Viable mouse thymocytes as a model system for studying the onset of hormone-induced cellular refractoriness

Biochimica etBiophysica Acta, 762 (1983) 355-365

355

Elsevier Biomedical Press BBA 11147

VIABLE M O U S E T H Y M O C Y T E S AS A M O D E L S Y S T E M FOR STUDYING T H E O N S E T OF H O R M O N E - I N D U C E D CELLULAR REFRACTORINESS YEHIEL ZICK, RACHEL CESLA and SHMUEL SHALTIEL *

Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot (Israel) (Received October 30th, 1982)

Key words: Desensitization model," Hormonal induction," cyclic AMP," Protein kinase; Adenylate cyclase; (Mouse thymocyte)

Mouse thymocytes are characterized as a model cellular system for studying the onset of hormone-induced cellular refractoriness (desensitization). This system has the following combination of useful features. (a) The cells can be isolated without the use of digestive enzymes, avoiding possible damage to surface receptors or to other exposed membranal constituents. (b) They can be kept viable for several hours, a period during which both stimulation and desensitization get well under way. (c) They can be stimulated by a variety of hormones which function via cAMP (/~-ag0nists, prostaglandin E 1 and specific thymic humoral factors). (d) Their desensitization is receptor-specific. (e) They can be readily ruptured under mild conditions so as to allow a physiologically relevant biochemical analysis of hormonal stimulation and desensitization. (f) The hormonal response of these cells can be monitored simultaneously by the activation of adenylate cyclase, by the intracellular level of cAMP, and by the activation of cAMP-dependent protein kinase (which functions as a metabolic sensor for cAMP). In this cellular system, desensitization does not involve processes such as the efflux of cAMP, the activation of cAMP-phosphodiesterase or the synthesis of a protein mediator. On the other hand, desensitization can be accounted for by a hormone-triggered inactivation of the adenylate cyclase system. The immediate desensitization of thymocytes is reversible and occurs without apparent loss of functional receptors. Continuous presence of hormone is shown to be required not only for triggering the chain of events which leads to the readily reversible desensitization, but also for the process which transfers the cells to the subsequent, 'locked' desensitized state.

Introduction Hormones that function via cAMP utilize a complex signal-transferring device to get their physiological message across the cell membrane.

* To whom correspondence should be addressed. Abbreviations: cAMP, adenosine (cyclic)-3',5'-phosphate; C, catalytic subunit of cAMP-dependent protein kinase; R, regulatory subunit of cAMP-dependent protein kinase; R2C2, cAMP-dependent protein kinase in its undissociated (inactive) form; R, hormone receptor, Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; Mes, 4-morpholinethanesulfonic acid. 0167-4889/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

This device includes a hormone receptor (facing outside the cell), the enzyme adenylate cyclase (facing inside) and at least one regulatory protein (denoted G / F or N) which, upon binding of the hormone to its receptor, couples (or 'engages') the receptor with the cyclase, causing the emission of the intracellular signal cAMP [1]. It is currently accepted that a major intracellular detector for cAMP is the enzyme cAMP-dependent protein kinase [2]. When cAMP binds to the regulatory protein (R2) of the inactive complex of this enzyme (R2C2) , the catalytic subunit (C) is released in an active form. The latter then proceeds to catalyse the phosphorylation of several proteins,

356 which in turn bring about the appropriate physiological response. The cellular response to hormonal stimulation is usually characterized by a 'spike-like' change in the level of intracellular cAMP, i.e., by a rapid rise followed by a slower decline in the level of the cyclic nucleotide [3-6]. The period of time after stimulation, during which the level of intracellular cAMP declines to basal values in spite of the fact that the cells are still exposed to an excess of the agonist, is termed 'the refractory period' and the cells which have temporarily lost their responsiveness to the hormone are then said to be 'refractory' or 'desensitized'. Desensitization is not only hormone-dependent (it lasts as long as the hormone is present), but can often be shown to occur as a hormone-specific phenomenon (i.e., it will occur with the hormone used for stimulation but not with a heterologous hormone, even if the latter also functions via cAMP). Basically different mechanisms have been proposed for the process(es) involved in cellular refractoriness. These include a decrease in the number of functional receptors on the cell surface [7], a hormone-induced increase in cAMP-phosphodiesterase activity [8,9], an efflux of cAMP across the cell membrane [10], a release of feedback inhibitors of adenylate cyclase [11,12], a synthesis of a 'refractory protein' [13] and finally a direct involvement of the multicomponent adenylate cyclase system [14-16] which is in charge of the biosynthesis of cAMP. Thus, the detailed molecular mechanism of desensitization is not yet established. In fact, it is not even clear how many cellular constituents are involved in the onset of desensitization, what their molecular properties are, where within the cell they are located or even whether the same mechanism operates in different tissues. In order to tackle these questions it is necessary to characterize a few experimental systems in which, on the one hand, the hormonal stimulation and desensitization proceed in intact cells (approaching in vivo conditions), and, on the other hand, it would be possible to rupture the cells delicately so as to allow a biochemical analysis of the cellular response in a physiologically relevant manner. This paper characterizes mouse thymocytes as a cellular system for studying the onset of cellular refractoriness.

Materials and Methods

Isolation of mouse thymocytes. Thymus glands were removed from C 3 H e B / B L F~ mice and transferred immediately to an RPMI-1640 buffer supplemented with 25 mM Hepes (pH 7.0) (buffer I) and kept at room temperature (22°C). The isolation of thymocytes from these glands was carried out as described previously [17,18]. All cell preparations used in these experiments contained over 90% viable cells, as judged by the standard Trypan blue staining. Assay of cAMP-dependent protein kinase and determination of its activity ratio, cAMP-dependent protein kinase was assayed as described previously [17,18]. The activity ratio or the state of activation of the enzyme was calculated from its activity measured in the absence of cAMP divided by its activity in the presence of 5 ~M cAMP. One unit of kinase activity is defined as that amount of enzyme which catalyzes (at 30°C) the transfer of 1 nmol 32p from [y-3zp]ATP onto histone H2b per min. Cell rupture and release of cAMP-dependent protein kinase. Samples of cells to be assayed for the state of activation of their cAMP-dependent protein kinase were spun down (15 s at 12000 × g) and their supernatant was discarded. The pellets (each containing 107 cells) were resuspended (2 min, 4°C) in 0.5 ml of buffer II, composed of 10 m M Tris/3 mM Mg(CH3COO)2/1 mM theophylline (pH 7.2). Under these hypotonic, low ionic strength conditions the cells are ruptured with concomitant release of over 70% of the cAMP-dependent protein kinase [18]. An aliquot (50/zl) of 1.5 M NaC1 in buffer II was then added to reverse any cytosol-to-nucleus translocation of the enzyme due to exposure to the low ionic strength medium [18]. After an additional incubation peroid of 1 min at 4°C, the hypotonized cell suspension was spun down (15 s at 1 2 0 0 0 × g ) and the supernatant was immediately assayed (within 45 s) for cAMP-dependent protein kinase. This soluble fraction (denoted '12000 × g supernatant') contains the cAMP-dependent protein kinase mostly in its undissociated form, i.e., it has a low activity ratio. It can therefore be used to detect free cAMP by its dissociation or the increase in its activity ratio. This kinase preparation was used in the

357

experiments described in Table I. The particular preparation used in these experiments contained a cAMP-dependent protein kinase activity of 0.05 and 0.26 units/ml when assayed in the absence and in the presence of 5 /tM cAMP, respectively. Asssay of cAMP phosphodiesterase. Phosphodiesterase activity was determined after rupture of the thymocytes (5- 107 cells) by suspending them in 0.5 ml of buffer II (without theophylline) for 2 min at 4°C. An aliquot (50/~1) of 1.5 M NaC1 in the same buffer was added (to adjust the ionic strength to physiological values) and the incubation was continued for 1 min more. The hypotonized cell suspension was centrifuged (12 000 x g, 15 s) and the supernatant thus obtained served as the enzyme source. Phosphodiesterase activity was assayed by the method of Thompson and Appleman [19] using 1 • 10 -5 M [8-3H]cAMP (final concentration) as a substrate. The reaction was initiated by addition of the enzyme (in this case the 12000 x g supernatant) to the reaction mixture. It was allowed to proceed for 30 min at 30°C. One unit of enzyme activity is defined as that amount which catalyzes (at 30°C) the conversion of 1 pmol cAMP to AMP per min. Under these conditions,

the reaction was shown to be linear with time (up to 30 min) and with cell concentration (up to 2- 108 hypotonized cells/ml). Assay of adenylate cyclase. Adenylate cyclase activity was determined following rupture of the thymocytes by suspension in buffer II (2 min at 4°C). The enzyme activity was assayed by measuring the formation of [32p]cAMP from [a-32p]ATP according to the procedure of Salomon [20]. When added, isoproterenol or prostaglandin E~ were at a concentration of 0.1 mM. The reaction was initiated by the addition of the enzyme (40/~1 of the hypotonized cell suspension, 108 cells/ml) and allowed to proceed for 10 min at 30°C. The reaction was stopped by adding 200 /~1 of a solution containing nonradioactive 1.3 mM cAMP, 45 mM ATP and 0.3 M HC1, then boiling the samples (5 min at 95°C). The samples were transferred immediately to an ice bath, an aliquot (100 /~1) of [8-3H]cAMP (20000 cpm) was added (as an internal standard) and the tubes were centrifuged (2 min, 12000 × g ) . Aliquots (300 /tl) of the supernatant were transferred into tubes with 150 ~1 of a solution comprising 0.25 M Tris/0.02 M EDTA (pH 12.5). The amount of [32p]cAMP formed was

TABLE I T H E ACTIVITY R A T I O OF c A M P - D E P E N D E N T PROTEIN K I N A S E M E A S U R E D IN VITRO IN T H E T H Y M O C Y T E SYSTEM IS N O T A R T I F I C I A L L Y I N C R E A S E D D U E TO A RELEASE OF cAMP U N A V A I L A B L E IN VIVO Samples (0.95 ml) of intact mouse thymocytes (1.1 • 107 cells/ml buffer I) were allowed to equilibrate for 90 min at 37°C. Aliquots (50 #1) of isoproterenol (2 m M in buffer I) or of buffer I alone were added and the incubation at 37°C was continued. After 0.5 rain the cells were spun down (15 s, 12000x g) and the pellets were hypotonized (2 rain at 4°C) in 0,5 ml buffer II (a and b), in buffer II supplemented with charcoal (10 m g / m l ) (c and d) or in a 12000 × g supernatant (e and f) (see Methods). Aliquots (50/~1) of 1.5 M NaCI in buffer II were added to each of the samples to adjust the ionic strength to physiological values, and the incubation at 4°C was continued for an additional minute. The samples were spun down (15 s at 12000× g) and 50 /~1 were removed and assayed for cAMP-dependent protein kinase activity in the absence or in the presence of 5 /zM cAMP. The values given in parenthesis (experiments e and f) are calculated by adding the activities measured in a and b to the activity values of the kinase added in the form of the 12000 × g supernatant. These would be the values expected if the exogenous cAMP-dependent protein kinase were not further dissociated due to release of sequestered cAMP upon hypotonic rupture of the cells. Pretreatment

(a) (b) (c) (d) (e) (f)

Buffer I Isoproterenol Buffer I Isoproterenol Buffer I Isoproterenol

Hypotonic rupture in:

buffer II buffer II buffer II + charcoal buffer II + charcoal 12 000 x g supernatant 12000 x g supernatant

cAMP-dependent protein kinase activity (units/ml) - cAMP

+ cAMP

0.24 0.58 0.25 0.56 0.30 (0.30) 0.64 (0.63)

0.63 0.71 0.75 0.77 1.00 (0.89) 1.04 (0.97)

Activity ratio

Rise in activity ratio

0.38 0.82 0.33 0.73 0.30 (0.34) 0.62 (0.65)

0.44 0.40 0.32 (0.31)

358 determined as described by Salomon [20]. Under the above conditions the reaction was shown to be linear with time and with cell concentration. Determination of intracellular and extracellular levels of cAMP. Intracellular cAMP was determined on cell pellets containing (2-5). 10 7 cells. The cells were ruptured (to release cAMP) by the addition of 0.2 ml of 0.1 M HC1 and boiling 3 min at 95°C [21]. Their cAMP was determined [22,23] using the assay kit provided by Amersham International. Hormonal stimulation of mouse thymocytes. Hormonal stimulation was carried out using freshly prepared viable thymocytes (0.95 ml, (1-5)-107 cells/ml buffer I) which had been allowed to equilibrate for 90-120 min at 37°C so as to ensure (prior to stimulation) that the intracellular cAMP was indeed at basal levels (cf. Ref. 17). Aliquots (50 /~1) of the appropriate agonist dissolved in buffer III (composed of 25 mM Hepes/125 mM NaC1 (pH 7.0)) were added, and the incubation at 37°C was continued for the indicated times. The cells were then spun down (15 s, 12000 × g), the supernatant was discarded, and the cAMP content or the various enzymatic activities were determined. Inhibition of protein synthesis by cycloheximide. Protein synthesis and its inhibition was monitored by the incorporation of [3SS]methionine. Thymocytes were isolated at 22°C as described above except for the fact that a methionine-free RPMI1640 medium (supplemented with 25 mM Hepes (pH 7.0), buffer IV) was used. The cells ((1-2). 108/ml), in 1 ml samples, were allowed to equilibrate for 90 min at 37°C (cf., previous paragraph), then spun down (1 min, 12000 × g) and the pellets obtained were each resuspended in 0.8 ml buffer IV. Aliquots (100 ~1) of either a cycloheximide solution (at the indicated concentration, in buffer IV) or of buffer IV alone were added to the cell suspensions. At the same time, each of the samples was supplemented also with 100 #1 of a 0.35 /~M solution of L-[35S]methionine (2. 105 c p m / p m o l ) in buffer IV, and was allowed to incubate (37°C). At the indicated times aliquots (100 ~1) were withdrawn, mixed with 20% trichloroacetic acid, dried and counted in 20 ml toluenebased scintillation fluid. The nonspecific adsorption of [35S]methionine onto the cell precipitates

was found to be negligible under our experimental conditions. Mice. Inbred C 3 H e B / B L F l mice were obtained from the Experimental Animal unit of the Weizmann Institute of Science. Materials. cAMP, ATP,GTP, prostaglandin E~, DL-isoproterenol, Dz-phenylephrine, glucagon. serotonin, cycloheximide, histone H2b, and bovine serum albumin were purchased from Sigma. [~,32p]ATP, [c~-32p]ATP, [8-3H]cAMP, and the cAMP assay kit (TRK 432) were from Amersham International. Histamine phosphate was obtained from Fischer Scientific Company. RPMI-1640 buffer supplemented with 25 mM Hepes (pH 7.0) was purchased from Gibco and Mes was obtained from Serva Feinbiochemica. The charcoal absorbent used in the experiments described in Table I was supplied by Amersham International with their cAMP assay kit mentioned above. Results and Discussion

Choice of the cellular system The choice of mouse thymocytes for studying the phenomenon of hormone-induced cellular refractoriness (desensitization) was based on the fact that these cells have the following features. (a) They can be isolated by a procedure [18] that does not involve the use of any digestive enzyme, thus avoiding the degradation or modification of cell surface constituents which may play an important role in desensitization. (b) These cells can be kept viable for several hours [18], a period of time which is long enough for both their hormonal stimulation and desensitization to get well under way. (c) These cells can be readily ruptured under mild conditions [18], making it possible to analyse the biochemical events associated with cellular refractoriness (see above). The hormonal response pattern of mouse thymocytes The response of mouse thymocytes to fl-agonists or prostaglandin E 1 is characterized by a rapid rise (within 30 s) in the intracellular level of cAMP, followed by a slower exponential decline in the level of this nucleotide. As seen in Fig. 1, the spike-like rise and fall in cAMP levels (panel A) is reflected in the activity of cAMP-dependent protein kinase (panel B) and, under the conditions of

359 --

I

I

I

I

I

A -

3o-

~i

~)

this experiment, can b e closely m o n i t o r e d b y the activity ratio of the e n z y m e (panel C). It should b e n o t e d that the rise a n d fall in the activity ratio of the kinase occurs within a c o n s t a n t p o p u l a t i o n of p o t e n t i a l l y active catalytic subunits, as i n d i c a t e d b y the fact that the total kinase activity m e a s u r e d in the presence of an excess of c A M P r e m a i n s essentially c o n s t a n t t h r o u g h o u t the experiment, m a i n t a i n i n g a level which is identical to that f o u n d in n o n - s t i m u l a t e d cells (Fig. 1B). This excludes the p o s s i b i l i t y that the rise in the activity ratio of the kinase involves previously u n a v a i l a b l e catalytic subunits. Similarly, it shows that the d e c l i n e in activity ratio is n o t due to some inactivation or i n s o l u b i l i z a t i o n of the enzyme.

1--

"

rocellulor

,o- I

-

500 •

o

~

400

--

~

"

Monitoring the hormonal response by the activity ratio of cAMP-dependent protein kinase

3oo

,,, :~ 2 0 0

Q.

~2

I00

O

I

I

[

I

I

t cl

0.8

i

0.7

x* 0.6

.2

0.5

0.4 0.3

I

0

l

I0

I

20

L

30

l

40

t

50

t

60

Time (rain) Fig. 1. The response of viable thymocytes to stimulation with isoproterenol as monitored by (A) the level of intracellular and extracellular cAMP, (B) the activity of cAMP-dependent protein kinase, (C) the activity ratio (state of activation), of cAMP-dependent protein kinase. Samples (0.9 ml) of thymocyte suspensions (5.5.107 cells/ml for (A) and 1.1.107 cells/ml for (B) and (C), all in buffer I) were pre-equilibrated for 90 min at 37°C. At the end of this preincubation (time zero) aliquots of 0.1 ml of either an isoproterenol solution (0.1 mM in buffer Ili) or buffer III alone were added to each sample. After an additional incubation (37°C) of each sample for the indicated time, the samples were spun down, saving the supernatants which were then used for determination of extracellular cAMP. The cells themselves were assayed for cAMP, for cAMP-depen-

In order to ascertain that, u n d e r the c o n d i t i o n s used in our e x p e r i m e n t a l system, the activity ratio which is m e a s u r e d in vitro after r u p t u r e of the cells a d e q u a t e l y reflects the in vivo state of activation of the enzyme, we carried out a few c o n t r o l experiments, which b e c a m e essential in view of the o b s e r v a t i o n s r e p o r t e d b y P a l m e r et al. [24]. It was a t t e m p t e d , first of all, to d e t e r m i n e whether the d i l u t i o n of the enzyme which occurs u p o n r u p t u r e of the cells causes b y itself a significant change in the activity ratio of the kinase (cf. Ref. 25). F o r this p u r p o s e we used the 12000 × g s u p e r n a t a n t o b t a i n e d after r u p t u r e of the t h y m o c y t e s a n d dissociated its c A M P - d e p e n d e n t p r o t e i n kinase b y including 1 • 10-7 M c A M P in it. This s u p e r n a t a n t was then diluted 50-fold a n d the activity ratio of its kinase was m o n i t o r e d for 5 rain. It was f o u n d that this ratio d i d not decrease significantly (less t h a n 15%) within the 3 rain that elapse between the h y p o t o n i z a t i o n of the t h y m o c y t e s a n d the as-

dent protein kinase, and for the activity ratio of this enzyme. Panel A: extracellular (&) and intracellular (O) cAMP content of isoproterenol-treated cells; extracellular (zx)and intracellular (O) cAMP content in control (buffer-treated) cells. Panel B: cAMP-dependent protein kinase activity in isoproterenoltreated cells (O, O) or control (buffer-treated) cells (zx, A) as measured in the absence (O, zx) or in the presence (O, A) of 5 #M cAMP. Panel C: activity ratio of cAMP-dependent protein kinase in isoproterenol-treated (O) or control (buffer-treated) cells (A)

360

say of the kinase (cf. Methods), in spite of the fact that the cAMP concentration in the diluted supernatant ( 2 . 1 0 -9 M) is considerably below the apparent K0. 5 for the cAMP-dependent activation of the enzyme ( 7 . 1 0 - S M [18]). This result is in agreement with the findings of Cherrington et al. [26]. Another question which we considered was whether, because of the hypotonic rupture of the cells, some cAMP that might have been sequestered in the intact cell is released, leading to an in vitro dissociation of cAMP-dependent protein kinase and to activity ratio values higher than those prevailing in the intact cell (cf. Ref. 24). We therefore stimulated the cells with isoproterenol and compared the activity ratio of their kinase after cell rupture in the absence or in the presence of charcoal (10 mg/ml). (This concentration of charcoal was shown to be sufficient to adsorb over 10-times the total intracellular content of cAMP in our samples.) Had the rise in activity ratio measured in vitro been partly due to the release (during cell rupture) of previously unavailable cAMP, such charcoal treatment would have curtailed the increase in the activity ratio of the kinase. However, as seen in Table I, the rise in kinase activity ratio (0.44) which occurs upon stimulation of the cells with isoproterenol is very similar to that obtained when the cell rupture was carried out in the presence of charcoal (0.40). This similarity could not be attributed to an adsorption of some released C subunits by the charcoal, since there is no significant difference in the total kinase activity measured with excess (5 /~M) cAMP, whether the hypotonic rupture was carried out in the presence of charcoal or not. In another set of experiments we attempted to detect any release (during cell rupture) of previously unavailable cAMP, using a preparation of undissociated cAMP-dependent protein kinase with a low activity ratio (0.21). As seen in Table I, when the rupture of the thymocytes was carried out in the presence of this exogenous kinase there was no evidence for a release of previously unavailable cAMP. In view of the above, it seems reasonable to assume that in our system and under our experimental conditions, the activation of cAMP-dependent protein kinase monitored in vitro (after cell rupture) adequately reflects the in vivo state of

activation of the enzyme and it can thus be taken as a detector for the changes in the intracellular level of cAMP. This is further corroborated by the fact that the change in the activity ratio of the kinase caused by a hormonal stimulation follows virtually the same kinetic pattern as the change in the intracellular level of cAMP (Fig. 1). It should be emphasized that while the endogenous cAMP-dependent protein kinase acts as a sensitive detector for cAMP, it will function only up to a certain cAMP level, a level which is limited by the total content of this kinase in the cell. However, since it is currently accepted that most, if not all, intracellular effects of cAMP are mediated through this kinase [27] then this limiting value is physiologically important, as it presumably reflects the maximally possible expression of intracellular cAMP levels.

The desensitization of thymocytes is receptor-specific It was previously shown [28] that the adenylate cyclase of thymocytes is not activated by serotonin, histamine or glucagon, while isoproterenol, epinephrine, prostaglandin E ~and the thymic humoral

T A B L E II THE RESPONSE OF MOUSE THYMOCYTES TO VARIO U S A G O N 1 S T S AS M E A S U R E D BY T H E A C T I V A T I O N O F T H E I R I N T R A C E L L U L A R c A M P - D E P E N D E N T PROTEIN KINASE Samples (0.95 ml) of i n t a c t m o u s e t h y m o c y t e s (I.1.107 c e l l s / m l buffer I) were allowed to equilibrate for 90 min at 37°C. A l i q u o t s (0.1 ml) of various agonists were a d d e d to give the i n d i c a t e d final concentration. After 0.5 min, the cells were spun d o w n (I 5 s, 12 000 × g ) and the cell pellets were h y p o t o n i z e d (2 m i n at 4°C). Agonist

Final concentration

Activity ratio

(M) Epinephrine Isoproterenol Prostaglandin E I Glucagon Phenylephrine Serotonin Histamine

1010 1010 10 1010 -

6 6 5 3 6 5 5

0.36 0.76 0.73 0.77 0.45 0.42 0.42 0.41

361 factor [29] b r i n g a b o u t a c o n s i d e r a b l e elevation of c A M P in these cells [29-32]. A s seen in T a b l e II, this specificity of response can also be d e m o n s t r a t e d at the level of c A M P - d e p e n d e n t p r o t e i n kinase, T h e s t i m u l a t i o n of intact m o u s e t h y m o cytes b y a n y one of the agonists in the latter group is i m m e d i a t e , a n d the activity ratio of the kinase reaches high values (over 0.7) within 0.5 min. S u b s e q u e n t l y the cells b e c o m e desensitized tow a r d s the stimulant. A s seen in Fig. 2, when the cells are triggered b y isoproterenol, then re-challenged with either the s a m e or with a h o m o l o g o u s agonist (epinephrine), the cells will not r e - r e s p o n d to it. T h e y will, however, b e s t i m u l a t e d b y a heterologous agonist such as p r o s t a g l a n d i n E j. Similarly, cells which have been s t i m u l a t e d b y prost a g l a n d i n E 1 will b e c o m e desensitized to this

r 08 -.[- P.--GE-2! _EPi_._ne~hi.....%e ÷

~_ 07 0,6 ~= a~ 0 5 -

I

~" O4 -~ ~

O3

'~

_

1

• Isp

i

. . . . . . . I

0

i

I

40

qEpi I

I

1

80

Time ( m i n )

Fig. 2. The desensitization of mouse thymocytes is hormonespecific. Samples (0.9 ml) of viable mouse thymocytes which had been allowed to equilibrate for 90 min at 37°C, were incubated with 50 /~1 of either an isoproterenol solution (0.2 mM in buffer III) (e) or of buffer III alone (©) which were added at time zero. At the indicated times the cell samples were spun down (15 s at 12000 x g), their cellular cAMP-dependent protein kinase was released, and its activity ratio was determined: At t = 10, 30 or 90 rain an attempt was made to restimulate the cells with isoproterenol (50 #1 of a 0.2 mM solution in buffer III) (A) using some of the cell samples, and at t = 90 rain a similar attempt was also made with epinephrine (11) and with prostaglandin E I (zx),in each case by addition of 50 #1 of a 0.2 mM solution in buffer IlL The immediate cellular response to these second challenges was measured after 0.5 min, by spinning the cells down and determining the activity ratio of their cAMP-dependent kinase. The upper dashed line represents the activity ratio of the kinase determined in freshly prepared intact thymocytes, 0.5 rnin after challenge with either epinephrine or prostaglandin E l (in both cases with 50 ttl of a 0.2 mM solution in buffer III). This value of activity ratio can be taken as a full response of the cells to these hormones under our experimental conditions. PGEI, prostaglandin El; Isp, isoproterenol; Epi, epinephrine.

agonist b u t will b e fully responsive to e p i n e p h r i n e or i s o p r o t e r e n o l (not shown).

Excluding some of the possible mechanisms for desensitization The fact that the desensitization of t h y m o c y t e s is a receptor-specific process a l r e a d y excludes the possibility that desensitization involves a general cellular paralysis or a d e p l e t i o n of some m e t a b o lites (e.g., G T P or A T P ) which are essential for the t r a n s d u c t i o n of the h o r m o n a l signal or for the p r o d u c t i o n of c A M P . F o l l o w i n g the intracellular as well as the extracellular levels of c A M P d u r i n g the course of the h o r m o n a l response ( s t i m u l a t i o n a n d desensitization) clearly shows (Fig. 1A) that even t h o u g h a certain a m o u n t of c A M P is released into the m e d i u m of the i s o p r o t e r e n o l - t r e a t e d cells, it is certainly not sufficient to account for the d r o p in intracellular c A M P levels. A n o t h e r possibility that was c o n s i d e r e d was that after h o r m o n a l s t i m u l a t i o n the cells either inactivate the h o r m o n e itself, or internalize it, or release into the m e d i u m an i n h i b i t o r that i m p a i r s the function of the hormone. These possibilities were excluded b y stimulating cells and, at various times during the process of desensitization, spinning t h e m d o w n a n d showing that the s u p e r n a t a n t was c a p a b l e of fully stimulating fresh cells (Fig. 3). A plausible m e c h a n i s m for desensitization could involve h o r m o n e - t r i g g e r e d activation of c A M P p h o s p h o d i e s t e r a s e [8,9], which could result in the lowering of c A M P levels a n d of the activity ratio of the kinase. However, no significant change was f o u n d in the overall (soluble) c A M P - p h o s p h o diesterase activity as it r e m a i n e d c o n s t a n t (at 17 + 2 u n i t s / 1 0 7 cells) in 12000 x g s u p e r n a t a n t s obt a i n e d from cells which h a d been s t i m u l a t e d for various d u r a t i o n s (up to 60 min) with isoproterenol. In fact these values were f o u n d to be very similar to those o b t a i n e d from cells which were n o t s t i m u l a t e d with the h o r m o n e . T h e r a p i d i t y of the onset of the refractory state in t h y m o c y t e s (cf. Fig. 1) a l r e a d y suggests that this process is n o t associated with a newly synthesized p r o t e i n m e d i a t o r . Indeed, when p r o t e i n synthesis was arrested (e.g., b y i n c u b a t i n g these cells with 10 3 M cycloheximide) the p a t t e r n of their response to h o r m o n e was f o u n d to be identical to that of n o n - t r e a t e d cells (not illustrated).

362

O3

I 0

I

I 4:

L

I 80

II

Time (min) Fig. 3. The extracellular medium of isoproterenol-treated cells which are already in the refractory state is capable of stimulating fresh cells to their maximal immediate response. Samples (0.95 ml) of viable mouse thymocytes which has been allowed to equilibrate for 90 min at 37°C (1.1.10’ cells/ml of buffer I) were incubated with 50 pl of either an isoproterenol solution (0.2 mM in buffer III) (0) or of buffer III alone (0). At the indicated times the cell samples were spun down (15 s at 12000 x g), the supernatants were saved. and the activity ratio of the cells in the pellets was determined. Pellets of fresh (untreated) thymocytes (1.10’ cells) were suspended, each in 1 ml obtained from a duplicate of the above-mentioned supernatants, and the activity ratio of the CAMP-dependent protein kinase in these cells was determined 0.5 min after suspension

Fig. 4. The response of thymocytes to stimulation by isoproterenol as monitored (after cell rupture) by the time-dependent changes in the activity of the adenylate cyclase. Samples (0.95 ml) of thymocytes (5.25’10’ cells/ml of buffer I) were allowed to equilibrate for 2 h at 37’C. At time zero the cells were stimulated by addition of 50 ~1 0.2 mM DL-isoproterenol (m) in buffer III or with buffer III alone as a control (0). At the indicated times, the cells were spun down and hypotonized (2 min, 4’C), and their adenylate cyclase activity was measured in the presence

(B) and in the absence

(0) of DL-isoproterenol

(0.1

mM).

(A).

Attributing the deJen.sitization of thymocytes to their adenylate cyclase system As seen in Fig. 4, the adenylate cyclase activity of thymocytes rises immediately after hormonal stimulation from approx. 10 to approx. 50 pm01 CAMP formed per min per lo6 cells. The cyclase activation is instantaneously attenuated, bringing the cyclase activity back to basal values, in spite of the fact that an excess of intact hormone is available to the cells. This activation and attenuation (measured in vitro after cell rupture) can thus account for the hormone-induced stimulation and desensitization of viable thymocytes. Moreover, this immediate attenuation is restricted to homologous hormones. For example, cells which are fully desensitized to isoproterenol (cf. A and C vs. B in Table III) are still capable of full cyclase activation in response to stimulation by prostaglandin E, (cf. D and E in Table III). It seems, therefore, that not only the desensitization of thymocytes but also their hormone specificity can be attributed to their adenylate cyclase system. This is supported by the fact that the processes of

stimulation and desensitization have identical hormone concentration dependencies (half maximal increase at approx. 60 nM), suggesting that the two processes are mechanistically linked and probably share a rate-limiting step. The above results are in agreement with several reports in the literature which showed (in other systems) that the hormonal activation of adenylate cyclase is agonist-specific and that this enzyme undergoes a hormone-induced desensitization [33,34]. The immediate desensitization in thymocytes is reversible and occurs without apparent loss in the number of functional receptors Several reports in the literature indicate that a change in receptor density on the cell surface (e.g., by internalization) is one of the ways through which the cell regulates its response to hormones [7,15], including catecholamines. However, it was pointed out [34] that these changes may serve primarily as a long-term regulatory mechanism, since from the kinetic point of view they do not adequately account for the rapid desensitization which occurs in some tissues [34]. As mentioned above, the onset of refractoriness

363 T A B L E III T H E DESENSITIZATION OF A D E N Y L A T E CYCLASE IN T H Y M O C Y T E S IS H O R M O N E - S P E C I F I C Samples (0.95 ml) of viable mouse thymocytes (5.25.107 cells/ml buffer I) were allowed to equilibrate for 90 min at 37°C. Aliquots (50 #1) of an isoproterenol solution (5 m M in buffer III) were added to some of the samples (columns C and E) in order to cause an in vivo cellular refractoriness. Buffer III alone was added to other samples (columns A, B and D) which served as controls. All samples continued to be incubated at 37°C. After 30 rain, the samples were spun down (15 s at 12000× g) and the cells in the pellets were hypotonized by suspension in buffer II (2 min at 4°C). Aliquots (40/.tl) were removed from these cell suspensions and assayed for adenylate cyclase activity in the presence of 10 # M GTP. (A) Basal values of adenylate cyclase in the cells (no hormone added). (B-E) Adenylate cyclase activities measured in the presence of either isoproterenol (B and C) or prostaglandin E I (D and E), each at a final concentration of 1.10 -4 M. Values given are the mean + S.E. of five experiments, each assayed in duplicate. The value of 100% activity was assigned in each experiment to the cyclase activity obtained from freshly prepared (non-treated) cells upon stimulation in vitro with isoproterenol. This reference cyclase activity varied in different experiments between 20 and 35 pmol cAMP formed/106 cells per 10 min. Adenylate cyclase activity

(%) A B C D E

41 5:10 100 525:6 1065:5 90+ 7

in mouse thymocytes occurs within a few minutes after hormonal stimulation (cf. Fig. 1). The experiment depicted in Fig. 5 shows that this immediate desensitization can be readily reversed. When thymocytes are stimulated with isoproterenol and allowed to desensitize (Fig. 5A), then washed extensively, their activity ratio drops back to basal levels (Fig. 5B). Upon rechallenge with fresh hormone (monitoring their immediate maximal response, Fig. 5C), it is found that their capability to fully respond to the hormone is regained, provided that their previous contact with the hormone was brief (less than approx. 20 min). Cells that have a longer contact period with the hormone, before being washed and restimulated, gradually lose their ability to respond to a secondary challenge (a loss of about 25% within 60 min, Fig. 5C), indicating a

/ I

,

I

,

I

,

I /

0 20 40 60 EXPOSUREPERIOD(mini I Washing, 70 rain ncubation, then

+Buffe/

_o.--

~<~-~08

B

~soproterenol

=~0.8

o~

>-+

>-+

~ _ 0.6 _>~ i-<

~-

_>~ i-<

~2 0.4

0.6

.

,

0 20 40 60 EXPOSURE PERIOD( m i n )

i

,

1

,

1

,

I

/

0 20 40 60 EXPOSURE PERIOD(min)

Fig. 5. The extent of resensitization of thymocytes already desensitized to isoproterenol depends on the contact period between the cells and the hormone. Samples (0.95 ml) of mouse thymocytes (2.2.107 cells/ml buffer I) were allowed to equilibrate for 90 min at 37°C. At the end of this period (time zero) aliquots (50 #1) of either an isoproterenol solution (0.2 m M in buffer III) ( i ) or buffer III alone ( i ) were added to each sample. After an additional incubation of each sample (37°C) for the indicated time, some of the cell samples were spun down (15 s at 12000x g) and the activity ratio of their cAMPdependent protein kinase was determined (panel A). The remaining cell samples were also spun down as above, washed twice more with 1 ml of buffer I and resuspended in 0.95 ml of the same buffer. After an additional incubation of 70 min at 37°C, the cells were rechallenged with an aliquot (50 #1) of a 0.2 m M solution of isoproterenol in buffer III (panel C), or buffer III alone (panel B). The cells were then further incubated for 0.5 min at 37°C, spun down, and the activity ratio of their cAMP-dependent protein kinase was determined.

partial loss of functional hormone receptors. The extent of resensitization thus depends on the contact period between the cells and the hormone during the primary stimulation. Yet thymocytes which were stimulated with isoproterenol, then extensively washed and allowed to stand (at 37°C) for either 70 rain or 180 min before rechallenge with the hormone, responded equally well to the secondary challenge. In both cases there was an abrupt rise (within 30 s) in the

364

activity ratio of their intracellular cAMP-dependent protein kinase to a value of 0.7. It seems, therefore, that the period of time which elapses between the primary and the secondary hormonal challenge does not by itself affect the extent of resensitization, if the cells are not exposed to the hormone during this period. Our results with the thymocyte system are compatible with the model proposed by Perkins and his collaborators [34,35], which suggests that catecholamine-specific desensitization involves at least two sequential steps: (a) A rapid, readily reversible conversion of the receptor (R) to a modified form (R') that no longer activates the catalytic subunit of adenylate cyclase, but still retains the ability to specifically bind the agonist. The system is then referred to as functionally uncoupled; (b) A slower conversion of the R' form to another form (R") which is neither capable of activating the catalytic subunit of the cyclase nor binds the agonist. The system can then be regarded as 'locked' in the desensitized state. The observations reported here indicate that a continuous exposure to the hormone is required not only for triggering the chain of events which leads to the readily reversible desensitized state but also for the process which transfers the cells to the 'locked' desensitized state.

Concluding remarks Using the model cellular system characterized in this paper, it is possible to stimulate and desensitize viable cells, rupture them under mild conditions, and analyze the biochemical consequences of these processes on three levels: the activation of adenylate cyclase, the intracellular cAMP, and the activation of cAMP-dependent protein kinase. The suitability of this system for studying the molecular events involved in the onset of hormone-induced desensitization had already been demonstrated in two recent reports from our laboratory [6,36]. These studies described chemical as well as physical means for selectively blocking the desensitization of the cells (while preserving their capacity to respond to hormonal stimuli), i.e., for experimentally dissecting hormonal stimulation from desensitization.

Acknowledgements This work was supported by a grant (No. 3131) from the U.S.-Israel Binational Science Foundation. We thank Mrs. Elana Friedman for excellent secretarial assistance. S.S. is the incumbent of the Hella and Derrick Kleeman Chair in Biochemistry, The Weizmann Institute of Science.

References 1 Rodbell, M. (1980) Nature 284, 17-22 2 Walsh, D.A., Perkins, J.P. and Krebs, E.G. (1968) J. Biol. Chem. 243, 3763-3765 3 Kakiuchi, S. and Rall, T.W. (1968) Mol. Pharmacol. 4, 367-378 4 Barber, R., Clark, R.B., Keely, L.A. and Butcher, R.W. (1978) Adv. Cyclic Nucleotide Res. 9, 507-516 5 Wessels, M.P., Mullikin, D. and Lefkowitz, R.J. (1978) J. Biol. Chem. 253, 3371-3373 6 Zick, Y., Cesla, R. and Shaltiel, S. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5967-5971 7 Tell, G.P., Haour, F. and Saez, J.M. (1978) Metabolism 27, 1566-1596

8 Manganiello, V.C. and Vaughan, M. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 269-273 9 Nemeck, G.M., Ray, K.P. and Butcher, R.W. (1979) J. Biol. Chem. 254, 598-601 10 Kelley, L.A. and Butcher, R.W. (1974) J. Biol. Chem. 249, 3098-3102 11 Ho, R.J. and Sutherland, E.W. (1971) J. Biol. Chem. 246, 6822-6827 12 Fain, J.N. and Shepherd, R.E. (1975) J. Biol. Chem. 250, 6586-6592 13 Terasaki, W.L., Brooker, G., DeVellis, J., Inglish, D., Hsu, C.Y. and Moylan, R.D. (1978) Adv. Cyclic Nucleotide Res. 9, 33-52 14 Ross, E.M., Haga, T., Howlett, A.C., Schwarzmeier, J., Schleifer, L.S. and Gilman, A.G. (1978) Adv. Cyclic Nucleotide Res. 9, 53-68 15 Lefkowitz, R.J. and Williams, L.T. (1978) Adv. Cyclic Nucleotide Res. 9, 1-17 16 Perkins, J.P., Johnson, G.L. and Harden, T.K. (1978) Adv. Cyclic Nucleotide Res. 9, 19-31 17 Zick, Y., Cesla, R. and Shaltiel, S. (1978) FEBS Lett. 90, 239-242 18 Zick, Y., Cesla, R. and Shahiel, S. (1979) J. Biol. Chem. 254, 879-887 19 Thompson, W.J. and Appleman, M.M. (1971) Biochemistry 10, 311-316 20 Salomon, Y. (1979) Adv. Cyclic Nucleotide Res. 10, 35-55 21 Yakir, Y., Kook, A.I., Schlesinger, M. and Trainin, N. (1977) Isr. J. Med. Sci. 13, 1191-1196 22 Gilman, A.G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312 23 Brown, B.L., Albano, J.D.M., Ekins, R.P., Sgherzi, A.M. and Tampion, W. (1971) Biochem. J. 121, 561-562

365 24 Palmer, W.K., McPherson, J.M. and Walsh, D.A. (1980) J. Biol. Chem. 255, 2663-2666 25 Corbin, J.D., Soderling, T.R. and Park, C.R. (1973) J. Biol. Chem. 248, 1813-1821 26 Cherrington, A.D., Assimacopoules, F.D., Harper, S.C., Corbin, J.D., Park, C.R. and Exton, J.H. (1976) J. Biol. Chem. 251, 5209-5218 27 Kuo, J.F. and Greengard, P. (1969) Proc. Natl. Acad. Sci. U.S.A. 64, 1349-1355 28 Makman, M.H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 885-889 29 Trainin, N. (1974) Physiol. Rev. 54, 272-315

30 Franks, D.J., MacManus, J.P. and Witfield, J.F. (1971) Biochem. Biophys. Res. Commun. 44, 1177-1183 31 Bach, M.A. (1975) J. Clin. Invest. 55, 1074-1081 32 Niaudet, P., Beaurain, G. and Bach, M.A. (1976) Eur. J. Immunol. 6, 834-836 33 Ross, E.M. and Gilman, A.G. (1980) Annu. Rev. Biochem. 49, 533-564 34 Perkins, J.P., Harden, T.K. and Harper, J.F. (1982) Handb. Exp. Pharmacol. 58/I, 187-224 35 Su, Y.F., Harden, T.K. and Perkins, J.P. (1979) J. Biol. Chem. 254, 38-41 36 Zick, Y., Cesla, R. and Shaltiel, S. (1982) J. Biol. Chem., 257, 4253-4259