Pituitary and hypothalamic hormones in normal and neoplastic adrenal medullae: biologically active corticotropin-releasing hormone and corticotropin

Regulatory Peptides, 18 (1987) 173 188 Elsevier

173

RPT 00603

Pituitary and hypothalamic hormones in normal and neoplastic adrenal medullae: biologically active corticotropin-releasing hormone and corticotropin Wendell E. Nicholson, G. Stephen DeCherney*, Richard V. Jackson** and David N. Orth Department ¢~/"Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, U.S.A. (Received 13 January 1987; revised version received and accepted 23 March 1987)

Summary Six normal and 8 neoplastic adrenal medullae were assayed for several immunoreactive (IR) proopiomelanocortin (POMC) and hypothalamic peptides. IR-POMC peptides were found in normal and tumor tissue in concentrations ranging from 0.0003 to 0.1% of those in pituitary. Their molecular sizes resembled those of pituitary intermediate lobe POMC peptides. No intact POMC was found. One pheochromocytoma contained fully bioactive IR-adrenocorticotropic hormone (IRACTH; Mr "-- 4~500) and an intermediate-sized (Mr ~ 10,000) IR-ACTH with ~ 69% bioactivity. Normal and tumorous medullae contained IR-corticotropin-releasing hormone (CRH) in concentrations ranging from 0.6 to 4% of those in hypothalamus except for one pheochromocytoma that contained 40 times that amount of IR-CRH, which was chromatographically indistinguishable from hypothalamic CRH and fully bioactive. IR-somatostatin and IR-growth hormone-releasing hormone were found in both tissue types, but IR-gonadotropin-releasing hormone and IRthyrotropin-releasing hormone (TRH) were not, although IR-histidyi-proline diketopiperazine, a putative TRH metabolite, was found. IR-arginine vasopressin was found in two normal medullae, but not in pheochromocytomas. Bioactive pituitary-hypothalamic peptide; Adrenal medulla

Correspondence." W.E. Nicholson. A4213 Vandcrbilt Medical Center North, Nashville, TN 37232, U.S.A. *Present address: Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, U.S.A. **Present address: Department of Medicine, University of Queensland, Grcenslopes Hospital, Brisbane, Australia 4120. 0167-0115,'87/503.50 ,~.'; 1987 Elsevier Science Publishers B.V. (Biomedical Division)

174 Introduction

Hypothalamic corticotropin-releasing hormone (CRH) stimulates anterior pituitary corticotrophs to secrete adrenocorticotropic hormone (ACTH) and other proopiomelanocortin (POMC)-derived peptides [1], ACTH stimulates adrenocortical cortisol secretion, and cortisol inhibits CRH and ACTH release. POMC and POMCderived immunoreactive (IR) peptides, including ACTH, the lipotropins (LPHs), ~melanocyte-stimulating hormone (~MSH) and/3-endorphin (fiEND), are also found in normal adrenal medulla and adrenomedullary tumors [2,3]. Furthermore, the adrenal medulla contains hypothalamic neuropeptides, including CRH [2,4], somatostatin (SRIH) [5], which inhibits ACTH release by pituitary tumor cells in culture [6], and arginine vasopressin (AVP) [7], which potentiates CRH action on anterior pituitary cells [8]. We examined extracts of normal and neoplastic human adrenal medullae to determine whether POMC-derived and hypothalamic peptides coexist in the same tissue, define further the nature of the POMC-derived peptides and ascertain whether IR-ACTH and IR-CRH in the adrenal medulla are bioactive.

Materials and Methods

Tissue colh, ction

Eight pheochromocytomas obtained at surgery were frozen immediately at - 70°C. No patient had signs or symptoms of hypersecretion of hormones other than catecholamines. Six normal adrenal medullae were obtained at surgery from 4 patients with Cushing's disease, one with primary aldosteronism and one who was pronounced brain dead after an accident and was having his kidneys removed for transplant. Medullae were immediately dissected from cortices and stored at -70°C. Tissue extraction

Tissues were weighed while frozen. For comparison of extraction efficiency of lR-POMC peptides and CRH from adrenomedullary tissues, portions of 8 pheochromocytomas and 1 normal medulla were quickly minced, and equal aliquots were extracted with water [9] or I N acetic acid/0.1 N HCI [10]. Aliquots of extracts were stored at -70°C and, prior to assay, each acid extract was lyophilized and reconstituted with RIA buffer [11]. Peptides and antisera

Synthetic hACTH, hLPH-(35 56) and :~MSH were provided by Ciba-Geigy (Basel, Switzerland). H u m a n / / L P H and h7LPH [hflLPH-(I 56)] were purified in our laboratory [12]. Synthetic hflEND, hCRH, oCRH and [TyrZ~Gly22]-oCRH-(I 22) were gifts of Dr. J. Rivier, synthetic b73MSH a gift of Dr. N. Ling and purified human NH2-terminal POMC-(I-76) peptide (hNT) a gift of Dr. M. Chr6tien. Synthetic hLPH-(39 56). oxytocin (OT), AVP, lysine vasopressin (LVP), z~MSH-tYee acid, desacetyl-:~MSH and hAc/3END were purchased from Peninsula Laboratories (Bel-

175 mont, CA). Anti-b73MSH, -hAcflEND, -~tMSH, and -oCRH sera were provided by Drs. N. Ling, G.P. Mueller, C. Oliver and W. Vale, respectively. Anti-AVP and -ACTH sera (IgG-AVP-1 and IgG-ACTH-1) were gifts of IgG Corporation (Nashville, TN).

Radioimmunoassays RIAs for CRH, AVP, and POMC-derived peptides were performed as described for ACTH [11], except that bovine serum albumin (BSA) was not added to the RIA buffer. Intraassay and interassay coefficients of variation for all of the RIAs were 7-11% and 10-15%, respectively. The features of each RIA are as follows. A 1 plus 1-day, 2-stage ACTH RIA used synthetic hACTH as standard and tracer and rabbit anti-ACTH serum IgG-ACTH-I [11], which is directed to ACTH-(5-18). A 2 plus l-day, 2-stage 73MSH RIA used synthetic b73MSH as standard and tracer and rabbit anti-b73MSH serum RB-294/5-29-80 [13], which is directed to b73MSH-(5-14), corresponding to hNT-(55-64); hNT was only 29% as potent, although h~,3MSH and b73MSH were equipotent. A 3-day, l-stage fiEND RIA used synthetic hfiEND as standard and tracer and rabbit anti-hfiLPH serum R2489/7 [14]. which is directed to hfiLPH-(80-89) and recognizes hfiLPH and h fiEND equally. A 1 plus l-day, 2-stage hAcfiEND RIA used synthetic hAcfiEND as standard and tracer and rabbit antihAcfiEND serum RN-20/7-8-83; hfiEND cross-reacts 0.5%. A ! plus l-day, 2-stage aMSH RIA used synthetic ~tMSH as standard and tracer and rabbit anti-:tMSH serum 2/6-10-72 [15]; the COOH-terminai VaI-NH2 is required, since neither ACTH nor :¢MSH free acid react, but desacetyl-~MSH is fully active. A 1 plus l-day, 2-stage LPH RIA used hLPH-(35 56) as standard and tracer and rabbit anti-hLPH(35 56) serum R2489/12 [1], which is directed to hLPH-(39 -55); hfiLPH is 46% as potent as hTLPH or hLPH-(35 56). A 3 plus 2-day, 2-stage CRH RIA used synthetic hCRH as standard, [Tyr2~,GlyZZ]-oCRH-(I-22) as tracer, and rabbit anti-[Tyr 22, Gly23] oCRH-(I-23) serum oC-24 [10], which is directed to CRH-(4-20) common to oC RH and hCRH. A 3-day, 1-stage AVP RIA used synthetic AVP as standard and tracer and rabbit anti-AVP serum IgG-AVP-! [16]; OT and LVP cross-react 0.06% and 33%, respectively. TRH and histidyl-proline diketopiperazine (His-Pro-DKP) RIAs were performed by Dr. 1.M.D. Jackson, using synthetic TRH and His-Pro-DKP, respectively, as standards and tracers [17,18]. His-Pro-DKP is not active in the TRH RIA, nor is TRH in the His-Pro-DKP RIA. The GnRH, G R H and SRIH RIAs were performed by Dr. W. Vale, using synthetic GnRH, hpGRH-(I-40) and SRIH, respectively, as standards and tracers [19,20], Gel exclusion chromatograph), Small Sephadex G-50 or G-IO0 column. Extracts equivalent to 0.1 g wet tissue were applied to a 0.9 × 57-cm column of Sephadex G-50 or G-100 (fine) resin (Pharmacia Fine Chemicals, Piscataway, N J), developed at 22°C in RIA buffer [11] and calibrated with 200 fmol of each reference compound. 1-ml fractions were stored at -70°C until assayed. Large Sephadex G-50 column. Pheochromocytoma extract (4.2 g wet tissue) was applied to a 5 × 90-cm Sephadex G-50 (fine) column developed at 4°C in 20 mM

176

NaCI containing 1% (v/v) acetic acid and 0.1% (w/v) BSA ( # A-6003, Sigma Chemicals, St. Louis, MO) and calibrated with 20 fmol ~2Sl-hACTH. 17-ml fractions were assayed for IR-ACTH. Sephacryl S-200 column. Fractions representing 70% of the high-molecular-weight IR-ACTH from the large Sephadex G-50 column were pooled, evaporated in a Speed Vac Concentrator (Savant Instruments, Hicksville, NY), redissolved in 12 ml 0.15 M NH4HCO3 and applied to a 2.6 × 90-cm Sephacryl S-200 (Pharmacia) column developed in 0.15 M NHaHCO3 at 4°C and calibrated with 20 fmol '2Sl-hACTH. Absorbance at 280 nm and IR-ACTH were measured in 5.2-ml fractions. Sephaeryl S-200 column under denaturing and reducing conditions. Fractions comprising the major peak of I R-ACTH from the Sephacryl S-200 column were pooled, evaporated and redissolved in 2 ml 6 M guanidine hydrochloride (Gdn • HCI) buffer (6 M G d n - HCI in 0.5 M Tris, pH 8.1, containing 1 mM EDTA and 10 mM dithiothreitol), heated to 50°C for 4 h and cooled to 4°C [21]; 1 ml was applied to a 1.6 × 90-cm Sephacryl S-200 column developed at 40C in Gdn • HC1 buffer containing 1 mM dithiothreitol and calibrated with 20, 25, and 20 fmol ~2SI-hACTH,-hflLPH, and -hPRL respectively. Gdn - HCI was removed from alternate 2-ml fractions using PD-10 columns (17-0851-01, Pharmacia) equilibrated in 5% (v/v) acetic acid containing 1 mg/ml BSA. The protein fraction was evaporated, redissolved in 1 ml RIA buffer [11] diluted !:10 with water, and subjected to ACTH RIA.

High-perfbrmance liquid chromatograph.v (HPLC) of tumor CRH Pur([ication o['tumor CRH. IR-CRH in a tumor extract was partially purified and concentrated by two extractions with silicic acid [22]; the pooled eluates were lyophilized and stored at -700C until HPLC. Oxidation ~[" CRH. 10 lag synthetic hCRH, were dissolved in 500/~1 0.1% (v/v) trifluoroacetic acid (TFA) in water, mixed with 100/~1 30% (w/v) H202 in water, allowed to react for 30 min at 4°C and applied directly to the HPLC column. HPLC. Samples of hCRH, oxidized hCRH, solvent blank, and partially purified tumor IR-CRH were sequentially analyzed by reverse-phase HPLC, using a 0.46 × 25-cm, 5-pm particle size, 3000 nm pore size, C~8 column (218TP54, Vydac, Hesperia, CA), 2 M600A pumps, an M660 gradient programmer, an M450 variable wavelength detector and an M730 data module (Waters, Milford, MA). The solvent system was 0.1% (v/v) TFA in water (solvent A) and 60% (v/v) acetonitrile in solvent A (solvent B). Gradient conditions were defined as percent acetonitrile. The column was equilibrated in 18% acctonitrilc, the sample was applied in solvent A, isocratic elution with 18% acetonitrile was continued for 10 min, and peptides were eluted with a 30-min linear 18 48% acctonitrile gradicnt at 1.2 ml/min; l-min fractions were stored at -700C, lyophilized and reconstituted in 1 ml RIA buffer [11].

CRH bioassay CRH bioactivity was determined in a column perifusion system consisting of dispersed rat anterior pituitary cells supported by Bio-Gel P-2 resin ( # 150.0150, BioRad Laboratories, Richmond. CA) [23], using synthetic o C R H as standard. Removal of catecholamines prior to CRH bioassav. Epinephrine stimulates ACTH

m

0.78 :t: 0.67 a 1.3 :t_ 1.0

57 7.4 0.37 I.I 0.08 0.10 0.04 0.02

10.1 + 7.0

5.3 ± 2.2 c

13 4.8 0.24 0.18 0.12 0.12 <0.02 < 0.02

4.2 24 b 2.1

12' 12 4.9 1.8 0.94 0.13

IR-y3MSH

1.5 + 1.2

14 8.4 1.2 0.31 0.21 0.21 < 0.06 < 0.06

8.9 -1:3.4

0.52

1.4 + 0.39

2.9 2.2 1.6 0.29 1.4 3.0 1.0 0.07

-

-

1.0

< 0.06 1.0

IR-AcflEND

< 0.06

17 18 14 3.2

IR-I~END

0.5 + 0.40

8.2 2.9 0.32 0.12 0.04 0.09 0.09 < 0.03

2.5 ± 1.6

0.90 10 4.2 0.04 0.09 < 0.03

IR-aMSH

4.1 ± 3.2

210 23 1.6 3.6 0.31 0.35 0.05 < 0.02

21 + 7.9

54 34 12 13 15 < 0.02

IR-LPH

Mean + S.E.M. For the purpose of calculating means and S.E.M.s, undetectable values were assumed to be half of the detection limit (e.g. < 0.02 fmol/mg wet tissue of IR-ACTH = 0.01 fmol/mg wet tissue). n All means and S.E.M. for pheochromocytomas exclude no. I.

'

Fmol of immunoreactive peptide per mg of wet tissue. b Not measured.

_

2 3 4 5 6 7 8

1

Pheochromocytoma no.

2 3 4 5 6

I

Normal medulla no.

IR-ACTH

lmmunoreactive pro-opiomelanocortin-related peptide concentrations in normal and neoplastic adrenomedullary tissues

TABLE I

178

release, an effect which is additive with that of CRH [24]. Therefore, catecholamines were removed from the tumor extract by PD-10 column chromatography at 22°C in 30 mM NaCI containing 1% (v/v) acetic acid and 0.1% (w/v) BSA. The protein fraction containing IR-CRH was lyophilized and reconstituted in 10 ml 1 mM HCI immediately prior to assay. Aliquots were appropriately diluted in perifusion medium. Catecholamine assay. Epinephrine, norepinephrine and dopamine were measured [25] with a commercial kit (CAT-A-KIT, NDS 0009-3000-04, Upjohn, Kalamazoo, MI).

A CTH hioassay Fractions comprising the two major peaks of IR-ACTH identified in the large Sephadex G-50 column effluent, were pooled, lyophilized, reconstituted to 10% of their original volumes with water and subjected to ACTH bioassay [26] and RIA.

Results

IR-peptide concentrations in tissue extracts POMC-derived peptides. Water [9] was 3.0 + 0.74 (S.E.M.) times as efficient as acid [10] for extracting IR-POMC-derived peptides from these tissues. The relative efficiency for extracting individual IR-POMC-derived peptides ranged from 1.8 + 0.85 for IR-fiEND to 6.6 + 2.8 for IR-LPH. Extracts of normal and neoplastic adrenal medullae generated parallel RIA displacement curves, indicating they contained materials that were immunologically similar to normal pituitary POMC products, including IR-ACTH, -~MSH, -LPH, -fiEND, -73MSH, and -AcflEND (Table I). The IR-ACTH content of each tissue correlated with those of IR-LPH, -fiEND and -',e3MSI-I (r = 0.74, 0.72, and 0.75 respectively, P < 0.01). ~MSH is derived from ACTH, and the combined IR-ACTH-plus-~MSH content of each tissue also correlated with those of IR-LPH, -fiEND and -73MSH (P < 0.05). The slope of the regression line for IR-ACTH-plus-~MSH vs IR-LPH was 5.0, indicating an excess of LPH vs ACTH-plus-~MSH, but the slopes for IR-ACTH-plus-~MSH vs IRfiEND and IR-73MSH were 0.84 and 1.6, respectively, not different from unity (P > 0.05). There was no correlation between tissue concentrations of IR-ACTH and IR-AcfiEND (r = 0.15, P > 0.1) or between IR-AcfiEND and either IR-fiEND or IR-~MSH (r = 0.06 and 0.04, respectively, P > 0.1). Hypothalamic peptMes. Water was 1.2 + 0.48 times as efficient as acid for extracting IR-CRH. Two normal and 4 neoplastic adrenal medullae contained IR-CRH (Table II). No tissue contained IR-CRH without detectable IR-ACTH; 5 contained IR-ACTH without detectable IR-CRH. There was no correlation between IR-CRH and IR-ACTH content in extracts that contained both (P > 0.5). IR-GRH and IR-SRIH were each found in 6 tissues; both were present in 3 (Table II). Neither IR-TRH nor IR-GnRH were detected in normal or neoplastic tissue, but IR-HisPro-DKP, a putative T R H metabolite [27], was found in both (Table II). IR-AVP was tbund in two normal mcdullae, but not in tumor tissue (Table II).

0.10

0.10

0.67

0.58

0.10

0.10

3

4

5

6

7

8

0.70

0.84

0.56

0

< 0.14

<0.14

< 0.14

< 0.14

<0.14

< 0.14

< 0.14

< 0.14

-

-

b

IR-AVP

0

< 2.8

< 2.8

< 2.8

< 2.8

<2.8

< 2.8

< 2.8

< 2.8

0

< 5.5

<2.8 <2.8

< 2.8

< 2.8

IR-TRH

261 + 67

2 I0

<34

160

440

520

440

270

34

75 + 25

69

34 34

170

66

1R-HP-DKW

0

< 0.09

<0.09

< 0.09

< 0.09

<0.09

< 0.09

< 0.09

< 0.09

0

<0.09

<0.09 <0.09

<0.09

< 0.09

IR-GnRH

6.1

0.70

0.46

1.8

0.46

0.33

0.59

1.1 + 0.8

< 0.33

<0.33

< 0.33

< 0.33

-

-

-

IR-GRH

120

98

37

67

56 a: 33

31 + 12

37

<24

< 24

< 24

<24

-

<24 37

-

IR-SRIH

= 0.05 f m o l / m g wet tissue).

All m e a n s a n d S . E . M . s f o r p h e o c h r o m o c y t o m a s e x c l u d e no. 1.

wet tissue o f I R - C R H

' IR-His-pro-DKP 't M e a n + S . E . M . F o r the p u r p o s e o f c a l c u l a t i n g m e a n s a n d S . E . M . s , u n d e t e c t a b l e values w e r e a s s u m e d to be h a l f o f the d e t e c t i o n limit (e.g., < 0.10 f m o l / m g

b Not measured.

F m o l o f i m m u n o r c a c t i v e p e p t i d e p e r m g o f wet tissue.

0.28 + 0 . 0 9 '

0.29

580

2

Pheochromoc y t o m a no. 1

0.10 <0.10

5 6

0.12 + 0.05 'j

0.10 <0.10

3 4

0.35"

<0.10

2

N o r m a l m e d u l l a no. 1

IR-CRH

I m m u n o r e a c t i v e hylm~thalamic h o r m o n e c o n c e n t r a t i o n s in n o r m a l a n d n e o p l a s t i c a d r e n o m e d u l l a r y tissues

T A B L E !1

180

A

Vo

hACTH

vt

Vo

hACTH

Vt

I

;

1

l

120 15

c

._o 3 0 0

A

36.0

C

g 10 8O

24 "rl--

t...-

54O

,~ ,oo 0

0

10

20

0 0 ~ 0 10 40 FRACTION NUMBER

30

20

30

0

40

Fig. 1. Sephadex G-50 (fine) gel exclusion chromatography of IR-ACTH extracted from two normal adrenal medullae (A) and 2 pheochromocytomas (B). In this figure and Figs. 3-6, the scales on the vertical axes, from left to right, indicate the concentrations of IR-peptide in the column fractions from normal medullae I (A---A) and 3 ( O - - O ) and pheochromocytomas 1 (11--11) and 2 ( O - - ¢ , ) , respectively. Open symbols indicate undetectable IR-peptide at the concentration plotted. Recoveries of IR-ACTH in the extracts applied to the column were 88, 106, 84 and 8 0 0 , respectively.

Apparent molecular sizes of IR-peptides POMC-derivedpeptides. Most of the I R - A C T H in normal tissues eluted in or just after the small Sephadex G-50 column's void volume, where POMC (Mr ~ 37,000) [28] would elute; very little eluted as hACTH (Fig. IA). Tumor IR-ACTH was more heterogeneous, consisting of POMC-, intermediate- and hACTH-sized materials (Fig. I B). The IR-ACTH in pheochromocytoma 1 eluted in 2 peaks from the large Sephadex G-50 column: peak 1 in and just after the void volume and peak 2 in the hACTH region (not shown). The IR-ACTH in Peak 1 eluted before pACTH from the Sephacryl S-200 column (Fig. 2A). When this material, Mr ~ 20,000, was rechromatographed on a Sephacryl S-200 column under denaturing and reducing conditions, it eluted with an apparent Mr "-- 10,000 (Fig. 2B). 0.4

,nt-~mt.

0.4

Jtst..~,m~

12

'@e hlllG NSA

2.5

L

¥

|

II~I"tIACTH

1

l

~

0.9 i

2.0 q~ Jt I I I

u~ 1.5

~

03

l

0.6 ~ 0.2

0.2--

0.3 ¢- Ol

O.I ~

l,

I.0

< 0.5 5O

40

50

61)

70

OO

9O

30

I00

40

50

60

70

80

90

tO0

FRACTION NUMBER

Fig. 2. A: Sephacryl S-200 gel exclusion chromatography of pooled high-molecular-weigh tumor IRACTH fractions from the large Sephadex G-50 column. (---), absorbance at 280 nm; ( O - - O ) IR-ACTH. Recovery of I R-ACTH applied to the column was 140%. B: Sephacryl S-200 gel exclusion chromatography of high-molecular-weight tumor I R-ACTH (fractions 66-73, A) under denaturing and reducing conditions. Recovery IR-ACTH applied to the column was 95%. The fractional elution volume (Vff~)of ] 2H-hPRL , -hflLPH and -hACTH relative to their respective molecular weights is shown in the inset.

181

Vo

"~ 24 .9

o E -r u') ~;

cI

t

16

8

& 0

, I0

0

i 20

1

60

:30

I

i 30

(1MS,~I

Vo

90

l

0

4O

20

I0 0 NUMBER

FRACTION

30

40

Fig. 3. Sephadex G-50 (fine) gel exclusion chromatography of IR-c(MSH extracted from a normal adrenal medulla (A) and 2 pheochromocytomas (B). Recoveries of IR-aMSH in the extracts applied to the column were 62, 61 and 75%, respectively.

IR-ccMSH was present in only one form, the size of standard ~MSH (Fig. 3). Ir-LPH in normal tissue consisted of 3 components similar in size to h/~LPH, h~LPH and hLPH-(39-56) (Fig. 4A). Tumor IR-LPH appeared to consist primarily of o/LPH, with a lesser amount eluting in the region of hLPH-(39-56) (Fig. 4B). The major IR-/~END in normal and tumor tissues was smaller than h/~END, apparently a fragment that retained immunoreactivity (Fig. 5); only a minor fraction appeared to be h/3END or hflLPH (Fig. 5). No POMC-sized IR-/~END was observed in the tumor extracts (Fig. 5B), but both normal medullae contained a minor POMC-sized component (Fig. 5A). IR-73MSH in each tissue consisted mostly of hNT-sized material (Fig. 6). A minor peak was found in each extract, but little or no material the size of POMC was observed. Hypothalamic peptides. 70% of tumor IR-CRH eluted from the small Sephadex G-50 column where standard hCRH would appear; the remainder eluted as smaller materials (Fig. 7A). No high molecular weight IR-CRH was observed. The reversephase HPLC elution profile of partially purified tumor IR-CRH revealed 2 materials: c .Q u o

750

Va I~PH I InLPH

A

Vo 150

I o

E

500

I

3600

(Bg-~5)hLPH

hBLPH

I ,yLP~ I* (S9-56)-

56O

o

v,

E

I00 2400

I

240 z

z

E

E

O nv.

0 r~

F-

t~

50

250

120

1200

I-o ..J

d-

& 0

0

I0

20

30

40

0

0

0

I0

2o

30

40

FRACTION NUMBER

Fig. 4. Sephadcx G-50 (fine) gel exclusion chromatography of IR-LPtt extracted from 2 normal adrenal medullae (A) and 2 pheochromocytomas (B). Recoveries of IR-LPH in the extracts applied to the column were 79, 93.99 and 80%, respectively.

182

c o

Vo h,~l.PH

B

hBEND

Vo h~LPH

"" 120

A

h.eE~O

Yt

vt

o =

2475

E Z

~

80

16

50

8

25

IO0 z

0

o

~ 4o

L.

i

00

I

I

I0

20

I

I0 40 FRACTION NUMBER

30

20

30

4O

0

Fig. 5. Sephadex G-50 (fine) gel exclusion chromatography of IR-/.CEND extracted from two normal adrenal medullae and (A) 2 pheochromocytomas (B). Recoveries of IR-//END in the extracts applied to the column were 78, 115.99 and 97%, respectively.

56% had the retention time of hCRH and the rest appeared to be oxidized hCRH (Fig. 7B), possibly a mixture, as mono- and dioxidized hCRH were not completely separated in this system. IR-hCRH was undetectable, < 2 pmol/fraction in the "blank' run which immediately preceded the application of the tumor sample to the HPLC column.

Bioassay of tumor IR-CRH The bioactive CRH content of the tumor extract was 55% (95% confidence limits, 40 75%) of its IR-CRH content (Fig. 8A) which corresponds to the HPLC estimate of non-oxidized, potentially fully bioactive, I R-CRH. The catecholamine concentration of the tumor extract was reduced prior to bioassay, so that 1.4 × 10 - 9 M IRCRH, the highest concentration perifused, contained 3.7 × 10-1o M epinephrine, less than that which is thought to be necessary to increase CRH-stimulated ACTH release [24].

A

i

75 A

Vo

hNT

Vt

1500

50

IO00

25

5OO

0 0

I0

20

30

40 FRACTION

0

B

IO NUMBER (

1225!

?

75 ~'5

20

30

40

0

Fig. 6. Sephadex G-100 gel exclusion chromatography of IR-),.~MSH extracted from a normal adrenal medulla (A) and 2 pheochromocytomas (B). Recoveries of IR-;,~MSH in the extracts applied to the column were 83.93 and 84%. respectively.

183

A

Ve hCRH

Vt

OXIDIZECL.... r....-.hCRH

" B

hCRH

rr I'"

/ / / / ....

/

hi t.D

/

25 '~

I.,Z hi

/

?

W

a,-

0

0

I0

20

~g)

40

0

I0

20

30

40

0 50

FRACTION NUMBER

Fig. 7. A: Sephadex G-50 (fine) gel exclusion chromatography of IR-CRH extracted from pheochromocytoma 1. Recovery of IR-CRH applied to the column was 74%. B: reverse-phase HPLC of partially purified IR-CRH extracted from pheochromocytoma I. Recovery of IR-CRH applied to the column was 84%. (---) % acetonitrile in the eluent.

Bioassay of tumor ACTH The tumor IR-ACTH that eluted as ACTH (peak 2, large Sephadex G-50 column) was fully bioactive (potency !!2% relative to standard hACTH; 95% confidence limits, 83-151%) (Fig. 8B). The higher molecular weight IR-ACTH (peak 1) appeared to be 69% (95% confidence limits, 52-92%) as bioactive as standard hACTH. Discussion

Tumors of the adrenal medulla have been associated with production of ACTH and other POMC peptides, thereby causing the ectopic ACTH syndrome [29]. Recently, normal human adrenal medulla was reported to contain IR-ACTH, -fiEND and -~tMSH [2,3]. Our results confirm this, establish the presence of IR-peptides derived from the NHE-terminal region of POMC and demonstrate that two forms of IR-ACTH in a pheochromocytoma are bioactive. Our data suggest that most of the IR-ACTH in normal medullae and pheochromocytomas is medium-sized and only a small fraction is M, 4500 ACTH itself. Others [2,3], found that about 35% of adrenomedullary IR-ACTH appeared to be M, 4500 ACTH. However, the adrenals were obtained at autopsy, so postmortem proteolysis may account for their findings. Other possible explanations include different extraction efficiencies, antiserum specificities and RIA standards. Our present and previous [30] data indicate that water is equal or superior to other solvents, including acid and acid-acetone, for extracting POMC-derived peptides from placental and adrenal tissue. The precise concentrations of POMC peptides in normal adrenal medulla remain to be established, but appear to be no more than 0.01 -0.1% of those in the anterior pituitary gland [31]. High-molecular-weight (M, 10,000) tumor IR-ACTH was almost fully bioactive. Mouse pituitary tumor POMC and medium-sized ACTH [32] and 'big' ACTH from human non-pituitary tumors [33] have little or no bioactivity. Mouse M, 13,000

184 70

40

B

3,5

60

/

t=

k

/k o _'-2

~3o <-> ZO iI:

5 I

I

I0

1(30 CRH

(fmol/ml)

I

K)O0

0,1

0.5

I

5

i t n 0 IO

ACTH (nq)

Fig. 8. A: I R - A C T i l released from dispersed rat anterior pituitary cells in the perifusion column system in response to graded doses of standard o C R H ( O - - - O ) or partially purified C R H extracted from pheochromocytoma 1 ( l l - - n ) , the concentration of which was determined by R1A. In the case of standard o C R H , I R - A C T H released is the total IR-ACTH under the response curve minus basal IR-ACTH released in control columns ( ~ 4 fmol,,10° cells • min). In the case of the extract of phcochromocytoma 1, the amount of I R - A C T H contained in each dilution of the extract was also subtracted. The points represent the mean of results obtained with two identical test columns and two identical control columns pcrifuscd in parallel: the brackets indicate the duplicates. B: Plasma corticosterone responses in acutely hypophysectomized rats to graded i.v. doses of synthetic h A C T H or partially-puritied I R - A C T H extracted from pheochromocytoma I. Each point represents the mean plasma corticostcrone concentration in 3 rats 10 min after injection; brackets indicate S.E.M. ( 0 - - 0 ) , h A C T H standard: ([5]--[3), tirst peak of tumor I R - A C T H eluting from large Sephadcx G-50 column: ( A - - - A ) , second peak of tumor I R - A C T H from the same column.

ACTH (glycosylated M, 4500 ACTH) is fully bioactive [32], but no comparable form has been identified in man [28]. M, 10,000 ACTH may be cleaved after injection into the bioassay rat into Mr 4500 ACTH, which then stimulates adrenal steroidogenesis. However, it is fully active within 10 min of its injection [26]. If it is metabolized equally rapidly after secretion in man, this distinction is physiologically irrelevant. We confirmed the presence of IR-~MSH in normal adrenal medulla and showed that it is present in most pheochromocytomas. Our data neither confirm nor contradict the conclusion that it is desacetyl-c~MSH in normal adrenal medulla [3]. Like Evans et al. [3], we found little IR-//END the size of/?LPH. Most of the IR-/~END was smaller than h/~END, suggesting it was a COOH-terminal fragment of [3END. The complementary COOH-terminal fragments of human :x- and 7-endorphin [i.e., h//END-(17- 31) and -(18-31)] could account for the smaller IR-/~END materials found in this study. Evans et al. [3] found that most of the IR-/~END in their normal human adrenal extract eluted in reverse-phase HPLC as h~END; about 25% eluted earlier and was not furthcr identified. We found IR-Ac/3END in 1 of 2 normal medullae we were able to assay and in all 8 pheochromocytomas. In the 4 extracts containing the most IR-/~END, the higher its concentration, the lower was the percent (31. 26, 21 and 0.3%) accounted for by IR-Ac/:~END (Table I).

185 Both normal and neoplastic adrenal medullae contained NH2-terminal POMC IR-peptides. Most of the IR-~,3MSH in them appeared to be hNT; little appeared as smaller peptides, unlike the pituitary intermediate lobe [34]. Much of the flLPH in normal medullae and most in pheochromocytomas appeared to be processed to 7LPH, and some (14-25%) to hLPH-(35-56) and/or -(39-56), peptides which are not found in adult human pituitary [35], which lacks an intermediate lobe, but which are found in ectopic ACTH-producing tumors [36]. Details of posttranslational processing of POMC in adrenal medullae are not known, but our findings, except the "~,3MSH data, suggest it resembles that in the pituitary intermediate lobe. Of interest is the presence of hypothalamic IR-peptides in the normal and neoplastic human adrenal medulla. IR-CRH was detected in half of the normal adrenals and all pheochromocytomas, confirming the reports of Suda et al. [2,4]. One of their tumors and pheochromocytoma 1 had higher IR-CRH concentrations than normal hypothalamus (15 + 4.7 fmol/mg; range, 7.6-29 fmol/mg) [2]. Tumor IR-CRH was bioactive and, considering the content of oxidized CRH, which contributes little bioactivity [37], appeared to have full potency. We eliminated the possibility that some of the apparent CRH bioactivity in the pheochromocytoma extract might be due to the high epinephrine concentration by removing most of the catecholamines prior to bioassay. We confirmed the presence of IR-AVP in normal human adrenal medulla. Higher levels (22 and 94 fmol/mg) than those we found were reported in two medullae by Ang and Jenkins [7], and we could not detect IR-AVP in any of our 8 pheochromocytomas, whereas their 2 tumors contained 11 and 12 fmol/mg, almost 100 times greater than our RIA's exclusion limit (Table II). They extracted their tissues in 1 M acetic acid, which may more efficiently extract AVP. We confirmed the presence of IR-SRIH in normal human adrenal medullae and pheochromocytomas [5] in concentrations similar to those previously reported in pheochromocytomas [38] and identified IR-GRH for the first time in extracts of both normal and neoplastic adrenal medullae. Neither IR-GnRH nor IR-TRH was found, but relatively high concentrations of IR-His-Pro-DKP, a putative metabolite of TRH [27] with bioactivities both similar and opposite to those of TRH [39], were found in virtually all tissues. The physiological function of none of the peptides found in the adrenal medulla, including the enkephalins [5], is known. However, hypothalamic peptides known to regulate POMC secretion by the anterior pituitary are found with POMC in the medulla. CRH, AVP and epinephrine are POMC secretagogues, the latter two acting synergistically and additively, respectively, with CRH [8,24]. In some patients with POMC-secreting pituitary tumors, but not in normal subjects, TRH also stimulates release of POMC peptides [40]. SRIH inhibits POMC peptide secretion by pituitary tumors in patients with Nelson's syndrome [41] and CRH-stimulated ACTH secretion by mouse pituitary tumor cells in culture [42], but has not been shown to regulate ACTH secretion by normal cells [42]. Thus, many of the hypothalamic factors regulating POMC peptide secretion are present in the medulla, and receptors for at least one of them, CRH, are also found there [43].

186

Acknowledgements W e t h a n k Mr. C . D . M o u n t , Ms. I. H e n d r y , Ms. T. C u r r y , a n d M r . E. M i t c h e l for their t e c h n i c a l a s s i s t a n c e , a n d Ms. S. G a d for t r a n s l a t i n g the m a n u s c r i p t . T h i s w o r k was

supported

in p a r t

by N a t i o n a l

Cancer

C A ! 1685. G . S . D . C . was s u p p o r t e d by N I C H D

Institute

Research

Grant

5-RO1-

Clinical Investigator Award 5KO8-

H D - 0 0 4 3 9 . R . V . J . is an A p p l i e d H e a l t h S c i e n c e s F e l l o w , N a t i o n a l H e a l t h a n d M e d i cal R e s e a r c h C o u n c i l o f A u s t r a l i a .

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