Eur. J. Biochem. 92, 189-196 (1978)
P-Adrenergic Receptors in Brown-Adipose Tissue. Characterization and Alterations during Acclimation of Rats to Cold Ludwik BUKOWIECKI, Nicole FOLLEA, Julien VALLIERES, and Jacques LEBLANC Laboratoires d’Endocrinologie Climatique, Dkpartment de Physiologie, Faculte de Medecine, Universite Lava1 (Received April 12, 1978)
1. The capacity of brown adipose tissue to respond calorigenically to catecholamines increases markedly during cold-acclimation of adult rats. To investigate this phenomenon, we have used the potent radioactive ligand ( -)-[3H]dihydroalprenolol to directly estimate the number, the density and the affinity of P-adrenergic receptors in brown adipose tissue membranes from control, coldexposed and cold-acclimated rats. 2 . Binding of ( -)-[3H]dihydroalprenolol to unfractionated membranes was rapid, stable, saturable and reversible. It displayed the affinity, specificity and stereoselectivity expected of binding to adenylate cyclase-coupled P-adrenergic receptors. P-Adrenergic agonists competed for binding sites with an order of potency typical of the P I subtype of adrenergic receptors: (-)-Isoproterenol > (-)-norepinephrine 2 (-)-epinephrine. Binding exhibited a remarkable stereoselectivity, the (-)-isomers of /3-adrenergic agonists and antagonists being 34 to 280 times more potent than the (+)-isomers in competing for ( -)-[3H]dihydroalprenolol binding sites. 3. Total interscapular brown adipose tissue of the adult, warm-acclimated rat contained 1.12 k 0.08 pmol of ( -)-[3H]dihydroalprenolol binding sites. During cold-acclimation, growth of the tissue was accompanied by a 4- 5-fold increase in the total number of receptor sites. However, this increase did not keep pace with the increase in brown adipose tissue cellularity (as estimated by total tissue DNA content), resulting in a 40-50% reduction in receptor density. The decrease in receptor density was associated with cold-exposure rather than with cold-acclimation. 4. The affinity of ( -)-[3H]dihydroalprenolol receptor sites was not significantly altered by coldacclimation. 5. The results of this study are interpreted as indicating that catecholamines released via activation of the sympathetic nervous system regulate both the density and the number of their own receptors in brown adipose tissue of cold-exposed animals. The development of the hyperadrenergic response of this thermogenic tissue during cold-acclimation may result from a marked organ hyperplasia associated with an increased number of a-adrenergic receptor sites and cannot be explained by alterations in receptor density or affinity.
When mammalian homeotherms are exposed to cold for prolonged periods of time, they develop an increased sensitivity to many metabolic and cardiovascular actions of catecholamines. In particular, the calorigenic effects of epinephrine or norepinephrine increase markedly during acclimation of adult rats to cold [l - lo]. This phenomenon occurs principally in brown adipose tissue and to a smaller extent in other tissues such as skeletal muscles [7]. It is generally accepted that it represents the metabolic basis of the increased capacity of cold-acclimated rats for nonshivering thermogenesis [l - 101. Abbreviation. CAMP, adenosine 3’: 5‘-monophosphate.
There is abundant evidence that catecholamines elicit heat production in brown adipose tissue via stimulation of a hormone-sensitive adenylate cyclase, resulting in a transient rise in cellular CAMP levels [11 - 151. This leads to a stimulation of lipolysis mediated by CAMP-dependent protein kinase [I 61 and to an increase in mitochondria1 oxygen consumption [17,18]. Experiments with CI and adrenergic agonists or antagonists support the concept that these effects are evoked primarily via p-adrenergic pathways [15, 19-22]. In contrast, little is known about the hormonal and cellular mechanisms inducing a marked enhancement of the capacity of brown adipose tissue to respond
190
calorigenically to catecholamines during cold-acclimation [6]. Although it has recently been demonstrated that catecholamines are able to regulate the number of their own receptors in several systems, the role of such receptors in determining the extent to which tissues can respond to catecholamines is still unclear [23,24]. A potential explanation for the hyperadrenergic response of brown adipose tissue in cold-acclimated rats would be an increased number or an increased affinity of P-adrenergic receptors which would, in turn, mediate the amplified response of this tissue. To test this hypothesis and to gain a better understanding of the early regulatory steps controlling the metabolism of brown adipose tissue, we used the potent radioactive ligand ( - )-[3H]dihydroalprenolol to directly identify and characterize p-adrenergic receptors in this thermogenic tissue [23,24]. Next, we evaluated the effect of cold-exposure and cold-acclimation on the number, the density and the affinity of these receptors. The results indicate that marked changes in the total number and in the density of (-)-[3H]dihydroalprenolol binding sites occur in brown adipose tissue during prolonged cold-exposure. EXPERIMENTAL PROCEDURE Materials ( -)-[3H]Dihydroalprenolo1 (48.6 Ci/mmol) was purchased from New England Nuclear. Drugs and chemicals were obtained from the following manufacturers: (-)-isoproterenol bitartrate, (-)-epinephrine bitartrate, (-)-norepinephrine bitartrate, ( k)-propranolol hydrochloride from Sigma ; (-)propanolol hydrochloride and ( + )-propranolol hydrochloride from Ayerst ; (+)-isoproterenol bitartrate, (+)-epinephrine bitartrate and (+)- norepinephrine bitartrate from Sterling-Winthrop; phentolamine from Ciba; phenoxybenzamine hydrochloride from Smith Kline and French Labs. All other reagents used were of the highest purity commercially available.
Methods
Female rats (Wistar), fed laboratory chow ad libitum, were killed by decapitation. Interscapular brown adipose tissue was quickly removed, placed in ice-cold 0.25 M sucrose/lO mM Tris-HC1 pH 7.4, and carefully trimmed of extraneous tissue. The washed pieces of tissue were blotted with a filter paper and weighed. The tissue was then minced with scissors in ice-cold 50 mM Tris-HCl/lO mM MgC12 pH 7.4 at 25 "C, homogenized with a prechilled glass PotterElvehjem homogenizer fitted with a teflon pestle, carefully filtered through four layers of cheesecloth,
P-Adrenergic Receptors in Rat Brown-Adipose Tissue
and diluted to a final protein concentration of 2.55 mg/ml. This preparation will be referred to as 'unfractionated membranes'. In some experiments semi-purified or 'crude' brown adipose tissue membranes were prepared as follows. The cleaned tissue was minced directly in ice-cold 0.25 M sucrose/lO mM Tris-HC1 pH 7.4, homogenized as above and centrifuged at 650 x g during 10 min. The layer of fat which rose to the top was carefully removed, the supernatant was decanted and centrifuged for 20 min at 40000 x g. The resulting pellet was washed twice in the same medium and the final pellet was resuspended in ice-cold incubation buffer at a concentration of 2.5 - 5 mg of protein/ml. Binding Assay
Unfractionated or 'crude' membranes (0.5 - 1 mg protein) were incubated in a total volume of 250 pl of incubation buffer (50 mM Tris-HCl/lO mM MgC12 pH 7.4 at 25°C) containing 13.1nM (-)-t3H]dihydroalprenolol (unless otherwise specified) for 20 min, at 25 "C. Incubations were terminated by rapid vacuum filtration of the mixture through Whatman GF/C glass fiber filters followed by three washes with 5 ml ice-cold buffer (50mM Tris-HCl/lOmM MgCl2 pH 7.4 at 4 "C). The filters were introduced into scintillation vials, dried and covered with 8 ml of scintillation fluid (4 g of Omnifluor/l of toluene). Bound (-)-[3H]dihydroalprenolol was determined by counting the radioactivity in a Beckman liquid scintillation spectrometer at 42 - 45 % efficiency. Specific binding was determined from the difference between total binding and binding in the presence of 10 pM-( f)-propranolol. Nonspecific binding usually ranged from 30 - 50 % of total binding. Values reported in all tables and figures refer to specific S.E.M. (represented by vertical binding. Means bars in the figures) were calculated from the means of triplicate values obtained from experiments performed on separate occasions with individual membrane preparations. Protein determinations were done by the method of Lowry et al. [25]and DNA was estimated fluorimetrically by a modification [26] of the Kissane and Robins method [27]. RESULTS Suturability and Affinity of (-)-[3H]DihydroalprenotoE Binding to Unjractionated and Fractionated Brown-Adipose-Tissue Membranes
One of the principal objectives of this study was to assess whether the number and the properties of brown adipose tissue P-adrenergic receptors would be altered by cold-acclimation. Since this tissue grows
191
L. Bukowiecki, N. Follea, J. Vallieres, and J. Leblanc
0' 0
[ (-)- [3H]Dihydroalprenolol] (nM)
4
8
12
16 20 24 T h e (rnin)
28
32
36
40
Fig. 1. Binding of ( -/-(3H]dihq~droalprenolol to unfvuctionated (0) and fracrionated ).( brown adipose tissue membranes as a function of' increusing concentrations qf ligand. Membranes were incubated with 13.1 nM (-)-[3H]dihydroalprenolol at 25°C in the presence and absence of 10 pM (+)-propranolol during 20 min. Each point represents the mean of triplicate determinations from two separate experiments in which fractionated and unfractionated membranes were prepared and incubated in parallel as described in Experimental Procedure
Fig. 2. Time course of binding of ( - ) - [ 3 H ] d i h ~ d r o a ~ ) r e n ~ ~ l o l (13.1 n M ) to unfractionated brown adipose tissue membranes. The time course of reversal of binding by 10 pM ( f)-propranolol after equilibrium of binding was reached, is also shown in the insert. Each value represents the mean S.E.M. of a minimum of three experiincnts performed in triplicate
considerably in cold-exposed adult rats, we felt that it would be useful to express possible alterations in the number of receptor sites, not only per unit of protein, but also per unit DNA, the amount of DNA being considered as an index of tissue cellularity. For this purpose, it was decided to attempt performing binding assays with unfractionated membranes containing brown adipose tissue nuclei. If feasible, this would enable us to estimate, in the same preparation, radioactive ligand binding capacity and DNA concentration. We have therefore compared, in preliminary experiments the binding characteristics of ( -)-[3H]dihydroalprenolol in unfractionated and fractionated membranes, prepared as described in Experimental Procedure, in order to determine whether membrane fractionation was necessary for the characterization ( -)-[3H]dihydroalprenolo1 binding sites in brown adipose tissue. It can be seen in Fig. 1 that saturability of membrane binding sites with ( -)- [3H]dihydroalprenolol was obtained in both types of preparation. This demonstrates that materials removed during the preparation of 'crude' membranes (nuclei, triglycerides, soluble enzymes, etc.), did not impede the saturation of receptor sites with the radioactive ligand. The specific activity of ( -)-[3H]dihydroalprenolol binding when expressed in femtomoles of radioactive ligand bound per mg of protein, was approximately threefold higher in fractionated than in unfractionated membranes, indicating that some purification of membrane-bound receptor sites had been achieved. Affinity of ( -)-[3H]dihydroalprenolol for its binding sites was similar in both preparations: the halfmaximal saturation of binding sites occurred at (-)-
[3H]dihydroalprenolo1concentrations of 70 - 80 nM. This value represents a first estimate of the dissociation constant, K d , for the interaction of ( -)-[3H]dihydroalprenolol with its brown adipose tissue receptor sites (vide infra). Considering these initial results, it was decided to further characterize the proprieties of (-)-['H]dihydroalprenolol binding in unfractionated membranes.
+
Kinetics of' ( - )-[3HJDihydroalpreizololBinding The time-course of binding of ( -)-t3H]dihydroalprenolol to unfractionated brown adipose tissue membranes is represented in Fig. 2. ( -)-[3H]Dihydroalprenolol binding was rapid, reaching equilibrium in less than 10 min and remained stable for at least 40 min. At equilibrium, the addition of 10 pM (&)propranolol resulted in a very rapid dissociation of bound radioactive ligand (insert of Fig. 2). The halftime of dissociation was less than 3 min. Specificity and Steveoselectivity -L3 H]Dihydroalprenolol Binding
of ( - )
The B-adrenergic antagonist, (-)-propranolol, potently competed for ( -)-[3H]dihydroalprenolo1 binding sites (Fig. 3 ) with a dissociation constant, K d , of 56 nM (Table 1). In contrast, the a-adrenergic antagonists, phentolamine and phenoxybenzamine, were totally ineffective in displacing the radioactive ligand from its binding sites, even at concentrations as high as 0.1 mM. Other B-adrenergic agonists also competed for ( -)-[3H]dihydroalprenolol binding sites with an
192
p-Adrenergic Receptors in Rat Brown-Adipose Tissue Table 1. Dissociation constants and stereoisomeric potency ratiosfor agonists and antagonists Specific ( -)-[3H]dihydroalprenolol binding sites were defined as the portion of total binding inhibited by 10 pM (*)-propranolol. Dissociation constants were calculated according to Cheng and Prusoff [38] from the equation: K d = E C ~ O[l/ SjK,,,,],where ECzo is the concentration of the competing agent which produces 50 % inhibition of specific binding, S is the concentration of (-)-[3H]dihydroalprenolol in the binding assay and K , is the dissociation constant of ( -)-[3H]dihydroalprenolol for its binding sites. Each value is the mean of at least three determinations performed in triplicate. ( - ) No inhibition of binding
+
-loglo Concentration of adrenergic antagonist (-log M)
Compound
Fig. 3. Inhibition OJ ( -)-(3H[dihydroalprenolol binding to unfructionated brown adipose tissue membranes by E and j? antagonists. Membranes were incubated in the presence of 13.1 nM (-)-[3H]dihydroalprenolol and various concentrations of E and p antagonists as indicated. 100% inhibition refers to binding observed in the presence of 10 pM (+)propranolol. Each value represents the mean of three experiments performed in triplicate. (0)(-)-Propranolol; (0)(+)-propranolol ; (A) phentolamine; (0)phenoxybenzamine
( - )-Propranolol ( -)-Isoproterenol ( -)-Norepinephrine
(+ )-Propranolol (+ )-Isoproterenol ( )-Norepinephrine
+
(+)-Epinephrine Phen t olaniine Phenoxybenzamine
O L
A
6 5 4 -loglo C o n c e n t r a t i o n o f P - a d r e n e r g i c agonist ( l o g M i
3
Fig. 4. Inhibition of ( - ) - [ 3 H ] d i h ~ d r o a ~ r e n obinding l ~ ~ l to unfractionated brown adiposr lissue memhranes by (-) and (+)-isomers of' p agonists. Experimental conditions were identical to those described in Fig. 3. (0)(-)-Isoproterenol; (A) (-)-norepinephrine; (0) (-)-epinephrine; (0) (+)-isoproterenol; (A) (+)-norepinephrine; (w) (+)-epinephrine
order of potency typical of the PI subtype of P-adrenergic receptors: (-)-isoproterenol (Kd = 210 nM) > (-)-norepinephrine (Kd = 2 pM) 2 (-)-epinephrine ( Kd = 4 pM) (Fig.4 and Table 1). Binding also displayed a remarkable stereoselectivity towards the (-)-isomers of j3-adrenergic agonists and antagonists, the (-)isomers being 34 to 280 times more effective than the corresponding (+)-isomers in competing for (-)[3H]-dihydroalprenolol binding sites (Fig. 3, 4 and Table 1). Effects of Cold-Acclimation on the Number, Density and Affinity of ( -)-[3H]Dihydroalprenolol Sites
During cold-acclimation of adult rats, interscapular brown adipose tissue underwent a remarkable hyper-
Stereoisomeric potency ratio : K d (+)-isomer/ Kd ( -)-isomer
PM
(-)-Epinephrine
100 r
Kd for inhibition of specific binding
0.056 0.210 2.0 4.0 3.9 59 68 183
69 280
w
46
-
trophy and hyperplasia as judged by the increases in total tissue wet weight (541 total protein content (820 %) and total DNA content (783 %) (Table 2). The total number of ( -)-[3H]dihydroalprenolol receptor sites also increased from 1.12 0.08 pmol in warmacclimated rats to 5.39 & 0.39 pmol in cold-acclimated rats, i.e. by 480 %. However, this augmentation was smaller than the increase in total tissue DNA content (or protein content), resulting in a 39% reduction of receptor density. The equilibrium dissociation constant, Kd, of ( -)-[3H]dihydroalprenolol for specific binding sites in brown adipose tissue membranes isolated from warm-acclimated and cold-acclimated animals was determined by measuring binding of ( -)-[3H]dihydroalprenolol in function of radioactive ligand concentration (Fig. 5A) and analyzing the data in the form of a Hill plot (Fig. SB). The density of (-)-[3H]dihydroaiprenolol binding sites was decreased by approximately 40 % in membranes isolated from coldacclimated rats a compared to warm-acclimated controls (Fig. 5A). The Kd values of ( -)-[3H]dihydroalprenolol binding in these two groups were 95 nM and 100nM, respectively, indicating that the affinity of the receptor sites was not significantly altered by the acclimation process (Fig. 5B).
x),
L. Bukowiecki, N. Follea, J . Vallieres, and J. Leblanc
193
Table 2. Effect of cold-acclimation on the number and on the density of specific ( -)-[3H]dihydr~afpren~)lo01 binding sites in interscapular brown ad@ose tissue membranes Specific binding was determined as described in Experimental Procedure in three replicate samples for each concentration of ( -)-[3H]dihydroalprenolol tested. Maximum binding was obtained from the saturation curves. Animals were acclimated to cold (5°C) or to warm (25 “C) during 3 weeks. There were 12 individual experiments at each temperature. The values of the cold-acclimated group are also presented as a percentage values of the warm-acclimated group Treatment
Animal weight
Tissue wet wt
g
mg ~ ~ 178 k 9 936 f 40
257 k 5 237 & 4
Warm-dcchmated Cold-acclimated
% ~
92
Cold-acclimated
3.5
_
_
Total protein
~~
820
,
r
1
0.0 0.4 0.8
1.2 1.6 2.0 2.4 log ,o[(-)- [3H]Dihydroalprenolol] t9
~ _ 0 36 k 0 03 2 85 & 0 15
~~~~
[ ( - ) -[3H]Dihydroalprenoiol] (nM) t0.8
DNA
_ _ _ 11 42 i 0 6 8 93 70 k 6 30
~
525
Total
Total binding
Density of binding sites
pmol
pmol/mg DNA
112 & 008 5 39 f 0 39
3 12 & 0 31 189+018 _
~
783
480
_
_
61
and density were associated with the progressive development of the cold-acclimated state or whether they might have occurred in consequence of the activation of sympathetic nervous system by coldexposure. For this purpose, the total number and the density of ( -)-[3H]dihydroalprenolol binding sites were measured in brown adipose tissue at various intervals during three weeks of continuous exposure to cold, since it is generally considered that such a period of time is required for maximal development of the calorigenic responses of the rat to catecholamines [l]. It can be seen in Fig.6 that the total number of ( - )-[3H]dihydroalprenolo1 binding sites increased progressively during cold-acclimation. However, this increase did not keep pace with the increase in total tissue DNA. Indeed, receptor density decreased by 20 % after only one day of cold-exposure, then dropped to 50 % at 4 days and remained at such levels during the rest of the acclimation period.
2.8
Fig. 5. Binding of’ ( - /-[3H]dihydroulprenolol to unjractionated brown adipose tissue membranes of warm-acclimated ( 0 ) and coldacclimated (@) rats as a function o j increasing concentrations of ligand. The animals and the experimental conditions were identical to those described in Table2. (B) Hill plot of the data in A. B represents the amount of ( -)-[3H]dihydroalprenolol bound at each given concentration of radioligand, whereas B,,, is the amount of ( -)-[3H]dihydroalprenolol bound at saturation. The lines were drawn by regression analysis of the data. The K d values were obtained from the intercept of the lines with the abscissa
Effects of Cold-Exposure on the Number and Density of ( - )-(3H]Dihydroalprenolol Binding Sites
This experiment was designed to assess whether the previously described changes in receptor number
DISCUSSION Since previous attempts to identify P-adrenergic receptors directly with radioactively labelled agonists were unsuccessful in brown adipose tissue [28] as in many other tissues [23,30], we used a potent P-adrenergic antagonist, ( -)-[3H]dihydroalprenolol [23,24], in order to characterize such receptors in brown adipose tissue of warm-acclimated and cold-acclimated rats. In the first part of this study, it was found that binding of this new radioactive ligand to brown adipose tissue membranes possessed the affinity, specificity and stereoselectivity expected of binding to P-adrenergic receptors in vitro [23,24]. Indeed, binding of ( -)-[3H]dihydroalprenolo1 was rapid, stable and saturable, indicating the presence of a finite number of
~
194
p-Adrenergic Receptors in Rat Brown-Adipose Tissue
r
1
600 r
1
1000
I 1
I
01
4
J
I
14 Time in cold ( d a y s )
21
Fig. 6. Binding of ( - )-[3Hldili~droalprt~nolol to unfractionated memhraries during cold-exposure ( 5 ‘ C ) of adult warm-acclimated (25 “ C ) rats, weighing 175-200 g when exposed to cold. Binding was estimated at saturating ( -)-[3H]dihydroalprenolol concentrations of 180 nM (see Fig. 5). Results werc expressed as a percentage of control warm-acclimated animals. Receptors density was expressed in picomoles of (-)-[3H]dihydroalprenolol bound per mg tissue DNA. Values at day 1, 4, 14 and 21 represent the mean of‘ values obtaincd respectively from 6, 4. 3 and 11 animals
receptor sites (Fig.1, 2 and 4). It was also rapidly reversible after the addition of an excess of (+)propranolol (Fig. 2). This agrees with physiological studies on isolated brown adipocytes which have shown the rapidity and the reversibility of catecholamine effects on cellular respiration [I 5,19,29]. Moreover, binding displayed high specificity and stereoselectivity (Fig. 3 and Table 1). The lack of influence of a-adrenergic antagonists on (- )-[3H]dihydroalprenolol binding is consistent with studies in vivo and in vitro which have suggested that a and fi receptors of the brown adipocyte membrane are distinct entities [21,22,31]. p-Adrenergic agonists competed for binding sites with an order of potency typical of fil-adrenergic receptors: (-)-isoproterenol > (-)-norepinephrine 2 (-)-epinephrine (Fig. 4 and Table 1) [23]. This is in good agreement with the pattern of the respiratory response observed with isolated hamster brown adipocytes [15]. Finally, the equilibrium dissociation constant for the interaction of (- )-norepinephrine with p-adrenergic receptors (Kd = 2 pM) appears to be in the same order of magnitude as the concentration of the hormone required to observe
half-maximal stimulation of adenylate cyclase in similarly prepared membranes [13]. During cold-acclimation, brown adipose tissue underwent a remarkable hyperplasia as judged by 7-8-fold increases in the total contents of tissue protein and DNA (Table 2 and Fig. 6). This hyperplasia might significantly contribute to the 4- 5-fold increase in the total capacity of this tissue for norepinephrine-stimulated oxygen consumption that slowly develops duringcold-acclimation [7,8]. It should be pointed out that the magnitude of the hyperplastic response of brown adipose tissue appears to vary with the age of the animals [17]. The present observations are in accord with previous results obtained by Thomson et al. [32], who used rats of approximately the same age as ours (8 to 9 weeks) when exposed to cold for the first time. The cold-induced hyperplasia of brown adipose tissue resulted in a 4-5-fold increase in the total number of ( - )-[3H]dihydroalprenolo1 binding sites (Table 2, Fig. 6). On the basis of previous ultrastructural studies [lo], it seems possible that this increase in receptor sites might occur via proliferation and differentiation of ‘progenitor’ cells, rather than via mitotic divisions of differentiated brown adipocytes. The fully differentiated brown adipose tissue appears to be a rather homogenous tissue with 80 % of tissue volume consisting of multilocular adipocytes [33]. However, the presence of various types of cells (unilocular or multilocular cells, fat ‘precursor’ cells, reticuloendothelial cells) has been described in warm-acclimated animals [lo, 321. Although it would be informative to assess in which type of cells the observed increase in receptor number occurs, methods for isolating and fractionating representative populations of brown adipose tissue cells have not yet been perfected [12, 34,361. Although the total number of ( -)-[3H]dihydroalprenolol binding sites was increased in cold-acclimated rats, their density was decreased by 40% (Table 2). A study of the variation in receptor density at various times after cold-exposure of the warmacclimated rats (Fig. 6), revealed that this decrease was not associated with cold-acclimation per se : it started after only 1 day of cold-exposure, was maximal after 4 days and remained at such levels during the rest of the acclimation period. It is therefore likely that the decrease of receptor density occurs in consequence of the intense sympathetic stimulation to which brown adipose tissue is submitted immediately after cold-exposure and that it is directly mediated by catecholamines. This interpretation is supported by the following observations. Prolonged exposure of several tissues to high levels of adrenergic agonists leads to a progressive desensitization of the adenylate cyclase complex to subsequent stimulation by these agents and this phenomenon appears to be mediated by a
L. Bukowiecki, N . Follea, J . Vallieres, and J. Leblanc
195
Table 3. Effect of cold-acclimation on catecholamine-srimulntionof adenylate cycluse activitv, C A M P levels, lipoljsis and cute oJ ouygen con.wmplion in hron.n adipose cissue Species
Tissue preparation
Parameter
Effect of cold-acclimation
Reference
Rat Rat
tissue slices isolated cells isolated cells tissue fragments
Rat
unfractionated membranes
decrease decrease decrease decrease decrease or no change depending on experimental conditions decrease
[391 1361
Hamster Rat
lipolysis lipolysis cAMP levels cAMP levels rate of oxygen uptake adenylate cyclase activity
fall in the number of adrenergic receptor sites [24]. The levels of catecholamines in rat arterial plasma increase 5 - 10-times immediately after cold-exposure and remain elevated as long as the animal is maintained in the cold [37]. Stimulation by catecholamines of adenylate cyclase activity, cAMP production, lipolysis and oxygen consumption, as measured in a variety of systems is decreased in brown adipose tissue of cold-acclimated animals (Table 3). Thus, everything happens as if catecholamines released by activation of the sympathetic nervous system induce a rapid decrease in brown adipose tissue receptor density resulting in a desensitization of the adenylate cyclase response to adrenergic stimulation. The hyperadrenergic response of brown adipose tissue that slowly develops during cold-acclimation cannot be explained by alterations in receptor density but may result, at least partially, from a marked hyperplasia associated with an increased number of b-adrenel-gic receptors. If this view is correct, brown adipose tissue, just as many other tissues, would be subject to catecholamine-induced desensitization [24], but would possess the particularity of increasing its total capacity for calorigenesis by a pronounced hyperplastic response to cold-exposure. This work was supported by grant no. MA-6018 of the Medical Research Council of Canada and by an establishment grant of the Conseil de la recherche en sailtk du QuPbec. A report of part of this work was presented at the Satellite Symposium of the XXVIIth International Congress of Physiological Sciences: The EEectors of Thermogenesis, Geneva, 1977. L. B. and J. V. are Scholars of the Conseil de la recherche en sanrk du QuPbec. J. L. is an Associate of the Medical Research Couiicil of Canada. The authors wish to thank Drs F. Labrie and M. G. Caron for assistance during the early part of these studies.
REFERENCES I. Depocas, F. (1960) Fed. Proc. Suppl. 19, 19-24. 2. LeBlanc, J., Villemaire, A . & Vallii-res, J. (1969) Arch. Physiol. Biochim. 77, 731 -741.
Inl.
[401 [41 I [I21
3. LeBlanc, J. & Villemaire, A. (1970) Am. J . Physiol. 218, 1742-1745. 4. LeBlanc, J., Vallikres, J. & Vachon, C. (1972) A m . J. Physiol. 222, 1043- 1048. 5. Himms-Hagen, J., Bukowiecki, L., Behrens, W. & Bonin, M . (1 972) in Environmental Physiology and Bioenergetics (Smith, R., ed.) pp. 127-133, Federation of American Societies for Experimental Biology, Bethesda. 6. Himms-Hagen, J. (1976) Annu. Rev. Physiol. 38, 315-351. 7. Foster, D. 0. & Frydman, M. L. (1978) Can. J . Physiol. Pharmacol. 56, 110-122. 8 . Jansky, L. (1973) B i d . Rev. 4, 85- 132. 9. Chaffee, R. R. I. & Roberts, J. C. (1971) Annu. Rev. Physiol. 33, 155-202. 10. Smith, R. & Horwitz, B. (1969) Physiol. Rev. 4Y, 330-425. 31. Beviz, A,, Lundholm, L. & Mohme-Lundholm, E. (1968) Brit. J . Pharmacol. Exp. Ther. 34, 198P- 199P. 12. Muirhead, M. & Himms-Hagen, J. (1971) Can. J . Biochem. 49, 802-810. 13. Skala, J., Hahn, P. & Braun, T. (1970) LIfe Sci. 9, 1201- 1212. 14. Skala, J., Novak, E., Hahn, P. & Drummond, G. 1. (1972) Int. J. Biochem. 3, 229-242. 15. Pettersson, B. & Vallin, I. (1976) Eur. J. Biochem. 62, 383-390. 16. Skala; J . (1977) J . Biol. C'hem. 252, 1064-1070. 17. Flatmark, T. & Pedersen, J. I . (1975) Biochim. Biophys. Acta, 416, 53- 103. 18. Nicholls, D. (1976) FEBS Lett. 61, 103- 110. 19, Fain, J. N., Jacobs, M . J . & Clement-Cormier, Y. (1973) Am. J . Physiol. 224, 346 - 351. 20. Heini. T. & Hull, D . (1966) J . PhJaiol. (Lond.) 197, 271 -283. 21. Flaim, K. E., Horwitz, B. A. & Horwitz, J. M. (1977) A m . J . Physiol. 232, RlOl -R109. 22. Fink, S. A. & Williams, J. A. (1976) Am. J . Physiol. 231, 700 - 706. 23. Lefkowitz, R. J., Limbird, L. E., Mukherjee, C. & Caron, M. (1976) Biochim. Biophys. Acta, 457, 1 - 39. 24. Lcfkowitz, R. J . (1978) Fed. Proc. 37, 123-129. 25. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J . (1951) J. Biol. Chem. 193, 265-275. 26. Switzcr, R. R. & Summer, C . K . (1971) Clin. ('him, Acta, 32, 203 -206. 27. Kissane, J . & Robbins, E. ( I 958) J. Biol. Chrnz. 233, 184 - 188. 28. Girardier, L., Seydoux, J. P., Giacobino, J . P. CG Chinet, A. ( 3 976) in Regulation of' Depres.sed Metabolism rind Thermogenesis (Jansky, L. & Musacchia, X. J., eds) pp. 196-212, C. C. Thomas, Springfield, 111. 29. Prusiner, S. B., Cannon, B. & Lindberg, 0. (1968) Lur. J . Biochem. 6, 15- 22. 30. Haber, E. & Wrenn, S . (1976) Phy.sio1. Rev. 56. 317-338. 31. IIorwitz, B. A.. Horwitz, J. M. & Smith, R. E. (1969) Ploc. Natl Acad. Sci. U . S . A . 64, 133-120.
196
L. Bukowiecki, N. Follea, J. Vallieres, and J. Leblanc: /l-Adrenergic Receptors in Rat Brown-Adipose Tissue
32. Thomson, J. F., Habeck, D. A,, Nance, S. L. & Beetham, K. L. (1969) J . Cell. Biol. 41, 312-334. 33. Barnard, T. (1977) Experientia (Basel) 39, 1124-1126. 34. Pettersson, B. (1976) Doctoral Thesis, University of Stockholm, Stockholm, Sweden. 35. Reference deleted. 36. Bertin, R. & Portet, R. (1976) Eur. J . Biochem. 69, 177-183. 37. Depocas, F. & Behrens, W. (1978) Experientia (Basel), in the press.
38. Cheng, Y. & Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108. 39. Dorigo, P., Maragno, I., Bressa, A. & Fassina, G . (1971) Biochem. Pharmacol. 20, 1201- 1211. 40. Hittelmdn, K. J., Bertin, R. & Butcher, R. W. (1974) Biochim. Biophys. Acta, 338, 398-407. 41. Friedli, C., Chinet, A. & Girardier, L. (1978) Experienliu (Basel) in the press.
L. Bukowiecki, N. Follea, J. Vallikres, and J. Leblanc, Labordtoires d’Endocrinologie Climdtique, Departement de Physiologie, Faculte de Medccine, Universite Laval, Cite Universitaire, Quebec, Canada, G1K 7P4
P-Adrenergic Receptors in Brown-Adipose Tissue. Characterization and Alterations during Acclimation of Rats to Cold Ludwik BUKOWIECKI, Nicole FOLLEA, Julien VALLIERES, and Jacques LEBLANC Laboratoires d’Endocrinologie Climatique, Dkpartment de Physiologie, Faculte de Medecine, Universite Lava1 (Received April 12, 1978)
1. The capacity of brown adipose tissue to respond calorigenically to catecholamines increases markedly during cold-acclimation of adult rats. To investigate this phenomenon, we have used the potent radioactive ligand ( -)-[3H]dihydroalprenolol to directly estimate the number, the density and the affinity of P-adrenergic receptors in brown adipose tissue membranes from control, coldexposed and cold-acclimated rats. 2 . Binding of ( -)-[3H]dihydroalprenolol to unfractionated membranes was rapid, stable, saturable and reversible. It displayed the affinity, specificity and stereoselectivity expected of binding to adenylate cyclase-coupled P-adrenergic receptors. P-Adrenergic agonists competed for binding sites with an order of potency typical of the P I subtype of adrenergic receptors: (-)-Isoproterenol > (-)-norepinephrine 2 (-)-epinephrine. Binding exhibited a remarkable stereoselectivity, the (-)-isomers of /3-adrenergic agonists and antagonists being 34 to 280 times more potent than the (+)-isomers in competing for ( -)-[3H]dihydroalprenolol binding sites. 3. Total interscapular brown adipose tissue of the adult, warm-acclimated rat contained 1.12 k 0.08 pmol of ( -)-[3H]dihydroalprenolol binding sites. During cold-acclimation, growth of the tissue was accompanied by a 4- 5-fold increase in the total number of receptor sites. However, this increase did not keep pace with the increase in brown adipose tissue cellularity (as estimated by total tissue DNA content), resulting in a 40-50% reduction in receptor density. The decrease in receptor density was associated with cold-exposure rather than with cold-acclimation. 4. The affinity of ( -)-[3H]dihydroalprenolol receptor sites was not significantly altered by coldacclimation. 5. The results of this study are interpreted as indicating that catecholamines released via activation of the sympathetic nervous system regulate both the density and the number of their own receptors in brown adipose tissue of cold-exposed animals. The development of the hyperadrenergic response of this thermogenic tissue during cold-acclimation may result from a marked organ hyperplasia associated with an increased number of a-adrenergic receptor sites and cannot be explained by alterations in receptor density or affinity.
When mammalian homeotherms are exposed to cold for prolonged periods of time, they develop an increased sensitivity to many metabolic and cardiovascular actions of catecholamines. In particular, the calorigenic effects of epinephrine or norepinephrine increase markedly during acclimation of adult rats to cold [l - lo]. This phenomenon occurs principally in brown adipose tissue and to a smaller extent in other tissues such as skeletal muscles [7]. It is generally accepted that it represents the metabolic basis of the increased capacity of cold-acclimated rats for nonshivering thermogenesis [l - 101. Abbreviation. CAMP, adenosine 3’: 5‘-monophosphate.
There is abundant evidence that catecholamines elicit heat production in brown adipose tissue via stimulation of a hormone-sensitive adenylate cyclase, resulting in a transient rise in cellular CAMP levels [11 - 151. This leads to a stimulation of lipolysis mediated by CAMP-dependent protein kinase [I 61 and to an increase in mitochondria1 oxygen consumption [17,18]. Experiments with CI and adrenergic agonists or antagonists support the concept that these effects are evoked primarily via p-adrenergic pathways [15, 19-22]. In contrast, little is known about the hormonal and cellular mechanisms inducing a marked enhancement of the capacity of brown adipose tissue to respond
190
calorigenically to catecholamines during cold-acclimation [6]. Although it has recently been demonstrated that catecholamines are able to regulate the number of their own receptors in several systems, the role of such receptors in determining the extent to which tissues can respond to catecholamines is still unclear [23,24]. A potential explanation for the hyperadrenergic response of brown adipose tissue in cold-acclimated rats would be an increased number or an increased affinity of P-adrenergic receptors which would, in turn, mediate the amplified response of this tissue. To test this hypothesis and to gain a better understanding of the early regulatory steps controlling the metabolism of brown adipose tissue, we used the potent radioactive ligand ( - )-[3H]dihydroalprenolol to directly identify and characterize p-adrenergic receptors in this thermogenic tissue [23,24]. Next, we evaluated the effect of cold-exposure and cold-acclimation on the number, the density and the affinity of these receptors. The results indicate that marked changes in the total number and in the density of (-)-[3H]dihydroalprenolol binding sites occur in brown adipose tissue during prolonged cold-exposure. EXPERIMENTAL PROCEDURE Materials ( -)-[3H]Dihydroalprenolo1 (48.6 Ci/mmol) was purchased from New England Nuclear. Drugs and chemicals were obtained from the following manufacturers: (-)-isoproterenol bitartrate, (-)-epinephrine bitartrate, (-)-norepinephrine bitartrate, ( k)-propranolol hydrochloride from Sigma ; (-)propanolol hydrochloride and ( + )-propranolol hydrochloride from Ayerst ; (+)-isoproterenol bitartrate, (+)-epinephrine bitartrate and (+)- norepinephrine bitartrate from Sterling-Winthrop; phentolamine from Ciba; phenoxybenzamine hydrochloride from Smith Kline and French Labs. All other reagents used were of the highest purity commercially available.
Methods
Female rats (Wistar), fed laboratory chow ad libitum, were killed by decapitation. Interscapular brown adipose tissue was quickly removed, placed in ice-cold 0.25 M sucrose/lO mM Tris-HC1 pH 7.4, and carefully trimmed of extraneous tissue. The washed pieces of tissue were blotted with a filter paper and weighed. The tissue was then minced with scissors in ice-cold 50 mM Tris-HCl/lO mM MgC12 pH 7.4 at 25 "C, homogenized with a prechilled glass PotterElvehjem homogenizer fitted with a teflon pestle, carefully filtered through four layers of cheesecloth,
P-Adrenergic Receptors in Rat Brown-Adipose Tissue
and diluted to a final protein concentration of 2.55 mg/ml. This preparation will be referred to as 'unfractionated membranes'. In some experiments semi-purified or 'crude' brown adipose tissue membranes were prepared as follows. The cleaned tissue was minced directly in ice-cold 0.25 M sucrose/lO mM Tris-HC1 pH 7.4, homogenized as above and centrifuged at 650 x g during 10 min. The layer of fat which rose to the top was carefully removed, the supernatant was decanted and centrifuged for 20 min at 40000 x g. The resulting pellet was washed twice in the same medium and the final pellet was resuspended in ice-cold incubation buffer at a concentration of 2.5 - 5 mg of protein/ml. Binding Assay
Unfractionated or 'crude' membranes (0.5 - 1 mg protein) were incubated in a total volume of 250 pl of incubation buffer (50 mM Tris-HCl/lO mM MgC12 pH 7.4 at 25°C) containing 13.1nM (-)-t3H]dihydroalprenolol (unless otherwise specified) for 20 min, at 25 "C. Incubations were terminated by rapid vacuum filtration of the mixture through Whatman GF/C glass fiber filters followed by three washes with 5 ml ice-cold buffer (50mM Tris-HCl/lOmM MgCl2 pH 7.4 at 4 "C). The filters were introduced into scintillation vials, dried and covered with 8 ml of scintillation fluid (4 g of Omnifluor/l of toluene). Bound (-)-[3H]dihydroalprenolol was determined by counting the radioactivity in a Beckman liquid scintillation spectrometer at 42 - 45 % efficiency. Specific binding was determined from the difference between total binding and binding in the presence of 10 pM-( f)-propranolol. Nonspecific binding usually ranged from 30 - 50 % of total binding. Values reported in all tables and figures refer to specific S.E.M. (represented by vertical binding. Means bars in the figures) were calculated from the means of triplicate values obtained from experiments performed on separate occasions with individual membrane preparations. Protein determinations were done by the method of Lowry et al. [25]and DNA was estimated fluorimetrically by a modification [26] of the Kissane and Robins method [27]. RESULTS Suturability and Affinity of (-)-[3H]DihydroalprenotoE Binding to Unjractionated and Fractionated Brown-Adipose-Tissue Membranes
One of the principal objectives of this study was to assess whether the number and the properties of brown adipose tissue P-adrenergic receptors would be altered by cold-acclimation. Since this tissue grows
191
L. Bukowiecki, N. Follea, J. Vallieres, and J. Leblanc
0' 0
[ (-)- [3H]Dihydroalprenolol] (nM)
4
8
12
16 20 24 T h e (rnin)
28
32
36
40
Fig. 1. Binding of ( -/-(3H]dihq~droalprenolol to unfvuctionated (0) and fracrionated ).( brown adipose tissue membranes as a function of' increusing concentrations qf ligand. Membranes were incubated with 13.1 nM (-)-[3H]dihydroalprenolol at 25°C in the presence and absence of 10 pM (+)-propranolol during 20 min. Each point represents the mean of triplicate determinations from two separate experiments in which fractionated and unfractionated membranes were prepared and incubated in parallel as described in Experimental Procedure
Fig. 2. Time course of binding of ( - ) - [ 3 H ] d i h ~ d r o a ~ ) r e n ~ ~ l o l (13.1 n M ) to unfractionated brown adipose tissue membranes. The time course of reversal of binding by 10 pM ( f)-propranolol after equilibrium of binding was reached, is also shown in the insert. Each value represents the mean S.E.M. of a minimum of three experiincnts performed in triplicate
considerably in cold-exposed adult rats, we felt that it would be useful to express possible alterations in the number of receptor sites, not only per unit of protein, but also per unit DNA, the amount of DNA being considered as an index of tissue cellularity. For this purpose, it was decided to attempt performing binding assays with unfractionated membranes containing brown adipose tissue nuclei. If feasible, this would enable us to estimate, in the same preparation, radioactive ligand binding capacity and DNA concentration. We have therefore compared, in preliminary experiments the binding characteristics of ( -)-[3H]dihydroalprenolol in unfractionated and fractionated membranes, prepared as described in Experimental Procedure, in order to determine whether membrane fractionation was necessary for the characterization ( -)-[3H]dihydroalprenolo1 binding sites in brown adipose tissue. It can be seen in Fig. 1 that saturability of membrane binding sites with ( -)- [3H]dihydroalprenolol was obtained in both types of preparation. This demonstrates that materials removed during the preparation of 'crude' membranes (nuclei, triglycerides, soluble enzymes, etc.), did not impede the saturation of receptor sites with the radioactive ligand. The specific activity of ( -)-[3H]dihydroalprenolol binding when expressed in femtomoles of radioactive ligand bound per mg of protein, was approximately threefold higher in fractionated than in unfractionated membranes, indicating that some purification of membrane-bound receptor sites had been achieved. Affinity of ( -)-[3H]dihydroalprenolol for its binding sites was similar in both preparations: the halfmaximal saturation of binding sites occurred at (-)-
[3H]dihydroalprenolo1concentrations of 70 - 80 nM. This value represents a first estimate of the dissociation constant, K d , for the interaction of ( -)-[3H]dihydroalprenolol with its brown adipose tissue receptor sites (vide infra). Considering these initial results, it was decided to further characterize the proprieties of (-)-['H]dihydroalprenolol binding in unfractionated membranes.
+
Kinetics of' ( - )-[3HJDihydroalpreizololBinding The time-course of binding of ( -)-t3H]dihydroalprenolol to unfractionated brown adipose tissue membranes is represented in Fig. 2. ( -)-[3H]Dihydroalprenolol binding was rapid, reaching equilibrium in less than 10 min and remained stable for at least 40 min. At equilibrium, the addition of 10 pM (&)propranolol resulted in a very rapid dissociation of bound radioactive ligand (insert of Fig. 2). The halftime of dissociation was less than 3 min. Specificity and Steveoselectivity -L3 H]Dihydroalprenolol Binding
of ( - )
The B-adrenergic antagonist, (-)-propranolol, potently competed for ( -)-[3H]dihydroalprenolo1 binding sites (Fig. 3 ) with a dissociation constant, K d , of 56 nM (Table 1). In contrast, the a-adrenergic antagonists, phentolamine and phenoxybenzamine, were totally ineffective in displacing the radioactive ligand from its binding sites, even at concentrations as high as 0.1 mM. Other B-adrenergic agonists also competed for ( -)-[3H]dihydroalprenolol binding sites with an
192
p-Adrenergic Receptors in Rat Brown-Adipose Tissue Table 1. Dissociation constants and stereoisomeric potency ratiosfor agonists and antagonists Specific ( -)-[3H]dihydroalprenolol binding sites were defined as the portion of total binding inhibited by 10 pM (*)-propranolol. Dissociation constants were calculated according to Cheng and Prusoff [38] from the equation: K d = E C ~ O[l/ SjK,,,,],where ECzo is the concentration of the competing agent which produces 50 % inhibition of specific binding, S is the concentration of (-)-[3H]dihydroalprenolol in the binding assay and K , is the dissociation constant of ( -)-[3H]dihydroalprenolol for its binding sites. Each value is the mean of at least three determinations performed in triplicate. ( - ) No inhibition of binding
+
-loglo Concentration of adrenergic antagonist (-log M)
Compound
Fig. 3. Inhibition OJ ( -)-(3H[dihydroalprenolol binding to unfructionated brown adipose tissue membranes by E and j? antagonists. Membranes were incubated in the presence of 13.1 nM (-)-[3H]dihydroalprenolol and various concentrations of E and p antagonists as indicated. 100% inhibition refers to binding observed in the presence of 10 pM (+)propranolol. Each value represents the mean of three experiments performed in triplicate. (0)(-)-Propranolol; (0)(+)-propranolol ; (A) phentolamine; (0)phenoxybenzamine
( - )-Propranolol ( -)-Isoproterenol ( -)-Norepinephrine
(+ )-Propranolol (+ )-Isoproterenol ( )-Norepinephrine
+
(+)-Epinephrine Phen t olaniine Phenoxybenzamine
O L
A
6 5 4 -loglo C o n c e n t r a t i o n o f P - a d r e n e r g i c agonist ( l o g M i
3
Fig. 4. Inhibition of ( - ) - [ 3 H ] d i h ~ d r o a ~ r e n obinding l ~ ~ l to unfractionated brown adiposr lissue memhranes by (-) and (+)-isomers of' p agonists. Experimental conditions were identical to those described in Fig. 3. (0)(-)-Isoproterenol; (A) (-)-norepinephrine; (0) (-)-epinephrine; (0) (+)-isoproterenol; (A) (+)-norepinephrine; (w) (+)-epinephrine
order of potency typical of the PI subtype of P-adrenergic receptors: (-)-isoproterenol (Kd = 210 nM) > (-)-norepinephrine (Kd = 2 pM) 2 (-)-epinephrine ( Kd = 4 pM) (Fig.4 and Table 1). Binding also displayed a remarkable stereoselectivity towards the (-)-isomers of j3-adrenergic agonists and antagonists, the (-)isomers being 34 to 280 times more effective than the corresponding (+)-isomers in competing for (-)[3H]-dihydroalprenolol binding sites (Fig. 3, 4 and Table 1). Effects of Cold-Acclimation on the Number, Density and Affinity of ( -)-[3H]Dihydroalprenolol Sites
During cold-acclimation of adult rats, interscapular brown adipose tissue underwent a remarkable hyper-
Stereoisomeric potency ratio : K d (+)-isomer/ Kd ( -)-isomer
PM
(-)-Epinephrine
100 r
Kd for inhibition of specific binding
0.056 0.210 2.0 4.0 3.9 59 68 183
69 280
w
46
-
trophy and hyperplasia as judged by the increases in total tissue wet weight (541 total protein content (820 %) and total DNA content (783 %) (Table 2). The total number of ( -)-[3H]dihydroalprenolol receptor sites also increased from 1.12 0.08 pmol in warmacclimated rats to 5.39 & 0.39 pmol in cold-acclimated rats, i.e. by 480 %. However, this augmentation was smaller than the increase in total tissue DNA content (or protein content), resulting in a 39% reduction of receptor density. The equilibrium dissociation constant, Kd, of ( -)-[3H]dihydroalprenolol for specific binding sites in brown adipose tissue membranes isolated from warm-acclimated and cold-acclimated animals was determined by measuring binding of ( -)-[3H]dihydroalprenolol in function of radioactive ligand concentration (Fig. 5A) and analyzing the data in the form of a Hill plot (Fig. SB). The density of (-)-[3H]dihydroaiprenolol binding sites was decreased by approximately 40 % in membranes isolated from coldacclimated rats a compared to warm-acclimated controls (Fig. 5A). The Kd values of ( -)-[3H]dihydroalprenolol binding in these two groups were 95 nM and 100nM, respectively, indicating that the affinity of the receptor sites was not significantly altered by the acclimation process (Fig. 5B).
x),
L. Bukowiecki, N. Follea, J . Vallieres, and J. Leblanc
193
Table 2. Effect of cold-acclimation on the number and on the density of specific ( -)-[3H]dihydr~afpren~)lo01 binding sites in interscapular brown ad@ose tissue membranes Specific binding was determined as described in Experimental Procedure in three replicate samples for each concentration of ( -)-[3H]dihydroalprenolol tested. Maximum binding was obtained from the saturation curves. Animals were acclimated to cold (5°C) or to warm (25 “C) during 3 weeks. There were 12 individual experiments at each temperature. The values of the cold-acclimated group are also presented as a percentage values of the warm-acclimated group Treatment
Animal weight
Tissue wet wt
g
mg ~ ~ 178 k 9 936 f 40
257 k 5 237 & 4
Warm-dcchmated Cold-acclimated
% ~
92
Cold-acclimated
3.5
_
_
Total protein
~~
820
,
r
1
0.0 0.4 0.8
1.2 1.6 2.0 2.4 log ,o[(-)- [3H]Dihydroalprenolol] t9
~ _ 0 36 k 0 03 2 85 & 0 15
~~~~
[ ( - ) -[3H]Dihydroalprenoiol] (nM) t0.8
DNA
_ _ _ 11 42 i 0 6 8 93 70 k 6 30
~
525
Total
Total binding
Density of binding sites
pmol
pmol/mg DNA
112 & 008 5 39 f 0 39
3 12 & 0 31 189+018 _
~
783
480
_
_
61
and density were associated with the progressive development of the cold-acclimated state or whether they might have occurred in consequence of the activation of sympathetic nervous system by coldexposure. For this purpose, the total number and the density of ( -)-[3H]dihydroalprenolol binding sites were measured in brown adipose tissue at various intervals during three weeks of continuous exposure to cold, since it is generally considered that such a period of time is required for maximal development of the calorigenic responses of the rat to catecholamines [l]. It can be seen in Fig.6 that the total number of ( - )-[3H]dihydroalprenolo1 binding sites increased progressively during cold-acclimation. However, this increase did not keep pace with the increase in total tissue DNA. Indeed, receptor density decreased by 20 % after only one day of cold-exposure, then dropped to 50 % at 4 days and remained at such levels during the rest of the acclimation period.
2.8
Fig. 5. Binding of’ ( - /-[3H]dihydroulprenolol to unjractionated brown adipose tissue membranes of warm-acclimated ( 0 ) and coldacclimated (@) rats as a function o j increasing concentrations of ligand. The animals and the experimental conditions were identical to those described in Table2. (B) Hill plot of the data in A. B represents the amount of ( -)-[3H]dihydroalprenolol bound at each given concentration of radioligand, whereas B,,, is the amount of ( -)-[3H]dihydroalprenolol bound at saturation. The lines were drawn by regression analysis of the data. The K d values were obtained from the intercept of the lines with the abscissa
Effects of Cold-Exposure on the Number and Density of ( - )-(3H]Dihydroalprenolol Binding Sites
This experiment was designed to assess whether the previously described changes in receptor number
DISCUSSION Since previous attempts to identify P-adrenergic receptors directly with radioactively labelled agonists were unsuccessful in brown adipose tissue [28] as in many other tissues [23,30], we used a potent P-adrenergic antagonist, ( -)-[3H]dihydroalprenolol [23,24], in order to characterize such receptors in brown adipose tissue of warm-acclimated and cold-acclimated rats. In the first part of this study, it was found that binding of this new radioactive ligand to brown adipose tissue membranes possessed the affinity, specificity and stereoselectivity expected of binding to P-adrenergic receptors in vitro [23,24]. Indeed, binding of ( -)-[3H]dihydroalprenolo1 was rapid, stable and saturable, indicating the presence of a finite number of
~
194
p-Adrenergic Receptors in Rat Brown-Adipose Tissue
r
1
600 r
1
1000
I 1
I
01
4
J
I
14 Time in cold ( d a y s )
21
Fig. 6. Binding of ( - )-[3Hldili~droalprt~nolol to unfractionated memhraries during cold-exposure ( 5 ‘ C ) of adult warm-acclimated (25 “ C ) rats, weighing 175-200 g when exposed to cold. Binding was estimated at saturating ( -)-[3H]dihydroalprenolol concentrations of 180 nM (see Fig. 5). Results werc expressed as a percentage of control warm-acclimated animals. Receptors density was expressed in picomoles of (-)-[3H]dihydroalprenolol bound per mg tissue DNA. Values at day 1, 4, 14 and 21 represent the mean of‘ values obtaincd respectively from 6, 4. 3 and 11 animals
receptor sites (Fig.1, 2 and 4). It was also rapidly reversible after the addition of an excess of (+)propranolol (Fig. 2). This agrees with physiological studies on isolated brown adipocytes which have shown the rapidity and the reversibility of catecholamine effects on cellular respiration [I 5,19,29]. Moreover, binding displayed high specificity and stereoselectivity (Fig. 3 and Table 1). The lack of influence of a-adrenergic antagonists on (- )-[3H]dihydroalprenolol binding is consistent with studies in vivo and in vitro which have suggested that a and fi receptors of the brown adipocyte membrane are distinct entities [21,22,31]. p-Adrenergic agonists competed for binding sites with an order of potency typical of fil-adrenergic receptors: (-)-isoproterenol > (-)-norepinephrine 2 (-)-epinephrine (Fig. 4 and Table 1) [23]. This is in good agreement with the pattern of the respiratory response observed with isolated hamster brown adipocytes [15]. Finally, the equilibrium dissociation constant for the interaction of (- )-norepinephrine with p-adrenergic receptors (Kd = 2 pM) appears to be in the same order of magnitude as the concentration of the hormone required to observe
half-maximal stimulation of adenylate cyclase in similarly prepared membranes [13]. During cold-acclimation, brown adipose tissue underwent a remarkable hyperplasia as judged by 7-8-fold increases in the total contents of tissue protein and DNA (Table 2 and Fig. 6). This hyperplasia might significantly contribute to the 4- 5-fold increase in the total capacity of this tissue for norepinephrine-stimulated oxygen consumption that slowly develops duringcold-acclimation [7,8]. It should be pointed out that the magnitude of the hyperplastic response of brown adipose tissue appears to vary with the age of the animals [17]. The present observations are in accord with previous results obtained by Thomson et al. [32], who used rats of approximately the same age as ours (8 to 9 weeks) when exposed to cold for the first time. The cold-induced hyperplasia of brown adipose tissue resulted in a 4-5-fold increase in the total number of ( - )-[3H]dihydroalprenolo1 binding sites (Table 2, Fig. 6). On the basis of previous ultrastructural studies [lo], it seems possible that this increase in receptor sites might occur via proliferation and differentiation of ‘progenitor’ cells, rather than via mitotic divisions of differentiated brown adipocytes. The fully differentiated brown adipose tissue appears to be a rather homogenous tissue with 80 % of tissue volume consisting of multilocular adipocytes [33]. However, the presence of various types of cells (unilocular or multilocular cells, fat ‘precursor’ cells, reticuloendothelial cells) has been described in warm-acclimated animals [lo, 321. Although it would be informative to assess in which type of cells the observed increase in receptor number occurs, methods for isolating and fractionating representative populations of brown adipose tissue cells have not yet been perfected [12, 34,361. Although the total number of ( -)-[3H]dihydroalprenolol binding sites was increased in cold-acclimated rats, their density was decreased by 40% (Table 2). A study of the variation in receptor density at various times after cold-exposure of the warmacclimated rats (Fig. 6), revealed that this decrease was not associated with cold-acclimation per se : it started after only 1 day of cold-exposure, was maximal after 4 days and remained at such levels during the rest of the acclimation period. It is therefore likely that the decrease of receptor density occurs in consequence of the intense sympathetic stimulation to which brown adipose tissue is submitted immediately after cold-exposure and that it is directly mediated by catecholamines. This interpretation is supported by the following observations. Prolonged exposure of several tissues to high levels of adrenergic agonists leads to a progressive desensitization of the adenylate cyclase complex to subsequent stimulation by these agents and this phenomenon appears to be mediated by a
L. Bukowiecki, N . Follea, J . Vallieres, and J. Leblanc
195
Table 3. Effect of cold-acclimation on catecholamine-srimulntionof adenylate cycluse activitv, C A M P levels, lipoljsis and cute oJ ouygen con.wmplion in hron.n adipose cissue Species
Tissue preparation
Parameter
Effect of cold-acclimation
Reference
Rat Rat
tissue slices isolated cells isolated cells tissue fragments
Rat
unfractionated membranes
decrease decrease decrease decrease decrease or no change depending on experimental conditions decrease
[391 1361
Hamster Rat
lipolysis lipolysis cAMP levels cAMP levels rate of oxygen uptake adenylate cyclase activity
fall in the number of adrenergic receptor sites [24]. The levels of catecholamines in rat arterial plasma increase 5 - 10-times immediately after cold-exposure and remain elevated as long as the animal is maintained in the cold [37]. Stimulation by catecholamines of adenylate cyclase activity, cAMP production, lipolysis and oxygen consumption, as measured in a variety of systems is decreased in brown adipose tissue of cold-acclimated animals (Table 3). Thus, everything happens as if catecholamines released by activation of the sympathetic nervous system induce a rapid decrease in brown adipose tissue receptor density resulting in a desensitization of the adenylate cyclase response to adrenergic stimulation. The hyperadrenergic response of brown adipose tissue that slowly develops during cold-acclimation cannot be explained by alterations in receptor density but may result, at least partially, from a marked hyperplasia associated with an increased number of b-adrenel-gic receptors. If this view is correct, brown adipose tissue, just as many other tissues, would be subject to catecholamine-induced desensitization [24], but would possess the particularity of increasing its total capacity for calorigenesis by a pronounced hyperplastic response to cold-exposure. This work was supported by grant no. MA-6018 of the Medical Research Council of Canada and by an establishment grant of the Conseil de la recherche en sailtk du QuPbec. A report of part of this work was presented at the Satellite Symposium of the XXVIIth International Congress of Physiological Sciences: The EEectors of Thermogenesis, Geneva, 1977. L. B. and J. V. are Scholars of the Conseil de la recherche en sanrk du QuPbec. J. L. is an Associate of the Medical Research Couiicil of Canada. The authors wish to thank Drs F. Labrie and M. G. Caron for assistance during the early part of these studies.
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L. Bukowiecki, N. Follea, J. Vallikres, and J. Leblanc, Labordtoires d’Endocrinologie Climdtique, Departement de Physiologie, Faculte de Medccine, Universite Laval, Cite Universitaire, Quebec, Canada, G1K 7P4
