Identification of cu-Adrenergic Receptors in Uterine Smooth Muscle Membranes by [3H]Dihydroergocryptine Binding*

(Received for publication, March 25, 1976)

LEWIS T. WILLIAMS,+ DEBRA MULLIKIN, AND ROBERT J. LEFKOWITZ§

From the Departments of Medicine, Biochemistry, Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710

[3H]Dihydroergocryptine, a potent a-adrenergic antagonist, was used to label smooth muscle membrane binding sites which have the characteristics expected of a-adrenergic receptors. Binding of [3H]dihydroergocryptine to rabbit uterine membranes was rapid and reversible with rate constants of 1.26 x lo1 M-I min ~’ and 0.034 min’ for the forward and reverse reactions, respectively. [3H]Dihy- droergocryptine binding was of high affinity, with an equilibrium dissociation constant (K,) of 8 to 10 nM. Binding was saturable with 0.14 to 0.17 pmol of [3H]dihydroergocryptine bound/mg of protein at maximal occupancy of the sites. No cooperative interactions among the sites were detected.

The specificity of the binding sites for a large number of adrenergic agonists and antagonists was identical with the specificity of cu-adrenergic responses to these agents. The cr-adrenergic agonist (-)-epinephrine competed for binding with a K,, of 0.23 PM. The order of potencies for several adrenergic agonists in competing for the binding sites was (~ )-epinephrine > (-)-norepinephrine > (-)-phenyleph- rine > (-)-isoproterenol in agreement with their cu-adrenergic potencies. A series of 19 phenylethylamine adrenergic agonists competed for binding in a manner paralleling their potencies as a-adrenergic agonists. a-Adrenergic antagonists such as phentolamine (K a = 15 nM) and phenoxybenzamine (Ka = 18 nM) potently competed for the binding sites. In contrast, P-adrenergic antagonists such as propranolol (K, = 27,000 nM) and practolol (K, > lo6 nM) did not have high affinity for the binding sites. A series of ergot alkaloids competed for [3H]dihydroergocryptine binding in a manner which paralleled their potencies as cu-adrenergic agents. Competition for binding sites by cu-adrenergic agonists and antagonists was a stereospecific process. The (-)-stereoisomers of epinephrine, norepinephrine, and ergotamine were at least 20- to 50.fold more potent than the corresponding (+)-stereoisomers. Compounds devoid of significant a-adrenergic activity, such as pyrocatechol, 3,4-dihydroxymandelic acid, normetanephrine, and n-lysergic acid, did not effectively compete for [3H]dihydroergocryptine binding sites. These rabbit uterine binding sites for [3H]dihydroergocryptine appear to have characteristics indistinguishable from those of the physiologically active oc-adrenergic receptors.

A variety of physiological processes are regulated by the order to elicit the observed (Y or p response. u-Adrenergic endogenous catecholamines, epinephrine and norepinephrine. responses include contraction of smooth muscle in vascular, Ahlquist (1) observed that the adrenergic responses to cate- uterine, and other tissues (2). Such responses are stimulated by cholamines could be divided into two distinct groups, 01 and 0, catecholamines with a typical potency series of epinephrine > based on the order of potency of a series of adrenergic agonists norepinephrine > phenylephrine > isoproterenol (1) and are in eliciting the responses. This classification was further blocked by specific “Lu-adrenergic” antagonists such as phen- strengthened by the observation that one group of competitive tolamine, phenoxybenzamine, and ergot alkaloids (3). Typical adrenergic antagonists specifically blocked cu-adrenergic re- @-adrenergic responses are smooth muscle relaxation and sponses, while another group of compounds specifically stimulation of cardiac contractility (2). Such responses are blocked /3-adrenergic responses. The remarkable specificity of stimulated by catecholamines with a typical potency series of each type of response suggested that catecholamines must isoproterenol > epinephrine >_ norepinephrine > phenyleph- interact with distinct 01 or p receptors in the target tissue in rine (1) and are blocked by specific “P-adrenergic” antagonists

such as proprl: :olol and dichlorisoproterenol (4, 5). *This study was supported by Health, Education and Welfare In recent years the molecular mechanism of P-adrenergic re-

Grant HL 16037 and by a grant-in-aid from the American Heart ceptor activation has been studied extensively. Many if not all Association with funds contributed in part by the North Carolina fl-adrenergic responses are mediated by intracellular cyclic Heart Association.

$ Student in the Medical Scientist Training Program supported by AMP, the formation of which is catalyzed by the enzyme National Institutes of Health Grant 5T 05-6 M01678. adenylate cyclase (6, 7). Adenylate cyclase appears to be

$ Established Investigator of the American Heart Association. localized in the plasma membrane (8) where it is coupled to the

6915

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6916 Identification of a-Adrenergic Receptors

fi-adrenergic receptors. Recently P-adrenergic receptors have been identified by binding studies using radioactively labeled /3-adrenergic antagonists such as (-)- [3H]dihydroalprenolol (g-12), (=t)- [3H]propranolol (13), and (+)-1251-hydroxybenzyl- pindolol (14). These techniques have provided a direct approach to studying the fl-adrenergic receptor as a physico- chemical entity in a variety of tissues.

In contrast with studies of the P-adrenergic system, little information has been reported on the molecular mechanism of a-receptor-mediated adrenergic stimulation. Some evidence has suggested that cu-adrenergic receptors may be coupled to processes which elevate intracellular cyclic GMP (15) or may be related to regulation of cation permeability of the plasma membrane (16). However, to date most of the reported information about the characteristics of (Y receptors has been inferred from measurement of physiological responses to adre- nergic agents, and no previous attempts have been successful in directly identifying the a-adrenergic receptor by binding techniques. Identification of a-adrenergic receptors by radioli- gand binding would require fulfillment of several criteria which are based on the known characteristics of a-adrenergic re- sponses.

1. The affinity of the radioligand for the binding sites should reflect the known biological potency of the ligand.

2. The time course of the binding of radioligand to receptor sites should be consistent with the rate at which the ligand elicits its physiological response. Similarly, if the physiological effect of the ligand is reversible, the binding of radioligand to receptor sites should be reversible.

3. Radioligand binding should reflect an ol-adrenergic speci- ficity. a-Adrenergic agonists and antagonists should compete for the binding sites in an appropriate order of affinity. Specific P-adrenergic agents should have a much lower affinity for the sites than cu-adrenergic agents. Compounds devoid of CY- adrenergic activity should not interact with the binding sites. Binding sites should exhibit stereospecificity toward adrener- gic agents since (-)-isomers of a-adrenergic agonists and antagonists are considerably more potent than (+)-isomers in physiological systems (2, 17, 18).

We have recently described a method for the direct identifi- cation of a-adrenergic binding sites in uterine smooth muscle membranes (19). The binding sites are identified by measuring the binding of the potent a-adrenergic antagonist, [3H]dihy- droergocryptine, to uterine membranes. We now report a detailed analysis of the kinetics, affinity, and specificity of the interaction of [3H]dihydroergocryptine with its binding sites in smooth muscle membranes from rabbit uterus. This approach provides a method for the direct study of the molecular nature of cu-adrenergic receptors.

MATERIALS AND METHODS

Radioligund- [3H]Dihydroergocryptine (Fig. 1) was chosen as the cu.adrenergic radioligand for this study for several reasons. First, the compound can be prepared by reduction of the double bond at position 9,lO of ergocryptine by established procedures (20). This reduction permits insertion of greater than 1 mol of tritium/mol of compound thus assuring high specific radioactivity of the product. A second reason for using [3H]dihydroergocryptine to identify a-adrenergic receptors was the high potency of dihydroergocryptine as an a- adrenergic antagonist (3, 21). Its apparent affinity for cu-adrenergic receptors as determined from pharmacological studies is greater than that of any other ergot alkaloid (21). [3HJDihydroergocryptine was prepared at New England Nuclear by catalytic reduction of ergocryp- tine (Sigma) using tritium gas and was purified by preparative thin layer chromatography. The tritiated compound was homogeneous and indistinguishable from authentic unlabeled dihydroergocryptine (San-

doz) by thin layer chromatography on silica gel plates using the following solvent systems: (A) chloroform:benzene:ethanol:ammonia (4:2:1:0: 1) and (B) chloroform:ethanol:acetic acid (9:5:1). In Solvent System A, authentic dihydroergocryptine (Sandoz) and [3H]dihydroer- gocryptine migrated with an R, of 0.68 while the parent compound, ergocryptine, migrated with an RF of 0.74. Similarly, in Solvent Sys- tem B the RF for authentic dihydroergocryptine (Sandoz) and [3H]- dihydroergocryptine was 0.73, whereas the RF for ergocryptine was 0.62. Elemental analysis of unlabeled dihydroergocryptine prepared under identical conditions as the radioactively labeled material (except for the substitution of hydrogen for tritium) was identical with that pre- dicted from the structural formula of the authentic compound. The ultraviolet spectrum of synthesized unlabeled compound was also identical with that of the authentic compound. The specific activity of [3H]dihydroergocryptine was 23.0 Ci/mmol determined by comparison of the radioactively labeled compound with authentic standards using ultraviolet spectroscopy. The compound was stable for up to 6 months when stored in the dark at -20” in ethanol. Immediately prior to using a fresh stock solution was prepared by adding the appropriate amount of [3H]dihydroergocryptine to an aqueous solution containing 5 mM HCl and 10% ethanol. This solution was diluted B-fold when added to the binding assay. Ethanol at concentrations in the range of 0.15 to 4% in the assay had no effect on specific binding (see below) of [3H]dihy- droergocryptine.

Membrane Preparation-Since catecholamine-induced contraction of uterine smooth muscle is an cu-adrenergic response, rabbit uteri were used as a source of a-adrenergic receptors. Uterine membranes were prepared from uteri from mature female New Zealand White rabbits weighing approximately 8 to 10 pounds each. In some experiments frozen rabbit uteri (type II, mature) obtained from Pel Freeze Biologicals Inc. and maintained at -70” were used as a source of smooth muscle. These uteri gave binding results indistinguishable from those obtained with membranes prepared from fresh tissue. Uteri were cleaned of fat, opened longitudinally, and stripped of endome- trium using a scalpel. Uteri were then minced and homogenized in ice cold buffer (0.25 M sucrose, 1 mM MgCl,, 5 mM Tris, pH 7.4) for four 10-s periods using a Tekmar tissue grinder at high speed. After filtration through a single layer of gauze, the homogenate was centrifuged at 400 x g for 10 min at 4” and the pellet was discarded. The supernatant was centrifuged at 28,000 x g for 10 min at 4”. The resulting pellet was washed twice in ice cold incubation buffer (10 rnM MgCl,, 50 mM Tris, pH 7.5) by resuspension and centrifugation at 28,000 x g for 10 min. The final pellet was resuspended in incubation buffer for use in the binding assay.

Binding Assay- [3H jDihydroergocryptine (8 nM unless otherwise specified) and uterine membranes (-4 mg/ml unless otherwise speci- fied) were incubated for 15 min (unless otherwise specified) at 25” with shaking in a total volume of 150 ~1 of incubation buffer. In binding competition experiments the competing agent was added directly to the incubation. Incubations were terminated by diluting 125.~1 incu- bation aliquots with 2 ml of incubation buffer (25”) followed by rapid filtration through Whatman GFC glass fiber filters. Filters were rapidly washed with 20 ml of incubation buffer (25’). This amount of washing did not reduce specific binding of [SH]dihydroergocryptine but significantly reduced the nonspecific binding of [‘Hldihydroergo- cryptine (see below). After drying, filters were counted in a Triton/ Toluene scintillation mixture at an efficiency of 40%.

Nonspecific binding is defined as binding which is not displaced by a high concentration (10 KM) of phentolamine, a potent ol-adrenergic antagonist which should occupy all of the oc-adrenergic receptor

CH3 CH3 \/

CH 0 CH2CH(CH&

FIG. 1. Structure of ergocryptine. [“HlDihydroergocryptine was pre- pared by catalytic reduction of ergocryptine at the double bond in position 9,lO (see “Materials and Methods”).

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Identification of a-Adrenergic Receptors 6917

binding sites. This concentration of phentolamine was arbitrarily chosen to estimate nonspecific binding since higher concentrations did not further inhibit binding. Specific or receptor binding is defined as total radioactivity bound minus nonspecific binding, and was generally 75 to 90% of the total counts bound to membrane protein. A small amount of [3H]dihydroergocryptine (0.5 to 0.6% of the total counts filtered) was also nonspecifically adsorbed to the glass fiber filters in the absence of protein. This “filter blank” was not displaced by phentolamine or other agents. “[‘HlDihydroergocryptine binding” in the figures refers to specific binding as defined above.

No degradation of [3H]dihydroergocryptine during the incubations could be detected. Membranes were incubated with [3H]dihydroergo- cryptine as described above and were centrifuged at 28,000 x g for 10 min. [3H]Dihydroergocryptine present in the supernatant was indistin- guishable chromatographically from fresh [3H]dihydroergocryptine. [3H]Dihydroergocryptine bound to the membrane-containing pellet was extracted from the membranes with ethanol, concentrated by evaporation, and was chromatographed. This material which had been previously bound to membranes was chromatographically indis- tinguishable from fresh [3H]dihydroergocryptine.

Calculation of Dissociation Constants for Competing Ligands-A number of adrenergic compounds were tested for their abilities to compete with [3H]dihydroergocryptine for the binding sites. The equilibrium dissociation constant, K,, for the interaction of each compound with the binding site was calculated from the concentration of each agent which caused 50% maximum displacement (EC,,) using the equation of Cheng and Prusoff (22):

K, = EC,,I[l t- PHE’lIK,,,,l

where [DHE] is the concentraion of (3H]dihydroergocryptine in the assay (8 nM), KDHE is the K, for dihydroergocryptine (8 nM) com- puted from equilibrium binding studies (Fig. 2), and EC,, is the con- centration of the competing compound which inhibits 50% of the [3H]dihydroergocryptine binding. Hence under these conditions K, = EC,,/2. An alternative calculation of these data as previously de- scribed (11) using the method of Rodbard and Lewald (23) resulted in calculated K, values which were within 20% of the values calculated by the method described above.

Compounds-( - )-Epinephrine bitartrate, (-)-norepinephrine mtartrate, (-)-isoproterenol bitartrate, (It)-octopamine hydrochlo- ride, dopamine hydrochloride, tyramine hydrochloride, (A)-dihydroxy- phenylalanine, dihydroxymandelic acid, (+)-normetanephrine, dihy- droergotamine, ergotamine tartrate, ergocryptine, ergocryptine sul- fate, ergonovine maleate, ergotamine, (*,)-propranolol hydrochloride,

and n-lysergic acid were from Sigma. Dihydroergotamine and ergo- cryptine and ergotamine tartrate were also obtained from Sandoz and gave results indistinguishable from those of the same compounds from Sigma. Dihydroergocryptine methane sulfonate, dihydroergocristine methane sulfonate, dihydroergocornine methane sulfonate, ergocro- nine hydrogen maleate were from Sandoz. (+)-Isoproterenol bitartrate, (+)-epinephrine bitartrate, (+)-norepinephrine bitartrate, (+)-deoxy- isoproterenol hydrochloride, (h)-ethylnorepinephrine, and (+)-cobe- frin hydrochloride were from Sterling-Winthrop Pharmaceuticals. Pyrocatechol was from Mann. Practolol hydrochloride was from Ayerst. Phenoxybenzamine, chlorpromazine hydra&ride, and tri- fluoperazine were from Smith, Kline and French. (+)-Dichloriso- proterenol hydrochloride and ( A)-epinephrine hydrochloride were from Lilly. Phentolamine hydrochloride and tolazoline hydrochloride were from Ciba-Geigy. Azapetine phsophate was from Hoffman-La Roche. Dibozane was from NcNeil Laboratories. Ethylnorphenylephrine (S-40032-7) was from Aldrich Chemicals. Metaraminol bitartrate was from Merck, Sharpe and Dohme. (*)-Cc-34 benzoate and (*)-CC-25 benzoate were from Phillips-Duphar, Netherlands. Yohimbine hydro- chloride was from Nutritional Biochemicals. Mephentermine sulfate was from Wyeth. Nylidrin was from USV Pharmaceutical Corp. (i-)-Isoxuprine hydrochloride was from Mead Johnson. Clonidine hydrochloride was from Boehringer Ingelheim, Ltd. Methoxamine hydrochloride was from Burroughs Welcome & Co.

Stock solutions of dihydroergocornine methane sulfonate, ergocor- nine hydrogen maleate, and ergocrystine sulfate were prepared at 6 x lo-’ M in 50% ethanol and serial dilutions were made in water. Dihydroergocryptine methane sulfonate stock solutions were made at 6 x lo-‘M in absolute ethanol and diluted to 6 x 10m5 M in 10% ethanol containing 5 rnM HCl. Further dilutions of dihydroergocryptine were made in water. Stock solutions of dihydroergotamine and ergocryptine were prepared at 6 x lo-’ M in absolute ethanol and diluted in water. Ergotaminine was dissolved at 6 x 10e3 M in glacial acetic and diluted to 6 x lOmE M in water for use in the assay. All other stock solutions were made in water. All stock solutions were prepared less than 2 h before use in the binding assay.

RESULTS

Number and Affinity of Binding Sites-The binding of [3H]dihydroergocryptine to rabbit uterine membranes was a saturable process (Fig. 2) with 0.14 pmol of [3H]dihydroergo- cryptine bound/mg of protein at saturation. Half-maximal saturation occurred at 8 nM [3H]dihydroergocryptine providing

. l 0

MB. .

OM * . I 10&O”Y

* 401

TI-

..Bli&,~wxl~N

*.

a02 . .

00 (12 ad a6 08 p”] D,H”mxX‘lxP”PTINL BO”NO,“M,

‘0 lo 20 30 40 50 60 70 80 90

[[“I-] DIHYDROERGOCRYPTINE](nM)

FIG. 2. Specific binding of [‘Hldihydroergocryptine to rabbit uter- tine binding to rabbit uterine membranes. The ratio (B/F) of bound ine membranes as a function of [3H]dihydroergocryptine concentra- [3H]dihydroergocryptine to free [3H]dihydroergocryptine is plotted as tion. Rabbit uterine membranes were incubated with indicated con- a function of the concentration of bound [3H]dihydroergocryptine. The centrations of [3H]dihydroergocryptine and binding was determined as slope of the plot, - l/K,, was determined by linear regression analysis described under “Materials and Methods.” Each value is the mean of (r = 0.90). The number of binding sites, n, is computed from the duplicate determinations. The experiment shown is representative of intercept of the plot with the abscissa. Each value is the mean of four such experiments. Inset, Scatchard plot of [‘Hldihydroergocryp- duplicate determinations.

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6918 Identification of Lu-Adrenergic Receptors

an estimate of the equilibrium dissociation constant, KD, for the interaction of [3H]dihydroergocryptine with the binding sites. A Scatchard plot (24) demonstrates a single order of sites with a KD of 10 nM (Fig. 2, inset). The intercept of this plot (Fig. 2, inset) with the abscissa provides an estimate of the number of binding sites in rabbit uterine membranes (n = 0.17 pmol/mg of protein). No evidence of cooperative interactions is apparent from Fig. 2 in agreement with a Hill plot (25) (not shown) of [3H]dihydroergocryptine binding which has a slope Of 1.09 (F = 0.99).

Kinetics of Binding-The binding of [Wldihydroergocryp- tine to uterine membranes was rapid (tH = 4.5 min) (Fig. 3) and reversible (Fig. 4) at 25”. Since the concentration of radioligand (8 nM) was much greater than the binding site concentration in the assay (0.76 nM), the forward reaction could be considered to be a pseudo-first order reaction which depends on the binding site concentration. Taking into account the reversible nature of the binding reaction, the reaction can be described by In [X,,/(X,, ~ X)] = k,, t where X is the amount of [3H]dihydroergocryptine bound at each time (t), and X,, is the amount bound at equilibrium (0.4 nM). Thus the line in Fig. 3 (inset) determined by linear regression analysis (F = 0.96) has a slope of 0.135 min. ’ which is the observed rate constant (k,,) for the reversible pseudo-first order reaction. The second order rate constant, k,, can be calculated from

k, = (kob - k,)/[DHE] = 1.26 x lo7 M ’ min-’

where 12, is the rate constant for the reverse (dissociation) reaction (Fig. 4) and [DHE] is the concentration of [3H]dihy- droergocryptine in the reaction mixture. The reverse reaction rate constant, k,, is 0.034 min-’ (determined by linear regres- sion analysis (Fig. 4)) (F = 0.97). The ratio k,@, = 3 nM is a kinetically derived estimate of the K, for the reaction of [3H]dihydroergocryptine with its binding site. This value is comparable to the K, (8 to 10 nM) determined by equilibrium studies (Fig. 2).

2 I . I z

04 t l . .

!

LO 1 I I1 I I I I I I 0 4 0 12 16 20 24 20

TIME (mwtes)

FIG. 3. [3H]Dihydroergocryptine binding to uterine membanes as a function of time. [3H]Dihydroergocryptine (8 nM) was incubated with uterine membranes (-4 mg/ml) for the indicated times at 25” and specific binding was determined as described under “Materials and Methods.” Each value is the mean of duplicate determinations. Inset, pseudo-first order kinetic plot of [3H]dihydroergocryptine binding. Data were used to determine X (amount of [3H]dihydroergocryptine bound at time t) and X,, (amount of [3H]dihydroergocryptine bound at equilibrium). This line, determined by linear regression analysis (r = 0.96), has a slope, k,,, equal to the observed rate constant for the pseudo-first order reaction. The forward rate constant, k,, for the second order reversible reaction between receptor and ligand is calculated from 12, = (kob k,)/[DHE] where k, is the first order rate constant for the reverse reaction (dissociation) and [DHE] is the concentration of [3H]dihydroergocryptine in the assay (8 nM).

Specificity of Binding-Adrenergic agonists competed for the [3H]diLydroergocryptine binding sites (Fig. 5) in an order of potency: (-)-epinephrine > (-0-norepinephrine > (-)- phenylephrine > > ( P)-isoproterenol. This potency order is identical with the well known order of potency for these agents in eliciting physiological ol-adrenergic responses (1, 2, 17). This order is in contrast with the potency order previously demon- strated for competition of the /3-adrenergic receptor: (-)-iso- proterenol, (-)-epinephrine 2 (-)-norepinephrine > > (-)- phenylephrine (9-14). The a-adrenergic agonists had high af- finity for the binding sites, the K, values for (-)-epinephrine and (-)-norepinephrine being 0.23 pM and 0.65 PM, respec- tively. These values from binding studies are comparable with

z ol , , , , , , , , , , , , 0 4 a 12 16 20 24 28

TIME (mmtes)

FIG. 4. Reversibility of [3H]dihydroergocryptine binding to uterine membranes. Membranes were incubated with [SH]dihydroergocryp- tine (8 nM) at 25” for 16 min after which a large excess of phentolamine (100 Jo M) was added. The time of phentolamine addition is defined in this figure as t = 0. At the indicated subsequent times, specific [3H]dihydroergocryptine binding was determined. Maximum binding is defined as the amount of binding just prior to the addition of phentolamine at t = 0. Each value shown is the mean of duplicate determinations from two separate experiments. Inset, first order rate plot of the dissociation of the receptor ligand complex. Data were used to construct a first order rate plot. The line, determined by linear regression analysis (r = 0.97), has a slope, k,, equal to the first order rate constant (0.034 min-‘). X,, refers to the amount of binding present immediately prior to the addition of lo-’ M phentolamine, X refers to the amount of binding present at each time after the addition of phentolamine

”

-i -4 -5 -4 -3

LOG,,[ADRENERGlC AGONIST] (M)

FIG. 5. Inhibition of [3H]dihydroergocryptine binding by adrenergic agonists. Uterine membranes were incubated with [3H]dihydroergo- cryptine in the absence and presence of the indicated agonists and specific binding was determined. “100% inhibition” refers to the inhibition of specific binding by 10e5 M phentolamine. Each value is the mean of duplicate determinations from two separate experiments.

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Identification of a-Adrenergic Receptors 6919

the equilibrium constants (0.8 PM and 4.0 PM, respectively) previously reported from physiological studies (17, 18) of these agents in stimulating smooth muscle preparations. The binding

sites are highly stereospecific (Fig. 5), the K,] values for the (+)-stereoisomers of epinephrine and norepinephrine being 22. and 30-fold higher than the K, values for the corresponding (-)-stereoisomers.

The endogenous catecholamines, ( - )-epinephrine and (-)-norepinephrine, belong to the structurally related phenyl- ethylamine group of compounds. A series of these compounds were tested for affinity for the [3H]dihydroergocryptine binding sites (Table I). Where possible, the K, values determined from binding studies were compared with potencies from physiologi- cal studies reported in the literature (Table I). The structure activity relationships which determine the affinities of these compounds for the [3H]dihydroergocryptine binding sites agree closely with previously observed structure activity relation- ships which determine the abilities of these compounds to elicit

cu-adrenergic responses.

Compounds which are structurally related to the catechola- mines but devoid of n-adrenergic physiological effects did not effectively compete for [3H]dihydroergocryptine binding sites. Pyrocatechol did not inhibit binding at a concentration of 10 fiM. Similarly, dihydroxyphenylalanine, a catecholamine pre- cursor, did not inhibit binding at 10 fiM. The catecholamine metabolites, 3,4-dihydroxymandelic acid and normetaneph- rine, were also ineffective in competing for the binding sites

(not shown). ol-Adrenergic antagonists (Fig. 6, Table II) potently com-

peted for the binding sites. The competitive antagonist phen-

tolamine competed with a K,, of 15 nM in good agreement with its reported Kr, (6 to 15 nM) as an a-adrenergic antagonist (26, 27). The antagonist phenoxybenzamine also potently competed for binding. When membranes were preincubated with phen- tolamine for 15 min and then washed, inhibition of [3H]dihy- droergocryptine binding was completely reversed. In contrast, preincubation with phenoxybenzamine produced persistent inhibition of binding after repeated membrane washing. This is

TABLE IA

Inhibition of [3H]dihydroergocryptine binding to uterine membranes by phenylethylamine agonists

Incubations were performed as described under “Materials and inhibition of binding from different experiments were usually within a Methods” in the absence and presence of four to five concentrations 10% range of the mean value. The potency of each compound relative (ranging from 1Om8 to 10m3 M) of the indicated phenylethylamine to epinephrine was computed as the ratio of the K,) for epinephrine to compounds. Values for inhibition of binding at each concentration the K, of the compound under consideration. Values for the relative were the means of duplicate determinations in two separate experi- potency of each compound in eliciting cu-adrenergic physiological ments. For each compound listed the percentage of inhibition of responses were taken from the literature where possible. The tissue control binding by each concentration of the compound was computed. used in each physiological study from the literature is shown in paren- Percentage values from two to four separate experiments were meaned theses. _ indicates no literature value could be found; [3H]DHE, and a plot of the mean values was used to determine the K, as 13Hldihydroergocryptine; VSM, vascular smooth muscle. described under “Materials and Methods.” Values for percentage of

H H H

RI +=j-{-+-‘4 Inhibition of 13HlDHE

CmPound

(-)Epinephrine

t-)Norepinephri”c

(+)Wordefrine

(*)Epinephrine

mpaminc

(*)Norepinephrine

(-lIsoprotere”ol

(+)Et~lnorepinephrine

(-)Phenylephrine

(‘Metarminol H

(+)Et~l”orpherWlephrine H

(+Jctopaminc OH

Tfrmine OH

~~)Deolyisoproterenol OH

(+zthoxanine* H

He,>hentemine H

(+)Ephedrine ”

“1 OH

OH

OH

OH

OH

OH

OH

OH

H

Binding

Potency “3 3 5 Relative to

h I-)&Anephrine

OH H CH3 0.23 1

OH H H 0.65 0.35

OH CH3 H 2.3 0.10

OH H CH3 5.0 0.046

H H H 21 0.011

OH H H 20.0 0.011

OH H CH(CH3)2 43 0.0053

OH CH2-cH3 H 100 0.0023

OH II 5 3.5 0.066

OH CH3 H 5.0 o ,046

OH H clip, 25.0 0.0092

OH H H 40 0.0058

H H H 75 0.0031

OH H CH(CH3$ 250 0.0092

OH CM3 H 7.5 0.031

H cli3.cs3** ai3 12.5 0.018

OH aI3 m3 49.0 0.0046

Potency Relative to

-)EDinwhrine

0.20 (rat vasdeferens)(l7 )

0.60 (cat YSM) (35)

0.03 (rabbit uterusx30 )

0.10 (G.P. ileum) (30)

0.08 (rat vasdeferens)(l7 )

0.062 (rat vasdeferens)C17)

0.047 C-at “asdeferens)C17)

0.008 (rat “asdefere”s)(l7)

0.002 (rabbit uterus)(30)

0.08 (rat vasdeferens)(19 )

0.2 (rabbit utems)~O)

(1002 (rabbit uterus)(30)

0.001 (rabbit uterus)00)

0.001 (rabbit uteW.)~O)

*In addition to the indicated substituents, methoxamine has methoxy substituents at positions 2 and 5 of the phenyl group. ** Two methyl groups are substituted on the a-carbon of mephentermine.

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6920 Identification of a-Adrenergic Receptors

TABLE IB

Inhibition of [3H]dihydroergocryptine binding to uterine membranes by phenylethylamine antagonists

Incubations, assays, and calculations were as described in the legend for Table IA.

(~mylidrill OH

tgcc-34 OH

(+)Cc-25 OH

()Dtcblortsoproterenol Cl

OH

OH

OH

OH

OB

CH3 -kH2-CH20

Y3 H -lT20-OH

H -&,+

Irn3 Ii -cIl

' CH 3

Ighibition of [ BIDHE Binding

%lJM)

Antagonism 0r Alpha-Adrenergic Response

%( al)

0.5 1.3 (rat vasdeferens) (18)

0.8 1.6 (rat vasdeferens) (18)

4.0

10.0 10.0 ke.t asdeferens) (11)

27 5.1 (rabbit VW) (29)

-9 -0 -7 -6 -5 -4 -3

LOG,, [ADRENERGIC ANTAGONIST] (M)

FIG. 6. Inhibition of [3H]dihydroergocryptine binding by adrenergic antagonists. Uterine membranes were incubated with [3H]dihydroer- gocryptine in the absence and presence of the indicated antagonists and specific binding was determined. “100% inhibition” refers to the inhibition of specific binding by 1Om5 M phentolamine. Each value represents the mean of duplicate determinations from two separate experiments.

consistent with the reported competitive reversible nature of phentolamine as an a-antagonist and the noncompetitive irreversible nature of phenoxybenzamine as an a-adrenergic antagonist (4). The specific competitive Lu-adrenergic antago- nists dibozane and yohimbine also competed for the binding sites with KD values of 0.13 fiM and 0.22 pM, respectively.

In contrast to the marked potency of these oc-adreneric antagonists in competing for the [3H]dihydroergocryptine binding sites, P-adrenergic antagonists such as propranolol, dichlorisoproterenol, and practolol competed for the sites only at very high concentrations (Fig. 6, Table II). These results agree well with the previously reported finding that in physio- logical studies P-adrenergic antagonists such as propranolol and dichlorisoproterenol weakly block a-adrenergic receptors with K, values of 7 pM and 5 pM, respectively (27).

A variety of ergot alkaloids were tested for their abilities to compete for the [3H]dihydroergocryptine binding sites. Affini- ties as assessed by binding studies (Table II) parallel closely

TABLE II

Inhibition of [3H]dihydroergocryptine binding to uterine membranes

by adrenergic antagonists

Incubations were performed as described under “Materials and Methods” in the absence and presence of four to six concentrations (ranging from lo-’ to lo-’ or lo-’ M) of the indicated adrenergic antagonists. Values for inhibition of binding at each concentration were the means of duplicate determinations in two separate experi- ments. For each compound listed the percentage of inhibition of control binding by each concentration of the compound was computed. Values from two to four separate experiments were meaned and a plot of the mean values was used to determine the K,. Values for percentage of inhibition of binding from different experiments were usually within a 10% range of the mean value.

Adrenergic antagonist

Dihydroergocryptine Dihydroergotamine Phentolamine Phenoxybenzamine Ergotamine Dihydroergocristine Dihydroergocornine Ergocornine Ergocryptine Dibozane Ergocristine Yohimbine Ergonovine Azapetine Chlorpromazine Trifluoperazine Tolazoline Ergotaminine (*)-Dichlorisoproterenol (*)-Propranolol Lysergic acid Practolol

Inhibition of [$H]DHECl hinding K,

la4

10 15 15 18 21 42 60

100 120 130 140 220 450 650 650

1,250 2,100

>>l,OOO 27,000 27,000

> 105 >106

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Identification of a-Adrenergic Receptors 6921

the potencies of these compounds as cu-adrenergic agonists or antagonists as demonstrated by physiological studies. Each of these compounds contains a lysergic acid moiety which alone has no known a-adrenergic activity and which does not compete for the [3H]dihydroergocryptine binding sites (Table II). Ergotamine, a potent a-adrenergic agent, competed for binding with a K, of 21 nM (Fig. 7A, Table II). Dihydroergota- mine, the oc-adrenergic antagonist prepared by reduction of ergotamine, had slightly higher affinity than ergotamine for the binding sites. The (+)-stereoisomer of ergotamine, which is called ergotaminine, did not have high affinity for the binding site (Fig. 7A) thus demonstrating the stereospecificity of the binding sites toward the ergot alkaloids as well as toward catecholamines (Fig. 7A). Ergonovine, an amine ergot alkaloid with a-adrenergic agonist activity (28) also competed for the binding sites. In the ergotoxin group of alkaloids (Fig. 7B) the dihydroergot alkaloids which are reduced at position 9, 10 were more potent in competing for the binding sites than the corresponding native ergot alkaloids. This is consistent with physiological studies (3, 21) which have shown that the reduced ergot alkaloids are more potent as ol-adrenergic antagonists than the native alkaloids. The ergot alkaloid demonstrating the highest affinity for the receptor was dihy- droergocryptine (K,- 10 nM). The KD for dihydroergocryptine determined by competition binding (Fig. 7B) agrees well with that determined by equilibrium binding studies with [3H]dihy- droergocryptine (Fig. 2). This further confirms the biological equivalence of [3H]dihydroergocryptine and dihydroergocryp- tine at the a-adrenergic receptors. Dihydroergocryptine was lo-fold more potent than ergocryptine (Fig. 7B).

I

n 201 c P/ / I t

0 B 100 80

60

40

20

0 1 lOG,,[ERGOT ALKALOID] (M)

FIG. 7. A, inhibition of [3H]dihydroergocryptine binding by several ergot alkaloids. Uterine membranes were incubated with [3H]dihydro- ergocryptine in the absence and presence of the indicated ergot alkaloids and specific binding was determined. “100% inhibition” refers to the inhibition produced by 10 ’ M phentolamine. Each value is the mean of duplicate determinations in two separate experiments. R, inhibition of [3H]dihydroergocryptine binding by the ergotoxin group of ergot alkaloids.

Several additional compounds with known activity as LY- adrenergic antagonists (3) competed for the [3H]dihydroergo- cryptine binding sites (Table II). Thus chlorpromazine, tri- fluoperazine, and azapetine inhibited binding with K, values in the range of 0.25 to 1.25 pM. In addition, the vasoactive amine serotonin competed for binding with a K, of 20 PM. Histamine had a low affinity for the binding sites (K, = 300 PM). Clonidine, an a-adrenergic agonist, inhibited binding with a K, of 0.26 WM.

DISCUSSION

These data demonstrate that the a-adrenergic antagonist, [3H]dihydroergocryptine, can be used to identify uterine smooth muscle binding sites which have the characteristics expected of physiological Lu-adrenergic receptors. Although the existence of a-adrenergic receptors has been postulated for over two decades (l), information about the receptor could only be inferred from physiological studies of the abilities of various agents to stimulate or block smooth muscle contraction. By contrast, the radioligand binding methods used here permit for the first time the direct study of the interaction of a-adrenergic agonists and antagonists with the receptor binding sites.

The binding sites identified with [3H]dihydroergocryptine appear to satisfy the criteria which must be met for identifica- tion of physiological ol-adrenergic receptors. The kinetics and affinity of the receptor ligand interaction are appropriate. There is remarkable agreement in the specificity of binding and the specificity of the a-adrenergic effects of the 50 compounds tested.

The group of structurally related phenylethylamine com- pounds demonstrated a distinct specificity in competing for [SH]dihydroergocryptine binding (Tables IA and IB). Several generalizations can be made about the structure-activity relationships which determine the affinities of these com- pounds for the binding sites.

Aromatic Ring Substituents-Compounds such as (-)-epi- nephrine and (-)-norepinephrine with hydroxyl substituents at positions 3 and 4 on the aromatic ring had higher affinity for the receptor than analogous compounds lacking one or both hydroxyl substituents (Table IA). The next most potent configuration with respect to phenyl substituents was the group with only a 3-hydroxyl substituent (phenylephrine, metaraminol, ethylnorphenylephrine). Methoxamine, which has methoxy groups at positions 3 and 5 was slightly less potent than the 3-hydroxy compounds. Methoxamine is of particular interest since physiologically it is a relatively specific (Y- adrenergic agonist and is not susceptible to catecholamine uptake processes (27, 29). Compounds with only a 4-hydroxy substituent (octopamine, tyramine, deoxyisoproterenol) had somewhat lower affinities as did the compounds with no ring substituents (ephedrine and mephentermine).

Alkyl Substituents on Amino Nitrogen-( -)-Epinephrine, which has a methyl substituent on the amino nitrogen had a slightly higher affinity than the corresponding compound with no substituent on the amino nitrogen (( -)-norepinephrine). A further increase in size of the amino nitrogen substituent to an isopropyl group (( -)-isoproterenol) markedly reduced the affinity for the binding site (Table IA). A comparable reduc- tion in the physiological potency of these compounds when the amino substituent is increased from a methyl to an isopropyl group has been reported (17, 18). Among the 3-hydroxy compounds a similar relationship is apparent with respect to increasing the size of the amino substituent, i.e. the compound

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6922 Identification of a-Adrenergic Receptors

with a methyl substituent on the amino nitrogen (phenyleph- rine) had a 7-fold higher affinity than the analogous compound with an ethyl group on the nitrogen (ethylnorphenylephrine). Similarly for the 4..hydroxylphenyl compounds, increasing the size of the amino substituent from hydrogen (octopamine) to isopropropyl (deoxyisoproterenol) resulted in a significant reduction in affinity. Hence the order of affinity of compounds with respect to alkyl substituents on the amino nitrogen appears to be CH, > H > CH, CH, > CH(CH,),. This is in agreement with the physiological potency order of these compounds as ol-adrenergic agonists (30).

Aralkyl Substituents on Amino Nitrogen-In physiological studies it has been demonstrated (17, 18) that substitution of large aralkyl groups on the amino nitrogen eliminates the activity of the compound as an a-adrenergic agonist, while enhancing its affinity as an a-adrenergic antagonist. The K, values derived from inhibition of [3H]dihydroergocryptine binding (Table IB) by these compounds are in close agreement with their reported K, values as a-adrenergic antagonists.

&Carbon Hydroxyl’Group-The highest affinity phenyleth- ylamine agonists contain a P-carbon hydroxyl group which is in the levo (-)-stereoconfiguration (e.g. (-)-norepinephrine)(Ta- ble IA). The presence of a P-hydroxyl in the dextro (+)-con- figuration ((+)-norepinephrine) or the absence of a P-hydroxyl group (dopamine) is accompanied by a 30-fold loss of affinity. This stereospecificity is also manifest in the physiological response to these agents (2, 17).

Several previous attempts by others to directly label a- adrenergic receptors using radioligands did not succeed in labeling sites which have the appropriate characteristics (31-34). Possible reasons for the success of the present study in contrast with previous attempts at a-receptor identification are as follows.

1. In general, previous attempts have utilized radioactively labeled ligands of relatively low specific radioactivity (-25 to 50 mCi/mmol). Thus in order to identify the true receptors, of which there are a very small number, high concentrations of ligand were required. At these high concentrations, nonspecific binding of ligands to high capacity low affinity nonreceptor sites presumably obscured binding to the small number of physiological high affinity receptors. The ligand used in the present study ( [3H]dihydroergocryptine) not only possesses high specific radioactivity (23 Ci/mmol) but has very high affinity for the ol-adrenergic receptor. Hence at the low concentrations of ligand used in the assays (-8 nM) specific o( receptor binding could be detected in the absence of significant nonspecific binding.

2. In several previous attempts to identify a-adrenergic receptors by radioligand binding, intact strips of tissue were used as a source of receptors (31, 32). The use of membrane preparations in the present study might allow the attainment of higher receptor site concentrations and may also eliminate some of the drug and hormone uptake processes which occur in intact tissue (29).

The techniques described in this report for the identification of cu-adrenergic receptors by [3H]dihydroergocryptine binding may also be of value in the study of cu-receptors in tissues other than uterine smooth muscle. In each tissue and species studied, however, a careful documentation of the specificity of binding should be undertaken. It has been reported that under appro- priate conditions in certain tissues ergot alkaloids interact with

pharmacological responses other than cu-adrenergic responses (4). Of particular interest in this regard, is that Davis et al.’ have demonstrated that [3H]dihydroergocryptine binds to sites in rat brain membranes which have a specificity pattern somewhat different from that of the rabbit uterine binding sites reported here. The specificity of the binding in the brain membranes is such as to suggest that either several receptors are being labeled (including cy receptors, dopamine receptors, and serotonin receptors) or that a receptor of hybrid specificity is involved. Hence, a careful study of binding specificity should precede application of these techniques to the study of a-adrenergic receptors in tissues other than rabbit uterine smooth muscle.

Acknowledgment-We are grateful to Ms. Donna Addison for her excellent assistance in preparation and typing of this manuscript.

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L T Williams, D Mullikin and R J Lefkowitzby [3H]dihydroergocryptine binding.

Identification of alpha-adrenergic receptors in uterine smooth muscle membranes

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