Cell Tissue Res (1982) 223:73-86 Cell and Tissue Research ~, Springer-Verlag 1982

Dibutyrylic cyclic AMP and theophylline inhibit proliferation of accessory cells in primary cultures of adrenomedullary cells

K. Unsicker and W. Ziegler*

Department of Anatomy and Cell Biology, Philipps-Universit~it Marburg, Marburg, Federal Republic of Germany

Summary. Studies on isolated adrenal chromaffin cells in primary cultures may be seriously hampered by the presence of non-chromaffin, mainly fibroblast- like cells, which always occur in dissociates of adrenal medullary tissue and often outnumber the chromaffin cells by the end of the first week of culture, when no measures are taken to control their proliferation. The present study offers a new means to inhibit effectively the proliferation of these accessory cells by treating the cultures with dibutyrylic cyclic AMP (dbcAMP, 0.1 or 0.01 mM) and equimolar amounts of the phosphodiesterase inhibitor theophylline. With this treatment cultures of young ra t adrenal chromaffin cells remain virtually free of accessory cells for two weeks of culture. Cultures of bov ine adrenomedullary cells retain their initial amounts of non-chromaffin cells, which largely depends upon whether the primary cell suspensions have undergone differential plating prior to seeding. Suppression of accessory cell proliferation with dbcAMP and theophylline is partly due to maintaining differentiation of cortical cells, which otherwise dedifferentiate into rapidly dividing fibroblast-like elements. However, a more direct action ofdbcAMP on accessory cells in terms of growth control is also conceivable. DbcAMP and theophylline in the doses applied do not impair the viability, ultrastructure and catecholamine-storing capacity of cultured chromaffin cells.

Key words: Adrenal medullary cells - Culture - Growth control of non- chromaffin cells - Dibutyrylic cyclic AMP - Theophylline

The adrenal medulla is a modified sympathetic ganglion with endocrine function that secretes catecholamines (CA) and opioid peptides. Isolated adrenal medullary cells maintained in culture have now become increasingly valuable tools to study

Send offprint requests to: Prof. K. Unsicker, Department of Anatomy and Cell Biology, Robert-Koch- StraBe 6, D-3550 Marburg, Federal Republic of Germany

* Present address: Strahlenbiologisches lnstitut der Universit~.t Mfinchen, Bavariaring 19, 8000 Mfinchen 2, Federal Republic of Germany

0302-766X/82/0223/0073/$02.80

74 K. Unsicker and W. Ziegter

important neuronal functions such as CA-synthesis and secretion (Hochman and Perlman 1976; Brooks 1977; Fenwick et al. 1978; Liang and Perlman 1979; Kilpatrick et al. 1980; Trifar6 and Lee 1980; Aunis and Garcia 1981), enzyme induction (Waymire et al. 1977; Kumakura et al. 1979), receptor regulation (Livett et al. 1979; Mizobe et al. 1979; Kumakura et al. 1980; Lemaire et al. 1981), opioid storage and release (Livett and Dean 1980; Livett et al. 1981; Wilson et al. 1981), and phenotypic plasticity (Unsicker and Chamley 1977; Unsicker et al. 1978, 1980; Livett et al. 1978a, b; Aunis et al. 1980; Tischler et al. 1980).

Such studies may be impeded by non-endocrine (synonyms: non-chromaffin, accessory) cells that may constitute up to 25 % of the total number of cells in primary bovine adrenal medullary cultures (Unsicker and Miiller 1981), consisting mainly of fibroblast-like elements. Various strategies including treatment with antimitotic agents (Kilpatrick et al. 1980), differential plating (Waymire et al. 1977; Kumakura et al. 1980; Unsicker and Miiller 1981) and ~-irradiation (Unsicker and Ziegler 1980; Ziegler et al. 1980) have been employed to eliminate accessory cells from these cultures. Each of these methods has its limitations. 5-fluoro- deoxyuridine, a widely used antimitotic agent, does not completely block proliferation of fibroblast-like cells in primary cultures of adrenal medulla (Kilpatrick et al. 1980). ~-Irradiation, although being highly effective and causing differentiation in terms of maintenance of the epinephrine/norepinephrine ratio rather than destruction of chromaffin cells (Miiller, Ziegler and Unsicker, unpublished), may bring about unexpected metabolic changes by ionization of molecules. Finally, purification of chromaffin cells by differential plating is accompanied by a large loss of cells and does not sufficiently reduce the amount of fibroblast-like cells in long-term cultures (Unsicker and Mtiller 1981).

Thus, at the present state of the development, new advances in the field of establishing and maintaining highly purified cultures of adrenal medullary cells are highly desired.

Materials and methods

Culture methods

Adrenal glands were obtained from 8 to 12 day-old rats (Hanover-Wistar strain) and adult cattle. After removing the cortex, adrenal medulla was enzymatically dissociated as previously described (Fenwick et al. 1978; see Unsicker et al. 1978, 1980, for details).

Isolated medullary cells of the rat adrenal were plated on collagen-coated coverslips in modified Rose chambers at an initial density of 50,000 or 100,000 cells, respectively, per chamber. Culture medium consisted of glucose-enriched medium 199, 20 % FCS and 20 lag/ml of gentamicin. A few cultures were supplemented with 50ng/ml nerve growth factor (NGF, 2.5S subunit, prepared from mouse submaxillary glands according to the method of Bocchini and Angeletti 1969). The cultures were run for 7 or 14 days, respectively, and divided into the following experimental groups: (1) controls; (2) medium containing 0.1 mM N 6, 02-dibutyryladenosine 3': 5'-phosphate, cyclic (dbcAMP; Serva, Heidelberg, F.R.G.) and an equimolar amount of the phosphodiesterase inhibitor theophylline (1,3- dimethylxanthine; Sigma, Munich, F.R.G.); (3) medium containing 0.01 mM dbcAMP and 0.01 mM theophylline. The experiments were performed at least in triplicates. At the end of the experiment the cultures were processed for catecholamine histochemistry or electron microscopy. For the identification of chromaffin and non-chromaffin cells, see below.

Bovine adrenal medullary cells were isolated using a modified version of the method described by Fenwick et al. (1978; see also Livett et al. 1976, 1978 a, b; Unsicker et al. 1980). Cells of the primary

Accessory cells in primary adrenomedullary cultures 75

suspension that contained 74.4 + 7.7 % chromaffin cells (Unsicker and Miiller 1981); (viability > 90 % as judged by trypan-blue exclusion) were either immediately plated at a density of 100,000 cells per dish on 35 nun Petri dishes (Falcon; coated with rat tail collagen or uncoated) or brought through four steps of differential plating that resulted in cultures containing an enriched fraction of chromaffln cells (89.5 + 5.4%) (Unsicker and Mfiller 1981). Both cultures prepared from crude cell suspensions and cultures obtained after differential plating were run for 7 or 14 days, respectively, and divided into the same experimental groups as cultures from rat adrenomedullary cells (see above). All cultures were run in triplicate and, at the end of the experiment, processed for catecholamine histochemistry or electron microscopy. Survival of chromaffin cells at the end of the experiment was determined by the trypan-blue dye-exclusion test and amounted to >90%, independent of the experimental group to which they belonged. Cultures were inspected daily by phase contrast optics, and photographs were taken with Agfapan 25 film.

Catecholamine histochemistry

After removal of the medium, cultures were incubated for 3 sec in ice-cold, buffered glyoxylic acid and processed according to De la Torre and Surgeon (1976).

Electron microscopy

Cultures grown on collagen were fixed for electron microscopy in 2.5% phosphate-buffered glutaraldehyde, washed in 0.1 M phosphate buffer, and postfixed with 2 % aqueous OsO4. After rapid dehydration cultures were embedded in Araldite (cf. Unsicker and Chamley 1977). Cells grown on plastic surfaces were removed using a "rubber policeman" and processed for electron microscopy in an identical fashion.

Cell counts

In cultures of rat adrenomedullary cells, where non-chromaffin cells were virtually absent after treatment with 0.1 mM dbcAMP and equimolar amounts of theophylline, no attempts were made to quantify the ratio of chromaffin to non-chromaffin cells. In one series of experiments the number of surviving chromaffin cells showing catecholamine-specific fluorescence were determined after 1 and 12days both in control chambers and in cultures treated with 0.1 mM dbcAMP and an equimolar amount of theophylline. Fields of 5 mm 2 were marked in each chamber, the cells were counted, and the total number of ceils per chamber was calculated.

In cultures of bovine adrenomedullary cells processed for catecholamine histochemistry, five randomly selected areas with a size of 0.125 mm 2 each were analyzed in triplicate sets of dishes both with fluorescence epiilluminescence (BG 3 or BG 12 (Schott) primary and Zeiss 47 + 50 secondary filters; Zeiss Universal microscope) and normal phase contrast illuminescence. Fluorescent chromaffln and non-fluorescent accessory cells were counted; figures obtained from the five areas were added, and the percentage of non-fluorescent cells in the triplicates was calculated as ~ + S.D. (Fig. 6a, b).

Results

Cultures o f rat adrenomedullary cells

W i t h i n the first 12 h a f te r p l a t i ng all a d r e n o m e d u l l a r y cells d i sp l ayed a r o u n d shape

a n d a re f rac t i le halo . A f t e r f l a t t en ing o u t S c h w a n n cells, co r t i ca l cells c h a r a c t e r i z e d

by an ep i t he lo id shape a n d la rge n u m b e r s o f cho les t e ro l d rop le t s , f ib rob las t - l ike p o l y m o r p h i c cells, a n d c h r o m a f f i n cells t ha t were read i ly iden t i f i ab le by the i r re f rac t i l e h a l o a n d the e x t e n s i o n o f p rocesses a f te r a d d i t i o n o f N G F (cf. U n s i c k e r et al. 1978) b e c a m e discernib le . In i t ia l ly , c h r o m a f f i n cells c o n s t i t u t e d 80 -90 % o f the

to ta l cell p o p u l a t i o n , wh ich was a lso ver i f i ed by c a t e c h o l a m i n e h i s t o c h e m i s t r y a n d e l ec t ron m i c r o s c o p y . Typ i ca l u l t r a s t r u c t u r a l fea tures o f co r t i ca l a n d c h r o m a f f i n

76 K. Unsicker and W. Ziegler

Fig. 1. Isolated rat adrenal cells cultured for a 12 h period. Both the chromaffin (ChC) and the cortical cells (CoC) display ultrastructural features typical of these cell types in vivo. After a two week-culture period and treatment with 0.I mM dbc AMP and theophylline, chromaffin and cortical cells still exhibit most of their characteristic features including chromaffin-storage vesicles (v), rough and smooth endoplasmic reticulum (rER, sER), tubular mitochondria (m), and lipid droplets (d). x 18,000

Accessory cells in primary adrenomedullary cultures 77

Fig. 2a, b. Phase-contrast micrographs of rat adrenal medullary cells cultured for 12 days a in the presence of nerve growth factor (NGF, 2.5 S, 50 ng/ml), and b with both NGF and 0.1 mM dbcAMP + theophylline. The main differences between both regimens of treatment concern (1) accessory fibroblast- like cells (J), which are abundant in the former case and virtually absent in the latter, and (2) the extension of neurite-like processes (p) from chromaffin cells (C), which is not seen under dbc AMP treatment (cf. Ziegler and Unsicker 1981). The inset shows cortical cells that have preserved their lipid droplets (arrows) in the dbcAMP-treated culture, x 400

cells inc luded tubu la r mi tochondr i a , smoo th endop lasmic re t iculum (sER) and the specific ca techo lamine-s to rage vesicles (Fig. 1). Wi th increasing t ime in cul ture cort ical cells d i sappea red and appa ren t l y a d o p t e d the m o r p h o l o g y o f f ibroblas t - like cells. The cholesterol d rople t s d i sappeared , tubu la r m i t o c h o n d r i a were replaced by cristae forms, and the sER was diminished. A t the end o f the first week o f cul ture small areas o f f ibroblas t - l ike cells were spread over the coversl ip. Af te r two weeks these cells fo rmed a con t inuous sheath, with chromaff in cells growing on top o f them (Fig. 2a).

T rea tmen t with 0.1 m M d b c A M P and an equ imola r a m o u n t o f theophyl l ine a lmos t comple te ly p reven ted the p ro l i fe ra t ion o f non-ch romaf f in cells in p r imary cul tures o f ra t adrena l medul lae . Af te r twelve days, only few f ibroblas t - l ike cells

78 K. Unsicker and W. Ziegler

Fig. 3a, b. Bovine adrenal medullary cells cultured for 4 days in the presence of 0.1 mM dbc AMP and theophylline (b) and without dbc AMP (a). Note destruction of fibroblast-like cells (]) by dbc AMP. Chromaffin cells (c). x 280

were found (Fig. 2b). Fibroblast-like cells were seen to cover a few scarce disseminated fields after two weeks, but never formed a confuent layer. Interestingly, cortical cells retained their typical light- and electron-microscopical appearance (cf. Fig. 2 b inset, Fig. 5a, b, bovine cultures). Chromaffin cells flattened out more readily, but were otherwise indistinguishable with phase contrast optics from untreated chromaffin cells. Ultrastructurally, most chromaffin cells appeared normal after one week of culture. In a few cases, chromaffin cells exhibited an increase in the amount of rough endoplasmic reticulum (rER) (cf. Fig. 5 a, inset, bovine cultures). Treatment of cultures with 0.01 mM dbcAMP plus theophylline also caused a clear suppression of accessory cell proliferation, which, however, was less pronounced than with 0.1 mM dbcAMP.

Counts of chromaffin cells performed at day 1 and day 12 on 12 cultures that had received 0.1 mM dbcAMP or were run as controls, gave the following results: after the first medium change the chambers still contained an average of 25,000 chromaffin cells (250cells per 5mm2), 50,000 cells having been injected 24h previously. In controls, chromaffin cells identified by catecholamine histofluores- cence decreased to approximately 12,500 at day 12. The corresponding figure for dbcAMP-treated cultures was 14,000 cells, suggesting that this treatment rather enhanced than decreased survival of chromaffin cells.

Treatment of chromaffin cells with dbcAMP caused a substantial inhibition of NGF-induced neurite outgrowth (Ziegler and Unsicker 1981). However, after withdrawal of dbcAMP, NGF-treated chromaffin cells extended neurites again, indicating that administration of dbcAMP had not altered their viability.

Fig.4a-d. Corresponding phase contrast (a, c) and fluorescence (catecholamine histofluorescence) micrographs (b, d) of cultures of bovine adrenal medullary cells after 7 days without dbc AMP (a, b) and in the presence of 0.1 mM dbc AMP and theophylline (c,d). Both types of cultures were grown on plastic surfaces (Falcon flasks) and had undergone differential plating prior to seeding. Note the different amounts of fibroblast-like cells depending upon dbc AMP treatment, x 480

Accessory cells in primary adrenomedullary cultures 81

NCC in*/, of

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Non-chromaffin cetls(NCC) in primary cultures of bovine o, drenat medutlae after one week

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Fig. 6a, h. Non-chromaffin cells (NCC) in primary cultures of bovine adrenal medulla after one week (a with differential plating, b without differential plating). Figures for the percentage ofnon-chromaffin cells present in the primary cell suspensions prior to seeding are given at the right of each figure. Note the different proportions of non-chromaffin cells depending on whether the cultures were grown on plastic or collagen surfaces

Fig. 5a--e. Electron micrographs of bovine adrenal cells cultured for 7days (a, b) in the presence of 0.1 mM dbc AMP and theophylline; c without dbc AMP. a Chromaffin cell showing normal ultrastructural features including catecholamine-storage vesicles (v) and rough endoplasmic reticulum (rER), which, in a few rare cases, displayed dilated cisternae (inset). b A cell exhibiting some ultrastructural characteristics o f a cortical cell, such as tubular mitochondria (m), lipid droplets (d), and smooth endoplasmic reticulum (sER). c Portion of a fibroblast-like cell in an untreated control culture with an abundance of free ribosomes, some rough endoplasmic reticulum and crista-type mitochondria (m). a, b x 10,000; inset and c x 16,000

82 K. Unsicker and W. Ziegler

Cultures of bovine adrenomedullary cells

24 h after plating the same types of cells as observed in cultures of rat adrenal medulla could be identified. However, there was a larger proportion of fibroblast- like cells, unless the primary cell suspension had been brought through a series of differential plating steps. Previous investigations had shown that non-chromaffin cells in primary cell suspensions amounted to 25.8 _+ 8.0 ~ (n = 8, determined by electron microscopy; Unsicker and Mfiller 1981). The corresponding figures for purified cultures that had been brought through four steps of differential plating were 10.2 _+ 2.8 ~ (n = 4). After one week, cultures that had undergone differential plating contained 45.5 + 1.3~ (n = 3) accessory cells, whereas 86.7_+ 4.6~ (n = 3) non-chromaffin cells were determined for unpurified cultures. These data apply to cultures that had been grown on plastic surfaces (Unsicker and Miiller 1981) and are in good agreement with cell counts performed in the present study showing that bovine adrenomedullary cells cultured without differential plating and kept on plastic Petri dishes for one week contained approxinately 90 ~ non- chromaffin cells (Fig. 6b). When grown on collagen surfaces proliferation of accessory cells was less extensive (73.9 _+ 10.2~; Fig. 6b), indicating that the surface of the culture dish plays a major role in the proliferation of accessory fibroblast-like cells. Treatment with 0.1 mM dbcAMP and theophylline con- siderably reduced the percentage of accessory cells present after one week (57.4_+ 3.4~o on plastic and 23+ 3.1~ on collagen surface; Fig. 4; Fig. 6b). Application of 0.01 mM dbcAMP had greater effects, if the cultures were grown on collagen instead of plastic (Fig. 6b).

Cultures that had undergone differential plating gave more satisfactory results concerning the amounts of non-chromaffin cells present after one week (Fig. 6a). As in cultures of rat adrenomedullary cells the inhibitory effect on accessory cell proliferation was due to enhanced maintainance of differentiation of cortical cells (Fig. 5 b, c). In addition, however, we also observed fibroblast-like cells that were severely damaged indicating a direct toxic effect of dbcAMP and theophylline (Fig. 3a, b). A few chromaffin cells showed an increase in the amount ofrER at the electron microscopic level (Fig. 5a, inset) as described above for cultured rat chromaffin cells; the cells were otherwise unaltered (Fig. 5a).

Discussion

Increased interest in the use of cultured adrenal chromaffin cells is due to the fact that they offer potential advantages to neurobiologists investigating transmitter synthesis, secretion, process formation and receptor regulation of neuronal cells (for references, see Introduction). An essential advantage of isolated chromaffin cells compared to neurons of the CNS is their homogeneity. However, non- chromaffin cells are always present in primary cultures of adrenal medullary cells and may seriously hamper studies of chromaffin cell function. Without any additives that inhibit the proliferative activity of non-chromaffin cells, these cells become predominant in one- and two week-old cultures. This has been well documented with bovine adrenomedullary cells (Unsicker and Mfiller 1981). In primary, unpurified cell suspensions non-chromaffin cells already constitute approximately 25 ~o of the total cell population. When kept on plastic surfaces these

Accessory cells in primary adrenomedullary cultures 83

cells amount to 80 ~o after one week. Even rigorous differential plating reduces the proportion of non-chromaffin cells after one week to only 45 ~. In primary cultures of dissociated adrenal medullae from the rat non-chromaffin cells are inherently less problematic, since (1) the early postnatal rat adrenal medulla contains a smaller proportion of accessory cells, and (2) the cultures are by routine grown on collagen- coated coverslips, where proliferation of accessory cells is much slower than on plastic (see below).

The present study offers a new alternative for inhibiting the proliferation of non-chromaffin cells in primary cultures of adrenal medullae by treating them with dbcAMP and theophylline. In addition, collagen-coated surfaces have been shown to reduce considerably the amount of non-chromaffin cells in cultures of bovine adrenal medulla. Cultures of young rat adrenomedullary cells remain almost free of accessory cells when treated with 0.1 mM dbcAMP plus theophylline even up to the end of the second week of culture. Bovine cell cultures retain approximately the quantity of accessory cells that was present at the beginning of the experiments (Fig. 6 a, b).

The numbers of accessory cells largely depend on whether the primary cell suspensions have been purified by differential plating.

The mechanism by which dbcAMP prevents overgrowth of chromaffin cell cultures by accessory cells may include several modes of action:

(1) We observed that dbcAMP maintained differentiation of adrenocortical cells and prevented their dedifferentiation into fibroblast-like cells as indicated by the persistence of mitochondria with tubular and vesicular cristae, sER membranes and cholesterol droplets. This observation is in accordance with the reported effects of cAMP and its analogs on the ultrastructure, histochemistry and steroidogenesis of rat adrenocortical cells in culture (O'Hare 1976; Armato et al. 1978; Armato and Andreis 1973; Schulster 1974). By maintaining adrenocortical cells in a differentiated state, a potential source of fibroblast-like cells can be effectively controlled.

(2) It is well documented that cAMP may act as a growth regulator of cell multiplication in numerous continuous nontransformed fibroblastic cell lines, such as 3T3 cells (Willingham 1976), and may slow down or arrest proliferation. It is conceivable that such an effect may also occur in cultured fibroblasts of adrenomedullary origin.

(3) We have found that dbcAMP and theophylline cause death of fibroblasts in cultures of bovine adrenomedullary cells. This observation is in line with an observation by Epstein et al. (1978; see, however, Kurz et al. 1979), who have reported death of normal human fibroblasts by these drugs.

In a report like ours it is important to establish not only the efficiency of the drugs used, but also their compatibility with the normal structure and function of the purified cell population, i.e., the chromaffin cells. Cell counts performed on cultures of rat adrenomeduUary cells suggest that cAMP increases rather than decreases survival of chromaffin cells. The cells store large amounts of catecholamines as indicated by their intense specific histofluorescence. Further- more, addition of NGF to rat chromaffin cells after dbcAMP withdrawal re- establishes neurite outgrowth indicating their unaltered viability. Electron micrographs of treated chromaffin cells show normal ultrastructural features and

84 K. Unsicker and W. Ziegler

p ropo r t i ons o f cell organelles, with only a few rare except ions where d i la ted cisternae o f rER were observed. H y p e r t h r o p h y and d i la t ion o f r E R profi les in cu l tured embryon ic chromaff in cells in response to 1 m M d b c A M P has been repor ted previously by Manue l id i s (1976). This h y p e r t r o p h y o f the rER p r o b a b l y reflects an increased p roduc t i on o f enzymes and pro te ins o f ch romaf f in vesicles. W a y m i r e et al. (1977) have assessed the effects o f 1 . 0 m M 8 Br c A M P on cu l tured bovine chromaff in cells and found an a lmos t 4.5 fold increase in the level o f ty ros ine hydroxylase act ivi ty within three days, while d o p a m i n e f l -hydroxylase was not influenced. Fu r the r analysis o f this p h e n o m e n o n ind ica ted tha t there was an increase in the n u m b e r o f tyros ine hydroxy lase molecules ra ther than an ac t iva t ion involving a l te ra t ions in the enzyme affini ty for ei ther subs t ra te or cofactor . W a y m i r e et al. (1977) also s ta ted tha t cAMP has no toxic side-effects on the cul tured chromaff in cells, bu t d id no t men t ion the pro l i fe ra t ion- inh ib i t ing effect u p o n accessory cells. In a s imilar series o f experiments , K u m a k u r a et al. (1979) demons t r a t ed a 55 ~ increase o f tyros ine hydroxy lase upon t r ea tmen t with 0.1 m M 8 Br cAMP.

In conclusion, the present results show that t r ea tment o f p r imary cul tures o f adrena l medu l l a ry cells with d b c A M P and theophyl l ine effectively cont ro l s p ro l i fe ra t ion o f f ibroblas t - l ike cells, which otherwise become the d o m i n a n t cel lular e lement in these cultures within one week. The ac c ompa ny ing effects o f the appl ied drugs upon chromaff in cells are small and imply enhanced di f ferent ia t ion ra ther than impa i rmen t o f s t ructure and function.

Acknowledgements. The authors wish to thank M. Johannsen, W. Lorenz and H. Reichert for excellent technical assistance. We are also grateful to Ch. Fiebiger and H. Schneider for the graphic and photographic work and to I. Ganski for editorial help. Nerve growth factor was a generous gift from PD Dr. U. Otten, Biocenter of the University, Basel, Switzerland. This work was supported by grants from Deutsche Forschungsgemeinscha ft

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Accepted November 3, 1981