β-adrenoceptor studies. 6. Further investigations on the hybrid nature of the rat adipocyte β-adrenoceptor

European Journal of Pharmacology, 63 (1980) 73--83 © Elsevier/North-Holland Biomedical Press

73

~-ADRENOCEPTOR STUDIES. 6. FURTHER INVESTIGATIONS ON THE HYBRID NATURE OF THE RAT ADIPOCYTE ~-ADRENOCEPTOR JAN DE VENTE, AALT BAST, LEENDERT VAN BREE and JOHAN ZAAGSMA

Department of Medicinal Chemistry, Section of Molecular Pharmacology, Free University, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands Received 21 June 1979, revised MS received 23 November 1979, accepted 3 January 1980

J. DE VENTE, A. BAST, L. VAN BREE and J. ZAAGSMA, ~-Adrenoceptor studies. Further investigations on the hybrid nature of the rat adipocyte ~-adrenoceptor, European J. Pharmacol. 63 (1980) 73--83. The nature of the rat adipocyte ~-adrenoceptor was studied in further detail using a selected series of tolamoltype ~-adrenoceptor antagonists. Isoprenaline antagonism by these compounds was evaluated on adipocytes, right atrium, left atrium and left hemidiaphragm of the rat. Adipocyte pA2 values were corrected for binding to bovine serum albumin determined separately for each antagonist. A strong correlation between adipocyte and diaphragm pA2 values was found with those antagonists which had an identical N-substituent but a different substitution pattern in the phenoxypropanolamine ring. This relationship was absent with those compounds which had the same 2-methylphenoxypropanolamine moiety but a different N-substituent. With the latter antagonists, however, adipocyte pA2 values correlated significantly with the two cardiac pA2 values. These results support the idea that the interaction site of the rat adipocyte ~-adrenoeeptor for the aromatic moiety of arylethanolamines and aryloxypropanolamines has ~2-characteristics whereas the alkanolamine side-chain interaction site has a ~x-nature. Finally, it was ascertained that the apparently dualistic (~1 and ~2) character of the rat adipocyte adrenoceptor is not due to the presence of both a ~1- and a ~2-receptor population on the fat cell plasma membrane. ~-Adrenoceptor characterization ~-Adrenoceptor antagonists

Adipocyte

1. Introduction On the basis of the relative potencies of a series of catecholamines, Lands et al. (1967) proposed a differentiation of ~-adrenoceptors into two subtypes, ~1 and ~2. Their correlation analysis indicated that the adipocyte receptor is very similar to the cardiac receptor (~1) but quite different from bronchial and vascular adrenoceptors (~2). In recent years, however, data have become available which indicate that there are differences between lipolytic and cardiac ~-adrenoceptors. Thus, a number of cardioselective antagonists block adrenergically induced lipolysis less than expected on the basis of their affinities towards cardiac ~l-receptors (Stanton, 1972; Goldberg et al., 1975; Harms

Right and left atrium

Diaphragm

and van der Meer, 1975; Kather and Simon, 1977). Moreover, the potency ratios of the stereoisomers of both the agonist noradrenaline and some fl-adrenoceptor antagonists were found to be significantly higher on cardiac preparations that on adipose tissue (Patil et al., 1971; De Santis et al., 1974; Harms et al., 1977) which also suggests a difference between the cardiac fl-adrenoceptor and the fat cell adrenoceptor. Using a series of aromatic bis(2-hydroxy-3isopropylaminopropyl) ethers with various substituents in the aromatic ring, we reported that. the potencies to antagonize the effects of isoprenaline on rat adipocyte fl-adrenoceptors were correlated with the potencies on guinea pig treacheal receptors but not on guinea pig right atrial receptors (Harms et al., 1974).

74

J. DE VENTE ET AL.

OH /~_~O-CH2-CH-CH2-NH-CH2-CH2-O~ Subseries A RI= 2-CH 3, 3-CH 3, 4-CH 3 2-OCH 3, 3-OCH 3, 4-OCH 3 2-CI, 3-CI 2-Cell s

R~ 4-CONH 2

Subseries B RI= 2-CH 3

R~ 2-CONH s, 3-CONH 2, 4-CONH 2 4-SO2NH 2 4-CONHCH 3 4-CONHNH s

Fig. 1. Chemical structures of the investigated analogues of tolamolol.

Since the agonists used by Lands et al. (1967) only differed as to the structure of the ethanolamine side-chain, it was tentatively suggested that the part of the fat cell fl-adrenoceptor which interacts with the aromatic moiety of agonists and antagonists closely resembles the corresponding part of the tracheal (f12) receptor while the part of the adipocyte receptor that interacts with the ethanolamine side-chain resembles the corresponding part of the cardiac fl-adrenoceptor. In order to investigate this working hypothesis in further detail, we looked for a series of compounds in which (1) the aromatic substitution pattern varied while the alkanolamine side-chain did not change and (2) the structure of the N-substituent changed but not that of the aromatic part of the molecule. Additionally, it was thought to be advantageous to have compounds with selectivity towards cardiac (/3~) and bronchial (f12) adrenoceptors present in both subseries. In 1973, Augstein et al. reported on a series of analogues of:tolamolol which seemed to serve this goal very well. The structure of the selected antagonists made available to us are given in fig. 1. We also took into account the possibil-

ity of different populations of adrenoceptors, both/3, and f12, being present on the adipocyte plasma membrane as an explanation of the observed dualistic receptor characteristics. In order to exclude possible interference by species differences we determined all in vitro fl-adrenoceptor blocking properties on rat organs: as the ill-reference systems, isolated left and right atria were used, the affinity for /32-adrenoceptors was estimated using the isolated left hemidiaphragm preparation whereas the antagonism of isoprenalineinduced lipolysis was determined using isolated adipocytes prepared from epididymal adipose tissue. The reference fll and f12 tissues were selected on the basis of evidence indicating that a homogenous or virtually homogenous adrenoceptor population was involved in the responses. Thus, whereas in the isolated cat heart a preferential blockade b y fl~-selective antagonists of the positive chronotropism induced by fl~-selective agonists as compared with nonselective or fl2-selective agonists (Carlsson et al., 1972; Carlsson, 1979) points to a heterogenous ~1- and fl2-receptor population, no such differentiated blockade was

RAT ADIPOCYTE ~-ADRENOCEPTOR CHARACTERIZATION

observed with rat atria (Johansson, 1973; Kramer and Zaagsma, unpublished observations). This was confirmed recently with radioligand binding studies of rat heart homogenates where two laboratories both detected one single class (~1) of adrenoceptors (Barnett et al., 1978; Hancock et al., 1979) although a third laboratory reported the presence of a minor amount (17%) of ~2adrenoceptors in combination with 83% of ~l-adrenoceptors (Minneman et al., 1979). Similarly, H~stmark and Horn (1975) found that, in rat diaphragm, there was no preferential antagonism by practolol of the noradrenaline response nor by butoxamine of the terbutaline-induced response. Protein binding is a variable which may interfere with the determination of the affinities of both agonists and antagonists towards adipocyte fl-adrenoceptors since bovine serum albumin has to be present in the medium as a free fatty acid acceptor. To the best of our knowledge, this variable has not been introduced in previous classification studies. Among the ~-adrenoceptor antagonists, pronounced differences in binding to bovine serum albumin have been reported (see, e.g. Appelgren et al., 1974; Zaagsma et al., 1977). We therefore decided to measure this parameter with all antagonists used in order to transform the observed pA2 values into 'true' pA2 values based on the free drug concentrations.

75

was 30 min with the cardiac preparations and 45--60 min with the skeletal muscle preparation. Epididymal adipose tissue was chopped and a fat cell suspension was prepared using collagenase digestion according to Rodbell (1964).

2.2. Right atrial fl-adrenoceptors Antagonism of the positive chronotropism caused by cumulative doses of isoprenaline was measured on spontaneously beating right atria suspended in a Krebs buffer of the following composition (mM): NaC1 117.5; KC1 5.6; MgSO4 1.18; CaC12 2.5; NaH2PO4 1.28; NaHCO3 25.0; glucose 5.5; gassed with 5% CO: in 02; pH 7.4. Contractions were recorded using an isometric Endevco Pixie transducer (8101). Resting tension was adjusted at 0.5 g and checked before each dose-response curve.

2.3. Left atrial {J-adrenoceptors Antagonism of isoprenaline-induced positive inotropism was determined using left atria suspended in the Krebs solution mentioned above. The organs were stimulated by punctate platinum electrodes with a distance of 2 mm, using square-wave pulses of 0.5 msec duration, a stimulus strength of 1.5 times the threshold voltage and a frequency of 6 Hz (Grass stimulator $88). Contractions were recorded as described above. Both the resting tension and the stimulus strength were checked before each dose-response curve.

2: Materials and methods

2.4. Skeletal muscle fJ-adrenoceptors

2.1. General

The left hemidiaphragm was mounted in a muscle holder equipped with thin platinum electrodes (distance 2 mm), constructed in such a way that:the mechanical contact with the phrenic nerve could be adjusted individually. The organ~bath contained a Ringer solution (Close, 1964) of the following composition (mM): NaC1 137; KC1 5; CaC12 2; MgC12 1; 'NaH:PO4 1; NaHCO3 11,9; glucose 11; aerated with 5% CO2 in 02; pH 7.1. The phrenic nerve was stimulated with square wave pulses of 2 msec duration, 0.1 Hz fre-

Male Wistar rats (140--180 g) fed ad libiturn, were used in all experiments. After the animals were killed, the organs were,rapidly excised and prepared. Right and left atria were sel~arated from the ventricles and mounted i n different, water-jacketed (37 ° C) organ baths i (20 ml). From the diaphragm, the left~ hemidiap~ragra with its phrenic nerve was prepared and mounted in a 50 ml organ bath also maintaine d at 37°C. Equilibration time

76

KCI 13,4 mM

J. DE VENTE ET AL.

isoprenaline 10-9M 3x10"9M 10"8M

3x10"8M 10"7M 3x10-7M

Fig. 2. Registogram showing the dose-dependent increase by isoprenaline of the twitch contractions of the partially depolarized rat left hemidiaphragm preparation.

quency and a voltage slightly above the value necessary to induce maximal twitch contractions. The contractions were registered isotonically with a preload of 0.5 g using a Hugo Sachs Hebel-Messvorsatz with and H F model preamplifier. After the equilibration period, 100 to 130 pl of a 25% KC1 solution was added gradually to cause partial depolarization and, after an initial transient increase, a reduction of the contraction amplitude to 10--30% of the original value. Cumulative dosages of isoprenaline induced a stepwise partial recovery of the contraction height (fig. 2).

2.5. Adipocyte {J-adrenoceptors Isolated adipocytes were suspended in a Krebs solution of the following composition (mM): NaC1 119.0; KC1 4.7; CaC12 2.5; MgSO4 1.2; KH2PO4 1.3; NaHC03 25.0; bovine serum albumin 0.14 (1%); pH 7.4. The cell suspension was incubated for 1 h in Teflon vessels with several concentrations of isoprenaline in the absence or the presence of the antagonist, using a D u b n o f f Incu-shaker at 37°C under an atmosphere of 5% CO2 in 02. Total F F A production was determined according to Ko and R o y e r (1967). All incubations within the same experiment were performed in duplicate or triplicate.

2.6. Protein binding Since the structure-activity relationships in protein binding of the tolamolol analogues will be the subject of a separate paper, the experimental protocol is only briefly outlined here. Binding to 1% bovine serum albumin in Krebs solution was studied by the equilibrium dialysis technique using.Teflon compartments separated b y cellulose acetate membranes (Technicon nr. 105--10). The composition of the Krebs solution was the same as used in the lipolysis experiments except that NaHCO3 was absent; instead the pH wad adjusted to 7.4 using NaOH. Dialysis was performed for 16--18 h at room temperature. Samples were taken from the albumin-free compartment, adjusted to pH 11.0 and extracted with 30% (v/v) cyclohexane in n-butanol. After centrifugation, a sample of the organic layer was taken and evaporated at 60°C. The residue was taken up in 70% methanol adjusted to pH 3 with HC1. Antagonist concentrations were determined using high pressure liquid chromatography (Waters Associates model ALC/GPC 204) with a /~-Bondapak C-18 column and 70% methanol at pH 3 as the eluant. The binding percentages determined for three different concentrations of each antagonist were plotted against the total concentra-

RAT ADIPOCYTE ~-ADRENOCEPTOR CHARACTERIZATION tions; from the regression line obtained b y the m e t h o d of least squares, the percent binding of the antagonist concentration used in the individual lipolysis experiments was derived. For the construction of the calibration lines, a set of antagonist concentrations were dialyzed simultaneously in the absence of albumin, thus precluding the possible influence of drug adsorption to cellulose acetate or to the Teflon surface. In separate experiments using tolamolol this adsorption was found to be negligible.

2.8. Drugs and chemicals

2. 7. Dose-response curves

3. Results

All isoprenaline concentrations were stabilized using 50 #g/ml of Na2S2Os. Stock solutions of the antagonists were prepared in dimethylsulfoxide (DMSO) and further diluted with water. The final concentrations of DMSO present during the experiments were found to have no influence at all on the position of the isoprenaline dose-response curves. Albumin with the same batch-number was used in both protein binding and in lipolysis experiments, since some differences in protein binding were found with different batches from the same supplier. With the atrial preparations t w o doseresponse curves with isoprenaline were performed first, the second one serving as the control curve. In case of diaphragm, only one curve was constructed before the antagonist was introduced. Preincubation times of the antagonists were 10 min in all cases; in separate experiments using tolamolol it was found that prolongation of the incubation period to 30 rain yielded identical results. The pA2 values of the antagonists were evaluated from the parallel shifts to the right according to Van Rossum (1963). All individual pA2 values obtained an adipocytes were corrected for protein binding. T o do this the free drug concentration belonging to the total concentration of antagonist added to the fat cell suspension was calculated from the binding data as indicated above and the pA2 value was reevaluated.

3.1.

77

All tolamolol analogues were kindly donated b y Pfizer Central Research (Sandwich, England); dl-isoprenaline sulphate was purchased from ACF (Amsterdam, The Netherlands); coUagenase, t y p e CLS, from Worthington (Freehold, N.J.); demineralized bovine serum albumin from Organon Teknika (Oss, The Netherlands). All other chemicals were of reagent grade.

All c o m p o u n d s appeared to be competitive antagonists of isoprenaline, both on cardiac and skeletal muscle and on adipocyte fi-adrenoceptors. The pA2 values obtained with the isolated organs and the adipocytes are listed in table 1. The pD2 values of isoprenaiine estimated from the control curves were 8.63 + 0 . 0 3 on r. atrium, 8.11 + 0,06 on 1. atrium, 8.27 + 0.03 on diaphragm and 7.58 + 0.05 on adipocytes, all of which are quite similar to the values reported previously (Harms et al., 1977). Comparison of cardiac and skeletal muscle pA2 values (table 1) reveals that in subseries A (compounds carrying various substituents (R1) in different positions of the phenoxypropanolamine ring and a 4-CONH: group in the second aromatic moiety) the lowest activity is found when the R, substituent is in the 4-position. Moving R1 to the 3-position invariably increased the affinity. With r. atrial fi-adrenoceptors the affinity was further increased when the same substituents were located in the 2-position; with I. atria and diaphragm no important differences were found between 2- and 3-substitution, however. With subseries B compounds, all having R1 = 2-CH3, b u t a different substitution (R2) in the second aromatic nucleus, moving the CONH2-group from the 4-position to the 3and 2-position decreased the p o t e n c y on

78

J. DE VENTE ET AL.

TABLE 1 pA2 values -+ S.E.M. for isoprenaline antagonism on right atrium, left atrium, left hemidiaphragm and adipocytes (both uncorrected and corrected for protein binding) of the rat. The nubmer of experiments is given in parentheses.

~_~-o- cH~-c~-c.~-.H-C~.-C.~O-~_.~ Compound No.

Substituents

R. atrium pA2

L. atrium pA2

Diaphragm pA2

Adipocytes-1 pA2

Adipocytes-2 pA2 (corrected)

8.28 -+ 0.09 (12) 8.25 -+ 0.08

7.02 -+ 0.05 (8) 7.10 _+ 0.08

6.75 + 0.05 (6) 6.76 + 0.06

6.87 -+ 0.05 (6) 6.84 + 0.05

R1

R2

1

3-Cl

4-CONH2

2

2-C1

4-CONH2

8.19 + 0.06 (12) 8.64 -+ 0.06

(10)

(10)

3

2-C6Hs

4-CONH2

7.67 -+ 0.09

7.67 + 0.04

8.16 _+ 0.10

7.02 _+0.10

7.51 -+ 0.09

4

4-OCH3 4-CONH2

(ii) 7.24 -+0.05

(14) 7.26+ 0.08

(I0) 5.62+ 0.08

(6) 4.97+ 0.04

(6) 5.07 -+0.04

5

3-OCH3

(7) 8.00 + 0.06

(10) 8.05 -+ 0.09

(8) 6.96 -+ 0.10

(6) 6.41 -+ 0.04

(6) 6.48 -+ 0.04

6

2-OCH3 4-CONH2

(9) 8.41 + 0.05 (8)

(12) 8.21-+0.04 (8)

(10) 6.89-+0.09 (9)

(6) 6.34+ 0.10 (6)

(6) 6.40 -+0.10 (6)

7

4-CH3

7.52 + 0.05

7.67 _+ 0.07

6.40 _+ 0.07

6.43 -+ 0.09

6.50 -+ 0.09

(6)

4-CONH2

4-CONH2

(9) 8

3-CHa

4-CONH2

8.17 -+ 0.05

9

2-CH3

4-CONH2 :

10

2-CH3

3-CONH2

11

2-CH3

2-CONH2

8.42 + 0.04 (16) 7.63 -+ 0.10 (7) 7.87 -+ 0.07

12

2-CH3

4-SO2NH2

13

2-CH3

4-CONHCH3

14

2-CH 3

4-CONHNH 2

(6)

(9) 7.76 -+ 0.05 (8) 8.50 -+ 0.04

(9) 7.92 -+ 0.06 (8)

(7)

(14)

8.10 + 0.07

(6)

(7)

8.20 -+ 0.05 (11) 7.39 -+ 0.06 (6) 7.70 -+ 0.06

(9)

7.01 -+ 0.07 (14) 7.55 -+ 0.08 (7) 7.74 -+ 0.13

(7)

7.41 -+ 0.05 (8) 8.34 -+ 0.07

(8)

7.31 -+ 0.10 (8) 7.48 -+ 0.09

(6)

7.82 -+ 0.08 (8)

cardiac fi-adrenoceptors but increased the pA2 values obtained on skeletal muscle; this resulted in a disappearance of ill-selectivity as present in tolamolol (compound 9). Inspection of the complete series reveals that all compounds with the R2-substituent in the 4-position possess fl,-selectivity with the exception of compound 3. Apparently the bulky phenyl group located in the ortho position of the phenoxypropanolamine ring is not well tolerated by cardiac ~-adrenoceptors

6.89 -+ 0.10

7.06 -+ 0.09 (7)

(5)

(7) 6.59 -+ 0.08

(6) 6.86 -+ 0.07 (13) 6.56 -+0.05 (6) 6.77 + 0.08

(6) 6.54 -+ 0.08 (7) 7.26 -+ 0.11

(6) 6.74 -+ 0.12 (7)

(5)

(7) 6.71 -+ 0.08

(6) 6.99 -+ 0.07 (13) 6.83 -+ 0.07 (6) 7.16 -+ 0.08

(6) 6.61 + 0.08 (7) 7.40 -+ 0.11

(6) 7.04 -+ 0.12 • (7)

whereas on diaphragm receptors a productive contribution in binding follows which increases affinity. Comparison of the above data with those of Augstein et al. (1973), who reported on the antagonism of isoprenaline-stimulated adenylate cyclase in rat heart and lung homogenates, showed the same activity pattern within subseries A when a particular R, substituent changed position. However, the differences between the affinities towards fi,-

RAT ADIPOCYTE

~-ADRENOCEPTOR

CHARACTERIZATION

and ~2-adrenoceptors f o u n d by these workers were mu ch lower and f our o f the c o m p o u n d s (5, 10, 11 and 12) had some ~2-selectivity whereas we f o u n d t h e m to be cardioselective or non-selective. Besides differences in the target systems, these discrepancies might also be c o n n e c t e d with the fact t h a t in the screening program o f the huge n u m b e r o f c o m p o u n d s studied by Augstein et al. (1973), m a n y substances were tested only once. E x c e p t f or tolamolol, this had been t he case with all c o m p o u n d s selected by us f or the present study. T h e pA2 values de t e r m i ne d on a d i p o c y t e ~-adrenoceptors, b o t h u n c o r r e c t e d and corrected f o r binding to 1% bovine serum are shown in th e last tw o columns o f table 1.

3.2. Correlation analysis Using the pA2 values listed in table 1, correlation analyses were p e r f o r m e d in order t o

79

characterize the a d i p o c y t e ~-adrenoceptor. Table 2 summarizes the results obtained with the com pl et e set o f c o m p o u n d s . As expected, a clear-cut correlation was f o u n d between isoprenaline-antagonism on right and left atrial ~-adrenoceptor~, and no correlation of the pA2 values measured on t he t w o cardiac preparations with the skeletal muscle values. A weak correlation was also f o u n d bet w een the t w o cardiac parameters and the antagonistic potencies on a d i p o c y t e ~-adrenoceptors which became even worse after i n t r o d u c t i o n o f t he corrected adi pocyt e values in the correlation analysis. In contrast, there was a significant correlation between a d i p o c y t e and diaphragm ~-adrenoceptor affinities which improved slightly on using ' t r u e ' adi pocyt e values. It appears t hat the latter correlation is actually due to the c o m p o u n d s which had the same nitrogen-substituent but variable substituents (R1) in t he p h e n o x y p r o p a n o l a m i n e ring: as shown in table 3, in subseries A the affinities towards fat cell receptors, n o t a b l y

TABLE 2 Correlation analysis of the PA2 values of the complete set of tolamolol-type~-adrenoceptorantagonists. Adipocytes-l: uncorrected PA2 values, based on total drug concentrations. Adipocytes-2: Corrected pA2 values based on free drug concentrations, n, number of compounds; r, correlation coefficient; s, level of significance of the correlation. Computations were performed by treating the two parameters involved as independent variables using the Pearson correlation test (Anderson, 1958). All compounds R , ~ oH

TABLE 3 Correlation analysis of the pA2 values of tolamolol derivatives differing in the nature and position of the substituent (RI) in the phenoxypropanolamine ring. Adipocytes-l: uncorrected pA2 values, based on total drug concentrations. Adipocytes-2: corrected PA2 values, based on free drug concentrations. For further details see heading of table 2. Subseries A

~ R ~

RI

OH

"~-

(%~'-- O-CHi"CIH-CH2-NH-CH2-CJ'I2-0 ~ - ~

PA2 values compared

n

r

R. atrium

I. atrium

14

0.919

R. atrium R. atrium R. atrium L. atrium L. atrium L. atrium Diaphragm Diaphragm

diaphragm adipocytes-1 adipocytes-2 diaphragm adipocytes-1 adipocytes-2 adipocytes-1 adipocytes-2

14 14 14 14 14 14 14 14

0.257 0.585 0.457 0.133 0.539 0.413 0.815 0.886

s

~0.001 0.188 0.014 0.050 0.325 0.023 0.071 ~0.001 ~0.001

O- CHiC.J-I-CHa-NH-CHiCH ~O~-f~.~CON H.

PA2 values compared

n

r

R. atrium R. atrium, :

I. atrium diaphragm

R. atrium R. atrium L. atrium L. atrium

adipocytes-1 adipocytes-2 diaphragm adipocytes-1

L. atrium

adipocytes-2

Diaphragm Diaphragm

adipocytes-1 adipocytes-2

9 9 9 9 9 9 9 9 9

0.946 0.399 0.603 0.493 0.427 0.677 0.556 0.861 0.925

s ~0.001 0.143 0.043 0.089 0.126 0.023 0.060 <~0.001 ~0.001

80

J. DE VENTE ET AL.

4. Discussion

TABLE 4 Correlation analysis of the pA2 values of tolamolol derivatives differing in the nature and position of the substituent (R2) in the N-phenoxyethyl ring. Adipocytes-l: uncorrected pA2 values, based on total drug concentrations. Adipocytes-2: corrected pA2 values, based on free drug concentrations. For further details see heading of table 2. Subseries B ~'~I

O- CHI- CH-CH a"NH- CHI" CH I" O

pA2 values compared R. atrium R. atrium R. atrium R. atrium L. atrium L. atrium L. atrium Diaphragm Diaphragm

1. atrium diaphragm adipocytes-1 adipocytes-2 diaphragm adipocytes-1 adipocytes-2 adipocytes-1 adipocytes-2

n

r

6 6 6 6 6 6 6 6 6

0.979 0.332 0.885 0.674 0.314 0.915 0.780 0.022 0.225

s <0.001 0.260 0.009 0.071 0.273 0.005 0.034 0.484 0.334

after correction for protein binding, correlate strongly with the pA2 values determined on the skeletal muscle preparation which relationship is completely absent in subseries B (table 4). Conversely, the data from the compounds in subseries B which all have an ortho-methyl group in the phenoxypropanolamine ring b u t a variable substitution pattern in the second phenyl nucleus, show that the two cardiac parameters correlate significantly with uncorrected adipocyte pA2 values. However, the significance of this relationship decreased after correction for albumin binding. As with the complete series of compounds, both in subseries A and B, the relationship between right and left atrial ~-adrenoceptor blocking potencies on the one hand and the lack of correlation between cardiac and skeletal muscle parameters on the other hand remained present.

The subdivision of fl-adrenoceptors into/3, and f12 as proposed by Lands et al. (1967) and worked o u t subsequently by the same group (see review by Arnold, 1972), was based on the relative potencies of up to 15 catecholamine agonists. Apart from the fact that both in vivo and in vitro data were included in their correlation analyses and that no precautions were taken to block extraneuronal and neuronal uptake processes (these factors have already been discussed by Furchgott, 1972) it should also be realized' that the use of catecholamines which only differ structurally in the ethanolamine side-chain, makes it impossible to detect differences in that part of the t w o receptors under study which interacts with the catechol group. Since all c o m p o u n d s have the same affinity towards this site of the two receptors, these possible receptor differences do n o t influence the quality of the correlation. Similarly, compounds with identical side-chains cannot be used to detect differences in the part of the receptors which interacts with the side-chain. These factors could be involved in the discrepancy between (1) the apparent relationship of adipocyte and cardiac ~-adrenoceptors reported b y Lands et al. (1967) with catecholamines and (2) the correlation between pA2 values on adipocyte and tracheal fl-adrenoceptors of a series of fl-adrenoceptor antagonists with different substitution patterns in the aromatic moiety, as found b y us (Harms et al., 1974). The implication of both observations would be that the adipocyte fl-adrenoceptor has a dualistic, i.e., fll and /32, character in such a way that the interaction site for the alkanolamine side-chain of agonists and antagonists corresponds with the site of the ill-receptor and that the site interacting with the aromatic moiety of fl-adrenoceptor ligands is similar to that of the/32-receptor. To test this working hypothesis, the present study involved a series of ~ a d r e n o c e p t o r antagonistic tolamolol analogues which could be divided into two subseries carrying either a

R A T ADIPOCYTE ~-ADRENOCEPTOR C H A R A C T E R I Z A T I O N

fixed nitrogen-substituent and a variable substitution pattern in the phenoxypropanolamine ring (subseries A) or a fixed 2-methylphenoxypropanolamine moiety and variation in the N-phenoxyethyl group (subseries B). If the fat cell fl-adrenoceptor indeed has the proposed dual 13, and j32 properties, the correlation analyses on subseries A would reveal this receptor to be j32 whereas j31-characteristics would be expected with subseries B. In order to make the interpretation of the analyses as reliable as possible the following precautions were taken (Furchgott, 1970, 1972): (1) by using only rat organs and adipocytes, the possible influence of species differences was excluded; (2) the apparent affinities of the antagonists towards adipocyte ~-adrenoceptors were corrected for binding to 1% of bovine serum albumin, which was present in the fat cell suspension; (3) because the performance of a dose-response curve on the diaphragm preparation takes a relatively long time, the antagonist preincubation time was set at 10 rain so as to be able to construct at least two curves in the presence of the antagonist without unnecessary ageing of the preparation. The same preincubation time was also used with the other preparations. In separate experiments using tolamolol it was found that this incubation period was sufficiently long to reach equilibrium in filling the receptor compartment since its prolongation to 30 min produced no detectable differences in isoprenaline antagonism on any of the target systems; (4) the concentration of antagonist in the bath solution did not change during the preincubation and the performance of the •dose-response curve. This was ascertained with tolamolol and compound 3 by measuring the concentrations after 0, 1, 10, 20, 60 and 90 min of incubation with the organs and the adipocytes, using the analytical procedure described above; the adipocytes were separated from the medium by centrifuging the suspension in the presence of dinonylphthalate (Gliemann et al., 1972). A significant decrease of the bath concentration could not be detected with any of the preparations: the

81

concentrations varied from 95 to 101% of the starting values. The results in tables 2, 3 and 4, substantially support the idea of a dualistic ill- and ~2-character of the adipocyte j3-adrenoceptor. In addition, they also show that even moderate differences in protein binding within a homologous series of compounds can significantly affect conclusions as to the nature of that receptor. The observation that with subseries A 'true' adipocyte pA2 values correlate very significantly with diaphragm pA2 values but badly with the two cardiac parameters, confirms that the binding site of adipocyte ~-adrenoceptors for the aromatic part of arylethanolamines and aryloxypropanolamines has ~2-characteristics. With subseries B compounds, any relationship with skeletal muscle pA2 values was completely absent, whereas the predicted relationship with cardiac (~1) pA2 values was clearly present when the apparent affinities for adipocyte j3-adrenoceptors were not corrected for protein binding; when adipocyte pA2 values were based on free drug concentrations, the quality of this relationship diminished, however. Although the latter finding seems to argue against the #3~-character of the alkanolamine side-chain binding site of the adipocyte receptor it should be realized that the compounds used possess large and bulky nitrogen substituents. The implication of this could be the involvement of additional forces with the receptor environment which may be different for cardiac and adipocyte ~-adrenoceptors. It is clear that the dualistic ~1- and fl2-c~aracter of the adipocyte receptor cannot*~be explained by the presence of two different populations of adrenoceptors, both ~ and ~2, on the fat cell membrane since (!) a non,selective agonist (isoprenaline) was used and (2) in both subseries A and subseries B the majority of antagonists are ~-selective except for compound 3 which has some ~32-selectivity and the nonselective derivatives 10 and 11. However, in view of recent evidence on the coexistence of j31- and ~2-adrenoceptor binding sites in the same tissue (Rugg et al., 1978;

82

Nahorski, 1979; Nahorski et al., 1979; Minneman et al., 1979) and on the participation of the two receptors in the same response (Carlsson et al., 1972; Ablad et al., 1974; Furchgott, 1976; Zaagsma and Oudhof, 1976; Zaagsma et al., 1979) we investigated the homogeneity of rat adipocyte adrenoceptors as well. With ( -- )-noradrenaline (~l-selective) and salbutamol (/~2-selective) as agonists, the /~l-selective antagonist practolol was found to have a pA2 of 4.46 + 0.14 (8) against ( - ) noradrenaline and 4.29 + 0.05 (7) against salbutamol, whereas values of 4.68 + 0.16 (4) and 4.74 + 0.11 (4), respectively, were found with ~2-selective H 35/25. Thus, no differentiated blockade could be detected with either of the antagonists demonstrating that the rat adipocyte ~-adrenoceptor population is homogenous. The same result was also obtained recently by Belfrage and Fredholm (1978) on rat fat cells whereas in dog fat cells they demonstrated that both ~1- and ~2-adrenoceptors were involved in lipolysis. In conclusion, the above data show that one single class of ~-adrenoceptors is present in rat fat cells which apparently have a dualistic, both fl, and ~2, character.

Acknowledgements The authors are much indebted to Dr. A.L. Ham and Dr. K.R. Adam (Pfizer-Central Research, Sandwich, England) for the generous supply of tolamolol and analogues.

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