Ccmp. Biocher?~. Phgsiol. Vol. 74C. No. I. pp. 41.-45. 1983 Printed in Great Britain.

0306.4492’83 010041-05501.00 0 0 19X3 Perg;imon Press Ltd

LACK OF FUNCTIONAL ANTILIPOLYTIC x2-ADRENOCEPTOR IN RAT FAT CELL:COMPARISON WITH

HAMSTER ADIPOCYTE

CHRISTIAN CARPENE’, MICHEL BERLAN’ and MAX LAFONTAN’

‘lnstitut de Physiologie. Universiti Paul Sabatier. ERA 412 CNRS, 2. Rue F. Magendie, 3 I400 Toulouse. France

‘Laboratoire de Pharmacologic MCdicale. FacultC de Mt-decine. 37. Alit-es Jules Guesde. 31000 Toulouse, France

Abstract-- I. The small population of [3H]clonidine binding sites in rat fat cell membranes do not have the characteristics of typical r,-adrenoceptors.

2. Clonidine (an r,-adrenoceptor agonist) has no antilipolytic effect on rat fat cells stimulated b) theophylline.

3. In contrast to the rat. [“Hlclonidine labels an a,-adrenoceptor in hamster fat cell membranes and clonidinc exerts a strong antilipolytic effect on theophylline-stimulated lipolysis.

INTRODUCTION

There is now evidence that both a,-adrenoceptors and a,-adrenoceptors are present in white fat cell membranes. The stimulation of the cc,-receptors pro- motes an increase in phosphatidylinositol turnover in the membranes followed by calcium movements (Gar- cia-Sainz et ul., 1980) while the stimulation of the a,-adrenoceptors leads to an inhibition of the lipoly- tic rates (Garcia-Sainz et al., 1980; CarpenC et al., 1980). The occurrence of an antilipolytic effect mediated by r,-adrenoceptors stimulation has been clearly demonstrated in fat cells from several species of mammals, including hamster (Schimmel et LI/.. 1980; Carpene et al., 1980), rabbit (Lafontan, 1981) dog (Berlan rt ul., 1982). and man (Lafontan & Ber- Ian, 1980; Burns et al., 1981).

Surprisingly, there is no clear evidence for the pres- ence of r,-adrenoceptors in rat adipocytes. In the literature, noticeable discrepancies exist concerning this problem.

m-adrenoceptor-mediated effects on lipolysis or CAMP accumulation have been shown in rat adipose tissue by Himms-Hagen (1970) and Lawrence & Larner (1977) whereas Birnbaumer & Rodbell (1969) and Burns & Langley (1971) did not find any r-adre- nergic response in this species. The major criticism of these experimental results concerns the indirect ex- perimental design used to demonstrate the occurrence of an g-adrenergic response. The potentiation of the lipolytic effect of adrenaline or noradrenaline by the r-adrenergic blocking drug, phentolamine. although commonly used by various investigators. is still ques- tionable when large amounts of blocking agent are used since the drug may impair the lipolytic effect of sympathomimetic amines without interacting with the adrenoceptors. Moreover, phentolamine, a non-selec- tive a-antagonist. interacts with both rl- and r,-adrenoceptor sites. More recently, radioligand binding studies have not clarified the matter at all. Using the x,- x,-antagonist ligand [“Hldihydroergo- cryptine ([“HIDHEC). Dax c’t (I/. (1981) have reported

that the sites do not exist in rat fat cell membranes whereas Giudicelli et al. (1981). using the same radio- ligand. found that [3H]DHEC does label an x-adre- noceptor on rat adipocytes.

Recently, we identified and characterized functional cc,-antilipolytic adrenoceptors in human fat cells by using the cc,-agonist ligand [“Hlclonidine (Berlan & Lafontan. 1980). In the present paper. we tried to obtain further information on the r-adrenoceptors of rat adipose tissue by using an experimental design which consists of comparing the [‘Hlclonidine bind- ing on rat fat cell membranes and the biological cffcct of clonidine estimated by the modification of the lipo- lytic rate in rirro. We also compared the results obtained in rat fat cells with those in hamster fat cells which possess functional r,-adrenergic receptors as shown by various recent investigations (Schimmel cl ul., 1980: Carpene et cl/.. 1980; Garcia-Sainz c’t (I/.. 1980).

MATERIALS AND METHODS

Adult male golden hamsters (~~~c.\oc,,ic,l,fl,.\ ~~urut~~.~l (Commercial Breeding Centre Evic Ceba. lone industrlcllc F-33290. Blanquefort, France) weighing between 100 and 145 g, and adult male Sprague Dawlcj rats weighing 40@430 g, were housed at 2@22 C and fed cltl lihituvl for at least 2 months before use. Another group of older rats (8-9 months, weighing 5OG55Og) was also used. The ani- mals w’ere killed by decapitation after okcrnlght fasting. Perirenal adipocytes were prepared by the action of a crude collagenase at a concentration of 1.7 mg ml in 6 ml of Krebs Ringer bicarbonate buffer containing 3.5”,, bovine serum albumin (KRBA) for IO 15 min. after which the adipocytes were washed 3 times in KRBA.

The isolated adipocytes (3&40 mg cell lipid) were dis- persed in plastic vials in 2 ml KRBA and 6 ltmol, ml glu- cose. Theophylline and other agents were added in por- tions of 20 itl just before starting the incubation proccdurc. After 90 min. an aliquot of the incubation medium was taken to determine glycerol release by Wieland’\ methods (1957). The metabolic activity is expressed in k!rnol of gly- cerol produced by 100mg total lipid determined gravi-

41

metrtcally after extractton according to the method of Dole & Mcincrt/. (1960).

Pcrircnal adipocytes from 5 or 6 hamsters or rats were prcparcd as described abole and washed three times in a lysing medium (MgCI?. 2mM; KHC03. 1 mM: ATP, 1 mM: Tris-HCI. 2 mM: pH 7.6). Crude adipoqte mem- branes wcrc pelleted by crntrifugation (3OOOg. 10 mtn. 4 C); washed twice in a buffer at pH 7.6 (Tris. 5 mM; EDTA. I mM) and finally suspended (3-4mg protein,ml) in an incubation medium (MgCI,. IO mM; Tris HCI. 50 mM: pH 7.5). They were used immediately in binding assays or stored at most for 1 week at -80 C. Protein was determined using the method of Low! (‘I tri. I 1951) with hoLine serum albumin as B standard.

Binding assays were carried out as described by Guel- laen cf rrl. (1976) with the following modification>: 100 111 of membrane suspension (20s 300 /q protein) was incubated for IO min at 25 C‘ with 100111 aqueous [‘Hlclonidtnc so- ution in 400 1’1 total volume of 50 mM Tris HCI, (pH 7.5) containing 10 mM MgCI,, Non-specific binding was deter- mined by the addition of 100 AIM phcntolaminc. At the end of the incubation, duplicate 150/tl aliquots were diluted in 4 ml of ice-cold bufl’er and filtered immediately undcl vacuum using Whatman GF’C‘ glass-tibre liltcrs. The filters Mere Bashed rapidly with two IO ml portions of incubation buffer at 4 C. dried at 50 C for 30 40 min and counted in mini\ials in a 4 ml AC’S scintillation mixture (Amcrsham Searle. Arlington Heights. Illinois. U.S.A.) with an efti- ciency of 40 45”,,. All values rcfcr to spccitic binding which represented 75 X0”,, (hamster) or 30 40”,, (rat) of the radio- activity retained on the filters. Treatment of the Ireceptor binding a\saq results wcrc conducted according to the gcn- cral prlnciplcs of Williams & L&on itz (I 97X).

Chemicals wcrc obtained from the follolcing sources: [“Hlclonidine (specific activity 22.2 f Ci mmol) from New England Nuclear. Boston. U.S.A.: clonidinc base from Boehrinper. Ingclheim West Germanq: yohimbtne base from HoudL: Labs. Paris, France: mcthoxamlnc from Well- come Foundation. London. England: Phentolaminc base

from (‘iba-Geig). Haslc. S\sit/erland: theoph>Iline from Bruneau Labs. Paris. France. Bovine serum albumins (frac- tion V) was obtained from Sigma. St. LouiT. (1.S.A. and crude coII;uzcnasc from the Worthincton BiochcmIcaI Cor- poration. Freehold. New Jcrseq. C.S.A. En?! mc\ came from BoehrinFcr. M;innheim. West Cicl-many

RESl’I.TS

C3H]clonidine binding to rat and hamster fat cell membranes was rapid. In both casts. half of the maxi- mal binding occurred in less than 2 min at 25 C. The association of the radioligand to its binding sites reached equilibrium within I@~ I2 min :md was stable

for at least 60min. The binding of [‘Hlclonidine has a linear relationship to the protein concentration used. Figure I shows the specific binding of [“H]clo-

nidine to hamster and rat cell membranes as a func- tion of the [3H]clonidine concentration (0.1P50 nM). Non-specific binding represented 25”,, of the total binding at 2 IOnM for hamster fat cell membranes and 65”,, of the total binding for rat cell membranes at 4-IOnM [‘Hlclonidine. The maximal specific binding (B,,,) was more than seven times higher in hamster fat cell membranes than in rat fat cell mem- branes (Table 1). Besides. the respective KD value. rep- resenting an estimate of the equilibrium dissociation constant. were respectively 3.52 nM and I .45 nM.

The specificity of the binding was also studied by measuring the competition of various r-adrenergic antagonists towards [3H]clonidine binding sites. In hamster fat cell membranes. the r-adrenergic antag- onists competed for the [3H]clonidine binding sites in an order or potency: phentolamine > yohimbine > prazosin (Table 2). This order is consistent with the order of potency previously described for these agents when an r,-adrenoceptor is labelled. especially in hamster (Garcia-Sainz et trl.. 1980) and man (Lafontan & Berlan. t 980) fat cell membranes. The same series of compounds were tested for affinity for the [-‘H]clo- nidine binding sites of the rat fat cell membranes. BJ contrast with the hamster. while phentolamine com- petes for the sites labelled by [“Hlclonidine with ;I

high efficiency (EC,,, = 0. I48 /IM) yohimbine com- petes for the site at higher concentrations which were identical to the concentrations of prazosin required (Table 2). The x,-antagonist yohimbine failed to dis- place C3H]clonidine bound on rat fat cell membranes at concentrations compatible with the delineation of ;I

“true” r+drenoceptor. In order to clear thcsc apparent discrepancies

revealed by radioligand binding studies. the erect of selected x-agonists on lipolysis was investigated. In human (Lafontan & Berlnn. 1980) and in hamster adi- pocytes (Carpene of rrl.. 1980) we have shown that the antilipolyticeffect ofclonidine on basal or theophylline- induced lipolysis was related to the involvement of an x,-adrcncrgic receptor. Concerning thcophylline, the mechanism by which this methylxanthine stimu- lates lipolysis is not clear. Theophylline prevents the degradation of cAMP by the inhibition of phospho- diesterase. This substance also acts by competing with adenosine for its membrane binding sites (Trost & Schwabc. 1981). Whatever the mechanism involved. theophylline allows the study of the antilipolytic effect of x2-adrenomimctic drugs.

Using the same cxpcrimental design, we investi- gated the clkct of clonidinc on theophylline-induced lipolysis in isolated adipocytes from hamster and rat. By these experiments. we attempted to determine whether the observed difference in the number of z-adrenoccptors in rat and hamster fat cell mcm-

i

rat

2 4 6 8 IO 15 20 30 40 50

[3H] Clmdine (nM I

Fig. 1. Specific bindlng of [‘H]clonidine to rat (0) and hamster (0) fat cell membrane as a function of [‘H]clo- nidine concentration. Values rcprcsent the mean of tripll- catc determinations from 5 6 pooled rat or hamster adi- pose tissues samples. Fat ccl1 “ghosts” were incubated for I() min at 25 C v,ith [2H]clonidine at 17 concentrations

ransing from 0. I to 50 nM.

a-adrenoceptor in rat and hamster fat cells 43

Table 1. Comparative [“Hlclonidine bindmg characteristics for ham. ster and rat fat cell membranes

Receptor density, B,,, (fmol/mg protein)

KII (nM)

Hamster 234 * 19 3.52 + 0.61 Rat 30 & 9 1.45 + 0.34

KI, and B,,, were calculated from lmear regression analysis of the Scatchard plot determined from saturation curves of specific binding (Fig. 1).

Values + SEM represent the mean of triplicate determinations from 5 6 pooled rat or hamster adipose tissue samples.

Table 2. Inhibition of specitic [3H]clonidine binding to hamster and rat fat cell membranes by various r-adrener-

gic antagonists

EC,,, for competing drug Hamster (5) Rat (3)

Pentolamine 0.190 + 0.047 0.148 + 0.052 Yohimbine 1.491 f 0.030 5.603 f 0.900 Prasozin 5.040 k 0.762 5.697 + 1.493

Adipocyte membranes were incubated wth 1OnM of [‘Hlclonidine alone and with increasing concentrations of competing r-antagonists (lo- “‘~10~’ M). EC,, values were determined from log-probability analysis of concen- tration-inhibition curves and refer to the concentration of each drug causing 50”,, inhibition of [JH]clonidinc bind- ing. Means are given i SEM. Numbers in parentheses refers to the number of assays. Each assay was carried out in duplicate.

branes had an influence on the r-adrenergic antilipo-

lytic effect.

The glycerol released in the presence or absence of various doses of theophylline is shown in Table 3. In basal conditions (without theophylline), the adipo- cytes from rats released less glycerol per 100 mg lipid than those of hamster (P < 0.01). It can also be seen that theophylline increased lipolysis in adipocytes from both species. Nevertheless, the increment of the lipolytic rate was different with regard to the concen- tration of theophylline used. With 0.33 mM theophyl- line. the lipolysis was stimulated about live-fold in hamster adipocytes. To obtain a similar increase of lipolysis with rat adipocytes, it was necessary to per- form the experiment with a ten-fold higher dose of theophylline (3.3 mM).

As shown in Fig. 2, clonidine caused a concentra- tion-dependent inhibition of the lipolytic response induced by theophylline in hamster adipocytes. The maximal antilipolytic effect was obtained with 5 and 50pM clonidine (about 7CrSO”,,). This effect was

competitively suppressed by the a,-adrenergic antag- onist yohimbine (for details see Carpene et rrl.. 1980). In comparison, clonidine had no effect on theophylline- induced lipolysis in rat adipocytes (Fig. 2) for any concentration of clonidine (0.05%50 /tM).

In order to test the possible involvement of a,-adrenoceptors in the control of lipolysis in rat adi- pocytes we analysed the effect of increasing concen- trations of the r,-adrenergic agonist drug, methox- amine, and we compared the response to that of ham- ster adipocytes. It can be seen (Fig. 2) that methox- amine, even at concentrations up to 50jtM, had no effect on theophylline-induced lipolysis in both rat and hamster adipocytes.

According to the biological response to catechol- amines on adipose tissue. the adrenergic receptors were classified into p. x1 and x2. The stimulation of lipolysis by catecholamines is mediated by P-adreno- ceptors. The role of a1 adrenoceptors has been widely studied in hamster adipocytes. The activation of these receptors increased cytosol calcium: this effect is pre- ceded by an increased incorporation of inorganic phosphorus ions into phosphatidylinositol (Garcia- Sainz uf rrl.. 1980). r,-adrenoceptors have also been described in isolated f:tt cells (Be&n & Lafontan. 1980; Burns ef ctl.. 1981). Antilipolytic sc,-effects are due to inhibition of adenylate cyclase (Aktories c’r r/l..

Table 3. Effect of various concentrations of theophylline on lipolysis in fat cells from the perirenal adipose tissue of hamster and rats

0

Theophylline

(mM) 0.33 I .65 3.3

Hamster adipocytes Rat adipocytes

0.63 f 0.07 (8) 3.2* f 0.4(6) lO.6* + 2.3 (6) 9.2t * 14.1 (7) 0.23 f 0.03 (7) 0.62* f 0.12(6) 1.251- & O.lO(6)

Lipolysis is expressed as pmol glycerol released per 100 mg cell lipid for 90 min incubation. Numbers in parentheses refer to the number of assays. Means are given k SEM.

* P < 0.01 significance of the difference between stimulated and basal values. Student’s t-test.

+ P < 0.001.

44 CHRISTIAN CARPEKE et cd.

Clomdme IpMl Methoxarnme (PM)

M5 0.5 5 IO $..““_ M5 U5 5 IO P-

l Hamster edipocytes

Rat adipocytes

Fig. 2. Effect of clonidine (left) and methoxamine (right) on theophylline-induced lipolysis in hamster (a) and rat (0) isolated fat cells. The adipocytes were incubated with 0.33 mM (hamster) or 3.3 mM (rat) theophylline m the presence of variable concentrations of clonidine and mcthoxamine. The results are expressed as the percentage inhibition of theophylline-induced lipolysis in the presence of correspondmy concentrations of cl-agonist. The SEMs are indicated by the bars and the number of assays is given for each curve. **P <: 0.01: ***i’ < 0.001 values significantly different from control valocs (thcophylline

alone) according to Student’s paired t-test.

1980; Kather & Simon, 1981) and there is now evi- dence for biological control of lipolysis by a,-adrenoceptors in the adipose tissue of various ani- mal species except the rat in which the presence of a,-adrcnoceptors remains uncert~~in.

The present results show that [“H]clonidine. known to be a potent agonist for r,-adrenoceptors. binds “specifically” on both hamster and rat fat cell membranes. However. the amount of [‘Hlclonidine bound was at least seven-fold higher in hamster than

in rat adipocyte membranes (Table I ). x2-adrenoceptors seem to be present in rat fat cell

membranes but in comparison with other animal spe- cies such as man, hamster and rabbit (unpublished data). the amount of labelled sites was very small. In all the species studied so far. [3H]clonidine allowed the identification of binding sites which have the physiological function expected, i.e. counteracting lipolvtic process. In hamster adipocytes, the large denaty of az-sites labelled by [‘H]clonidine (234 fmol/mg protein) was responsible for the strong inhibitory effect of clonidine when the lipolytic rate was measured (Fig. 2).

In rat adipocytes, the inhibition of theophylline- induced lipolysis by clonidine does not occur: this suggests a lack of x,-mediated alltilipolytic effect in these cells. Garcia-Sainz et trl. (1980) did not find any modifications of CAMP levels with x-agonists in rat fat cells. There are at least two hypotheses which can be proposed. First of all it is possible that the small number of r,-adrenoceptors (detectable in rat fat cell membranes by binding methods) do not affect the lipolytic process and hence no ~lntilipolytjc effect can be observed. The second hypothesis is relevant to recent studies of coupling systems between adreno- ccptor and adenylate cyclase in membranes. As reported by Aktories rt rd. (1980) and Kather & Simon (1981) the inhibition of tipolysis in adipocytes involves adenylatc cyclase inhibition. Moreover, the

guanine nucleotide seems to play an important regu- latory effect on receptor binding. GTP in particular ensures the connection between the hormone receptor and the catalytic moiety of the adenylate cyclase (Lef- kowitz & Hoffman, 1980). Taking this model into ;ICCOLltlt the coupling system linked to the

x2-adrcnoceptor ma! be impaired in rat adipocytes.

So, the inhibitory x2-effect cannot occur when the lipolytic rate is measured.

Nevertheless, the low potency of yohimbine in dis- placing [3H]clonidine from its binding sites would not be an ar&ument in favonr of a true x1-adrenoceptor (Table 2). We agree with the conclu- sion of Giudicelli et trl. (1981) who postulate that the existence of adrenoceptor in rat fat cells is doubtful.

Using [“HIDHEC. an x,- y(2 adrenoceptor ligand. they found specific binding to rat fat cell membranes. The binding was displaced by low concentrations of prazosin (,~~-antagonist) and by hig.h concentrations of yohimbine (x,-antagonist) suggesting specific label- ling of the a,-adrenoceptors. Nevertheless, ,x,-agonists failed to compete with C3H]DHEC. So, the R sites labelled by 13HJDHEC on rat fat ccl1 membranes exhibit abnormal characteristics for an x-adrenocep- tnr. It is not to be excluded that [3H]clonidine binds to an abnormal structure. the stimul~ltion of which is unable to provoke a biological response in the rat adipocyte.

With regard to the r,-adrenergic response, the fat cell size and agcing are factors which must be con- sidered if we refer to rabbit (Lafontan, 1979) and dog (Be&n (‘1 LII., 1982) adipocytes. In these two species. ageing and obes$ promote a concoi~lit~~nt increase of r-adrenergic antlllpolytic effect. From our results age- ing and obesity are not the Factors which induce the appearance of the x,-adrencrgic response in the rat since no antilipolytic etrect was observed in the adipo- cytes from old rats (IX--20 months) (not shown).

In conclusion. [“I-I]clonidine binds to rat and ham-

r-adrenoceptor in rat and hamster fat cells

ster fat cell membranes, Rat adipocytes contain a small amount of [3H]clonidine binding sites com- pared to the hamster and other species. However, in rat adipocytes. biological inhibition of lipolysis is not detectable. The lack of biological response could be related to the abnormalities revealed by the binding studies carried out on the fat cell membrane of this species.

J. & FAIN J. N. (1980) Role of alpha, adrenoceptors in the turnover of phosphatidylinositol and of alpha, ad- rcnoceptors in the regulation of cyclic AMP accumu- lation in hamster adipocytes. Lift Si. 27, 953-961.

GIUDI(‘~LLI Y.. THOTAKIJRA N. R.. LACASA D.. P~CQUERY R. & ALGI B. (1981) Direct binding studies do not sup- port the existence of true r-adrenoreceptors in rat white fat cells. Bi~cIwm Bkydys. Re.s. Cornmrn. 100, 621 -628.

GLI.LI_~~:u G.. YATr:s-Aoc;f.Rel:t.K M.. VAIJQLIELIN G.. STROSH~RG D. & HANOLINE J. (1976) Characterization with [“H]dihydrocrgocryptinc of the z-adrenergic recep- tor of the hepatic plasma membrane. J. ho/. Cherw 253, 1114-1120.

45

.4~,krto~~/rdilc,~)1~,~1f~ -This work was supported by grants from CNRS (ERA 412 and ATP: Pharmacologic des reccp- teurs des neuromediateurs). We thank Mrs Dauzats for her exccllemt technical assistance.

REFERENCES

A~TORI~S K.. SCHI!LTZ G. & JAKO~S K. H. (1980) Regu- lation of adenylate cyclase activity in hamster adipo- cytes. Inhibition by prostaglandins, r-adrenergic agonists and nicotinic acid. :\‘cl~~/l!.,~-S~,/~,~~j~,[~~,~l,~~/.s. Ar(,/l. c’sp. Ptrth. Phtrrrt1trk. 312. 167m 173.

BTRI.AX M. & LAFOXTAN M. (1980) Identification of r,-adrenergic receptors in human fat cell membranes by [“Hlclonidine binding. Eur. J. Phrrrmc. 67, 481~ 484.

BIXRLAX M.. CARI’IZN~ C.. LAFOU~AN M. & DAX TRAN L. (1982) Alpha,-adrenergic antilipolytic effect in dog fat cells: incidence of obesity and adipose tissue locallration. Hor,,~. I\c,f~lh. Rcs. 14, 257m 260.

BIRNBAI.MFR L. & RODB~LL M. (1969) Adenyl cyclase in fat cells. II. Hormone receptors. J. hiol. Churn. 244, 3477-3482.

B[jRNs T. W. & LANGLFY P. E. (1971) Differential effects of r and /I adrenergic blocking agents on basal and stimu- lated lipolysis of human and rat isolated adipose tissue cells. Phtrrmrrc. Rcs. Commrn. 3, 27 I-277.

BURNS T. W.. LANGLEY P. E.. TERRY B. E.. BYLC~~;II 0. t3.. HOFFMAN B. B., THARP M. D.. LEFKOWIT~ R. S.. GARUA- SAIYXZ J. A. & FAIU J. N. (1981) Pharmacological charac- terizations of adrenergic receptors in human adipocytes. J. c,litl. Inrcst. 67, 467 475.

CAfcfvxf: C.. LAFO~~AN M. & BERI.AN M. (1980) A postayn- aptic r,-receptor: the alpha-adrenergic receptor of ham- ster white fat cells. Eu/xvi<,ntin 36, 1413 1414.

DAX E. M.. PARTILLA J. S. & GRI,GI.RMAN R. I. (1981) Mcchanlsm of the age-related decrease of epincphrine- stimulated lipolysis in isolated rat adipocytes: /I-adrener- gic receptor binding. adenylate cyclase activity. and cyc- lic AMP accumulation. J. Lipid. Re.\. 22, 934 943.

DOLF V. P. & Mrl~~v H. (1960) Micro-determination of long cham fatty acids In plasma and tissues. .1. hiol. Ch<vn. 235, 2529 -2599.

GAKCIA-SAINT J. A., HOFFMAW B. B., Lrs Y.. LEFKOWITZ R.

HlhlMs-HAc;f:v J. (1970) Adrencrgic receptors for metabolic response in a&pose tissue. E‘r~d. Pwc~. 29, 1388-1401.

KAT~~I‘R H. & SIMON B. (1981) Adrenoceptor of the alphaz-subtype mediating inhibition of the human fat

cell adcnylate cyclaac. Ew. J. c,lin. In{ c\t. I I. 11 1-l 14. L,\~ONTAU M. (1979) Intxhition of cpinephrinc-induced

lipolysis in isolated R hite adipocytes of ageing rabbits by

mcreascd alpha-adrcnerglc responsiveness. .I. Lipid. Re.\. 20. 208 -2 16.

LAFONI~U M. (19X1) Alpha-adrenergic responses in rabbit uhitc rat cells: the inBuence of obesity and food restric-

tion. J. Lipid. Rtc\. 22, 1084 1093. LA~OUTAK M. & B~RLA\ M. (1980) Evidence for the

rz-nature of the x-adrenergic receptor inhibiting lipolysis in human fat cells. ELI,- J. Phtrrmtrc. 66, X7-93.

LAFONTAN M. & BFRI.AN M. (1981) Alpha-adrenergic receptors and the regulation of lipolysis in adipose tissue. T~ot~l,\ Phurnmc. Sci. 2, l26- 129.

LAWRENCE J. C. & LARNEK L. (1977) Evidence for alpha-

adrenergic activation of phosphorylase and inactivation of glycogen synthase in rat adipocytes. Mol. Pharmrrc. 13. 106C1075.

Lt.tKowfTz R. J. & HOFFMA\ B. B. (IYXO) Neu directions in adrencrgic receptor research -Part I. Tr(v1rl.5 Phtrmtrc~. Si. II, 314 318.

LOWRY 0. H.. Rosf-frRoL’c;ff N. J.. FARR A. L. & RA>I)ALL R. J. (1951) Protein measurement with the Folin-phenol reagent. J. hid. C/IW~I. 193. 265-275.

SCHIVUEI. R. J.. S~KIO R. & MCMAHON K. K. (1980) r-adrcnergic inhibition of cyclic AMP accumulation in

hamster adipocytes. Similarity of receptor with r,-adrencrgic roccptors. Bioc~him hiopl11,~ .4ctu. 632,

544 552. TKOS~ T. & SCH\~AIII. U. (198 1) Adenosine receptors in fat

cells. Identification by ( - )-Nb-[‘Hlphenylisopropylade-

nosine binding. !Lf~j/. Phwuc. 19, 228-235. WII:LAUI) 0. (1957) Eine en7ymatische methode /.ur Bes-

timmung van glycerin. Biodwm Z. 239, 313-319. W11.r IAMS L. T. & LI.~KOM’ITZ R. J. (Eds) (197X) Rcccptor.s

Birdiwg Stutlic,.\ ii1 .4rlwwyic~ Phtr,-n~rrc,o/oc/~. Raven Press. New York.