Life Sciences, Vol. 26, pp. 1343-1347 Pergamon Press Printed in the U.S.A.

THE ROLE OF DOMPERIDONE IN THE REGULATION OF PROLACTIN RELEASE IN RATS

Hiroshi Kato, Toshio Fujino, ShoJiro Aramaki, Masahiro Koresawa, Saburo Yamashita and Tadashi Torigoe

Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, 1144 Kogushi, Ube, 755 Japan

(Received in final form February 20, 1980)

Summary

Effects of domperidone, a dopamine antagonist, on prolactin release in female rats were studied. Oral administration of domperidone for 14 days caused a significant increase in serum prolactin levels in mature female rats. The routes by which domperidone exerted its effects on prolactin release were studied by a in vitro incubation system using rat pituitary tissues. Pituitary halves were incubat- ed with (i) domperidone, (2) dopamine, (3) dopamine plus domperi- done, (4) hypothalamic extracts from rats which had been treated with control meal (control hypothalamic extract), (5) control hypo- thalamic extract plus domperidone, and with (6) hypothalamic extract from rats which had been treated with domperidone for 14 days (domperidone-treated hypothalamic extract). Pituitary halves, when incubated alone, released a significant amount of prolactin into the incubation medium after 24 hours incubation, which was com- pletely inhibited by dopamine or control hypothalamic extract. The addition of domperidone could not reverse the inhibitory effect of dopamine or control hypothalamic extract. On the other hand, domperidone-treated hypothalamic extract showed no inhibitory effects on prolactin release. These results indicated that domperidone could increase serum prolactin levels in female rats by acting primarily at the hypothalamus.

Prolactin release from the pituitary is regulated mainly by a prolactin inhibiting factor (PIF) from the hypophysiotrophic area (I, 2, 3). Although little information is available at present concerning the nature of the physiological PIF, it is, at least, certain that dopamine and dopamine agonists show potent PIF activities° Recently, an anti-emetic drug, domperidone was found to be a potent dopamine antagonist (4), and to overcome apomorphine- suppressed prolactin release from cultured pituitary cells of rats (5). There are also some indirect evidences that this drug stimulates prolactin secretion in rats. Namely, daily administration of domperidone (70 mg/day for 14 days, p.o.) into female rats caused a marked development in the mammary gland and the milk secretion (Hara et al., unpublished findings). The purposes of the present studies are to further confirm the effect of domperidone on prolactin release in rats and to investigate where this drug acts to stimulate prolactin release.

Methods

Animals: Mature female Sprague-Dawely rats were housed in a temperature

O024-3205/80/161343-Q5502,0Q/Q Copyright (c) 1980 Pergamon Press Ltd

1344 Domperidone and Prolactin Secretion Vol. 26, No. 16, 1980

and light-controlled room (lights on 06:00 - 20:00 h) with food and water sup- plied ad libitum. Two groups of l0 rats each were given 70 mg/day of domperi- done (p.o.) or control meal every day for 14 days. The mean of body weights was adjusted to 288 g in each group, ranging 271 - 307 g in the drug-treated and 273 - 308 g in the control groups. In the morning (between 09:00 - ii:00 h) of 15th day (15 days after the beginning of the drug administration), all rats were killed by decapitation. Hypothalami and pituitaries were removed and stored at -20 °C. Blood samples were allowed to clot and centrifuged at 4 °C. Serum samples were stored at -20 °C until assay.

Hypothalamic extract: The hypothalamus and the pituitary of each rat were placed into a glass homogenizer and homogenized in 1.5 ml of Eagle's MEM. The homogenate was centrifuged at 1,800 x g for 30 minutes at 4 °C and the supernatant was removed. The hypothalamic extracts of domperidone treated rats and of control rats were subsequently referred to as "domperidone-treated hypothalamic extract" and "control hypothalamic extract", respectively.

Pituitary incubation: Mature female donor rats, weighing 214 - 250 g, were decapitated and the pituitaries were removed and hemisected longitudinal- ly. Each pituitary half was washed once with saline and placed in a 10-ml culture tube containing i ml of Eagle's MEM. After 30 minutes of pre-incuba- tion, i ml of fresh medium containing the testing materials, which were desig- nated in Experiment i, 2, 3 and 4, was added into each tube. The tubes were agitated gently, and 0.3 ml of medium sample was removed from each tube as the controls, which was immediately frozen at -20 °C. Incubation was then con- tinued at 37 °C under constant gassing with 95% air-5% CO 2 for 24 hours. After the %ncubation, tubes were centrifuged at 500 x g for 5 minutes, and the supernatant was removed and stored at -20 °C until assay.

Experiment I: The possibility that domperidone, alone, might directly stimulate prolactin release at the pituitary level was tested, by adding i ml of medium containing various concentrations of domperidone.

Experiment 2: The possibility that domperidone might interfere with the inhibitory effect of dopamine at the pituitary level was tested, by adding i ml of medium containing dopamine (final concentration: 500 ng/ml) and various concentrations of domperidone.

Experiment 3: The possibility that domperidone might modify the response of the pituitary to the physiological PIF activities was tested, by adding 0.5 ml of 1:5 dilution of control hypothalamic extract (which had been proven to be a minimum and sufficient amount to inhibit prolactin release from the pituitary in our experimental conditions) plus 0.5 ml of medium containing various concentrations of domperidone.

Experiment 4: The possibility that domperidone might act to change PIF activities at the hypothalamus was tested, by adding i ml of i:i0 dilution of domperidone-treated hypothalamic extract or control hypothalamic extract.

Prolactin levels were determined using a radioimmunoassay kit supplied by NIAMDD, and expressed in terms of a purified rat prolactin reference standard (NIAMDD Rat Prolactin RP-I), Domperidone was supplied by Janssen Research Laboratory (Belgium), through the courtesy of Kyowa Hakko Kogyo Co. Ltd. (Japan). Student's t-test was used to determine the significance of difference between experimental groups.

Results

Serum prolactin levels in rats which had been treated with domperidone for 14 days were significantly (p <0.05) higher than those of control rats (Table 1). Table 2 shows the changes in prolactin release from the pituitaries which were incubated with various testing materials. When the pituitaries were incubated alone, prolactin levels in the medium were significantly (p < 0.05)

Vol. 26, No. 16, 1980 Domperidone and Prolactin Secretion 1345

increased, and the addition of domperidona did not show any additional effects on prolactin release. Prolactin release from the pituitary was significantly inhibited by dopamine or control hypothalamic extract, and again, the addition of domperidone did not reverse the inhibitory effects of dopamine or control hypothalamic extract on prolactin release. On the other hand, domperidone- treated hypothalamic extract did not show any inhibitory effects on prolactin release from the pituitary. High doses of domperidone rather caused decreases in prolactin release.

TABLE i

Effect of domperidone on serum prolactin levels in female rats. Rats were given 70 mg/day of domperidone for 14 days and killed by decapitation. Values represent the mean + SEM.

Treatment No. of Rats Serum Prolactln Level, ng/ml

Domperidone i0 499 + 88.8 *

Control i0 44.0 + 6.4

• p < 0.001 versus control group.

TABLE 2

Effects of domperidone, dopamine, control hypothalamic extract or domperidone-treated hypothalamic extract on prolactin release from the pituitary. Pituitary halves from normal donor female rats were incubated alone, or with various testing materials for 24 hours. Values represent the mean + SEM.

Treatments No. of Rats

Prolactin in Medium

Pre-incubation ng/ml

After Incubation ng/ml

Control 6 943 ~ 272 3,923 Domperidone 0.5 Fg/ml 6 1,033 ~ 135 3,567

1.0 ~g/ml 6 1,026 ~ 200 3,407 5.0 pg/ml 6 916 ~ 250 2,250

Dopamine 500 ng/ml 6 1,260 ~165 928 Dopamine 500 ng/ml

plus Domperidone i pg/ml 5 1,390 ~ 330 904 i0 pg/ml 5 990 + 254 544

Control Hypothalamic Extract 7 1,057 ~ 268 829 Control Hypothalamic Extract

plus Domperldone 5 pg/ml 7 1,200 ~ 307 412 Domperidone-treated

Hypothalamic Extract 6 1,350 ~ 295 2,483

1,295 * + 631 + 613

624 + 181 **

+ 362 ¥ 303 + 352

+ 208

326

* p ~0.05 versus pre-lncubation value; ** p <0.05 versus control group; *** p (0.001 versus control hypothalamic extract group.

1346 Domperidone and Prolactin Secretion Vol. 26, No. 16, 1980

Discussion

The present results demonstrated that domperidone increased serum prolac- tin levels in rats, and that this drug would act primarily at the hypothalamus to increase prolactin secretion. The role of domperidone in affecting at the hypothalamus is not clear yet. However, domperidone was reported to be a potent dopamine antagonist (4), and some investigators have reported that dopamine exerted its inhibitory effect on prolactin secretion at the hypothala- mus (6, 7), although there are some evidences which suggest the direct action of dopamine at the pituitary (8, 9, i0, ii, 12). It is likely therefore that domperidone may act at the hypothalamus and affect on prolactin secretion through the dopaminergic mechanism. On the other hand, domperidone was repor- ted to overcome the apomorphine-suppressed prolactin secretion from cultured pituitary cells (5). In the present studies, domperidone did not reverse the inhibitory effect of dopamine or control hypothalamic extract at the pituitary level (Table 2). The discrepancy between these two observations may reside in the pituitary tissues used for each experiment or in the doses or kinds of drugs. Namely, the freshly prepared normotopic pituitary was used in the present studies, while the established cultured cells were used in the other (5). It was interesting to note that TRH (thyrotropin releasing hormone) pro- duced a significant increase in prolactin release from the ectopic pituitary tissues which had been transplanted under kidney capsule for 30 days, while it was not the case with the normotopic pituitary tissue (13). There might be some differences in the nature of cells between the established cultured cells and the normotopic pituitary tissues. Doses of domperidone used in the present studies were based on our pilot study, in which intravenous injections of 2 or 4 mg/kg (body weight) domperidone produced significant increases in serum prolactin levels in female rats. Since high doses of domperidone (e.g., 5 or i0 pg/ml) rather inhibited prolactin release from the pituitary (Table 2), it might be necessary to use much smaller doses of domperidone to study the direct effect of this drug at the pituitary. However, the present data would at least indicate that domperidone could act via the hypothalamus.

The previous findings that domperldone did not cross the blood-braln barrier (4) would not be controversy to the present result that domperidone would act via the hypothalamus, because it had been reported that the hypotha- lamus was supplied by vessels arising from the basement of the brain (14). Domperidone would therefore enter the hypothalamus from the general circula- tion. Further studies regarding the acting site of domperidone in the hypo- thalamus are now under way in our laboratory.

Acknowledgements

We are indebted to Dr. I. Rothchild, Case Western Reserve University, Cleveland, Ohio, for the advice and encouragement. We are also grateful to Dr. A.F. Parlow, Harbor General Hospital, Torrance, CA, and the NIAMDD Rat Pituitary Distribution Program for supplying the materials for the rat prolac- tin radioimmunoassay. Domperidone was kindly provided by Janssen Research Lab- oratory, Belgium, through the courtesy of Kyowa Hakko Kogyo Co. Ltd., Japan.

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