In vitro effect of dopamine and pimozide on human chorionic gonadotropin secretion CHAMEL

MACARON

MILOSLAVA

KYNCL

OLUFUNSHO BERNARD JOHN Chicago,

FAMUYIWA HALPERN

BREWER Ninois

In human placental explants cultured in vitro, dopamine inhibited human chorionic gonadotropin (hCG) secretion into the culture media. In the control flasks, the level of hCG secretion was 751 2 215 mlU/gm of tissue. When 1 mM of dopamine was added, hCG levels decreased to 321 i- 57.6 mlU/gm of tissue (n = 6, P < O.l)--5 and 10 mM of dopamine significantly inhibited hCG secretion. In contrast, 1 mM of pimozide enhanced hCG secretion by 1.8-fold cornpared to control levels (1,707 i 343 versus 3,117 2 0.005). This in vitro effect on hCG is similar to the effect of dopamine and pimozide on hCS secretion by placental explants. (AM. J. OBST’ET. GYNECOL. 132499, 1979.)

regulation of human chorionic gonadotropin (hCG) synthesis and release by normal placenta, choriocarcinoma cells, and tumors ectopically producing hCG remains largely unknown. hCG is secreted by the syncytiotrophoblast during pregnancy,’ and its secretion peaks at 7 to 10 weeks’ gestation. Later on, the serum concentration starts to fall at a relatively constant level for the rest of the gestation.’ This pattern reflects placental hCG secretion since the tissue content of hCG per unit of wet weight is greatest during the first trimester and declines thereafter.* The placental hCG subunits follow qualitatively the same profile as that of intact hCG. However, an excess of the alpha subunit of hCG in relation to immunoreactive hCG does THE

From the Dlepartnlent and Chicago Med’ical

of Medicine, School.

Edgewater

Hospital

Supported by General Research Support Grant BR-5366 from the General Research Support Branch, Division of “Research Facilities and Resources, and by Grant ROl I CA121 09 )frorn the United States Public Health Service, National &tit&es of Health. Receivedfor

publication

Accepted December

May

31, 1978.

14, 1978.

Reprint requests: Dr. Charnel Macaron, Department oj Medicine, Stern Building, Room 204, 1619 West Edgewater, Edgewater Hospital and Chicago Medical School, Chicago, lllinois 60660.

0002-9378/79/200499+04$00.40/O

0

1979

The

C. V. Mosby

Co.

occur during the last two trimesters3 In addition to normal placenta, choriocarcinoma cells and many nontrophoblastic tumors secrete intact hCG as well as its alpha and beta subunits. 4-6 Although the physiologic factors regulating the synthesis and release of hCG are unknown, many pharmacologic actions have been identified. Thus, in human term placenta cultured in vitro, hCG secretion can be markedly stimulated by dibutyrylcyclic adenosine monophosphate (dbcAMP)’ and minimally by sodium butyrate.8 Similarly, the synthesis of hCG and hCG-a! in choriocarcinoma cell lines has been enhanced by dbcAMP and theophylline but not by butyrate. 8, 9 In sharp contrast, the nontrophoblastic tumors did not respond to dbcAMP, but a dramatic stimulation occurred with butyrate.*s ’ Therefore it seems that the regulation of hCG and its subunits differs in the normal or malignant trophoblast from the nontrophoblastic tumors. In other pharmacologic manipulations, actinomycin D in low concentrations” and methotrexate” stimulated hCG production by choriocarcinoma cell lines. Somatostatin failed to suppress hCG secretion and release in JAR cell lines.‘* In addition, high levels of epidermal growth factor receptor have been detected in term placenta,13 and recently Benveniste and associatesn showed that epidermal growth factor stimulates hCG secretion by human choriocarcinoma cell lines. 499

Control Dopamine Fig. 1. Dopamine culture.

inhibition

of secretion

of hCG

into

control

We have previously shown that, in short-term placental cultures, dopamine inhibits human chorionic somatomammotropin (hCS) release, and that pimozide which is a dopaminergic receptor blocking agent can stimulate hCS secretion into the media.‘” The present investigation was undertaken to assess the effects of dopamine and pimozide on hCG and to determine whether their action is specific for hCS and not for hCG.

methods We used the in vitro organ culture oi‘ placental explants as described by Handwerger and associates.7 Freshly delivered term human placentas were obtained and washed with distilled water; then small fragments of vilii were cut from several quadrants and washed in Hanks balanced salt solution (Gibco). Fight to ten pieces were randomly selected and put in 10 ml culture flasks containing 5 ml of I5% fetal caif serum 199. No antibiotics were added because the incubation period was short. The flasks were loosely capped and were placed in an atmosphere of 95% Q2 and 5% COZ in a Dubnoff metabolic incubator at 39” C and shaken at 80 cycles/ minute. There was an initial preincubation period of 2 hours after which the medium was changed and either dopamine, pimozide, or both were added to the appropriate flasks. Dopamine (molecular weight 189.6) was dissolved in medium 199, while pimozide (molecular weight 461.54) was dissolved in absolute ethanol and added in a volume not exceeding 300 ~1. The same amount of alcohol was added to the control flask in the pimozide experiments. The control and experimental flasks were incubated (in triplicate) for 3 hours, after which the medium was collected and stored frozen at

-20” C. Under these experimentai conciitions, oxidation of dopamine in the 5 and 10 mM flasks does occur as evidenced by the black color of the incubation medium. The fragments of each Aask were blotted gently on a filter paper, and wet weight was determined (range 140 to 250 mg). ad~oimmun~as§a~. hCG was assayed by doubleantibody radioimmunoassay.‘* Each sample was assayed in duplicate and each experiment was analyzed in a singie run to decrease interassay variation. The intraand interassay coefficients of vatiation were 6% and 9.7%, respectively. The results are expressed as milli-International Units of KG per gram of tissue per I ml of incubate. Statistics. Student’s t test for @ired data was used for statistical analysis. Effect of dopamine. Dopamine inhibits the secretion of hCG into the culture media (Fig, l). The results represent the mean and standard error (SE) of six experiments. In the control flasks, the mean (ISI?) of hCG secreted during the 3 hours was 751 i 21.5 mILJ/gm of tissue. When 1 mM dopamine was added, hCG levels decreased to 321 t 57.6 mIU/gm of tissue (P < 0.1). In one of the six experimenats, the decrements in hCG levels were minimal, which accounts for the lack of statistical significance (I’ < 0. I). A concentration of 5 mM also inhibited hCG secretion (273 + 5’7.6 mIUigm tissue) (P < 0.03). Increasing the concentration of dopamine to 10 mM had no further effect (260.4 i 47) (P Effects of pimozide alone a use of 1 mM pimozide; a dopaminergic receptorblocking agent, stimulated hCG secretion into the culture media (Fig. 2). In the control flasks, the meax% (*SE) concentration of hCG was 1,707 mIU ZY 343igm of tissue; when pimozide was added, an increment of I .8-fold occurred as detected by a mean of 3, I I9 r’ 478 (P < 0.005). Again in these experiments the inhibitory effect of 5 mM dopamine was noted by a mean of 1,352.6 XL 310. When I mM pimozide was added immediately after 5 mM dopamine, the stimulatory effect of pimozide, via control, persisted, although the effect was not statistically significant,

The present study demonstrates -hat dopamine and/or its metabolites inhibit hCG secretion by- the placental explants in organ tissue culture. This effect seems to be more pronounced at 5 mM than at I mM concentrations. Furthermore, pimozide; which blocks the dopaminergic receptors, has an appropriate effect and stimulates hCG release into the culture media.

Volume Number

135 4

Thus

it

pimozide

Effect

appears on hCG

that

the

secretion

on hCS secretion and mechanisms underlying

actions are

of similar

therefore the action

dopamine to their

are nonspecific. of dopamine

the

enzymes

required

for

the

on hCG

The remain

;

dopa-

minergic pathway are present in the placenta is not known. Second, dopaminergic drugs such as alpha bromoergocryptin (CB-154) have been used therapeutically in the treatment of the amenorrhea-galactorrhea syndrome, and1 pregnancies -occurred while CB-154 was continued.‘* Thus, it appeared that dopamine stimulate rather than inhibit hCG or hCS secretion; nevertheless, the in vitro effects described above may be different from the in vivo results. On the other hand, hCG is similar in its structure and biological activity to luteinizing hormone (LH); therefore, they may beh,ave similarly in regard to neurotransmitter act.ion. Thus, Lachelin and colleaguesrv have shown that a dopamine infusion can inhibit LH and prolactin release in women. This in vivo effect is similar to the h(CG and hCS responses to dopamine in our vitro system. A drug which stimulates hCG secretion in vivo would be benefical at one point in the management of trophoblastic disease. A stimulation test performed at the end of the therapy might differentiate the patients who are in complete remission from those still at risk of relapse. Thus, the use of additional therapy in the latter patients might prevent the 4% relapse which occurs

secretion

501

PC 0.005

Q) 3500

Furtlhermore we are puzzled by this observation for many reasons. First, the placenta, like the pituitary, is not an innervated organ, but it has the capacity to syntlhesize neurotransmitters like acetylchoWhether

and pimozide

and effects

unknown.

line.”

of dopamine

n=6

3000

Pc0.I

.t- 2500 E m 2000 3 2

T

PcO.05

T

1500

Ill

1000

2 -E

500 Control

Fig. 2. Pimozide media.

Dopamine 5mM

stimulation

-

-

Pimozide ImM

of secretion

Dopamine Pim&ide

of hCG

into

culture

after primary remission is attained. The resting cells may be unable to secrete hCG or may produce minimal amounts not detected by our present methods. We assume that an increase in hCG levels will presumably indicate

a higher

probability

of relapse

and

that

a fail-

ure of stimulation would indicate a cure. Whether dopamine and pimozide will have the same effects on choriocarcinoma

cell

lines

is currently

under

inves-

tigation. We are grateful to McNeil Laboratories for providing the pimozide, to Dr. R. Hussa for many helpful suggestions, and to Mrs. Lynda Mottl for technical assistance.

REFERENCES

1. Dreskin,

2.

3.

4.

5.

6.

R. B., Spicer, S. S., and Greene, W. B.: Cytochemical studies of the maternal surface of the syncytiotrophoblast of human early and term placenta, J. Histochem. Cytochem. l&862, 1970. Diczfalusy, E.: Chorionic gonadotrophin and oestrogens in the huma.n placenta, Acta Endocrinol. (Kbh.) (Suppl) 12:, 1953. Vaitukaitis, J. L.: Changing placental concentrations of human chorionic gonadotropins and its subunits during gestation, J. Clin. kndocrinol Metab. 38:755, 1974. I Kohler, P. O., and Bridson, W. E.: Isolation of hormone-producing clonal lines of human choriocarcinoma, J. Clin. Endocrinol. Metab. 32:683, 1971. Tashjian, A, H., Jr., Weintraub, B. D., Barowsky, N. J., Rabson, A. S., and Rosen, S. W.; Subunits of human chorionic gonadotropin: Unbalanced synthesis and secretion by clonal cell strains derived from a bronchogenic carcinoma, Proc. Natl. Acad. Sci. U.S.A. 70:1419, 1973. Lieblich, J. M., Weintraub, B. D., Rosen, S. W., Shosh, N. K., and Cox, IR. P.: Secretion of the hCG alpha subunits and hCG by Hela strains, Nature (Lond.) 265:746, 1977.

7. Handwerger, S., Barrett, J., Tyrey, L., and Schomberg, D.: Differential effect of cyclic adenosine monophosphate on the secretion of human chorionic gonadotropin, J. Clin. Endocrinol. Metab. 36: 1268, 1973. 8. Hussa, R. O., PatilPo, R. A., Ruckert, A. C. F., and Scheuermann. K. W.: Effects of butyrate and dibutyryl cyclic AMP on hCG-secreting trophoblastic and nontrophoblastic cells, J. Clin. Endocrinol. Metab. 46:69, 1978. 9. Chou, J. Y., Robinson, J. C., and Wang, S. S.: Effects of sodium butyrate on synthesis of human chorionic gonadotropin in trophoblastic and non-trophoblastic tumors, Nature (Lond.) 268:543, 1977. 10. Hussa, R. O., Patillo, R. A., Delfs, E., and Mattingly, R. F.: Actinomycin D stimulation of hCG production by human choriocarcinoma, Obstet. Gynecol. 42:65 1, 1973. 11. Speeg, K. V., Jr., Azizkhan, J. C., and Stromberg, K.: The stimulation by methotrexate of human chorionic gonadotropin and placental alkaline phosphatase in cul_ _ tured choriocarcinoma cells. 12. Macaron, C., Kyncl, M., Rutzky, L., Halpern B., and Brewer, J.: Failure of somatostation to affect hCS and

502

Macaron

el a;.

hCG secretion in vitro. J. Clin. Endocrinol. Metab. In press. 13. O’Keife, E., Hollenberg, M. D., and Cuatrecasas, P.: Epidermal growth factor characteristics of specific binding in membranes from liver, placenta and other target tissues, Arch. Biochem. Biophys. 164~518, 1974. 14. Benveniste, R., Speeg, K. V., Carpenter, G., Cohen, S., tindner, J., and Rabinowitz, D.: Epidermal growth factor stimulates secretion of human chorionic gonadotropin by cuhured human choriocarcinoma cells, J. Clin. Endoninol. Metab. 46:169, 1978, I5 Macaron, C., Famuyiwa, O., and Singh, S. P.: In vitro effects of dopamine and pimozide on human chorionic somatommatratropin (hCS) secretion, J. Clin. Endocrinol. Metab. In mess.

16. Halpern, B., Eckman, T. R,, and DoEkari, R. E.: Sevenhour radioimmunoassay of human chorionic goaadotropin, AM. J. OBSTET. ~PNECOL. 93:223, 196.5. 17. Rama Sastrv. B. V,. Qlubadewo. T., Harbison. R. D.. and Schmidt, D! !I?.: Human piacentaici7oIinergic system. Occurrence. distribution and variation with gestational age of acetylcholine in human placenta, Biochem. Pharmacol. 25:425, 1976, 18. Child, D. F.. Gordon, I-I., Mashiter, K., andjophn, G. F.: Pregnancv, prolactin and pituitary tumor, Br. Med. Jo 4537, 1975. 19. Lacbelin, G. C. L., Leblanc, II.: and Yen, S. S, 6.: The inhibitory effect of dopamine agonists on LH release in women, J. Clin. Endocrinol. Metab. 44~728, 1977.

In vitro effect of dopamine and pimozide on human chorionic gonadotropin secretion.

In vitro effect of dopamine and pimozide on human chorionic gonadotropin secretion CHAMEL MACARON MILOSLAVA KYNCL OLUFUNSHO BERNARD JOHN Chicago,...
372KB Sizes 0 Downloads 0 Views