0021-972X/91/7305-1093$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society

Vol. 73, No. 5 Printed in U.S.A.

The Effect of Dynorphin on Placental Pulsatile Human Chorionic Gonadotropin Secretion in Vitro E. R. BARNEA, R. ASHKENAZY, AND Y. SARNE Feto-Placental Unit, Rappaport Institute, Technion, Haifa, Israel; and Department of Physiology (Y.S.), Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

administration there was a substantial increase in hCG secretion. The involvement of opioid receptors in mediating the effect of DYN on hCG release was demonstrated by using naloxone, an opioid receptor antagonist. Coadministration of DYN, 10"11 mol/L, and naloxone, 10~10 mol/L, reduced markedly the effect of DYN. This was followed by a delayed increase in hCG secretion. Furthermore, des-tyrosine-DYN, the nonopioid derivative of DYN was 1000 times less potent in stimulating hCG release than DYN. The involvement of DYN in physiological control of placental hCG secretion is suggested. (J Clin Endocrinol Metab 73:1093-1098, 1991)

ABSTRACT. Using in vitro methods we have studied the effect of dynorphin (DYN) a natural k opioid receptor ligand upon hCG secretion in the first trimester placenta. In superfusion, where we have recently reported that hCG secretion is episodic, we found that the addition of 1-min pulses of DYN had a significant stimulatory effect upon pulsatile hCG secretion. This effect was seen at concentrations of 10~8 mol/L-10"11 mol/L. Higher doses (10~6 mol/L) and lower doses (10~12 mol/ L) were ineffective. A 10-min administration was more effective than the 1-min pulses. Prolonged administration (90 min) caused an initial increase in pulsatile hCG secretion which was followed by a decrease to control values. However, upon stopping the prolonged opiate


PIOID receptors have been identified in the syncytiotrophoblast brush border membrane of the human placenta (1). They are mainly of the K subtype, and have been well characterized at term (2). The placenta is a rich source of a variety of opioid peptides, some of which have been also shown to be produced locally (3). The opioid peptides which were demonstrated in placenta are: /3-endorphin (4, 5), enkephalin (6), and dynorphin (DYN) (7). DYNs are the main endogenous ligand for the k opioid receptors in the human placenta (8). The available information about the effects of opiates upon placental endocrine function remains limited. Opioid drugs increased placental lactogen production at term in vitro (9). Opiates were also shown to modulate acetylcholine release from the placenta (10). No consistent effects of opiates on hCG secretion were previously found in vitro. In first trimester explant cultures, high doses of morphine (10~7 M) caused an increase in hCG secretion after 3 but not after 1-h incubation. On the other hand, no effect was observed after such a treatment at term (11). At term, stimulatory effects of the K agonist ethylkeReceived August 13, 1990. Address correspondence and requests for reprints to: E. R. Barnea, M.D., FACOG, Director, Feto-Placental Endocrine Unit, Rappaport Institute, Technion, P.O. Box 9697, Rh Efron, Haifa, Israel. Supported by a grant from the Israel Health Ministry Chief Scientist (to E.R.B.).

tocyclazocine (EKC), 10 6 mol/L, on potassium induced hCG release were observed; however, DYN, the natural K agonist had no significant effect (8). Furthermore, EKC and DYN (8) were tested only in one high concentration. It was assumed that it would reveal all possible effects of these compounds. In order to determine the whole spectrum of activity of the ligands, their effect should be tested in a wide range of concentrations. In the present work we aimed to study the effect of DYN 1-13 upon first trimester placental function as reflected by hCG secretion. We used our novel superfusion system where hCG was found to be secreted in distinct pulses every 18-23 min (12), and which is resembling the pattern present in vivo in women (13). Our data show that DYN 1-13 augmented the release of hCG in the first trimester in vitro. This effect was opioid receptor dependent, as it was blocked by the opiate antagonist naloxone and was not mimicked by the nonopioid analog des-Tyr-DYN.

Materials and Methods Materials DYN 1-13 and des-Tyr-DYN were purchased from Sigma (St. Louis, MO). Naloxone hydrochloride was a generous gift of Du Pont (Wilmington, DE). Kits for MAIA clone measurements of hCG were purchased from Serono, Rehovot, Israel.


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Dulbecco's modified Eagle's medium (DMEM) was purchased from Beit Ha-Emek, Beit Ha-Emek, Israel. Placental preparations Fifteen 8- to 11-week-old placentas were studied. After obtaining informed consent, elective pregnancy terminations by vacuum curettage were performed at the Rambam Medical Center. These patients were healthy and were not using any medication. After the procedure, samples were placed on ice. Small placental fragments were dissected out in a sterile fashion and placed in a large volume of 0.9% NaCl solution to remove all blood. Subsequently, the tissue was rinsed several times in DMEM containing antibiotic solution composed of 5000 U penicillin, 50 ng streptomycin, and 10,000 U fungizone (amphotericin B)/ml. Static culture Static explant culture was carried out as we have previously described (14). Incubation was carried out with 30 mg (wet weight) placental tissue (6-12 replicate dishes per treatment or vehicle-treated control group) in 1 ml medium with or without opioid ligands for 24 h at 37 C in a gas mixture of 5% CO2 and 95% air. After incubation the media were removed and saved for assay at -20 C. Placental tissue was saved for protein determination by the method of Lowry et al. (15). In this model there was a linear increase in hCG secretion for the duration of the culture. Tissue viability was assessed by progressive glucose consumption and vital staining. Superfusion culture The method of placental superfusion was recently reported (12). A multichannel peristaltic pump and a fraction collector (model 272, ISCO, Durham, NC) were used to study the shortterm dynamics of hCG secretion. The explants were placed into the culture chambers and HEPES (18 mmol/L)/DMEM solution was washed through in an atmosphere of 5% CO2 and 95% air at 37 C. The experiments were conducted for 120-150 min. One milliliter samples were collected every 2.4 min. In each experiment, one channel served as control and four served as experimental channels. At given intervals, a pulse of test agent was given through a peristaltic pump equipped with a digital flowmeter (ISMATECH DD, Chicago, IL).

JCE & M • 1991 Vol 73 • No 5

Results One-minute pulses of DYN 1-13 10~10 mol/L caused a significant stimulatory effect upon hCG secretion, compared to spontaneous pulsatile patterns seen in untreated controls (Fig. 1). The increase induced by a single pulse of DYN 1-13 was of varying amplitude. This stimulatory effect was achieved by adding DYN 1-13 at the concentration range of 10~8 mol/L-10"11 mol/L. No effect was seen, however, at very low (10~12 mol/L) and at high (10~6 mol/L) concentrations. Figure 2 shows the calculated dose-dependent effect of DYN 1-13 upon hCG secretion, compared to controls. After 1-min pulses of the opioid peptide the maximal amplitude was noted at 10~9 mol/L concentrations. The dependency of the stimulatory effect of DYN 113 on the duration of the drug administration was examined. The 1-min pulse of DYN 1-13 10"10 mol/L caused an increase in hCG which lasted for 10-12 min. Administration of the opioid peptide for 10 min caused an increased hCG secretion which lasted for about 20 min. This difference was statistically significant (P < 0.05) as calculated by the area under the curve. A subsequent 1-min pulse yielded a response similar to that obtained before the 10-min administration (Fig. 1). Similar results were obtained at 10~8 mol/L and 10~9 mol/L. No effect upon hCG secretion was seen while giving DYN 1-13 for only 10 sec (data not shown). When first trimester explants were perfused for 90 min with DYN 1-13, a rapid increase in hCG secretion was

o—o control — dyn 1-13 10






RIA Measurements of hCG in the media of explants or superfusion were made using standard RIA techniques as previously described (12). For a given placenta, hCG measurements for all collected samples were conducted in the same assay. Intraassay variability for hCG was 1.7%. Data for hCG were expressed as MIU per mg protein.

o o 0

10 20 30 40 50 60 70 80 90 100110120130 I (D

| dyn 1-13 pulse

Statistics Statistical analysis was performed using Student's t test for area under curve and P < 0.05 was considered as statistically significant. Results reported represent data generated in at least three different placentas.

I (i)


I . C°)


I («)

FIG. 1. The effect of 10~10 mol/L DYN 1-13 pulses upon spontaneous hCG secretion. One-minute pulses of DYN 1-13 were given at 18, 48, and 108 min. A 10-min pulse was added at 78 min. Similar results were obtained in eight other placental explant superfusions, giving four pulses per channel.

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o u.

2 0 0 ••

o o




5010 -12




10 - 9





added for 24 h induced only a slight (19%) nonsignificant increase in hCG secretion compared to controls (data not shown). In order to evaluate the involvement of opioid receptors in the stimulatory effect of DYN 1-13, the opiate antagonist naloxone was used. High doses of naloxone (10~9 mol/L) given for 10 min stimulated the release of hCG (data not shown)). A 10~10 mol/L naloxone pulse given for 10 min, caused no effect upon administration, but led to a delayed large peak of secreted hCG (Fig. 4A). Ten minutes of perfusion with naloxone 10~10 mol/L prevented the expected opioid induced stimulatory effect of DYN 1-13 10~n mol/L, given concomitantly for 10

DYN 1-13mol/L

panel A

FIG. 2. Dose-dependent effect of DYN 1-13 upon hCG secretion. Results are taken 2.4 min after the administration of a 1-min pulse of DYN 1-13. Data are expressed as percentage of the control channel. Results depicted were generated in at least five placentas each, run in two channels giving four pulses per channel. *, P < 0.05.


dyn 1 - 1 3 10

- 9


o — o CONTROL •


(10~ 1 0 mol/L)




VAA 10



\ 25





minutes panel B o—o CONTROL •—»DYN 1-13(10 550 f

10 20 30 40 50 60 70 80 90 10011012Q130140150 II

(90) II dyn 1 - 1 3


FiG. 3. The effect of administration of 10"9 mol/L DYN 1-13 for 90 min upon pulsatile hCG secretion. For the first 30 min there was a significant increase which was followed by a return to baseline. Upon stopping DYN 1-13 administration there was a significant increase in hCG secretion. The response to 1-min pulse of DYN 1-13 given at 135 was maintained.

initially seen which lasted for about 20 min. Then it was followed by a decrease to baseline. However, when DYN 1-13 administration was stopped, a highly significant increase in spontaneous hCG secretion was seen with several successive peaks lasting for a total of 30 min compared to the control channel. A subsequent 1-min pulse of DYN 1-13 given at 135 min also showed the expected significant stimulatory effect upon hCG secretion (Fig. 3). The effect of DYN 1-13 10~9 mol/L upon hCG secretion in placental explants after 24 h of static incubation was also examined. In static cultures the opioid peptide

M)+ NALOXONE (10" 10M)

c *a> "o a.

o o 80


minutes dyn 1-13



(10) dyn 1-13 + naioxone

FIG. 4. A, Lack of effect on hCG secretion during administration of naloxone 10"10 mol/L for 10 min. Upon stopping naloxone administration there was a significant increase in hCG secretion. B, The stimulatory effect upon hCG secretion by DYN 1-1310"11 mol/L was blocked by concomitant administration of naloxone 10~10 mol/L for 10 min at 50 min. A major delayed increase was however noted after stopping the compounds administration.

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panel a oQYN 1-13 •DYN 2 - 1 3


10 20 30 40 50 60 70 80 90 100 110 120 i i 10"11(1min) 10" n (2min)

i i E o o 30 |dyn 2-13 pulse

JCE & M • 1991 Vol73«No5






FIG. 5. A, Lack of effect of des-Tyr-DYN (DYN 2-13) (KT11 and 10"10 mol/L) {closed circles) given for 1 or 2 min upon hCG secretion compared to DYN 1-13 (open circles). B, The stimulatory effect of 10~8 mol/L des-Tyr-DYN pulses upon hCG secretion. One-minute pulses of des-Tyr-DYN were added at 18, 48.

min (Fig. 4B). However, it led to a large increase in hCG secretion after stopping naloxone administration (Fig. 4B). Another criterion for determining the opioid nature of DYN 1-13 effect is its comparison with the nonopioid analog des-Tyr-DYN. Des-Tyr-DYN had no stimulatory effect in doses equivalent to active doses of DYN 1-13 (10- n -10- 10 mol/L) (Fig. 5A). However, at a 1000 times higher dose (10~8 mol/L) des-Tyr-DYN had, in some cases, a significant stimulatory effect on pulsatile hCG secretion (Fig. 5B).

Regulation of placental and other endocrine organs secretion by opioid peptides has been recently suggested (16). In the present study we provide evidence that DYN 1-13, which binds preferentially to the k opioid receptor, has a significant stimulatory effect upon pulsatile hCG secretion in superfusion of first trimester placental explants. The stimulatory effect was dose dependent at the 10~n-10~8 mol/L range of concentrations with no effect produced at very low (10~12 mol/L) concentrations. This stimulatory effect is mediated through opioid receptors since it was markedly reduced by a low concentration (10"10 mol/L) of naloxone. A delayed effect of DYN 113 was noted' after such concomitant administration. Furthermore, the nonopioid analog des-Tyr-DYN, was not effective in 10~n-10~10 mol/L concentration, and a thousand times higher dose was required in order to stimulate release. The mechanism by which DYN stimulates hCG secretion is not clear yet. Opioid ligands are known to either block calcium current or inhibit adenylate cyclase. Either effect might explain our unpublished observation that DYN 1-13 also inhibits the secretion of progesterone from placental explants. Since this steroid was shown to block the release of hCG, it is tempting to suggest that DYN stimulates hCG secretion by removing an ongoing paracrine block by progesterone. This hypothesis is currently under investigation in our laboratory. At very high concentration (10~6 mol/L) DYN 113 has no effect on hCG secretion. The biphasic effect of DYN 1-13 in our study illustrates the importance of testing a wide range of doses, and might explain the disagreement with previous results on the lack of effect of DYN (8), which had been tested in a single concentration. That an inhibitory effect of DYN 1-13 might mask the stimulatory effect of the peptide is also indicated by the findings with a prolonged administration of DYN 113. Thus, 90-min application caused an initial increase in release, which was followed by a decrease to baseline levels. When the pulse was stopped a high amplitude hCG pulse was followed, indicating that lack of effect during the long pulse was not due to depleted stores of hCG. Also the response to a subsequent DYN 1-13 pulse was maintained suggesting that no down-regulation of the opioid receptors occurred during the prolonged pulse. The stimulatory effect by high doses of the opiate antagonist naloxone could indicate the presence of an on-going inhibitory effect of endogenous opioid ligand, whereby the inhibition of the inhibitor caused the release of hCG in superfusion. The finding that des-Tyr-DYN, which does not bind to opioid receptors, had stimulatory effects in some cases

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EFFECT OF DYN ON THE PLACENTA suggests that at high doses DYN action may be exerted through nonopioid mechanisms (18). An alternative explanation is that at high doses impurities present in desTyr-DYN could have caused the observed stimulatory response. Previously a single class of opioid receptors, of the k subtype, was identified in placenta at term (17). However, whether the receptors in the first trimester are similar to that previously reported at term is not established. The possible inhibitory presence of other opioid receptors (fi, 8), which might mediate other effects (by high DYN 1-13 concentrations), is suggested by our experiments with jS-endorphins (unpublished observations). We found that low concentrations (10~9 mol/L) of 0endorphin inhibited the release of hCG in static cultures. /3-Endorphin exhibits a very low affinity towards the k receptor, suggesting that this inhibitory effect is mediated by either n or 8 opioid receptors. These receptors can be activated by high concentration of DYN 1-13. The concomitant activation of a stimulatory process (through k receptors) and an inhibitory process (through either /* or 8 receptors) may lead to the biphasic effect of DYN, where very high concentration have no effect upon hCG secretion. Other endocrine functions of the placenta are also regulated by opiates. Belisle et al. (9) have shown that EKC increases human placental lactogen secretion at term in vitro. The possible presence of a heterogenous population of opiate receptors in the human placenta points to the importance of using highly specific opioid drugs in such investigations. Most conventional opiates are not highly specific and, at high concentrations, might act through various opiate receptors. This may explain the findings by Cemerikic et al. (11) who have shown that another opiate alkaloid, morphine, had a differential effect upon hCG secretion, stimulatory in the first trimester while having no effect at term. Lack of effect of DYN 1-13 in static cultures may be a result of desensitization of the opioid receptors after prolonged exposure to the drug. Recently, we have shown that other placental regulatory factors, like epidermal growth factor that controls hCG secretion, are also less effective when they are tested in static cultures of explants in the first trimester (19), which points to the importance of dynamic measurements of hCG secretion, such as were used in our current study. Our data confirms and extends previous observations on opiate alkaloids to indicate that also natural opioids i.e. DYN have a significant stimulatory effect upon hCG secretion, and this takes place at very low, i.e. physiological concentrations similar to those present in human placenta (20). These effects became clearly evident using the superfusion model, where a continuous washing of the explants takes


place, probably eliminating the local para/autocrine effect of internal opiates and/or other compounds, and thereby enabling the examination of externally added DYN.

References 1. Valette A, Pontonnier G, Cros J. Evidence for a stereospecific [3H] etorphine binding in human placenta. FEBS Lett. 1979;103: 362-5. 2. Porthe G, Valette A, Moisand A, Tafani M, Cros J. Localization of human placental opiate binding sites on the syncitial brush border membrane. Life Sci. 1982;31:2647-54. 3. Liotta AS, Krieger DT. In vitro biosynthesis and comparative posttranslational processing of immunoreactive precursor corticotropin//3-endorphin by human placental and pituitary cells. Endocrinology. 1980;106:1504-ll. 4. Nakai Y, Nakao K, Oki S, Imura H. Presence of immunoreactive /Mipotropin and /3-endorphin in human placenta. Life Sci. 1978;23:2013-8. 5. Odagiri E, Sherrel BJ, Mount CD, Nicholson WE, Orthe DN. Human placental immunoreactive corticotropin, lipotropin, and /3endorphin: evidence for a common precursor. Proc Natl Acad Sci USA. 1979;76:2027-31. 6. Sastry BVR, Barnwell SL, Tayeb OS, Jansoh VE, Owens LK. Occurrence of methionine enkephaline in human placental villus. Biochem Pharmacol. 1980;29:475-8. 7. Lemaire S, Valette A, Chouinard L, et al. Purification and identification of multiple forms of dynorphin in human placenta. Neuropeptides. 1983;3:181-91. 8. Valette A, Tafani M, Porthe G, Pontonnier G, Cros J. Placental kappa binding site: interaction with dynorphin and its possible implication in hCG secretion. Life Sci. 1983;33:523-6. 9. Belisle S, Petit A, Gallo-Payet N, Bellabarba D, Lehoux JG, Lemaire S. Functional opioid receptor sites in human placentas. J Clin Endocrinol Metab. 1988;66:283-9. 10. Ahmed MS, Horst MA. Opioid receptors of human placental villi modulate acetylcholine release. Life Sci. 1986;39:535-40. 11. Cemerikic B, Genbacev 0, Sulovic V, Beaconsfield R. Effect of morphine on hCG release by first trimester human trophoblast in vitro. Life Sci. 1988;42:1773-9. 12. Barnea ER, Kaplan M. Spontaneous, gonadotropin releasing hormone-induced, and progesterone-inhibited pulsatile secretion of human chorionic gonadotropin in the first trimester placenta in vitro. J Clin Endocrinol Metab. 1989;69:215-7. 13. Owens OM, Ryan KJ, Tulchinsky D. Episodic secretion of human chorionic gonadotropin in early pregnancy. J Clin Endocrinol Metab. 1981;53:1307-9. 14. Barnea ER, Fares F, Kol S. Stimulatory effect of prolactin in human placenta progesterone secretion at term in vitro, possible inhibitory effect upon estradiol secretion. Placenta. 1989;10:37-43. 15. Lowry OH, Rosebrough NA, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951; 193:265— 74. 16. Bardin CW, Chen CLC, Morris PL, et al. Propiomelanocortinderived peptides in testis, ovary, and tissues of reproduction. Recent Prog Horm Res. 1987;43:l-27. 17. Porthe G, Valette A, Cros J. Kappa opiate binding sites in human placenta. Biochem Biophys Res Commun. 1981;101:l-6. 18. Walker JM, Moises HC, Coy DH, Baldrighi G, Akil H. Nonopiate effects of dynorphin and Des-Tyr-Dynorphin. Science. 1982;218:1136-8.

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19. Barnea ER, Kaplan M, Feldman D, Morrish D. The dual effect of epidermal growth factor upon placental hCG secretion. J Clin Endocrinol Metab. 1990;71:923-28.

20. Ahmed MS, Laura WR, Randall BS, et al. Human placental opioid peptides: correlation to the route of delivery. Am J Obstet Gynecol. 1986;155:703-6.

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The effect of dynorphin on placental pulsatile human chorionic gonadotropin secretion in vitro.

Using in vitro methods we have studied the effect of dynorphin (DYN) a natural k opioid receptor ligand upon hCG secretion in the first trimester plac...
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