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Production of “ectopic” vasoactive intestinal peptide-like immunoreactivity in normal human chromaffin cell cultures
Life Sciences, Vol. 37, pp= 1881-1886 Printed in the U.S.A.
Pergamon Press
PRODUCTION OF "ECIDPIC" VASOACTIVE INTESTINAL PEPTIDE-LIKE I~I~K)REACFIVITY IN NO~4AL HUMAN CHROMAFFIN CELL CULTURES Arthur S. Tischler I , ging ~. Lee 2, Robert L. ~erlman 3, Donna Costopoulos~ Stephen R. Bloom i. Department of Pathology, Tufts University School of f~dicine, 136 Harrison Avenue Boston, ~ 02111 USA 2. Department of Medicine Hammersmith Hospital, Royal Postgraduate Medical School, Ducane Road, London WI20HS ENGLAND 3. Department of Physiology and Biophysics, University of Illinois College of ~dicine, Chicago, IL 60680 USA (Received in final form September 3, 1985) Summary Vasoactive intestinal peptide-like immunoreactivity (VIPLI) is not detectable in normal adult human chrcmaffin cells in vivo, but was demonstrated in cultured chromaffin cells from two normal adults after 22 days in vitro. Cellular content of VIPLI was markedly increased in the presence of nerve qrowth factor, which also stimulated neurite outgrowth. Catecholanine content decreased in the same cultures, and was not regulated in parallel with VIPLI. The amounts of VIPLI in normal human chromaffin cells in culture are comparable to those previously reported in human pheochromocytcma cell cultures. Theoretical models have attributed production of ectopic peptides by pheochromocytomas and other tumors to "immaturity" of tumor cells. Our findings, however, indicate that neither neoplasia nor cellular immaturity is a prerequisite for ectopic peptide production. Ectopic neuropeptides produced by normal chrcmaffin cells which undergo neuronal differentiation are of potential clinical importance in patients receiving autologous chrom~ffin cell transplants for Parkinsons' disease. Vasoactive intestinal peptide (VIP) is a regulatory peptide present in a n~nber of different types of neurons in the central and peripheral nervous systems (16). VIP is generally undetectable in normal adrenal chromaffin cells of humans and other mammals (ii). Human adrenal pheochrcmocytomas, however, can produce abundant "ectopic" VIP capable of causing a severe diarrheal syndrome (2,17,8,26). Neoplastic chromaffin cells frem human pheochromocytomas exhibit outgrowth of long, neurite-like processes in vitro (21), despite the absence of such process in vivo, and we have recently demonstrated that the acquisition of neuron-like morphology is accompanied by onset or marked enhancement of production of VIP-like immunoreactivity (VIPLI) (22). Both neurite outgrowth and production of VIPLI by pheochromocytQma cells tend to be increased by nerve growth factor (~F), and decreased by the corticosteroid dexamethasone (22). ~brmal adult human chrc~naffin cells in culture resemble their neoplastic counterparts in that they also exhibit process outgrowth which is stimulated by NGF and diminished by dexamethasone (19,20). The present investigation was undertaken to determine whether these normal cells are able to produce VIPLI, and whether changes in content of VIPLI are acccmDanied by changes in catecholamine stores. 0024-3205/85 $3.00 4 .00 Copyright (c) 1985 Pergamon Press Ltd.
1882
"Ectopic" VIP in Human Cromaffin Cells
Vol. 37, No. 20, 1985
Materials and Methods Normal adrenal glands were obtained from 69 year old (case i) and 49 year old (case 2) men who underwent radical nephrectomy for kidney tLrnors. Adrenal medullary tissue was dissected away from the cortex as previously described (19), minced, and dissociated by incubation for ].5 hr in collagenase (Worthington, 450 units/ml) followed by collagenase plus trypsin (0.25%) for an additional i hr. The dissociated cells were suspended in ~ C o y ' s 5Amediu~ with 20% fetal bovine ser~n and 50 ug/ml gentamycin. Replicate aliquots of dissociated cells from each adrenal were plated in collagen-coated (3) 35 mm tissue culture dishes (Corning) in routine medium or in medium supplemented with 2.5Smouse saliva_r~ gland NGF (14) (100ng/ml) and/or dex@nethasone disodium phosphate (10-JMi. Cytosine arabinoside (Sigma, I0 ~M) was added to all cultures to suppress fibroblast proliferation. Additional sets of identical cell aliquots were harvested immediately to be assayed for VIPLI and foE catecholamines. The cells from adrenal #i were plated at a density of 2XI0 cells/dish, and those from adrenal #2 at approximately 5XI0 q cells/dish. Cultures were incubated at 37°C in a watersaturated atmosphere of 95% air and 5% CO?, with medium changes 3 times per week. A final mediL~n change was performed 24 hours before harvest. On day 22 in vitro, both cell extracts and culture medium were harvested from each dish to be assayed for VIPLI, and cell extracts were again harvested for catechol~nine assays. In addition, process outgrowth and chromaffin cell survival under the different sets of culture conditions were quantitated in some. cultures by means of strip counts performed with a phase contrast microscope (20). For the harvests at day 22, the median was removed from each dish, supplemented with aprotinin (Sigma, final concentration 500 kallikrein inhibitor units per ml), boiled for 5 rain, frozen and lyophilzed. The cells were washed three times in Hank's balanced salt solution, scraped from the culture dishes in ice-cold, aprotinin-supDlemented phosphate-buffered saline, pH 7.2, and sonicated. Aliquots were taken for catechola~ine determinations and supplemented with HCI04 (final concentration 0.4N) and ethylenedi~nine tetraacetate (EDTA, final concentration imM). The romainder was boiled for 5 rain, supplemented with radioimmunoassay grade bovine sert~ albu~nin (Sigma, final concentration 1.5%), and centrifuged at 12,000 X g for 5 min in an EDpendorf microcentrifuge. The supernatant was then frozen and lyophilized. For the harvests on day 0, the dissociated cells in suspension were centrifuged at 300X~ for 5 min, washed 3 times in Hank's balanced salt solution, and then treated identically to the cells on day 22. VIPLI was measured in reconstituted lyophilized cell extracts and medium by a previously described radioimmunoassay using whole molecule-directed antibody against synthetic porcine VIP (13). The detection limit of the assay was 0.4 fmol/tube, and the interassay variation was 99%. Chromategraphic analyses were also performed on extracts of NGF-treated cells from both adrenals using a Sephadex G50 s u ~ f i n e col~nn (0.9X60om) calibrated with dextran blue, cytochrome c, and Na I as molecular size markers. The col~nn was eluted with 0.06M phosphate buffer, DH 7.0, containing 0.~% human ser~n albumin and 0.2M NaCI, and 0.9-ml fractions were collected at 4 C. Catecholamines were measured by liquid chromatography with electrochemical detection (7). Resu] ts Chromaffin cells were readily recoqnized (19,20) as polygonal, phase-dark epithelial-like cells which usually formed clusters, and which to varying degrees gave rise to thin, branchinq, varicose processes similar to those of cultured neurons. On day 22 in vitro, chromaffin cells in cultures from both adrenals exhibited processes TFi(~ure, a and b). As previously reported (19,
Vol. 37, No. 20, 1985
"Ectopic" VIP in Human 6romaffin Cells
1883
20), process outgrowth was stimulated by NGF and diminished by dexamethasone, and the different culture conditions caused no significant differences in chrcmaffin cell survival (20). At least 80% of all cells in the cultures appeared to be chromaffin cells, and the remainder were fibroblast-like (Figure, a & b). VIPLI was not detectable in cells from either adrenal at the time of plating, but was present in both cells and medium in all cultures at day 22 i n vitro. NGF-treated cultures of adrenals 1 and 2 respectively contained approximately 3 and 23 times as much total VIPLI as those without additives. When subjected to chromatographic analysis, virtually all of this VIPLI coeluted with a synthetic VIP standard (Figure, c). The VIPLI from the cultures also paralleled authentic VIP in displacement of tracer from antibody.
6
4 .= E o. 2
I'0
2'0
3'0
4'0
5'0
Fraction number
A&B, Representatiw~ phase contrast photomicrographs of live cultures from adrenal #2 at day 21 in vitro (X64). A, Culture in routine medium; B, culture in medium with NGF. Chromaffin cells occur predominantly in clusters, which show varying degroes of process outgrowth. Rare fibroblast-like cells are also present. Thirty two percent of chrcmaffin cells from adrenal #I formed processes in routine medium, vs. 75% in medium with NGF. Twenty two percent of chromaffin cells from adrenal #2 formed processed in routine medium, vs. 90% in mediL#n with NGF, 16% with dexanethasone, a~d 62% with NGF plus dexamethasone. Differences in p r e s s outgrowth between all conditions were statistically significant in X-tests (rejection limit FA.05). At least 365 cells were scored for each culture condition. C. Sephadex gel chromatographic profile of VIPLI in pooled cell extracts from NGF-treated cultures of adrenal #i. Virtually all of the VIPLI in both cases coeluted in single peaks with a synthetic ---I-VIP standard.
1884
"Ectopic" VIP in Human Cromaffin Cells
The amounts were comparable cells (22). In levels of VIPLI
of VIPLI t~ those contrast were not
Vol. 37, No. 20, 1985
in cultures of normal chromaffin cells in this study previously reported in cultures of pheochromocytoma to our findings with pheochrc~ocytoma cells, however, markedly affected by dexanethasone (Table i )
TABLE 1 - Eff,~cts of culture conditions on VIP-like immunoreactivity in cells and medium Immunoreactive VIP*
Adrenal # __Da~_q
Day 22
~ 1
2
cells(fmol/dish)
ND
medit~n(fmol/dish/24h)
-
Additives
1650+! 10 106+
2
cells(fmol/dish)
ND
46+
4
medium(fmol/dish/24h)
--
9+
1
NGF (100ng/ml) 4668+323 128+
Dexa~_~hasone (i0 ~M) 1893+245
5
114+
1070+ 79 51+
NGF + Dexamethasone
1
4543+85
3
132+ 7
66+
3
930+48
15+
0
54+ 1
* Values represent Mean + SEM of 4 culture dishes (adrenal i), or 3 cultur~ dishes (adrenal 2). Cultures from adrenals 1--and 2 respectively contained approximately 1.7X]0" and 1.9X10- chromaffin cells per dish at day 22, reflecting different plating densities..NGF did not affect chromaffin cell survival in either case, and dexanethasone did not affect survival of cells from adrenal 2. Cell survival in dexamethasone-treated cultures was not quantitated for adrenal I. Culturing cells in medium with no additives tends to increase VIP-like immunoreactivity, whether calculated per dish or per cell, a~l this increase is markedly enhanced by NGF. In contrast, dexamethasone exerts little or no effect. ND = [~t Detectable.
In contrast to the large increases in VIPLI, catecholanine content in the sane culture dishes decreased to less than 10% of initial levels per dish in cultures with no additives. Concurrently, there were marked decreases in the ratios of epinephrine (E) to noreDhinephrine (ME). Dexamethasone and/or NGF produced only small increases in total catecholamine content, without large changes in E/NE ratios (Table 2).
TABLE 2 - Effects of culture conditions on cellular catecholamine content* Adrenal #
Catechola~aine Content (~mol/dish)* Day O
Day 21
No additives i.
Dexameth@sone (10~M)
NGF + Dexamethasone
E
16434+500
72+ 8
112+ 5
92+ 4
194+ 5
NE
2816+ 50
930+22
1492+72
1042+39
1382+74
DA Total
2.
NGF (100ng/ml)
244+ 15
159+12
363+30
300+26
591+ 6
19494
1161
1967
1434
2167
E
1845
16
39
52
30
NE
245
8
15
22
23
[~%
50
27
35
38
44
Total
2149
51
89
112
97
* Values for E, NE, and [I~ represent Mean + SEM of 4 culture dishes (adrenal I) or mean of 2 dishes (adrenal 2). In contrast to increases in ir~munoreactive VIP, (Table i) concentrations of catecholamines in the same cultures decrease markedly after 22 days in vitro, and are only minimally affected by NGF. Dexamethasone and/or NGF variably affect content of individual catecholamines and produce small increases in total catecholamine content, without markedly altering E/NE ratio.
Vol. 37, No. 20, 1985
"Ectopic" VIP in Human Cromaffin Cells
1885
Discussion Our findings suggest that capacity for VIP production is a characteristic that is latent in normal adult human chromaffin cells as well as in many human pheochromocytomas, and is expressed in response to environmental signals. Quantitative differences which we observed in content of VIPLI between cultures from two different individuals after normalization for differences in plating density may reflect intrinsic individual variability or experimental factors. The latter might include different ages or batches of medium or sert~, or the different plating densities per se. In general, signals which favor production of VIPLI also favor neurite outgrowth in monolayer culture. The fact that dexanethasone decreased neurite outgrowth without affecting levels of VIPLI, however, is consistent with other evidence that neurite outgrowth and VIP production can probably bedissociated (9,22). It is of interest that recent studies of bovine chromaffin cells provide further evidence for at least some correlation between promotion of neurite outgrowth and of VIP production. Although adult bovine chromaffin cells in culture are known to form processes (12), process outgrowth from these cells is considerably less extensive than in human chromaffin cell cultures, and is not stimulated by NGF (15,24). Adult bovine chromaffin cells have also recently been reported to produce VIPLI in culture (5.6). The amount of VIPLI reported in these cells, however, is two orders of magnitude less than that in NGF-treated htlnan chromaffin cells. Effects of NGF on VIPLI in bovine chromaffin cell cultures have not been reported. The minimal effects of dexamethasone on catecholanine content in this study are scmewhat surprising in view of the reported ability of corticosteroids to increase the content or rate of production of E in other normal chromaffin cells in vitro (4,18,23). Dexamethasone, however, also failed to increase E/NE ratios in cultures of 5 of 6 human pheochromocytomas studied by us, but increased the ratio 14-fold in cultures of the 6th tumor (22). We have studied catecholamines in cultures of two additional normal adult human adrenals, and have observed no effect of dexanethasone on E/NE ratio in one case, and a 5-fold increase in the other (A.S. Tischler and R.L. Perlman, unpublished data). Our observation with both normal and neoplastic human chromaffin cells suggest that the ability of dexamethasone to increase E/NE ratio might be dependent on the presence of as yet unknown permissive factors. Theoretical models accounting for the production of ectopic peptides by t~nors have related this phenomenon to "immaturity" or "incomplete differentiation" of tt~nor cells (1,25). The finding that normal adult human chromaffin cells can produce large quantities of an ectopic peptide, however, suggests that these models need to be modified for tumors arising form some types of cells in which maintenance of a "mature" phenotype is environmentdependent. An additional implication of this finding relates to current attempts to transplant chromaffin cells to the brain to replace catecholaminergic neurons in patients with Parkinson's disease (I0). It must now be ascertained that the transition from chromaffin cell to neuronal phenotype in vivo is not accompanied by production of physiologically inappropriate peptide neurotransmitters. Acknowledgements This research was supported by NIH grants CA27808, CA37370 and HL29025, and the Medical Research Council. The authors thank Ms. S. Tsymbalov for technical assistance.
1886
"Ectopic" VIP in Human Cromaffin Cells
Vol. 37, No. 20, 1985
References I. S.B. BAYLIN and G. MENDELSOHN, Endocrine Rev. 1 45-77 (1980). 2. P. BER~I~D, J. LEBRAS, J. PRODEL, A.C. VIGUER, P. CURET and J. GRELL~, J. Radiol. 61 121-124 (1980) 3. M.B. BORNSTEIN, Lab. Invest 7 134-137 (1958). 4. R.E. COUPLAND and J.D.B. MACDOUC9%LL, J. Endocrinol. 36 317-324 (1966). 5. L.E. EIDEN, R.L. ESKAY, J. SCOTT, H. POLLARD and A.J. HOTCHKISS, Life Sci. 33 687-693 (1983). 6. L.E. EIDEN, P. GIRAUD, A. HOTCHKISS and M.J. BROWNSTEIN, In: Regulato ~ Pe__ptides: from Molecular Biolog_/ to Function, (E. COSTA and H. TRABUCCI, eds.) 387-395, Raven Press, NY (1982). 7. R.E. ERNY, M.W. BEREZO and R.L. PERLMAN, J. Biol. Chem. 256 1335-1339 (1981). 8. I. GOZES, D.T. O ' C ( ~ O R and F.E. BLOOM, Reg. Peptides 6 111-119 (1983). 9. J. HASSOUN, G. ~K3NGES, P. GIRAUD, J.F. HENRY, C. CHARPIN, H. PAYAN and M. TOGA, Am. J. Pathol. 114 56-63 (1984). 10. G. KOLATA, Science 217 342- (1982). ii. R.I. LINNOILA, R.P. DIAUGUSTINE, A. HERV(~EN and R.J. rIILLER, Neuroscience 5 2247-2259 (1980). 12. B.G. LIVE'I~, D.M. DEAN and G.M. BRAY, Abst. Soc. Neurosci. 4 592- (1978). 13. S.J. MITCHELL and S.R. BLOCM, Gut 19 1043-1048 (1978). 14. W.C. MOBLEY, A. SCHENKER and E.M. SHOOTER, Biochemistry 15 5543-5551. 15. K.W. NAUJOKS, S. KORSCHING, H. ROHRER and H. THOENEN, Develop. Biol. 92 365-379 (1982). 16. S.I. SAID, In: Vasoactive Intestinal Peptid__ee, Raven Press, NY (1982). 17. S.I. SAID, J. Clin. Endocrinol. 5 201S (1976). 18. G. TEITEL~tAN, T.H. JOH, D. PARK, M. BRODSKY and D.J. REIS, Develop. Biol. 89 450-459 (1982). 19. A.S. TISCHLER, R.A. DELELLIS, B. BIALES, G. NUNNEMACHER, V. CARABBA and H.J. WOLFE, Lab. Invest. 43 399-409 (1980). 20. A.S. TISCHLER, R.A. DELELLIS, V.W. SLAYIDN, M. BLOUNT and H.J. WOLFE, Lab. Invest. 48 13- (1983). 21. A.S. TISCHLER, M.A. DICHTER, B. BL~LES, R.A. DELELLIS and H.J. WOLFE, Science 192 902-904 (1976). 22. A.S. TISCHLER, Y.C. LEE, R.L. PERLMAN, D. C(36TOPOULOS, V.W. SLAYTON and S.R. BLOOM, J. Neurosci 4 1398-1404 (1984). 23. A.S. TISCHLER, R . L PERLr~%N, G. NUNNF~ACHER, G.M. MORSE, R.A. DELELLIS and H.J. WOLFE, Cell Tissue Res. 225 525-542 (1982). 24. K. UNSICKER and H.D. HOFFHAH, Develop. Brain Res. 7 41-52 (1983). 25. J. URIEL, In: Cancer, A Comprehensive Treatise, Vol. 3 (F.F. BECKER , ed.) 21-55, Plen~n Press, NY (1975). 26. J.V. VERNER and A.B. ~KgRRISON Arch. Intern. Med. 133 492-500 (1958).
Pergamon Press
PRODUCTION OF "ECIDPIC" VASOACTIVE INTESTINAL PEPTIDE-LIKE I~I~K)REACFIVITY IN NO~4AL HUMAN CHROMAFFIN CELL CULTURES Arthur S. Tischler I , ging ~. Lee 2, Robert L. ~erlman 3, Donna Costopoulos~ Stephen R. Bloom i. Department of Pathology, Tufts University School of f~dicine, 136 Harrison Avenue Boston, ~ 02111 USA 2. Department of Medicine Hammersmith Hospital, Royal Postgraduate Medical School, Ducane Road, London WI20HS ENGLAND 3. Department of Physiology and Biophysics, University of Illinois College of ~dicine, Chicago, IL 60680 USA (Received in final form September 3, 1985) Summary Vasoactive intestinal peptide-like immunoreactivity (VIPLI) is not detectable in normal adult human chrcmaffin cells in vivo, but was demonstrated in cultured chromaffin cells from two normal adults after 22 days in vitro. Cellular content of VIPLI was markedly increased in the presence of nerve qrowth factor, which also stimulated neurite outgrowth. Catecholanine content decreased in the same cultures, and was not regulated in parallel with VIPLI. The amounts of VIPLI in normal human chromaffin cells in culture are comparable to those previously reported in human pheochromocytcma cell cultures. Theoretical models have attributed production of ectopic peptides by pheochromocytomas and other tumors to "immaturity" of tumor cells. Our findings, however, indicate that neither neoplasia nor cellular immaturity is a prerequisite for ectopic peptide production. Ectopic neuropeptides produced by normal chrcmaffin cells which undergo neuronal differentiation are of potential clinical importance in patients receiving autologous chrom~ffin cell transplants for Parkinsons' disease. Vasoactive intestinal peptide (VIP) is a regulatory peptide present in a n~nber of different types of neurons in the central and peripheral nervous systems (16). VIP is generally undetectable in normal adrenal chromaffin cells of humans and other mammals (ii). Human adrenal pheochrcmocytomas, however, can produce abundant "ectopic" VIP capable of causing a severe diarrheal syndrome (2,17,8,26). Neoplastic chromaffin cells frem human pheochromocytomas exhibit outgrowth of long, neurite-like processes in vitro (21), despite the absence of such process in vivo, and we have recently demonstrated that the acquisition of neuron-like morphology is accompanied by onset or marked enhancement of production of VIP-like immunoreactivity (VIPLI) (22). Both neurite outgrowth and production of VIPLI by pheochromocytQma cells tend to be increased by nerve growth factor (~F), and decreased by the corticosteroid dexamethasone (22). ~brmal adult human chrc~naffin cells in culture resemble their neoplastic counterparts in that they also exhibit process outgrowth which is stimulated by NGF and diminished by dexamethasone (19,20). The present investigation was undertaken to determine whether these normal cells are able to produce VIPLI, and whether changes in content of VIPLI are acccmDanied by changes in catecholamine stores. 0024-3205/85 $3.00 4 .00 Copyright (c) 1985 Pergamon Press Ltd.
1882
"Ectopic" VIP in Human Cromaffin Cells
Vol. 37, No. 20, 1985
Materials and Methods Normal adrenal glands were obtained from 69 year old (case i) and 49 year old (case 2) men who underwent radical nephrectomy for kidney tLrnors. Adrenal medullary tissue was dissected away from the cortex as previously described (19), minced, and dissociated by incubation for ].5 hr in collagenase (Worthington, 450 units/ml) followed by collagenase plus trypsin (0.25%) for an additional i hr. The dissociated cells were suspended in ~ C o y ' s 5Amediu~ with 20% fetal bovine ser~n and 50 ug/ml gentamycin. Replicate aliquots of dissociated cells from each adrenal were plated in collagen-coated (3) 35 mm tissue culture dishes (Corning) in routine medium or in medium supplemented with 2.5Smouse saliva_r~ gland NGF (14) (100ng/ml) and/or dex@nethasone disodium phosphate (10-JMi. Cytosine arabinoside (Sigma, I0 ~M) was added to all cultures to suppress fibroblast proliferation. Additional sets of identical cell aliquots were harvested immediately to be assayed for VIPLI and foE catecholamines. The cells from adrenal #i were plated at a density of 2XI0 cells/dish, and those from adrenal #2 at approximately 5XI0 q cells/dish. Cultures were incubated at 37°C in a watersaturated atmosphere of 95% air and 5% CO?, with medium changes 3 times per week. A final mediL~n change was performed 24 hours before harvest. On day 22 in vitro, both cell extracts and culture medium were harvested from each dish to be assayed for VIPLI, and cell extracts were again harvested for catechol~nine assays. In addition, process outgrowth and chromaffin cell survival under the different sets of culture conditions were quantitated in some. cultures by means of strip counts performed with a phase contrast microscope (20). For the harvests at day 22, the median was removed from each dish, supplemented with aprotinin (Sigma, final concentration 500 kallikrein inhibitor units per ml), boiled for 5 rain, frozen and lyophilzed. The cells were washed three times in Hank's balanced salt solution, scraped from the culture dishes in ice-cold, aprotinin-supDlemented phosphate-buffered saline, pH 7.2, and sonicated. Aliquots were taken for catechola~ine determinations and supplemented with HCI04 (final concentration 0.4N) and ethylenedi~nine tetraacetate (EDTA, final concentration imM). The romainder was boiled for 5 rain, supplemented with radioimmunoassay grade bovine sert~ albu~nin (Sigma, final concentration 1.5%), and centrifuged at 12,000 X g for 5 min in an EDpendorf microcentrifuge. The supernatant was then frozen and lyophilized. For the harvests on day 0, the dissociated cells in suspension were centrifuged at 300X~ for 5 min, washed 3 times in Hank's balanced salt solution, and then treated identically to the cells on day 22. VIPLI was measured in reconstituted lyophilized cell extracts and medium by a previously described radioimmunoassay using whole molecule-directed antibody against synthetic porcine VIP (13). The detection limit of the assay was 0.4 fmol/tube, and the interassay variation was 99%. Chromategraphic analyses were also performed on extracts of NGF-treated cells from both adrenals using a Sephadex G50 s u ~ f i n e col~nn (0.9X60om) calibrated with dextran blue, cytochrome c, and Na I as molecular size markers. The col~nn was eluted with 0.06M phosphate buffer, DH 7.0, containing 0.~% human ser~n albumin and 0.2M NaCI, and 0.9-ml fractions were collected at 4 C. Catecholamines were measured by liquid chromatography with electrochemical detection (7). Resu] ts Chromaffin cells were readily recoqnized (19,20) as polygonal, phase-dark epithelial-like cells which usually formed clusters, and which to varying degrees gave rise to thin, branchinq, varicose processes similar to those of cultured neurons. On day 22 in vitro, chromaffin cells in cultures from both adrenals exhibited processes TFi(~ure, a and b). As previously reported (19,
Vol. 37, No. 20, 1985
"Ectopic" VIP in Human 6romaffin Cells
1883
20), process outgrowth was stimulated by NGF and diminished by dexamethasone, and the different culture conditions caused no significant differences in chrcmaffin cell survival (20). At least 80% of all cells in the cultures appeared to be chromaffin cells, and the remainder were fibroblast-like (Figure, a & b). VIPLI was not detectable in cells from either adrenal at the time of plating, but was present in both cells and medium in all cultures at day 22 i n vitro. NGF-treated cultures of adrenals 1 and 2 respectively contained approximately 3 and 23 times as much total VIPLI as those without additives. When subjected to chromatographic analysis, virtually all of this VIPLI coeluted with a synthetic VIP standard (Figure, c). The VIPLI from the cultures also paralleled authentic VIP in displacement of tracer from antibody.
6
4 .= E o. 2
I'0
2'0
3'0
4'0
5'0
Fraction number
A&B, Representatiw~ phase contrast photomicrographs of live cultures from adrenal #2 at day 21 in vitro (X64). A, Culture in routine medium; B, culture in medium with NGF. Chromaffin cells occur predominantly in clusters, which show varying degroes of process outgrowth. Rare fibroblast-like cells are also present. Thirty two percent of chrcmaffin cells from adrenal #I formed processes in routine medium, vs. 75% in medium with NGF. Twenty two percent of chromaffin cells from adrenal #2 formed processed in routine medium, vs. 90% in mediL#n with NGF, 16% with dexanethasone, a~d 62% with NGF plus dexamethasone. Differences in p r e s s outgrowth between all conditions were statistically significant in X-tests (rejection limit FA.05). At least 365 cells were scored for each culture condition. C. Sephadex gel chromatographic profile of VIPLI in pooled cell extracts from NGF-treated cultures of adrenal #i. Virtually all of the VIPLI in both cases coeluted in single peaks with a synthetic ---I-VIP standard.
1884
"Ectopic" VIP in Human Cromaffin Cells
The amounts were comparable cells (22). In levels of VIPLI
of VIPLI t~ those contrast were not
Vol. 37, No. 20, 1985
in cultures of normal chromaffin cells in this study previously reported in cultures of pheochromocytoma to our findings with pheochrc~ocytoma cells, however, markedly affected by dexanethasone (Table i )
TABLE 1 - Eff,~cts of culture conditions on VIP-like immunoreactivity in cells and medium Immunoreactive VIP*
Adrenal # __Da~_q
Day 22
~ 1
2
cells(fmol/dish)
ND
medit~n(fmol/dish/24h)
-
Additives
1650+! 10 106+
2
cells(fmol/dish)
ND
46+
4
medium(fmol/dish/24h)
--
9+
1
NGF (100ng/ml) 4668+323 128+
Dexa~_~hasone (i0 ~M) 1893+245
5
114+
1070+ 79 51+
NGF + Dexamethasone
1
4543+85
3
132+ 7
66+
3
930+48
15+
0
54+ 1
* Values represent Mean + SEM of 4 culture dishes (adrenal i), or 3 cultur~ dishes (adrenal 2). Cultures from adrenals 1--and 2 respectively contained approximately 1.7X]0" and 1.9X10- chromaffin cells per dish at day 22, reflecting different plating densities..NGF did not affect chromaffin cell survival in either case, and dexanethasone did not affect survival of cells from adrenal 2. Cell survival in dexamethasone-treated cultures was not quantitated for adrenal I. Culturing cells in medium with no additives tends to increase VIP-like immunoreactivity, whether calculated per dish or per cell, a~l this increase is markedly enhanced by NGF. In contrast, dexamethasone exerts little or no effect. ND = [~t Detectable.
In contrast to the large increases in VIPLI, catecholanine content in the sane culture dishes decreased to less than 10% of initial levels per dish in cultures with no additives. Concurrently, there were marked decreases in the ratios of epinephrine (E) to noreDhinephrine (ME). Dexamethasone and/or NGF produced only small increases in total catecholamine content, without large changes in E/NE ratios (Table 2).
TABLE 2 - Effects of culture conditions on cellular catecholamine content* Adrenal #
Catechola~aine Content (~mol/dish)* Day O
Day 21
No additives i.
Dexameth@sone (10~M)
NGF + Dexamethasone
E
16434+500
72+ 8
112+ 5
92+ 4
194+ 5
NE
2816+ 50
930+22
1492+72
1042+39
1382+74
DA Total
2.
NGF (100ng/ml)
244+ 15
159+12
363+30
300+26
591+ 6
19494
1161
1967
1434
2167
E
1845
16
39
52
30
NE
245
8
15
22
23
[~%
50
27
35
38
44
Total
2149
51
89
112
97
* Values for E, NE, and [I~ represent Mean + SEM of 4 culture dishes (adrenal I) or mean of 2 dishes (adrenal 2). In contrast to increases in ir~munoreactive VIP, (Table i) concentrations of catecholamines in the same cultures decrease markedly after 22 days in vitro, and are only minimally affected by NGF. Dexamethasone and/or NGF variably affect content of individual catecholamines and produce small increases in total catecholamine content, without markedly altering E/NE ratio.
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Discussion Our findings suggest that capacity for VIP production is a characteristic that is latent in normal adult human chromaffin cells as well as in many human pheochromocytomas, and is expressed in response to environmental signals. Quantitative differences which we observed in content of VIPLI between cultures from two different individuals after normalization for differences in plating density may reflect intrinsic individual variability or experimental factors. The latter might include different ages or batches of medium or sert~, or the different plating densities per se. In general, signals which favor production of VIPLI also favor neurite outgrowth in monolayer culture. The fact that dexanethasone decreased neurite outgrowth without affecting levels of VIPLI, however, is consistent with other evidence that neurite outgrowth and VIP production can probably bedissociated (9,22). It is of interest that recent studies of bovine chromaffin cells provide further evidence for at least some correlation between promotion of neurite outgrowth and of VIP production. Although adult bovine chromaffin cells in culture are known to form processes (12), process outgrowth from these cells is considerably less extensive than in human chromaffin cell cultures, and is not stimulated by NGF (15,24). Adult bovine chromaffin cells have also recently been reported to produce VIPLI in culture (5.6). The amount of VIPLI reported in these cells, however, is two orders of magnitude less than that in NGF-treated htlnan chromaffin cells. Effects of NGF on VIPLI in bovine chromaffin cell cultures have not been reported. The minimal effects of dexamethasone on catecholanine content in this study are scmewhat surprising in view of the reported ability of corticosteroids to increase the content or rate of production of E in other normal chromaffin cells in vitro (4,18,23). Dexamethasone, however, also failed to increase E/NE ratios in cultures of 5 of 6 human pheochromocytomas studied by us, but increased the ratio 14-fold in cultures of the 6th tumor (22). We have studied catecholamines in cultures of two additional normal adult human adrenals, and have observed no effect of dexanethasone on E/NE ratio in one case, and a 5-fold increase in the other (A.S. Tischler and R.L. Perlman, unpublished data). Our observation with both normal and neoplastic human chromaffin cells suggest that the ability of dexamethasone to increase E/NE ratio might be dependent on the presence of as yet unknown permissive factors. Theoretical models accounting for the production of ectopic peptides by t~nors have related this phenomenon to "immaturity" or "incomplete differentiation" of tt~nor cells (1,25). The finding that normal adult human chromaffin cells can produce large quantities of an ectopic peptide, however, suggests that these models need to be modified for tumors arising form some types of cells in which maintenance of a "mature" phenotype is environmentdependent. An additional implication of this finding relates to current attempts to transplant chromaffin cells to the brain to replace catecholaminergic neurons in patients with Parkinson's disease (I0). It must now be ascertained that the transition from chromaffin cell to neuronal phenotype in vivo is not accompanied by production of physiologically inappropriate peptide neurotransmitters. Acknowledgements This research was supported by NIH grants CA27808, CA37370 and HL29025, and the Medical Research Council. The authors thank Ms. S. Tsymbalov for technical assistance.
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Vol. 37, No. 20, 1985
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