D2-dopamine agonists inhibit adenosine 3′ :5′-cyclic monophosphate (cAMP) production in human term trophoblastic cells

Life Sciences, Vol. 55, No. 20, pp. 1545-1552, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/94 $6.00 + .00

Pergamon

0024-3205(94)00315-7

D2-DOPAMINE AGONISTS INHIBIT ADENOSINE 3' : 5'-CYCLIC MONOPHOSPHATE (cAMP) PRODUCTION IN HUMAN TERM TROPHOBLASTIC CELLS C.Vaillancourt, A. Petit, and S. B~lisle. Department of Obstetrics and Gynecology, University of Montreal, Centre de Recherche, Hopital Ste-Justine, Montreal, Quebec, Canada, H3T 1C5. (Received in final form September 7, 1994)

Summary We previously reported that dopamine (DA) acted via D2-dopamine receptors on human trophoblastic cells to inhibit basal and hormonestimulated secretion of human placental lactogen (hPL). We also described that these DA effects were coupled with inhibition of calcium influx. The present study examines the interaction of placental D2dopamine receptor with adenylate cyclase (AC). Incubations of isolated human term trophoblastic cells with R(-)-propylapomorphine (NPA), (+)PPHT, and bromocriptine (3 different D2 agonists) led to time- and dosedependent inhibitions of cAMP production as determined by measuring the conversion of [2-3H]-ATP into [2-3H]-cAMP. The maximal inhibition was reached after 15 min of incubation and was 33 _+ 1 (SE) %, 29 + 3 % and 31 + 1 % for bromocriptine (10-5 M), NPA (10-7 M) and (+)-PPHT (10-8 M) respectively. However, the time- and dose-dependent curves were biphasic with NPA and (_+)-PPHT and the inhibition of cAMP production was abolished at higher agonist concentrations or after time incubations longer than 15 min. These inhibitions were receptor specific since they were reversed by spiperone and haloperidol, two specific dopamine antagonist, and by butaclamol (mix D2/Dl-dopamine antagonists) but not by c~- and I~-adrenergic, D1- and D4-dopaminergic, and 5-HT2-serotonergic antagonists. The results reported here suggest that human placental D2 receptors interact with AC to inhibit its activity. Also, bromocriptine seems a better agonist for the characterization of dopaminergic effects on human placenta. Key Words: placenta, D z - d o p a m i n e , a d e n o s i n e 3 ' : ' 5-cyclic m o n o p h o s p h a t e ( c A M P )

Address requests for reprints to : Dr Serge Belisle, Department of Obstetrics and Gynecology, Centre de Recherche, H6pital Ste-Justine, 3175 Chemin COte SainteCatherine, Montreal, Quebec, Canada, H3T 1C5.

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The presence of dopamine (DA) in the human placenta was first described by Kubota et al (1). We recently reported that, the major effect of DA was the inhibition of human placental lactogen (hPL) release from human trophoblastic cells (2,3) and that this effect was mediated at least by the inhibition of calcium influx through D2dopamine receptors (3). We also characterised the presence of high affinity D2binding sites in membrane preparations (4) and trophoblastic cells (5) of human term placenta. It is now-well established, that the D2-dopamine receptors (6,7,8) reduce adenylyl cyclase (AC) activity (for review see 10,11), and block enhancement of cAMP levels by other agents in lactotrophs cells of anterior pituitary (8,9), melanotrophs cells of intermediate lobe (12), and striatum (13). Moreover, an endocrine analogy between the human placenta and hypothalamic-pituitary axis was proposed several years ago (14-16). However, studies on the involvement of cAMP in the endocrine control of human placental functions have yielded conflicting results (for review see 17) : some groups found no hPL response to cAMP (18,19), while others reported either a stimulatory effect (20-23) or an inhibitory one (24). We have recently investigated the role of AC as a potential cellular mediator of DA-inhibited hPL release. In an initial study, we have previously observed a stimulation of cAMP production by DA (2). We have also demonstrated that this activity of DA was mainly due to interaction with I~-adrenergic receptors rather than interaction with specific dopaminergic receptors (2). To extend our findings we have studied in this report the effect of specific D2-dopamine agonists on cAMP production by human trophoblastic cells. The results suggest that human placental D2 receptors interact with AC. Materials and methods

[2-3H]-adenine (specific activity : 29 Ci/mmol) was purchased from Amersham (Oakville,Ont., Canada). B r o m o c r i p t i n e methanesulfonate, S-(+)-butaclamol, dopamine (DA), ketanserin, 2-(N-phenethyI-N-propyl) amino-5-hydroxytetralin ( (+)PPHT ), prazosin, R(-)-propylapomorphine (NPA), (+)-propranolol, and R-(+)scherring 23390 were obtained from Research Biochemicals Inc (Natick, MA) while clozaril (clozapine) was a gift from Sandoz Canada Inc (Dorval, Quebec, Canada). ATP, cyclic AMP, bacitracin, bovine serum albumin (BSA), DNase I (type IV), forskolin, haloperidol, 3-isobutyl-1 -methylxanthine (IBMX), phenylmethylsulfonylfluoride (PMSF), Percoll, and spiperone were purchased from Sigma (St-Louis, MO) while trypsin (type III) was purchased from Boehringer Mannheim Canada (Laval, Quebec,Canada), Minimum Essential Medium-Eagle's Salts (MEM), Ham's F-10 medium, foetal bovine serum (FBS), L-glutamine and penicillin (10,000 U/ml) - streptomycin (10,000 #g/ml) were bought from Gibco Laboratories (Burlington, Ontario, Canada). Dowex AG 50W-X8 (200-400 mesh) hydrogen form, and neutral alumina AG 7 (100-200 mesh) were from Bio-Rad (Richmond, CA). Cell susoensions This project was approved by the Human Subject Research Committee of our institution. Placentas were freshly obtained from women who delivred normal infants at term (38-41 weeks gestation). Placentas were brought to the laboratory immersed in ice-cold 150 mM NaCI and processed within 30 min as previously described (25) . In brief, trophoblast was separated from the amnion and chorion, minced, washed with ice-cold 150 mM NaCI, and digested at 37°C with Minimum Essential Medium Eagle's Salts (MEM) supplemented with 0.25% trypsin, 500 U/ml DNase I, 200 U/ml

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penicillin, 200 Bg/ml streptomycin, and 2 mM L-glutamine buffered with NaHCO3, pH 7.4, for 6-8 periods of 10 min each. The supernatants of each digestion were collected, filtred, pooled, and washed in MEM without enzymes by centrifugation at 150 x g for 8 min Erythrocytes were removed by further centrifugation at 800 x g for 15 min over a 60% Percoll barrier. Cells were then suspended in the same medium without enzymes, and washed by centrifugation at 150 x g for 8 min. Cells were resuspended in Ham's F-10 supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 Bg/ml streptomycin (pH 7.4) at a concentration of 1 x 106 cells/ml. Cells were used after a 24 hour resting period. Cell viability, estimated before and after experiments by trypan blue exclusion, was greater than 90%. Accumulation of cyclic AMP (cAMP) Intracellular cAMP production was determined by measuring the conversion of [2-3H]ATP into [2-3H]-cAMP, according to the method previously described by Salomon (26) with minor modifications. In short, cells (4 x 106 cells/ml) were incubated for 1 hour at 37°C in Ham's F-10 supplemented with 200 U/ml penicillin, and 200 Bg/ml streptomycin (pH 7.4) containing 4 I.tCi/ml [2-3H]-adenine. Cells were then washed with PBS (0.45 mM CaCI2, 2.7 mM KCI, 1.5 mM KH2PO4, 150 mM NaCI, 0.5 mM MgCI2, 2.5 mM Na2HPO4), pH 7.4, supplemented with 1 mg/ml of each glucose, BSA, and bacitracin, 0.1 mM PMSF, 1 mM MgCI2, and 1 mM IBMX. They were further preincubated in the same PBS buffer for 30 min at 37°C. Except when otherwise stated, the incubations with different drugs were performed at 37°C for 15 min and were stopped by the addition of 10% trichloroacetic acid (TCA) at a final concentration of 5%. [2-3H]-ATP and [2-3H]-cAMP biosynthetized from [2-3H]adenine were separated by chromatography on Dowex AG 50W-X8 (200-400 mesh) hydrogen form and alumina AG7 (100-200 mesh), cAMP formation is expressed as : % conversion = [ [2-3H]-cAMP / ( [2-3H]-cAMP + [2-3H]-ATP) ] X 100 Results

Effect of D2-dopamine agonists on

placental cAMP

production.

We have compared the time- and dose-dependent curves of NPA, (+)-PPHT, and bromocriptine, three D2-dopamine agonists, in isolated human trophoblastic cells. These studies were conducted in the presence of 1 mM IBMX which inhibits phosphodiesterase activity. Thus, the measured cAMP production reflects changes in the rate of synthesis of cAMP rather than changes in its degradation. Figure 1 shows the kinetics of inhibition of cAMP production in response to 10-7M NPA, 10-8M (+)-PPHT, and 10-6M bromocriptine. The inhibition of cAMP production was maximal after 15 min exposure to 10-8M (+)-PPHT and 10-6M bromocriptine. The inhibition reached a plateau and was maintained at 77 % of control (23 % inhibition) after 30min exposure to bromocriptine while the inhibition did not reach a plateau and increased up to 85 % of control (15 % inhibition) after 30 min exposure to (+)-PPHT. Prolonged incubation (more than 30 min) did not lead to further inhibition of basal cAMP production in trophoblastic cells. In contrast to that observed with (+)-PPHT and bromocriptine, we have observed that NPA (10-7 M) diminished intracellular cAMP levels (55 % of control) within only 5 min incubation, followed by a rapid increase, cAMP level still remained inhibited at 67 % of control (33 % inhibition) after 15 min exposure to 10-7 M NPA and inhibition was lost after longer incubation time. Therefore subsequent incubations were all realized at 37°C for 15 min.

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lOO

o

NPA

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100

100q

75

75-

75

BROMOCRIPTINE

a.

"A"

,<

50

"'X"

,

50

15

30

15

TIME (rain)

30

15

TIME (min)

Fig.

30

TIME (min)

1

Kinetics of inhibition of c A M P p r o d u c t i o n by D 2 - d o p a m i n e a g o n i s t s in h u m a n trophoblastic cells. Human trophoblastic cells were prepared as described in Materials and Methods. Cells (4x106 cells/ml) were labelled with 4 pCi/ml of [2-3 H]-adenine, washed, and preincubated 30 rain at 37°C in PBS (pH 7.4) supplemented with 1 mg/ml of each BSA, glucose, bacitracin, and IBMX. Cells were further incubated at 37 °C with 10 ,7 M NPA, 10 .8 M (+)-PPHT, or 10-6 M bromocriptine. The incubations were stopped at different times, and cAMP production was measured as described in Materials and Methods. Results, expressed as % of control, are the mean + SE of triplicate determinations with 3 different placentas. Control at time 0 = 0.533 _+ 0 . 0 6 1 % conversion ATP into cAMP. * p< 0.01 vs control by A N O V A followed by Fisher's PLSD comparison test.

Z

110 "

110

100,1

1 O0

110"

1001

90

o.~

8o

60

90

80

80

70

70

60 -11

-9 NPA (LOG M )

-7

-5

60 -1t

-9

(~)-PPHT ( LOG M )

Fig.

-7

-5

-11

-9

BROMOCRIPTINE

*7

( LOG M ]

2

Dose-dependent e f f e c t of D 2 - d o p a m i n e a g o n i s t s on c A M P p r o d u c t i o n in h u m a n trophoblastic cells. Human trophoblastic cells were isolated, labeled washed, and preincubated as described in legend of Figure 1. Cells were further incubated 15 min at 37°C with increasing concentrations of NPA, (_+)-PPHT ( 10 -11 to 10 -6 M ), or bromocriptine ( 10-11 to 10 5 M). cAMP production was measured as described in Materials and Methods. Results, expressed as % of control, are the mean + SE of triplicate determinations with 3 different placentas. Control after 15 rain incubation= 0.840 + 0.227 % conversion ATP into cAMP.

-5

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Figure 2 shows the dose-dependent inhibitory effect of NPA and (+)-PPHT (10 -11 to 10 -6 M), and bromocriptine (10-11 to 10-5 M) on basal cAMP production in human trophoblastic cells. The maximal inhibition after exposure to 10-5M bromocriptine reached 67 + 1 % of control (33 % inhibition) with ECso of about lnM. This inhibition was maintained at higher agonist concentrations. In contrast to that observed with bromocriptine, dose-response curves were biphasic in presence of NPA and (+)-PPHT. The maximal inhibition was 71 _+4 % and 69 + 1 % of control (29 and 3 1 % inhibition) at 10-7M NPA, and 10-8M (_+)-PPHT respectively. These inhibitions were lost at higher concentrations.

Pharmacology of D2 dopaminergic inhibition of placental cAMP production. The pharmacological specificity of the inhibitory effect of D2dopaminergic agonists (10-7M NPA, 10-8M (_+)-PPHT,and t0-6M bromocriptine) on cAMP production in human trophoblastic cells is shown in Figure 3 and TABLE I. Figure 3 demonstrates that the specific D2-dopamine antagonist spiperone (10-5M) and the mix D2/Dl-dopamine antagonist (+)-butaclamol (10-5M) abolished the inhibition elicited by D 2 agonists on cAMP production. The specificity of inhibitory effects of D2 agonists on cAMP formation was further analysed using bromocriptine (the more stable agonist) and several receptor antagonists. TABLE I shows that haloperidol (10-5M), spiperone (10-5M), and (+)-butaclamol (10-5M), three relatively selective antagonists of D2 dopaminergic receptor, blocked the inhibitory effect of bromocriptine on basal cAMP production, whereas, even at high concentration (10-6 M), clozapine and scherring 23390 (specific D4- and Dl-antagonists), propranolol and prazosin (~- and c~-adrenergic specific antagonists) and ketanserin (a potent 5HT2-serotonergic antagonist) did not affect the D2-dopamine inhibition of cAMP production in human trophoblastic cells.

J-

z

w

Fig. 3 Specificity of D2-dopaminergic agonist effects on cAMP production in human trophoblastic cells. Human trophoblastic cells were isolated, labeled washed, and

preincubated as described in legend of Figure 1. Cells were further incubated 15 min without (control) or with 10-7M NPA, 10-8M (_+)-PPHT,or 10.6 M bromocriptine in combination with 10-5 M spiperone or butaclamol, cAMP production was measured as described in Materials and Methods. Results, expressed as % of control, are the mean _+SE of triplicate determinations with 3 different placentas. Control after 15 min incubation = 0.930 + 0.126 % conversion ATP into cAMP. * p< 0.01 vs control by ANOVAfollowed by Fisher's PLSD comparison test.

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TABLE I Specificity of bromocriptine effect on cAMP production in human trophoblastic cells

cAMP PRODUCTION (% of control) 78 ± 3

Bromocriptine alone

+ ANTAGONISTS

Receptor subtype

haloperidol spiperone (+)-butaclamol

02 D2>5-HT2 D2/D1

91 + 2 95+7 102±7

scherring 23390 clozapine

81 D4

74+5 85+1

ketanserin

5-HT2

78±3

(_+)-propranolol prazosin

~-A ~-A

81 + 2 79+4

-'X-3(*

Human trophoblastic cells were isolated, labeled washed, and preincubated as described in legend of Figure 1. Cells were further incubated 15 min without (control) or with 10 .6 M bromocriptine in combination with 10 .5 M of different antagonists, cAMP production was measured as described in Materials and Methods. Results, expressed as % of control, are the mean _+SE of quadruplicate determinations with 3 different placentas. Control after 15 min incubation = 0.935 + 0.126 % conversion ATP into cAMP. * p< 0.05 vs Bromocriptine alone by ANOVA followed by Fisher's PLSD comparison test.

Discussion

Our results demonstrate that D2-dopamine agonists inhibit human placental cAMP production. Furthermore, in light of our previous observations indicating that dopamine (DA) inhibited human placental lactogen (hPL) release by human trophoblastic cells (2), our present data suggest a role for adenylyl cyclase (AC) as a cellular mediator of this DA effect. In this study we have compared the time- and dose-dependent curves of NPA, (_+)-PPHT and bromocriptine, three D2-dopamine agonists. We observed that the three agonists inhibited basal cAMP production with maximal inhibition of about 30% similar to that observed in other tissues (9, 13). However, we observed that the time- and dose-dependent curves were biphasic with NPA and (_+)-PPHT, since the inhibition of cAMP production was abolished at higher concentration of agonists or after incubation longer than 15 min. These biphasic profiles of inhibition could be explained by 1) a toxic effect of these drugs or their metabolites (rapid oxydation), 2) a non-specific effect on other receptors types (e.g. c~-

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and 13-adrenergic and 5-HT-serotonergic) (5, 25), and 3) a rapid desensitization of the receptor. Our results do not support one hypothesis more than another. Together, the results suggest that NPA and (+)-PPHT were not enough stable analogues for the charaterization of dopaminergic effects on human placenta. However, the time- and dose-dependent studies demonstrated that bromocriptine seems a better agonist for the characterization of placental dopaminergic effects since, in contrast to that observed with NPA and (+)-PPHT, inhibition of cAMP production by bromocriptine was maintained at high agonist concentrations and for longer incubation times. The pharmacological study of the inhibitory effect of D2-dopaminergic agonists on human placental cAMP production demonstrated that this inhibition was specific for D2-receptors ; the effects of D2 agonists were completely reversed by spiperone, a specific D2-dopamine antagonist, and by (+)-butaclamol, a mix D2/Dl-dopamine antagonist. Moreover, the pharmacological profile of the inhibitory effect of bromocriptine on placental cAMP production clearly demonstrated that this inhibition was receptor specific since bromocriptine effect was completely reversed by haloperidol, a highly specific D2-dopamine antagonist. D1- and D4-dopamine antagonists (scherring 23390 and clozapine), c~1- and !5-adrenergic antagonists (prazosin and (+)-propranolol), and 5-HT2-serotonergic antagonist (ketanserin) even at higher concentrations (10-5M) did not significantly affect bromocriptine inhibition of human placental cAMP production. The effects of cAMP in endocrine control of human placental functions have yielded conflicting results. The results presented in this study support a role for cAMP as second messager controlling hPL secretion by the placenta since receptors that mediated cAMP inhibition also mediated hPL release inhibition (2) and that receptors inducing cAMP production also stimulated hPL release (22). In conclusion, the data presented in this study reinforce the functional correlation existing between the cytotrophoblast-syncytiotrophoblast and the hypothalamo-pituitary axis, since in these two systems, DA-agonists inhibited cAMP production via the D2-receptor subtypes.

Acknowledgements We thank Hugues Beaudry for technical assistance. This work was supported by a grant from Medical Research Council of Canada (MRC) and Biop~dia Inc and studentship to C.V. from MRC Canada.

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