Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

62

Biochimica el Biophysica Acta, 451 (1976} 62--71 © Elsevier/North-Holland Biomedical Press

BBA 28085 EFFECTS OF CHOLERAGEN ON HORMONAL RESPONSIVENESS OF ADENYLATE CYCLASE IN HUMAN FIBROBLASTS AND RAT FAT CELLS

VINCENT C. MANGANIELLO, CHRISTOPHER J. LOVELL-SMITHand MARTHA VAUGHAN Laboratory o f Cellular Metabolism, National Heart and Lung Institute, National Institutes o f Health, Bethesda, Md. 20014 (U.S.A.)

(Received April 21st, 1976}

Summary Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E j also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E~ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 pM isoproterenol is increased and approaches that produced by 5.6 pM prostaglandin E~. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 pM prostaglandin E~ is reduced by as much as 50%, the concentration of prostaglandin Ej required to induce a maximal increase in cyclic AMP is 1/10 that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin El can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells. In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.

63 Introduction Choleragen enhances adenylate cyclase activity and increases cyclic AMP content in a variety of tissues [1]. In addition, there have been several reports of alterations in hormonal responsiveness of adenylate cyclases associated with choleragen activation. In some instances it has been found that the sensitivity and/or maximal responses to catecholamines or peptide hormones are increased [2,3]. On the other hand, it has also been reported that the choleragen-actirated cyclase of cultured melanoma cells was not responsive to melanocytestimulating hormone which markedly activated adenylate cyclase preparations from control cells [4]. The effects of choleragen on hormonal responsiveness in intact cells have n o t been extensively studied and it appears that adenylate cyclase activity as assayed in broken cell systems often does not reflect the behavior of the enzyme in the intact cells. We undertook, therefore, to compare the effects of choleragen on hormonal responsiveness of adenylate cyclase in intact cells and in cell-free preparations. As reported below, the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E1 were altered independently during incubation with choleragen b u t differences in responsiveness to the two agonists were not demonstrable in assays of adenylate cyclase from control or choleragen-treated cells. Incubation of fat cells with choleragen did not alter the accumulation of cyclic AMP produced by a maximally effective concentration of isoproterenol in intact cells or adenylate cyclase preparations. Methods Human foreskin fibroblasts were maintained in monolayer cultures in 250 ml Falcon flasks (Falcon Plastics) in Eagle's basal medium supplemented with Earle's salts, 20% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin and 100 pg/ml streptomycin (Grand Island Biol. Co., Grand Island, N.Y.) [5]. Cells were removed from the flasks by brief (<5 min) treatment with trypsin (a 0.125% trypsin solution in Dulbecco's phosphate-buffered saline without Ca ~÷ and Mg 2÷ (Grand Island Biol. Co.). Subcultures initiated with approx. 2.5 • 10 s cells in Optilux Tissue Culture dishes (100 × 20 mm) that contained 10 ml growth medium were incubated at 37°C in an Airflow CO2 incubator (National Appliance Co., Portland, Oreg.) as described previously [5] for 7 days (medium changed on days 3 and 6). Such confluent cultures (approx. 3 • 106 ceils per dish) were used for all experiments. After removal of growth medium, the cells were washed twice and incubated for 30 min at 37°C in Hanks balanced salt solution. Choleragen was then added, and the incubation continued. In some experiments both control and choleragen-treated cells were incubated with isoproterenol for 10 min or prostaglandin E~ for 15 min. To terminate the incubation, medium was quickly removed by aspiration and 1.5 ml cold 5% trichloroace'tic acid was added to the tissue culture dishes. Cyclic AMP was extracted and purified as described previously [6] and assayed by the protein binding method of Gilman [7]. Protein content of the dishes was determined as previously described [5]. For measurement of adenylate cyclase activity, the medium was removed and cells washed

64 twice with cold 20 mM Tris • HC1, pH 8.0/1 mM MgC12/1 mM dithiothreitol. The cells were scraped from dishes with a rubber policeman and homogenized in a Dounce homogenizer in 2 ml of the same buffer. Portions of homogenates (<75 pg protein) were incubated at 30°C for 10 min in a final volume of 75 pl containing 3.5 mM ATP (approx. 2 • 106 cpm [3H]ATP), 6.6 mM MgC12, 33 mM Tris. HCL pH 8.0, 0.9 mM cyclic AMP, 0.3 mM dithiothreitol, 200 pg bovine serum albumin, 16.7 mM sodium phosphoenolpyruvate, and 16 pg pyruvate kinase (including 5.5 pmol (NH4)2SO4). The reaction was terminated by addition of 0.5 ml of 1% sodium dodecyl sulfate solution containing 7.0 mM ATP, 2.5 mM cyclic AMP and 10 mM Tris buffer pH 7.4. The reaction mixture was transferred to AG50W-X8 (100--200 mesh) columns (0.5 × 3.0 cm) and cyclic AMP eluted in 3.0 ml as described by Krishna et al. [8]. After two precipitations with ZnSO4 and Ba(OH)z, the supernatants were transferred to columns (0.8 × 2.5 cm) of AG 1X2 (100--200 mesh). The columns were washed with 10 ml water and 10 ml 0.003 M HC1; cyclic AMP was eluted with 6 ml of 0.03M HC1. One portion of each eluate was used for radioassay of cyclic [3H]AMP and another for adsorbance at 259 nm, to assess and correct for recovery of cyclic AMP. Enzyme activity was constant in the standard assay system for 15--20 min over the range of protein concentrations used. Isolated epididymal fat cells were prepared from NIH Osborne-Mendel rats by the m e t h o d of Rodbell as described previously [9], using Krebs Ringer bicarbonate buffer with bovine serum albumin, 40 mg/ml, and 5 mM glucose. The cells were washed three times, and samples incubated, with shaking, in the presence or absence of choleragen. Samples of fat cells plus medium were removed at various times for the extraction, purification, and assay of cyclic AMP [9]. Fat cell weight was determined by filtering a portion of the suspension of fat cells on a tared Millipore filter {0.45 pM). To measure adenylate cyclase activity, after incubation of cells with or without choleragen, cells were washed once in Krebs Ringer medium, then three times in 0.25 M sucrose with 10 mM Tris • HC1, pH 7.4. Cells were suspended in 5 ml of an ice-cold solution of 0.25 M sucrose/10 mM Tris, pH 7.4, and homogenized in a 7 ml Dounce homogenizer. Homogenates were centrifuged at 100 000 × g for 40 min. The fat cake was removed with a spatula, the supernatant decanted and the sides of the tube wiped dry. The particulate fraction (100 000 × g pellet) was suspended in cold 0.25 M sucrose/10 mM Tris, pH 7.4, and dispersed with a Dounce homogenizer. Samples (<100 pg protein) were assayed in duplicate immediately for adenylate cyclase activity. Protein was determined by the m e t h o d of Lowry et al. [10], using bovine serum albumin as standard. Prostaglandins were the gift of Dr. J. Pike of The Upjohn Co., Kalamazoo, Mich. Solutions of l(+)-isoproterenol (Sterling Winthrop Res. Inst., Rensselaer, N.Y.) were prepared from the bitartrate salt. Cyclic [3H]AMP (22.1 Ci/mmol) and [3H]ATP (approx. 10--33 Ci/mmol) were purchased from New England Nuclear, Boston, Mass. Cholera toxin (Lot 0172) was prepared under contract for the National Institute of Allergy and Infectious Diseases (NIAID) by Dr. R. Finkelstein of the University of Texas Southwestern Medical School, Dallas, Texas, and supplied to us by Dr. Carl E. Miller (NIAID). The toxin was diluted in the recommended buffer and stored at --70°C. The toxin concentrations reported were calculated assuming a molecular weight of 80 000 [11].

65

Results

Human fibroblasts Incubation of confluent fibroblasts with 0.025--25 nM choleragen produced large increases in their cyclic AMP content {Fig. 1). With the higher concentrations of choleragen the increase in cyclic AMP content was demonstrable after 20 min and reached a maximum by or before 90 min; with the lower concentrations, the increase in cyclic AMP was demonstrable only after 45 min. Incubation with theophylline for 10 min did not increase the cyclic AMP content of control cells and increased that of cells that had been incubated for 90 min with 25 nM choleragen by little more than 100% (data not shown). Maximally effective concentrations of isoproterenol (2 #M) and prostaglandin E1 (5.6 gM) produce large increases in the cyclic AMP content of the human fibroblasts and in confluent cultures the effect of prostaglandin E~ is much greater than that of isoproterenol [5]. After incubation of confluent fibroblasts with 25 nM choleragen for 30 min (during which time choleragen itself raised the cyclic AMP content), the increment in cyclic AMP caused by 2 #M isoproterenol was markedly increased and approached that induced by 5.6 #M prostaglandin E1 (Fig. 2). In cells incubated for longer periods with choleragen (in high concentration) the response to prostaglandin E, was reduced and often was actually lower than that in control cells (Fig. 2, also Fig. 3, 4 and 5A). Enhancement of the isoproterenol effect by choleragen also tend-

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F i g . 1. E f f e c t o f c h o l e r a g e n o n c y c l i c A M P ( c A M P ) c o n t e n t o f f i b r o b l a s t s . C e l l s w e r e i n c u b a t e d w i t h o r w i t h o u t c h o l e r a g e n a s i n d i c a t e d . V a l u e s r e p r e s e n t the m e a n c y c l i c A M P c o n t e n t o f d u p l i c a t e i n c u b a t i o n s . F i g . 2. E f f e c t o f e h o l e r a g e n o n r e s p o n s e s o f f i b r o b l a s t s t o i s o p r o t e r e n o ] a n d p r o s t a g l a n d i n E l . C e l l s w e r e incubated without (...... ) or with (o e ) 2 5 n M c h o l e r a g e n . A t t h e i n d i c a t e d t i m e s , c o n t r o l cells (D . . . . . . ~, ~ . . . . . . ~ ) and c h o l e r a g e n - t r e a t e d cells ( a ' m, 4 A) w e r e i n c u b a t e d w i t h i s o p r o t e r e n o l f o r 1 0 m i n o r p r o s t a g l a n d i n E l ( P G E 1 ) f o r 1 5 r a i n . E a c h p o i n t is t h e m e a n c y c l i c A M P ( c A M P ) c o n tent (lower panel) or the mean increase in cyclic AMP induced by isoproterenol or prostaglandin E I (upper panel) in duplicate incubations.

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Fig. 4. E f f e c t s o f i s o p r o t e r e n o l a n d P r o s t a g l a n d i n E 1 o n c y c l i c A M P ( c A M P ) c o n t e n t o f c o n t r o l a n d chole r a g e n - t r e a t e d f i b r o b l a s t s w i t h o u t or w i t h 25 nM c h o l e r a g e n f o r 9 0 rain. A t t h i s t i m e c o n t r o l cells (.~) a n d c h o l e r a g e n - t r e a t e d cells ( $ ) w e r e i n c u b a t e d w i t h 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 s o f i s o p r o t e r e n o l f o r 10 r a i n o r p r o s t a g l a n d i n E 1 ( P G E 1 ) f o r 15 ,uin. 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 i n c r e a s e in c y c l i c A M P c o n tent i n d u c e d by i s o p r o t e r e n o l or prostaglandin E l duplicate incubations. The cyclic AMP c o n t e n t of c o n t r o l cells w a s 6 . 4 a n d o f c h o l e r a g e n - t r e a t e d cells 2 5 1 p m o l ] m g p r o t e i n .

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Fig. 5. E f f e c t o f c h o l e r a g e n o n r e s p o n s e s o f f i b r o b l a s t s t o i s o p r o t e r e n o l and p r o s t a g l a n d i n E 1 ( P G E 1 ). Cells w e r e i n c u b a t e d w i t h o u t or w i t h the i n d i c a t e d c o n c e n t r a t i o n s o f c h o l e r a g e n for 9 0 r a i n . A t t h i s t i m e , i n E x p t . A, c o n t r o l and c h o l e r a g e n - t r e a t e d cells w e r e i n c u b a t e d w i t h 2 . 0 / ~ M i s o p r o t e r e n o l (~) f o r 1 0 mirl or 5.6 p M p r o s t a g l a n d i n E l (~) f o r 1 5 rain. In E x p t . B, c o n t r o l and c h o l e r a g e n - t r e a t e d cells w e r e i n c u b a t e d w i t h 2 . 0 p M i s o p r o t e r e n o l (o) f o r 1 0 r a i n o r 0 . 5 6 /~M p r o s t a g l a n d i n E 1 (~) f o r 1 5 rain. E a c h p o i n t repres e n t s t h e m e a n c y c l i c A M P ( c A M P ) c o n t e n t ( l o w e r p a n e l s ) o r t h e m e a n i n c r e a s e in c y c l i c A M P i n d u c e d b y i s 0 p r o t e r e n o l o r p r o s t a g l a n d i n E 1 ( u p p e r p a n e l s ) in d u p l i c a t e i n c u b a t i o n s .

ed to be less than maximal when cells were incubated for prolonged periods with high concentrations of the toxin (Fig. 2, also 5B). Cyclic AMP accumulation was usually determined 10 min after the addition of isoproterenol and 15 min after the addition of prostaglandin El. As shown in Fig. 3, the time course of cyclic AMP accumulation in response to prostaglandin E1 and isoproterenol was not altered b y cholera toxin. In the choleragentreated cells, the alterations in responsiveness to isoproterenol and prostaglandin El were observed 5 min after the addition of these agents (Fig. 3). The concentration of isoproterenol which produced a maximal increment in cyclic AMP content was similar in control and choleragen-treated cells but the shape of the dose response curve was apparently altered by choleragen treatment (Fig. 4A). Although the response to 5.6 pM prostaglandin E1 was lower in choleragen-treated cells than in control cells, the concentration of prostaglandin El required to produce a maximal increase in cyclic AMP content was approx. 1/10 that necessary in the control cells (Fig. 4B). As shown in Fig. 5, alterations in the responsiveness to isoproterenol and prostaglandin El were demonstrable when fibroblasts were incubated with concentrations of choleragen ranging from 0.0025 to 25 nM. After incubation of the fibroblasts for 90 min with choleragen adenylate cyclase activity was markedly increased as shown in Table I b u t responsiveness to isoproterenol or prostaglandin El was not enhanced. Even with the adenylate cyclase from control fibroblasts, however, the effects of these agents did not parallel their effects on cyclic AMP accumulation in the intact cells.

68 TABLE I EFFECTS OF CHOLERAGEN

ON FIBROBLAST ADENYLATE

CYCLASE ACTIVITY

F i b r o b l a s t s w e r e i n c u b a t e d w i t h or w i t h o u t 25 nM c h o l e r a g e n f o r 9 0 rain. Cells f r o m 5 d i s h e s w e r e c o m b i n e d , h o m o g e n i z e d in 2 m l o f m e d i u m ( 2 0 m M Tris • HC1, p H 8 . 0 / 1 m M MgC12/1 m M d i t h i o t h r e i t o l ) , a n d a s s a y e d i n d u p l i c a t e as d e s c r i b e d in M e t h o d s . V a l u e s r e p o r t e d r e p r e s e n t t h e m e a n o f v a l u e s f r o m d u p l i c a t e pools of 5 dishes ± one-half the range. Additions in a s s a y

Adenylate cyclase activity ( p m o l / m g protein per rain)

None I s o p r o t e r e n o l , 1.3 /~M 100 pM P r o s t a g l a n d i n E l , 0 . 3 7 /~M 11 /~M N a F , 5.3 rnM

Control

Choleragen-activated

6 50 62 16 74 41

76 100 100 91 110

+ 0.5 ± 6 ± 10 ± 1,5 t 4 ±

5

4 10 + 0 + 11 * 9 ~ 10

6 8 +-

5

T A B L E II EFFECT OF CHOLERAGEN

ON THE RESPONSE OF FAT CELLS TO ISOPROTERENOL

F a t cells w e r e i n c u b a t e d f o r 4 h w i t h o r w i t h o u t 0 . 6 2 nM c h o l e r a g e n , a n d t h e n f o r a n a d d i t i o n a l 5 rain w i t h o r w i t h o u t 1 p M i s o p r o t e r e n o l . V a l u e s r e p r e s e n t m e a n ± S.E. (n = 3) c y c l i c A M P c o n t e n t o f cells p l u s medium (from three experiments). Additions

Cyclic AMP c o n t e n t ( p m o l / m g dry weight)

None Isoproterenol, 1 pM

46 | ~" >-

0

Control

•

Choleragen, 062nM

C o n t r o l cells

Choleragen-treated

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0.155 ± 0.05 0.475 + 0.08

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120

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TIME (mini Fig. 6. E f f e c t o f c h o l e r a g e n or c y c l i c A M P ( c A M P ) c o n t e n t o f f a t cells. F a t cells w e r e i n c u b a t e d w i t h o u t or w i t h 0 . 6 2 nM e h o l e r a g e n . A t t h e i n d i c a t e d t i m e s , 1 . 0 r a m t h e o p h y l l i n e w a s a d d e d t o s o m e s a m p l e s o f c h o l e r a g e n - t r e a t e d cells a n d t h e i n c u b a t i o n s c o n t i n u e d f o r 5 m 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 c y c l i c AMP content of duplicate incubations.

69

Rat fat cells When fat cells were incubated with 0.62 nM choleragen no increase in cyclic AMP was demonstrable after 30, 60 or 90 min. After 180 min the cyclic AMP content was increased only four- to five-fold. (As assessed at 180 min, 0.62 nM was a maximally effective concentration of choleragen.) When theophylline was added for the last 5 min of the incubation period, an increase in cyclic AMP content of the fat cells was demonstrable after 60 min of incubation with choleragen (Fig. 6). Theophylline added to cells incubated for 3 h with choleragen increased their cyclic AMP content almost 100 fold. Incubation of fat cells with choleragen did not increase the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol (Table II). After incubation of rat fat cells with choleragen for 3 h adenylate cyclase activity was increased from 1.2 -+ 0.1 to 4.1 + 0.3 pmol cyclic AMP/mg protein per 10 rain (mean +S.E., n = 7). In these experiments, 100 pM isoproterenol (a maximally effective concentration) enhanced cyclase activity in both control (5.4 + 0.6) and choleragen activated preparations (8.6 + 0.8 pmol cyclic AMP/ mg protein per 10 min). The increment in activity of adenylate cyclase from the choleragen-treated cells produced by 100 pM isoproterenol was not significantly different (100 + 6.8%, mean -+ S.E.) from that of paired cyclase preparations from control cells. Discussion

Diverse effects of choleragen on the hormonal responsiveness of adenylate cyclases from several different types of cells have been reported [2--4]. Adenylate cyclase activity and the effects of hormones on it in broken cell systems often appear, however, n o t to reflect quantitatively the situation in the intact cell. We undertook, therefore, to assess the effects of choleragen on cyclic AMP accumulation in intact cells in response to hormones and to compare these with the effects of choleragen on the hormonal responses of the adenylate cyclase directly assayed. In the human fibroblasts isoproterenol and prostaglandin El cause rapid and dramatic accumulation of cyclic AMP. In rapidly dividing cells the maximal effects of the two agonists are of similar magnitude. In confluent cultures, on the other hand, the magnitude of the response to isoproterenol is much decreased whereas the prostaglandin E1 effect is relatively little altered and if anything increased somewhat [ 5]. Thus, the capacities of the flbroblasts to respond to isoproterenol and to prostaglandin El are apparently independently regulated during growth in subculture [ 5]. As reported here, the responses of the cells to isoproterenol and to prostaglandin E I can also be altered independently b y choleragen. Incubation of confluent cultures with choleragen in relatively low concentration or for periods of less than 1 h (in addition to raising the cyclic AMP content of the cells) increased the magnitude of the response to maximally effective concentrations of isoproterenol b y 100--300%. Incubation with choleragen in high concentration (25 nM) for 90 min caused somewhat less enhancement of the maximal isoproterenol effect and decreased the response to 5.6 pM prostaglandin El, sometimes by more than 50%. Nevertheless the responses to low concentrations of either isoproterenol or prostaglandin El were increased under these conditions.

70

The enhancement of isoproterenol responsiveness that was observed in the intact fibroblasts after choleragen treatment was not demonstrable when adenylate cyclase activity was assayed in homogenates. This was perhaps not surprising since in homogenates of control cells the marked differences in responsiveness to isoproterenol and to prostaglandin E~ characteristic of the intact cells were not observed. In the fat cells in contrast to the fibroblasts the increases in cyclic AMP induced b y catecholamines or by choleragen were relatively small in the absence of theophylline. This suggests that either phosphodiesterase activity in fat cells is sufficient to compensate for increases in cyclase activity produced by choleragen or that increases in cyclase activity are accompanied by increases in phosphodiesterase activity. We have, in fact, previously demonstrated that agents which increase cyclic AMP content of fat cells can increase the activity of a particulate low Km phosphodiesterase [9]. It has recently been reported that choleragen increases b o t h adenylate cyclase and phosphodiesterase activities in the pineal [12]. We similarly have found increased phosphodiesterase activity in particulate fractions isolated from fat cells exposed to choleragen for 3 h (unpublished observations). No enhancement of the isoproterenol response was observed in choleragentreated fat cells. Likewise, the increase in adenylate cyclase activity produced by a maximally effective concentration of isoproterenol was not demonstrably different in particulate fractions from control and choleragen-treated cells. The reasons for the differences between our findings and those of other workers who have found increased responsiveness of the fat cell cyclase to isoproterenol after choleragen activation [13] are not readily apparent, although in our studies the lack of effect of choleragen on the isoproterenol response was observed in intact cells also. The fact that the hormonal responsiveness of the fibroblast adenylate cyclase in homogenates did not reflect that of enzyme in the intact cells serves simply as another unnecessary reminder that, whether due to instability of the complex cyclase system, conditions of assay, methods of enzyme preparation or other causes, the activity assayed in broken cells may or may n o t reflect in any quantitative sense the behavior of the enzyme in the intact cell. Acknowledgements We thank Mrs. Betty Horn, Mrs. Ferol Lieberman and Miss Sally Stanley for excellent technical assistance. References 1 2 3 4 5 6 7

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