Molecularand Cellular Endocrinology, 12 (1978) 167-176 0 Elsevier/North-Holland Scientific Publishers, Ltd.

INFLUENCE OF BROMOERGOCRYPTINE PROLACTIN

167

ON ESTROGEN-MODULATED

RECEPTORS OF MOUSE MAMMARY

GLAND

Nandini A. SHETH ‘, Shubhada S. TIKEKAR ‘, Kamal J. RANADIVE ’ and Anil R. SHETH 2 1 Biology Division, Cancer Research Institute, Tata Memorial Centre, and 2 Institute for Research in Reproduction

Received

12 June

Modulation

(ICMR), Parel, Bombay 400012,

1978; accepted

of specific

binding

14 July

India

1978

of prolactin

to murine

mammary

gland as a

response to treat-

ment with various doses of estradiol benzoate (EB) was studied. Measurements of serum and pituitary levels of prolactin were also carried out simultaneously. Estradiol benzoate at a dose rat prolactin (rPRL) to mammary of 5 pg significantly increased the binding of 1251-labelled gland concomitant with increased serum prolactin levels in ovariectomized mice. Administration of bromoergocryptine along with EB resulted in decreased serum prolactin levels as well as binding of prolactin to breast tissue. It thus appears that the influence of estradiol on binding of prolactin to mammary gland is mediated primarily via its property of enhancing serum prolactin concentration apart from its possible direct effect at the target tissue level. Keywords:

prolactin

receptors;

estrogen

effect;

mammary

gland; bromoergocryptine.

Hormonal control of development and growth of mammary gland is well recog nized. Prolactin and estrogens are two key hormones having a significant action on growth of normal (Cowie, 1974; Denamur, 1971) and neoplastic (Welsch and Nagasawa, 1977) mammary gland. However, interaction of these hormones modulating the response of each other is not yet very well understood. In the present report we describe the dose-related effects of estrogen on prolactin receptors on mammary glands of ovariectomized mice. Whether this action of estrogen is directly on the mammary gland or mediated through increased release of pituitary prolactin was studied using bromoergocryptine, a specific inhibitor of prolactin secretion.

MATERIALS

AND METHODS

Materials Chemical.

Highly purified rPRL, a gift from NIAMDD, National Institute of Health, Bethesda, U.S.A., was used for binding studies. Radioimmunoassay kit for

168

N.A. Sheth et al.

mPRL, a gift from Dr. Y.N. Sinha, Scripps Lab., LaJolla, Calif., U.S.A., was used for estimation of the levels of serum and pituitary prolactin. Carrier-free 12’1 was obtained from the Radiochemical Centre, Amersham, U.K. Bromoergocryptine (CB-154) was a gift from Sandoz Laboratories, Sweden. Biological.

Virgin female mice of C3H(Jax) strain obtained from the Animal Colony of our Institute were ovariectomized at the age of 4-5 months and divided into 8 groups. Each group consisted of 7-10 mice. 7 days after ovariectomy the mice were given daily S.C. injections for 8 days as follows:

Group Group Group Group Group Group Group Group

1: 2: 3: 4: 5: 6: 7: 8:

0.1 ml sesame oil (control) 5 pg EB in 0.1 ml sesame oil 10 pg EB in 0.1 ml sesame oil 25 /*g EB in 0.1 ml sesame oil 0.1 ml sesame oil t 200 pg bromoergocryptine 5 ,ug EB in 0.1 ml sesame oil + 200 pg bromoergocryptine 10 pugEB in 0.1 ml sesame oil t 200 1.18bromoergocryptine normal intact mice at diestrus.

Methods

Mice were killed by decapitation 16 h after the last injection. Blood was collected from the decapitated trunk and allowed to clot at 4°C. Serum was separated by centrifugation and stored at -20°C until radioimmunoassayed for circulating mPRL. Pituitary from each mouse was immediately excised and weighed on a Sartorius balance, and stored in cold 0.01 M phosphate buffer (pH 7.0) containing 0.14 M sodium chloride (PBS) at -20°C until radioimmunoassayed for mPRL. Breast tissue of individual mice was dissected out, minced finely in cold condition, homogenized in a Potter Elvehjem homogenizer in cold PBS and made up to 15% tissue concentration with cold PBS. The whole homogenate was used as such after removal of fat by centrifugation in cold at 4’C. Protein was estimated in whole homogenate by Lowry’s method (1951) and adjusted so as to have 1 mg protein per incubation tube for all binding experiments throughout the present study. Iodination.

Iodination of rPRL and mPRL as described in our earlier report (Sheth et al., 1974) was carried out essentially according to the method of Greenwood et al. (1963) as modified by Midgley (1966). The specific activity of labelled hormone ranged from 80 to 100 Ci/g. To discover the extent of hormone damaged during iodination, radioiodinated hormone was precipitated by the specific antibody to,.it as supplied by NIAMDD, Bethesda, U.S.A., and by Dr. Y.N. Sinha, La Jolla, Calif., U.S.A.

Radioimmunoassay.

All assays were carried out by the double antibody technique as described by Midgley (1966). Purified mPRL was used as reference standard and for iodination. Specific antiserum to mPRL was used as first antibody.

Estrogen effect on prolactin receptors of mammary gland

169

Binding procedure. The following method of binding was employed in our assay system unless otherwise specified. Aliquots of tissue homogenate representing 1 mg of protein were incubated with [lz5 I]rPRL in a final volume of 0.5 ml made up with PBS at 37°C for 60 min in a Dubnoff metabolic shaker (80 strokes/min). At the end of the incubation period the contents of each tube were diluted to 3.5 ml with PBS and centrifuged at 800g for 30 min at 4°C. The pellets were washed twice with 3 ml of PBS. The tubes were drained off and the amount of radioactive hormone bound to the pellet was determined by gamma-ray spectrometry. Tubes containing only PBS-gel (0.1% gel in PBS) in place of homogenate served as controls for determining nonspecific adherence of radioactive material to the glass. All determinations were carried out at least in duplicates. The results are expressed as percentage of total added counts which were bound to the pellet. Parallel incubations were carried out in the presence of an excess of unlabelled oPRL (2 pg/ml or 1 pg/ tube). Radioactivity bound in the presence of excess of unlabelled oPRL was subtracted from total binding in order to get the displaceable binding which was considered as specific binding.

RESULTS Results of a number of experiments carried out under different conditions revealed that in the presence of an excess of cold ovine prolactin 70-80% of prolactin bound to receptors was displaceable (specific), whereas 20-30% could not be displaced (nonspecific). Therefore specific and nonspecific binding were determined in all experimental groups and results are expressed as specific binding. All the results on specific binding were expressed per mg of protein. As shown in table 1, the specific binding of prolactin to mammary gland and serum prolactin level remained comparable in normal and ovariectomized (15 days after ovariectomy) mice, whereas pituitary of ovariectomized mice was depleted of its prolactin content.

Table 1 to mammary gland Specific binding of [ *2SI]rPRL and pituitary of normal and ovariectomized mice

and concentrations

per mg of protein

a) Denotes statistical significance groups when P < 0.001.

between

0.952 91.6 2.17 pituitary

in serum

Castrated

Normal % specific binding Serum (ng/ml) Pituitary (pg/mg)

of prolactin

* 0.1 f 4.4 f 0.16

PRL concentration

0.970 94.0 0.806 of normal

c 0.2 A 6.7 * 0.07 a) and castrated

N.A. Sheth et al,

10

5

0.0 DOSES

OF

ESTRADIOL

25

BENZOATE

(Bg)

Fig. 1, Effect of graded doses of estradiol on the specific binding of l2 SI-labelIed rat prolactin (rPRL) to mammary gland (1 mg protein) of ovariectomized mice. *, ** Denotes statistical significance when P < 0.001 between ovariectomized control and 5 ng dose and P < 0.002 between 5 ng and 10 ng doses respectively.

2 i= 0 a g a P b. 0

01 c

1oc )-

-L 0

DOSES

OF

5

ESTRADIOL

Fig. 2. Effect of &radio1 on serum prolactin significance between ovariectomized control

IO BENZOATE

25 (.rug

)

levels in ovariectomized mice. * Denotes statistical and estradiol-treated groups when P < 0.01.

Estrogen effect on prolactin receptors of mammary gland

171

Fig. 1 shows that percentage specific binding of [“‘I]rPRL to mammary gland was highest in mice treated with 5 pg EB. The binding was increased to 267% when control value was considered as 100%. At higher doses of 10 or 25 pg EB, specific binding of prolactin decreased as compared to that in 5 pg EB and remained comparable to that in ovariectomized control mice. Since EB is known to have its direct effect on prolactin release and synthesis, the concentrations of prolactin were measured in serum as well as pituitary. As shown in fig. 2, serum prolactin concentration was highest (247 ng/ml) in mice treated with 5 ,ug EB, whereas it decreased to some extent in 10 and 25 1.18EB injected mice. However, at all the dose levels EB treatment resulted in an increase in serum concentration over that in ovariectomized control mice. Fig. 3 shows that doses of 5, 10 or 25 pg EB evoked a progressive increase in pituitary prolactin concentration. The increase in PRL concentration was 4-12fold over ovariectomized control. The increase in pituitary prolactin could be due either to suppressed release of prolactin and/or to enhanced synthesis. However, it may be noted that pituitary as well as serum prolactin concentrations were increased in mice treated with 5 pg EB, thereby implying that EB treatment at a 5 pg dose promotes both synthesis and release of pituitary prolactin to a great extent, while injections of 10 and 25 pg EB resulted primarily in increased synthesis and to a small extent in release.

*

OOSES

OF

ESTRAOIOL

Fig. 3. Effect of estradiol on * Denotes statistical significance when P < 0.001.

BENZOATE

pituitary between

(,4(P)

prolactin concentration ovariectomized control

in ovariectomized and estradiol-treated

mice. groups

N.A. Sheth et al.

,-

t

co

164(ryo~

200

-

-

200

-

200

Fig. 4. Effect of bromoergocryptine (CB-154) on specific binding of ’ 251-labelled rat prolactin (rPRL) to mammary gland of control and estradiol-treated ovariectomized mice. Each tube contained homogenate equivalent to 1 mg protein. * Denotes statistical significance when P < 0.05.

Z K w

a

200

I )c

ESTl?AOIOL(A~) 0 CB-154

Fig. 5. Effect ovariectomized

* I“L ‘I

(Co)

-

0

5

I

200

-

200

5

10 10

-

200

of bromoergocryptine (CB-154) on serum prolactin levels in estradiol-treated mice. * Denotes statistical significance when P < 0.01.

Estrogen effect on prolactin receptors of mammary gland

f

t

173

*

L

10 10 CO-l94(F9

1

0

200

0

200

0

200

Fig. 6. Effect of bromoergocryptine (CB-154) on pituitary prolactin concentration treated ovariectomized mice. * Denotes statistical significance when P < 0.002.

in estradiol-

The increase in specific [ “‘I]rPRL binding was found to be correlated to serum prolactin levels. This indicates that the effect of EB on prolactin receptor could perhaps be through its action on pituitary prolactin release. Hence, the effect of EB on binding in the presence of lowered serum prolactin was studied using bromoergocryptine, a potent inhibitor of prolactin release. Fig. 4 shows that treatment with bromoergocryptine brought down the specific binding of [12’I]rPRL to breast tissue in ovariectomized control as well as in EBtreated mice, demonstrating that EB in the presence of lowered levels of prolactin did not increase the binding capacity of mammary gland for [12’I]rPRL. Fig. 5 shows that treatment with bromoergocryptine brought down the serum prolactin levels in all the groups. In addition to being a potent inhibitor of prolactin release in ovariectomized control mice, bromoergocryptine showed the capacity to counteract the effect of EB on prolactin release. The pituitary prolactin concentration in bromoergocryptine-treated mice remained unaltered as compared to the respective control group (fig. 6). Even if the release of prolactin in serum (fig. 5) was inhibited, the concentration of pituitary prolactin was not increased. It thus seems that synthesis of pituitary prolactin is also inhibited by bromoergocryptine.

174

N.A. Sheth et al.

DISCUSSION Studies on prolactin receptors under controlled hormonal conditions have been mainly performed on liver (Posner et al., 1974, 1975; Gelato et al., 1975; Costlow et al., 1975), rabbit mammary gland (Djiane and Durand, 1977) and on 7,12dimethylbenzanthracene-induced mammary tumors (Bradley et al., 1975; Kledzik et al., 1976; Costlow et al., 1976a, b). We extended these studies on murine mammary gland and showed that EB treatment at a lower dose level (5 pg) increased specific binding of [ ‘*‘I]rPRL to mammary gland of ovariectomized mice, along with an increase in serum prolactin concentration. EB is known to have its direct effect on breast tissue and indirect effect through its action on pituitary prolactin release. Hence, the effect of EB in the presence of lowered levels of prolactin was studied by simultaneous treatment of EB and bromoergocryptine to ovariectomized mice. Bromoergocryptine brought down the circulating levels of prolactin concurrent with reduced specific binding, indicating that EB in the presence of lowered levels of prolactin does not increase binding. It thus appears that an increase in serum prolactin raises its own specific binding to the target site and vice versa. Exogenous estrone or estradiol has been shown to increase specific binding of prolactin to liver tissue of ovariectomized or normal rats (Posner et al., 1974; Gelato et al., 1975). Induction of prolactin receptors by prolactin itself in liver tissue of hypophysectomized rats is demonstrated by pituitary implants in kidney capsule (Posner et al., 1975) or by exogenous treatment of prolactin (Costlow et al., 1975). EB treatment at higher doses (10 and 25 pg*g)decreased the specific binding of [12’I]rPRL and serum prolactin concentrations in a dose-related manner as compared to the maximum rise caused in both these entities by EB at the lower dose of 5 pg. However, as compared to ovariectomized control group, EB treatment at higher doses did induce a slight but significant rise in serum prolactin but did not accordingly enhance the binding. Perhaps a very high threshold level of serum prolactin is required to induce increased prolactin binding. Alternatively, EB at higher doses interferes with the action of prolactin at the target site, which is reflected in receptor levels. An increase in prolactin binding to mammary gland of pseudopregnant rabbit by exogenous oPRL (100 IU) has been shown by Djiane and Durand (1977). However, they have also shown that a lower dose of prolactin (12.5 IU) was ineffective during short-term treatment. Our results are similar to the observation of Kledzik et al. (1976) that large doses of estrogen interfere with prolactin binding to DMBA-induced mammary tumors. However, an increase in binding by a low dose of estrogen as shown in the present study was not evident in their studies. Perhaps the response of a mammary tumor is different from that of normal breast tissue. Bradley et al. (1975) have also reported that in vitro treatment of estrogen decreases prolactin binding to slices of normal and neoplastic mammary tissue. Reduction in prolactin binding to rabbit mammary gland by progesterone and to responsive mammary tumors by androgens has been reported (Costlow et al., 1976a; Djiane and Durand, 1977).

Estrogen effect on prolactin receptors of mammary gland

175

Gelato et al. (1975) have shown that ovariectomy causes a significant decrease in prolactin binding to liver tissue. It should be noted that these studies were carried out 31 days after ovariectomy and probably binding decreases by that period after ovariectomy. The serum levels of prolactin were, however, not studied by these workers. In contrast, in the present experiments, ovariectomy (15 days) did not reduce prolactin binding to breast tissue. It is interesting to note that serum prolactin levels did not decrease during this period. Perhaps liver responds earlier and to a greater extent than mammary gland. Our studies emphasize the significance of .simultaneous estimation of serum and pituitary prolactin along with prolactin binding studies. Our results indicate that EB at all the dose levels used increases the pituitary as well as serum prolactin concentrations above that observed in ovariectomized controls. Stepwise increases in pituitary prolactin levels in response to increasing doses of estrogen have been reported previously by several workers (Grosvenor and Turner, 1960; Folley and Malpress, 1948; Chen and Meites, 1970). According to earlier workers (Folley and Malpress, 1948) large doses of estrogen have been shown to inhibit serum prolactin, whereas our findings are in agreement with those reported by Chen and Meites (1970). It seems that the level of prolactin receptors on the target tissue is very sensitive to hormonal environments. There is also evidence that nonhormonal factors such as theophylline (Sheth et al., 1977) and polyamines (Sheth et al., 1976) could also influence the prolactin binding to target tissue. Studies on modulation of receptor levels would provide important information as to how the peripheral sensitivity to hormones is controlled.

ACKNOWLEDGEMENTS We are highly grateful to NIAMDD, Bethesda, U.S.A., for the gift of rPRL and oPRL; to Dr. Y.N. Sinha, Scripps Clinic and Research Foundation, La Jolla, Calif., U.S.A., for the RIA kit of mPRL; and to Dr. H. Weidmann and Dr. H. Fridli, Sandoz Ltd., Hanover, for bromoergocryptine.

REFERENCES Bradley, C.J., Campbell, G.A., Marshall, S., Meites, .I. and Coolings, W. (1975) Fed. Proc. 34, 343. Chen, C.L. and Meites, J. (1970) Endocrinology 86,503-505. Costlow, M.E., Buschow, R.A. and McCuire, W.L. (1975) Life Sci. 17,1457-1465. Costlow, M.E., Buschow, R.A. and McGuire, W.L. (1976a) Cancer Res. 36,3324-3329. Costlow, M.E., Buschow, R.A. and McGuire, W.L. (1976b) Cancer Res. 36, 3941-3943. Cowie, A.T. (1974) In: Mammary Cancer and Neuroendocrine Therapy, Ed.: B.A. Stoll (Butterworth, London) ch. 1, pp. 3-24.

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Denamur, R.J. (1971) J. Dairy Res. 38,237-264. Djiane, J. and Durand, P. (1977) Nature (London) 266,641-643. Folley, S.J. and Malprcss, F.H. (1948) In: The Hormones, Eds.: G. Pincus and K.V. Thiman (Academic Press, New York and London) ch. 16, pp. 745-805. Gelato, M., Marshall, S., Boudreau, M., Bruni, J., Campbell, G.A. and Meites, J. (1975) Endocrinology 96, 1292-1296. Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1963) Biochem. J. 89, 1144123. Grosvenor, C.E. and Turner, C.W. (1960) Endocrinology 66, 96-99. Kledzik, G.S., Bradley, C.J., Marshall, S., Campbell, G.A. and Meites, J. (1976) Cancer Res. 36, 3265-3268. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265275. Midgley Jr., A.R. (1966) Endocrinology 79, 10-18. Posner, B.I., Kelly, P.A. and Friesen,H.G. (1974) Proc. Natl. Acad. Sci. U.S.A. 71,2407-2410. Posner, B.I., Kelly, P.A. and Friesen, H.G. (1975) Science 188,57-59. Sheth, N.A., Ranadive, K.J. and Sheth, A.R. (1974) Eur. J. Cancer 10,653-660. Sheth, N.A., Tikekar, S.S., Ranadive, K.J. and Sheth, A.R. (1976) (IRCS) Med. Sci. 4,323. Sheth, N.A., Tikekar, S.S., Sheth, A.R. and Ranadive, K.J. (1977) Endokrinologie 70, 169175. Welsch,C.W. and Nagasawa, H. (1977) Cancer Res. 37,951-963.