Effects of medroxyprogesterone acetate on serum prolactin levels and liver prolactin binding capacity in the rat

Pharmacological Research Communications, Vol. 20, No. 8, 1988 719

EFFECTS OF MEDROXYPROGESTERONE ACETATE ON SERUM PROLACTIN LEVELS AND LIVER

PROLACTIN BINDING CAPACITY IN THE RAT

Giampiero Muccioli, Silvia Racca, Silvana Ricci Gamaloro

and Francesco Di Carlo

Institute of Pharmacology, Faculty of Medicine, University of Turin,

Corso Raffaello 30, 10125 Turin, Italy

Received in fina/ form 2 May 1988

SUMMARY

Modifications in liver prolactin (PRL) receptor levels and serum PRL

concentration induced by administration of medroxyprogesterone acetate

(MPA) were investigated in rats of both sexes. MPA induced a reduction both

of the levels of PRL in the serum and of liver FRL receptors in the female

rat. The reduction of the number of PRL receptors caused by MPA was rapid

and almost complete after I0 days of treatment and appeared earlier than

that of serum PRL levels. Furthermore the MPA-induced decrease in PRL

receptors was specific, since insulin binding to the same liver membranes

was not affected. MPA given simultaneously with oestradiol (which increases

both the number of liver FRL receptors and the serum PRL levels in the male

rats) was able to counteract the increase in PRL binding induced by

oestradiol. On the contrary, the oestrogen-induced increase in serum PRL

was not affected by MPA treatment. Similar results were obtained using

tamoxifen, a well known antioestroEenic druE. In conclusion~ our results

show that the reduction of PRL receptor idvels induced by MPA in rat liver

is specific, not correlated to serum FRL concentration, and sep~s to depend

on the antioestrogenic activity of the drug.

Ke> words: medroxyprogesterone, serum prolact,in, prolactin receptors

INTRODUCTION.

In a previous research we have demonstrated that a long-term

administration of high doses of medroxyprogesterone acetate (MPA) in fe-~le

0031-6989/88/080719-121503.0010 © 1988 The Italian Pharmacological Society

720 Pharmacologma/Resea~h Communmation~ ~,~2~ No. & 1988

rats was able to reduce the number of oestrogen receptors in target tissues

and to change the structure of organs (ovaries, adrenals and pituitary

gland), which are directly or indirectly a source of oestrogens (Di Carlo

et al., 1984). Furthermore, we hav~ observed that MPA decreased prolactin

(PRL) plasma levels and the number of PRL receptors in the rat liver and

ovarian membranes (Di Carlo et al., 1984). Since PRL receptor levels in

different target tissues are regulated by circulating levels of PRL (Posner

et al., 1975; Di Carlo and Muccioli, 1981; Barkey et al., 1985; Barash et

al., 1986) and/or oestrogens (Posner et al., 1974; Kelly et al., 1975;

Waters et al., 1978; Amit et al., 1983; Muccioli et al., 1985), we have now

investigated wh~ther the reduction of PRL receptor levels induced by MPA in

the liver is related to its hypoprolactinaemic or to its antioestrogenlc

activity.

MATERIALS AND METHODS

Drugs and Hormones

Medroxyprogesterone acetate (Depo-Provera) was obtained from Upjohn

Italiana S.p.A., Milan (Italy). Tamoxifen was provided by ICI-Pharma, Milan

(Italy) and suspended in 0.5Z carboxymethylcellulose. Oestradiol (Sigma

Chemicals, USA) was dissolved in a small volume of ethanol and diluted in

corn oil. Controls were treated with an equivalent volume of the vehicle

that was used to administer oestradiol. In a preliminary study we observed

that this vehicle did not influence plasma PRL levels and liver PRL binding

sites.

Highly purified ovine PRL (oPRL) for radioiodination (NIDDK-oPRL-I-2;

Pharmaco/ogicalResea~hCommunication~Vo~2~ No.~ 1988

35 I.U./mg) and oPRL for competition studies (NIDDK-oPRL 16; 30.5 I.U./mg)

were kindly supplied by the Pituitary Hormone Distribution Program of

NIDDK. Porcine insulin (pINS: 24 I.U./mg) was purchased from Sigma Chem.,USA.

721

Animals and treatments

Male and female (in proestrus) Sprague-Dawley rats, 130-150 g, were

housed at constant temperature (200C) under fixed 12 h light and dark

cycles with free access to food and water. Animals were treated with MPA,

oestradiol or tamoxifen as reported in the results. All rats were killed by

decapitation 24 h after the last treatment and their livers were removed

and stored at -200C for less than two weeks prior to membrane

preparation. Trunk blood samples were also collected, centrifuged and the

serum frozen at -20=C until analyzed for PRL. Female control rats were

killed at proestrus in order to avoid changes in basal plasma PRL levels

and liver PRL binding capacity.

Membrane preparation

Livers were homogenized in 0.3 M sucrose (I0 vol) at 4°C, using a

Polytron PT-10 homogenizer. Microsomal membranes, obtained as previously

described (Muccioli et al., 1984), were used for hormone binding studies.

The protein content was determined by the method of Lowry et al. (1951).

Hormone iodination

Ovine PRL was iodinated ( 12~-oPRL; spec. act. 47-56 uCi/~g) using the

method of Bolton and Hunter (1973) as previously reported (Di Carlo and

722 Pharmaco~g~alResearch Commun~ation~VoL 2~ No.~ 1888

Muccioli, 1981) in which radioactive iodine is introduced by reaction of

free aminogroups of the protein with N-succinimidyl 3(4-hydroxy-,5-12SI

iodophenyl) propionate (Amersham International). 125I-porcine insulin (125I

pINS; spec.act. 99 ~Ci/gg) and rat PRL (125I-rPRL; spec. act. 42 ~Ci/ug)

were purchased from New England Nuclear, Boston, ,MA, USA.

Hormone bindin~ studies

Prolactin and insulin binding were determined as described in our

previous studies (Muccioli et al., 1984; 1985). Each membrane preparation

(0.2 mg-protei n) was incubated in triplicate with approximately 70,000 cpm

of 1251-oPRL or 50,000 cpm of 1251-pINS in a final volume of 0.5 ml assay

buffer (25 mM Tris-HCl, i0 mM MgC~, 0.1% bovine serum albumin, pH 7.4).

Similar triplicate incubations for each membrane sample containing 1 ~g

unlabelled oPRL or pINS were used for determination of non specific

binding. After 16 h of incubation at 20°C for PRL binding or 48 h at 4°C

125 for pINS binding, bound and free 1-hormone were separated by low-speed

centrifugation. The supernatant was decanted and the radioactivity bound to

membranes was counted in a Packard auto-gamma counter. Specific binding was

calculated as the difference between binding in the absence or presence of'

1 ~g/tube unlabelled hormones. Specific binding was expressed as percentage

of the total counts added to each tube for 0.2 mg membrane protein.

Scatchard analysis (Scatchard, 1949) of the PRL binding was also

performed by transformation of binding data from the competition studies

with increasing concentrations of unlabelled hormone (0-I00 ng/tube) mixed

with a fixed amount of tracer.

Pharmacolog~alResearch Commun~ation~ VoL,2~ No.& 1988

Prolactin assay

Serum PRL levels were determined by a double antibody RIA as

previously described (Di Carlo et al., 1984) using materials kindly

supplied by the NIDDK Pituitary Hormone Distribution Program. The

intraassay variations were less than 5% while the interassay variations

were around 9%.

Statistical analysis

All results are expressed as group arithmetic means and standard

deviation of the means. Statistical analysis was carried out by one-way

analysis of variance. Means were compared using the Newman-Keul's multiple-

range test.

RESULTS

Table i shows the effects of MPA administration, as a function of the

days of treatments, on the specific binding of PRL and insulin to liver

membranes and on serum PRL concentrations of female rats. A significant

inhibition of PRL specific binding appeared already after the first MPA

administration. This effect further increased after 3 and I0 days of

treatment and then tended to plateau. In contrast, the binding of insulin

to the same membrane preparation showed no significant difference between

control and MPA-treated animals.

The same treatments caused also a reduction in serum PRL levels.

However, the effects of MPA on this parameter appeared later than that on

PRL receptors, being significant only after 10 days of treatment.

723

--L

724 Pharmacological Resear~ch Communications, Vol. 20, No. 8, 1988

TABLE i. Effect of medroxyprogesterone acetate (MPA) on specific binding of

125I-oPRL and 125I-insulin to liver membranes (% total radioactivity per

0.2 mg protein) and serum prolactin concentration (ng/ml) of female rats.

The animals were treated with MPA (15 mg/kg/day i.m.) for various days as

indicated. All rats (4 per group) were killed 24 h after the last

treatment. Values are means ± S.D. Per cent changes from control values are

shown in parentheses.

Days of 1251-Hormone bound

treatment Prolactin Insulin

Serum prolactin

0

1

3

I0

30

I0.0 + 0.7 6.1 + 0.7 17.9 + 2.3

4.6 + 0.8 (-54z)~'~', 5.7 Z 0.5 16.0 + 4.9 (-llZ)

2.2 +_ 0.7 (-78%)':'* 5.8 + 0.Z 13.6 _+ 8.8 (-24%)

1.9 +_ 0.6 (-81%) ~'~ 5,9 +_ 0.7 5.6 _+ 1.3 (-69%) ~'~

1.5 + 0.6 (-857.) ~'~* 6.0 + 0.7 3.2 ± 0.5 (-82%) *='~

*p<0.05; **p<O.Ol vs controls

To determine whether the reduction in PRL binding was the result of a

decrease in receptor number or a change in affinity of the receptor ligand

125 complex, the binding of I-oPRL to liver membranes from controls and

animals treated with MPA for i or 30 days was analyzed in the presence of

increasing concentrations of unlabelled oPRL (Fig. i). In the liver

membranes from control animals a proportional displacement with increasing

amounts of the unlabelled hormone was observed. Such displacement was less

evident with the membranes taken from animals treated with MPA for I or 30

days. The inset to Fig. I shows the Scatchard analysis of the data

calculated from the corresponding displacement curves. No evident

difference in dissociation constant (Kd) was observed in the livers between

Pharmacolog/ca/ Research Commun/cat/ons, Vo/. 20,/Vo, 8, 1988 725

A

(3 Z

0 m ..J n" D. 0

I

U3 OI

8 B/F

0"45 7

\ ' ! ' I I "--I

30 60 90 120

oPRL b o u n d (frnol/'rng)

10 ° 10 ~ 10 2

U N L A B E L L E D o P R L ( n g / m l )

I 10 3

Fi~.l- Specific binding of 1251-oPRL to membranes from female rats

untreated (o) or treated with MPA (15 mg/kg/day i.m.) for 1 (@) or 30 days

( • ), expressed as a function of unlabelled oPRL concentrations. Binding

was determined at a membrane protein concentration of 0.2 mg/tube and

expressed as a percentage of the total 1251-oPRL added. The corresponding

Scatchard plot is shown in the inset.

controls and rats treated with MPA for i or 30 days (Kd were: 3.2, 3.8 and

3.7xI0"i~, respectively), whereas the number of binding sites varied,

showing an apparent decrease in the animals treated with MPA.

To assess whether the effects of MPA on PRL receptor levels were

mediated by its antioestrogenic activity, we have used male rats since in

the animals of this sex the liver PRL binding is very low and may be

726 Pharmacological Research Communications, Vol. 20, No. 8, 1988

TABLE 2. Effect of medroxyprogesterone acetate (MPA) and tamoxifen (TAM) on

prolactin specific binding (% of total radioactivity per 0.2 mg protein)

and on serum prolactin concentration (ng/ml) of male rats treated with

vehicle (controls) or with oestradiol (E21 MPA and TAM were given alone or

simultaneously with E 2 for 10 days. All rats were killed 24 h after the

last treatment.

Values are means ± S.D, of 5 animals.

Group Treatment Liver Prolactin Bound Serum Prolactin

A Controls 0.84 + 0.15 16.4 + 3.2

B MPA (15 mg/kg/day

i.m. for i0 days)

C TAM (i mg/kg/day

os for i0 days)

D E 2 (0.25 mg/kg/day

s.c. for I0 days)

E MPA : as in group B

+E 2 : as in group D

F TAM : as in group C

+E 2 : as in group D

0.89 + 0.27 9.4 + 1.4"

0.82 + 0.39 19.6 + 7.5 m

9 .53 + 2 . 0 0 * * 8 7 . 2 +11"*

2L59 + 0.44 °* 90.0 +14"*

3 .40 + 1.~00~ 94 .6 +17"* m

*p<O.05; **p<O.Ol vs controls

°p<O.Ol vs E2-treated animals

easily increased by oestradiol administration (Posner et al., 1974).

Indeed, Table 2 shows that in rats given oestradiol for 10 days the mean

serum PRL concentration increased more than 5 times. In the same animals a

125 remarkable increase (more than i0 times) in the specific binding of l-

oPRL was also observed. When MPA was given simultaneously with oestradiol,

the increase in PRL binding was almost completely inhibited. A similar

result was obtained by using tamoxifen, a well known antioestrogenic drug

(Katzenellenbogen and Ferguson, 1975). On the contrary, the oestrogen-

Pharmaco~gica/Resea~h Commun~ation~VoL 2~ No.& 1988

induced increases in serum PRL were not affected by MPA (or tamoxlfen)

treatment.

727

DISCUSSION

The results of the present investigation confirm that the

administration of MPA induces a reduction both of levels of PRL in the

serum and of liver PRL receptor in the female rat. The reduction of the PRL

receptor concentration caused by MPA is rapid and almost complete after 10

days of treatment and appears earlier than that of plasma PRL levels.

Furthermore, the MPA-induced decrease in PRL receptors is specific, since

insulin binding to the same membrane preparation is not affected. Our data

suggest that the down-regulation of PRL receptors induced by MPA is not

related to prolactinaemia. In other words, MPA seems to be able to act

independently on liver PRL receptors ~nd on pituitary gland. This last

effect may be due to a decrease in the rate of PRL synthesis rather than to

a decrease in hormone release by the pituitary cells. Indeed, we have

previously observed a marked reduction of PRL-producing cells in the

pituitary gland of MPA-long term treated female rats (Di Carlo et al.,

1984). Various possibilities exist with regard to the level at which MPA

may act when affecting the hepatic PRL receptor concentration. For

instance, the secretion of the pituitary PRL receptor-inducing factor,

whose release is influenced by steroid hormones (Norstedt et al., 1980;

Norstedt and Mode, 1982; Gustafsson et al., 1980; 1983), might be affected

by MPA administration at the level of the hypothalamo-pituitary axis. It is

also possible that MPA could reduce the secretion of growth hormone (GH),

728

which, on the contrary, is able to induce an increase in the number

liver PRL receptors (Baxter et al., 1984; Norstedt et al., 1984).

Alternativel} since oestrogens exert an important role in the control of

PRL receptors [n rat liver (for review see Waters et al., 1978; Gustafsson

et al., 1983; Di Carlo and Muccioli, 1988), MPA could decrease PRL

receptor levels via its manifold antioestrogenic activity (Di Carlo et al.,

1984). This hypothesis is also supported by the fact that MPA, like

tamoxifen, is able to prevent the increase in the number of hepatic PRL

Pharmacological Research Communications, Vol. 20, No. 8, 1988

of

receptors induced by oestradiol in male rats. However, a direct action of

MPA on liver PRL receptors cansot be excluded. In contrast, it is unlikely

that the effects of MPA on PRL receptor levels can depend on the androgenic

activity of this drug, which appears only at doses much higher

(approximately 150 mg/kg) than that used in the present study (Mowszowicz

et al., 1974; Bullock et al., 1978).

In conclusion our results show that the reduction of PRL receptor

levels induced by MPA in rat liver is specific, not correlated to serum PRL

concentration, and seems to depend on the antioestrogenic activity of the

drug.

ACKNOWLEDGMENTS

We thank the NIDDK Pituitary Hormone Distribution Program and Dr. A.F.

Parlow for providing oPRL and rat PRL kit, as well es Dr. C. Ghe' for his

skilful technical assistance. We also gratefully acknowledge Mrs. A.

B i a v a t l f o r typing the m a n u s c r i p t .

AMIT T., (1983).

Pharmaco~gicalRese~ch Communwation~,Vo~ 2~ No. ~ 1988

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