Eur. J. Biochem. 102, 203-210 (1979)

High Number of Higli-Affinity Binding Sites for (-)- [3H]Dihydroalprenolol on Isolated Hamster Brown-Fat Cells A Study of the P-Adrenergic Receptors

Petr SVOBODA, Jan SVARTENGREN, Marek SNOCHOWSKI, Josef HOUSTEK, and Barbara CANNON

Institute of Physiology, Czechoslovak Academy of Sciences, Prague; Wenner-Gren Institute, University of Stockholm; and Chemistry Department, Karolinska Institute, Stockholm

(Received June 18, 1979)

The beta-adrenergic receptors of hamster brown adipocytes have been characterised by binding of the radioactive ligand (-)-[3H]dihydroalprenolol, directly to isolated intact cells in suspension. The brown fat cell contains 57000 specific and saturable binding sites which have a dissociation constant ( K d ) for [3H]dihydroalprenolo1 of 1.4 nM as determined by Scatchard analysis. The kineti- cally derived Kd, determined from forward and reverse rate constants, is 5 nM. Both of these values are in agreement with the dissociation constant (Kd = 2.2 nM) for alprenolol, determined from competition studies with [3H]dihydroalprenolo1 in these cells. Beta-adrenergic agonists com- peted for the specific binding sites with a typical b,-adrenergic specificity. The order of potency of agonists agrees well with the ability of these agents to stimulate respiration in isolated brown adipocytes: 50 % stimulation of respiration occurs with apparently less than 10 % occupancy of binding sites.

Both the high affinity and high number of specific binding sites of [3H]dihydroalprenolol in brown fat cells presumably reflect the generally accepted dominating role of catecholamines in the regulation of brown fat metabolism and non-shivering thermogenesis.

The primary stimulus for induction of heat produc- tion by brown adipose tissue is presumably binding of catecholamine molecules to b-adrenergic receptors situated in the plasma membrane [l]. Therefore, the elucidation of the detailed mechanism of such phenom- ena as cold adaptation and hibernation needs, among other things, delineation of catecholamine/brown- adipocyte interaction at the molecular level. The potent beta-adrenergic antagonist (-)-[3H]dihydroal- prenolol, has been used here to characterise and quantify the specific binding sites in isolated brown adipocytes. It was found that as many as 57000 receptors are present on a single hamster brown fat cell. Some of these results have been presented else- where [2-41.

MATERIALS AND METHODS

Isolation of Broicn Adipocytes

Adipocytes from the pooled brown adipose tissue of golden hamsters (Mesocricetus auratus) maintained

at 21 'C were prepared by a slight modification of the collagenase method of Pettersson and Vallin [5]. Krebs/Ringer phosphate buffer had the following composition: 110.9 mM NaCl, 1.4 mM KH2P04, 3.8 mM NaH2P04, 16.7 mM Na2HP04, 1.4 mM MgS04, 1.5 mM CaCI2, 10 mM fructose and 10 mM glucose, pH 7.4. In addition, during cell preparation 40/;; bovine serum albumin fraction V was present. The method was modified such that undigested tissue fragments after the second incubation were incubated for another 25 min under the same conditions. Adipo- cytes from the filtrates after the second and third incubations were combined, washed twice by flotation at 0 - 4 "C in Krebs/Ringer phosphate buffer plus 4 7;) bovine serum albumin and kept in the cold for 12- 16 h. Aggregated material was then removed by re- peated filtration through silk cloth and the cell sus- pension was washed once more. Any material which aggregated during standing was removed immediately before the assay of (-)-[3H]dihydroalprenolol binding. The number of cells per ml was determined by counting a diluted suspension in a Biirker chamber and the

204 (-)-[3H]Dihydroalprenolol Binding Sites on Isolated Hamster Brown-Fat Cells

fraction of broken cells was estimated by staining with 0.1 % Alcian blue. This was routinely less than 10%.

Measurement of (-) -(3H]Dihydroalprenolol Binding

Isolated brown fat cells were incubated with various concentrations of (-)- [3H]dihydroalprenolo1 (specified in legends to figures) in 0.3 ml Krebs/Ringer phosphate buffer plus the concentrated cell suspension. After 15 min at 37 ‘C, the incubation mixture was diluted with 2 ml ice-cold Krebs/Ringer phosphate buffer and immediately filtered through a single Whatman GFC glass-fiber filter under very low vacuum, equivalent to a flow rate of 50- 60 ml/min. As soon as the diluted mixture was completely filtered, the filter was washed with 25 ml of cold Krebs/Ringer phosphate buffer at the same flow rate. The filter was then dried slowly and taken for determination of radioactivity using a scin- tillation mixture of toluene/Triton X-200 (2/1, v/v) plus 2.5-diphenyloxazole (5 gil), in an Intertechnique SL30 liquid scintillation spectrometer.

Binding data were analyzed according to Scatchard [6] and dissociation constant (Kd & SE), number of binding sites (Bmax) and 95% confidence interval for B,,, were calculated using a Wang programmable electronic calculator (model 720). All data presented on specific binding were obtained after subtraction of non-specific binding from total binding. This non- specific binding was calculated from parallel incuba- tions containing a 100-fold excess of unlabelled alpre- nolol in addition to the labelled ligand, as was sug- gested by Chamness and McGuire [7]. The non- specific binding accounted for l0-20%, of total binding (less than 1.5 ”/, of total radioactivity offered) at 0.15 - 0.3 nM (-)-[3H]dihydroalprenolol, which were the lowest radioactive ligand concentrations used. This number increased to 75-80% when the ligand concentration was increased to 20 nM. Direct proportionality existed between the non-specific bind- ing and the total (- )- [3H]dihydroalprenolo1 concen- tration in the media.

For kinetic experiments, small changes in the procedure were made. For determination of the asso- ciation rate, the brown fat cells (300000 cells/ml) were incubated with 5 nM (-)-[3H]dihydroalprenolol in a total volume of 2.5 ml at 37°C. The reaction was terminated by addition of 0.3-ml aliquots of the reac- tion mixture to 2 ml of cold Krebs/Ringer phosphate buffer at various time intervals up to 10 min. Thus sample was filtered as above. For measurement of the rate of dissociation of the reaction, ‘zero time’ was taken as the time of addition of 1000-fold excess of unlabelled alprenolol to a cell suspension preincubated for 10 min with 5 nM (-)-[3H]dihydroalprenolol, and aliquots sampled at various time intervals as above.

O.vygen Uptake

Oxygen uptake rates were measured at 37 ‘C using a Clark-type oxygen electrode in a Perspex chamber of 1 ml capacity, as previously described [8].

Materials

(-)-[3H]Dihydroalprenolol (specific radioactivity 1.8 TBq/mmol) was purchased from New England Nuclear Chemicals GmbH (Dreieich, F.R.G.). Other compounds used in this study were : (-)-alprenolol, (-)-isoproterenol bitartate, (-)-epinephrine bitartate, (-)-norepinephrine bitartate and crude collagenase (Sigma, St Louis, Mo., U.S.A.), (*)-propranolol (ICI, Macclesfield, England), (+)-norepinephrine bitartate (Pharmacia, Uppsala, Sweden) and bovine serum albumin (fraction V) (Miles, Kankakee, Ill., U.S.A.). All other chemicals were of the highest purity com- mercially available.

RESULTS

Binding of (-)-[3H]Dihydroalprenolol as Function of Radioactive Ligand Concentration

The affinity of cellular receptors for (-)-[3H]- dihydroalprenolol can be determined from binding experiments performed at thermodynamic equilib- rium. Incubation of brown adipocytes with increasing concentrations of (-)-[3H]dihydroalprenolol (0.15 - 10 nM) for 15 min at 37°C showed that specific binding is a saturable process with approximately 80 fmol ligand bound/l O 6 cells at apparent saturation (Fig. 1 A). Half-maximum saturation occurred at 0.6- 0.7 nM, providing an estimate of the equilibrium dissociation constant Kd for (-)- [3H]dihydroalprenolo1 binding. For more accurate determination of both dissociation constant (Kd) and maximum number of binding sites (B,,J the results were expressed as a Scatchard plot [6] (Fig. 1 B).

The Scatchard plot was linear with a correlation coefficient r = 0.96. As evidenced by the linearity of the plot there is no indication of cooperative inter- actions between receptors. This linearity also verifies that at 15 min the binding reaction has substantially come to equilibrium. The calculated dissociation constant from this experiment was 1.08 & 0.16 nM and saturation of receptor sites was achieved at 92 fmol (-)-[3H]dihydroalprenolol bound/l O6 cells, which corresponds to 61 000 receptors/cell.

The Kd and B,,, values were determined in seven cell preparations in all. The results are summarised in Table 1. The value for Kd determined here is in good agreement with the kinetically derived value and the value for alprenolol calculated from com-

P. Svoboda. J . Svartengren, M. Snochowski, J. HouStZk, and B. Cannon 205

0 K) 100 Bound (fmol)

I i

Fig. 1. ( /-(3H]Dihydroalprenolol binding to isolated brown fat ce1l.r us u function ojradioligund concentration. (A) Specific binding as a function of the total concentrations of (-)-[3H]dihydroalprenolol. 125000 cells were incubated in a final volume of 0.37 ml, as de- scribed in Materials and Methods. Each value shown is the mean of determinations from triplicate incubations. Reproducibility of the triplicate samples was k 5 % . The experiment shown is represen- tative of seven such experiments performed with different cell preparations and different sets of( -)-[3H]dihydroalprenolol concen- trations. (B) Scatchard plot of specific (-)-[3H]dihydroalpren~lol binding to isolated brown adipocytes. The results of A were calculated according to Scatchard [6] as described in Materials and Methods. The maximum binding capacity (Bmax) was determined as the intercept with the ordinate. Bound = the amount of (-)-[3H]- dihydroalprenolol specifically bound to 10' cells. Free = the differ- ence between thc total amount of (-)-[3H]dihydroalprenolol present in the incubation medium and the total amount of (-)-[3H]dihydro- alprenolol bound, both specifically and non-specifically

petition studies with (-)-[3H]dihydroalprenolol (vide influ). Alprenolol and dihydroalprenolol have been shown to react identically [9]. The Kd and total number of binding sites remained strikingly constant within the concentration range 175000- 700000 cells/ ml. This indicated that the experimental conditions were optimal during the binding assay, since a direct proportionality between the total number of cells and total specific binding existed. However, when the cell density was increased over lo6 cells/ml, severe aggre- gation occurred and (-)-[3H]dihydroalprenolol bind- ing was depressed (not shown).

Kinetics of (-) -[3HjDihydroalprenolol Binding to Isolated Brown Fat Cells

In an attempt to confirm the low Kd value deter- mined from equilibrium studies, kinetic measure- ments were undertaken. Specific binding of (-)-[3H]- dihydroalprenolol to isolated brown adipocytes was rapid, reaching equilibrium within 5 min at 37 'C (Fig.2A). Kinetic data from Fig.2A can be used to calculate the rate constant kl for the reaction

R + L + R L

where L represents (-)-[3H]dihydroalprenolol and R the free b-adrenergic receptor (vide infra).

The slope kobs of the line in Fig. 2 B (0.40 min-') is an estimate of the observed forward rate constant for a pseudo-first-order reversible reaction. The treat- ment of the data as a pseudo-first-order reaction is valid [lo, 111, since the concentration of receptors as determined from the equilibrium binding studies (about 0.02 nM) is very much lower than the concen- tration of (-)-[3H]dihydroalprenolo1 (5 nM).

The dissociation rate of (-)-[3H]dihydroalprenolol from its binding sites (Fig.3A) was measured by

Table 1. Comparison of dissociation constants and total numbers of' binding sites in seven different experiments with isolated hamster brown

The parameters have been evaluated from Scatchard plot analysis. The bottom row is the means + standard error, calculated from the seven results of K d and B,,,

fat cells

Preparation x Cell Kd (i- SE) density

Bmax 95 confidence interval (binding sites/per cell) of Em,,

cells/ml nM

328 40 1 297 375 252 565 406

1.08 0.16 61 000 0.97 k 0.16 52 000 1.28 k 0.17 59000 1.94 k 0.41 74000 2.62 k 0.56 57000 1.35 & 0.41 55000 0.62 + 0.09 41 000

52000- 81000 39 000 - 109 000 51000- 80000 58 000 - 127 000 41000-103000 33000- 129000 33000- 58000

._ ~ ~ ~- _. - ~

Mean k SE 1.41 + 0.25 57000 k 3800

206 (- )-[3H]Dihydroalprenolol Binding Sites on Isolated Hamster Brown-Fat Cells

25 I

A

0 0 1 2 3 4 5 6 7 8 9 10

Time (mini

Fig. 2. Time course of specific binding of (-)-[3H]dih~droalprenolol to isolated brown adipocytes. (A) Binding of (-)-[3H]dihydroalpre- nolol as a function of time. Specifically bound (-)-[3H]dihydro- alprenolol was determined as described in Materials and Methods. (B) Pseudo-first-order rate plot of specific binding of (-)-[3H]- dihydroalprenolol. Data were taken from the results presented in Fig. I A. RL,, = concentration of specifically bound (-)-[3H]-di- hydroalprenolol at equilibrium. RL = concentration of bound (-)-[3H]dihydroalprenolol a t the stated times

adding an excess of unlabelled alprenolol to an equili- brated mixture of (-)-[3H]dihydroalprenolol and brown adipocytes. Dissociation at 37 "C was rapid, following first-order kinetics with an initial rate constant, k2 [Eqn (I)], of 0.29 min-' (Fig. 3B). Within 5 min after addition of excess unlabelled alprenolol, more than 80 % of the specifically bound (-)-[3H]dihydroalpre- nolol was dissociated at 37 "C. At 4 "C, dissociation was much slower (not shown), requiring over 10 min for 10% of the bound (-)-[3H]dihydroalprenolol to dissociate. The low rate of dissociation at low tem- peratures permitted the assay of specific binding by the method used here.

When data from five cell preparations were used to determine kobsr a value 0.25 k 0.04 min-' was ob- tained ( r = 0.7). Data from four cell preparations were used to calculate k2 and a value 0.12 & 0.04 min-' was found ( r = - 0.6). From these values the second- order rate constant kl [Eqn (l)] was then computed as

= 2.6 ( f 1.6) x 10' min-' . M- ' . (2) kobs- k2 [LI

kl =

I 04

B

10 Time (min)

b

0- 0 1 2 3 4 5 6 7 8 9 1 0

Time (min)

Fig. 3. Dissociation of (-)-[3H]dihydroalpreno101 from brown fa t cells. (A) Dissociation of (-)-[3H]dihydroalprenolol as a function of time. The experiment was performed as described in Materials and Methods. Maximum binding refers to the amount of (-)-[3H]- dihydroalprenolol bound at equilibrium, just prior to the addition of alprenolol at time 0. The data derive from duplicate incubations of one cell suspension. (B) First-order plot of dissociation of (-)-[3H]dihydroalprenolol from brown adipocytes. Data were taken from the results in Fig.3A and calculated similarly to those in Fig.2B. The slope of the line is an estimate of the dissociation rate constant kZ at 37 "C

The values of kl and k2 were used for calculation of the dissociation constant

(3) k2 ki

K d = - = 5 f 4 n M .

Displacement of [ H]Dihydroalprenolol by 8-Adrenergic Antagonists and Agonists. Specijicity of the Binding Reaction

The influence of various competitors on the specific binding of (-)-[3H]dihydroalprenolo1 in isolated brown adipocytes is presented in Fig. 4. To estimate the relative potency of these competitors in displacement of [3H]dihydroalprenolo1 from the specific binding sites, the relative binding affinity was calculated from Fig. 4 as the ratio between the concentration of un- labelled alprenolol yielding 50 % displacement (EC&), and that of a competitor yielding the same displace- ment (Table 2). Beta-adrenergic agonists competed for [3H]dihydroalprenolo1 binding sites with affinities

P. Svoboda, J . Svartengren, M. Snochowski, J. HouStEk, and B. Cannon 207

log [Cornpetitor]/M

Fig. 4. Inhibition of (-)-(3H]dihydroalprenolol binding to isolated brown adwocytes by adrenergic antagonists and agonists. Isolated brown fat cells were incubated with 5 nM (-)-[3H]dihydroalprenolol in the presence or absence of the indicated concentrations of various competitors, as described in Materials and Methods. 100 % inhibi- tion was equivalent to that observed with 500 nM alprenolol. The results presented are the average of triplicate determinations with a given cell suspension. The displacement studies for various com- petitors were evaluated from five different cell preparations. (0) (-)-Alprenolo1 ; (m) (+)-propranolol; (0) (-)-isoproterenol (isopropyl- norepinephrine); (A) (-)-norepinephrine; (0) (-)-epinephrine; (A) (+)-norepinephrine

Table 2. Competition ,for [3H]dihydroalprenoioi binding sites by different antagonists and agonists EC;o is the concentration of competitor yielding 50 % displacement of (-)-[3H]dihydroalprenolo1. Kd is calculated from Eqn (4), i.e. IG = EC;o/l + (5/1.41) nM

Competitor Relative EC;o Kd binding affinity

nM ________ ~-

(-)-Alprenolo1 1.000 10 2.2 (+)-Propranolol 0.333 30 6.6 (-)- Isoproterenol 0.083 120 26 (-)-Norepinephrine 0.013 800 176

(+)-Norepinephrine 0.0007 14500 3190 (-)-Epinephrine 0.010 1000 220

significantly lower than the antagonists. The order of potency was (-)-isoproterenol > (-)-norepinephrine 2 (-)-epinephrine. This order of potency, charac- teristic for the P1-subtype of adrenergic receptors [12] is identical with the potency series of adrenergic agonists previously reported for stimulation of respi- ration in brown adipocytes [5] . The relative binding affinity of the (+)-steroisomer of norepinephrine was less than 0.06 of the (-)-stereoisomer, indicating the stereospecificity of the binding reaction.

The EC& values from above have been used for calculation of apparent equilibrium dissociation con- stants (KA) (Table 2) for the interaction of antagonists and agonists with the binding sites, according [13] to the equation

(4)

where Kd is the independently determined dissociation constant for (-)-[3H]dihydroalprenolol from the Scat- chard plot analysis (& = 1.41 nM, see Table 1).

The calculated values of KA for agonists have been compared in Table 3 with the physiologically active concentrations of adrenergic agonists for stimulation of respiration in intact brown adipocytes. In order to determine these values under conditions similar to those used for calculation of KA, a series of oxygen- consumption measurements were performed in the presence of 5 nM alprenolol. From the dose-response curves (Fig. 5 ) of these experiments, the values for agonist concentration giving 50 stimulation of respi- ration (ECib) were obtained. Using Eqn (4), apparent dissociation constants (KA’) were then computed. In Table 3, these values have been compared with those obtained from Pettersson and Vallin [ 5 ] , who mea- sured respiratory stimulation in the absence of added antagonists. Good agreement is observed between the two sets of values. Thus, calculation of apparent dissociation constants from ECso values, using Eqn (4), seems to be valid.

Table 3. Potencies of p-adrenergic agonists for competition with [3H]dihydroalprenolol binding sites and for stimulation of respiration in intact brown adipocytes KA (agonist) = dissociation constant determined from competition studies (Fig. 4 and Table 2). EC& = concentration of agonist needed for 50 %stimulation of respiration in isolated cells incubated with 5 nM alprenolol (Fig. 5). KY = dissociation constant determined from EC&, i.e. Ki’ = EC&/1 + (5/1.41) nM. EC& is derived from the results of Pettersson and Vallin [5]

Agonist & (agonist) EC& (Fig. 5) KY ECTo [51 Occupancy for 50 % stimulation of respiration [14]

nM

(-)-Isoproterenol (-)-Norepinephrine (-)-Epinephrine

26 11 2.4 2 8.5 176 77 16.9 6 8.8 220 100 22.0 9 9.0

208 (--)-[3H]DihydroalprenoloI Binding Sites on Isolated Hamster Brown-Fat Cells

100

al + E 50

-10 -9 -8 -7 -6 -5 log [Agonist] / M

Fig. 5. Respiratory rare us CI function of ugonist concentrution in isolatc.d brown adipocytes. 5 nM (-)-[3H]dihydroalprenolol was incubated with brown fat cells at 37 C in Krebs/Ringer phosphate buffer in the presence of increasing concentrations of (-)-iso- proterenol (A), (-)-norepinephrine (0) or (-)-epinephrine (0). 2.5 x lo5 cells/nil were used for determination of respiratory rate (see Materials and Methods)

Further, it is evident from Table 3 that the con- centration of agonist giving 50 % stimulation of respi- ration (K&' and ECfo) is much smaller than the concen- tration needed for 50 occupancy of available binding sites (KA). According to the relationship described by De Meyts [14]

- ( 5 )

Kd ' Y = - KA + KA'

it is possible to calculate the percentage (j) of recep- tors needed to elicit a half-maximal stimulation of respiration. These values are also given in Table 3. As seen, less than 10% occupancy is required.

DISCUSSION

Numerous attempts to identify directly /-adrener- gic receptors using radioactively labelled /-adrenergic antagonists as receptor ligands have been published recently [9,10,15 - 221. However, these studies were mostly confined to avian and amphibian erythrocytes and to isolated mammalian plasma membranes [lo, 15,17,19,20]. Relatively few studies have been devoted to the investigation of intact mammalian cells [22,23].

In this study, (-)-[3H]dihydroalprenolol was used for characterisation of the adrenergic receptors in isolated intact brown fat cells. These cells represent an experimentally advantageous system, since a direct evaluation of hormone-receptor interaction by binding studies may be performed simultaneously with mea-

surements of the final biological response (= respira- tion) to the adrenergic stimulus.

Using two independent methods, we have deter- mined the apparent dissociation constant for (-)-[3H]- dihydroalprenolol binding to brown adipocytes and found it to be in the range 1 - 5 nM. This value agrees well with the apparent dissociation constant for alprenolol (2.2 nM), calculated from our competition studies (dihydroalprenolol and alprenolol have earlier been reported to react identically [9]).

Further, as seen in Table 3, the use of this low Kd value for calculation of the expected KA' for respiratory stimulation by catecholamines gives results that agree very well with those obtained experimentally (EC&). This would not have been the case if a much higher Kd for alprenolol binding had been obtained.

Thus, a receptor with a Kd for alprenolol of about 1.4 nM is probably responsible at least for the physio- logical response. Such a low Kd is also in good agree- ment with dissociation constants reported for various cell types: 1.2 nM for frog erythrocyte membranes [24] and 5 nM for frog erythrocytes 191, 2.8 nM for turkey erythrocyte membranes [25] and 8 nM for intact turkey erythrocytes 1171, 15 - 19 nM for white fat membranes [lo, 181 and 19 nM for rat pineal gland

However, in a recent article, a rather high Kd (95 nM) for alprenolol has been determined for the /3-receptor in brown fat homogenates [20]. This value was determined from titrations with alprenolol up to concentrations of 240 nM. We found (not shown) that at high concentrations of dihydroalprenolol only a non-specific (non-saturable, low-affinity) type of bind- ing was measured. In such experiments the high- affinity binding site described here was difficult to observe as it had already been saturated at low con- centrations of (-)-[3H]dihydroalprenolol. We find it nonetheless feasible that the binding of agonists to a binding site with a Kd for alprenolol below 10 nM is responsible for the physiological response (respira- tory stimulation) and that this' alprenolol-receptor represents the physiologically active /-receptor.

The values obtained here for the number of specific binding sites per cell (57000) are considerably higher than most previously reported for other kinds of cells (500-4000) [9,15,17,21]. It should be mentioned in this respect that an artificially high maximum number of binding sites will be measured when high-capacity, non-specific binding is not clearly differentiated from low-capacity, specific binding. This problem is evident from the experimental definition of specific binding : it is defined as the difference between binding of the radioactive ligand in the absence (total binding) and presence (non-specific) of an excess of unlabelled analogue. As was discussed by Chamness and McGuire [7], very high concentrations of unlabelled analogue

[261.

P. Svoboda, J . Svartengren, M. Snochowski, J. HouStEtk, and B. Cannon 209

may compete with both high-affinity and low-affinity binding sites and artificially high values for specific binding may result. In the case of brown fat cells, 100-fold excess of unlabelled alprenolol was found to be optimal for detection of all criteria for identification of specific binding to the receptor: affinity, saturability, reversibility, specificity and stereospecificity. Simi- larly, Malchoff and Marinetti [17] used 100-fold excess of unlabelled ligand for the measurement of non- specific binding of [3H]dihydroalprenolo1 on turkey erythrocytes. Therefore, the range of 41 000- 74000 receptors/cell obtained here seems to reflect the true capacity of the brown adipocytes for specific binding of [3H]dihydroalprenolo1. Usage of 1000-fold excess of alprenolol or propranolol(l0 yM range of concen- trations), which were frequently used in other systems [lo, 13,lX-211 resulted in the case of brown fat cells in B,,, values as high as lo6 receptors/cell, and in a very high Kd. Saturability and other criteria of specific binding could not be demonstrated under these con- ditions.

The specificity of the adrenoreceptors was of the PI-subtype. The relative potency of adrenergic agonists in competition with [3H]dihydroalprenolo1 binding sites and in stimulation of respiration in intact brown adipocytes was the same. This indicates that binding of [3H]dihydroalprenolol occurs to the physiological P-adrenergic receptor. In a recent study on brown fat homogenates [20], a concentration of 13 nM dihydro- alprenolol was used for competition studies for the reported dihydroalprenolol binding site with a Kd of 95 nM. From these values and the E G O values for the competitors obtained experimentally, the authors cal- culated very high apparent K i values for the respec- tive competing agents. If one speculates that at 13 nM the high-affinity site which we report here has been saturated and therefore use the Kd value we have ob- tained for this site, the KA values then calculated for the competing agents are in excellent agreement with the ones we report here. It is perhaps therefore pos- sible that at 13 nM the authors are studying the high-affinity site in our report, and not the site with a Kd of 0.95 nM. Further work should clarify this point.

As seen from Table 3, for half-maximum respira- tion to occur, a receptor occupancy of less than 10 %, is required. This may be interpreted to mean that brown fat cells have ‘spare receptors’. In the absence of spare receptors, the ECso for norepinephrine stimu- lation of respiration would be the same as the Kd for the receptor itself: 176 nM; the existence of spare receptors leads to an apparent Kd (ECSO) of about 6 nM. Further, if this high sensitivity had been ob- tained, not by having spare receptors, but by having receptors endowed with a low Kd themselves, the sensitivity to changes in norepinephrine concentration would have been lower.

Conclusion

The brown fat cell appears to possess specific [3H]dihydroalprenolo1 binding sites which, uniquely among other adrenergic systems, combine extremely high total capacity with high affinity. It may reflect the generally accepted dominating role of catechol- amines in regulation of brown fat metabolism and non-shivering thermogenesis. This finding, together with the marked respiratory response of isolated cells to catecholamine stimulus, support the view that isolated brown fat cells represent a good experimental system for the simultaneous evaluation of hormone- receptor interaction and the corresponding biological response.

This work was supported in part by a grant from the Swedish Natural Science Research Council (B 41 74-loo), and has been carried out within the framework for collaboration between the Royal Swedish Academy of Sciences and the Czechoslovak Academy of Sciences. The authors are grateful to Professor Olov Lindberg and Dr Jan Nedergaard for helpful discussions.

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P. Svoboda and J . HouSttk, Fysiologicky Ustav, Ceskoslovenska Akademia Vtd, BudEjovicka 1083, CS-142-20 Praha, Czechoslovakia

J. Svartengren and B. Cannon*, Wenner-Grens Institut, Stockholms Universitet, Norrtullsgatan 16, S-I 13 45 Stockholm, Sweden

M. Snochowski, Kemiska Institutionen, Karolinska Institutet, Fack, S-104 01 Stockholm, Sweden

* To whom correspondence should be addressed.