0013-7227/90/1261-0565102.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 126, No. 1 Printed in U.S.A.

/?-Adrenergic Receptors in the Rat Mammary Gland during Pregnancy and Lactation: Characterization, Distribution, and Coupling to Adenylate Cyclase BIANCA MARCHETTI, MICHEL-A FORTIER, PATRICK POYET, NICOLLE FOLLEA, GEORGES PELLETIER, AND FERNAND LABRIE Medical Research Council Group in Molecular Endocrinology, Research Center, Laval University Medical Centre, Quebec Gl V 4G2, Canada; and the Department of Pharmacology, Medical School, University of Catania (B.M.), 95125 Catania, Italy

The radioautographic localization of [125I]CYP reveals the presence of specific 0-adrenergic receptors in the epithelial cells, alveoles, ducts, as well as adipocytes. [126I]CYP binding shows a 2- to 3-fold increase during pregnancy. Such a result correlates with parallel increases in stimulation of adenylate cyclase activity, the cytosolic progesterone receptor concentration, as well as plasma 17/3-estradiol and progesterone levels. At parturition, a sharp decline in /?-adrenergic receptor concentration is observed, a finding concomitant with a drop in progesterone receptor levels as well as plasma estradiol and progesterone concentrations. During midlactation, /8-adrenergic receptors reach their maximal levels. The presence of specific /3-adrenergic receptors functionally coupled to the adenylate cyclase system and the marked changes in receptor capacity and distribution measured during the different physiological states of the mammary gland suggest that the mmmary /3-adrenergic receptors are highly sensitive to changes in the hormonal milieu and provide a mechanism for a direct catecholaminergic influence on mammary gland growth and differentiation. (Endocrinology 126: 565-574,1990)

ABSTRACT. To investigate a possible role of catecholamines in mammary gland growth and differentiation, we have studied the characteristics of a specific /3-adrenergic receptor population during the different reproductive phases of the rat mammary gland, namely pregnancy and lactation. The functional response to mammary /3-adrenergic receptor stimulation was assessed by measurement of adenylate cyclase activity during the same physiological states of the gland. [126I]Cyanopindolol (CYP) binds specifically to membranes prepared from lactating mammary glands. Scatchard analysis of the binding data shows the presence of a single class of high affinity sites, with an apparent Kd value of 25.0 ± 0.4 pM and a binding capacity of 32.5 ± 1.2 fmol/ mg protein in lactating mammary glands at random stages of lactation. The order of potency of a series of agonists to compete for [126I]CYP binding is consistent with the interaction with a /82-subtype receptor. The binding of [125I]CYP to mammary glands also shows a marked stereoselectivity; the (—)isomers of isoproterenol and propranolol are more potent than their respective enantiomers.

/^ATECHOLAMINES have been shown to stimulate V_y adenylate cyclase activity in the mammary gland of different species, including the rat, mouse, guinea pig, and cow (1). Besides the variety of cellular processes in which it has been implicated (2), the second messenger cAMP is thought to be an important growth promoter for mouse, rat, and human mammary epithelia (3-6) and for a variety of other cell types (7). cAMP levels increase in parallel with mammary gland growth during pregnancy (8, 9), and they are elevated in several breast carcinomas (10). Isoproterenol stimulates cell surface 0adrenergic receptors and the adenylate cyclase system in a variety of cell types and has been shown to induce mammary epithelia cell division in vitro (3) and stimulate localized mammary end-bud development in ovariectomized animals (6). Rat mammary gland cAMP levels have been reported

to decrease with the onset of lactation and increase at weaning (8, 9, 11). Various in vitro studies (12-18) using organ cultures of rat, pig, or mouse mammary glands have shown the ability of high doses of cAMP and its derivatives as well as phosphodiesterase inhibitors to inhibit enzymes associated with lipogenesis and depress the synthesis of milk proteins (12-18). In analogy with other paired exocrine glands, a sympathetic innervation regulates the tone of ductal smooth muscles and blood vessels in the rat mammary gland (19, 20). Furthermore, in lactating rats, the sympatheticadrenal system is activated after stimulation of suckling, and catecholamines are released after suckling or electrical stimulation of the mammary nerve in lactating rats (21). Although a growing body of experimental evidence has accumulated in the last 10 yr supporting an important role played by the direct catecholaminergic innervation of various endocrine glands (including the ovaries, testis, and prostate) (22-30), no available information exists on

Received July 5,1989. Address all correspondence and requests for reprints to: Dr. Bianca Marchetti, Ph.D., Department of Pharmacology, Medical School University of Catania, 6 Avenue Doria, 95125 Catania, Italy. 565

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the catecholaminergic control of mammary gland growth, differentiation, and activity. This seems of particular interest in view of the accumulating data underlying the complex and potentially important regulatory networks involving, besides the classical neuroendocrine hormones, a variety of neuropeptides, neurotransmitters, growth factors, and other paracrine regulators in the control of reproductive glands function (30). The aim of the present study was, then, firstly, to characterize and localize the /3-adrenergic receptor population in the mammary gland of virgin, gestating, and lactating rats, and secondly, to determine if a correlation exists between changes in the /3-adrenergic receptoradenylate cyclase system and alterations in cytosolic progesterone receptors within the mammary gland as well as with plasma estrogen, progesterone, and PRL concentrations during pregnancy and lactation.

Materials and Methods Animals Sprague-Dawley [Crl:CD(SD)Br] virgin (nulliparous, at random days of the estrous cycle) as well as pregnant and lactating female rats obtained from Charles River (Canada, Inc., St. Constant, Quebec, Canada) were housed in a temperature (22 ± 2 O - and light (14-h light, 10-h dark cycle; lights on at 05O0 h)-controlled room and received Purina rat chow (RalstonPurina, St. Louis, MO) and water ad libitum. The pregnant rats (8-10/group) were killed on day 10 or 16 of gestation. The day of parturition was designated day 1 of lactation, and animals (bearing at least 6-8 pups) were killed on days 1, 3, 10, and 20 of lactation. Mothers were kept with their pups until death. Membrane preparation Membranes from virgin, gestating, and lactating mammary glands were prepared from right and left cervical, thoracic, abdominal, and inguinal mammary glands. Tissue was homogenized in 10 vol (wt/vol) 0.25 M sucrose and 25 mM Tris-HCl (pH 7.5), using a Polytron PT-10 homogenizer (Brinkmann Instruments, Canada) at a setting of 5 for 3 10-sec periods, with an interval of 10 sec for cooling. The homogenate was then centrifuged at 600 X g for 10 min. The supernatant was carefully collected and centrifuged at 105,000 X g for 60 min in a Beckman L5-65 centrifuge (Beckman, Palo Alto, CA), using a 50-Ti rotor. Pellets were resuspended in assay buffer (25 mM Tris-HCl, pH 7.5; 1:20, wt/vol), and /3-adrenergic receptors were assayed in the membrane preparation as described previously (27, 31). Cytosol preparation Tissue was homogenized in 5 vol (wt/vol) buffer A [25 mM Tris-HCl, 1.5 mM EDTA (disodium salt), 10 mM a-monothioglycerol, 10% glycerol, and 1.5 mM sodium molybdate, pH 7.4] using a Polytron PT-10 homogenizer as described above. The homogenate was then centrifuged at 105,000 X g for 60 min, and the progesterone receptor assays were performed with freshly prepared cytosol as described previously (27, 31). The

Endo • 1990 Vol 126 • No 1

protein concentration was measured according to the method of Lowry et al. (32), using BSA as standard. Progesterone receptor assay [3H]R5020(6,7[3H]17,21-dimethyl-19-nor-pregna-4,9-diene3,20-dione; 87 Ci/mmol) and the corresponding unlabeled steroid were purchased from New England Nuclear (Boston, MA). After evaporation of the solvent under a stream of nitrogen, the labeled steroid was dissolved in buffer A. [3H]R5020 binding was measured using the dextran-coated charcoal adsorption technique; 0.1-ml aliquots of cytosol preparations were incubated with 0.1 ml 16 nM [3H]R5020 (200,000 cpm) and 100 nM triamcinolone acetonide in the presence or absence of a 100fold excess of the unlabeled steroid for 18-22 h at 0-4 C as previously described (33). Unbound steroid was then removed by incubation for 15 min at 0-4 C with 0.3 ml 0.5% Norit-A0.05% Dextran T-70 in buffer B [1.5 mM EDTA (disodium salt), 10 mM monothioglycerol, and 10 mM Tris-HCl, pH 7.4] and centrifugation at 3,000 x g for 15 min. Aliquots of the supernatant (0.3 ml) were col-lected after the addition of 4 ml Formula-963 scintillation liquid (New England Nuclear), and the radioactivity was measured in a Beckman counter at a counting efficiency of 32%. Receptor ^I]CYP

assay

[125I]CYP was purchased from New England Nuclear at a specific activity of 2000 Ci/mmol. In the standard assay, [125] CYP binding was measured by triplicate incubation for 180 min at room temperature of 100 fi\ membrane preparation, 250 125 Ml buffer (25 mM Tris-HCl, pH 7.5), and 100 *d [ I]CYP in the presence or absence of 50 /il of the indicated unlabeled drug. The reaction was stopped by the addition of 0.5 ml bovine 7-globulin (0.1%, vol/vol) in Tris-buffer and 1.0 ml of a solution of 24% (wt/vol) polyethylene glycol (PEG-6000) (27, 31). The assay tube was then mixed vigourously for 10 sec before standing for 5 min and centrifuged during 20 min at 3000 x g. The supernatant was discarded, and the radioactivity in the pellet was counted. Calculation of affinity and number of P^IJCYP-binding sites Binding data were analyzed with a Hewlett-Packard calculator (model 9845, Palo Alto, CA), using a program based on model II of Rodbard and Lewald (34). ED50 values for displacement of [125I]CYP binding by the various drugs were calculated using a weighted iterative nonlinear least square regression (35). Data are expressed in the figures as a percentage of the tracer bound at the zero dose (B/Bo) after subtraction of nonspecific binding measured in the presence of 10"7 M (—)propranolol. Apparent Kx values for displacement of [125I]CYP binding were calculated according to the equation Kd = ED50/ (1 + S/K) (36), where S represents the concentration of [125I] CYP in the assay, Kd is the apparent dissociation constant of [125I]CYP determined by Scatchard analysis (37), and ED50 is the concentration of the drug producing 50% displacement of specific binding. Statistical significance was assessed according to the multiplerange test of Duncan-Kramer (38).

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0-ADRENERGIC RECEPTORS IN RAT MAMMARY GLAND Determination of adenylate cyclase activity Adenylate cyclase activity was measured according to the method of Fortier et al. (39), by enzymatic conversion of [a32 P]ATP into [32P]cAMP in homogenates of rat mammary glands obtained at the indicated physiological stages. The mammary tissue was minced in 20 vol (wt/vol) homogenization buffer CHB (0.001 M EGTA, 0.05 M HEPES, and 10% dimethylsulfoxide, pH 7.6) at 4 C and homogenized as described above. The homogenate was filtered through a nylon mesh (0.16 mm2) and centrifuged at 800 X g for 15 min at 4 C. The supernatant was centrifuged at 40,000 X g for 20 min, and the pellet was washed twice in CHB. The final pellet was resuspended in CHB to obtain a protein concentration ranging from 1.0-1.5 mg/ml and was immediately used for the assay. Enzymatic activity was measured as described for rat myometrial tissue (39) and cells in culture (40) in the presence of Mg2* (20 mMJ, GTP (300 ^M) or its nonhydrolyzable analog guanylylimidodiphosphate (GPP; 300 nM), and the indicated increasing concentrations of isoproterenol (1 nM to 100 ^ M )The maximal activity was calculated from dose-response curves using a weighted iterative nonlinear least square regression (35). The threshold stimulation was estimated using two-way analysis of variance. Autoradiographic localization of f1251] CYP-binding

sites

Frozen sections were cut at a 15-^m thickness and mounted onto gelatin-coated microscope slides by thawing. Sections were dehydrated under vacuum at 4 C for 18 h and, if not immediately used, were stored at —70 C. For receptor localization, all slides were processed in parallel under the same standard conditions (time of exposure, incubation, and development). The slides were brought to room temperature, preincubated for 30 min in O.I M Mg Tris-HCl buffer, pH 7.6, and incubated for 2 h in the same buffer containing 100,000 cpm/ml [125I] CYP in the presence or absence of 10"6 M (—)propranolol as previously described (27). After rinsing, the sections were fixed in 2.5% glutaraldehyde for 10 min, dried at room temperature, and placed against Ultrafilm (LKB, Rockville, MD) or coated in liquid chromatographic emulsion (Kodak NTB2, Eastman Kodak, Rochester, NY). Plasma estrogen, progesterone, and PRL measurements Plasma steroids were extracted with ether and separated on LH-20 columns before measurement by RIA as previously described (41). Plasma PRL was measured by double antibody RIA using rat hormones and rabbit antisera (NIDDK Rat Pituitary Program). Analysis of RIA data was performed using a program based on model II of Rodbard and Lewald (34). Statistical significance was measured according to the multiple range test of Dunkan-Kramer (38).

567

(Durham, NC). Other drugs and reagents were commercially available. Nuclear tract emulsion (type TB-2) was purchased from Eastman Kodak.

Results Kinetics of P25I]CYP binding to lactating mammary glands The /3-adrenergic antagonist [125I]CYP specifically binds to membranes prepared from lactating rat mammary glands obtained from animals at random stages of lactation. In fact, at room temperature half-maximal binding is achieved at 30 min of incubation, while nearmaximal specific binding values are reached between 120-180 min (not shown). At the concentration of the ligand routinely used in the assay (0.06 nM), binding is linear up to 3 mg protein/ml, thus in the standard assay a protein concentration of 0.8-1 mg/ml was used. Equilibrium studies of P2bI]CYP binding As can be seen in Fig. 1A, [125I]CYP binds to a single class of high affinity sites in membranes from rat mammary glands of lactating rats; half-maximal binding is reached at a [125I]CYP concentration of 0.028 nM, while near-maximal binding is obtained at 0.090 nM. Nonspecific binding increases linearly to reach approximately 48% of the total binding at the highest concentration of ligand used (0.165 nM). Scatchard analysis of the data (Fig. 2B) indicates a Kd value of 25.0 ± 0.4 pM, with a value for number of sites of 32.5 ± 1.2 fmol/mg protein in mammary gland tissue obtained from animals at random stages of lactation. Specificity of P25I]CYP binding As shown in Fig. 2 and Table 1, agonists compete for [125I]CYP binding with the following order of potency: zinterol > (—)isoproterenol > (—)epinephrine » (—)norepinephrine » (+) isoproterenol in the rat lactating mammary gland. This relative order of potency is typical of a j82-subtype receptor (36, 37). Specificity of binding was confirmed by the potency of a series of selective fii as well as /?2-adrenergic agonists and antagonists (Table 1). [125I]CYP binding in the rat normal mammary tissue displays a marked stereoselectivity; (—)propranolol is much more potent than its respective pharmacologically less active enantiomer (+)propranolol. Similarly, (-)isoproterenol is more potent than isoproterenol (Table 1).

Materials

fi-Adrenergic receptor levels in the mammary gland of virgin, pregnant, and lactating rats

The following compounds were gifts: metoprolol (Ciba-Geigy, Summit, NJ), zinterol (Mead-Johnson), L- and D-propranolol (Ayerst Research Laboratories, Montreal, Quebec, Canada), and butoxamine (Burroughs-Wellcome, Research Triangle Park, NC). Practolol was kindly provided by Dr. M. G. Caron

After characterization of specific /3-adrenergic receptors in the mammary gland of lactating animals at random stages of lactation, we measured /?-adrenergic receptor concentration and distribution during the physiolog-

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Endo • 1990 Vol 126 • No 1

LACTATING MAMMARY GLAND

B

0.075-1

KD =25pM BMAX = 32.5 fmol / mg protein

1.0-

0.5-

0

.025

.05

.075

.1

.125

.15

o-

0

.01

.02

.03

.04

.05

125

[

FREE (nM)

I] CYP BOUND (pmol / mg protein)

125

FlG. 1. Equilibrium kinetics of [ I]CYP binding to membranes prepared from lactating mammary gland. Membranes were incubated for 180 min at 21-22 C with [125I]CYP in the absence (•; total binding) or presence (O; nonspecific binding) of 0.1 ^M (—)propranolol. Specific binding (•) is indicated as the difference between total and nonspecific binding. Incubation was performed in triplicate. B, Scatchard analysis of the specific binding data. The free ligand concentration is the total radioactivity added minus the nonspecific binding. Results shown are the mean ± SEM of triplicate determinations. TABLE 1. Kd values of various drugs for the inhibition of specific [126I] CYP binding in rat ovarian and mammary gland membrane preparations

LACTATING MAMMARY GLAND ZINTEROL O O ( - ) ISOPROTERENOL A - A ( - ) EPINEPHRINE A - A ( - ) NOREPINEPHRINE ( + ) ISOPROTERENOL

Kd value (nM) Competitor

(nM)

Agonists Zinterol (—)Isoproterenol (—)Epinephrine (—)Norepinephrine (+)Isoproterenol Antagonists (-)Propranolol (+)Propranolol Butoxamine Metoprolol Practolol

10090-

o

80-

h-

70-

z o o LL

o

605040-

o z

30-

z

20-

Q

CD

Ovary

Lactating mammary gland

25 ± 50 ± 420 ± 10,000 ± 25,000 ±

2 (3) 9 (3) 40 (3) 670 (2) 6740 (3)

15 ± 2 (4) 42 ± 11 (4) 128 ± 27 (5) 2,130 ± 290 (4) 18,750 ± 5600 (4)

0.6 ± 60 ± 1,500 ± 2,000 ± 40,000 ±

0.06 (2) 6 (3) 200 (3) 350 (3) 8,600 (3)

2.2 ± 195 ± 1,460 ± 2,130 ± 21,970 ±

0.5 (3) 28 (4) 170 (5) 105 (5) 7,500 (4)

Apparent kd values for displacement of [125I]CYP binding were calculated according to the equation Kd = ED60/(l + S/K) (36), where S represents the concentration of [125I]CYP in the assay, K is the apparent dissociation constant of [125I]CYP determined by Scatchard analysis (37), and ED6o is the concentration of the drug producing 50% displacement of specific binding. Values are the mean ± SE; the number of separate experiments is in parentheses.

100J

-10

-9

-8

-7

-6

-5

-4

AGENT CONCENTRATION (LOG M) FIG. 2. Effects of increasing concentrations of a series of unlabeled /3adrenergic agonists on [125I]CYP binding to membranes prepared from lactating mammary glands. One hundred percent (control) specific binding was 32 fmol/mg protein and represented 65% of the total binding.

ical growth and differentiation of the gland, namely pregnancy and lactation. To gain information on the role of hormonal priming of the mmmary gland during pregnancy and lactation, a group of nulliparous (virgin) rats at random stage of the estrous cycle were included in the

study. As illustrated in Fig. 3 from Scatchard analysis of the data, a value for [125I]CYP-binding sites of 8.2 ± 0.6 fmol/mg protein was measured, with an apparent Kd of 10.0 ± 0.4 pmol in virgin animals. During pregnancy, the number of [125I]CYP-binding sites doubled (with values of 16.2 ± 0.8 and 14.1 ± 0.6 fmol/mg protein being reached at 10 and 16 days gestation, respectively; Fig. 3A). On the first day of lactation (Fig. IB), the 0adrenergic receptor concentration (7.9 ± 0.4 fmol/mg protein) returned to values similar to those measured in virgin animals (Fig. 3B). During midlactation, an 8-fold

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0-ADRENERGIC RECEPTORS IN RAT MAMMARY GLAND

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MAMMARY GLAND

1O O LACTATION (1 day) A - A LACTATION (10 days) LACTATION (20 days)

o-a VIRGIN GESTATION (10 days) GESTATION (16 days) .75-

FIG. 3. Scatchard analysis of [125I]CYP binding to membranes prepared from virgin (A), pregnant (A; days 10 and 16), and lactating (B; days 1,10, and 20) rats. Results shown are the mean ± SEM of triplicate determinations. Data where no error is shown have a SEM smaller than the symbol used.

LLJ LLJ DC

HI ID

.05z> O

O CD

CD

.25-

.005

.01

.015 [

increase in /3-adrenergic receptor concentration (65.9 ± 6.0 fmol/mg protein), was observed. Toward the end of lactation, on the other hand, /3-adrenergic receptor levels decreased to 18.1 ± 0.8 fmol/mg. Autoradiographic localization of P25ljCYP'-binding sites in virgin animals as well as during pregnancy and lactation X-Ray autoradiograms clearly demonstrate a weak reaction of /3-adrenergic receptors in mammary glands from virgin animals (Fig. 4A). During pregnancy, labeling becomes much stronger (Fig. 4B), whereas on the first day of lactation a decrease in labeling intensity was observed (Fig. 4C). Ten days after the beginning of lactation, the autoradiographic reaction was very intense (Fig. 4D). At the light microscopic level, it can be seen in Fig. 5A that the silver grains are associated with secretory cells. When [125I]CYP was incubated in the presence of 10"6 M (—)propanolol, the autoradiographic reaction was almost completely abolished (Fig. 5B). Adenylate cyclase activity Adenylate cyclase activity was at its highest level (P < 0.01) under both basal and stimulated conditions on day 16 of gestation. Although there was no significant effect of lactation on basal or GTP-induced enzymatic activity, the GTP analog GPP caused a higher stimulation of adenylate cyclase activity during lactation than in mammary tissue from virgin animals. In the presence of GTP, isoproterenol-induced adenylate cyclase activity was unchanged on day 16 of pregnancy, but was increased on days 3 and 10 of lactation (Fig. 6 and Table 2). As observed for the number of /?-adrenergic receptors (Fig.

125

.02

0

.02

.04

.08

I] CYP BOUND (pmol / mg protein)

3), maximal isoproterenol-induced enzymatic activity was measured on day 10 of lactation. Mammary gland cytosolic progesterone receptors and plasma estradiol, progesterone, and PRL levels in virgin animals and during pregnancy and lactation As shown in Table 3 and in agreement with previous reports (42, 43), the levels of progesterone receptors in the mammary gland cytosol show a marked increase during pregnancy (from 29.0 ± 4.0 fmol mg protein in virgin animals to 78.0 ± 15.0 fmol/mg protein and 86.0 ± 18.0 fmol/mg protein at 10 and 16 days of pregnancy, respectively). The day of parturition (day 1 of lactation) is characterized by a sharp decline in progesterone receptor concentration (11.0 ± 2.0 fmol/mg protein; P < 0.01). At midlactation, levels are undistinguishable from those in virgin animals, while an increase is again observed toward the end of lactation. In accordance with previously published data in the rat and mouse (42, 43), we found that plasma progesterone levels are high during pregnancy, decline sharply at parturition, increase again during midlactation, and fall at the end of lactation (Table 3). 17,3-Estradiol levels also change considerably in the different stages of pregnancy and lactation. Estrogen levels increase to their maximal values during pregnancy, decline sharply at parturition, and increase again toward the end of lactation. As observed previously (44), plasma PRL levels remain low during pregnancy, but increase sharply on the day of parturition and remain high during lactation (Table 3).

Discussion The present study shows that the rat mammary gland contains specific and high affinity 0-adrenergic receptors

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/3-ADRENERGIC RECEPTORS IN RAT MAMMARY GLAND

Endo • 1990 Voll26«Nol

B

VIRGIN

PREGNANCY (10 days)

LACTATION (1 day )

LACTATION (10 days)

FIG. 4. X-Ray autoradiograms showing distribution of [126I]CYP-binding sites in mammary gland sections from virgin (A), pregnant day 10 (B), lactating day 1 (C), and lactating day 10 (D) rats. Note the marked increase in [125I]CYP binding during pregnancy (B) and midlactation (D).

of the j82 subtype functionally coupled to the adenylate cyclase cAMP system. The radioautographic localization of [125I]CYP binding revealed the presence of receptors in the epithelial cells, alveoles, ducts, and adipocytes. The marked changes in 0-adrenergic receptor concentration and distribution during pregnancy and lactation suggest that this receptor population is highly sensitive to changes in the hormonal milieu, thus providing a mechanism for a direct catecholaminergic influence on mammary growth, differentiation, and activity. The present data extend previous findings (45), de-

scribing a jS-adrenergic receptor population in epithelial cell membranes prepared from lactating mammary glands and acini, using [3H]dihydroalprenolol as radioligand. [125I]CYP binding was found to reach equilibrium between 120-180 min of incubation at room temperature, to be saturable, and to display a typical j82-adrenergic specificity. In fact, the ability of a series of agonists to compete for [125I]CYP binding showed an order of potency characteristic of a /^-subtype adrenergic receptor: zinterol > (—)isproterenol > (—)epinephrine » (—)norepinephrine (46, 47). The binding also displayed a

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/3-ADRENERGIC RECEPTORS IN RAT MAMMARY GLAND

FIG. 5. Light microscopic autoradiographic localization of /3-adrenergic receptors in a mammary gland of a pregnant (10 days) rat. A, Total binding. Silver grains are overlying secretory cells. B, Nonspecific binding. Only a few grains can be observed. Magnification, X800.

marked stereoselectivity; the (—)isomers of agonists and antagonists were much more potent than the respective (+)isomers. The high potency of the /32-selective drugs (the agonist zinterol and the antagonist butoxamine) and the low affinity of /^-selective drugs, such as practolol, in competing for [125I]CYP binding support the /32 nature of the mammary gland adrenergic receptor (48). Such findings are in agreement with /32-receptor specificity in rat cerebellum (49) as well as in rat lung (50), ovarian tissue (24), dispersed Leydig cells (28), intermediate lobe of the pituitary gland (51), mammary tumors induced by DMBA administration (31), and other tissues (52). While the development and differentiation of the mammary gland are under the control of a variety of hormones and growth factors, including PRL, estrogens, progesterone, adrenal steroids, insulin, GH, and thyroid hormones (53, 54), the present results suggest that circulating or locally released catecholamines might partic-

571

ipate in this phenonemon. Depending on its developmental state, the mammary gland undergoes marked changes relative to its epithelial, connective, and adipose tissue composition. Mammary gland growth in preparation for lactation (mammogenesis) is characterized by a proliferation of epithelial elements of the alveoli and formation of lobular alveolar structures (53). At the end of gestation, further changes occur in the whole gland, including additional growth and proliferation, as well as dilatation of the lumina with secretory material for initiation of milk secretion and lactogenesis (53). The autoradiographic localization of mammary /3-adrenergic receptors shows an intense reaction in mammary glands from pregnant animals, when the mammary epithelium is known to become 5-7 times more sensitive to insulin, cortisol, and PRL than similar tissue from virgin animals (55, 56). At the same time, adenylate cyclase activity was also at its highest level, being 3 times more active than that in virgin rats in the presence of GPP. However, the maximal response to isoproterenol was not significantly increased, thus suggesting that the enzyme was not efficiently coupled with jS-adrenergic receptors. A significant increase in /3-adrenergically induced adenylate cyclase activity was, however, observed during lactation. The progesterone receptor concentration as well as plasma estradiol and progesterone levels were markedly elevated during pregnancy (42, 43), thus representing factors potentially responsible for the observed stimulation of /3-adrenergic receptor number. Indeed, in rabbit myometrial cells in culture, we have found the estradiol to progesterone ratio to be critical in regulation of the /?adrenergic response (57). It is of interest that variations in the estradiol to progesterone ratio, as observed during pregnancy, have profound effects on carcinogen-induced mammary tumors. In fact, the hormonal milieu of pregnancy is known to markedly increase the growth of mammary neoplasms induced by DMBA administration (58, 59). In analogy with our observation in the rat mammary gland, in the guinea pig myometrium, /?2-adrenergic receptors have been found to increase during gestation and to decline in the early postpartum period (60). Recently, Legrand and co-workers (61) have shown that high levels of myometrial /?2-adrenergic receptors are maintained throughout gestation, while 6 h before delivery, the number of /?2-adrenergic binding sites coupled to the adenylate cyclase system fell dramatically (—75%). In fact, the present data show, using either membrane binding assays or in vitro autoradiography, a sharp decline in mammary 0-adrenergic receptors on the day of parturition. Such an effect is accompanied by a marked increase in the plasma PRL concentration and a marked decline in plasma estradiol and progesterone levels as well as in the levels of progesterone receptors.

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FIG. 6. Changes in mammary gland adenylate cyclase activity during pregnancy and lactation. Enzymatic activity was measured using Mg2"1" [32P]ATP as a substrate in the absence (basal; A) or presence of GPP (300 fiM; B), GTP (300 MM; C), and GTP plus isoproterenol (10 /xM). The net hormone response to isoproterenol (GTP plus isoproterenol minus GTP alone) is illustrated in D. Results represent the mean ± SEM of three different experiments using four animals per group. Activity on day 16 of gestation is higher (P < 0.01) in A, B, and C. **, Isoproterenol response is higher (P < 0.05) on day 10 of lactation than in all other groups. *, On day 2, response is higher (P < 0.05) than in virgin animals and rats on day 16 of gestation.

50 -

50 -

45 -

45 -

40 -

40 -

35 -

35 -

30 -

30 -

25 -

25 -

20 -

20 -

15 -

15 -

10 -

10 -

5-

50

0 GEST 16

LACT 2

LACT

10

LACT 20

m

LACT 2

LACT

10

LACT 20

LACT 2

LACT

10

LACT 20

LACT 2

LACT

10

LACT 20

35 -

25 20 15 -

5VIRG

GEST 16

PHYSIOLOGICAL STATE

TABLE 2. Parameters of isoproterenol-induced adenylate cyclase activity in mammary glands from virgin, pregnant, and lactating rats

Virgin Pregnant, day 16 Lactating, day 3 Lactating, day 10 Lactating, day 20

GEST 16

30 -

0

Sensitivity

T

40

10 -

Groups

Endo • 1990 Vol 126 • No 1

(nM)°

Apparent Vmai (% over GTP)

100 1 1 1 1

73 ± 1.5 58 ± 1.5 99 ± 2 133 ± 11 105 ± 9

Dose responses were measured in the presence of GTP (300 ^M) and increasing concentrations (from 1 nM to 100 /uM) of isoproterenol. The parameters are derived from three different dose-response curves, using four animals per group. Vmai, Maximal velocity. 0 Sensitivity was determined by threshold stimulation, defined as the smallest agonist concentration that increased activity significantly (P < 0.05) over that in presence of GTP alone.

The density of j8-adrenergic receptors is known to be modulated by steroid hormones in a variety of tissues (62). However, according to the species or tissue examined, the roles of progesterone and estradiol in the regulation of the density of adrenergic receptors seem to be different. On the other hand, it is possible that among the factors contributing to its control, circulating or locally released catecholamines could be involved. In the uterus, there is a large increase in the release of norepinephrine from sympathetic nerve terminals associated with a decline in the synthesis of the amine at parturition. The local accumulation of norepinephrine near the postsynaptic membrane has been suggested as a possible down-regulatory mechanism responsible for the decrease in the number of /3-adrenergic receptors observed at this

VIRG

GEST 16

PHYSIOLOGICAL STATE

time (60). Indeed, agonist-dependent desensitization of /3-adrenergic receptors is a well recognized phenomenon (62). Concerning the mammary gland, to our knowledge there are no available studies describing changes in catecholamine content within the mammary glands during pregnancy and lactation. Nevertheless, it appears that catecholamines are released into the circulation in response to the suckling stimulus, and it has been suggested that the tone of the sympathetic nervous system may be influenced or even regulated by mammary stimulation as a consequence of suckling or milking (21). It is then possible that the variations in catecholamine levels in the circulation or locally released catecholamines at the mammary gland level might in part be responsible for the observed changes in the number of /3-adrenergic binding sites. As an example, as suckling continues, catecholamine release has been shown to be reduced (21), thus providing a possible mechanism for an increase in /?-adrenoceptors observed during mid- and late lactation. Moreover, 0-adrenergic receptors could be modulated by changes in steroid hormones, PRL, and other undetermined factors, acting locally at the mammary gland level. In summary, the present data show parallel changes in the autoradiographic localization and concentration of /3-adrenergic receptors functionally coupled with adenylate cyclase in the rat mammary gland during pregnancy and lactation. In view of the positive correlation observed between /3-adrenergic and progesterone receptor concentrations as well as plasma estrogens and mammary

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/3-ADRENERGIC RECEPTORS IN RAT MAMMARY GLAND

573

TABLE 3. Specific binding of [3H]R5020 to mammary gland cytosol as well as plasma progesterone, 17/8-estradiol, and PRL levels in virgin female rats and during pregnancy and lactation [3H]R5020 binding (fmol/mg protein)

Group Virgin Pregnancy, 10 days Pregnancy, 16 days Lactation, 1 day Lactation, 10 days Lactation, 20 days

29.0 ± 78.0 ± 86.0 ± 11.0 ± 31.0 ± 45.0 ±

4.0 15.0° 18.0" 2.0° 6.0 11.0*

17/3-Estradiol (pg/ml)

Progesterone (ng/ml)

PRL (ng/ml)

19.8 ± 2.5 54.6 ± 8.2° 57.2 ± 5.5° 18.7 ± 2.0 29.7 ± 3.8 34.3 ± 4.5°

12.4 ± 0.9 57.6 ± 5.9° 95.0 ± 6.5° 19.1 ± 2.5 86.4 ± 11.2° 19.6 ± 1.0

4.6 ± 2.2 ± 3.6 ± 130 ± 180 ± 198 ±

0.6 0.8 1.2 25° 28° 32°

Progesterone receptor assays were performed with freshly prepared cytosols from virgin, pregnant, and lactating mammary glands. Values represent the mean ± SEM (8-10 animals/group). 0 P < 0.01 compared to virgin animals. 6 P < 0.05 compared to virgin animals.

growth during pregnancy, it is tempting to speculate that circulating or locally released catecholamines may directly participate in the process of growth and differentiation of the mammary gland.

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574

0-ADRENERGIC RECEPTORS IN RAT MAMMARY GLAND

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Endo • 1990 Vol 126 • No 1

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