Expression of α-adrenoceptors in a human transformed lymphoblastoid cell line

Journal of Neuroimmunology, 29 (1990) 165-172 Elsevier

165

JNI 00987

Expression of a-adrenoceptors in a human transformed lymphoblastoid cell line Enri S. Borda, Maria M.E. de Bracco, Claudia Perez Leir6s and Leonor Sterin-Borda Centro de Estudios Farmacolbgicosy de Principios Naturales (CEFAPRIN), CONICET and Instituto de Investigaciones Hematolbgicas, Academia Nacional de Medicina, 1414 Buenos Aires, Argentina

(Received 28 December 1989) (Revised, received 9 April 1990) (Accepted 10 April 1990)

Key words: a-Adrenoceptor; Lymphocyte, transformed; Raji cell line; Cell growth; Binding study; Inotropic effect

Summary The presence of a-adrenergic receptors (absent in normal lymphocytes) has been demonstrated in transformed human lymphocytes of the Raji cell line. Binding properties of fl-adrenergic receptors were similar to those reported for normal lymphocytes. A single population of a2-adrenergic receptors was characterized in intact Raji lymphoblasts by binding and saturation assays with the a2-adrenergic antagonist yohimbine. Competition curves with [3H]yohimbine indicate the presence of typical a2-adrenoceptors. Reaction of Raji with the a2-adrenergic agonist clonidine (10 -6 M) stimulated their growth rate. In contrast, the al-adrenergic agonist methoxamine (10 -6 M) had no effect. Previous work indicates that Raji can actively produce thromboxanes (TX) and that these decreased atrium contractility. In agreement with these results and with the binding studies, it is now shown that clonidine stimulation enhanced the negative inotropic effects of Raji on isolated rat atria. This reaction was prevented by incubation of Raji with yohimbine (10 -6 M) but not with the al-adrenergic antagonist prazosin (10 -6 M) or the/3-adrenergic antagonist propranolol (10 -7 M). The biologic effect of Raji on rat atria was probably due to production of cyclooxygenase metabolites of arachidonic acid, because it was blocked by preincubation of the cells with the cyclooxygenase inhibitors indomethacin (10 -6 M) and aspirin (10 -4 M) or the thromboxane synthetase inhibitors nictindol (10 -s M) and imidazole (10 -4 M). The presence of a-adrenoceptors on transformed Raji cells may play a role in the regulation of cell growth and biologic activity, since a2-adrenerglc stimulation can change the balance of the intracellular signals involved in triggering cell division and function.

Introduction

Address for correspondence: Enri S. Borda, M.D., CEFAPRIN, Serrano 665, 1414 Buenos Aires, Argentina.

The functional activity of normal lymphoid cells may be modulated by different hormones and neurotransmitters (Bourne et al., 1974; Besedov-

0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

166

sky et al., 1979; Felten et al., 1985). Thus, as a consequence of the interaction of the lymphocyte fl-adrenoceptors with norepinephrine, the intracellular concentration of cyclic AMP (cAMP) increases, and this in turn modulates cell function (Strom et al., 1977; Kammer, 1988). In contrast, a-agonist binding to specific a-adrenoceptors triggers a series of reactions that cause reduction of adenylate cyclase activity and increased Ca 2+ concentration (Schultz et al., 1982). Through their regulatory action on second messenger concentration (Spiegel, 1987), a-adrenergic receptors may also be involved in the control of cell activity. Functional studies suggest a role for a-adrenoceptor activation in the modulation of antibody response (Hadden et al., 1970; Besedovsky et al., 1981). Thus, the al-agonist methoxamine increased IgM production, while the a2-agonist clonidine suppressed it in primary splenocyte cultures (Sanders and Munson, 1985). Using direct binding assays, the presence of a-adrenoceptors has been demonstrated in platelets (Newman et al., 1978), their occurrence is uncertain in monocytes (Lappin and Whaley, 1982) and although guinea pig splenocytes possess a2-adrenoceptors (McPherson and Summers, 1982) they have not been documented in normal human lymphocytes (Casale and Kaliner, 1984). Since neoplastic transformation may induce the expression of membrane receptors and markers that are absent in normal resting cells (Gahrton et al., 1987), it is important to determine if a-adrenoceptors may be expressed in transformed lymphoblastoid cells and contribute to the stimulation of cell growth. In lymphoproliferative diseases, such as chronic lymphocytic leukemia (CLC), malignant lymphocyte clones are expanded and the normal mechanisms of control of cell growth fail. Transformed CLC lymphocytes are functionally and phenotypically different from normal human lymphocytes (Gale and Foon, 1984). In a previous communication, we have shown that CLC lymphocytes and cells from the transformed lymphoblastoid Raji line (Raji) were able to synthesize thromboxanes (TX) (Finiasz et al., 1989) in contrast to normal resting lymphocytes (Awara et al., 1986). Apparently, TX were involved in the regulation of Raji cell growth and could induce changes in rat atrium contractility

(Finiasz et al., 1989). As it is known that a-adrenoceptor stimulation can trigger the activation of phospholipases that in turn leads to increased synthesis of both lipoxygenase and cyclooxygenase metabolites (Lapetina, 1982), in this study we investigated the occurrence of a-adrenoceptors in Raji cells and we analyzed the consequences of a-adrenergic stimulation on the biological activity of these cells. We will demonstrate that Raji lymphoblasts express both a 2- and fl-adrenoceptors. The expression of fl-adrenoceptors is similar to that of normal lymphocytes, while a2-adrenergic receptors predominate in these cells and their reaction with specific agonists stimulates cell growth and modifies their function.

Materials and methods

Drugs Stock solutions of yohimbine (a2-adrenergic antagonist), indomethacin (cyclooxygenase inhibitor) and irnidazole (thromboxane synthetase inhibitor) (Sigma Chemical Company); prazosin (al-adrenergic antagonist) (Knoll Lab.); ( - ) - p r o pranolol (fl-adrenergic antagonist) (Ayerst); clonidine (a2-adrenergic agonist) (Boehringer); methoxamine (al-adrenergic agonist) (Burroughs Wellcome) and nictindole (thromboxane synthetase inhibitor) (L-8027, Labaz) were prepared in distilled water and fresh dilutions were used at the concentrations cited in the text. Raft cell culture Raji lymphoblastoid cells were maintained in continuous culture in RPMI 1640 tissue culture medium (RPMI; Gibco, U.S.A.) supplemented with 10% fetal calf serum (FCS, Gibco) (RPMIFCS) as described by Pearson and Orr (1976) at an initial cell concentration of 105/ml. Cultures were set up in triplicate in tissue culture flasks (Coming) and kept in a humidified CO2 incubator. Analysis of Raji cell growth was done by counting daily the viable Raji cells in a Neubauer chamber. To evaluate the effect of clonidine and methoxamine on cell growth, cultures were set up including either drug (10 -6 M) at day 0. For inhibition experiments, cells were pretreated with

167 yohimbine (10-6 M) for 30 min at room temperature before the addition of clonidine. Inhibition of clonidine-stimulated cell growth was performed by adding the drugs to Raji cell cultures on day 0.

Binding assays Cells collected on day 3 of culture were washed 3 times in RPMI without FCS (RPMI) at room temperature and the concentration for each assay system was adjusted. Assay conditions were tested in preliminary experiments where time, temperature and cells were varied. In the case of a-adrenoceptors, 25 ° C and 30 min of incubation were chosen, while 20 min and 37 ° C were the optimal conditions for fl-adrenergic binding. All tests were done in RPMI (550/~1, final volume). [3H]Yohimbine ([3H]YOH, specific activity 76 C i / m m o l ) and [3H]prazosin ([3H]PZN, specific activity 82 C i / m m o l ) were used and binding studies were performed with 2 × 106 ceils and 0.5-20 nM of the radioligand. Specific binding was defined as that displaceable by 5 x 10-5 M cold yohimbine or prazosin, and was about 60% at 5 nM of [3H]YOH. In the case of fl-adrenergic binding, 3 × 106 cells were incubated with 0.3-6 nM [3H]dihydroalprenolol (( - )-[3H]DHA, specific activity 95 C i / m m o l ) in RPMI in the presence or not of 10 -5 M ( - ) - p r o p r a n o l o l to assess specific binding. In c o m p e t i t i o n experiments with [3H]YOH, cells were incubated with 9 nM of the radioligand and increasing concentrations of unlabelled agonists and antagonists. In all cases, to stop the reaction, samples were diluted in 4 ml pH 7.4 phosphate-buffered saline (PBS) at 25 ° C and filtered on Whatman G F / C under mild pressure. Filters were washed with 12 ml of the same buffer, placed into vials and counted in Triton-toluene scintillation cocktail in a Beckman LSC-100 counter with approximately 40% efficiency. Equilibrium dissociation constants ( K d) and the maximal number of binding sites (Bmax) were calculated from saturation assays and Scatchard analysis.

Isolated rat atrium preparations Male albino rats of the Wistar strain weighing between 220 and 250 g were used. The animals were killed by decapitation. The entire hearts were excised quickly and placed in Petri dishes filled

with a modified Krebs-Ringer-bicarbonate solution (KRB) composed as follows (mM): Na ÷ 145; K ÷ 6.02; Ca 2+ 1.22; Mg 2+ 1.33; C1- 126; HCO3 25.3; SO4z- 1.33; PO43- 1.20; glucose 5.5. The atria were separated from the ventricles, dissected, attached to a glass holder and immersed in 15 ml KRB gassed with 5% CO 2 in 02 and equilibrated for 60 rain. A constant resting tension of 750 mg was applied and the contractile activity of the isolated atria was recorded with an oscillograph coupled to transducers as previously described (Sterin-Borda et al., 1983). The isometric developed tension (IDT) and the frequency of the contractile cycles ( F ) were taken as basal reference for the action of Raji cells harvested after 72 h of culture, suspended at 8 × 106/ml, pretreated with clonidine (10 -6 M) for 15 min at 37°C, washed 3 times and resuspended in RPMI. For inhibition studies, Raji (8 x 106/ml) were preincubated for 20 rain with the appropriate dilutions of the inhibitors and antagonists before the addition of clonidine. Raji were added to the organ bath in a 0.5 ml volume and their final concentration in contractility studies was 2.6 × 105/ml and was selected on the basis of previous studies. RPMI medium (0.5 ml) had no effect per se upon contractility of atria (Finiasz et al., 1989).

Statistics After analysis of variance, means were compared by the Student-Newman-Keuls (SNK) test. Differences between means were considered significant if P was equal or less than 0.05.

Results

Identification lymphoblasts

of adrenergic receptors in Raji

Preliminary assays were done to establish the presence of fl-adrenergic receptors on Raji cells. Raji cells displayed a population of/3-adrenoceptors with binding properties similar to those described for non-transformed lymphocytes ( K d 1.2 _+ 0.2 nM; Bmax 1.9 +_ 0.4 fmol/106 cells), consistent with about 1200 binding sites/cell. In order to identify a-adrenoceptors on Raji cells, binding studies were carried out with selective Ot1- and a2-radioligands. In agreement with

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Fig. l. [3H]Yohimbine (YOH) binding to intact gaji cells. (A) Time course of the association reaction (©) and the dissociation reaction (e). For the association reaction, 2)< 10 6 Raji cells/ml and 2.4 nM of [3H]YOH were carried at 37°C for different times and the specific binding was determined in the presence of 5 )<10-s M yohimbine. For the dissociation reaction, at 20 rain, 5 ×10 -5 M yohimbine was added (indicated by arrow) and the specific binding was determined at the different times shown. (B) Cell concentration dependence of [3H]YOH binding. (C) Effect of temperature (T) on [3H]YOH specific binding. 2 )<106 Raji cells/ml were incubated with 2.4 nM of [3H]YOH for 20 rain at different T. Binding data are mean ± SE of four experiments done in duplicate. the f u n c t i o n a l studies, [3H]prazosin, a specific a]adrenergic a n t a g o n i s t ( 0 . 5 - 5 0 n M ) failed to show a n y specific b i n d i n g at all t e m p e r a t u r e s a n d cell c o n c e n t r a t i o n s tested (data n o t shown). O n the other h a n d , [3H]yohimbine, a k n o w n selective a 2antagonist, specifically a n d reversibly b o u n d to Raji cells i n a t e m p e r a t u r e - a n d c o n c e n t r a t i o n - d e p e n d e n t m a n n e r (Fig. 1). S a t u r a t i o n assays a n d Scatchard analysis (Fig. 2A a n d B) d e m o n s t r a t e d a single p o p u l a t i o n of a2-receptors o n i n t a c t cells with K d 5.4 + 0.7 n M a n d Bm~, 4.2 + 0.9 f m o l / 1 0 6 cells (2538 + 541 receptors/cell), C o m p e t i t i o n curves (Fig. 3) showed a n order of p o t e n c y typical for a2-adrenergic receptors ( H o f f m a n et al., 1979).

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Effect of clonidine-stimulated Raft cells on isolated atria Because we had previously shown that Raji cells had a negative inotropic effect on rat atria, probably by release of thromboxanes during the bioassay (Finiasz et al., 1989), we investigated if clonidine binding to Raji a2-adrenoceptors affected their biologic activity. To eliminate any direct effect of clonidine- or a-adrenoceptor antagonists on rat atria, Raji were washed before addition to the organ bath. Frequency ( F ) remained unchanged, but the contractile tension (IDT) decreased in both cases. The magnitude of

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Fig. 4. Effect of a-adrenergic agonists on Raji cell growth. Cultures of Raji cells were set up in triplicate at 105/ceUs/ml in RPMI-FCS as described in Materials and Methods. Clonidine (10 -6 M) or methoxamine (10 -6 M) were included at day 0 of culture. Cell growth was evaluated by counting viable Raji cells each 24 h in a Nenbauer chamber. (e) Raji alone; (o) Raji+clonidine; (/,) Raji+methoxamine. Mean +SE are given; number of experiments in parentheses. P < 0.05 between Raji alone or Raji + methoxamine vs. Raji + clonidine at 96 h (SNK test).

the culture period stimulated their growth rate. In contrast, methoxamine (an az-adrenoceptor agonist) had no significant effect on Raji cell growth (Fig. 4). Neither agent altered cell viability at the concentration used. Furthermore, clonidine had no stimulatory effect on cell growth if cells had been preincubated with yohimbine (10 -6 M). Likewise, if the culture was carried out in the presence of imidazole (10 -5 M) or indomethacin (10 -6 M), clonidine stimulation of cell growth was prevented (cells/ml, Raji + clonidine 96 h: 15,800 + 1100; Raji + imidazole + clonidine 96 h: 9300 + 875; Raji + indomethacin + clonidine 96 h: 9660 + 940). Neither imidazole nor indomethacin had a significant effect on the growth of Raji cells without clonidine (cells/ml, Raji 96 h: 11,600 +

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Fig. 5. Effect of clonidine-stimulated Raji cells on tension and frequency of atria. Rat atria were incubated for 15 min in the presence of 2.5 × 10S/ml Raji cells alone (open bar); Raji cells pretrcatcd with 10 -6 M clonidine for 15 rain, 37°C and washed (hatched bar) or Raji cells blocked (20 min, 37°C) with 10 -6 M yohimbinc (filled bar), 10 -6 M prazosin (striped bar) or 10 -7 M propranolol (cross-hatched bar) before addition of clonidinc. Changes in tension (IDT, %) referred to basal values (490 + 40 mg) were calculated and mean + SE arc shown. Differences between Raji alone vs. (Raji+clonidine), and (Raji + yohimbine + clonidine) vs. (Raji + clonidine) were statistically significant ( P < 0.001, SNK test). The inhibitors, at the concentrations used, had no effect on basal IDT. The numbers in parentheses indicate number of preparations tested. Basal frequency was 1405:7 and remained unchanged in all experimental conditions tested.

170 TABLE 1 I N F L U E N C E OF INHIBITORS OF A R A C H I D O N I C A C I D METABOLISM ON THE INOTROPIC N E G A T I V E EFFECT T R I G G E R E D BY C L O N I D I N E - T R E A T E D RAJI CELLS Raft cells (0.5 ml of an 8 x 1 0 6 / m l cell suspension) were incubated during 15 rain with 10 -6 M clonidine alone or in the presence of 10 6 M indomethacin (indo); 10 -4 M aspirin (ASA); 10 -6 M nictindol (L-8027) or 10 -4 M imidazole and then washed. Cells were preincubated during 20 min with the blockers before the addition of clonidine, as described in Materials and Methods. Values are means 5: SE; n = number of preparations tested. Drugs

IDT (% of inhibition)

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- 25.7 + 4.2 - 1.3 + 2.1 - 3.2 5:6.1 - 2.3 + 5.0 - 2.1 + 3.2

9 4 4 4 4

P < 0.001 between Raji + clonidine alone vs. Raji + clonidine in the presence of inhibitors (SNK test).

inhibition was significantly greater with clonidine-treated Raji than with Raji alone. To determine whether a2-, a 1- or fl-adrenoceptors were involved in the reaction, Raft cells were preincubated for 20 min with yohimbine ( 1 0 - 6 M ) , prazosin ( 1 0 - 6 M) or propranolol (10 -7 M) respectively, before the addition of clonidine. The results shown in Fig. 5 demonstrate that inhibition of Raji az-adrenoceptors with yohimbine completely blunted the effect of Raji + clonidine on IDT. The a 1- and fl-adrenoceptor blockers (prazosin and propranolol) had no effect on the contractile tension (Fig. 5). An active cyclooxygenase and tromboxane synthetase system was necessary to generate the inhibitory activity of clonidine-treated Raji, since their preincubation with indomethacin ( 1 0 - 6 M ) , aspirin ( 1 0 - 4 M ) nictindol (10 -5 M) or imidazole ( 1 0 - 4 M ) eliminated the negative inotropic effect of Raji + clonidine (Table 1).

Discussion

The results of this study demonstrate that cells from a transformed human lymphoblastoid line

(Raji) express a-adrenergic receptors that cannot be recognized in normal human lymphocytes (Casale and Kaliner, 1984). In contrast, the occurrence of fl-adrenergic receptors is similar both in quality and number to that reported in the literature for non-transformed lymphocytes (Meurs et al., 1982). Raji cells displayed a single population of az-adrenergic receptors, as demonstrated in binding studies with a selective a2-radiolabelled antagonist, [3H]yohimbine, as well as by the order of potency of a-adrenergic agonists and antagonists. These receptors showed an equilibrium dissociation constant that was rather higher than the one previously described for other tissues (Motulsky et al., 1980) and approximately 2500 receptors/cell. Even though the order of potency indicated by the competition curves is consistent with that of an a2-adrenoceptor, the values are higher than those expected for a typical one. A similar discrepancy between K d values of lymphocyte muscarinic cholinergic receptors and those described for other tissues has already been documented (Adem et al., 1986; Costa et al., 1988). The appearance of a2-receptors in Raji cells seems to be related to metabolic changes rather than to an artifact, since the occurrence and quality of the B-adrenoceptors proved to be similar to that reported for resting, non-transformed lymphocytes (Meurs et al., 1982). Viable cells are an interesting working system because the study of their characteristic metabolic regulation of transformed vs. normal cells. The results of culture studies performed in the presence of the az-adrenergic agonist clonidine (Fig. 4) indicate that binding of az-agonists to Raji cell adrenoceptors could modulate the rate of cell growth. On the other hand, methoxamine had no effect on Raft cell growth, suggesting that prevalence of functional cz2-adrenoceptors in Raji cells. Apparently, stimulation of Raji cell growth by clonidine required the synthesis of cyclooxygenase metabolites of arachidonic acid, since the continuous presence of indomethacin or imidazole prevented the clonidine effect. We had previously shown that, in contrast with normal lymphocytes, Raji lymphoblasts actively produced thromboxanes (Finiasz et al., 1989) and that these arachidonic acid metabolites could affect heart contractility reducing the contractile

171 tension. Binding of a2-adrenergic ligands to their specific r e c e p t o r s on R a j i e n h a n c e d the negative i n o t r o p i c effect o f these cells on the h e a r t tissue. T h e specificity o f the r e a c t i o n was c o n f i r m e d because p r e i n c u b a t i o n of Raft with the a 2 - a d r e n o c e p t o r a n t a g o n i s t y o h i m b i n e e l i m i n a t e d their effect, while p r a z o s i n a n d p r o p r a n o l o l were inactive (Fig. 5). P r o d u c t i o n of t h r o m b o x a n e s b y clonid i n e - s t i m u l a t e d R a j i was p r o b a b l y involved, since i n h i b i t i o n of c y c l o o x y g e n a s e activity b y ind o m e t h a c i n a n d aspirin, or a d d i t i o n of the t h r o m b o x a n e synthetase i n h i b i t o r s n i c t i n d o l a n d imidazole, also a b r o g a t e d their b i o l o g i c effect ( T a b l e 1). Thus, it m a y be p r o p o s e d that a z - a d r e n o c e ptors on the surface of these t r a n s f o r m e d cells m a y trigger m e t a b o l i c events related to the c o n t r o l of cell growth a n d b i o l o g i c action. It is k n o w n that a z - a d r e n o c e p t o r s t i m u l a t i o n decreases the activity of a d e n y l a t e cyclase. C o n s e q u e n t l y the levels of the second messenger c A M P are r e d u c e d a n d this m a y p r o v i d e a signal for cell a c t i v a t i o n ( S t r o m et al., 1977; K r a m e r , 1988). D e c r e a s e d c A M P m a y f a v o r the a c t i v a t i o n of p h o s p h o l i p a s e s increasing the availability of a r a c h i d o n i c acid f r o m e n d o g e nous sources which in turn can be m e t a b o l i z e d to biologically active p r o d u c t s b y the l i p o x y g e n a s e or c y c l o o x y g e n a s e p a t h w a y s ( L a p e t i n a , 1982). Therefore, it can be p o s t u l a t e d that the p r e s e n c e of a2-adrenergic r e c e p t o r s on R a j i l y m p h o b l a s t o i d cells is an i m p o r t a n t factor in the r e g u l a t i o n of their p h y s i o l o g i c a l behavior.

Acknowledgements W e gratefully a c k n o w l e d g e the e x p e r t technical assistance of Ms. M a r t a F e l i p p o a n d Elvita Vanucchi. W e are also i n d e b t e d to Dr. M a r t a R. F i n i a s z for helpful discussion a n d advice. This w o r k was d o n e with funds f r o m C O N I C E T P I D 9180/85, 3 0 2 5 0 0 0 / 8 8 a n d 3066900/88.

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