Absence of D1 dopamine receptors that stimulate prolactin release in the rat pituitary

European Journal of Pharmacology, 142 (1987) 425-429 Elsevier

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EJP 00978

Absence of D 1 dopamine receptors that stimulate prolactin release in the rat pituitary D. Cocchi *, S. Ingrassia, L. Rusconi, I. Villa and E.E. Mfiller Department of Pharmacology, Universityof Milan, Via Vanvitelli 32, 20129 Milan, Italy

Received 12 May 1987, accepted 4 August 1987

It has been shown recently that SKF 38393-A (2,3,4,5-tetrahydro-7,8-dihydroxy-l-phenyl-lH-3-benzazepine-7-ol),a D-1 receptor agonist, possesses a prolactin-releasing effect in the rat, though the pituitary or central nervous system location of the receptors involved has not been clarified. The aim of our study was to elucidate this point. SKF 38393-A administered to freely moving adult female and male rats induced a striking, short-lived increase of basal prolactin levels. The prolactin stimulatory effect of SKF 38393-A was counteracted by pretreatment with SCH 23390, a D-1 receptor blocker. SKF 38393-A (10-11-10-6 M) added to monolayer primary cultures of anterior pituitary cells from rats of both sexes failed to modify prolactin release. At higher concentrations (10-5-10 -4 M) the drug induced a slight inhibition of prolactin release. Similarly, SKF 38393-A failed to stimulate adenylate cyclase activity in anterior pituitary membranes from rats of both sexes at low concentrations, while it inhibited enzyme activity at higher concentrations (10-5-10 -3 M). The latter effect was counteracted by concomitant addition of the antagonist of D-2 receptors, 1-sulpiride. These data demonstrate that: (1) the anterior pituirary does not contain D-l-dopamine receptors (positively coupled to adenylate cyclase) stimulatory to prolactin release; (2) the striking prolactin-releasing effect of SKF 38393-A in the rat is due to activation of extra-pituitary D-1 dopamine receptors; (3) SKF 38393-A, at high concentrations, is capable of activating pituitary D-2 receptors. Prolactin; Dopamine receptors; Primary pituitary cultures

1. Introduction It has b e e n shown recently b y Sailer a n d S a l a m a (1986) that systemic a d m i n i s t r a t i o n to rats of the selective agonist for the D-1 type of d o p a m i n e receptors, S K F 38393-A (2,3,4,5-tetrahydro-7,8-dihydroxy-l-phenyl-1 H-3-benzazepine-7-ol) produces in rats d o s e - d e p e n d e n t elevations in p l a s m a p r o l a c t i n c o n c e n t r a t i o n s . This effect is c o m p l e t e l y reversed b y the D-1 a n t a g o n i s t S C H 23390. This study, however, d i d n o t d e t e r m i n e w h e t h e r

* To whom all correspondence should be addressed.

the D-1 r e c e p t o r s s t i m u l a t o r y to p r o l a c t i n release were l o c a t e d on the p i t u i t a r y or in the central n e r v o u s system (CNS). T h e a i m of o u r w o r k was to clarify this point.

2. Materials and methods M a l e a n d female S p r a g u e - D a w l e y rats ( N o s s a n , Corezzana), weighing 200-300 g, were used in these experiments. Laboratory chow and bottled tap w a t e r were a v a i l a b l e at all times. T h e a n i m a l s were h o u s e d 5 p e r cage with 14 h l i g h t / 1 0 h darkness.

0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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2.1. In vivo experiments 2.1.1. Surgery The day before the in vivo experiments, silastic indwelling atrial cannulas (0.6 x 0.11 cm) were implanted into male and female rats through the right external jugular vein. Surgery was performed under light anesthesia with tribromoethanol (200 mg/kg i.p.). 2.1.2. Experimental procedure The day of the experiment, polyethylene tubing extensions (20 cm) were attached to the atrial cannulas in order to obtain samples (0.3 ml) while the rats moved freely. After two baseline samples were collected ( - 3 0 and 0 min), test drugs were injected through the implanted cannula and further blood samples were collected 15, 30 and 60 min later. Samples were centrifuged at 3500 r.p.m. for 15 min and plasma was separated and stored at - 2 0 °C until the time of radioimmunoassay. 2.2. In vitro experiments 2.2.1. Primary pituitary cultures Rat primary pituitary cultures were prepared from anterior pituitary glands of male and female rats. The animals were decapitated in the morning and the anterior lobes of the pituitary were quickly removed, washed with Ca2+-free medium, cut into small pieces with a sterile scalpel then enzymatically dissociated. The enzymatic dispersion was made in Ca2+-free medium with 1% glucose, 0.1% albumin and 0.25% trypsin (Difco Labs) at 37 °C for 1.5 h. The dispersed cells were plated at 4 × 105 cells/ml in Ham's F-10 medium (Flow Laboratories) supplemented with 15% horse serum, 2.5% fetal calf serum, and antibiotics (streptomycin and penicillin, 100 U/ml). The cells were incubated at 37°C in 95% air with 5% CO2 for 3-4 days before the experiments. 2.2.2. Experimental procedure On the day of the experiment the attached cells were washed three times with Krebs Ringer medium buffered with HEPES 25 mM (KRH) containing 1% glucose, 0.1% albumin and ascorbic acid. The cells were incubated with different drugs

in a randomized way for 2 h. At the end of the incubation the media were collected and immediately frozen.

2.3. Prolactin radioimmunoassay The prolactin in plasma and media was measured with materials provided by NIADDK (Bethesda, MD). The data are expressed in terms of r-PRL-RP-3. The intraassay coefficient of variation was 4.5% and the interassay variation was 11%. The sensitivity of the method was 0.5 ng/ml.

2.4. Adenylate cyclase assay The anterior pituitaries were rapidly dissected out and removed after decapitation of the rats. The tissue was homogenized in 20 volumes of 1 nM Tris maleate buffer (pH 7.4) containing 1 mM EGTA using a glass-Teflon Potter homogenizer. The homogenate was filtered through gauze, centrifuged at 20 000 x g for 10 min and the pellet was resuspended to give a final concentration of about 1 mg protein/ml. The samples were then incubated for 5 min at 30°C in 0.1 ml assay mixture containing 80 mM Tris-maleate buffer pH 7.4; 0.15 mM a-32p ATP (200 d.p.m./pmol); 1.5 mM Mg C12; 1 mM cyclic AMP; 0.2 mM EGTA; 7 mM phosphocreatine; 20 U / m l creatine phosphokinase; 0.01 mM GTP; 0.1 mg bovine serum albumin and tissue protein (30-70 /~g/sample). SKF 38393-A and DA were dissolved in distilled water and were added to pituitary homogenates at stepwise increased concentrations (10-8-10 -3 M and 10-7-10 -4 M, respectively). 1-Sulpiride (RV 12309, Ravizza, Milan) was added at the concentration of 10-5 M. Isolation and measurement of a-32p cAMP were performed according to the method of Salomon et al. (1974). Protein concentration was evaluated with the micromethod of Bradford (1976).

2.5. Statistical analysis Statistical evaluation of the results was made by means of Dunnett's t-test for multiple comparisons.

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3. Results

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Administration of SKF 38393-A (5 m g / k g i.v.) induced a striking increase in the plasma prolactin levels of freely moving female and male rats (fig. 1). Fifteen minutes after drug administration, the peak rise in plasma prolactin was four times higher in female than in male rats, though the baseline prolactin levels were not significantly different in the two groups (fig. 1, left and right panels). A dose of SKF 38393-A of 2.5 mg/kg i.v. also increased significantly the basal prolactin levels in male rats (data not shown). In male rats, pretreatment with SCH 23390 (0.25 m g / k g i.v., 30 rain before) significantly reduced the stimulatory effect of SKF 38393-A (P < 0.05 vs. SKF 38393-A alone at 15 rain and P < 0.01 at 30 min). Addition of SKF 38393-A (10-11-10 -6 M) to primary cultures of rat anterior pituitary cells failed to alter the prolactin concentrations in the medium (fig. 2). SKF 38393-A significantly decreased the release of prolactin from the pituitary cells in culture only at the two highest concentrations used (10-s-10 -4 M) (fig. 2). In the same experiment, TRH (10 -7 M) induced a two-fold increase and DA (10 -8 M) a striking decrease (A decrement = 80%) of prolactin in the medium. To exclude that the ifi vivo prolactin-releasing effect of SKF 38393-A might have been due to partial

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Fig. 1. Effect of SKF 38393-A (5 m g / k g i.v.) on plasma prolactin levels of untreated female (left panel) and male (right panel) freely moving rats and of male rats pretreated with SCH 23390 (0.25 m g / k g i.v.) 30 rain before the injection of SKF 38393-A (right panel). Each point represents the mean _+ S.E.M. of 4-7 determinations made in duplicate. * P < at least 0.05 vs. placebo-treated rats. = P < 0.05 vs. SKF 38393-A-treated rats.

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Fig. 3. Effect of S K F 38393-A and D A with or without l-sulpiride on adenylate cyclase activity in anterior pituitary membranes from rats of both sexes. * P < 0.05 vs. basal values; • * P < 0.01 vs. basal values; ° P < at least 0.05 vs. D A or SKF 38393-A alone.

428 alter the striking prolactin release inhibitory effect of DA (10 -8 M); (controls 1173 ___39; DA 232 + 23; DA + SKF 38393-A 267 + 30 ng/ml; DA vs. DA + SKF 38393-A, NS). SKF 38393-A (10-8-10 -s M) did not increase adenylate cyclase activity in pituitary membranes of rats of both sexes (fig. 3, upper panel). At high concentrations (10-5-10-3 M) the drug induced a slight but significant decrease of basal enzyme activity (P < 0.05 vs. controls). This inhibition was, however, strikingly less than that evoked by DA (10-7-10 -4 M) under the same experimental conditions (fig. 3, lower panel). The inhibitory effect on adenylate cyclase activity of SKF 38393-A at high concentrations and DA was counteracted by coincubation with sulpiride.

4. Discussion

The observation that the D-1 agonist SKF 38393-A increases plasma prolactin in the rat (Sailer and Salama, 1986) has potential relevance for both basic and clinical research. First, it suggests the existence of a reciprocal interaction between pituitary D-1 and D-2 receptors in the regulation of prolactin secretion, as recently shown for brain D-1 and D-2 receptors which regulate striatal DA metabolism (Sailer and Salama, 1985). Second, experimentally induced or spontaneous conditions leading to supraphysiological DA concentrations in the hypophyseal portal vessels would also cause the activation of pituitary D-1 receptors that normally have a lower affinity for DA than D-2 receptors (Enjalbert and Bockaert, 1983). The possibility that feedback regulation of prolactin secretion through hypothalamic DA release (Moore and Johnston, 1982) could also involve potential activation of stimulatory pituitary D-1 receptors would open new perspectives for current understanding of the etiopathogenesis of prolactin-secreting tumors (Camanni and Miiller, 1984). With this in mind, we were prompted to investigate the possible pituitary location of the D-1 receptor that stimulates prolactin release. Our study showed that, as previously reported by Sailer and Salama (1986), SKF 38393-A 5 m g / k g i.v.

was a potent though short-lived prolactin releaser, its action being more manifest in female than in male rats. It induced a significant increase of prolactin levels in male rats also at a lower dose (2.5 m g / k g i.v.). Pretreatment with the D-1 antagonist SCH 23390 reduced the prolactin-releasing effect of SKF 38393-A, which would indicate the specificity of its effect at D-1 receptors. Reportedly, SCH 23390, in addition to blocking D-1 receptor sites, interacts with brain serotonin-2 (5HT-2) receptors (Bischoff et al., 1986). However, the possibility that SCH 23390 inhibited the SKF 38393-induced increase in prolactin through an interaction with 5HT-2 receptors appears to be unlikely. In fact, administration of the 5HT-2 antagonists pirenperone (Meltzer et al., 1983) and ketanserin (S.G. Celia and E.E. Miiller, unpublished results) itself increases prolactin secretion in male rats. Though our in vivo findings confirmed previous observations of Sailer and Salama (1986), the in vitro studies failed to provide evidence for the pituitary location of D-1 receptors stimulatory to prolactin release. SKF 38393-A, even at high concentrations, did not increase prolactin secretion from monolayer cultured pituitary ceils and, at 10-5-10 -4 M, it decreased prolactin secretion. The possibility that procedures related to the dispersion of pituitary cells would have destroyed DA receptors on the lactotrophs is unlikely, since the cells exhibited a proper responsiveness to TRH and DA stimulation. Results obtained with SKF 38393-A on prolactin secretion in vitro have their counterpart in its effects on pituitary adenylate cyclase. Despite the reported ability of SKF 38393-A to stimulate DA-sensitive adenylate cyclase in homogenates of rat caudate (Setler et ai., 1978), in our study it failed to do this in homogenates of anterior pituitary membranes. This provides further evidence that there are no dopamine D-1 receptors positively linked to adenylate cyclase in the pituitary of normal rats. However, SKF 38393-A at very high concentrations induced a slight but significant decrease of adenylate cyclase activity in the pituitary, and this effect was blocked by the D-2 receptor antagonist 1-sulpiride. This finding indicates that SKF 38393A at high concentrations can interact with the D-2

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receptors which are negatively coupled to adenylate cyclase (Giannattasio et al., 1981) and are physiologically stimulated by DA released into the hypophyseal portal capillaries (Enjalbert and Bockaert, 1983). This view was supported by the observation that SKF 38393-A at the same high concentrations significantly decreased prolactin secretion in vitro (see above). Apart from being a D-1 receptor agonist SKF 38393-A might have the property of partially antagonizing pituitary DA receptors, thus providing an alternative mechanism for its prolactin-releasing effect in vivo. However, this possibility was ruled out by the failure of SKF 38393-A to counteract in vitro the DA-induced inhibition of prolactin release. It can thus be said that the prolactin-stimulatory effect of SKF 38393-A in the rat is the result of activation of extrapituitary D-1 receptors. In this context, it is noteworthy that the rat hypothalamus, in clear contradistinction to the striatum, contains a relatively high ratio of D-1 to D-2 receptors (Leibowitz et al., 1982). In particular, receptor density is high in the arcuate nucleus and in the median eminence, the latter area containing a significant amount of DA-stimulated adenylate cyclase activity (Makman et al., 1980). The functional relevance of these CNS D-1 receptors to the stimulatory control of prolactin secretion is presently unknown. It can be hypothesized that they are located on neurons elaborating peptides endowed with prolactin-releasing properties, viz. vasoactive-intestinal polypeptide or TRH. These possibilities are currently being investigated in our laboratory.

Acknowledgements The gifts of SKF 38393-A by Smith Kline & French Labs. and of SCH 23390 by Dr. Ongini (Essex Italia) are greatly appreciated. Miss Mafia Lupo provided secretarial assistance.

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