Comp. Biochem. Physiol. Vol. 106C, No. 3, pp. 125-132, 1993 Printed in Great Britain

Pergamon Press Ltd

BINDING OF AGONISTS AND ANTAGONISTS TO THE PORCINE ADIPOSE TISSUE j?-ADRENERGIC

RECEPTOR(S)

HARRY J. MERSMANN, KAMIL AKANBI, ALLA SHPARBER and RONALD L. MCNEEL USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine,

Houston, TX 77030, U.S.A. (Tel. 713-798-7128; Fax 713-798-7130)

(Received 21 June 1993; accepted for publication 6 August 1993)

Abstract-l. Affinities of agonists for porcine adipose tissue j?-adrenergic receptors, determined by competitive ligand binding with ‘H-dihydroalprenolol to crude adipose tissue membranes in uifro, varied from 50 times > to 25 times < than isoproterenol. Affinities for antagonists varied from 8 times > to 1000 times < propranolol.

2. Receptor affinity was not related to the ability to stimulate or inhibit lipolysis, or to the agonist or antagonist purported receptor subtype specificity.

3. Modeling of l&and-binding data indicated more than one binding site for several ligands. The assignment of B-adrenergic subtypes to the individual binding sites was unclear because this would depend on the individual ligands used to establish binding sites.

INTRODUCTION

Properties of the porcine adipose tissue /?-adrenergic receptor have been ascertained primarily by study of the metabolic pathways modulated by the receptor(s). Much of this information was obtained by the study of lipolysis (Mersmann et al., 1974; Mersmann, 1984a,b, 198’7; Liu et al., 1989; Liu and Mills, 1989), with additional efforts directed toward anabolic path- ways, particularly de nova fatty acid biosynthesis (Liu et al., 1989; Mersmann 1989). Properties of a receptor may be determined more directly by using ligand binding as an experimental approach. Ligand binding to the porcine adipose tissue @-adrenergic receptor was first reported in Bockien et al. (1986); the tech- nique was used to measure receptor affinity and number in several types of pigs. Details of receptor- binding and of properties of the porcine adipose tissue ~-adrener~c receptor(s) have been recently published (Coutinho et al., 1992; Mersmann and McNeel, 1992a,b; Spurlock et al., 1993a,b).

Pharmacological approaches to the study of /?- adrenergic receptor(s) in porcine adipose tissue using tissue metabolic activity, or the more direct approach of ligand binding have not clearly indicated the receptor subtype(s) present in this tissue; nor have they allowed prediction of the ability of an adrenergic agonist to change plasma-free fatty acids or glycerol concentration when infused intravenously

Abbreviations used: B,,,,, maximum binding of ligand; DHA, dihydroalprenolol; EGTA, ethyleneglycol-bis (B- aminoethyl) N,N,N’,N’-tetraacetic acid, K+,, concen- tration of competive ligand to yield 50% inhibition of the binding of 2.5 nM DHA; &, dissociation constant for DHA, Ki, inhibitory constant calculated according to Cheng and Prusoff (1973).

(Mersmann, 1986, 1987). Because the first step for any agonist or antagonist to modulate a receptor- mediated function is to bind to the receptor, we have measured ligand binding to the porcine adipose tissue b-adrenergic receptor(s) with a variety of agonists and antagonists in an attempt to define the receptor subtypes present in this cell. We also compared binding affinity to the ability of the agonist or antagonist to stimulate or inhibit lipolysis in vitro. These ligand-binding approaches did not clarify receptor subtype(s), nor were they predictive of lipolytic activity in vitro or in viva

MATERIALS AND METHODS

Castrated male crossbred pigs (Sus domesticus; Dekalb terminal cross hybrids; major breeds are Duroc, Hampshire, Landrace, and Yorkshire) were purchased from a local commercial producer. They were maintained at this laboratory for 1 to 25 d, given free access to water and a standard corn-soybean meal ration, and weighed 20-32 kg when killed with a captive bolt gun and exsanguinated. The experimen- tal protocol was approved by the Bayfor College of Medicine Institutional Animal Care and Use Com- mittee.

Subcutaneous adipose tissue (combined upper and middle layers) was obtained from the dorsal neck region, transported to the laboratory (< lOmin), sliced and digested with collagenase to yield isolated adipocytes. Details of tissue transport, slicing and adipocyte isolation have been described (Mersmann and Hu, 1987; Mersmann and McNeel, 1992a).

Isolated adipocytes were lysed in hypotonic medium that contained 7.5 mM EGTA (ethylenegly col-bis (/I-aminoethyl) N,N,N’,N’-tetraacetic acid)

725

126 H. J. MERSMANN et al.

Table 1. Competitive binding of agonists

Compound K, nMt

Specificity’ nt Mean SD Dose range (M)

Isoproterenol 1+2 6 280 161 7.56 x IO-‘O-7.56 x IO-’ Norepinephrine 1+2 2 I7347 10879 9.40 x lo-‘0-9.40 x 10-4 Epinephrine 1+2 2 9993 8566 9.00 x lo-‘0-9.00 x lo-’ Fenoterol 2 2 917 534 7.81 x IO-‘O-7.81 x 10m4 Metaproterenol 2 2 4665 1525 5.74 x lo-‘O-5.74 x 10-4 Terbutaline 2 2 5415 3493 5.44 x 1om’0-5.44 x 10-4 Salbutamol (Albuterol) 2 2 2619 2092 5.20 x lo-‘O-5.20 x 10m4 Quinterenol 2 2 6446 297 I 9.38 x IO-‘O-9.38 x 1O-4 Zinterol 2 2 68.5 39.1 7.18 x IO-‘O-7.18 x IO-’ Clenbuterol 2 2 4.68 1.13 9.52 x 10-‘0-9.52 x lo-’ Tazolol 1 2 72.0 53.7 1.16 x 10~9~l.16 x lo-’ Dobutamine I 2 2500 1209 8.88 x lo-‘9-8.88 x IO_’ Dopamine 2 2959 2288 1.58 x 10~9-l.58 x 10-j Catechol 2 t 2.73 x W-2.73 x IO-”

*Purported /7-adrenergic receptor subtype specificity. tAbbreviations: n = number of experiments, each with tissue from a different animal.

K, = inhibitory constant calculated according to Cheng and Prusoff (1973); it represents 50% inhibition of the binding of 2.5 nM )H-DHA.

$35% inhibition at 2.73 x IO-’ M.

and 0.8 mM phenylmethyl-sulfonyl fluoride as protease inhibitors. The pellet obtained by centrifu- gation at 30,OOOg for 30 min was used as a crude plasma membrane source (ghosts) for ligand-binding studies; details of ghost preparation have been de- scribed (Mersmann and McNeel, 1992a). Ghosts were frozen in liquid nitrogen and stored at -70°C until used for binding experiments. The preparation was thawed rapidly at 37”C, maintained in an ice bath, used within 30 min of thawing to initiate binding, and only thawed once.

Dihydroalprenolol (DHA’) was used as the triti- ated ligand (dihydroalprenolol hydrochloride, leoo- [propyl- 1 ,2,3-3H] from NEN Research Products, DuPont, Wilmington, DE) to measure equilibrium binding of the ligand or competitive binding by an agonist or antagonist, in the crude preparation of plasma membranes. Approximately 200 pg of the crude membrane protein was incubated in a total volume of 150 ~1 at 25°C for 30min. Equilibrium saturation binding was measured by variation of the DHA concentration. Competitive ligand binding was determined with a constant concentration of DHA (2.5 nM); the competitive ligand was varied over the

dose ranges indicated in Tables 1 and 2. Minimal dose curves (Tables l-5) varied the competitive ligand by orders of magnitude, whereas extended dose curves (Tables 3,6 and 7) varied the competitive ligand using intermediate points (e.g. 10d9 M, 2.5 x 10m9 M, 5 x 10m9 M, 10e8 M, etc.). Details of the ligand-binding procedures have been described (Mersmann and McNeel, 1992a,b). The binding affinity for DHA’ (& = equilibrium dissociation con- stant or the concentration to yield 50% saturation of specific binding) and receptor number (B,,,) were obtained by computer analysis using the McPherson (1985) modification of the nonlinear curve- fitting program, LIGAND (Munson and Rodbard, 1980). The competitive binding constants (IC,, = concentration of competitor to yield 50% inhibition of the binding of 2.5 nM DHA) for the various agonists and antagonists were obtained by computer analysis of the data using the EBDA portion of LIGAND. The Ki or equilibrium dissociation con- stant for a competing ligand was calculated from the IC,, by the formula of Cheng and Prusoff (1973) using an average K,, for DHA of 3.6 nM. Most competitive ligand-binding studies were designed to study three

Table 2. Comoetitive bindinn of antagonists

Compound

Propranolol

K, nMt Specificity* nt Mean SD Dose range (M)

1+2 7 3.51 3.35 1.01 x lo-“-1.01 x 10-s Alpienolol 1+2 2 2.91 0.70 1.05 x 10~“-1.05 x IO_ 5 Pindolol I+2 2 1.19 1.36 1.21 x lo-“-1.21 x 10-s Butoxamine 2 2 1965 321 9.87 x IO-‘O-9.87 x IO-’ H35/25 2 2 771 318 1.25 x IO-‘-l.25 x IO-’ ICI 118.551 2 I 80.3 9.55 x lo- ‘O-9.55 x 10-4 CGP 12,177 2 2 0.91 I 0.618 9.49 x lo-‘Z-9.49 x 10-6 Practolol 1 3 1760 818 1.14 x 10-9-1.14 x 10-j Atenolol I 2 4573 2167 I.13 x 10-9-1.13 x 10-j Metoprolol I 2 1848 1094 7.19 x lom’0-7.19 x 10-4 ICI 89,406 1 2 50.3 52.4 8.48 x IO-‘O-8.48 x lO-4 CGP 20,712 1 3 29.8 7.7% 5.09 x lo- ‘Z-5.09 x lomd

*Purported fl-adrenergic receptor subtype specificity. tAbbreviations: n = number of experiments, each with tissue from a different animal;

K, = inhibitory constant calculated according to Cheng and Prusoff (1973); it represents 50% inhibition of the binding of 2.5 nM lH-DHA.

IInhibition = 53.3 (SD = 9.3) % of propranolol at 5.09 x IOm4M.

Adipose tissue /I-adrenergic receptors 721

Table 3. Comoarison of minimal and extended dose-resoonse curves

GO* Compound Minimal Extended

ICI 118,551 600 (16) 328 (12) H35/25 1514 (60) I370 (25) Fenoterol 913 (53) 870 (37)

1733 (30) 1706(18) Atenolol 80,505 (29) 60,226(18) ICI 89,406 495 (23) 334(10)

13 (144) 48 (43) Metoprolol 3653 (56) 1330 (29)

1395 (38) 1535 (24) 972 (25) l312(14) 968 (18) 922 (13)

1022 (26) 1215(18)

SlOpe’ Minimal Extended

0.93 (8) 0.84 (5) I .08 (38) 1.05(15) 0.67 (22) 0.64(16) 0.82 (17) 0.77 (IO) I .37 (37) 1.07(13) I.71 (38) 1.14(S) 0.41 (31) 0.62 (15) 0.69 (22) 0.53 (8) 0.63 (13) 0.67 (8) 0.63 (9) 0.77 (7) 0.70 (7) 0.68 (5) 0.82 (12) 0.85 (9)

Number of concentrations used in dose-response curve for competi- tive binding. Minimal curve (min) was represented by variation of dose range indicated in Tables I and 2 by orders of magnitude (lO-9 M, IO-* M, etc.) whereas, the extended curve (ext) was represented by variation of the same dose range with inter- mediate points (10m9 M, 2.5 x 10m9 M, 0.5 x 10m9 M, lOm8 M, 2.5 x 10-s M, etc.).

‘The II+,, or the concentration to yield 50% inhibition of binding of 2.5 nM ‘H-DHA and its (coefficient of variation) as well as the slope of the inhibition curve slope (coefficient of variation) were obtained from the EBDA portion of the LIGAND pro- gram (McPherson, 1985).

ligands plus a standard ligand with the same ghost preparation on the same day. Thus, the Ki obtained for the ligands could be related to the K, for the standard obtained on the same ghost preparation on the same day to eliminate some of the variation. The standard was ( + )-propranolol for antagonists and ( - )-isoproterenol (ISO) for agonists. The extended competitive binding curves were further analyzed by the ligand portion of LIGAND to give the K1 and K, (the Ki for a high and low affinity binding site, respectively) and the proportion of each of the bind- ing sites detected.

Compounds were obtained from Boehringer-Ingel- heim (fenoterol: HBr, metaproterenol : 1/2H, SO,, clenbuterol : HCl), Burroughs Wellcome (butoxam-

ine: HCl), Ciba-Geigy (metoprolol : tartarate, terbu- taline: 1/2HzS0,, CGP 12,177, CGP 20,712), Glaxo (salbutamol = albuterol), Hassle (H35/25 : HCl, metroprolol : tartarate), Imperial Chemical Industries (atenolol, ICI 118551: HCl, ICI 89,406), Lilly (dobu- tamine: HCl), Mead-Johnson (zinterol : HCl), Pfizer (quinterenol : HCl), Sandoz (pindolol), Schering (al- buterol = salbutamol), Sigma [L-alprenolol: tartrate, catechol, dopamine: HCl, ( - )-epinephrine bitar- trate, ( - )-isoproterenol : bitartrate, ( - )-norepi- nephrine bitartrate, rn,-propranolol : HCl], and Syntex (tazolol : HCl). All compounds were generous gifts from the companies mentioned, except those purchased from Sigma.

RESULTS AND DISCUSSION

DHA binding properties

Equilibrium saturation curves were generated for DHA binding to ghost preparations from seven different pigs. The mean (SD; range) for the seven preparations was: protein = 2.17 (0.63; 1.67-3.45) mg/ml; K,, = 3.86 (1.63; 1.61 to 4.37) nM; B,,,,, = 263 (76; 191-383) fmol/mg protein. The estimated standard error for determination of an individual Kd was approximately 400% and that for the B,,,,, was approximately 25%.

Membrane stability

Ghost preparations from five pigs were assayed and then frozen in liquid nitrogen, stored at -70°C thawed rapidly at 37°C and placed on ice; a pooled preparation of equal portions of each ghost prep- aration was assayed on days 7, 14, 28, and 84 of storage at -70°C. Assay by generation of DHA saturation curves produced a mean (SD) for the K,, of 3.4 (0.5) nM and for the B,,,,, of 280 (23) fmol/mg

Table 4. Relative potency and efficacy for ligand binding and stimulation of lipolysis by agonists

Compound

Clenbuterol Zinterol Tazolol Isoproterenol Fenoterol Salbutamol (albuterol) Dobutamine Epinephrine Dopamine Metaproterenol Terbutaline Quinterenol Norepinephrine Catechol

Speciticityt

2 2 I

1+2 2

2 I

1+2 2 2 2 2

1+2

Potency Efficacy

Binding Lipolysis’ Binding Lipolysis’ Mean SD Without Theo With Theo Mean Without Theo With Theo

0.020 0.001 nr 5.95 100 <20 72 0.276 0.281 nr 0.34 IO0 <20 89 0.289 0.228

It; xl 100 <20 <20

1.0 100 100 100 4.09 2.25 40 63 100 80 100

8.75 5.30 2.22 100 c30 85 10.03 3.10 9: I8 100 30 100 14.6 10.20 30 NT 100 100 NT 18.9 13.50 126 100 <20 76 19.6 6.30 loo”’ 126 100 60 100 19.6 19.50 430 I54 100 60 100 22.6 4.90 163 100 <20 64 26.2 II.10 1: NT 100 100 NT

i nr 1856 1 t20 61

Abbreviations: Theo = theopbylline; nr = not resolved because maximal response too low to generate an interpretable dose-response curve; NT = not tested. Potency = the binding affinity expressed as K, (Table I) relative to the K, for isoproterenol in the same experiment or the ED~ for lipolysis relative to the ED,, for isoproterenol in the same experiment; Efficacy = the extent of inhibition of binding of ‘H-DHA relative to that of isoproterenol or the extent of stimulation of lipolysis relative to isoproterenol.

*The lipolysis data are from Mersmann, 1984a,b, 1987 (without Theo) and Mersmann, 1992 (with Theo). tpurported B-adrenergic receptor subtype specificity. 135% inhibition at IO-’ M.

728 H. J. MERSMANN et al.

Table 5. Relative potency and efficacy for l&and binding and inhibition of lipolysis by antagonists

Potency Efficacy

Binding Lipolysis’ Lipolysis’ Compound Specificityt Mean SD Without Theo With Theo Binding Without Theo With Theo

Pindolol If2 0.128 0.097 0.5 0.055 100 100 100 CGP 12,177 2 0.398 0.325 NT 0.008 100 NT 68 Alprenolol 1+2 0.925 0.288 1.3 0.67 loo 100 100 Propranolol 1+2 1.0 1 .oo 1.0 100 100 100 100 CGP 20,712$ I 5.59 3.82 NT 19.0 53 NT 82 ICI 89,406 I 5.64 3.30 NT 0.293 100 NT 77 ICI 118,551 2 58.6 52.0 8.1 100 100 100 100 H35/25 2 302 68 1758 4.4 100 88 100 Metoprolol 1 349 148 10 5.9 100 100 100 Butoxamine 2 791 26 888 11.1 100 67 100 Practolol 1 1032 103 61 10.1 100 100 100 Atenolol 1 1162 839 27 8.7 100 100 100

Abbreviations: Theo = Theophylline; NT = not tested; Potency = the binding affinity expressed K, (Table 1) relative to the K, for isoproterenol in the same experiment or the ED~ for lipolysis relative to the ED~ for isoproterenol in the same experiment; Efficacy = the extent of inhibition of binding of ‘H-DHA relative to that of isoproterenol or the extent of inhibition of lipolysis relative to propranolol.

*The lipolysis data are from Mersmann, 1984a,b, 1987 (without Theo) and Mersmann, 1992 (with Theo). TPurported /3-adrenergic receptor subtype specificity. IBinding by DHA was inhibited only 50% by CGP 20,712 (Table 2).

protein. The greatest difference from the day 0 value for I$,,,, was an 18% decrease at 14 d; however, there was no trend for decrease with storage time. Our experience with numerous ghost preparations has been that the E(d does not change with storage under these conditions for up to at least three months; the B,,,,, may decrease up to 15% over three months, but this is not totally clear because the assay variation is at least this great.

a K, for clenbuterol of approximately 150 nM with membrane preparations from 100 kg pigs; we observed a K, of approximately 5 nM; we have no explanation for the discrepancy between labora- tories. Examination of the data in Table 1 indicates no apparent relationship between the K, and the purported receptor subtype specificity of the ligand.

Competitive binding-agonists (Table 1) Competitive binding-antagonists (Table 2)

Isoproterenol was the standard agonist used in every ligand-binding experiment for agonists; the mean isoproterenol K, for six different membrane preparations was 280 nM. All agonists tested totally inhibited DHA binding to the residual or non- specific binding level observed with 10m5 M or greater concentration of isoproterenol. Catechol with no ethanolamine side chain, was only a partial inhibitor of DHA binding. A few agonists had greater affinity, or potency (smaller K,) for the receptor than isoproterenol (e.g. zinterol, clenbuterol, and tazolol), but most agonists had less affinity (greater Ki) than isoproterenol. Spurlock et al. (1993b) indicated

Propranolol was the standard antagonist used in every ligand-binding experiment for antagonists; the mean propranolol Ki for seven different membrane preparations was 3.5 nM. All antagonists, except CGP 20,712, totally inhibited DHA binding to the residual or non-specific binding level observed with 10e6M or greater concentration of propranolol. A few antagonists had greater affinity for the receptor than propranolol (e.g. alprenolol, pindolol and CGP 12,177), but most had less affinity than propranolol. Examination of the data in Table 2 indicates no apparent relationship between the K, and the pur- ported receptor subtype specificity of the ligand.

Table 6. Extended competitive receptor-binding dose-response curves for purported fi,- or &selective adrenereic active comoounds

Competitor Specificity*

ICI 118,551 2 H 35/25 2 Fenoterol 2

Atenolol I ICI 89.406 1

One-site model

K,. nM ssq. 150 750 768 1656

1109 607 744 3048

24,277 3054 36 2699

116 1002 1.7 19.713

Two-site model

K,, nM K2, nM K, , sites, % ssq.

192 554,381 99 14,534 396 2308 74 874 67 3241 38 48 6.93 2840 53 100

785 60,934 25 98 0.025 68 40 961

NFt 0.034 83 57 464

Each competition curve was run with 2.5 nM ‘H-DHA. Competitor dose-response curves had approxi- mately IS points extending over the dose range indicated in Tables 1 and 2 (for example, 10~~’ M. 2.5 x 10m9 M, 5 x 10m9 M, IO-” M, 2.5 x IO-” M, etc). The data were analyzed with the McPherson (1985) version of LIGAND. S.Sq. = sum of squares for data fit to one- or two-site model.

*Purported p-adrenergic receptor subtype specificity. tNF = will not fit 2-site model (either ill-conditions or fits K, = 0).

Adipose tissue fi-adrenergic receptors

Table 7. Metoprolol competitive iirrand bindinn with DHA

729

One-site model Two-site model

DHA, nM K,, nM ssq. K,, nM K2, nM K,, sites, % ssq.

7.2 204 4222 0.89 I213 51 153 472 2658 22 3535 51

3.6 994 1963 14 2314 41 185 1.8 838 486 14 1264 24 172 1.35 491 3378 99 3063 53 114 0.9 698 797 28 1194 33 275

The DHA concentration was varied from 0.9 to 7.2 nM. Extended metoprolol (purported &-specific adrenergic antagonist) competitive ligand-binding dose-response curves were generated at each DHA concentration (metoprol&l used at IO-‘M, 10m8M, 2.5 x IO-* M 5 x 10-s M, lo-‘M, 2.5 x IO-‘M. etc. to IO-’ M or 17 concentrations). The data were analyzed with the McPherson (1985) version of LIGAND. SSa. = sum of squares for data fit to one- or two-site model.

Extended co~~tit~ue binding curves

Computer simulation studies (M. D. MacNeil and H. J. Mersmann, unpublished observations) suggested that minimal dose-response curves with six points varied by orders of magnitude (e.g. 1W9M, IO-* M, etc.) are not as accurate as extended dose-response curves, with 11 points varied over the same concentration range (e.g. 10m9 M, 5 x 10m9 M, lO-8 M, 5 x IO-* M, etc.). The dete~ination of the minimal and maximal values of the dose-response curves was less accurate, but the greatest inaccuracies were for the determination of the slope and the 50% response point of the dose-response curve, the EDGE or IC,,. The intermediate doses increase the number of data points used most extensively to determine the slope and midpoint, so that these two variables are better estimated. Using a one-site model (in the EBDA portion of LIGAN~McPherson, 1985) to fit competitive ligand-binding data for the porcine adipose tissue j?-adrenergic receptor (Table 3), the values for the lcSO and slope were not dramatically altered in most cases by use of an extended (e.g. 10m9M, 2.5 x 10m9M, 5 x 10m9 M, 10-‘M, etc.), compared to a minimal (e.g. 10e9M, 10-‘M, etc.) dose-response curve. However, the coefficient of variation for the estimation of the IC, and for the slope was approximately 50% less with an ex- tended, compared to a minimal curve. The minimal dose-response curves are adequate to survey a variety of compounds for relative activity; more accurate determinations of the IC,, or K, for a compound of particular interest must use the extended format. Furthermore, any attempt to decipher multiple bind- ing sites must use the extended format.

Association of ligand binding and lipolysis

Because isoproterenol was used as a standard agonist in every ligand-binding experiment, the po- tency (Ki or concentration for 50% inhibition of binding of 2.5 nM DHA) and efficacy (the extent of inhibition of DHA binding) can each be set to 1.0, so that the potency and efficacy of each of the other agonists in the experiment may be related to iso- proterenol. The use of these relative potencies and

efficacies eliminated some of the ex~~mental and animal variation. A similar approach has been used in the past for lipolysis experiments (Mersmann et al., 1974; Mersmann 1984a,b, 1987, 1992). The data in Table 4 indicate the relative potency and efficacy for ligand binding to the receptor, and for lipolysis measured in the absence and in the presence of theophylline. In a few cases (e.g. zinterol or metaproterenol~, the relative affinity for the receptor approximates the relative potency of that agonist to stimulate lipoiysis. On the other hand, agonists that were bound to the receptor with high affinity (K, < isoproterenol Ki) did not necessarily stimulate lipolysis at low concentration. For example, clen- buterol had 50 times greater affinity for the receptor than isoproterenol, but partially stimulated lipolysis only in the presence of theophylline, and then was 6 times less potent than isoproterenol. Tazolol was bound to the receptor with 3 times greater affinity than isoproterenol, but did not stimulate lipolysis with or without theophylline present in the incu- bation medium. Epinephrine and norepinephrine had relative binding potencies similar to their relative potency for stimulation of lipolysis.

Experiments with antagonists used propranolol as a standard antagonist so that relative potency and efficacy of ligand binding could be determined (Table 5). Three antagonists (pindolol, CGP 12,177, and alprenolol) were more potent than propranolol (ligand-binding K, < than K, for propranolol); these same three antagonists were also quite potent for the inhibition of lipolysis (although CGP 12,177 was quite potent for the inhibition of lipolysis, it was only a partial antagonist for lipolysis, i.e. it did not totally inhibit or was less efficacious than propranolol). In general, the potency for ligand binding did not necessarily predict the potency for inhibition of lipolysis (e.g. ICI 89,406 was 5 times less potent than propranolol for ligand binding but 3 times more potent for inhibition of lipolysis). Except for CGP 20,712 (lipolysis was also not totally inhibited), all antagonists completely inhibited binding of DHA (100% efficacy). Two antagonists that totally inhibited ligand binding, CGP 12,177 and ICI 89,406, did not totally inhibit lipolysis; CGP 20,712 did

730 H. J. MERSMANN et al.

not totally inhibit ligand binding or lipolysis. Overall, neither binding potency nor efficacy necessarily predicted the ability to inhibit lipolysis.

Subtypes of the /I-adrenergic receptor

Previous attempts to determine the b-adrenergic receptor subtype(s) present in porcine adipose tissue by measuring lipolysis were unsuccessful (Mersmann, 1984a,b, 1987, 1992). Initially, we measured lipolysis in a basic incubation medium containing Krebs- Ringer bicarbonate buffer, glucose, ascorbate, and bovine serum albumin; a dose-response curve was generated for the agonist of interest. For antagonists, a similar approach was used, with generation of a dose-response curve in the presence of a fixed concentration of isoproterenol. Using this approach, most agonists stimulated rat adipose tissue lipolysis in vitro, but there were only a very limited number of agonists that stimulated porcine adipose tissue lipol- ysis in vitro (Mersmann, 1984a,b, 1987). Measure- ment of lipolysis gave no indication that the porcine adipose tissue /3-adrenergic receptors were predomi- nantly of the /I,- or &subtype. Subsequent studies indicated that agonists that stimulated porcine adipose tissue lipolysis increased tissue CAMP con- centration, and those that did not stimulate lipolysis did not cause an increase in tissue CAMP concen- tration (Hu et al., 1987). Acute infusion into pigs of selected fl-adrenergic agonists indicated that, for the most part, agonists that were active in vitro were active in vivo; inactive agonists in vitro were inactive in vivo, except for two agonists (clenbuterol and zinterol) that partially increased plasma-free fatty acids and glycerol, but had little activity in vitro (Mersmann, 1987). Antagonists were used also in these studies in vitro and in vivo. Most antagonists totally inhibited isoproterenol-stimulated lipolysis, but again, there was no indication of predominance of a particular fi-adrenergic receptor subtype. Exper- iments with antagonists in viuo also did not clarify receptor subtypes in this tissue.

Liu et al. (1989) demonstrated that some potential agonists for the porcine adipose tissue /I-adrenergic receptor were active when theophylline was included in the incubation medium, but were inactive in a basic incubation medium without theophylline in vitro. Theophylline presumably antagonized the inhibitory adenosine receptor and/or inhibited CAMP-phospho- diesterase. The former mechanism is supported by the fact that addition of adenosine deaminase (destroys adenosine) to the incubation medium allowed the expression of agonistic effects as did theophylline; however, adenosine deaminase was not as effective as theophylline, regardless of the concentration used, suggesting that theophylline acts at sites in addition to the adenosine receptor (Mersmann, 1989). Many of the agonists that did not stimulate porcine adipose tissue lipolysis in the basic incubation medium in vitro, stimulated lipolysis when theophylline was included in the incubation medium (Mersmann,

1992). Studies of lipolysis measured in the presence of theophylline have not clarified the /I-adrenergic receptor subtype(s) present in porcine adipose tissue, and the results were no better at predicting the effects of acute infusion in vivo than incubation of the potential agonists in the basic medium. For example, clenbuterol and zinterol were not active in the basic lipolysis system in vitro, but stimulated lipolysis in vitro in the presence of theophylline, and elevated plasma-free fatty acids and glycerol to some extent when infused in vivo. The implication is that lipolysis measurements in the presence of theophylline in vitro predict lipolysis activity in vivo. However, quinterenol was inactive in the basic system in vitro and in vivo,

but stimulated lipolysis in vitro in the presence of theophylline.

Perusal of the values (Tables 1 and 2) or the relative potencies (Tables 4 and 5) for the purported /I,- or &specific agonists and antagonists indicates no suggestion of a prevalence of b,- or &adrenergic receptors (i.e. the dissociation constants were not distributed according to the purported specificities of the competitive ligands). This is the same conclusion drawn previously from data obtained by measure- ment of the stimulation or inhibition of lipolysis in porcine adipose tissue in vitro (Mersmann, 1984a,b, 1987, 1992).

We examined three compounds, purportedly specific for the &adrenergic receptor (ICI 118,551, H35/25, and fenoterol), using competitive ligand binding with extended dose-response curves (Table 6) to maximize the detection of multiple binding sites. Coutinho et al. (1992) previously measured competi- tive ligand binding (radiolabeled ligand was “‘I-pin- dolol) in porcine adipocyte ghosts using extended dose-response curves to study receptor subtypes of the /I-adrenergic receptor. They used ICI 118,551 as the competitive ligand to detect purported P,-adren- ergic receptors; it inhibited ligand binding in a linear fashion (one-site model) with a Ki of 110 nM. Our data generated with ICI 118,551 also fit a one-site model with a K, of 157 nM; it very poorly fits a two-site model as indicated by the large sum of squares and K2 value of 5 x 10m4 M (greater than the highest concentration used in the experiment). However, with the two other purported fl,-specific competitive ligands (H35/25 and fenoterol) our ob- servations were more complex (Table 6) because modeling of competitive binding data suggested more than one binding site. The fit for H35/25 was slightly better for the two-site than for the one-site model (sum of squares decreased 50%) whereas, the fit for fenoterol was considerably better for the two-site than for the one-site model.

Coutinho et al. (1992) found that competitive ligand-binding data generated with ICI 89,406, a purportedly /I,-specific adrenergic antagonist, could be analyzed best by a two-site model with Kis of 2 and 57 nM. The K, for the ICI 89,406 low-affinity site, 57 nM was similar to the one-site model K, of 110 nM

Adipose tissue j?-adrenergic receptors 731

for ICI 118,551. The authors indicated that the high-affinity site for ICI 89,406 represented a receptor subtype not detected by ICI 118,551, whereas, the low-affinity site for ICI 89,406 represented another receptor subtype also detected by ICI 118,55 1. For two ghost preparations, we found (Table 6) the high-affinity site for ICI 89,406 had a Ki < 1 nM and the low-affinity site had a K, of approximately 90 nM; thus, the K, for ICI 89,406 was similar to the one-site K, for ICI 118,551 (157 nM), and similar to the observations of Coutinho et al. (1992). Data obtained for ICI 89,406 with the third ghost preparation would not fit a two-site model.

Data obtained with atenolol, another purported &-specific adrenergic antagonist fit a two-site model better than a one-site model (Table 6). Competitive ligand binding for metoprolol, a third purported b,-specific adrenergic antagonist (Table 7), was measured at DHA concentrations from 0.9 to 7.2 nM. If the radioligand used in competitive bind- ing studies (DHA) had decided specificity for one receptor subtype, the binding to the other subtype would be expected to diminish as the concentration of radiohgand was decreased (McGonigle et al., 1986). Metoprolol data all analyzed better with a two-site than a one-site model (Table 7). The K, and K, for the two sites did not vary in a systematic manner as the DHA concentration was decreased; nor were either K, or K2 eliminated, suggesting that the preference of DHA for a particular binding site was minimal. Coutinho et al. (1992), using the approach of McGonigle, indicated that the radio- ligand, ‘2SI-pindolol, had approximately three times greater affinity for the ICI 89,406 low-affinity site than for the high-affinity site. We did not have access to appropriate computer programs to analyze our data using the approach of McGonigle et al. (1986).

Because ICI 89406 is usually designated a /I,- specific adrenergic antagonist, Coutinho et al. (1992) concluded that the high affinity site (low K,) measured the &adrenergic receptor. The site with the larger K, for ICI 89,406 was also measured by ICI 118,551, a purported P,-specific adrenergic antagonist (K, for ICI 118,551 and the K, for ICI 89,406 were numeri- cally similar); consequently, this site was designated the &adrenergic receptor. It is our contention that the conclusion drawn regarding receptor subtypes in porcine adipose tissue depends on the specific com- pounds selected to represent the /I,- and fi,-subtypes of the adrenergic receptor(s). It is only fortuitous that the K, for ICI 118,551 and the K, for ICI 89,406 are similar, so that it was concluded by Coutinho et al. (1992) that they detected a j,-specific site, whereas, the high-affinity site detected by CI 89,406 was designated a /3,-specific site. Choice of other purported p,- or fi,-specific adrenergic agonists or antagonists would have yielded different conclusions (note the range of the K, and K2 values in Tables 6 and 7). Using the assumptions that /j’,- and /$-adren- ergic receptors were being detected by ICI 89,406,

Coutinho indicated the proportion of /I,- to &adren- ergic receptor binding sites was approximately 45: 55.

With metoprolol, we observed about 50% of each of two receptor types; however, several of the analyses indicated fewer high-affinity sites (Table 7). With the other compounds, there was no consistent pattern of distribution for the two binding sites (Table 6).

P,-Adrenergic receptors

An atypical or /I,-adrenergic receptor is found in adipose tissue from some species (Strosberg, 1992; Langin et al., 1991). An unusual property of this receptor is that it is stimulated by the /I-adrenergic antagonist, CGP 12,177; this antagonist acts as a partial or sometimes full agonist (Emorine et al., 1989; Langin et al., 1991; Scarpace and Matheny, 1991). In our laboratory (unpublished observations), lipolysis by rat epididymal adipose tissue fragments pooled from several rats was partially stimulated by CGP 12,177 in vitro; pooled perirenal fat pad frag- ments were also stimulated by this compound, but to a lesser extent than those from the epididymal depot. In subcutaneous adipose tissue slices obtained from two pigs, CGP 12,177 partially stimulated lipolysis in vitro; however, adipose tissue from a third pig obtained from a different source did not respond to CGP 12,177. These data suggest that &-adrenergic receptors may be present in porcine adipose tissue. The b-adrenergic antagonist, CGP 12,177, binds to the rat (Langin et al. 1991) as well as the pig (Table 2) adipose tissue /I-adrenergic receptor(s) with high affinity. The high affinity binding of CGP 12,177 is probably to /II-or b,-adrenergic receptors (Langin et al., 1991). Alprenolol or DHA, the radiolabeled ligand used by us, may not detect the /Y,-adrenergic receptor in binding studies. Porcine adipose tissue lipolytic activity stimulated by isoproterenol was only partially antagonized by CGP 12,177, suggesting that the tissue may contain more than one type of p-adrenergic receptor. Additional suggestions for a &-or atypical receptor in porcine adipose tissue are the high concentrations of propranolol and ICI 118,551 required to inhibit lipolysis. Both rat (Langin et al., 1991) and pig (Mersmann, 1992) adipocytes require approximately 1O-6 M propanolol and 1O-5 M ICI 118,551 to inhibit lipolysis 50%. Unfor- tunately, we do not have access to a compound such as BRL 37,344, a purported &-specific adrenergic agonist. This compound is approximately IO times more potent than isoproterenol for stimulation of rat adipose tissue lipolysis in vitro (Langin et al., 1991). Messenger RNA, specific for the &adrenergic recep- tor, has been detected in adipose tissues of the rat and mouse (Strosberg, 1992). The mRNA for a /&-adren- ergic receptor in human adipose tissue may (Stros- berg, 1992) or may not (Thomas and Liggett, 1993) be present. Overall, our data suggest the possibility for functional &adrenergic receptors in porcine adipose tissue. However, because the /Y-adrenergic receptor(s) in porcine adipose tissue have such unique

732 H. J. MERSMANN et al.

properties for stimulation of lipolysis by norepi- nephrine analogues, it is difficult to clearly interpret these suggestions.

CONCLUSION

Our ligand-binding data do not clarify the presence of multiple /I-adrenergic receptor subtypes in porcine adipose tissue. Modeling of the data obtained from competitive ligand binding for some agonists and antagonists suggests more than one receptor subtype. It is not clear whether there are two or more receptor subtypes present; it is even less clear how the suggested multiple receptor subtypes relate to more classical /II- and j,-adrenergic receptors, or to the /I,-adrenergic receptor.

Acknowledgements-This work is a publication of the USDA/ARS Children’s Nutrition Research Center, Depart- ment of Pediatrics, Baylor College of Medicine, Houston, Texas. This project has been funded in part with federal funds from the U.S. Department of Agriculture, Agricul- tural Research Service under Cooperative Agreement num- ber 58-6250-l-003. The contents of this publication do not necessarily reflect the views or policies of the U.S. Depart- ment of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement from the U.S. Government.

Part of these data was presented at the American Society of Animal Science meeting at Spokane, WA, June 1993 (J. Anim. Sci. 71 (Supplement 1);. 1993, Abstract No. 182.

We thank J. Cunnineham. H. Asberrv, and F. Biggs for care of animals, S. P. Smith’ for secretarial assistance, and S. Charboneau for editorial guidance.

REFERENCES

Bocklen E., Flad S., Muller E. and von Faber H. (1986) Comparative determination of /I-adrenergic receptors in muscle, heart and backfat of Pietrain and Large White pigs. Anim. Prod. 43, 335-340.

Cheng Y. and Prusoff W. H. (1973) Relationship between the-inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I,,) of an enzymatic reaction. Biochem. Pharmac. 22, 3099-3108.

Coutinho L. L., Bergen W. G., Merkel R. A. and Smith II C. K. (1992) Quantitative characterization of /I-adrener- gic receptor subtypes in porcine adipocytes. Comp. Bio- them. Physiol. lOlC, 481485.

Emorine L. J., Marullo S., Briend-Sutren M.-M., Patey G., Tate K., Delavier-Klutchko C. and Strosberg A. D. (1989) Molecular characterization of the human &adren- krgic receptor. Science 245, 1118-1121. _

Hu C. Y., Novakofski J. and Mersmann H. J. (1987) Hormonal control of porcine adipose tissue fatty acid release and cyclic AMP concentration. J. Anim. Sci. 64, 1031-1037.

Langin D., Portillo M. P., Salulnier-Blache J. S. and Lafontan M. (1991) Coexistence of three /I-adrenoceptor subtypes in white fat cells of various mammalian species. European J. Pharmac. 199, 291-301.

Lui C. Y., Boyer J. L. and Mills S. E. (1989) Acute effects of /I-adrenergic agonists on porcine adipocyte metabolism in vitro. J. Anim. Sci. 67, 293&2936.

Liu C. Y. and Mills S. E. (1989) Determination of the affinity of ractopamine and clenbuterol for the fl-adreno- ceptor of the porcine adipocyte. J. Anim. Sci. 67, 2937-2942.

McGonigle P., Neve K. A. and Molinoff P. (1986) A quantitative method of analyzing the interaction of slightly selective radioligands with multiple receptor sub- types Molec. Pharmac. 30, 329-337.

McPherson G. A. (1985) Analysis of radioligand binding experiments: a collection of computer programs for the IBM PC. J. Pharmac. Meth. 14, 213-228.

Mersmann H. J. (1984a) Adrenergic control of lipolysis in swine adipose tissue. Camp. Biochem. Physiol. 77C, 143-53.

Mersmann H. J. (1984b) Specificity of B-adrenergic control of lipolysis in swine adipose tissue. Camp. Biochem. Physiol. 77C, 3942.

Mersmann H. J. (1986) Comparison of plasma free-fatty- acid and blood glycerol concentrations with measurement of lipolysis in porcine adipose tissue in vitro. J. Anim. Sci. 63, 757-769.

Mersmann H. J. (1987) Acute metabolic effects of adrener- gic agents in swine. Am. J. Physiol. 252, E85-E95.

Mersmann H. J. (1989) Inhibition of porcine adipose tissue lipogenesis by. /I-adrenergic agonists. Camp.* Biochem. Physiol. 94C, 619623.

Mersmann H. J. (1992) Regulation of porcine adipose tissue lipolytic activity by adrenergic agonists and antagonists. Camp. Biochem. Physiol. 102C, 407411.

Mersmann H. J., Brown L. J., Underwood M. C. and Stanton H. C. (1974) Catecholamine-induced lipolysis in swine. Camp. Biochem. Physiol. 47B, 263-270.

Mersmann H. J. and Hu C. Y. (1987) Factors affecting measurement of glucose metabolism and lipolytic rates in porcine adipose tissue slices in vitro. J. Anim. Sci. 64, 148-164.

Mersmann H. J. and McNeel R. L. (1992a) p-Adrenergic receptor binding in crude porcine adipose tissue plasma membranes. J. Anim. Sci. 70, 781-786.

Mersmann H. J. and McNeel R. L. (1992b) Ligand binding to the porcine adipose tissue /I-adrenergic receptor. J. Anim. Sci. 70, 787-797.

Munson P. J. and Rodbard D. (1980) LIGAND: a versatile computerized approach for the characterization of ligand binding systems.. Analyf. Biochem. 107, 220-239.

Scarnace P. J. and Mathenv M. (1991) Adenylate cyclase _ agonist properties of CGP-12177A in’brown fat: evidence for atypical b-adrenergic receptors. Am. J. Physiol. 260, (Endocrinol. Metab. 23): E226E23 1.

Spurlock M. E., Cusumano J. C. and Mills S. E. (1993a) ( - )-[‘HI-Dihydroalyprenolol binding to fi-adrenergic ,receptors in porcine adipose tissue and skeletal muscle membrane preparations. J. Anim. Sci. 71, 1778-1785.

Spurlock M. E., Cusumano J. C. and Mills S. E. (1993b) The affinity of ractopamine, clenbuterol, and L-644,969 for the /I-adrenergic receptor population in porcine adipose tissue and skeletal muscle membrane. J. Anim. Sci. 71, 206lL2065.

Strosberg A. D. (1992) Biotechnology of P-Adrenergic Receptors. Molec. Neurobiol. 4, 21 l-250.

Thomas R. F. and Liggett S. B. (1993) Lack of &-adrenergic receptor mRNA expression in adipose and other meta- bolic tissues in the adult human. Molec. Pharmac. 43, 3433348.