293

Biochimica et Biophysica Acta, 543 (1978) 293--304

© Elsevier/North-Holland Biomedical Press

BBA 28669 ZINC ACCUMULATION AND METABOLISM IN PRIMARY CU L T U RE S OF A D U L T R AT L I V E R CELLS R E G U L A T I O N BY GLUCOCORTICOIDS

M A R K L. F A I L L A a n d R O B E R T J. C O U S I N S *

Department o f Nutrition, Rutgers, The State University o f New Jersey, New Brunswict¢, N.J. 08903 (U.S.A.) (Received March 13th, 1978)

Summary Adult rat liver parenchymal cells were isolated by the collagenase perfusion technique and cultured as a m o n o l a y e r for up to 20 h. The quant i t y of zinc accumulated from the extracellular envi r onment was significantly increased by adding physiological concentrations of certain glucocorticosteroids to the medium. The degree of stimulation was directly related to glucocorticoid p o t e n c y . Sex steroids, certain peptide h o r m o n e s and prostaglandins E~ and F ~ did n o t influence zinc accumulation. Control cells exhibited a decline of zinc accumulation after 4 h in culture although u p tak e processes were still operative. When dexamethasone, the most p o t e n t glucocorticoid used, was present in the medium the cells accumulated zinc at a linear rate greater than that seen in control cells, for at least 20 h. The dexamethasone-induced stimulation of zinc accumulation was relatively specific since 4SCa, 14C-labelled amino acids and [3SS]cystine accumulation was n o t influenced by the h o r m o n e . A lag of 4 h was observed before an effect of d e x a m e t h a s o n e on zinc accumulation could be detected. Moreover, the hormone-stimulated phase of accumulation was blocked when the cells were simultaneously incubated with either a c t i nom yci n D or cycloheximide. The additional c o m p l e m e n t of zinc accumulated by the dexamethasone-treated cells was localized in the cytosol fraction. Gel filtration and ion-exchange chromatography confirmed that this additional cytosol zinc was bound to metallothionein. [35S]Cystine was incorporated into metallothionein in h o r m o n e - t r e a t e d cells indicating th at the protein was synthesized de novo during periods of enhanced zinc accumulation.

* To whom reprint requests should be addressed.

294 Introduction Mammals maintain their body c o n t e n t of zinc primarily through homeostatic control of intestinal absorption of this essential trace element. Once absorbed zinc distribution among tissues and fluids can be altered by certain dietary changes and stresses such as starvation, chronic liver disease, bacterial infection and neoplasia [1]. While these changes have been attributed to u n k n o w n control mechanisms [2], it seems likely that such fluxes might be under hormonal control, at least in part. Indeed, the pituitary-gonadal axis has been shown to regulate zinc accumulation by and distribution within the male reproductive tract [1,3,4]. Likewise, the serum zinc concent rat i on appears to be altered in response to changes in pituitary and/or adrenal activity [5--7]. Although the liver is the major organ involved in accumulating and storing an available pool o f zinc [1,8], possible hor m onal influences on hepatic zinc metabolism and homeostasis have not been investigated in detail. Recently, we initiated studies of hepatic zinc metabolism using primary cultures of adult rat liver parenchymal cells. It was found that hepat ocyt es concentrated zinc from the extracellular environment by a t e m p e r a t u r e and energy-dependent process that required reactive sulfhydryl groups and was saturated at physiological levels of the metal [9]. It was also observed that the quantity of zinc accumulated was influenced by the presence of certain hormones in the culture medium. In this r epor t we have characterized the stimulatory affect of specific glucocorticosteroids on zinc accumulation by hepatocytes. We have also found that elevation of the intracellular level of this metal results in de novo synthesis of metallothionein, a zinc storage protein. Materials and Methods

Isolation and maintenance o f liver parenchymal cells. Male Sprague-Dawley rats (175--300 g) were fed a standard, natural diet that contained 50 ppm zinc ad libitum and were given free access to tap water. Animals were anesthetized with p en to b ar b ito l (60 mg/kg, intraperitoneally) and surgery was initiated between 1300 and 1500 h. Liver cells were isolated by a modification of the collagenase perfusion technique of Berry and Friend [10] that has been described in detail [9]. The cells (viability 88--95%) were suspended at 1 • 106 cells/ml in M199 supplemented with 15 mM N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (HEPES), 10 mM N-tris ( h y d r o x y m e t h y l ) m e t h y l - 2 amino-ethanesulfonic acid (TES), 5 mM NaHCO3, 0.5 mM pyruvate, streptom y c in sulfate ( 1 0 0 p g / m l ) , penicillin G ( 6 0 g g / m l ) and insulin ( l p g / m l ) (henceforth referred to as M199m), plus 10% fetal calf serum. This medium contained approximately 10 pM zinc. 2-ml aliquots of cell suspension were placed in collagen-coated 60-mm plastic culture dishes. Viable parenchymal cells were purified by selective a t t a c h m e n t to the dish surface, during a 60--75 min preliminary incubation at 37°C [9,11]. After the medium and unattached cells were removed by aspiration, the surface of the dish was washed twice with 5 ml of HEPES-buffered Hanks' salts solution, pH 7.4, and 3.8 ml fresh M199m containing 10% fetal calf serum was added. Radioisotopes and/or h o r m o n e s were added at times indicated in the t e xt a nd/or legends.

295 Zinc accumulation. The accumulation of zinc from the medium by the parenchymal cells was studied according to the following protocols. The cells were either incubated overnight in medium containing 6SZn (50 nCi/dish) or following overnight incubation, the cells were exposed to fresh medium containing 6SZn (100 nCi/dish) for periods up to 3 h. The methods used to remove nonspecifically bound zinc from the cell surface and for the quantitation of accumulated zinc by liquid scintillation counting have been described [9]. Protein concentration was determined by the microbiuret procedure [ 12] using bovine serum albumin as the standard. Distribution of accumulated zinc and amino acids in cell cytosol. Ceils were incubated in medium either with or without dexamethasone and containing 65Zn (100 nCi/dish) for 20 h. The radioactive medium was removed and the cell surface washed twice with 5 ml ice-cold 10 mM HEPES-buffered, Hanks' salts solution, pH 7.4. Control and dexamethasone-treated cells were scraped from the surface of 15 replicate dishes each in a total of 4.5 ml ice-cold 10 mM Trisacetate, pH 8.0. The suspended cells were disrupted by 20 up-down strokes with a motor-driven, glass-Teflon Potter-Elvehjem homogenizer. Cellular particulate matter was removed by centrifugation at 166000 × g for 60 min. The supernatant was applied to a 2.6 × 55 cm column packed with Sephadex G-75 and previously equilibrated with 10 mM Tris-acetate buffer, pH 8.0. The column was standardized with blue dextran, rat liver metallothionein and tryptophan. The fractions (5 ml) were eluted at a rate of 25 ml/h. Similarly, cells were incubated in medium containing either [3H]glycine, [3H]lysine and [3H]serine (total activity, 250 nCi/dish) or 65Zn (100 nCi/dish) and [35S]cystine (250 nCi/dish) to test the affect of dexamethasone on metallothionein synthesis in vitro. The 166000 × g supernatant was prepared and fractionated by gel filtration chromatography as described above. Fractions that eluted in the metallothionein region were pooled and further resolved by ion-exchange chromatography [13]. Fractions were measured simultaneously for 6SZn and 3sS content by differential liquid scintillation spectrometry [14]. Materials. Chemicals and materials were obtained from the following sources: collagenase (CLS, type II), Worthington Biochemical Co.; fetal calf serum and M199, GIBCO; cycloheximide, prostaglandins E2 and F2~, insulin, growth hormone, triiodothyronine, steroid hormones and pentobarbitol, Sigma Chemical Co.; ACTH, U.S. Biochemical Corp.; carrier-free 6SZn and SgFe, 4SCa, L-[U-14C]histidine, L-[35S]cystine, L-[3-3H]serine, [2-3H]glycine, L-4,5[n-3H]lysine and [U-14C]protein hydrolysate, Amersham/Searle Corp. Results

Hormonal stimulation o f zinc accumulation The quantity of zinc accumulated by primary cultures of rat liver parenchymal cells was significantly increased by the addition of certain adrenal glucocorticosteroids to the incubation medium (Table I). Degree of stimulation was directly related to glucocorticoid potency, viz., dexamethasone > prednisolone, prednisone > corticosterone [15]. That the increased ability of hepatocytes to accumulate Zn was specifically enhanced by these hormones was demonstrated by the failure of other adrenal corticosteroids, as well as male and

296 TABLE

I

EFFECT

OF HORMONES

ON ZINC ACCUMULATION

BY LIVER

PARENCHYMAL

CELLS

C e l l s w e r e p r e p a r e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . A f t e r s e l e c t i v e a t t a c h m e n t the cells were washed and incubated in M199m + 10% fetal calf serum containing 6SZn (50 nCi/dish) with the indicated hor-

for 16 h, and the quantity of zinc accumulated w a s m e a s u r e d . I n s u l i n w a s a d d e d t o a ll c u l t u r e s a t a concentration o f 1 p g / m l ( a p p r o x . 1 6 5 n M ) . V a l u e s r e p r e s e n t t h e m e a n ~ S . E . M . o f a t Least 4 r e p l i c a t e plates each from 2 separate experiments. mones

Hormone

added

nmol Zn accumulated/ mg protein

Accumulation alone (as%)

Insulin Cortieosteroids a Aldosterone

1.98 ~ 0.04

100

1.94 ~ 0.05

98

Corticosterone

2.33 t 0.03

118 d

Cortisone Desoxycortisone Dexamethasone

2 . 0 4 _+ 0 . 0 6 1.89 ~ 0.03 4.08 • 0.05

103 95 206 d

Hydrocortisone Prednisolone Prednisone

2.07 + 0.10 3.00 • 0.10 2.72 * 0.08

105 151 d 137 d

1.98,

0.06

100

1.98 ~ 0.04 1.98 ~ 0.02 1.95 * 0.04

100 100 98

Sex steroids a Androsterone Estradiol Mestranol

Methyl-testosterone Progesterone Testosterone

Polypeptide

hormones

1.94 ~ 0.05

98

1.99 + 0.09

101

relative to insulin

b

Adrenocorticotropin

1.92 + 0.08

97

Calcitonin

2 . 0 2 ,: 0 . 0 3

102

Growth

2.06 ÷ 0.04

104

hormone c

Other hormones

Prostaglandin

E2

1.92 : 0.05

97

Prostaglandin

F2a

1 . 8 3 ~_ 0 . 0 8

92

1.97 • 0.03

99

Triiodothyronine a Added

at a concentration

b Added

at a concentration

c Added

at a concentration

d Significantly

different

o f 1 0 - 7 M. o f 1 0 - 8 M. o f 1 0 - 6 M.

at P ~ 0.001.

female sex hormones, peptide hormones and prostaglandins, to stimulate Zn uptake. Similar results were obtained whether the hormones and the radioisotope were simultaneously added to the medium prior to overnight incubation or hormone-treated overnight cultures were pulse-labelled the following day. Elimination of the hormone from the medium did not alter the quantity of zinc accumulated during pulse-labelling studies once cultures had been incubated with the stimulatory glucocorticoids overnight. It has been suggested that leukocytic endogenous mediator, a family of lipoproteinaceous, hormone-like substances directly stimulates hepatic accumulation of plasma zinc and iron during the early stages of acute infections [2,16]. Intraperitoneal administration of 1 ml of a standard preparation of leukocytic endogenous mediator reduced plasma zinc levels by 66% (from 150 to 51 pg/ 100 ml) within 5 h. However, the addition of leukocytic endogenous mediator to the culture medium (5 pl/dish) failed to enhance cell zinc accumulation during 6- and 18-h incubations. Likewise, addition of leukocytic endogenous

297 c

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F i g . 1. I n f l u e n c e o f d e x a m e t b a s o n e concentration on zinc accumulation b y liver p a r e n c h y m a l c e l l s . Culwere incubated in medium containing specific concentrations of dexamethasone. The quantity of zinc accumulated b y t h e c e l l s d u r i n g a 16 h incubation (o o) w a s m e a s u r e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . S i m i l a r l y , a f t e r a 16 h i n c u b a t i o n in t h e p r e s e n c e o f t h e v a r i o u s c o n c e n t r a t i o n s of dexam e t h a s o n e , o t h e r c u l t u r e s w e r e w a s h e d t w i c e w i t h HEPES-buffered Hanks' s a l t s s o l u t i o n a n d i n c u b a t e d f o r 3 h in m e d i u m w i t h o u t d e x a m e t h a s o n e (e o). T h e q u a n t i t y o f z i n c a c c u m u l a t e d b y t h e e n d o f t h i s 3 h p u l s e p e r i o d w a s m e a s u r e d . E a c h v a l u e is t h e m e a n o f 3 p l a t e s f r o m e a c h o f 2 s e p a r a t e e x p e r i m e n t s .

tures

F i g . 2. T h e a c c u m u l a t i o n o f z i n c b y c u l t u r e s o f liver p a r e n c h y m a l c e l l s as a f u n c t i o n o f l e n g t h o f i n c u b a tion. Cultures were incubated without dexamethasone (e --) o r w i t h 10 -7 M d e x a m e t h a s o n e (© o). Z i n c a c c u m u l a t i o n w a s m e a s u r e d at s p e c i f i c t i m e s as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . Each value represents the mean of 3 plates from each of 2 separate experiments. Dx = d e x a m e t h a s o n e .

mediator to culture medium with dexamethasone did n o t further enhance dexamethasone-mediated stimulation of zinc accumulation by parenchymal cells.

Characteristics of dexamethasone stimulation of zinc accumulation The influence of hormone concentration on dexamethasone-stimulated accumulation of zinc during overnight (16 h) and short-term (3 h) incubations in medium with 6SZn is shown in Fig. 1. In both cases, 50% of the maximal response was observed in cultures containing an initial hormone concentration of approximately 5 . 1 0 -9 M, while at concentrations of 3 . 1 0 - S M or greater, maximal stimulation was achieved. Thus, isolated cells respond to concentrations of this synthetic glucocorticoid equivalent to physiological levels [15] of corticosterone. The dexamethasone c o n t e n t of the medium in subsequent studies was 1 • 10 -7 M. Recently isolated parenchymal cells began to accumulate zinc from the medium immediately (Fig. 2). The rate at which control cultures accumulated the metal declined within 4 h and net accumulation ceased by 16 h. Replacement of "stale" medium with fresh medium containing 6SZn at the same specific activity failed to elevate the quantity of radioisotope associated with the cells. Thus, the cessation of zinc accumulation did n o t result from nutrient depletion and/or physiochemical alterations of the culture medium. When dexamethasone was present in the incubation medium, the cells continued to accumulate the metal at a linear rate t h r o u g h o u t the incubation period (Fig. 2). The failure of cells maintained in dexamethasone-free medium to continue to increase their cellular c o n t e n t of zinc might result from cessation of zinc

298

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F i g . 3. T h e a c c u m u l a t i o n o f z i n c w i t h t i m e b y l i v e r p a r e n c h y m a l c e l l s p r e v i o u s l y c u l t u r e d i n m e d i u m w i t h or w i t h o u t 10 -7 M d e x a m e t h a s o n e f o r 16 h. C o n t r o l (e-e) a n d d e x a m e t h a s o n e - t r e a t e d ( c - o) cultures were next incubated for periods up to 3 h in dexametbasone-free medium and the quantity of z i n c a c c u m u l a t e d m e a s u r e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . E a c h v a l u e r e p r e s e n t s t h e m e a n o f 5 r e p l i c a t e dishes f r o m each o f 2 separate e x p e r i m e n t s . Dx = d e x a m e t h a s o n e .

transport processes. To test this possibility overnight cultures were exposed to medium containing 6SZn for periods up to 3 h (Fig. 3). The results demonstreted that control cells did retain the ability to take up zinc at a rate of 4.5 pmol zinc/min per mg cell protein. However, dexamethasone-treated cells accumulated the metal at twice this rate (8.7 pmol zinc/min per mg cell protein). Thus, dexamethasone appears to affect both the uptake rate and the quantity of zinc accumulated by hepatocytes. When control and dexamethasone-treated cells were prelabelled with 6~Zn and incubated in medium without the radioisotope, the same quantity of 6SZn was transferred to the medium by both cultures. This result suggests that the hormone does not affect exitexchange processes. The stimulatory effect of dexamethasone on hepatocyte zinc accumulation

T A B L E II COMPARISON OF EFFECT OF DEXAMETHASONE O N 65 Z n , 4 5 C a ' 1 4 C . L A B E L L E D A M I N O A C I D S , [14C]HISTIDINE AND [35S]CYSTINE ACCUMULATION BY LIVER PARENCHYMAL CELLS C e l l s w e r e p r e p a r e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . A f t e r s e l e c t i v e a t t a c h m e n t t h e c e l l s w e r e w a s h e d a n d i n c u b a t e d i n M 1 9 9 m + 1 0 % f e t a l c a l f s e r u m c o n t a i n i n g i n s u l i n (1 ~ g / m l ) f o r 1 6 h a n d t h e q u a n t i t y o f zinc a c c u m u l a t e d was m e a s u r e d . R a d i o i s o t o p e s were a d d e d ( 5 0 n C i / d i s h ; 4 5 C a at 5 0 0 n C i / d i s h ) at the start of the incubation. Values represent the mean ± S.E.M. of 4 replicate plates each from 2 separate experiments. Radioisotope

65Zn 45Ca 14C.labelled amino acids [35 S ] C y s t i n e [14C]Histidine

% radioactivity accumulated/rag

cell p r o t e i n

Control cultures

Dexamethasone-treate d cultures

Dexamethasone / control

4.09 0.05 2.73 1.57 3.93

7.81 0.06 2.78 1.62 3.91

1.91 1.20 1.02 1.03 1.00

+ + ± ~ +

0.05 0.002 0.04 0.07 0.08

+ + ± ± +

0.08 0.003 0.06 0.01 0.05

299 appears to be highly specific for this metal and is not due to a general increase in the permeability of the plasma membrane (Table II). Incubation of cultures in medium with h o r m o n e failed to enhance the uptake of either calcium or a mixture of ~4C-labelled amino acids. Also, the accumulation of [~4C]histidine and [3SS]cystine, the two amino acids suggested to have a role in tissue zinc accumulation [17,18], was not stimulated by dexamethasone. Finally, ~gFe was not taken up by either control or dexamethasone-treated cells, possibly due to protease contamination of the collagenase preparation which has been shown to adversely affect transferrin receptors on the cell surface [ 19]. Pulse-labelling studies (3 h) were conducted in dexamethasone-free medium with cells previously incubated in the presence of the hormone for varying times. After a 4 h exposure to dexamethasone the cells accumulated 23% more zinc than control cells, while a 2 h exposure was without effect. These data suggest that the stimulation of accumulation by the hormone requires the synthesis and/or assembly of specific components. As shown by the data in Table III both actinomycin D and cycloheximide blocked the dexamethasonemediated stimulation of zinc accumulation. However, neither drug adversely affected the quantity of zinc accumulated during a pulse-labelling study with cells that had been incubated overnight in medium containing dexamethasone. Together these results show that the stimulation of zinc accumulation by dexamethasone represents an induction process. Cytosol distribution o f zinc in control and dexamethasone-treated cells As shown above (Table I, Fig. 1), incubation of liver parenchymal cells in medium containing dexamethasone increased zinc accumulation two-fold. Similarly, the a m o u n t of zinc accumulated in vitro in the cytosol fraction of steroid-treated cells was 2.3-fold greater than that from control cells. Sephadex G-75 chromatography of the cytosol from both groups of cells is shown in Fig. 4. Cytosol from control cells fractionated into 2 major zinc-containing peaks representing high molecular weight, zinc-containing proteins, and a low

MT

Vo 1

i

10 ~+Dx

~ 0 x E ~ c

8 6 4 2 0 10

20

30 40 50 Fraction Number

60

70

F i g . 4. G e l - f i l t r a t i o n c h r o m a t o g r a p h y o f c y t o s o l f r o m 65 Z n - l a b e l l e d c o n t r o l a n d d e x a m e t h a s o n e - t r e a t e d l i v e r p a r e n c h y m a l cells. C u l t u r e s w e r e i n c u b a t e d f o r 2 0 h i n m e d i u m c o n t a i n i n g 6 5 Z n a n d w i t h o u t (¢ . ) o r w i t h (o o) d e x a m e t h a s o n e . Cells f r o m 15 c u l t u r e d i s h e s were p o o l e d , h o m o g e n i z e d a n d the c y t o s o l f r a c t i o n p r e p a r e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e c y t o s o l was a p p l i e d to a c a l i b r a t e d c o l u m n o f S e p h a d e x G - 7 5 a n d e l u t e d w i t h 0 . 0 1 M T r i s • HC1 b u f f e r ( p H 8 . 6 ) , a n d t h e q u a n t i t y o f 65 Z n i n e a c h f r a c t i o n w a s m e a s u r e d . D x = d e x a m e t h a s o n e .

300 10 ?

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200 ,

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k

cz ~

L

2

![

150

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40

60

Fraction

Number

80

F i g . 5. P u r i f i c a t i o n o f z i n c - t h i o n e i n f r o m d e x a m e t h a s o n e - t r e a t e d l i v e r p a r e n c h y m a l cells. C e l l s w e r e i n c u bated for 20 h in medium containing dexamethasone, as w e l l as 6 5 Z n a n d [ 3 5 S ] c y s t i n e . C y t o s o l w a s p r e pared and chromatographed o n S e p h a d e x G - 7 5 as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . F r a c t i o n s t h a t eluted in the same region as standard rat liver metallothionein were pooled and further purified on DEAESephadex A-25. The column (1.5 × 30 cm) was eluted with a linear gradient (total volume 250 ml) of 20--200 mM Tris/acetate, pH 7.4, at a rate of 20 ml/h. Two-ml fractions were collected and assayed for both 65Zn (o o ) a n d 3 5 S (© o). T h e A a n d B f o r m s o f z i n c - t h i o n e i n w e r e e l u t e d a t f r a c t i o n s Nos. 41--51 and 72--89, respectively.

molecular weight zinc-binding fraction that was eluted in an identical fashion to the zinc-binding protein, metallothionein. The elution profile of cytosol zinc from dexamethasone-treated cells was identical, except that most of the additional zinc accumulated was found associated with the lower molecular weight species corresponding to metallothionein. When cells were incubated with or w i t h o u t dexamethasone in medium containing [3SS]cystine and 6SZn, neither the quantity of 3sS accumulated (Table ,ID nor the percentage associated with the cytosol fraction was significantly altered. 3sS from the control cell cytosol eluted from Sephadex G-75 exclusively with fractions corresponding to high molecular weight proteins and to small peptides and amino acids. While the 3sS and 65Zn contents of these two fractions were virtually identical, radioactivity was also incorporated into material corresponding to metallothionein in cytosol from dexamethasonetreated cells. Both the 6SZn- and 3SS-labelled metallothionein-like species isolated from these cells were resolved into the A and B forms of metallothionein when further purified on DEAE-Sephadex (Fig. 5). These data demonstrate that de novo synthesis of the zinc-binding protein metallothionein occurred in cultures of parenchymal cells incubated in the presence of dexamethasone. However, it does not establish whether the hormone (a) induced metallothionein synthesis which subsequently results in enhanced zinc accumulation, or (b) stimulates zinc accumulation which in turn induces metallothionein biosynthesis. The following data suggest that the latter alternative is more likely. First, elevation of the zinc concentration of dexamethasone-free medium to 98 pM increased the quantity of the metal accumulated by hepatocytes 2.5-fold (Table IV). [3H]Glycine, [3H]lysine and [3H]serine, three amino acids that account for approximately 30% of the total residues found in rat liver metallothionein [20], were incorporated into metallothionein-containing fractions as determined by gel filtration chromatography. Thus, metallothionein synthesis in cultures exposed to high levels of the metal

301 T A B L E III EFFECT OF ACTINOMYCIN D AND CYCLOHEXIMIDE ON ACCUMULATION OF ZINC BY LIVER PARENCHYMAL CELLS

DEXAMETHASONE-STIMULATED

C e l l s w e r e p r e p a r e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . A f t e r s e l e c t i v e a t t a c h m e n t t h e c e l l s w e r e w a s h e d a n d i n c u b a t e d i n M 1 9 9 m + 1 0 % f e t a l c a l f s e r u m f o r 4 h. T h i s m e d i u m w a s r e m o v e d a n d r e p l a c e d w i t h f r e s h m e d i u m c o n t a i n i n g 65 Z n ( 1 0 0 n C i / d i s h ) w i t h a n d w i t h o u t d e x a m e t h a s o n e . W h e r e i n d i c a t e d , a c t i n o m y c i n D (1 p g / m l ) o r c y c l o h e x i m i d e ( 1 0 p g / m l ) , b o t h i n 1 0 0 % e t h a n o l , o r a n e q u i v a l e n t v o l u m e o f 1 0 0 % e t h a n o l (in c o n t r o l c u l t u r e s ) was a d d e d . All c u l t u r e s were i n c u b a t e d for an a d d i t i o n a l 6 h a n d the quantity of zinc a c c u m u l a t e d was m e a s u r e d . Values r e p r e s e n t the m e a n ± S.E.M. of 5 replicate plates each from 2 separate experiments. Condition of incubation

nmol Zn accumulated/mg

Control + cycloheximide + actinomycin D

protein

-- dexamethasone

+ dexamethasone

1 . 0 9 ~- 0 . 0 3 1.01 + 0.05 0.96 ± 0.03

1.41 ± 0.03 0 . 9 6 -+ 0 . 0 2 0.99 z 0.02

did n o t require the addition of dexamethasone to the medium. Secondly, only a minimal amount of 3H-labelled metallothionein was detected when cultures were incubated overnight in serum-free medium (1 pM zinc; see ref. 9) containing dexamethasone. By increasing the zinc concentration of medium with dexamethasone to a physiological level, 10 pM, the quantity of metallothionein synthesized significantly increased. Finally, when dexamethasone-treated cells previously incubated in serum-free medium were exposed to 10 gM zinc for 6 h, the newly accumulated metal was found associated with a low molecular weight complex smaller than metallothionein [9]. Consequently, it seems that the intracellular level of zinc must be sufficiently elevated before synthesis of metallothionein occurs.

T A B L E IV EFFECT OF ZINC CONCENTRATION AND M ETA LLOTHIONEIN-SYNTHESIS

OF MEDIUM

ON CELLULAR

ACCUMULATION

OF METAL

C e i l s w e r e i n c u b a t e d i n M 1 9 9 m + 1 0 % f e t a l c a l f s e r u m w i t h i n s u l i n (1 p g / m l ) a n d t h e i n d i c a t e d c o n c e n t r a t i o n o f z i n c f o r 2 0 h. D e x a m e t h a s o n e w a s n o t a d d e d t o t h e m e d i u m . T o d e t e r m i n e t h e q u a n t i t y o f z i n c a c c u m u l a t e d 65 Z n ( 2 0 0 n C i / d i s h ) w a s a d d e d t o t h e m e d i u m . I n v i t r o s y n t h e s i s o f m e t a l l o t h i o n e i n w a s s t u d i e d by i n c u b a t i n g c e l l s i n m e d i u m c o n t a i n i n g [ 3 H ] g l y c i n e , [ 3 H ] l y s i n e a n d [ 3 H ] s e r i n e ( t o t a l a c t i v i t y , 2 5 0 n C i / d i s h ) . C y t o s o l p r e p a r a t i o n and f r a c t i o n a t i o n on S e p h a d e x G - 7 5 was i d e n t i c a l to t h a t d e s c r i b e d in the legend to Figure 4 and Materials and Methods. Each value for zinc accumulation represents the mean zS.E.M, of 3 dishes from each of 2 separate experiments. Zn concentration (pM)

nmol Zn accumulated/ mg protein

% c y t o s o l 3H in metallothionein fractions

10 22 44 98

2.25 3.01 3.99 5.56

0.67 ND * ND * 8.35

• ND, not determined.

~ ! ~ z

0.04 0.04 0.06 0.17

302 Discussion Primary cultures of non-dividing h e p a t o c y t e s are a useful model system for studying a wide variety of metabolic processes in liver [21,22] including those regulated by h o r m o n e s [23--29]. Our results dem onst rat e that the ability of liver cells in primary culture to accumulate zinc from the extracellular environm e n t can be increased by the addition of physiological levels of certain adrenal glucocorticosteroids to the culture medium (Table I). Stimulation of accumulation was directly correlated with glucocorticoid pot ency. A m a x i m u m increase of 106% was observed in the presence of dexamethasone, while corticosterone, the principal glucocorticoid in the rat, increased zinc uptake by 18%. Serum corticosterone concentrations exhibit circadian r h y t h m i c i t y and are sensitive to stress. Non-stressed rats had corticosterone levels of 1--5 pg/100 ml at 1000 h, as determined by competitive protein-binding assay [30], while the serum corticosterone of rats used in these experiments averaged 70 pg/100 ml at time of killing. Consequently, the qua nt i t y of zinc accumulated by cultures may have been influenced by residual levels of endogenous corticosterone, as well as glucocorticoids present in fetal calf serum. Together these factors may have decreased the maximal response of cultures to h o r m o n e s added to the incubation medium. Dexamethasone-treated liver cells accumulated extracellular zinc t h r o u g h o u t a 20 h incubation, while in control cells net accumulation declined after 4 h and ceased by 16 h, even though influx and efflux processes were maintained (Figs. 2 and 3). It is clear that the d e x a m e t h a s o n e effect required a lag of about 4 h before differential accumulation was observed. Therefore, it is likely, since both actinomycin D and cycloheximide blocked the dexamethasone effect on zinc accumulation, that this induction process is similar to other glucocorticoid-mediated processes that require selective transcription [31,32[. Our results are very similar to those reported by Cox on the effecLs of h y d r o c o r t i s o n e and prednisone on HeLa cell zinc uptake [33,34]. Moreover, the dexamethasoneinduced stimulation of zinc: accunmlation appears to be specific for the metal, since neither Ca 2+ nor amino acid uptake was affected (Table II). F u r t h e r m o r e , these data offer direct evidence in support of earlier suggestions that the adrenals have a critical role in regulating zinc distribution in intact animals [5,61. Gel filtration of the cytosol from dexamethasone-treated cells labelled with either 6SZn (Fig. 4) or 6SZn and [3SS]cystine support the concept that these cells synthesized metallothionein in response to dexamethasone. Furt her purification of the labelled metallothionein preparations by ion-exchange chrom a t o gr ap h y (DEAE-Sephadex, A-25) resolved the protein into its characteristic A and B forms [ 13,20]. Both forms c oc hr om a t ographed with standard rat liver metallothionein. Since 3~S-labelled metallothionein was not detected in the control cells, it is likely that this protein was induced de novo, either directly or indirectly, by the glucocorticoid treatment. In vivo metallothionein synthesis has been shown to be regulated in rat liver by a mechanism that increases the a m o u n t of poly(A)-containing metallothionein-mRNA that is associated with liver polysomes during initial phases of elevated zinc status [35,36]. Hepatic levels o f metallothionein have also been shown to increase significantly

303

following ingestion of diet containing normal quantities of zinc [37] or during fasting [20,37]. Thus, metallothionein formation is directly correlated with hepatic zinc accumulation under physiological conditions. Similarly, in our studies metallothionein synthesis was induced in response to dexamethasone when phsyiological levels of zinc were present in the culture medium. The agent that is directly responsible for initiating events leading to the induction of metallothionein has not been defined. However, it is important to note that metallothionein-like proteins have been identified in established cell lines derived from human skin epithelium [38], pig kidney [39] and pig liver [40] when the cells were exposed to Cd 2+ for long periods. More recently, de novo synthesis of metallothioneins A and B has been found in primary cultures of rat hepatocytes [41,47] and in a clonal line of rat hepatoma cells [42] that were exposed to cadmium. These data suggest that zinc and cadmium are able to influence the biosynthesis of metallothionein directly. In addition, the data presented herein with isolated liver cells suggest that the zinc content of cells must be sufficiently elevated before metallothionein will be synthesized. Therefore, it is likely that glucocorticoids have a role in regulating the synthesis of this intracellular zinc storage protein in animals by affecting the cellular level of the metal. In conclusion, our data clearly demonstrate the certain adrenal corticosteroids may have an essential role in the regulation of hepatic zinc metabolism. It has been suggested that the regulation of mammalian metabolism may well depend upon the mediating influence of hormones on selective accumulation, losses and/or intracellular redistribution of substrates and cofactors, including metal ions [43]. Considering the well d o c u m e n t e d requirement for zinc as a c o m p o n e n t of numerous zinc-metalloenzymes [44], as a regulator of the activity of some enzymes [44--46] and for the structural integrity and function of cell organelles [46], it is possible that this metal has a role in glucocorticoidmediated alterations of hepatic metabolic processes. Acknowledgements The authors wish to thank Melody J. Mascenik and Kenneth T. Smith for technical assistance and Nancy Shoenfeld and Dr. James H. Leathem for determining serum corticosterone concentrations. This work was supported by NIH Research Grants AM 18555 and AM 20485 from the National Institute of Arthritis, Metabolism and Digestive Diseases, and a Biomedical Sciences Support Grant from Rutgers University. This is a paper of the Journal Series, New Jersey Agricultural Experiment Station. The following materials were received as generous gifts: leukocytic endogenous mediator from Dr. Philip Sobicinsky, calcitonin from Dr. Irwin Clark and actinomycin D from Merck, Sharpe and Dohme. References 1 U n d e r w o o d , E. ( 1 9 7 7 ) in T r a c e E l e m e n t s in H u m a n a n d A n i m a l N u t r i t i o n , 4 t h e d n . , pp. 1 9 6 - - 2 4 2 , A c a d e m i c Press, N e w Y o r k 2 Beisel, W.R., P e k a r e k , R.S. a n d W a n n e m a c h e r , R.W. ( 1 9 7 6 ) in T r a c e E l e m e n t s in H u m a n H e a l t h a n d Disease (Prasad, S.S., e d . ) , Vol. I, p p . 8 7 - - 1 0 6 , A c a d e m i c Press, N . Y .

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