Potentiation by steroids of the β-adrenergic agent-induced stimulation of cyclic AMP in isolated mouse thymocytes

Biochimica et Biophysica Acta, 762 (1983) 315-324 Elsevier Biomedical Press

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BBA 11139

POTENTIATION BY STEROIDS OF THE/3-ADRENERGIC AGENT-INDUCED STIMULATION OF CYCLIC AMP IN ISOLATED MOUSE THYMOCYTES SYLVIE DURANT, DOMINIQUE DUVAL and FRAN(~O1SE HOMO-DELARCHE I N S E R M UT, Physiology and Pharmacology, H@ital Necker, 161, rue de SOvres, 75015 Paris (France)

(Received July 2nd, 1982) (Revised manuscript received December 20th, 1982)

Key words: cyclic A M P stimulation," Dexamethasone," fl-Adrenergic agent; (Mouse thymocyte)

There is an increasing amount of evidence suggesting that glucocorticoids may modulate the responsiveness of various cell types to ~-adrenergic agents. In some systems, it has been shown, in addition, that steroids potentiate the elevation of cAMP induced by catecholamines. Little is known however of the mechanism underlying steroid action. We have studied this 'permissive action' in isolated thymocytes which have specific receptor sites for both glucocorticoids and fl-adrenergic agents. The glucocorticoid compound dexamethasone did not alter intracellular cAMP level but markedly enhanced the stimulation produced by isoproterenol. This effect was instantaneous and was still measurable at 10- 7 M dexamethasone. A similar potentiating action was observed in the presence of corticosterone but also in the presence of sex steroids. Determination of fl-receptors after cell preincubation in the presence of dexamethasone showed that rapid alterations in fl-receptors are not involved in this permissive action. Experiments done in the presence of the calcium chelator, ethyleneglycol bis(fl-aminoethyl ether)-N,N'-tetraacetic acid, suggest that dexamethasone action could be related to a modification of calcium mobilization.

Introduction

It appears that, under certain conditions, steroids are a prerequisite for the full expression of other hormonal activities, particularly those mediated through a stimulation of cyclic AMP (cAMP). Most of these so-called permissive effects of steroids correspond to classical receptor-mediated actions of steroid hormones. Indeed, they always require a lag period before emergence of an effect and are blocked by inhibitors of RNA and protein synthesis. Exton et al. [1] showed, for example, that the stimulation of glucose production by glucagon and epinephrine was impaired in liver from adrenalectomized rats and that normal Abbreviation: EGTA, ethyleneglycol bis(/3-aminoethyl ether)N, N'-tetraacetic acid. 0167-4889/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

stimulation could be restored within 60 min following glucocorticoid infusion. Several explanations have been proposed to account for these permissive effects of steroids but the situation remains unclear. The regulation of fl-adrenergic receptors in response to modulation of the circulating levels of steroid hormones may in part play a role in this phenomenon [2-5]. It was also suggested that corticosteroids could modulate intracellular cAMP action, decreasing its hydrolysis by the phosphodiesterase enzymes. Indeed, several authors demonstrated an increase in phosphodiesterase activity following adrenalectomy in various target tissues [6], whereas Ross et al. [7] reported a decreased enzyme activity in HTC cells grown for 24 h in the presence of dexamethasone. On the other hand, the experimental results of Exton et al. [1] showed that the level of cAMP

316 elicited by glucagon stimulation in the liver of adrenalectomized animals was similar and even higher than that in intact animals. Several authors, thus, pointed out that steroids are not required for normal cAMP accumulation but may be necessary for maintenance of normal cell responsiveness to the hormonal stimuli [8,9]. In addition, Liu and Greengard [10] showed that cortisol treatment specifically decreased 32p incorporation in a rat liver cytosolic protein, whose phosphorylation is also regulated by a cAMP-dependent protein kinase. They therefore suggested that this protein may represent a common intermediate in steroid and cyclic nucleotide action. Finally, Rasmussen and Tenenhouse [11] postulated that steroids could modulate cyclic nucleotide action by controlling intracellular calcium concentration. Besides these long term permissive effects, several authors have reported the ability of steroids to provoke in vitro rapid permissive action on intracellular cAMP concentrations. Tolone and coworkers [12] showed that hydrocortisone, which does not affect the basal level of cAMP, markedly potentiates the elevation of cyclic nucleotide induced by epinephrine in isolated intraperitoneal mast cells. Similar effects were also observed in normal human lymphocytes by Lee and Reed [13], Mendelsohn et al. [14] and Marone et al. [15]. The mechanism of these rapid permissive effects of corticosteroids remained essentially unknown but did not correspond to the classical action of steroid hormones. We have therefore studied the effect of glucosteroids, alone or in combination with isoproterenol, on the intracellular cAMP content in isolated mouse thymocytes. These cells which are known to represent glucocorticoid target cells [16,17] are also sensitive to the action of catecholamines [ 18-20]. The purpose of our study was to determine the optimal conditions for the observation of these rapid permissive actions and to investigate the mechanisms involved in their action.

Material and Methods Reagents. Dexamethasone, corticosterone, 17flestradiol, testosterone, progesterone and pros-

taglandin E 2 were purchased from Sigma. Stock solutions (10-2-10 -3 M) were prepared in absolute ethanol and diluted to the appropriate concentration before each experiment. The final ethanol concentration was less than 1%, a concentration which did not modify cell viability. The various fl-agonists or antagonists and the beef heart phosphodiesterase were from Sigma. All solutions were prepared fresh before use. Cyclic AMP antiserum and [~25I]cAMP (1500-2000 C i / m m o l ) were purchased from Institut Pasteur (Paris, France). [3H]Dihydroalprenolol (42 C i / mmol) was obtained from Amersham International, U.K. Minimum essential medium and supplements were obtained from Gibco biocult. All other reagents were of the highest available purity. Animals. Female C57 B L / 6 1nice, 6-8 weeks old, 15-20 g body weight were purchased from Iffa-Credo (France). The animals were adrenalectomized under pentobarbitone anesthesia at least 2 weeks before experiments and kept with free access to 0.9% saline solution and standard diet. Isolation of thymocytes. The procedure of cell isolation has been described in detail elsewhere and was carried out at 20°C [21]. The cells were adjusted to 2- 10 7 cells/ml in minimum essential medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine and 1% nonessential amino acid solution. The minimum essential medium used in our experiments was designed for cell suspensions and did not contain calcium ions (Gibco Biocult ref 138). The cell viability was determined at the end of the isolation procedure by Trypan blue exclusion and was always greater than 95%. Determination of cyclic AMP content. The determination of intracellular cAMP was made by radioimmunoassay [22] according to the method described by Steiner et al. [23]. The cell suspensions were incubated at 37°C in a 95% air/5% CO 2 atmosphere with the various agents tested, but in the absence of inhibitors of phosphodiesterase. At various intervals after drug addition the reaction was terminated by addition of 1 ml ethanol and boiling for 6 min. After centrifugation at 400 × g for 30 min, the supernatants were immediately frozen and lyophilized. The extracts were resuspended in 50 mM sodium acetate buffer (pH 6.2) and acetylated according to Frandsen

317

and Krishna [24] in the presence of acetic anhydride and triethylamine. 100-/~1 aliquots of each sample were then incubated overnight at 4°C in the presence of tracer (7000-10000 cpm of [125I]cAMP) and of 100/~1 antiserum (diluted 1 to 20 000). The separation of bound and free radioactivity was performed using ice-cold ethanol and rabbit serum as carrier. After centrifugation at 400 × g for 15 min the amount of radioactivity collected in the pellet was determined. The validity of the assay was verified by the following observations: the amount of cAMP measured was a linear function of the number of cells per sample up to 108 cells/ml; the yield of the extraction and acetylation steps determined by addition of known amounts of exogenous cAMP were close to 100% (results not shown); in addition, incubation of the samples for 1 h at 37°C in the presence of purified beef heart phosphodiesterase (EC 3.1.4.17, 0.5 U / t u b e ) destroyed more than 98% of the measurable cAMP. Under our experimental conditions the sensitivity of the method was 0.01 pmol/ml. Binding experiments. The determination of fladrenergic receptors was made on crude membrane preparations using [3 H]dihydroalprenolol as radioligand [25]. The cell suspension was first centrifuged at 250 x g for 5 min and the cell pellet resuspended in 5 mM Tris buffer (pH 7.8)/10 mM MgC12. After homogenization using a Branson B12 sonifier (three periods of 5 s at maximal intensity), the homogenate was centrifuged at 400 x g for 10 min. The supernatant was centrifuged again at 40000 x g for 15 min. The membrane pellet was finally resuspended in 50 mM Tris buffer (pH 7.8)/10 mM MgC12/0.1% ascorbic acid and carefully homogenized by several passages through a syringe needle. All this preparation was carried out at 0 - 4 °. Aliquots of the membrane suspension (0.1-0.2 mg protein) were then incubated at 30°C in the presence of increasing concentrations of 3H-labeled ligand (1-20 nM). Nonspecific binding was determined by parallel incubation in the presence of 10/~M (+)-propranolol. Incubations were terminated by addition of 10 vols. of ice-cold Tris buffer and filtration through Whatman G F / C filters. The filters were washed three times with 20 ml ice-cold Tris buffer, dried and counted by liquid scintillation spectrometry.

Protein determination. Determination of protein content was performed according to Lowry et al. [26] using bovine serum albumin as standard. Results

Optimal conditions for the determination of basal cAMP content In preliminary experiments the evolution of the basal level of cAMP was determined as a function of time following isolation and resuspension of the cells at 37°C in minimal essential medium. As shown in Fig. 1, this level was extremely high immediately after isolation but declined gradually to reach a plateau after 60 min incubation. In all the subsequent experiments the cells were thus preincubated for 60 min at 37°C before drug addition. It should be noted that even under these experimental conditions the intracellular level of cAMP greatly varied from one experimental to another. The range of basal values in 52 experimental samples (four to six thymuses were pooled in each experiment) is represented in Fig. 2. In about 60% of these experiments, the basal cAMP content was below 1 pmol/107 cells, in good agreement with other reports [20,27,28]. In some experiments,

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L-ISOPROTERENOL CONCENTRATION(M) Fig. 4. Dose-response curve of the isoproterenol-elicitedstimulation of cAMP content. The cAMP content was measured by radioimmunoassay at various times followingisoproterenol addition. Each value expressed as stimulation ratio represents the mean of duplicate determinations in a typical experiment, taken at the peak response, which in this experiment is reached 3 min after fl-agonist stimulation, In this experiment basal cAMP content was 1.1 pmol/107 cells. Similar results were obtained in five experiments.

Kinetics of cAMP accumulation after stimulation by &oproterenol As shown in Fig. 3, 1 0 - 6 M L-isoproterenol i n d u c e d a very rapid increase of c A M P c o n t e n t which reached a m a x i m u m 1-3 m i n following drug a d d i t i o n a n d which, in the absence of phosphodiesterase inhibitors, rapidly declined to basal level after 4 5 - 6 0 rain incubation. P r e i n c u b a t i o n of the cells in the presence of 10 5 M p r o p r a n o l o l completely abolished the stimulatory effect of 1 0 - 6 M isoproterenol, thus suggesting that this effect is mediated through a n interaction with specific B-receptors (data not shown).

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TIME (minutes) Fig. 3. Time course of the stimulation of cAMP content by isoproterenol 10-6 M. The ceils (107 cells/ml) were first preincubated at 37°C for 60 min before the addition of the drug. At various intervals following isoproterenol addition, the cAMP content was determined by radioimmunoassay. Each value represents the mean of duplicate determinations and is expressed as stimulation ratio (i.e., cAMP content in the presence of the drug over basal cAMP content). Result of a typical experiment: basal cAMP value was 1.3 pmol/107 cells. Similar patterns of stimulation were obtained in 30 experiments.

Dose response curve of the isoproterenol-induced stimulation of cAMP content The effect of increasing c o n c e n t r a t i o n s of isoproterenol, in the range 1 0 - ] ° - 1 0 -3 M, was d e t e r m i n e d 3 m i n after drug addition. As shown in Fig. 4, elevation of c A M P c o n c e n t r a t i o n only became significant at 10-7 M and increased a b r u p t l y between 10 -7 a n d 10 -5 M to reach a plateau level above 10 -5 M. This p a t t e r n of stimulation corresponds to that described in other target tissues [29,30],

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Fig. 5. Potentiation of the isoproterenol-induced stimulation of cAMP content by dexamethasone. The cells (107 cells/ml) were incubated in the presence of 1 0 - r M isoproterenol (O), steroid (O) or isoproterenol plus steroid (A). In these experiments the 1 0 - S M glucocorticoid was added 10 rain before isoproterenol (A). At various intervals following isoproterenol addition the content of cAMP was determined by radioimmunoassay. Each value expressed as stimulation ratio represents the mean of duplicate determinations in a typical experiment with a basal cAMP value of 1.35 pmol/107 cells. Similar results were obtained in 23 experiments.

Effect of steroids on isoproterenol-induced stimulation of cAMP We first demonstrated that none of the steroids tested (10-5-10 -7 M) induced any modification of the basal level of cAMP (data not shown). a) Effect of dexamethasone. As shown in Fig. 5, 10 -5 M dexamethasone preincubated with the cells for l0 min before 10 -6 M isoproterenol addition markedly potentiated cAMP elevation. It should be noted that steroid treatment did not significantly alter the kinetics of cAMP production but increased the stimulation ratio observed in the presence of isoproterenol. We have attempted to determine the time-course of the dexamethasone action. In some, but not all, of these experiments the effect of dexamethasone was seen almost instantaneously following addition and gradually decreased with the time of preincubation (Fig. 6). It is important to note that during a 30 min incubation period in the absence of any stimulus, the thymocytes partially lost their responsiveness to fl-adrenergic stimulation. A similar emergence of refractoriness was observed in rat

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larly performed under these experimental conditions (10 min preincubation with steroid before isoproterenol addition). In the following experiments we have measured the effects of increasing doses of dexamethasone on the accumulation of cAMP elicited by various concentrations of isoproterenol. As shown in Fig. 7, there is a progressive stimulation of cAMP content with increasing concentrations of L-isoproterenol, reaching a maximum at 10 -5 M as already described in Fig. 4. It appears however that the effect of dexamethasone greatly varied according to the concentration of isoproterenol tested. At submaximal concentrations (i.e., 10 - 7 and 10 -6 M ) the action of dez~amethasone markedly increased with increasing concentration of steroid up to 10 -4 M. In contrast, at 10-SM isoproterenol the action of dexamethasone is no longer visible whatever the concentration of steroid tested (Fig. 7). We have also tested the effect of ethanol on either the basal cAMP content or the stimulation induced by isoproterenol. Even at the highest concentration tested (1%), ethanol did not alter these parameters (data not shown). In addition, we demonstrated that dexamethasone did not change the

pattern of cAMP release in the incubation medium (results not shown). b) Effect of corticosterone. We have also tested the action of corticosterone, the major circulating glucocorticoid in rodents, on the isoproterenol-induced stimulation of cAMP content. As shown in Table I, corticosterone exhibited a potentiating effect comparable to that of dexamethasone. c) Effect of sex steroids. The sex steroids (tested at 1 0 - 7 M in three experiments) were able to potentiate the action of isoproterenol (10 6 M), although progesterone appeared more potent than testosterone and 17fl-estradiol, as shown by the stimulation ratio (control, 3.56; progesterone, 24.20; testosterone, 8.50; 17fl-estradiol, 9.10). These results thus indicate that the potentiating action of steroids is not specific for glucocorticoid compouds and suggest that it may represent a non-genomic action of steroid molecules.

Characterization of fl-adrenergic receptors in thymocyte membranes One of the possible sites of steroid action may be the fl-receptor itself. Indeed, several authors have reported a modulation of fl-receptors following hormonal manipulation [2,5,31]. Given the rapidity of the steroid action it appears unlikely

TABLE I E F F E C T OF 10 -5 M C O R T I C O S T E R O N E ON L-ISOPROT E R E N O L - I N D U C E D S T I M U L A T I O N OF cAMP CONTENT The experimental procedure was similar to that described in the legend of Fig. 5. The action of isoproterenol ( + corticosterone) was measured at 0.5, 1, 3 and 6 min following isoproterenol addition and expressed as stimulation ratio. In this experiment, the basal cAMP value was 0.64 pmol/107 cells. Similar results were obtained in three experiments. Time (min)

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that it could alter either receptor synthesis or degradation, but it could well modify the affinity of the receptors for the agonist ox lead to an unmasking of unoccupied binding sites. We have thus studied the characteristics of [3H]dihydroalprenolol binding in thymocyte membranes. The kinetics of association of 10 -9 M [3H]dihydroalprenolol to thymocyte membrane preparations at 30°C is shown in Fig. 8. The specific binding increased rapidly with time to reach a plateau level 10-20 rain after the beginning of the incubation. In all subsequent experiments the samples were thus incubated during 20 min at 30°C. The association of the tracer with its membrane binding sites appeared readily reversible upon addition of unlabeled (+)-propranolol or following dilution of the sample in a large volume of buffer (data not shown). Determination of the specific binding of increasing concentrations of [3H]dihydroalprenolol showed that saturation was achieved at concentrations close to 8-10 nM, whereas the affinity of these binding sites, calculated according to Scatchard [32] was 3.4 nM (Fig. 9). The number of specific binding sites per cell averaged 400 sites. The specificity of these binding sites was studied by competition experiments: the ability of increas-

ing concentrations of an unlabeled competitor to displace 5 • 10 - 9 M [3H]dihydroalprenolol from its binding sites being representative of its affinity for the receptors. The relative order of affinity among the compounds tested was the following: isoproterenol > alprenolol > epinephrine > norepinephrine > phentolamine > clonidine. The ( - )-isomers of these compounds exhibited as least 10-fold more affinity for the binding sites than the ( + ) forms. These characteristics are therefore compatible with the binding of [3H]dihydroalprenolol to specific f12 receptors on mouse thymocyte membranes, which is in good agreement with others reports [33,34]. On the other hand, we have studied the effect of cell preincubation, in the presence of dexamethasone, on membrane fl receptors. Under conditions where dexamethasone potentiated the action of isoproterenol (i.e., 10 min of cell preincubation with 1 0 - S M dexamethasone), no significant changes in receptor number (control, 418 + 117; dexamethasone, 383 + 59 sites/cell) or affinity ( K D control, 3.38 ___1.2 nM; K D dexamethasone, T A B L E II E F F E C T O F C A L C I U M ON D E X A M E T H A S O N E PERMISSIVE E F F E C T The cells isolated from intact animals were preincubated for 60 rain at 37°C in either minimal essential medium alone or containing 1 m M E G T A before addition of 1 0 - 6 M isoproterenol with or without 1 0 - S M dexamethasone. At various intervals after drug addition cAMP contents were determined in parallel in the two sets of samples. Each value represents the mean of duplicate determinations in a typical experiment and is expressed as stimulation ratio. The values of basal cAMP content were 1.28 and 1.25 pmol/107 cells, respectively, in control medium and in medium containing 1 m M EGTA. Similar results were obtained in 4 distinct experiments. Time (min)

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322 3.58 + 0.6 nM) was observed in isolated thymocyte membranes in four experiments. Effect of EGTA on dexamethasone permissive action Calcium ion has been shown to play a critical role in the control of cAMP production and degradation either directly or through its interaction with calcium binding protein [35-38]. Recent experiments have shown, in addition, that calcium removal or excess may considerably alter the pattern of catecholamine stimulation [39]. We have thus studied the permissive action of dexamethasone in the presence of the chelating agent, EGTA. In these experiments, the cell suspensions were preincubated during 60 rain at 37°C, before the beginning of the experiment, either in minimal essential medium, (calcium concentration, determined by atomic absorption spectrometry, was about 2 . 1 0 -5 M) or in minimum essential medium supplemented with 1 mM EGTA. The two samples were then stimulated by isoproterenol in the presence or absence of dexamethasone. As shown in Table II, whereas addition of EGTA did not alter the pattern of fl-adrenergic-induced increase of cellular cAMP content it completely abolished the permissive action of dexamethasone. However, attempts made to overcome the action of EGTA by addition of divalent cations such as Ca 2+ or Mg 2+ were unsuccessful. Discussion

Our results clearly established that glucocorticoids, but also sex steroids, are able to potentiate the isoproterenol-elicited increase of cAMP content in isolated thymocytes. The demonstration and the study of this rapid permissive action of steroids appear critically dependent upon the experimental conditions. Our results, together with those of Zick et al. [28] and Fredholm and coworkers [40], strongly suggested that the isolation process by itself enhanced the intracellular cAMP content, thus leading to the necessity of leaving the cells in a resting state before experiment. We have also attempted to maintain as constant as possible several factors such as anesthesia, medium composition and pH and experimental temperature which may explain alterations in cell

responsiveness [41-43]. In addition, as the thymus, a well known target tissue for glucocorticoids and catecholamines, is very sensitive to stress, our experiments were made using adrenalectomized animals. Despite these precautions, the basal level of cAMP greatly varied from one experiment to another, thus suggesting that other factors such as the age or the batch of the mice might also be important. Moreover, we have shown that the responsiveness of the cells to the action of isoproterenol decreased during in vitro incubation, a phenomenon which may in part explain the variability observed from one experiment to another. Although similar rapid permissive effects of glucocorticoids and sex steroids have been observed in mast cells or in human lymphocytes [12-25], the mechanisms of these effects remain unknown. Nevertheless, in our experimental model, the steroid potentiation does not appear to be a general phenomenon as we have shown that dexamethasone does not alter the stimulation of cAMP elicited by prostaglandin E 2 (results not shown). Given the rapidity of this steroid action, which is hard to reconcile with a mechanism requiring gene activation, as well as the fact that the permissive action of glucocorticoids is not abolished in the presence of inhibitors of macromolecular synthesis [12,15] it has been suggested that they could represent nongenomic, nonreceptor-mediated glucocorticoid action. This assumption is reinforced by the demonstration that sex steroids which do not bind to specific receptors in thymus cells [47] also produced similar permissive effects. In order to investigate the possible sites of steroid action we have tested the hypothesis that steroids may alter the fl-agonist action by modulation of its interaction with membrane receptors. Our results showed, however, that preincubation in the presence of dexamethasone, under conditions allowing the manifestation of the permissive effect of the drug, does not change significantly the binding of [3H]dihydroalprenolol to lymphocyte membranes. These results, together with those obtained by Tolone et al. [12] in rat mast cells, make the hypothesis of fl-receptor alteration unlikely. Among the hypotheses proposed to explain the permissive action of glucosteroids a potential in-

323 h i b i t o r y effect of these drugs o n phosphodiesterase activity has received widespread consideration. However, in vitro d e t e r m i n a t i o n s of phosphodiesterase activity showed that i n h i b i t i o n of e n z y m e activity in the presence of glucosteroids was only observed at c o n c e n t r a t i o n s higher than 10 -4 M [16,46]. U n d e r these conditions, the percent i n h i b i t i o n did n o t exceed 10-25%, whereas p o t e n t i a l i s a t i o n of isoproterenol action in intact cells can be observed at 10-7_ 10-6 M dexamethasone. Nevertheless, these results do n o t exclude that steroids m a y m o d u l a t e in vivo phosphodiesterase activity, p r o b a b l y through an indirect mechanism. The d e m o n s t r a t i o n that the chelating agent, E G T A , almost abolishes the permissive effect of d e x a m e t h a s o n e m a y suggest that the calcium ion is involved in steroid action. A l t h o u g h we were unable to overcome chelator action by a d d i t i o n of divalent cations, this a s s u m p t i o n is nevertheless reinforced b y our recent d e m o n s t r a t i o n that dexa m e t h a s o n e is able to acutely interfere with calcium transport in the same target cells [48]. This, however, does n o t clarify the possible sites of steroid actions as calcium, in its ionic form or associated with c a l c i u m - b i n d i n g proteins, has been shown to m o d u l a t e several of the steps involved in the control of c A M P m e t a b o l i s m [44,45]. The characteristics of the steroid permissive action would appear to favour a n effect on the coupling between fl-receptors a n d adenylate cyclase rather than a n action on the phosphodiesterase activities, b u t this hypothesis r e m a i n to be further investigated.

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