European Journal of Pharmacology, 218 (1992) 205-211
205
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52557
Partial dopamine 0 2 receptor agonists antagonize prolactin-regulating D 2 receptors in a transfected clonal cell line ( G H 4 Z R 7 ) C h r i s t e r N i l s s o n a n d Elias E r i k s s o n Department of Pharmacology, University of G6teborg, P.O.B. 33031, S-400 33 G6teborg, Sweden Received 16 January 1992, revised MS received 16 April 1992, accepted 5 May 1992
(-)-(3-Hydroxyphenyl)-N-n-propylpiperidine ((-)-3-PPP) and transdihydrolisuride (terguride, TDHL) are partial dopamine D 2 receptor agonists displaying low intrinsic activity at normosensitive postsynaptic dopamine D 2 receptors in brain while activating prolactin-regulating dopamine D 2 receptors in male rats with an efficacy close to that of full dopamine D 2 receptor agonists. In the present study we examined the effects of these partial dopamine D 2 receptor agonists on prolactin release in vitro. For this purpose prolactin-producing tumour cells which have been transfected with the dopamine D 2 receptor cDNA and hence which express a dopamine D e receptor (short isoform) that is functionally active with respect to inhibition of adenylate cyclase and prolactin release (GH4ZR7; Albert et al., J. Biol. Chem. (1990) 265, 2098) were used. While the full dopamine D 2 receptor agonists, quinpirole, (+)-(3-hydroxyphenyl)-N-n-propylpiperidine ((+)-3-Ppp) and dopamine induced a dose-dependent suppression of vasoactive intestinal peptide (VIP)-induced prolactin release from G H 4 Z R 7 , both (-)-3-PPP and terguride were inactive. Furthermore, the prolactin-suppressive effects of dopamine and quinpirole were significantly antagonized by pretreatment with (-)-3-PPP and terguride. It is concluded that partial dopamine D 2 receptor agonists, which activate male lactotroph dopamine Dz receptors in vivo, may antagonize dopamine D 2 receptors on GH4ZR 7 cells. There were similar results from an experiment using monolayers of anterior pituitary cells from male rats. Thus, in this in vitro preparation (+)-3-PPP suppressed spontaneous prolactin release while (-)-3-PPP again was inactive. Possible explanations, are offered for the ineffectiveness of partial dopamine D 2 receptor agonists with respect to the suppression of prolactin release in vitro. G H 4 Z R 7 cells; Cell culture; Anterior pituitary; Prolactin; Dopamine D 2 receptors; Partial agonists; Transdihydrolisuride;
( + )3-PPP (( + )-3-(3-hydroxyphenyl)-N-n-propylpiperidine); (In vitro)
I. Introduction D o p a m i n e D 2 receptors situated on pituitary lactotrophs exert an inhibitory influence on the release of prolactin in many different species. Administration of full dopamine D e receptor agonists to rats thus induces a decrease in serua-n levels of prolactin. A decrease in prolactin release is also observed when dopamine or dopamine D e receptor agonists are added to the medium surrounding monolayers of rat anterior pituitary ceils in culture (Foord et al., 1983), enabling investigations of dopamine D E receptor mechanisms in vitro. However, the use of pituitary lactotrophs for this purpose is marred by various methodological problems; for example, paracrine interactions between lactotrophs and other pituitary cells may influence the results obtained (Denef, 1986).
Correspondence to: C. Nilsson, Department of Pharmacology, University of G6teborg, P.O.B. 33031, S-400 33 G6teborg, Sweden. Tel. 46.31.853 415, fax 46.31.414 008.
Prolactin-producing tumour cell lines have proved very useful for investigations of various cellular mechanisms involved in prolactin secretion. However, in contrast to normal lactotrophs, these tumour cells generally do not express functionally active dopamine D e receptors (Cheung et al., 1983; Cronin et al., 1980; Day and Hinkle, 1988; Faure et al., 1980); consequently, they have not been suitable for studies on dopamine D 2 receptor function. Recently, however, Albert and coworkers successfully transfected the dopamine D 2 receptor c D N A into a prolactin-producing cell line ( G H g C t) normally not responding to dopamine, hence producing a stable cell line ( G H 4 Z R 7 ) expressing a dopamine D e receptor that is functionally active with respect to inhibition of both adenylate cyclase activity and prolactin release (Albert et al., 1990). The G H g Z R 7 line, and other prolactin-secreting transfectants expressing the dopamine D E receptor (McChesney et al., 1991), are likely to become important tools for the screening of putative dopamine D e receptor agonists and antagonists, as well as for studies on dopamine D 2 receptor function and regulation.
206 Partial agonists at dopamine D 2 receptors, such as ( - )-3-(3-hydroxyphenyl)-N-n-propylpiperidine (( - )-3PPP, preclamol) and transdihydrolisuride (terguride, TDHL), have been increasingly studied in the past ten years. Dopamine autoreceptors are generally activated by these compounds while normosensitive postsynaptic dopamine D 2 receptors are antagonized (Arnt and Hyttel, 1984; Carlsson et al., 1987; Clark et al., 1985). Consequently, it has been suggested that partial dopamine D 2 receptor agonists may be beneficial in the treatment of schizophrenia (Carlsson, 1988). Moreover, since they activate postsynaptic dopamine D 2 receptors that are up-regulated due to denervation (Arnt and Hyttel, 1984; Carlsson and Eriksson, 1988), they have also been suggested to be effective for the treatment of Parkinson's disease (Carlsson, 1986; Filipova et al., 1988). With respect to prolactin-regulating dopamine D 2 receptors in male rats, partial dopamine D 2 receptor agonists behave almost as full agonists. Thus, (-)-3PPP and terguride have been shown to decrease serum prolactin levels in male rats in vivo with an efficacy very close to that of full agonists (Eriksson et al., 1983; Wachtel and Dorow, 1983). Consequently, it has been concluded that lactotroph dopamine D 2 receptors in male rats are characterized by a responsiveness greater than that of postsynaptic dopamine D 2 receptors in brain and comparable to that of dopamine autoreceptors. If GH4ZR 7 cells are to be used for the screening of dopamine D z receptor agonists and for studies of dopamine D 2 receptor function, the degree of intrinsic activity displayed by partial dopamine D 2 receptor agonists on the dopamine D z receptors of these cells is an issue of considerable interest. In the present experiments we therefore compared the effect of various doses of dopamine 0 2 receptor agonists with high (dopamine, quinpirole, (+)-3-PPP) and low (terguride, (-)-3-PPP) intrinsic efficacy, respectively, on VIP-induced prolactin release from GH4ZR 7 cells. For comparison, the effects of a full and a partial dopamine D 2 receptor agonist on spontaneous prolactin release from male rat anterior pituitary cells in monolayer were also examined.
2. Materials and methods
2.1. GH4ZR 7 cell experiments Cells from a GH4C 1 clone transfected with the dopamine D 2 receptor cDNA (GH4ZR 7) (Albert et al., 1990) were provided by Dr Olivier Civelli, The Vollum Institute for Advanced Biomedical Research (Oregon Health Science University, Portland, Oregon, USA). Stock cultures (monolayers) were maintained in
Nunclon T-25 flasks (Nunc A / S , Roskilde, Denmark) with approximately 10 ml of Ham's F-10 medium supplemented with 3.7 g / l sodium bicarbonate, 15% horse serum, 2.5% fetal calf serum, 100 U / m l penicillin G + 100 /zg/ml streptomycin (PeSt), 0.6 /~g/ml amphotericin B, and 1 mM l-glutamine (= F-10 ÷) and incubated at 37°C in a water-saturated atmosphere of 5% CO 2 in air. The total medium was changed every 4th to 5th day. Four to five days before the experiment, GHnZR 7 cells were seeded in Nunclon 16-mm 24-multiwell plates in an approximate amount of 100000 cells in 500 ~1 of the same medium as in stock growth. Immediately before the experiment, the ceils were washed twice (15 min each time) with Phosphate Buffered Saline Solution (PBSS). Test drugs were added as a solution in pre-warmed, pre-CO2-equilibrated Earle's Balanced Salt Solution (EBSS) supplemented with 0.2% bovine serum albumin (BSA). When two test drugs were to be given (i.e. 'antagonist' +'agonist') to the same wells, they were added at an interval of 5 min. The prolactin secretagogue, vasointestinal peptide (VIP, culture grade, 300 nM) dissolved in 50 ~1 of the same medium was always added 5 min after the last test drug (i.e. the 'agonist'). After 30 min of incubation with VIP, the supernatants were gently pipetted from the wells, centrifuged (300 × g; 5 min) in order to remove any remaining cells, and frozen for subsequent rat prolactin RIA analysis. The same drug treatment was always given to at least three different wells. All wells in one treatment group were never situated on the same 24-multiwell plate. Since different plates sometimes differed in baseline and VIP-induced prolactin levels, before statistical calculations, the value for each well was expressed as percent of the value for control wells (given VIP only) on the same plate. 2.2. Rat anterior pituitary cell culture experiments Male Sprague-Dawley rats (ALAB, Sollentuna, Sweden) (150-200 g) were killed by decapitation and the anterior portion of the pituitary glands was immediately dissected out. Mechanical chopping and pasteur pipette triturition in sterile Ca 2+- and Mg2+-free EBSS, followed by enzymatic dissociation (trypsin EDTA) were used to prepare a suspension of approximatively 1.5 x 106 viable cells/ml EBSS. Cell viability was 95% as judged from trypan blue exclusion. The cells were resuspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 3.7 g / l sodium bicarbonate, 15% horse serum, 2.5% fetal calf serum, 100 U / m l penicillin G + 100 /zg/ml streptomycin (PeSt), 0.6 tzg/ml amphotericin B, and 1 mM l-glutamine (= DMEM+), and seeded in Nunclon 16-mm 24-multiwell plates (500/~1 DMEM+; 500 000 cells) for incuba-
207 PRL % of VIP 120 110
PRL % of VIP
t
100
120" 110" l
90
90"
80
80"
70
70.
60
60"
50
50"
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I
l
100
I
VIP 0.0005
i
0.005
i
i
0.05
0.5
i
i
5
50
40
i
!
[DA]p.M
i
VIP
Fig. 1. Effect of various doses of dopamine on VIP-induced prolactin secretion in G H 4 Z R 7 cells. Each point represents the means from three different wells_+ S.E.M. Mean absolute prolactin concentration for VIP = 18.3 n g / m l ; cells not given VIP = 6 0 ± 2 % of VIP (not shown). The effect of dopamine was significantly correlated to dose ( R = 0 . 9 1 ; P
205
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52557
Partial dopamine 0 2 receptor agonists antagonize prolactin-regulating D 2 receptors in a transfected clonal cell line ( G H 4 Z R 7 ) C h r i s t e r N i l s s o n a n d Elias E r i k s s o n Department of Pharmacology, University of G6teborg, P.O.B. 33031, S-400 33 G6teborg, Sweden Received 16 January 1992, revised MS received 16 April 1992, accepted 5 May 1992
(-)-(3-Hydroxyphenyl)-N-n-propylpiperidine ((-)-3-PPP) and transdihydrolisuride (terguride, TDHL) are partial dopamine D 2 receptor agonists displaying low intrinsic activity at normosensitive postsynaptic dopamine D 2 receptors in brain while activating prolactin-regulating dopamine D 2 receptors in male rats with an efficacy close to that of full dopamine D 2 receptor agonists. In the present study we examined the effects of these partial dopamine D 2 receptor agonists on prolactin release in vitro. For this purpose prolactin-producing tumour cells which have been transfected with the dopamine D 2 receptor cDNA and hence which express a dopamine D e receptor (short isoform) that is functionally active with respect to inhibition of adenylate cyclase and prolactin release (GH4ZR7; Albert et al., J. Biol. Chem. (1990) 265, 2098) were used. While the full dopamine D 2 receptor agonists, quinpirole, (+)-(3-hydroxyphenyl)-N-n-propylpiperidine ((+)-3-Ppp) and dopamine induced a dose-dependent suppression of vasoactive intestinal peptide (VIP)-induced prolactin release from G H 4 Z R 7 , both (-)-3-PPP and terguride were inactive. Furthermore, the prolactin-suppressive effects of dopamine and quinpirole were significantly antagonized by pretreatment with (-)-3-PPP and terguride. It is concluded that partial dopamine D 2 receptor agonists, which activate male lactotroph dopamine Dz receptors in vivo, may antagonize dopamine D 2 receptors on GH4ZR 7 cells. There were similar results from an experiment using monolayers of anterior pituitary cells from male rats. Thus, in this in vitro preparation (+)-3-PPP suppressed spontaneous prolactin release while (-)-3-PPP again was inactive. Possible explanations, are offered for the ineffectiveness of partial dopamine D 2 receptor agonists with respect to the suppression of prolactin release in vitro. G H 4 Z R 7 cells; Cell culture; Anterior pituitary; Prolactin; Dopamine D 2 receptors; Partial agonists; Transdihydrolisuride;
( + )3-PPP (( + )-3-(3-hydroxyphenyl)-N-n-propylpiperidine); (In vitro)
I. Introduction D o p a m i n e D 2 receptors situated on pituitary lactotrophs exert an inhibitory influence on the release of prolactin in many different species. Administration of full dopamine D e receptor agonists to rats thus induces a decrease in serua-n levels of prolactin. A decrease in prolactin release is also observed when dopamine or dopamine D e receptor agonists are added to the medium surrounding monolayers of rat anterior pituitary ceils in culture (Foord et al., 1983), enabling investigations of dopamine D E receptor mechanisms in vitro. However, the use of pituitary lactotrophs for this purpose is marred by various methodological problems; for example, paracrine interactions between lactotrophs and other pituitary cells may influence the results obtained (Denef, 1986).
Correspondence to: C. Nilsson, Department of Pharmacology, University of G6teborg, P.O.B. 33031, S-400 33 G6teborg, Sweden. Tel. 46.31.853 415, fax 46.31.414 008.
Prolactin-producing tumour cell lines have proved very useful for investigations of various cellular mechanisms involved in prolactin secretion. However, in contrast to normal lactotrophs, these tumour cells generally do not express functionally active dopamine D e receptors (Cheung et al., 1983; Cronin et al., 1980; Day and Hinkle, 1988; Faure et al., 1980); consequently, they have not been suitable for studies on dopamine D 2 receptor function. Recently, however, Albert and coworkers successfully transfected the dopamine D 2 receptor c D N A into a prolactin-producing cell line ( G H g C t) normally not responding to dopamine, hence producing a stable cell line ( G H 4 Z R 7 ) expressing a dopamine D e receptor that is functionally active with respect to inhibition of both adenylate cyclase activity and prolactin release (Albert et al., 1990). The G H g Z R 7 line, and other prolactin-secreting transfectants expressing the dopamine D E receptor (McChesney et al., 1991), are likely to become important tools for the screening of putative dopamine D e receptor agonists and antagonists, as well as for studies on dopamine D 2 receptor function and regulation.
206 Partial agonists at dopamine D 2 receptors, such as ( - )-3-(3-hydroxyphenyl)-N-n-propylpiperidine (( - )-3PPP, preclamol) and transdihydrolisuride (terguride, TDHL), have been increasingly studied in the past ten years. Dopamine autoreceptors are generally activated by these compounds while normosensitive postsynaptic dopamine D 2 receptors are antagonized (Arnt and Hyttel, 1984; Carlsson et al., 1987; Clark et al., 1985). Consequently, it has been suggested that partial dopamine D 2 receptor agonists may be beneficial in the treatment of schizophrenia (Carlsson, 1988). Moreover, since they activate postsynaptic dopamine D 2 receptors that are up-regulated due to denervation (Arnt and Hyttel, 1984; Carlsson and Eriksson, 1988), they have also been suggested to be effective for the treatment of Parkinson's disease (Carlsson, 1986; Filipova et al., 1988). With respect to prolactin-regulating dopamine D 2 receptors in male rats, partial dopamine D 2 receptor agonists behave almost as full agonists. Thus, (-)-3PPP and terguride have been shown to decrease serum prolactin levels in male rats in vivo with an efficacy very close to that of full agonists (Eriksson et al., 1983; Wachtel and Dorow, 1983). Consequently, it has been concluded that lactotroph dopamine D 2 receptors in male rats are characterized by a responsiveness greater than that of postsynaptic dopamine D 2 receptors in brain and comparable to that of dopamine autoreceptors. If GH4ZR 7 cells are to be used for the screening of dopamine D z receptor agonists and for studies of dopamine D 2 receptor function, the degree of intrinsic activity displayed by partial dopamine D 2 receptor agonists on the dopamine D z receptors of these cells is an issue of considerable interest. In the present experiments we therefore compared the effect of various doses of dopamine 0 2 receptor agonists with high (dopamine, quinpirole, (+)-3-PPP) and low (terguride, (-)-3-PPP) intrinsic efficacy, respectively, on VIP-induced prolactin release from GH4ZR 7 cells. For comparison, the effects of a full and a partial dopamine D 2 receptor agonist on spontaneous prolactin release from male rat anterior pituitary cells in monolayer were also examined.
2. Materials and methods
2.1. GH4ZR 7 cell experiments Cells from a GH4C 1 clone transfected with the dopamine D 2 receptor cDNA (GH4ZR 7) (Albert et al., 1990) were provided by Dr Olivier Civelli, The Vollum Institute for Advanced Biomedical Research (Oregon Health Science University, Portland, Oregon, USA). Stock cultures (monolayers) were maintained in
Nunclon T-25 flasks (Nunc A / S , Roskilde, Denmark) with approximately 10 ml of Ham's F-10 medium supplemented with 3.7 g / l sodium bicarbonate, 15% horse serum, 2.5% fetal calf serum, 100 U / m l penicillin G + 100 /zg/ml streptomycin (PeSt), 0.6 /~g/ml amphotericin B, and 1 mM l-glutamine (= F-10 ÷) and incubated at 37°C in a water-saturated atmosphere of 5% CO 2 in air. The total medium was changed every 4th to 5th day. Four to five days before the experiment, GHnZR 7 cells were seeded in Nunclon 16-mm 24-multiwell plates in an approximate amount of 100000 cells in 500 ~1 of the same medium as in stock growth. Immediately before the experiment, the ceils were washed twice (15 min each time) with Phosphate Buffered Saline Solution (PBSS). Test drugs were added as a solution in pre-warmed, pre-CO2-equilibrated Earle's Balanced Salt Solution (EBSS) supplemented with 0.2% bovine serum albumin (BSA). When two test drugs were to be given (i.e. 'antagonist' +'agonist') to the same wells, they were added at an interval of 5 min. The prolactin secretagogue, vasointestinal peptide (VIP, culture grade, 300 nM) dissolved in 50 ~1 of the same medium was always added 5 min after the last test drug (i.e. the 'agonist'). After 30 min of incubation with VIP, the supernatants were gently pipetted from the wells, centrifuged (300 × g; 5 min) in order to remove any remaining cells, and frozen for subsequent rat prolactin RIA analysis. The same drug treatment was always given to at least three different wells. All wells in one treatment group were never situated on the same 24-multiwell plate. Since different plates sometimes differed in baseline and VIP-induced prolactin levels, before statistical calculations, the value for each well was expressed as percent of the value for control wells (given VIP only) on the same plate. 2.2. Rat anterior pituitary cell culture experiments Male Sprague-Dawley rats (ALAB, Sollentuna, Sweden) (150-200 g) were killed by decapitation and the anterior portion of the pituitary glands was immediately dissected out. Mechanical chopping and pasteur pipette triturition in sterile Ca 2+- and Mg2+-free EBSS, followed by enzymatic dissociation (trypsin EDTA) were used to prepare a suspension of approximatively 1.5 x 106 viable cells/ml EBSS. Cell viability was 95% as judged from trypan blue exclusion. The cells were resuspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 3.7 g / l sodium bicarbonate, 15% horse serum, 2.5% fetal calf serum, 100 U / m l penicillin G + 100 /zg/ml streptomycin (PeSt), 0.6 tzg/ml amphotericin B, and 1 mM l-glutamine (= DMEM+), and seeded in Nunclon 16-mm 24-multiwell plates (500/~1 DMEM+; 500 000 cells) for incuba-
207 PRL % of VIP 120 110
PRL % of VIP
t
100
120" 110" l
90
90"
80
80"
70
70.
60
60"
50
50"
40
I
l
100
I
VIP 0.0005
i
0.005
i
i
0.05
0.5
i
i
5
50
40
i
!
[DA]p.M
i
VIP
Fig. 1. Effect of various doses of dopamine on VIP-induced prolactin secretion in G H 4 Z R 7 cells. Each point represents the means from three different wells_+ S.E.M. Mean absolute prolactin concentration for VIP = 18.3 n g / m l ; cells not given VIP = 6 0 ± 2 % of VIP (not shown). The effect of dopamine was significantly correlated to dose ( R = 0 . 9 1 ; P
