0013-7227/91/1282-0917$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 2 Printed in U.S.A.
Galanin Secretion from Anterior Pituitary Cells in Vitro Is Regulated by Dopamine, Somatostatin, and Thyrotropin-Releasing Hormone* JAMES F. HYDE AND BRIAN K. KELLERt Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084
somatostatin (5 nM) in both female and male cells. The pattern of PRL release in response to dopamine, TRH, and somatostatin was similar to that observed for galanin, regardless of the sex of the pituitary donor. Although galanin has been localized in somatotrophs, 5 nM GH-releasing hormone (GRF) failed to alter galanin release in male as well as female pituitary cells; GH secretion was significantly increased by GRF. LHRH (5 nM) and CRF (5 nM) failed to alter galanin release in vitro. We conclude that in estrogen-exposed pituitary cells obtained from male and ovariectomized Fischer 344 rats: 1) galanin secretion is inhibited by dopamine and somatostatin, and stimulated by TRH; 2) GRF, LHRH, and CRF do not regulate galanin release in these cells; and 3) the profile of the regulated pathway for galanin release suggests that the primary location of galanin is the lactotroph, probably within secretory granules. (Endocrinology 128: 917-922, 1991)
ABSTRACT. Lactotrophs, somatotrophs, and thyrotrophs have been shown to contain immunoreactive galanin. furthermore, estrogen stimulates galanin mRNA and peptide levels in the rat anterior pituitary, particularly within lactotrophs. To determine whether galanin is released from the anterior pituitary in a regulated manner, we used cultured pituitary cells from male and ovariectomized Fischer 344 rats implanted with estrogen-containing capsules. Anterior pituitary cells (5 x 105 cells/ well) were challenged (0.5-3 h) with hypothalamic factors known to regulate anterior pituitary hormone secretion, and medium galanin levels were measured by RIA. In female pituitary cells, galanin secretion was inhibited by dopamine (10 and 100 nM) and stimulated by TRH (20 and 100 nM). Although galanin release was significantly lower in male pituitary cells, dopamine and TRH inhibited and stimulated galanin secretion, respectively. Medium galanin levels were also significantly reduced by
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ALANIN is distributed throughout the central nervous system in the rat and is found in high concentrations in the hypothalamus and posterior pituitary (1, 2). Normal male and ovariectomized female rats contain nearly undetectable levels of galanin mRNA in the anterior pituitary (3). Estrogen treatment, however, dramatically increases galanin gene expression and peptide synthesis in the anterior pituitary (3, 4). Thus, the sex differences in the galanin content of the anterior pituitary observed in adult rats are most likely the result of increased levels of circulating estrogens in the female rat (5, 6). Recently, the localization of galanin within specific populations of anterior pituitary cells has been investigated. Immunocytochemical studies have identified galanin in somatotrophs and thyrotrophs in normal male
rats (7) as well as in lactotrophs in normal and estrogentreated female rats (7, 8). In addition, galanin mRNA has been localized in lactotrophs by in situ hybridization (9). Although the physiological roles of galanin in the anterior pituitary and hypothalamus are presently unclear, the central administration of galanin results in increased plasma levels of PRL, GH, and TSH (10). Galanin stimulates GH secretion directly from male (11) and GH3/B6 pituitary cells (12). Alternatively, galanin may elevate GH levels by altering GH-releasing hormone (GRF) or somatostatin release (13). The increase in PRL release is probably due to the inhibition of dopamine (DA) release from the median eminence (14) or is the result of other hypothalamic interactions (15, 16). Using dispersed anterior pituitary cells obtained from estrogen-treated Fischer 344 rats, the objectives of this study were to 1) determine whether hypothalamic factors known to alter anterior pituitary hormone release also regulate galanin secretion, 2) evaluate whether the effects of these factors on anterior pituitary hormone release were similar to their effects on galanin, and 3) compare galanin release from male and female rat anterior pitui-
Received August 20,1990. Address requests for reprints to: Dr. James F. Hyde, Department of Anatomy and Neurobiology, University of Kentucky College of Medix cine, 800 Rose Street, Lexington, Kentucky 40536-0084. * This work was supported by Grant IN-163 from the American Cancer Society and the University of Kentucky Medical Center Research Fund. t Supported by a University of Kentucky College of Medicine Dean's Research Fellowship.
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REGULATION OF GALANIN SECRETION FROM PITUITARY CELLS
tary cells. We are now reporting that galanin secretion is inhibited by physiological concentrations of DA and somatostatin, and stimulated by TRH. These effects are very comparable to those on PRL. Although galanin release is significantly lower from male pituitary cells, its regulation appears similar to that in females.
Materials and Methods Animals Male and female 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 libitum. To increase galanin gene expression in the anterior pituitary, all animals were implanted with capsules containing 17/3-estradiol (Sigma Chemical Co., St. Louis, MO) for 2 weeks. The capsules were made with Silastic tubing (id, 0.020 in.; od, 0.037 in.; Dow-Corning Corp., Midland, MI) and measured 1 cm in length. The capsules were implanted sc using Brevital (Eli Lilly Co., Indianapolis, IN) anesthesia (45 mg/kg, ip). Female rats were ovariectomized at the time of capsule implantation. Anterior pituitary cell culture Anterior pituitary cells were dispersed as previously described (17). After removing the neurointermediate lobe, the anterior pituitaries were cut into small pieces and incubated with 0.2% trypsin (Worthington Biochemical Corp., Freehold, NJ) for 30 min. After treatment with DNase (Sigma Chemical Co.) and lima bean trypsin inhibitor (Worthington), the cells were dispersed by gentle trituration. Cells (5 X 105/ well) were cultured in 24-well plates (Costar, Cambridge, MA) with Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) containing 10% horse serum, 2.5% fetal bovine serum, antibiotics, and 1 nM 17/3-estradiol. The cells were maintained in a water-saturated atmosphere of 5% CO2-95% air at 37 C. After 4 days in culture, the cells were washed three times for 30 min with serum-free medium 199 (Gibco) containing 0.1% BSA. The cells were then incubated for 0.5-3 h in medium 199BSA alone or medium 199-BSA containing test substances. After incubation, the medium was collected and stored at -20 C until assayed for hormone content. Generally, triplicate wells were used for each test substance in every pituitary cell preparation. The effects of test substances on hormone secretion were examined in a minimum of three individual experiments. TRH, rat GRF, LHRH, rat CRF, and somatostatin-(1-14) were obtained from Peninsula Laboratories, Inc. (Belmont, CA). DA (Sigma) solutions were prepared in 0.1 mM ascorbic acid just before introduction to pituitary cells. Ascorbic acid alone had no effect on galanin release. Hormone determinations and data analysis PRL, LH, and GH concentrations were determined in triplicate by NIDDK RIA kits, using rat PRL RP-3, rat LH RP-3,
Endo • 1991 Voll28«No2
and rat GH RP-2 as reference preparations. The between- and within-assay coefficients of variation were less than 10%. Galanin concentrations were determined in triplicate by RIA (18) using a rabbit-generated rat galanin antiserum (SB4) prepared by Dr. Steven M. Gabriel (Mount Sinai School of Medicine, New York, NY). No significant molar cross-reactivities were evident with rat GRF, LHRH, somatostatin, arginine vasopressin, neurotensin, rat PRL, rat GH, rat LH, ACTH, bradykinin, or neuropeptide-Y. Slight molar cross-reactivities were observed with CRF, bombesin, substance-P, and physalamine (Gabriel, S. M., personal communication). The antiserum was used at a final tube dilution of 1:100,000. Iodinated rat galanin and synthetic rat galanin for standards were purchased from Peninsula Laboratories. The limit of sensitivity of the RIA was 2.5 pg/tube, and total binding typically represented 25-30% of the initial radioactivity added. Nonspecific binding was less than 3%. The between- and within-assay coefficients of variation were 9.7% and 7.6%, respectively. Data are expressed as the mean ± SE. Statistical analyses were performed by analysis of variance, followed by NewmanKeuls multiple range test where appropriate (19).
Results Effects of estradiol on plasma PRL levels and pituitary cell number After 2 weeks of estrogen treatment, plasma PRL levels were elevated in both female and male Fischer 344 rats; PRL levels were higher in females (Table 1). The pituitaries were enlarged in both sexes, and this effect of estrogen was reflected in the number of anterior pituitary cells per gland. In association with the higher plasma PRL levels, female anterior pituitary glands exposed to high levels of estrogen contained significantly more cells than comparably treated male glands (Table 1). Time course of galanin secretion in vitro As shown in Fig. 1, galanin secretion from anterior pituitary cells obtained from estrogen-treated ovariectomized rats was detectable within 30 min. Galanin levels increased significantly (P < 0.01) at each time examined TABLE 1. Effect of estradiol (E2) on plasma PRL levels and anterior pituitary (AP) cell number in Fischer 344 rats
OVEX Male OVEX + E2 Male + E2
PRL (ng/ml)
Cells (X1O6)/AP gland
10.2 ± 1.6 12.7 ± 2.1 913.2 ± 89.50'6 141.1 ± 14.2°
2.8 ± 0.26 3.0 ± 0.29 a b
14.5 ± 0A5 -
6.2 ± 0.15°
Male (n = 5/group) and ovariectomized (OVEX; n = 5/group) Fischer 344 rats were implanted (sc) with or without a 17/3-estradiolcontaining capsule for 2 weeks. PRL levels were determined by RIA, and cell counts were made using a hemocytometer. Each value represents the mean ± SE. " Significantly greater than rats not treated with E2 (P < 0.05). b Significantly greater than male rats treated with E2 (P < 0.05).
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REGULATION OF GALANIN SECRETION FROM PITUITARY CELLS
Effects of DA and TRH on galanin and PRL secretion
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co
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FIG. 1. Time course of galanin release from pituitary cells obtained from estrogen-treated ovariectomized Fischer 344 rats. Medium galanin levels from wells containing 500,000 cells were measured at various intervals during a 180-min incubation period. Each value represents a mean ± SE of three to six experiments.
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DA significantly (P < 0.01) inhibited galanin release from pituitary cells obtained from estrogen-treated ovariectomized rats in a concentration-dependent manner (Fig. 2). Galanin release was suppressed 56% and 72% by 10 nM and 100 nM DA, respectively, during the 3-h incubation. TRH, on the other hand, significantly (P < 0.05) stimulated galanin release in a concentration-dependent manner. Figure 2 also shows that the changes in PRL secretion induced by DA and TRH paralleled those in galanin. PRL release was inhibited 34% and 68% by 10 and 100 nM DA, respectively, in female pituitary cells. TRH induced a concentration-dependent increase in PRL release (Fig. 2). Galanin release from anterior pituitary cells obtained from estrogen-treated male rats was also inhibited by DA (Fig. 3). Compared to that in female pituitary cells, basal galanin release was significantly (P < 0.01) lower in male cells, and 100 nM DA caused a 63% inhibition of galanin release. TRH (100 nM) increased galanin secretion from male pituitary cells (P < 0.05). As illustrated in Fig. 3, basal PRL release, similar to that of galanin, was lower {P < 0.01) in male pituitary cells, and PRL levels were significantly (P < 0.01) reduced by 100 nM DA (34% of control) and stimulated (217% of control) by 100 nM TRH.
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3 eoo
180 min
30 min
a> o
0
o o 600
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£
r
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§ 0.5). Similar to cells obtained from female rats, somatostatin (5 nM) caused a 79% inhibition (P < 0.01) of galanin release from male pituitary cells, while 5 nM GRF, LHRH, and CRF had no effect (data not shown). PRL release was also significantly inhibited by 5 nM somatostatin in female (69% inhibition) and male (83% inhibition) pituitary cells (data not shown). Effects of GRF and somatostatin on GH secretion GRF (5 nM) induced a 188% and 105% increase in GH secretion from female and male pituitary cells, respectively (Fig. 5). Both basal and GRF-stimulated GH re1400
o o o °o
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in
^3>
600 400 200
GRF
ss
LHRH
CRF
FIG. 4. Effects of somatostatin (SS), GRF, LHRH, and CRF on galanin release from pituitary cells obtained from estrogen-treated ovariectomized Fischer 344 rats. Pituitary cells were exposed to each hormone (5 nM) for 180 min. Control wells (C) were exposed to medium 199 alone. Each value represents a mean ± SE of four to six experiments. 300 »
250 200
150 100
T 50
GRF
SS
GRF
SS
FIG. 5. Effects of somatostatin (SS) and GRF on GH release from pituitary cells obtained from estrogen-treated ovariectomized (•) and male W Fischer 344 rats. Pituitary cells were exposed to medium 199 alone (control; C), 5 nM SS, or 5 nM GRF for 180 min. Each value represents a mean ± SE of three to six experiments.
Endo • 1991 Voll28«No2
lease were significantly (P < 0.05) higher in male pituitary cells. Somatostatin (5 nM) significantly (P < 0.05) inhibited GH secretion in both female (34%) and male (65%) pituitary cells (Fig. 5). In contrast, DA had no effect (P > 0.5) on GH release from either male or female pituitary cells. LH release was increased approximately 10-fold in both male and female pituitary cells by 5 nM LHRH (data not shown). Medium TSH and ACTH concentrations were not measured. Discussion These studies demonstrate that galanin secretion from anterior pituitary cells is inhibited by physiological concentrations of DA and somatostatin, and stimulated by TRH. In these estrogen-treated cells, the profile of galanin release mirrored the secretion of PRL, suggesting that galanin is primarily localized within lactotrophs. Furthermore, the regulation of galanin release by known hypophysiotropic hormones argues in favor of the compartmentalization of galanin within secretory granules. These results also raise the possibility of the colocalization of galanin and PRL within the same secretory granules. This is in contrast to a recent report suggesting that galanin is localized in the Golgi region and unavailable for export in estrogen-treated Fischer 344 pituitary cells (8). In this study Fischer 344 rats were treated with estradiol to maximize the potential for detecting galanin release. This strain of rat is exquisitely sensitive to estrogen; pituitary tumors are evident within 2 weeks of exposure to high levels of estrogen (20). The hyperplasia of the pituitary is only partially accounted for by an increase in the number of lactotrophs, which may represent 80% of the pituitary cells (21, 22). To sustain elevated galanin gene expression, 1 nM 17/3-estradiol was included in the culture medium. However, the addition of estradiol was not essential to detect galanin release (Hyde, J., and B. K. Keller, unpublished observation). The low levels of estrogen in the culture medium (8.3 pM, as determined by RIA) due to the horse and fetal bovine sera or estrogen sequestered in the pituitary cells during the exposure to estradiol in vivo appear to be sufficient to maintain high levels of galanin release in vitro. DA is the major physiological inhibitor of PRL release (23, 24). Estrogen has been shown to decrease the number of pituitary DA receptors (25) and reduce the ability of DA to inhibit PRL secretion in vitro (26). Moreover, PRL secretion is much greater in pituitary cell cultures obtained from estrogen-treated Fischer 344 rats (22, 27). In the present study DA remained an effective inhibitor of PRL and galanin release in spite of prolonged exposure to estradiol. It is presently not known whether estradiol
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REGULATION OF GALANIN SECRETION FROM PITUITARY CELLS alters the regulation of galanin secretion in vitro. The ability of somatostatin to inhibit GH secretion is well documented. PRL release is also inhibited, by somatostatin under certain conditions. For example, somatostatin lowers plasma PRL levels in the estrogenprimed male rat (28). In addition, exposure of lactotrophs to estrogen in vitro increases the number of somatostatin receptors and confers the ability of somatostatin to inhibit PRL secretion (29). Thus, the reduction of galanin secretion by somatostatin may represent primarily an inhibition of release from the estrogen-exposed lactotrophs rather than from somatotrophs. This hypothesis is strengthened by the failure of GRF to stimulate galanin release in spite of increased levels of GH. However, the lack of effect of GRF does not rule out the possibility that GRF stimulates galanin release. Galanin has been localized in somatotrophs in male and female rats (7, 9). The galanin localized within the GH-containing cells may represent only a small portion of the total pituitary galanin in estrogen-exposed cells, and the high basal rate of galanin release, presumably from the hyperplastic lactotrophs, may, thus, mask any affect of GRF. It is also possible that estrogen alters the regulation of galanin secretion from somatotrophs. The stimulatory effect of TRH on galanin release most likely represents an increase in secretory activity from the lactotrophs. TRH has been long known to have a direct effect on lactotrophs to stimulate PRL release (30). Although galanin is affected by thyroid hormones (9, 31) and has been localized in thyrotrophs (7, 9), these cells represent only a small percentage of the total number of cells in the estrogen-treated Fischer 344 rat model (8, 22). Therefore, the contribution of galanin release from thyrotrophs is probably minimal. To date, galanin has not been localized in LH/FSH- or ACTH-containing cells. Thus, it is not surprising that LHRH and CRF had no effect on galanin release. Galanin and PRL release were significantly lower from male rat pituitary cells. Estrogen induces pituitary tumor formation and hyperprolactinemia in male Fischer 344 rats; however, these effects are much less pronounced than those in females (20). Although galanin gene expression appears to be lower in the estrogen-treated male pituitary cells, galanin is regulated identically at the level of secretion in both male and female pituitary cells. Moreover, the profiles of galanin and PRL release from male pituitary cells parallel one another. We postulate that estradiol induces galanin gene expression and peptide synthesis within the lactotrophs of the male Fischer 344 rat. The need to coordinate galanin release from the anterior pituitary is not apparent. It has been suggested that galanin may have an autocrine or paracrine role in the pituitary gland (7, 9), but compelling evidence for
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such a role is lacking. It is also tempting to speculate that galanin may act as a growth factor and contribute to pituitary tumor formation, but the present and past data offer no definitive support for this hypothesis. The coregulation of PRL and galanin release suggests that under certain physiological conditions, galanin may serve to modulate the actions of PRL locally or on peripheral target organs. Hopefully, future studies will elucidate the role of galanin secreted by the anterior pituitary. In conclusion, galanin release from anterior pituitary cells in vitro is regulated by DA, somatostatin, and TRH. GRF, LHRH, and CRF failed to alter galanin secretion. Although the Fischer 344 rat was used in this study, pituitary cells obtained from ovariectomized SpragueDawley rats treated with estrogen-containing capsules for 2 weeks also secreted galanin in vitro, and the release of galanin was inhibited by DA (Hyde, J., and B. K. Keller, unpublished data). Thus, it appears that galanin release from nontumorous pituitary cells is also dynamically regulated. The regulation of galanin secretion by hypothalamic hormones poses new questions about the role of galanin within the anterior pituitary as well as its subcellular localization. Acknowledgments We thank Dr. Steven M. Gabriel for generously supplying us with the rat galanin antiserum. We are indebted to the National Hormone and Pituitary Program, NIDDK, University of Maryland School of Medicine for the PRL, GH, and LH RIA kits.
References 1. Skofitsch G, Jacobowitz DM 1986 Quantitative distribution of galanin-like immunoreactivity in the rat central nervous system. Peptides 7:609-613 2. Palkovits M, Rokaeus A, Antoni FA, Kiss A 1987 Galanin in the hypothalamo-hypophyseal system. Neuroendocrinology 46:417423 3. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, Chin WW 1988 Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 85:7408-7412 4. Vrontakis ME, Peden LM, Duckworth ML, Friesen HG 1987 Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA. J Biol Chem 262:16755-16758 5. Gabriel SM, Kaplan LM, Martin JB, Koenig JI 1989 Tissuespecific sex differences in galanin-like immunoreactivity and galanin mRNA during development in the rat. Peptides 10:369-374 6. Gabriel SM, Koenig JI, Kaplan LM Galanin-like immunoreactivity is influenced by estrogen in peripubertal and adult rats. Neuroendocrinology 51:168-173 7. Steel JH, Gon G, O'Halloran DJ, Jones PM, Yanaihara N, Ishikawa H, Bloom SR, Polak JM 1989 Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression. Histochemistry 93:183-189 8. Hsu DW, El-Azouzi M, Black PMcL, Chin WW, Hedley-White ET, Kaplan LM 1990 Estrogen increases galanin immunoreactivity in hyperplastic prolactin-secreting cells in Fischer 344 rats. Endocrinology 126:3159-3167 9. O'Halloran DJ, Jones PM, Steel JH, Gon G, Giaid, A, Ghatei MA, Polak JM, Bloom SR 1990 Effect of endocrine manipulation on
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REGULATION OF GALANIN SECRETION FROM PITUITARY CELLS
anterior pituitary galanin in the rat. Endocrinology 127:467-475 10. Ottlecz A, Snyder GD, McCann SM 1988 Regulatory role of galanin in control of hypothalamic-anterior pituitary function. Proc Natl Acad Sci USA 85:9861-9865 11. Gabriel SM, Milbury CM, Nathanson JA, Martin JB 1988 Galanin stimulates rat pituitary growth hormone secretion in vitro. Life Sci 42:1981-1986 12. Drouhault R, Guerineau N, Corcuff J-B, Vacher AM, Vilayleck N, Mollard P 1990 Galanin evokes cytosolic Ca2+ transients and hormone release from GH3/B6 pituitary cells. J Endocrinol Invest [Suppl 2] 13:21 13. Ottlecz A, Samson WK, McCann SM 1986 Galanin: evidence for a hypothalamic site of action to release growth hormone. Peptides 7:51-53 14. Nordstrom O, Melander T, Hokfelt T, Bartfai T, Goldstein M 1987 Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence. Neurosci Lett 73:2126 15. Koshiyama H, Kato Y, Inoue T, Murakami Y, Ishikawa Y, Yanaihara N, Imura H 1987 Central galanin stimulates pituitary prolactin secretion in rats: possible involvement of hypothalamic vasoactive intestinal polypeptide. Neurosci Lett 75:49-54 16. Koshiyama H, Shimatsu A, Kato Y, Assadian H, Hattori N, Ishikawa Y, Tanoh T, Yanaihara N, Imura H 1990 Galanin-induced prolactin release in rats: pharmacological evidence for the involvement of alpha-adrenergic and opioidergic mechanisms. Brain Res 507:321-324 17. Hyde JF, Murai I, Ben-Jonathan N 1987 The rat posterior pituitary contains a potent prolactin-releasing factor: studies with perifused anterior pituitary cells. Endocrinology 121:1531-1539 18. Gabriel SM 1989 Temporal relationships between 17-beta-estradiol, luteinizing hormone and galanin during sexual maturation in the female rat. Soc Neurosci Abstr 15:1084 19. Zar JH 1974 Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, p 151 20. Wiklund J, Wertz N, Gorski J 1981 A comparison of estrogen
21. 22. 23. 24. 25. 26.
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effects on uterine and pituitary growth and prolactin synthesis in F344 and Holtzman rats. Endocrinology 109:1700-1707 Hymer WC, Motter KA 1988 Heterogeneity in mammotrophs prepared from diethylstilbestrol-induced prolactinomas. Endocrinology 122:2324-2338 Phelps C, Hymer WC 1983 Characterization of estrogen-induced adenohypophyseal tumors in the Fischer 344 rat. Neuroendocrinology 37:23-31 Ben-Jonathan N 1985 Dopamine: a prolactin-inhibiting hormone. Endocr Rev 6:564-589 Ben-Jonathan N, Arbogast LA, Hyde JF 1989 Neuroendocrine regulation of prolactin release. Prog Neurobiol 33:399-447 Heiman ML, Ben-Jonathan N 1982 Rat anterior pituitary dopaminergic receptors are regulated by estradiol and during lactation. Endocrinology 111:1057-1060 West B, Dannies PS 1980 Effects of estradiol on prolactin production and dihydroergocryptine-induced inhibition of prolactin production in primary cultures of rat pituitary cells. Endocrinology 106:1108-1113 Lloyd RV, Coleman K, Fields K, Nath V 1987 Analysis of prolactin and growth hormone production in hyperplastic and neoplastic rat pituitary tissues by the hemolytic plaque assay. Cancer Res 47:1087-1092 Cooper GR, Shin SH 1981 Somatostatin inhibits prolactin secretion in the estradiol primed male rat. Can J Physiol Pharmacol 59:1082-1088 Kimura N, Hayafuji C, Konagaya H, Takahashi K 1986 17/3Estradiol induces somatostatin (SRIF) inhibition of prolactin release and regulates SRIF receptors in rat anterior pituitary cells. Endocrinology 119:1028-1036 Tashjian Jr AH, Barowsky NJ, Jensen DK 1971 Thyrotropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem Biophys Res Commun 43:516-523 Hooi SC, Koenig JI, Gabriel SM, Maiter D, Martin JB 1990 Influence of thyroid hormone on the concentration of galanin in the rat brain and pituitary. Neuroendocrinology 51:351-356
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Vol. 128, No. 2 Printed in U.S.A.
Galanin Secretion from Anterior Pituitary Cells in Vitro Is Regulated by Dopamine, Somatostatin, and Thyrotropin-Releasing Hormone* JAMES F. HYDE AND BRIAN K. KELLERt Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084
somatostatin (5 nM) in both female and male cells. The pattern of PRL release in response to dopamine, TRH, and somatostatin was similar to that observed for galanin, regardless of the sex of the pituitary donor. Although galanin has been localized in somatotrophs, 5 nM GH-releasing hormone (GRF) failed to alter galanin release in male as well as female pituitary cells; GH secretion was significantly increased by GRF. LHRH (5 nM) and CRF (5 nM) failed to alter galanin release in vitro. We conclude that in estrogen-exposed pituitary cells obtained from male and ovariectomized Fischer 344 rats: 1) galanin secretion is inhibited by dopamine and somatostatin, and stimulated by TRH; 2) GRF, LHRH, and CRF do not regulate galanin release in these cells; and 3) the profile of the regulated pathway for galanin release suggests that the primary location of galanin is the lactotroph, probably within secretory granules. (Endocrinology 128: 917-922, 1991)
ABSTRACT. Lactotrophs, somatotrophs, and thyrotrophs have been shown to contain immunoreactive galanin. furthermore, estrogen stimulates galanin mRNA and peptide levels in the rat anterior pituitary, particularly within lactotrophs. To determine whether galanin is released from the anterior pituitary in a regulated manner, we used cultured pituitary cells from male and ovariectomized Fischer 344 rats implanted with estrogen-containing capsules. Anterior pituitary cells (5 x 105 cells/ well) were challenged (0.5-3 h) with hypothalamic factors known to regulate anterior pituitary hormone secretion, and medium galanin levels were measured by RIA. In female pituitary cells, galanin secretion was inhibited by dopamine (10 and 100 nM) and stimulated by TRH (20 and 100 nM). Although galanin release was significantly lower in male pituitary cells, dopamine and TRH inhibited and stimulated galanin secretion, respectively. Medium galanin levels were also significantly reduced by
G
ALANIN is distributed throughout the central nervous system in the rat and is found in high concentrations in the hypothalamus and posterior pituitary (1, 2). Normal male and ovariectomized female rats contain nearly undetectable levels of galanin mRNA in the anterior pituitary (3). Estrogen treatment, however, dramatically increases galanin gene expression and peptide synthesis in the anterior pituitary (3, 4). Thus, the sex differences in the galanin content of the anterior pituitary observed in adult rats are most likely the result of increased levels of circulating estrogens in the female rat (5, 6). Recently, the localization of galanin within specific populations of anterior pituitary cells has been investigated. Immunocytochemical studies have identified galanin in somatotrophs and thyrotrophs in normal male
rats (7) as well as in lactotrophs in normal and estrogentreated female rats (7, 8). In addition, galanin mRNA has been localized in lactotrophs by in situ hybridization (9). Although the physiological roles of galanin in the anterior pituitary and hypothalamus are presently unclear, the central administration of galanin results in increased plasma levels of PRL, GH, and TSH (10). Galanin stimulates GH secretion directly from male (11) and GH3/B6 pituitary cells (12). Alternatively, galanin may elevate GH levels by altering GH-releasing hormone (GRF) or somatostatin release (13). The increase in PRL release is probably due to the inhibition of dopamine (DA) release from the median eminence (14) or is the result of other hypothalamic interactions (15, 16). Using dispersed anterior pituitary cells obtained from estrogen-treated Fischer 344 rats, the objectives of this study were to 1) determine whether hypothalamic factors known to alter anterior pituitary hormone release also regulate galanin secretion, 2) evaluate whether the effects of these factors on anterior pituitary hormone release were similar to their effects on galanin, and 3) compare galanin release from male and female rat anterior pitui-
Received August 20,1990. Address requests for reprints to: Dr. James F. Hyde, Department of Anatomy and Neurobiology, University of Kentucky College of Medix cine, 800 Rose Street, Lexington, Kentucky 40536-0084. * This work was supported by Grant IN-163 from the American Cancer Society and the University of Kentucky Medical Center Research Fund. t Supported by a University of Kentucky College of Medicine Dean's Research Fellowship.
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REGULATION OF GALANIN SECRETION FROM PITUITARY CELLS
tary cells. We are now reporting that galanin secretion is inhibited by physiological concentrations of DA and somatostatin, and stimulated by TRH. These effects are very comparable to those on PRL. Although galanin release is significantly lower from male pituitary cells, its regulation appears similar to that in females.
Materials and Methods Animals Male and female 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 libitum. To increase galanin gene expression in the anterior pituitary, all animals were implanted with capsules containing 17/3-estradiol (Sigma Chemical Co., St. Louis, MO) for 2 weeks. The capsules were made with Silastic tubing (id, 0.020 in.; od, 0.037 in.; Dow-Corning Corp., Midland, MI) and measured 1 cm in length. The capsules were implanted sc using Brevital (Eli Lilly Co., Indianapolis, IN) anesthesia (45 mg/kg, ip). Female rats were ovariectomized at the time of capsule implantation. Anterior pituitary cell culture Anterior pituitary cells were dispersed as previously described (17). After removing the neurointermediate lobe, the anterior pituitaries were cut into small pieces and incubated with 0.2% trypsin (Worthington Biochemical Corp., Freehold, NJ) for 30 min. After treatment with DNase (Sigma Chemical Co.) and lima bean trypsin inhibitor (Worthington), the cells were dispersed by gentle trituration. Cells (5 X 105/ well) were cultured in 24-well plates (Costar, Cambridge, MA) with Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) containing 10% horse serum, 2.5% fetal bovine serum, antibiotics, and 1 nM 17/3-estradiol. The cells were maintained in a water-saturated atmosphere of 5% CO2-95% air at 37 C. After 4 days in culture, the cells were washed three times for 30 min with serum-free medium 199 (Gibco) containing 0.1% BSA. The cells were then incubated for 0.5-3 h in medium 199BSA alone or medium 199-BSA containing test substances. After incubation, the medium was collected and stored at -20 C until assayed for hormone content. Generally, triplicate wells were used for each test substance in every pituitary cell preparation. The effects of test substances on hormone secretion were examined in a minimum of three individual experiments. TRH, rat GRF, LHRH, rat CRF, and somatostatin-(1-14) were obtained from Peninsula Laboratories, Inc. (Belmont, CA). DA (Sigma) solutions were prepared in 0.1 mM ascorbic acid just before introduction to pituitary cells. Ascorbic acid alone had no effect on galanin release. Hormone determinations and data analysis PRL, LH, and GH concentrations were determined in triplicate by NIDDK RIA kits, using rat PRL RP-3, rat LH RP-3,
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and rat GH RP-2 as reference preparations. The between- and within-assay coefficients of variation were less than 10%. Galanin concentrations were determined in triplicate by RIA (18) using a rabbit-generated rat galanin antiserum (SB4) prepared by Dr. Steven M. Gabriel (Mount Sinai School of Medicine, New York, NY). No significant molar cross-reactivities were evident with rat GRF, LHRH, somatostatin, arginine vasopressin, neurotensin, rat PRL, rat GH, rat LH, ACTH, bradykinin, or neuropeptide-Y. Slight molar cross-reactivities were observed with CRF, bombesin, substance-P, and physalamine (Gabriel, S. M., personal communication). The antiserum was used at a final tube dilution of 1:100,000. Iodinated rat galanin and synthetic rat galanin for standards were purchased from Peninsula Laboratories. The limit of sensitivity of the RIA was 2.5 pg/tube, and total binding typically represented 25-30% of the initial radioactivity added. Nonspecific binding was less than 3%. The between- and within-assay coefficients of variation were 9.7% and 7.6%, respectively. Data are expressed as the mean ± SE. Statistical analyses were performed by analysis of variance, followed by NewmanKeuls multiple range test where appropriate (19).
Results Effects of estradiol on plasma PRL levels and pituitary cell number After 2 weeks of estrogen treatment, plasma PRL levels were elevated in both female and male Fischer 344 rats; PRL levels were higher in females (Table 1). The pituitaries were enlarged in both sexes, and this effect of estrogen was reflected in the number of anterior pituitary cells per gland. In association with the higher plasma PRL levels, female anterior pituitary glands exposed to high levels of estrogen contained significantly more cells than comparably treated male glands (Table 1). Time course of galanin secretion in vitro As shown in Fig. 1, galanin secretion from anterior pituitary cells obtained from estrogen-treated ovariectomized rats was detectable within 30 min. Galanin levels increased significantly (P < 0.01) at each time examined TABLE 1. Effect of estradiol (E2) on plasma PRL levels and anterior pituitary (AP) cell number in Fischer 344 rats
OVEX Male OVEX + E2 Male + E2
PRL (ng/ml)
Cells (X1O6)/AP gland
10.2 ± 1.6 12.7 ± 2.1 913.2 ± 89.50'6 141.1 ± 14.2°
2.8 ± 0.26 3.0 ± 0.29 a b
14.5 ± 0A5 -
6.2 ± 0.15°
Male (n = 5/group) and ovariectomized (OVEX; n = 5/group) Fischer 344 rats were implanted (sc) with or without a 17/3-estradiolcontaining capsule for 2 weeks. PRL levels were determined by RIA, and cell counts were made using a hemocytometer. Each value represents the mean ± SE. " Significantly greater than rats not treated with E2 (P < 0.05). b Significantly greater than male rats treated with E2 (P < 0.05).
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REGULATION OF GALANIN SECRETION FROM PITUITARY CELLS
Effects of DA and TRH on galanin and PRL secretion
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FIG. 1. Time course of galanin release from pituitary cells obtained from estrogen-treated ovariectomized Fischer 344 rats. Medium galanin levels from wells containing 500,000 cells were measured at various intervals during a 180-min incubation period. Each value represents a mean ± SE of three to six experiments.
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DA significantly (P < 0.01) inhibited galanin release from pituitary cells obtained from estrogen-treated ovariectomized rats in a concentration-dependent manner (Fig. 2). Galanin release was suppressed 56% and 72% by 10 nM and 100 nM DA, respectively, during the 3-h incubation. TRH, on the other hand, significantly (P < 0.05) stimulated galanin release in a concentration-dependent manner. Figure 2 also shows that the changes in PRL secretion induced by DA and TRH paralleled those in galanin. PRL release was inhibited 34% and 68% by 10 and 100 nM DA, respectively, in female pituitary cells. TRH induced a concentration-dependent increase in PRL release (Fig. 2). Galanin release from anterior pituitary cells obtained from estrogen-treated male rats was also inhibited by DA (Fig. 3). Compared to that in female pituitary cells, basal galanin release was significantly (P < 0.01) lower in male cells, and 100 nM DA caused a 63% inhibition of galanin release. TRH (100 nM) increased galanin secretion from male pituitary cells (P < 0.05). As illustrated in Fig. 3, basal PRL release, similar to that of galanin, was lower {P < 0.01) in male pituitary cells, and PRL levels were significantly (P < 0.01) reduced by 100 nM DA (34% of control) and stimulated (217% of control) by 100 nM TRH.
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§ 0.5). Similar to cells obtained from female rats, somatostatin (5 nM) caused a 79% inhibition (P < 0.01) of galanin release from male pituitary cells, while 5 nM GRF, LHRH, and CRF had no effect (data not shown). PRL release was also significantly inhibited by 5 nM somatostatin in female (69% inhibition) and male (83% inhibition) pituitary cells (data not shown). Effects of GRF and somatostatin on GH secretion GRF (5 nM) induced a 188% and 105% increase in GH secretion from female and male pituitary cells, respectively (Fig. 5). Both basal and GRF-stimulated GH re1400
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GRF
ss
LHRH
CRF
FIG. 4. Effects of somatostatin (SS), GRF, LHRH, and CRF on galanin release from pituitary cells obtained from estrogen-treated ovariectomized Fischer 344 rats. Pituitary cells were exposed to each hormone (5 nM) for 180 min. Control wells (C) were exposed to medium 199 alone. Each value represents a mean ± SE of four to six experiments. 300 »
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GRF
SS
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SS
FIG. 5. Effects of somatostatin (SS) and GRF on GH release from pituitary cells obtained from estrogen-treated ovariectomized (•) and male W Fischer 344 rats. Pituitary cells were exposed to medium 199 alone (control; C), 5 nM SS, or 5 nM GRF for 180 min. Each value represents a mean ± SE of three to six experiments.
Endo • 1991 Voll28«No2
lease were significantly (P < 0.05) higher in male pituitary cells. Somatostatin (5 nM) significantly (P < 0.05) inhibited GH secretion in both female (34%) and male (65%) pituitary cells (Fig. 5). In contrast, DA had no effect (P > 0.5) on GH release from either male or female pituitary cells. LH release was increased approximately 10-fold in both male and female pituitary cells by 5 nM LHRH (data not shown). Medium TSH and ACTH concentrations were not measured. Discussion These studies demonstrate that galanin secretion from anterior pituitary cells is inhibited by physiological concentrations of DA and somatostatin, and stimulated by TRH. In these estrogen-treated cells, the profile of galanin release mirrored the secretion of PRL, suggesting that galanin is primarily localized within lactotrophs. Furthermore, the regulation of galanin release by known hypophysiotropic hormones argues in favor of the compartmentalization of galanin within secretory granules. These results also raise the possibility of the colocalization of galanin and PRL within the same secretory granules. This is in contrast to a recent report suggesting that galanin is localized in the Golgi region and unavailable for export in estrogen-treated Fischer 344 pituitary cells (8). In this study Fischer 344 rats were treated with estradiol to maximize the potential for detecting galanin release. This strain of rat is exquisitely sensitive to estrogen; pituitary tumors are evident within 2 weeks of exposure to high levels of estrogen (20). The hyperplasia of the pituitary is only partially accounted for by an increase in the number of lactotrophs, which may represent 80% of the pituitary cells (21, 22). To sustain elevated galanin gene expression, 1 nM 17/3-estradiol was included in the culture medium. However, the addition of estradiol was not essential to detect galanin release (Hyde, J., and B. K. Keller, unpublished observation). The low levels of estrogen in the culture medium (8.3 pM, as determined by RIA) due to the horse and fetal bovine sera or estrogen sequestered in the pituitary cells during the exposure to estradiol in vivo appear to be sufficient to maintain high levels of galanin release in vitro. DA is the major physiological inhibitor of PRL release (23, 24). Estrogen has been shown to decrease the number of pituitary DA receptors (25) and reduce the ability of DA to inhibit PRL secretion in vitro (26). Moreover, PRL secretion is much greater in pituitary cell cultures obtained from estrogen-treated Fischer 344 rats (22, 27). In the present study DA remained an effective inhibitor of PRL and galanin release in spite of prolonged exposure to estradiol. It is presently not known whether estradiol
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REGULATION OF GALANIN SECRETION FROM PITUITARY CELLS alters the regulation of galanin secretion in vitro. The ability of somatostatin to inhibit GH secretion is well documented. PRL release is also inhibited, by somatostatin under certain conditions. For example, somatostatin lowers plasma PRL levels in the estrogenprimed male rat (28). In addition, exposure of lactotrophs to estrogen in vitro increases the number of somatostatin receptors and confers the ability of somatostatin to inhibit PRL secretion (29). Thus, the reduction of galanin secretion by somatostatin may represent primarily an inhibition of release from the estrogen-exposed lactotrophs rather than from somatotrophs. This hypothesis is strengthened by the failure of GRF to stimulate galanin release in spite of increased levels of GH. However, the lack of effect of GRF does not rule out the possibility that GRF stimulates galanin release. Galanin has been localized in somatotrophs in male and female rats (7, 9). The galanin localized within the GH-containing cells may represent only a small portion of the total pituitary galanin in estrogen-exposed cells, and the high basal rate of galanin release, presumably from the hyperplastic lactotrophs, may, thus, mask any affect of GRF. It is also possible that estrogen alters the regulation of galanin secretion from somatotrophs. The stimulatory effect of TRH on galanin release most likely represents an increase in secretory activity from the lactotrophs. TRH has been long known to have a direct effect on lactotrophs to stimulate PRL release (30). Although galanin is affected by thyroid hormones (9, 31) and has been localized in thyrotrophs (7, 9), these cells represent only a small percentage of the total number of cells in the estrogen-treated Fischer 344 rat model (8, 22). Therefore, the contribution of galanin release from thyrotrophs is probably minimal. To date, galanin has not been localized in LH/FSH- or ACTH-containing cells. Thus, it is not surprising that LHRH and CRF had no effect on galanin release. Galanin and PRL release were significantly lower from male rat pituitary cells. Estrogen induces pituitary tumor formation and hyperprolactinemia in male Fischer 344 rats; however, these effects are much less pronounced than those in females (20). Although galanin gene expression appears to be lower in the estrogen-treated male pituitary cells, galanin is regulated identically at the level of secretion in both male and female pituitary cells. Moreover, the profiles of galanin and PRL release from male pituitary cells parallel one another. We postulate that estradiol induces galanin gene expression and peptide synthesis within the lactotrophs of the male Fischer 344 rat. The need to coordinate galanin release from the anterior pituitary is not apparent. It has been suggested that galanin may have an autocrine or paracrine role in the pituitary gland (7, 9), but compelling evidence for
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such a role is lacking. It is also tempting to speculate that galanin may act as a growth factor and contribute to pituitary tumor formation, but the present and past data offer no definitive support for this hypothesis. The coregulation of PRL and galanin release suggests that under certain physiological conditions, galanin may serve to modulate the actions of PRL locally or on peripheral target organs. Hopefully, future studies will elucidate the role of galanin secreted by the anterior pituitary. In conclusion, galanin release from anterior pituitary cells in vitro is regulated by DA, somatostatin, and TRH. GRF, LHRH, and CRF failed to alter galanin secretion. Although the Fischer 344 rat was used in this study, pituitary cells obtained from ovariectomized SpragueDawley rats treated with estrogen-containing capsules for 2 weeks also secreted galanin in vitro, and the release of galanin was inhibited by DA (Hyde, J., and B. K. Keller, unpublished data). Thus, it appears that galanin release from nontumorous pituitary cells is also dynamically regulated. The regulation of galanin secretion by hypothalamic hormones poses new questions about the role of galanin within the anterior pituitary as well as its subcellular localization. Acknowledgments We thank Dr. Steven M. Gabriel for generously supplying us with the rat galanin antiserum. We are indebted to the National Hormone and Pituitary Program, NIDDK, University of Maryland School of Medicine for the PRL, GH, and LH RIA kits.
References 1. Skofitsch G, Jacobowitz DM 1986 Quantitative distribution of galanin-like immunoreactivity in the rat central nervous system. Peptides 7:609-613 2. Palkovits M, Rokaeus A, Antoni FA, Kiss A 1987 Galanin in the hypothalamo-hypophyseal system. Neuroendocrinology 46:417423 3. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, Chin WW 1988 Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 85:7408-7412 4. Vrontakis ME, Peden LM, Duckworth ML, Friesen HG 1987 Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA. J Biol Chem 262:16755-16758 5. Gabriel SM, Kaplan LM, Martin JB, Koenig JI 1989 Tissuespecific sex differences in galanin-like immunoreactivity and galanin mRNA during development in the rat. Peptides 10:369-374 6. Gabriel SM, Koenig JI, Kaplan LM Galanin-like immunoreactivity is influenced by estrogen in peripubertal and adult rats. Neuroendocrinology 51:168-173 7. Steel JH, Gon G, O'Halloran DJ, Jones PM, Yanaihara N, Ishikawa H, Bloom SR, Polak JM 1989 Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression. Histochemistry 93:183-189 8. Hsu DW, El-Azouzi M, Black PMcL, Chin WW, Hedley-White ET, Kaplan LM 1990 Estrogen increases galanin immunoreactivity in hyperplastic prolactin-secreting cells in Fischer 344 rats. Endocrinology 126:3159-3167 9. O'Halloran DJ, Jones PM, Steel JH, Gon G, Giaid, A, Ghatei MA, Polak JM, Bloom SR 1990 Effect of endocrine manipulation on
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anterior pituitary galanin in the rat. Endocrinology 127:467-475 10. Ottlecz A, Snyder GD, McCann SM 1988 Regulatory role of galanin in control of hypothalamic-anterior pituitary function. Proc Natl Acad Sci USA 85:9861-9865 11. Gabriel SM, Milbury CM, Nathanson JA, Martin JB 1988 Galanin stimulates rat pituitary growth hormone secretion in vitro. Life Sci 42:1981-1986 12. Drouhault R, Guerineau N, Corcuff J-B, Vacher AM, Vilayleck N, Mollard P 1990 Galanin evokes cytosolic Ca2+ transients and hormone release from GH3/B6 pituitary cells. J Endocrinol Invest [Suppl 2] 13:21 13. Ottlecz A, Samson WK, McCann SM 1986 Galanin: evidence for a hypothalamic site of action to release growth hormone. Peptides 7:51-53 14. Nordstrom O, Melander T, Hokfelt T, Bartfai T, Goldstein M 1987 Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence. Neurosci Lett 73:2126 15. Koshiyama H, Kato Y, Inoue T, Murakami Y, Ishikawa Y, Yanaihara N, Imura H 1987 Central galanin stimulates pituitary prolactin secretion in rats: possible involvement of hypothalamic vasoactive intestinal polypeptide. Neurosci Lett 75:49-54 16. Koshiyama H, Shimatsu A, Kato Y, Assadian H, Hattori N, Ishikawa Y, Tanoh T, Yanaihara N, Imura H 1990 Galanin-induced prolactin release in rats: pharmacological evidence for the involvement of alpha-adrenergic and opioidergic mechanisms. Brain Res 507:321-324 17. Hyde JF, Murai I, Ben-Jonathan N 1987 The rat posterior pituitary contains a potent prolactin-releasing factor: studies with perifused anterior pituitary cells. Endocrinology 121:1531-1539 18. Gabriel SM 1989 Temporal relationships between 17-beta-estradiol, luteinizing hormone and galanin during sexual maturation in the female rat. Soc Neurosci Abstr 15:1084 19. Zar JH 1974 Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, p 151 20. Wiklund J, Wertz N, Gorski J 1981 A comparison of estrogen
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effects on uterine and pituitary growth and prolactin synthesis in F344 and Holtzman rats. Endocrinology 109:1700-1707 Hymer WC, Motter KA 1988 Heterogeneity in mammotrophs prepared from diethylstilbestrol-induced prolactinomas. Endocrinology 122:2324-2338 Phelps C, Hymer WC 1983 Characterization of estrogen-induced adenohypophyseal tumors in the Fischer 344 rat. Neuroendocrinology 37:23-31 Ben-Jonathan N 1985 Dopamine: a prolactin-inhibiting hormone. Endocr Rev 6:564-589 Ben-Jonathan N, Arbogast LA, Hyde JF 1989 Neuroendocrine regulation of prolactin release. Prog Neurobiol 33:399-447 Heiman ML, Ben-Jonathan N 1982 Rat anterior pituitary dopaminergic receptors are regulated by estradiol and during lactation. Endocrinology 111:1057-1060 West B, Dannies PS 1980 Effects of estradiol on prolactin production and dihydroergocryptine-induced inhibition of prolactin production in primary cultures of rat pituitary cells. Endocrinology 106:1108-1113 Lloyd RV, Coleman K, Fields K, Nath V 1987 Analysis of prolactin and growth hormone production in hyperplastic and neoplastic rat pituitary tissues by the hemolytic plaque assay. Cancer Res 47:1087-1092 Cooper GR, Shin SH 1981 Somatostatin inhibits prolactin secretion in the estradiol primed male rat. Can J Physiol Pharmacol 59:1082-1088 Kimura N, Hayafuji C, Konagaya H, Takahashi K 1986 17/3Estradiol induces somatostatin (SRIF) inhibition of prolactin release and regulates SRIF receptors in rat anterior pituitary cells. Endocrinology 119:1028-1036 Tashjian Jr AH, Barowsky NJ, Jensen DK 1971 Thyrotropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem Biophys Res Commun 43:516-523 Hooi SC, Koenig JI, Gabriel SM, Maiter D, Martin JB 1990 Influence of thyroid hormone on the concentration of galanin in the rat brain and pituitary. Neuroendocrinology 51:351-356
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