Effect of Adrenergic Agonists on Human Chorionic Gonadotropin Release by Human Trophoblast Cells Obtained from First Trimester Placenta
Summary The cultured syncytiotrophoblast cells from human first trimester placenta were used to determine the effect of adrenergic agonists on human chorionic gonadotropin (hCG) production in vitro. Beta-adrenergic agonists isoproterenol, ritodrine and isoxsuprine increased the hCG release during the 2 h incubation period, however, alpha-agonists norepinephrine and phenylephrine and a beta1-agonist dobutamine had no effect. The effect of isoproterenol was blocked by propranolol and butoxamine, but less efficiently by phentolamine and atenolol. These results indicate that placental hCG production can be modulated by stimulation of beta-, possibly beta2-adrenoceptors but not by alpha-adrenoceptors. Key words Human Chorionic Gonadotropin - Cultured Placental Cells - Beta2-Adrenoceptor - Ritodrine
Introduction It has been demonstrated that human chorionic gonadotropin (hCG) biosynthesis in placental explants (Handwerger, Barrett, Tyrey and Schomberg 1973; Haning, Choi, Kiggens, Kuzma and Summerville 1982; Hilf and Merz 1985) and placental cells (Zeitler, Markoff and Handwerger 1983; Feinman, Kliman, Caltabiano and Strauss 1986; Zhou, Yuen and Leung 1987; Ulloa-Aguirre, August, Golos, Kao, Sakuragi, Kliman and Strauss 1987; Rodway, Zhou, Benoit, Yuen and Leung 1988) is stimulated by cyclic nucleotides. The placenta has been reported to contain beta adrenergic receptors and catecholamine sensitive adenylate cyclase (Whitsett and Johnson 1979; Whitsett, Johnson, Noguchi, Darovec-Beckerman and Costello 1980). Therefore, there is a possibility that catecholamines may stimulate hCG secretion of placenta through increasing placental cAMP content. However, direct demonstration of such effect of catecholamines is not known.
Horm. metabol. Res. 22 (1990) 188-191 © Georg Thieme Verlag Stuttgart • New York
The purpose of the present study was to show adrenergic agonists enhanced hCG production by placental cells maintained in the culture and to determine the type of adrenergic receptors involved by using several specific adrenergic agonists and antagonists for alpha-, beta-, betaiand beta2-adrenoceptors (Weiner 1985a; Weiner 1985b). Materials and Methods Chemicals Adrenergic agonists and antagonists used are: isoproterenol HC1 (Sigma Co., MO), dl-norepinephrine (Noradrenaline injection®, Sankyo Co., Tokyo), isoxsuprine HC1 (Duvadilan injection®, Daiichi Seiyaku Co., Tokyo), phenylephrine HC1 (Neosynesin injection®, Kowa Co., Nagoya), dobutamine HC1 (Dobutex injection®, Shionogi and Co., Osaka), ritodrine HC1 (Utemerine injection®, Kissei Pharmaceut. Co., Matsumoto), phentolamine mesylate (Regitin injection®, Ciba-Geigy Japan, Tokyo), propranolol HC1 (Inderal injection®, ICI Pharma, Osaka) atenolol (ICI Pharma, Osaka), and butoxamine HC1 (Burroughs Wellcome Co., NC). Hyaluronidase, collagenase, trypsin inhibitor (soybean), DNAse (type 1A), bovine serum albumin (fraction V, BSA) were purchased from Sigma Co. (St. Louis, MO). Medium 199 and fetal calf serum (FCS) were purchased from Gibco Labs. (NY). Other drugs were obtained from commercial sources. Two kinds of media were used for the culture: buffer A contains medium 199 with 25 mM HEPES, 30 mM NaHCO3 and 10% FCS at pH 7.4 and buffer B is the medium where FCS of buffer A was replaced with 10 % BSA. Preparation of trophoblast
cells
This study was approved by the College Human Ethics Research Committee. Trophoblast cells were prepared from fresh placentas, which were obtained from legal abortion at 7 to 12 weeks with informed consent (Iwashita, Watanabe, Adachi, Oohira, Shinozaki, Takeda and Sakamoto 1989). Briefly, the villous tissue was separated from connective tissue and minced on ice. The villi were incubated with collagenase (0.2 mg/ml), hyaluronidase (0.12 mg/ml), trypsin inhibitor (0.02 mg/ml), DNAse (0.02 mg/ml) in buffer B at 37 °C for 30 min in a shaking water bath. The mixture was filtered through nylon mesh (150 urn) and the digestion of the residue was repeated once. The pooled filtrate was centrifuged at 800 x g for 10 min. The precipitated cells were suspended in buffer A at a concentration of 2 x 105 cells/ml. Cell viability estimated by trypan blue exclusion was more than 90 %. The freshly prepared cells suspended in buffer A were plated in 22 mm culture plates (Corning, NY) at 2 x 105 cells/ml/well and maintained for 48 h in a 37 °C incubator with 5 %
Received: 7 Apr. 1989
Accepted: 31 July 1989
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N. Oike1, M. Iwashita2, T. Muraki1, T. Nomoto1, Y. Takeda3 and S. Sakamoto2 1 Department of Pharmacology, 2the Maternal and Perinatal Center, and 3Department of Gynecology and Obstetrics, Tokyo Women's Medical College, Kawadacho, Shinjuku-ku, Tokyo, Japan
Adrenergic Agonists and Placental hCG
Horm. metabol. Res. 22(1990) 189
C02-95 % air. On day of stimulation, the cells were washed twice with 1 ml of buffer B. After addition of 1 ml of buffer B with or without drugs dissolved in 10 ul of saline to the placental cells, the plates were returned to the CO2 incubator. After incubation, the medium was removed and was stored at -20 °C until analysis for hCG.
Results The hCG release into the medium occurred in 2 phases during the 4 h of incubation of cultured trophoblast
Fig. 1 Time course of effect of isoproterenol on the release of hCG from cultured trophoblast cells. Trophoblast cells were cultured for 1,2 and 4 h in the absence (control) and presence of 10~4 M isoproterenol. Each value and vertical bar represent mean ± SE of duplicate incubation from 5 separate experiments. * P < 0.05 vs control (2 h) (Student's t-test)
Fig. 2 Concentration-dependent effect of adrenergic agonists on the hCG release of cultured trophoblast cells. Trophoblast cells were incubated with or without drugs of indicated concentration for 2 h. The results are expressed as % of control (hCG release in the absence of drug additions) which was 38.7 ± 8 . 3 mill/well (mean ± SE, n = 20). Each value and vertical bar represents mean ± SE of duplicate incubations from separate 5 experiments. *P < 0.05, **P < 0.01 vs10~ 7 Mof the same drug (Dunnetttest).
Table 1 Effect of adrenergic antagonists on the hCG release induced by beta adrenergic agonists. Trophoblast cells were cultured with or without additions of drugs of indicated concentrations for 2 h. The results are expressed as % of control (the hCG release without drug additions) which was 37.4 + 6.8 mill/well (mean ± SE, n = 5). The results are mean ± SE of duplicate incubations from 5 separate
Table 2 Effect of atenolol and butoxamine on the hCG release induced by isoproterenol. Trophoblast cells were cultured with or without additions of drugs for 2 h. The results are expressed as % of control (the hCG release without drug additions) which was 53.4 ± 9.0 mill/well (mean + SE, n = 5). The results are the mean ± SE of duplicate incubations from 5 separate experiments.
GYnpriments
Additions
hCG release (% of control) 4
Isoproterenol (10~ M) MSO) ISO + propranolol (10~6M) ISO + propranolol (10" 4 M) ISO + phentolamine(10"5M) ISO + phentolamine(10"4M) Ritodrine (10_4M)(RIT) RIT + propranolol (10"5M) RIT + propranolol (10~4M)
235.4 ± 26.6 133.0 ± 16.2** 122.8 ± 6 . 0 * 217.0 ± 16.0 157.8 ± 9.6* 185.2 ± 9.0 157.2 ±17.8 120.8 ± 10.1*
**P < 0.01 vs isoproterenol (10 4 M) (Student's t-test); *P < 0.05 vs ritodrine (10~4 M) (Student's t-test).
hCG release (% of control)
Additions 4
Isoproterenol (10" M) (ISO) ISO + atenolol (10"5M) ISO + atenolol (10"4M) ISO + butoxamine (10"5M) ISO + butoxamine (10~4M)
154.6 ± 7.5 145.4 ± 12.2 157.5 ±14.4 127.0 ± 4.7 102.6 ± 6.8*
*P < 0.001 vs isoproterenol (Student's t-test).
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cells, in the absence of added adrenergic agonists, the release of hCG into the medium occurred within 1 h after the medium change followed by a slow rate of hCG release (Fig. 1). The addition of 10 -4 M isoproterenol accelerated the hCG release during the first 2 h, however, hCG concentration in the medium showed a plateau thereafter. Since the effect of isohCG assay proterenol was significantly found at 2 h incubation, we deterhCG in the medium was measured by an enzyme im- mined the effects of sympathomimetic drugs by 2 h incubation munoassay kit (Mochida Co., Tokyo) using monoclonal antibody in the following experiments. Fig. 2 shows the dose effect of specific for beta subunit of hCG. various adrenergic agonists. Beta agonists such as isoproterenol, ritodrine and isoxsuprine increased the hCG release in a concentration-dependent fashion between 10" -10" M, Statistics however, alpha-agonists such as norepinephrine and phenyleValues are represented as mean + SE of the indi- phrine and a betai-agonist dobutamine had no effect. At the cated number of separate experiments, each performed with a different trophoblast cell preparation in duplicate. Statistical analysis of the results was performed using Student's t-test, Aspin-Welch test or Dunnett (nonparametric) test.
N. Oike, M. Iwashita, T. Muraki, T. Nomoto, Y. TakedaandS. Sakamoto
highest concentration examined (10 M), the potencies of the drugs were isoproterenol > ritodrine > isoxsuprine > dobutamine, norepinephrine, phenylephrine in that decreasing order when evaluated by Student's t-test or Aspin-Welch test (P < 0.05). This result suggests that beta-adrenergic agonists, except dobutamine, but not alpha adrenergic agonists increased the hCG release from the trophoblast cells obtained from thefirsttrimester placenta. To further characterize the adrenoceptors involved in the hCG release, we examined the effect of beta and alpha adrenergic blockers on the release of hCG induced by beta adrenergic agonists (Table 1). A nonselective beta blocker propranolol inhibited the stimulatory effect of isoproterenol (10~4 M) at both 10~5 and 10~4 M, whereas phentolamine, an alpha-blocker, showed an inhibition at the higher concentration (10 -4 M). The effect of ritodrine was also antagonized by the same concentration of propranolol. These results suggest the involvement of beta-adrenoceptors in the effect of isoproterenol to release hCG. Our result that a beta2-agonist ritodrine increased the hCG release but a betai agonist dobutamine did not, may suggest the involvement of beta2-adrenoceptors in the hCG release. To confirm the role of beta2adrenoceptors in the hCG release, we examined the inhibition of the effect of isoproterenol by atenolol (betai-blocker) and butoxamine (beta2-blocker) (Table 2). Although there was no difference between atenolol and butoxamine in the inhibition of the isoproterenol effect at one-tenth the concentration of isoproterenol, 10" M butoxamine, but not 10" M atenolol, antagonized the hCG release induced by 10" M isoproterenol, suggesting the involvement of beta2-adrenoceptors again. Discussion This study shows that the addition of beta but not alpha-adrenergic agonists to the placental cells maintained in the culture significantly increases the accumulation of hCG in culture medium. Isoproterenol was the most potent, followed by ritodrine and isoxsuprine. The response to isoproterenol was dose-related and was blocked by the betaadrenergic antagonist propranolol more efficiently than by the alpha antagonist phentolamine. These results indicate that the sympathomimetic drugs increased the hCG release by activating the beta-adrenergic receptors. The result that a beta2-agonist ritodrine increased the hCG release but a betai-agonist dobutamine did not, and that the effect of isoproterenol was more effectively blocked by butoxamine than by atenolol, may suggest that beta2-adrenoceptors are involved in the hCG release elicited by adrenergic agonists.
sent study. Therefore, most probably isoproterenol directly activates the beta2-adrenoceptors on the syncytiotrophoblasts, stimulates the cAMP production, and thereby increases the hCG release. A previous study showed that ritodrine increased the cAMP content of the placenta and thatjthis effect was most marked in the first trimester placenta (Cemerikic, Genbacev and Sulovic 1985). Therefore, beta agonists such as ritodrine and isoxsuprine may increase the hCG release through the same mechanism. Beta adrenergic agonists are used for preventing preterm labor by eliciting the relaxation of uterine muscle through acting on beta2-adrenoceptors (Barden, Peter and Merkatz 1980; Caritis, Carson, Greebon, McCormick, Edelstone and Mueller-Heubach 1982; Caritis 1988). Also, beta-adrenergic agonists increased the progesterone production of the term placental cells and this effect may be mediated by increased intracellular cAMP (Caritis, Hirsch and Zeleznik 1983). Our study revealed that the mechanism of beta agonists to increase the hCG production is similar to the case of progesterone production found in the term placental cells. Petraglia, Lim and Vale (1987) reported that epinephrine stimulated the secretion of luteinizing hormonereleasing hormone (LHRH) from cultured human placental cells through beta-adrenoceptors. In addition, it has been reported that LHRH stimulated the hCG release from the cultured human syncytiotrophoblast (Belisle, Guevin, Bellabarba and Lehoux 1984; Iwashita etal. 1989). It is considered that the cytotrophoblast releases LHRH which in turn activates the syncytiotrphoblast to induce the hCG production (Kohdr and Siler-Kohdr 1978). Therefore, there is a possibility that the hCG release induced by beta-agonists may be mediated by production of LHRH by placental cells. However, this would be less likely because almost all the cytotrophoblast was transformed into syncytiotrophoblast within 48 h after plating when we examined the effect of adrenergic drugs (Kliman, Nestler, Sermasi, Sangerand Strauss 1986). In summary, this study demonstrates that betaadrenergic agonists increase the hCG production of cultured syncytiotrophoblast cells through activating beta2-adrenoceptors. Further study is needed to clarify whether or not LHRH may play a role in the hCG effect of beta agonists. References
Barden, T. P., J. B. Peter, I. R. Merkatz: Ritodrine hydrochloride: a betamimetic agent for use in preterm labor. Obstet. Gynecol. 56: 1-6(1980) Barnett, D. B., N. Cook, S. R. Nahorski: Heterogeneity of P-adrenoceptor subtypes in the human placenta. J. Auton. Pharmacol. 2: It was demonstrated that both subtypes of beta 103-110(1982) adrenoceptors exist in the human placenta (Bamett, Cook and Belisle, S., J.-F. Guevin, D. Bellabarba, J.-G. Lehoux:Luteinizing hormone-releasing hormone binds to enriched human placental memNahorski 1982) and that catecholamines stimulate placental branes and stimulates in vitro the synthesis of bioactive human adenylate cyclase by acting on the beta-adrenoceptors (Satoh chorionic gonadotropin. J. Clin. Endocrinol. Metab. 59: 119-126 and Ryan 1971; Whitsett et al. 1980). Cyclic AMP and its (1984) analogs stimulate the hCG production in the human placental Caritis, S. N., D. Carson, D. Greebon, M. McCormick, D. I. Edelstone, tissue and cell cultures (Handwergeret al. 1973; Haninget al. E. Mueller-Heubach: A comparison of terbutaline and ethanol in 1982; Hilf and Merz 1985; Zeitler, Markoff and Handwerger the treatment of preterm labor. Am. J. Obstet. Gynecol. 142:183— 1983; Feinman etal. 1986; Zhou, Yuen and Leung 1987; Ulloa190(1982) Aguirre et al. 1987; Rodway et al. 1988). In a preliminary ex- Caritis, S. N., R. P. Hirsch, A. J. Zeleznik: Adrenergic stimulation of placental progesterone production. J. Clin. Endocrinol. Metab. periment, we found that isoproterenol increased the cAMP 56:969-972(1983) content of the cultured human placental cells used in the pre-
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Horn, metabol. Res. 22 (1990)
Horm. metabol. Res. 22 (1990)
Caritis, S. N.: A pharmacologic approach to the infusion of ritodrine. Satoh, K., K. J. Ryan: Adenyl cyclase in the human placenta. Biochim. Am. J. Obstet. Gynecol. 158:380-384 (1988) Biophys. Acta 244:618-624 (1971) Cemerikic, B., O. Genbaiev, V. Sulovic: In vitro effect of ritodrine on Weiner, N.: Norepinephrine, epinephrine and the sympathomimetic cAMP concentration in human placentas of different gestational amines. In: The Pharmacological Basis of Therapeutics, 7th Ed.; age. Exp. Clin. Endocrinol. 85:223-227 (1985) Gillman, Goodman, Rail and Murad, Ed. Macmillan, New York Feinman, M. A., H. J. Kliman, S. Caltabiano, J. F. Strauss III: 8(1985a), pp. 145-180 Bromo-3',5'-adenosine monophosphate stimulates the endocrine Weiner, N.: Drugs that inhibit adrenergic nerves and block adrenergic activity of human cytotrophoblasts in culture. J. Clin. Endocrinol. receptors. In: The Pharmacological Basis of Therapeutics, 7th Metab. 63:1211-1217(1986) Ed.; Gillman, Goodman, Rail and Murad, Ed. MacMillan, New Handwerger, S., J. Barrett, L. Tyrey, D. Schomberg: Differential effect York(1985b), pp. 181-214 of cyclic adenosine monophosphate on the secretion of human Whitsett, J.A..C.L. Johnson: Adenylate cyclase from human decidua placental lactogen and human chorionic gonadotropin. J. Clin. parietalis. Am. J. Obstet. Gynecol. 133:479-483 (1979) Endocrinol. Metab. 36:1268-1270(1973) Whitsett, J. A., C. L. Johnson, A. Noguchi, C. Darovec-Beckerman, M. Haning, R. V. Jr., L. Choi, A. J. Kiggens, D. L. Kuzma, J. W. Summer- Costello: |3-Adrenergic receptors and catecholamine-sensitive ville: Effects of dibutyryl adenosine 3',5'-monophosphate, adenylate cyclae of the human placenta. J. Clin. Endocrinol. luteinizing hormone-releasing hormone, and aromatase inhibitor Metab. 50:27-32 (1980) on simultaneous outputs of progesterone, 17fl-estradiol, and Ulloa-Aguirre, A., A. M. August, T. G. Golos, L.-C. Kao, N. Sakuragi, human chorionic gonadotropin by term placental explants. J. Clin. H. J. Kliman, J. F. Strauss III: 8-Bromo-adenosine 3',5'-monoEndocrinol. Metab. 55:213-218 (1982) phosphate regulates expression of chorionic gonadotropin and fiHilf, G., W. E. Merz: Influence of cyclic nucleotides on receptor bindbronectin in human cytotrophoblasts. J. Clin. Endocrinol. Metab. ing, immunological activity, and microheterogeneity of human 64:1002-1009(1987) choriogonadotropin synthesized in placental tissue culture. Mol. Zeitler, P., E. Markoff, S. i/andwerg«7Characterization of the syntheCell. Endocrinol. 39:151-159(1985) sis and release of human placental lactogen and human chorionic Iwashita, M., M. Watanabe, T. Adachi, A. Oohira, Y. Shinozaki, Y. gonadotropin by an enriched population of dispersed placental Takeda, S. Sakamoto: Effect of gonadal steroids on cells. J. Clin. Endocrinol. Metab. 57:812-818 (1983) gonadotropin-releasing hormone stimulated human chorionic Zhou, F., B. H. Yuen, P. C. K. Leung: Effects of adenosine 3',5'-cyclic gonadotropin release from trophoblast cells. Placenta 10: 103monophosphate, forskolin, and cholera toxin on hormone pro112(1989) duction in human term placental cells. Am. J. Obstet. Gynecol. Kliman, H. J., J. E. Nestler, E. Sermasi, J. M. Sanger, J. F. Strauss III: 156:420-424(1987) Purification, characterization and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118: 1567-1582(1986) Requests for reprints should be addressed to: Kohdr, G. S., T. Siler-Kohdr:Loca\ization of luteinizing hormone-releasing factor in the human placenta. Fertil. Steril. 29: 523-526 Nobuko Oike, M. D. (1978) Department of Pharmacology Petraglia,F.,A. T. W.Lim, W. Vale:Adenosine 3',5'-monophosphate, Tokyo Women's Medical College prostaglandins, and epinephrine stimulate the secretion of imKawadacho 8-1, Shinjuku-ku munoreactive gonadotropin-releasing hormone from cultured Tokyo 162 (Japan) human placental cells. J. Clin. Endocrinol. Metab. 65:1020-1025 (1987) Rodway, M., F. Zhou, J. Benoit, B. H. Yuen, P. C. K. Leung /Differential effects of 8-bromo-cyclic AMP on human chorionic gonadotropin (hCG), progesterone and estrogen production by term placental cells. Life Sci. 43:1451-1458 (1988)
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Adrenergic Agonists and Placental hCG
Summary The cultured syncytiotrophoblast cells from human first trimester placenta were used to determine the effect of adrenergic agonists on human chorionic gonadotropin (hCG) production in vitro. Beta-adrenergic agonists isoproterenol, ritodrine and isoxsuprine increased the hCG release during the 2 h incubation period, however, alpha-agonists norepinephrine and phenylephrine and a beta1-agonist dobutamine had no effect. The effect of isoproterenol was blocked by propranolol and butoxamine, but less efficiently by phentolamine and atenolol. These results indicate that placental hCG production can be modulated by stimulation of beta-, possibly beta2-adrenoceptors but not by alpha-adrenoceptors. Key words Human Chorionic Gonadotropin - Cultured Placental Cells - Beta2-Adrenoceptor - Ritodrine
Introduction It has been demonstrated that human chorionic gonadotropin (hCG) biosynthesis in placental explants (Handwerger, Barrett, Tyrey and Schomberg 1973; Haning, Choi, Kiggens, Kuzma and Summerville 1982; Hilf and Merz 1985) and placental cells (Zeitler, Markoff and Handwerger 1983; Feinman, Kliman, Caltabiano and Strauss 1986; Zhou, Yuen and Leung 1987; Ulloa-Aguirre, August, Golos, Kao, Sakuragi, Kliman and Strauss 1987; Rodway, Zhou, Benoit, Yuen and Leung 1988) is stimulated by cyclic nucleotides. The placenta has been reported to contain beta adrenergic receptors and catecholamine sensitive adenylate cyclase (Whitsett and Johnson 1979; Whitsett, Johnson, Noguchi, Darovec-Beckerman and Costello 1980). Therefore, there is a possibility that catecholamines may stimulate hCG secretion of placenta through increasing placental cAMP content. However, direct demonstration of such effect of catecholamines is not known.
Horm. metabol. Res. 22 (1990) 188-191 © Georg Thieme Verlag Stuttgart • New York
The purpose of the present study was to show adrenergic agonists enhanced hCG production by placental cells maintained in the culture and to determine the type of adrenergic receptors involved by using several specific adrenergic agonists and antagonists for alpha-, beta-, betaiand beta2-adrenoceptors (Weiner 1985a; Weiner 1985b). Materials and Methods Chemicals Adrenergic agonists and antagonists used are: isoproterenol HC1 (Sigma Co., MO), dl-norepinephrine (Noradrenaline injection®, Sankyo Co., Tokyo), isoxsuprine HC1 (Duvadilan injection®, Daiichi Seiyaku Co., Tokyo), phenylephrine HC1 (Neosynesin injection®, Kowa Co., Nagoya), dobutamine HC1 (Dobutex injection®, Shionogi and Co., Osaka), ritodrine HC1 (Utemerine injection®, Kissei Pharmaceut. Co., Matsumoto), phentolamine mesylate (Regitin injection®, Ciba-Geigy Japan, Tokyo), propranolol HC1 (Inderal injection®, ICI Pharma, Osaka) atenolol (ICI Pharma, Osaka), and butoxamine HC1 (Burroughs Wellcome Co., NC). Hyaluronidase, collagenase, trypsin inhibitor (soybean), DNAse (type 1A), bovine serum albumin (fraction V, BSA) were purchased from Sigma Co. (St. Louis, MO). Medium 199 and fetal calf serum (FCS) were purchased from Gibco Labs. (NY). Other drugs were obtained from commercial sources. Two kinds of media were used for the culture: buffer A contains medium 199 with 25 mM HEPES, 30 mM NaHCO3 and 10% FCS at pH 7.4 and buffer B is the medium where FCS of buffer A was replaced with 10 % BSA. Preparation of trophoblast
cells
This study was approved by the College Human Ethics Research Committee. Trophoblast cells were prepared from fresh placentas, which were obtained from legal abortion at 7 to 12 weeks with informed consent (Iwashita, Watanabe, Adachi, Oohira, Shinozaki, Takeda and Sakamoto 1989). Briefly, the villous tissue was separated from connective tissue and minced on ice. The villi were incubated with collagenase (0.2 mg/ml), hyaluronidase (0.12 mg/ml), trypsin inhibitor (0.02 mg/ml), DNAse (0.02 mg/ml) in buffer B at 37 °C for 30 min in a shaking water bath. The mixture was filtered through nylon mesh (150 urn) and the digestion of the residue was repeated once. The pooled filtrate was centrifuged at 800 x g for 10 min. The precipitated cells were suspended in buffer A at a concentration of 2 x 105 cells/ml. Cell viability estimated by trypan blue exclusion was more than 90 %. The freshly prepared cells suspended in buffer A were plated in 22 mm culture plates (Corning, NY) at 2 x 105 cells/ml/well and maintained for 48 h in a 37 °C incubator with 5 %
Received: 7 Apr. 1989
Accepted: 31 July 1989
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N. Oike1, M. Iwashita2, T. Muraki1, T. Nomoto1, Y. Takeda3 and S. Sakamoto2 1 Department of Pharmacology, 2the Maternal and Perinatal Center, and 3Department of Gynecology and Obstetrics, Tokyo Women's Medical College, Kawadacho, Shinjuku-ku, Tokyo, Japan
Adrenergic Agonists and Placental hCG
Horm. metabol. Res. 22(1990) 189
C02-95 % air. On day of stimulation, the cells were washed twice with 1 ml of buffer B. After addition of 1 ml of buffer B with or without drugs dissolved in 10 ul of saline to the placental cells, the plates were returned to the CO2 incubator. After incubation, the medium was removed and was stored at -20 °C until analysis for hCG.
Results The hCG release into the medium occurred in 2 phases during the 4 h of incubation of cultured trophoblast
Fig. 1 Time course of effect of isoproterenol on the release of hCG from cultured trophoblast cells. Trophoblast cells were cultured for 1,2 and 4 h in the absence (control) and presence of 10~4 M isoproterenol. Each value and vertical bar represent mean ± SE of duplicate incubation from 5 separate experiments. * P < 0.05 vs control (2 h) (Student's t-test)
Fig. 2 Concentration-dependent effect of adrenergic agonists on the hCG release of cultured trophoblast cells. Trophoblast cells were incubated with or without drugs of indicated concentration for 2 h. The results are expressed as % of control (hCG release in the absence of drug additions) which was 38.7 ± 8 . 3 mill/well (mean ± SE, n = 20). Each value and vertical bar represents mean ± SE of duplicate incubations from separate 5 experiments. *P < 0.05, **P < 0.01 vs10~ 7 Mof the same drug (Dunnetttest).
Table 1 Effect of adrenergic antagonists on the hCG release induced by beta adrenergic agonists. Trophoblast cells were cultured with or without additions of drugs of indicated concentrations for 2 h. The results are expressed as % of control (the hCG release without drug additions) which was 37.4 + 6.8 mill/well (mean ± SE, n = 5). The results are mean ± SE of duplicate incubations from 5 separate
Table 2 Effect of atenolol and butoxamine on the hCG release induced by isoproterenol. Trophoblast cells were cultured with or without additions of drugs for 2 h. The results are expressed as % of control (the hCG release without drug additions) which was 53.4 ± 9.0 mill/well (mean + SE, n = 5). The results are the mean ± SE of duplicate incubations from 5 separate experiments.
GYnpriments
Additions
hCG release (% of control) 4
Isoproterenol (10~ M) MSO) ISO + propranolol (10~6M) ISO + propranolol (10" 4 M) ISO + phentolamine(10"5M) ISO + phentolamine(10"4M) Ritodrine (10_4M)(RIT) RIT + propranolol (10"5M) RIT + propranolol (10~4M)
235.4 ± 26.6 133.0 ± 16.2** 122.8 ± 6 . 0 * 217.0 ± 16.0 157.8 ± 9.6* 185.2 ± 9.0 157.2 ±17.8 120.8 ± 10.1*
**P < 0.01 vs isoproterenol (10 4 M) (Student's t-test); *P < 0.05 vs ritodrine (10~4 M) (Student's t-test).
hCG release (% of control)
Additions 4
Isoproterenol (10" M) (ISO) ISO + atenolol (10"5M) ISO + atenolol (10"4M) ISO + butoxamine (10"5M) ISO + butoxamine (10~4M)
154.6 ± 7.5 145.4 ± 12.2 157.5 ±14.4 127.0 ± 4.7 102.6 ± 6.8*
*P < 0.001 vs isoproterenol (Student's t-test).
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cells, in the absence of added adrenergic agonists, the release of hCG into the medium occurred within 1 h after the medium change followed by a slow rate of hCG release (Fig. 1). The addition of 10 -4 M isoproterenol accelerated the hCG release during the first 2 h, however, hCG concentration in the medium showed a plateau thereafter. Since the effect of isohCG assay proterenol was significantly found at 2 h incubation, we deterhCG in the medium was measured by an enzyme im- mined the effects of sympathomimetic drugs by 2 h incubation munoassay kit (Mochida Co., Tokyo) using monoclonal antibody in the following experiments. Fig. 2 shows the dose effect of specific for beta subunit of hCG. various adrenergic agonists. Beta agonists such as isoproterenol, ritodrine and isoxsuprine increased the hCG release in a concentration-dependent fashion between 10" -10" M, Statistics however, alpha-agonists such as norepinephrine and phenyleValues are represented as mean + SE of the indi- phrine and a betai-agonist dobutamine had no effect. At the cated number of separate experiments, each performed with a different trophoblast cell preparation in duplicate. Statistical analysis of the results was performed using Student's t-test, Aspin-Welch test or Dunnett (nonparametric) test.
N. Oike, M. Iwashita, T. Muraki, T. Nomoto, Y. TakedaandS. Sakamoto
highest concentration examined (10 M), the potencies of the drugs were isoproterenol > ritodrine > isoxsuprine > dobutamine, norepinephrine, phenylephrine in that decreasing order when evaluated by Student's t-test or Aspin-Welch test (P < 0.05). This result suggests that beta-adrenergic agonists, except dobutamine, but not alpha adrenergic agonists increased the hCG release from the trophoblast cells obtained from thefirsttrimester placenta. To further characterize the adrenoceptors involved in the hCG release, we examined the effect of beta and alpha adrenergic blockers on the release of hCG induced by beta adrenergic agonists (Table 1). A nonselective beta blocker propranolol inhibited the stimulatory effect of isoproterenol (10~4 M) at both 10~5 and 10~4 M, whereas phentolamine, an alpha-blocker, showed an inhibition at the higher concentration (10 -4 M). The effect of ritodrine was also antagonized by the same concentration of propranolol. These results suggest the involvement of beta-adrenoceptors in the effect of isoproterenol to release hCG. Our result that a beta2-agonist ritodrine increased the hCG release but a betai agonist dobutamine did not, may suggest the involvement of beta2-adrenoceptors in the hCG release. To confirm the role of beta2adrenoceptors in the hCG release, we examined the inhibition of the effect of isoproterenol by atenolol (betai-blocker) and butoxamine (beta2-blocker) (Table 2). Although there was no difference between atenolol and butoxamine in the inhibition of the isoproterenol effect at one-tenth the concentration of isoproterenol, 10" M butoxamine, but not 10" M atenolol, antagonized the hCG release induced by 10" M isoproterenol, suggesting the involvement of beta2-adrenoceptors again. Discussion This study shows that the addition of beta but not alpha-adrenergic agonists to the placental cells maintained in the culture significantly increases the accumulation of hCG in culture medium. Isoproterenol was the most potent, followed by ritodrine and isoxsuprine. The response to isoproterenol was dose-related and was blocked by the betaadrenergic antagonist propranolol more efficiently than by the alpha antagonist phentolamine. These results indicate that the sympathomimetic drugs increased the hCG release by activating the beta-adrenergic receptors. The result that a beta2-agonist ritodrine increased the hCG release but a betai-agonist dobutamine did not, and that the effect of isoproterenol was more effectively blocked by butoxamine than by atenolol, may suggest that beta2-adrenoceptors are involved in the hCG release elicited by adrenergic agonists.
sent study. Therefore, most probably isoproterenol directly activates the beta2-adrenoceptors on the syncytiotrophoblasts, stimulates the cAMP production, and thereby increases the hCG release. A previous study showed that ritodrine increased the cAMP content of the placenta and thatjthis effect was most marked in the first trimester placenta (Cemerikic, Genbacev and Sulovic 1985). Therefore, beta agonists such as ritodrine and isoxsuprine may increase the hCG release through the same mechanism. Beta adrenergic agonists are used for preventing preterm labor by eliciting the relaxation of uterine muscle through acting on beta2-adrenoceptors (Barden, Peter and Merkatz 1980; Caritis, Carson, Greebon, McCormick, Edelstone and Mueller-Heubach 1982; Caritis 1988). Also, beta-adrenergic agonists increased the progesterone production of the term placental cells and this effect may be mediated by increased intracellular cAMP (Caritis, Hirsch and Zeleznik 1983). Our study revealed that the mechanism of beta agonists to increase the hCG production is similar to the case of progesterone production found in the term placental cells. Petraglia, Lim and Vale (1987) reported that epinephrine stimulated the secretion of luteinizing hormonereleasing hormone (LHRH) from cultured human placental cells through beta-adrenoceptors. In addition, it has been reported that LHRH stimulated the hCG release from the cultured human syncytiotrophoblast (Belisle, Guevin, Bellabarba and Lehoux 1984; Iwashita etal. 1989). It is considered that the cytotrophoblast releases LHRH which in turn activates the syncytiotrphoblast to induce the hCG production (Kohdr and Siler-Kohdr 1978). Therefore, there is a possibility that the hCG release induced by beta-agonists may be mediated by production of LHRH by placental cells. However, this would be less likely because almost all the cytotrophoblast was transformed into syncytiotrophoblast within 48 h after plating when we examined the effect of adrenergic drugs (Kliman, Nestler, Sermasi, Sangerand Strauss 1986). In summary, this study demonstrates that betaadrenergic agonists increase the hCG production of cultured syncytiotrophoblast cells through activating beta2-adrenoceptors. Further study is needed to clarify whether or not LHRH may play a role in the hCG effect of beta agonists. References
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Horn, metabol. Res. 22 (1990)
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Adrenergic Agonists and Placental hCG
