Peptides.Vol. 13, pp. 1201-1206, 1992

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Regulation of Galanin Secretion From Pituitary Cells In Vitro by Estradiol and GHRH ANTHONY

HEMMER

A N D J A M E S F. H Y D E l

Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, K Y 40536 R e c e i v e d 4 J u n e 1992 HEMMER, A. AND J. F. HYDE. Regulationofgalanin secretionfrom pituitary cellsin vitroby estradioland GHRH. PEPTIDES 13(6) 1201-1206, 1992.--The effects of estradiol and growth hormone-releasing hormone (GHRH) on galanin release from anterior pituitary cells were examined in vitro. 17-/3-Estradiol(0.001-10 riM) increased galanin secretion from anterior pituitary cells in a concentration-dependent manner. Estradiol (10 riM) increased galanin release 300 and 600% from pituitary cells of ovariectomized and male rats, respectively. Immunocytochemical studies demonstrated that estradiol (10 nM) increased the number of galanin-containing cells twofold after 4 days in culture. Growth hormone-releasing hormone (1 and 10 riM) increased and SRIF ( 1 and 10 nM) decreased galanin release from pituitary cells ofovariectomized and male rats. We conclude that estradiol increases galanin release by a direct effect on pituitary cells, in part by increasing the number of pituitary cells synthesizing galanin. In addition, GHRH stimulates galanin release when estradiol levels are low. Galanin Estradiol Anterior pituitary gland

GHRH

Somatostatin

Prolactin

THE neuropeptide galanin is widely distributed in the peripheral and central nervous systems of a variety of species (1,3,22). In the rat brain, the highest concentration of galanin is in the hypothalamus (15,17), where specific galanin binding sites have also been identified (14). In addition, intracerebroventricular administration of galanin alters the secretion of anterior pituitary hormones including prolactin (PRL) and growth hormone (GH) (12,16). These observations have led to the hypothesis that hypothalamic galanin serves a role as a hypophysiotropic factor. Galanin is also present in the rat anterior pituitary gland. Galanin is localized in somatotrophs and thyrotrophs of male rats, as well as lactotrophs ofdiestrous female rats (23). Estrogen dramatically and specifically increases galanin mRNA and peptide levels in the rat anterior pituitary gland (10,25), especially within lactotrophs (5,6). Moreover, galanin is secreted from estrogen-treated anterior pituitary cells in vitro (8). Dopamine, somatostatin, and TRH regulate galanin and PRL release from estrogen-exposed pituitary cell cultures in a similar fashion, whereas GHRH, LHRH, and CRH did not alter galanin release (8). Galanin and PRL are colocalized within the same secretory granules after estrogen treatment, thus providing a morphological basis for the coregulation of their secretion (6). Although it is clear that estrogen has marked effects on pituitary galanin in vivo, the in vitro effects of 17-/3-estradiol on galanin have not been examined. Furthermore, the inability of G H R H to stimulate galanin secretion in vitro in previous studies

Growth hormone

Pituitary cells

may have been due to the continued presence ofestradiol in the culture medium (8). The objectives of this study were to 1. examine the in vitro effects of 17-~3-estradiol on galanin secretion from pituitary cells obtained from ovariectomized and male rats, 2. determine if 17-/3-estradiol alters the number of pituitary cells expressing immunoreactive galanin in vitro, and 3. test if G H R H regulates galanin secretion from pituitary cells not exposed to high levels of estrogen. We now report that 17-/3-estradiol augments galanin release in vitro in a sex- and dose-dependent manner, and increases the number of pituitary cells containing immunoreactive galanin twofold. In addition, G H R H stimulates galanin secretion from pituitary cell cultures maintained in medium containing low levels of estradiol. METHOD

Animals Female (n = 35) and male (n = 30) Fischer 344 rats (Harlan Industries, Indianapolis, IN), weighing 125-150 g, were used in this study. The rats were housed under controlled temperature and lighting conditions (lights on from 0700-1900 h). Food and water were available ad lib. Female rats were ovarieetomized using sodium methohexital (Brevital; Eli Lilly Co., Indianapolis,

t Requests for reprints should be addressed to Dr. James F. Hyde, Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536-0084.

1201

1202 IN) anesthesia (45 mg/kg, IP) 14-21 days before preparing anterior pituitary cell cultures.

Anterior Pituitary Cell Culture and Hormone Release Experiments Anterior pituitary ceils from ovariectomized and male rats were prepared as previously described (8). Each batch of dissociated pituitary ceils used in a single hormone release experiment was prepared from a pool of three anterior pituitaries, and four to five pools were used for each experiment. Trypsindispersed anterior pituitary cells (5 × 105 cells/well) were cultured in 24-well plates (Costar, Cambridge, MA) with phenol red-free Dulbecco's Modified Eagle's Medium (DMEM; Sigma Chemical Co., St. Louis, MO) containing 15% gelded horse serum (Hazelton Biologics, Inc., Lenexa, KS), and antibiotics (GIBCO BRL, Grand Island, NY). Phenol red was omitted from all culture media due to its potential estrogenic activity (2). The cells were maintained in a water-saturated atmosphere of 5% CO2-95% air at 37°C. To examine the effects of estradiol, pituitary cells were cultured in medium containing 0-10 rtM 17-13-estradiol. 17-aEstradiol (10 nM) had no effect on galanin release (data not shown). Hormone release was assessed after 4 days in culture. The cells were first washed three times for 30 min with phenol red-free medium 199 (Sigma) containing 0.1% BSA. The cells were then incubated for 3 h in medium 199-BSA. To examine the effects of GHRH and somatostatin, pituitary cells were cultured in DMEM without added estradiol for 4 days. After three washes in medium 199-BSA, the cells were incubated for 3 h in medium 199-BSA alone or medium 199-BSA containing 1 or I0 nM somatostatin(l-14) or rat G H R H (Peninsula Laboratories, Inc., Belmont, CA). After a 3-h incubation, the medium was collected and stored at - 2 0 ° C until assayed directly for hormone content. Triplicate wells were used for each treatment in every pituitary cell preparation.

lmmunocytochemistry for Galanin in Pituitary Cell Cultures Pituitary cells (3 × 10S/well) from ovariectomized Fischer 344 rats were cultured in 4-well microslides (Thomas Scientific, Swedesboro, NJ) with phenol red-free DMEM containing 15% gelded horse serum and antibiotics. Medium in two of the wells on each slide also contained 17-/~-estradiol (10 nM). After 4 days in culture, the cells were fixed with a solution of 3% paraformaldehyde-7.5% picric acid in 0.1 M phosphate buffer (pH 7.4) for 15 min. After washing the cells with 0.5 M phosphate-buffered saline (PBS; pH 7.4) three times (20 min each), the cells were treated with normal goat serum (1:30) for 30 rain. The cells were then incubated overnight (4°C) in a rabbit-generated rat galanin antiserum (1:25,000; Peninsula Laboratories, Inc.). On the following day, the cells were washed with PBS (three times for 10 min) and incubated with biotinylated goat anti-rabbit IgG serum for 30 min at room temperature. After washing the cells with PBS, freshly prepared ABC reagent (Vector Laboratories, Inc., Budingame, CA) was added to the cells for 60 min. Finally, a solution of 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) was added to the cells for 3 rain. After washing again in PBS, the cells were counterstained with Ehrlich's hematoxylin, dehydrated, and coverslipped. For each pituitary cell preparation, only single pituitary ceils were counted. Pituitary cell clusters were not included in the analysis due to the uncertainty ofimmunostaining. At least 1000 cells for each treatment were counted in each pituitary cell preparation (n = 5). Immunostaining was not observed after

HEMMER AND HYDE 1. preequilibration of the galanin antiserum with an excess of synthetic rat galanin (10 /zg/ml; 3.2 #M) 24 h before immunocytochemistry, 2. incubation in either primary or secondary antiserum alone, and 3. incubation with normal rabbit serum substituted for the primary antiserum.

High Performance Liquid Chromatography (HPLC) The specificity of the galanin radioimmunoassay was examined by HPLC. Immunoreactive galanin-like peptides in anterior pituitary cell culture medium were characterized by HPLC with a Dynamax C-18 reverse-phase column (1 × 25 cm; Rainin Instr. Co., Woburn, MA). The HPLC system utilized a dual piston pump (PM-38, Bioanalytical Systems, Inc., West Lafayette, IN) and a gradient programmer (model 2360; Isco, Lincoln, NE). A linear gradient of 20-50% acetonitrile in 0. ! % trifluoroacetic acid over 60 min (2.5 ml/min) was used to elute the peptides. Fractions (2.5 ml) were collected, lyophilized, and stored at - 2 0 ° C until assayed in duplicate for galanin-like immunoreactivity by RIA.

Hormone Determinations and Data Analysis Galanin levels were determined by radioimmunoassay using rabbit-generated rat galanin antiserum, iodinated rat galanin, and synthetic rat galanin for standards from Peninsula Laboratories, Inc. The antiserum was used at a final dilution of 1:i00,000. Total specific binding typically represented 25-30% of the total radioactivity added. The limit of sensitivity was 1 pg/tube, and the between- and within-assay coefficients of variation were 10.5% and 8.1%, respectively. Prolactin and GH levels were determined in triplicate with NIDDK radioimmunoassay kits using rat PRL RP-3 and rat GH RP-2 as the reference preparations. The between- and within-assay coefficients of variation were less than 10% for the PRL and GH assays. Data are expressed as the mean ___ SE. Statistical analyses were performed by an analysis of variance (ANOVA), followed by Newman-Keuls multiple range test where appropriate. The Mann-Whitney test was used to analyze the immunocytochemistry data (28). RESULTS

Characterization of Secreted Galanin-Like lmmunoreactivity in Pituitary Cell Culture Medium by HPLC The profile of galanin-like immunoreactivity in anterior pituitary cell culture medium is shown in Fig, 1. Under the HPLC conditions described, synthetic rat galanin(1-29) eluted as a single peak. The major peak ofgalanin-like immunoreactivity coeluted with synthetic galanin, and represented 68-87% of the total immunoreactivity.

Effects of 17-B-Estradiol on Galanin Secretion Four days of exposure to varying concentrations of 17-/~estradiol caused increases of galanin release from pituitary ceils obtained from both ovariectomized and male rats (Fig. 2). Twoway ANOVA showed significant effects of both sex, F(I, 20) = 210.12, p < 0.001, and estradiol concentration, F(5, 20) = 140.98, p < 0.001, on galanin release. Cells from ovariectomized rats released significantly more galanin than cells from male rats, and estradiol increased galanin release in a concentration-

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0.25, failed to alter PRL secretion (Fig. 6). The patterns of G H and PRL release from pituitary cells of ovariectomized rats were similar to those of males (data not shown). DISCUSSION The effects of estradiol on galanin gene expression in the hypothalamus and anterior pituitary gland of the rat have been studied in vivo. Galanin mRNA levels in the pituitary gland are elevated within several hours after acute injection of estradiol and return to normal by 24 h (25). Chronic exposure to estradiol (i.e., subcutaneous capsules) leads to a persistent elevation of pituitary galanin m R N A and peptide levels (5,9,26), which decline only if the estradiol capsule is removed (5). In contrast to the pituitary, hypothalamic galanin m R N A and peptide levels appear to be only modestly increased by estradiol (4,5). In diestrous female rats, galanin is localized solely within somatotrophs and thyrotrophs of male and ovariectomized rats (6,23). Moreover, galanin has been localized in a subpopulation of PRL-containing c e l l s when estradiol is present (5,6). Although the effects of estradiol on PRL gene expression in vitro and in vivo are well known (13,20,21,24), it is not clear at the present time how estradiol increases galanin gene expression, or if a common mechanism within lactotrophs coregulates PRL and galanin gene expression. We have shown that galanin secretion from pituitary ceils obtained from estrogen-treated rats is regulated by hypothalamic factors (8). Dopamine and somatostatin inhibit galanin secretion, whereas TRH stimulates galanin release. Prolactin release from estradiol-exposed cells is regulated in a similar manner (8), suggesting that these hypothalamic factors regulate galanin release from lactotrophs. Moreover, we have extended these observations to show that dopaminergic agents also regulate galanin gene expression in vivo (9). To study the regulation ofgalanin release by estradiol in vitro, we used pituitary cells from animals with less exposure to endogenous estrogens, namely male and ovari-

ectomized rats. Furthermore, the pituitary cells were cultured under conditions in which estradiol levels were low. In this way, the numbers of lactotrophs synthesizing and secreting galanin should be minimized. Exposure to estradiol for 4 days in vitro increased galanin release from pituitary cells in a concentration-dependent manner. In addition, galanin release from cells obtained from male rats was consistently lower than from cells of ovariectomized rats. Previously, we observed a similar relationship after in vivo treatment with estradiol (8). In agreement with previous reports (19,27), PRL release was also increased by the estradiol treatments. Furthermore, pituitary cells from ovariectomized rats released more PRL. Estradiol could be increasing galanin release by either 1) further increasing galanin mRNA/peptide levels in the same cells synthesizing galanin in the absence of estradiol, or 2) inducing galanin mRNA/peptide levels in cells previously not synthesizing the peptide. We favor the second possibility due to the fact that galanin and PRL are only colocalized when estradiol levels are high. Immunocytochemical analysis of pituitary cell cultures indicated that the number of galanin-containing pituitary cells increase twofold after 4 days of exposure to estradiol in vitro. This suggests that estradiol recruits new pituitary cells, possibly lactotrophs, to synthesize and secrete galanin. However, we cannot exclude the possibility that the increased number ofgalanincontaining pituitary cells is due to higher levels of galanin production that previously were below the limit of immunodetection. Nevertheless, the data demonstrate that estradiol directly increases galanin synthesis and release in pituitary cells. Growth hormone-releasing hormone, LHRH, and CRH failed to alter galanin release when pituitary cells from estrogentreated Fischer 344 rats were used in a previous study (8). Galanin has not been localized in either gonadotrophs or corticotrophs; thus, it is not surprising that LHRH and CRH have no effect on galanin release. However, galanin has been localized within secretory granules of somatotrophs of ovariectomized and male rats (6). Galanin is primarily associated with lactotrophs when estrogen levels are high (5,6). Thus, the large number (up to 80%) of lactotrophs found in pituitaries of estrogen-treated Fischer 344 rats (18) may have masked any effects of G H R H

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