Bovine adrenal chromaffin cells: high-yield purification and viability in suspension culture

Journal of Neuroscience Methods, 7 (1983) 329-351 Elsevier Biomedical Press

32q

Bovine adrenal chromaffin cells' high-yield purification and viability in suspension culture Jack C. Waymire, William F. Bennett, Richard Boehme, Linda Hankins, Katrina Gilmer-Waymire and John W. Haycock Department o/NeurohiologO"and A naton~v. Unicer~i(v (ff Te vas Medical School. Box 2070& ttou~um, l \ ' 77025 (U.S.A.) (Received March 30th, 1982) (Accepted September 14th, 1982)

Kt'v words': bovine adrenal chromaffin cells

suspension culture

collagenase-containing medium

A method for purifying chromaffin cells from adult, bovine, adrenal medullae and the techniques for maintaining the cells in suspension culture for at least 14 days are presented. Perfusion of medullae with a collagenase-containing medium produced a cell fraction that contained, in addition to chromaffm cells, a significant percentage of non-chromaffin cells. These cells were found to attach more rapidl\' than chromaffin cells to glass and tissue-culture plasticware. Using this property, we devised a selective plating procedure that yielded - 1-2 × 10 s chromaffin cells per adrenal medulla at a purity of 95~7:/ or higher. On the basis of catecholamine levels and enzyme activities, suspension (as opposed to monolavert cultures were chosen to further investigate their potential as a model system for the regulation of adrenergic function. In contrast to chromaffin cells cultured in monolayer, chromaffin cells m suspension had a more rounded appearance and formed multicellular aggregates with time in culture. Ver,, fev, neurite-like structures, commonly observed in monolayer cultures, were present in the suspension cultures. Also, inhibitors of mitosis were not necessary to prevent overgrowth by non-chromaffin cells as therc was little or no cell division in the suspension cultures. Catecholamine levels were relatively stable for at least 2 weeks, although a gradual decline in epinephrine occurred after day 5. Unlike other enzymes involved in catecholamine metabolism, phenylethanolamine N-methyl transferase activity declined significanth v~illa time in culture in parallel to the gradual loss of epinephrine. In addition, both oxygen consumption and amino acid incorporation into proteins were relatively stable. Thus, the primary suspension cultures of adult, bovine chromaffin cells seem to offer several advantages for studying long-term regulation of chromaffin cell function and provide a stable source of adrenergic cells for examining short-term regulatory processes.

Introduction In studying regulatory mechanisms involved in neurosecretory function in catecholaminergic cells in situ, one is confronted by both the complexity of other ongoing physiological interactions and the diversity of cell types present in a given organ. Various attempts have been made to avert these complications by establishing suitable in vitro conditions. For example, perfused sympathetic organs, brain slices. 0165-0270/83/0000-0000/$03.00

1983 Elsevier Science Publishers

330 synaptosomes, continuous cell lines and, more recently, sympathetic tissue in organ and cell culture have contributed considerably towards our understanding of the synthesis and secretion of ct~techolamine~. Each system offers its particular advantages. Cultured sympathetic tissues allow the greatest and most direct control of the extracellular environment and eliminate many indirect interactions. However, such (primary) cultures are usually explants of fetal tissue and thus present the disadvantages of cell heterogeneity, limited quantities of tissue, and immaturity in terms of cell differentiation. Although the use of clonal cell lines eliminates these problems, it is often difficult to distinguish whether the regulation of already differentiated function or the regulation of differentiation per se is being investigated le.g., Waymire et al., 1978). A preparation of easily purified, homogeneous, differentiated cells that would sustain their differentiated functions in primary culture would obviate such difficulties. Several laboratories have reported the successful isolation of functional chromaffin cells from adult, mammalian adrenal medulla (Douglas et al., 1967: Hochman and Perlman, 1976; Brooks, 1977: Schneider et al., 1977; Waymire et al., 1977: Fenwick et al., 1978; Kumakura et al., 1979: Role and Perlman, 1980; kemaire et al., 1981), and some have successfully cultured these cells in monolayer for periods of time from several days to several weeks (Hersey and DiStefano, 1979; Livett et al., 1979: Mizobe et al., 1979; Aunis et al., 1980; Kilpatrick et al., 1980; Trifaro and Lee, 1980; Unsicker et al., 1980; Wilson and Viveros, 1981). However, placing chromaffin cells in monolayer culture is not without effect on the morphology and metabolism of the cells. The present paper reports methods for the dissociation and purification of chromaffin cells in high yield. In addition, these cells can be maintained in suspension culture for periods of time sufficient to examine both short- and long-term regulation of chromaffin cell function. Preliminary reports from this laboratory (Waymire et al., 1976, 1977) and two studies that utilized the present techniques (Kumakura et al., 1979, 1980) have appeared previously.

Materials and Methods Materials

Most chemicals and reagents were purchased from Mallinckrodt and Sigma Chemicals (St. Louis, MO). Polyethylene tubing (Intramedic) was from Clay Adams (Parsippany, N J), silastic tubing from Dow Corning (Midland, MI), and nylon monofilament mesh cloth from McMaster-Carr (Santa F6 Springs, CA) and Small Parts, (Miami, FL). Culture media, penicillin, streptomycin and animal sera (except as noted) were obtained from Gibco (Grand Island, NY). With the exception of the prescription flasks used for selective plating, all glassware with which the cells came in contact was coated with either Siliclad (Clay Adams) or 2% (v/v) dichlorodimethylsilane in heptane.

331

Solutions Hanks' A (pH 7.2 at 4°C): Hanks' balanced salts, 0.1% (w/v) bovine serum albumin, 100 U / m l penicillin, and 0.10 m g / m l streptomycin; Hanks' B: Hanks" A with 1% bovine serum albumin, instead of 0.1%: PBS (pH 7.2 at 4°(_'): 137 mM NaC1, 8.1 mM N a 2 H P O 4, 2.7 mM KCI, 1.4 mM KH_~PO4: buffer 1 (pH 7.2 at 37°(_'): 150 mM NaC1, 30 mM NaPO 4, 10 rnM D-glucose, 10 mM 1-ascorbic acid, 5 mM KCI, 3 mM CaC12, 2 mM MgSO a, 2 mM ethylene glycol bis(h-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA): buffer 2 (ptt 6.2 at 24°('): 150 mM NaCI, 15 mM 4-(2-hydroxyethyl)-l-piperazine ethane sulfonic acid (HEPES), 5.5 mM D-glucose, 1.9 mM K2HPO 4, 1.5 mM CaCI~, 1.0 mM MgSO 4, 0.5 mM i_-ascorbic acid, 0.5 mM EGTA: buffer 3 (pH 7.2 at 37°('): 150 mM Na('l, 30 mM HEPES, 11 mM D-glucose, 4 mM KCI, 1.5 mM CaCI,, 1 mM Mg acetate, 1 mM I~-ascorbic acid. All Hanks" solutions were gassed with 5c~: ('():/95c~ air (Lindc, Houston, TX). Dissociation of the adrenal medul& Bovine adrenal glands were obtained at a local slaughterhouse within 20 3{) min after the animals were killed. Most of the superficial fat was trimmed a~ay and the glands were placed in a plastic bag c)n ice. In the laboratory, the gland surfaces \~cre thoroughly rinsed with 95% ethanol, carefully trimmed of all remaining fat and connective tissue, and rinsed again with ethanol. All subsequent procedures ~ere performed with sterilized materials and, with the exception of centrifugations, in a laminar flow hood. The adrenal cortex, with the exception of a small collar around the adreno-lumbar vein, was dissected away and a perfusion catheter inserted and secured with surgical thread. The construction and insertion of the perfusion catheter is extremely important for obtaining effective digestion. The catheter, polyethylene tubing with a small glas~ plug in the end, had a series of holes (needle punctures) in the terminal 2.5 cm to permit a more uniform perfusion along the length of the medulla. Thus. the catheter was always inserted at least 2.5 cm into the medulla. The most successful digestions were obtained from those glands in which the catheter was inserted almost :ill the way to the distal tip of the medulla. Up to 8 perfusion catheters were connected via Silastic tubmg to 2 Mastcrflex peristaltic pump heads (Cole Parmer, Chicago. IL). Four or 8 glands x~ere routinely prepared in this fashion. Upon cannulation, individual glands were placed in l lanks" A on ice and transiently perfused to facilitate cooling. This and all sub:,equcnt perfusions were performed in a retrograde manner. After all glands had been cannulated, they were transferred to fresh Hanks' A on ice and perfused ~ith recirculation at 10 m l / m i n / g l a n d for 15 min. The glands were transferred to another beaker, perfused (without recirculation) with 200 ml of cold Hanks" A (to remove red blood cells), and then perfused (recirculating, ~ 30°C) vdth tlanks" B containing 70 units collagenase/ml (Sigma, type 1, -0.()259~ (w/v), depending upon lot no.) in a volume of 35 ml/gland. It should be noted that this amount of collagenase is over an order of magnitude lower than that used in some othcr dissociations on the basis of reported %s (w/v) because in our experience, the \ields

332 arc substantially lower at higher collagenase concentrations. After 2-2.5 h of perfusion at 20 m l / m m / g l a n d , the catheters were removed, and the now puffy and swollen glands were disrupted by agitation with hemostats a n d / o r a stainless-steel egg beater. (Since these studies were completed, we have found that inclusion of 5 mM HEPES (pH 7.4) in the Hanks' B approximately doubles the yields reported below.) The resulting dispersion (initial digest) was filtered through a stainless-steel strainer and 88 ptm mesh nylon filter (arranged in series). Additional Hanks' B was rinsed through both to bring the volume to 45 ml/gland. The filtrate was transferred to 40 ml conical centrifuge tubes (Pyrex 8142, 15 m l / t u b e ) and spun at 50 g × 5 min. The cloudy supernatant was aspirated and the pellets were resuspended in 15 ml Hanks' B per tube. The suspensions were centrifuged at 50 g × 3 rain, and the pellets were suspended in H a m ' s F-12 medium supplemented with 1/10th vol. newborn calf serum ( F I 2 / N C S ) . After centrifugation (50 g × 3 min), the pellets were resuspended in F 1 2 / N C S , combined, and filtered through a 66 p,m mesh nylon filter to remove the filamentous material that usually formed upon the second addition of the F I 2 / N C S . (Occasionally this filamentous material formed during earlier centrifugation steps of the procedure. Filtration through a 66/~m mesh nylon was utilized whenever this occurred.) If the relative amount of debris was still substantial at this point, the last rate centrifugation step was repeated 1 or 2 more times. Viability was assessed by trypan blue exclusion, and the cells were diluted with F I 2 / N C S to 6 × 105 viable cells/ml. The yield at this stage was typically 2 -4 × 10 ~ cells/medulla (see Fig. 1A). This suspension is referred to as the initial cell fraction.

('ell separation by selectit,e platin~¢ Because a substantial number of the cells in the initial cell fraction were not chromaffin cells (see below), a method was adopted to selectively eliminate the non-chromaffin cells. This procedure (similar to that used by Yaffe (1968) to eliminate fibroblasts from embryonic chick myoblasts) exploits the tendency of non-chromaffin cells to adhere more rapidly than chromaffin cells to untreated glass or tissue-culture plastic. The initial cell fraction was transferred to either 32 oz. prescription bottles or 250 ml plastic, tissue-culture flasks (Falcon no. 3042, Oxnard, CA) at 2 × 107 cells/30 m l / 1 3 0 cm ~ surface area. After 6 h at 37°C in a 95% air/5% CO 2 atmosphere, the non-attached (chromaffin) cells were decanted away from the attached cells. The cell suspensions were pooled, diluted to 2 × 105 cells/ml, and transferred to plastic petri dishes for suspension culture. This suspension is referred to as the chromaffin cell fraction. The viability of the cells at this point is always greater than 90% (usually 95%). And, over the past 5 years, 30--60% of the viable cells in the initial cell fraction have been recovered in the chromaffin cell fraction. The conditions of the selective plating have been particularly important for the success of this procedure and were determined empirically. A particularly important variable is the ratio of cells to surface area. For example, given the tendency of the cells to collect toward the center of round dishes, the optimal cell/surface area ratio for round dishes could be much lower than the optimal value given above.

333

Cu[ture conditions A variety of media (e.g. Ham's F-12, Eagle's Basal Medium, Dulbecco's Modified Eagle, BGJ, McCoy's 5A, Trowell TS) and sera (fetal and newborn, bovine and horse, various suppliers) were examined for their influence on cell viability, enzyme activities and catecholamine levels with time in culture (not presented). Although F-12, McCoy's or Trowell's medium in combination with either fetal or newborn calf serum all produced satisfactory results, Ham's F-12 medium supplemented with 1/10th vol. newborn calf serum (heat inactivated. Gibco) in a 95¢~ air/5~7 ('O, atmosphere (water saturated) at 37°C was chosen for routine maintenance of chromaffin cells on the basis of cost, availability and overall performance. As a rule, Eagle's or BGJ medium and fetal or newborn horse serum produced less than satisfactory results. When cells were maintained at 2 × 105 cells/ml or less over a 2-week period in culture, the nutrient medium was not replaced. In monolayer cultures in which inhibition of cell division was desired (see below) to keep cell number from increasing, cytosine arabinoside (6/xg/ml) was included in the culture medium.

ttistochemical analyses Dichromate staining and formaldehyde-induced fluorescence (FIE) ~ere evahiated in cells plated onto either collagen-coated or tissue-culture plastic dishes. The dichromate reaction was used to stain both epinephrine (E) and norepmephfine (NE) (e.g. Hillarp and HOkfelt, 1953). The medium was aspirated from plated cells and replaced with 100 mM sodium chromate-dichromate sohition (pH 6.0). After 24 h at room temperature, the cells were rinsed twice with 0.991 (w//v) Na('l and covered with 100% glycerol for examination. The relative number of cells exhibiting the characteristic brown staining was quantified visually with a Wild M40 inverted phase-contrast light microscope. Formaldehyde-induced fluorescence of E and NE (e.g. Corrodi and J(msson. 1967) was produced by replacing the culture medium from plated cells with a solution of formaldehyde (3.7%, w / v ) in 0.32 M sucrose. After 1 rain, the plates were dried for 5 rain with a 400 W hair dryer from a distance of about 20 cm and incubated for 3 h at 80°C in a closed vessel containing paraformaldehyde pellets and an atmosphere 85% saturated with water. Fluorescence was viewed on a Zeiss fluorescence microscope.

Electron microscopy Cells in suspension culture were pelleted (40 g × 4 rain) and suspended in Ham's F-12 containing 4% (v/v) glutaraldehyde (2 h, room temperature). The celb, ~ere pelleted and the pellets were incubated in Caulfield's buffer containing 4% glutaraldehyde (2 h), 1% (w/v) osmium tetroxide (in Caulfield's buffer, 2 h) and 0.5% (w/v) uranyl acetate (in 50 mM veronal buffer: 2 h). Tile samples were then rinsed, dehydrated in a graded series of ethanol solutions and embedded in Epon/Araldite.

334 Respiratoo, activity Suspension cultures were pelleted (40 g × 4 rain) and the cells were suspended in PBS containing 10 mM D-glucose (air saturated, 3 ml). Oxygen consumption of the chromaffin cells (4 x 10 ~ cells in 3 ml) was monitored with a Clark-type oxygen electrode (Yellow Springs, Model 53) at 37°C. Oxygen consumption was assessed over a 60 min period in the presence and absence of treatments. Values for medium (and treatments, as appropriate) were subtracted. A drenergic enzyme activities After various durations in culture, cells were pelleted (40 g × 4 min), rinsed in PBS, repelleted, and freeze-thawed 3 times in 15 mM KCI. Aliquots were assayed for enzyme activity and protein. Tyrosine hydroxylase (TH: EC 1.14.16.2), aromatic-L-amino acid decarboxylase (AADC; EC 4.1.1.28), dopamine beta-hydroxylase (DBH: EC 1.14.17.1), catechol O-methyltransferase (COMT; EC 2.1.1.6), and monoamine oxidase (MAO: EC 1.4.3.4) activities were measured as previously described (Waymire and Gilmer-Waymire, 1978) with the exception that 1.5 mM CuSO4 was found to provide optimal DBH activity. Phenylethanolamine N-methyltransferase (PNMT: EC 2.1.1.28) was assayed according to the procedure of Axelrod (1962) using methyl-[3H]S-adenosyl methionine (New England Nuclear) as methyl donor and normetanephrine as substrate. Catecholamine biosynthesis Catecholamine synthesis rates in intact cells were measured by analyzing the rate of 14CO2 evolution from exogenous L-[1-1aC]tyrosine. Cells were pelleted and washed twice by centrifugation (40 g × 4 min) and resuspension in buffer 1. Aliquots (approximately 106 cells in 0.1 ml) of the cell suspensions were distributed to reaction tubes on ice, and the reaction was initiated by addition of 100/~1 of buffer 1 containing L-[l-lac]tyrosine (new England Nuclear, 50 mCi/mmol, 20 ~M final) and placement of the tubes in a shaking water bath (37°C). The tyrosine is accumulated and hydroxylated, and the resulting L-[1-JaC]DOPA is decarboxylated to form dopamine and ~4CO2. Thus, the rate of dopamine synthesis is proportional to 14CO2 production. At the end of a 10 min incubation, 0.2 ml of 10% (w/v) trichloroacetic acid was added to terminate dopamine synthesis and drive the ~4CO2 out of solution. Released CO~ was collected and counted as previously described (Waymire et al., 1971). Evolution of ~4CO2 was proportional to cell number up to at least 2 x 10 6 cells and was linear for at least 30 min. Catecholamine levels NE and E levels in 0.1 M perchloric acid extracts of the cells were measured (Laverty and Taylor, 1968) after isolation on alumina microcolumns (Gauchy et al., 1976). Aliquots of the column eluates were oxidized at pH 4 and pH 7.4. Recoveries were greater than 90%; therefore, no corrections were made.

335 Catecholamine secretion N E and E efflux from the purified chromaffin cells was determined using previously described procedures (Haycock et al., 1978). Pelleted cells were rinsed and resuspended in buffer 2. Matched aliquots (1-2 × 10 ~ cells) were applied to filter units and then rinsed simultaneously with buffer 2. After three 30 s rinses, the matched filter units received either buffer 2 or buffer 2 supplemented with 0.1 mM acetylcholine (ACh) or 0.1 mM ACh plus 1 mM EGTA. After 30 s of unit gravity perfusion, residual buffer was rapidly collected by vacuum and both the filtrate and filter were acidified (0.4 M perchloric acid, 1 mM EDTA, 1 mM sodium metabisulrite, final). NE and E levels in the filtrate and in the acid eluate from the filters were determined as described above. In situ protein synthesis Chromaffin cells were pelleted and then rinsed and resuspended in buffer 3. A ~4C-labeled protein hydrolysate (Amersham CFB-25 in 1 ml buffer 3) was added to each chromaffin cell suspension (8 × 106 cells in 4 ml) to give a final radiochemical concentration of 10 ~ C i / m l . After incubation for 2 b (37°C1 with gentle shaking, the cells were pelleted, rinsed in buffer 3 and resuspended in 1 ml of 25 mM Tris-t-tCI pH 7.8, 2 mM EDTA, 2 mM dithiothrcitol, 2.5 mM phenylmethylsulfonyl fluoride (prepared immediately before use). The suspension was frozen and thawed twice, an equal volume of twice concentrated sodium dodecyl sulphate (SDS) sample buffer was added, and the samples were heated at 95°C for 2 rain. Aliquots representing 1.5 × 106 cells were subjected to SDS-polyacrylamide electrophoresis on a 7.5 20~ polyacrylamide slab gel as previously described (Kelly and kuttges, 1975). The gels were stained with Coomassie blue R, dried and exposed to Trimax X D L fihn (3M) for 5 days. Other aliquots were assayed for protein and radioactivity after removal of SDS (Zaman and Verwilghen, 1979) and precipitation with deoxycholate and trichloroacetic acid (Peterson, 1977).

Results Characteristics of the initial cell fraction Initially, after perfusion with collagenase-containing medium and disruption of the adrenal medullae by agitation, the initial digest consisted of cells ranging in size from 5 to over 30/~m in diameter (including some erythrocytes) and a large amount of fibrous and other particulate debris. Filtration and rate centrifugation steps eliminated the majority of erythrocytes and most non-cellular material, and the resulting initial cell fraction contained - 2 4 × 10 s cells per gland. As shown in Table 1, substantial amounts of catecholamines and T H activity were present in the initial cell fraction. Cell viability, as judged by trypan blue exclusion, was routinely 90-95%. Phase-contrast microscopy revealed that the initial cell fraction comprised at least 3 morphologically distinguishable cell populations (Figs. 1A and 2): smooth, phase-bright 20-30 ~m diameter cells; rough or surface-granular, less phase-bright,

33~, TABLE 1 ISOLATION OF C H R O M A F F t N ('ELt.S The medullae from 7 adrenal glands were dissected as described in Methods. Four medullae ,acre dissociated and the initial cell fraction and the purified chromaffin cell fraction were prepared as described in Methods. The remaining 3 medullae were minced and homogenized in 0.32 M sucrose (adrenal medulla homogenate). Cell counts, catecholamine levels and tyrosine hydroxylase activity were determined as described in Methods. Similar results were obtained in the 2 other cell preparations in which these particular dependent measures were determined. Values represent the mean :- S.I). of triplicate determinations. {.'ell yield Icells/gland)

Catecholamines Total

I{

(rimot/mg

(c;)

Tyrosme hydroxylase activity (nmol/mg protein/h)

protein) Adrenal medulla homogenate Initial cell fraction Chromaffin cell fraction

4 x 10 s 2 × 10 ~

580 + 50 410 _+ 15 860_+ 30

62 60 61

20 + 5 12 _+6 19 + 7

20-30/~m diameter cells; and occasional clumps of 8-10 ~na diameter cells. Placement of the initial cell fraction in monolayer culture revealed a further distinction of cell types. One population of cells rapidly attached and flattened on either glass, tissue-culture plastic or collagen-coated surfaces within 2-3 h (Fig. 1B). A second population also attached to collagen within 2-3 h but took more than 6-12 h to plate down on either glass or tissue-culture plastic, a large proportion of the attachment being to the already attached, flattened cells. The more slowly attaching cells did not rapidly flatten on any surface, but retained their smooth, phase-bright appearance and were usually clumped with cells of similar surface morphology (Fig. 1B). Histochemical analysis demonstrated that not all cells from the initial cell fraction reacted positively for catecholamines. Dichromate staining and FIF both indicated that only the smooth, non-flattening, slowly attaching cells were catecholamine positive (e.g. Fig. IC). Approximately 70% of the cells in the initial cell fraction exhibited FIF and 65% gave positive dichromate reactions (Table 2). Electron microscopy of the cells also indicated, on the basis of cells containing granules, that at best only 60-70% of the cells in the initial cell fraction were chromaffin cells (Table 2). (In our earlier studies (Waymire et al., 1976), as low as 35% of the cells were identifiable as chromaffin, presumably as a function of overdigestion.) Fig. 3A illustrates a NE-containing chromaffin cell, an E-containing chromaffin celt and an adrenal cortical cell found in the initial cell fraction.

Enrichment of chromaffin cells in the chromaffin cell fraction Both the histochemical and ultrastructural data indicated that the proportion of chromaffin cells in the initial cell fraction was at best 70%. In the experiment

337

® Fig. 1. Photomicrographs of dissociated cells before (initial cell fraction, A C) and after (chromaffin cell fraction, D- F) selective plating. Cell concentration ,*as 6 x 10 > cells/ml (A ('1 before selectixc plating. The concentration of supernatant cells (D F) v, as reduced to 2x l0 s cells/ml b\' selective plating. A: freshly isolated initial cell fraction displaying a range of cell sizes. B: initial cell fraction 24 h afTcr plating on rat Tail collagen-coated petri dish. (': initial cell fraction. Treated as in B, viewed under ultraviolet illumination after treatment to display FIF as described in Methods. D: chromaffin cell fraction, purified from the initial cell fraction shown in A. E: chromaffin cell fraction in monolayer culture as m B (noTe the absence of flattened, phase-dark cells). F: chromaffin cell fraction in monolaver culture as m ('. The calibration bar represent 50 /tin for all panels.

r e p o r t e d in T a b l e 2, 14% h a d t h e u l t r a s t r u c t u r a l a p p e a r a n c e of c o r t i c a l cells (Fig. 3 A ) w h i l e t h e r e m a i n d e r of t h e n o n - c h r o m a f f i n cells s h o w e d n e i t h e r c h r o m a f f i n n o r c o r t i c a l cell u l t r a s t r u c t u r e . If, as i n d i c a t e d b y t h e h i s t o c h e m i c a l r e s u l t s , t h e s m o o t h , p h a s e - b r i g h t cells w e r e c h r o m a f f i n cells, it w a s r e a s o n e d t h a t t h e i r r e l a t i v e l y slow a d h e r e n c e to glass o r t i s s u e - c u l t u r e p l a s t i c s u r f a c e s c o u l d b e e x p l o i t e d to i s o l a t e t h e s e cells in s u s p e n s i o n a f t e r t h e n o n - c h r o m a f f i n cells h a d a t t a c h e d . A s i l l u s t r a t e d in Fig. 4 a n d s u m m a r i z e d in T a b l e 2, t h e m a j o r i t y o f t h e n o n - c h r o m a f f i n cells d i d a d h e r e to a g l a s s o r t i s s u e - c u l t u r e p l a s t i c s u r f a c e d u r i n g t h e first - 3 h o f c u l t u r e . T h i s p l a t i n g p e r i o d r e d u c e d t h e n u m b e r o f r o u g h ( o r s u r f a c e - g r a n u l a r ) cells a n d e l i m i n a t e d t h e s m a l l , c l u m p e d cells f r o m t h e m e d i u m (Fig. ID). W h e n t h e n o n - a t t a c h e d cells w e r e p l a t e d o n t o a c o l l a g e n s u r f a c e , t h e y e x h i b i t e d a p h a s e - b r i g h t

33~

Fig. 2. Phase-contrast photomicrograph of the initial cell fraction in suspension. Two cell types, distinguished by their surface morphologies, were detected in the initial cell fraction, r. the rough, or surface-granular cells which had slightly larger diameters than the smooth, phase-bright cells. 'lhe calibration bar represents 20 ,ttm.

TABLE 2 P U R I F I C A T I O N OF C H R O M A F F I N CELLS WITH SELECTIVE PLATING Cells ( ] 0 6 in 5 ml) taken either before (initial cell fraction) or after (chromaffin cell fraction) selective plating were seeded into collagen-coated 60 mm tissue-culture dishes and incubated for 24 h (37°('). Attached cells and cells showing either dichromate staining or FIF were determined in 10 low-magnification fields from each of 3 dishes. Matched cell samples (not plated onto collagen-coated surfaces) were pelleted and processed for electron microscopy as described in Methods, Granule-containing cells in the initial and chromaffin cell fractions were determined in 3 separate cell pellets. For each cell pellet, 10 low-magnification micrographs containing 8 or more cells each were analyzed in sections cut perpendicular to the direction of centrifugation. The data presented represent the mean +_S.D. of median values from the 3 samples in each category. The entire experiment was repeated once, with similar results.

Initial cell fraction Chromaffin cell fraction

DichromateF1Fpositive positive (% of total cells±S.E.M.)

Granulecontaining

62 ± 20 95 ± 5

65 ± 28 96 ± 10

69 _+22 93 ± 8

33'~ appearance (Fig. 1E) and F I F (Fig. IF). Also, about 95% of the cells remaining in suspension contained electron-dense granules characteristic of chromaffin cells (Table 2) and the specific activity of both TH and catecholamines was higher in the chromaffin cell fraction than in the initial cell fraction (Table 1). This selective plating of the initial cell fraction thus provided a one-step purification of the chromaffin cells and produced a cell suspension in which over 90 95% of the cells were chromaffin cells. As pointed out in the Methods section, however, several variables can dramatically influence the purification and yield of this procedure.

Chromaffin cell viability in suspension culture Numerous laboratories have established that chromaffin cells in monolayers are relatively stable for several weeks in culture (see Introduction). Based on trypan blue exclusion and FIF, the cells in the chromaffin cell fraction were also viable in culture as monolayers on a collagen surface. But, unless inhibitors of mitosis were present (Mizobe et al., 1979), the amitotic chromaffin cells (Viola-Magni, 1966) in the monolayer cultures were rapidly overgrown by a dividing population of non-chromaffin cells. However, in that most cell types will not divide in culture unless permitted to flatten by attachment to a substrate (Folkman and Moscona, 1978), we explored the possibility of preserving the purity of the chromaffin cell fraction in culture by maintaining the cells under conditions unfavorable to attachment. It was found that plastic petri dishes (non-tissue-culture) failed to promote the attachment of any of the cells in the chromaffin cells fraction even without agitation. Thus, suspension culture of the chromaffin cell fraction was achieved simply by seeding the cells in this type of dish. As shown in Fig. 5, one characteristic of the chromaffin cells in suspension culture is their tendency to aggregate with time in culture. After a few hours in culture, small trains of cells were observed which, by 24 h, became quite long (Fig. 5A) and, by 48 h, coalesced into loose aggregates (Fig. 5B). The aggregates increased in size and compactness with further time in culture (Fig. 5C). This phenomenon persisted in the presence or absence of 6 /.Lg/ml cytosine arabinoside, in high (3 raM) or low (0.3 mM) CaCl 2, and under still or gyrating conditions. The ultrastructure of cells in the suspension cultures was similar to cells from either intact (Coupland, 1965) or dissociated (Fenwick et al., 1978) adrenal medullae. This ultrastructural appearance was maintained for at least 2 weeks in culture. Comparison of recently dissociated chromaffin cells with those purified and in culture for 1, 7 or 14 days revealed few qualitative differences (Fig. 3A-D). In addition to the typical chromaffin granules, pseudopodia, invaginations, and an occasional cilium were also observed. Numerous examples of close appositions between aggregated cells were observed (Fig. 3A, C, D), and large extracellular spaces resembling canaliculi were distributed between aggregated cells (Fig. 3C). Occasionally, cells with dilated endoplasmic reticulum were observed after 2 weeks in culture, but normal ultrastructure was still predominant in the majority of cells (Fig. 3D). Throughout the 2-week period in suspension culture, only a small percentage (roughly 5%) of cells lacking chromaffin granules were found in the

ii ii~,~,~ii~i,~~

i ¸¸

i !i i ¸

341

illustrates an aggregate of chromaffin cells with both NE- and E-containing chromaffin cells and open spaces resembling canaliculi (c) that are commonly found between cells in otherwise close apposition. D: chromaffin ceil fraction (14 days) illustrates an aggregate of chromaffin cells, some of which have storage granules with dense, contracted cores and dilated endoplasmic reticulum, whereas others appear normal (E). Horizontal calibration bars represent 1 ~m.

342

chromaffin cell fraction, despite the absence of mitotic inhibitors. Thus, because the small population of non-chromaffin cells not eliminated by selective plating did not proliferate in suspension, a relatively pure population of chromaffin cells was total ~= 120 O9 _J

uJ o 80 c3 W I (D ,< ~ 40' .<

0

4

8

12

PLATING DURATION (h} Fig. 4. Attachment rates of cells in the initial cell fraction. Cells from the initial ce!l fraction were

incubated (37°C) in Falcon tissue-culture flasks (no. 3042) as described in Methods. After the times indicated, the non-attached cells were decanted and the attached cells were either counted or stained with dichromate and then counted. Two culture flasks were evaluated for each condition, and 4 low-magnification microscopic fields were counted from each flask. The values represent the average of the median cell count from the 4 fields.

maintained in the suspension cultures in the absence of antimitotic drugs. As measures of 'general' cell function, oxygen consumption, protein levels, cell nuclei number, and protein synthesis were determined over a two-week period in culture. Table 3 shows that basal oxygen consumption gradually increased during the second week in culture. This appeared to be at the expense of the stimulation of respiration by either 55 mM KCt or 100 ktM carbachol seen during the first week.

Fig. 5. Aggregation of the chromaffin cells in suspension culture. Phase-contrast micrographs of the chromaffin cells in suspension culture after 24 h (A), 48 h (B), and 120 h (C). All calibration bars represent 40/~m.

343 TABLE 3 OXYGEN CONSUMPTION IN SUSPENSION CULTURES OF ('HROMAFFIN ('Et.I,S After isolation of the chromaffin cell fraction, cells were maintained in suspension culture for the times indicated in the table prior to harvesting and measurement of oxygen consumption, as described in Methods. Oxygen consumption was estimated from % saturation values relative to 4.9 ffl O : / m l . Values represent the average of 2 determinations, which varied by less than 10g, /*1 O_, c o n s u m e d . b / l l ) ~ cells. Additions

Days in culture 1

None KCI (55 raM) Carbachol (100 ffM)

3

5

7

7

I1

6

9

14 15

14 14

12 13

14 14

9

13

14 14 14

12 13 14

P r o t e i n levels r e m a i n e d r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e 2 w e e k s (Fig, 6) as d i d cell n u c l e i n u m b e r / c u l t u r e ( n o t s h o w n ) . P r o t e i n s y n t h e s i s b y t h e i n t a c t c h r o m a f f i n cells w a s a s s e s s e d a f t e r 1 a n d 14 d a y s in c u l t u r e . N o q u a n t i t a t i v e d i f f e r e n c e s in i n c o r p o r a tion of the ~4C-labeled amino acids into trichloroacetic acid insoluble material were o b s e r v e d ( n o t p r e s e n t e d ; see a l s o K i l p a t r i c k et al., 1980). I n a d d i t i o n , e l e c t r o p h o r e s i s o f t h e p r o t e i n s o n S D S - p o l y a c r y l a m i d e s l a b gels (Fig. 7) r e v e a l e d n o q u a l i t a t i v e d i f f e r e n c e s e i t h e r in t o t a l p r o t e i n s ( S t a i n ) o r in p r o t e i n s s y n t h e s i z e d d u r i n g t h e 2 h i n c u b a t i o n (14C). Adrenergic enzyme activities, catecholamine biosynthesis, catecholamine levels,and c a t e c h o l a m i n e s e c r e t i o n w e r e e m p l o y e d as i n d i c e s of ' d i f f e r e n t i a t e d ' cell f u n c t i o n . A s s h o w n in T a b l e 4, t h e a c t i v i t i e s o f e n z y m e s i n v o l v e d in c a t e c h o l a m i n e m e t a b o l i s m ,

8

u]

<

_J

0 ~ 0 ----- 0 *'"--- 0 ~

toO~X O ~

b_

0 "--'~-~0

o - -

o

n0repi..

°~o

~o

epi. \

0.4

~.o~O

0.2 x__x~X

6 ~

~){-,

~

~

X--

X ~ X

)

~

~" U] m

X

1'1

1'~

DAYS IN CULTURE Fig. 6. Cell catecholamine and protein levels in suspension cultures of the chrornaffin cell fraction. Chromaffin cells (106 cells in 5 ml F12/NCS) were pelleted, suspended in PBS, repelleted and then evaluated for protein (crosses) and catecholamines (E, solid circles, epi.; NE, open rectangles, norepi.: E+ NE, open circles, total) after suspension culture for the times indicated in the figure. Each value is the mean of 3 determinations. S.E.M. were 10-20% of the mean in most cases. Day 0 values were obtained from cells in the chromaffin cell fraction within 2 h after selective plating.

344 with the e x c e p t i o n of P N M T . v~ere m a i n t a i n e d d u r i n g 2 weeks of s u s p e n s i o n c u l t u r e o f the cells. P N M T a c t i v i t y d e c r e a s e d after d a y 5 to a p l a t e a u by d a y 9 al - 209: of that o r i g i n a l l y p r e s e n t . A l t h o u g h C O M T a c t i v i t y was p r e s e n t in the initial digest ( d a t a not p r e s e n t e d ) , C O M T a c t i v i t y was not d e t e c t e d in e i t h e r freshly p u r i f i e d or in c u l t u r e d cells ( T a b l e 4). T h e cells also t n a i n t a i n e d their ability to s y n t h e s i z e

Fig. 7. In situ protein synthesis in suspension cultures of the chromaffin cell fraction. Chromaffin cells were harvested after 1 or 14 days in suspension culture and incubated (2 h, 37°C) with [ 14C]amino acids as described in Methods. The samples were subjected to SDS-polyacrylamide electrophoresis, stained with Coomassie blue R, dried and then autoradiographed. The protein staining is shown on the right and the autoradiograms from these lanes are shown on the left. Molecular weight ( x 1 0 3) standards are indicated on the left: 96, rabbit muscle phosphorylase B; 68, bovine serum albumin; 42, rabbit muscle creatine phosphokinase; 29, carbonic anhydrase; and 13, horse heart cytochrome c.

345 FABLE 4 VIABILITY OF D I F F E R E N T I A T E D F U N C T I O N IN SUSPENSION C U L T U R E Chromaffin cells, after selective plating, were maintained in suspension culture as described in Methods. After primary culture for the indicated durations, the cells were harvested and assaved for the ~arious enzyme activities or intact catecholamine biosynthesis rates as also described in Methods. Values represent the average of triplicates from a single preparation (S.E.M. 10--20% of the mean), lx~o other cell preparations gave similar results. All values are expressed in terms of n m o l / m g prot e i n/ h, I)uc Io lhe aggregation of the cells with time in culture, cell counts were not possible, ttov, e,cer, because cell p r o t e i n / c u l t u r e does not vary in the suspension cultures during this period of time 1,see Fig. 6L the values listed here may be converted to nmol/11,) ~' c e l l s / h by dividing bv 10. , not determined: n.d., not detected less than 2 m n o l / m g p r o t e i n / h . Days in culture

0 1 3 5 7 9 11 13

Enzyine activities

l)opamine synthesis

TH

AADC

DBI-t

PNMT

('OM'I

MAO

20 26 30 33 29 30 29 29

161) 210 220 280 220 210 230 260

1 300 2 000 2 000 1 800 1 600 1500 1 500 1 5011

1.2 1.4 1.4 1.3 0.5 0.3 0.3 1,).3

n.d.

22/) 26/) 241t 320 2811 27// 321,/ 320

n.d.

n.d.

3.2 3.4 4.2 4.2 4.1 4.1 5.1 4.8

catecholamines from exogenous k-[l-HC]tyrosine. This implies that. in addition to the requisite enzyme activities, tyrosine accumulation and pterin cofactor levels were also maintained. Catecholamine levels decreased to approximately 70¢5~ of origimtl TABLE 5 C A T E C H O L A M I N E EFFLUX A N D SECRETION BY C H R O M A F F I N CI~LLS Chromaffin cells, after selective plating, were maintained m suspension culture as described m Methods and harvested after primary culture for the indicated durations. Catecholamine efflux ~<~s delernained a~, described in Methods. Values represent the average of duplicate samples from a single cell preparation. Similar results were obtained from duplicate samples in another cell preparation. The numbers in parentheses are the differences in efflux between samples exposed to ACh and ACh plus E(}IA, and therefore represent the calcium-dependent secretion produced by ACh. Values for the initial time poinl were obtained from chromaffin cell suspensions 2 h after selective plating. Time in culture (days)

Catecholamine efflux Epinephrine -

0 3 7 14

2 4 4 5

Norepinephrine

ACh + EGTA 3 4 3 3

A('h 10 8 9 12

(7) (4) (6) (7)

A('h + EGTA 3 5 7 6

3 5 7 6

A('h 15 13 14 15

(12) (8) (7) (9)

346

levels by day 13 (Fig. 6) due specifically to a decline in E levels. This may be due to the temporally parallel decline in PNMT (Table 4). Table 5 presents basal and ACh-stimulated efflux of E and NE. As with oxygen consumption, basal efflux tended to increase during the culture period although a concomitant decrease in the stimulation of release by ACh was not apparent. The fractional, basal efflux (2-7% of total stores) was consistently higher than previously reported for monolayer cultures, whereas the ACh-stimulated efflux was smaller. However, given the relatively shorter collection period in the present studies, the rate of release stimulated by ACh, when calculated as %/rain, was comparable if not higher. More importantly, ACh produced a relatively reproducible increase in catecholamine efflux throughout a 14-day culture period, and the effects of ACh were prevented by addition of EGTA to the medium. ACh-induced release of N E tended to be higher (on a percentage basis) than that of E, as also seen with nicotine in monolayer cultures (Livett et at., 1981), although the reasons for this remain unclear. Discussion

The present studies define techniques for the isolation and purification of functional chromaffin cells from adult, bovine adrenal medullae. The yield of purified chromaffin cells with the present technique (1-2 × l0 s cells/gland) is much higher than with most of the previously reported methods ( - 1 0 7 cells/gland) (Brooks, 1977; Fenwick et al., 1978; Schneider et al., 1977; Unsicker and Mtiller. 1981; but see Kilpatrick et al., 1980) whereas the purity compares favorably with the highest reported values (Unsicker and Mtiller, 1981). The present studies also establish the viability of these cells for 2 weeks in primary, suspension culture. Not only are these latter data prerequisite for studying long-term regulatory processes in the cells, but they also establish the chromaffin cells as a source of adrenergic cells for studying short-term regulatory processes during the 2-week period in suspension culture. A prominent difference among techniques for dissociating medullary cells is the proportion of non-chromaffin cells in the initial cell fraction. These non-chromaffin cells are probably a mixture of fibroblasts, satellite cells and adrenal cortical cells. One of the first studies, in which collagenase digestion of very carefully dissected medullary slices was utilized, reported an extremely pure initial cell fraction (Brooks, 1977). The yields, however, were quite low. More recent studies, utilizing either whole medulla perfusion (Unsicker and Mfiller, 1981; Wilson and Viveros, 1981) or slice incubation (Role and Perlman, 1980), have reported anywhere from 20 to 70% chromaffin cells in the initial cell fraction in agreement with the 40-70% that we have observed. In that chromaffin cells constitute more than 90% of the cells in adult mammalian adrenal medullae (R. Coupland, personal communication), it seems reasonable to conclude that the dissected medullae contain a substantial amount of adrenal cortical cells a n d / o r the digestion procedure compromises a substantial number of chromaffin cells.

347 Two different approaches have been used to purify chromaffin cells--density gradient centrifugation (Livett et al., 1979: Role and Perlman, 1980: l_~emaire et al., 1981) and selective plating (Waymire et al., 1977: Unsicker and MOiler, 1981). Density gradient centrifugation presumably relies upon physical differences such as size and bouyant density, whereas selective plating relies upon a functional difference, i.e. the tendency of a given cell type to attach to glass or tissue-culturc plastic surfaces. The selective plating technique results in a slightly greater purity of the chromaffin cell fraction than does the Percoll or Metrizamide method (approximately 95% (Table 2; Unsicker and Mtiller, 1981)vs 85% (Kilpatrick el al., 1980) and 90% (Role and Perlman. 1980)). Because the 2 methods of purification rely upon different characteristics of the cells, perhaps some combination of the 2 methods would provide even better purification. It is clear, however, that in order to obtain a preparation of chromaffin cells at high purity, some method of purification is required after isolation of the initial cell fraction. Unsicker and M011er (1981) have also reported a selective plating procedure for purifying chromaffin cells. Hox~ever. a single plating produced only an 80%, pure chromaffin cell population, e~en though the ratio of cells to plating area was similar to ours: thus the reasons for the differences in purity are not immediately obvious. Three methodological differences which may explain this difference are the digestion conditions, the selective plating medium, and the shape of the plating surface. For example, in comparison to Unsicker and MOiler (1981), we use over a 10-fold-lower collagenase concentration (by weight) at a lower temperature, our plating medium was lower in calcium and serum, and rectangular plating surfaces were used (see Methods).

Characteristics of the purified ehromaffin cells in suspensio#t culture After purification, chromaffin cells were cultured in plastic petri dishes (non-tissue-culture, to which the cultured cells do not adhere) and maintained routinely for about 2 weeks. New, or additional, culture medium was not appareqtly required for the maintenance of the chromaffin cells in vitro for at least 2 weeks. This observation is consistent with those of Wilson and Viveros (1981). They reported that less frequent changes in culture medium resulted m more stable catecholamine levels and protein content. The chromaffin cells in suspension culture were judged to be stable on the following bases: (1) cell protein content was constant: (2) all but one of the enzymes involved in catecholamine metabolism were relatively constant: (3) oxygen consumption rates were maintained; (4) NE levels were constant (with a slight but stead', loss of E over the culture period, see below); (5) cell number was maintained m apparent absence of cell division: and (6) amino acid incorporation was neither quantitatively nor qualitatively changed with time in culture. In terms of surface morphology, the chromaffin cells in suspension culture maintained a smooth, rounded appearance throughout the culture period, although they invariably exhibited increasing cell cell contact. By about the fourth day. the cultures consisted almost entirely of complex aggregates (Fig. 5). Although the aggregation interfered with the determination of cell number, it did not appear to

348 adversely affect chromaffin cell function. The aggregation may even help maintain cell function (see below). Uhrastructural[y, the chromaffin cells were relativel) normal over the culture period. Some abberations of the endoplasmic reticulum. similar to those described by Unsicker et al. (1980), were observed in a small percentage of cells towards the end of the culture period. The chromaffin cells also maintained viability in culture from a functional standpoint. Catecholamine biosynthesis and receptor-mediated stimulus-secretion coupling were relatively constant over the 2-week culture period. Although not yet evaluated, the gradual increase in basal oxygen consumption rates may reflect an alteration in the integrity of the chromaffin cells and compensatory increase in sodium-potassium ATPase activity. This is consistent with the decrease in the ability of depolarizing treatments to increase oxygen consumption and the concomitant gradual increase in basal catecholamine biosynthesis rates.

Comparison of suspension and mono&ver cultures of puri/Ted chromaffin cells Although numerous laboratories have characterized monolayer cultures of adrenal chromaffin cells, only a few reports of long-term suspension cultures have appeared (Waymire et al., 1977; Kumakura et al., 1979, 1980), all of which utilized essentially the techniques reported in the present paper. In terms of comparison to the suspension cultures, the data of Kilpatrick et al. (1980); Unsicker and Mtiller (1981); and Wilson and Viveros (1981) seem to be the most relevant on the basis of either similarity in preparation a n d / o r determination of chromaffin cell purity. Both culture systems seem to maintain physiological viability in terms of catecholamine levels, enzymes and secretion. And, although catecholamine biosynthesis has not been previously reported for any of the chromaffin cell cultures, maintenance of the biosynthetic enzymes suggests that biosynthesis rates would be similarly maintained in monolayer cultures. Another similarity between monolayer and suspension cultures is the time-dependent decrease in P N M T activity. According to Hersey and DiStefano (1979), maintenance of P N M T levels should depend upon the amount of contamination by cortical cells a n d / o r amount of relevant glucocorticoids in the serum supplement. Thus, in their monolayer cultures of chromaffin cells that were deficient in cortical cells, P N M T activity decreased rapidly, whereas the presence of glucocorticoids or cortical cells delayed the decrease. On the other hand, Kilpatrick et al. (1980) did not see a substantial drop in P N M T activity until well after the first week in culture. But, approximately 30% of the cells in the cultures were cortical cells (Kilpatrick et al., 1980). To an extent, then, a relatively early decrease in P N M T activity is indicative of effective elimination of cortical cells during the purification. There are, however, a substantial number of differences between monolayer and suspension cultures of chromaffin cells. Chromaffin cell morphology is markedly different between the 2 types of culture. In contrast to the rounded, aggregated cells described above, chromaffin cells that have adhered to culture dishes tend to flatten and extend 'neurite'-like processes. And, for the most part, they are not in contact with other cells (see also Unsicker et al., 1980). In monolayer cultures, catecholamine

t4c#

levels drop substantially over the first 2-3 days in cuhure (Kilpatrick et al., 1980: Wilson and Viveros, 1981), in temporal correspondence to the establishment of 'neurite + morphology. And, unlike the selective loss of E with time bv chronlaffin cells in suspension, this large initial loss occurs for both E and NE: thus, the bases for the loss of catecholamines in the two culture systems tire probably not related. Whether this difference reflects a relationship between catecholamine loss and cell shape, adhesion to the substratum or lack of cell cell interaction is unkno\vn at present. Another obvious difference between monolaver and suspension culture of the chromaffin cell fractions involves the need for inhibitors of mitosis. Ahhough the chromaffin cell preparations may be relatively pure immediatel\ after purification. contaminating 'fibroblast'-like cells are capable of overgrowing monolaver cultures unless inhibitors such as fluorodeoxyuridine and cytosine arabinoside are included in the culture medium (e.g. kivett et al., 1981), In fact, Wilson and Vi\eros (IONI) report that cell division and a net protein increa,,,e occur in their monoluver cultures despite the presence of 10 >M fluorodeoxvuridine. We find that such inhibitors :trc unnecessary in suspension cultures in that the non-transfornled, cultured cells are essentially amitotic unless allowed to attach to a subststrate (Folkman and Moscona. 1978). Consequently, if the suspension cultures are transferred to tissue-culture dishes, all of the cells do eventually attach and the non-chromaffin cells begin to divide and become the major cell type in the culture. Given the differences between the rnonolayer and suspension cultures, the qucstion arises as to which culture system best constitutes a model for adrcnergic function. At present, the answer depends upon the particular functional aspect being addressed. For example, aside from the possibility that some of the cells arc diverting their cellular energies to neurite extension, the study of stimulu,, secretion coupling seems to be facilitated by monolayer cultures. The cells necd not be removed from the culture dish while washes and treatments are applied. This gentler treatment may be responsible for the relatively lower basal catecholanfine efflux from monolayer cultures (Livett et al., 1981: Trifar(+ and Lee, t980), ltux~cvcr, studies of catecholamine biosynthesis, storage, and turnovcr would he best carried out in cells that have a stable, well-regulated catecholamine metabolism, and ~l morphology more closely resembling that of cells in the intact adrenal gland, i.e. the suspension cultures. These experimental value judgements will evcntuallx be ~upplanted with a better notion of what an "ideal' model system for adrencrgic Iunctit+n should be. Perhaps side-by-side comparisons of suspension vs monolaxer cultures of the chromaffin cells will provide such a notion.

Acknowledgements This research was supported by grants from the USPHS (NS 11016 to ,I.C.W.), NSF (BNS 8041105 to W.F.B.) and Muscular Dystrophy Association (to W.F.B.).

35~

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