dopaminergic receptors in the anterior pituitary · pdf filedopaminergic receptors and...

THE .JOU~NAL OF Bmr.ocrca~ CHEMISTRY Vol 253, No 7, Issue of April 10, pp. 2244-2253, 1978

Prrnted ,n U.S.A.

Dopaminergic Receptors in the Anterior Pituitary Gland CORRELATION OF [:‘H]DIHYDROERGOCRYPTINE BINDING WITH THE DOPAMINERGIC CONTROL OF PROLACTIN RELEASE*

(Received for publication, July 25,1977)

MARC G. CARON,+ MICH~LE BEAULIEU,~ VINCENT RAYMOND,~ BERNARD GAGN~, JACQUES DROUIN,§ ROBERT J. LEFKOWITZ,~ AND FERNAND LABRIE/~

From the Medical Research Council Group in Molecular Endocrinology, Le Centre Hospitalier de 1’Universite Laval, Quebec GlV 4G2 and Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

The ergot alkaloid [“Hldihydroergocryptine, a potent dopaminergic agonist, has been used to study binding sites in bovine anterior pituitary membranes. One function of the anterior pituitary gland, prolactin secretion, as we show is under typical dopaminergic control and was measured in uitro in another series of experiments using rat anterior pituitary cells in primary culture in order to establish a correlation between binding and a biological function.

The dopaminergic specificity of ligand binding and the biological process was demonstrated by the fact that agonists competed for [“Hldihydroergocryptine binding and inhibited prolactin release with an identical order of potency: apormorphine > dopamine > epinephrine 2 norepinephrine >> isoproterenol. Ergot alkaloids, which behave as agonists in this system, potently inhibited prolactin release from pituitary cells to about the same extent as

dopamine (80 to 90% of basal levels) and were potent competitors of [“Hldihydroergocryptine binding (K,, 0.2 to 0.5 4. Dopaminergic antagonists competed for [“HI- dihydroergocryptine binding in bovine anterior pituitary membranes in parallel to their ability to reverse either dopamine or dihydroergocornine inhibition of prolactin release from rat anterior pituitary cells.

[3HlDihydroergocryptine binding fulfilled another crite- rion of specific receptor sites in that binding to the anterior pituitary sites was saturable with an apparent dissociation constant (K,) of 2.2 + 0.6 nu and a mean saturation value of 0.32 + 0.02 pmol/mg of protein.

The close correlation which exists between the properties

* This work was supported in part by the Medical Research Council of Canada (MA-56131 and the National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Scholar of the Medical Research Council of Canada. Author to whom reprint requests should be addressed. Present address, De- partment of Medicine, Box 3287, Duke University Medical Center, Durham, N.C. 27710.

8 Recipient of a Studentship from Medical Research Council of Canada.

ll Investigator, Howard Hughes Medical Institute. (/ Associate of the Medical Research Council of Canada.

of [“Hldihydroergocryptine binding to pituitary membrane receptors and characteristics of modulation of prolactin secretion by dopaminergic agents suggests that [“Hldihydroergocryptine binding sites are associated with mammotrophs and that these dopaminergic binding sites modulate prolactin secretion.

The diverse physiological effects of catecholamines appear to be mediated by three distinct types of receptors. These are known as a- and p-adrenergic and dopaminergic receptors. Each receptor type is characterized by a distinct order of binding affinities of agonist and antagonist agents (1, 2). In the case of the cy- and p-adrenergic receptors, it has been possible to correlate binding data of specific radioactive ligands with a proximate effect of receptor occupancy, smooth muscle contraction (3, 4), or catecholamine-stimulated K+ efllux (5, 6) and adenylate cyclase activation (7-121, respectively.

Correlation of binding of specific ligands to the dopaminergic receptor with a physiological or biochemical response have been less successful. In the brain, where most dopaminergic binding studies have been performed (13-161, dopamine receptors appear to be coupled to adenylate cyclase (2, 17, 18). However, the potency of dopaminergic antagonists in inhibiting dopamine-sensitive adenylate cyclase activity did not always correlate well with the ability of all classes of compounds to compete with [Z<H]haloperidol or [:‘Hldopamine binding (19-25).

Prolactin secretion from the anterior pituitary gland appears to be under constant hypothalamic control (261. Accu- mulating evidence suggests that dopamine may be the main or even the only inhibitory substance involved (27-30). Dopa- minergic agents have been shown to modulate prolactin secretion by a direct action at the anterior pituitary level in vivo and in vitro (31,321 suggesting the presence of specific receptor sites for dopamine in the anterior pituitary.

Since adenohypophyseal cells in primary culture proved to be a very precise system for studying control of the secretion of many pituitary hormones (34-361, we have used this model in the present report in an attempt to correlate properties of

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Dopaminergic Receptors and Prolactin Release 2245

putative dopamine receptor binding sites with the specificity of action of a large series of dopaminergic agonists and antagonists on prolactin release.

Our original goal was to study control of prolactin secretion and binding of a specific dopaminergic ligand in the rat anterior pituitary system. In preliminary experiments, we have been able to demonstrate binding of [3Hldihydroergo- cryptine to rat anterior pituitary membranes or whole cells in culture which appears to possess features of dopaminergic specificity. However, due to the very small amount of membrane protein which can be derived from rat anterior pituitary, the relatively low concentration of receptors in these preparations and the modest specific activity of the labeled ligand, it was felt that the bovine anterior pituitary would be a preferable tissue to carry out such detailed studies of binding to dopaminergic receptors.

We report that the characteristics of binding of the ligand [JH]dihydroergocryptine to bovine anterior pituitary membranes are typical of binding to a dopamine receptor. In addition, the binding data are shown to be in excellent agreement with the characteristics of the prolactin response to dopaminergic agents in rat anterior pituitary cells in primary culture. Thus, the ability of a wide variety of dopaminergic agonists and antagonists to interact with [“Hldihydroergocryptine binding sites in pituitary membranes correlates well with the relative potency of these agents for the dopaminergic response.

EXPERIMENTAL PROCEDURES

Dopamine, (- )-epinephrine, (- )-norepinephrine, (- j-isoproterenol, L-dihydroxyphenylalanine, a-methyl-dopal (?I-dihydroxyman- delic acid, (2).normetanephrine, ergocryptine, triiodothyronine, tetraiodothyronine, estradiol benzoate, y-aminobutyric acid, and various other chemicals were obtained from Sigma. 5a-Dihydrotes- tosterone was from Steraloids. The following compounds were gifts: (+)- and (-)-butaclamol and (-)-propranolol (Ayerst Laboratories); OI- and /3-flupenthixol and cis- and trons-thiothixene (Lundbeck); (t)-ephedrine (Lilly); (?I-phenylephrine (Sterling-Wintrop); apomorphine (Merk Frosst); haloperidol (McNeil); pimozide, clozapine, and clonidine (Roussel UCLAF); chlorpromazine and other phenothiazines (Smith, Kline & French!; phentolamine (CIBA); dihydroergocornine, dihydroergocryptine, and CB-154 (Z-bromo-o-ergocryptine) (Sandoz); morphine (May and Baker, Toronto); neurotensin (Bachem Inc.); substance P (Beckman); thyrotropin-releasing hormone (Peninsula Labs); a-melanocyte-stimulating hormone (CIBA); p-endorphin (Dr. C. H. Li, University of California, San Francisco) and somatostatin (Dr. D. H. Coy, Tulane University, New Orleans). Dulbecco’s modified Eagle’s medium and sera were obtained from Grand Island Biologicals Co.

rsHIDihydroergocryptine was prepared at New England Nuclear by catalytic reduction of ergocryptine with tritium gas using palla- dium as the catalyst to specific activities of 21.3 and 24.1 Ci/mmol in two different batches. Properties and stability of this ligand as a marker for ol-adrenergic receptors in uterus have been described previously (4). In the bovine anterior pituitary system [“Hldihy- droergocryptine was found to be very stable. When the labeled ligand was bound to pituitary membranes, dissociated by dilution, lyophilized, and then chromatographed on thin layer silica gel plates in the solvent system benzene/ethanol (78:22), radioactivity co-chromatographed with fresh tritiated as well as authentic unlabeled material (data not shown). [“HlDihydroergocryptine previously ex- posed to pituitary membranes could also bind to fresh membranes with the exact same efficiency as new ligand. Thus, ligand which had been bound to and dissociated from pituitary membranes was chemically and biologically intact. Furthermore, the biological activity of Y’Hldihydroergocryptine appeared to be identical with that of unlabeled material. Thus, as shown in Fig. lB, increasing

’ The abbreviations used are: L-dopa, L-dihydroxyphenylalanine; DHEC, dihydroergocryptine; CB-154, 2-bromo-a-ergocryptine; Hepes, 4-(2.hydroxyethyll-1-piperazineethanesulfonic acid.

concentrations of tritiated and unlabeled dihydroergocryptine led to almost superimposable inhibition of prolactin release.

MATERIALS AND METHODS

Preparation of Anterior Pituitary Cells - Adult female Sprague- Dawley rats at random stages of the estrous cycle and weighing 150 to 300 g (obtained from Canadian Breeding Farms, St. Constant, Quebec) were used for the preparation of primary cultures of anterior pituitary cells. Cells were prepared as described (34, 37). Briefly, anterior pituitary glands (usually 50 in each experiment) were cut into small fragments and first digested for 90 min at room temperature under constant agitation in 10 ml of 0.1% hyaluronidase (Sigma), 0.35% collagenase (Worthington), and 3% bovine serum albumin in 137 rnM NaCl, 5 rnM KCl, 0.7 rnM Na,HPO,, 10 rnM glucose, 25 rnM Hepes buffer, pH 7.2 at 23” (Hepes buffer). The suspension was then centrifuged at 50 x g for 5 min at room temperature and the pellet resuspended in 10 ml of 0.25% Viokase in Hepes buffer for a further 30-min incubation. The suspension of well dispersed cells was then washed by centrifugation through a layer of 4% bovine serum albumin in Hepes buffer. The cells were further washed four times before plating (5 to 7 x lo5 cells) in Falcon Petri dishes (35 x 10 mm) in 1.5 ml of Dulbecco’s modified Eagle’s medium containing 10% horse serum and 2.5% fetal calf serum (dextran- coated charcoal adsorbed), nonessential amino acids, 50 units/ml of penicillin, and 50 pg/ml of streptomycin. Dextran-coated charcoal adsorbed sera were prepared by overnight incubation of the sera at 4” with 1% charcoal (Norit A) and 0.1% Dextran T 70 obtained from Fisher and Pharmacia, respectively. This treatment was found to remove more than 99% of added 17 /3-[:‘Hlestradiol and to lower the sera content of I:lHlestradiol to undetectable levels as measured by radioimmunoassays (10.05 pg/ml). Cells usually formed monolayers after 1 day in culture under an atmosphere of water-saturated 95% air, 5% CO, at 37”.

Incubation Procedure-Cells were washed four times with Dul- becco’s modified Eagle’s medium without serum and the incubation performed in triplicate for 2 or 3 h after addition of the substance(s) to be tested. Catecholamines and apomorphine were usually tested in the presence of 0.2 mM sodium metabisulfite and 0.1% ascorbic acid, respectively. At the end of incubation, medium was spun at 1000 x g for 5 min at 2-4” and the supernatant kept at -20” until assayed.

Prolactin Assays and Calculations - Prolactin was measured in duplicate by double antibody radioimmunoassay (38) using rat prolactin I-l and rabbit antisera (anti-rat-prolactin-S-3) kindly sup- plied by Dr. A. F. Parlow for the National Institute of Arthritis and Metabolic Diseases, Rat Pituitary Hormone Program. Data were calculated and analyzed with a Hewlett-Packard calculator, model 9930, using a program based on model II of Rodbard and Lewald (39). Statistical significance was determined according to the multi- ple range test of Duncan-Kramer (40). Dose-response curves and 50% effective doses (ED,,J were calculated using a weighted iterative nonlinear least squares regression (41). All data are presented as mean ?S.E. of duplicate measurements of triplicate dishes.

Bovine Anterior Pituitary Membrane Preparations- Fresh pituitary glands were obtained from a local slaughterhouse (Abattoir St. Charles, Quebec). The anterior lobes were freed of intermediate and posterior lobes and connective tissues within 10 min after death of the animals, were frozen immediately on dry ice and then kept at -90” and thawed before use. All further operations were performed at O-4”. Anterior pituitary glands were minced and homogenized in 6 volumes (w/v) of 0.25 M sucrose, 25 rnM Tris/HCl, 2 mM MgCl,, pH 7.4 at 4”, using a motor-driven glass-Teflon homogenizer. The homogenate was filtered through two layers of cheesecloth and centrifuged at 300 x g for 10 min in an International PR-6000 centrifuge (swinging buckets) in 50-ml conical tubes. The supernatant was then collected while the loosely packed pellet was rehomog- enized in the same buffer and recentrifuged twice. Pooled superna- tants were then centrifuged at 30,000 x g for 20 min in a Sorvall RC2B. The supernatant fluid was discarded. The top brown layer of the pellet containing plasma membranes and other organelles was mechanically separated from the bottom white layer (which almost exclusively corresponds to pituitary secretory granules) and washed once more in the same buffer. The final brown pelleted material was resuspended in buffer without sucrose and stored frozen at -90 until assayed. Prior to an experiment, membrane material was thawed, washed once with the no-sucrose buffer and resuspended in an appropriate volume of 25 rnM Tris/HCl, 2 rnM MgCl,, pH 7.4 at 25”.


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2246 Dopaminergic Receptors and Prolactin Release

In experiments in which fractionation of the membrane preparation was performed, the top layer of the 30,000 x g pellet collected after the first wash was suspended in buffer and adjusted to a density of 1.18 with sucrose before layering in a five-layer discontinu- ous sucrose gradient of densities 1.14, 1.16, 1.18, 1.20, and 1.22 (42). Gradients were centrifuged at 40,000 x g for 2.5 h in a Beckman L- 65 (SW 27 rotor). Each layer was collected, diluted with buffer and pelleted at 30,000 x g for 20 min. After resuspension in incubation buffer (25 rnM Tris/HCl, 2 rnM MgCl,, pH 7.4 at 25”) fractions were ready to be assayed.

Binding Assays- 13H]Dihydroergocryptine binding to anterior pituitary membranes was assessed by a rapid filtration technique (431. 13H]Dihydroergocryptine was generally incubated with 0.7 to 2 mg of pituitary membrane protein for 60 min at 25” in a medium containing 25 mM Tris/HCl, 2 mM MgCl,, pH 7.4 at 25”, in a total volume of 500 ~1. At the end of the incubation, samples were diluted with 2 to 3 ml of cold washing buffer (25 mM Tris/HCl, 2 mM MgCl*, pH 7.4 at 4”) and rapidly filtered through Whatman (GF/C) glass fiber filters under reduced pressure. Filters were quickly washed with aliquots (3 x 5 ml) of washing buffer. The filtration and washing procedure took less than 10 s. This procedure provides a good estimation of equilibrium binding since, as shown under Results,” dissociation was much slower than the filtration time. Filters were immediately placed into scintillation vials, stored overnight to become translucent, and then radioactivity counted with 10 ml of Aquasol (New England Nuclear) with an efficiency of 42 to 45%.

In all experiments, the amount of 13H]dihydroergocryptine non- specifically bound or trapped in membranes and filters was determined by performing parallel incubations in the presence of 10 PM (+)-butaclamol, a potent dopaminergic antagonist. Concentrations of (+)-butaclamol higher than 10 PM did not displace appreciably greater amounts of 13H]dihydroergocryptine. (+)-Butaclamol was used to determine nonspecific binding since it was felt that its high affinity for the dopaminergic receptor, its specificity, and its stability under the assay conditions were all desirable. “Specific” binding determined as total binding minus “nonspecific” binding usually ranged from 60 to 70% of total binding at the concentrations of 13H]dihydroergocryptine routinely used (7 to 9 nM1. In all experiments, the nonspecific binding value (except where stated) has been substracted from total binding and results are presented as specific binding. In all experiments, determinations were done in triplicate and results presented are usually the average or representative of several experiments (n = 2 to 5). In figures, brackets represent standard error of the mean and where no bracket is shown, standard error of the mean was smaller than the symbol used.

13HlDihydroergocryptine-binding activity in bovine anterior pituitary membranes were stable to freeze-thawing and could be stored for long periods (1 to 2 months) at -90” without any loss of activity.

RESULTS

Specificity of Catecholaminergic Modulation of Prolactin Release from Rat Anterior Pituitary Cells in Primary Culture

Agonists - Fig. IA shows the inhibitory effect of increasing concentrations of various catecholamines and analogs on prolactin release during a Z-h incubation of rat anterior pituitary cells in primary culture. As measured by the concentration giving a 50% inhibition of hormone release by an agent the following order of potency was obtained: dopamine (35 nM) > epinephrine (420 nM) 2 norepinephrine (540 nM) while the (Y- and P-agonists phenylephrine and isoproterenol were without effect up to 10 PM. Apomorphine, the prototype of dopaminergic agonists (44), was approximately 10 times more potent than dopamine as an inhibitor of prolactin release with an ED,,, value of 3 nM (Table I). At high concentrations, apomorphine and the catecholamines led to a maximal 85 to 90% inhibition of basal prolactin release. Serotonin, a neurotrans- mitter potentially involved in the control of prolactin secretion in uiuo (46, 47) was a very weak inhibitor of prolactin release in cells in culture (Table I). The inhibition of prolactin release by catecholamines showed the expected stereoselectivity with

(-)-isomers being about 8-fold more potent than (+)-isomers (Table I). Ergot alkaloids which are thought to act mainly through a dopaminergic mechanism on prolactin secretion in vivo (48) accordingly showed potent dopaminergic agonist activities in this in vitro system. It can be seen in Fig. 1B that dihydroergocornine and dihydroergocryptine inhibited prolac-

A I T -

w DOPAMINE

- (-) lSOPROTEllENOt

bo (-1 PHENYLEPHAINE

0 , 10-e 10“ 10-b 10-S

CATECHOLAMINE CONC. (M)

B &--o OlHYDAOERGOCORNlNE ceo lllHYOROEAGOCRYPTlNE

T H [3H] OIHYOROFRGOCRYPTINE

IC

,lI J -11 -10 -9 -8 -7

CONCENTRATION LOG (M) FIG. 1. Effect of increasing concentrations of (A) various cate-

cholamines and analogs and (B) dihydroergocornine, dihydroergocryptine, and 13H]dihydroergocryptine on the release of prolactin in rat anterior pituitary cells in primary culture. Four days after plating, cells were incubated for (A) 2 h and @I) 3 h in the presence of the indicated drug concentrations. Prolactin is expressed in nanogram equivalents of prolactin standard RP-1 from National Institute of Arthritis and Metabolic Diseases Rat Pituitary Hormone Pro- gram.


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nM

Agonists Apomorphine 3 Dopamine 35 (~ )-Epinephrine 420 ( + )-Epinephrine 3,100 (-)-Norepinephrine 540 (+)-Norepinephrine 4,200 (-)-Isoproterenol Clonidine Phentolamine >10,000 Propranolol Dihydroergocryptine 0.2 Dihydroergocornine 0.4 Ergocryptine 0.2 2Bromo-a-ergocryptine 2.9

Antagonists (+ )-Butaclamol 0.3 (~ I-Butaclamol >l,OOO a-Flupenthixol 2.4 /3-Flupenthixol 330 cis-Thiothixene 4.6 trans.Thiothixene 44 Haloperidol” 0.7 Pimozide” 0.3 Fluphenazine 0.6 Triflupromazine n.t. Perphenazine n.t. Chlorpromazine 28 Serotonin n.t. Cyproheptadine” 14 Methysergide” 190

” Indicates that when tested these antagonists were found to also have agonistic activity as judged by their ability to inhibit prolactin release. The followingK,, values were obtained: haloperidol (>lO,OOO nM), pimozide (27 nM1, cyproheptadine (500 nM), and methysergide (>lO,OOO nM). K,, values for antagonists activity of these agents were calculated from the concentrations giving 50% reversal of inhibition of prolactin release by an agent. At these concentrations pimozide, cyproheptadine, and methysergide had significant agonist activity and did not reverse the dopaminergic inhibition of prolactin completely. Note that not all antagonists were tested for agonist activity. __ indicates effect of agent was too weak to calculate a valid K,] value. n.t. = not tested. Several catecholamine analogs,

tin release to about the same extent as dopamine (80 to 90%) but with a potency about 100 times greater. 2-Bromo-a-ergocryptine (CB-154), a compound used as an inhibitor of prolactin secretion in hyperprolactinemia in humans (49, 50) was

also tested. It was somewhat less potent with an ED,,, value of


TABLE I

Apparent dissociation constants of dopammergic agents in anterior pituitary

Apparent dissociation constants (KJ of dopaminergic agonists tration of the agent giving 50% reversal of the inhibitory action of and antagonists and other compounds for t,heir effects on prolactin dopamine or dihydroergocornine. For the effects of agonists and release in anterior pituitary cells in primary culture and their antagonists on YHldihydroergocryptine binding, K,, values were ability to compete for 1:‘Hldihydroergocryptine binding to bovine calculated according to the relation K,, = I&,/l + S/K (45). In this anterior pituitary membranes. K,, values for agonistic activity were equation, S represents the concentration of labeled ligand present in taken directly as the concentration of an agent (41) giving 50% the assay mixture. K represents the apparent dissociation constant (ED,,,) of maximal inhibition of prolactin release by that agent. For (K,,) of F’Hldihydroergocryptine for the binding sites as estimated antagonistic activity, compounds were tested in the presence of 3 nM by equilibrium or kinetic analysis (2.2 nM was used). IC,,, is the dihydroergocornine or 50 nM dopamine and apparent dissociation concentration of an agent giving 50% competition of specific binding. constant (K,,) values were calculated according to the equation K,, Relative potencies for agonists and antagonists were calculated in = IC,,,/(l + S/K) (45). In this equation, S is the concentration of reference to dopamine: K,, of dopamine/K,, of agent) x 100. Thus dopaminergic agonist, K is the ED,,, value of dopamine or dihydroer- arbitrarily dopamine was chosen as having a relative potency of 100. gocornine for inhibition of prolactin release, and IC,,, is the concen-

Agents

Prolactin release from ;rk&erior pituitary cells in [“HIDHEC binding to b;wfln;uhrmr pituitary mem-

K., Relative ootencv K,, Relative wtencv

TLM

1,170 78 628 100 490 100

8.33 1,700 28.8 1.13 16,500 2.97 6.48 3,200 15.3 0.83 15,000 3.27

>100,000 <0.5 >100,000 co.5

<0.35 3,200 15.3 >100,000 co.5

17,500 4.7 10,400 8,750 10.8 4,530

17,500 6.5 7,540 1,210 24 2,040

11,700 1.3 37,700 c3.5 >100,000 <0.5

1,460 6.7 7,310 10.6 1,600 30.6

760 27 1,810 79.5 1,300 37.7

5,000 12 4,080 11,700 33 1,480 5,830 3.4 14,400

n.t. 9.1 5,380 n.t. 30 1,630

125 44 1,110 n.t. 23,000 2.13

250 230 213 18.4 1,100 44.5

precursors, and metabolites were tested for their ability to compete for binding. Ephedrine, L-dopa, a-methyl-dopa, and c-t)-dihydroxy- mandelic acid did not interact significantly with the binding sites below 100 ELM. Normetanephrine inhibited binding by 50% at 100 PM. Various other agents having a presumable influence on dopaminergic systems in uiuo have been screened for possible interactions with the receptor sites. None of the following drugs tested had any effect at 10 to 100 PM: morphine, p-endorphin, thyrotropin-releasing hormone, triiodothyronine, ol-melanocyte-stimulating hormone, 17&estradiol, 5a-dihydrotestosterone, substance P, neurotensin, somatostatin, and y-aminobutyric acid.

2.9 nM (Table I). As mentioned, [oH]dihydroergocryptine was fully biologically active since it was equipotent with unlabeled dihydroergocryptine in inhibiting prolactin release.

Antagonists -To further study the specificity of this process we took advantage of the precision of the in vitro system to


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*O” H (+) BIJTACLAMOL A w (-1 BUTACLAMOL

H a-FLUPENTHIXOL B - CIS-THIOTHIXENE c

T m-a p- FLUPENTHIXOL o-a Tram-THIOTHIXENE

ANTAGONISTS CONC LOG (M)

FIG. 2. Ability of various dopaminergic antagonists and their enantiomers to reverse the inhibition of prolactin release induced by 3 nM dihydroergocornine in rat anterior pituitary cells in primary culture. 0, basal prolactin secretion; 0, prolactin secretion in the presence of 3 nM dihydroergocornine. Increasing concentrations of

examine the ability of various dopaminergic antagonists to reverse the inhibition of prolactin release induced by dihydroergocornine (3 nM) or dopamine (50 nM). As previously observed (Fig. IL?), 3 nM dihydroergocornine decreased basal prolactin release by more than 75% (Fig. 2, A, B, 0. This inhibitory action of 3 nM dihydroergocornine could be progres- sively and completely reversed by the addition of increasing concentrations of the potent neuroleptic (+)-butaclamol (K, = 0.3 nM) whereas its pharmacologically inactive enantiomer, (-)-butaclamol, had only slight activity at 10 PM (Fig. 2A). In agreement with their relative dopaminergic blocking activities in pharmacological tests cis-thiothixene and cu-flupenthixol were approximately lo- and loo-fold more potent than trans-thiothixene and /I-flupenthixol in reversing the dihydroergocornine-induced inhibition of prolactin release (Fig. 2, B and C). Comparable results were obtained when 50 nM dopamine was used to inhibit prolactin release (data not shown).

Several other compounds were tested for their effects on prolactin release. Thus, phenothiazines such as fluphenazine and chlorpromazine were able to reverse the inhibition of prolactin release caused by 3 nM dihydroergocornine (Table I). This is consistent with their known pharmacological activity (U-53).

Mixed Agonist-Antagonists- When assayed in the presence of 3 nM dihydroergocornine the classical dopaminergic antagonists pimozide and haloperidol led to a reversal of the inhibitory effect of the ergot alkaloid at relatively low concentrations (K, = 0.3 nM and 0.7 nM, Table I). However, at concentrations above 10 nM and 1 PM both pimozide and haloperidol caused a decrease of prolactin release characteris- tic of dopaminergic agonist activity. The dopaminergic agonist activity of these compounds was confirmed when the neuroleptics were tested alone, a 45% inhibition of prolactin release being measured at 10 PM haloperidol whereas pimozide inhibited prolactin release with potency similar (ED,, = 25 nM)

the indicated compounds were all tested in the presence of 3 no dihydroergocornine during a 2-h incubation. Prolactin is expressed in nanogram equivalents of prolactin standard RP-1 from National Institute of Arthritis and Metabolic Diseases Rat Pituitary Hormone Program.

to that of dopamine itself (35 nM) (Table I). In view of reported actions of serotonergic agents in the control of prolactin secretion in uivo (46, 471, it was of interest to study the effect of such agents directly at the pituitary level on prolactin release. Cyproheptadine and methysergide, both putative serotonergic antagonists, were found to be quite potent dopaminergic antagonists (Table I) but to also show weak partial agonist properties above 100 nM (Table I).

The order of potency of catecholaminergic agonists in inhibiting prolactin release and the specificity of the interaction of antagonists with this process clearly suggest that prolactin release is affected directly at the pituitary level via a dopaminergic process.

[3HIDihydroergocryptine Binding to Bovine Anterior Pitui- tary Membranes

In order to correlate the biological activity of the various dopaminergic agents with their interactions with putative dopaminergic receptors we develop a direct ligand-binding technique using the potent dopaminergic agonist [3H]dihydroergocryptine. Binding to bovine anterior pituitary membranes was found to fulfill the expected criteria of specific binding namely kinetic, saturability, and specificity.

Kinetics of Binding- Binding of [3H]dihydroergocryptine to pituitary sites was time- and concentration-dependent. At concentrations of 8 to 10 nM, [3H]dihydroergocryptine binding reached equilibrium between 40 to 60 min of incubation at 25” (Fig. 31, whereas when lower ligand concentrations were used (0.2 to 0.5 nM), equilibrium was obtained after 2 to 3 h. Data presented in Fig. 3 have been replotted in the inset and a second order association rate constant k, = 7.2 + 1.1 x lo6 Me’

min-’ was calculated. Similar k, values were obtained at low ligand concentrations.

Fig. 4 shows that after the addition of 10 PM (+)-butaclamol to an incubation mixture at equilibrium specifically bound 13H1dihydroergocryptine completely dissociated after 12 h with


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30 r

A------- 0 5 10 15 10 TIME (min)

0 I 0 20 40 60 80 100 120

TIME (min)

FIG. 3. Time comae of [3H]DHEC binding to bovine anterior pituitary membranes. Membrane preparations (200 ~1, 2.58 mg/ml) which were equilibrated at 25” for 10 min, were incubated at 25” with [3H]DHEC (8.7 nM) in a total volume of 500 ~1 containing 25 mM Tris/HCl, pH 7.4, 2 rnM MgClp. The reaction was started by the addition of membranes. Specific binding (picomoles per mg) was determined by subtraction of binding which was not competed for by 10 PM (+)-butaclamdl (nonspecific binding) from total binding. Nonspecific binding (not shown) varied linearly with time from 0.15 pmol/mg at 0 min to 0.25 pmol/mg after 120 min of incubation. Results shown are means ? standard error of triplicate determinations from an experiment which is representative of four comparable experiments. Inset, estimation of second order rate constant for [3HlDHEC binding to pituitary membrane preparations. R is the concentration of unoccupied binding sites at given times; R,, the free receptor concentration at time zero, estimated by equilibrium saturation experiments; DHEC, the concentration of free PHIDHEC, which was determined by subtracting bound PHIDHEC concentration (DHEC,J. The linearity of this plot is consistent with a second order reaction (54). The slope provides an estimate of the association rate constant (k,). In the experiment shown, k, = 5.9 x lo6 M-’ min-I. In four comparable experiments, an average k, value of 7.2 + 1.1 x 10b M-’ min-’ was obtained.

a half-time of about 3 h. A first order dissociation rate constant Kz of 4.4 ? 1.0 x 10e3 min-’ (n = 2) was obtained. As evidenced by the open circles, specific [aH]dihydroergocryptine binding to pituitary membranes was stable for at least 12 h at 25 indicating that apparent dissociation is a true reflection of ligand dissociation from binding sites.

The ratio k&r (0.6 nM) of the rate constants at 25” provides an estimate of the equilibrium dissociation constant (K,) for the interaction of [3H]dihydroergocryptine with the pituitary binding sites which is in good agreement with that derived from equilibrium binding data (Fig. 5).

Equilibrium Studies of[3H]Dihydroergocryptine Binding- As shown in Fig. 5 (left) specific binding of VHldihydro- ergocryptine increased with increasing concentrations of free [aH]dihydroergocryptine and was saturable. Nonspecific binding increased linearly from 20 to 25% of total binding at low concentrations of ligsnd (-5 nM) to reach -75% of total binding at the highest concentrations used. Repre- sented in Fig. 5 (right) are data of specific binding plotted according to the method of Scatchard (55). Since the Scatchard plot yielded a straight line (56), these results are consistent with there being a single class of PHldihydroergocryptine binding sites. As calculated from the slope of the line, a KD value of 2.2 + 0.6 nM was obtained from the pooled data of five

:. j ZZ :~~+;~~:::l’/~

6 8 10 12 TIME (hours)

FIG. 4. Dissociation of [“HIDHEC from binding sites in bovine anterior pituitary membranes. A membrane preparation (3.5 mg of protein/ml) was equilibrated at 25” with 7.4 nM [“HIDHEC for 60 min. At time zero, (+)-butaclamol was added to the incubation mixture to a final concentration of 10 PM. At indicated times, 500- ~1 aliquots were removed, filtered through GF/C glass fiber filters, and washed rapidly to determine [“HIDHEC bound. Specific binding (0-O) after addition of 10 PM (+)-butaclamol was obtained by subtraction from total binding of the value of nonspecific binding which was estimated in a separate incubation mixture containing 10 ELM (+)-butaclamol from the beginning of the incubation period. This nonspecific binding (not shown) increased linearly with time from 0.125 pmol/mg at time zero to 0.225 pmol/mg at 12 h. Specific binding (O-O) was also monitored during the dissociation period. Inset, specific VHIDHEC binding after addition of 10 KM (+)-buta- clam01 is plotted here as per cent of initial binding (10 to lOO%, log scale) uersus time of dissociation. Initial binding (0.240 pmol/mg) refers to the amount of [3HlDHEC bound at equilibrium prior to addition of (+)-butaclamol. The slope of the line provided an estimate of the dissociation rate constant k2 at 25”. In the experiment shown, kz = 3.3 x 10e3 min-I. Results shown are means f standard error of triplicate determinations and the experiment is representative of two.

experiments. In these experiments, a mean saturation value of 0.32 r 0.02 pmol/mg of protein was obtained.

Specificity and Affinity of Interaction of Dopaminergic and Other Agents with P7HIDihydroergocryptine Binding Sites

Agonists- As shown in Fig. 6, agonists competed for [3H]dihydroergocryptine binding with the following order of potency: apomorphine > dopamine > epinephrine 2 norepinephrine >> isoproterenol = clonidine. This relative order of potency is typical of a dopaminergic process (2) and closely resembles the potency of these agonists in inhibiting prolactin secretion from rat anterior pituitary cells in culture. Ergot alkaloids which act as potent dopaminergic agonists on prolactin secretion (Fig. H?, Table I, 57-59) were also potent competitors of [“Hldihydroergocryptine binding. These ergot compounds competed for binding of [JHldihydroergocryptine to about the same extent as agonists such as apomorphine and dopamine (Fig. 6) and antagonists such as (+)-butaclamol (Fig. 7A), thus, suggesting that these compounds interacted with the same sites.

Antagonists- [“HlDihydroergocryptine binding sites in bovine anterior pituitary displayed marked stereoselectivity toward various neuroleptics and their isomers. Thus, (+)- butaclamol, Lu-flupenthixol, and cis-thiothixene which possess potent pharmacological dopamine-blocking activity (51-53, 60) were 2 to 5 orders of magnitude more effective than their


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r.. “0

I L \ , 1 10 IS ?o 25 a Oo 02 06

[FRfl OW] IY [souro ontcl R:

FIG. 5. A, binding of [“HIDHEC to bovine anterior pituitary membranes as a function of increasing concentrations of ligand. Membrane preparations were incubated with increasing concentrations of FHIDHEC (0.35 to 30 nM) for 180 min. A longer incubation period was chosen to ensure that binding at low ligand concentrations had reached equilibrium. Specific FH]DHEC binding was measured as described under “Experimental Procedures” with the exception that samples containing greater than 10 nM FHIDHEC were rapidly washed with an additional 20 to 40 ml of washing buffer to remove excess unbound ligand. Specific binding (0-O) was calculated from the difference between total and nonspecific (10 PM (+I-butaclamol) binding. Saturation binding was 0.32 f 0.02 pmol/ mg (n = five experiments). B, Scatchard plot of FH]DHEC binding. Results from the experiment shown in A have been plotted here. An apparent dissociation constant K, = 1.6 nM was obtained in this experiment. A mean apparent K, value of 2.2 + 0.6 nM was obtained from five such experiments. Results shown in A and B are means 2 standard error of triplicate determinations from a representative experiment. Data where no bracket is shown indicate that the standard error of the mean was smaller than the symbol used.

- APOMORPHlNE - OOPAMINE - k)EPINEPHRINE - I-)NOREPINEPHRINE - k)ISOPAOTEAENOL - CLONIOINE

(AGONIST) M toclo

FIG. 6. Competition of various agonists for [3HlDHEC binding to bovine anterior pituitary membranes. FHIDHEC (8.2 nM) binding was determined as described under “Experimental Procedures” except that membranes were pretreated at 25” for 10 min with 0.1% ascorbic acid and 10 PM pargyline. One hundred per cent or control specific binding was 0.185 f 0.009 pmobmg (n = 3). Results shown are means 2 standard error of the means of three experiments determined in triplicate.

respective pharmacologically less active enantiomers in competing for binding (Fig. 7A).

Various other compounds which possess dopamine antagonist activity in viva were tested for their ability to compete for ?Hldihydroergocryptine binding. Fig. 7B shows that haloper-

c---o B-WJPENTHIXOL - a-FLIJPENTHIXOL G---O kMJTACLAMOL

_ m--a (+)BUTACLAMOL - TAANS-THIOTHIXENE - CIS-THIOTHIXENE

(ANTAGONIST) M to6,o

9 -8 -7 -6 -5 -4 -3

(ANTAG~NW M tori,,

FIG. 7. A, competition of various dopaminergic antagonists and their enantiomers for FHIDHEC binding to bovine anterior pituitary membranes. L3HIDHEC (14.1 nM) binding was determined as described under “Experimental Procedures” in the presence and ab- sence of various concentrations of these agents. Control binding was 0.234 2 0.005 pmol/mg. B, competition of various antagonists for FHIDHEC binding. FHIDHEC was present at 14.4 nM for pimozide and clozapine and 12.2 nM for the remaining drugs. One hundred per cent specific binding was 0.163 f 0.012 pmol/mg. Results shown are means of triplicate determinations for each compound and this experiment is representative of two.

idol reduced specific [3H]dihydroergocryptine binding with high affinity to about the same extent as (+)-butaclamol. Pimozide and chlorpromazine as well as other phenothiazines (Table I) also competed for binding with relatively high affinity whereas clozapine inhibited binding with somewhat lower potency. The cu-adrenergic antagonist phentolamine was a relatively weak competitor of 13H1dihydroergocryptine binding in pituitary membranes. Propranolol, a selective P-adrenergic antagonist, did not compete for binding below 100 pM.

Other Agents- Serotonergic agents have been found to stimulate prolactin secretion in viuo (47), we have examined


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TABLE II

Distribution of [3Hldihydroergocryptine-binding activity m adenohypophyseal subcell&r fractions

Bovine pituitaries were homogenized in 6 volumes of 0.25 M

sucrose, 25 nnv~ Tris/HCl, 2 rn~ MgCl,, pH 7.4, at 4” before fractionation by differential and isopycnic centrifugation as described under “Experimental Procedures.” The various layers of the sucrose gradient were removed and assayed for binding after having been washed with 25 mM TrislHCl, 2 mM MgCl,, pH 7.4, at 25”. Morpho- logical and some biochemical characteristics of these various puri- fied fractions of bovine anterior pituitary have been studied previously in this laboratory by Poirier et al. (42). Binding is expressed as femtomoles per mg of protein. Results are typical of three experiments and are expressed as mean t S.E. of triplicate determinations from one experiment.

Fraction [“HlDihvdroeraocrvptine binding

fmollmg protein Homogenate Interface 1.14-1.16 Interface 1.16-1.18 Interface 1.18-1.20 Interface 1.20-1.22 Pellet (secretory granules)

14 -c 14 597 i 2 335 k 11

108 ? 3 38 -c 1

0

the ability of serotonin and two serotonergic antagonists, methysergide and cyproheptadine, to compete for binding. Serotonin was a very weak competitor whereas cyproheptadine and methysergide competed for binding with K,, values of 0.23 and 11 FM, respectively (Table I). The interaction of these compounds with [“Hldihydroergocryptine was comparable to their ability to inhibit or antagonize the dopaminergic inhibition of prolactin secretion and to compete for [“Hlhaloperidol binding sites in brain (19).

Several other catecholamine analogs, precursors, and metabolites were tested for competition of binding and found to have little or no activity (listed in Table I).

Subcellular Distribution of [“HJDihydroergocryptine Binding

Binding of [:‘Hldihydroergocryptine to bovine pituitary membranes was studied in various fractions prepared by stepwise sucrose density gradient (42) centrifugation. The binding activity present at interfaces of density 1.14 to 1.16 and 1.16 to 1.18 of the stepwise sucrose density gradient constituted about 80% of the total activity present in all fractions. The specific activity of binding in these two fractions was increased by 5 to &fold over starting material (Table II). The distribution of [“Hldihydroergocryptine binding in these fractions was found to closely parallel the distribution of adenylate cyclase, 5’-nucleotidase, and magnesium and sodium-potassium ion-activated ATPase activities obtained by Poirier et al. (42) using a similar procedure suggesting the likely association of the binding sites with the plasma membrane fraction of bovine pituitary cells.

DISCUSSION

Ergot alkaloids are known to interact with a variety of drug receptors including cu-adrenergic, serotonergic, and dopaminergic receptors. [“HlDihydroergocryptine has proved a useful ligand for studying a-adrenergic receptor binding sites in membranes derived from uterine smooth muscle (3, 41, parotid acinar cells (5, 61, and human platelets (61). In membranes from brain, binding of the ligand is more complex appearing to label more than one type of receptor sites (62) but under certain conditions u-adrenergic receptors can be exclusively labeled with [:‘H]dihydroergocryptine (63).

The present study indicates that in anterior pituitary membranes, where a-adrenergic receptors could not be detected under the conditions used [“Hldihydroergocryptine appears to selectively and exclusively label dopaminergic sites. In the anterior pituitary system, haloperidol and (+)-butaclamol have been found to be, respectively, 300 and 2500 times more potent in competing for [“Hldihydroergocryptine binding than the cu-adrenergic antagonist phentolamine whereas in cu-adrenergic systems, the reverse situation is observed. Similarly, whereas apomorphine and dopamine are more potent than epinephrine and norepinephrine in competing for binding in this system, epinephrine and norepinephrine were the more potent agonists in cu-adrenergic systems (4). As summarized in Table I, the order of potency of a large variety of drugs in competing for [“Hldihydroergocryptine binding in adenohypophyseal membranes closely parallels their ability to elicit inhibition or reversal of inhibition of prolactin secretion in whole anterior pituitary cells in culture.

As shown by the studies with the rat anterior pituitary cells in culture the pharmacology of the control of prolactin release

was found to be typical of a dopaminergic process. This was demonstrated: 1) by the order of potency of agonists in inhibiting prolactin release (apomorphine > dopamine > epinephrine 2 norepinephrine >> isoproterenol = phenylephrine), 2) by the high affinity of antagonists such as haloperidol, pimozide, and various phenothiazines for this process, and 3) by the high degree of stereoselectivity (Table I) and the order of potency of antagonists such as the neuroleptics butaclamol, flupenthioxol, and thiothixene. Many previous investigations performed in uitro using intact pituitaries (29, 64-68) and in uiuo (28, 69-71) have clearly shown a direct inhibitory effect of dopamine on prolactin release at the anterior pituitary level. The present experiments performed in cells in culture extend those observations and demonstrate the specificity of the dopaminergic action. In preliminary experiments, we have found that the pharmacology of prolactin control appears to be the same in the presence of thyrotropin-releasing hormone, yet thyrotropin-releasing hormone does not evoke a major change in prolactin release in these cells because prolactin levels are already very high.

The high degree of precision of the cultured cell system used has permitted the somewhat unexpected findings that several

compounds so far classified only as dopaminergic antagonists, (e.g. haloperidol and pimozide) (44, 721, do in fact have mixed agonist-antagonist properties. The inhibitory effect (agonist activity) of high concentrations of haloperidol are in agreement with previous in vitro findings (67, 73) but may represent nonspecific effects. The presumed serotonergic specific antagonists methysergide and cyproheptadine were also found to modulate prolactin release both as agonists and antagonists in addition to their ability to interact with [“Hldihy- droergocryptine binding sites in pituitary membranes. There- fore, interaction of these compounds with the dopaminergic receptors in anterior pituitary could offer an explanation for the in uiuo observations of a stimulation of plasma prolactin levels after administration of these compounds (46, 47). These data show that the control of prolactin release in anterior pituitary cells in primary culture, beside its own intrinsic interest, offers a precise model for studying dopaminergic agonistic and antagonistic activities of compounds.

As mentioned above [JH]dihydroergocryptine binding to bovine anterior pituitary membranes displayed the specificity expected of a dopaminergic process and the relative potencies of more than 30 agents in competing for binding was identical


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with their ability to modulate prolactin release from cultured cells. However, dissociation constant (K,,) values for all agents calculated from their effects on prolactin release were somewhat lower than those calculated for their interaction with the [:‘H]dihydroergocryptine binding sites (2 to 20-fold). These differences could possibly be due to differences in the two species used in these studies. More likely, however, these differences might be due to the use of high concentrations of protein in our binding assays. Indeed, because of the relatively

low concentration of receptor in these membranes and the modest specific radioactivity of our tracer [“Hldihydroergo- cryptine, high concentrations of membrane protein (1.0 to 2.5 mg/ml) had to be used to obtain reasonable levels of specific binding (3000 to 6000 cpm/assay). Recent work of Brown et al. (12) on the binding of l’Y]hydroxybenzylpindolol to turkey erythrocyte membranes has shown that estimated K,, values were directly dependent on the concentrations of receptor protein used in the assays. Using protein concentrations from 60 to 300 pg/ml, they showed variations of estimated K,, values by 8- to lo-fold. Since binding assays were performed at room temperature while prolactin release was measured at 37”, it is also possible that this difference of assay temperature has played a role.

Previous studies of dopaminergic receptors in membranes derived from brain have utilized either the antagonist [“Hlhaloperidol (14-19) or the agonists [“Hldopamine (14-16) and [“Hlapomorphine (74). As noted, above, in no previous study, has it been possible to observe a correlation of binding to putative dopaminergic receptors with a specific dopaminergic biological response as it has been done in the present investigation. Marked differences have been observed in the relative affinities of dopaminergic agonists and antagonists for [“Hlhaloperidol versus [:‘H]dopamine binding sites in brain membranes (19). Dopaminergic antagonists are relatively more potent in competing for [:‘Hlhaloperidol whereas agonists are more potent toward [:‘H]dopamine binding. It has therefore been proposed that the two ligands label, respectively, “antagonist” and “agonist” states of the dopamine receptors (19).

Data presented in this study are of particular interest with regard to this model. Dihydroergocryptine, as shown here, behaves as a full agonist, causing the same maximum inhibition of prolactin secretion as dopamine. Therefore, according to the two-state model, agonist should compete with much higher potencies than antagonists for [:‘HJdihydroergocryptine

binding sites. However, antagonists were found to have very high potency in competing for [“Hldihydroergocryptine binding. In fact, binding affinities of both agonists and antagonists for [“Hldihydroergocryptine binding sites resemble more closely those obtained for competition with [:‘H]haloperidol than [:‘Hldopamine in brain studies (19). These observations suggest that, at least in the pituitary, the two-state (agonist versus antagonist) model of dopamine receptors might not fit the experimentally obtained data.

Several reports have previous presented supportive evidence that specific dopamine receptors might be present in pituitary (75-79). However, the studies reported here represent the first direct study and characterization of these binding sites in anterior pituitary and they represent the first study in which binding of a dopaminergic ligand has been correlated with a dopaminergic physiological process. More- over, the close correlation observed between the properties of [“Hldihydroergocryptine binding sites with those of the dopaminergic inhibition of prolactin release strongly suggest that

these sites correspond to the physiologically relevant dopamine receptors which regulate prolactin secretion. The present data indicate that the anterior pituitary gland, beside its own intrinsic interest, should represent a useful model for detailed study of the mechanisms of dopaminergic action. In fact, changes of [“Hldihydroergocryptine binding to the dopaminergic receptor can be correlated with an easily accessible and highly precise parameter of biological activity, prolactin release in cells in culture. Such a model of dopamine action

has not been previously available and should be useful for a better understanding of the mechanisms controlling dopamine receptor-mediated actions.

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REFERENCES

Wolfe, B. B., Harden, T. K., and Molinoff, P. B. (1977) Anna. Rev. Pharmacol. Toxicol. 17, 575-604

Iversen, L. L., Horn, A. S., and Miller, R. J. (1975) in Pre- and Postsvna~tic Recenters (Usdin. E.. and Bunnev. W. E.. eds)

” 1

pp. 207-243, Marcel Dekker, New ‘York ” Lefkowitz, R. J., and Williams, L. T. (1976) Science 192, 791-793 Williams, L. T., .Mullikin, D., and Lefkowitz, R. J. (1976) J. Biol.

Chem. 251, 6915-6923 Strittmatter. W. J.. Davis. J. N.. and Lefkowitz. R. J. (1977) J.

Biol. Chem. 252, 5472-5471 ’ Strittmatter, W. J., Davis. J. N.. and Lefkowitz. R. J. (1977) J.

Biol. Chem. 252, k478-5482 Levitzki, A., Atlas, D., and Steer, M. L. (1974) Proc. Natl. Acad.

Sci. U. S. A. 71, 2773-2776 Lefkowitz, R. J., Mukherjee, C., Coverstone, M., and Caron, M.

G. (1974) Biochem. Biophys. Res. Commun. 60, 703-709 Aurbach, G. D., Fedak, S. A., Woodward, C. J., Palmer, J. S.,

Hauser, D.. and Troxler. F. (1974) Science 186. 1223-1224 Mukherjee, C’., Caron, M. G., Coverstone, M., and LeIkowitz, R.

J. (1975) J. Biol. Chem. 250, 4869-4876 Maguire, M. E., Wiklund, R. A., Anderson, H. J., and Gilman,

A. G. (1976) J. Biol. Chem. 251, 1221-1231 Brown. E. M.. Rodbard. D.. Fedak. S. A.. Woodard. C. J.. and

Aurbach, G: D. (1976) J. Viol. Ciem. 261, 1239-1246 Creese, I., Burt, D. R., and Snyder, S. H. (1975) Life Sci. 17,

993-1002 Seeman, P., Chau-Wong, M., Tedesco, J., and Wang, K. (1975)

Proc. Natl. Acad. Sci. U. S. A. 72, 4376-4380 Burt, D. R., Enna, S., Creese, I., and Snyder, S. H. (1975) Proc.

Natl. Acad. Sci. U. S. A. 72, 4655-4659 Burt, D. R., Creese, I., and Snyder, S. H. (1976) Mol. Pharmacol.

12, 631-638 Kebabian, J. W., Petzold, G. L., and Greengard, P. (19723 Proc.

Natl. Acad. Sci. U. S. A. 69, 2145-2149 Greengard, P. (1975) in Advances in Cyclic Nucleotide Research

(Drummond, G. I., Greengard, P., “and Robison, G. A., eds) Vol. 5, pp. 585-601, Raven Press, New York

Burt, D. R., Creese, I., and Snyder, S. H. (1976) Mol. Pharmacol. 12, 800-812

Creese, I., Burt, D. R., and Snyder, S. H. (1976) Science 192, 481-483

Miller, R. J., Horn, A. S., and Iversen, L. L. (1974) Mol. Pharmacol. 10, 759-762

Clement-Cormier, Y. C., Kebabian, J. W., Petzold, G. L., and Greengard, P. (1974) Proc. Natl. Acad. Sci. U. S. A 71, 1113- 1117

Karobath, M., and Leitich, H. (1974) Proc. N&Z. Acad. Sci. U. 5’. A. 71, 2915-2918

Brown, J. H., and Makman, M. H. (197315. Neurochem. 21,477- 479

Iversen, L. L. (1975) Science 188, 1084-1089 Meites, J., Nicoll, C. S., and Talwaker, P. K. (1963) in Advances

in Neuroendocrinology (Nalbandov, A. V., ed) Vol. 9, p. 238, University of Illinois Press. Urbana

Bishop, W.; Fawcett, C. P., ‘Krulich, L., and McCann, S. M. (1972) Endocrinology 91, 643-656

Takahara, J., Arimira, A., and Schally, A. V. (1974) Endocri- nology 95, 462-465

Clemens, J. A., Shaar, C. J., Smalstig, E. B., Bach, N. J., and Kornfeld, E. C. (1974) Endocrinology 94, 1171-1176

Ben-Jonathan, N., Oliver, C., Weiner, H. J., Mical, R. S., and


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


31.

32.

33.

34.

35. 36.

37.

38.

39.

40. 41. 42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

Porter, J. C. (1977) Endocrznolo~y 100, 452-458 MacLeod, R. M., and Lehmeyer, J. E. (1974) Endocrinology 94,

1077-1085 Ojeda, S. R., Harms, P. G., and McCann, S. M. (1974) Endocri-

nology 94, 1650-1657 Takahara. J.. Arimura. A.. and Schallv. A. V. (19741 Endocri-

nology 45, 1490-1494 ”

Labrie. F.. Pelletier, G., Lemav, A., Borgeat, P., Barden, N., Dupont, A., Savary, M., Cote, J., and Boucher, R. (1973) Karolinska Symp. Res. Methods Reprod. Endocrinol. 301-340

Drouin, J., and Labrie, F. (1976) Endocrinology 98, 1528-1534 Drouin, J., Lagace, L., and Labrie, F. (1976) Endocrinology 99,

1477-1481 Vale, W., Grant, G., Amoss, M., Blackwell, R., and Guillemin,

R. (1972) Endocrinology 91, 562-572 Drouin. J.. De Lean. A.. Rainville. D.. Lachance. R.. and

Labrie, $. (1976) Endocrinology 98,514~521 Rodbard, D., and Lewald, J. E. (1970) Karolinska Symp. Res

Methods Reprod. Endocrinol. 79 Kramer, C. Y. (1956) Biometrics 12, 307 Rodbard, D. (1974) Endocrinology 94, 1427-1437 Poirier, G., De Lean, A., Pelletier, G., Lemag, A., and Labrie,

F. (1974) J. Biol. Chem. 249, 316-322 Mukherjee, C., Caron, M. G., Mullikin, D., and Lefkowitz, R. J.

(1975) Mol. Pharmacol. 12, 16-31 Anden, N. E., Rubenson, A., Fuxe, K., and Hokfelt, T. (1967)5.

Pharmacol. 19, 627-629 Cheng, Y., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22,

3099-3108 Subramanian, M. G., and Gala, R. R. (1976) Endocrinology 98,

842-848 Kordon, C., Blake, C. A., Terkel, J., and Sawyer, C. H. (1973/

74) Neuroendocrinology 13, 213-223 Clemens, J. A. (1974) in Hypothalamus and Endocrine Functions

(Labrie. F.. Meites. J.. and Pelletier. G.. eds) DD. 283-301. Plenum’ Press, New’York

II

Thorner. M. 0.. McNeillv. A. S.. Hagan, C., and Besser, G. M. (1974)‘Br. Med. J. 2, 419-422 _

Del Poso, E., Varga, L., Schutz, K. D., Kunzig, H. J., Marbach, P., Lopez de1 Campo, G., and Eppenberger, U. (1975) Obstet. Gynecol. 46, 539-543

59.

60.

61.

62.

63. 64.

65.

66.

67.

68.

69. 70.

71.

72.

73. MacLeod, R. M. (1976) in Frontiers in Neuroendocrinology, (Martini, L., and Ganong, W. F., eds) Vol. 4, pp. 169-194, Raven Press. New York

74. Moller-Nielson, I., Paderson, V., Nymark, M., Franck, K. F.,

Boeck, V., Fjalland, B., and Christensen, A. V. (1973) Acta Pharmacol. Toxicol. 33, 353-362

Weissman, A. (1974) in The Phenothiazines and Structurally Related Drugs (Forrest, I. S., Carr, C. J., and Usdin, E., eds) pp. 471-480, Raven Press, New York

75. 76.

77.

Seeman, P., Lee, T., Chau-Wong, M., Tedesco, J., and Wong, T. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 4354-4358

MacLeod, R. M. (1975) Endocrinology 96, Suppl. 94 Brown, G. M., Seeman, P., and Lee, T. (1976) Endocrinology 99, -.

1407-1410 Caron, M. G., Raymond, V., Lefkowitz, R. J., and Labrie, F.

(1977) Fed. Proc. 36, 287 Petersen, P. V., Nielsen, I. M., Pedersen, V., Jorgensen, A., 78. MacLeod, R. M. (1977) Fed. Proc. 36, 287

and Lanssen, N. (1977) in Psychotherapeutic Drugs (Usdin, 79. Cronin, M., Robert, J., and Weiner, R. (1977) Fed. Proc. 36, 287

54.

55. 56. 57.

58.

E., and Forest, I. S., eds) Part II, pp. 827-867, Marcel Dekker, New York

Butler, J. N., and Bobrow, D. G. (1965) The Calcu1u.s ofChem- istry, pp. 77-78, W. A. Benjamin, Inc., New York

Scatchard, G. (1949) Ann. N.Y. Acnd. Sci. 51, 660-672 Rodbard, D. (1973) Adu. Ezp. Med. Biol. 36, 289-326 Fldckiger, E., and Wagner, H. R. (1968) Experimentia 21 , 1130-

1131 Fuxe, K., Agnati, L. F., Covrodi, H., Everett, B. J., Hokfelt, T.,

Lofstrom, A., and Ungerstedt, U. (1975) Adu. Neural. 9, 223- 242

Del Pozo, E., Brun Del Re, R., Varga, L., and Friesen, H. (1972) J. Clin. Endocnnol. Metah. 35, 768-771

Bruderlin, F. T., Humber, L. G., and Voith, K. (1975) J. Med. Chem. 18, 185-188

Newman, K. D., Williams, L. T., and Lefkowitz, R. J. (1978) J. Clin. Inuest. 61, 395-402

Davis, J. N., Strittmatter, W. J., Hoyler, E., and Lefkowitz, R. J. (1977) Brain Res. 132, 327-336

Greenberg, D. A., and Snyder, S. H. (1977) Life Sci. 20, 927-932 MacLeod. R. M.. Fontham. E. H.. and Lehmever. J. E. (1970)

Neuroendocrinblogy 6, 283-284 ”

Koch, Y., Lu, K.-H., and Meites, J. (1970) Endocrinology 87, -_ 673-675

MacLeod, R. M., and Lehmeyer, J. E. (1974) Endocrinology 94, 1077-1085

Quijada, M., Illner, P., Krulich, L., and McCann, S. M., (1973/ 74) Neuroendocrinology 13, 151-163

Hill, M. K., MacLeod, R. M., and Orcutt, P. (1976) Endocrinol- ogy 99, 1612-1617

Lu, K.-H., and Meites, J. (19721 Endocrinology 91, 868-872 Donoso, A. O., Bishop, W., and McCann, S. M. (1973) Proc. Sot.

Exp. Biol. Med. 143, 360-363 Donoso, A. O., Banzan, A. M., and Barcaglioni, J. C. (19741

Neuroendocrinology 15, 236-239 MacLeod, R. M., and Lehmeyer, J. E. (1974) Cancer Res. 34,

345-350


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LabrieM G Caron, M Beaulieu, V Raymond, B Gagné, J Drouin, R J Lefkowitz and F

release.[3H]dihydroergocryptine binding with the dopaminergic control of prolactin

Dopaminergic receptors in the anterior pituitary gland. Correlation of

1978, 253:2244-2253.J. Biol. Chem.

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