Brain Research, 537 (1990) 227-232 Elsevier

227

BRES 16191

fl-Endorphin responses to different serotonin agonists: involvement of corticotropin-releasing hormone, vasopressin and direct pituitary action Gyorgy Bagdy 1, Aldo E. Calogero 3, Katalin Szemeredi a, M. Teresa Gomez 3, Dennis L. Murphy 2, George P. Chrousos 3 and Philip W. Gold 1 1Clinical Neuroendocrinology Branch and 2Laboratory of Clinical Sciences, National Institute of Mental Health, 3Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health and ~Hypertension and Endocrinology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892 (U.S.A.)

(Accepted 24 July 1990) Key words: Serotonin; Serotonin-lA receptor; Serotonin-lC receptor; Serotonin-2 receptor; Corticotropin-releasing hormone; Vasopressin; fl-Endorphin; Hypothalamus; Pituitary

Activation of serotonergic neurotransmission has been shown to increase plasma fl-endorphin-like immunoreactivity (fl-End-LI). To study the mechanism(s) of this action, we measured the effects of 3 potent serotonin (5-HT) agonists with different structures and 5-HT receptor binding profiles in conscious unrestrained Sprague-Dawley rats in vivo and in dispersed anterior pituleytes in vitro. The 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5-HTlc agonist, m-chlorophenylpiperazine (m-CPP), and the 5-HT2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), all markedly increased fl-End-LI in plasma in vivo. All 3 responses were blocked by dexamethasone pretreatment. Pituitary stalk transection (PST), as well as pretreatment with rabbit serum hyperimmune against rat corticotropin-releasing hormone (CRH, TS-6) completely abolished fl-End-LI response to 8-OH-DPAT and attenuated the responses by about 60% to DOI. Responses to m-CPP were markedly attenuated in PST rats, but pretreatment with TS-6 had no significant effect. To examine whether vasopressin (AVP) might be involved in the CRH neutralizing antibody-resistant fl-End-LI responses after m-CPP and DOI, we measured AVP concentrations after each agonist, m-CPP, but not DOI or 8-OH-DPAT, significantly elevated circulating AVP levels. As a proof of direct pituitary effect, DOI markedly stimulated fl-End-LI release from the anterior pituitary cell culture preparation in vitro. It was approximately as potent as CRH in the picomolar range, m-CPP was much less effective than DOI, while 8-OH-DPAT did not stimulate fl-End-LI release in vitro. Thus, the present findings suggest that the 5-HT1A agonist 8-OH-DPAT causes plasma fl-End-LI increases in vivo by stimulating hypothalamic CRH secretion. The mechanism of fl-End-LI response to the 5-HTlc agonist m-CPP is a complex phenomenon including mainly AVP-mediated and direct pituitary actions. The 5-HT2 agonist DOI may elicit fl-End-LI responses by CRH-mediated and direct pituitary effects. INTRODUCTION Serotonergic neurotransmission is a principal regulator of corticotropin-releasing h o r m o n e (CRH), arginine vasopressin (AVP), and adrenocorticotropin ( A C T H ) secretion 3'6-s'11'19'24. Serotonin (5-HT) releasing agents, uptake inhibitors and precursors also increase the concentration of circulating fl-endorphin-like immunoreactivity (fl-End-LI) in the plasma that originates mainly from the anterior pituitary 6"21,22. Both 5-HT 1 and 5-HT 2 receptor agonists have been shown to increase circulating levels of fl-End-LI is. However, studies about the mechanism of these actions are still lacking. Although both C R H and A V P have been shown to be released after serotonergic stimuli, there is no evidence whether C R H , or other hypothalamic factors mediate the fl-End-LI response after different 5-HT agonists ~5'21. Although a direct

pituitary action of 5-HT has been documented ~9'23, the type of 5-HT receptor for this action is not known. In the present study we used 3 potent 5-HT agonists, the 5-HT~A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin ( 8 - O H - D P A T ) , the 5-HT 2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ( D O I ) , and the 5H T l c agonist, m-chlorophenylpiperazine (m-CPP). All have previously been shown to produce marked, dosedependent increases in plasma A C T H and corticosterone 3. To study the mechanisms involved in the elevation of fl-End-LI concentrations, we administered these 5-HT agonists to conscious male S p r a g u e - D a w l e y rats with pituitary stalk transection (PST) or intact animals pretreated with hyperimmune C R H rabbit serum (TS-6) or dexamethasone ( D E X ) . To study the possible role of A V P in fl-End-LI secretion, we measured plasma A V P concentrations after the administration of these

Correspondence: G. Bagdy, Clinical Neuroendocrinology Branch, NIMH, NIH Clinical Center, 10-3S231, Bethesda, MD 20892, U.S.A.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

228 5 - H T agonists. To study the direct pituitary actions of the drugs we used dispersed rat a n t e r i o r pituicytes in culture. O u r study shows that activation of different 5 - H T r e c e p t o r s s t i m u l a t e s f l - E n d - L I release by different mecha n i s m s , a n d that activation of the 5 - H T I A receptors is the o n l y m e c h a n i s m that seems capable of increasing flE n d - L I c o n c e n t r a t i o n in p l a s m a t h r o u g h a specific C R H mediated mechanism. MATERIALS AND METHODS Animals and surgical procedure Experiments were performed on intact (Taconic Farms, Germantown, NY) or PST and sham-operated (Zivic North, Zelienopole, PA) male Sprague-Dawley rats weighing 350-430 g. Animals had free access to food and water, and were housed 5 per cage under constant conditions with a 12:12 h light-dark cycle (lights on at 06.00 h). At least 2 days before drug challenge, polyethylene cannulae (PE-50) were implanted in the left femoral artery and vein as described earlier4. Cannulae were exited subcutaneously at the back of the neck. After surgery, animals were housed individually. This system enabled us to obtain repeated blood samples from conscious, unrestrained animals. Experimental protocol and drug administration Animals were randomly assigned to groups for treatment with different 5-HT agonists and pretreatments with anti-rat CRI-I rabbit serum (TS-6), normal rabbit serum (NRS), DEX or saline. Each animal was challenged with only one drug. TS-6 was raised in our laboratory as previously described 13. Its titer was 1:80,000 at 30% substrate binding. TS-6 showed no cross-reactivity (less than 0.01%) with the following peptides: luteinizing hormone-releasing hormone, growth hormone-releasing hormone, thyrotropin-releasing hormone, somatostatin, substance P, porcine vasoactive intestinal polypeptide, neuropeptide Y, human ACTH-(1-39), a-melanoeytestimulating hormone (a-MSH), t-End, dynorphin-(1-17), AVP and oxytocin. All experiments were performed between 10.00 and 14.30 h. Baseline samples of blood were obtained a few minutes before administration of NRS, TS-6, DEX or saline (all i.v. at -240 min) and 4 h later just before the administration of the 5-HT agonists (time 0). m-CPP hydrochloride, 8-OH-DPAT methylbromide, and DOI hydrochloride (Research Bioehemicals, Inc., Wayland, MA) were administered into the femoral vein in a volume of 0.5 mi/kg b. wt. during a period of 10 s. Blood samples (750/A) were collected into prechilled polyethylene tubes containing EDTA by free flow through the intra-arterial cannula. Blood from age- and breedmatched halothane-anesthetized donor rats was returned immediately to the animals. Blood samples were refrigerated and centrifuged immediately; aliquots of plasma were removed and stored at -80 °C until the assay. Primary anterior pituitary cell culture After decapitation, anterior pituitaries from male SpragueDawley rats were removed from the sella turcica and placed in 0.5% bovine serum albumin (BSA) Dulbecco's modified Earle's medium (DMEM). Pituitaries were then minced onto a petri dish and transferred to a tube containing DMEM with 0.3% collagenase, 0.1% hyaluronidase, and 0.1% DNAase in a shaking water bath at 37 °C for 15 rain. After mechanical dispersion, pituicytes were centrifuged for 10 min at 750 rpm at room temperature, and the pellet was dispersed once more. The pellet was washed twice with DMEM containing 0.1% BSA. After centrifugation, the pellet was reconstituted in the DMEM containing 10% fetal calf serum, aprotinin, ascorbic acid, penicillin, streptomycin, and fungi,zone. Cell viability, tested by Trypan blue exclusion, was always greater than 95%. The cells were plated into a multiwell petri dish at a

density of 105 cells/well. After 48 h of incubation at 37 °C with a 7.5% CO 2 atmosphere, the medium was replaced. The next day, cells were washed with medium without fetal calf serum and treated with vehicle, and different concentrations of 5-HT agonists or rat CRH for 4 h. Each measurement was made in triplicate in 3-4 different cultures. After the experiments, the media were collected and stored at -80 °C for the radioimmunoassay (RIA). Hormone measurements Plasma samples for t-End measurements were extracted on SEP-PAK C-18 cartridges (Water, Milford, MA), according to methods described earlier 26, The recovery rate of t-End added to hormone-free plasma by this extraction procedure was 65%. RIA procedures for plasma or medium were carried out according to methods previously described2'9. Corresponding samples of all different treatment groups were run in the same assays. The antibody, anti-t-End (developed in our laboratory), used at a final dilution of 1:90,000 (assay volume of 0.3 ml) bound 37 +_ 2.6% of 125I-labeled/~-End. It showed no cross-reactivity (less than 0.01%) with prolactin, growth hormone, ACTH, a-MSH, CRH, dynorphin(1-13), Met-enkephalin, Leu-enkephalin and a-neoendorphin. The cross-reactivity with o-fl-lipotropin was 8.8%. Non-specific binding of the assay was 4.1 +_ 1.2%. The detection limit was 2.5 _+ 0.8 pg per assay tube. The intra-assay coefficient of variation was 2.4 _+ 1.4%. Vasopressin was measured in duplicates by RIA following extraction from 200/~l aliquots of plasma using microcolumns of Amberlite CG-50 resin (Sigma Chemicals, St. Louis, MO, U.S.A.) 2s. Statistical methods A repeated measures analysis of variance (ANOVA) followed by Fisher's post-hoe test (Stat View 512+, Brain Power, Inc., Calabasas, CA, U.S.A.) was used to compare treatment groups for differences in changes from baseline resulting from 5-HT agonist drug treatments. Data are expressed as means + S,E.M. Each group contained 5-7 animals. Area under the curve (AUC) was calculated by integration of the measured parameters in conventional units and time of testing in minutes. AUC net from the baseline (Net AUC) was calculated as the difference between total AUC and basal AUC (baseline x length of the testing). The effects of 5-HT agonists on pituitary fl-End-LI secretion were calculated as percent fl-End-LI increase above baseline, applying the following formula: Afl-End-LI (%) = stimulated fl-End-LI concentration/ basal fl-End-LI concentration × 100. Statistical evaluation was performed using one-way ANOVA, followed by Fisher's test. A P-value less than 0.05 defined statistical significance.

RESULTS R e s p o n s e s to all 3 agonists were b l o c k e d by p r e t r e a t m e n t with d e x a m e t h a s o n e (200 # g / r a t , 4 h p r i o r to the challenge), suggesting that the origin of t h e circulating f l - E n d - L I after these drugs is t h e a n t e r i o r p i t u i t a r y (Fig. 1). To e x a m i n e w h e t h e r these r e s p o n s e s w e r e m e d i a t e d by h y p o t h a l a m i c factors o r were direct at t h e pituitary level, we a d m i n i s t e r e d the agonists to P S T o r s h a m - o p e r a t e d rats (Fig. 2). f l - E n d - L I r e s p o n s e s to 8 - O H , D P A T were c o m p l e t e l y a b o l i s h e d , a n d r e s p o n s e s to D O I a n d m - C P P were significantly, b u t n o t e n t i r e l y (by 5 8 % a n d b y 7 0 % , respectively) b l u n t e d , suggesting that w h e r e a s f l - E n d - L I r e s p o n s e to 8 - O H - D P A T was e n t i r e l y m e d i a t e d b y h y p o t h a l a m i c factors, those to D O I a n d m - C P P were partially d e p e n d e n t o n these factors.

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