368

Biochimica et Biophysica Acta, 381 (1975) 368--376 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27587 [3 H] N O R E P I N E P H R I N E BINDING BY RAT GLIAL CELLS IN C U L T U R E LACK OF C O R R E L A T I O N BETWEEN BINDING AND A D E N Y L A T E CYCLASE ACTIVATION

JOEL PREMONT, PHILIPPE BENDA and SERGE JARD Laboratoire de Physiologie CeUulaire, Coll~ge de France 75231, Paris Cedex 05 (France)

(Received July 29th, 1974)

Summary Subcellular fractions prepared from rat glial cells in culture (clonal line C6 ) were used in an a t t e m p t to characterize the adrenergic receptor involved in adenylate cyclase activation. Both [3 H] norepinephrine binding and enzyme activation were measured under identical experimental conditions. Binding sites for norepinephrine could be detected; their main characteristics were: apparent Km: 4 " 10 -6 M, maximal capacity: 20 pmol/mg protein. Their stereospecificity towards structually related drugs was found to be different from the stereospecificity of the receptor involved in adenylate cyclase activation. Thus, 3 - m e t h o x y d o p a m i n e (a competitive inhibitor of norepinephrine for adenylate cyclase activation), phenylephrine (a partial adrenergic agonist) and the blocking agent propranolol were unable to compete with [3H]norepinephrine for binding. On the other hand, several molecules like dopa bearing a catechol group and which are unable to interact with the adenylate cyclase as agonists or competitive inhibitors strongly inhibited [ 3 H] norepinephrine binding. As in several other systems so far studied, the presence on the glial cell's membrane of a large number of "catechol-binding sites" makes it difficult to characterize the ~-adrenergic receptor.

Introduction Several attempts to identify the fi-adrenergic receptor from binding studies of radioactive catecholamines [ 1--10] (Lacombe, M.L. and Hanoune, J., personal communication) have been partly unsuccessful. On a large variety of intact target cells or subcellular fractions in which a fi-adrenergic receptor-mediated

369

adenylate cyclase activation was demonstrable (liver [1,3,4] (Lacombe, M.L. and Hanoune, J., personal communication), heart [5,6], fat cells [ 2 ] , spleen capsule [7] and t u r k e y erythrocytes [8,9] ), it was claimed that at least a large fraction of the detected binding sites could be the receptors involved in adenylate cyclase activation [1,3--10] (Lacombe, M.L. and Hanoune, J., personal communication). However, the characteristics of radioactive binding failed to completely satisfy criteria usually retained for the identification of a pharmacological receptor (time course of binding compatible with the time course of response, reversibility, satisfactory correspondence between apparent dissociation constants deduced either from dose--response curve or dose-binding curve, identical stereospecificity ~or binding and response). Recently, it was shown that tumoral rat glial cells from the clone C6 responded to ~-adrenergic agonists by a tremendous (up to 600-fold) increase in intracellular cyclic AMP [11,12] ; it was further demonstrated that particulate fractions prepared from these cells contained an adenylate cyclase activity highly sensitive to catecholamines [13]. In the present study, the possibility was investigated that these tumoral and partially differentiated glial cells could constitute a convenient biological material for the identification of the/3-adrenergic receptor. The binding of [3 H] norepinephrine and activation of the membrane-bound adenylate cyclase were measured on the same preparations and under identical experimental conditions. The binding and activation processes were compared with respect to their relative time course, reversibility, apparent affinity for adrenergic agonists and stereospecificity. Methods Material Cells from the clonal line C6 derived from chemically induced glial t u m o r in the rat [14,15] were grown in a HAM F.10 medium complemented with 10% foetal calf serum. They were seeded at a density of 5 • l 0 s per flask (area 75 cm 2 ) and usually collected 7 days later at the end of the logarithmic phase to osmotic lysis at 0°C in Tris-HC1, pH 8.0, 25 mM EDTA-Tris, pH 8.0, 5 mM (106 cells per 100 pl). The lysate was gently homogenized using an Elvejehm glass potter equipped with a Teflon pestle. The subcellular fraction sedimenting in 10 min at 10 000 X g was collected and resuspended in the homogenization medium (0.4--3 mg protein/ml). This fraction was found [13] to contain 80% of basal adenylate cyclase activity and 96% of isoproterenol-sensitive activity. Usual methods for the preparation of pure plasma membrane fractions all led to a sharp decrease in responsiveness to ~-adrenergic agonists. Both binding experiments and adenylate cyclase assays were performed with the crude particulate fraction referred to as " e n z y m e " in the following description. The enzyme was frozen immediately after preparation; it could be stored in liquid nitrogen for several weeks without any loss in adenylate cyclase activity and responsiveness to catecholamines.

Chemicals Adenosine 5'-triphosphate (sodium salt) and cyclic 3'-5'-adenosine mono-

370 phosphate (cyclic AMP) were purchased from Sigma, neutral A12 03 from Woelm, creatine kinase and phosphocreatine from Boehringer. D,L-Isoproterenol, D,L-epinephrine, L-norepinephrine, L-dopa, dopamine, L-tyrosine, L-tryptophane, L-serotonine, 3-methoxydopamine and phenylephrine were obtained from Calbiochem and the blocking agents propranolol and phenoxybenzamine from Sigma. The catecholamines and derivatives were dissolved in water together with equal amounts of ascorbic acid immediately before testing.

Radiochernicals [a-32p]ATP (900--1200 Ci/M), cyclic [3H]AMP (19 Ci/mM) and D,L-[ 3 H] norepinephrine (5--10 Ci/mM) were purchased from Commissariat h l'Energie Atomique (Saclay, France) and L-[7 -3 H] norepinephrine (15 Ci/mM) from the Radiochemical Centre (Amersham, England). Adenylate eyclase assay Adenylate cyclase activity was measured by the conversion of [0/-32 P]ATP into cyclic [32 p] AMP in experimental conditions found to be optimal for activation by/3-adrenergic agonists [3]. The incubation medium (final volume 100 pl) contained 100 mM Tris--HC1, pH 8.0, 4.5 mM MgC12,0.1 mM ATP, 0.2 pCi [a -3z p] ATP, 1 mM cyclic AMP, 15 mg/ml phosphocreatine, 5 mg/ml creatine kinase and various amounts of the tested compounds. The reaction was initiated by the addition of enzyme (10--75 pg protein) and allowed to proceed for 10 rain at 37°C. It was stopped by cooling and dilution of the radioactive precursor with a large excess of unlabelled ATP. Cyclic [32 p] AMP was separated by filtration on dry A12 03 columns according to Ramachandran and Lee [16] and counted by liquid scintillation in 8 ml of Bray's scintillation medium [17]. Cyclic [3 H] AMP was used for the determination of cyclic AMP recovery. Control experiments validating the experimental method used have been previously described [18]. Adenylate cyclase activities are expressed in pmol cyclic AMP formed/mg protein per 10 min. All determinations were performed in duplicate. Proteins were determined according to the method of Lowry et al. [19] using bovine serum albumin as a standard. [3 H] Norepinephrine-binding assay The incubation medium for binding assays (volume 100 pl) was identical to that used for adenylate cyclase assay except for the omission of labelled ATP and the addition of [3H] norepinephrine (10 -6 M, 0.5--1.5 pCi except when otherwise specified). The binding reaction was initiated by the addition of enzyme and allowed to proceed for 10 min at 37°C. At the end of the incubation period, 75/al of the reaction mixture were rapidly layered at the top of a small polyethylene tube {diameter 4 mm, length 40 mm) containing 350 p l of 36% sucrose dissolved in 100 mM Tris--HC1, pH 8.0, 3 mM MgC12 at 0°C. The tube was immediately centrifugated at maximal speed in a Wifug microcentrifuge (maximal speed reached within 15 s). After freezing in liquid nitrogen the lower part of the tube (5 mm from the bottom) was cut and its content solubilized in 0.5 ml soluene at 60°C for 20 min before counting by liquid scintillation in 8 ml of Bray's solution. Aliquots from the incubation medium were submitted to the same treatment before counting. All the experimental

371 series included the determination of non-specific binding, i.e. residual radioactivity binding in the presence of a large excess (1 mM) of unlabelled norepinephrine. Specific binding (see below) is defined as the difference between total and non-specific binding. Results are expressed in terms of norepinephrine b o u n d (pmol/mg protein). Control experiments indicated that: (1) the final centrifugation led to a complete recovery of the particulate material layered at the top of the sucrose solution; (2) blank values (amount of radioactivity recovered at the b o t t o m of the tube after layering an aliquot from the incubation medium w i t h o u t enzyme) were negligible (less than 0.05%, of total radioactivity in the tube); (3) the amount of specifically b o u n d radioactivity present in intermediate sections from the centrifuge tube represented less than 10% of that recovered at the b o t t o m of the tube. It was not modified by the further addition of 10 -3 M unlabelled norepinephrine immediately before centrifugation. It thus appeared that the determination of the binding c o m p o n e n t under study is not deeply altered by partial dissociation during the final separation of free from b o u n d radioactivities. Results

Pharmacological characterization of the glial cells ~-adrenergic receptor A typical dose-response curve of rat glial cells adenylate cyclase stimulated by increasing amounts of L-norepinephrine is illustrated in Fig. 1. Threshold dose was a b o u t 10 -s M and maximal activation (about 7-fold increase in activity) obtained at 10 -4 M. The apparent Km value (concentration of norepinephrine eliciting half the maximal stimulation) was 4 • 10 -7 M. Such dose--response curves were determined for the different c o m p o u n d s tested. The Km values and maximal activities are given in Table I. Isoproterenol was more p o t e n t than norepinephrine and epinephrine. This observation, together with the previous demonstration that the fi-blocking agent propranolol but n o t the s-blocking agent phenoxybenzamine was able to inhibit the isoproterenol-induced adenylate cyclase activation, clearly indicate that the glial

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372

TABLE I STEREOSPECIFICITY CHOLAMINES

OF

RAT

GLIAL

CELLS

ADENYLATE

CYCLASE

ACTIVATION

BY C A T E -

A p p a r e n t K m v a l u e s w e r e d e d u c e d f r o m d o s e - - r e s p o n s e c u r v e s s i m i l a r t o t h o s e d e s c r i b e d i n Fig. 1 f o r n o r e p i n e p h r i n e . M a x i m a l a c t i v a t i o n is e x p r e s s e d i n p e r c e n t o f t h a t i n d u c e d b y n o r e p i n e p h r i n e w h i c h w a s u s e d as t h e r e f e r e n c e c o m p o u n d f o r e a c h i n d i v i d u a l e x p e r i m e n t .

Name of compound

Apparent value K m (pM)

Maximal activation (percent of norepinephrine)

Norepinephrine Epinephrine Isoproterenol Phenylephrine Dopamine 3-Methoxydopamine Dopa Tyrosine Metanephrine Normetanephrine Tryptamine Tyramine Serotonin

0.4--1.2* 1.0 0.1--0.15 100 41 65* * * --------

100 100 100 46.0 37.5 5 (10 -4 5 ( 1 0 -4 0 ( 1 0 -3 0 ( 1 0 -5 0 ( 1 0 -5 0 (10 -4 0 (10 -4 0 (10 -4

* Extreme values measured. ** M a x i m a l c o n c e n t r a t i o n t e s t e d . *** This value (apparent Ki) was deduced from the competition

M)* * M) M) M) M) M) M) M)

e x p e r i m e n t d e s c r i b e d i n Fig. 2.

cell adrenergic receptor is of the fl-type. Dopamine and phenylephrine behave like partial agonists with low affinity as compared to isoproterenol. Competition experiments indicated that these t w o agents are able to interact with the ~-adrenergic receptor. The 3-methoxyderivative of dopamine which was inactive per se was found to be a pure competitive inhibitor of norepinephrine (Fig. 2). All the other drugs tested were inactive. //

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Fig. 2. ( r i g h t ) . E f f e c t o f 3 - m e t h o x y d o p a m i n e on adenylate cyclase activation by norepinephrine. Adenylate cyclase a c t i v i t y was m e a s u r e d as i n d i c a t e d u n d e r M e t h o d s in t h e p r e s e n c e of t h e i n d i c a t e d a m o u n t s o f n o r e p i n e p h r i n e a d d e d t o t h e i n c u b a t i o n m e d i u m e i t h e r a l o n e (A) o r t o g e t h e r w i t h a c o n s t a n t a m o u n t o f 3-methoxydopamine ( 1 0 -3 M ) (z~). T h e K i o f 3 - m e t h o x y d o p a m i n e for the fl-adrenergic receptor was c a l c u l a t e d f r o m t h e o b s e r v e d s h i f t i n a p p a r e n t K m f o r n o r e p i n e p h r i n e : K i = 1 0 -3 X K m ] K m - - K m . T h e e s t i m a t e d v a l u e w a s 6 . 5 • 1 0 -5 M.

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Fig. 3. T i m e c o u r s e o f n o r e p i n e p h r i n e b i n d i n g to r a t glial cells. T h e s p e c i f i c c o m p o n e n t of n o r e p i n e p h r i n e b i n d i n g w a s m e a s u r e d as i n d i c a t e d u n d e r M e t h o d s : t h e c o n c e n t r a t i o n o f r a d i o a c t i v e D , L - n o r e p i n e p h r i n e in t h e i n c u b a t i o n m e d i u m w a s 2 .10 -6 M a n d t h e t e m p e r a t u r e 3 7 ° C . V a l u e s a r e t h e m e a n (+-S.D.) o f t h r e e determinations. Fig. 4. R e v e r s i b i l i t y of n o r e p i n e p h r i n e b i n d i n g t o r a t glial cells. R a t glial cell m e m b r a n e s w e r e f i r s t i n c u b a t e d f o r 1 0 r a i n i n t h e p r e s e n c e o f 6 • 10"TM t r i t i a t e d L - n o r e p i n e p h r i n e b e f o r e u n l a b e l l e d n o r e p i n e p h r i n e ( 1 0 -3 M) w a s a d d e d to t h e i n c u b a t i o n m e d i u m . T o t a l n o r e p i n e p h r i n e b i n d i n g w a s m e a s u r e d as a f u n c t i o n o f t h e t i m e w h i c h h a d e l a p s e d b e t w e e n d i l u t i o n a n d final c e n t r i f u g a t i o n (see M e t h o d s ) . T h e e x p e r i m e n t w a s p e r f o r m e d a t 0 ° C ; e a c h v a l u e is t h e m e a n o f t h r e e d e t e r m i n a t i o n s . T h e r a t e c o n s t a n t f o r t h e d i s s o c i a t i o n r e a c t i o n w a s 0 . 5 3 m i n -1 .

Characterization o f norepinephrine-binding sites on glial cells Glial cells were able to bind norepinephrine. The binding process was found to be very rapid; when the enzyme was incubated in the presence of 2 . 1 0 -6 M tritiated norepinphrine at 37°C for increasing periods of time from 2.5 min to 45 min, the a m o u n t of b o u n d radioactivity was already maximal after 2.5 min incubation (the shortest possible period of time separating the addition of membranes to the incubation medium from the beginning of final contrifugation (Fig. 3)). By lowering the temperature of incubation from 37 to 0°C it was not possible in our experimental conditions to demonstrate the time dependency of norepinephrine binding. Unlabelled norepinephrine (10 -3 M) was able to induce a time-dependent inhibition of radioactivity binding to membranes previously incubated for 10 min in presence of 6 • 10 -~ M tritiated norepinephrine (Fig. 4). Residual b o u n d radioactivity at equilibrium (see Fig. 4) corresponded fairly well to the non-specific binding c o m p o n e n t defined as the a m o u n t of radioactivity b o u n d b y the enzyme incubated in presence of labelled and unlabelled norepinephrine (10 -3 M) added together (see Methods). Specific norepinephrine bindings is thus reversible. The half-life of the norepinephrine--receptor complex is a b o u t 1 min (1.3 rain). From the latter figure the estimated rate constant for norepinephrine--receptor complex dissociation was 0.53 rain-'. Specific norepinephrine binding was saturable when increasing norepinephrine concentration in the incubation medium (Fig. 5). Maximal binding capacity was a b o u t 20 pmoles/mg protein and the apparent K m , 4 " 1 0 -6 M. These t w o parameters were n o t aHected by varying temperature of the incubation medium from 37 to 0°C. Assuming that norepinephrine binding is a reversible process involving a homogeneous population of independent receptor sites it is possible to estimate the association rate constant K1 from the experi-

374

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mental determinations of apparent K m (k_, /kl ) and dissociation rate constant k , (see above). The estimated value kl = 1.3 • 10 +s M-' min -1 can account for the observed binding time course (see Fig. 3). Thus, using a norepinephrine concentration of 2 • 10 .6 M, the expected half-time for reaching the equilibrium value would be less than 1 min.

Stereospecificity of norepinephrine-bin ding sites Structural requirements for the a t t a c h m e n t of catecholamines to the norepinephrine-binding sites were deduced from the determination of the relative abilities of several structurally related drugs to inhibit radioactive norepinephrine binding (Table II). Among the tested compounds all those bearing a catechol m o i e t y (norepinephrine, isoproterenol, dopamine and dopa) were able to inhibit tritiated norepinephrine binding to a similar maximal extent. Their respective apparent Km values were very close to that of norepinephrine itself. Among these four compounds, only three were able to activate the adenylate cyclase; dopa which exhibits high affinity for the norepinephrine-binding sites T A B L E I1 STEREOSPECIFICITY

OF RAT GLIAL CELLS NOREPINEPHRINE-BINDING

SITES

Name of compound

Apparent K m for compound* / Apparent K m for norepinephrine

Maximal inhibition (percent maximal inhibition induced by unlabelled norepinephrine)

Norepinephrine Isoproterenol Dopamine Dopa Phenylephrine 3-Methoxydopamine Propanolol Phenoxy benzamine Tyrosine

1.00 1.45 1.00 0,58 ------

100 103 111 127 0 0 0 0 0

* C a l c u l a t e d as t h e r a t i o o f t h e c o n c e n t r a t i o n s o f u n l a b e l l e d n o r e p i n e p h r i n e c o m p o u n d l e a d i n g to 50% i n h i b i t i o n o f r a d i o a c t i v e n o r e p i n e p h r i n e b i n d i n g .

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amounts

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of unlabelled

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in percent

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Fig. 7 (right). Effect of propranolol on norepinephrine binding. Specific norepinephrine binding was m e a s u r e d in t h e p r e s e n c e o f t h e i n d i c a t e d a m o u n t s of p r o p a n o l o l a n d t r i a t e d L - n o r e p i n e p h r i n e : 2 • 1 0 -6 M (~); 5 • 10 -7 M (o); 2.5 • 10 -7 M (~).

was completely inactive and did n o t inhibit the response to the active agonists. On the other hand, phenylephrine which was found to be a partial agonist and 3-methoxydopamine which behaves like a competitive inhibitor of norepinephrine for the ~-adrenergic receptor did not significantly inhibit [3 H] norepinephrine binding; the ~-blocking agent propanolol was also inactive (Figs 6, 7). Discussion

From the above results, it is clear that the radioactive norepinephrinebinding sites detected on rat glial cell membranes can hardly be identified as the ~-adrenergic receptors involved in adenylate cyclase activation: (1) apparent Km for binding was about ten times higher than the apparent Km for activation determined under identical experimental conditions. (2) The stereospecificity of the norepinephrine-binding sites is strikingly different from the stereospecificity of the fi-adrenergic receptor as deduced from an analysis of structural requirements for adenylate cyclase activation. As in the other systems so far studied the detected binding sites are able to interact with compounds possessing a 3-4-dihydroxyphenolic moiety but devoid of biological activity on the adenylate cyclase system. The stereospecificity of the fi-adrenergic receptor is much more pronounced than that of the catechol-binding sites; thus the ~-adrenergic receptor exhibit a marked stereospecificity towards (+)- and (--)-isomers of catecholamines [20]. The observed norepinephrine binding by rat glial cells does not seem to be involved in the norepinephrine-transport system present in these cells. Thus, control experiments indicated that tyrosine, which has no affinity for the catechol-binding sites, inhibits the uptake of radioactive norepinephrine by glial cells. As recently suggested by Cuatrecasas et al. [2] the catechol-binding sites could be related to the e n z y m e catchol-O-methyltransferase. The difficulties encountered for the detection of a c o m p o n e n t of norepinephrine binding in-

376

volved in adenylate cyclase activation deserve further comment. If one assumes that the density of fl-adrenergic receptors on the cell membrane of glial cells is comparable to the density of several other well-characterized hormonal receptors such as insulin on fat cells [21], glucagon on hepatocytes (2 pmol/mg) [22], or vasopressin on pig kidney medulla (1 pmole/mg) [23] it is obvious that the binding of norepinephrine to the ~-adrenergic receptor would represent a fraction of total binding (less than 10%), t o o small to be adequately detected. On the other hand, as pointed o u t by Cuatrecasas et al. [2], we can expect the half-life of the hormonal receptor complex to be very short as compared to the values obtained for peptidic hormones exhibiting much higher affinities for their specific receptors. It is thus n o t possible to exclude that almost a complete dissociation of the norepinephrine--receptor complex occurred during the course of the separation procedure (see Methods). From the comparison of the relative stereospecificities of the glial cell ~-adrenergic receptor and catecholbinding sites (Tables I and II) it appears that in this system phenylephrine and 3-methoxydopamine could constitute useful ligands for a further attempt to characterize the fl-receptor provided the possibility of obtaining them in a radioactive form of sufficiently high specific activity. The rat glial cell catecholbinding sites are most probably homologous to those detected on several other systems; maximal binding capacity and apparent affinity determined on glial cells fell in the range of values obtained on others tissues [1--10] (Lacombe, M.L. and Hanoune, J., personal communication). However, in contrast with what was observed with liver, heart and fat cells membranes [1], norepinephrine attachment to glial cells catechol receptors was easily reversible. In addition, the time course of norepinephrine binding to rat glial cells was found to be much more rapid than in other systems, in which 5--10 or even 60 min incubation in the presence of radioactive catecholamines is needed to reach an equilibrium. References 1 T o m a s i V., K o r e t z , S., R a y , T . K . , D u n n i c k , J. a n d M a r i n e t t i G . V . ( 1 9 7 0 ) B i o c h i m . B i o p h y s A c t a 211, 31--42 2 C u a t r e c a s a s , P., Tell, G . P . E . , S i c a , V., P a r i k h , I. a n d C h a n g , K . J . ( 1 9 7 4 ) N a t u r e 2 4 7 , 9 2 - - 9 7 . 3 M a r i n e t t i , G . V . , R a y , T . K . a n d T o m a s i , V. ( 1 9 6 9 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3 6 , 1 8 5 - - 1 9 3 4 Dunnick, J.K. and Marinetti G.V. (1971) Biochim. Biophys. Acta 249,122--134 5 L e f k o w i t z , R . J . a n d Haber~ E. ( 1 9 7 1 ) P r o c . N a t l . A c a d . Sci. U.S. 6 8 , 1 7 7 3 - - 1 7 7 7 6 L e f k o w i t z , R . J . , S h a r p , G . W . 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[3H] norepinephrine binding by rat glial cells in culture. Lack of correlation between binding and adenylate cyclase activation.

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