0013-7227/90/1261-0575$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society

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

Hormonal Regulation of /?-Adrenergic Receptors in the Rat Mammary Gland during the Estrous Cycle and Lactation: Role of Sex Steroids and Prolactin BIANCA MARCHETTI AND FERNAND LABRIE Medical Research Council Group in Molecular Endocrinology, Research Centre, Laval University Medical Centre, Quebec, Gl V 4G2 Canada; and the Department of Pharmacology, Medical School, University of Catania, 95125 Catania, Italy

ABSTRACT. To gain further knowledge on the role of ovarian hormones in the regulation of mammary /9-adrenergic receptors, virgin animals were killed during the various phases of the estrous cycle as well as after ovariectomy and treatment with sex steroids. /3-Adrenergic receptor levels fluctuate in the rat mammary gland during the estrous cycle, with higher receptor numbers during the proestrous and estrous phases of the cycle. Ovariectomy caused an almost 50% loss of/3-adrenergic receptor concentration in the mammary gland of virgin rats. Treatment of ovariectomized animals with 170-estradiol or progesterone alone or in combination for 3 weeks induced a marked increase in /3-adrenergic receptor concentration, while administration of the androgen dihydrotestosterone did not modify mammary fjadrenergic binding sites. While levels of /3-adrenergic receptors in control lactating animals (10 days of lactation) were elevated, chronic treatment with the dopaminergic-mimetic agent 2a-bromoergocryptine

T

HE MAMMARY gland is the target of complex and multiple hormonal influences that impinge on cell growth and differentiation. The normal development, proliferation, and differentiation of the mammary gland during adolescence, the menstrual cycle, pregnancy, and lactation depend upon a prescribed and well programed sequence of interactions between steroids, thyroid and lactogenic hormones, as well as a variety of growth factors (1). Among this wide range of substances, steroid hormones are well known to modulate tissue sensitivity in a variety of systems, including the mammary gland (1-3), and to alter the adrenergic response, possibly through changes in the number of adrenergic receptors (4-6). Estrogens stimulate a number of myometrial receptors, including a-adrenergic receptors (7-12), while /3-adrenergic receptors have been reported to remain unchanged (13), increase (10, 14), or decrease (15) after steroid administration.

(CB-154; for 7 days) reduced /3-adrenergic receptor concentration. Castration of lactating animals decreased /3-adrenergic receptor number to approximately 30% of the value in intact controls, while combined withdrawal of circulating ovarian hormones and inhibition of plasma PRL levels caused an almost complete inhibition of /3-adrenergic receptor concentration. Scatchard analysis of the binding data revealed that the observed alterations in /3-adrenergic receptors resulted from changes in the number of /3-adrenergic binding sites, with no change in binding affinities. The present findings indicate that the /3adrenergic receptor population of the rat mammary gland is under the control of ovarian hormones and PRL and suggest that circulating or locally released catecholamines could interact with sex steroids and PRL in the regulation of mammary gland growth, differentiation, and activity. {Endocrinology 126: 575581, 1990)

The marked stimulation of the /?-adrenergic receptoradenylate cyclase system in the rat mammary gland observed during pregnancy and lactation prompted us to examine 1) the physiological regulation of mammary /3adrenergic receptors during the estrous cycle, 2) the effect of sex steroid hormone withdrawal and replacement, and 3) the possible modulatory role exerted by PRL during lactation.

Materials and Methods Animals Sprague-Dawley [Crl:CD(SD)Br] virgin as well as lactating female rats purchased from Charles River (Canada, Inc., St. Constant, Quebec, Canada) were housed in a temperature (22 ± 2 C)- and light (10-h dark cycles; lights on at 0500 h)controlled room and received Purina rat chow (Ralston-Purina, St. Louis, MO) and water ad libitum. The stage of the estrous cycle was evaluated by daily vaginal smears, and only animals showing at least two consecutive 4-day cycles were used. Groups (5-6 animals) of estrous, diestrous day 1, diestrous day 2, and proestrous rats were killed between 0900-1000 h. Lactating rats

Received July 5, 1989. Address all correspondence and requests for reprints to: Dr. Bianca Marchetti, Department of Pharmacology, University of Catania Medical School, 6 Avenue Doria, 95125 Catania, Italy. 575

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(8-10/group), were killed on day 10. The day of parturition was designated day 1 of lactation. Treatments Virgin female rats were ovariectomized under light ether anesthesia and received for 3 weeks twice daily sc injections of the following steroids: 17/3-estradiol (E2; 1.125 ^g), progesterone (P; 2 mg), and dihydrotestosterone (DHT; 250 /xg), alone or in combination, in 0.2 ml 1% gelatin-0.9% NaCl. The compounds were first dissolved in a small volume of ethanol before further dilution with 1% gelatin-0.9% NaCl. Groups of lactating animals (2-3 days of lactation) underwent ovariectomy or daily treatment with the dopaminergic agonist 2a-bromoergocryptine (CB-154; 500 ng), or both, and were killed after 1 week of treatment. At the end of treatment period, the animals were killed by decapitation, the blood was collected into heparinized tubes and centrifuged, and the plasma was stored at —20 C for hormone measurements. The right and left cervical, thoracic, abdominal, and inguinal mammary glands were quickly removed, cleaned of fat and connective tissue, frozen on dry ice, and stored at -80 C for RRAs. Membrane preparation Mammary glands were individually 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 three periods of 10 sec each, 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 using a 50-Ti rotor. Pellets were resuspended in assay buffer (25 mM Tris-HCl, pH 7.5; 1:20, wt/ vol), and #-adrenergic receptors were measured in the membrane preparation as described previously (16). Receptor-r^Ucyanopindolol (^IJCYP) assay [125I]CYP was purchased from New England Nuclear (Boston, MA) at a specific activity of 2000 Ci/mmol. Nonspecific binding was estimated from the amount of [125I]CYP bound in the presence of 0.1 nM propranolol. Saturation analysis experiments were carried out with 10-12 concentrations of [125I]CYP (1-200 pM). Competition studies were performed with a range of 8-12 concentrations of selected adrenergic agents in the presence of approximately 30 pM [125I]CYP. A total volume of 500 n\ was incubated to equilibrium for 180 min. To terminate the incubation, 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-60O0) were added to each tube (16), shaken vigourously during 10 sec before standing for 5 min, and centrifuged for 20 min at 3000 X g. The supernatant was discarded, and the radioactivity in the pellet was counted. Protein concentration was measured according to the method of Lowry et al. (17) using BSA as a standard. Calculation of affinity and number of P2t'I]'CYP'-binding sites

Binding data were analyzed with a Hewlett-Packard calculator (model 9845, Palo Alto, CA), using a program based on

Endo • 1990 Vol 126 • No 1

model II of Rodbard (18). ED50 values for displacement of [125I] CYP binding by the various drugs were calculated using a weighed iterative nonlinear least square regression (19). Apparent Ki values for displacement of [125I]CYP binding were calculated according to the equation Kd = ED5o/(l + S/K) (20), where S represents the concentration of [125I]CYP in the assay, K is the apparent dissociation constant of [125I]CYP determined by Scatchard analysis (21), and ED50 is the concentration of the drug causing 50% displacement of specific binding. Statistical significance was assessed according to the multiple range test of Duncan-Kramer (22). Materials The following compounds were gifts: metoprolol (Ciba-Geigy, Summit, NJ), zinterol (Mead-Johnson), L- and D-propranolol (Ayerst Reasearch Laboratories, Montreal, Quebec, Canada), butoxamine (Burroughs-Wellcome, Research Triangle Park, NC), and 2a-bromoergocryptine (Sandoz, Basel, Switzerland). Practolol was kindly provided Dr. M. G. Caron (Durham, NC). The different steroids were obtained from Steraloids (Wilton, NH).

Results Mammary ^-adrenergic receptor levels during the rat estrous cycle 0-Adrenergic receptors show marked fluctuations during the various phases of the rat estrous cycle. Initial binding studies, performed with a saturating dose (0.1 nM) of [125I]CYP, indicated that the high affinity specific [125I]CYP binding was highest during estrous (10.5 ± 0.7 fmol/mg protein) and proestrous (11.8 ± 0.9 fmol/mg protein) and declined by approximately 50% during the diestrous phases of the cycle (5.8 ± 0.5 and 6.2 ± 0.6 fmol/mg protein on diestrous days I and II, respectively). Then, saturation analysis experiments were carried out to determine whether the observed changes reflected alterations in receptor numbers or binding affinities. When 10-12 concentrations of [125I]CYP (1-200 pM) were added to mammary gland membranes prepared from estrous, diestrous day I, diestrous day II, and proestrous animals, Scatchard analysis of the data revealed that the observed modifications in the /3-adrenergic receptor concentration were the result of changes in the number of binding sites, rather than alterations in binding affinities. In fact, significantly (P < 0.01) higher binding capacities were measured during estrus (12.8 ± 1.0 fmol/mg protein) and proestrus (13.5 ± 1.2 fmol/mg protein) than on diestrous days I (7.6 ± 0.7 fmol/mg protein) and II (6.9 ± 0.4 fmol/mg protein), with no significant changes in Kd values, ranging between 14.518.3 pM (Table 1). To discriminate whether the physiological fluctuations in plasma sex steroids during the estrous cycle could change the predominant j82-adrenergic receptor popula-

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HORMONAL MODULATION OF MAMMARY 0-ADRENERGIC RECEPTORS TABLE 1. Characteristics of 0-adrenergic receptors in the mammary gland during the rat estrous cycle /3-Adrenergic receptors Groups

B mai (fmol/mg protein)

(pmol)

12.8 ± 1.0 7.6 ± 0.7" 6.9 ± 0.4° 13.5 ± 1.2

14.5 ± 0.7 16.5 ± 0.5 18.3 ± 1.0 18.5 ± 0.9

Estrous Diestrous day I Diestrous day II Proestrous

Kd

Mammary gland membranes were prepared from estrous, diestrous day I, diestrous day II, and proestrous virgin female rats and incubated with increasing concentrations (1-200 pM) of [126I]CYP in the absence or the presence of 0.1 fiM (—)propranolol. Experimental groups consisted of five or six animals. Results represent the mean ± SE of two or three separate experiments. Bmax, Binding capacity. " P < 0.01 compared to estrous and proestrous groups.

tion characterized in lactating and pregnant animals, competiton studies using various subtype-selective adrenergic compounds were performed. As observed in Table 2, our data confirm the predominant representation of the j82-adrenergic receptor subpopulation, with no qualitative variations during the various physiological states of the gland. In fact, at each stage of the ovarian cycle, the most potent competitor was zinterol, followed by (—)isoproterenol, (—)epinephrine, and (—)norepinephrine. The prevalent ^-nature of the adrenergic receptor was further confirmed by the finding that the compounds (zinterol and butoxamine) selective for the j82 adrenergic-receptor were remarkably more potent in displacing [125I]CYP binding to mammary glands than agents more selective for /3X-adrenergic receptors (such as practolol) at each stage of the ovarian cycle (Table 2). Effect of ovariectomy and treatment with sex steroids on [125IJCYP binding to mammary gland membrane preparations of virgin animals As observed in Fig. 1, intact female rats at random stages of the estrous cycle have a concentration of [125I]

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CYP-binding sites of 12.0 ± 2.9 fmol/mg protein. Three weeks after ovariectomy, [125I]CYP-binding sites decreased by approximately 50% (6.8 ± 1.2 fmol/mg protein; P < 0.01), while treatment for 3 weeks of ovariectomized rats with E2 causes a marked increase in [1251] CYP binding to 49.5 ± 12.1 fmol/mg protein (P < 0.01). Treatment with P also caused an increase (P < 0.01) in /3-adrenergic binding sites to 36.8 ± 9.4 fmol/mg protein. The combination of the two steroids led to a further increase in /3-adrenergic receptor levels (65 ± 15 fmol/ mg protein; P < 0.01); on the other hand, administration of the androgen DHT did not modify [125I]CYP binding in ovariectomized animals (Fig. 1). In analogy to what we observed during the estrous cycle, when performing saturation analysis in membranes prepared from the different experimental groups, the data revealed that the observed changes in /3-adrenergic receptor levels were the result of modifications in the binding capacity after either ovariectomy (4.8 ± 0.7 fmol/mg protein) or replacement of OVX animals with E2 (60.7 ± 7.8 fmol/mg protein), P (48.5 ± 8.4 fmol/mg protein), or E2 + P (74.5 ± 12.6 fmol/mg protein), with no significant changes in Kd values, ranging between 1620 pM (Table 3). Effects of ovariectomy and treatment with CB-154 alone or in combination on P^IJCYP binding to mammary glands of lactating rats Inhibition of the circulating plasma PRL concentration in lactating (2-3 days of lactation) animals was achieved by daily administration of CB-154. As observed in Fig. 2, 0-adrenergic receptors are at a high level (55.0 ± 4.9 fmol/mg protein) after 10 days of lactation. Daily treatment with CB-154 caused a marked fall in both [125I]CYP binding to 22.0 ± 1.9 fmol/mg protein (P < 0.01) and the plasma PRL concentration, from control

TABLE 2. Kd values of various drugs for the inhibition of specific [125I]CYP binding in rat mammary gland prepared from intact virgin estrous, diestrous day I, diestrous day II, and proestrous rats Kd (nM)

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

Estrous

Diestrous day I

Diestrous day II

Proestrous

14 ± 2 39 ± 8 170 ± 20 3,480 ± 510

17 ± 2 45 ± 11 240 ± 25 4,550 ± 490

15 ± 1 54 ± 9 150 ± 30 2,980 ± 300

12 ± 2 50 ±12 230 ± 20 3,740 ± 400

3.2 ± 0.4 1,560 ± 270 4,700 ± 680 33,300 ± 8,000

4.2 ± 0.5 2,540 ± 300 3,870 ± 290 28,000 ± 9,000

4.5 ± 0.5 1,2000 ± 190 3,000 ± 450 25,000 ± 5,000

2.9 ± 1,700 ± 3,900 ± 36,000 ±

0.6 340 560 9,000

ED60 values for displacement of [125I]CYP binding by the various drugs were calculated using a weighed iterative nonlinear least square regression (19). Apparent Kd values were calculated according to the equation Kd = ED50/(l + S/K) (20). Values are the mean ± SE of two or three separate experiments.

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HORMONAL MODULATION OF MAMMARY /3-ADRENERGIC RECEPTORS

578

75-

Endo • 1990 Voll26«Nol

LACTATING MAMMARY GLAND

D INTACT [ill OVX [III E2 gPROG 62 E2 + PROG K3DHT

60 HI

T **

E E £ S>

CONTROL (lOd LACTATION) CB-154 (500 »g b.i.d.) OVX OVX+CB-154

6 cr 50-



o

I

25-

a z o CD

0

FlG. 1. Effects of ovariectomy (OVX) and treatment of OVX animals for 3 weeks with E2, P (PROG), and DHT, alone or in combination, on [126I]CYP binding to mammary gland membrane preparations. Membrane preparations were incubated with a saturating concentration (0.1 nM) of [125I]CYP in the presence or absence of 0.1 /*M propranolol for 180 min at room temperature. Values represent the mean ± SEM (8-10 animals/group). **, P < 0.01 compared to OVX animals. TABLE 3. Characteristics of mammary /3-adrenergic receptors after ovariectomy (OVX) and replacement with different hormones (8-Adrenergic receptors Groups Intact OVX OVX + E2 OVX + P OVX + E2 + P

Bmai (fmol/mg protein) 12.9 ± 4.8 ± 60.7 ± 48.4 ± 74.5 ±

1.5° 0.7 7.8° 8.4° 12.6°

Kd (pmol) 16.2 ± 0.6 17.3 ± 0.7 20.5 ± 1.3 20.0 ± 0.8 20.5 ± 0.9

Saturation analysis experiments were carried out with 10-12 concentrations (1-200 pM) of [125I]CYP added to mammary gland membranes prepared from the different experimental groups in the absence or the presence of 0.1 pM (—)propranolol. Groups consisted of five or six animals. Results are the mean of two or three separate experiments. 0 P < 0.01 compared to OVX.

levels of 195 ± 37 to 5.8 ± 0.8 ng/ml (data not shown). While castration alone caused a nearly 30% decrease in /3-adrenergic receptor levels (38.0 ± 3. fmol/mg protein; P < 0.01), the combination of ovariectomy and CB-154 led to an approximately 70% decrease in [125I]CYP binding to 12.7 ± 1.7 fmol/mg protein (P < 0.01). Figure 3 shows the Scatchard analysis of the data, indicating a marked decrease in /3-adrenergic receptors from a control level of 69.9 ± 1.80 fmol/mg protein to 33.2 ± 0.60 and 48.8 ± 0.70 fmol/mg protein after daily treatment with CB-154 or ovariectomy alone, respectively. A more pronounced decrease in [125I]CYP binding was observed after the combined withdrawal of ovarian hormones and plasma PRL concentration to 22.8 ± 0.6

40

20-

i

FIG. 2. Effects of ovariectomy (OVX) and/or treatment of intact animals with the dopaminergic agonist CB-154 on [125I]CYP binding to membranes prepared from lactating mammary glands. Values represent the mean ± SEM (8-10 animals/group). **, P < 0.01 compared to OVX animals.

fmol/mg protein. On the other hand, the Kd values calculated from the slopes of these lines were similar in intact (21.1 ± 0.4 pM), CB-154-treated (19.2 ± 0.5 pM) and ovariectomized and CB-154-treated (22.3 ± 0.4 pM)treated animals, while a slightly lower value (14.4 ± 0.4 pM) was measured in ovariectomized animals.

Discussion The present data show that the /3-adrenergic receptor in rat mammary gland exhibits distinctive quantitative variations during different stages of the estrous cycle, accompanied by a predominant representation of the j82receptor subtype. Furthermore, estrogens and P as well as PRL exert a potent modulatory effect on the mammary /3-adrenergic receptor concentration. While further studies are required to determine whether such alterations in receptor binding are accompanied by functional changes in coupled activation, the parallel stimulation of adenylate cyclase activity and /3adrenoceptors during pregnancy and midlactation (22a), would suggest that dramatic alterations of plasma sex steroids and PRL concentrations are, indeed, correlated to the changes in the /3-adrenergic receptor-adenylate cyclase system within the rat mammary gland. Thus, it is tempting to speculate that circulating and/or locally released catecholamines could interact with sex steroids and PRL to regulate the sensitivity of the mammary gland to hormones and growth factors known to physio-

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HORMONAL MODULATION OF MAMMARY /3-ADRENERGIC RECEPTORS LACTATING MAMMARY GLAND Z - Z CONTROL (10d LACTATION) • - • CB-154 (500 ug b.i.d.) C—C OVX OVX+CB-154

BOUND (pmol / mg protein)

FIG. 3. Scatchard analysis of [125I]CYP binding to membranes prepared from mammary glands of lactating (10 days of lactation) animals treated with CB-154, ovariectomized (OVX), or ovariectomized and treated with CB-154. Specific binding is indicated as the difference between total and nonspecific binding in the presence of 0.1 /*M (—)propranolol. Results are the mean ± SEM of three experiments. Data where no error is shown have a SEM smaller than the symbol used.

logically influence mammary gland function. It is of interest to mention that in cycling rats the myometrial content of /?2-adrenergic receptors is elevated duringproestrus and estrus (14). Furthermore, castration reduces the density of uterine /32-adrenergic receptors, while treatment of castrated rats with estrogens prevents the inhibitory effect of castration (14). The acute administration of P in ovariectomized rats increases myometrial /?2-adrenergic receptor concentration (10). The hormonal milieu of pregnancy has also been shown to increase the levels of j82-adrenergic receptors within the myometrium (23, 24), and P has been shown to play an important role in the myometrial /32-adrenergic receptor increase (25). The present data show a similar high sensitivity of the mammary 02-adrenergic receptor population to physiological changes in circulating sex steroid levels. The /?adrenergic receptor concentration is, in fact, increased during the phases of maximal activity of both estrogens and P, namely the proestrous and estrous phases of the cycle, while significantly lower levels are measured during the follicular (diestrous days I and II) phases. The role of sex steroid hormones in the regulation of these receptors is further substantiated in ovariectomized rats,

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in which castration results in a reduction in mammary /3-adrenergic receptor content, while treatment of ovariectomized animals with estradiol and P caused a marked stimulation of /3-adrenergic receptor numbers. Such alterations in receptor concentration during the estrous cycle as well as after castration and hormone replacement were the result of changes in the number of j8adrenergic binding sites, with no significant modifications in binding affinities. While Perkins and co-workers (26) have recently reported that specific ovarian /3-adrenergic receptor subtypes as well as content vary significantly during the follicular and luteal phases of the swine estrous cycle, in the rat mammary gland of virgin, pregnant, and lactating animals (22a), the observed changes in receptor concentration were accompanied by a predominant representation of the /?2-adrenergic receptor subpopulation, suggesting that the marked alterations in circulating sex steroid hormones accompanying such physiological conditions do not affect the differential expression of 0adrenergic receptor subtypes. In dimethylbenz(a)nthracene-induced rat mammary tumors, the recently characterized /32-adrenergic receptor subpopulation (27, 28) is also highly sensitive to the hormonal milieu, since ovariectomy reduced by 50% the number of [125I]CYP-binding sites, and replacement with estrogens, alone or in combination with P, causes a marked stimulation in the number of adrenergic binding sites, with no changes in binding affinity (27, 28). While the present study confirms the important modulatory role exerted by sex steroid hormones in the dynamic regulation of /5-adrenergic receptors (5), the biochemical mechanism(s) of these regulatory effects is unknown at present, and further studies are required to determine whether the steroid hormones may be affecting the activity of the genes for the /9-adrenergic receptors in the rat mammary gland. The present results indicate the importance of the sex steroid hormonal milieu during lactation in the modulation of mammary /3-adrenergic receptor concentration. In fact, altough the plasma estrogen concentration is low during midlactation, plasma P levels are markedly elevated (22a), representing, therefore, a potential factor involved in the marked increased in ^-adrenergic receptor levels in the mammary gland of the lactating rat. The mammary gland is also recognized as a primary target for PRL, and PRL receptors fluctuate throught pregnancy and lactation in the rabbit mammary gland (29). The present data obtained in lactating animals after daily treatment with the potent dopaminergic agonist CB-154 support an important role of circulating plasma PRL levels in the regulation of /3-adrenergic receptor concentration within the mammary gland. We have recently observed (28) that endogenous hy-

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perprolactinemia induced in dimethylbenz(a)nthracenetreated animals sharply increases both /32-adrenergic and P receptor concentrations within the tumors as well as tumor growth (28). In fact, besides regulating normal mammary gland growth, PRL has a well recognized role in the development and progression of mammary neoplasms in mice and rats (30). In this context, it seems worthwhile to mention that the hyperprolactinemia accompanying the aging process can be tumorigenic, and that the spontaneous mammary tumors of aging (18- to 22-month-old) Sprague-Dawley rats contain a 15- to 20fold higher concentration of specific /32-adrenergic receptors compared to the values measured in the mammary gland of virgin rats (27). Although little is known about the mechanism(s) by which PRL can modulate adrenergic receptor number, the anterior pituitary hormone has been shown to control the level of a number of peptide hormone receptors, including LH/hCG (31), LHRH (32), and catecholaminergic (33) receptors. Since estrogens are well known to stimulate PRL secretion, it is likely that the hyperprolactinemia resulting from the daily treatment of ovariectomized virgin rats with a high dose (2.5 /ug/day) of E2 is at least in part responsible for the important increase in the /3-adrenergic receptor concentration observed after steroid treatnent (32). It also seems likely that changes in circulating levels of PRL can synergize with the plasma E2 and/or P concentrations to modulate the number of /3-adrenergic binding sites. In the rat ovary the increase in follicular norepinephrine content (34) has been suggested as a potential factor in determining the proestrous fall in ovarian 0-adrenergic receptor content (35). During development, the changes in distribution of /32-adrenergic receptors in the different compartments of the prepubertal rat ovary (16) and their marked sensitivity to changes in noradrenergic ovarian tone have led to the speculation that a neural efferent system might be involved in the adjustement of ovarian responsiveness to stimulation by the gonadotropins via changes in adrenergic receptor content and distribution (16). Similarly, the mammary gland is innervated by the sympathetic nervous system, and changes in the sympathetic-adrenal tone have been described during lactation (36). While the physiological significance of the mammary ^-adrenergic receptor is presently unknown, it seems tempting to speculate that changes in central and/or peripheral catecholaminergic activity accompanying various physiological situations (such as puberty, the estrous cycle, pregnancy, and lactation) might influence mammary gland function via changes in /3-adrenergic receptor concentration. Nevertheless, it is known that /?-adrenergic receptors can be influenced by hormones other than catechol-

Endo • 1990 Voll26«Nol

amines, e.g. thyroid hormones, glucocorticoids, sex steroids, and other factors (5). For example, there is evidence that gonadotropins can induce increases in the number of 0-adrenergic binding sites in rat granulosa cells (37). Moreover, Aguado and Ojeda (35) have clearly demonstrated that /3-adrenergic receptors in granulosa cells may be under heterologous regulation by LH, PRL, and corticosterone, although only FSH and corticosterone are capable of facilitating the P response to the /?adrenergic receptor agonist. In rat ovarian thecal-interstitial cells, norepinephrine amplifies hCG-stimulated androgen production (38). Furthermore, an interaction between catecholestrogens, a major group of estrogen metabolites, catecholamines, and gonadotropins at the ovarian level has been proposed (39, 40) as an important regulatory mechanism during the follicular development. Of interest, catecholestrogens together with their catabolizing enzyme, catechol-Omethyltranseferase, are locally formed in both normal and neoplastic mammary tissues (41-44), thus providing a potential paracrine/autocrine regulatory mechanism. In summary, a complex and potentially important local regulatory network involving catecholamines, gonadal steroids, and PRL participates in the dynamic control of mammary gland ^-adrenergic receptor function, thus providing an anatomical substrate for a direct interaction of catecholamines with other hormonal factors in the regulation of mammary gland development, growth, and activity.

References 1. Topper YJ, Freeman CS 1980 Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049 2. Banerjee MR 1976 Responses of mammary cells to hormones. Int Rev Cytol 47:1 3. Bolander FF 1984 Enhanced endocrine sensitivity of mouse mammary glands: hormonal requirements for induction and maintenance. Endocrinology 115:630 4. Hoffman BB, Lavin TN, Lefkowitz RJ, Ruffolo RR 1981 Alphaadrenergic receptor subtypes in rabbit uterus: mediation of myometrial contractions and regulation by estrogens. J Pharmacol Exp Ther 219:290 5. Stiles GL, Caron MG, Lefkowitz RJ 1984 /J-Adrenergic receptors: biochemical mechanisms of physiological regulation. Physiol Rev 61:661 6. Elliot JM, Peters JR, Grahame-Smith DC 1980 Oestrogen and progesterone change the binding characteristics of j8-adrenergic and serotonin receptors on rabbit platelets. Eur J Pharmacol 66:22 7. Ichida S, Tokunaga H, Oda Y, Fujita A, Hata T 1983 increase of serotonin receptors in rat uterus induced by estradiol. J Biol Chem 58:1343 8. Schirar A, Capponi A, Catt KJ 1980 Regulation of uterine angiotensin II receptors by estrogen and progesterone. Endocrinology 106:5 9. Nissenson R, Flouret G, Hechter O 1978 Opposing effects of estradiol and progesterone on oxytocin receptors in rabbit uterus. Proc Natl Acad Sci USA 75:2044 10. Kano T 1982 Effects of estrogen and progesterone on adrenoceptors and cyclic nucleotides in rat uterus. Jpn J Pharmacol 32:535 11. Williams LT, Lefkowitz RJ 1977 Regulation of rabbit myometrial

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Hormonal regulation of beta-adrenergic receptors in the rat mammary gland during the estrous cycle and lactation: role of sex steroids and prolactin.

To gain further knowledge on the role of ovarian hormones in the regulation of mammary beta-adrenergic receptors, virgin animals were killed during th...
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