85

Biochirnica et Biophysica Acta, 585 (1979) 85--93

© Elsevier/North-HollandBiomedicalPress

BBA 28910 BETA-ADRENERGIC RECEPTOR DESENSITIZATION IN RAT ADIPOCYTE MEMBRANES

YVES GIUDICELLI, BRIGITTE AGLI and DANII~LELACASA Service de Biochimie, Centre Hospitalier Intercomrnunal, 78303 Po/ssy Cddex, and Facultd de Mddecine, Paris-Ouest (France)

(Received September 1st, 1978) Key words: ~-Adrenergic receptor; Desensitization; (Adipocyte membrane)

Summary 1. The ability of fi-adrenergic agonists to inactivate the fi-adrenergic receptors of rat white adipocytes has been investigated using (--)-[3H]dihydroalprenolol as the ~-adrenergic ligand. 2. When adipocyte membranes are successively exposed to (--)-isoproterenol at 25 or 4°C, repeatedly washed and then incubated with [3H]dihydroalprenolol, 40--60% of the ~-receptors are lost. 3. This loss (receptor desensitization) is rapid (half-life = 5--10 rain), is concentration-dependent and is associated with a fall in the maximal receptor number with no significant change in the affinity of the remaining receptors for dihydroalprenolol and isoproterenol. Decreased binding is also accompanied by decreased ability of isoproterenol to stimulate adenylate cyclase. 4. Fat cell ~-receptor desensitization is stereospecific and characteristic of a ~-receptor mediated process, the order of potency of agonists in inducing desensitization being (--)-isoproterenol > (--)-norepinephrine > (--)-epinephrine > (+)-isoproterenol. 5. Filipin and 5'-guanylylimidodiphosphate, a nucleotide which reduces the receptor affinity for the agonists, both prevent and reverse the desensitization process, suggesting that this process is related to a poorly and slowly reversible high affinity binding of the agonist. 6. The possible role played by fi-receptor desensitization in the regulation of catecholamine-mediated process in adipocytes and in the catecholamine resistance of fat cells exposed to catecholamines is discussed.

Abbreviation: Gpp(NH)p,

5~-guanylylimldodiphosphate.

86 Introduction

During the last three years, numerous studies have provided evidence that insulin [1], growth hormone [2], angiotensin [3] and catecholamines [4] can act as regulatory factors in modulating the number of their respective specific membranous receptors; the higher' the concentration of hormone, the less the number of its receptors. In the case of catecholamines, this phenomenon called 'desensitization' has been shown to apply to /~-adrenergic receptors of some catecholamine target cells such as frog erythrocytes [5--7], rat pineal cells [8,9] and human astrocytoma cells [10], but not to those of other cells such as turkey erythrocytes and reticulocytes [11 ]. In a recent study, it was reported that fat cells from patients with phaeochromocytoma are resistant to catecholamines, i.e. elicit a weak lipolytic response to catecholamines [12]. As catecholamine-induced desensitization of fi-adrenergic receptors does not appear to be a general feature for all catecholamine-sensitive cells, and because of the lack of information concerning adipocytes, we have now determined whether the fi-adrenergic receptors of rat fat cell membranes are or are not desensitizable by their agonists. The present data show clearly that exposure of unfractionated fat cell membranes to the potent ~-adrenergic agonist isoproterenol leads, indeed, to a rapid fall in the apparent number of fl-adrenergic receptor binding sites in the membrane. Materials and Methods

Materials (--)-[3H]Dihydroalprenolol (spec. act., 39 Ci/mmol) was prepared by the Radiochemical Centre (Amersham, U.K.). (+)-Alprenolol was a gift of Laboratoires Lematte et Boinot (France). Other compounds used in this study were: (--)-isoproterenol bitartrate, (--)-epinephrine bitartrate and (--)-norepinephrine bitartrate (Sigma Chemical Co., U.S.A.); Instagel (Packard Instruments, U.S.A.); crude bacterial collagenase type CLS 45 DO 06, specific activity, 157 units per mg (Worthington Biochemical Corp., U.S.A.); bovine serum albumin, fraction V, fatty acid poor (Calbiochem Inc., U.S.A.) and Gpp(NH)p (Boehringer, Mannheim, F.R.G.). Filipin was kindly supplied by Dr. G.B. Whitfield from Upjohn Co. (U.S.A.). The (+)-stereoisomers of catecholamines were a generous gift of Sterling-Winthrop (U.S.A.). Preparation of 'crude' adipocyte membranes Male Wistar CF rats (225--250 g), fed ad libitum, were killed by decapitation. Epididymal fat pads (25--30 g) from 15--20 rats were pooled and isolated fat cells prepared as previously described [13]. Isolated fat cells were suspended in Medium I (0.25 M sucrose/1 mM EDTA/10 mM Tris-HC1, pH 7.4) added with 3 mM ATP and 3 mM MgC12 and disrupted by six aspirations through a swinny filter holder fitted with a stainless steel photo-etched support (XX 30 01200, Millipore, U.S.A.), as described by Avruch and Wallach [14]. After breakage, the suspension was immediately centrifuged at 20 000 × g for 3 rain at 20°C in a Sorvall RC 5 centrifuge SM-24 rotor. The floating fat cake

87 and the infranatant were discarded and the resulting pellet was redispersed in Medium I. After centrifugation at 20 000 × g for 20 min at 4°C, the pellet was resuspended in Medium II (10 mM MgC12/50 mM Tris-HC1) and recentrifuged at 20 000 × g for 20 min at 4°C. The final pellet {'crude' membranes) was resuspended in Medium II, resulting in a suspension containing 3--4 mg o f protein/ ml which was immediately used in the binding assays. Recovery of crude membrane protein was 20--22% of the whole protein content of the fat cell. Protein was determined b y the m e t h o d of Lowry et al. [15].

Preliminary incubation with isoproterenol Membrane suspension (1 ml) was incubated for different periods at 25 or 4°C in the presence or absence of isoproterenol (0.1--100 pM) in Medium II added with ascorbic acid (final concentration 1/10 000, w/v). At the end of incubation, the membranes were diluted to 10 ml with Medium II and centrifuged at 30 000 × g for 15 min. The membranes were washed t w o more times in the same way prior to their use for the binding assays. The washing procedures were performed at 4°C and t o o k a b o u t 45 min. In separate experiments, we have found that these procedures were sufficient to remove completely the radioactivity b o u n d to the membrane after a preliminary incubation with 20 nM (--)-[3H]dihydroalprenolol. In addition, adenylate cyclase activity of membranes first exposed to 100 pM isoproterenol and then subjected to these washing procedures returned to control levels after the wash.

Binding assays and adenylate cyclase determination ~-Adrenergic receptors were assessed b y measuring the binding of (--)-[3H]dihydroalprenolol according to a modification of the m e t h o d o f Williams et al. [16]. Membrane (150--175 pg) was usually incubated with 20 nM (--)-[3H]dihydroalprenolol in a total volume of 75 t~l of incubation buffer {Medium II) for 8 min with shaking at 37°C. Incubations were terminated b y adding 1 ml of ice-cold incubation buffer, followed b y rapid vacuum filtration of the suspension through a Whatman GFC glass-fiber filter. Filters were rapidly washed with two 2.5-ml portions of ice-cold buffer. Under these conditions, filtration and washing procedures required less than 30 s. Filters were dried and added to 10 ml scintillation cocktail (Instagel, Packard, U.S.A.) and c o u n t e d in a Kontron MR 300 spectrometer. Non-specific binding (background, non-specific adsorption to filters and non-specific adsorption to membrane protein) was determined b y measuring the radioactivity retained on filters when incubations were performed in the presence of a large excess {20 pM) of (~)-alprenolol. Nonspecific adsorption to the filter was less than 0.4% of total counts filtered and was n o t affected b y the presence of high concentrations of unlabeled ligands. The nonspecific adsorption to membrane-protein averaged 20--25% o f the counts specifically b o u n d to adipocyte membranes. The binding values reported refer to specific binding determined b y subtracting the non-specific binding from the total counts bound. Adenylate cyclase activity was determined as previously described [17].

88 Results

When crude adipocyte membranes were exposed for different times to 10 pM isoproterenol at 25°C, washed and then incubated for the determination of fl-adrenergic receptors, the apparent number of ~-adrenergic receptors fell in a time-dependent fashion (Fig. 1). This process was fairly rapid, since the halflife for the decrease in (--)-[3H]dihydroalprenolol binding was 5--10 min and the process was essentially complete by 20 min. When incubations were performed at 4°C in the presence of 100 HM isoproterenol, the magllitude of the desensitizable process was not significantly different from that found after incubation at 25¢'C with the same concentration of agonist (percent decrease in the apparent n u m b e r of receptors at 4 and 25°C: 50 !: 7 and 57 :~ 12, respectively). As shown in Fig. 2, desensitization of the fl-adrenergic receptors was dependent on the concentration of the fi-agonist added in the preincubation medium. However, even at the concentration of 100 pM isoproterenol, the maximum decrease in the apparent number of receptors did not exceed 60% of those of the control. Half-maximal desensitization occurred at 0.5 ~M isoproterenol, a concentration which corresponds closely to that which causes half-maximal occupancy of (--)-[3H]dihydroalprenolol binding sites (Fig. 4) and half-maximal stimulation of adenylate cyclase in rat fat cell membranes [16]. When isoproterenol-incubated membranes were studied over a range of [3H]dihydroalprenolol concentrations (Fig. 3), Scatchard analysis [18] of the bind-

DESENSI T| ZAT| ON 100

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I

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5 10

I

I

I

20

30

45

TIME OF PREINCUBATIONWIIH 10 ~M tSOP~TERENOL

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ISOPROTERENOLCONCENTRATIONIN PREINCU~TION (-LOGIo M )

( MIN )

I.'ig. 1. T i m e - c o u r s e o f i s o p r o t e r e n o l - i n d u e e d r e d u c t i o n in ( - - ) - [ 3 H i d i h y d r o a l p r e n o i o l b i n d i n g sites o f rat a d i p o c y t c m e m b r a n e s . C r u d e m e m b r a n e s w e r e p r e i n c u b a t e d w i t h or w i t h o u t 1 0 / z M ( - - ) - i s o p r o t e r e n o l at 4 (m) or 2 5 ° C (~]) f o r the i n d i c a t e d p e r i o d s . M e m b r a n e s w e r e t h e n w a s h e d a n d a s s a y e d for (--)-[ 3 H J d i h y d r o a l p r e n o l o l b i n d i n g as d e s c r i b e d u n d e r Material a n d M e t h o d s . R e s u l t s are e x p r e s s e d as p e r c e n t o f c o n t r o l b i n d i n g ( 0 . 2 4 5 + 0 . 0 1 8 p m o l / m g p r o t e i n ) a n d r e p r e s e n t t h e m e a n of t r i p l i c a t e d e t e r m i n a t i o n s . Fig. 2. C o n c e n t r a t i o n d e p e n d e n c e o f ( - - ) - i s o p r o t e r c n o l - i n d u c e d r e d u c t i o n in ( - - ) - [ 3 H ] d i h y d r o a l p r e n o l o l b i n d i n g sites o f r a t a d i p o c y t e m e m b r a n e s . C r u d e m e m b r a n e s w e r e p r e i n c u b a t e d w i t h or w i t h o u t t h e indic a t e d c o n c e n t r a t i o n s o f ( - - ) - i s o p r o t e r e n o l for 4 5 rain a t 2 5 ° C . Membrazlcs w e r e t h e n w a s h e d a n d a s s a y e d f o r ( - - ) - | 3 H ] d i h y d r o a l p r e n o l o l b i n d i n g as d e s c r i b e d u n d e r Material and M e t h o d s . Values s h o w n r e p r e s e n t the m e a n t S . E . o f t r i p l i c a t e d e t e r m i n a t i o n s .

89

ing data showed that the decreased receptor binding was associated with a fall in receptor number with no significant change in the binding affinity of the remaining receptors for dihydroalprenolol (dissociation constants, KD, in control and desensitized membranes: 12 and 10 nM, respectively). Similarly, data in Fig. 4 show that there was no significant change in the apparent binding affinity of the remaining receptors for isoproterenol after desensitization ( g D values of 0.3 and 0.5 gM in control and desensitized membranes, respectively). When adipocyte membranes that were preincubated with isoproterenol and washed were tested for adenylate cyclase activity (Table I), a decrease in catecholamine-sensitive activity was observed, an effect which was rather slight compared with the magnitude of the decrease in the fl-adrenergic receptor num-

O,020

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0,i

0,2

(-)- [3HiDIHYDROALPRENOLOLBOUND ( PMOL/ MG)

KD(ISO)IN CONTROL= 0,25 pM

N -8

-7

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-3

LOGIo ( ISOPROTERENOL)

Fig. 3. S c a t c h a r d analysis o f (--)-[ 3 H ] d i h y d r o a l p r e n o l o l b i n d i n g as a f u n c t i o n o f (--)-[ 3 H ] d i h y d r o a l p r e n o lol c o n c e n t r a t i o n in c o n t r o l a n d d e s e n s i t i z e d ' c r u d e ' r a t a d i p o c y t e m e m b r a n e s . C r u d e m e m b r a n e s w e r e p r e i n c u b a t e d in t h e p r e s e n c e (~ o) o r a b s e n c e ( e "-) o f 1 0 0 # M ( - - ) - i s o p r o t e r e n o l f o r 2 0 m i n at 2 5 ° C , w a s h e d a n d a s s a y e d f o r ( - - ) - [ 3 H ] d i h y d r o a l p r e n o l o l b i n d i n g . D i s s o c i a t i o n c o n s t a n t s , K D , w e r e d e r i v e d f r o m t h e r e c i p r o c a l o f t h e slopes a n d m a x i m a l n u m b e r o f b i n d i n g sites, N , f r o m t h e abscissa i n t e r cepts. E a c h p o i n t r e p r e s e n t s t h e m e a n o f t r i p l i c a t e d e t e r m i n a t i o n s . F o r line n o (isoproterenol present): K D = 1 0 nM; N = 0 . 1 2 p m o l / m g p r o t e i n ; R = 0 . 9 5 . F o r line • ~" ( i s o p r o t e r e n o l a b s e n t ) : K D = 1 2 nM, N = 0 . 2 6 p m o l / m g p r o t e i n ; R = 0 . 9 6 . Fig. 4. I n f l u e n c e o f d e s e n s i t i z a t i o n o n t h e d i s p l a c e m e n t c u r v e s o f ( - - ) - [ 3 H ] d i h y d r o a l p r e n o l o l b i n d i n g t o adipocyte membranes by (--)oisoproterenoL Crude rat adipocyte membranes were preincubated with (~ . . . . . . ~) or w i t h o u t ( • -') 1 0 0 / ~ M ( - - ) - i s o p r o t e r e n o l a t 4 ° C f o r 4 5 m i n . M e m b r a n e s w e r e t h e n w a s h e d a n d i n c u b a t e d w i t h 2 0 n M ( - - ) - [ 3 H ] d i h y d r o a l p r e n o l o l a n d w i t h incxeasing c o n c e n t r a t i o n s o f (--)-isoproterenol for 8 min. The a m o u n t of [ 3 H ] d i h y d r o a l p r e n o l o l specifically b o u n d was t h e n determ i n e d . R e s u l t s are e x p r e s s e d as p e r c e n t o f t h e a m o u n t b o u n d in t h e a b s e n c e o f i s o p r o t e r e n o l ( 0 . 2 4 3 a n d 0 . 1 1 4 p m o l / m g o f p r o t e i n in c o n t r o l a n d d e s e n s i t i z e d m e m b r a n e s , r e s p e c t i v e l y ) . E a c h v a l u e is t h e m e a n o f quadruplicate determinations. Dissociation constants K D were calculated according to the m e t h o d of Cheng and Prusoff [24] by the following equation: K D=ECS0/I+

[DHA] KD(DHA ) '

w h e r e ECso is t h e c o n c e n t r a t i o n o f a g o n i s t w h i c h c a u s e d 50% of m a x i m a l d i s p l a c e m e n t , [ D H A ] , t h e c o n c e n f r a t i o n o f l a b e l e d l i g a n d p r e s e n t in t h e assay a n d K D ( D H A ) , t h e d i s s o c i a t i o n c o n s t a n t o f [ 3 H ] d i h y d r o a l p r e n o l o l d e r i v e d f r o m e q u i l i b r i u m studies.

90 TABLE I INFLUENCE OF ADIPOCYTE MEMBRANE PREINCUBATION CATECHOLAMINE-STIMULATED ADENYLATE CYCLASE

WITH (--)-ISOPROTERENOL

ON

M e m b r a n e s w e r e p r e i n c u b a t e d at 4°C in t h e a b s e n c e o r p r e s e n c e o f 1 0 0 DM ( - - ) - i s o p r o t e r e n o l . M e m b r a n e s w e r e t h e n w a s h e d as d e s c r i b e d in Materials and M e t h o d s b e f o r e to be a s s a y e d for a d e n y l a t e c y c l a s e . N u m b e r s in p a r e n t h e s e s i n d i c a t e t h e n u m b e r o f dete~cminations. E a c h value r e p r e s e n t s t h e m e a n ± S.E. * P 0.001. Preincubation

A d e n y l a t e c y c l a s e in r e s p o n s e to ( - - ) - i s o p r o t e r e n o l (% a b o v e basal a c t i v i t y )

Control (--)-Isoproterenol (100 pM)

2 9 1 _+ 43 ( 1 5 ) 1 9 0 ± 28 * ( 1 5 )

ber. Under the same conditions, however, fluoride-sensitive activity was unaffected. Fat cell t-adrenergic receptor desensitization by adrenergic agonists was found to be a ill-receptor mediated process [19]: in fact, as shown in Table II, the order of potency of agonists in inducing an apparent fall in receptor number was (--)-isoproterenol > (--)-norepinephrine > (--)-epinephrine. Receptor desensitization was stereospecific, the (--)-stereoisomers being 100--1000 times more potent than the (+)-stereoisomers in decreasing the receptor binding site number (data not shown). Filipin (500 pg/ml), a polyene antibiotic which was shown to induce an uncoupling o f l-adrenergic receptors and adenylate cyclase in frog erythrocytes [6], caused a complete inhibition of isoproterenol-induced decrease in receptor number (Table III). The presence of 100 ~M Gpp(NH)p during the preliminary incubation o f membranes also prevented (although to a lesser extent than did filipin) the reduction in receptor number induced by isoproterenol (Table III). Gpp(NH)p was also able to reverse partially the decrease in receptor number induced by isoproterenol: this reversal, which was partial (50--60%) with 100 ~M Gpp(NH)p, was almost complete with a ten-fold higher concentration (data not shown). As this nucleotide has been recently reported to induce a shift in the affinity of the l-adrenergic receptors of erythrocyte membranes for their agonists [20], it seemed of interest to determine whether such an effect could or not apply to the fat cell receptors as well. By testing the influence of 100

TABLE II COMPARATIVE ACTIVITIES OF ~-ADRENERGIC MEMBRANOUS ~-ADRENERGIC RECEPTORS

AGONISTS IN DESENSITIZING

RAT FAT CELL

F a t cell m e m b r a n e s w e r e p r e i n c u b a t e d w i t h or w i t h o u t ( c o n t r o l ) 1 0 0 ~ M o f t h e d i f f e r e n t c a t e c h o l a m i n e s o for 4 5 m i n at 4 C. M e m b r a n e s w e r e t h e n w a s h e d and a s s a y e d f o r d i h y d r o a l p r e n o l o l b i n d i n g . Each value is t h e m e a n ± S.E. o f f o u r d e t e r m i n a t i o n s . * P ~ 0 . 0 0 1 ; ** 0 . 0 2