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Adenosine 3′:5′-cyclic monophosphate (cAMP) is not the mediator of kappa opiate effect on human placental lactogen release
Life Sciences, Vol. 49, pp. 465-472 Printed in the U.S.A.
Pergamon Press
ADENOSINE 3':5'-CYCLIC MONOPHOSPHATE (cAMP) IS NOT THE MEDIATOR OF KAPPA OPIATE EFFECT ON HUMAN PLACENTAL LACTOGEN RELEASE A. Petit 1, N. Gallo-Payet~, J-G. Lehoux 2, D. Bellabarba 2, and S. B~lisle' Department of Obstetric and Gynecology, University of Montreal, Montreal (QuObec), Canada1, and Faculty of Medecine, University of Sherbroohe, Sherbrooke (Quebec), Canada2. (Received in final form June 6, 1991)
Summary We previously reported that kappa opiates stimulated the release of human placental lactogen (hPL) from human placental cells. In this study, we investigated the role of adenylate cyclase as a potential cellular mediator of such an effect. Incubations with ethylketocyclazocine (EKC) led to a time- and dose-dependent inhibition of adenylate cyclase activity. The maximal inhibition was 45 + 5% of control value after 15 rain exposure to 107M EKC. This inhibition was reversed by opiate antagonist naloxone and was specific to kappa opiate type. Preincubation of human trophoblastic cells with 0.1 I~g/ml Islet-Activating-Protein (lAP; also called pertussis toxin) did not modify basal adenylate cyclase activity but abolished the inhibition of adenylate cyclase activity by EKC, indicating that the effect of opiates on cAMP production was mediated by an lAP-sensitive GTP binding protein. Also, lAP stimulated basal hPL release; the control levels were 22,4 ng/ml and 46,5 ng/ml without and with lAP respectively. However, the EKC-stimulated hPL levels were unchanged by preincubation with lAP. This difference in cAMP and hPL response in lAP-treated cells suggested that the opiate receptors are not directly coupled to adenylate cyclase. This hypothesis was confirmed by 1) experiments on placental membranes showing that in absence of the cytoplasmic elements (membranes only), EKC had no effect on membrane adenylate cyclase and 2) experiments on placental cells showing that dibutyryl-cAMP (dbcAMP) stimulated hPL release. The presence of kappa opiate binding sites has been demonstrated in membrane preparations (1-3), perifusion slices (4), and cultured cells (5) of human placentas. These receptors have been solubilized (6,7) and partially purified (8-10). The receptor appears to be a glycoprotein with a molecular weight of 63,000 daltons, even if the purified preparation did not attain homogeneity (10). The role and action mechanisms of this receptor on placental functions are not well known. In a previous report, we observed that kappa opiate agonists stimulated human placental lactogen (hPL) release from cultured trophoblastic cells (5). More recently, Porth~ et ai reported that the human placental kappa opiate receptor was associated with a GTP binding protein Address requests for repnnts to: Dr Serge Bdlisle, Department of Ob~etdcs and Gynecology, University of Montrdal, Quebec, Canada, H3C 3J7 0 0 2 4 - 3 2 0 5 / 9 1 $ 3 . 0 0 + .00 Copyright (c) 1991 Pergamon Press plc
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(11). In light of the latter suggestion that cAMP could stimulate hPL release in human placenta (12-14), we postulated that opiate receptors could be coupled to a GTP binding protein, thus modulating cAMP production and stimulating hPL release. In the present study, we demonstrate that the opiate-stimulated human placental lactogen release is not due to a direct modulation of adenylate cyclase by receptor/ transducerleffector interactions. METHODS [2-~H]-adenine (S.A.: 15.5 Ci/mmol) was purchased from New England Nuclear (Boston, MA) while [SH]-cAMP and [(x-3=P]-ATP were purchased from Amersham (Oakville, Ont., Canada). The following hormones and analogues were used: (D-Ala 2, NMe-Phe 4, Gly-olS)enkephalin (DAGO) and (D-Ser=-Leu-Thr ~) enkephalin (DSLET) [Peninsula Laboratories, San Carlos, CA], naloxone [New England Nuclear], U-50,488H [Upjohn, Montr(~al, Qu6bec, Canada], Dynl., [gift of Dr Simon Lemaire], ethylketocyclazocine (EKC) [Steding Winthrop Research Institute, Renssaler, NY], and isoproterenol [Research Biochemicals Incorporated, Natrick, MA]. Trypsin (type III), DNase I (type IV), bovine serum albumin (BSA), Islet-Activating- Protein (lAP), isobutylmethylxanthine (IBMX), adenosine deaminase (type VII), creatine phosphokinase (type I), myokinase, phosphocreatine, and forskolin were purchased from Sigma (St-Louis, MO). Antibiotics (10 000 U/ml Penicillin - 10 000 I~g/ml Streptomycin) and Minimum Essential Medium-Earle's salts (MEM) were bought from Gibco Laboratories (Burlington, Ontario, Canada) while Ham's F-10 was obtained from Flow Laboratories (Richmond, CA). Dowex AG 50W-X8 (200-400 mesh) hydrogen form, and neutral alumina AG 7 (100-200 mesh) were from Bio-Rad (Richmond, CA). Percoll and hPL RIA kits were obtained from Pharmacia (Dorvai, Quebec, Canada). Culture of trophoblastic cells Tissues from placentas were prepared according to a technique we previously described (5). Placental tissue was separated from the amnion and chorion, minced, washed with ice-cold NaCI 0.9%, and digested with Minimum Essential Medium Earle's salts (MEM) supplemented with 0.25% trypsin, 500 U/ml DNase I, 200 units/ml penicillin, and 200 I~g/ml streptomycin buffered with NaHCO3 (pH 7.4), for 8-10 periods of 10 min each at 37°C. The supematant of each digestion was collected, filtered, 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 over a 60% Percoll barrier. The cells were then suspended in Ham's F-10 supplemented with 10% fetal bovine serum, 200 units/ml penicillin, and 200 I~g/ml streptomycin (pH 7.4) in 35 xl0 mm Falcon dishes (no. 3001, Becton Dickinson, Oxnard, CA) at a concentration of 750 000 cells in 1.5 ml. The cells were cultured at 37°C for 48 h under an atmosphere of 95% air and 5% CO2. The medium was changed after 24 h incubation and cell viability was assessed by trypan blue dye exclusion. Preparation of the placental membranes Placental cells were prepared as described above. The cells were resuspended by scraping with a policeman in 50 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1 mM PMSF and left in ice for 15 rain. The cells were then homogenized with teflon potter and centrifuged 15 min at 600 x g to remove cellular debris. The supematant was centrifuged at 25000 x g for 15 min. The pellet was resuspended in 50 mM Tris-HCI (pH 7.4) 5 mM EDTA, 0,1 mM PMSF and kept frozen in liquid nitrogen until used. Acqumulation of cAMP Accumulation of cAMP was measured by the conversion of [3H]-ATP into [3H]-cAMP as previously described (15) with minor modifications. In brief, cells were incubated 1 h at 37°C in Ham's F-10 medium supplemented with 10 % fetal calf serum with 2.5 Ci/ml [2-SH]-adenine. Cells were then washed with 2 x 2 ml PBS (0.45 nM CaCI 2, 2.7
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mM KCI, 1.5 mM KH=PO4, 150 mM NaCI, 0.5 mM MgCI=, 2.5 mM Na~HPO4), pH 7.4, and preincubated 1 h at 37~C in PBS supplemented with 1 mg/ml BSA, 1 mg/ml glucose, and 1 mM IBMX. The incubations were performed at 37°C and stopped by aspiration of the buffer and addition of 1 ml 5% perchloric acid to dishes. [2-3H]- ATP and [2-3HI-cAMP biosynthetized from [2-SH]-adenine were separated by chromatography on Dowex AG 50W-X8 (200-400 mesh) hydrogen form and alumina AG 7 (100-200 mesh). Adenylate cyclase assay Adenylate cyclase activity was measured on placental membranes (25-35 I~g protein) as previously described (15, 16). The incubation medium (100 I~1) was composed of: 50 mM Tris-HCI (pH 7,4), 1 mM IBMX, 1 mM cAMP, 0.25 mM ATP, 0.07 I~Ci [3H]-cAMP, 10 t~Ci/ml [o~-~P]-ATP 1 mg/ml phosphocreatine, 5 U/ml adenosine deaminase, 36 U/ml creatine kinase, 1 mM MgCI2, and 10SM GTP. The reaction was initiated by addition of membranes, conducted for 15 min at 30°C and stopped by addition of 400 ILl of 2% SDS. Column recoveries of [3H]-cAMP varied from 80 to 90%. All determinations were performed in triplicate and the coefficient of variation was less than 10% in replicate samples. Hormone assays and statistics Human placental lactogen release was estimated by determination of content in extracellular milieu of placental cell incubations. Cells were incubated 1 h at 37 C in PBS supplemented with 1 mg/ml BSA, 1 mg/ml glucose, and 1 mM IBMX. The milieu was collected and kept frozen at -20 C until assayed. Extracellular hPL content was measured by RIA from Pharmacia. The sensitivity of the test was 50 ng/ml and the cross-reactivity of antibody used was < 0.5% and < 0.06% for hGH and PRL respectively. Statistical evaluation of the results was done by Student's t test. RESULTS The effects of EKC on cAMP production by human trophoblastic cells are shown in figures 1 and 2 whereas figure 3 shows the effect of lAP on this activity and hPL release. In preliminary studies, we determined that EKC inhibited cAMP production in a time-dependent manner; the inhibition of cAMP production was significant (p < 0.05) after 5 min exposure to EKC and was maximal (45:1: 5%) after 15 min (not shown). Prolonged incubations did not lead to a further inhibition of adenylate cyclase activity. Therefore, all incubations were realized at 37°C for 15 min. Figure 1 shows the dose-dependent inhibitory effect of EKC on cAMP production. The inhibition was significant (p < 0.05) with as low as 10"°M EKC and was maximal with 107M EKC (45 + 5 %). This inhibition was significantly (p < 0.01) reversed by the addition of the antagonist naloxone to 75% of basal value. Naloxone alone at 105M had no effect on cAMP accumulation. EKC also inhibited forskolin-stimulated cAMP production (result not shown). The specificity of the inhibitory effect of EKC was demonstrated in figure 2. This figure shows that the kappa opiate agonists U-50,488H (488H) and Dyn,.,s have the same inhibitory effect than EKC on cAMP production whereas DAGO and DSLET that are respectively mu and delta agonists did not affect placental cAMP production. Preincubation of trophoblastic cells with lAP did not affect basal cAMP production but the inhibitory effect of EKC was reversed (Fig. 3A). However, lAP induced a significant increase (2 fold) of the basal hPL release without changing the EKC stimulated levels (Fig. 3B). Figure 4 shows that EKC and dbcAMP stimulated hPL release by cultured human trophoblastic cells 2 and 3.3 fold respectively (p < 0.01). Forskolin (not shown) also stimulated hPL release in these cells. Figure 5 shows the effect of EKC on adenylate cyclase activity on trophoblastic membrane preparations. EKC had no effect on cyclase activity whereas forskolin and isoproterenol significantly
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increased this activity, the level of cAMP increasing from 152 fmol/min/mg prot. in control conditions to 320 and 368 fmol/min/mg prot. when incubated with 10"M forskolin and 105M isoproterenol respectively (p < 0.05). Also, EKC had no effect on forskolin-and isoproterenol-stimulated placental adenylate cyclase activity.
Vol. 49, No. 6, 1991
Opiates & Human Placental Lactogen Release
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469
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Opiates & Human Placental
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Vol, 49, No. 6, 1991
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DISCUSSION Our results demonstrate that in human placental cells kappa opiates induce inhibition of cAMP production via a G,-protein sensitive to lAP and stimulate hPL release by a mechanism which is uncoupled to cAMP production. The specificity of opiate activity for kappa type was in good agreement with data from our previous binding studies and hPL release experiments (5). The sensitivity of opiate effects on cAMP production to lAP suggests that the human placental opiate receptor is coupled to a G~-Iike GTP binding protein. The stimulation of basal hPL release in lAP treated cells suggests that basal hPL release is under a constant inhibitory effect mediated by a G= inhibitory GTP binding protein. A large number of hormones and neurotransmitters has been found to modify cAMP level (for review see 17). However, nothing is known on the role of these molecules on hPL release. Thus, the mechanism of tonic inhibition of hPL remain unknown. However, the differences in cAMP production and hPL release suggest that the effect of EKC on hPL release is not directly coupled to adenylate cyclase activity inhibition. Also, we observed a stimulatory effect of forskolin and dbcAMP on hPL release. The experiments on membrane preparations are in good agreement with this conclusion since EKC was without effect in the absence of intracellular components. The stimulatory effects of forskolin and isoproterenol demonstrated that the adenylate cyclase pathway was still intact in our membrane preparations. This result is in good agreement with previous results demonstrating the absence of kappa opiate effects on basal and forskolin-stimulated adenylate cyclase activity in the guinea pig cerebellum (18). However, an inhibitory effect of opiates was found in rabbit cerebellum (18) and rat spinal cord (19) suggesting differential effect of opiates depending on opiate type, species and tissues studied. Moreover, conflicting results were reported concerning opiate effects on the same tissue of the same specie (18,20). The putative intracellular component responsible for inhibiton of cAMP production may be cGMP since cGMP is known to play a role in opiate analgesia (for review see 21) and cGMP-stimulated phosphodiesterases can inhibit cAMP production (22). it would seem that cAMP is not the mediator of hPL response to kappa opiates in human placenta. Since the hydrolysis of phosphatidylinositol(4,5)P= is not implicated in the hPL response to opiates (personal unpublished data) and since the ionic environment seems to be important in the modulation of hPL release (23,24), it will be important to investigate the other aspects of kappa opiate effects, such as cGMP (20) and ionic mechanisms (24-26) to clearly understand the mechanisms of action of kappa opiates in hPL response in human term trophoblastic cells.
ACKNOWLEDGEMENTS This work was supported by a grant from the Medical Research Concil of Canada. We thank Mrs Paulette Mercier and Marie-Claude Gaudreau for technical assistance.
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A. VALE'R'E, G. PONTONNIER and J. CROS. FEBS lett. 103362-365 (1979). A. VALETTE, J.M. RENI~, G. PONTONNIER and J. CROSo Biochem. PharmacoL 2._992657-2662 (1980). G. PORTHI~, A. VALET'rE and J. CROSo Biochem. Biophys. Res. Commun. 101 1-6 (1981).
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A. VALETTE, M. TAFANI, G. PORTHi~, G. PONTONNIER and J. CROS. Life Sci. 3._335.23-526 (1983). S. BELISLE, A. PETIT, N. GALLO-PAYET, D. BELLABARBA, J.-G. LEHOUX and S. LEMAIRE. J. Clin. Endocrinol. Metab. 6._66283-289 (1988). M.S. AHMED, W.L. BYRNE and W.A. KLEE. Placenta. Suppl.3115-121 (1981). M.S. AHMED. Membr. Biochem. 535-47 (1983). M.S. AHMED and A.G. CAVINATO. Trophoblast Res. 2279-287 (1987). A. VALE'I-I'E, P. ROUGE, E. COULAIS, G. PONTONNIER, J. CROS and E.J. SIMON. Ufe Sci. 40535-540 (1987). M.S. AHMED, D.-H. ZHOU, A.G. CAVlNATO, and D. MAULIK. Life Sci. 4.~4 861-871 (1989). G. PORTHE, B. FRANCE'S, B. VERRIER, J. CROS, and J.-C. MEUNIER. Life Sci. 4._33559-567 (1988). F. WELSCH. Res. Comm. Chem. Pathol. Pharmacol. 2._4468-73 (1986). I. HARMAN, A. COSTELLO, A. SANE, and S. HANDWERGER. Endocrinology 12159-63 (1987). Y.Q. WU, E.V. JORGENSEN, and S. HANDWERGER. Endocrinology 123 1879-1884 (1987). Y. SALOMON, C. LONDOS, and M. RODBELL. Anal. Biochem. 5.._.88541-548 (1974). M. TENCI~ and A. PETIT. Mol. Cell. Endocrinol. 6._33111-119 (1989). K.H. JAKOBS. Mol. Cell. Endocrinol. 1....66147-156 (1979) J. POLASTRON, M.-J. BOYER, Y. QUERTERMONT, J.P. THOUVENOT, J,-C. MEUNIER, and P. JAUZAS. J. Neurochem. 5..~4562-570 (1990). B. ATTALI, D. SAYA, and Z. VOGEL. J. Neurochem. 5.__22360-369 (1989). C.S. KONROY and S.R. CHILDERS. Mol. Pharmacol. 3._66627-633 (1989). R.E. WEST and R.J. MILLER. Brit. Med. Bull. 3...9953-58 (1983). R.T. MACFARLAND, B.D. ZELUS, and J.A. Beavo. J. Biol. Chem. 266136-142 (1991) V.J. CHOY and W.B. WATKINS. J. Endocdnol. 6.99349-358 (1976). Z. HOCHBERG, T. BICK, R. PERLMAN, M. LAHAV, and D. BARZILAI. Mol. Cell. Endocrinol. 3.__77359-362 (1984). R.A. NORTH. Trends in NeuroSci. 9114-117 (1986). I. MCFADZEN. Neuropeptides 1_! 173-180 (1988).
Pergamon Press
ADENOSINE 3':5'-CYCLIC MONOPHOSPHATE (cAMP) IS NOT THE MEDIATOR OF KAPPA OPIATE EFFECT ON HUMAN PLACENTAL LACTOGEN RELEASE A. Petit 1, N. Gallo-Payet~, J-G. Lehoux 2, D. Bellabarba 2, and S. B~lisle' Department of Obstetric and Gynecology, University of Montreal, Montreal (QuObec), Canada1, and Faculty of Medecine, University of Sherbroohe, Sherbrooke (Quebec), Canada2. (Received in final form June 6, 1991)
Summary We previously reported that kappa opiates stimulated the release of human placental lactogen (hPL) from human placental cells. In this study, we investigated the role of adenylate cyclase as a potential cellular mediator of such an effect. Incubations with ethylketocyclazocine (EKC) led to a time- and dose-dependent inhibition of adenylate cyclase activity. The maximal inhibition was 45 + 5% of control value after 15 rain exposure to 107M EKC. This inhibition was reversed by opiate antagonist naloxone and was specific to kappa opiate type. Preincubation of human trophoblastic cells with 0.1 I~g/ml Islet-Activating-Protein (lAP; also called pertussis toxin) did not modify basal adenylate cyclase activity but abolished the inhibition of adenylate cyclase activity by EKC, indicating that the effect of opiates on cAMP production was mediated by an lAP-sensitive GTP binding protein. Also, lAP stimulated basal hPL release; the control levels were 22,4 ng/ml and 46,5 ng/ml without and with lAP respectively. However, the EKC-stimulated hPL levels were unchanged by preincubation with lAP. This difference in cAMP and hPL response in lAP-treated cells suggested that the opiate receptors are not directly coupled to adenylate cyclase. This hypothesis was confirmed by 1) experiments on placental membranes showing that in absence of the cytoplasmic elements (membranes only), EKC had no effect on membrane adenylate cyclase and 2) experiments on placental cells showing that dibutyryl-cAMP (dbcAMP) stimulated hPL release. The presence of kappa opiate binding sites has been demonstrated in membrane preparations (1-3), perifusion slices (4), and cultured cells (5) of human placentas. These receptors have been solubilized (6,7) and partially purified (8-10). The receptor appears to be a glycoprotein with a molecular weight of 63,000 daltons, even if the purified preparation did not attain homogeneity (10). The role and action mechanisms of this receptor on placental functions are not well known. In a previous report, we observed that kappa opiate agonists stimulated human placental lactogen (hPL) release from cultured trophoblastic cells (5). More recently, Porth~ et ai reported that the human placental kappa opiate receptor was associated with a GTP binding protein Address requests for repnnts to: Dr Serge Bdlisle, Department of Ob~etdcs and Gynecology, University of Montrdal, Quebec, Canada, H3C 3J7 0 0 2 4 - 3 2 0 5 / 9 1 $ 3 . 0 0 + .00 Copyright (c) 1991 Pergamon Press plc
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(11). In light of the latter suggestion that cAMP could stimulate hPL release in human placenta (12-14), we postulated that opiate receptors could be coupled to a GTP binding protein, thus modulating cAMP production and stimulating hPL release. In the present study, we demonstrate that the opiate-stimulated human placental lactogen release is not due to a direct modulation of adenylate cyclase by receptor/ transducerleffector interactions. METHODS [2-~H]-adenine (S.A.: 15.5 Ci/mmol) was purchased from New England Nuclear (Boston, MA) while [SH]-cAMP and [(x-3=P]-ATP were purchased from Amersham (Oakville, Ont., Canada). The following hormones and analogues were used: (D-Ala 2, NMe-Phe 4, Gly-olS)enkephalin (DAGO) and (D-Ser=-Leu-Thr ~) enkephalin (DSLET) [Peninsula Laboratories, San Carlos, CA], naloxone [New England Nuclear], U-50,488H [Upjohn, Montr(~al, Qu6bec, Canada], Dynl., [gift of Dr Simon Lemaire], ethylketocyclazocine (EKC) [Steding Winthrop Research Institute, Renssaler, NY], and isoproterenol [Research Biochemicals Incorporated, Natrick, MA]. Trypsin (type III), DNase I (type IV), bovine serum albumin (BSA), Islet-Activating- Protein (lAP), isobutylmethylxanthine (IBMX), adenosine deaminase (type VII), creatine phosphokinase (type I), myokinase, phosphocreatine, and forskolin were purchased from Sigma (St-Louis, MO). Antibiotics (10 000 U/ml Penicillin - 10 000 I~g/ml Streptomycin) and Minimum Essential Medium-Earle's salts (MEM) were bought from Gibco Laboratories (Burlington, Ontario, Canada) while Ham's F-10 was obtained from Flow Laboratories (Richmond, CA). Dowex AG 50W-X8 (200-400 mesh) hydrogen form, and neutral alumina AG 7 (100-200 mesh) were from Bio-Rad (Richmond, CA). Percoll and hPL RIA kits were obtained from Pharmacia (Dorvai, Quebec, Canada). Culture of trophoblastic cells Tissues from placentas were prepared according to a technique we previously described (5). Placental tissue was separated from the amnion and chorion, minced, washed with ice-cold NaCI 0.9%, and digested with Minimum Essential Medium Earle's salts (MEM) supplemented with 0.25% trypsin, 500 U/ml DNase I, 200 units/ml penicillin, and 200 I~g/ml streptomycin buffered with NaHCO3 (pH 7.4), for 8-10 periods of 10 min each at 37°C. The supematant of each digestion was collected, filtered, 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 over a 60% Percoll barrier. The cells were then suspended in Ham's F-10 supplemented with 10% fetal bovine serum, 200 units/ml penicillin, and 200 I~g/ml streptomycin (pH 7.4) in 35 xl0 mm Falcon dishes (no. 3001, Becton Dickinson, Oxnard, CA) at a concentration of 750 000 cells in 1.5 ml. The cells were cultured at 37°C for 48 h under an atmosphere of 95% air and 5% CO2. The medium was changed after 24 h incubation and cell viability was assessed by trypan blue dye exclusion. Preparation of the placental membranes Placental cells were prepared as described above. The cells were resuspended by scraping with a policeman in 50 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1 mM PMSF and left in ice for 15 rain. The cells were then homogenized with teflon potter and centrifuged 15 min at 600 x g to remove cellular debris. The supematant was centrifuged at 25000 x g for 15 min. The pellet was resuspended in 50 mM Tris-HCI (pH 7.4) 5 mM EDTA, 0,1 mM PMSF and kept frozen in liquid nitrogen until used. Acqumulation of cAMP Accumulation of cAMP was measured by the conversion of [3H]-ATP into [3H]-cAMP as previously described (15) with minor modifications. In brief, cells were incubated 1 h at 37°C in Ham's F-10 medium supplemented with 10 % fetal calf serum with 2.5 Ci/ml [2-SH]-adenine. Cells were then washed with 2 x 2 ml PBS (0.45 nM CaCI 2, 2.7
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mM KCI, 1.5 mM KH=PO4, 150 mM NaCI, 0.5 mM MgCI=, 2.5 mM Na~HPO4), pH 7.4, and preincubated 1 h at 37~C in PBS supplemented with 1 mg/ml BSA, 1 mg/ml glucose, and 1 mM IBMX. The incubations were performed at 37°C and stopped by aspiration of the buffer and addition of 1 ml 5% perchloric acid to dishes. [2-3H]- ATP and [2-3HI-cAMP biosynthetized from [2-SH]-adenine were separated by chromatography on Dowex AG 50W-X8 (200-400 mesh) hydrogen form and alumina AG 7 (100-200 mesh). Adenylate cyclase assay Adenylate cyclase activity was measured on placental membranes (25-35 I~g protein) as previously described (15, 16). The incubation medium (100 I~1) was composed of: 50 mM Tris-HCI (pH 7,4), 1 mM IBMX, 1 mM cAMP, 0.25 mM ATP, 0.07 I~Ci [3H]-cAMP, 10 t~Ci/ml [o~-~P]-ATP 1 mg/ml phosphocreatine, 5 U/ml adenosine deaminase, 36 U/ml creatine kinase, 1 mM MgCI2, and 10SM GTP. The reaction was initiated by addition of membranes, conducted for 15 min at 30°C and stopped by addition of 400 ILl of 2% SDS. Column recoveries of [3H]-cAMP varied from 80 to 90%. All determinations were performed in triplicate and the coefficient of variation was less than 10% in replicate samples. Hormone assays and statistics Human placental lactogen release was estimated by determination of content in extracellular milieu of placental cell incubations. Cells were incubated 1 h at 37 C in PBS supplemented with 1 mg/ml BSA, 1 mg/ml glucose, and 1 mM IBMX. The milieu was collected and kept frozen at -20 C until assayed. Extracellular hPL content was measured by RIA from Pharmacia. The sensitivity of the test was 50 ng/ml and the cross-reactivity of antibody used was < 0.5% and < 0.06% for hGH and PRL respectively. Statistical evaluation of the results was done by Student's t test. RESULTS The effects of EKC on cAMP production by human trophoblastic cells are shown in figures 1 and 2 whereas figure 3 shows the effect of lAP on this activity and hPL release. In preliminary studies, we determined that EKC inhibited cAMP production in a time-dependent manner; the inhibition of cAMP production was significant (p < 0.05) after 5 min exposure to EKC and was maximal (45:1: 5%) after 15 min (not shown). Prolonged incubations did not lead to a further inhibition of adenylate cyclase activity. Therefore, all incubations were realized at 37°C for 15 min. Figure 1 shows the dose-dependent inhibitory effect of EKC on cAMP production. The inhibition was significant (p < 0.05) with as low as 10"°M EKC and was maximal with 107M EKC (45 + 5 %). This inhibition was significantly (p < 0.01) reversed by the addition of the antagonist naloxone to 75% of basal value. Naloxone alone at 105M had no effect on cAMP accumulation. EKC also inhibited forskolin-stimulated cAMP production (result not shown). The specificity of the inhibitory effect of EKC was demonstrated in figure 2. This figure shows that the kappa opiate agonists U-50,488H (488H) and Dyn,.,s have the same inhibitory effect than EKC on cAMP production whereas DAGO and DSLET that are respectively mu and delta agonists did not affect placental cAMP production. Preincubation of trophoblastic cells with lAP did not affect basal cAMP production but the inhibitory effect of EKC was reversed (Fig. 3A). However, lAP induced a significant increase (2 fold) of the basal hPL release without changing the EKC stimulated levels (Fig. 3B). Figure 4 shows that EKC and dbcAMP stimulated hPL release by cultured human trophoblastic cells 2 and 3.3 fold respectively (p < 0.01). Forskolin (not shown) also stimulated hPL release in these cells. Figure 5 shows the effect of EKC on adenylate cyclase activity on trophoblastic membrane preparations. EKC had no effect on cyclase activity whereas forskolin and isoproterenol significantly
468
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increased this activity, the level of cAMP increasing from 152 fmol/min/mg prot. in control conditions to 320 and 368 fmol/min/mg prot. when incubated with 10"M forskolin and 105M isoproterenol respectively (p < 0.05). Also, EKC had no effect on forskolin-and isoproterenol-stimulated placental adenylate cyclase activity.
Vol. 49, No. 6, 1991
Opiates & Human Placental Lactogen Release
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469
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DISCUSSION Our results demonstrate that in human placental cells kappa opiates induce inhibition of cAMP production via a G,-protein sensitive to lAP and stimulate hPL release by a mechanism which is uncoupled to cAMP production. The specificity of opiate activity for kappa type was in good agreement with data from our previous binding studies and hPL release experiments (5). The sensitivity of opiate effects on cAMP production to lAP suggests that the human placental opiate receptor is coupled to a G~-Iike GTP binding protein. The stimulation of basal hPL release in lAP treated cells suggests that basal hPL release is under a constant inhibitory effect mediated by a G= inhibitory GTP binding protein. A large number of hormones and neurotransmitters has been found to modify cAMP level (for review see 17). However, nothing is known on the role of these molecules on hPL release. Thus, the mechanism of tonic inhibition of hPL remain unknown. However, the differences in cAMP production and hPL release suggest that the effect of EKC on hPL release is not directly coupled to adenylate cyclase activity inhibition. Also, we observed a stimulatory effect of forskolin and dbcAMP on hPL release. The experiments on membrane preparations are in good agreement with this conclusion since EKC was without effect in the absence of intracellular components. The stimulatory effects of forskolin and isoproterenol demonstrated that the adenylate cyclase pathway was still intact in our membrane preparations. This result is in good agreement with previous results demonstrating the absence of kappa opiate effects on basal and forskolin-stimulated adenylate cyclase activity in the guinea pig cerebellum (18). However, an inhibitory effect of opiates was found in rabbit cerebellum (18) and rat spinal cord (19) suggesting differential effect of opiates depending on opiate type, species and tissues studied. Moreover, conflicting results were reported concerning opiate effects on the same tissue of the same specie (18,20). The putative intracellular component responsible for inhibiton of cAMP production may be cGMP since cGMP is known to play a role in opiate analgesia (for review see 21) and cGMP-stimulated phosphodiesterases can inhibit cAMP production (22). it would seem that cAMP is not the mediator of hPL response to kappa opiates in human placenta. Since the hydrolysis of phosphatidylinositol(4,5)P= is not implicated in the hPL response to opiates (personal unpublished data) and since the ionic environment seems to be important in the modulation of hPL release (23,24), it will be important to investigate the other aspects of kappa opiate effects, such as cGMP (20) and ionic mechanisms (24-26) to clearly understand the mechanisms of action of kappa opiates in hPL response in human term trophoblastic cells.
ACKNOWLEDGEMENTS This work was supported by a grant from the Medical Research Concil of Canada. We thank Mrs Paulette Mercier and Marie-Claude Gaudreau for technical assistance.
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