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Basic fibroblast growth factor promotes transmitter storage and synthesis in cultured chromaffin cells
Developmental Brain Research, 65 (1992) 211-216 ~) 1992 Elsevier Science Publishers B.V. All fights reserved. 0165-3806/92/$05.00
211
BRESD 51405
Basic fibroblast growth factor promotes transmitter storage and synthesis in cultured chromaffin cells Klaus Unsicker and Reiner Westermann Department of Anatomy and Cell Biology, University of Marburg, Marburg (ER.G.) (Accepted 8 August 1991)
Key words: Neurotrophic factor; Adrenal medulla; Basic fibroblast growth factor; Catecholamine; Enzyme; Chromaffin cell culture; Rat
We have studied the effects of basic fibroblast growth factor (bFGF), which occurs in the adrenal medulla, on the survival, morphological phenotype, storage capacity for catecholamines and induction of the synthesizing enzymes tyrosine hydroxyla~e (TH) and phenylethanolamine-N.methyltransferase (PNMT) of cultured chromaffin cells from young postnatal rats. Basic FGF (40 n~/ml), like nt~,rvegrowth factor (NGF; 40 ng/ml) prevented a drastic numerical decrease of chromaffin cells over a 4-day c~iture period, but, in contras~ to NGF, did not induce neurite outgrowth, unless the cells were maintained for 7 days. Basic FGF was also more effective than~NGF in maintaining the initial storage capacity for catecholamines, and even increased it under certain culture conditions (laminin instead of polyornitbine, or 200 ng instead of 40 ng/ml). Basic FGF and NGF did not induce TH and PNMT activities beyond their initial levels, but parti~JUyprevented the reduction of TH activity seen after 4 days in culture. Based on the present data and the previously reported greater in vitro survival and transmitter stability of older chmmaflin cells, which contain bFGF, and the relative instability of young postnatal chromaffin cells, which express no or very low levels of bFGF until 8 days postnatally, but respond to it, we hypothesize that bFGF is an important autocrine/ paracrine maintenance factor for adult chromaffih cells.
INTRODUCTION Basic and acidic fibroblast growth factors (bFGF, aFGF) are members of a multigene family with widespread distribution in mesoderm- and neuroectodermderived tissues and a broad spectrum of functions including cell division and induction and maintenance of differentiation t9'27. FGFs occur in the central and peripheral nervous systemzT'2s'3t. They have a variety of well-documented in vitro effects on neurons and glial cells, but it is not clear to what extent such effects reflect the physiological repertoire of FGF functions in the nervous system. A major handicap in proposing roles for FGFs in unlesioned tissues is the enigma of their release; bFGF and aFGF lack conventional signal peptides t and it is not clear, whether and how they may be released from intact cells. Basic FGF also occurs in the neuron-like chromaffln cells of the adrenal medulla 3'7's'3°. Immunoreactivity for bFGF is absent to sparse in the developing rat adrenal medulla until the end of the first postnatal week, but increases during the second and third week and further in adult animals7,s. With regard to putative functions of adrenal medullary bFGF, Blottner et al.3 have convinc-
ingly demonstrated that bFGF installed into a medullectomized adult rat adrenal gland prevents death of neurons in the ipsilateral intermediolateral column of the spinal cord, which provide the cholinergic innervation of adrenal chromaffin cells. This observation suggested that the chromaffin bFGF might act, directly or indirectly, as a neurotrophic maintenance factor for preganglionic neurons. Basic FGF (and aFGF) is also a mitogen and differentiation factor for embryonic rat chromaffin precursor cells, induces neurite outgrowth from these cells and confers NGF dependence upon the phenotypically shifted cells4'xs. Whether this sequale reflects the physiological steps of determinative and differentiative events that channel uncommitted sympathoadrenal precursor cells into sympathetic neurons remains to be shown. The present investigation tests the hypothesis that adrenal medullary bFGF may be a maintenance factor for chromaffin cells and was prompted by the observation that bFGF containing adult chromaflin cells possess a remarkable stability in terms of survival and transmitter storage in culture 11'25'32, whereas cells cultured from young postnatal rats, which lack bFGF, exhibit poc~,r survival and a drastically reduced capacity for storage and synthesis of catecholamines 16'24.
Correspondence: K. Unsicker, Department of Anatomy and Cell Biology, University of Marburg, Robert-Koch-Str. 6, D-3550 Marburg, ER.G. Fax: (49) (6421) 285783.
212 MATERIALS AND METHODS
Chemicals, reagents, additives, and growth factors Hanks' balanced salt solution without Ca 2+ and Mgz+ (CMF). Dulbecco's modified Eagle's medium (DMEM), penicillin G, and glutamine were purchased from Flow Laboratories. Meekenheim, ER.G. Noradrenaline, adrenaline, and HCIO4 were from Hoechst; t-tyrosine and ascorbic acid from Merck; L-3,4-dihydroxyphenylalanine (DOPA) and 6,7-dimethyltetrahydropterine from Calbiochem. C~tolase was purchased from Boehringer, Mannheim, dopamine from Serva, Tris-(hydroxymethyl)-aminomethanol from Roth, collagevase (type II) from Worthington, trypsin from ICN Pharmaceuticals, poly-L-a-ornithine HBr (PORN), deoxyepinephrine, o-benzylhydroxylamine, S-adenosylmethionine, dithiothreitol and bovine serum albumin (BSA; Cohn fraction V) from Sigma, and Sephadex from Pharmacia. All chemicals were of analytical grade. NGF was isolated from submaxillary glands of male mice and further purified on Sephadex G-75 as described 9. Recombinant human bFGF was a generous gift from PROGEN, Heidelberg (Dr. W. Knoerzer). Laminin (LN) was from Bethesda Research Laboratories.
Animal.~ Young postnatal Hanover-Wistar rats were obtained from Ivanovas Laboratories, Kislegg, and used at the 8th day after birth.
Celt cultures Cell cultures were set up in 24-well (16 mm diameter) or 96-well plates (6 mm diameter). Wells were coated for 1 h with PORN solution (0.1 mg/ml in 15 raM borate buffer, pH 8.4), washed twice with sterile water and once with DMEM. Alternatively, wells were coated with PORN and LN. LN was dissolved in DMEM and applied overnight at 15 ltg/ml. Pretreated culture wells received 250 ld (24-well plates ) or 50 td (96-well plates) of culture medium (DMEM containing NaHCO~ to 44.4 raM, glutamine to 2 raM, 100 U/ml of penicillin O, 0.25% BSA and the NI supplements prepared as described"* and 250 ~d'~° of cell suspension (25,000 and 1000, respectively, ehromaffin cells), Isolated chromaffin cells were prepared as nreviouslv described by Unsicker et al. '2 with minor modifications"~a. Chromaffin cells were large, rounded in shape and phase-bright and their identity was verified by staining with antibodies to dopamine//-hydroxylase (DBHJ-). Non-chrvmaffin cells amounted to about 15% after 4 days in culture. NC3F was presented at 40 ng/ml, and bFOF at 40 or 200 ng/ml, Cultures were incubated for 8 h, 4 or 7 days at 37°C and 5% CO.,/ 95% air in a humidified atmosphere. Medium was changed once or twice, respectively. Numbers of surviving ehromaffin cells were determined in glutaraldehyde-fixed cultures by phase contrast optics and verified in parallel cultures by staining for DBH.
Quantitative aeterminations of catecholamines and activities of tyrosine hydrn.ryla.~e (TH) and phenylethanolamine N-methyltrans]'erase (PNMT) in chromaffin cells Catecholamines, TH and PNMT activities were quantified as previously described in detail1~-~6 using cultures grown in 24-well plates. At 8 h or 4 days, respectively, numbers of surviving chromuffin cells were determined by counting within two diametrical stripes in each well covering 20% of the total surface area, Cell numbers were also determined in parallel cultures that had been processed for DBH immunocytochemistry in order to distinguish chromaffin and non-chromaffin cells. Then, culture medium was replaced by 300 td of ice-cold 20 mM sodium acetate buffer, pH 6.0 co~:taining0.1% Triton X-100, Cells were vigorously triturated, vortex-mixed, and 100-/d aliqots were processed for analysis of dopamine, noradrenaline, and adrenaline, whilst the remaining solution was stored at -80°C for measurements of enzyme activities. Determinations of catecholamines were performed in triplicate after deproteinization with HCIO4 by high-performance liquid chromatograph;~ (HPLC) with amperometric detection l~. N-Methyl-
dopamine was used as an internal standard. Samples for determining TH and PNMT activities were gel-filtered at 6°CTM, and DOPA and adrenaline, respectively, formed by enzymatic reaction from exogenously added tyrosine and noradrenaline, respectively, were measured by HPLC and amperometric detection.
Statistics Data were subject to a one.way analysis of variance and expressed as means _+ S.E.M. Differences were considered to be significant with P < 0.02. RESULTS
Survival and neurite outgrowth of cultured chromaffin cells Table I and Fig, 1 present quantitative and qualitative data with respect to survival and neurite growth, In confirmation of previous results -'6 approximately half of the seeded cells were attached to P O R N or P O R N / L N substrata after 8 h. A b o u t two-thirds of them survived over a period of 4 days on either substrate in the absence of trophic factors. N G F (40 ng/ml) doubled that number. In the presence of b F G F (40 ng/ml) numbers of chromaffin cells after 4 days matched those counted at 8 h. The simultaneous administration of b F G F and N G F did not further increase cell numbers. Acidic F G F , even when applied at 400 ng/ml, maintained as many chromaffin cells as N G E Basic F G F was also effective, when bound to PORN, and, at 80 ng per well, s u p p o r t e d an equal number of cells as N O F and a F G E T h e r e were about 2000 non-chromaffin cells present at 8 h, and their absolute number remained constant irrespectively of whether F G F s were present o r not. As 7reported previously "~'22-'4 N G F elicited neurite outgrowth from cultured chromaffin cells. In contrast to two previous investigations 4'ts, F G F s did not increase the p r o p o r t i o n of neurite-bearing cells during the initial 4-day period, and induced modest neuritic growth between days 4 and 7 (Fig. 1). When combined with NGF, b F G F did not enhance the percentage of cells with neurites as c o m p a r e d to N G F alone until day 4,
Storage of catecholamines and activities of TH and P N M T Table II relates the survival data to the changes in catecholamine content and proportions of d o p a m i n e , noradrenaline and adrenaline. Absolute amounts and relative proportions of catecholamines at 8 h were identical to values reported previously t5.16. W h e n chromaffin cells were grown on P O R N for 4 days, total catecholamines d r o p p e d to about 40% of their original level. N G F (40 ng/ml) modestly decreased these losses, whereas an identical dose of b F G F fully prevented them. A t 200 rig/ ml, b F G F even increased catecholamines above their initial value by approximately 50%. LN as a substrate maintained the storage capacity of chromaffin cells for
213 TABLE I Survival, neurite growth and development of non-chromaffin cells in 4-day cultures of rat adrenal chromaffin cells from 8-day-old rats: comparison of the effects of NGF, bFGF and aFGF Results are given as means _+ S.E.M.; n = 6. Surviving chromaffin cells
12 h 4 days without factors + NGF (40 ng/ml) + bFGF (40 ng/ml) + aFGF (400 ng/ml) + bFGF (80 ng/ml) bound to polyornithine + bFGF (40 ng/ml) + NGF (40 ng/ml)
Neurite.bearing cells as % of surviving cells
Non.chromaffin cells
on polyornithine
on laminin
+ 696 4- 427a 4- 700 a'b 4- 748b 4- 325a'b
0 0.8 4- 0.2 9.6 4- 1.0 1.0 + 0.2 ~<1
0 l.O ± 0.1 10.5 _+ 1.3 1.0 _.+ 0.2 ~<1
1,950 1,820 1,733 1,790
7,426 __ 590a'b
0.9 4- 0.2
n.d.
n.d.
11.0 4- 1.8
n.d.
12,137 3,948 8,032 11,600 7,850
12,080 + 918b
10.5 4- 1.2
4- 285 _ 315 4- 503 + 221
n.d., not determined ap < 0.02 compared to data for cells attached at 8 h after seeding. bp < 0.02 compared to data for cells surviving after 4 days in cultures without factors.
catecholamines without a d d e d N G F or b F G F . E i t h e r factor caused a small increase of cellular c a t e c h o l a m i n e s , which was m o r e pronounced with b F G F ( + 3 1 % ) t h a n with N G F ( + 5 % ) .
C u l t u r i n g chromaffin cells for 4 days resulted in a drastic r e d u c t i o n of T H a n d P N M T activities (Table III) as c o m p a r e d to the initial levels d e t e r m i n e d at 8 h ( - 7 7 % a n d - 6 5 % , respectively, o n P O R N ; - 6 1 % and - 5 6 % , re-
Fig. 1. Phase contrast micrographs of cultures established from 8-day-old rat adrenal glands after 4 days (A,B and C) and 7 days (D), respectively. Cultures received NGF (B, 40 ng/ml), bFGF (C and D, 40 ng/ml) or no trophic supplements (A). Neuritic growth was obvious with NGF (B), but very modest with bFGF even after 7 days (D).
214 TABLE II Catecholamine (CA) content per cell and relative amounts of noradrenaline, adrenaline and dopamine in mol % in cultures from &day-old rat chromaffin cells: effects of NGF, bFGF,, and the substrates polyornithine (PORN) and laminin Results are given as means + S.E.M.; n = G. Total CA (fmollcell) 8h 25.3 ± 0.9 PORN 4 days without factors 10.0 + 0.5a + NGF (40 ng/ml) 15.0 + 1.0 a'b + bFGF (40 ng/ml) 26.9 + 1.6b'~ + bFGF (200 ng/ml)37.6 + 2,2 a'b'c Laminin 4 days without factors 22.4 4- 1.1b'c + NGF (40 ng/mi) 26.6 4- 0 . 9 b'c + bFGF (40 ng/ml) 33.3 + 1.7a'b'c
Noradrenaline Noradrenaline Adrenaline (fmol/cell) (mol %) (fmollcell) 4.6 _+ 0.1 2.3 4- 0.2a 4.3 4- 0 . 2 b 8.9 "1" 0 . 6 a'b'c 14.4 4- 0.8a'b'~
Adrenaline (mol %)
Dopamine (fmol/cell)
Dopamine (moi %)
18.2 -+ 0.1
20.1 -+ 0.7
79.4 + 0.2
0.6 + 0.1
23.0 + 1.8 28.7 - 1.1a'b 33.1 -- 2 . 5 a'b 38.3 4- 2.3a'b'c
6.1 + 0.3a 9.1 -4- 0 . 6 a'b 15.7 4- 0.8 a'b-c 18.1 4- 0 . 9 b'c
61.0 +'3.2 e 50.7 + 4.2a 58.4 4- 3 . 1 a'b'c 48.1 4- 2.5~
1.6 1.6 2.3 5.1
+ 0.1a + 0.1a 4- 0.2~'~ + 0.3a'b'¢
16.0 4- 0.P 10.7 -4- 0 . 7 a.b 8.6 4- 0 . 7 e'b 13.5 4- 1.0a
13.9 _+ 0.4a'b 15.1 4- 0.5 a'b 17.6 4- 0.8b'~
62.1 4- 1.7a 56.8 + 2.0a 52.9 -+ 2.3~
1.8 + 0.1a 2.0 --+ 0.1a 2.7 4- 0 . 2 a'b'c
8.0 '4" 0.6 a'b 7.5 4- 0 . 4 a'b 8.1 4- 0.5 a'b
6.7 -+ 0.6a'b 29.9 4- 2.5a 9.5 4- 0.3b'~ 35.7 4- 1.3 a'b 13.0 4- 0.8a'b'~ 39.0 + 2.3~'b
2.4 + 0.2
~P < 0.02 compared to data for cells attached at 8 h after seeding, bp < 0.02 compared to data for cells surviving after 4 days in culture without factors on polyomithine (PORN). ~P < 0.02 compared to data for cells surviving after 4 days with NGF on PORN.
spectively, on LN). N G F (40 ng/ml) and b F G F (40 or 200 ng/ml) partially prevented these reductions with regard to TH, but not PNMT activity. DISCUSSION Our results are consistent with the notion that b F G F may act as a maintenance factor for chromaffin cells. They are also in line with our previous observation that cell culture media conditioned by adult bovine chroma-
TABLE Ill Effects of NGF and bFGF on TH and PNMT activities Results are given as means :1: S,E.M,: n = 4.
8h PORN 4 days without factors + NGF (40 ng/ml) + bFGF (40 ng/ml) + hFGF (200 ng/ml) t.aminin 4 days without factors + NGF (40 ng/ml) + bFGF(40 n~ml)
TH tfmol/DOPA/. h/cell)
PNMT (fmo! adrenaline h/cell)
94.7 + 1.7
31,4 + 0.5
21.3 + 2.3" 40.5 + 3.1"'b 44.8 :l: 5.6a'b 53.5.4-7.7e,b
10.9 :l: 1.6" 14.7 + 2.8s 12,2 + 1.6a 12,8 4- 2.1a
36.7 + 4,7e 54.5 + 5.1a'b 50.0.4-4.9a.b
13.9 + 1,9e 12.8 + 1,8~ 10.7 ± 0,5a
"P < 0.02 compared to data for cells attached at 8 h after seeding. b p < 0.02 compared to data for cells surviving after 4 days in culture without factors on polyornithine (PORN).
flin cells that might contain b F G F (see below) support survival of young rat chromaffin cells2s. Basic F G F clearly prevents or diminishes the losses in the storage capacity of the cells for catecholamines and activity of the rate-limiting enzyme of catecholamine synthesis, TH. At high doses or in combination with LN, b F G F even increases catecholamine storage above the initial level. Since this increase in storage capacity is not accompanied by an induction of T H activity, it possibly reflects an effect on storage organelles or basal release, Dexamethasone has also been shown to enhance catecholamine storage of chromaffin cells cultured from early postnatal rats without elevating T H activityts. Although activity of PNMT, the adrenaline synthesizing enzyme, was not affected by bFGF, the factor prevented the culture-mediated losses of adrenaline as well as those of noradrenaline suggesting that both the adrenaline- and the noradrenaline-storing subpopulations of chromaffin cells are maintained, It is conceivable, therefore, that both subpopulations of chromaffi~ cells possess receptors for FGF, although in rat b F G f immunoreactivity seems to be restricted to the noradrePergic subpopulations. Alternatively, the effects of FG1~ might be indirect and mediated by non-chromaffin ~'ells. Although n u m b e r s of non-chromaffin cells remained constant over 4 days even in the presence of FGF, thus reducing the probability of a mitogenic effect, the possibility has to be considered that F G F enhanced the output of trophic factors supporting chromaffin cells. The maintenance effect of F G F on chromaffin cells also included numerical stability. I n view of the documented mitogenic effect of a F G F on postnatal 4 and
215 b F G F on embryonic rat chromaffin (precursor) cells ms effects on cell number observed by us could result from a combination of cell proliferation, death, and survival. It should be noted, however, that F O F does not seem to promote chromaffin cell survival in long-term cultures 4. Neurite outgrowth in response to b F G F and aFOF was very modest in our experiments, even after 1 week. While a lack of effect of a F G F might be due to the absence of exogenous heparin 4, the small effect of bFGF is in contrast to reports that used embryonic rat chromaffin precursor cells TM or immortalized precursor cells 2 and difficult to explain, but can possibly be attributed to differences in culture conditions and developmental state of the cells. Beyond the description of a supportive effect of FGF on catecholamine storage and synthesis o f early postnatal chromaffin cells in culture, our study may provide cues for understanding the physiological roles of bFGF in chromaffin cells. The postnatal appearance and increase in b F G F immunoreactivity in rat chromaffin cells s suggest roles for differentiated rather than embryonic states. Stability of adolescent and adult chromaffin cells in culture has frequently been reported H'24,2s'32, while neonatal and early postnatal cells from the first postnatal week (rat) seem to be more difficult to maintain and keep differentiated in terms of transmitter synthesis ~6'24, unless being supported by glucocorticoids s'~7. We suggest that F G F may assure differentiative stability of chromaffin cells, possibly by autocrine and para-
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crine mechanisms, in the adult adrenal medulla. Since b F G F immunoreactivity and biological activity has been found in chromaffin vesicles 3'3°, release of the material by exocytosis seems possible. However, cytosolic b F G F 3° may also be released by alternative pathways ¢r be effective without leaving the cell I. Although FGF can be stored in and presented to cells by basement membranes 6, it has not been detected in basement membranes of the adrenal medulla TM. Since factors other than FGFs, such as glucocorticoids5'2° and ciliary neurotrophic factor (CNTF 16) also occur in the microenvironment of or within chromaffin cells 29 and effectively promote differentiation of chromaffin cells in culture, it is conceivable that the stability of this cell type, in vivo, is controlled by multiple auto- and paracrine humoral factors. The inverse temporal relationship between increasing F G F expression by chromaffin cells during postnatal development and decreasing frequency of cell divisions l°'2t with age does not support a role of FGF in the control of chromaffin cell proliferation, unless FGF would be anti-mitogenic, as has recently been shown for several neuroblastoma cell lines 13.
Acknowledgements. We thank, Heidi Hlaziaty, Heike ReichertPreibsch and Heidi Schneider for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Un 34/11) and Deutsche Krebshilfe-Dr. Mildred ScheelStiftung.
8 Grothe, C. and Un~icker, K., Immunocytochemical mapping of basic fibrohlast gro~vth factor in the developing and adult rat adrenal gland, Histochemistry, 94 (1990) 141-147. 9 Hofmann, H.D. and Unsicker, K., Characterization and partial purification of a novel neuronotrophic factor from bovine seminal vesicle, J. Neurochem., 48 (1987) 1425-1433. 10 Jurecka, W., Lassmann, H. and H0randner, H., The proliferation of adrenal medullary cells in newborn and adult mice, Cell Ti3s. Res., 189 (1978) 305-312. 11 Kilpatrick, D.L., Ledbetter, F.H., Carson, K.A., Kirshner, A.G., Slepetis, R. and Kirshner, N., Stability of bovine adrenal medulla cells in culture, J. Neurochem., 35 (1980) 679-692. 12 Lietzke, R. and Unsicker, K., Tetanus toxin binding to different morphological phenotypes of cultured rat and bovine adrenal medullary cells, Neurosci. Lett., 38 (1983) 233-238. 13 Ltldecke, O. and Unsieker, K., Heterogeneity of human neumblastoma cell lines in their proliferative responses to basic FGF, NGF and EGF, Cancer Res., submitted. 14 Milller, T.H. and Un~;icker, K., High-performance liquid chto-. matography with electrochemical detection as a highly efficient tool for studying catecholaminergic systems. I. Quantification of noradrenaline, adrenaline and dopamine in cultured a~,'enal medullary cells, J. Neurosci. Meth., 4 (1981) 36-52. 15 Miiller, T.H. and Unsicker, K., Nerve growth factor and dexamethasone modulate ;ynthesis and storage of catecholamines in cultured rat adrenat medullary cells: dependence on postnatal age, J. Neurochem., 46 (1986) 516-524. 16 Seidl, K., Manthorpe, M., Varon, S. and Unsicker, K., Differential effects of nerve growth factor and ciliary neuronotrophic
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211
BRESD 51405
Basic fibroblast growth factor promotes transmitter storage and synthesis in cultured chromaffin cells Klaus Unsicker and Reiner Westermann Department of Anatomy and Cell Biology, University of Marburg, Marburg (ER.G.) (Accepted 8 August 1991)
Key words: Neurotrophic factor; Adrenal medulla; Basic fibroblast growth factor; Catecholamine; Enzyme; Chromaffin cell culture; Rat
We have studied the effects of basic fibroblast growth factor (bFGF), which occurs in the adrenal medulla, on the survival, morphological phenotype, storage capacity for catecholamines and induction of the synthesizing enzymes tyrosine hydroxyla~e (TH) and phenylethanolamine-N.methyltransferase (PNMT) of cultured chromaffin cells from young postnatal rats. Basic FGF (40 n~/ml), like nt~,rvegrowth factor (NGF; 40 ng/ml) prevented a drastic numerical decrease of chromaffin cells over a 4-day c~iture period, but, in contras~ to NGF, did not induce neurite outgrowth, unless the cells were maintained for 7 days. Basic FGF was also more effective than~NGF in maintaining the initial storage capacity for catecholamines, and even increased it under certain culture conditions (laminin instead of polyornitbine, or 200 ng instead of 40 ng/ml). Basic FGF and NGF did not induce TH and PNMT activities beyond their initial levels, but parti~JUyprevented the reduction of TH activity seen after 4 days in culture. Based on the present data and the previously reported greater in vitro survival and transmitter stability of older chmmaflin cells, which contain bFGF, and the relative instability of young postnatal chromaffin cells, which express no or very low levels of bFGF until 8 days postnatally, but respond to it, we hypothesize that bFGF is an important autocrine/ paracrine maintenance factor for adult chromaffih cells.
INTRODUCTION Basic and acidic fibroblast growth factors (bFGF, aFGF) are members of a multigene family with widespread distribution in mesoderm- and neuroectodermderived tissues and a broad spectrum of functions including cell division and induction and maintenance of differentiation t9'27. FGFs occur in the central and peripheral nervous systemzT'2s'3t. They have a variety of well-documented in vitro effects on neurons and glial cells, but it is not clear to what extent such effects reflect the physiological repertoire of FGF functions in the nervous system. A major handicap in proposing roles for FGFs in unlesioned tissues is the enigma of their release; bFGF and aFGF lack conventional signal peptides t and it is not clear, whether and how they may be released from intact cells. Basic FGF also occurs in the neuron-like chromaffln cells of the adrenal medulla 3'7's'3°. Immunoreactivity for bFGF is absent to sparse in the developing rat adrenal medulla until the end of the first postnatal week, but increases during the second and third week and further in adult animals7,s. With regard to putative functions of adrenal medullary bFGF, Blottner et al.3 have convinc-
ingly demonstrated that bFGF installed into a medullectomized adult rat adrenal gland prevents death of neurons in the ipsilateral intermediolateral column of the spinal cord, which provide the cholinergic innervation of adrenal chromaffin cells. This observation suggested that the chromaffin bFGF might act, directly or indirectly, as a neurotrophic maintenance factor for preganglionic neurons. Basic FGF (and aFGF) is also a mitogen and differentiation factor for embryonic rat chromaffin precursor cells, induces neurite outgrowth from these cells and confers NGF dependence upon the phenotypically shifted cells4'xs. Whether this sequale reflects the physiological steps of determinative and differentiative events that channel uncommitted sympathoadrenal precursor cells into sympathetic neurons remains to be shown. The present investigation tests the hypothesis that adrenal medullary bFGF may be a maintenance factor for chromaffin cells and was prompted by the observation that bFGF containing adult chromaflin cells possess a remarkable stability in terms of survival and transmitter storage in culture 11'25'32, whereas cells cultured from young postnatal rats, which lack bFGF, exhibit poc~,r survival and a drastically reduced capacity for storage and synthesis of catecholamines 16'24.
Correspondence: K. Unsicker, Department of Anatomy and Cell Biology, University of Marburg, Robert-Koch-Str. 6, D-3550 Marburg, ER.G. Fax: (49) (6421) 285783.
212 MATERIALS AND METHODS
Chemicals, reagents, additives, and growth factors Hanks' balanced salt solution without Ca 2+ and Mgz+ (CMF). Dulbecco's modified Eagle's medium (DMEM), penicillin G, and glutamine were purchased from Flow Laboratories. Meekenheim, ER.G. Noradrenaline, adrenaline, and HCIO4 were from Hoechst; t-tyrosine and ascorbic acid from Merck; L-3,4-dihydroxyphenylalanine (DOPA) and 6,7-dimethyltetrahydropterine from Calbiochem. C~tolase was purchased from Boehringer, Mannheim, dopamine from Serva, Tris-(hydroxymethyl)-aminomethanol from Roth, collagevase (type II) from Worthington, trypsin from ICN Pharmaceuticals, poly-L-a-ornithine HBr (PORN), deoxyepinephrine, o-benzylhydroxylamine, S-adenosylmethionine, dithiothreitol and bovine serum albumin (BSA; Cohn fraction V) from Sigma, and Sephadex from Pharmacia. All chemicals were of analytical grade. NGF was isolated from submaxillary glands of male mice and further purified on Sephadex G-75 as described 9. Recombinant human bFGF was a generous gift from PROGEN, Heidelberg (Dr. W. Knoerzer). Laminin (LN) was from Bethesda Research Laboratories.
Animal.~ Young postnatal Hanover-Wistar rats were obtained from Ivanovas Laboratories, Kislegg, and used at the 8th day after birth.
Celt cultures Cell cultures were set up in 24-well (16 mm diameter) or 96-well plates (6 mm diameter). Wells were coated for 1 h with PORN solution (0.1 mg/ml in 15 raM borate buffer, pH 8.4), washed twice with sterile water and once with DMEM. Alternatively, wells were coated with PORN and LN. LN was dissolved in DMEM and applied overnight at 15 ltg/ml. Pretreated culture wells received 250 ld (24-well plates ) or 50 td (96-well plates) of culture medium (DMEM containing NaHCO~ to 44.4 raM, glutamine to 2 raM, 100 U/ml of penicillin O, 0.25% BSA and the NI supplements prepared as described"* and 250 ~d'~° of cell suspension (25,000 and 1000, respectively, ehromaffin cells), Isolated chromaffin cells were prepared as nreviouslv described by Unsicker et al. '2 with minor modifications"~a. Chromaffin cells were large, rounded in shape and phase-bright and their identity was verified by staining with antibodies to dopamine//-hydroxylase (DBHJ-). Non-chrvmaffin cells amounted to about 15% after 4 days in culture. NC3F was presented at 40 ng/ml, and bFOF at 40 or 200 ng/ml, Cultures were incubated for 8 h, 4 or 7 days at 37°C and 5% CO.,/ 95% air in a humidified atmosphere. Medium was changed once or twice, respectively. Numbers of surviving ehromaffin cells were determined in glutaraldehyde-fixed cultures by phase contrast optics and verified in parallel cultures by staining for DBH.
Quantitative aeterminations of catecholamines and activities of tyrosine hydrn.ryla.~e (TH) and phenylethanolamine N-methyltrans]'erase (PNMT) in chromaffin cells Catecholamines, TH and PNMT activities were quantified as previously described in detail1~-~6 using cultures grown in 24-well plates. At 8 h or 4 days, respectively, numbers of surviving chromuffin cells were determined by counting within two diametrical stripes in each well covering 20% of the total surface area, Cell numbers were also determined in parallel cultures that had been processed for DBH immunocytochemistry in order to distinguish chromaffin and non-chromaffin cells. Then, culture medium was replaced by 300 td of ice-cold 20 mM sodium acetate buffer, pH 6.0 co~:taining0.1% Triton X-100, Cells were vigorously triturated, vortex-mixed, and 100-/d aliqots were processed for analysis of dopamine, noradrenaline, and adrenaline, whilst the remaining solution was stored at -80°C for measurements of enzyme activities. Determinations of catecholamines were performed in triplicate after deproteinization with HCIO4 by high-performance liquid chromatograph;~ (HPLC) with amperometric detection l~. N-Methyl-
dopamine was used as an internal standard. Samples for determining TH and PNMT activities were gel-filtered at 6°CTM, and DOPA and adrenaline, respectively, formed by enzymatic reaction from exogenously added tyrosine and noradrenaline, respectively, were measured by HPLC and amperometric detection.
Statistics Data were subject to a one.way analysis of variance and expressed as means _+ S.E.M. Differences were considered to be significant with P < 0.02. RESULTS
Survival and neurite outgrowth of cultured chromaffin cells Table I and Fig, 1 present quantitative and qualitative data with respect to survival and neurite growth, In confirmation of previous results -'6 approximately half of the seeded cells were attached to P O R N or P O R N / L N substrata after 8 h. A b o u t two-thirds of them survived over a period of 4 days on either substrate in the absence of trophic factors. N G F (40 ng/ml) doubled that number. In the presence of b F G F (40 ng/ml) numbers of chromaffin cells after 4 days matched those counted at 8 h. The simultaneous administration of b F G F and N G F did not further increase cell numbers. Acidic F G F , even when applied at 400 ng/ml, maintained as many chromaffin cells as N G E Basic F G F was also effective, when bound to PORN, and, at 80 ng per well, s u p p o r t e d an equal number of cells as N O F and a F G E T h e r e were about 2000 non-chromaffin cells present at 8 h, and their absolute number remained constant irrespectively of whether F G F s were present o r not. As 7reported previously "~'22-'4 N G F elicited neurite outgrowth from cultured chromaffin cells. In contrast to two previous investigations 4'ts, F G F s did not increase the p r o p o r t i o n of neurite-bearing cells during the initial 4-day period, and induced modest neuritic growth between days 4 and 7 (Fig. 1). When combined with NGF, b F G F did not enhance the percentage of cells with neurites as c o m p a r e d to N G F alone until day 4,
Storage of catecholamines and activities of TH and P N M T Table II relates the survival data to the changes in catecholamine content and proportions of d o p a m i n e , noradrenaline and adrenaline. Absolute amounts and relative proportions of catecholamines at 8 h were identical to values reported previously t5.16. W h e n chromaffin cells were grown on P O R N for 4 days, total catecholamines d r o p p e d to about 40% of their original level. N G F (40 ng/ml) modestly decreased these losses, whereas an identical dose of b F G F fully prevented them. A t 200 rig/ ml, b F G F even increased catecholamines above their initial value by approximately 50%. LN as a substrate maintained the storage capacity of chromaffin cells for
213 TABLE I Survival, neurite growth and development of non-chromaffin cells in 4-day cultures of rat adrenal chromaffin cells from 8-day-old rats: comparison of the effects of NGF, bFGF and aFGF Results are given as means _+ S.E.M.; n = 6. Surviving chromaffin cells
12 h 4 days without factors + NGF (40 ng/ml) + bFGF (40 ng/ml) + aFGF (400 ng/ml) + bFGF (80 ng/ml) bound to polyornithine + bFGF (40 ng/ml) + NGF (40 ng/ml)
Neurite.bearing cells as % of surviving cells
Non.chromaffin cells
on polyornithine
on laminin
+ 696 4- 427a 4- 700 a'b 4- 748b 4- 325a'b
0 0.8 4- 0.2 9.6 4- 1.0 1.0 + 0.2 ~<1
0 l.O ± 0.1 10.5 _+ 1.3 1.0 _.+ 0.2 ~<1
1,950 1,820 1,733 1,790
7,426 __ 590a'b
0.9 4- 0.2
n.d.
n.d.
11.0 4- 1.8
n.d.
12,137 3,948 8,032 11,600 7,850
12,080 + 918b
10.5 4- 1.2
4- 285 _ 315 4- 503 + 221
n.d., not determined ap < 0.02 compared to data for cells attached at 8 h after seeding. bp < 0.02 compared to data for cells surviving after 4 days in cultures without factors.
catecholamines without a d d e d N G F or b F G F . E i t h e r factor caused a small increase of cellular c a t e c h o l a m i n e s , which was m o r e pronounced with b F G F ( + 3 1 % ) t h a n with N G F ( + 5 % ) .
C u l t u r i n g chromaffin cells for 4 days resulted in a drastic r e d u c t i o n of T H a n d P N M T activities (Table III) as c o m p a r e d to the initial levels d e t e r m i n e d at 8 h ( - 7 7 % a n d - 6 5 % , respectively, o n P O R N ; - 6 1 % and - 5 6 % , re-
Fig. 1. Phase contrast micrographs of cultures established from 8-day-old rat adrenal glands after 4 days (A,B and C) and 7 days (D), respectively. Cultures received NGF (B, 40 ng/ml), bFGF (C and D, 40 ng/ml) or no trophic supplements (A). Neuritic growth was obvious with NGF (B), but very modest with bFGF even after 7 days (D).
214 TABLE II Catecholamine (CA) content per cell and relative amounts of noradrenaline, adrenaline and dopamine in mol % in cultures from &day-old rat chromaffin cells: effects of NGF, bFGF,, and the substrates polyornithine (PORN) and laminin Results are given as means + S.E.M.; n = G. Total CA (fmollcell) 8h 25.3 ± 0.9 PORN 4 days without factors 10.0 + 0.5a + NGF (40 ng/ml) 15.0 + 1.0 a'b + bFGF (40 ng/ml) 26.9 + 1.6b'~ + bFGF (200 ng/ml)37.6 + 2,2 a'b'c Laminin 4 days without factors 22.4 4- 1.1b'c + NGF (40 ng/mi) 26.6 4- 0 . 9 b'c + bFGF (40 ng/ml) 33.3 + 1.7a'b'c
Noradrenaline Noradrenaline Adrenaline (fmol/cell) (mol %) (fmollcell) 4.6 _+ 0.1 2.3 4- 0.2a 4.3 4- 0 . 2 b 8.9 "1" 0 . 6 a'b'c 14.4 4- 0.8a'b'~
Adrenaline (mol %)
Dopamine (fmol/cell)
Dopamine (moi %)
18.2 -+ 0.1
20.1 -+ 0.7
79.4 + 0.2
0.6 + 0.1
23.0 + 1.8 28.7 - 1.1a'b 33.1 -- 2 . 5 a'b 38.3 4- 2.3a'b'c
6.1 + 0.3a 9.1 -4- 0 . 6 a'b 15.7 4- 0.8 a'b-c 18.1 4- 0 . 9 b'c
61.0 +'3.2 e 50.7 + 4.2a 58.4 4- 3 . 1 a'b'c 48.1 4- 2.5~
1.6 1.6 2.3 5.1
+ 0.1a + 0.1a 4- 0.2~'~ + 0.3a'b'¢
16.0 4- 0.P 10.7 -4- 0 . 7 a.b 8.6 4- 0 . 7 e'b 13.5 4- 1.0a
13.9 _+ 0.4a'b 15.1 4- 0.5 a'b 17.6 4- 0.8b'~
62.1 4- 1.7a 56.8 + 2.0a 52.9 -+ 2.3~
1.8 + 0.1a 2.0 --+ 0.1a 2.7 4- 0 . 2 a'b'c
8.0 '4" 0.6 a'b 7.5 4- 0 . 4 a'b 8.1 4- 0.5 a'b
6.7 -+ 0.6a'b 29.9 4- 2.5a 9.5 4- 0.3b'~ 35.7 4- 1.3 a'b 13.0 4- 0.8a'b'~ 39.0 + 2.3~'b
2.4 + 0.2
~P < 0.02 compared to data for cells attached at 8 h after seeding, bp < 0.02 compared to data for cells surviving after 4 days in culture without factors on polyomithine (PORN). ~P < 0.02 compared to data for cells surviving after 4 days with NGF on PORN.
spectively, on LN). N G F (40 ng/ml) and b F G F (40 or 200 ng/ml) partially prevented these reductions with regard to TH, but not PNMT activity. DISCUSSION Our results are consistent with the notion that b F G F may act as a maintenance factor for chromaffin cells. They are also in line with our previous observation that cell culture media conditioned by adult bovine chroma-
TABLE Ill Effects of NGF and bFGF on TH and PNMT activities Results are given as means :1: S,E.M,: n = 4.
8h PORN 4 days without factors + NGF (40 ng/ml) + bFGF (40 ng/ml) + hFGF (200 ng/ml) t.aminin 4 days without factors + NGF (40 ng/ml) + bFGF(40 n~ml)
TH tfmol/DOPA/. h/cell)
PNMT (fmo! adrenaline h/cell)
94.7 + 1.7
31,4 + 0.5
21.3 + 2.3" 40.5 + 3.1"'b 44.8 :l: 5.6a'b 53.5.4-7.7e,b
10.9 :l: 1.6" 14.7 + 2.8s 12,2 + 1.6a 12,8 4- 2.1a
36.7 + 4,7e 54.5 + 5.1a'b 50.0.4-4.9a.b
13.9 + 1,9e 12.8 + 1,8~ 10.7 ± 0,5a
"P < 0.02 compared to data for cells attached at 8 h after seeding. b p < 0.02 compared to data for cells surviving after 4 days in culture without factors on polyornithine (PORN).
flin cells that might contain b F G F (see below) support survival of young rat chromaffin cells2s. Basic F G F clearly prevents or diminishes the losses in the storage capacity of the cells for catecholamines and activity of the rate-limiting enzyme of catecholamine synthesis, TH. At high doses or in combination with LN, b F G F even increases catecholamine storage above the initial level. Since this increase in storage capacity is not accompanied by an induction of T H activity, it possibly reflects an effect on storage organelles or basal release, Dexamethasone has also been shown to enhance catecholamine storage of chromaffin cells cultured from early postnatal rats without elevating T H activityts. Although activity of PNMT, the adrenaline synthesizing enzyme, was not affected by bFGF, the factor prevented the culture-mediated losses of adrenaline as well as those of noradrenaline suggesting that both the adrenaline- and the noradrenaline-storing subpopulations of chromaffin cells are maintained, It is conceivable, therefore, that both subpopulations of chromaffi~ cells possess receptors for FGF, although in rat b F G f immunoreactivity seems to be restricted to the noradrePergic subpopulations. Alternatively, the effects of FG1~ might be indirect and mediated by non-chromaffin ~'ells. Although n u m b e r s of non-chromaffin cells remained constant over 4 days even in the presence of FGF, thus reducing the probability of a mitogenic effect, the possibility has to be considered that F G F enhanced the output of trophic factors supporting chromaffin cells. The maintenance effect of F G F on chromaffin cells also included numerical stability. I n view of the documented mitogenic effect of a F G F on postnatal 4 and
215 b F G F on embryonic rat chromaffin (precursor) cells ms effects on cell number observed by us could result from a combination of cell proliferation, death, and survival. It should be noted, however, that F O F does not seem to promote chromaffin cell survival in long-term cultures 4. Neurite outgrowth in response to b F G F and aFOF was very modest in our experiments, even after 1 week. While a lack of effect of a F G F might be due to the absence of exogenous heparin 4, the small effect of bFGF is in contrast to reports that used embryonic rat chromaffin precursor cells TM or immortalized precursor cells 2 and difficult to explain, but can possibly be attributed to differences in culture conditions and developmental state of the cells. Beyond the description of a supportive effect of FGF on catecholamine storage and synthesis o f early postnatal chromaffin cells in culture, our study may provide cues for understanding the physiological roles of bFGF in chromaffin cells. The postnatal appearance and increase in b F G F immunoreactivity in rat chromaffin cells s suggest roles for differentiated rather than embryonic states. Stability of adolescent and adult chromaffin cells in culture has frequently been reported H'24,2s'32, while neonatal and early postnatal cells from the first postnatal week (rat) seem to be more difficult to maintain and keep differentiated in terms of transmitter synthesis ~6'24, unless being supported by glucocorticoids s'~7. We suggest that F G F may assure differentiative stability of chromaffin cells, possibly by autocrine and para-
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Acknowledgements. We thank, Heidi Hlaziaty, Heike ReichertPreibsch and Heidi Schneider for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Un 34/11) and Deutsche Krebshilfe-Dr. Mildred ScheelStiftung.
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