504
Biochimica et Biophysica Acta, 541 (1978) 504--514 © Elsevier/North-Holland Biomedical Press
BBA 28585
ADRENOCORTICOTROPIN-INDUCED UNRESPONSIVENESS IN CULTURED ADRENAL TUMOR CELLS
ERIC L I F R A K and RODNEY M. WISHNOW
Veterans Administration Hospital, Long Beach, Calif, 90822, and the Departments of Medicine and Microbiology of the University of California College of Medicine, Irvine, Calif 92664 (U.S.A.) (Received November 21st, 1977)
Summary Our results demonstrate that adrenocorticotropin (ACTH)-induced refractoriness occurs in cultured adrenal t u m o r cells. Cells became 85% refractory to ACTH-induced cyclic AMP formation in 20 min and the effect persisted if the hormone remained in the incubation medium. Refractory cells gradually regained hormone-specific responsiveness within 24 h if cultures were incubated in fresh media containing serum. The observed effect is hormone specific since cyclic AMP could not induce unresponsiveness to ACTH. The addition of ACTH plus inhibitors of protein synthesis partially reversed hormone-specific refractoriness. However, preincubation with cycloheximide or diphtheria toxin led to superinduction of ACTH-induced cyclic AMP formation. These experiments suggest that unresponsiveness, following hormonal activation of adrenal cells~ may be related to a deCrease in hormone-specific binding sites or to synthesis of an adenylate cyclase inhibitor.
Introduction Adrenocorticotropin (ACTH) interacts with a specific receptor on the cell membrane of cultured mouse Y-1 adrenal t u m o r cells to stimulate adenosine 3':5'-monophosphate (cyclic AMP), steroidogenesis, and a change in morphology from flattened to spherical shaped cells [1--3]. The addition of ACTH to adrenal cells leads to a rapid accumulation of intracellular cyclic AMP which reaches a maximum in about 15 min and then begins to decline toward the basal level despite the continued presence of the hormone [4,5]. This phenomenon has been observed in a variety of cell types including liver, thyroid,
Abbreviation: ACTH, adrenocorticotropin.
505 n e u r o b l a s t o m a l y m p h o m a and fibroblasts and has been called hormoneinduced unresponsiveness, desensitization, refractoriness or tachyphylaxis [6--13]. Refractoriness may effectively modulate cyclic AMP activity during periods of prolonged exposure to hormones and may thus limit energetically wasteful cyclic AMP generation. There have been several suggested mechanisms which might contribute to refractoriness in a number of cells including (a) induction of phosphodiesterase [14--16] ; (b) secretion of cyclic AMP [17] ; (c) synthesis of an antagonist to adenylate cyclase activity [18--19] and (d) reduction in the number or affinity of hormone receptors [20--23]. In this study we have demonstrated that ACTH rapidly induces an unresponsive state in cultured adrenal cells. The effect is reversible, hormone specific, and is unrelated to induction of phosphodiesterase or secretion of cyclic AMP. Inhibitors of protein or RNA synthesis do not appear to reverse ACTH-induced refractoriness. Materials and Methods
Cell culture procedures. Y-1 adrenal t u m o r cells were maintained in monolayer culture in Eagle's minimum essential medium with Earle's salts, containing 12.5% horse serum, 2.5% fetal calf serum, 1 mM L-glutamine, 10 units penicillin/ml and 10 pg streptomycin/ml. The cells were grown in 60 X 15 mm Petri dishes (Falcon plastics) at 37°C in a humidified atmosphere of 5% CO2 in air. Subculturing procedures and the fluorometric steroid assay have been described [24]. The a m o u n t of cyclic AMP in the cells and in the medium was measured after extraction with ethanol by the Gilman competitive binding assay [4,25,26]. Protein was determined by the m e t h o d of Lowry et al. [27]. Incubation procedures. Duplicate confluent cultures were washed twice with warm (37°C) Eagle's minimal essential medium. Then, warm minimal essential medium or minimal essential medium with 13% fetal calf serum containing ACTH (10 munits/ml) was added for various time intervals. The media were removed for extracellular cyclic AMP determination and 3.0 ml ethanol was added to the cells before determining intracellular cyclic AMP. In the time course of ACTH unresponsiveness, ACTH was added for 5, 10, 20 min and 2 h and 7 h intervals, then removed. Then the cells were washed twice and 2.0 ml minimal essential medium containing ACTH or without hormone was added for an additional 20 min incubation. Both intracellular and extracellular cyclic AMP was determined on duplicate plates. In each experiment, basal cyclic AMP and maximum ACTH-stimulated cyclic AMP were determined after a 20 min incubation in cultures not previously exposed to ACTH. Recovery from ACTH unresponsiveness. Confluent cultures were incubated in 2.0 ml of minimal essential medium containing ACTH (10 munits/ml) for 1 h. The medium was then removed and the cells were washed twice with 2.0 ml warm minimal essential medium. Next, 2.0 ml of fresh warm minimal essential medium was added to the cells, which were incubated for various times for recovery. The cells were again washed twice with minimal essential: medium and 2.0 ml of the medium containing ACTH (10 munits/ml) or the medium without hormone was added at each time interval for an additional 20 min incubation. Cyclic AMP was determined in the cells and in the medium
506 at the end of 20 min for each plate. Control cultures for ACTH (10 munits/ml) added for 20 min and with minimal essential medium alone were also run. A parallel group of cultures were incubated with growth medium (minimal essential medium supplemented with 12.5% horse serum, 2.5% fetal calf serum and 1.0 mM L-glutamine) during the recovery period, and cyclic AMP determined. Effect of pre-incubation with cyclic AMP. Confluent cultures were incubated in 2.0 ml of minimal essential medium containing 2.5 mM cyclic AMP. At 2 h the medium was removed, the cells were washed 10 times with 2.0 ml warm minimal essential medium and then fresh medium with ACTH (10 munits/ml), or medium without hormone was added for 20 min incubation and total cyclic AMP measured in quadruplicate cultures. Effect of inhibitors on ACTH-induced cyclic AMP. ACTH (10 munits/ml) or ACTH plus cycloheximide (28 pg/ml) was added to confluent adrenal cells for 30 min to 7 h and cyclic AMP determined intracellularly and in the media in duplicate cultures. Controls with cycloheximide alone or Eagle's minimal essential medium were also assayed. In the preincubation experiment, cycloheximide (28 t~g/ml) was added for 15 min to 6 h and then ACTH was added for 20 min incubation before measuring cyclic AMP. Cultures incubated with cycloheximide alone for as long as 6 h were also assayed. Control cultures incubated with ACTH or minimal essential medium alone were also assayed after a 20 min incubation. The inhibitors cycloheximide (28 pg/ml), and puromycin (200 pg/ml) in 2.0 ml of warm minimal essential medium containing ACTH (10 munits/ml) were incubated with duplicate confluent adrenal cultures for 2 h. Diphtheria toxin (200 pg/ml) was added in 2.0 ml of warm minimal essential medium to cultures 2 h prior to the addition of ACTH (10 munits/ml) for an additional 2 h. Then the medium was removed and the cells washed twice and fresh media containing the same reagents or without ACTH were added for an additional 20 rain incubation before measuring cyclic AMP. Control cultures incubated with ACTH or minimum essential medium for 20 min were also assayed. In the pretreatment experiment cycloheximide or puromycin was added for 2 h and diphtheria toxin 4 h. Then ACTH was added to the cultures for 20 min and total cyclic AMP determined. Control cultures were also run with inhibitor alone. Actnomycin D (5 pg/ml) or cordycepin (20 pg/ml) plus ACTH (10 munits/ ml) was incubated with Y-1 cells for 3--8 h. The medium was then removed, the cells washed twice and fresh medium with ACTH, or minimal essential medium with the same inhibitor was added for 20 min before measuring cyclic AMP. Control cultures incubated with ACTH or minimal essential medium alone were assayed after a 20 min incubation. Cultures were also preincubated with actinomycin D or cordycepin for 1--6 h before addition of ACTH for 20 rain. Control cultures were also run with inhibitor alone. Reagents. The following reagents were used in tissue culture incubations: porcine ACTH (Armour, Chicago, Ill.), purified diphtheria toxin (gift of Dr. J. Collier of UCLA), cyclic AMP, sodium salt, actinomycin D, and cordycepin (3'-deoxyadenosine) (Sigma, St. Louis, Mo.), puromycin dihydrochloride (United States Biochemical Corporation, Cleveland, Ohio), and cycloheximide (Upjohn, Kalamazoo, Mich.). Tritiated cyclic AMP (37.7 Ci/mmol; New Eng-
507 land Nuclear, Boston, Mass.) and cyclic AMP binding protein (Calbiochem, LaJolla, Calif.) were used in determination of cyclic AMP. Results Effect o f A C T H on intracellular and extracellular cyclic AMP accumulation and steroidogenesis. Fig. 1 shows that the addition of ACTH to confluent adrenal monolayer cultures leads to a rapid increase in b o t h intracellular and extracellular cyclic AMP. The intracellular cyclic AMP begins to decline toward the basal level after 30 min b u t the extracellular cyclic AMP concentration reaches a maximum 4 h after exposure to ACTH (10 munits/ml). At 30 min, 70% of the total cyclic AMP, is outside the adrenal cells and at 4 h more than 95% is. This figure also demonstates that steroid release into the tissue culture medium continues linearly despite the early peak in intracellular cyclic AMP concentration. In this system, since most of the cyclic AMP was rapidly released into the medium, both intracellular and extracellular cyclic AMP were measured in subsequent experiments and results were expressed as total cyclic AMP. Fig. 2 demonstrates the effect of minimal essential medium supplemented
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Fig. 1. T i m e c o u r s e o f i n t r a e e l l u l a r a n d e x t r a c e U u l a r cyclic AMP f o r m a t i o n a n d s t e r o i d o g e n e s i s . A d r e n a l cells w e r e i n c u b a t e d at 3 7 ° C in Eagle's m i n i m a l essential m e d i u m c o n t a i n i n g A C T H ( 1 0 m u n i t s / m l ) f o r v a r i o u s t i m e intervals. T h e cyclic AMP values are e x p r e s s e d as p m o l cyclic A M P / r a g cellular p r o t e i n a n d t h e s t e r o i d values as p g / m g cellular p r o t e i n . E a c h p o i n t r e p r e s e n t s t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s o n t w o s e p a r a t e cultures.
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F i g . 2. E f f e c t o f s e r u m o n A C T H - i n d u c e d intracellular cyclic AMP concentration. A d r e n a l cells w e r e incubated at 37°C in minimal;-essential medium (MEM) in the presence and absence of 13% fetal calf serum (FCS), containing ACTH (10 munits/ml) for various time intervals before intracellular cyclic AMP w a s m e a s u r e d . R e s u l t s a r e e x p r e s s e d as i n t r a e e l l u l a r c y c l i c A M P / r a g c e l l u l a r p r o t e i n . E a c h p o i n t r e p r e s e n t s the mean of duplicate determinations on two separate cultures.
with 13% fetal calf serum on the intracellular cyclic AMP response to ACTH stimulation. In the presence of serum the peak or maximum intracellular cyclic AMP concentration was approximately 50% of the cyclic AMP level in ACTHtreated adrenal tumor cells incubated without fetal calf serum. Despite the difference in cyclic AMP response shown in this figure, serum had no detectable effect on steroidogenesis. Since sera are k n o w n to contain phosphodiesterese and other factors which effect cyclic nucleotide metabolism [6], the following incubations were carried out in the absence of serum unless specifically stated to the contrary. Time course o f A CTH unresponsiveness. Confluent cultures of adrenal tumor cells were incubated with a maximal stimulating dose of ACTH (10 munits/ml)
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F i g . 3. T i m e c o u r s e o f A C T H - i n d u c e d u n r e s p o n s i v e n e s s . A d r e n a l cells w e r e i n c u b a t e d a t 3 7 ° C in m i n i m a l essential medium containing ACTH (10 munits/ml) for various time intervals, washed twice, then incub a t e d i n f r e s h m e d i u m w i t h A C T H ( 1 0 m u n i t s / m l ) (Q o ) o r in f r e s h m e d i u m a l o n e (~ . . . . . . ~) f o r 2 0 r a i n b e f o r e m e a s u r i n g i n t r a c e l l u l a r a n d e x t r a c e l l u l a r c y c l i c A M P . R e s u l t s a r e e x p r e s s e d as % t o t a l cyclic AMP response to an initial 20 rain incubation with ACTH. Each point represents the mean of two separate experiments with each point determined on duplicate cultures.
509 for 5 min to 7 h. Following the initial incubation, the cells were washed twice, then incubated with ACTH in fresh medium for an additional 20 min and total cyclic AMP measured. Fig. 3 shows a time-dependent decrease in the total cyclic AMP response of adrenal cells to a second exposure to ACTH. Cells initially incubated with ACTH for 5 min were 70% responsive, but cells preincubated with ACTH for 20 min were 15% as responsive as control cultures which had not previously been exposed to ACTH. Cells incubated for up to 7 h with ACTH in serum-free medium remained maximally refractory to a second exposure to ACTH even if ACTH was increased to 100 munits/ml. In addition, if the adrenal cells were washed 10 times before restimulation there was no reversal of ACTH-induced unresponsiveness. This experiment also demonstrated that washing cells twice with warm media effectively removed ACTH since cyclic AMP levels after washing fell to basal levels. Recovery from ACTH unresponsiveness. The time course of recovery of ACTH responsiveness after a 1 h incubation of adrenal cells with ACTH was compared in serum-supplemented and unsupplemented minimal essential medium (Table I). If cells were treated with ACTH for 1 h, washed twice, and incubated in minimal essential medium for 23 h prior to restimulation with ACTH, they were 60% as resPonsive as control cultures. In contrast, if cells were incubated in serum-supplemented minimal essential medium, then ACTH responsiveness returned more rapidly and completely. At the end of 8 h, the cyclic AMP response in cells allowed to recover in serum-supplemented medium was 60% of control cultures and at 24 h the cells regained their normal ACTH response. Specificity of hormonal unresponsiveness. Since cyclic AMP mediates the ACTH response it seemed possible that it could also induce ACTH unresponsiveness. In order to test this hypothesis, cells were incubated with 2.5 mM cyclic AMP for 2 h, washed 10 times, and ACTH was added for 20 min before measuring total cyclic AMP. The cyclic AMP concentration in duplicate cultures, following preincubation with cyclic AMP, was 520 pmol/mg protein compared to 505 pmol/mg protein in control cultures incubated with ACTH for 20 min. This experiment demonstrated that incubation with cyclic AMP does not render cells refractory to ACTH stimulation. A control showed t h a t
TABLE I RECOVERY
FROM ACTH-INDUCED UNRESPONSIVENESS
A d r e n a l cells w e r e i n c u b a t e d w i t h A C T H ( 1 0 m u n i t s / m l ) f o r 1 h, w a s h e d t w i c e , t h e n i n c u b a t e d w i t h s e r u m - s u p p l e m e n t e d or u n s u p p l e m e n t e d m i n i m a l essential m e d i u m f o r 1 - - 2 3 h. A t various t i m e s d u p l i c a t e c u l t u r e s w e r e w a s h e d t w i c e , i n c u b a t e d w i t h A C T H ( 1 0 m u n i t s / m l ) or m i n i m a l e s s e n t i a l m e d i u m a l o n e for 20 r a i n and t o t a l c y c l i c A M P w a s d e t e r m i n e d . R e s u l t s are e x p r e s s e d as a % t o t a l c y c l i c A M P c o m p a r e d to a n i n i t i a l 20 m i n s t i m u l a t i o n w i t h A C T H . Time (h)
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Fig. 4. E f f e c t o f c y c l o h e x i m i d e ( C H ) on A C T H - i n d u c e d t o t a l cyclic AMP. A d r e n a l cells were i n c u b a t e d w i t h m e d i u m c o n t a i n i n g A C T H (10 m u n i t s / m l ) or A C T H plus c y c l o h e x i m i d e (28 # g / m l ) for 30 rain to 7 h b e f o r e i n t r a c e l l u l a r a n d e x t r a c e l l u l a r cyclic AMP was assayed. E a c h p o i n t r e p r e s e n t s t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s on t w o s e p a r a t e cultures.
the washing procedure was effective in reducing the total cyclic AMP almost to the basal concentration. Previously, we have demonstrated that preincubation of adrenal cells with ACTH did not reduce cholera toxin-induced cyclic AMP formation [28]. Effect o f cycloheximide on time course o f A C T H stimulation o f adrenal cells. Fig. 4 compares the effect of the addition of ACTH and ACTH plus cycloheximide on cyclic AMP formation. If adrenal cells are incubated with ACTH plus cycloheximide there is a linear increase in total cyclic AMP for 6 h, while in cells incubated with ACTH the total cyclic AMP concentration decreases after 3 h. This dose of cycloheximide was sufficient to completely inhibit ACTH-induced steroidogenesis and did not alter basal cyclic AMP concentration. In order to test whether or not new protein synthesis was required for ACTH-induced refractoriness of Y-1 cells, the effect of various inhibitors of protein synthesis was evaluated. If adrenal cells are incubated with ACTH plus cycloheximide, ACTH plus puromycin, or ACTH plus diphtheria toxin, there appears to be a partial inhibition of ACTH-induced refractoriness (Fig. 5A). However, Fig. 5B demonstrates that incubation of adrenal cells with cycloheximide for 2 h or diphtheria toxin for 4 h prior to the addition of ACTH led to super-induction of total cyclic AMP formation. This figure also shows that puromycin has a slight inhibitory effect on cyclic AMP formation. Fig. 6 shows the effect of incubating adrenal cells with cycloheximide for 0.25--6 h before addition of a maximal stimulating dose of ACTH for a 20 min incubation. Preincubation with cycloheximide leads to a time-dependent increase in ACTHinduced total cyclic AMP concentration. If Y-1 cells were incubated with ACTH plus actinomycin D or ACTH plus cordycepin for as long as 8 h, washed, and fresh medium with the same reagents added for a 20 min incubation, the total cyclic AMP concentration was the same as cells treated with ACTH alone. These results demonstrate that inhibition of RNA synthesis does not reverse ACTH-induced refractoriness in
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Fig. 5. A. E f f e c t o f p r o t e i n s y n t h e s i s i n h i b i t o r s o n A C T H - i n d u c e d t o t a l c y c l i c A M P f o r m a t i o n . A d r e n a l cells w e r e i n c u b a t e d w i t h c y c l o h e x i m i d e ( C H , 28 # g / m l ) or p u r o m y c i n (PM 2 0 0 p g / m l ) p l u s A C T H ( 1 0 m u n i t s ] m l ) f o r 2 h. D i p h t h e r i a t o x i n ( D T , 2 0 0 p g / m l ) w a s p r e i n c u b a t e d with" a d r e n a l cells f o r 2 h p r i o r to t h e a d d i t i o n o f A C T H ( 1 0 m u n i t s / m l ) f o r an a d d i t i o n a l 2 h. A f t e r t h e initial i n c u b a t i o n , t h e cells w e r e w a s h e d t w i c e , a n d f r e s h m e d i u m c o n t a i n i n g t h e s a m e i n h i b i t o r w i t h o r w i t h o u t A C T H w a s a d d e d f o r 20 r a i n b e f o r e t o t a l c y c l i c A M P w a s m e a s u r e d . R e s u l t s are e x p r e s s e d as a % t o t a l c y c l i c A M P c o m p a r e d t o an initial 20 r a i n s t i m u l a t i o n w i t h A C T H . E a c h b a r r e p r e s e n t s t h e m e a n o f t w o d i f f e r e n t e x p e r i m e n t s w i t h d u p l i c a t e c u l t u r e s . B. E f f e c t o f p r e i n c u b a t i o n w i t h p r o t e i n s y n t h e s i s i n h i b i t o r s o n A C T H - i n d u c e d c y c l i c A M P . A d r e n a l cells w e r e i n c u b a t e d w i t h m i n i m a l e s s e n t i a l m e d i u m c o n t a i n i n g c y c l o h e x i m i d e ( 2 8 p g / m l ) , p u r o m y c i n ( 2 0 0 ttg/ml) f o r 2 h, or d i p h t h e r i a t o x i n ( 2 0 0 p g / m l ) f o r 4 h, t h e n A C T H w a s a d d e d f o r 20 r a i n b e f o r e m e a s u r i n g i n t r a c e l l u l a r a n d e x t r a c e l l u l a r c y c l i c A M P . C o n t r o l c u l t u r e s w e r e also r u n w i t h inhibitor alone. 260o.. 220•~ 1 8 0 >.¢_) j 140I.F° t 0 0 o
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Fig. 6. T i m e c o u r s e o f p r e i n c u b a t i o n w i t h c y c l o h e x i m i d e ( C H ) o r A C T H - i n d u c e d c y c l i c AMP. A d r e n a l cells w e r e i n c u b a t e d w i t h c y c l o h e x i m i d e ( 2 8 # g / m l ) f o r 1 5 m i n to 6 h, t h e n A C T H ( 1 0 m u n i t s / m l ) w a s a d d e d f o r 20 m i n a n d t o t a l c y c l i c AMP d e t e r m i n e d (o o). C o n t r o l c u l t u r e s w i t h ' c y c l o h e x i m i d e ( 2 8 p g / m l ) a l o n e (~ . . . . . . o) w e r e also a s s a y e d . R e s u l t s are e x p r e s s e d as % t o t a l c y c l i c A M P c o m p a r e d to an i n i t i a l 20 r a i n s t i m u l a t i o n w i t h A C T H . E a c h p o i n t r e p r e s e n t s t h e m e a n o f t w o s e p a r a t e e x p e r i m e n t s with duplicate cultures.
512
adrenal cells. In contrast to the cycloheximide effect, preincubation with actinomycin D or cordycepin for as long as 6 h did not alter ACTH-induced cyclic AMP production. Discussion Our results demonstrate that maximal hormone-specific refractoriness occurs 20 min after exposure of adrenal cells to ACTH. At that time the cells became 85% refractory, intracellular cyclic AMP levels were near maximum, and cells had changed from a flattened to a spherical shape. Hormonal unresponsiveness was not due to loss of ACTH activity since media from unresponsive cells could maximally stimulate cells that had not been exposed to ACTH. Neither extensive washing of adrenal cells nor the addition of fresh ACTH (100 munits/ml} reversed the loss of ACTH unresponsiveness. Ho and Sutherland [19] found that washing could remove a soluble antagonist of adenylate cyclase released into the media by fat cells. If induction of phosphodiesterase were responsible for hormone-induced refractoriness, then incubation with ACTH should render cells refractory to cholera toxin. However, previously we have demonstrated that ACTH refractory cells can be maximally stimulated by cholera toxin [28]. Recently, a similar effect of cholera toxin has been shown in isoproterenol-stimulated lymp h o m a cells [12]. In addition, incubation with cyclic AMP itself does not lead to ACTH unresponsiveness. In neuroblastoma cells that lack phosphodiesterase, hormone-specific unresponsiveness can also be demonstrated [29]. Although induction of phosphodiesterase has been implicated as a cause of refractoriness in several cell systems, it does not appear to be responsible for refractoriness in adrenal cell cultures [14,16]. The time course and specificity of hormone-induced refractoriness suggest that ACTH must interact with its membrane receptor and stimulate adenylate cyclase before refractoriness can be detected. 5 min after exposure of adrenal cells to ACTH, there is a detectable increase in cyclic AMP and cells are 30% less responsive than control cultures to restimulation by ACTH. Recent evidence with isolated cell membranes suggests that the loss of receptors is responsible for hormone-induced refractoriness. Mukherjee et al. [20] have shown that in purified frog erythrocyte plasma membranes, isoproterenol decreases the number of functional ~-adrenergic receptors and 5'-guanylimidodiphosphate and other purine nucleotides reverse this effect. The OS-3 adrenal t u m o r mutant appears to bind ACTH, but cannot be stimulated by the hormone [2]. However, this m u t a n t can be stimulated by cholera toxin and cyclic AMP [28]. Specific binding studies with this strain may confirm whether or not receptor occupancy plus adenylate cyclase activation are necessary for ACTH-induced refractoriness. In serum-supplemented medium the maximal intracellular cyclic AMP response to ACTH was reduced by approximately 50%. A similar effect of serum has been shown in fibroblasts and thyroid cells [6,8]. However, since refractoriness occurred in the absence of serum in our system, a serum factor could not be implicated in the observed effect. On the other hand, the more rapid recovery of ACTH responsiveness in cells incubated in serum-supple-
513 mented media compared to serum-free media suggests that new protein synthesis is necessary for recovery from ACTH-induced refractoriness. The partial reversal of refractoriness induced when protein synthesis inhibitors were added simultaneously with ACTH may be independent of the presence of ACTH, since if cells were incubated with cycloheximide for 1--6 h before exposure to ACTH there was a time-dependent increase in cyclic AMP. Super-induction of ACTH-stimulated cyclic AMP accumulation may be due to inhibition of synthesis of an inhibitor of adenylate cyclase or decreased synthesis of phosphodiesterase. In fibroblasts, a 2 h pretreatment with cycloheximide led to a 60% greater cyclic AMP accumulation with prostaglandin E~ [30]. In thyroid cells, a supramaximal cyclic AMP response to thyrotropin plus cycloheximide has also been demonstrated [8]. In glial tumor cells, both inhibitors of R N A synthesis and protein synthesis appear to partially reverse catecholamine-induced refractoriness [29]. In our system, actinomycin D and cordycepin had no effect on ACTHinduced unresponsiveness or super-induction suggesting that the template for the synthesis of the cycloheximide-sensitive inhibitory protein may have a long half-life. In adrenal tumor cells, hormone-induced unresponsiveness may be important for cell proliferation since cells become resistant to ACTH-induced growth inhibition in the refractory state [31]. Acknowledgments This research was supported by NIH grant no. CA 16868, and by the Medical Research Programs, Long Beach Veterans Administration Hospital. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
B u o n a s s i s i , V., S a t o , G. and C o h e n , A.I. ( 1 9 6 2 ) Proc. Natl. A c a d . Sei. U.S. 4 8 , 1 1 8 4 - - 1 1 9 0 S c h i m m e r , B.P. ( 1 9 7 2 ) J. Biol. C h e m , 2 4 7 , 3 1 3 4 - - 3 1 3 8 K w a n , C.N. and W i s h n o w , R.M. ( 1 9 7 4 ) In V i t r o 1 0 , 2 6 8 - - 2 7 3 K w a n , C.N. and W i s h n o w , R.M. ( 1 9 7 4 ) E x p . Cell Res. 8 7 , 4 3 6 - - 4 3 8 K o w a l , J. and Srinivasan, S. ( 1 9 7 5 ) E n d o c r i n o l . Res. C o m m u n . 2, 6 5 - - 8 6 C h l a p o w s k i , F . J . , K e l l y , L . A . and B u t c h e r , R,W. ( 1 9 7 5 ) in A d v a n c e s in Cyclic N u c l e o t i d e R e s e a r c h , ( G r e e n g a r d , P. and R o h i s o n , G . A . , eds.), Vol. 6, p p . 2 4 5 - - 3 3 8 , R a v e n Press, N e w Y o r k De R u b e r t i s , F . R . and Craven, P. ( 1 9 7 6 ) J. Clin. Invest. 57, 4 3 5 - - 4 4 3 R a p o p o r t , B. and A d a m s , R . J . ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 6 6 5 3 - - 6 6 6 1 S h u r n a n , S.J., Z o r , U., C h a y o t h , R. and Field, J.B. ( 1 9 7 6 ) J. Clin. Invest. 57, 1 1 3 2 - - 1 1 4 1 R a p o p o r t , B. ( 1 9 7 5 ) E n d o c r i n o l o g y 9 8 , 1 1 8 9 - - 1 1 9 7 S c h u l t z , J. ( 1 9 7 5 ) J. N e u r o c h e m . 2 4 , 4 9 5 - - 5 0 1 S h e a r , M., Insel, R . A . , M e l m a n , K . L . and C o f f i n o , P. ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 7 5 7 2 - - 7 5 7 6 F r a n k l i n , T.J., Morris, W.P. a n d T w o s e , P.A. ( 1 9 7 5 ) Mol. P h a r m a c o l . 1 1 , 4 8 5 - - 4 9 1 M a n g a n i e l l o , V. and V a u g h a n , M. ( 1 9 7 2 ) P r o c . Natl. Acad. Sci. U.S. 6 9 , 2 6 9 - - 2 7 3 D ' A r m i e n t o , M., J o h n s o n , R . and Pastan, I. ( 1 9 7 2 ) P r o c . Natl. A c a d . Sci. U.S. 6 9 , 4 5 9 - - 4 6 2 S c h w a r t z , J . P . and P a s s o n n e a u , J . V . ( 1 9 7 4 ) P r o c . Natl. Acad. Sci. U.S. 71, 3 8 4 4 - - 3 8 4 8 K e l l y , L . A . and B u t c h e r , R.W. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 3 0 9 8 - - - 3 1 0 2 H o , R . J . and S u t h e r l a n d , E.W. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 6 8 2 2 - - 6 8 2 7 H o , R . J . and S u t h e r l a n d , E.W. ( 1 9 7 5 ) P r o c . Natl. Acad. Sci. U.S. 7 2 , 1 7 7 3 - - 1 7 7 7 M u k h e r j e e , C., C a r o n , M.F. a n d L e f k o w i t z , R . J . ( 1 9 7 5 ) Proc. Natl. Acad. Sci. U.S. 72, 1 9 4 5 - - 1 9 4 9 L e f k o w i t z , R . J . , M u l l i k i n , D., W o o d , C.L., G o r e , T.B, and M u k h e r j e e , C. ( 1 9 7 6 ) J. Biol. C h e m . 1 5 , 5295--5303 K e b a b i a n , J.W., Z a t z , M., R o m e r o , J . A . and A x e l r o d , J. ( 1 9 7 5 ) P r o c . Natl. Acad. Sci. U.S. 7 2 , 3 7 3 5 - 3739 H s u e h , A.J.W., D u f a u , M.L. and Cart, K . J . ( 1 9 7 7 ) Proc. Natl. A c a d . Sci. U.S. 7 4 , 5 9 2 - - 5 9 . 5
514 24 25 26 27 28 29 30
W i s h n o w , R . M . , Feist, P. a n d G l o w a l l a , M.B. ( 1 9 7 4 ) A r c h . B i o e h e m . B i o p h y s . 1 6 1 , 2 7 5 - - 2 8 0 Tsang, C.P.W., L e h o t a y , D.C. a n d M u r p h y , B.E.P. ( 1 9 7 2 ) J. Clin. E n d o c r i n o l . M e t a b . 35, 8 0 9 - - 8 1 7 G i l m a n , A . G . ( 1 9 7 0 ) Proe. Natl. A c a d . Sci. U.S. 6 7 , 3 0 5 - - 3 1 2 L o w r y , O . H . , R o s e b r o u g h , N.J., F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J, Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 W i s h n o w , R . M . , L i f r a k , E. a n d C h e n , C, ( 1 9 7 6 ) J . I n f e c t , Dis. 1 3 3 , $ 1 0 8 - - $ 1 1 4 De Vellis, J. a n d B r o o k e r , G. ( 1 9 7 4 ) S c i e n c e 1 8 6 , 1 2 2 1 - - 1 2 2 3 M a k m a n , M . H . , D v o r k i n , B. a n d K e e h n , E. ( 1 9 7 4 ) in C o n t r o l o f P r o l i f e r a t i o n in A n i m a l Cells (Clark. s o n , B. a n d B a s c r g a , R . , eds.), p p . 6 4 9 - - 6 6 3 , C o l d S p r i n g H a r b o r L a b o r a t o r y , N e w Y o r k 31 H o r n s b y , P.J. a n d Gill, G . N . ( 1 9 7 7 ) J. Clin. Invest. 6 0 , 3 4 2 - - 3 5 2
Biochimica et Biophysica Acta, 541 (1978) 504--514 © Elsevier/North-Holland Biomedical Press
BBA 28585
ADRENOCORTICOTROPIN-INDUCED UNRESPONSIVENESS IN CULTURED ADRENAL TUMOR CELLS
ERIC L I F R A K and RODNEY M. WISHNOW
Veterans Administration Hospital, Long Beach, Calif, 90822, and the Departments of Medicine and Microbiology of the University of California College of Medicine, Irvine, Calif 92664 (U.S.A.) (Received November 21st, 1977)
Summary Our results demonstrate that adrenocorticotropin (ACTH)-induced refractoriness occurs in cultured adrenal t u m o r cells. Cells became 85% refractory to ACTH-induced cyclic AMP formation in 20 min and the effect persisted if the hormone remained in the incubation medium. Refractory cells gradually regained hormone-specific responsiveness within 24 h if cultures were incubated in fresh media containing serum. The observed effect is hormone specific since cyclic AMP could not induce unresponsiveness to ACTH. The addition of ACTH plus inhibitors of protein synthesis partially reversed hormone-specific refractoriness. However, preincubation with cycloheximide or diphtheria toxin led to superinduction of ACTH-induced cyclic AMP formation. These experiments suggest that unresponsiveness, following hormonal activation of adrenal cells~ may be related to a deCrease in hormone-specific binding sites or to synthesis of an adenylate cyclase inhibitor.
Introduction Adrenocorticotropin (ACTH) interacts with a specific receptor on the cell membrane of cultured mouse Y-1 adrenal t u m o r cells to stimulate adenosine 3':5'-monophosphate (cyclic AMP), steroidogenesis, and a change in morphology from flattened to spherical shaped cells [1--3]. The addition of ACTH to adrenal cells leads to a rapid accumulation of intracellular cyclic AMP which reaches a maximum in about 15 min and then begins to decline toward the basal level despite the continued presence of the hormone [4,5]. This phenomenon has been observed in a variety of cell types including liver, thyroid,
Abbreviation: ACTH, adrenocorticotropin.
505 n e u r o b l a s t o m a l y m p h o m a and fibroblasts and has been called hormoneinduced unresponsiveness, desensitization, refractoriness or tachyphylaxis [6--13]. Refractoriness may effectively modulate cyclic AMP activity during periods of prolonged exposure to hormones and may thus limit energetically wasteful cyclic AMP generation. There have been several suggested mechanisms which might contribute to refractoriness in a number of cells including (a) induction of phosphodiesterase [14--16] ; (b) secretion of cyclic AMP [17] ; (c) synthesis of an antagonist to adenylate cyclase activity [18--19] and (d) reduction in the number or affinity of hormone receptors [20--23]. In this study we have demonstrated that ACTH rapidly induces an unresponsive state in cultured adrenal cells. The effect is reversible, hormone specific, and is unrelated to induction of phosphodiesterase or secretion of cyclic AMP. Inhibitors of protein or RNA synthesis do not appear to reverse ACTH-induced refractoriness. Materials and Methods
Cell culture procedures. Y-1 adrenal t u m o r cells were maintained in monolayer culture in Eagle's minimum essential medium with Earle's salts, containing 12.5% horse serum, 2.5% fetal calf serum, 1 mM L-glutamine, 10 units penicillin/ml and 10 pg streptomycin/ml. The cells were grown in 60 X 15 mm Petri dishes (Falcon plastics) at 37°C in a humidified atmosphere of 5% CO2 in air. Subculturing procedures and the fluorometric steroid assay have been described [24]. The a m o u n t of cyclic AMP in the cells and in the medium was measured after extraction with ethanol by the Gilman competitive binding assay [4,25,26]. Protein was determined by the m e t h o d of Lowry et al. [27]. Incubation procedures. Duplicate confluent cultures were washed twice with warm (37°C) Eagle's minimal essential medium. Then, warm minimal essential medium or minimal essential medium with 13% fetal calf serum containing ACTH (10 munits/ml) was added for various time intervals. The media were removed for extracellular cyclic AMP determination and 3.0 ml ethanol was added to the cells before determining intracellular cyclic AMP. In the time course of ACTH unresponsiveness, ACTH was added for 5, 10, 20 min and 2 h and 7 h intervals, then removed. Then the cells were washed twice and 2.0 ml minimal essential medium containing ACTH or without hormone was added for an additional 20 min incubation. Both intracellular and extracellular cyclic AMP was determined on duplicate plates. In each experiment, basal cyclic AMP and maximum ACTH-stimulated cyclic AMP were determined after a 20 min incubation in cultures not previously exposed to ACTH. Recovery from ACTH unresponsiveness. Confluent cultures were incubated in 2.0 ml of minimal essential medium containing ACTH (10 munits/ml) for 1 h. The medium was then removed and the cells were washed twice with 2.0 ml warm minimal essential medium. Next, 2.0 ml of fresh warm minimal essential medium was added to the cells, which were incubated for various times for recovery. The cells were again washed twice with minimal essential: medium and 2.0 ml of the medium containing ACTH (10 munits/ml) or the medium without hormone was added at each time interval for an additional 20 min incubation. Cyclic AMP was determined in the cells and in the medium
506 at the end of 20 min for each plate. Control cultures for ACTH (10 munits/ml) added for 20 min and with minimal essential medium alone were also run. A parallel group of cultures were incubated with growth medium (minimal essential medium supplemented with 12.5% horse serum, 2.5% fetal calf serum and 1.0 mM L-glutamine) during the recovery period, and cyclic AMP determined. Effect of pre-incubation with cyclic AMP. Confluent cultures were incubated in 2.0 ml of minimal essential medium containing 2.5 mM cyclic AMP. At 2 h the medium was removed, the cells were washed 10 times with 2.0 ml warm minimal essential medium and then fresh medium with ACTH (10 munits/ml), or medium without hormone was added for 20 min incubation and total cyclic AMP measured in quadruplicate cultures. Effect of inhibitors on ACTH-induced cyclic AMP. ACTH (10 munits/ml) or ACTH plus cycloheximide (28 pg/ml) was added to confluent adrenal cells for 30 min to 7 h and cyclic AMP determined intracellularly and in the media in duplicate cultures. Controls with cycloheximide alone or Eagle's minimal essential medium were also assayed. In the preincubation experiment, cycloheximide (28 t~g/ml) was added for 15 min to 6 h and then ACTH was added for 20 min incubation before measuring cyclic AMP. Cultures incubated with cycloheximide alone for as long as 6 h were also assayed. Control cultures incubated with ACTH or minimal essential medium alone were also assayed after a 20 min incubation. The inhibitors cycloheximide (28 pg/ml), and puromycin (200 pg/ml) in 2.0 ml of warm minimal essential medium containing ACTH (10 munits/ml) were incubated with duplicate confluent adrenal cultures for 2 h. Diphtheria toxin (200 pg/ml) was added in 2.0 ml of warm minimal essential medium to cultures 2 h prior to the addition of ACTH (10 munits/ml) for an additional 2 h. Then the medium was removed and the cells washed twice and fresh media containing the same reagents or without ACTH were added for an additional 20 rain incubation before measuring cyclic AMP. Control cultures incubated with ACTH or minimum essential medium for 20 min were also assayed. In the pretreatment experiment cycloheximide or puromycin was added for 2 h and diphtheria toxin 4 h. Then ACTH was added to the cultures for 20 min and total cyclic AMP determined. Control cultures were also run with inhibitor alone. Actnomycin D (5 pg/ml) or cordycepin (20 pg/ml) plus ACTH (10 munits/ ml) was incubated with Y-1 cells for 3--8 h. The medium was then removed, the cells washed twice and fresh medium with ACTH, or minimal essential medium with the same inhibitor was added for 20 min before measuring cyclic AMP. Control cultures incubated with ACTH or minimal essential medium alone were assayed after a 20 min incubation. Cultures were also preincubated with actinomycin D or cordycepin for 1--6 h before addition of ACTH for 20 rain. Control cultures were also run with inhibitor alone. Reagents. The following reagents were used in tissue culture incubations: porcine ACTH (Armour, Chicago, Ill.), purified diphtheria toxin (gift of Dr. J. Collier of UCLA), cyclic AMP, sodium salt, actinomycin D, and cordycepin (3'-deoxyadenosine) (Sigma, St. Louis, Mo.), puromycin dihydrochloride (United States Biochemical Corporation, Cleveland, Ohio), and cycloheximide (Upjohn, Kalamazoo, Mich.). Tritiated cyclic AMP (37.7 Ci/mmol; New Eng-
507 land Nuclear, Boston, Mass.) and cyclic AMP binding protein (Calbiochem, LaJolla, Calif.) were used in determination of cyclic AMP. Results Effect o f A C T H on intracellular and extracellular cyclic AMP accumulation and steroidogenesis. Fig. 1 shows that the addition of ACTH to confluent adrenal monolayer cultures leads to a rapid increase in b o t h intracellular and extracellular cyclic AMP. The intracellular cyclic AMP begins to decline toward the basal level after 30 min b u t the extracellular cyclic AMP concentration reaches a maximum 4 h after exposure to ACTH (10 munits/ml). At 30 min, 70% of the total cyclic AMP, is outside the adrenal cells and at 4 h more than 95% is. This figure also demonstates that steroid release into the tissue culture medium continues linearly despite the early peak in intracellular cyclic AMP concentration. In this system, since most of the cyclic AMP was rapidly released into the medium, both intracellular and extracellular cyclic AMP were measured in subsequent experiments and results were expressed as total cyclic AMP. Fig. 2 demonstrates the effect of minimal essential medium supplemented
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Fig. 1. T i m e c o u r s e o f i n t r a e e l l u l a r a n d e x t r a c e U u l a r cyclic AMP f o r m a t i o n a n d s t e r o i d o g e n e s i s . A d r e n a l cells w e r e i n c u b a t e d at 3 7 ° C in Eagle's m i n i m a l essential m e d i u m c o n t a i n i n g A C T H ( 1 0 m u n i t s / m l ) f o r v a r i o u s t i m e intervals. T h e cyclic AMP values are e x p r e s s e d as p m o l cyclic A M P / r a g cellular p r o t e i n a n d t h e s t e r o i d values as p g / m g cellular p r o t e i n . E a c h p o i n t r e p r e s e n t s t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s o n t w o s e p a r a t e cultures.
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with 13% fetal calf serum on the intracellular cyclic AMP response to ACTH stimulation. In the presence of serum the peak or maximum intracellular cyclic AMP concentration was approximately 50% of the cyclic AMP level in ACTHtreated adrenal tumor cells incubated without fetal calf serum. Despite the difference in cyclic AMP response shown in this figure, serum had no detectable effect on steroidogenesis. Since sera are k n o w n to contain phosphodiesterese and other factors which effect cyclic nucleotide metabolism [6], the following incubations were carried out in the absence of serum unless specifically stated to the contrary. Time course o f A CTH unresponsiveness. Confluent cultures of adrenal tumor cells were incubated with a maximal stimulating dose of ACTH (10 munits/ml)
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F i g . 3. T i m e c o u r s e o f A C T H - i n d u c e d u n r e s p o n s i v e n e s s . A d r e n a l cells w e r e i n c u b a t e d a t 3 7 ° C in m i n i m a l essential medium containing ACTH (10 munits/ml) for various time intervals, washed twice, then incub a t e d i n f r e s h m e d i u m w i t h A C T H ( 1 0 m u n i t s / m l ) (Q o ) o r in f r e s h m e d i u m a l o n e (~ . . . . . . ~) f o r 2 0 r a i n b e f o r e m e a s u r i n g i n t r a c e l l u l a r a n d e x t r a c e l l u l a r c y c l i c A M P . R e s u l t s a r e e x p r e s s e d as % t o t a l cyclic AMP response to an initial 20 rain incubation with ACTH. Each point represents the mean of two separate experiments with each point determined on duplicate cultures.
509 for 5 min to 7 h. Following the initial incubation, the cells were washed twice, then incubated with ACTH in fresh medium for an additional 20 min and total cyclic AMP measured. Fig. 3 shows a time-dependent decrease in the total cyclic AMP response of adrenal cells to a second exposure to ACTH. Cells initially incubated with ACTH for 5 min were 70% responsive, but cells preincubated with ACTH for 20 min were 15% as responsive as control cultures which had not previously been exposed to ACTH. Cells incubated for up to 7 h with ACTH in serum-free medium remained maximally refractory to a second exposure to ACTH even if ACTH was increased to 100 munits/ml. In addition, if the adrenal cells were washed 10 times before restimulation there was no reversal of ACTH-induced unresponsiveness. This experiment also demonstrated that washing cells twice with warm media effectively removed ACTH since cyclic AMP levels after washing fell to basal levels. Recovery from ACTH unresponsiveness. The time course of recovery of ACTH responsiveness after a 1 h incubation of adrenal cells with ACTH was compared in serum-supplemented and unsupplemented minimal essential medium (Table I). If cells were treated with ACTH for 1 h, washed twice, and incubated in minimal essential medium for 23 h prior to restimulation with ACTH, they were 60% as resPonsive as control cultures. In contrast, if cells were incubated in serum-supplemented minimal essential medium, then ACTH responsiveness returned more rapidly and completely. At the end of 8 h, the cyclic AMP response in cells allowed to recover in serum-supplemented medium was 60% of control cultures and at 24 h the cells regained their normal ACTH response. Specificity of hormonal unresponsiveness. Since cyclic AMP mediates the ACTH response it seemed possible that it could also induce ACTH unresponsiveness. In order to test this hypothesis, cells were incubated with 2.5 mM cyclic AMP for 2 h, washed 10 times, and ACTH was added for 20 min before measuring total cyclic AMP. The cyclic AMP concentration in duplicate cultures, following preincubation with cyclic AMP, was 520 pmol/mg protein compared to 505 pmol/mg protein in control cultures incubated with ACTH for 20 min. This experiment demonstrated that incubation with cyclic AMP does not render cells refractory to ACTH stimulation. A control showed t h a t
TABLE I RECOVERY
FROM ACTH-INDUCED UNRESPONSIVENESS
A d r e n a l cells w e r e i n c u b a t e d w i t h A C T H ( 1 0 m u n i t s / m l ) f o r 1 h, w a s h e d t w i c e , t h e n i n c u b a t e d w i t h s e r u m - s u p p l e m e n t e d or u n s u p p l e m e n t e d m i n i m a l essential m e d i u m f o r 1 - - 2 3 h. A t various t i m e s d u p l i c a t e c u l t u r e s w e r e w a s h e d t w i c e , i n c u b a t e d w i t h A C T H ( 1 0 m u n i t s / m l ) or m i n i m a l e s s e n t i a l m e d i u m a l o n e for 20 r a i n and t o t a l c y c l i c A M P w a s d e t e r m i n e d . R e s u l t s are e x p r e s s e d as a % t o t a l c y c l i c A M P c o m p a r e d to a n i n i t i a l 20 m i n s t i m u l a t i o n w i t h A C T H . Time (h)
1 4 7 23
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Fig. 4. E f f e c t o f c y c l o h e x i m i d e ( C H ) on A C T H - i n d u c e d t o t a l cyclic AMP. A d r e n a l cells were i n c u b a t e d w i t h m e d i u m c o n t a i n i n g A C T H (10 m u n i t s / m l ) or A C T H plus c y c l o h e x i m i d e (28 # g / m l ) for 30 rain to 7 h b e f o r e i n t r a c e l l u l a r a n d e x t r a c e l l u l a r cyclic AMP was assayed. E a c h p o i n t r e p r e s e n t s t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s on t w o s e p a r a t e cultures.
the washing procedure was effective in reducing the total cyclic AMP almost to the basal concentration. Previously, we have demonstrated that preincubation of adrenal cells with ACTH did not reduce cholera toxin-induced cyclic AMP formation [28]. Effect o f cycloheximide on time course o f A C T H stimulation o f adrenal cells. Fig. 4 compares the effect of the addition of ACTH and ACTH plus cycloheximide on cyclic AMP formation. If adrenal cells are incubated with ACTH plus cycloheximide there is a linear increase in total cyclic AMP for 6 h, while in cells incubated with ACTH the total cyclic AMP concentration decreases after 3 h. This dose of cycloheximide was sufficient to completely inhibit ACTH-induced steroidogenesis and did not alter basal cyclic AMP concentration. In order to test whether or not new protein synthesis was required for ACTH-induced refractoriness of Y-1 cells, the effect of various inhibitors of protein synthesis was evaluated. If adrenal cells are incubated with ACTH plus cycloheximide, ACTH plus puromycin, or ACTH plus diphtheria toxin, there appears to be a partial inhibition of ACTH-induced refractoriness (Fig. 5A). However, Fig. 5B demonstrates that incubation of adrenal cells with cycloheximide for 2 h or diphtheria toxin for 4 h prior to the addition of ACTH led to super-induction of total cyclic AMP formation. This figure also shows that puromycin has a slight inhibitory effect on cyclic AMP formation. Fig. 6 shows the effect of incubating adrenal cells with cycloheximide for 0.25--6 h before addition of a maximal stimulating dose of ACTH for a 20 min incubation. Preincubation with cycloheximide leads to a time-dependent increase in ACTHinduced total cyclic AMP concentration. If Y-1 cells were incubated with ACTH plus actinomycin D or ACTH plus cordycepin for as long as 8 h, washed, and fresh medium with the same reagents added for a 20 min incubation, the total cyclic AMP concentration was the same as cells treated with ACTH alone. These results demonstrate that inhibition of RNA synthesis does not reverse ACTH-induced refractoriness in
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Fig. 5. A. E f f e c t o f p r o t e i n s y n t h e s i s i n h i b i t o r s o n A C T H - i n d u c e d t o t a l c y c l i c A M P f o r m a t i o n . A d r e n a l cells w e r e i n c u b a t e d w i t h c y c l o h e x i m i d e ( C H , 28 # g / m l ) or p u r o m y c i n (PM 2 0 0 p g / m l ) p l u s A C T H ( 1 0 m u n i t s ] m l ) f o r 2 h. D i p h t h e r i a t o x i n ( D T , 2 0 0 p g / m l ) w a s p r e i n c u b a t e d with" a d r e n a l cells f o r 2 h p r i o r to t h e a d d i t i o n o f A C T H ( 1 0 m u n i t s / m l ) f o r an a d d i t i o n a l 2 h. A f t e r t h e initial i n c u b a t i o n , t h e cells w e r e w a s h e d t w i c e , a n d f r e s h m e d i u m c o n t a i n i n g t h e s a m e i n h i b i t o r w i t h o r w i t h o u t A C T H w a s a d d e d f o r 20 r a i n b e f o r e t o t a l c y c l i c A M P w a s m e a s u r e d . R e s u l t s are e x p r e s s e d as a % t o t a l c y c l i c A M P c o m p a r e d t o an initial 20 r a i n s t i m u l a t i o n w i t h A C T H . E a c h b a r r e p r e s e n t s t h e m e a n o f t w o d i f f e r e n t e x p e r i m e n t s w i t h d u p l i c a t e c u l t u r e s . B. E f f e c t o f p r e i n c u b a t i o n w i t h p r o t e i n s y n t h e s i s i n h i b i t o r s o n A C T H - i n d u c e d c y c l i c A M P . A d r e n a l cells w e r e i n c u b a t e d w i t h m i n i m a l e s s e n t i a l m e d i u m c o n t a i n i n g c y c l o h e x i m i d e ( 2 8 p g / m l ) , p u r o m y c i n ( 2 0 0 ttg/ml) f o r 2 h, or d i p h t h e r i a t o x i n ( 2 0 0 p g / m l ) f o r 4 h, t h e n A C T H w a s a d d e d f o r 20 r a i n b e f o r e m e a s u r i n g i n t r a c e l l u l a r a n d e x t r a c e l l u l a r c y c l i c A M P . C o n t r o l c u l t u r e s w e r e also r u n w i t h inhibitor alone. 260o.. 220•~ 1 8 0 >.¢_) j 140I.F° t 0 0 o
Y
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Fig. 6. T i m e c o u r s e o f p r e i n c u b a t i o n w i t h c y c l o h e x i m i d e ( C H ) o r A C T H - i n d u c e d c y c l i c AMP. A d r e n a l cells w e r e i n c u b a t e d w i t h c y c l o h e x i m i d e ( 2 8 # g / m l ) f o r 1 5 m i n to 6 h, t h e n A C T H ( 1 0 m u n i t s / m l ) w a s a d d e d f o r 20 m i n a n d t o t a l c y c l i c AMP d e t e r m i n e d (o o). C o n t r o l c u l t u r e s w i t h ' c y c l o h e x i m i d e ( 2 8 p g / m l ) a l o n e (~ . . . . . . o) w e r e also a s s a y e d . R e s u l t s are e x p r e s s e d as % t o t a l c y c l i c A M P c o m p a r e d to an i n i t i a l 20 r a i n s t i m u l a t i o n w i t h A C T H . E a c h p o i n t r e p r e s e n t s t h e m e a n o f t w o s e p a r a t e e x p e r i m e n t s with duplicate cultures.
512
adrenal cells. In contrast to the cycloheximide effect, preincubation with actinomycin D or cordycepin for as long as 6 h did not alter ACTH-induced cyclic AMP production. Discussion Our results demonstrate that maximal hormone-specific refractoriness occurs 20 min after exposure of adrenal cells to ACTH. At that time the cells became 85% refractory, intracellular cyclic AMP levels were near maximum, and cells had changed from a flattened to a spherical shape. Hormonal unresponsiveness was not due to loss of ACTH activity since media from unresponsive cells could maximally stimulate cells that had not been exposed to ACTH. Neither extensive washing of adrenal cells nor the addition of fresh ACTH (100 munits/ml} reversed the loss of ACTH unresponsiveness. Ho and Sutherland [19] found that washing could remove a soluble antagonist of adenylate cyclase released into the media by fat cells. If induction of phosphodiesterase were responsible for hormone-induced refractoriness, then incubation with ACTH should render cells refractory to cholera toxin. However, previously we have demonstrated that ACTH refractory cells can be maximally stimulated by cholera toxin [28]. Recently, a similar effect of cholera toxin has been shown in isoproterenol-stimulated lymp h o m a cells [12]. In addition, incubation with cyclic AMP itself does not lead to ACTH unresponsiveness. In neuroblastoma cells that lack phosphodiesterase, hormone-specific unresponsiveness can also be demonstrated [29]. Although induction of phosphodiesterase has been implicated as a cause of refractoriness in several cell systems, it does not appear to be responsible for refractoriness in adrenal cell cultures [14,16]. The time course and specificity of hormone-induced refractoriness suggest that ACTH must interact with its membrane receptor and stimulate adenylate cyclase before refractoriness can be detected. 5 min after exposure of adrenal cells to ACTH, there is a detectable increase in cyclic AMP and cells are 30% less responsive than control cultures to restimulation by ACTH. Recent evidence with isolated cell membranes suggests that the loss of receptors is responsible for hormone-induced refractoriness. Mukherjee et al. [20] have shown that in purified frog erythrocyte plasma membranes, isoproterenol decreases the number of functional ~-adrenergic receptors and 5'-guanylimidodiphosphate and other purine nucleotides reverse this effect. The OS-3 adrenal t u m o r mutant appears to bind ACTH, but cannot be stimulated by the hormone [2]. However, this m u t a n t can be stimulated by cholera toxin and cyclic AMP [28]. Specific binding studies with this strain may confirm whether or not receptor occupancy plus adenylate cyclase activation are necessary for ACTH-induced refractoriness. In serum-supplemented medium the maximal intracellular cyclic AMP response to ACTH was reduced by approximately 50%. A similar effect of serum has been shown in fibroblasts and thyroid cells [6,8]. However, since refractoriness occurred in the absence of serum in our system, a serum factor could not be implicated in the observed effect. On the other hand, the more rapid recovery of ACTH responsiveness in cells incubated in serum-supple-
513 mented media compared to serum-free media suggests that new protein synthesis is necessary for recovery from ACTH-induced refractoriness. The partial reversal of refractoriness induced when protein synthesis inhibitors were added simultaneously with ACTH may be independent of the presence of ACTH, since if cells were incubated with cycloheximide for 1--6 h before exposure to ACTH there was a time-dependent increase in cyclic AMP. Super-induction of ACTH-stimulated cyclic AMP accumulation may be due to inhibition of synthesis of an inhibitor of adenylate cyclase or decreased synthesis of phosphodiesterase. In fibroblasts, a 2 h pretreatment with cycloheximide led to a 60% greater cyclic AMP accumulation with prostaglandin E~ [30]. In thyroid cells, a supramaximal cyclic AMP response to thyrotropin plus cycloheximide has also been demonstrated [8]. In glial tumor cells, both inhibitors of R N A synthesis and protein synthesis appear to partially reverse catecholamine-induced refractoriness [29]. In our system, actinomycin D and cordycepin had no effect on ACTHinduced unresponsiveness or super-induction suggesting that the template for the synthesis of the cycloheximide-sensitive inhibitory protein may have a long half-life. In adrenal tumor cells, hormone-induced unresponsiveness may be important for cell proliferation since cells become resistant to ACTH-induced growth inhibition in the refractory state [31]. Acknowledgments This research was supported by NIH grant no. CA 16868, and by the Medical Research Programs, Long Beach Veterans Administration Hospital. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
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