Characteristics of β-adrenergic receptors in isolated cells and in crude membranes of brown adipose...

BIOCHIMIE. 1984, 66, 573-578

Characteristics of [ -adrenergic receptors in isolated cells and in crude membranes of brown adipose tissue.

Claude SENAULT, V~ronique LE C O M T E and Ren6 PORTET.

Laboratoire d'Adaptation Energdtique ~ i'Environnement (Ecole Pratique des Hautes Etudes). ERA CNRS N"413, !!, Place Marcelin Berthelot. 75231 Paris Cedex 05. France.

(Refue h" 26-6-1984. acceptt;e apres r~h'ixion h' 12.9-19,'~4).

R6sum6 - - Pour tenter d'expliquer la diminution de la sensibilit6 aux catdcholamines observde in vitro, dans le tissu adipeux brun du rat adapt6 au froid, nous avons dtudld comparativement les rdcepteurs [$-adrdnergiques dans les cellules isoldes el dans une prdparation membranmre non purifide ~ partir du tissu adipeux brun interscapulaire. La liaison du I SHt dihydroalprenolol prdsente les m~mes caractdristiques dans les 2 0 ' p e s de prdparations. Les expdriences de comp6tition de la liaison du I JHI dihydroaiprenolol p~r les [$-agor, is:es ont permis la caractdri- sation de rdcepteurs adrdnergiques de o,pe [$1 avec une plus faible affinit6 des agonistes [$ pour ies sites de liaison du 13HI dihydroaiprenolol clans les membranes que dans les cellules, L'adaptation au froid provoque, clans les cellules isoldes seulement, une diminution de 41% des sites de liaison du 13H I dihydroalprenolol ainsi qu'une augmentation de I'affinit6 des agonistes [$-adrdnergiques.

On conclut que la diminution du nombre des r~cepteu~rs [$-adrdnergiques peut ~tre un facteur. au niveau de I~nteraclion hormone r6ceptcur, de la rdgulation de la transmission de Faction biologique, responsab!e de la diminution de la rdponse aux calecholamines induite par le .froid. Les cellules isoldes paraissent constituer un matdriel phls approprid qu'une fraction membranaire non purifide pour 6tudier ce phdnom~ne de ddsensibilisation.

Mots=cl~s : adaptaiio~ at, ftoid / tiss~ adipe,.x ~ u n / recel~teurs i~-adr~nergiques / I~H 1 dihydroalprenoiol.

Summary - - In relation to decreased metabolic ,~ensitiviO, to catechoiamines observed, in vitro, in brown fa t o f cold-acclimated rats. [$-adrenergic receptors were studied in isolated cells al:~l ;.n a crude membrane preparation from rat interscapular brown adipose tis.~'ue. [ SH l dih),droalpreno- !ol bindin~ Z'a'; the same characteristics in both ti,n,~,- e f p::;paratim~, compeli~ion studies o f lSH l dihydroalprenoloi binding led to the characte,rization o f [$1 subtype adrenergic receptors with a lower affiniO, o f [$-adrenergic agonists for i~HI dihydroalprenolol binding sites in membranes than that found in isolated cells. Cold acclirnation produced, in isolated cells only. a decrease o f 41% in the I ZHl dihydroalprenolol binding sites and a [$-adrenergic agonist affinity increase.

It is concluded that [$-adrenergic receptor decrease could be a factor, at the hormone receptor interaction level, in the regulation o f the transmission o f biological action responsible for the cold-induced decrease in catecholamine responsiveness in brown adipose tissue. For a study of the desensitization process in brown fat. isolated cells seem to offer certain advantages over a crude membrane preparation.

Key-words : cold acclimation i ~-adrenergic receptors / brown adipose tissue / ( - - ) - - t~Xi dihydroaiprenolol.

574 Claude Senaul t and coll.

It is now general ly admitted that brown fat serves as a sympathet ica l ly controlled heat-gene- rating organ that can mainta in normal body temperature via nonshiver ing thermogenesis . E 'o rep i rephr ine released by the sympathet ic nerve endings is responsible for activation of the tissue [1]. However the mechanism by which the neurotransmit ter increases the rate o f the 02 consumpt ion has not been fully elucidated. Nevertheless, there is substantial evidence to show that no rep inephr ine elicits heat product ion via the [I-adrenergic receptor- cAMP pathway [2].

Moreover several reports indicate cold-induced decreases in norepinephr ine s t imulated cAMP formation [3], adenylate cyclase activity [4], l ipolysis [3, 5], and oxygen consumpt ion [6].

The aim of our investigation is to identify [3 receptors in isolated cells and in crude membrane of brown adipose tissue and to determine whether this cold- induced desensit ization occurs at the level of the [3 receptors, through a decrease in number a n d / o r aff ini ty of [3 receptors.

Materials and methods

i) Animals • All the experiments were performed on 13 week-old male Long-Evans rats acclimated to constant temperature of either 23 °C (control group) or 5 °C (cold-acclimated group) for 4 weeks. The animals were maintained on a daily 12 h dark/light cycle and given commercial rat chow and tap water ad libitumo

2) Isolation o f brown fa t cells : Rats were killed by decapitation and interscapular brown adipose tissue immediately removed. Brown fat cells were isolated by the coilagenase digestion method, as described by Senauit et al. [7], with minor modifications. At the end of the collagenase incubation and dilution by Krebs Ringer phosphate buffer containing 4% albumin (KRPA), cells were allowed to settle out at the surface in a plastic tube, for 20 minutes at room temperature and for 40 minutes at 4"C. Cells were washed wish KRPA and stored at 4°C for 60 minutes. Aggregated material was removed by filtration through silk cloth. Cells were cou~ted~ using a Mallassez cell in the cell-suspension, diluted with trypan blue, and used immediately in the binding assay. Yield of isolated cells was lower than 10% ( 2 4.106 cells from ! g tissue). In the two groups, muitilocular cells were contaminated by unilocular cells, more numerous in the control group than in the cold-acclimated group. The viability of the isolated cells was controlled by measuring their cytochrome oxydase activity [8]. Cytochrome oxydase activity was 0.290 _+ 0.030gmol O2,min/106 cells in control rats, and 0.400 _+ 0.040 p.mol O2/min/106 cells in cold-acclimated rats, respectively.

3) Preparation of crude membranes from brown adipose tissue : Minced brown fat pads were homogeni- zed at 0°C in a Potter Thomas homogenizer with a teflon pestle (8 strokes, 2000 rev/min), and prepared according to Bukowiecki's method [9]. The final pellet was suspended in 50raM Tris-HCl, 10raM MgCl2, pH 7.4, at a protein concentration of 7 mg/ml, and filtered. The total yield of membrane protein from the cold-acclimated animals was on average twice as high as that from controls. 5" Nucleotidase activity was 13.4 _ 1.2 nmol P min -t mg -I protein in control and 8.2 + 0.9 nmoi P min-I mg-~ in cold-acclimated rats.

Binding assay

Isolated brown adipocytes : 3 to 7.104 cells were incubated with 0.5 nM to 10nM (--)]3HI dihydroalprenolol (l~HI DHA) (40 Ci/mol) in a total volume of 0.390 ml of 0.7 % albumin Krebs Ringer phosphate buffer, according to the method of Svoboda et al. [10], at 37°C for 8 min. In each experiment total and non specific binding to brown adipocytes was determined by measuring the amount of radioactivity retained on filter in the absence and in the presence of 100 lxM (--) isoproterenol or 2011M (--) alprenoloi. Non specific binding accounted for 30 to 60 % of total binding.

Competition experiments were performed in a similar way with 3 nM [~HI DHA and various concentrations of adrenergii: ligands. Ascorbic acid 0.2 mM was added to the incubation medium to prevent possible oxidation of agonists. The kinetic experiments were performed as single incubations at 37°C. The total volume at the start (time zero) was 2.5 ml containing 0.450 ml cell suspension (_- 1.106 cell/ml) and 5 nM 13HI DHA. 0.3 ml aliquots were taken at different times and the reaction was stopped by addition of these aliquots to 2 ml ice-cold Krebs-phosphate and filtration as in equilibrium binding studies.

Measurements o f l3HI dihydroalprenolol binding to crude membranes : The experiments on I~HI DHA binding to crude membranes were performed in the same way as those with isolated cells. However, the Krebs-Ringer buffer was remplaced by Tris buffer (50 mM Tris-HCl, l0 mM Mg,_ ~:, pH 7.4 at 37 °C). Non specific binding accounted ,'c, 70 % at ! nM of total binding.

Materials

(--)-- I~HI dihydroalprenolol (40Ci/nmol) was purchase from New England Nuclear. Bacterial crude collagenase (Type II 125 U/mg), bovine serum albumin, fraction V (poor in fatty acids), (--) isoproterenoi bitartrate, ( + ) isoproterenol bitartrate, (--) norepine- phdne bitartrate and (--) epinephrine bitartrate were

~-adrenergic receptors in cold-acclimated rats 575

obtained from Sigma, (--) alprenolol was a gift from Ciba-Geigy laboratory. All other reagents were of standard analytic purity.

Statistical methods

Student's test was used to determine the statistical significance of the difference between groups; a P value < 0.05 was considered significant.

Resul t s

Association kinetics o f l3H I dihydroalprenoloi binding to brown adipocytes and crude membranes

In brown adipocytes and crude membranes, the association of 13H I DHA was very rapid. 50 % were bound within one minute, and equilibrium was reached at 10 minutes and was stable for up to 10 minutes.

Number and affinity o f 13H I dihydroalprenolol binding sites

a) Isolated cells.

The incubation of isolated cells with increasing concentrations of 13H I DHA showed that specific 13HI DHA bipding was a saturable and high affinity process. The results of a representative experiment are shown in Figure i A. Binding reached apparent saturation at - l0 nM

L~HI DHA. The Scatchard plot analysis of the binding data resulted in a linear plot : r = 0.98, P < 0.001 indicating a single class of binding sites. Hill plot analysis gave a slope of 0.91 supporting the absence of cooperativity.

b) Crude membranes

Specific 13HI DHA binding to crude membrane was also a saturable process. A representative experiment on 13HI DHA binding to crude membranes is shown in Figure lB. We observed similar binding properties to those observed in isolated cells: high affinity of 13HI DHA binding, Kj --- 2 nM, a single class of binding sites, with absence of cooperativity (ni l = I).

Comparisons of results obtained in six or seven experiments in isolated cells and crude membranes are summarized in Table !. K~ was very similar in the two preparations. It was calculated there were -- 50,000 receptors per cell in controls. We have estimated (unpublished data) that !.!06 cells contain about 1.5 mg membrane protein. Bmax in crude membrane was calculated to be 40 f m o l / 1 0 6 cells.

Relative potency o f agonists in competition for specific 13H I dihydroalprenoloi binding

a) Isolated cells [~-adrenergic agonists competed for the

13H I DHA binging sites in the following order of potency : ( - - ) isoproterenol > ( - - ) norepinephr|ne >I ( - - )epinephr ine demonstrating that 13HI DHA did bind to q, adrenergic receptors.

TABLE ! Dissociation constant and total number of binding sites in isolated cells and in crude membrane fractions from brown adipose tissue. The parameters were evaluated by Scatchard plot analysis of specific I-~H i dihydroalprenolol binding data.

The values are means +_ SEM

Isolated cells Crude membranes

Controls Cold-acclimated Controls Cold-acclimated n 6 7 6 6 K~(nM) 1.9 _+ 0.6 2.2 _+ 0.2 2.1 + 0.3 2.7 _+ 0.5 Bm~, 0.083 + 0.009 0.049 + 0.008* 0.029 _+ 0.003 0.033 _+ 0.007

pmol / 10 ~ cells pmol / m g proteins 2.3 _+ 0.4 4.6 _+ 1.5

pmol / pmol P min

Receptors per cell 49,800 + 4,770 29,400 +_ 4,800*

* Sign;qca~tly different from controls (P < 0.05)

576 Claude Senauit and coll.

®

50

"= 40 e=- ,,_..

~ N 30 ~ ' ~ g 2o

mg i --;-- 20 lo

5

10

I ,

5 (-) I3HI-DHA (nM)

0

0

:1.26nM

20 40 60 80

( - ) 13HI-DHA bound (fmol/106 cells)

I

10

20

~ = 1 5

,=1:o .-v.- ca.

- - E 10

_®

--- 5 KO =2.

~ 2.70nM

/ / '°1 -k\

lo 20 so ( - ) 13HI-DHA bound (fmol/mg proteins)

I I I i

2 3 4 5 1-) 13HI-DHA (nM)

FIG. 1. -- Specific I SHI dihydroalprenolol binding to brown adipose tissue from control and cold-acclimated rats as a function of increasing concentration of ligand.

Binding experiments were performed as described in Methods. Each value represents the mean of triplicate determinations: the experiments shown are representative of 6 experiments with different cell prepara t ions (A) or crude membrane~ (BL Inserts : Scatchard plot analysis of specific I~H I dihydroalprc.~olol binding. The data of the binding curve were calculated according to Scatchard [19]. The slope of the plot l/Kd was determined by linear regression analysis. The maximum binding capacity, Bmj, was obtained as the intercept with the abscissa.

o o Controls z ?, Cold acclimated

The stereospecificity of binding was tested with the ( + ) stereoisomer of isoproterenol which is 100 times less potent than (--) stereoisomer in the displacement of 13HI DHA binding (Table II).

b) Crude membranes The same order of potency of 13 adrenergic

agonists in competing for 13HI DHA binding sites was found in crude membranes.

The ECs0 (concentration of competitor yielding 50% displacement of 13HI DHA) values from

displacement studies were used for calculations of apparent equilibrium dissociation constants (K~), according to the equation of Cheng and Pru- soft [11].

Similar K~ values were observed for antagonists in isolated cells and crude membranes but for agonists these values were lower in isolated cells than in crude membranes, only significant for (--) isoproterenol and (--) epinephrine in cold-acclimated group. K~ for norepinephrine in cells was of the same order than half maximally

~l-adrenergic receptors in cold-acclimated rats 577

T A B L E I I

K'd values of adrenergic antagonists and agonists for inhibition of specific 13HI dihydroalprenolol binding to isolated cells and crude membrane fractions from adipose tissue. K'~ values were determined according to the equation o.f Cheng

and Prusoff [l l]

Isolated cells Crude membranes

Controls Cold-acclimated Controls Cold-acclimated nM nM nM nM

(--) alprenolol 19 + 3 (2) (--) isoproterenol 100 _+ 19 (4) (--) norepinephrine 1660 + 195 (5) (--) epinephrine 1623 + 274 (4)

(+) isoproternol

I I + 5 (2) 61 _+ 6 (8)*

1055 + 294 (5) 769 + 262 (7)

5640 ( ! )

14 _ 5 (4) 5 + ! (4) 769 __ 121 (5) ° 1296 + 415 (7)"

2237 + 432 (3) 2120 _ 640 (3) 1783 _+ 99 (3) 1952 _ 409 (4) 8760 ( ! ) 9868 ( i )

* Significantly different from controls. • Significantly different between cells and membranes.

effective concentration of this hormone in stimulation cyclic AMP production (K act) [3].

Effects o f cold acclimation on the number and affinity o f J 3HI dihydroalprenolol binding sites

Specific 13H I DHA binding to brown adipocytes from cold-acclimated rats was lower than controls. The maximal number of specific 13H I DHA binding sites per cell unit was significantly decreased (29,000 receptors per cell). L ! . . . . . . . . . ~,I-A ~£ '~---- '~ ,° . A~" 3 I ] ~,.,~,~,,~, t .~ d~lmty t,~ I n l DHA binding was not modified (Table I).

b) Crude membranes

No change was observed following cold act.A- mation, either in maximal number of specific l aHI DHA binding sites expressed per mg protein, or per pmol P rain -I (5' nucleotidase activity), or in the affinity of 13HI DHA binding (Table I).

Effects o f cold-acclimation on the potency o f agonists in competition with specific 13HI dihydroalprenoloi binding

a) Isolated cells

In the cold-acclimated group it was observed that binding displacement curves were shifted to the left, indicating an increase in agonist affinity to specific 13HI DHA binding sites. The .~ of agonists was significantly decreased only for ( - - ) isoproterenoi in the cold-acclimated group (Ta- ble It).

b) Crude membranes

No change was observed in the potency of agonists in competition with specific 13HI DHA binding (Table II).

Discussion

!) Study of the binding of 13HI DHA on isolated cells and on crude membranes showed [3Hi DHA binding with the same characteristics in both types of preparation • saturability between 5 and 10nM, fast kinetics, high affinity, and Scatchard analysis showed one class of receptors. Study of isolated cells gave an estimate of -- 50,000 receptors per cell unit. These data are comparable to those obtained by Svoboda et al. [10] on the hamster, and by Kurahashi and Kuroshima [12] on the rat. However in a study of brown adipose tissue homogenate in the rat Bokowiecki et ai. [9] found values of Bm~, per mg protein, and of K~ higher than ours. These differences can be explained by the fact that their study was made with very high concentrations of 13HI DHA, unappropriate for the determination of high affinity binding sites.

2) [I-adrenergic receptor characterization.

Competition of 13HI DHA binding by [I agonists leads to the characterization o~ 151 subtype adrenergic receptors in isolated cells as well as in crude membranes. However a difference appea- red between the 2 preparations • a lower affinity of I~-agonists for 13H I DHA binding sites in crude

578 Claude Senaul t and coll.

membranes than in isolated cells; significant for (--) isoproterenoi. This lower level of agonist affinity in membrane may be due either to the incubation medium, which is different in the two experiments (although ascorbic acid was added in the two cases), or to the loss of cellular integrity that could modify receptors accessibility. It should also be noted that in brown adipose tissue only 35 % of total cells are brown adipocytes [7], and thus crude membrane preparation contains proteins proceeding from cellular fractions from cells other than brown adipocytes (nervous cells, endothelial cells). This could add other receptors to the preparation.

Such differences in (--) isoproterenol affinity were also found by Mohell et al. [13] in a study of identification of 13~ adrenergic receptors in crude membranes and isolated cells from hamster brown adipose tissue. On the contrary, on other types of cells, the apparent binding affinity of I~- adrenergic receptor agonists has been shown to be lower in intact cells than in membrane preparations [14].

3) Effect of cold acclimation.

In isolated cells, cold acclimation led to a decrease in the number of 13HI DHA binding sites. These results agree with Kurahashi [12] who found a decrease of 45% in the number of [3HI DHA sites in cold-acclimated rats. As has been observed after incubation of cells in the presence of agonists [15, 16] chronic cold-induced adrenergic stimulation of brown adipose tissue wuu~u lead to a desensitization of the adenylate cyclase system. This could explain the decrase in metabolic sensitivity to catecholamines in vitro [3, 4, 5, 6] in brown adipose tissue of cold-acclimated rats. Desensitization was observed only in isolated brown adipocytes in which the maximal number of sites per cell was decreased. According to Chang and Costa [17] during desensitization, the I] adrenergic receptors transiocate from plasma membrane to cytoplasm. In the same way, in our crude membrane preparation from tissue homogenate, internalized receptors may be i-eco- vered and again become accessible to the radio ligand.

However the regulation of the number of adrenergic receptors represents only one level of control. According to Lefkowitz [18] another step in the regulation of the receptor appears to be the formation of a transient coupled form of the receptor with the guanine nucleotide regulatory protein which has a high affinity for agonist. Desensitization led to an uncoupling of the receptors with adenylate cyclase. This could be

due to an alteration in the receptor, in th~ regulatory protein or in the membrane environe. ment (GTP presence) in which they normall 3 interact. Cold acclimation could induce modifi. cations of the interaction between the receptor ant the nucleotide regulatory protein, resulting in change in the transmission of the message [20 despite the increased sensitivity of brown adipo. cytes to [~-agonists.

In conclusion, crude membrane preparatio~ and isolated adipocytes from brown adipose tissue demonstrate approximatively the same high affinity state of the I~1 adrenergic receptors, but tc study the desensitization process in cold-accli. mation we seem it more convenient to utilize isolated cells than crude membrane preparation.

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5. Dorigo, P., Maragno, I., Bressa, A. & Fassina, G. ( i 971 ) Biochem. Pharmacol.. 20, 1201-121 l.

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,,.~ v'~n°~n:,, v . , , . , , , , , a-,. & ~',"~,D~'" ,.,.o.'-" c, [ l:,u.,j'"~L~" J. o~ol.n'-" Lnem.,"' z4u,"'~ 3392-3398.

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i O. Svoboda, P., Svartengren, J., Snochowski, M., Hofist6k, J. & Cannon, B. (1979) Eur. J. Biochem., 102, 203-210.

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! 5. Lefkowitz, R.J., (1978) Fed. Proc., 37, ! 23- ! 29. 16. Giudicelli, Y., Agli, B. & Lacasa, D. ~!979), Bio-

chim. Biophys. Acta, 585, 85-93. 17. Chang, D.M. & Costa, E. (1979) Proc. Natl. Acad.

Sci. USA. 76, 3024-3028. 18. Lefkowitz, R.J., Caron, M.G., Michel, T. & Stadel,

J.M. (1982) Fed. Proc.. 41, 2664-2670. 19. Scatchard, G. (1949) Ann. N.Y. dcad. Sci., 51,

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Characteristics of β-adrenergic receptors in isolated cells and in crude membranes of brown adipose...

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