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The rat lipolytic β-adrenoceptor: Studies using novel β-adrenoceptor agonists
European Journal of Pharmacology, 100 (1984) 309-319
309
Elsevier
THE RAT L I P O L Y T I C [3-ADRENOCEPTOR: S T U D I E S U S I N G N O V E L [3-ADRENOCEPTOR AGONISTS
CAMPBELL WILSON *, SHELAGH WILSON, VALERIE PIERCY, MATTHEW V. SENNITT and JONATHAN R.S. ARCH Beecham Pharmaceuticals Research Division, Great Burgh, Epsom, Surrey, KT18 5XQ, U.K.
Received 12 July 1983, revised 8 December 1983, accepted 24 January 1984
C. WILSON, S. WILSON, V. PIERCY, M.V. SENNITT and J.R.S. ARCH, The rat lipolytic fl-adrenoceptor: studies using nooel fl-adrenoceptor agonists, European J. Pharmacol. 100 (1984) 309-319. ECs0 and relative intrinsic activity values were obtained for isoprenaline, fenoterol, salbutamol, prenalterol and three new fl-adrenoceptor agonists, BRL 28410, BRL 35113 and BRL 35135 on rat white adipocyte lipolysis, rat atrial rate and tension, rat uterus tension and guinea-pig tracheal tension. Fenoterol and salbutamol were selective for tracheal and uterine responses, prenalterol was selective for atrial responses, but BRL 28410, BRL 35113 and BRL 35135 were selective for the adipocyte lipolytic response, p A 2 values for propranolol, practolol, ICI 118,551 and sotalol were obtained on adipocytes, atria and trachea, pA 2 values for propranolol and sotalol were much lower on adipocytes than on atria or trachea. The pA 2 value for practolol was lower on adipocytes than on atria and the pA 2 value for ICI 118,551 was lower on adipocytes than on trachea. Both agonist and antagonist studies therefore suggest that the rat adipocyte lipolytic receptor does not fit into the current fll/flE-adrenoceptor classification. fl-Adrenoceptor agonists
fl-Adrenoceptor antagonists
1. Introduction
The nature of the receptor mediating the lipolytic response of rat white adipose tissue to /3adrenoceptor agonists is a matter of dispute. Lands et al. (1967a) proposed a subdivision of fi-adrenoceptors into fil- and fiE-Subtypes and they classified the rat adipose tissue fi-adrenoceptor as fil since lipolytic activity in the rat correlated with cardiac stimulant activity in the rabbit for a series of catecholamines. Subsequent work using selective fil- and fiE-adrenoceptor agonists and antagonists has not confirmed this classification. Jolly et al. (1978) found that the selective fiEadrenoceptor agonist salbutamol was lipolytic in the rat whereas the selective fil-adrenoceptor agonist tazolol was not and therefore suggested that the lipolytic receptor was of the f/E-Subtype. More comprehensive studies have been carried out by Harms, Zaagsma and coworkers (Harms et al., * To whom all correspondence should be addressed. " 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Lipolysis
1974; De Vente et al., 1980) who showed that antagonism of lipolysis could correlate with either fil-adrenoceptor antagonism or fiE-adrenoceptor antagonism depending on the structural type of the antagonist used. They proposed a hybrid receptor with characteristics of both the fil" (affinity for the alkanolamine side chain of fi-adrenoceptor ligands) and fiE- (affinity for the aromatic moiety of fi-adrenoceptor ligands) adrenoceptor subtypes. Tan and Curtis-Prior (1983) have recently supported this proposal. The possibility that a heterogeneous ill- plus fiE-adrenoceptor population was involved has been refuted, since the selective flladrenoceptor antagonists practolol and betaxolol have similar pA E values for antagonism of the lipolytic responses to noradrenaline (fil-selective) and salbutamol or fenoterol (f/E-selective). Similarly, pA E values for the selective fiE-adrenoceptor antagonists H 3 5 / 2 5 and ICI 118,551 do not differ for noradrenaline and salbutamol or fenoterol (De Vente et al., 1980; Belfrage and Fredholm, 1978; Bojanic et al., 1984).
310 OH
CH 3
I
I
OH
CF3
CO2H BRL 28410
OH CH 3 I CHCH2NHCH
OHI ~H 3 CHCH~NHCHCH~
1
CO2H
CH 3
I
CHCH2NHCHCH2
OCH2CO2CH3
BRL 35135
BRL 35113
OH
'
CH O
CH~
NH
OH I
~H 3
OH 4
7H3
CHCH2NH~--CH3
CHCH2NH~H
~H
OHI OH
CH 3 O
PRENALTEROL
H
FENOTEROL
CH2OH
SALBUTAMOL
ISOPRENALINE
Fig. 1. Structures of fl-adrenoceptor agonists.
Although the precise nature of the rat adipose tissue lipolytic fl-adrenoceptor has proved difficult to ascertain within the current fll/fl2-adrenoceptor classification (Stanton, 1972; H a r m s et al., 1974; H a r m s and Van der Meer, 1975; H a r m s et al., 1977; De Vente et al., 1980; H a r m s et al., 1982), the hypothesis of a discrete receptor subtype remains tenuous in the absence of agonists or antagonists selective for this receptor. In the present study, the activity of three novel arylethanolamine fl-adrenoceptor agonists has been examined on several responses mediated by fl-adrenoceptors: BRL 28410, BRL 35113 and BRL 35135 (fig. 1), unlike other fl-adrenoceptor agonists, demonstrate selectivity for adipose tissue lipolysis supporting the view that the rat lipolytic fl-adrenoceptor is atypical.
2. Materials and methods
2.1. Rat left and right atria Separated left and right atria were removed from male Sprague-Dawley rats (250-350 g) by the
method of Broadley and Lumley (1977) and mounted on a perspex tissue holder-electrode. The atria were immersed in a glass tissue bath containing Krebs-bicarbonate solution (composition in millimolar: NaC1 118.4; KC1 4.7; CaC12. 2 H 2 0 2.5; N a H C O 3 25.0; M g S O 4 . 7 H 2 0 1.2; glucose 11.7; K H z P O 4 - 2 H 2 0 1.2) gassed with 5% CO 2 in O 2 (pH 7.4). Bath temperature was maintained at 32°C by means of a water jacket connected to a Churchill circulator. Each atrium was attached by a cotton thread to a Dynamometer UF1 isometric transducer from which tension recordings were obtained. An initial diastolic tension of 0.6 g was applied to both atria. The left atrium was paced at 1 Hz, 3 ms duration at threshold + 50% voltage by means of an SRI 6050 stimulator. Rate recordings from the spontaneously beating right atrium were obtained from the tension signal using an Ormed 4521 ratemeter unit. All traces were displayed on an Ormed M19 chart recorder. The tissues were washed several times during an initial 30 min stabilization period. After ensuring steady baseline rate and tension recordings, the tissues were exposed to a supramaximal concentration (10 6 M) of isoprenaline. The tissues were
311 again washed several times until steady baselines were obtained and then one cumulative log concentration-response curve to the test agonist in half log units (Van Rossum, 1963) was performed. Agonist responses were measured as the increase in rate of the right atrium and increase in tension of the left atrium over pre-agonist levels. In experiments where isoprenaline was the test agonist, the maximal tension response at the end of the concentration-response curve was 1.20 + 0.07 (mean +S.E.M., n = 4) times larger than the initial response to the 10 -6 M concentration. Therefore in experiments using other agonists, the initial tension response to 10 - 6 M isoprenaline was multiplied by 1.2 and responses to each concentration of test agonist were expressed as a percentage of this value. No sensitization of the rate response to isoprenaline was observed and so responses to the test agonist were simply expressed as a percentage of the initial response to 10 -6 M isoprenaline. Experiments involving antagonist drugs were of similar format, except that after washout of the initial isoprenaline dose, the tissues were equilibrated for 30 min with the antagonist before commencing the concentration-response curve to the test agonist. This 'between preparations' design was used rather than the more common 'within preparations' design because a long washout time was required for BRL 28410 and BRL 35135. Thus more than one concentration-response curve per preparation was impractical. Preliminary experiments with fenoterol as agonist using the two different experimental designs produced identical pA 2 values for propranolol. 2.2. Rat uterus
Female Sprague-Dawley rats (120-160 g) were brought into oestrus by the injection of diethyl stilboestrol 0.1 mg kg -1 i.p. 24 h prior to the experiment. The animals were killed and the two uterine horns removed. Each horn was divided longitudinally into two thus providing four preparations from each animal, and each preparation was used for a separate experiment. The preparations were placed in glass tissue baths containing gassed Krebs-bicarbonate solution at 32°C and tension was recorded as for the atria. Resting
tension of 0.2 g was maintained throughout an initial 30 min stabilization period by washing and stretching the tissues. The bathing medium was then exchanged for a solution in which all Na ÷ ions were replaced by the K ÷ ion equivalent (composition in mM: KC1 118.4; CaC12. 2 H 2 0 2.5; K H C O 3 25.0; M g S O a - 7 H 2 0 1.2; glucose 11.7; K H 2 P O 4 1.2). This induced immediate contraction of the tissues followed by a gradual relaxation which stabilized after 3-10 min at approximately 2 g developed tension. One concentration-response curve to the test agonist was then performed. The procedure was similar to that for atria except that the maximal response to isoprenaline was obtained after and not before the test agonist curve. Responses were measured as the degree of relaxation below the initial level and expressed as a percentage of the isoprenaline maximum. 2.3. Guinea-pig trachea
Zig-zag tracheal preparations were prepared from male Dunkin-Hartley guinea-pigs (400-500 g) by the method of Emmerson and Mackay (1979). The trachea was divided into two preparations and these were used for separate experiments. Each preparation was mounted in a glass tissue bath containing gassed Krebs-bicarbonate solution at 37°C. Tension was recorded as described for the atria. An initial resting tension of 1 g was applied and the tissues were left for 1.5 h during which time intrinsic tone developed, stabilizing at approximately 2 g tension. One concentration-response curve to the test agonist was then performed as described for the uterus. Responses were measured as the degree of relaxation below the initial level and expressed as a percentage of the isoprenaline maximum. Experiments using an antagonist were of similar format except that the antagonist was present in the bathing medium throughout the experiment. 2.4. Rat white adipocytes
White adipocytes were prepared from the epididymal fat pads of male Sprague-Dawley rats (250300 g) according to the method of Rodbell (1964).
312 Briefly, the fat pads were chopped coarsely and incubated with collagenase (8 mg per g of tissue) in pregassed, modified Krebs-bicarbonate solution (composition in mM: NaC1 118•4; KC1 4.7; CaC12 • 2 H 2 0 1.25; N a H C O 3 25.0; MgSO 4- 7 H 2 0 1.2; glucose 20.0; K H 2 P O 4. 2 H 2 0 1.2; bovine serum albumin fraction V 4% w / v ) at p H 7.4. The digestion was carried out for one hour in sealed plastic tubes in a shaking water bath maintained at 37°C. The resultant cell suspension was filtered through plastic gauze, the fat cells were allowed to float up and the infranatant was removed. The cells were washed twice and diluted to a final volume of 5 ml per g of tissue. The cells were stirred to maintain a homogeneous suspension whilst 450 /,1 portions were removed for incubation with the agonist-antagonist combination in a final volume of 500/,1. Incubations were carried out in sealed plastic tubes pregassed with 5% CO 2 in 02 maintained at 37°C by means of a shaking water bath. The reaction was stopped after 30 min by addition of 200 t*l of 10% w / v trichloroacetic acid and the resultant precipitate was separated by centrifugation (2000 × g; 15 min). Aliquots of supernatant were removed for glycerol determination according to the method of Garland and Randle (1962). The lipopytic response of the cells was determined by measuring the amount of glycerol released during incubation of the cells with a range of agonist concentrations in the absence or presence of antagonist. Each agonist concentration was used in triplicate within a single concentration-response curve, and the mean value was used for calculation of results• Three separate agonist concentration-response curves were obtained from each batch of cells. In experiments where ECs0 and relative intrinsic activity values (see below) for different agonists were compared, a different agonist was used in each of the concentration-response curves. In experiments where pA 2 values (see below) for antagonists were obtained, one concentration-response curve was for the agonist alone and the other two curves were for the agonist in the presence of two different concentrations of the antagonist. Thus agonist concentration ratios were always obtained within one batch of cells. In addition, a control incubation (no agonist present)
and a maximal incubation (10 -5 M isoprenaline present) were carried out for each batch of cells• Glycerol production in the controls was subtracted from that in agonist-stimulated incubations and all responses were expressed as a percentage of the response to 10 5 M isoprenaline.
2.5. Calculation of ECso, relative intrinsic activity and pA e values The ECs0 values given in the text are expressed as 50% of the isoprenaline maximum response and are the geometric means (n >/4) of individual experiments with 95% confidence limits in parentheses. Relative intrinsic activities (RIA) are arithmetic means (n > 4) of the maximal responses to the test agonists from individual experiments relative to isoprenaline ( = 1.0). Partial agonists are defined as agonists whose RIA value is significantly (P < 0.05) less than unity. Agonist concentration ratios (CR) were calculated at 50% of the agonist's own maximum response, pA 2 values (Arunlakshana and Schild, 1959) were obtained from the point of intersection on the concentration axis of the regression line when log ( C R - 1) was plotted against log molar antagonist concentration. In all experiments three antagonist concentrations were used. The exception was that pA 2 values for propranolol on atria with BRL 28410 as agonist were calculated using only one propranolol concentration, since BRL 28410 had activity on atria at only very high concentrations. The pA 2 values in this case were calculated from the formula pA 2 = log (CR - 1) log [antagonist]. The slopes and regression coefficients of all Schild plots did not significantly differ from 1.0 (P > 0.05, Student's t-test) unless otherwise stated.
2.6. Drugs and reagents used BRL 28410, BRL 35113 and BRL 35135 hydrobromide were synthesized in these laboratories. The other drugs used were diethyl stilboestrol (Sigma), fenoterol hydrobromide (Boehringer Ingelheim), ICI 118, 551 hydrochloride (ICI), ( + ) isoprenaline hydrochloride (Sigma), metanephrine hydrochloride (Sigma), practolol (ICI), prenalterol
313
hydrochloride (AB Hassle), (+)-propranolol hydrochloride (ICI), salbutamol sulphate (Leiras), sotalol hydrochloride (Glaxo) and tropolone (Aldrich). Bovine serum albumin fraction V (Sigma), adenosine deaminase (Sigma) and collagenase (from clostridium) (Boehringer Mannheim) were used in lipolysis experiments. All drugs stock solutions were freshly prepared in distilled water and agonist serial dilutions made in 0.9% NaC1. All isoprenaline solutions included ascorbic acid (approximately 1 mg ml-1).
2a. The potency order in terms of ECs0 values was isoprenaline (1.3(1.2-1.4) x 10 - 9 M) > prenalterol (2.9(1.9-4.2) X 10 -8 M) >/fenoterol (4.8(2.0-11.0) x 10 -8 M)>~ BRL 35135 (5.4(3.8-7.6)x 10 8 M) > salbutamol (9.1(3.8-21.8) x 10 -7 M) > BRL 28410 (8.7(4.7-16.2)x10 -5 M) and BRL 35113. Prenalterol (RIA = 0.82 + 0.03), salbutamol (0.91 + 0.03), BRL 28410 (0.69 + 0.04) and BRL 35113 (0.44 + 0) were partial agonists.
3.2. Stimulation of atrial tension Tension responses of the corresponding left atria to the same agonists are shown in fig. 2b. Although agonists were less potent than for rate responses, the potency order remained the same, i.e. isoprenaline (4.2(3.3-5.3) x 10 -9 M ) > prenalterol (5.2(2.7-10.0) x 10 -8 M) >/fenoterol
3. Results
3.1. Stimulation of atrial rate Rate responses of rat isolated right atria to seven fl-adrenoceptor agonists are shown in fig. 100--
I a,
t.u cO 5OO O. CO O 100-
1
I
I
I
I
I
I
I
I
I
I
I
l
I
I
I
I
I
I
I
10
10
10
AI
5O-
1~3_9
1-8 10 1-7 10 1-6 10 1-5 10 [AGONIST] (g)
1104
Fig. 2. Mean (n >~ 4) concentration-response curves to isoprenaline (I), fenoterol (A), salbutamol (41~), prenalterol (e), BRL 28410 (zx), BRL 35113 ( ~ ) and BRL 35135 (1:3)on: (a) rat right atrial rate, (b) rat left atrial tension, (c) rat uterus, (d) guinea-pig trachea and (e) rat white adipocyte lipolysis. Responses are expressed as a percentage of the m a x i m u m response to isoprenaline as described in the text.
11(33
103
O.
10
[AGONIST] (g)
1104
10 3
314 (6.7(4.5-9.9) x 10 8 M) >/ B R L 35135 (9.4(4.718.7) x 10 8 M) > salbutamol (3.4(2.0-5.8) x 10 -6 M) > B R L 28410 (1.9(0.8-4.5) x 10 4 M) and B R L 35113. Prenalterol (0.91 + 0.03), B R L 35135 (0.81 + 0.05), salbutamol (0.86 + 0.04), B R L 28410 (0.68 + 0 . 0 7 ) and B R L 35113 (0.30 + 0.12) were partial agonists.
3.3. Relaxation of uterus Relaxation of K + - c o n t r a c t e d rat uterus preparations by the same agonists is shown in fig. 2c. The potency order differed from that on atria and was as follows: isoprenaline (3.1(2.4-4.1)x 10 - 9 M ) > f e n o t e r o l (1.7(0.63-4,6) x 1 0 - 8 M ) > salbutamol (2.4(1.7-3.5) x 10 -8 M) > B R L 35135 > B R L 28410 (9.6(5.9-15.0)x 10 6 M ) > B R L 35113 (1.2(0.53-2.6) X 1 0 - s M) > prenalterol. B R L 28410 (0.82 + 0.01) and B R L 35113 (0.86 + 0.04) were partial agonists. B R L 35135 and prenalterol produced variable responses in this tissue making estimation of ECs0 and R I A values difficult. B R L 35135 produced a shallow biphasic concentrationresponse curve. Prenalterol was an agonist only at very high concentrations, and in some preparations produced contraction of the tissue.
3. 4. Relaxation of trachea The agonist p o t e n c y order for relaxation of guinea-pig intrinsic tone tracheal preparations (fig. 2d) was similar to that observed on uterus except for increased p o t e n c y of prenalterol. The p o t e n c y order was isoprenaline (2.3(1.1-3.9) x 10 - 9 M) > fenoterol (3.3(1.8-6.2) X 10 - 9 M) > salbutamol (2.6(1.2-5.6)x 10 -8 M ) > B R L 35135 (3.4(1.5-7.8) x 10 -8 M ) > prenalterol > B R L 28410 (1.8(1.42.3) x 10 5 M ) > B R L 35113 (2.1(1.2-3.8) x 10 s M). B R L 35135 ( 0 . 8 5 _ 0.05), prenalterol (0.46 + 0.08) and B R L 35113 (0.93 + 0.02) were partial agonists.
3.5. Stimulation of adipocyte lipolysis The agonist p o t e n c y order for adipocyte lipolysis (fig. 2e) differed from that observed on left and right atria or uterus and trachea. The order was isoprenaline (1.3(1.0-1.5)x 10 -8 M ) > B R L 35135
(1.6(0.9-2.0) X 10 8 M) > fenoterol (4.6(4.3-4.8) x 10 7 M) > B R L 35113 (8.7(5.0-15.0) x 10 -7 M) > B R L 28410 (3.7(2.4-5.9) x 10 6 M) > salbutamol (1.2(0.49-2.7) x 1 0 - s M) > prenalterol. B R L 35135 (0.91 + 0.03), B R L 35113 (0.88 + 0.04), B R L 28410 (0.86 + 0.02), salbutamol (0.84 + 0.05) and prenalterol (0.14 + 0.06) were partial agonists. Selectivity indices for lipolytic responses over atrial rate responses and for lipolytic responses over tracheal responses were calculated for each agonist relative to isoprenaline (table 1). The values > 1 for B R L 28410, B R L 35113 and B R L 35135 demonstrate selectivity for lipolytic over atrial or tracheal responses. Fenoterol and salbutamol demonstrated selectivity for tracheal over lipolytic responses and prenalterol was selective for atrial over lipolytic responses (values < 1). Selectivity indices were calculated using ECs0 values obtained at 50% of the agonist's own maxim u m response, since 50% of the isoprenaline maxim u m response was not always attained. This procedure m a y in fact under-estimate the selectivity of B R L 28410, B R L 35113 and B R L 35135. Higher selectivity indices for these c o m p o u n d s are obtained when it is possible to use ECs0 values at 50% of the isoprenaline m a x i m u m response. Atrial, uterine and tracheal responses were measured at a time when they were maximal for each TABLE 1 Indices of selectivity for fl-adrenoceptor agonists relative to isoprenaline. Values > 1 indicate relative selectivity for lipolysis over atrial or tracheal responses, while values < 1 indicate the reverse. Selectivity values have been calculated as: (agonist ECs0 (% own max.) for atria or trachea)/ (agonist ECs0 (% own max.) for lipolysis) (isoprenaline ECso for atria or trachea)/ (isoprenaline ECso for lipolysis) Agonist Isoprenaline Fenoterol Salbutamol Prenalterol BRL 28410 BRL 35113 BRL 35135
Lipolysis Atrial rate
Trachea
1 0.91 0.82 0.036 112 160 33
1 0.038 0.026 0.42 30 121 7.1
315 TABLE 2
3.6. Antagonist studies
The relative potencies of isoprenaline, BRL 28410 and BRL 35135 for stimulation of glycerol release from adipocytes over the 0-30 and 10-30 rain time periods.
pA 2 values for fl-adrenoceptor antagonists were obtained on adipocytes, left atrium and trachea (table 3). Irrespective of the agonist used, pA 2 values for propranolol were some 2 log units lower on adipocytes than on left atrium or trachea. The pA 2 value for antagonism of the lipolytic effect of isoprenaline by propranolol was not affected by removal of adenosine by inclusion of adenosine deaminase (1/~g m l - 1 ) in the incubation medium. Practolol, a ill-selective antagonist, was a less effective antagonist on adipocytes than on left atrium, although the slope of the Schild plot in the former was less than unity. Similarly, ICI 118,551 a fiE-selective antagonist, was a less effective antagonist on adipocytes than on trachea. Sotalol, which like propranolol is non-selective between illand fl2-adrenoceptors, but in contrast to propranolol is hydrophilic (Hachisu and Koeda, 1980), was also a less effective antagonist on adipocytes than for /31- or fl2-adrenoceptor mediated responses in other tissues. A further study was undertaken to test whether differences in experimental conditions could explain why pA 2 values obtained on adipocytes were lower than those obtained on left atrium and trachea. Only lipolytic experiments included bovine serum albumin and it has been shown that albumin may bind fl-adrenoceptor antagonists reducing their effective concentration (Zaagsma et al., 1977). Furthermore insulin contained within albumin may reduce receptor affinity for antagonists (Bertholet et al., 1981). However, when the albumin concentration was reduced to 0.1% (and incubation time to 20 min), the pA 2 value for propranolol with isoprenaline as agonist was 6.7, which is similar to the value of 6.6 when the standard 4% albumin was used. Moreover, when insulin was removed from the albumin (4%) by charcoal adsorption, a pA 2 value of 6.6 for propranolol was again obtained. The effect of antagonist pre-incubation on pA 2 value was also studied since pre-incubation was used in all but the lipolysis experiments. The standard procedure for lipolysis was followed except that the adipocytes were pre-incubated for 30 rain at 37°C with propranolol 10 -6 M before addition of the isoprenaline. A pA z value of 7.0 was obtained.
Relative rates of glycerolrelease Isoprenaline 10 -5 M Isoprenaline 10-7 M BRL 28410 10-5 M BRL 35135 2×10 -s M
0-30 min 100 a 52 60 48
10-30 min 100 b 53 65 56
a Rate of glycerol release = 6.8 #mol/min per g adipose tissue. b Rate of glycerol release = 8.7 #mol/min per g adipose tissue. concentration of agonist, whereas lipolytic responses were measured over the first 30 rain of exposure to the agonist. This exposure time was chosen for the lipolysis experiments because preliminary experiments showed that rates of lipolysis in the presence of isoprenaline, BRL 35135 and BRL 28410 were close to linear over the first 30 rain, except for some lag during the first 10 min. Furthermore, in preliminary experiments the relative potencies of isoprenaline, BRL 35135 and BRL 28410 were similar over the 0-30 and 10-30 min periods (table 2). To investigate the possibility that the selectivity of the BRL compounds was due to their antagonizing the antilipolytic effect of adenosine released from the fat cells (Fredholm, 1978), control experiments were performed in the presence of adenosine deaminase at a concentration of 1 /~g m l - 1. We have found that this concentration causes maximal stimulation of lipolysis. Addition of adenosine deaminase caused similar shifts to the left in the dose-response curves for isoprenaline (5-fold shift) and BRL 28410 (3-fold shift), which suggests that antagonism of the adenosine receptor by BRL 28410 cannot account for its selectivity as a stimulant of lipolysis. To investigate the possibility that uptake and metabolism may have reduced the potency of isoprenaline in adipocytes, experiments were performed which included metanephrine, an extraneuronal uptake inhibitor, or tropolone, a catechol-O-methyl transferase inhibitor, in the incubations. Neither metanephrine 5 × 10 -5 M nor tropolone 10 -4 M produced a shift to the left of the isoprenaline concentration-response curve.
316 TABLE 3 pA 2 values for fl-adrenoceptor antagonists on rat white adipocyte lipolysis, rat left atrial tension stimulation and guinea-pig intrinsic tone tracheal relaxation. Response Lipolysis
Agonist Isoprenaline Fenoterol BRL28410 BRL 35113 BRL 35135
Propranolol 6.6 6.3 6.2 6.2 6.2
Practolol 4.9 d
Atrial t e n s i o n
Isoprenaline Fenoterol BRL 28410 BRL 35135
8.7 8.2 8.7 8.8
6.5
Isoprenaline Fenoterol BRL 28410
8.3
4.5 "
Trachea
8.6
ICI 118,551 5.8 5.5 5.2
Sotalol 5.6 4.8 4.3
6.3 6.8 b,~
6.7 6.8 6.5 8.7 b 8.0 d
a From Hachisu and Koeda (1980). b From O'Donnell and Wanstall (1980). ~Using guinea-pig atrial rate. d Slope of Schild plot < 1 (P < 0.05).
4. Discussion Characterisation of the rat lipolytic adrenoceptor in terms of the fll/fl2 subdivision has proved difficult (Stanton, 1972; Harms et al., 1974; H a r m s and Van Der Meer, 1975; H a r m s et al., 1977; De Vente et al., 1980; H a r m s et al., 1982; Bojanic et al., 1984). This task has now been facilitated by the availability of three novel fl-adrenoceptor agonists, BRL 28410, BRL 35113 and BRL 35135, which demonstrate selectivity for the rat adipocyte lipolytic response. Strong evidence that the novel compounds stimulate the lipolytic response by interaction with the fl-adrenoceptor has come from the finding that fl-adrenoceptor antagonists have similar pA 2 values for antagonism of responses to the novel compounds and traditional fl-adrenoceptor agonists (table 3). There appears to be no a-adrenoceptor involvement in lipolysis in the rat (Guidicelli et al., 1981), and the antagonism of the response to fl-adrenoceptor agonists by fl-adrenoceptor antagonists is stereoselective (Harms et al., 1977; Fassina, 1966). The lipolytic response to the novel fl-adrenoceptor agonists, in common with that to the s t a n d a r d fl-adrenoceptor agonists, was inhibited in a competitive manner by fl-adrenoceptor antagonists (except for practolol). Results from both agonist
and antagonist experiments therefore suggest that lipolysis in the rat is mediated by fl-adrenoceptors. However, the data are inconsistent with the response being mediated either by adrenoceptors of the ill-subtype, of the fla-subtype or a heterogeneous population of both subtypes. Both rate and tension responses of rat atria to fl-adrenoceptor agonists are mediated by adrenoceptors of the ill-subtype (Bryan et al., 1981). The low RIA and high ECs0 values of BRL 28410 and BRL 35113 in these tissues therefore indicate poor fll-agonist activity. BRL 35135 was more potent than BRL 28410 and BRL 35113 in these tissues, but was not more potent than the fl2-selective agonist fenoterol. Relaxation of guinea-pig trachea by fl-adrenoceptor agonists involves predominantly fl2-adrenoceptors, although a small proportion of fll-adrenoceptors has been demonstrated by radioligand binding (Engel et al., 1982) and pharmacological studies (Furchgott, 1976). Relaxation of the rat uterus is also mediated by fl2-adrenoceptors (Lands et al., 1967b). Consistent with this classification, the fiR-selective agonists fenoterol and salbutamol were much more potent on trachea and uterus than on atria, whereas the fl2-selective agonist prenalterol exhibited the reverse selectivity. BRL 28410 and BRL 35113 had higher RIA on trachea
317
and uterus than on atria but remained poor agonists in terms of ECs0 values. BRL 35135 was slightly more potent on trachea than on atria but the response on uterus was difficult to interpret. It is possible that higher concentrations of BRL 35135 produced relaxation not mediated by fl2-adrenoceptors in this tissue. Isoprenaline, which is non-selective between ill" and fl2-adrenoceptors, was a less potent stimulant of lipolytic responses than of responses mediated by ill- or fl2-adrenoceptors. This difference was not due to a more avid extra-neuronal uptake or metabolism of isoprenaline in adipocytes since inhibition of both uptake 2 and catechol-O-methyltransferase by metanephrine and tropolone respectively did not potentiate the lipolytic response to isoprenaline. Prenalterol was a much weaker agonist on adipocytes than on atria and fenoterol and salbutamol were much weaker agonists on adipocytes than on uterus and trachea. These low agonist potencies could be explained by a smaller receptor reserve in adipocytes than in the other tissues. However, this explanation cannot account for the selectivity demonstrated by BRL 28410, BRL 35113 and BRL 35135. These three agonists were more potent, in terms of ECs0 values, on adipocytes than on atria, uterus or trachea. When expressed relative to isoprenaline, this selectivity was considerable (table 1). Selective agonist studies therefore suggest that the rat adipocyte lipolytic receptor differs from that in rat atria, rat uterus or guinea-pig trachea. Antagonist studies support this hypothesis. Propranolol, which is non-selective between ill- and fl2-adrenoceptors, was considerably less effective in antagonizing lipolytic responses than atrial or tracheal responses whichever agonist was used. Propranolol has been shown to bind to bovine serum albumin in vitro (Zaagsma et al., 1977) and this could lower its apparent pA 2 value. However, in the present studies, reduction of the albumin concentration from 4% to 0.1% had little influence on the pA 2 value and thus albumin binding cannot explain the difference in pA 2 values for propranolol between lipolysis and the other responses. Furthermore, the pA 2 value for sotalol, a nonselective fl-adrenoceptor antagonist which does not bind to albumin due to its hydrophilic nature
(Hachisu and Koeda, 1980), was also much lower for inhibition of lipolysis than reported for inhibition of other fl-adrenoceptor mediated responses. Bertholet et al. (1981) reported that insulin, which is present in variable amounts in all commercially available bovine serum albumin preparations, can reduce fl-adrenoceptor antagonist pA 2 values. The presence of insulin in lipolysis experiments cannot however account for the pA 2 differences, since the pA 2 value for propranolol was unaltered when albumin in which insulin had been removed by charcoal adsorption was used. Propranolol pA 2 values may also be reduced if insufficient time is allowed for equilibration with the tissue before addition of the agonist (Grana et al., 1972). In the present experiments atria and trachea were incubated for 30 min with antagonist before exposure to agonist, whereas adipocytes received agonist and antagonist simultaneously. Pre-incubation of adipocytes for 30 min with propranolol yielded a slightly higher pA 2 value than when there was no pre-incubated. However, a much larger increase in pA 2 value than that observed would be required to account for the pA 2 difference between adipocytes and other tissues. Both ill- and fl2-selective antagonists were also relatively poor inhibitors of the lipolytic response. The pA 2 value for practolol (flcselective) was much lower on adipocytes than on atria and the pA 2 value for ICI 118,551 (fl2-selective)was much lower on adipocytes than on trachea. Therefore, irrespective of ill- or fl2-selectivity, fl-adrenoceptor antagonists were poor inhibitors of fl-adrenoceptor agonist stimulated lipolysis. These results cannot be explained by the different experimental procedure used in lipolysis and must therefore indicate the involvement of a non ill- or fl2-adrenoceptor. The hybrid fll/fl2 receptor proposed by Harms et al. (1974) and De Vente et al. (1980) adequately explains the results obtained by these authors. This model however, does not readily explain the present results. It is difficult to envisage a lipolytic receptor with ill- and fl2-adrenoceptor characteristics when, for example, BRL 35113 is a poor agonist at both ill- and fl2-adrenoceptors yet is a potent agonist at the lipolytic fl-adrenoceptor. It is possible that the rat lipolytic fl-adrenoceptor is structurally more primitive and less complex than
318 fl~- or fl2-adrenoceptors, as suggested by Bertholet et al. (1981). I n other species, the lipolytic fl-adrenoceptor m a y or m a y n o t be similar to that in the rat. A heterogeneous p o p u l a t i o n of ill- plus flz-adrenoceptors has been d e m o n s t r a t e d in hamster (Bjorgell a n d Belfrage, 1982) a n d dog (Belgrage a n d F r e d h o l m , 1978). The lipolytic receptor in swine has not b e e n clearly characterized (Hruska, 1981). f l - A d r e n o c e p t o r a n t a g o n i s t pA2 values for guineapig adipocyte responses were lower t h a n atrial or tracheal responses (Bertholet et al., 1981) although the difference for p r o p r a n o l o l was m u c h lower t h a n f o u n d in the rat (this paper). I n man, high p A 2 values for non-selective, fla-selective a n d fl2selective a d r e n o c e p t o r a n t a g o n i s t s were f o u n d on lipolysis, suggesting a mixed fll plus f12 p o p u l a t i o n ( W e n k e o v a et al., 1976). However, the correlation of p A 2 values for a n u m b e r of fl-adrenoceptor a n t a g o n i s t s b e t w e e n h u m a n a n d rat lipolytic responses has suggested a similarity of the receptor type in b o t h species ( H a r m s et al., 1982). fl-Adrenoceptors m e d i a t i n g other metabolic responses have not b e e n clearly characterized. A h r e n a n d L u n d q u i s t (1981) showed that the fl-adrenoceptor regulating i n s u l i n secretion in vivo in the m o u s e does not fit into the /~1///~2 subdivision. B R L 35135 is a p o t e n t i n s u l i n secretagogue in vivo (M.A. Cawthorne, personal c o m m u n i c a t i o n ) as is B R L 26830, the p a r e n t ester of BRL 28410 ( C a w t h o r n e et al., 1982). Therefore it m a y be that the atypical fl-adrenoceptor which mediates lipolysis in the rat is similar to that involved in i n s u l i n secretion or other metabolic responses. I n conclusion, synthesis of a novel series of f l - a d r e n o c e p t o r agonists has e n a b l e d further characterization of the fl-adrenoceptor m e d i a t i n g lipolysis in the rat. Both agonist a n d a n t a g o n i s t studies have shown this receptor to be distinct from either the ill- or flz-adrenoceptor subtype. I n v o l v e m e n t of this receptor type in lipolysis in other species or i n v o l v e m e n t in other metabolic responses r e m a i n s to be the determined.
Acknowledgements The authors would like to thank Miss C. Duffett, Mr. R. Ellis, Mr. M. Khan and Mrs. V. Thody for their technical
assistance in this work, and Miss C. Saunders for typing the manuscript. They would also like to thank the companies concerned for gifts of drugs used.
References Ahren, B. and I. Lundquist, 1981, Effects of selective and non-selective fl-adrenergic agents on insulin secretion in vivo, European J. Pharmacol. 71, 93. Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 48. Belfrage, E. and B.B. Fredholm, 1978, Rat and dog fat cell fl-adrenoceptors are different, Abstracts 7th International Congress of Pharmacology, IUPHAR, Paris, 1978, p. 553. Bertholet, A., M. Milavec, R. Waite and H.O. Schild, 1981, A study of fl-adrenoceptors in the guinea-pig, Postgrad. Med. J. 57, Suppl. 1, 9. Bjorgell, P. and P. Belfrage, 1982, Characteristics of the lipolytic fl-adrenergic receptors in hamster adipocytes, Biochim. Biophys. Acta 713, 80. Bojanic, D., J.D. Jansen, S.R. Nahorski and J. Zaagsma, 1984, Atypical nature of rat adipocyte fl-adrenoceptors: evidence from lipolysis and cAMP production, Br. J. Pharmacol. 81, Suppl. 4P. Broadley, K.J. and P. Lumley, 1977, Selective reserpine-induced supersensitivity of the positive inotropic and chronotropic responses to isoprenaline and salbutamol in guineapig isolated atria, Br. J. Pharmacol. 59, 51. Bryan, L.J., J.J. Cole, S.R. O'Donnell and J.C. Wanstall, 1981, A study designed to explore the hypothesis that beta-I adrenoceptors are 'innervated' receptors and beta-2 adrenoceptors are 'hormonal' receptors, J. Pharmacol. Exp. Ther. 216, 395. Cawthorne, M.A., M.V. Sennitt, S.A. Smith, D.L. Simson and J.R.S. Arch, 1982, Anti-hyperglycaemic action of BRL 26830A in animal models of diabetes, Abstracts of 'Lessons From Animal Diabetes', Jerusalem, 1982, p. 131. De Vente, J., A. Bast, L. van Bree and J. Zaagsma, 1980, fl-Adrenoceptor studies. 6. Further investigations on the hybrid nature of the rat adipocyte fl-adrenoceptor, European J. Pharmacol. 63, 73. Emmerson, G. and D. Mackay, 1979, The zig-zag tracheal strip, J. Pharm. Pharmacol. 31,798. Engel, G., D. Hoyer and U. Bretz, 1982, Biochemical identification of subclasses of adrenoceptors in guinea pig, rat and human lung, Therapie 37, 541. Fassina, G., 1966, Effects of lipomobilisation of the fl-adrenergic blocking drugs, propranolol and INPEA, J. Pharm. Pharmacol. 18, 399. Fredholm, B.B., 1978, Local regulation of lipolysis in adipose tissue by fatty acids, prostaglandins and adenosine, Med. Biol. 56, 249. Furchgott, R.F., 1976, Postsynaptic adrenergic receptor mechanisms in vascular smooth muscle, in: Vascular Neuroeffector Mechanisms, eds. J.A. Bevan et al. (Karger, Basel) p. 131.
319 Garland, P.B. and P.J. Randle, 1962, A rapid enzymatic assay for glycerol, Nature 196, 987. Grana, E., O. Cattarini Mastelli, A. Luchelli and M.G. Santagostino, 1972, The selectivity of fl-adrenergic compounds. II. Studies on the mimetic compound salbutamol and the lytic compound practolol, I1 Farmaco Ed. Sci. 27, 842. Guidicelli, Y., N.R. Thotakuru, D. Lacasa, R. Pecquery and B. Agli, 1981, Direct binding studies do not support the existence of true a-adrenoceptors in rat white fat cells, Biochem. Biophys. Res. Commun. 100, 621. Hachisu, M. and T. Koeda, 1980, Study on the pharmacological actions of fl-adrenoceptor blockers with reference to their physico-chemical properties, J. Pharm. Dyn. 3, 183. Harms, H.H., J. De Vente and J. Zaagsma, 1982, fl-Adrenoceptor blocking agents and lipolysis, Br. J. Clin. Pharmacol. 13, 181s. Harms, H.H. and J. Van Der Meer, 1975, lsoprenaline antagonism of cardioselective fl-adrenoceptor blocking agents on human and rat adipocytes, Br. J. Clin. Pharmacol. 2, 311. Harms, H.H., J. Zaagsma and J. De Vente, 1977, Differentiation of fl-adrenoceptors in right atrium, diaphragm and adipose tissue of the rat, using stereoisomers of propranolol, alprenolol, nifenalol and practolol, Life Sci. 21, 123. Harms, H.H., J. Zaagsma and B. Van de Wal, 1974, fl-Adrenoceptor studies. III. On the fl-adrenoceptors in rat adipose tissue, European J. Pharmacol. 25, 87. Hruska, R.L., 1981, Adrenergic control of swine adipose tissue lipolysis, Fed. Proc. 40, 225. Jolly, S.R., J.J. Lech and L.A. Menahan, 1978, Comparison of isoproterenol, salbutamol and tazolol as lipolytic agents with isolated rodent adipocytes, Biochem. Pharmacol. 27, 1885.
Lands, A.M., A. Arnold, J.P. McAuliff, F.P. Luduena and J.G. Brown, 1967a, Differentiation of receptors activated by sympathomimetic amines, Nature 214, 597. Lands, A.M., F.P. Luduena and H.J. Buzzo, 1967b, Differentiation of receptors responsive to isoproterenol, Life Sci. 6, 2241. O'Donnell, S.R. and J.C. Wanstall, 1980, Evidence that ICI 118,551 is a potent, highly beta2-selective adrenoceptor antagonist and can be used to characterize beta-adrenoceptor populations in tissues, Life Sci. 27, 671. Rodbell, M., 1964, Metabolism of isolated fat cells. 1. Effects of hormones on glucose metabolism and lipolysis, J. Biol. Chem. 239, 375. Stanton, H.C., 1972, Selective metabolic and cardiovascular fl-adrenoceptor antagonism in the rat, Arch. Int. Pharmacodyn. 196, 246. Tan, S. and P.B. Curtis-Prior, 1983, Characterization of the beta-adrenoceptor of the adipose cell of the rat, Int. J. Obesity, 7, 409. Van Rossum, J.M., 1963, Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters, Arch. Int. Pharmacodyn. 143, 299. Wenkeova, J., E. Kuhn and M. Wenke, 1976, Some adrenergic fl-blocking agents affecting lipolysis in human adipose tissue in vitro, European J. Pharmacol. 37, 91. Zaagsma, J., L. Meems and M. Boorsma, 1977, fl-Adrenoceptol studies. 4. Influence of albumin on in vitro fl-adrenocepto~ blocking and antiarrhythmic properties of propranolol. pindolol, practolol metoprolol, Naunyn-Schmiedeb. Arch. Pharmacol. 298, 29.
309
Elsevier
THE RAT L I P O L Y T I C [3-ADRENOCEPTOR: S T U D I E S U S I N G N O V E L [3-ADRENOCEPTOR AGONISTS
CAMPBELL WILSON *, SHELAGH WILSON, VALERIE PIERCY, MATTHEW V. SENNITT and JONATHAN R.S. ARCH Beecham Pharmaceuticals Research Division, Great Burgh, Epsom, Surrey, KT18 5XQ, U.K.
Received 12 July 1983, revised 8 December 1983, accepted 24 January 1984
C. WILSON, S. WILSON, V. PIERCY, M.V. SENNITT and J.R.S. ARCH, The rat lipolytic fl-adrenoceptor: studies using nooel fl-adrenoceptor agonists, European J. Pharmacol. 100 (1984) 309-319. ECs0 and relative intrinsic activity values were obtained for isoprenaline, fenoterol, salbutamol, prenalterol and three new fl-adrenoceptor agonists, BRL 28410, BRL 35113 and BRL 35135 on rat white adipocyte lipolysis, rat atrial rate and tension, rat uterus tension and guinea-pig tracheal tension. Fenoterol and salbutamol were selective for tracheal and uterine responses, prenalterol was selective for atrial responses, but BRL 28410, BRL 35113 and BRL 35135 were selective for the adipocyte lipolytic response, p A 2 values for propranolol, practolol, ICI 118,551 and sotalol were obtained on adipocytes, atria and trachea, pA 2 values for propranolol and sotalol were much lower on adipocytes than on atria or trachea. The pA 2 value for practolol was lower on adipocytes than on atria and the pA 2 value for ICI 118,551 was lower on adipocytes than on trachea. Both agonist and antagonist studies therefore suggest that the rat adipocyte lipolytic receptor does not fit into the current fll/flE-adrenoceptor classification. fl-Adrenoceptor agonists
fl-Adrenoceptor antagonists
1. Introduction
The nature of the receptor mediating the lipolytic response of rat white adipose tissue to /3adrenoceptor agonists is a matter of dispute. Lands et al. (1967a) proposed a subdivision of fi-adrenoceptors into fil- and fiE-Subtypes and they classified the rat adipose tissue fi-adrenoceptor as fil since lipolytic activity in the rat correlated with cardiac stimulant activity in the rabbit for a series of catecholamines. Subsequent work using selective fil- and fiE-adrenoceptor agonists and antagonists has not confirmed this classification. Jolly et al. (1978) found that the selective fiEadrenoceptor agonist salbutamol was lipolytic in the rat whereas the selective fil-adrenoceptor agonist tazolol was not and therefore suggested that the lipolytic receptor was of the f/E-Subtype. More comprehensive studies have been carried out by Harms, Zaagsma and coworkers (Harms et al., * To whom all correspondence should be addressed. " 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Lipolysis
1974; De Vente et al., 1980) who showed that antagonism of lipolysis could correlate with either fil-adrenoceptor antagonism or fiE-adrenoceptor antagonism depending on the structural type of the antagonist used. They proposed a hybrid receptor with characteristics of both the fil" (affinity for the alkanolamine side chain of fi-adrenoceptor ligands) and fiE- (affinity for the aromatic moiety of fi-adrenoceptor ligands) adrenoceptor subtypes. Tan and Curtis-Prior (1983) have recently supported this proposal. The possibility that a heterogeneous ill- plus fiE-adrenoceptor population was involved has been refuted, since the selective flladrenoceptor antagonists practolol and betaxolol have similar pA E values for antagonism of the lipolytic responses to noradrenaline (fil-selective) and salbutamol or fenoterol (f/E-selective). Similarly, pA E values for the selective fiE-adrenoceptor antagonists H 3 5 / 2 5 and ICI 118,551 do not differ for noradrenaline and salbutamol or fenoterol (De Vente et al., 1980; Belfrage and Fredholm, 1978; Bojanic et al., 1984).
310 OH
CH 3
I
I
OH
CF3
CO2H BRL 28410
OH CH 3 I CHCH2NHCH
OHI ~H 3 CHCH~NHCHCH~
1
CO2H
CH 3
I
CHCH2NHCHCH2
OCH2CO2CH3
BRL 35135
BRL 35113
OH
'
CH O
CH~
NH
OH I
~H 3
OH 4
7H3
CHCH2NH~--CH3
CHCH2NH~H
~H
OHI OH
CH 3 O
PRENALTEROL
H
FENOTEROL
CH2OH
SALBUTAMOL
ISOPRENALINE
Fig. 1. Structures of fl-adrenoceptor agonists.
Although the precise nature of the rat adipose tissue lipolytic fl-adrenoceptor has proved difficult to ascertain within the current fll/fl2-adrenoceptor classification (Stanton, 1972; H a r m s et al., 1974; H a r m s and Van der Meer, 1975; H a r m s et al., 1977; De Vente et al., 1980; H a r m s et al., 1982), the hypothesis of a discrete receptor subtype remains tenuous in the absence of agonists or antagonists selective for this receptor. In the present study, the activity of three novel arylethanolamine fl-adrenoceptor agonists has been examined on several responses mediated by fl-adrenoceptors: BRL 28410, BRL 35113 and BRL 35135 (fig. 1), unlike other fl-adrenoceptor agonists, demonstrate selectivity for adipose tissue lipolysis supporting the view that the rat lipolytic fl-adrenoceptor is atypical.
2. Materials and methods
2.1. Rat left and right atria Separated left and right atria were removed from male Sprague-Dawley rats (250-350 g) by the
method of Broadley and Lumley (1977) and mounted on a perspex tissue holder-electrode. The atria were immersed in a glass tissue bath containing Krebs-bicarbonate solution (composition in millimolar: NaC1 118.4; KC1 4.7; CaC12. 2 H 2 0 2.5; N a H C O 3 25.0; M g S O 4 . 7 H 2 0 1.2; glucose 11.7; K H z P O 4 - 2 H 2 0 1.2) gassed with 5% CO 2 in O 2 (pH 7.4). Bath temperature was maintained at 32°C by means of a water jacket connected to a Churchill circulator. Each atrium was attached by a cotton thread to a Dynamometer UF1 isometric transducer from which tension recordings were obtained. An initial diastolic tension of 0.6 g was applied to both atria. The left atrium was paced at 1 Hz, 3 ms duration at threshold + 50% voltage by means of an SRI 6050 stimulator. Rate recordings from the spontaneously beating right atrium were obtained from the tension signal using an Ormed 4521 ratemeter unit. All traces were displayed on an Ormed M19 chart recorder. The tissues were washed several times during an initial 30 min stabilization period. After ensuring steady baseline rate and tension recordings, the tissues were exposed to a supramaximal concentration (10 6 M) of isoprenaline. The tissues were
311 again washed several times until steady baselines were obtained and then one cumulative log concentration-response curve to the test agonist in half log units (Van Rossum, 1963) was performed. Agonist responses were measured as the increase in rate of the right atrium and increase in tension of the left atrium over pre-agonist levels. In experiments where isoprenaline was the test agonist, the maximal tension response at the end of the concentration-response curve was 1.20 + 0.07 (mean +S.E.M., n = 4) times larger than the initial response to the 10 -6 M concentration. Therefore in experiments using other agonists, the initial tension response to 10 - 6 M isoprenaline was multiplied by 1.2 and responses to each concentration of test agonist were expressed as a percentage of this value. No sensitization of the rate response to isoprenaline was observed and so responses to the test agonist were simply expressed as a percentage of the initial response to 10 -6 M isoprenaline. Experiments involving antagonist drugs were of similar format, except that after washout of the initial isoprenaline dose, the tissues were equilibrated for 30 min with the antagonist before commencing the concentration-response curve to the test agonist. This 'between preparations' design was used rather than the more common 'within preparations' design because a long washout time was required for BRL 28410 and BRL 35135. Thus more than one concentration-response curve per preparation was impractical. Preliminary experiments with fenoterol as agonist using the two different experimental designs produced identical pA 2 values for propranolol. 2.2. Rat uterus
Female Sprague-Dawley rats (120-160 g) were brought into oestrus by the injection of diethyl stilboestrol 0.1 mg kg -1 i.p. 24 h prior to the experiment. The animals were killed and the two uterine horns removed. Each horn was divided longitudinally into two thus providing four preparations from each animal, and each preparation was used for a separate experiment. The preparations were placed in glass tissue baths containing gassed Krebs-bicarbonate solution at 32°C and tension was recorded as for the atria. Resting
tension of 0.2 g was maintained throughout an initial 30 min stabilization period by washing and stretching the tissues. The bathing medium was then exchanged for a solution in which all Na ÷ ions were replaced by the K ÷ ion equivalent (composition in mM: KC1 118.4; CaC12. 2 H 2 0 2.5; K H C O 3 25.0; M g S O a - 7 H 2 0 1.2; glucose 11.7; K H 2 P O 4 1.2). This induced immediate contraction of the tissues followed by a gradual relaxation which stabilized after 3-10 min at approximately 2 g developed tension. One concentration-response curve to the test agonist was then performed. The procedure was similar to that for atria except that the maximal response to isoprenaline was obtained after and not before the test agonist curve. Responses were measured as the degree of relaxation below the initial level and expressed as a percentage of the isoprenaline maximum. 2.3. Guinea-pig trachea
Zig-zag tracheal preparations were prepared from male Dunkin-Hartley guinea-pigs (400-500 g) by the method of Emmerson and Mackay (1979). The trachea was divided into two preparations and these were used for separate experiments. Each preparation was mounted in a glass tissue bath containing gassed Krebs-bicarbonate solution at 37°C. Tension was recorded as described for the atria. An initial resting tension of 1 g was applied and the tissues were left for 1.5 h during which time intrinsic tone developed, stabilizing at approximately 2 g tension. One concentration-response curve to the test agonist was then performed as described for the uterus. Responses were measured as the degree of relaxation below the initial level and expressed as a percentage of the isoprenaline maximum. Experiments using an antagonist were of similar format except that the antagonist was present in the bathing medium throughout the experiment. 2.4. Rat white adipocytes
White adipocytes were prepared from the epididymal fat pads of male Sprague-Dawley rats (250300 g) according to the method of Rodbell (1964).
312 Briefly, the fat pads were chopped coarsely and incubated with collagenase (8 mg per g of tissue) in pregassed, modified Krebs-bicarbonate solution (composition in mM: NaC1 118•4; KC1 4.7; CaC12 • 2 H 2 0 1.25; N a H C O 3 25.0; MgSO 4- 7 H 2 0 1.2; glucose 20.0; K H 2 P O 4. 2 H 2 0 1.2; bovine serum albumin fraction V 4% w / v ) at p H 7.4. The digestion was carried out for one hour in sealed plastic tubes in a shaking water bath maintained at 37°C. The resultant cell suspension was filtered through plastic gauze, the fat cells were allowed to float up and the infranatant was removed. The cells were washed twice and diluted to a final volume of 5 ml per g of tissue. The cells were stirred to maintain a homogeneous suspension whilst 450 /,1 portions were removed for incubation with the agonist-antagonist combination in a final volume of 500/,1. Incubations were carried out in sealed plastic tubes pregassed with 5% CO 2 in 02 maintained at 37°C by means of a shaking water bath. The reaction was stopped after 30 min by addition of 200 t*l of 10% w / v trichloroacetic acid and the resultant precipitate was separated by centrifugation (2000 × g; 15 min). Aliquots of supernatant were removed for glycerol determination according to the method of Garland and Randle (1962). The lipopytic response of the cells was determined by measuring the amount of glycerol released during incubation of the cells with a range of agonist concentrations in the absence or presence of antagonist. Each agonist concentration was used in triplicate within a single concentration-response curve, and the mean value was used for calculation of results• Three separate agonist concentration-response curves were obtained from each batch of cells. In experiments where ECs0 and relative intrinsic activity values (see below) for different agonists were compared, a different agonist was used in each of the concentration-response curves. In experiments where pA 2 values (see below) for antagonists were obtained, one concentration-response curve was for the agonist alone and the other two curves were for the agonist in the presence of two different concentrations of the antagonist. Thus agonist concentration ratios were always obtained within one batch of cells. In addition, a control incubation (no agonist present)
and a maximal incubation (10 -5 M isoprenaline present) were carried out for each batch of cells• Glycerol production in the controls was subtracted from that in agonist-stimulated incubations and all responses were expressed as a percentage of the response to 10 5 M isoprenaline.
2.5. Calculation of ECso, relative intrinsic activity and pA e values The ECs0 values given in the text are expressed as 50% of the isoprenaline maximum response and are the geometric means (n >/4) of individual experiments with 95% confidence limits in parentheses. Relative intrinsic activities (RIA) are arithmetic means (n > 4) of the maximal responses to the test agonists from individual experiments relative to isoprenaline ( = 1.0). Partial agonists are defined as agonists whose RIA value is significantly (P < 0.05) less than unity. Agonist concentration ratios (CR) were calculated at 50% of the agonist's own maximum response, pA 2 values (Arunlakshana and Schild, 1959) were obtained from the point of intersection on the concentration axis of the regression line when log ( C R - 1) was plotted against log molar antagonist concentration. In all experiments three antagonist concentrations were used. The exception was that pA 2 values for propranolol on atria with BRL 28410 as agonist were calculated using only one propranolol concentration, since BRL 28410 had activity on atria at only very high concentrations. The pA 2 values in this case were calculated from the formula pA 2 = log (CR - 1) log [antagonist]. The slopes and regression coefficients of all Schild plots did not significantly differ from 1.0 (P > 0.05, Student's t-test) unless otherwise stated.
2.6. Drugs and reagents used BRL 28410, BRL 35113 and BRL 35135 hydrobromide were synthesized in these laboratories. The other drugs used were diethyl stilboestrol (Sigma), fenoterol hydrobromide (Boehringer Ingelheim), ICI 118, 551 hydrochloride (ICI), ( + ) isoprenaline hydrochloride (Sigma), metanephrine hydrochloride (Sigma), practolol (ICI), prenalterol
313
hydrochloride (AB Hassle), (+)-propranolol hydrochloride (ICI), salbutamol sulphate (Leiras), sotalol hydrochloride (Glaxo) and tropolone (Aldrich). Bovine serum albumin fraction V (Sigma), adenosine deaminase (Sigma) and collagenase (from clostridium) (Boehringer Mannheim) were used in lipolysis experiments. All drugs stock solutions were freshly prepared in distilled water and agonist serial dilutions made in 0.9% NaC1. All isoprenaline solutions included ascorbic acid (approximately 1 mg ml-1).
2a. The potency order in terms of ECs0 values was isoprenaline (1.3(1.2-1.4) x 10 - 9 M) > prenalterol (2.9(1.9-4.2) X 10 -8 M) >/fenoterol (4.8(2.0-11.0) x 10 -8 M)>~ BRL 35135 (5.4(3.8-7.6)x 10 8 M) > salbutamol (9.1(3.8-21.8) x 10 -7 M) > BRL 28410 (8.7(4.7-16.2)x10 -5 M) and BRL 35113. Prenalterol (RIA = 0.82 + 0.03), salbutamol (0.91 + 0.03), BRL 28410 (0.69 + 0.04) and BRL 35113 (0.44 + 0) were partial agonists.
3.2. Stimulation of atrial tension Tension responses of the corresponding left atria to the same agonists are shown in fig. 2b. Although agonists were less potent than for rate responses, the potency order remained the same, i.e. isoprenaline (4.2(3.3-5.3) x 10 -9 M ) > prenalterol (5.2(2.7-10.0) x 10 -8 M) >/fenoterol
3. Results
3.1. Stimulation of atrial rate Rate responses of rat isolated right atria to seven fl-adrenoceptor agonists are shown in fig. 100--
I a,
t.u cO 5OO O. CO O 100-
1
I
I
I
I
I
I
I
I
I
I
I
l
I
I
I
I
I
I
I
10
10
10
AI
5O-
1~3_9
1-8 10 1-7 10 1-6 10 1-5 10 [AGONIST] (g)
1104
Fig. 2. Mean (n >~ 4) concentration-response curves to isoprenaline (I), fenoterol (A), salbutamol (41~), prenalterol (e), BRL 28410 (zx), BRL 35113 ( ~ ) and BRL 35135 (1:3)on: (a) rat right atrial rate, (b) rat left atrial tension, (c) rat uterus, (d) guinea-pig trachea and (e) rat white adipocyte lipolysis. Responses are expressed as a percentage of the m a x i m u m response to isoprenaline as described in the text.
11(33
103
O.
10
[AGONIST] (g)
1104
10 3
314 (6.7(4.5-9.9) x 10 8 M) >/ B R L 35135 (9.4(4.718.7) x 10 8 M) > salbutamol (3.4(2.0-5.8) x 10 -6 M) > B R L 28410 (1.9(0.8-4.5) x 10 4 M) and B R L 35113. Prenalterol (0.91 + 0.03), B R L 35135 (0.81 + 0.05), salbutamol (0.86 + 0.04), B R L 28410 (0.68 + 0 . 0 7 ) and B R L 35113 (0.30 + 0.12) were partial agonists.
3.3. Relaxation of uterus Relaxation of K + - c o n t r a c t e d rat uterus preparations by the same agonists is shown in fig. 2c. The potency order differed from that on atria and was as follows: isoprenaline (3.1(2.4-4.1)x 10 - 9 M ) > f e n o t e r o l (1.7(0.63-4,6) x 1 0 - 8 M ) > salbutamol (2.4(1.7-3.5) x 10 -8 M) > B R L 35135 > B R L 28410 (9.6(5.9-15.0)x 10 6 M ) > B R L 35113 (1.2(0.53-2.6) X 1 0 - s M) > prenalterol. B R L 28410 (0.82 + 0.01) and B R L 35113 (0.86 + 0.04) were partial agonists. B R L 35135 and prenalterol produced variable responses in this tissue making estimation of ECs0 and R I A values difficult. B R L 35135 produced a shallow biphasic concentrationresponse curve. Prenalterol was an agonist only at very high concentrations, and in some preparations produced contraction of the tissue.
3. 4. Relaxation of trachea The agonist p o t e n c y order for relaxation of guinea-pig intrinsic tone tracheal preparations (fig. 2d) was similar to that observed on uterus except for increased p o t e n c y of prenalterol. The p o t e n c y order was isoprenaline (2.3(1.1-3.9) x 10 - 9 M) > fenoterol (3.3(1.8-6.2) X 10 - 9 M) > salbutamol (2.6(1.2-5.6)x 10 -8 M ) > B R L 35135 (3.4(1.5-7.8) x 10 -8 M ) > prenalterol > B R L 28410 (1.8(1.42.3) x 10 5 M ) > B R L 35113 (2.1(1.2-3.8) x 10 s M). B R L 35135 ( 0 . 8 5 _ 0.05), prenalterol (0.46 + 0.08) and B R L 35113 (0.93 + 0.02) were partial agonists.
3.5. Stimulation of adipocyte lipolysis The agonist p o t e n c y order for adipocyte lipolysis (fig. 2e) differed from that observed on left and right atria or uterus and trachea. The order was isoprenaline (1.3(1.0-1.5)x 10 -8 M ) > B R L 35135
(1.6(0.9-2.0) X 10 8 M) > fenoterol (4.6(4.3-4.8) x 10 7 M) > B R L 35113 (8.7(5.0-15.0) x 10 -7 M) > B R L 28410 (3.7(2.4-5.9) x 10 6 M) > salbutamol (1.2(0.49-2.7) x 1 0 - s M) > prenalterol. B R L 35135 (0.91 + 0.03), B R L 35113 (0.88 + 0.04), B R L 28410 (0.86 + 0.02), salbutamol (0.84 + 0.05) and prenalterol (0.14 + 0.06) were partial agonists. Selectivity indices for lipolytic responses over atrial rate responses and for lipolytic responses over tracheal responses were calculated for each agonist relative to isoprenaline (table 1). The values > 1 for B R L 28410, B R L 35113 and B R L 35135 demonstrate selectivity for lipolytic over atrial or tracheal responses. Fenoterol and salbutamol demonstrated selectivity for tracheal over lipolytic responses and prenalterol was selective for atrial over lipolytic responses (values < 1). Selectivity indices were calculated using ECs0 values obtained at 50% of the agonist's own maxim u m response, since 50% of the isoprenaline maxim u m response was not always attained. This procedure m a y in fact under-estimate the selectivity of B R L 28410, B R L 35113 and B R L 35135. Higher selectivity indices for these c o m p o u n d s are obtained when it is possible to use ECs0 values at 50% of the isoprenaline m a x i m u m response. Atrial, uterine and tracheal responses were measured at a time when they were maximal for each TABLE 1 Indices of selectivity for fl-adrenoceptor agonists relative to isoprenaline. Values > 1 indicate relative selectivity for lipolysis over atrial or tracheal responses, while values < 1 indicate the reverse. Selectivity values have been calculated as: (agonist ECs0 (% own max.) for atria or trachea)/ (agonist ECs0 (% own max.) for lipolysis) (isoprenaline ECso for atria or trachea)/ (isoprenaline ECso for lipolysis) Agonist Isoprenaline Fenoterol Salbutamol Prenalterol BRL 28410 BRL 35113 BRL 35135
Lipolysis Atrial rate
Trachea
1 0.91 0.82 0.036 112 160 33
1 0.038 0.026 0.42 30 121 7.1
315 TABLE 2
3.6. Antagonist studies
The relative potencies of isoprenaline, BRL 28410 and BRL 35135 for stimulation of glycerol release from adipocytes over the 0-30 and 10-30 rain time periods.
pA 2 values for fl-adrenoceptor antagonists were obtained on adipocytes, left atrium and trachea (table 3). Irrespective of the agonist used, pA 2 values for propranolol were some 2 log units lower on adipocytes than on left atrium or trachea. The pA 2 value for antagonism of the lipolytic effect of isoprenaline by propranolol was not affected by removal of adenosine by inclusion of adenosine deaminase (1/~g m l - 1 ) in the incubation medium. Practolol, a ill-selective antagonist, was a less effective antagonist on adipocytes than on left atrium, although the slope of the Schild plot in the former was less than unity. Similarly, ICI 118,551 a fiE-selective antagonist, was a less effective antagonist on adipocytes than on trachea. Sotalol, which like propranolol is non-selective between illand fl2-adrenoceptors, but in contrast to propranolol is hydrophilic (Hachisu and Koeda, 1980), was also a less effective antagonist on adipocytes than for /31- or fl2-adrenoceptor mediated responses in other tissues. A further study was undertaken to test whether differences in experimental conditions could explain why pA 2 values obtained on adipocytes were lower than those obtained on left atrium and trachea. Only lipolytic experiments included bovine serum albumin and it has been shown that albumin may bind fl-adrenoceptor antagonists reducing their effective concentration (Zaagsma et al., 1977). Furthermore insulin contained within albumin may reduce receptor affinity for antagonists (Bertholet et al., 1981). However, when the albumin concentration was reduced to 0.1% (and incubation time to 20 min), the pA 2 value for propranolol with isoprenaline as agonist was 6.7, which is similar to the value of 6.6 when the standard 4% albumin was used. Moreover, when insulin was removed from the albumin (4%) by charcoal adsorption, a pA 2 value of 6.6 for propranolol was again obtained. The effect of antagonist pre-incubation on pA 2 value was also studied since pre-incubation was used in all but the lipolysis experiments. The standard procedure for lipolysis was followed except that the adipocytes were pre-incubated for 30 rain at 37°C with propranolol 10 -6 M before addition of the isoprenaline. A pA z value of 7.0 was obtained.
Relative rates of glycerolrelease Isoprenaline 10 -5 M Isoprenaline 10-7 M BRL 28410 10-5 M BRL 35135 2×10 -s M
0-30 min 100 a 52 60 48
10-30 min 100 b 53 65 56
a Rate of glycerol release = 6.8 #mol/min per g adipose tissue. b Rate of glycerol release = 8.7 #mol/min per g adipose tissue. concentration of agonist, whereas lipolytic responses were measured over the first 30 rain of exposure to the agonist. This exposure time was chosen for the lipolysis experiments because preliminary experiments showed that rates of lipolysis in the presence of isoprenaline, BRL 35135 and BRL 28410 were close to linear over the first 30 rain, except for some lag during the first 10 min. Furthermore, in preliminary experiments the relative potencies of isoprenaline, BRL 35135 and BRL 28410 were similar over the 0-30 and 10-30 min periods (table 2). To investigate the possibility that the selectivity of the BRL compounds was due to their antagonizing the antilipolytic effect of adenosine released from the fat cells (Fredholm, 1978), control experiments were performed in the presence of adenosine deaminase at a concentration of 1 /~g m l - 1. We have found that this concentration causes maximal stimulation of lipolysis. Addition of adenosine deaminase caused similar shifts to the left in the dose-response curves for isoprenaline (5-fold shift) and BRL 28410 (3-fold shift), which suggests that antagonism of the adenosine receptor by BRL 28410 cannot account for its selectivity as a stimulant of lipolysis. To investigate the possibility that uptake and metabolism may have reduced the potency of isoprenaline in adipocytes, experiments were performed which included metanephrine, an extraneuronal uptake inhibitor, or tropolone, a catechol-O-methyl transferase inhibitor, in the incubations. Neither metanephrine 5 × 10 -5 M nor tropolone 10 -4 M produced a shift to the left of the isoprenaline concentration-response curve.
316 TABLE 3 pA 2 values for fl-adrenoceptor antagonists on rat white adipocyte lipolysis, rat left atrial tension stimulation and guinea-pig intrinsic tone tracheal relaxation. Response Lipolysis
Agonist Isoprenaline Fenoterol BRL28410 BRL 35113 BRL 35135
Propranolol 6.6 6.3 6.2 6.2 6.2
Practolol 4.9 d
Atrial t e n s i o n
Isoprenaline Fenoterol BRL 28410 BRL 35135
8.7 8.2 8.7 8.8
6.5
Isoprenaline Fenoterol BRL 28410
8.3
4.5 "
Trachea
8.6
ICI 118,551 5.8 5.5 5.2
Sotalol 5.6 4.8 4.3
6.3 6.8 b,~
6.7 6.8 6.5 8.7 b 8.0 d
a From Hachisu and Koeda (1980). b From O'Donnell and Wanstall (1980). ~Using guinea-pig atrial rate. d Slope of Schild plot < 1 (P < 0.05).
4. Discussion Characterisation of the rat lipolytic adrenoceptor in terms of the fll/fl2 subdivision has proved difficult (Stanton, 1972; Harms et al., 1974; H a r m s and Van Der Meer, 1975; H a r m s et al., 1977; De Vente et al., 1980; H a r m s et al., 1982; Bojanic et al., 1984). This task has now been facilitated by the availability of three novel fl-adrenoceptor agonists, BRL 28410, BRL 35113 and BRL 35135, which demonstrate selectivity for the rat adipocyte lipolytic response. Strong evidence that the novel compounds stimulate the lipolytic response by interaction with the fl-adrenoceptor has come from the finding that fl-adrenoceptor antagonists have similar pA 2 values for antagonism of responses to the novel compounds and traditional fl-adrenoceptor agonists (table 3). There appears to be no a-adrenoceptor involvement in lipolysis in the rat (Guidicelli et al., 1981), and the antagonism of the response to fl-adrenoceptor agonists by fl-adrenoceptor antagonists is stereoselective (Harms et al., 1977; Fassina, 1966). The lipolytic response to the novel fl-adrenoceptor agonists, in common with that to the s t a n d a r d fl-adrenoceptor agonists, was inhibited in a competitive manner by fl-adrenoceptor antagonists (except for practolol). Results from both agonist
and antagonist experiments therefore suggest that lipolysis in the rat is mediated by fl-adrenoceptors. However, the data are inconsistent with the response being mediated either by adrenoceptors of the ill-subtype, of the fla-subtype or a heterogeneous population of both subtypes. Both rate and tension responses of rat atria to fl-adrenoceptor agonists are mediated by adrenoceptors of the ill-subtype (Bryan et al., 1981). The low RIA and high ECs0 values of BRL 28410 and BRL 35113 in these tissues therefore indicate poor fll-agonist activity. BRL 35135 was more potent than BRL 28410 and BRL 35113 in these tissues, but was not more potent than the fl2-selective agonist fenoterol. Relaxation of guinea-pig trachea by fl-adrenoceptor agonists involves predominantly fl2-adrenoceptors, although a small proportion of fll-adrenoceptors has been demonstrated by radioligand binding (Engel et al., 1982) and pharmacological studies (Furchgott, 1976). Relaxation of the rat uterus is also mediated by fl2-adrenoceptors (Lands et al., 1967b). Consistent with this classification, the fiR-selective agonists fenoterol and salbutamol were much more potent on trachea and uterus than on atria, whereas the fl2-selective agonist prenalterol exhibited the reverse selectivity. BRL 28410 and BRL 35113 had higher RIA on trachea
317
and uterus than on atria but remained poor agonists in terms of ECs0 values. BRL 35135 was slightly more potent on trachea than on atria but the response on uterus was difficult to interpret. It is possible that higher concentrations of BRL 35135 produced relaxation not mediated by fl2-adrenoceptors in this tissue. Isoprenaline, which is non-selective between ill" and fl2-adrenoceptors, was a less potent stimulant of lipolytic responses than of responses mediated by ill- or fl2-adrenoceptors. This difference was not due to a more avid extra-neuronal uptake or metabolism of isoprenaline in adipocytes since inhibition of both uptake 2 and catechol-O-methyltransferase by metanephrine and tropolone respectively did not potentiate the lipolytic response to isoprenaline. Prenalterol was a much weaker agonist on adipocytes than on atria and fenoterol and salbutamol were much weaker agonists on adipocytes than on uterus and trachea. These low agonist potencies could be explained by a smaller receptor reserve in adipocytes than in the other tissues. However, this explanation cannot account for the selectivity demonstrated by BRL 28410, BRL 35113 and BRL 35135. These three agonists were more potent, in terms of ECs0 values, on adipocytes than on atria, uterus or trachea. When expressed relative to isoprenaline, this selectivity was considerable (table 1). Selective agonist studies therefore suggest that the rat adipocyte lipolytic receptor differs from that in rat atria, rat uterus or guinea-pig trachea. Antagonist studies support this hypothesis. Propranolol, which is non-selective between ill- and fl2-adrenoceptors, was considerably less effective in antagonizing lipolytic responses than atrial or tracheal responses whichever agonist was used. Propranolol has been shown to bind to bovine serum albumin in vitro (Zaagsma et al., 1977) and this could lower its apparent pA 2 value. However, in the present studies, reduction of the albumin concentration from 4% to 0.1% had little influence on the pA 2 value and thus albumin binding cannot explain the difference in pA 2 values for propranolol between lipolysis and the other responses. Furthermore, the pA 2 value for sotalol, a nonselective fl-adrenoceptor antagonist which does not bind to albumin due to its hydrophilic nature
(Hachisu and Koeda, 1980), was also much lower for inhibition of lipolysis than reported for inhibition of other fl-adrenoceptor mediated responses. Bertholet et al. (1981) reported that insulin, which is present in variable amounts in all commercially available bovine serum albumin preparations, can reduce fl-adrenoceptor antagonist pA 2 values. The presence of insulin in lipolysis experiments cannot however account for the pA 2 differences, since the pA 2 value for propranolol was unaltered when albumin in which insulin had been removed by charcoal adsorption was used. Propranolol pA 2 values may also be reduced if insufficient time is allowed for equilibration with the tissue before addition of the agonist (Grana et al., 1972). In the present experiments atria and trachea were incubated for 30 min with antagonist before exposure to agonist, whereas adipocytes received agonist and antagonist simultaneously. Pre-incubation of adipocytes for 30 min with propranolol yielded a slightly higher pA 2 value than when there was no pre-incubated. However, a much larger increase in pA 2 value than that observed would be required to account for the pA 2 difference between adipocytes and other tissues. Both ill- and fl2-selective antagonists were also relatively poor inhibitors of the lipolytic response. The pA 2 value for practolol (flcselective) was much lower on adipocytes than on atria and the pA 2 value for ICI 118,551 (fl2-selective)was much lower on adipocytes than on trachea. Therefore, irrespective of ill- or fl2-selectivity, fl-adrenoceptor antagonists were poor inhibitors of fl-adrenoceptor agonist stimulated lipolysis. These results cannot be explained by the different experimental procedure used in lipolysis and must therefore indicate the involvement of a non ill- or fl2-adrenoceptor. The hybrid fll/fl2 receptor proposed by Harms et al. (1974) and De Vente et al. (1980) adequately explains the results obtained by these authors. This model however, does not readily explain the present results. It is difficult to envisage a lipolytic receptor with ill- and fl2-adrenoceptor characteristics when, for example, BRL 35113 is a poor agonist at both ill- and fl2-adrenoceptors yet is a potent agonist at the lipolytic fl-adrenoceptor. It is possible that the rat lipolytic fl-adrenoceptor is structurally more primitive and less complex than
318 fl~- or fl2-adrenoceptors, as suggested by Bertholet et al. (1981). I n other species, the lipolytic fl-adrenoceptor m a y or m a y n o t be similar to that in the rat. A heterogeneous p o p u l a t i o n of ill- plus flz-adrenoceptors has been d e m o n s t r a t e d in hamster (Bjorgell a n d Belfrage, 1982) a n d dog (Belgrage a n d F r e d h o l m , 1978). The lipolytic receptor in swine has not b e e n clearly characterized (Hruska, 1981). f l - A d r e n o c e p t o r a n t a g o n i s t pA2 values for guineapig adipocyte responses were lower t h a n atrial or tracheal responses (Bertholet et al., 1981) although the difference for p r o p r a n o l o l was m u c h lower t h a n f o u n d in the rat (this paper). I n man, high p A 2 values for non-selective, fla-selective a n d fl2selective a d r e n o c e p t o r a n t a g o n i s t s were f o u n d on lipolysis, suggesting a mixed fll plus f12 p o p u l a t i o n ( W e n k e o v a et al., 1976). However, the correlation of p A 2 values for a n u m b e r of fl-adrenoceptor a n t a g o n i s t s b e t w e e n h u m a n a n d rat lipolytic responses has suggested a similarity of the receptor type in b o t h species ( H a r m s et al., 1982). fl-Adrenoceptors m e d i a t i n g other metabolic responses have not b e e n clearly characterized. A h r e n a n d L u n d q u i s t (1981) showed that the fl-adrenoceptor regulating i n s u l i n secretion in vivo in the m o u s e does not fit into the /~1///~2 subdivision. B R L 35135 is a p o t e n t i n s u l i n secretagogue in vivo (M.A. Cawthorne, personal c o m m u n i c a t i o n ) as is B R L 26830, the p a r e n t ester of BRL 28410 ( C a w t h o r n e et al., 1982). Therefore it m a y be that the atypical fl-adrenoceptor which mediates lipolysis in the rat is similar to that involved in i n s u l i n secretion or other metabolic responses. I n conclusion, synthesis of a novel series of f l - a d r e n o c e p t o r agonists has e n a b l e d further characterization of the fl-adrenoceptor m e d i a t i n g lipolysis in the rat. Both agonist a n d a n t a g o n i s t studies have shown this receptor to be distinct from either the ill- or flz-adrenoceptor subtype. I n v o l v e m e n t of this receptor type in lipolysis in other species or i n v o l v e m e n t in other metabolic responses r e m a i n s to be the determined.
Acknowledgements The authors would like to thank Miss C. Duffett, Mr. R. Ellis, Mr. M. Khan and Mrs. V. Thody for their technical
assistance in this work, and Miss C. Saunders for typing the manuscript. They would also like to thank the companies concerned for gifts of drugs used.
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