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Ontogenesis of chromogranin A and B and catecholamines in rat adrenal medulla
Brain Research, 478 (1989) 41-46 Elsevier
41
BRE 14180
Ontogenesis of chromogranin A and B and catecholamines in rat adrenal medulla M. Schober, R. Fischer-Colbrie and H. Winkler Department of Pharmacology, Universityof lnnsbruck, lnnsbruck (Austria) (Accepted 12 July 1988) Key words: Ontogenesis; Adrenal medulla; Chromogranin A; Chromogranin B; Catecholamine
The ontogenesis of chromogranins A and B and catecholamines was investigated for rat adrenal medulla. The chromogranins, the major secretory peptides of chromaffin granules, were characterized by one- and two-dimensional immunoblotting. Chromogranin B appeared identical in fetal and postnatal adrenals. On the other hand a significant portion of chromogranin A immunoreactivityin fetal adrenals was present in a component which was identified as a proteoglycan form of chromogranin A. In adult adrenals this proteoglycan chromogranin A was different and much less prominent. Total chromogranins and catecholamines increased parall¢ly from the 17th prenatal day to the adult stage. However in early periods of development chromaffin granules are likely to contain relatively higher catecholamine levels than adult granules, At the end of the gestational period both the ratio of adrenaline to noradrenaline and of chromogranin A vs B increases sharply. These results establish that from the 17th prenatal day onward fetal adrenal glands contain chromaffin granules which are filled with both the major secretory peptides and catecholamines. The relative composition of the so cretory content (adrenaline vs noradrenaline and chromogranin A vs chromogranin B) changes at the end of the gestational periot l,, parallel with the development of the corticosteroid-producing adrenal cortex. INTRODUCTION The adrenal chromaffin cells are derived embryologically from the neural crest. During and after migration to the adrenal anlage they undergo differentiation from primitive sympathetic cells and pheochromoblasts to mature chromaffin cells 4. The characteristic subcellular organelle of these latter cells is the chromaffin granule storing the hormones of adren~:l medulla i.e. the catecholamines 37'38. As shown in a thorough study at the ultrastructural level 8 the first chromaffin granules during ontogeny appear in the adrenal an!age of the rat at day 12 of gestation 5"7'21. Immunohistochemical studies revealed that the enzymes involved in catecholamine synthesis, i.e. tyrosine hydroxylase and dopamine fl-hydroxylase can be first demonstrated at day 14 (refs. 30, 32, 33). At about the same time catecholamines can be detected !'33. In addition to catecholamines chromaffin granules contain several neuropeptides, including enkephalins and neuropeptide Y and a family of acidic pro-
teins, the so-called chromogranins as. In primate adrenal medulla there is a parallel ontogeny of enkephalins and dopamine fl-hydroxylase 34. There is also an early appearance of somatostatin-like reactivity in avian adrenals, however the co-localization with catecholamines in chromaffin granules was not established '5. On the ontogenic appearance and properties of chromogranins no previous data are available. The chromogranins are the major soluble proteins of catecholamine-storing vesicles representing more than 90% of the total secretory peptides 35'3s. There are two families of these acidic proteins (for nomenclature see ref. 6), the chromogranins A and B. Iffrat adrenal about equal amounts of these two families are present ~°'~2. Recent studies have established that the synthesis of chromogranin A depends on corticosteroids. Thus in hypophysectomized rats levels of adrenal chromogranin A decline by 80% (ref. 24) and so does its mRNA 13. In the present study we investigated the ontogenic development of chromogranin A and B in rats in
Correspondence: H. Winkler, Department of Pharmacology, Universityof Innsbruck, Peter Mayr Str. 1, A-6020 Innsbruck, Austria. 0006-8993/89/$03.50 © 1989Elsevier Science Publishers B.V. (Biomedical Division)
42 comparison with catecholamines. We attempted to answer the following questions. (1) Do chromogranins arid catecholamines app.~ar together in the developing adrenal medulla? (2) Do the levels of chromogranin A and adrenaline which might both depend on the presence of corticosteroid rise parallely? (3) Are chromogranin A and B in fetal adrenal identical to the proteins in the adult organ? MATERIALS AND METHODS
Animals Sprague-Dawley rats (obtained from Versuchstierzuchtanstalt Himberg, Austria, at an age of 90 days) were maintained under standa:d conditions of temperature and lighting (12-h light-dark cycle, 21 °C, commercial diet and water ad libitum). Postnatal rats were killed by decapitation, the adrenal glands were excised, decapsulated, frozen on dry-ice and stored at -70 °C until further use. To obtain fetal adrenals of defined gestational age females were caged overnight with males (1-5:1-2) and vaginal smears examined for the presence of sperm the next morning at 09.00 h. The day a positive smear was obtained was taken as the first prenatal day. The day of spontaneous delivery was termed as day 0. Fetuses were removed from the uterus immediately after decapitation of the pregnant rats and kept in ice-cold water until dissection of adrenals. It took about 30 min to collect the adrenals of one litter. Excised adrenal glands were frozen on dry-ice and kept at -70 °C until preparation of homogenates. It was not possible to dissect adrenals from fetuses removed before the 17th prenatal day in a reliable way.
Analytical ,~says Frozen adrenal glands (26 adrenals for prenatal stages, 6 for day 0 and 1-week-old rats; from 3 to 1 for later stages) were homogenized in 100-300/~l 5 mM Tfis-succinate buffer, pH 5.9 (pH 7.3 for chondroitinase ABC experiments) by ultrasonication (3 times for 3 s) with a Branson B-30 sonifier. Chromogranins A and B were determined by a dot-immunobinding assay 16. Aliquots of adrenal homogenates (70/~l for prenatal ages and day 0 and accordingly less for postnatal ages) were dissolved in sample buffer 16without Triton X-100 but plus sodium dodecyl sulfate (1% final concentration) in a volume of 140/~l. After boil-
ing for 5 min 10 #1 of Triton X-100 (7% final concentration) was added followed by ultrasonication for 4 s. Antisera against rat chromogranins A and B (dilution 1:200) were prepared as described n. As a blocking reagent 2% lipid-free instant milk plus 2% normal goat serum was used. In a typical assay 6 serial dilutions of each adrenal homogenate (ranging from 10 to 0.4 ~tl for fetal adrenals and 3.5 to 0.06/~! for postnatal ones) were spotted in a total volume of 20 ~tl. Catecholamines (1-4 ~1 of the adrenal homogenates) were separated by reversed-phase high-performance liquid chromatography and quantified with an electrochemical detector 29.
Electrophoresis One- and two-dimensional electrophoresis 17,2° were performed as described in detail 22. Immunoblots with antisera against rat chromogranins A and B (diluted 1:200 (ref. 11)) were done following the protocol of Burnette 2 with some minor modifications 14. For digestion with chondroitinase ABC adrenal homogenates were boiled for 5 min 22 and spun for 20 min at 12,000 gmax. The supernatant was incubated with 0.06 units enzyme for 15 min at 37 °C, boiled for 5 min and loaded onto the gels. RESULTS
Characterization of chromogranins by immunoblotting Chromogranins were characterized in adrenals of fetuses and postnatal rats by immunoblotting. The 17th prenatal day was the earliest time point for which sufficient adrenal tissue could be collected. Fig. 1 presents the results obtained by one-dimensional immunoblotting. The bands reacting for chromogranin B appear very similar throughout the preand postnatal period moving as a close doublet. However at the earliest stage (17th prenatal day) only the slowest moving band seems dominant. For chromogranin A more significant changes can be seen. In the prenatal period and at birth a significant, slow moving component (M r 92,000 Da) is present which is apparently absent at later stages. It was considered that this band represented a proteoglycan form of chromogranin A 6.9. Therefore the soluble (heat-stable) adrenal extract was incubated with chondroitinase
43
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Fig. 1. ][mmunoblottingof adrenal extracts. Adrenal homogenates (3-26/~1)from fetal (17th, 19thprenatal day) and postnatal (2w, 2 weeks postnatal; ad, adult, 4-6-month-old) rats were subjected to one-dimensional electrophoresis followed by immunoblotting with antisera against chromogranin A (anti-Ch A) and chromogranin B (anti-Ch B). In the immunoblots for chromogranin A samples incubated before electrophoresis with chondroitinase ABC (+) are compared with untreated ones (-).
4 I
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ABC to digest the glycosaminoglycan side chains. As shown in Fig. 1 after this treatment the slowest moving band has disappeared demonstrating its proteoglycan nature. In the adult animal the proteoglycan band apparently is also present, but moves very close to the chromogranin A band. It disappears after enzyme treatment. This is further illustrated by the results of two-dimensional analysis (see Fig. 2). The slightly more acidic proteoglycan band is clearly visible in fetal extracts (see Fig. 2a) and disappears after enzyme treatment (see inset). In the adult adrenals the proteoglycan moves close to chromogranin A (see Fig. 2b).
Quantitative measurements of chromogranins A, B and catecholamines Results of quantitative immunodotting for chromogranin A and B are shown in Fig. 3. The levels of total chromogranin (A + B) and catecholamines at various age points are expressed as percentages of the adult (4-6 months) levels. The figure demonstrates that the two curves increase in a parallel fashion. However at the earlier stage there are some differences in the percentage levels. Thus, at the 17th prenatal day the catecholamines represented 0.089 +
-22
Fig. 2. Two-dimensional immunoblotting of adrenal homogenares. Adrenal heat-stable extracts of newborn (a) and adult rats (b) were subjected to two-dimensional electrophoresis followed by .;mmunobiotting with antisera against chromogranin A. The insets show the relevant region of immunoblots obtained after digesting the sample with chondroitinase ABC. A, chromogranin A; PG-A, proteoglycan form of chromogranin A.
0.012% (n - 3, mean + S.E.M.) of the adult level, total chromogranins only 0.038 + 0.007% (n - 6). Within the few days leading to birth these levels have already risen to 1.06 + 0.13% (n = 5) for catecholamines and to 0.63 + 0.065% (n -- 11) for the chromogranins. A more detailed presentation of the early steps of development is given in Fig. 4. Apparently there are two patterns of development. The levels of noradrenaline and chromogranin B increase little at first and then start to rise. On the other hand both adrenaline and chromogranin A exhibit a fast rise from the 17th day onwards. This is further illustrated by comparing the ratios of adrenaline to noradrenaline versus the ratios of chromogranin A to B during development
44 10 • total chromooranins
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Fig. 3. Levels of total catecholamines and chromogranins during ontogeny. The figure represents the adrenal levels of cate-
cholamines and chromogranins (A + B) from the 17th prenatal day onwards as percentage of the adult levels (100%). The scale is logarithmic. The S.E.M. of the experimental points (n = 4-13) was smaller than 20%.
(see Table I). During the prenatal period the adrenaline/noradrenaline ratio rises sharply by a factor of more than 10 (from 0.038 to 0.55) and then increases more slowly. The chromogranin AJB ratio also increases steeply before birth from 0.78 to its highest value of 2.78. In the postnatal development it slowly declines. DISCUSSION Previous studies on fetal rat adrenals have indicated that catecholamines L33 and dopamine fl-hydroxylase 3a can be demonstrated in the adrenal medulla starting from the i5th prenatal day. At this time point chromaffin granules can already be seen in histological sections s. The present study provides the first quantitative data for the prenatal ontogeny of the two major components of chromaffin granules, i.e. catecholamines and chromogranins. It should be emphasized that in adrenal medulla these two components are specifically confined to chromaffin granules and therefore present excellent markers of granule development 35. The levels of these two secretory constituents increase in parallel from the prenatal period to the adult level. What do these results establish for the prenatal biogenesis of chromaffin granules? Already at the 17th prenatal day the chro-
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Fig. 4. Levels of noradrenaline, adrenaline and chromogranin A and B during ontogeny. The figure represents the adrenal levels of noradrenaline (NA), adrenaline (A), chromogranin A (Ch A) and chromogranin B (Ch B) from the 17th prenatal day onwards as percentage of the adult levels (100%). The scale is logarithmic. The S.E.M. of the experimental points (n = 4-13) was smaller than 20%.
maffin granules in the adrenal contain the major secretory peptides, i.e. the chromogranins, and are filled with catecholamines. Two other possible mechanisms of prenatal biogenesis of chromaffin granules (for a discussion of biogenesis in adult animals see ref. 36) can now be excluded: (1) formation of chromaffin granules containing only catecholamines, but not the major secretory peptides, the chromogranins; (2) formation of granules containing secretory peptides but which are not yet able to take up catecholamines. Thus already in prenatal chromaffin TABLE I Catecholamines and chromogranins in adrenals during ontogeny
The table presents ratios for adrenaline/noradrenaline (A/NA) and for chromogranin A to B (A/B) relative to adult rats. The results are expressed as mean values _+S.E.M. (n = 3-15). Stage
A/NA
A/B
Prenatal day 17 Prenatal day 19 Prenatal d/ly 21 Birth 2 weeks 4 weeks Adult
0.038 + 0.0026 0.18 + 0.024 0.44 + 0.022 0.55 + 0.049 0.88 + 0.064 0.76 + 0.053 1.00 + 0.085
0.78_+0.083 1.68 + 0.24 2.78 + 0.22 2.13 + 0.21 2.58 + 0.35 1.67 + 0.17 1.00 +_0.042
45 granules the uptake system for catecholamines functions well. Up till now this had only been established for the postnatal period in very thorough studies by $10tkin 25-2~. This author also concluded that chromaffin granules in the postnatal period are 'overstuffed' with catecholamines. This seems already the case in the prenatal period, since catecholamines represent a higher percentage (0.09%) of the adult level than chromogranins (0.04%). The most likely explanation is that the turnover of granules is low, since at that time the adrenal is not yet functionally connected to the splanchnic nerve 2s, therefore granules can accumulate catecholamines for a longer time. Chromogranins in fetal adrenals have not been characterized previously. Apparently fetal chromogranin B is identical to the adult one, although at the earliest stage the proteolytic processing 3s may be slightly less advanced, since only the proprotein is present. For chromogranin A a more significant difference was detected. In the prenatal period significant amounts of an immunoreactive band migrating in electrophoresis slower than chromogranin A were present. By digestion experiments with chondroitinase ABC thi~ component could be identified as a proteoglycan form 9 of chromogranin A. A small amount of this proteoglycan-chromogranin A is also present in adult adrenals, however it is less significant and moves more closely to the chromogranin A band. Why does fetal adrenal medulla contain significant amounts of proteoglycan-chromogranin A? It might be an indication of the developmental relationship between chromaffin and nervous tissue. In rat brain the proteoglycan form of chromogranin A is a significant component which is in contrast to peripheral endocrine tissue (Wohlfarter et ai., in preparation). Thus one can suggest that this special form of chromogranin A declines during ontogenesis when the more endocrine phenotype of chromaffin cells of the adrenal medulla is finally established. In vitro a reverse change in the phenotype of chromaffin cells can be seen in culture, when neurite outgrowth is induced by certain treatments 18. We predict that this is REFERENCES 1 Bohn, M.C., Goldstein, M. and Black, I.B., Role of glucocorticoids in expression of the adrenergic phenotype in rat embryonic adrenal gland, Dev. Biol., 82 (1981) 1-10. 2 Burnette, W.N., 'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detec-
accompanied by an increase in the levels of the proteoglycan form of chromogranin A. During the prenatal development the ratio of chromogranin A to B rises at about the same time as the adrenaline/noradrenaline ratio. This latter finding is in agreement with previous studies 23.33. In addition it is already known that the rise in adrenaline coincides with an increase in the levels of phenylethanolamineN-methyltransferase 19. Already in an early study it was suggested that these phenomena are correlated with the concomitant development of the adrenal cortex 33. Finally it was demonstrated at the molecular level that the biosynthesis of phenylethanolamine-N-methyltransferase is increased by the presence of glucocorticoids 3'31"4°.This links up nicely with the increase in the chromogranin A/B ratio since we have already shown for adult rats ~hat the synthesis of chromogranin A and its mRNA, but not that of chromogranin B 13'24 depends on cortisone. Apparently late in the prenatal stage the cortex starts to produce corticosteroids which induce in the adrenal medulla the biosynthesis of both phenylethanolamine-Nmethyltransferase and chromogranin A. Thus our study has established that during prenatal development of rat adrenals chromaffin granules are synthesized which contain both catecholamines and the major secretory peptides, i.e. chromogranin A and B. Whereas chromogranin B is identical to the adult one, a higher portion of chromogranin A in the fetal period is present in the proteoglycan form. Both the adrenaline/noradrenaline and the chromogranin A/B ratios increase just before birth which is consistent with the concept that both the synthesis of adrenaline and that of chromogranin A are induced or increased by corticosteroids. Thus during development the relative composition of the 'secretory cocktail '39 contained in chromaffin granules changes. ACKNOWLEDGEMENTS This study was supported by the Dr. Legerlotz stirtung and by the Fonds zur F6rderung der Wissenschaftlichen Forschung (Austria). tion with antibody and radioiodinated protein A, Analyt. Biochem., 112 (1981) 195-203. 3 Ciaranello, R.D., Regulation of phenylethanolamine Nmethyitransferase synthesis and degradation, Mol. Pharmacol., 14 (1978) 478-489. 4 Coupland, R.E., The development and fate of catecholamine secreting endocrine cells. In H. Parvez and S. Parvez (Eds.), Biogenie Amines in Development, Elsevier,
40 Amsterdam, 1980, pp. 3-28. 5 Daikoku, S., Kinutani, M. and Sako, M., Development of the adrenal medullary cells in rats with reference to synaptogenesis, Cell Tiss. Res., 179 (1977) 77-86. 6 Eiden, L.E., Huttner, W.B., Mallet, J., O'Connor, D.T., Winkler, H. and Zanini. A., A nomenclature proposal for the chromogranin/secretogranin proteins, Neuroscience, 21 (1987) 1019-1021. 7 EI-Maghraby, M. and Lever, J.D., Typification and differentiation of medullary cells in the developing rat adrenal. A histoehemieal and electron microscopic study, J. Anat., 131 (1980) 103-120. 8 Eifvin, L.G., The development of the secretory granules in the rat adrenal medulla, J. Ultrastruct. Res., 17 (1967) 45-62. 9 Falkensammer, G., Fischer-Colbrie, R. and Winkler, H., Biogenesis of chromaffin granules: incorporation of sulfate into chromogranin B and into a proteoglycan, J. Neurochem., 45 (1985) 1475-1480. 10 Fischer-Colbrie, R. and Frischenschlager, I., Immunological characterization of secretory proteins of chromaffin granules: chromogranins A, chromogranins B and enkephalin-containing peptides, J. Neurochem., 44 (1985) 1854-1861. 11 Fischer-Colbrie, R. and Schober, M., Isolation and characterization of chromogranins A, B and C from bovine chromaffin granules and a rat pheochromocytoma, J. Neurochem., 48 (1987) 262-270. !2 Fischer-Colbrie, R., Hagn, C. and Schober, M., Chromogranins A, B and C: widespread constituents of secretory vesicles, Ann. N. Y. Acad. Sci., 493 (1987) 120-134. 13 Fischer-Colbrie, R., Iacangelo, A. and Eiden, L.E., Neural and humorai factors separately regulate neuropeptide Y, enkephalin, and chromogranin A and B mRNA levels in rat adrenal medulla, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) in press. 14 Fischer-Coibrie, R., Lassmann, H., Hagn, C. and Winkler, H., Immunological studies on the distribution of chromogranin A and B in endocrine and nervous tissues, Neuroscience, 16 (1985) 547-555. 15 Garcia-Arraras, J.E., Chanconie, M. and Fontaine-Perus, J., In vivo and in vitro development of somatostatin-likeimmunoreactivity in the peripheral nervous system of quail embryos, J. Neurosci., 4 (1984) 1549-1558. 16 Jahn, R., Schiebler, W. and Greengard, P., A quantitative dot-immunobinding assay for proteins using nitrocellulose membrane filters, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1684-1687. 17 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 227 (1970) 680-685. 18 LiveR, B.G., Adrenal medullary chromaffin cells in vitro, Physiol. Rev., 64 (1984) 1103-1161. 19 Margolis, F.L., Roffi, J. and Jost, A., Norepinephrine methylation in fetal rat adrenals, Science, 154 (1966) 275-276. 20 O'Farrell, P.H., High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem., 250 (1975) 4007-4021. 21 Ratzenhofer, M. and Miiller, O., Ultrastructure of adrenal medulla of the prenatal rat, J. Embryol. Exp. Morphol., 18 (1967) 13-25. 22 Schober, M., Fischer-Colbrie, R., Schmid, K.W., Bussolati, G,, O'Connor, D.T. and Winkler, H., Comparison of chromogranins A, B and secretogranin II in human adrenal medulla and pheochromocytoma, Lab. Invest., 57 (1987) 385-391. 23 Shepherd, D.M. and West, G.B., Noradrenaline and the
suprarenal medulla, Br. J. PharmaeoL, 6 (1951) 665-674. 24 Siemen, M., Schober, M., Fischer-Colbrie, R., Scherman, D., Sperk, G. and Winkler, H., Rat adrenal medulla: levels of chromogranins, enkephalins, dopamine fl-hydroxylase and of the amine transporter are changed by nervous activity and hypophysectomy, Neuroscience, 22 (1987) 131-139. 25 Slotkin, T.A., Maturation of the adrenal medulla. I. Uptake and storage of amines in isolated storage vesicles of the rat, Biochem. Pharmacol., 22 (1973) 2023-2032. 26 Slotkin, T.A, Maturation of the adrenal medulla. II. Content and properties of catecholamines storage vesicles of the rat, Biochem. Pharmacol., 22 (1973) 2033-2044. 27 Slotkin, T.A., Maturation of the adrenal medulla. III. Practical and theoretical considerations of age-dependent alterations in kinetics of incorporation of catecholamines and non-catecholamines, Biochem. Pharmacol., 24 (1975) 89-97. 28 Slotkin, T.A., Development of the sympathoadrenal axis. In P.M. Gootman (Ed.), Developmental Neurobiology of the Autonomic Nervous System, Humana, Clifton, NJ, !986, pp. 69-96. 29 Sperk, G., Berger, M., H6rtnagl, H. and Hornykiewicz, O., Kainic-acid induced changes of serotonin and dopamine metabolism in the rat striatum and substantia nigra of the rat, Eur. J. Pharmacol., 74 (1981) 279-286. 30 Teitelman, G., Baker, H., Joh, T.H. and Reis, D.J., Appearance of catecholamine-synthesizing enzymes during development of rat sympathetic nervous system: possible role of tissue environment, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 509-513. 31 Teitelman, G., Joh, T.H., Park, D., Brodsky, M., New, M. and Reis, D.J., Expression of the adrenergic phenotype in cultured fetal adrenal medullary cells: role of intrinsic and extrinsic factors, Dev. Biol., 89 (1982) 450-459. 32 Verhofstad, A.A.J., H6kfelt, T., Goldstein, M., Steinbusch, H.W.M., Joosten, H.W.J., Appearance of tyrosine hydroxylase, aromatic amino-acid decarboxylase, dopamine /~-hydroxylase and phenylethanolamine N-methyltransferase during the ontogenesis of the adrenal medulla, Cell Tiss. Res., 200 (1979) 1-13. 33 Verhofstad, A.A.J., Coupland, R.E., Parker, T.R. and Goldstein, M., Immunohistochemical and biochemical study on the development of the noradrenaline- and adrenaline-storing cells of the adrenal medulla of the rat, Cell Tiss. Res., 242 (1985) 233-243. 34 Wilburn, L.A., Goldsmith, P.C., Chang, K.-J. and Jaffe, R.B., Ontogeny of enkephalin and catecholamine-synthesizing enzymes in the primate fetal adrenal medulla, J. C!in. Endocrinol. MetaboL, 63 (1986)974-980. 35 Winkler, H., The composition of adrenal chromaffin granules: an assessment of controversial results, Neuroscience, 1 (1976) 65-80. 36 Winkler, H., The biogenesis of adrenal chromaffin granules, Neuroscience, 2 (1977) 657-683. 37 Winkler, H. and Carmichael, S.W., The chromaffin granule. In A.M. Poisner and J.M. Trifaro (Eds.). The Secretory Granule, Elsevier, Amsterdam, 1982, pp. 3-79. 38 Winkler, H., Apps, D.K. and Fischer-Colbrie, R., The molecular function of adrenal chromaffin granules: established facts and unresolved topics, Neuroscience, 18 (1986) 261-290. 39 Winkler, H., Sietzen, M. and Schober, M., The life cycle of catecholamine.storing vesicles, Ann. N.Y. Acad. Sci., 493 (1987) 3-19. 40 Wurtman, R.J. and Axelrod, J., Control of enzymatic synthesis of adrenaline in the adrenal medulla by adrenal cortical steroids, J. Bicn'. Chem., 241 (1966) 2301-2305.
41
BRE 14180
Ontogenesis of chromogranin A and B and catecholamines in rat adrenal medulla M. Schober, R. Fischer-Colbrie and H. Winkler Department of Pharmacology, Universityof lnnsbruck, lnnsbruck (Austria) (Accepted 12 July 1988) Key words: Ontogenesis; Adrenal medulla; Chromogranin A; Chromogranin B; Catecholamine
The ontogenesis of chromogranins A and B and catecholamines was investigated for rat adrenal medulla. The chromogranins, the major secretory peptides of chromaffin granules, were characterized by one- and two-dimensional immunoblotting. Chromogranin B appeared identical in fetal and postnatal adrenals. On the other hand a significant portion of chromogranin A immunoreactivityin fetal adrenals was present in a component which was identified as a proteoglycan form of chromogranin A. In adult adrenals this proteoglycan chromogranin A was different and much less prominent. Total chromogranins and catecholamines increased parall¢ly from the 17th prenatal day to the adult stage. However in early periods of development chromaffin granules are likely to contain relatively higher catecholamine levels than adult granules, At the end of the gestational period both the ratio of adrenaline to noradrenaline and of chromogranin A vs B increases sharply. These results establish that from the 17th prenatal day onward fetal adrenal glands contain chromaffin granules which are filled with both the major secretory peptides and catecholamines. The relative composition of the so cretory content (adrenaline vs noradrenaline and chromogranin A vs chromogranin B) changes at the end of the gestational periot l,, parallel with the development of the corticosteroid-producing adrenal cortex. INTRODUCTION The adrenal chromaffin cells are derived embryologically from the neural crest. During and after migration to the adrenal anlage they undergo differentiation from primitive sympathetic cells and pheochromoblasts to mature chromaffin cells 4. The characteristic subcellular organelle of these latter cells is the chromaffin granule storing the hormones of adren~:l medulla i.e. the catecholamines 37'38. As shown in a thorough study at the ultrastructural level 8 the first chromaffin granules during ontogeny appear in the adrenal an!age of the rat at day 12 of gestation 5"7'21. Immunohistochemical studies revealed that the enzymes involved in catecholamine synthesis, i.e. tyrosine hydroxylase and dopamine fl-hydroxylase can be first demonstrated at day 14 (refs. 30, 32, 33). At about the same time catecholamines can be detected !'33. In addition to catecholamines chromaffin granules contain several neuropeptides, including enkephalins and neuropeptide Y and a family of acidic pro-
teins, the so-called chromogranins as. In primate adrenal medulla there is a parallel ontogeny of enkephalins and dopamine fl-hydroxylase 34. There is also an early appearance of somatostatin-like reactivity in avian adrenals, however the co-localization with catecholamines in chromaffin granules was not established '5. On the ontogenic appearance and properties of chromogranins no previous data are available. The chromogranins are the major soluble proteins of catecholamine-storing vesicles representing more than 90% of the total secretory peptides 35'3s. There are two families of these acidic proteins (for nomenclature see ref. 6), the chromogranins A and B. Iffrat adrenal about equal amounts of these two families are present ~°'~2. Recent studies have established that the synthesis of chromogranin A depends on corticosteroids. Thus in hypophysectomized rats levels of adrenal chromogranin A decline by 80% (ref. 24) and so does its mRNA 13. In the present study we investigated the ontogenic development of chromogranin A and B in rats in
Correspondence: H. Winkler, Department of Pharmacology, Universityof Innsbruck, Peter Mayr Str. 1, A-6020 Innsbruck, Austria. 0006-8993/89/$03.50 © 1989Elsevier Science Publishers B.V. (Biomedical Division)
42 comparison with catecholamines. We attempted to answer the following questions. (1) Do chromogranins arid catecholamines app.~ar together in the developing adrenal medulla? (2) Do the levels of chromogranin A and adrenaline which might both depend on the presence of corticosteroid rise parallely? (3) Are chromogranin A and B in fetal adrenal identical to the proteins in the adult organ? MATERIALS AND METHODS
Animals Sprague-Dawley rats (obtained from Versuchstierzuchtanstalt Himberg, Austria, at an age of 90 days) were maintained under standa:d conditions of temperature and lighting (12-h light-dark cycle, 21 °C, commercial diet and water ad libitum). Postnatal rats were killed by decapitation, the adrenal glands were excised, decapsulated, frozen on dry-ice and stored at -70 °C until further use. To obtain fetal adrenals of defined gestational age females were caged overnight with males (1-5:1-2) and vaginal smears examined for the presence of sperm the next morning at 09.00 h. The day a positive smear was obtained was taken as the first prenatal day. The day of spontaneous delivery was termed as day 0. Fetuses were removed from the uterus immediately after decapitation of the pregnant rats and kept in ice-cold water until dissection of adrenals. It took about 30 min to collect the adrenals of one litter. Excised adrenal glands were frozen on dry-ice and kept at -70 °C until preparation of homogenates. It was not possible to dissect adrenals from fetuses removed before the 17th prenatal day in a reliable way.
Analytical ,~says Frozen adrenal glands (26 adrenals for prenatal stages, 6 for day 0 and 1-week-old rats; from 3 to 1 for later stages) were homogenized in 100-300/~l 5 mM Tfis-succinate buffer, pH 5.9 (pH 7.3 for chondroitinase ABC experiments) by ultrasonication (3 times for 3 s) with a Branson B-30 sonifier. Chromogranins A and B were determined by a dot-immunobinding assay 16. Aliquots of adrenal homogenates (70/~l for prenatal ages and day 0 and accordingly less for postnatal ages) were dissolved in sample buffer 16without Triton X-100 but plus sodium dodecyl sulfate (1% final concentration) in a volume of 140/~l. After boil-
ing for 5 min 10 #1 of Triton X-100 (7% final concentration) was added followed by ultrasonication for 4 s. Antisera against rat chromogranins A and B (dilution 1:200) were prepared as described n. As a blocking reagent 2% lipid-free instant milk plus 2% normal goat serum was used. In a typical assay 6 serial dilutions of each adrenal homogenate (ranging from 10 to 0.4 ~tl for fetal adrenals and 3.5 to 0.06/~! for postnatal ones) were spotted in a total volume of 20 ~tl. Catecholamines (1-4 ~1 of the adrenal homogenates) were separated by reversed-phase high-performance liquid chromatography and quantified with an electrochemical detector 29.
Electrophoresis One- and two-dimensional electrophoresis 17,2° were performed as described in detail 22. Immunoblots with antisera against rat chromogranins A and B (diluted 1:200 (ref. 11)) were done following the protocol of Burnette 2 with some minor modifications 14. For digestion with chondroitinase ABC adrenal homogenates were boiled for 5 min 22 and spun for 20 min at 12,000 gmax. The supernatant was incubated with 0.06 units enzyme for 15 min at 37 °C, boiled for 5 min and loaded onto the gels. RESULTS
Characterization of chromogranins by immunoblotting Chromogranins were characterized in adrenals of fetuses and postnatal rats by immunoblotting. The 17th prenatal day was the earliest time point for which sufficient adrenal tissue could be collected. Fig. 1 presents the results obtained by one-dimensional immunoblotting. The bands reacting for chromogranin B appear very similar throughout the preand postnatal period moving as a close doublet. However at the earliest stage (17th prenatal day) only the slowest moving band seems dominant. For chromogranin A more significant changes can be seen. In the prenatal period and at birth a significant, slow moving component (M r 92,000 Da) is present which is apparently absent at later stages. It was considered that this band represented a proteoglycan form of chromogranin A 6.9. Therefore the soluble (heat-stable) adrenal extract was incubated with chondroitinase
43
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Fig. 1. ][mmunoblottingof adrenal extracts. Adrenal homogenates (3-26/~1)from fetal (17th, 19thprenatal day) and postnatal (2w, 2 weeks postnatal; ad, adult, 4-6-month-old) rats were subjected to one-dimensional electrophoresis followed by immunoblotting with antisera against chromogranin A (anti-Ch A) and chromogranin B (anti-Ch B). In the immunoblots for chromogranin A samples incubated before electrophoresis with chondroitinase ABC (+) are compared with untreated ones (-).
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ABC to digest the glycosaminoglycan side chains. As shown in Fig. 1 after this treatment the slowest moving band has disappeared demonstrating its proteoglycan nature. In the adult animal the proteoglycan band apparently is also present, but moves very close to the chromogranin A band. It disappears after enzyme treatment. This is further illustrated by the results of two-dimensional analysis (see Fig. 2). The slightly more acidic proteoglycan band is clearly visible in fetal extracts (see Fig. 2a) and disappears after enzyme treatment (see inset). In the adult adrenals the proteoglycan moves close to chromogranin A (see Fig. 2b).
Quantitative measurements of chromogranins A, B and catecholamines Results of quantitative immunodotting for chromogranin A and B are shown in Fig. 3. The levels of total chromogranin (A + B) and catecholamines at various age points are expressed as percentages of the adult (4-6 months) levels. The figure demonstrates that the two curves increase in a parallel fashion. However at the earlier stage there are some differences in the percentage levels. Thus, at the 17th prenatal day the catecholamines represented 0.089 +
-22
Fig. 2. Two-dimensional immunoblotting of adrenal homogenares. Adrenal heat-stable extracts of newborn (a) and adult rats (b) were subjected to two-dimensional electrophoresis followed by .;mmunobiotting with antisera against chromogranin A. The insets show the relevant region of immunoblots obtained after digesting the sample with chondroitinase ABC. A, chromogranin A; PG-A, proteoglycan form of chromogranin A.
0.012% (n - 3, mean + S.E.M.) of the adult level, total chromogranins only 0.038 + 0.007% (n - 6). Within the few days leading to birth these levels have already risen to 1.06 + 0.13% (n = 5) for catecholamines and to 0.63 + 0.065% (n -- 11) for the chromogranins. A more detailed presentation of the early steps of development is given in Fig. 4. Apparently there are two patterns of development. The levels of noradrenaline and chromogranin B increase little at first and then start to rise. On the other hand both adrenaline and chromogranin A exhibit a fast rise from the 17th day onwards. This is further illustrated by comparing the ratios of adrenaline to noradrenaline versus the ratios of chromogranin A to B during development
44 10 • total chromooranins
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Fig. 3. Levels of total catecholamines and chromogranins during ontogeny. The figure represents the adrenal levels of cate-
cholamines and chromogranins (A + B) from the 17th prenatal day onwards as percentage of the adult levels (100%). The scale is logarithmic. The S.E.M. of the experimental points (n = 4-13) was smaller than 20%.
(see Table I). During the prenatal period the adrenaline/noradrenaline ratio rises sharply by a factor of more than 10 (from 0.038 to 0.55) and then increases more slowly. The chromogranin AJB ratio also increases steeply before birth from 0.78 to its highest value of 2.78. In the postnatal development it slowly declines. DISCUSSION Previous studies on fetal rat adrenals have indicated that catecholamines L33 and dopamine fl-hydroxylase 3a can be demonstrated in the adrenal medulla starting from the i5th prenatal day. At this time point chromaffin granules can already be seen in histological sections s. The present study provides the first quantitative data for the prenatal ontogeny of the two major components of chromaffin granules, i.e. catecholamines and chromogranins. It should be emphasized that in adrenal medulla these two components are specifically confined to chromaffin granules and therefore present excellent markers of granule development 35. The levels of these two secretory constituents increase in parallel from the prenatal period to the adult level. What do these results establish for the prenatal biogenesis of chromaffin granules? Already at the 17th prenatal day the chro-
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Fig. 4. Levels of noradrenaline, adrenaline and chromogranin A and B during ontogeny. The figure represents the adrenal levels of noradrenaline (NA), adrenaline (A), chromogranin A (Ch A) and chromogranin B (Ch B) from the 17th prenatal day onwards as percentage of the adult levels (100%). The scale is logarithmic. The S.E.M. of the experimental points (n = 4-13) was smaller than 20%.
maffin granules in the adrenal contain the major secretory peptides, i.e. the chromogranins, and are filled with catecholamines. Two other possible mechanisms of prenatal biogenesis of chromaffin granules (for a discussion of biogenesis in adult animals see ref. 36) can now be excluded: (1) formation of chromaffin granules containing only catecholamines, but not the major secretory peptides, the chromogranins; (2) formation of granules containing secretory peptides but which are not yet able to take up catecholamines. Thus already in prenatal chromaffin TABLE I Catecholamines and chromogranins in adrenals during ontogeny
The table presents ratios for adrenaline/noradrenaline (A/NA) and for chromogranin A to B (A/B) relative to adult rats. The results are expressed as mean values _+S.E.M. (n = 3-15). Stage
A/NA
A/B
Prenatal day 17 Prenatal day 19 Prenatal d/ly 21 Birth 2 weeks 4 weeks Adult
0.038 + 0.0026 0.18 + 0.024 0.44 + 0.022 0.55 + 0.049 0.88 + 0.064 0.76 + 0.053 1.00 + 0.085
0.78_+0.083 1.68 + 0.24 2.78 + 0.22 2.13 + 0.21 2.58 + 0.35 1.67 + 0.17 1.00 +_0.042
45 granules the uptake system for catecholamines functions well. Up till now this had only been established for the postnatal period in very thorough studies by $10tkin 25-2~. This author also concluded that chromaffin granules in the postnatal period are 'overstuffed' with catecholamines. This seems already the case in the prenatal period, since catecholamines represent a higher percentage (0.09%) of the adult level than chromogranins (0.04%). The most likely explanation is that the turnover of granules is low, since at that time the adrenal is not yet functionally connected to the splanchnic nerve 2s, therefore granules can accumulate catecholamines for a longer time. Chromogranins in fetal adrenals have not been characterized previously. Apparently fetal chromogranin B is identical to the adult one, although at the earliest stage the proteolytic processing 3s may be slightly less advanced, since only the proprotein is present. For chromogranin A a more significant difference was detected. In the prenatal period significant amounts of an immunoreactive band migrating in electrophoresis slower than chromogranin A were present. By digestion experiments with chondroitinase ABC thi~ component could be identified as a proteoglycan form 9 of chromogranin A. A small amount of this proteoglycan-chromogranin A is also present in adult adrenals, however it is less significant and moves more closely to the chromogranin A band. Why does fetal adrenal medulla contain significant amounts of proteoglycan-chromogranin A? It might be an indication of the developmental relationship between chromaffin and nervous tissue. In rat brain the proteoglycan form of chromogranin A is a significant component which is in contrast to peripheral endocrine tissue (Wohlfarter et ai., in preparation). Thus one can suggest that this special form of chromogranin A declines during ontogenesis when the more endocrine phenotype of chromaffin cells of the adrenal medulla is finally established. In vitro a reverse change in the phenotype of chromaffin cells can be seen in culture, when neurite outgrowth is induced by certain treatments 18. We predict that this is REFERENCES 1 Bohn, M.C., Goldstein, M. and Black, I.B., Role of glucocorticoids in expression of the adrenergic phenotype in rat embryonic adrenal gland, Dev. Biol., 82 (1981) 1-10. 2 Burnette, W.N., 'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detec-
accompanied by an increase in the levels of the proteoglycan form of chromogranin A. During the prenatal development the ratio of chromogranin A to B rises at about the same time as the adrenaline/noradrenaline ratio. This latter finding is in agreement with previous studies 23.33. In addition it is already known that the rise in adrenaline coincides with an increase in the levels of phenylethanolamineN-methyltransferase 19. Already in an early study it was suggested that these phenomena are correlated with the concomitant development of the adrenal cortex 33. Finally it was demonstrated at the molecular level that the biosynthesis of phenylethanolamine-N-methyltransferase is increased by the presence of glucocorticoids 3'31"4°.This links up nicely with the increase in the chromogranin A/B ratio since we have already shown for adult rats ~hat the synthesis of chromogranin A and its mRNA, but not that of chromogranin B 13'24 depends on cortisone. Apparently late in the prenatal stage the cortex starts to produce corticosteroids which induce in the adrenal medulla the biosynthesis of both phenylethanolamine-Nmethyltransferase and chromogranin A. Thus our study has established that during prenatal development of rat adrenals chromaffin granules are synthesized which contain both catecholamines and the major secretory peptides, i.e. chromogranin A and B. Whereas chromogranin B is identical to the adult one, a higher portion of chromogranin A in the fetal period is present in the proteoglycan form. Both the adrenaline/noradrenaline and the chromogranin A/B ratios increase just before birth which is consistent with the concept that both the synthesis of adrenaline and that of chromogranin A are induced or increased by corticosteroids. Thus during development the relative composition of the 'secretory cocktail '39 contained in chromaffin granules changes. ACKNOWLEDGEMENTS This study was supported by the Dr. Legerlotz stirtung and by the Fonds zur F6rderung der Wissenschaftlichen Forschung (Austria). tion with antibody and radioiodinated protein A, Analyt. Biochem., 112 (1981) 195-203. 3 Ciaranello, R.D., Regulation of phenylethanolamine Nmethyitransferase synthesis and degradation, Mol. Pharmacol., 14 (1978) 478-489. 4 Coupland, R.E., The development and fate of catecholamine secreting endocrine cells. In H. Parvez and S. Parvez (Eds.), Biogenie Amines in Development, Elsevier,
40 Amsterdam, 1980, pp. 3-28. 5 Daikoku, S., Kinutani, M. and Sako, M., Development of the adrenal medullary cells in rats with reference to synaptogenesis, Cell Tiss. Res., 179 (1977) 77-86. 6 Eiden, L.E., Huttner, W.B., Mallet, J., O'Connor, D.T., Winkler, H. and Zanini. A., A nomenclature proposal for the chromogranin/secretogranin proteins, Neuroscience, 21 (1987) 1019-1021. 7 EI-Maghraby, M. and Lever, J.D., Typification and differentiation of medullary cells in the developing rat adrenal. A histoehemieal and electron microscopic study, J. Anat., 131 (1980) 103-120. 8 Eifvin, L.G., The development of the secretory granules in the rat adrenal medulla, J. Ultrastruct. Res., 17 (1967) 45-62. 9 Falkensammer, G., Fischer-Colbrie, R. and Winkler, H., Biogenesis of chromaffin granules: incorporation of sulfate into chromogranin B and into a proteoglycan, J. Neurochem., 45 (1985) 1475-1480. 10 Fischer-Colbrie, R. and Frischenschlager, I., Immunological characterization of secretory proteins of chromaffin granules: chromogranins A, chromogranins B and enkephalin-containing peptides, J. Neurochem., 44 (1985) 1854-1861. 11 Fischer-Colbrie, R. and Schober, M., Isolation and characterization of chromogranins A, B and C from bovine chromaffin granules and a rat pheochromocytoma, J. Neurochem., 48 (1987) 262-270. !2 Fischer-Colbrie, R., Hagn, C. and Schober, M., Chromogranins A, B and C: widespread constituents of secretory vesicles, Ann. N. Y. Acad. Sci., 493 (1987) 120-134. 13 Fischer-Colbrie, R., Iacangelo, A. and Eiden, L.E., Neural and humorai factors separately regulate neuropeptide Y, enkephalin, and chromogranin A and B mRNA levels in rat adrenal medulla, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) in press. 14 Fischer-Coibrie, R., Lassmann, H., Hagn, C. and Winkler, H., Immunological studies on the distribution of chromogranin A and B in endocrine and nervous tissues, Neuroscience, 16 (1985) 547-555. 15 Garcia-Arraras, J.E., Chanconie, M. and Fontaine-Perus, J., In vivo and in vitro development of somatostatin-likeimmunoreactivity in the peripheral nervous system of quail embryos, J. Neurosci., 4 (1984) 1549-1558. 16 Jahn, R., Schiebler, W. and Greengard, P., A quantitative dot-immunobinding assay for proteins using nitrocellulose membrane filters, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1684-1687. 17 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 227 (1970) 680-685. 18 LiveR, B.G., Adrenal medullary chromaffin cells in vitro, Physiol. Rev., 64 (1984) 1103-1161. 19 Margolis, F.L., Roffi, J. and Jost, A., Norepinephrine methylation in fetal rat adrenals, Science, 154 (1966) 275-276. 20 O'Farrell, P.H., High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem., 250 (1975) 4007-4021. 21 Ratzenhofer, M. and Miiller, O., Ultrastructure of adrenal medulla of the prenatal rat, J. Embryol. Exp. Morphol., 18 (1967) 13-25. 22 Schober, M., Fischer-Colbrie, R., Schmid, K.W., Bussolati, G,, O'Connor, D.T. and Winkler, H., Comparison of chromogranins A, B and secretogranin II in human adrenal medulla and pheochromocytoma, Lab. Invest., 57 (1987) 385-391. 23 Shepherd, D.M. and West, G.B., Noradrenaline and the
suprarenal medulla, Br. J. PharmaeoL, 6 (1951) 665-674. 24 Siemen, M., Schober, M., Fischer-Colbrie, R., Scherman, D., Sperk, G. and Winkler, H., Rat adrenal medulla: levels of chromogranins, enkephalins, dopamine fl-hydroxylase and of the amine transporter are changed by nervous activity and hypophysectomy, Neuroscience, 22 (1987) 131-139. 25 Slotkin, T.A., Maturation of the adrenal medulla. I. Uptake and storage of amines in isolated storage vesicles of the rat, Biochem. Pharmacol., 22 (1973) 2023-2032. 26 Slotkin, T.A, Maturation of the adrenal medulla. II. Content and properties of catecholamines storage vesicles of the rat, Biochem. Pharmacol., 22 (1973) 2033-2044. 27 Slotkin, T.A., Maturation of the adrenal medulla. III. Practical and theoretical considerations of age-dependent alterations in kinetics of incorporation of catecholamines and non-catecholamines, Biochem. Pharmacol., 24 (1975) 89-97. 28 Slotkin, T.A., Development of the sympathoadrenal axis. In P.M. Gootman (Ed.), Developmental Neurobiology of the Autonomic Nervous System, Humana, Clifton, NJ, !986, pp. 69-96. 29 Sperk, G., Berger, M., H6rtnagl, H. and Hornykiewicz, O., Kainic-acid induced changes of serotonin and dopamine metabolism in the rat striatum and substantia nigra of the rat, Eur. J. Pharmacol., 74 (1981) 279-286. 30 Teitelman, G., Baker, H., Joh, T.H. and Reis, D.J., Appearance of catecholamine-synthesizing enzymes during development of rat sympathetic nervous system: possible role of tissue environment, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 509-513. 31 Teitelman, G., Joh, T.H., Park, D., Brodsky, M., New, M. and Reis, D.J., Expression of the adrenergic phenotype in cultured fetal adrenal medullary cells: role of intrinsic and extrinsic factors, Dev. Biol., 89 (1982) 450-459. 32 Verhofstad, A.A.J., H6kfelt, T., Goldstein, M., Steinbusch, H.W.M., Joosten, H.W.J., Appearance of tyrosine hydroxylase, aromatic amino-acid decarboxylase, dopamine /~-hydroxylase and phenylethanolamine N-methyltransferase during the ontogenesis of the adrenal medulla, Cell Tiss. Res., 200 (1979) 1-13. 33 Verhofstad, A.A.J., Coupland, R.E., Parker, T.R. and Goldstein, M., Immunohistochemical and biochemical study on the development of the noradrenaline- and adrenaline-storing cells of the adrenal medulla of the rat, Cell Tiss. Res., 242 (1985) 233-243. 34 Wilburn, L.A., Goldsmith, P.C., Chang, K.-J. and Jaffe, R.B., Ontogeny of enkephalin and catecholamine-synthesizing enzymes in the primate fetal adrenal medulla, J. C!in. Endocrinol. MetaboL, 63 (1986)974-980. 35 Winkler, H., The composition of adrenal chromaffin granules: an assessment of controversial results, Neuroscience, 1 (1976) 65-80. 36 Winkler, H., The biogenesis of adrenal chromaffin granules, Neuroscience, 2 (1977) 657-683. 37 Winkler, H. and Carmichael, S.W., The chromaffin granule. In A.M. Poisner and J.M. Trifaro (Eds.). The Secretory Granule, Elsevier, Amsterdam, 1982, pp. 3-79. 38 Winkler, H., Apps, D.K. and Fischer-Colbrie, R., The molecular function of adrenal chromaffin granules: established facts and unresolved topics, Neuroscience, 18 (1986) 261-290. 39 Winkler, H., Sietzen, M. and Schober, M., The life cycle of catecholamine.storing vesicles, Ann. N.Y. Acad. Sci., 493 (1987) 3-19. 40 Wurtman, R.J. and Axelrod, J., Control of enzymatic synthesis of adrenaline in the adrenal medulla by adrenal cortical steroids, J. Bicn'. Chem., 241 (1966) 2301-2305.