Coexistence of three β-adrenoceptor subtypes in white fat cells of various mammalian species

European Journal of Pharmacology, 199 (1991) 291-301 0 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991004380

291

EJP 51924

Dominique Langin, Maria P. Portillo, Jean-Skbastien Saulnier-Blache and Max Lafontan INSERM Unitt! 317, Institut de Physiologic, Unicersire Paul Sabatier, Rue Franqois Magendie, 31400 Toulouse, France

Received 7 June 1990, revised MS received 5 April 1991, accepted 9 April 1991

The nature of the P-adrenoceptors (P-ARs) of the white fat cells of five mammalian species (rat, hamster, rabbit, dog and humans) was reassessed. The coexistence of at least three p-ARs on the fat cell (except human) was demonstrated. Comparative binding and lipolysis studies were performed, using recently synthesized compounds selective for the atypical P-AR of the rat brown fat cell and of the rat colon. PI- and &-ARs have previously been identified in all the mammalian white fat cells using [‘251jcyanopindolol ([‘25]CYP) or [3H]dihydroalprenolo1. In addition to these receptors, we now demonstrated the existence of a third P-AR directly involved in adrenergic-mediated lipolysis, and identified it in the white fat cells of the most commonly studied animal species, except humans. This receptor is not detected by the classically used P-antagonist radioligands, explaining the discrepancies in reports on the nature of the /?-ARs of the adipose tissue. Pharmacological delineation of the third type of B-AR-induced lipolysis showed this receptor to be rather similar to the previously proposed atypical P-AR of brown and white rat fat cells. Its pharmacological properties were clarified, using new selective full agonists and partial agonists also acting as non-selective &/&-antagonists. The limits of [‘25]CYP as a radioligand were reported and the usefulness of BRL 37344, (*)-CGP 12177 and phenylethanolaminotralines derivatives (having an atypical p-activity on intestinal motility) as major tools usable for atypical P-AR activation was demonstrated. Moreover, confirming our previous results about the nature of the p-ARs (p,- and p,-ARs) located in the fat cells of women (Maurigge et al., J. Lipid Res.. 1987, 17, 1561, no atypical P-AR-mediated lipolysis was identified in abdominal adipose tissue from healthy women. The possible differences and similarities between this receptor and the recently cloned &-AR are discussed.

P-Adrenoceptors (atypical); Lipolysis; P-Adrenoceptor subtypes; Adipocytes; BRL 37344; Phenylethanolaminotetralines

1. Introduction

It is widely recognized that two /?-adrenoceptor

(P-AR) subtypes, defined as &- and &-subtype, are involved in the generation of the physiological effects of catecholamines (Lands et al., 1967). The presence of p,- and &-ARs in human fat cells, has been clearly established. The lipolytic responses and binding assays are well correlated, the receptors fitting the standard classification of Lands et al. (1967) (Lacasa et al., 1986; Maurigge et al., 1988). However, the putative existence of additional i.e. ‘atypical’ P-AR in brown and white fat cells of the rat has been proposed. The atypical nature of the rat brown and white fat cell P-AR (neither 8, or &) was established mostly from the

Correspondence to: M. Lafontan, INSERM U-317, Institut de Physi- ologie, Universite Paul Sabatier, Rue Franqois Magendie, 31400 Toulouse. France.

pharmacological analysis of the P-adrenergic-mediated metabolic response (i.e. oxygen consumption. lipolysis, adenylate cyclase activity and cyclic AMP accumula- tion) (De Vente et al., 1980; Tan and Curtis-Prior, 1983; Wilson et al., 1984; Arch et al., 1984; Bojanic et al., 1985; Arch, 1989; Zaagsma and Nahorski, 1990). Most of the binding studies indicated that the P-ARs located on the rat brown and white adipocyte mem- brane were predominantly &, although smaller amounts of &-AR have also been described by various groups, whereas some functional studies show only one subtype (Bojanic and Nahorski, 1983; Rothwell et al., 1985; Levin and Sullivan, 1986; Bahouth and Malbon, 1988; Muzzin et al., 1988a, b). A detailed review of the various aspects of the question was recently published by Zaagsma and Nahorski (1990). The existence of atypical P-ARs has also been proposed in the heart and in the ileum and colon of the rat; these receptors could mediate the agonist properties of some well known &/&-AR antagonists such as pindolol an’

nds (Bond and Clarke, 1988; Kaumann, i and Manara, 1990).

major step foonvard in this pharmacological debate made when a human gene that encodes a

clearly different from the previously cloned +R genes (Frielle et al., 1987; Kobilka et

aI., 1987) and referred to as the &-AR gene, was isolated from a human genomic library (Emorine et al., 1989). The mRNA products of the &-AR gene were identified in mouse white fat cells. The functional pharmacological delineation of the &-AR transfected into CHO cells showed some similarities with &-AR obtained in rat white and brown fat cells. In particular, a compound selective for the rat brown fat cell &AR (A&t et al., 1984), BRL 37344, stimulated with high potency both the atypical &AR-mediated lipolytic re- sponse and the &-AR-mediated cyclic AMP accumu- lation in rat white fat cells and transfected CHO cells, respectively (Hollenga and Zaagsma, 1989; Emorine et al., 1989). However, when results of functional studies are compared, some discrepancies remain between the expressed &AR in CHO celis and that mediating the responses of the rat fat ceil.

Since genetic methods have now clearly shown the existence of at least three genes coding for three p- ARs, several important questions should be addressed. First, it is necessary to refine the delineation of the exact pharmacological profile of the receptor(s) in the tissues where the presence of atypical P-ARs was previously suspected. Moreover, the existence of possi- ble species-specific differences and the possibility of the existence of multiple atypical P-ARs cannot yet be excluded. In an attempt to find an answer to some of these essential questions, the nature of the white fat cell &ARs in various mammals was re-examined, using a compara:ive approach to generalize the concept so far applicable only to the rat.

The &lipolytic responses of the fat cells of five species (rat, hamster, rabbit, dog and human) were explored with different P-AR agonists and antagonists reported as being selective or not for the atypical &ARs of the tissues in which their presence was suspected (white and brown rat fat cells, colon smooth muscle). The putative lipolytic potencies of BRL 37344 and recently described phenylethanolaminotralines (PEAT) (Manara and Bianchetti, 1989; Bianchetti and Manara, 1990) were compared to those of isopro- terenoi, the standard P-agonist. Moreover, ( + )-CGP 12177, a known potent &/&-antagonist on human fat cells, which was however recently reported to cause noradrenaline-like stimulation of oxygen consumption in hamster brown fat cells (Mohell and Dicker, 1389) and to be a putative P-AR partial agonist in the heart (Kunnann, 1989), was also used. Parallel binding stud- ies were performed on the various fat cell membranes to type the sites involved in the action of these agents.

Several derivatives having partial agonist effects on the atypical P-AR were also defined in rat and hamster fat cells.

2. Materials and methods

2.1. Isolation of adipocytes and lipolysis measurements

Animal and human studies were done in agreement with the INSERM guidJines. Adipose tissue was ob- tained from animals which had been fasted overnight before tissue sampling. Male golden hamsters (Mes- ocricetus auratus) (90-110 g), male New Zealand rab- bits (2.0-2.2 kg) and male Wistar rats (200-210 g) were killed by cervical dislocation. Perirenal and epididymal fat deposits were dissected out and pooled for adipocyte isolation. Fat biopsies (omental adipose tissue) from male mongrel dogs (lo-14 kg) were taken immediately after the induction of general anesthesia with pento- barbital. Human subcutaneous adipose tissue samples were obtained from premenauposal women (34 rt 3.1 years old) undergoing plastic surgery. The patients were healthy and none had any identified metabolic or endocrinological disorder, received pharmacological treatment or performed any bout of exercise or train- ing. The mean body mass index (the ratio: weight (kg): height’ cm’)) was 26.4 + 1.1 (n = 10). After surgical excision, the human adipose tissue was quickly trans- ported to the laboratory In sterile physiological saline (0.9% NaCl, 5 mM HEPES, 6 mM glucose, pH 7.5).

Adipocytes were isolated according to the method of Rodbell (1964) with minor modifications. The adi- pose tissue was cut into small pieces and incubated for 30-40 min at 37 a C under vigourous shaking in a cell culture medium consisting of a mixture (1: 1) of Dul- becco modified Eagle’s medium (DME H16) and Ham’s F12 medium supplemented with 15 mM NaHCO,, (pH 7.5) (Gibco Labs.) containing 35 mg/ml bovine serum albumin, 6 mM glucose (H16-F12 buffer) and 1 mg/ml collagenase. Krebs-Ringer buffer was replaced by the H16-F12 buffer in order to minimize lysis of the adipocytes. After collagenase digestion, the adipocytes were tieparated from the stroma-vascular fraction by flotation and washed three times in H16-F12 buffer.

Lipolytic activity was measured by including isolated adipocytes (20-30 mg of total ceil lipid) in 1 ml of H16-F12 buffer containing the agents to be tested. After 90 min of incubation at 37 o C, the reaction was stopped in ice and an aliquot of the incubation medium (200 ~1) was taken to determine glycerol released in the incubation buffer by the method of Wieland (1957). The lipid content of the incubation vials was deter- mined gravimetrically after extraction according to the method of Dole and Meinertz (1960).

The partial agonism of ( f I-CGP 12177 was studied

293

according to the standard technique for the exploration of the dualistic effect of a partial agonist as described by Bowman and Rand (1980). The dose-dependent iipoiytic response to (+ )-CGP 12177 was tested in the presence of increasing concentrations of the full ago- nists, BRL 37344 and isoproterenoi, the physiological agonist, noradrenaline, and a iipoiytic peptide (corti- cotropin) which acts through specific receptors inde- pendent of the P-AR.

Antagonist potencies were evaluated by calculation of their pA, values. Concentration-response curves for agonists were made in the presence of four to six different concentrations of antagonists. Schiid plots were constructed according to Aruniakshana and Schild (1959) and the pA, values were calculated for each concentration of antagonist according to the expres- sion: pA, = -log ([antagonistl/DR - 1) when the slopes of the Schiid plot did not differ from unity (Bowman and Rand, 1980). However, if the Schiid plot had a biphasic character, the pA, values were extrapo- lated from the linear part of the Schiid plot curves defined at higher concentrations of antagonists. Schiid plots were calculated from the individual experiments and average pA, values are given.

2.2. Crude membrane preparation

Crude membranes were prepared by iysis of the adipocytes in a hypotonic medium containing 2 mM Tris-HCI, 2.5 mM MgCi,, 1 mM KHCO,, 100 PM EGTA (35 mOsM, pH 7.5) and the following protease inhibitors: ieupeptin (1 pg/mi), benzamidine (100 PM) and phenyimethyisuifonyi fluoride (100 PM). Adipo- cytes were iysed at 25 o C in order to minimize trapping of membranes in the coalescing fat cake. Ceil ghosts were separated from the fat cake by certrifugation (40000 X g, 15 min at 25 ’ C). The pellet was resus- pended in 1 ml iysing medium and immediately frozen at - 80 o C until use.

Thawed crude membranes were diluted in a large volume (30 ml) of 50 mM Tris-HCi, 5 mM EDTA, pH 7.4 and centrifuged (40000 x g, 10 min at 4” C). The pellet was washed once in Tris-Mg2+ buffer (50 mM Tris-HCI, 0.5 mM MgCi,, pH 7.5) followed by a second centrifugation. The resulting pellet was finally resus- pended in the required volume of Tris-Mg2+ buffer and immediatciy used for measurements of binding capacity.

2.3. Binding studies

[ 12’1]( - )-Cyanopindoioi ([ iz51]CYP) binding studies were performed at 37 o C for 60 min (Engei et al., 1981; Hoyer and Engel, 1983). The incubation medium con- sisted of 50 ~1 of radioiigand and 50 pi of membrane suspension made up to a final volume of 200 pi with

Tris-Mg 2+ buffer. At the end of the incubation period, the suspensions were filtered through GF/C Whatman filters (soaked for 60 min in 0.01% poiyethyieneimine) on a Miliipore manifold sampling unit. The tubes and filters were washed with 20 ml of Tris-Mg2+ buffer. The radioactivity retained on the filters were counted in a Packard gamma counter (efficiency: 80%).

Specific binding was calculated as the difference between total and non-specific binding which was de- fined in the presence of 100 PM isoproterenol. For the saturation studies, the [‘2511CYP concentrations used ranged from 4 to 200 pM for the definition of high affinity binding sites (&- and P,-sites). Binding param- eters were determined using LIGAND, a non-linear curve-fitting program (Munson and Rodbard, 1980). Protein concentration was measured according to Lowry et al. (1951) using bovine serum albumin as standard. All data are given as means + S.E.M.

2.4. Drugs and chemicals

[‘2511CYP (2000 Ci/mmoi) and [3H]dihydroaipre- nolo1 (85 Ci/mmoi) came from New England Nuclear (Boston, MA) and from Amersham (Les Uiis, France), respectively. BRL 37344 and derivatives were generous gifts from Dr. M.A. Cawthorne (Beecham Pharmaceu- ticals, Epsom, UK). (+)-CGP 12177 and related com- pounds, ( + )- and ( - )-cyanopindoioi, were generous gifts from Dr. K. Scheibii (Ciba-Geigy, Basie, Switzer- land) and Dr. D. Hoyer (Sandoz Ltd., Basie, Switzer- land), respectively. Isoproterenol, propranoloi, ai- prenoioi, noradrenaline, ACTH and bovine serum ai- bumin (fraction V\ were from Sigma Co. (St. Louis, MO); bisoproioi from Merck (Darmstadt, FRG); ICI 118551 from ICI Ltd. (Macciesfieid, UK). Pindoioi and bupranoioi came from Sandoz Ltd. (Basie, Switzer- land) and from Sanoi (Monheim, FRG), respectively. The phenyiethanoiaminotetraiines (SR 58611A, SR 58612A and SR 58825) were kindly given by Drs. A. Bianchetti and L. Manara (Sanofi-Midi Group, Miiano, Italy). Coiiagenase and enzymes for glycerol assays came from Boehringer (Mannheim, FRG). All other chemicals were reagent grade of the best quality avail- able.

3. Results

3.1. Comparison of the lipolytic effects of BRL 37344, ( + )-CGP 12177 and isoproterenol on fat cells from fiue species

Concentration-response curves for the effects of BRL 37344, ( f )-CGP 12177 and isoproterenoi on hu- man, rat and hamster fat ceils are depicted in fig. 1. All the concentration-response curves were expressed reia-

5 ._ 50 50 b 5 E .- 3j .o S 6

Q

,Q II ICI 9 9 7 5 s

3 - tog BRL 37344 (MI - log CGP 12177 (M) 967654

0

10 9 1 6 s 4

-log lsoproterenol (M)

Fig. I. Comparison of the lipolytic effect of BRL 37344 (A), ( + KGP 12177 (B) and isoproterenol (0 on human ( ), rat (0) and hamster (a) fat

cells. Isolated fat cells (20-25 mg total lipid) were incubated in 1 ml H16-F12 cell culture medium containing 35 g/l bovine serum albumin and 6 mM glucose. Lip&is was measured by determination of glycerol release in the presence of increasing concentrations of various compounds UXi1 nM to 0.1 mM). The results are expressed as percent of maximal stimulation of the lipolysis induced by (- )-isoproterenol (10 pM). The values are means + S.E. from 5 to 10 separate experiments performed in duplicate. Mean pD, values and intrinsic activities are given in table 1

for all the species.

tive to the (- )-isoproterenol maximum which was equivalent to that reached with the highest BRL 37344 concentrations or with forskolin (10 PM). Although they have different intrinsic activities, BRL 37344 and (+_)_CGP 12177 were both lipolytic on the white fat cells of all the experimental animal species tested (rat, hamster, dog and rabbit) (table 1). In most of the species studied, the pD, values for BRL 37344 were higher than those for isoproterenol. The relative order of potency in all the species except man and rabbit was: BRL 37344 1 isoproterenol > ( rt )-CGP 12177 (ta-

ble 1). In man, BRL 37344 and (+I-CGP 12177 had no lipolytic effect on isolated subcutaneous fat cells while isoproterenol exerted a Iipolytic action (fig. 1).

3.2. Discrepancies between the binding affinities md the action of the lipolytic agem

Binding assays were carried out to determine if the parameters of [‘251]CYP binding (B,,, and K, values) on the fat cell membranes from the various species used under our working conditions fitted previously

rABLE 1

Stimulation of lipolysis by BRL 37344, isoproterenol and ( + I-CGP 12177 in white fat cells of various species. White fat cells were isolated by collagenase digestion. Lipolysis was assayed by determination ol’ glycerol release in the presence of increasing concentrations of agonists (0.02 nM-0.1 mM). Intrinsic activities (LA.) of BRL 37344 and ( j, )-CGP 12177 were expressed as a percentage of the maximal lipolytic effect initiated by t - )-isoproterenol (taken as reference). The lipolytic effect promoted by BRL 37344 was equivalent to that of isoproterenol while the effect of (t KGP 12177 was rather variable according to the species. The results were expressed as pD, values; the pD, values of the agonists (-log EC& were estimated from each concentration-response curve. The results (pD, values) are means*S.E. of S-10 separate expeiiments. An average value is given for LA. (value I corresponds to the LA. equivalent to that of isoproterenol, lower activities are expressed as fractions of the maximal action). ND. not determined (no agonist effect).

Human Rat Hamster Rabbit

Dog

BRL 37344

PD,

ND IX.55 * 0.07 8.76kO.17 6.69 + 0.05 8.01 + 0.08

LA.

0 1 1 0.90 0.97

Isoproterenol

PDF

7.32fO.18 7.67 f 0.06 7.30 + 0.10 7.00+0.27 7.74kO.21

LA.

1 1 1 1 I

( f )-CGP 12177

PDZ

ND 5.86 f 0.09 6.30 f 0.08 5.71 kO.17 6.72 f 0.09

LA.

0 0.36 0.76 0.25 0.65

295

published data. [‘2sI]CYP was used firstly for P-site definition (both pi- and p,-sites) in heart and lung membranes of guinea pig (Engel et al., 1981; Hoyer and Engel, 1983). The number of [1251]CYP binding sites (B values), defined within a low concentration range o??~‘I]CYP (4-200 PM), varied according to the species: 105 + 18, 95 f 14, 81+ 2, 31+_ 3 and 251 f 75 fmol/mg protein in human, rat, hamster, rabbit and dog fat cell membranes respectively. It was not signifi- candy different from that defined with [3H]dihydroal- prenolol on the same membrane batches (not shown) and agreed with previously published data obtained with ]‘2sIlCYP or other ligands. Moreover, the Ku values for [‘2511CYP were very similar in the various species (i.e. 27 f 3, 18 &- 4, 21 f 2, 19 f 4 and 39 + 4 pM for human, rat, hamster, rabbit and dog fat cell membranes, respectively). The coexistence of pi- and P,-ARs has previously been described in all the mam- mal white fat cells investigated (Lacasa et al., 1986; Mauriege et al., 1988; Engel et al., 1981; Bojanic and Nahorski, 1983; Bahouth and Malbon, 1989; Valet et al., 1989; Saulnier Blache et al., 1990). The &- and P2-AR numbers were clearly different in each species but the presence of both subtypes is not questionable in any of the white fat cells used. Nevertheless, since [‘251]CYP was generally treated as a non-selective radi- oligand whereas it is actually considered as being 2-fold selective for P2-AR, it cannot be excluded that the distortion in the estimation of the relative proportion of each kind of site could be considerable, as recently discussed by various investigators (Neve et al., 1986; Nanoff et al., 1987; Schiitz et al., 1988). Although this problem merits consideration, it is considered to be outside the scope of the present paper.

It was essential to demonstrate that the sites la- belled at the lowest concentrations of [12511CYP were different from those involved in the lipolytic action of BRL 37344 and (&I-CGP 12177. Inhibition of [‘251]CYP binding by BRL 37344, (rt)-CGP 12177 and isoproterenol was carried out on fat cell membranes in parallel with the lipolytic assays; typical competition curves (using 50 pM [‘251]CYP to label the sites) are shown for the rat (fig. 2). It was impossible to calculate accurate IC,, values and to give pIC,, values. In the absence of GTP or GppNHp in the assay buffer, the displacement curves with isoproterenol and probably BRL 37344 are complex to analyse since there are four components, i.e. high-affinity and low affinity states of p,- and P2-AR. Although it was impossible to express accurate binding parameters, the relative order of po- tency of the agents for the displacement of [12sIl- CYP binding (( + I-CGP 12177 > isoproterenol > BRL 37344) was found on human fat cell membranes and in those of the four other species.

To summarize, the fl,/P,-antagonist, (+ 1CGP 12177, exhibited the highest affinity for these sites; this

A

BRL 37344

lsoproterenol

CGP 12177

, I , , I , 1 ,

11 IO 9 9 7 6 5 4

- log Agonists (Ml

I1 IO 9 8 7 6 5 4 -log Agonists (M)

Fig. 2. Comparison of the lipolytic responses (A) and the displace- ment of [‘%]CYP binding from rat adipocyte membranes (B) initi- ated by BRL 37344 (a), (- kisoproterenol (0) and (+ )-CGP 12177 ( @ 1. Lipolysis is expressed as pmol of glycerol released per 100 mg total lipid over 90 min. Binding data is expressed as a percentage of [‘251]CYP specific binding. A fiied concentration of 50 pM of [““I]CYP was used for the binding assays. This experiment is repre- sentative of the data obtained in three others. Similar assays were performed on fat cell membranes of various species and very similar

competition curves with [‘zI]CYP were obtained.

result is in agreement with its high potency to inhibit isoproterenol-induced lipolysis (p,- and p,-AR-media- ted) in human fat cells (Mauriege et al., 1988). What- ever the species, BRL 37344 had a very weak affinity, as compared to that of isoproterenol for the sites labelled by 50 pM [‘251]CYP. The rank order from binding assays (( + )-CGP 12177 > isoproterenol > BRL 37344) is the complete opposite of that defined in the functional assays in non-human species (BRL 37344 > isoproterenol > ( & )-CGP 12177).

Further experiments described here were limited to rat and hamster adipocytes since the response of the fat cells to the lipolytic agents was quite similar (how- ever rabbit fat cells were less responsive) in the four mammalian species (table 1).

32. .~n~~~~tiisna of rite action of the lipolytic agents in rat and hamster fat cdls

It is clear, from their absence of effect on human fat cells, that BRL 37344 and (+)-CGP 12177 exert their lipolytic action in the other species without interacting on typicai &- and &-AR sites. The nature of the receptor activated by BRL 37344 and (&)-CGP 12177 was studied in more detail in rat and hamster white fat cells using the pharmacological antagonism of their lipolytic effects by various selective or non-selective @antagonists. The experiments were first performed with rat fat cells, used as reference used by most previous investigators (De Vente et al., 1980; Tan and Curtis-Prior, 1983; Wilson et al., 1984), to validate the analysis performed under our working conditions. First, ICI 118551 (&-antagonist) and bisoprolol (p,- antagonist) were used to counteract the lipolytic effects of BRL 37344 and ( t_ )-CGP 12177 in rat fat ceils. At the lowest concentrations of bisoprolol (1 nM-10 PM) no noticeable displacement to the right of the BRL 37344 concentration-response curve was seen; a clear shift to the right was only observed with 50 and 100 yM bisoprolol. The Schild plot was biphasic and pA, values were calculated with the highest concentrations of bisoprolol used (10-100 PM). The experiments with ICI 118551 demonstrated antagonism of BRL 37344- induced lipolysis from 10 to 100 PM; the slope of the Schild plot was not significantly different from unity ((1.12 -t 0.06). The results were very similar with ( + )- CGP 12177 (table 2). Experiments were performed on hamster fat cells with the &antagonist (bisoprolol) alone. The clearest shift to the right of the dose-re-

TABLE 2

Antagonism of the lipolytic action of BRL 37344 and (k )-CGP 12177 in rat (RI and hamster (H) fat cells by various selective (bisoprolol and ICI 118551) and non-selective (propranolol and bupranolol) @-antagonists. Increasing concentrations of antagonists were used to counteract the lipolytic actions of BRL 37344 and tk )-CGP 12177. pA, values were calculated for each antagonist according to the expression: pA, = - log[antagonist]/DR - 1 as de- scribed in Materials and methods. Details of Schild plot values and interpretations are given in Results. Values are means+S.E. of n separate experiments. ND, not determined.

n pA z values

Bisoprolol t&-antagonist)

ICI 118551 t&-antagonist)

Propranolol (/3&antagonist)

Bupranolol

t&-& and putative atypical /3-antagonist)

BRL 37344

4 6.250.1 td, 3 5.9 + 0.08 (H)

4 5.4kO.l (R) 3 6.3kO.l (R) 3 6.1kO.l (H) 3 7.3kO.l (RI 3 7.510.1 (H)

( f )-CGP 12177

5850.1 (R) 6.OkO.l (HI

6.OkO.l (R) ND ND ND ND

sponse curve of BRL 37344 only occurred, as with rat fat cells, at the highest concentrations of bisoproIol(50 and 100 PM) and the S,hild plot was clearly biphasic. Results similar to those for rat fat cells were obtained with hamster fat cells (table 2). Although extensive PA, calculations were not performed on rabbit and dog fat cells, bisoprolol, was a weak antagonist of the action of BRL 37344 (not shown), as in the rat and the hamster fat cell.

The similarity between the p-sites was also con- firmed by the following results: propranolol (/3,/&- antagonist) was at least 100 times less potent as a blocker of BRL 37344-induced lipolysis in rat and hamster fat cells than as a blocker of p,- or &-ARs in tissues containing a well characterized /3,- or &AR population (table 2). Bupranolol, which was reported to be a putative probe for the presence of an atypical P-AR in the heart (Kaumann, 1989), was the most efficient blocker of the lipolytic effects of BRL 37344 in hamster and rat fat cells (table 2). This thus demon- strates that the pharmacological profile of the P-recep- tor involved in the genesis of lipolytic effects and activated by BRL 37344 and ( + )-CGP 12177 in rat and hamster fat cells, does not correspond to that of the classical /3r- and &-A&.

3.4. Demonstration of the partial agonist effects of f k)- CGP 12177 at the atypical &AR

Interestingly, (+)-CGP 12177 which is a j?r/&- antagonist in human fat cells (Mauri2ge et al., 1988) was a partial stimulator of lipolysis (variable intrinsic activities) in the fat cells of all the species used except the human (table 1). It was postulated that this effect could be associated to a partial agonism by this com- pound at the putative atypical P-AR. Its partial atypi- cal #I-agonism was confirmed for the rat and hamster fat cells with a standard technique as described in Materials and methods. (f )-CGP 12177 partially an- tagonized the lipolytic effect of BRL 37344. The results obtained with hamster fat cells are depicted in fig. 3; similar results were obtained with rat fat cells. This was confirmed when 10 PM (+)-CGP 12177 was used in the presence of 1 PM of isoproterenol or 10 PM noradrenaline (in the presence of an cY,-antagonist i.e. 10 PM of RX 821002); both concentrations promoting maximal stimulation of lipolysis (equivalent to that induced by 1 PM BRL 37344). The partial antagonism promoted by (k )CGP 12177 (10 PM) was not different from that observed with BRL 37344 suggesting the action of f&)-CGP 12177, isoproterenol and nor- adrenaline on the same receptor site i.e. the atypical P-AR, which is also stimulated by BRL 37344 (fig. 4). There was no effect of (& )-CGP 12177 on corti- cotropin-induced (ACTH-induced) lipolysis (fig. 4) which operates via a different receptor, confirming the

0’ I

7 6 5 4 -log CGP 12177 (Ml

:

i

Fig. 3. Dose-response curve of ( f I-CGP 12177 on hamster fat cells in the absence (01 or presence of increasing concentrations of BRL 37344 (0). Lipolysis is expressed as pmol of glycerol release per 100 mg total lipid over 90 min. The data from this experiment are

representative of those obtained in two others.

specificity of action of ( +)-CGP 12177 OF stypical P-AR sites.

3.5. Demonstration of the lipolytic effect of various full and partial agonists for the atypical P-AR

Improved pharmacological delineation of the atypi- cal &AR is needed. The search for full and partial agonists was extended to several other compounds, using hamster white fat cells. These compounds were BRL derivatives active on brown fat cells (Arch et al.,

. . I!

NS

COP BRL NA IS0 ACTH

Fig. 4. Partial antagonism of the lipolytic effects of BRL 37344 (BRL). noradrenaline (NA), isoproterenol (ISO) and ACTH in iso- lated hamster fat cells by (5 )-CGP 12177. White columns: ( rt )-CGP 12177 (10 FM) was incubated alone (in the absence) or in the presence of 1 PM BRL 37344, 10 PM noradrenaline 1 PM isopro- terenol or 0.1 pg/ml ACTH. Black columns represent the action of the lipolytic agents in the absence of (+ )-CGP 12177. The values are means*S.E. from four different experiments performed in dupli- cate. ** indicates a significant difference at P < 0.01 (Studen?’ paired t-test). NS indicates the absence of a significant differ-

ence.

‘TABLE 3

Stimulation of ]iPolysis by PI /&-antagonists, phenylethanolamino- tetralines (PEAT) and BRL derivatives on hamster white fat cells. Fat cells were isolated by collagenase digestion. Lipoiysis was mea- sured by determination of glycerol release in the presence of increa+ ing COnCCntratiOIIS of various compounds (0.01 nM-0.1 mM). EC, values kor . :ntration of the lipolytic agent causing 50% stimulation of lipolysis) were calculated from the concentration-response curves and pD, values (-log EC.& of the agonists were determined, Al] concentration-response curves were expressed relative to ( - )-isopro- tereno]. ( - )-CGP 12177 was not available. PEAT formulas are given by Bianchetti and Manara (1990). For PEAT derivatives, the first letter (R or S) indicates the contiguration of the phenylethanolamine chiral carbon and the second that of the tetraline part of the moiety. The results are meansf S.E. of three to four separate experiments run in duplicate. ND, not determined (owing to very weak stimula- tion).

Percentage of maximum ( - )-isoprotereno] lipolytic effect

PD,

( - )-Cyanopindolol 62+ 9 7.12kO.08 ( + Kyanopindolol 61rf: 7 5.77+0.18 ( + )-Pindolol 14+ 2 ND ( f )-Alprenolol <5 ND ( * )-Oxprenolol 15* 5 6.05 f 0.03 ( _+ )-BRL 37344 109_+ 4 8.76kO.17 ( f )-BRL 28410 79+ 9 6.41 + 0.03 ; + I-BRL 26830 102+ I3 6.03+0.11 ( + J-CGP 12388 59_+ 3 5.75kO.11 ( f )-CGP 12177 76+ 3 6.30 f 0.08 (+ )-CGP 12177 28k 2 4.62 k 0.09 (+ KGP20712 <5 ND SR 58611 (RS) 98+ 5 7.28+0.11 SR 58612 (RR) 99+ 3 6.94kO.11 SR 58825 (SR) 0 ND

1984), fiI/&-AR blockers possessing non-conventional partial agonist properties in the heart (Kaumann, 1989) and the recently described PEAT having a potent inhibition action on intestinal motility (Bianchetti and Manara, 1990) and possible lipolytic actions (Manara and Bianchetti, 1989).

The stereoselectivity of the P-AR site stimulated by these agents was clearly demonstrated by the use of (+ )- and (+ )-CGP 12177 (the (- ) form of CGP 12177 was not available) and (- )- and (+ )-cyanopindolol enantiomers (p,/p,-blockers) (table 3). BRL deriva- tives acted with the rank order of potency (BRL 37344

> l3RL 28410 > BRL 26830) previously described for the activation of lipolysis in rat brown fat cells (Arch et al., 1984). PEAT derivatives also exerted lipolytic ac- tions and were full agonists like BRL 37344 and isopro- terenol in all the fat cells (except human). They were selected on the basis of their high efficiency on rat colon motor activity (Bianchetti and Manara, 1990). The lipolytic action of the RR isomer ;SR 58612A) and the RS isomer (SR 58611A) was quite similar while the SR form (SR 5SS25A) was totally ineffective (table 3).

e stereoselectivity of action of cyanopindolol and CGp ]?I77 enantiomers was also observed with rat and dog fat cells (not shown). In fact the slight differ- ence concerned the partial agonists ( - )-cyanopindolol and (+ )_CGP 12177, wnich had a weaker lipolytic action than on hamster fat cells (18 +_ 5 and 34 +_ 7% of maxImal lipolytic effect of ( - I-isoproterenol respec- tively: n = 4). In addition. as in hamster fat cells, the strictly similar order of potency of BRL and PEAT derivatives (depicted in table 3) was also found in rat fat cells (BRL 3734-t > SR 5861lA > SR 5S612A > BRL 28-110 > BRL 26830 > SR 58825A). It is notice- able that none of these agents were lipolytic in human fat cells even at the highest concentrations (10 PM).

Furthermore. it was confirmed that the lipolytic effects of the putative atypical P-AR agonist were not due to lipolysis induced by mechanisms other than those involving P-ARs (e.g. inhibition of CAMP-depcn- dent phosphodiesterase activity). Propranolol and bupranolol (10 PM). which counteracted the BRL 373-4-t-induced lipolysis, did not block the Iipolytic ef- fect of several inhibitors of the CAMP-dependent phos- phodiesterase (cilostamide, IBMX and RP 55462 (Saulnier-Blache et al.. 1989a)). Moreover, BRL 37344 and (_t !-CGP !?177 were not lipolytic in human fat cells. where CAMP-dependem phosphodiesterase is present (Kuribayashi et al., 1987). If these compounds were active on CAMP-dependent phosphodiesterase, they would stimulate lipolysis in all fat cells. Pindolol and [“IICYP have been reported to interact with serotoninergic receptors (Hoyer et al., 1985). However, in hamster and rat fat cells, serotonin did not induce lipolysis and did not inhibit [“‘IICYP binding whatever the concentrations of radioligand used. Finally, the lack of effect of putative atypical &agonists in human fat cells which contain a large number oflvrARs (Galitzky et al.. 1989; Lafontan et al., 1989) cannot be attributed to u,-agonist antilipolytic properties since BRL 37344 an+ ( f )-CGP 12177 c’;d not modify adeno- sine deaminase-activated lipolysis in human fat cells. Moreover, in hamster fat cells which also possess a large number of functional (Y,-ARs (Saulnier-Blache et al., 1989b). the putative atypical P-agonists have a high P-efficiency and no cr,-antilipolytic effect. Their lipoly- tic effects are not antagonized by RX 821002 (Saulnier-Blache et al., 1989b), a selective (Y?- antagonist.

3.6. Attempt at identification of atypical P-AR with / !2511CYP

[““IJCYP was used to label the P,-ARs in trans- fected CHO cells; the calculated dissociation constant for the ligand at this site expressed in CHO cells was at least 10 times greater than that defined for /3,- and &-ARs (Emorine et al., 1989). It was postulated at the

outset that, if the adipocyte atypical P-AR was a member of a family of the recently cloned &-AR, [“‘I]CYP could be a possible radioligand if used at concentrations higher than those usually required for the identification of the classical /3,- and P,-ARs. Although (- I-CYP is a partial atypical P-AR agonist and exhibits a rather low potency in our system (table 3), [““I]CYP binciillg studies were attempted at higher ligand concentrations of [““I]CYP) (from 100 to 4OOG pM) on the fat cell membranes of different species. Non-specific [ ““IICYP binding, defined with 100 PM isoproterenol, was very high in rat and hamster fat cells (more than 85% at 4000 pM) and it was impossible to assess any binding parameter (K, and B,, values) accurately. This failure is probably explainable by the very poor binding conditions in the fat cell membranes at the higher concentrations of [‘251]CYP and the re- sults of any further studies with this radioligand and fat cell membranes from the species used in the present experiments will remain highly questionable.

4. Discussion

This study both showed that atypical P-ARs are not limited to the brown and white fat cells of the rat but are more generally distributed in fat cells of various species, and suggests that three separate P-AR sub- types (&-, &- and atypical s-j exist in white fat cells (except probably in human fat cells). BRL 37344, the compound having selectivity for rat brown fat cell lipolysis and respiration, as compared to typical p,- and &-AR-mediated responses of atrium and trachea, is more potent than isoproterenol in rat, hamster and dog fat cells (table 1). Moreover, it is demonstrated that the &//3,-antagonist, (k)-CGP 12177, exerts a lipolytic action which is explainable by its partial ago- nism at the atypical P-AR (fig. 3). The lipolytic action of these agents is weakly antagonized by the selective &-antagonist, bisoprolol, as shown in rat and hamster fat cells. The pA, values for antagonism of lipolysis (table 2) were lower than those classically reported for tissues reported to contain typical /3,- or /3,-ARs i.e. pA, values ranging from 7.5 to 8.7. This result suggests that the lipolytic action of these compounds is not mediated essentially by the PI-AR described in rat fat cells in binding studies (Bahouth and Malbon, 1988).

During completion of the present study, another P,-antagonist CGP 20712A was used to investigate the characteristics of rat adipocyte lipolysis induced by isoproterenol and BRJ, 37344 (Hollenga and Zaagma, 1989). The results obtained with BRL 37344 in the present study with a different agonist (( & )-CGP 12177) and a different selective @,-antagonist (bisoprolol)), agree fully with the conclusions reached by the group of Zaagsma (Hollenga and Zaagsma, 1989). The au-

299

thors concluded from their functional approach, that there was a minor participation of &-AR in the induc- tion of isoproterenol-induced lipolysis and claimed that BRL 37344 acted solely through the atypical P-AR in rat fat cells. Thus, since we were in full agreement with this interpretation, we felt it unnecessary to discuss further the functional analysis of the rat fat cell. It is important to verify that these conclusions can also be extended to the hamster fat cell which exhibited a very similar responsiveness in-our study.

Some species-specific differences in the atypical P-AR could exist, but our results establish clearly and for the first time, that BRL 37344, the compound acting through the atypical receptor, is a potent agent on all the non-human mammalian white fat cells stud- ied. Although extended pA, calculations were not performed for rabbit and dog fat cells, since bisoprolol behaved as in the rat and the hamster fat cell and was a weak antagonist of the action of BRL 37344, it seems reasonable to propose that the atypical /#-AR of these species is not strikingly different. This conclusion is also supported by the results obtained with ( f )-CGP 12177. It was clearly established that this compound is a partial agonist of the atypical P-AR in hamster and rat fat cells (figs. 3 and 4). Although its potency was variable according to species, it acts on the same atypi- cal P-AR as found from the pA, values obtained in rat and hamster fat cells. It should be remembered that the maximum response to a partial agonist depends on the extent of receptor reserve; thus changes in (k )- CGP 12177 potency from one tissue to the other (inter- specific differences shown in table 1) may vary accord- ing to the extent of atypical /?-AR reserve and do not necessarily indicate the presence of atypical P-AR isotypes. Changes in intrinsic activities of partial ago- nists are not associated with any comparable variation of the response to full agonist derivatives as assessed by minor interspecies changes in BRL 37344 potencies (table 1). Utilization of a partial agonist could aid in the evaluation of variability in receptor reserve be- tween different adipose tissues; (&)-CGP 12177 could be a valuable tool for further studies. The (- XGP 12177 eniantomer (not available when the studies were performed) is expected to have a stronger action since the (+ )- and (+)-CGP 12177 effect was clearly stere- oselective (table 3).

The lack of effect of BRL 37344 and (+I-CGP 12177 in the human subcutaneous fat cells used in the present study is also an excellent argument in support of the atypical P-AR specificity of these compounds and to demonstrate the uniqueness of human fat cells as opposed to those of all the other mammals used in the present study. These data are in agreement with our previous investigations (Lacasa et al., 1986; Mauriege et al., 1988) into the nature of the P-AR in the subcutaneous fat cells of women (i.e. Pi-AR and

&-AR) but it cannot yet be excluded that differences could exist according to the anatomical location of the fat deposits. During the completion of the present ‘work, weak stimulation by BRL 37344 was reported for human adipocytes (< 20% of isoproterenol maximal lipolytic effect at 100 PM BRL 37344). The calculated EC& value was well above (Hollenga et ,I., 1990) the EC,, values reported herein (in the nanomolar range in non-human adipocytes). We have also observed some very weak stimulation of lipolysis by BRL 37344 in adipose tissue from patients with associated hormona] disorders and smaller fat deposits than those studied in the present experiments. For the moment, it is uncer- tain that the lipolytic effect of BRL 37344 on human adipocytes can be related to stimulation of an atypical P-AR. Since there is a large heterogeneity among patients and between fat deposits (Mauriege et al., 19871, further studies are required to delineate physio- logical and/or pathological changes in a putative atypi- cal &AR-mediated response in human fat cells. It cannot be excluded that human fat cells contain a population of atypical P-ARs on which BRL 37344 and ( f )-CGP 12177 are partial agonists or which are weakly coupled to/or uncoupled from G, proteins, and unde- tectable by a partial agonist ligand such as [‘251]CYP.

Previous binding studies with [‘251]CYP and i3H]di- hydroalprenolol (Lacasa et al., 1986; Mauriege et al., 1988; Engel et al., 1981; Bojanic and Nahorski, 1983; Bahouth and Malbon, 1989; Valet et al., 1989; Saulnier-Blache et al., 1990) have demonstrated that, under the binding conditions used classically, only &- and &-AR binding sites are detected in various species. Moreover it was shown here that the labelled p,- and &-ARs are not the sites involved in the generation of the lipolytic effects of BRL 37344 or f+ XGP 12177 (fig. 2). It can be proposed that tire specific action of these agents in rat, hamster, dog and rabbit adipocytes is linked to the presence of an atypical P-AR (as shown in functional assays) which is not identified at the concentrations of [‘*‘I]CYP normally used to label the classical &- and p,-ARs. The question was re- cently addressed using brown fat cells of the rat (Muz-

zin et al., 1988; Mohell and Nedergaard, 1990). Our attempt to identify atypical P-AR with higher [‘LiIICYP concentrations was unsuccessful. It is easily under- standable that, as partial agonists for the atypical P- AR, [iZ51]CYP and [3H]CGP 12177 cannot represent the best opportunity for accurate atypical P-AR site determination in the species studied here. The search for a selective and high-affinity antagonist radioligand for the atypical P-AR site is an important goal for the near future.

Delineation of receptor properties based only on potencies of agonists can be considered to be of limited value, however, in the absence of any specific antago- nist agent for the atypical P-AR, it was decided to

action of various compounds R-mediated lipolytic responses. The te: (i) demonstration of the stereose-

~cctivie of partial agonist derivatives having PI/P?- hikxking potencies with cyanopindolol and CGP 12 177 cn~~~t~~~~~~~ (table 2): (ii) definition of the potencies of

RL dcriv:jtives which had the same rank order of ~~>tenc\c defined for the atypical receptor of the rat bro\vn fat cells (Arch et al., 19S-I): two behaved as full agonists (table 2): (iii) demonstration that PEAT

ivatives. a new class of @agonists having preferen- intestinal action and high selectivity for the atypical

p-AR of rat colon (IManara et al., 1989; Bianchetti and Manara. 1990). are full agonists of the fat cell P-AR with the same relative order of potency and stereose- Iectivity as defined for the inhibition of intestinal motil- ity. The results confirm the atypical nature of the &AR of the white fat cell and suggest that, on the basis of PEAT effects, this receptor could show great similarities with the atypical P-AR of intestinal smooth muscle.

It cannot ye? be stated whether the &-AR ex- pressed in CHO cells (Emorine et al., 1989) corre- sponds to the atypical P-AR ic the fat cells of mam- mals. Although they have some common atypical prop- erties, the cloned /3,-AR and those of fat cells show numerous discrepancies. This aspect requires further validation since P,-ARs could have been overexpressed in CHO cells. Moreover. the transducing system in CHO cells involved in the P-AR-mediated events could also be very different from that of fat cells and intro- duce discrepancies which could be independent of the nature of the receptor protein.

Since the atypical P-AR appears to play a major role in the control of lipolysis in various animal species, it is certain that most previous studies on fat cells need total reassessment in terms of the role, efficiency and specificity of the @-ARs. Most of the investigators focused only on PI- and p,-sites identified with stan- dard ligands ([“Hldihydroalprenolol, [““I]CYP) and missed the atypical P-AR component. Moreover, it cannot be excluded that other P-AR-mediated cellular events could also be specifically due to this receptor family. To extend these results beyond adipose tissue and colon muscles, it should be emphasized that there is a rather good correlation between the partial agonist effects of several P,/&-antagonists on the white fat cell and their ‘intrinsic sympathomimetic activity’ de- fined in the heart (Kaumann, 1989).

To conclude, not only have we confirmed the results of Previous work on rat fat.cells. but this study showed that, using an approach associating functional assays and [‘“IICYP binding studies, the presence of an atypical P-AR is not limited as previously supposed to the white and brown fat cell of the rat but has a more general distribution in mammalian fat cells. The coex-

istence of three separate P-AR subtypes (except in human fat cells) which probably mediate the same lipolytic function is demonstrated. The limits of [“‘ICYP as a radioligand are delineated and BRL 37344, (_+ J-CGP 12177 and PEAT derivatives (having an activity on intestinal motricity) are shown to be major tools usable for exploration of atypical P-AR function in varic us species and various physiological or pathological situations.

Acknowledgements

The au!hors would like to thank Michele Dauzats and Franfoise Esclapez for expert technical assistance and Antonia Berg6 for care of the animals. They also thank Dr. A. Bianchetti and L. Manara (Sanofi-Midy group, Milaslo, Italy) for the phenylethanolaminotetra- line derivalives and the communication of their paper which was still in press. Dr. M. Cawthorne (Beecham Pharmaceuticals. Epsom, LIK). 6. Holloway (ICI Ltd.. Macclesfield, UK), D. Hoyer (Sandoz, Basle. CH) and K. P. !-Leibli (Ciba-Geigy, Basle, CH) are gratefully acknowledged for the various compounds kindly provided, with the appropriate technical comments for the utilization. Dominique Lan- gin was supported by the National Institute of Agronomic Research (INRA-CRZV Theix, 63122 Ceyrat. France). Maria P. Portillo, Uni- versity of Navarra, was a recipient of a fellowship from the Spanish Government. Parts of the results contained in the present paper have been published in abstract form at the Satellite Symposium of the Xlth international Congress of Pharmacology on Pharmacology of Adrenergic Receptors held in Manchester (27-29th June, 1990), UK and the 6th International Congress on Obesity held in Kobi (21-26th October. 19901, Japan.

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