Proc. Nati Acad. Sci. USAVol. 79, pp. 4823-4827, August 1982Neurobiology

Development of the adrenergic phenotype: Increase in adrenalmessenger RNA coding for phenylethanolamine-N-methyltransferase

(epinephrine/ontogeny/translation/gene expression/cell differentiation)

E. SABBAN*t, M. GOLDSTEIN*, M. C. BOHNt, AND I. B. BLACK:Departments of *Psychiatry and tCell Biology, New York University School of Medicine, 550 First Avenue, New York, New York 10016; and*Division of Developmental Neurology, Cornell University Medical College, 515 East 71st Street, New York, New York 10021

Communicated by Michael Heidelberger, May 6, 1982

ABSTRACT Mechanisms regulating the developmental in-crease in the activity of adrenal phenylethanolamine-N-methyl-transferase (PNMTase), an index of the adrenergic phenotype,were examined. Immunotitration indicated that the increase incatalytic activity in rat adrenal from birth to adulthood was at-tributable to increased numbers of PNMTase molecules, not en-zyme activation. To determine whether the ontogenetic increasein PNMTase protein was associated with elevation ofmRNA cod-ing for PNMTase, cell-free translation was performed on totalcellular mRNA obtained from adrenals at different ages. Trans-lation in wheat-germ and reticulocyte lysate systems, followed byimmunoprecipitation of the PNMTase product, NaDodSO4 gelelectrophoresis, and fluorography, showed an 8-fold increase inthe proportion of specific PNMTase mRNA relative to total mRNAin rat adrenals from birth to adulthood. Moreover, bovine adrenalmedullae exhibited a 100-fold increase in PNMTase mRNA levelsbetween embryonic life and adulthood. Consequently, the onto-genetic increase in adrenal PNMTase appears to be due to a de-velopmental rise in specific mRNA coding for the protein.

The adrenal medulla is a particularly useful model for definingthe factors regulating expression and differentiation of the ad-renergic phenotype because it is easily accessible, subject tohormonal influences, and expresses all the enzymes involvedin epinephrine biosynthesis (1, 2). In the rat embryo at 13.5 daysof gestation, medullary precursor cells, which have migratedfrom neural crest to adrenal primordium, contain the noradren-ergic enzymes tyrosine hydroxylase (EC 1.14.16.20) and do-pamine-,B-hydroxylase (EC 1.14.17.1) (3-5). However, phenyl-ethanolamine-N-methyltransferase (PNMTase; EC 2.1.1.28),the epinephrine-forming enzyme, is not detectable until 17.5days of gestation, indicating that adrenergic differentiation oc-curs 4 days later than noradrenergic expression (4-6). Althoughadrenal glucocorticoid hormones are required for the devel-opmental increase of PNMTase (6, 7), underlying molecularmechanisms are not known.We find that the ontogenetic rise in PNMTase catalytic ac-

tivity is accompanied by a parallel rise in enzyme protein. Todetermine whether the increase in numbers of PNMTase mol-ecules is attributable to elevated levels of mRNA coding forthe enzyme, we assayed rat and bovine adrenal mRNA forPNMTase in an in vitro translation system.

MATERIALS AND METHODSPreparation of mRNA. Total cellular mRNA was prepared

from the entire adrenal gland of Sprague-Dawley rats (HilltopAnimals, Scottdale, PA) and from bovine (Holstein) adrenal

medullae. The bovine adrenals were sent fresh on ice from FlowLaboratories (McLean, VA) and the medullae were dissectedafter arrival. For the isolation ofmRNA, total cellular RNA wasextracted with guanidine hydrochloride either by a modificationof Cox's method (8, 9) or according to Liu et al. (10). The firstmethod was used for initial experiments with bovine and ratadrenals; the second method was found to be more convenient,the mRNA was comparably active, and thus, it was used to com-pare PNMTase mRNA at various ages. The total RNA recoveredper gram of tissue was similar at all ages examined. In all ex-periments, the mRNA was purified by oligo(dT)-cellulose chro-matography (11) and the poly(A)-RNA was used for translationexperiments. The A260/A2w ratio was at least 1.95.

Cell-Free Translations. The mRNA was translated with[35S]methionine in a wheat-germ extract system at 25°C (12) orin a nuclease-treated rabbit reticulocyte lysate system at 370C(13), as described (9). After incubation, an aliquot (5 ,ul) wastaken to measure the formation of trichloroacetic acid-precip-itable protein (14) and the remainder was used for immuno-precipitation.

Immunoprecipitation ofPNMTase. The [35S]methionine-la-beled PNMTase was immunoprecipitated by a previously de-scribed (9) modification of Goldman and Blobel's method (15)and applied to a 6-12% gradient NaDodSO4/polyacrylamideslab gel. Gel electrophoresis (16) was run according to a mod-ification of the procedure of Maizel (17). The distribution ofradioactive polypeptides and 14C molecular weight markers(Bethesda Research Laboratories) was determined by fluorog-raphy with sodium salicylate (18) on prefogged Kodak X-OmatAR5 film.

Radioactivity in the PNMTase immunoprecipitate was esti-mated by two methods. In the first, the x-ray pattern obtainedby exposure ofthe gel to prefogged film was scanned on aZeinehsoft laser scanning densitometer (Biomed). The relative amountsof PNMTase synthesized in vitro were obtained by integrationof areas under those peaks that corresponded to the molecularweight of PNMTase. In the second method, the radioactivebands were eluted according to Albanese and Goodman (19) andradioactivity was determined in 20 ml of Hydrofluor (NationalDiagnostics). A region of the gel without radioactive bands onthe x-ray film was used as a blank.

Assay of PNMTase Activity. PNMTase catalytic activity wasassayed by using minor modifications (20) ofdescribed methods(21). Protein was determined by the method ofLowry et al. (22).

Immunotitration. PNMTase immunotitration was performedby minor modifications of published methods (23). Adrenalswere homogenized in 0.005 M Tris at pH 7.4, containing 1 mMdithiothreitol, trasylol at 100 units/ml, 0.2% bovine serum al-

Abbreviation: PNMTase, phenylethanolamine-N-methyltransferase.

4823

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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bumin, and 0.2% Triton X-100. Homogenates were centrifugedat 6,000 X g for 10 min, and 40 Aul of the supernatant fractionwas incubated with 20 /ul of specific PNMTase antiserum, pre-immune serum, or a combination thereof. After 1 hr of incu-bation at room temperature with 5 sec of vortexing every 15min, the mixture was centrifuged at 48,000 X g for 10 min.PNMTase catalytic activity was assayed in 20-/il aliquots of thesupernatant fraction in duplicate.

Histology. To determine volume parameters, rat adrenalglands from rats at ages 1, 15, and 60 days (n = 4 at each age)were fixed by immersion in Bouin's fixative and embedded inparaffin. Serial 10-,um sections were stained with hematoxylin/eosin and projected with a Bausch and Lomb projector at x50for day 1 and day 15 and at X20 for day 60; every fifth sectionwas traced and the areas of cortex and medulla were measuredwith an electronic planimeter (Numonics). The total volumesof the adrenal cortex and medulla were calculated by integra-tion of these areal measurements.Immunofluorescence was performed on sections of bovine

adrenal with Coons' method (24). Slices of bovine adrenal glandwere fixed in 4% buffered paraformaldehyde and cryoprotectedin 30% sucrose, and 10-pum sections were prepared for cryo-tomy. Preimmune serum was used as a control and in all casesgave negative staining.

RESULTSOntogeny of PNMTase. During development of the rat ad-

renal there is a dramatic increase in PNMTase catalytic activity(7, 20, 25). To assess whether this is a result of increasedPNMTase molecule numbers or activation of preexistent en-zyme, immunotitration of PNMTase from newborn and adultrats was performed (Fig. 1). Increasing amounts of antiserumwere added to a fixed amount of enzyme antigen in solution.The adult preparation was diluted by a factor of 17 to approxi-mate the concentration of PNMTase catalytic activity in the

20

18 *\a-o Adult

16 ° o-o Neonate

14

. 12

810

10'Ix

-

E.

15 30 45 60 75 90 0.5 1.0 1.5 2.0 50 75 100 1Incubation time, min Mg(OAc)2, mM KOAc, mM

DTotal Immunoprecipitates

Mg(OAc'2, mM KOAc. mM-- v 0.4 0.8 1.2 1.6 2.0 50 70 90 112 1355

Mr

68,000-

43,000 -i.

25,700 -

18,400 -..12,300 bi

I..

FIG. 2. Characterization of protein synthesis directed by mRNAfrom bovine adrenal medullary cells in a wheat-germ cell-free system.(A) Time course of [35S]methionine incorporation into trichloroaceticacid-precipitable protein. Reactions were at 25TC in the presence of 1.6mM Mg2+/112 mM K+ and in the presence (o) of or absence (o) ofmRNA at 20 pg/ml. (B) Effect of Mg2+ on protein synthesis. (C) Effectof K+ on protein synthesis. (D) NaDodSO4 gel electrophoresis of totaltranslation (- and + indicate absence and presence of mRNA, re-spectively) and immunoprecipitation with anti-PNMTase antisera atvarious Mg2+ and K+ concentrations. The arrows indicate the newlysynthesized Mr 32,000 polypeptide.

neonatal preparation. Immunotitration of neonatal and adultPNMTase yielded slopes that did not differ, suggesting that thepostnatal increase in PNMTase activity is attributable to in-creased numbers ofenzyme molecules. To determine, in turn,

Rat CowMr

92,500 -

68,000 -

43,000 -

2 4 6 8Antiserum added, A.l

10

FIG. 1. Immunotitration of PNMTase activity from adult and neo-natal adrenal gland. Adrenals were removed from adult rats 50-60days of age and from neonates on the day of birth. To obtain approx-imately equivalent enzyme activities, one pair of adult adrenals washomogenized in 2.890 ml of buffer, whereas one pair of neonatal ad-renals was homogenized in 171.5 ul of buffer. Anti-PNMTase anti-serum was used at a final dilution of 1:12. PNMTase activity is ex-pressed per 40 kil of homogenate supernatant for neonatal and adultadrenals. Each point represents the mean of duplicate determinations.

25,700-

18,400 -12,300 -

FIG. 3. NaDodSO4 gel electro-phoresis of PNMTase immunopre-cipitates. 3SILabeledPNMTase wassynthesized in a wheat-germ trans-lation system with poly(A)-mRNAat 20 uSg/ml from bovine adrenalmedulla or total rat adrenals andwas immunoprecipitated with anti-sera prepared to the homologousenzyme. The arrows indicate theMr 35,000 and Mr 32,000 polypep-tides in rat and cow, respectively.

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'I

<0

x

S.

z

0d 5 10 15 20 25Poly(A)-mRNA, ug/ml

Total ImmunoprecipitatesDay 1 Adult Day 1 Adult Day 15 Adult Preimmune

Mr

92,500-68,000-

xzI

1E

asco

FIG. 4. Effect of poly(A)-mRNA concentrationon the cell-free syn-thesis of PNMTase. Rat adrenal poly(A)-mRNA was translated in thewheat-germ cell-free system. An aliquot (2 suI) was taken to determinethe trichloroacetic acid-precipitable protein and.the remainder (98 1.l)was immunoprecipitated 'with antiserum against PNMTase and pu-rified by NaDodSO4 gel electrophoresis. The 'S-labeled PNMTasewas detected on x-ray film by fluorography. The film was aligned tothe gel and the band corresponding to PNMTase was eluted and quan-titated by scintillation spectroscopy.

whether this increase in enzyme is accompanied by a corre-sponding increase in mRNA coding for PNMTase, we assessedthe levels of PNMTase mRNA during development.

Translational Systems. Optimal conditions for translation ofPNMTase mRNA and for immunoprecipitation were initiallyestablished with bovine adrenal medullary mRNA. Translationin a wheat-germ extract was linear for almost 60 min (Fig. 2A).The Mg2e and K+ optimafor translation were 1.2mM Mg(OAc)2and 112 mM KOAc, respectively (Fig. 2 B and C). However,1.6 mM Mg(OAc)2 was preferable for translation of PNMTasemRNA, as shown in Fig. 2D. The newly synthesized polypep-tide had an apparent Mr of 32,000.

These conditions were used for translation of rat adrenalmRNA. Immunoprecipitation with antisera to rat PNMTaseyielded a [35S]methionine-labeled polypeptide with an apparentMr of 35,000 (Fig. 3); Similar results were obtained with therabbit reticulocyte lysate translation system.To determine whether the immunoprecipitated PNMTase

was, in fact, proportional to the amount of PNMTase mRNApresent, varying concentrations of rat adrenal poly(A)-mRNAwere used. Total translational activity and immunoprecipitablePNMTase were quantitated. Increased translation with increas-ing concentrations of adrenal poly(A)-mRNA was paralleled byincreased immunoprecipitable'PNMTase (Fig. 4). In each case,the PNMTase recovered was -0.1% of the total translationproduct.

Ontogeny of Rat PNMTase mRNA. Developmental changesin PNMTase mRNA were investigated by isolating poly(A)-

~~~;bte

43,000 M"

25,700-

18,400- -

FIG. 5. Developmental change in PNMTase mRNA.in the rat ad-renal. NaDodSO4 gel electrophoresis of total translation products andthe immunoprecipitates obtained with rabbit anti-rat PNMTase anti-serum using mRNA from adrenals of rats at day 1 or day 15 of life iscompared to adults. The arrows indicate the Mr 35,000 polypeptide.

mRNA from rat adrenals on postnatal days 1 and 15 and fromadults. The total translation products (Fig. 5) were strikinglysimilar, although not identical. (For example, a polypeptide ofMr 65,000 was greatly enriched in the translation of newbornmRNA.) To define developmental changes, an equal amountof total translation- products at each age was immunoprecipi-tated with antisera to PNMTase. Whereas immunoprecipitatesfrom adult mRNA exhibited a distinct PNMTase band, the com-parable band was less intense at day 15 and barely detectablein the day 1 adrenals (Fig. 5). Quantitation of this change byelution of radioactive bands and scintillation spectroscopy in-dicated that PNMTase mRNA was enriched -4-fold in the adultcompared to the 15-day adrenal, whereas the newborn (day 1)was barely above background. Quantitation by densitometrygave comparable results for the increase in PNMTase mRNAfrom day 15 to adulthood and revealed an 8-fold increase fromday 1 to adulthood (Table 1).

Because the entire rat adrenal gland was used in these stud-ies, the total poly(A)-RNA fraction contained both cortical andmedullary RNA. However, PNMTase is localized specificallyto the medulla. To determine how the medulla changes in sizerelative to the cortex during development, we. measured corticaland medullary volumes at days 1, 15, and 60. Although bothdivisions grew considerably, medullary volume, expressed aspercentage of the total adrenal, remained relatively constant(Table 1).

Ontogeny of Bovine PNMTase mRNA. To eliminate thecomplicating factor of the cortex, we isolated bovine (Holstein)adrenal medullae from third-trimester embryonic, newborn,and adult animals. In embryos and neonates, there was a gra-

Table 1. Comparison of development of rat adrenal size, PNMTase activity, and PNMTase translation product

Quantitation ofPNMTase immunoprecipitate,

% of adultCortical Medullary (Medullary volume/ PNMTase Elutionvolume, volume, adrenal volume) activity, and scintillation

Age mm3* n 3* x 100 nmol/pair/hr* Densitometry spectroscopyDay 1 0.45 ± 0.02 0.029 ± 0.001 6 0.26 ± 0.01 12 OtDay 15 1.95 ± 0.13 0.19 ± 0.02 9 4.3 ± 0.2 29 25Day 60 (Adult) 13.5 ± 0.5 0.8 ± 0.1 6 10.4 ± 0.3 100 100* Mean ± SEM.t Not significantly different from background.

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4826 Neurobiology: Sabban et al.

? :;4~~~~~~~~~~~~~~~~~~~~~~~~~*6..

'4'4~~~~~~~~~~~~~4

FIG. 6. Immunocytochemical demonstration of PNMTase in bovine adrenal of third-trimester embryo (A; x35), newborn (B; x 100), and adult(C; x 100). Note that in fetal and newborn adrenals, brightly stained cells in the medulla (m) are located adjacent or close to the unstained cortex(cx), whereas in the adult most medullary cells are brightly stained (only medulla shown for adult).

dient of PNMTase immunoreactivity in the medulla withbrightly stained cells located adjacent to the cortex and lightlystained cells located internally (Fig. 6). In the. adult adrenal,most medullary cells were brightly stained for PNMTase.PNMTase catalytic activity and translation of PNMTase

mRNA, as a proportion of total translation products, increased.markedly with age. Translation ofPNMTase was extremely lowin embryos and increased 50&fold in the neonate and 100-foldin the adult (Fig. 7; Table 2).

DISCUSSIONPrevious work has indicated that PNMTase, a specific index ofthe adrenergic phenotype, is initially expressed in the rat ad-renal medulla late in fetal life (4-6). Although initial expressionof PNMTase apparently occurs independently of high levels ofglucocorticoids, the subsequent ontogenetic increase in cata-lytic activity and immunocytochemical reactivity is criticallydependent on these steroid hormones. (6, 7). The present stud-ies demonstrate that the developmental rise in rat adrenalPNMTase activity is due. to an increase in numbers of enzymemolecules and that development involves an increase in mRNA

TotalSE LE N A

Ml'

92,700 --

184000-Mm

12,300

Immunoprecipitates

SE LE N A

_ _

FIG. 7. Developmental change in PNMTase mRNA in the bovinemedulla. NaDodSO4 gel electrophoresis profile of total translationproducts and immunoprecipitates with goat anti-bovine PNMTaseantiserum, with mRNA from bovine adrenal medulla of two third-trimester embryos that differed slightly in size: small embryo (SE) andlarge embryo (LE); newborn calf (N); and adult cow (A). The arrowindicates the Mr 32,000 polypeptide.

coding for PNMTase. Moreover, PNMTase mRNA increased,during development of the bovine adrenal medulla, confirmingresults obtained in the rat whole adrenal.

Although the immunotitration studies demonstrated that thepostnatal rise in rat adrenal PNMTase activity is attributable toan increase in. specific enzyme protein and not to activation ofpreexistent enzyme molecules, it is not clear whether this in-crease reflects increased PNMTase per cell, increased cells,. orboth.. It is likely that the observed increase between postnatalday 15 and adulthood is due to an increased amount of enzymeper cell because division of medullary cells has neared comple-tion by day i5 (26). In either case, the increased enzyme proteinmayi be due to elevated rates of PNMTase synthesis. or de-creased degradation (or both).One mechanism that might underlie increased PNMTase

synthesis involves an ontogenetic increase in mRNA coding forPNMTase. To investigate this possibility, we used the methodof cell-free translation to assess changes in PNMTase mRNAduring development. Although such systems are sensitive tovariations in salt concentrations and cannot be used to comparerelative amounts of different mRNAs (27), they have been suc-cessful in evaluating varying amounts ofthe same-mRNA duringcell development (28). Direct quantitation ofmRNA followingin vitro translation has confirmed the reliability of this method(29).A number of precautions were taken to ensure that devel-

opmental changes in mRNA detected did faithfully reflect theamounts ofPNMTase mRNA present. First, optimal conditionsfor' translation of PNMTase mRNA were established (Figs. 2and 3). Second, control experiments indicated that increasingamounts of poly(A)-RNA in the translation system yielded anearly linear increase in the PNMTase precipitated (Fig. 4).Third, total' cellular mRNA was employed to ensure that weassessed the total mRNA and not merely a potential subpopu-

Table 2. PNMTase development in bovine adrenal medullaPNMTase activity, Quantitation of

pmol/lyg of PNMTase translationAge protein/hr* product, % of adultt

Third-trimesterembryos* 0.90 ± 0.14 1-3

Newborn 1.8 ± 0.4 55Adult 11.7 ± 1.2 100

* Mean ± SEM.t Densitometric method.* Slaughterhouse specimens for which precise ages were not available.

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Proc. Natl. Acad. Sci. USA 79 (1982) 4827

lation functionally active on polysomes. Fourth, the apparentmolecular weight of the synthesized, precipitated PNMTase of35,000 in the rat and 32,000 in the cow are in agreement withothers (30-32). [We have not yet identified an additional poly-peptide, with apparent Mr of 48,000 (Fig. 5), which inconsis-tently appeared in immunoprecipitates of rat adrenal prepara-tions. ] Fifth, two independent methods were used to quantitatethe PNMTase translation product, laser densitometry and scin-tillation spectroscopy, yielding comparable results. Finally, de-velopmental comparisons were made only within single exper-iments to ensure comparable efficacy of immunoprecipitationand translational efficiency.Our results indicate that rat adrenal poly(A)-mRNA becomes

enriched for PNMTase message during development. Conse-quently, the ontogenetic increase of PNMTase in vivo appearsto be due to increased specific mRNA leading to enhancedPNMTase synthesis. This increase may be due to increased spe-cific mRNA per cell or an increased number of cells producingmessage (or both). However, interpretation is complicated bythe fact that assays in the rat contained both cortex and medulla.Nevertheless, because the ratio of medullary and cortical vol-umes was unchanged during development (Table 1), it is un-likely that the observed increases in PNMTase mRNA were dueto decreases in cortical total poly(A)-mRNA. To investigate thispoint, we assayed PNMTase mRNA from pure medullary prep-arations of bovine adrenals at different ages. Bovine medullaryPNMTase mRNA increased during development as in the rat,suggesting that this is a generalized phenomenon. However,in the bovine system, the increases in PNMTase activity andin mRNA were not completely parallel: message increases priorto enzyme activity.

The developmental increase of PNMTasemRNA could resultfrom a decreased rate ofdegradation (8, 33-35). Alternately, thebiosynthesis of PNMTase mRNA may be increased throughseveral different mechanisms, including (i) stimulation of geneexpression (36), (ii) enhanced processing of heterogeneous nu-clear RNA, increasing the poly(A)-mRNA coding for PNMTase,or (iii) increased export of the mRNA into the cytoplasm (37).

Regardless of underlying mechanisms, our observationsdemonstrate that the developmental increase in adrenomedul-lary PNMTase is accompanied by increased PNMTase mRNA.Since the development of PNMTase is glucocorticoid-depen-dent, it is possible that glucocorticoids regulate the develop-mental increase in PNMTase mRNA. This suggestion is con-sistent with the observation that glucocorticoids increasespecific mRNA for tyrosine hydroxylase in pheochromocytoma(38), for tyrosine aminotransferase and tryptophan oxygenasein rat liver (39, 40), for phosphoenolpyruvate carboxykinase inrat kidney (41), and for growth hormone in cultured pituitarycells (42). Moreover, since glucocorticoids affect the develop-ment of PNMTase in extraadrenal loci, such as sympatheticganglia (20, 21, 43), a generalized influence of these hormoneson PNMTase mRNA in various locations cannot be excluded.The authors acknowledge the excellent technical assistance of Mrs.

Dana Straka, Ms. Patricia Passeltiner, Ms. Elise Grossman, and Mrs.Bettye Mayer. The generosity of Drs. Mitchell Sayare and Victor Fried-rich in providing the laser densitometer and electronic planimeter wasalso gratefully appreciated. This work was supported by National In-stitutes of Health Grants NS 06400, NS 10259, and HD 12108, NationalInstitute of Mental Health Grant 02717, and grants from the Dys-autonomia Foundation, the National Foundation-March of Dimes, andthe Cerebral Palsy Association.

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