it is not apparent whether they employed freezing and thawing in their procedure. In any case, for obtaining pure membranes of chromaffin granules it seems advisable first to isolate chromaffin granules over 1.8 M sucrose (Schneider, 1972) and then to purify their membranes through a sucrose density gradient.

Soluble- and membrane-bound constituents of chromaffin granules.

After lysis of the granules and removal of the membranes catecholamines and nucleotides are quantitatively recovered in the supernatant which is consistent with their presence in the content of these organelles (Hillarp & Nilson, 1954). A major part of the proteins is also soluble and can therefore be assigned to the same compartment. For bovine granules Hillarp (1958c) reported that 77% of the total proteins were soluble. Further values are 70% (Winkler, Ziegler & Strieder, 1966), 76.8% (Taugner, 1971a) and 83% (Schneider, 1972) giving a mean value of 77%. Helle (1971a) found that the percentage of soluble protein under different conditions varied from 57 to 85%. A high proportion of soluble proteins is also typical for chromaffin granules of other species, e.g. those of horse (74%), pig (57%: Winkler et al., 1966) and human phaeochromocytoma (63%: see Winkler & Smith, 1972).

As far as the enzymes are concerned most of them are confined to the membrane, i.e. ATP-ase (Banks, 1965), cytochrome b-561 (Banks, 1965; Flatmark, Terland & Helle, 1971), Nadh: oxidoreductase (Flatmark et al., 1971) and phosphatidylinositol kinase (Phillips, 1973; Muller & Kirshner, 1975). Dopamine β-hydroxylase, on the other hand, is found both in the content and in the membrane. In bovine granules about 50% of the total activity is recovered in the membrane (Belpaire & Laduron, 1968; Winkler et al., 1970; Helle, 1971a; Schneider, 1972), whereas in other species this figure varies (rabbit: 42%; rat: 74%; Kirshner & Viveros, 1970; human phaeochromocytoma: 35%; Stone, Kirshner, Reynolds & Vanaman, 1974).

The major part (64%) of the calcium in chromaffin granules is released by hypotonic lysis (Borowitz, 1967) and seems therefore to be contained within these vesicles. Similarly, at least some of the mucopolysaccharides are present in the soluble content of the granule since ³⁵S-sulfate labels this compartment rather specifically (Baumgartner et al., 1974). Furthermore, ³⁵S-labelled mucopolysaccharides can be secreted from the adrenal medulla together with the catecholamines (Margolis et al., 1973).

Lipids of chromaffin granules: constituents of the membrane and or of the content?

This is a particularly controversial subject since the divergence of opinions ranges from lipids are only membrane components to a major part of the lipids is present in soluble lipoproteins. This latter view has been put forward by Mylroie & Koenig (1971; see also Koenig, 1974). The latter author’s usual procedure for isolating soluble lipoproteins from chromaffin granules consists of treating isolated granules with Triton X-100 and ultrasonication followed by centrifugation for 30 min (g-value not given). Under these conditions about 82% of the total phospholipids together with 68% of the proteins appear soluble. However, the authors claim that even without ultrasonication and Triton, 40–65% of the protein and 30–61% of the phospholipids are recovered in the soluble fraction. Taugner & Wähler (1974a) also state that 50% of the lipids belong to the soluble phase. However, since their claim is not based on a direct measurement but on a calculation depending on complete recovery, it is impossible to evaluate their data.

Helle (1968) has also claimed that the soluble proteins (chromogranins: see below) of chromaffin granules are lipoproteins. In her first paper on this subject (Helle, 1968) she reports that the soluble proteins contain 0.2–0.4 μmol protein-bound phosphate. However, in a later paper (Helle, 1971b) she states that the phosphate (0.2 μmol/mg protein) bound to the major soluble component (chromogranin Aws) was not extractable by lipid solvents. These results hardly therefore support the lipoprotein nature of the soluble proteins since chromogranin A comprises 40% of these proteins (see below). As far as the distribution of phospholipids between the soluble content and the membranes are concerned, Helle has published several results. In one paper 80% of the phospholipids are reported to sediment with the insoluble residue (Helle 1971a). However, in a later paper (Helle, 1973a) she finds that the phospholipid content of the soluble phase varies with the treatment (see Table 1 of Helle 1973a). The soluble phase of granules lysed by dialysis against hypotonic buffer contained no phospholipids (S1D, her Table 1) whereas the soluble phase obtained only by lysis and centrifugation contained about 12% of the total phospholipids (calculated from her Table 1: Sl). In any case these results do not support those of Koenig (1974) but do agree with our own experiments. More than 90% of the phospholipids were recovered in the insoluble residue obtained by lysis of the granules, freezing and thawing and high speed centrifugation (Winkler et al., 1970). For pig granules a value of 99% was reported (Winkler, 1969). However, these granules were subjected to freezing-thawing which was not used by Mylroie & Koenig (1971). Therefore, experiments under the conditions used by the latter authors were performed (Winkler et al., 1972a). When the suspension of lysed granules was centrifuged for 30 min at 180,000 g, 13.3% of the phospholipids remained in the supernatant; however this was further reduced to 1.4% when centrifugation time was extended to 60 min.

In conclusion, as far as the sedimentability of phospholipid in preparations of lysed granules is concerned the results of Mylorie & Koenig (1971) are not supported by two other research groups. I would like to suggest that the high degree of phospholipid solubilisation reported by these authors is due either to the use of ultrasonication and the detergent Triton and/or to the application of insufficient centrifugal force.

This, however, is not yet the complete story