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In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase
343
Biochimica et Biophysica Acta, 585 (1979) 3 4 3 - - 3 5 9 © Elsevier/North-Holland Biomedical Press
BBA 28963
IN V I T R O C H A R A C T E R I Z A T I O N OF SKELETAL MUSCLE ~-ADRENERGIC R E C E P T O R S COUPLED TO A D E N Y L A T E CYCLASE
N. B O J J I R E D D Y and W. K I N G E N G E L
Medical Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20205 (U.S.A.) (Received O c t o b e r 12th, 1978)
Key words: ~-Adrenergic receptor; Adenylate cyclase; Dihydroalprenolol; Receptor site; (Skeletal muscle; Plasmalemma)
Summary [~H]Dihydroalprenolol, a potent ~-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ~-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed by [3H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [3H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small b u t consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [3H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions. The binding of [3H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K~)and dissociation (K2) rate constants of 2.21 • 107 M -~ • min -1 and 3.21 • 10 -1 min -1, respectively, were obtained. The dissociation constant (Kd) of 15 nM for [3H]dihydroalprenolol obtained from saturation binding data closely agreed with the K d derived from the ratio of dissociation and association rate constants (K2/ K,). Several ~-adrenergic agents k n o w n to be active on intact skeletal muscle also c o m p e t e d for [3H]dihydrolaprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [aH]dihydroalprenolol binding sites with a p o t e n c y of isoprotereAbbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2' ethanesulfonic acid; EGTA, ethyleneglycolbis(~-aminoether)N',N'-tetraacetic acid ; GMP-PNP° 5'-guanylyl imidodiphosphate.
344
nol ~ epinephrine ~ norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (--)-isomers being more potent than (+)-isomers. Phenylephrine, an a-adrenergic agonist, showed no effect either on [3H]dihydroalprenolol binding or on adenylate cyclase. Known fl-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [3H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The Kj values for the antagonists determined from inhibition of [3H]dihydroalprenolol binding agreed closely with the Ki values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [3H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the fi-adrenergic receptor.
Introduction Catecholamines such as epinephrine and isoproterenol influence glycogenolysis, [1], Na + and K ÷ transport [2--4], and twitch-contraction [5] of mammalian skeletal muscle. These effects are believed to be mediated by interaction of catecholamines with the ~-adrenergic receptors. However, the site of localization of ~-receptors and the mechanism(s) of drug-receptor interactions in skeletal muscle have not been fully defined. Available evidence regarding the presence of ~-receptors in skeletal muscle has been derived mostly by indirect studies such as by studying the effect of fl-adrenergic agents on glycogen metabolism or muscle contraction. Since catecholamines regulate several muscle functions [ 5 ], it was felt important to identify the ~-adrenergic receptor sites and study their molecular properties. In homogenized tissue, the ~-receptor activity is manifested mostly in the form of adenylate cyclase, since that enzyme, as in the intact tissue, responds to ~-adrenergic drugs [6]. In most tissues, adenylate cyclase is localized in plasma membranes [6]. Accordingly, the isolated plasma membranes of tissues that respond to catecholamines have been used extensively to delineate the properties of fl-receptor-adenylate cyclase interactions [6--9]. Recently, considerable progress has been made for direct identification of fl-receptors coupled to adenylate cyclase in isolated membranes through the use of binding studies with radioactive fl-adrenergic agents of high specific activity [ 10--13 ]. Previous studies from our laboratory [14--16] indicated that a particulate fraction enriched with surface membranes could be isolated from homogenates of rat and human skeletal muscle. Those membranes contain high levels of adenylate cyclase and the enzyme responds to catecholamines with fl-adrenoceptor specificity [17]. The present study is an attempt to characterize further the skeletal muscle /3-adrenoceptors through direct labelling of the receptor sites with [3H]dihydroalprenolol, a potent/3-adrenergic antagonist, which has been successfully used to identify the fl-adrenoceptors in other systems [8]. The data indicate that [3H]dihydroalprenolol, like the catecholamine-sensitive adenylate cyclase, is predominantly localized in a plasmalemmal fraction. The characteristics of [3H]dihydroalprenolol binding to plasmalemma, including kinetics, drug
345 specificity and correlation with adenylate cyclase, are described. Some of these studies have appeared in an abstract [18]. Materials and Methods
Materials [3H]Dihydroalprenolol (32 Ci/mmol) and cyclic [G-3H]AMP (38 Ci/mmol) were purchased from New England Nuclear (Boston, MA). [a-32p]ATP (10-20 Ci/mmol), [7-32P]ATP (20--30 Ci/mmol), GMP-PNP, and neutral grade alumina were obtained from International Chemicals and Nuclear Co. (Irvine, CA). (+)-Epinephrine, (+)-isoproterenol were purchased from Winthrop Laboratories (New York, N.Y.). (--)- and (+)-alprenolol (Hassle Pharmacuticals, Sweden), and (--)- and (+)-propranolol (Ayerst Laboratories, Canada) were kindly donated by those companies. The sources of other materials were those previously described [16,17]. Isolation of muscle membranes Gastrocnemius-plantaris muscle complex of male Sprague-Dawley rats (200-250 g, Zivic-Miller Labs, Allison Park, PA) was used to prepare different membranes. Skeletal muscle plasmalemma, sarcoplasmic reticulum and mitochondria were prepared essentially as described previously [16], except that in all preparative reagents EDTA was omitted and Tris-HC1 buffer was substituted with 10 mM HEPES, pH 7.5. It was found that the specific activity of adenylate cyclase and its activation b y catecholamines were greater in the membranes prepared with these modifications than in those prepared by earlier procedure. However, these preparative changes did not alter the other properties of different membranes which were previously described in considerable detail [16]. Further purification of plasmalemma on discontinuous sucrose gradients was as described earlier [16], except that the sucrose gradients were prepared in 5 mM HEPES, pH 7.5. The plasmalemmal subfractions settled at sucrose interfaces of 0.6--0.8 M (PLF1), 0.8--1.0 M (PLF2), 1.0--1.2 M (PLF3) and 1.2--1.4 M (PLF4) were collected, diluted to 0.3 M sucrose with 5 mM HEPES, pH 7.5, and sedimented at 100 000 × g for 60 rain. The pellets were resuspended in 0.3 M sucrose/5 mM HEPES, pH 7.5. All samples (in aliquots of 0 . 2 - 0 . 4 ml) were frozen in liquid nitrogen and stored at --70°C until use. [3H]Dihydroalprenolol, adenylate cyclase and (Na ÷ + K+)-ATPase activities were stable for at least t w o months under those conditions. [3H] Dihydroalprenolol binding assay Muscle membranes (50--100 ~g protein) were incubated in siliconized glass tubes (12 × 75 mm) with 10 mM MgC12/50 mM Tris-HC1, pH 7.5 and indicated concentrations of [3H]dihydroalprenolol (approx. 45 000 d p m / p m o l ) in a total volume of 200 pl. In standard assays [3H]dihydroalprenolol was at 30 nM. Drugs whose effects on the binding were to be studied were freshly prepared in 50 mM Tris-HC1, pH 7.5 and added in 20 pl volume to the assay medium. The reaction was for 5 min at 37°C, unless mentioned otherwise. After 5 min, the reaction mixture was diluted with 2 ml of a cold (2°C) 'buffer medium' (10
346 mM MgC12/50 mM Tris-HC1, pH 7.5) and was immediately filtered through a Millipore filter (EHWP, 0.5 pm pore size) under vacuum pressure (30 sample manifold, Millipore Corporation). The filter was washed with an additional 15 ml of same cold buffer medium. The complete filtration procedure took about 15 s. The filters were dried, dissolved in 10 ml of Instabray (Yorktown Research, S. Hackensack, NJ), and counted in a Packard liquid scintillation spectrometer (Model 2650) at a 3H counting efficiency of 42%. In parallel experiments, the a m o u n t of [3H]dihydroalprenolol absorbed to Millipore filters in the absence of membranes or the a m o u n t nonspecifically bound to membranes in the presence of excess (100 ~M) nonradioactive (--)alprenolol was determined. Such binding was about 0.01% of the total radioactivity added to the incubation medium, and it was subtracted from appropriate total binding values to derive 'specific binding'. Only such 'specific binding' data are presented in the results. It was found that nonspecific binding of [3H]dihydroalprenolol was higher if the assays were carried out in polypropylene (Falcon) or glass tubes than for the assays performed in siliconized glass tubes.
Adenylate cyclase assay The enzyme activity was estimated as previously described [17]. The assay medium, in a total volume of 100 pl, comprised 50 mM Tris-HC1, pH 7.5, 0.5 mM [a-32P]ATP (20--30 dpm/pmol), 5 mM MgC12, 10 mM KC1, 10 mM theophylline, 15 mM creatine phosphate, 0.5 units of creatine phosphokinase, 40-60 ~g of membrane protein and other agents whose effects were to be studied. The reaction was for 15 rain at 37°C. The cyclic [32P]AMP produced was separated by a modified [17] column chromatographic procedure of Salomon et al. [19]. (Na ÷ + K+)-A TPase assay The ATPase activity was determined using [~,-32P]ATP as substrate and by measuring by Cerenkov radiation the 32pi produced as described earlier [16]. In the present study, (Na ÷ + K+)-ATPase was defined as the difference between the total activity (5 mM Mg2+/100 mM Na+/20 mM K ÷) in the absence and presence of 0.1 mM ouabain. Protein content of muscle membranes was estimated by the method of Lowry et al. [20] using bovine serum albumin as a standard. Results
Subcellular distribution o f [3H]dihydroalprenolol binding Since the ~-adrenergic receptors are known to be closely associated with adenylate cyclase, we have determined [3H]dihydroalprenolol binding and adenylate cyclase activity in different muscle fractions to evaluate their interrelationship. The skeletal muscle consists of three major membrane components, viz. plasmalemma, sarcoplasmic reticulum and mitochondria. Working with human skeletal muscle, we have previously established that the three fractions were distinctly different one from another in certain biochemical and morphological properties, and that cross-contamination was negligible under our isolation pro-
347
cedure [16]. Similar differences were also observed in the membrane fractions isolated from rat skeletal muscle used in the present study. The pattern of (Na++ K+)-ATpase distribution (Table I) clearly illustrates the difference between plasmalemma and the other muscle membranes. The enzyme was predominantly localized in plasmalemma, while it was undetectable or at a very insignificant level in sarcoplasmic reticulum and mitochondria (Table I). The subcellular distribution of fi-adrenergic receptor sites in skeletal muscle was examined by measuring specific [3H]dihydroalprenolol binding to plasmalemma, sarcoplasmic reticulum and mitochondria (Table I). When measured in the presence of 30 nM [3H]dihydroalprenolol, the binding to partially purified plasmelamma was about four times, and that to sucrose gradient purified plasmalemmal subfraction 1 (PLF,) about 10 times greater than the binding to sarcoplasmic reticulum or mitochondria, suggesting the prevalence of greater density of fi-adrenergic receptor sites in plasmalemma. The level of [3H]dihydroalprenolol binding to subcellular fractions of muscle closely corresponded with the level of stimulation of adenylate cyclase in those membranes by catecholamines, which are believed to activate the enzyme by interacting with ~-adrenergic receptor. (--)-Isoproterenol (10 ~M) induced about 3-fold activation of adenylate cyclase in plasmalemma, while it enhanced the enzyme in sarcoplasmic reticulum by only 60%, and that in mitochondria by 80%, over their respectively basal activities (Table I). The relative yields of protein and adenylate cyclase in different fractions were similar to those previously obtained with human skeletal muscle [16]. The co-purification of [3H]dihydroalprenolol binding and adenylate cyclase was evident when plasmalemma was subjected to further purification on sucrose gradients (Table I). The highest specific activities of [3H]dihydroalprenolol binding and adenylate cyclase were observed in a subfraction which settled at the sucrose interTABLE I CORRELATION OF [3H]DIHYDROALPRENOLOL BINDING WITH ADENYLATE CYCLASE (Na ÷ + K+)-ATPase A C T I V I T I E S IN T H E S U B C E L L U L A R F R A C T I O N S O F S K E L E T A L M U S C L E
AND
Preparation o f subcellular f r a c t i o n s and e n z y m e assays w e r e as d e s c r i b e d u n d e r M e t h o d s . PL, plasmaleraraa; P L F I - - P L F 4 , p l a s m a l e r a m a l s u b f r a c t i o n s o b t a i n e d b y s u c r o s e - g r a d i e n t c e n t r i f u g a t i o n ; S R , sarcop l a s m i c r e t i c u l u m . [ ~ H ] d i h y d r o a l p r e n o l o l ( [ J H ] D H A ) b i n d i n g w a s m e a s u r e d u n d e r standard i n c u b a t i o n c o n d i t i o n s . GTP and ( - - ) - i s o p r o t e r e n o l w e r e p r e s e n t at 1 0 p M e a c h in assay o f a d e n y l a t e c y c l a s e . V a l u e s are m e a n s o f d u p l i c a t e d e t e r m i n a t i o n s on six p r e p a r a t i o n s ± S . D . Muscle fraction
[3H]DHA
binding (pmol bound] mg protein)
A d e n y l a t e c y c l a s e ( p m o l c y c l i c AMP f o r m e d / r a g P r o t e i n per rain)
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0.01 0.02 0.02 0.04 0.06 0.02 0.02 0.01
4 ± 0.2 2 4 _+ 2 . 6 18-+ 2 . 0 32 ± 4.4 6 8 _+ 1 4 18 ± 4 . 0 8 ± 1.2 6 ± 0.8
12 34 26 86 180 30 14 8
± 1.8 ± 4 ± 3.4 + 5.8 ± 16 ± 8.0 + 2.0 ± 0.6
16 60 68 204 680 II0 36 14
± ± ± ± ± ± + ±
4 8 12 18 56 16 4 2
0.0 0.0 0.6 11.6 44.0 8.6 3.2 1.8
± ± ± ± ± ±
0.2 1.8 6.0 2.4 1.6 0.4
348
face of 0.6--0.8 M (corresponding to a sucrose density (g/cm 3) of 1.078--1.104 at 20°C). [3H]Dihydroalprenolol binding and the enzyme activity gradually decreased in the subfractions PLF:--PLF4 which settled at greater sucrose densities (Table I). GTP, and its analog GMP-PNP (10 gM), markedly enhanced the responsiveness of adenylate cyclase in each muscle membrane fraction to isoproterenol, and the highest specific activities were found in plasmalemma (Tables I and II). GTP alone caused marginal but consistent stimulation {20--40% above basal) of adenylate cyclase (Table II). The effect of GMP-PNP was more pronounced than that of GTP, both in the absence and in the presence of isoproterenol. These results are in accordance with findings in other tissues [21]. In agreement with our earlier findings on human muscle [17], the guanyl nucleotides potentiated the stimulating effect of {--)-isoproterenol on rat muscle plasmalemmal adenylate cyclase without altering the apparent Km for (--)-isoproterenol (data not shown). The highest specific activity of adenylate cyclase in each membrane fraction was obtained with NaF (10 mM) as a stimulant (Table II). NaF-stimulated enzyme activity in purified plasmalemmal fraction (PLF~) was 2.3--3.4 nmol cyclic AMP produced per mg protein per min in six different preparations. The enzyme activity values obtained in this study are significantly greater than those previously reported for rat [22], human [16] and rabbit [23] muscle plasma membranes. Thus, the muscle plasmalemma as prepared in the present study are highly purified in terms of their enrichment with adenylate cyclase, and hence provided asuitable material to evaluate the properties of adenylate cyclase and the associated ~-adrenergic receptor.
Properties of [3H]dihydroalprenolol binding to plasmalemma The specific binding of [3H]dihydroalprenolol was linear between 25 to 300 pg of plasmalemmal protein. The total binding of [3H]dihydroalprenolol to partially-purified plamalemma and sucrose-density-purified plasmalemmal subfraction 1, PLF1, respectively, was two and five times greater than the non-
T A B L E II ACTIVATION OF ADENYLATE C Y C L A S E BY N a F A N D G U A N Y L N U C L E O T I D E S MEMBRANE FRACTIONS OF SKELETAL MUSCLE
IN DIFFERENT
M e m b r a n e p r e p a r a t i o n a n d a s s a y o f a d e n y l a t e c y c l a s e w e r e as d e s c r i b e d i n M e t h o d s . M e m b r a n e f r a c t i o n a b b r e v i a t i o n s were s a m e as i n T a b l e I. V a l u e s are m e a n s o f six p r e p a r a t i o n s ,+ S . D . G M P - P N P , 5 ' - g u a n y l y l imidodiphosphate. Additions
Adenylate per rain) Membrane fraction:
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349 specific binding. Since the yield of PLF~ was very low (approx. 0.15 mg/g muscle wet weight), it became difficult to compare several aspects of [3H]dihydroalprenolol binding with adenylate cyclase from the same preparation. Most of the present studies were, therefore, carried out on partiaUy-purified plasmalemma. Preliminary experiments of saturation binding and selected drug specifity studies on partially-purified plasmalemma and PLF~ fractions revealed that those properties were qualitatively similar in both the fractions.
Saturability of binding sites The specific binding of [3H]dihydroalprenolol as a function of its concentration (Fig. 1) indicated that the binding sites were saturable. Apparent saturation of binding sites seemed to occur at a [3H]dihydroalprenolol concentration of about 50 nM. Determination of binding at concentrations of [3H]dihydroalprenolol greater than 50 nM was made difficult because of very high nonspecific binding. The half-maximal binding, as revealed by the double-reciprocal plot (Fig. 1), occurred at 12.5 nM, which provided an estimate of apparent dissociation constant (Kd). The Scatchard [24] plot (Fig. 2, left) of the saturation binding data (Fig. 1) yielded a straight line, suggesting the presence of a single
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Fig. 1. Binding of [ 3 H ] d i h y d r o a l p r e n o l o l (DHA) as a function of its c o n c e n t r a t i o n to plasmalemma. Inset shows the double-reciprocal p l o t of the same data. Values are means of four e x p e r i m e n t s each assayed in duplicate.
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group of binding sites with a K d of 15.4 ± 3.2 nM (n = 4). At apparent saturation, the receptor concentration in partially-purified plasmalemma was 0.33 pmol of [3H]dihydroalprenolol b o u n d per mg protein {Fig. 2, left). A receptor concentration of 1.6 pmol per mg protein was obtained for sucrose~lensity purified plasmalemmal subfraction 1 (PLF1) (data not shown). To test the nature of cooperativity of binding sites, the equilibrium binding data was analyzed by the Hill equation [25]. The Hill plot (Fig. 2, right) also gave a straight line with a Hill coefficient of 1.007 ± 0.105 (n = 4), indicating neither positive nor negative cooperativity. K i n e t i c s o f [ 3 H ] d i h y d r o a l p r e n o l o l binding
Under our assay conditions, the binding of [3H]dihydroalprenolol to plasmalemma was a rapid process. The binding reached equilibrium within 5 min, with half-maximal binding occurring at 1.5 min (Fig. 3a). After reaching a maximum, the binding slightly decreased when incubations were carried out for periods greater than 5 min (Fig. 3a). During prolonged incubation, the drugreceptor complex probably undergoes a spontaneous dissociation and reassociation resulting in a new steady state. To overcome that, we routinely conducted the binding experiments for 5 min. From the binding data up to 5 min incubation, an association rate constant (K~) of (2.21 ± 0.20) • 107 M -1 • min -1 (n = 3) was calculated from the equation [26] g~ = [1/t(a -- b)] ln[b(a - - x ) / a ( b - - x ) ] ;
where a is the initial concentration of [3H]dihydroalprenolol in the assay, b is receptor concentration (0.33 from Scatchard plot) and x is the a m o u n t of [3H]dihydroalprenolol specifically bound at time t. The drug-receptor complex readily dissociated when (--)-propranolol was added to the incubation medium after [3H]dihydroalprenolol binding reached its maximum (Fig. 3b). Similar dissociation could also be obtained by 1000fold dilution of incubation medium with Tris-HC1 buffer lacking propranolol (data n o t shown). The dissociation of radioligand from its receptor moiety was
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Fig. 3.a. T i m e c o u r s e o f a s s o c i a t i o n o f [ 3 H ] d i h y d r o a l p r e n o l o l ( D H A ) t o p l a s m a l e m m a . S t a n d a r d b i n d i n g c o n d i t i o n s w e r e e m p l o y e d e x c e p t i n c u b a t i o n t i m e w h i c h w a s v a r i e d as i n d i c a t e d . V a l u e s are m e a n s of t h r e e e x p e r i m e n t s , b. D i s s o c i a t i o n o f [ 3 H ] d i h y d r o a l p r e n o l o l f r o m its r e c e p t o r as a f u n c t i o n o f t i m e a n d t e m p e r a t u r e . P l a s m a l e m m a w e r e e q u i l i b r a t e d w i t h 3 0 nM [ 3 H ] d i h y d r o a l p r e n o l o l a t 3 7 ° C f o r 4 rain. Duplicate a l i q u o t s ( 1 0 0 pl) w e r e s a m p l e d f o r [ 3 H ] d i - h y d r o a l p r e n o l o l b i n d i n g as d e s c r i b e d in Materials a n d Methods and the remaining reaction mixture was equilibrated at required t e m p e r a t u r e for an additional 2 min. The dissociation was initiated at time zero by adding (--)-propranolol, maintained at appropriate t e m p e r a t u r e , to a final c o n c e n t r a t i o n o f 10 pM. A l i q u o t s (100/~1) w e r e t h e n w i t h d r a w n in d u p l i c a t e a t i n d i c a t e d t i m e s to d e t e r m i n e [ 3 H ] d i h y d r o a l p r e n o l o l b i n d i n g . C o n t r o l b i n d i n g r e f e r s t o t h e a m o u n t of [ 3 H ] d i h y d r o a l p r e n o l o l b o u n d p r i o r t o t h e a d d i t i o n of p r o p r a n o l o l . T h e e x p e r i m e n t w a s r e p e a t e d o n t w o d i f f e r e n t p r e p a r a t i o n s w i t h essentially similar results.
temperature dependent (Fig. 3b). It was rapid at 37°C, moderate at 25°C, and minimum at 0°C. Typical half-lives for the drug-receptor complex were 2, 8 and 18 min at 37, 25 and 0°C, respectively. The time required for haif-maximal dissociation at 37°C (2 min) closely resembled the time required for halfmaximal association (1.5 min, Fig. 3a). From the data at 37°C, a dissociation rate constant (K2) of (3.21 + 0.8) • 10 -1 min -1 (n = 2) was obtained from the equation K2 = ( l / t ) In (a/x); where a is the a m o u n t of [3H]dihydroalprenolol initially b o u n d (i.e., prior to the addition of propranolol to initiate dissociation) and x is the a m o u n t of [3H]dihydroalprenolol remain b o u n d at time t (i.e., after the addition of propranolol). The ratio of K2/K~ provided an independent K d of 14.5 nM for [3H]dihydroalprenolol; which agreed closely with the K d obtained b y the Scatchard analysis.
Selectivity and stereospecificity of [3H]dihydroalprenolol binding The specificity of [3H]dihydroalprenolol binding to plasmalemma was examined by studying the abilities of different fl-adrenergic agonists and antagonists in competing for the binding sites. Of the catecholamines tested, (--)-isoproterenol was the most p o t e n t inhibitor of [3H]dihydroalprenolol binding with an ICso of 0.07 pM (Fig. 4). The ICso values for (--)-epinephrine and (--)-norepinephrine were 0.5 and 60 gM, respectively. Thus, the potency sequence of agonists in competing for [3H]dihydroalprenolol binding sites was (--)-isoproterenol > (--)-epinephrine > (--)-norepinephrine. Such an order of
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CATECHOLAMINE (-log M) Fig. 4. E f f e c t s o f d i f f e r e n t c a t e c h o l a m i n e s o n p l a s m a l e m m a l a d e n y l a t e c y c l a s e (left) a n d [ 3 H ] d i h y d r o a l p r e n o l o l ( D H A ) b i n d i n g (right). Values are m e a n s of d u p l i c a t e analysis of t h r e e d i f f e r e n t p r e p a r a t i o n s .
potency is characteristic of fi~-adrenergic receptors [27]. Furthermore, isoproterenol and epinephrine showed considerable stereospecificity in that (--)-isomers were more p o t e n t than their corresponding (+)-isomers. The ICso values for (+)-isoproterenol and (+)-epinephrine were a b o u t 15 and 70 t~M, respectively (Fig. 4). The order of potency with which different catecholamines competed for [3H]dihydroalprenolol binding closely resembled the order of p o t e n c y with which they stimulated the plasmalemmal adenylate cyclase (Fig. 4). On a molar basis, (--)-isoproterenol was a more potent stimulator than (--)-epinephrine or (--)-norepinephrine. The apparent ICso values for the activation of adenylate cyclase were 0.09 t~M for (--)-isoproterenol, 0.65 t~M for (--)-epinephrine, and 80 pM for (--)-norepinephrine. Activation of adenylate cyclase by catecholamines was stereospecific with most of the potency residing in (--)-isomers (Fig. 4). An a-adrenergic agonist, (--)-phenylephrine, showed no significant effect either on [3H]dihydroalprenolol binding or on adenylate cyclase (Fig. 4). Similarly phentolamine, a p o t e n t a-blocker, showed no effect (data not shown). To characterize further the fi-receptor in skeletal muscle membranes, the effect of fi-adrenergic antagonists on [3H]dihydroalprenolol binding and adenylate cyclase were investigated. Propranolol and alprenolol are known to antagonize the fi-adrenergic stimulation of adenylate cyclase by isoproterenol. The nature of antagonist effect on the enzyme could be evaluated by its ability to alter the dose response to isoproterenol [8,28]. As can be seen from Fig. 5, proporanolol effectively shifted the dose vs. response curve of isoproterenol towards right. Moreover, the (--)-propranolol was more effective than (+)-propranolol. Similar results were obtained with (--)- and (+)-isomers of alprenolol (data not shown). Schild [29] plot (Fig. 6) yielded straight lines, suggesting a competitive nature of propranolol inhibition. The intercept of the lines with the abcissa of Schild plot provides an apparent K i value [29] for each antagonist. They varied 8--11 nM for (--)-propranolol, 600--900 nM for (+)-propra-
353
• CONTROL o 0,01vM "~ '= 0.10vM ~. (-) PROPRANOLOL Q 1,00gMJ
l~r
CONTROL 0.10~M'~ 1.00vM ~'(+) PROPRANOLOL o lO.OOgMJ
u.i
(-) ISOPROTERENOL (-Log M) Fig. 5. A n t a g o n i s m of ( - - ) - i s o p r o t e r e n o l s t i m u l a t i o n of p l a s m a l e m m a l a d e n y l a t e c y c l a s e b y s t e r e o i s o m e r s o f p r o p r a n o l o l . D o s e - r e s p o n s e of a d e n y l a t e c y c l a s e to ( - - ) - i s o p r o t e r e n o l w a s d e t e r m i n e d w i t h o u t or w i t h i n d i c a t e d f i x e d c o n c e n t r a t i o n s o f (--)- or ( + ) - p r o p r a n o l o l . T h e a c t i v i t y o b t a i n e d w i t h 10/~M (--)-isoprot e r e n o l , in t h e a b s e n c e o f a n t a g o n i s t , w a s c o n s i d e r e d as m a x i m a l . V a l u e s are m e a n s o f t h r e e e x p e r i m e n t s .
nolol, 10--14 nM for (--)-alprenolol, and 800--1100 nM for (+)-alprenolol in three such experiments. Propranolol and alprenolol were approximately equipotent in competing for
/ 3
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1
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8
7
6
5
~-ADRENERGICANTAGONIST(.-logM) Fig. 6. Schild p l o t s o f t h e d a t a s h o w n in Fig. 5. C R = c o n c e n t r a t i o n r a t i o o f ( - - ) - i s o p r o t e r e n o l n e e d e d to o b t a i n similar h a l f - m a x i m a l a c t i v i t y in t h e p r e s e n c e a n d a b s e n c e o f a given c o n c e n t r a t i o n o f (--)- or ( + ) o p r o p r a n o l o l . E n z y m e r e s p o n s e a t 10 #M ( - - ) - i s o p r o t e r e n o l w i t h o u t p r o p r a n o l o l w a s c o n s i d e r e d m a x i m u m , a n d t h e e q u i p o t e n t c o n c e n t r a t i o n s o f i s o p r o t e r e n o l r e q u i r e d f o r 50% m a x i m a l r e s p o n s e in t h e a b s e n c e a n d p r e s e n c e of a n t a g o n i s t w a s d e t e r m i n e d a n d u s e d to c a l c u l a t e C R . T h e slope o f e a c h line w a s a b o u t 1, i n d i c a t i n g t r u e c o m p e t i t i v e a n t a g o n i s m b y p r o p r a n o l o l .
354 [3H]dihydroalprenolol binding sites (Fig. 7). Like catecholamines, the fi-blockers also exhibited considerable stereospecificity. The ICso values of (--)-propranolol and (--)-alprenolol for inhibiting [3H]dihydroalprenolol binding were a b o u t the same, and this value was 40 nM. Corresponding ICso values for the inhibition of adenylate cyclase were 20 nM for (--)-propranolol and 30 nM for (--)-alprenolol (Fig. 7). A b o u t 120--170-fold higher concentrations of (+)-isomers of those fi-blockers were needed to cause an effect similar to that of (--)-isomers on [3H]dihydroalprenolol binding or on adenylate cyclase activity. The ICso values of different competing agents for the binding sites mentioned above may be higher than their actual K i values, because the labeled ligand concentration (30 nM) used in these studies was greater than its Kd, as well as the receptor concentration [30]. It is, however, possible to derive actual K i values for different agonists and antagonists from their ICso values using the equation [30] K i = ICso/(1 + S/Kd), where S is the [3H]dihydroalprenolol concentration used in the assay and K d is the dissociation constant (15 nM). The K i value thus derived was 11.7 nM for both (--)-alprenolol and (--)-propranolol. These K i values closely agreed with the K i values determined from studies on the inhibition of isoproterenol stimulation of adenylate cyclase (Fig. 6). It may also be noted that the K~ of (--)-alprenolol was very similar to the K d obtained from saturation binding (Fig. 2) or kinetic data (Fig. 3) of [3H]dihydroalprenolol binding. Effect o f different agents on [3H]dihydroalprenolol binding ATP (1 mM), GTP (0.1 mM), GMP-PNP (0.1 mM), EDTA (1 mM), and EGTA (1 mM) did n o t affect the binding of [3H]dihydroalprenolol to skeletal muscle plasmalemma (data not shown). Ca 2÷ showed differential effects on adenylate cyclase and the binding (Fig. 8). Enzyme activity (basal, isoproterenol- and NaF-stimulated) was progressively inhibited with increasing concentrations of Ca 2÷, with 50% inhibition occurring at a b o u t 0.08 mM. In contrast,
)
40
0
10
9
8
7
6
5
4
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9
8
7
6
5
4
/~-ADRENERGIC ANTAGONIST (-log M) Fig. 7. Effects o f s t e r e o i s o m e r s o f p r o p r a n o l o l and alprenolol o n (--)-isoproterenol ( 1 0 pM)-stimu]ated a d e n y l a t e cyclase (left) and [ 3 H ] d i h y d r o a l p r e n o l o l ( D H A ) binding (right). V a l u e s are m e a n s of three experiments.
355
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80
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60
60
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40
40
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~
20
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0 tLI ..I
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0 w J
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CaCl 2 (raM] Fig. 8. E f f e c t s o f Ca 2+ o n b a s a l (o), 1 0 / a M ( - - ) - i s o p r o t e r e n o l - s t i m u l a t e d (o) a n d 1 0 m M N a F - s t i m u l a t e d (~) a d e n y l a t e c y c l a s e , a n d o n [ 3 H ] d i h y d r o a l p r e n o l o l ( D H A ) b i n d i n g (e). V a l u e s axe m e a n s o f f o u r e x p e r i ments.
[aH]dihydroalprenolol binding remain unchanged. Ca 2÷ appears to inhibit the enzyme by acting directly on the catalytic site of enzyme since it inhibited uniformly the basal, NaF- and isoproterenol-stimulated activities (Fig. 8). Discussion Catecholamines, like several polypeptide hormones, are thought to elicit physiological responses in target tissues by interacting with specific membranebound receptors [6]. Considerable attention has, therefore, been given to identify different hormone-receptors to gain insight into the mode of hormonereceptor interactions at the molecular level [31]. One of the ways of identifying the receptor for a given hormone is by following the direct binding to isolated membranes of a radioligand known to be active at that receptor. In such studies, the radioligand binding should possess selectivity and specificity known for that receptor from other biochemical and physiological investigations. The properties of ~-adrenergic receptors in different tissues have been extensively characterized through pharmacological and physiological studies [6]. With the availability of highly specific radioactive ~-adrenergic agents in recent years, it is now feasible to identify the ~-adrenergic receptors by direct binding assays [ 10--13]. The characteristics of radioligand binding to fi-adrenergic receptor are generally compared with the properties of adenylate cyclase, since that enzyme is closely associated with fi-receptor functions in many tissues [8,9,32]. The present study indicated that [3H]dihydroalprenolol, a potent ~-adrenergic antagonist, could be used to identify in vitro the ~-adrenergic receptors of skeletal muscle that are coupled to the adenylate cyclase system. The ~-adrenergic receptor sites in fractionated skeletal muscle, as revealed by [3H]dihydroalprenolol binding, were localized predominantely in plasmalemma. [3H]dihydroalprenolol binding was also observed in sarcoplasmic reticulum and mito-
356 chondria b u t it was only one-fifth to one-tenth of that found in plasmalemmal fractions. The level of [3H]dihydroalprenolol binding to different membrane fractions closely correlated with level of adenylate cyclase. Thus the plasmalemmal fraction which contained the highest level of (--)-isoproterenol-stimulated adenylate cyclase also displayed the highest level of [3H]dihydroalprenolol binding. We have previously indicated the possibility that our skeletal muscle plasmalemmal fraction may contain the plasmalemma of other intramuscular cells, particularly those of blood vessels [16]. As yet, there is no way to distinguish the plasmalemma of muscle cells from that of blood vessels. It is, therefore, possible that a certain portion of [3H]dihydroalprenolol binding and its properties observed in skeletal muscle plasmalemmal preparation could also be due to the blood-vessel cell plasmalemma. Our qualitative autoradiographic studies (Lavenstein, B., Engel, W.K., R e d d y , N.B. and Carroll, S., unpublished results) with 125I-labelled h y d r o x y b e n z y l pindolol, another /3-adrenergic antagonist, indicated considerable grain density in blood-vessels of rat muscle sections, especially vessels of the arterial tree, suggesting a contribution of blood-vessel cell plasmalemma to skeletal muscle plasmalemmal fraction. Quantitative assessment of the a m o u n t of binding to vascular cell plasmalemma vis-a-vis skeletal muscle cell plasmalemma would be very important, b u t subcellular fraction studies do n o t yet allow that, and quantitation, even internal quantitation, of autoradiographs, if n o t done in compulsive detail, is fraught with sources of error, including problems of the threshold of silver reduction in relation to density of b o u n d isotope and time of emulsion exposure and development (i.e., 'contrast'). Cell separation before subcellular fractionation would be needed. At this stage we simply point out the need for an awareness of 'the blood-vessel problem' in interpreting our data and those of others who biochemically treat muscle, and other organs, as a 'black b o x ' consisting only of parenchymal cells. The [3H]dihydroalprenolol binding observed in sarcoplasmic reticulum and mitochondria appear to be due to the binding truly occurring to those membranes, and not due to contamination by plasmalemma. The lack of cross-contamination was evident from the (Na ÷ + K+)-ATPase studies. That enzyme was recovered only in plasmalemma and it was n o t detectable in sarcoplasmic reciculum or mitochondria. Entman et al. [33] have shown that specific [3H]dihydroalprenolol binding occurs in sarcoplasmic reticulum of cardiac muscle. Recently Caswell et al. [34] have reported specific [3H]dihydroalprenolol binding to transverse tubules of rabbit skeletal muscle. These authors [34] purified a T-tubule fraction from a microsomal preparation comparable to our sarcoplasmic reticulum, which supports our observation that sarcoplasmic reticulum contains catecholamine-sensitive adenylate cyclase and fl-adrenergic receptor. Caswell et al. [34] claimed that adenylate cyclase was present only in T-tubule b u t n o t in the terminal cisternae. In contrast, Raible et al. [35] reported that adenylate cyclase was localized in terminal cisternae b u t not in T-tubule. Thus the precise localization of cyclase within sarcoplasmic reticulum remains unclear. In any case, since we observed catecholamine-sensitive adenylate cyclase and fl-adrenergic receptor binding in plasmalemma, sarcoplasmic reticulum and mitochondria, it is possible that in skeletal muscle the catechol-
357
amines could initiate their actions at the cell surface and/or at intracellular loci. The characteristics of [3H]dihydroalprenolol binding to skeletal muscle plasmalemma fulfilled several criteria required for the identification of adenylate cyclase-coupled B-adrenoceptors [8,9]. The binding was rapid and reversible. At 37°C the time required for half-maximal association and dissociation was a b o u t 2 min. In an earlier study we found that propranolol could abolish the isoproterenol-induced activation of human plasmalemmal adenylate cyclase within 2 min of its addition to the reaction medium [17]. The rapidity of [3H]dihydroalprenolol association and dissociation suggest that the binding is occurring to those receptor sites closed linked to adenylate cyclase. Binding of [3H]dihydroalprenolol in plasmalemma occurred to a single group of receptor sites and it was saturable, indicating a finite number of binding sites. The apparent Kd for [3H]dihydroalprenolol was a b o u t 15 nM which is within the range of K d values reported for other tissues [9]. A Hill coefficient of 1.007 for [3H]dihydroalprenolol binding suggested that there is neither positive nor negative cooperative interaction among the B-receptor binding sites in skeletal muscle. Hill coefficients of around one have also been reported in fl-adrenoceptor binding studies with other membranes [9]. However, Lefkowitz and co-workers [8], who extensively characterized the [3H]dihydroalprenolol binding in frog erythrocytes and rat adipocytes, obtained Hill coefficients of significantly less than one, and they suggested that negative cooperativity exists among the B-receptor sites. The nature of cooperativity thus seems to vary in different tissues. Several ~-adrenergic agents known to be active on intact skeletal muscle competed for [3H] dihydroalprenolol binding sites with essentially similar selectivity and stereospecificity. For example, catecholamines are known to enhance glycogenolysis in skeletal muscle with a p o t e n c y sequence of isoproterenol epinephrine > norepinephrine [36,37]. A similar order of p o t e n c y with isomeric specificity (--) isomers > (+) isomers) was reported in studies of catecholamine effects on twitch~contraction, even though catecholamines induce differential effects on fast- and slow-twitch muscle [5]. Catecholamines have also been shown to enhance Na + efflux in skeletal muscle [2--4] with isomeric specificity [3]. Propranolol abolished the catecholamine effects on giycogenolysis [37], twitch-contraction [5], and Na + effiux [3,4], again with isomeric specificity. The results obtained in the present study on [3H]dihydroalprenolol binding and adenylate cyclase closely agree with the aforementioned physiological studies. However, the concentrations of agonists and antagonists needed to cause half-maximal effect on [3H]dihydroalprenolol or adenylate cyclase activity in the present study are a b o u t t w o orders of magnitude greater than those reported for glycogenolysis [37], twitch-contraction [5], and Na + fluxes [3,4]. This may be due to changes that the receptor and enzyme molecules undergo during several preparatory steps involved in the isolation of muscle membranes. Alternatively, partial occupation of receptor sites by agonists and antagonists might be sufficient to cause greater physiological response than that required to alter adenylate cyclase as measured in our assay. Furthermore, ouabain inhibits the catecholamine induced Na ÷ transport in intact skeletal muscle [3,4], whereas ouabain showed no effect on the plasmalemmal adenylate cyclase (Reddy, N.B., unpublished data). Thus, mechanisms other than
358
cyclic AMP may be involved in mediating different physiological effects by catecholamines in skeletal muscle. In conclusion, we have shown that fl-adrenergic receptors in skeletal muscle can be identified by direct binding assays. The present investigation constitutes a detailed study of the properties of skeletal muscle fl-adrenoceptors under in vitro conditions. The methods and values established in this study will be useful in analyzing the possible alterations of fl-adrenoceptor-associated functions in several pathophysiological states of skeletal muscle in animal models and in human neuromuscular diseases. For example, denervated skeletal muscle exhibits greater physiological sensitivity to catecholamines [5]. Our preliminary studies of rat skeletal muscle [38] indicated that the plasmalemmal fl-adrenergic receptor density, determined by 12SI-labelled hydroxybenzyl pindolol binding, increases about two-fold following denervation, while the adenylate cyclase decreases; suggesting that denervation causes uncoupling of receptor-enzyme interaction. We have also found differences in the fl-receptor binding and catecholamine sensitivity of adenylate cyclase in fast-twitch and slow-twitch skeletal muscle of rat [39]. Thus, the studies on fl-adrenoreceptors may be helpful in further delineating the 'neurotrophic' regulatory mechanisms as well as their involvement in certain neuromuscular disorders. Acknowledgements The authors wish to thank Ms. K.L. Oliver for technical assistance. This work is supported in part by Muscular Dystrophy Assn., Inc. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Gross, S.R. and Mayer, S.E. (1975) Metabolism 24, 3 6 9 - - 3 8 0 Hays, E.T., n w y e r , T.M., Horowicz, P. and Swift, J.G. (1974) Am. J. Physiol. 227, 1340--1347 Rogus, E.M., Cheng, L.C. and Zierlier, K. (1977) Biochim. B i o p h y s . A c t a 4 6 4 , 3 4 7 - - 3 5 5 Clausen, T. and F l a t m a n , J. (1977) J. Physiol. 270, 363--414 Bowman, W.C. and Nott, M.W. (1969) Pharmacol. Rev. 21, 27--72 Robison, G.A., Butcher, R.W. and Sutherland, E.W. (1971) Cyclic AMP, pp. 145--231, A c a d e m i c Press, New Yo rk Birnbaumer, L. (1973) Biochim. B i o p h y s . A c t a 300, 129--158 Lefkowitz, R.J., Limbixd, L.E., Mukherjee, C. and Caron, M.G. (1976) B i o e h i m . B i o p h y s . A c t a 457, 1--39 Wolfe, B.B., Harden, T.K. and Molinoff,, P.B. (1977) Annu. Rev. Pharmacol. Roxicol. 17, 575---604 Lefkowitz, R.J., Mukherjee, C., Coverstone, M. and Caron, M.G. (1974) B i o c h e m . B i o p h y s . Res. Cornm a n . 60, 704--709 Levitzki, A., Arias, D. and Steer, M.L. (1974) Proc, Natl. A c a d . Sci. U.S. 71, 2773--2776 Aurbach, G.D., Fedak, F.A., Woodward, C.J., Palmer, J.S., Hauser, D. and Troxler, F. (1974) Science 186, 1 2 2 3 - - 1 2 2 4 Lefkowitz, R.J. and Williams, L.T. (1977) Proc. Natl. A c a d . Sci. U.S. 74, 515--519 Festoff, B.W. and Engle, W.K. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2 4 3 5 - - 2 4 3 9 Red dy, N.B., Engel, W.K. and F e s t o f f , B.W. (1976) B i o c h i m . B i o p h y s . A c t a 433, 365--382 Red dy, N.B., Oliver, K.L., Festoff, B.W. and Engle, W.K. (1978) Biochim. B i o p h y s . A c t a 540, 371-388 Red dy, N.B., Oliver, K.L., Festoff, B.W. and Engel, W.K. (1978) B i o c h i m . B i o p h y s . A c t a 540, 389-401 R e d d y , N.B. and Engel, W.K. (1977) Fed. Proc. 3 6 , 5 8 4 Salomon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biochem. 58, 541--548 Lo wry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Rodben, M., Lin, M.C., Salomon, Y., Londos, C., Harwood, J.P., Martin, B., Rendell, M. and Berman, M. (1975) Adv. Cyclic N u c l e o t i d e Res. 5, 3--29
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22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Festoff, B.W., Oliver, K.L. and Reddy, N.B. (1977) J. Membrane Biol. 3 2 , 3 3 1 - - 3 4 3 Severson, D.L., D r u m m o n d , G.I. and Sulakhe, P.V. (1972). J. Biol. Chem. 247, 2949--2952 Scatchard, G. (1949) Ann. N,Y. Acad. Sci. 5 1 , 6 6 0 - - 6 7 2 Hill, A.V. (1910) J. Physiol. 4 0 , 1 9 0 - - 2 2 4 Bylund, D.B. and Snyder, S.H. (1976) Mol. Pharmacol. 12, 568--580 Lands, A.M., Arnold, A., McAuliff, J.P., Luduena, F.P. and Brown, T.G. (1967) Nature 214, 597--598 Birnbaumer, L., Pohl, S.L. and Kaumann, A.J. (1975) Adv. Cyclic Nucleotide Res. 4 , 2 3 9 - - 2 8 1 Schild, H.O. (1949) Br. J. Pharmacol. 4, 277--288 Cheng, Y.-C. and Prusoff, W.H. (1973) Biochem. Pharmacol. 22, 3 0 9 9 - - 3 1 0 8 Levey, G.S., ed. (1976) Hormone-receptor i n t e r a c t i o n : Molecular aspects, pp. 1--431, Marcel Dekker Inc., New Yo rk Maguire, M.E., Ross, E.M. and Gilman, A.G. (1977) Adv. Cyclic Nucleotide Res. 8, 1--85 E n t m a n , M.L., Bornet, E.P., Pitts, B.J.R. and Schwartz, A. (1977) Fed. Proc. 3 6 , 3 4 6 Caswell, A.H., Baker, S.P., Boyd, H., Potter, L.T. and Gaxcia, M. (1978) J. Biol. Chem. 253, 3049-3054 Raible, D.G., Cutler, L.S. and Rodan, G.A. (1978) FEBS Lett. 85, 149--152 Ellis, S., Davis, A.H. and Anderson, H.L., Jr. (1955) J. Pharmacol. Exp. Ther. 115, 120--125 Gross, S.R., Mayer, S.E. and Longshore, M.A. (1976) J. Pharmacol. Exp. Ther. 198, 526--538 R e d d y , N.B. and Engel, W.K. (1978) Neurology 2 8 , 3 9 9 - - 4 0 0 Oliver, K.L., Reddy, N.B. and Engel, W.K. (1977) Neurosci. Abstr. 3, 536
Biochimica et Biophysica Acta, 585 (1979) 3 4 3 - - 3 5 9 © Elsevier/North-Holland Biomedical Press
BBA 28963
IN V I T R O C H A R A C T E R I Z A T I O N OF SKELETAL MUSCLE ~-ADRENERGIC R E C E P T O R S COUPLED TO A D E N Y L A T E CYCLASE
N. B O J J I R E D D Y and W. K I N G E N G E L
Medical Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20205 (U.S.A.) (Received O c t o b e r 12th, 1978)
Key words: ~-Adrenergic receptor; Adenylate cyclase; Dihydroalprenolol; Receptor site; (Skeletal muscle; Plasmalemma)
Summary [~H]Dihydroalprenolol, a potent ~-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ~-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed by [3H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [3H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small b u t consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [3H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions. The binding of [3H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K~)and dissociation (K2) rate constants of 2.21 • 107 M -~ • min -1 and 3.21 • 10 -1 min -1, respectively, were obtained. The dissociation constant (Kd) of 15 nM for [3H]dihydroalprenolol obtained from saturation binding data closely agreed with the K d derived from the ratio of dissociation and association rate constants (K2/ K,). Several ~-adrenergic agents k n o w n to be active on intact skeletal muscle also c o m p e t e d for [3H]dihydrolaprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [aH]dihydroalprenolol binding sites with a p o t e n c y of isoprotereAbbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2' ethanesulfonic acid; EGTA, ethyleneglycolbis(~-aminoether)N',N'-tetraacetic acid ; GMP-PNP° 5'-guanylyl imidodiphosphate.
344
nol ~ epinephrine ~ norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (--)-isomers being more potent than (+)-isomers. Phenylephrine, an a-adrenergic agonist, showed no effect either on [3H]dihydroalprenolol binding or on adenylate cyclase. Known fl-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [3H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The Kj values for the antagonists determined from inhibition of [3H]dihydroalprenolol binding agreed closely with the Ki values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [3H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the fi-adrenergic receptor.
Introduction Catecholamines such as epinephrine and isoproterenol influence glycogenolysis, [1], Na + and K ÷ transport [2--4], and twitch-contraction [5] of mammalian skeletal muscle. These effects are believed to be mediated by interaction of catecholamines with the ~-adrenergic receptors. However, the site of localization of ~-receptors and the mechanism(s) of drug-receptor interactions in skeletal muscle have not been fully defined. Available evidence regarding the presence of ~-receptors in skeletal muscle has been derived mostly by indirect studies such as by studying the effect of fl-adrenergic agents on glycogen metabolism or muscle contraction. Since catecholamines regulate several muscle functions [ 5 ], it was felt important to identify the ~-adrenergic receptor sites and study their molecular properties. In homogenized tissue, the ~-receptor activity is manifested mostly in the form of adenylate cyclase, since that enzyme, as in the intact tissue, responds to ~-adrenergic drugs [6]. In most tissues, adenylate cyclase is localized in plasma membranes [6]. Accordingly, the isolated plasma membranes of tissues that respond to catecholamines have been used extensively to delineate the properties of fl-receptor-adenylate cyclase interactions [6--9]. Recently, considerable progress has been made for direct identification of fl-receptors coupled to adenylate cyclase in isolated membranes through the use of binding studies with radioactive fl-adrenergic agents of high specific activity [ 10--13 ]. Previous studies from our laboratory [14--16] indicated that a particulate fraction enriched with surface membranes could be isolated from homogenates of rat and human skeletal muscle. Those membranes contain high levels of adenylate cyclase and the enzyme responds to catecholamines with fl-adrenoceptor specificity [17]. The present study is an attempt to characterize further the skeletal muscle /3-adrenoceptors through direct labelling of the receptor sites with [3H]dihydroalprenolol, a potent/3-adrenergic antagonist, which has been successfully used to identify the fl-adrenoceptors in other systems [8]. The data indicate that [3H]dihydroalprenolol, like the catecholamine-sensitive adenylate cyclase, is predominantly localized in a plasmalemmal fraction. The characteristics of [3H]dihydroalprenolol binding to plasmalemma, including kinetics, drug
345 specificity and correlation with adenylate cyclase, are described. Some of these studies have appeared in an abstract [18]. Materials and Methods
Materials [3H]Dihydroalprenolol (32 Ci/mmol) and cyclic [G-3H]AMP (38 Ci/mmol) were purchased from New England Nuclear (Boston, MA). [a-32p]ATP (10-20 Ci/mmol), [7-32P]ATP (20--30 Ci/mmol), GMP-PNP, and neutral grade alumina were obtained from International Chemicals and Nuclear Co. (Irvine, CA). (+)-Epinephrine, (+)-isoproterenol were purchased from Winthrop Laboratories (New York, N.Y.). (--)- and (+)-alprenolol (Hassle Pharmacuticals, Sweden), and (--)- and (+)-propranolol (Ayerst Laboratories, Canada) were kindly donated by those companies. The sources of other materials were those previously described [16,17]. Isolation of muscle membranes Gastrocnemius-plantaris muscle complex of male Sprague-Dawley rats (200-250 g, Zivic-Miller Labs, Allison Park, PA) was used to prepare different membranes. Skeletal muscle plasmalemma, sarcoplasmic reticulum and mitochondria were prepared essentially as described previously [16], except that in all preparative reagents EDTA was omitted and Tris-HC1 buffer was substituted with 10 mM HEPES, pH 7.5. It was found that the specific activity of adenylate cyclase and its activation b y catecholamines were greater in the membranes prepared with these modifications than in those prepared by earlier procedure. However, these preparative changes did not alter the other properties of different membranes which were previously described in considerable detail [16]. Further purification of plasmalemma on discontinuous sucrose gradients was as described earlier [16], except that the sucrose gradients were prepared in 5 mM HEPES, pH 7.5. The plasmalemmal subfractions settled at sucrose interfaces of 0.6--0.8 M (PLF1), 0.8--1.0 M (PLF2), 1.0--1.2 M (PLF3) and 1.2--1.4 M (PLF4) were collected, diluted to 0.3 M sucrose with 5 mM HEPES, pH 7.5, and sedimented at 100 000 × g for 60 rain. The pellets were resuspended in 0.3 M sucrose/5 mM HEPES, pH 7.5. All samples (in aliquots of 0 . 2 - 0 . 4 ml) were frozen in liquid nitrogen and stored at --70°C until use. [3H]Dihydroalprenolol, adenylate cyclase and (Na ÷ + K+)-ATPase activities were stable for at least t w o months under those conditions. [3H] Dihydroalprenolol binding assay Muscle membranes (50--100 ~g protein) were incubated in siliconized glass tubes (12 × 75 mm) with 10 mM MgC12/50 mM Tris-HC1, pH 7.5 and indicated concentrations of [3H]dihydroalprenolol (approx. 45 000 d p m / p m o l ) in a total volume of 200 pl. In standard assays [3H]dihydroalprenolol was at 30 nM. Drugs whose effects on the binding were to be studied were freshly prepared in 50 mM Tris-HC1, pH 7.5 and added in 20 pl volume to the assay medium. The reaction was for 5 min at 37°C, unless mentioned otherwise. After 5 min, the reaction mixture was diluted with 2 ml of a cold (2°C) 'buffer medium' (10
346 mM MgC12/50 mM Tris-HC1, pH 7.5) and was immediately filtered through a Millipore filter (EHWP, 0.5 pm pore size) under vacuum pressure (30 sample manifold, Millipore Corporation). The filter was washed with an additional 15 ml of same cold buffer medium. The complete filtration procedure took about 15 s. The filters were dried, dissolved in 10 ml of Instabray (Yorktown Research, S. Hackensack, NJ), and counted in a Packard liquid scintillation spectrometer (Model 2650) at a 3H counting efficiency of 42%. In parallel experiments, the a m o u n t of [3H]dihydroalprenolol absorbed to Millipore filters in the absence of membranes or the a m o u n t nonspecifically bound to membranes in the presence of excess (100 ~M) nonradioactive (--)alprenolol was determined. Such binding was about 0.01% of the total radioactivity added to the incubation medium, and it was subtracted from appropriate total binding values to derive 'specific binding'. Only such 'specific binding' data are presented in the results. It was found that nonspecific binding of [3H]dihydroalprenolol was higher if the assays were carried out in polypropylene (Falcon) or glass tubes than for the assays performed in siliconized glass tubes.
Adenylate cyclase assay The enzyme activity was estimated as previously described [17]. The assay medium, in a total volume of 100 pl, comprised 50 mM Tris-HC1, pH 7.5, 0.5 mM [a-32P]ATP (20--30 dpm/pmol), 5 mM MgC12, 10 mM KC1, 10 mM theophylline, 15 mM creatine phosphate, 0.5 units of creatine phosphokinase, 40-60 ~g of membrane protein and other agents whose effects were to be studied. The reaction was for 15 rain at 37°C. The cyclic [32P]AMP produced was separated by a modified [17] column chromatographic procedure of Salomon et al. [19]. (Na ÷ + K+)-A TPase assay The ATPase activity was determined using [~,-32P]ATP as substrate and by measuring by Cerenkov radiation the 32pi produced as described earlier [16]. In the present study, (Na ÷ + K+)-ATPase was defined as the difference between the total activity (5 mM Mg2+/100 mM Na+/20 mM K ÷) in the absence and presence of 0.1 mM ouabain. Protein content of muscle membranes was estimated by the method of Lowry et al. [20] using bovine serum albumin as a standard. Results
Subcellular distribution o f [3H]dihydroalprenolol binding Since the ~-adrenergic receptors are known to be closely associated with adenylate cyclase, we have determined [3H]dihydroalprenolol binding and adenylate cyclase activity in different muscle fractions to evaluate their interrelationship. The skeletal muscle consists of three major membrane components, viz. plasmalemma, sarcoplasmic reticulum and mitochondria. Working with human skeletal muscle, we have previously established that the three fractions were distinctly different one from another in certain biochemical and morphological properties, and that cross-contamination was negligible under our isolation pro-
347
cedure [16]. Similar differences were also observed in the membrane fractions isolated from rat skeletal muscle used in the present study. The pattern of (Na++ K+)-ATpase distribution (Table I) clearly illustrates the difference between plasmalemma and the other muscle membranes. The enzyme was predominantly localized in plasmalemma, while it was undetectable or at a very insignificant level in sarcoplasmic reticulum and mitochondria (Table I). The subcellular distribution of fi-adrenergic receptor sites in skeletal muscle was examined by measuring specific [3H]dihydroalprenolol binding to plasmalemma, sarcoplasmic reticulum and mitochondria (Table I). When measured in the presence of 30 nM [3H]dihydroalprenolol, the binding to partially purified plasmelamma was about four times, and that to sucrose gradient purified plasmalemmal subfraction 1 (PLF,) about 10 times greater than the binding to sarcoplasmic reticulum or mitochondria, suggesting the prevalence of greater density of fi-adrenergic receptor sites in plasmalemma. The level of [3H]dihydroalprenolol binding to subcellular fractions of muscle closely corresponded with the level of stimulation of adenylate cyclase in those membranes by catecholamines, which are believed to activate the enzyme by interacting with ~-adrenergic receptor. (--)-Isoproterenol (10 ~M) induced about 3-fold activation of adenylate cyclase in plasmalemma, while it enhanced the enzyme in sarcoplasmic reticulum by only 60%, and that in mitochondria by 80%, over their respectively basal activities (Table I). The relative yields of protein and adenylate cyclase in different fractions were similar to those previously obtained with human skeletal muscle [16]. The co-purification of [3H]dihydroalprenolol binding and adenylate cyclase was evident when plasmalemma was subjected to further purification on sucrose gradients (Table I). The highest specific activities of [3H]dihydroalprenolol binding and adenylate cyclase were observed in a subfraction which settled at the sucrose interTABLE I CORRELATION OF [3H]DIHYDROALPRENOLOL BINDING WITH ADENYLATE CYCLASE (Na ÷ + K+)-ATPase A C T I V I T I E S IN T H E S U B C E L L U L A R F R A C T I O N S O F S K E L E T A L M U S C L E
AND
Preparation o f subcellular f r a c t i o n s and e n z y m e assays w e r e as d e s c r i b e d u n d e r M e t h o d s . PL, plasmaleraraa; P L F I - - P L F 4 , p l a s m a l e r a m a l s u b f r a c t i o n s o b t a i n e d b y s u c r o s e - g r a d i e n t c e n t r i f u g a t i o n ; S R , sarcop l a s m i c r e t i c u l u m . [ ~ H ] d i h y d r o a l p r e n o l o l ( [ J H ] D H A ) b i n d i n g w a s m e a s u r e d u n d e r standard i n c u b a t i o n c o n d i t i o n s . GTP and ( - - ) - i s o p r o t e r e n o l w e r e p r e s e n t at 1 0 p M e a c h in assay o f a d e n y l a t e c y c l a s e . V a l u e s are m e a n s o f d u p l i c a t e d e t e r m i n a t i o n s on six p r e p a r a t i o n s ± S . D . Muscle fraction
[3H]DHA
binding (pmol bound] mg protein)
A d e n y l a t e c y c l a s e ( p m o l c y c l i c AMP f o r m e d / r a g P r o t e i n per rain)
(Na ÷ + K+)ATPase ( # r a o l Pi
Basal
released/rag p r o t e i n per h )
Iso-
Iso-
proterenol
protcrenol + GTP
Horaogenate Mitochondria SR PL PLF PLF PLF PLF
1 2 3 4
0.03 ± 0.07 ± 0.05_+ 0.16 + 0.62 ± 0.14 ± 0.08 ± 0.05 ±
0.01 0.02 0.02 0.04 0.06 0.02 0.02 0.01
4 ± 0.2 2 4 _+ 2 . 6 18-+ 2 . 0 32 ± 4.4 6 8 _+ 1 4 18 ± 4 . 0 8 ± 1.2 6 ± 0.8
12 34 26 86 180 30 14 8
± 1.8 ± 4 ± 3.4 + 5.8 ± 16 ± 8.0 + 2.0 ± 0.6
16 60 68 204 680 II0 36 14
± ± ± ± ± ± + ±
4 8 12 18 56 16 4 2
0.0 0.0 0.6 11.6 44.0 8.6 3.2 1.8
± ± ± ± ± ±
0.2 1.8 6.0 2.4 1.6 0.4
348
face of 0.6--0.8 M (corresponding to a sucrose density (g/cm 3) of 1.078--1.104 at 20°C). [3H]Dihydroalprenolol binding and the enzyme activity gradually decreased in the subfractions PLF:--PLF4 which settled at greater sucrose densities (Table I). GTP, and its analog GMP-PNP (10 gM), markedly enhanced the responsiveness of adenylate cyclase in each muscle membrane fraction to isoproterenol, and the highest specific activities were found in plasmalemma (Tables I and II). GTP alone caused marginal but consistent stimulation {20--40% above basal) of adenylate cyclase (Table II). The effect of GMP-PNP was more pronounced than that of GTP, both in the absence and in the presence of isoproterenol. These results are in accordance with findings in other tissues [21]. In agreement with our earlier findings on human muscle [17], the guanyl nucleotides potentiated the stimulating effect of {--)-isoproterenol on rat muscle plasmalemmal adenylate cyclase without altering the apparent Km for (--)-isoproterenol (data not shown). The highest specific activity of adenylate cyclase in each membrane fraction was obtained with NaF (10 mM) as a stimulant (Table II). NaF-stimulated enzyme activity in purified plasmalemmal fraction (PLF~) was 2.3--3.4 nmol cyclic AMP produced per mg protein per min in six different preparations. The enzyme activity values obtained in this study are significantly greater than those previously reported for rat [22], human [16] and rabbit [23] muscle plasma membranes. Thus, the muscle plasmalemma as prepared in the present study are highly purified in terms of their enrichment with adenylate cyclase, and hence provided asuitable material to evaluate the properties of adenylate cyclase and the associated ~-adrenergic receptor.
Properties of [3H]dihydroalprenolol binding to plasmalemma The specific binding of [3H]dihydroalprenolol was linear between 25 to 300 pg of plasmalemmal protein. The total binding of [3H]dihydroalprenolol to partially-purified plamalemma and sucrose-density-purified plasmalemmal subfraction 1, PLF1, respectively, was two and five times greater than the non-
T A B L E II ACTIVATION OF ADENYLATE C Y C L A S E BY N a F A N D G U A N Y L N U C L E O T I D E S MEMBRANE FRACTIONS OF SKELETAL MUSCLE
IN DIFFERENT
M e m b r a n e p r e p a r a t i o n a n d a s s a y o f a d e n y l a t e c y c l a s e w e r e as d e s c r i b e d i n M e t h o d s . M e m b r a n e f r a c t i o n a b b r e v i a t i o n s were s a m e as i n T a b l e I. V a l u e s are m e a n s o f six p r e p a r a t i o n s ,+ S . D . G M P - P N P , 5 ' - g u a n y l y l imidodiphosphate. Additions
Adenylate per rain) Membrane fraction:
None NaF (10raM) GTP (10 #M) GMP-PNP (10 ~M) Isoproterenol (10 pM) + GMP-PNP (10 pM)
PL
34 620 46 394 528
± 8 ,+ 1 6 0 ,+ 4 _+ 6 2 ,+ 8 4
cyclase (pmol
cyclic AMP formed/rag protein
PLF l
SR
Mitochondria
80,+ 12 2800,+ 600 1 0 4 ,+ 1 6 9 0 8 ,+ 5 4 1 8 9 6 ,+ 4 4 0
20,+ 4 340,+ 80 2 6 ,+ 5 180-+ 14 2 8 4 ,+ 5 0
2 5 -+ 7 1 2 0 ,+ 3 0 2 9 ,+ 8 6 0 -+ 1 2 9 4 ,+ 8
349 specific binding. Since the yield of PLF~ was very low (approx. 0.15 mg/g muscle wet weight), it became difficult to compare several aspects of [3H]dihydroalprenolol binding with adenylate cyclase from the same preparation. Most of the present studies were, therefore, carried out on partiaUy-purified plasmalemma. Preliminary experiments of saturation binding and selected drug specifity studies on partially-purified plasmalemma and PLF~ fractions revealed that those properties were qualitatively similar in both the fractions.
Saturability of binding sites The specific binding of [3H]dihydroalprenolol as a function of its concentration (Fig. 1) indicated that the binding sites were saturable. Apparent saturation of binding sites seemed to occur at a [3H]dihydroalprenolol concentration of about 50 nM. Determination of binding at concentrations of [3H]dihydroalprenolol greater than 50 nM was made difficult because of very high nonspecific binding. The half-maximal binding, as revealed by the double-reciprocal plot (Fig. 1), occurred at 12.5 nM, which provided an estimate of apparent dissociation constant (Kd). The Scatchard [24] plot (Fig. 2, left) of the saturation binding data (Fig. 1) yielded a straight line, suggesting the presence of a single
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group of binding sites with a K d of 15.4 ± 3.2 nM (n = 4). At apparent saturation, the receptor concentration in partially-purified plasmalemma was 0.33 pmol of [3H]dihydroalprenolol b o u n d per mg protein {Fig. 2, left). A receptor concentration of 1.6 pmol per mg protein was obtained for sucrose~lensity purified plasmalemmal subfraction 1 (PLF1) (data not shown). To test the nature of cooperativity of binding sites, the equilibrium binding data was analyzed by the Hill equation [25]. The Hill plot (Fig. 2, right) also gave a straight line with a Hill coefficient of 1.007 ± 0.105 (n = 4), indicating neither positive nor negative cooperativity. K i n e t i c s o f [ 3 H ] d i h y d r o a l p r e n o l o l binding
Under our assay conditions, the binding of [3H]dihydroalprenolol to plasmalemma was a rapid process. The binding reached equilibrium within 5 min, with half-maximal binding occurring at 1.5 min (Fig. 3a). After reaching a maximum, the binding slightly decreased when incubations were carried out for periods greater than 5 min (Fig. 3a). During prolonged incubation, the drugreceptor complex probably undergoes a spontaneous dissociation and reassociation resulting in a new steady state. To overcome that, we routinely conducted the binding experiments for 5 min. From the binding data up to 5 min incubation, an association rate constant (K~) of (2.21 ± 0.20) • 107 M -1 • min -1 (n = 3) was calculated from the equation [26] g~ = [1/t(a -- b)] ln[b(a - - x ) / a ( b - - x ) ] ;
where a is the initial concentration of [3H]dihydroalprenolol in the assay, b is receptor concentration (0.33 from Scatchard plot) and x is the a m o u n t of [3H]dihydroalprenolol specifically bound at time t. The drug-receptor complex readily dissociated when (--)-propranolol was added to the incubation medium after [3H]dihydroalprenolol binding reached its maximum (Fig. 3b). Similar dissociation could also be obtained by 1000fold dilution of incubation medium with Tris-HC1 buffer lacking propranolol (data n o t shown). The dissociation of radioligand from its receptor moiety was
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Fig. 3.a. T i m e c o u r s e o f a s s o c i a t i o n o f [ 3 H ] d i h y d r o a l p r e n o l o l ( D H A ) t o p l a s m a l e m m a . S t a n d a r d b i n d i n g c o n d i t i o n s w e r e e m p l o y e d e x c e p t i n c u b a t i o n t i m e w h i c h w a s v a r i e d as i n d i c a t e d . V a l u e s are m e a n s of t h r e e e x p e r i m e n t s , b. D i s s o c i a t i o n o f [ 3 H ] d i h y d r o a l p r e n o l o l f r o m its r e c e p t o r as a f u n c t i o n o f t i m e a n d t e m p e r a t u r e . P l a s m a l e m m a w e r e e q u i l i b r a t e d w i t h 3 0 nM [ 3 H ] d i h y d r o a l p r e n o l o l a t 3 7 ° C f o r 4 rain. Duplicate a l i q u o t s ( 1 0 0 pl) w e r e s a m p l e d f o r [ 3 H ] d i - h y d r o a l p r e n o l o l b i n d i n g as d e s c r i b e d in Materials a n d Methods and the remaining reaction mixture was equilibrated at required t e m p e r a t u r e for an additional 2 min. The dissociation was initiated at time zero by adding (--)-propranolol, maintained at appropriate t e m p e r a t u r e , to a final c o n c e n t r a t i o n o f 10 pM. A l i q u o t s (100/~1) w e r e t h e n w i t h d r a w n in d u p l i c a t e a t i n d i c a t e d t i m e s to d e t e r m i n e [ 3 H ] d i h y d r o a l p r e n o l o l b i n d i n g . C o n t r o l b i n d i n g r e f e r s t o t h e a m o u n t of [ 3 H ] d i h y d r o a l p r e n o l o l b o u n d p r i o r t o t h e a d d i t i o n of p r o p r a n o l o l . T h e e x p e r i m e n t w a s r e p e a t e d o n t w o d i f f e r e n t p r e p a r a t i o n s w i t h essentially similar results.
temperature dependent (Fig. 3b). It was rapid at 37°C, moderate at 25°C, and minimum at 0°C. Typical half-lives for the drug-receptor complex were 2, 8 and 18 min at 37, 25 and 0°C, respectively. The time required for haif-maximal dissociation at 37°C (2 min) closely resembled the time required for halfmaximal association (1.5 min, Fig. 3a). From the data at 37°C, a dissociation rate constant (K2) of (3.21 + 0.8) • 10 -1 min -1 (n = 2) was obtained from the equation K2 = ( l / t ) In (a/x); where a is the a m o u n t of [3H]dihydroalprenolol initially b o u n d (i.e., prior to the addition of propranolol to initiate dissociation) and x is the a m o u n t of [3H]dihydroalprenolol remain b o u n d at time t (i.e., after the addition of propranolol). The ratio of K2/K~ provided an independent K d of 14.5 nM for [3H]dihydroalprenolol; which agreed closely with the K d obtained b y the Scatchard analysis.
Selectivity and stereospecificity of [3H]dihydroalprenolol binding The specificity of [3H]dihydroalprenolol binding to plasmalemma was examined by studying the abilities of different fl-adrenergic agonists and antagonists in competing for the binding sites. Of the catecholamines tested, (--)-isoproterenol was the most p o t e n t inhibitor of [3H]dihydroalprenolol binding with an ICso of 0.07 pM (Fig. 4). The ICso values for (--)-epinephrine and (--)-norepinephrine were 0.5 and 60 gM, respectively. Thus, the potency sequence of agonists in competing for [3H]dihydroalprenolol binding sites was (--)-isoproterenol > (--)-epinephrine > (--)-norepinephrine. Such an order of
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CATECHOLAMINE (-log M) Fig. 4. E f f e c t s o f d i f f e r e n t c a t e c h o l a m i n e s o n p l a s m a l e m m a l a d e n y l a t e c y c l a s e (left) a n d [ 3 H ] d i h y d r o a l p r e n o l o l ( D H A ) b i n d i n g (right). Values are m e a n s of d u p l i c a t e analysis of t h r e e d i f f e r e n t p r e p a r a t i o n s .
potency is characteristic of fi~-adrenergic receptors [27]. Furthermore, isoproterenol and epinephrine showed considerable stereospecificity in that (--)-isomers were more p o t e n t than their corresponding (+)-isomers. The ICso values for (+)-isoproterenol and (+)-epinephrine were a b o u t 15 and 70 t~M, respectively (Fig. 4). The order of potency with which different catecholamines competed for [3H]dihydroalprenolol binding closely resembled the order of p o t e n c y with which they stimulated the plasmalemmal adenylate cyclase (Fig. 4). On a molar basis, (--)-isoproterenol was a more potent stimulator than (--)-epinephrine or (--)-norepinephrine. The apparent ICso values for the activation of adenylate cyclase were 0.09 t~M for (--)-isoproterenol, 0.65 t~M for (--)-epinephrine, and 80 pM for (--)-norepinephrine. Activation of adenylate cyclase by catecholamines was stereospecific with most of the potency residing in (--)-isomers (Fig. 4). An a-adrenergic agonist, (--)-phenylephrine, showed no significant effect either on [3H]dihydroalprenolol binding or on adenylate cyclase (Fig. 4). Similarly phentolamine, a p o t e n t a-blocker, showed no effect (data not shown). To characterize further the fi-receptor in skeletal muscle membranes, the effect of fi-adrenergic antagonists on [3H]dihydroalprenolol binding and adenylate cyclase were investigated. Propranolol and alprenolol are known to antagonize the fi-adrenergic stimulation of adenylate cyclase by isoproterenol. The nature of antagonist effect on the enzyme could be evaluated by its ability to alter the dose response to isoproterenol [8,28]. As can be seen from Fig. 5, proporanolol effectively shifted the dose vs. response curve of isoproterenol towards right. Moreover, the (--)-propranolol was more effective than (+)-propranolol. Similar results were obtained with (--)- and (+)-isomers of alprenolol (data not shown). Schild [29] plot (Fig. 6) yielded straight lines, suggesting a competitive nature of propranolol inhibition. The intercept of the lines with the abcissa of Schild plot provides an apparent K i value [29] for each antagonist. They varied 8--11 nM for (--)-propranolol, 600--900 nM for (+)-propra-
353
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(-) ISOPROTERENOL (-Log M) Fig. 5. A n t a g o n i s m of ( - - ) - i s o p r o t e r e n o l s t i m u l a t i o n of p l a s m a l e m m a l a d e n y l a t e c y c l a s e b y s t e r e o i s o m e r s o f p r o p r a n o l o l . D o s e - r e s p o n s e of a d e n y l a t e c y c l a s e to ( - - ) - i s o p r o t e r e n o l w a s d e t e r m i n e d w i t h o u t or w i t h i n d i c a t e d f i x e d c o n c e n t r a t i o n s o f (--)- or ( + ) - p r o p r a n o l o l . T h e a c t i v i t y o b t a i n e d w i t h 10/~M (--)-isoprot e r e n o l , in t h e a b s e n c e o f a n t a g o n i s t , w a s c o n s i d e r e d as m a x i m a l . V a l u e s are m e a n s o f t h r e e e x p e r i m e n t s .
nolol, 10--14 nM for (--)-alprenolol, and 800--1100 nM for (+)-alprenolol in three such experiments. Propranolol and alprenolol were approximately equipotent in competing for
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~-ADRENERGICANTAGONIST(.-logM) Fig. 6. Schild p l o t s o f t h e d a t a s h o w n in Fig. 5. C R = c o n c e n t r a t i o n r a t i o o f ( - - ) - i s o p r o t e r e n o l n e e d e d to o b t a i n similar h a l f - m a x i m a l a c t i v i t y in t h e p r e s e n c e a n d a b s e n c e o f a given c o n c e n t r a t i o n o f (--)- or ( + ) o p r o p r a n o l o l . E n z y m e r e s p o n s e a t 10 #M ( - - ) - i s o p r o t e r e n o l w i t h o u t p r o p r a n o l o l w a s c o n s i d e r e d m a x i m u m , a n d t h e e q u i p o t e n t c o n c e n t r a t i o n s o f i s o p r o t e r e n o l r e q u i r e d f o r 50% m a x i m a l r e s p o n s e in t h e a b s e n c e a n d p r e s e n c e of a n t a g o n i s t w a s d e t e r m i n e d a n d u s e d to c a l c u l a t e C R . T h e slope o f e a c h line w a s a b o u t 1, i n d i c a t i n g t r u e c o m p e t i t i v e a n t a g o n i s m b y p r o p r a n o l o l .
354 [3H]dihydroalprenolol binding sites (Fig. 7). Like catecholamines, the fi-blockers also exhibited considerable stereospecificity. The ICso values of (--)-propranolol and (--)-alprenolol for inhibiting [3H]dihydroalprenolol binding were a b o u t the same, and this value was 40 nM. Corresponding ICso values for the inhibition of adenylate cyclase were 20 nM for (--)-propranolol and 30 nM for (--)-alprenolol (Fig. 7). A b o u t 120--170-fold higher concentrations of (+)-isomers of those fi-blockers were needed to cause an effect similar to that of (--)-isomers on [3H]dihydroalprenolol binding or on adenylate cyclase activity. The ICso values of different competing agents for the binding sites mentioned above may be higher than their actual K i values, because the labeled ligand concentration (30 nM) used in these studies was greater than its Kd, as well as the receptor concentration [30]. It is, however, possible to derive actual K i values for different agonists and antagonists from their ICso values using the equation [30] K i = ICso/(1 + S/Kd), where S is the [3H]dihydroalprenolol concentration used in the assay and K d is the dissociation constant (15 nM). The K i value thus derived was 11.7 nM for both (--)-alprenolol and (--)-propranolol. These K i values closely agreed with the K i values determined from studies on the inhibition of isoproterenol stimulation of adenylate cyclase (Fig. 6). It may also be noted that the K~ of (--)-alprenolol was very similar to the K d obtained from saturation binding (Fig. 2) or kinetic data (Fig. 3) of [3H]dihydroalprenolol binding. Effect o f different agents on [3H]dihydroalprenolol binding ATP (1 mM), GTP (0.1 mM), GMP-PNP (0.1 mM), EDTA (1 mM), and EGTA (1 mM) did n o t affect the binding of [3H]dihydroalprenolol to skeletal muscle plasmalemma (data not shown). Ca 2÷ showed differential effects on adenylate cyclase and the binding (Fig. 8). Enzyme activity (basal, isoproterenol- and NaF-stimulated) was progressively inhibited with increasing concentrations of Ca 2÷, with 50% inhibition occurring at a b o u t 0.08 mM. In contrast,
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7
6
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9
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/~-ADRENERGIC ANTAGONIST (-log M) Fig. 7. Effects o f s t e r e o i s o m e r s o f p r o p r a n o l o l and alprenolol o n (--)-isoproterenol ( 1 0 pM)-stimu]ated a d e n y l a t e cyclase (left) and [ 3 H ] d i h y d r o a l p r e n o l o l ( D H A ) binding (right). V a l u e s are m e a n s of three experiments.
355
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CaCl 2 (raM] Fig. 8. E f f e c t s o f Ca 2+ o n b a s a l (o), 1 0 / a M ( - - ) - i s o p r o t e r e n o l - s t i m u l a t e d (o) a n d 1 0 m M N a F - s t i m u l a t e d (~) a d e n y l a t e c y c l a s e , a n d o n [ 3 H ] d i h y d r o a l p r e n o l o l ( D H A ) b i n d i n g (e). V a l u e s axe m e a n s o f f o u r e x p e r i ments.
[aH]dihydroalprenolol binding remain unchanged. Ca 2÷ appears to inhibit the enzyme by acting directly on the catalytic site of enzyme since it inhibited uniformly the basal, NaF- and isoproterenol-stimulated activities (Fig. 8). Discussion Catecholamines, like several polypeptide hormones, are thought to elicit physiological responses in target tissues by interacting with specific membranebound receptors [6]. Considerable attention has, therefore, been given to identify different hormone-receptors to gain insight into the mode of hormonereceptor interactions at the molecular level [31]. One of the ways of identifying the receptor for a given hormone is by following the direct binding to isolated membranes of a radioligand known to be active at that receptor. In such studies, the radioligand binding should possess selectivity and specificity known for that receptor from other biochemical and physiological investigations. The properties of ~-adrenergic receptors in different tissues have been extensively characterized through pharmacological and physiological studies [6]. With the availability of highly specific radioactive ~-adrenergic agents in recent years, it is now feasible to identify the ~-adrenergic receptors by direct binding assays [ 10--13]. The characteristics of radioligand binding to fi-adrenergic receptor are generally compared with the properties of adenylate cyclase, since that enzyme is closely associated with fi-receptor functions in many tissues [8,9,32]. The present study indicated that [3H]dihydroalprenolol, a potent ~-adrenergic antagonist, could be used to identify in vitro the ~-adrenergic receptors of skeletal muscle that are coupled to the adenylate cyclase system. The ~-adrenergic receptor sites in fractionated skeletal muscle, as revealed by [3H]dihydroalprenolol binding, were localized predominantely in plasmalemma. [3H]dihydroalprenolol binding was also observed in sarcoplasmic reticulum and mito-
356 chondria b u t it was only one-fifth to one-tenth of that found in plasmalemmal fractions. The level of [3H]dihydroalprenolol binding to different membrane fractions closely correlated with level of adenylate cyclase. Thus the plasmalemmal fraction which contained the highest level of (--)-isoproterenol-stimulated adenylate cyclase also displayed the highest level of [3H]dihydroalprenolol binding. We have previously indicated the possibility that our skeletal muscle plasmalemmal fraction may contain the plasmalemma of other intramuscular cells, particularly those of blood vessels [16]. As yet, there is no way to distinguish the plasmalemma of muscle cells from that of blood vessels. It is, therefore, possible that a certain portion of [3H]dihydroalprenolol binding and its properties observed in skeletal muscle plasmalemmal preparation could also be due to the blood-vessel cell plasmalemma. Our qualitative autoradiographic studies (Lavenstein, B., Engel, W.K., R e d d y , N.B. and Carroll, S., unpublished results) with 125I-labelled h y d r o x y b e n z y l pindolol, another /3-adrenergic antagonist, indicated considerable grain density in blood-vessels of rat muscle sections, especially vessels of the arterial tree, suggesting a contribution of blood-vessel cell plasmalemma to skeletal muscle plasmalemmal fraction. Quantitative assessment of the a m o u n t of binding to vascular cell plasmalemma vis-a-vis skeletal muscle cell plasmalemma would be very important, b u t subcellular fraction studies do n o t yet allow that, and quantitation, even internal quantitation, of autoradiographs, if n o t done in compulsive detail, is fraught with sources of error, including problems of the threshold of silver reduction in relation to density of b o u n d isotope and time of emulsion exposure and development (i.e., 'contrast'). Cell separation before subcellular fractionation would be needed. At this stage we simply point out the need for an awareness of 'the blood-vessel problem' in interpreting our data and those of others who biochemically treat muscle, and other organs, as a 'black b o x ' consisting only of parenchymal cells. The [3H]dihydroalprenolol binding observed in sarcoplasmic reticulum and mitochondria appear to be due to the binding truly occurring to those membranes, and not due to contamination by plasmalemma. The lack of cross-contamination was evident from the (Na ÷ + K+)-ATPase studies. That enzyme was recovered only in plasmalemma and it was n o t detectable in sarcoplasmic reciculum or mitochondria. Entman et al. [33] have shown that specific [3H]dihydroalprenolol binding occurs in sarcoplasmic reticulum of cardiac muscle. Recently Caswell et al. [34] have reported specific [3H]dihydroalprenolol binding to transverse tubules of rabbit skeletal muscle. These authors [34] purified a T-tubule fraction from a microsomal preparation comparable to our sarcoplasmic reticulum, which supports our observation that sarcoplasmic reticulum contains catecholamine-sensitive adenylate cyclase and fl-adrenergic receptor. Caswell et al. [34] claimed that adenylate cyclase was present only in T-tubule b u t n o t in the terminal cisternae. In contrast, Raible et al. [35] reported that adenylate cyclase was localized in terminal cisternae b u t not in T-tubule. Thus the precise localization of cyclase within sarcoplasmic reticulum remains unclear. In any case, since we observed catecholamine-sensitive adenylate cyclase and fl-adrenergic receptor binding in plasmalemma, sarcoplasmic reticulum and mitochondria, it is possible that in skeletal muscle the catechol-
357
amines could initiate their actions at the cell surface and/or at intracellular loci. The characteristics of [3H]dihydroalprenolol binding to skeletal muscle plasmalemma fulfilled several criteria required for the identification of adenylate cyclase-coupled B-adrenoceptors [8,9]. The binding was rapid and reversible. At 37°C the time required for half-maximal association and dissociation was a b o u t 2 min. In an earlier study we found that propranolol could abolish the isoproterenol-induced activation of human plasmalemmal adenylate cyclase within 2 min of its addition to the reaction medium [17]. The rapidity of [3H]dihydroalprenolol association and dissociation suggest that the binding is occurring to those receptor sites closed linked to adenylate cyclase. Binding of [3H]dihydroalprenolol in plasmalemma occurred to a single group of receptor sites and it was saturable, indicating a finite number of binding sites. The apparent Kd for [3H]dihydroalprenolol was a b o u t 15 nM which is within the range of K d values reported for other tissues [9]. A Hill coefficient of 1.007 for [3H]dihydroalprenolol binding suggested that there is neither positive nor negative cooperative interaction among the B-receptor binding sites in skeletal muscle. Hill coefficients of around one have also been reported in fl-adrenoceptor binding studies with other membranes [9]. However, Lefkowitz and co-workers [8], who extensively characterized the [3H]dihydroalprenolol binding in frog erythrocytes and rat adipocytes, obtained Hill coefficients of significantly less than one, and they suggested that negative cooperativity exists among the B-receptor sites. The nature of cooperativity thus seems to vary in different tissues. Several ~-adrenergic agents known to be active on intact skeletal muscle competed for [3H] dihydroalprenolol binding sites with essentially similar selectivity and stereospecificity. For example, catecholamines are known to enhance glycogenolysis in skeletal muscle with a p o t e n c y sequence of isoproterenol epinephrine > norepinephrine [36,37]. A similar order of p o t e n c y with isomeric specificity (--) isomers > (+) isomers) was reported in studies of catecholamine effects on twitch~contraction, even though catecholamines induce differential effects on fast- and slow-twitch muscle [5]. Catecholamines have also been shown to enhance Na + efflux in skeletal muscle [2--4] with isomeric specificity [3]. Propranolol abolished the catecholamine effects on giycogenolysis [37], twitch-contraction [5], and Na + effiux [3,4], again with isomeric specificity. The results obtained in the present study on [3H]dihydroalprenolol binding and adenylate cyclase closely agree with the aforementioned physiological studies. However, the concentrations of agonists and antagonists needed to cause half-maximal effect on [3H]dihydroalprenolol or adenylate cyclase activity in the present study are a b o u t t w o orders of magnitude greater than those reported for glycogenolysis [37], twitch-contraction [5], and Na + fluxes [3,4]. This may be due to changes that the receptor and enzyme molecules undergo during several preparatory steps involved in the isolation of muscle membranes. Alternatively, partial occupation of receptor sites by agonists and antagonists might be sufficient to cause greater physiological response than that required to alter adenylate cyclase as measured in our assay. Furthermore, ouabain inhibits the catecholamine induced Na ÷ transport in intact skeletal muscle [3,4], whereas ouabain showed no effect on the plasmalemmal adenylate cyclase (Reddy, N.B., unpublished data). Thus, mechanisms other than
358
cyclic AMP may be involved in mediating different physiological effects by catecholamines in skeletal muscle. In conclusion, we have shown that fl-adrenergic receptors in skeletal muscle can be identified by direct binding assays. The present investigation constitutes a detailed study of the properties of skeletal muscle fl-adrenoceptors under in vitro conditions. The methods and values established in this study will be useful in analyzing the possible alterations of fl-adrenoceptor-associated functions in several pathophysiological states of skeletal muscle in animal models and in human neuromuscular diseases. For example, denervated skeletal muscle exhibits greater physiological sensitivity to catecholamines [5]. Our preliminary studies of rat skeletal muscle [38] indicated that the plasmalemmal fl-adrenergic receptor density, determined by 12SI-labelled hydroxybenzyl pindolol binding, increases about two-fold following denervation, while the adenylate cyclase decreases; suggesting that denervation causes uncoupling of receptor-enzyme interaction. We have also found differences in the fl-receptor binding and catecholamine sensitivity of adenylate cyclase in fast-twitch and slow-twitch skeletal muscle of rat [39]. Thus, the studies on fl-adrenoreceptors may be helpful in further delineating the 'neurotrophic' regulatory mechanisms as well as their involvement in certain neuromuscular disorders. Acknowledgements The authors wish to thank Ms. K.L. Oliver for technical assistance. This work is supported in part by Muscular Dystrophy Assn., Inc. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Gross, S.R. and Mayer, S.E. (1975) Metabolism 24, 3 6 9 - - 3 8 0 Hays, E.T., n w y e r , T.M., Horowicz, P. and Swift, J.G. (1974) Am. J. Physiol. 227, 1340--1347 Rogus, E.M., Cheng, L.C. and Zierlier, K. (1977) Biochim. B i o p h y s . A c t a 4 6 4 , 3 4 7 - - 3 5 5 Clausen, T. and F l a t m a n , J. (1977) J. Physiol. 270, 363--414 Bowman, W.C. and Nott, M.W. (1969) Pharmacol. Rev. 21, 27--72 Robison, G.A., Butcher, R.W. and Sutherland, E.W. (1971) Cyclic AMP, pp. 145--231, A c a d e m i c Press, New Yo rk Birnbaumer, L. (1973) Biochim. B i o p h y s . A c t a 300, 129--158 Lefkowitz, R.J., Limbixd, L.E., Mukherjee, C. and Caron, M.G. (1976) B i o e h i m . B i o p h y s . A c t a 457, 1--39 Wolfe, B.B., Harden, T.K. and Molinoff,, P.B. (1977) Annu. Rev. Pharmacol. Roxicol. 17, 575---604 Lefkowitz, R.J., Mukherjee, C., Coverstone, M. and Caron, M.G. (1974) B i o c h e m . B i o p h y s . Res. Cornm a n . 60, 704--709 Levitzki, A., Arias, D. and Steer, M.L. (1974) Proc, Natl. A c a d . Sci. U.S. 71, 2773--2776 Aurbach, G.D., Fedak, F.A., Woodward, C.J., Palmer, J.S., Hauser, D. and Troxler, F. (1974) Science 186, 1 2 2 3 - - 1 2 2 4 Lefkowitz, R.J. and Williams, L.T. (1977) Proc. Natl. A c a d . Sci. U.S. 74, 515--519 Festoff, B.W. and Engle, W.K. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2 4 3 5 - - 2 4 3 9 Red dy, N.B., Engel, W.K. and F e s t o f f , B.W. (1976) B i o c h i m . B i o p h y s . A c t a 433, 365--382 Red dy, N.B., Oliver, K.L., Festoff, B.W. and Engle, W.K. (1978) Biochim. B i o p h y s . A c t a 540, 371-388 Red dy, N.B., Oliver, K.L., Festoff, B.W. and Engel, W.K. (1978) B i o c h i m . B i o p h y s . A c t a 540, 389-401 R e d d y , N.B. and Engel, W.K. (1977) Fed. Proc. 3 6 , 5 8 4 Salomon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biochem. 58, 541--548 Lo wry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Rodben, M., Lin, M.C., Salomon, Y., Londos, C., Harwood, J.P., Martin, B., Rendell, M. and Berman, M. (1975) Adv. Cyclic N u c l e o t i d e Res. 5, 3--29
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