corticotropin dependency on zinc ions

Vol. 46, No. 3, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CORTICOTROPIN DEPENDENCY ON ZINC IONS

Arthur Flynn, William H. Strain and Walter J. Pories

Trace Element Center, Cleveland Metropolitan General Hospital

Case Western Reserve Universi ty School of Medicine, Cleveland, Ohio 44109

Received December 21, 1971

Summary: The steroidogenic ac t i v i t y of cort icotropin in rat adrenal sl ices i n v i t r o has been studied with regard to the dependency of ac t i v i t y on zinc ion binding. Chelation of available zinc by diethyldithiocarbamate stopped the production of cort icosterone, which was reversed by the addit ion of zinc. Cort icotropin dependency on zinc ions is proposed, with the binding s i te of zinc being at the glutamate moiety, as suggested by thin layer chromatography.

The essent ia l i t y of zinc in hormonal function has been proposed for the

action of many endocrine systems. Numerous studies on adrenocorticotropic

hormone, glucagon, gonadotrophins, growth hormone and insu l in have implicated

zinc in the i r ac t i v i t y , but l i t t l e evidence has been presented to d i rec t l y

l ink th is metal to any of these hormones. The recent report of the correla-

t ion between cort icotropin (ACTH) and zinc under traumatic conditions in the

rat suggests essent ia l i t y of the metal for adenohypophyseal-adrenal cortex

function ( I ) . A more de f in i t i ve study, however, is necessary to understand

the strong posi t ive correlat ion between zinc and ACTH and to show dependency.

Considerable c l in ica l and experimental evidence has accumulated to in-

dicate a functional role for zinc in ACTH. Early reports (2,3,4) on the

addit ion of zinc, as Zn3(P04) 2 or Zn(OH) 2, to preparations of ACTH pointed

to increased longevity and ac t i v i t y . C l i n i ca l l y , Sandstead et al (5), in

dealing with zinc def ic ient dwarfs, reported that exogenous doses of ACTH

e l i c i ted l i t t l e adrenal response. After oral zinc sulfate therapy, 50 mg

t i d , a marked response could be measured, in fer r ing a close relat ionship be-

tween the p i t u i t a r y hormone ACTH and zinc. Experimentally, studies on whale

ACTH by Holtermann et al (6) have more closely related ACTH and zinc by

Copyright © 1972, by Academic Press, Inc.

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VoI. 46, No. 3, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

indicat ing a dependency. Recently, var iat ions in rat ACTH have been shown to

correlate very strongly with serum zinc changes ( I ) . Rats stressed by hypo-

tensive episodes and allowed to recover, showed f luc tuat ing ACTH leve ls ,

mimiced by serum zinc. Yet, an invest igat ion to test the dependency of ACTH

on zinc ions has not been reported.

A study was designed to examine the ef fects of zinc on ACTH a c t i v i t y in

v i t ro , free from the problem of c i rcu la t ing zinc levels. The isolated adre-

nals allowed for control and comparison not only of systemic zinc, but also

the ef fects of a zinc chelator, diethyldithiocarbamate (DDC), and Zn3(P04) 2

supplementation. By measuring the cort icosterone production of the adrenals

in v i t ro , i t was possible to test the hypothesis that zinc is essential to

the ac t i v i t y of ACTH.

The re lat ionship between ACTH, DDC and zinc was tested in the adrenals

of male and female adult Sprague-Dawley rats; the animals weighed 410-500

gm, and were divided into contro l , chelate control , ACTH-chelate, ACTH-zinc

and ACTH-chelate/zinc groups. Rats were fed a d ie t of Ralston Purina rodent

chow ad l i b (Zn - 58 ppm) and had a mean serum zinc level of 150±6 mcg% and

a mean serum copper level of 1.18±0.3 mcg%. Normal rat serum values are

Zn = 147 mcg% and Cu = 1.20 mcg% (7,8).

Al l animals were anesthesized with 3.5 mg/kg pentabarbital sodium and a

transverse abdominal incis ion made for ease of adrenalectomy. The adrenals

were removed, cleansed of excess t issue and weighed to the nearest 0.01 mg.

Each adrenal was quartered and weighed again and placed in a 5.0 ml p las t ic

vial with appropriate preincubation mixtures. The incubation methods of

Saffaran and Schally (9) were modified for the addit ion of DDC and Zn3(P04) 2

solut ions. Table 1 gives the regimen followed in processing the adrenals.

Corticosterone production was measured in methylene chloride extracts of

incubation mixtures by the f luorometr ic method of Si lber et al ( I0) and com-

pared with a t issue blank. Means and s ta t i s t i ca l comparisons were made based

on the cort icosterone levels.

Incubation procedures were modified so that a known amount of ACTH, 1.0

uni t of ACTH, was added to each f lask during the incubation period. Acthar

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VoI. 46, No. 3, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1

REGIMEN USED IN PREINCUBATION AND INCUBATION OF RAT ADRENAL QUARTERS IN VITRO

G r o u p Preincubation (2 hr) Incubation (2 hr)

Control 1.5 ml Krebs Ringer 1.4 ml Krebs Ringer 0.I ml ACTH ( I .0 uni t )

Chelate 1.4 ml Krebs Ringer 1.4 ml Krebs Ringer Control 0.I ml 0.01 M DDC 0.I ml ACTH ( I .0 uni t)

ACTH 1.5 ml Krebs Ringer 1.3 ml Krebs Ringer Chelate 0. I ml ACTH ( I .0 uni t )

0.I ml 0.01 M DDC

ACTH 1.5 ml Krebs Ringer 1.3 ml Krebs Ringer zinc 0.I ml ACTH ( I .0 uni t )

0.I ml Zn3(P04) 2

ACTH 1.5 ml Krebs Ringer 1.2 ml Krebs Ringer Chelate 0.2 ml ACTH-Chelate

Zinc 0. I ml ACTH ( I .0 uni t ) 0.I ml Zn3(P04) 2 0.I ml 0.01M DDC

TABLE 2

ZINC CONTENT PER UNIT OF COMMERCIALLY AVAILABLE CORTICOTROPIN (ACTH)

SOURCE ZINC CONTENT (PPM/UNIT)

Armour Pharmaceutical Co. Acthar - 40 units

Parke, Davis & Co. ACTH - 40 units

Upjohn Co. Cort icotropin - 25 units

Nut r i t iona l Biochemical Co. Cort icotropin - 200 units/5 cc

ClBA-Geigy Synacthen - 25 mg B 1-24 ACTH

2.85

3.60

3.83

9.93

0.97*

* Zinc content is in ppm/mg.

(Armour Pharmaceutical Co,) was used because i t contained the lowest zinc

content of commercially avai lable ACTH, as is shown in Table 2. The chelator

DDC was made in a 0.01M solut ion and zinc supplementation was 195 ppm

Zn3(P04) 2 in a 0. I ml volume. Al l solut ions were made with double d i s t i l l e d ,

deionized water. A combination of chelate and zinc supplementation was made

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to test the inh ib i to ry e f fec t of DDC on ACTH and i f zinc supplementation could

restore ac t i v i t y . Therefore, pr ior to incubation ACTH solut ion was mixed with

an equal volume of DDC solut ion and allowed to stand for a minimum of 5.0

minutes, Zn3(P04) 2 solut ion was then added during incubation in an attempt to

overr ide chelat ion.

A s t a t i s t i c a l comparison was computed. A 1 x 4 analysis of variance

based on cort icosterone production was calculated fo r the f i ve groups. A

Student-t test was then used fo r ident i fy ing s ign i f i can t di f ferences among

combinations of means.

2.8

2.1

1.4

0.7

TISSUE BLANK

u~

÷I

CONTROL

+

n CHELATE ACTH ACTH CONTROL CHELATE ZINC

I

I i I I

ACTH CHELATE/ZINC

FIGURE 1

THE EFFECTS OF ZINC CHELATION AND SUPPLEMENTATION

ON ACTH ACTIVITY IN ISOLATED RAT ADRENAL QUARTERS (N= 50; n= I0)

The ef fects of zinc chelat ion and supplementation and the i r re la t ionsh ip

to ACTH a c t i v i t y are presented in Figure I . The cort icosterone production

above t issue blank demonstrates s ign i f i can t di f ferences (F = 2.81; df = 4/45;

p< 0.05) wi th in the f i ve test groups. The Student-t test indicated that the

ACTH-chelate group had a s ign i f i can t mean decrease in cort icosterone produc-

t ion (0.71 mg/g), over the control ( t = 1.92; df = I / 8 ; p< 0.05) (2.60 mg/g),

chelate control ( t = 1.86; df = I / 8 ; p<0.05) (2.43 mg/g), ACTH-zinc ( t = 2.05;

df = I / 8 ; p<0.05) (2.79 mg/g) and ACTH-chelate/zinc ( t = 1.85; df = I / 8 ;

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p<O.05 (2.46mg/g). No s ign i f i can t di f ferences were found among the remain-

ing combination of group means and zinc supplementation appeared to have no

e f fec t on ac t i v i t y .

De f in i t i ve studies on the re la t ionship of zinc to ACTH a c t i v i t y have

been lacking although the discussion is not a recent development. In 1952,

the binding of mineral elements to the ACTH molecule was measured polarograph-

i c l y by Carr et al ( I I ) . Zinc and copper complexes were shown to increase

a c t i v i t y , whereas manganese, cobalt and nickel complexes had no e f fec t . Dis-

cussion of ACTH-zinc complexes continued, (2,3,4) with more emphasis being

placed on increased a c t i v i t y , but not on essen t ia l i t y of metals in ACTH

funct ion.

The absence of cort icosterone production in the ACTH-chelate group gives

an indicat ion that a metal is necessary for ACTH ac t i v i t y . Corticosterone

production of the chelate control group showed DDC had l i t t l e e f fec t on the

adrenal cortex, for cort icosterone production was comparable to control val-

ues. The sequential chelation and zinc supplementation in the ACTH-chelate/

zinc mixture demonstrated no inh ib i to ry action on the part of DDC. The

action of DDC was l imited to the chelat ion of the metal zinc ions. The sup-

plementation of zinc to the incubation mixture appeared to have no s i g n i f i -

cant e f fec t on the ACTH-corticosterone re la t ionsh ip . Al l commercial prepara-

tions of ACTH tested, however, had high levels of zinc. Although, copper

does complex with ACTH, the copper levels of ACTH preparations are extremely

low when compared with zinc, for this study Acthar (Armour Pharmaceutical Co.)

had a copper content of 0.00015 ppm/unit ac t i v i t y ,

The binding s i te of zinc in ACTH can be c l a r i f i e d by studying the crystal

structure of metal amino acid complexes. Since only copper and zinc complexes

of ACTH have increased a c t i v i t y , the s i te of action may be an amino acid that

binds both metals in a s imi la r fashion. Only glutamic acid binds copper and

zinc in the same manner, although four amino acids of the ACTH molecule com-

plex with zinc (12). The glutamatozinc ( I I ) and glutamatocopper ( I I ) s t ruc-

tures have corresponding un i t - ce l l dimensions which d i f f e r by only 1% and

show st ructura l s i m i l a r i t y , but not isomorphism (13,14). Metal l igand bonds

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in the Zn(l l)-glutamate complex are markedly more stable than in the Cu( l l ) -

glutamate, suggesting zinc's natural posit ion in ACTH (12). We, therefore,

compared the zinc content of Synacthen, a 81-24 ACTH, with the zinc content

of other commercially available ACTH. Synacthen contains only one glutamate,

while f u l l chain ACTHs contain four glutamates, Table 2 shows the correspond-

ing low zinc levels for Synachten, 0.97 Dpm/mg.

Thin layer chromotography was used to substantiate the binding s i te of

zinc. Chromatographs were made of an acid hydrolyzed ACTH sample using

P H E N Y LALAN I N E -- :;:= -- ~--~ ;= -=~. ~__--_~

TY ROS I NE --- :.-._-:_- =--.-.----~ .,-.-":.~----~ ALAN I N E --. . . . . =~___=~. . . . . . .=~..~_~.

GLUTAMI C ACID ~ ;==- '~ -~ ~ - _ ~ L ~ _ Z I N C

HISTIDINE-. ---- ~--~ ~---'-'~

I < / a b

Rf

lO0

i

- - 50

- -Or ig in

Figure 2. Tracing of chromatographs of amino acids of ACTH. a. stained with ninhydrin for v isual izat ion of amino acids, and b. counterstained with oL{2-[~-(2-Hydroxy-5-sulfophenylazo) benzylidene]-hydrazino}benzoic acid for the v isual izat ion of zinc.

s i l i ca coated mylar plates and a butanol:acetic acid:water solvent. Figure

2a demonstrates the amino acid separation as visualized with ninhydrin.

Figure 2b is the same plate counterstained with o-{2-[~(2-Hydroxy-5-sulfo-

phenylazo)benzylidene]-hydrazino}benzoic acid, which is speci f ic for zinc

ions. The major stain for zinc coincides with the area known to possess the

glutamic acid segment of the ACTH sample. Accordingly, both crystal struc-

ture and thin layer chromatography support the glutamate-zinc complex.

The early work on zinc-ACTH ac t i v i t y (2,3,4,11), present chelation studies,

the crystalographic s im i la r i t i es between copper and zinc glutamates, and the

thin layer chromatographs are strong indications of zinc-ACTH interact ion.

1118


This research was aided in Dart by a grant from the Cleveland Foundation.

Synacthen was supplied by Dr. J.J. Chart of CIBA-GEIGY Pharmaceutical Co.

References

l ,

2.

3. 4. 5.

6 ,

7.

,

9 I0 I I 12 13 14

Flynn, A., Pories, W.J., Strain, W.H. and H i l l , J r . , O.A. Science 173: 1035 (1971). Homan, J.D., Overbeek, G.A., Neuteling, J.P.J. , Booij, C.J. and van der Vies, J. Lancet i : 541 (1954). Greene, R. ~Vaughan-Morgan, J. ib id. p. 545. den Oudsten, S.A., van Leeuwen, L. and Coers, R.J. ib id. p. 547. Sandstead, H.H., Prasad, A.S., Farid, Z., Schulert, A., Miale, A., Basi l ly, S. and Darby, W.J. in Zinc Metabolism, A.S. Prasad, Ed. (C.C. Thomas, Springfield, I I I . , 1966), p. 304. Holtermann, H., Heier, A. and Bergh, H. Lancet i i : 1308, 1952. Luecke, R.W., Ruskan, B.E. and Baltzer, B.V. in Trace Element Metabolism in Animals, C.F. Mi l ls , Ed. (Livingston, London, i970), ~.--471. Mi l ls , C.F. and Murray, G. J. Sci. Food Agr. 9:547 (1960). Saffaran, M. and Schally, A.V. Endbcrinology -56:523 (1955). Silber, R.H., Bush, R.D. and Oslapas, R. Cl~n.-C-hem. 4:278 (1958). Carr, J.E., Conn, J.B. and Wartman, T.G. Science 116:566 (1952). Freeman, H.C. Advances in Protein Chemistr~ 22:257 (1967). Gramaccioli, C.M. Acta Cryst. 21:600 (1966). Gramaccioli, C.M. add Marsh,~R.E__Acta Cryst. 2_]I: 594 (1966).

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corticotropin dependency on zinc ions

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