0013-7227/90/1273-1129$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 127, No. 3 Printed in U.S.A.
Growth Hormone and Prolactin Secretion in Cultured Somatomammotroph Cells* YOICHI KASHIO, PIOTR CHOMCZYNSKI, THOMAS R. DOWNS, AND LAWRENCE A. FROHMAN Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
cells by adenylate cyclase-related agents ((Bu)2 cAMP and forskolin) was comparable to that for GH secretion in mature somatotrophs but much greater than that of PRL release in mature lactotrophs. Stimulation of GH and PRL release in Po cells by protein kinase C-related agents (diacylglycerol and phorbol ester) was also similar to that observed for GH release from mature pituitary cells, whereas minimal or undetectable effects were observed on PRL release from mature cells. The results indicate that the Po somatomammotropic cell line possesses receptors, second messenger systems, and secretory characteristics of both somatotrophs and lactotrophs, although where differences exist, there is more resemblance to somatotrophs. They also demonstrate that the responses to each of the agents studied are bihormonal and appear to be regulated by a common mechanism. (Endocrinology 127: 1129-1135, 1990)
ABSTRACT. A somatomammotropic cell line (Po) derived from adult rat pituitaries has been maintained in culture for 2 yr. Secretion of GH and PRL by this cell line has been studied in response to hypophysiotropic peptides known to affect the release of both hormones as well as agents that affect second messenger systems in an attempt to characterize the stimulussecretion mechanisms used by these cells. GH and PRL release during short term (4 h) incubations of Po cells and primary cultures of dispersed rat pituitary cells was initially measured in response to GRF, TRH, vasoactive intestinal peptide (VIP), and SRIF. In Po cells, the minimal effective dose of each of the hypophysiotropic peptides was comparable with respect to GH and PRL secretion. The effects of TRH and VIP were similar to those in freshly dispersed cells with respect to PRL release, whereas those of GRF and SRIF were less potent with respect to GH release. The stimulation of GH and PRL release in P o
W
E HAVE recently described a pituitary cell line derived from primary cultures of dispersed adult male rat anterior pituitary cells by repeated and selective subculturing (1). Immunofluorescent staining revealed that virtually all of the cells contained GH, whereas 72% also contained PRL, and nine separate clones, obtained by limiting dilution of the cell line, each secreted GH and PRL, although in markedly varying ratios. These somatomammotroph cells differ from the pituitary tumor-derived cell lines GC, GH3, and GH4 in that they do not grow to confluence in culture, do not produce tumors after syngeneic transplantation, and maintain responsiveness to GRF. We have proposed that these cells may represent progenitor cells that can differentiate into somatotrophs and lactotrophs, although such transformations have yet to be demonstrated. Similar suggestions have been made by others on the basis of electron microscopic findings in somatomammotrophs (2) and bihormonal secretory patterns using the hemolytic plaque
assay (3). In an attempt to characterize the control of hormone secretion in somatomammotroph cells, we have studied the role of the hypothalamic hormones [GRF, TRH, vasoactive intestinal peptide (VIP), and somatostatin (SRIF)] known to affect secretion of GH and PRL in adult somatotrophs or lactotrophs. GRF and VIP exert their actions primarily through the activation of a cAMP-dependent protein kinase (4-9), whereas SRIF acts, at least in part, by inhibiting cAMP production (5, 6). In contrast, TRH action is mediated primarily by a protein kinase C-dependent pathway (10-12). We therefore also examined the effects of dibutyryl (Bu)2cAMP and forskolin to assess the adenylate cyclase system and those of phorbol myristate acetate (PMA) and diacylglycerol (DAG), activators of protein kinase C. The same agents were evaluated in primary cultures of dispersed anterior pituitary cells (from which the somatomammotrophs were derived) to compare the responses in developmental and mature cells.
Received March 23, 1990. Address all correspondence and requests for reprints to: Dr. Lawrence Frohman, Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, 231 Bethesda Avenue, M.L. #547, Cincinnati, Ohio 45267. * These studies were supported in part by USPHS Grant DK-30667.
Materials and Methods Pituitary cell line culture The origin of the pituitary cell line (herein designated as Po) has been previously described (1). Cells were maintained in a-
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SOMATOMAMMOTROPH GH AND PRL SECRETION
minimum essential medium (aMEM, Gibco, Grand Island, NY) supplemented with 10% horse serum (Sigma, St. Louis, MO), gentamicin (25 Mg/ml, Gibco) and nystatin (25 U/ml, Gibco) at 37 C in a water-saturated atmosphere containing 5% CO2. The culture medium was changed every 2 days. For experiments performed to study acute hormone release, cells were plated at a density of 1 to 2 x 105 cells/16-mm well and grown as monolayer cultures in aMEM supplemented with 10% horse serum, 50 nM hydrocortisone, and 50 fig/m\ gentamicin for 48 h. The cells were rinsed and then incubated for 4 h in 1 ml aMEM containing 0.1% BSA (Metrix, Chicago, IL) and specific test substances. The medium was then aspirated, and any floating cells were removed by centrifugation at 100 X g for 10 min at 4 C. The cell count was repeated at the end of the incubation (1). Each experimental protocol was performed from 2 to 5 times. Hormone secretion, expressed as the quantity of GH or PRL released during the 4-h incubation per 105 cells, is presented as the mean ± SE of quadruplicate incubations from a single representative experiment. Rat pituitary cell primary culture Primary monolayer cultures of dispersed pituitary cells were prepared as previously described (13, 14). After a 3-day culture period in aMEM containing 10% horse serum and 25 fig/ml gentamicin, the medium was removed and the cells washed twice with aMEM containing 0.1% BSA. The incubation medium and duration of the experimental period were identical to that described previously for the Po cells. Hormone measurements and data analysis GH and PRL were measured by RIA in duplicate as previously described (15, 16). Rat GH Reference Preparation (RP)1 and rat PRL RP-1 served as reference standards. Samples from each experiment were measured in a single assay with intra- and interassay coefficients of variation of 8.6% and 14.4% (GH) and 9.0% and 11.4% (PRL), respectively. The difference between group means was determined by analysis of variance using Dunnet's modified method for multiple groups compared with a single control after logarithmic transformation of the raw data to normalize variance. Drugs Rat (r) GRF (Peninsula Laboratories, Inc., Belmont, CA) was initially dissolved in 5 mM acetic acid containing 0.1% BSA. Porcine VIP(1o-28) (Peninsula), TRH (Abbott Laboratories, Chicago, IL), SRIF (Sigma), and (Bu)2cAMP (SchwartzMann, Orangeburg, NY) were dissolved in 0.05 M PBS containing 1% BSA and forskolin (Calbiochem, La Jolla, CA) in absolute ethanol. The latter was subsequently diluted so that the final concentration of ethanol in the incubation medium was less than 0.1%. PMA (Sigma) and l-oleoyl-2-acetyl-snglycerol (a synthetic DAG; Sigma) were initially dissolved and stored in dimethylsulfoxide (DMSO) and diluted so that the final concentration of DMSO in the incubation medium was less than 0.1%.
Results Effect of rGRF on GH and PRL release rGRF stimulated both GH and PRL release in pituitary somatomammotroph (Po) cells (Fig. 1). A significant
E n d o • 1990 Vol 127 • No 3
increase in GH release occurred at a concentration of 10~9 M and a maximal response was observed at 10~8 M to 10~7 M in several experiments. No stimulation of GH release was detected at 10"10 M rGRF in three separate incubations (data not shown). The maximum response constituted a 96% increase over basal hormone release. Basal GH release during a 4-h incubation period represented 33% of initial cell content (six experiments). rGRF also stimulated PRL release in P o cells at similar concentrations with a maximal increase of 198% over basal levels. Basal PRL release during the 4-h incubation period represented 109% of initial cell content. Twelve separate incubations were performed in which GRF was used as a stimulus. GH secretion was significantly increased in eight experiments but not in the other four. No explanation is presently available for the lack of consistency of the response to GRF, which stands in contrast to that of other stimuli, the effects of which were always reproducible. In primary pituitary cell cultures (PPC), in contrast, basal GH release represented 2% of cell content. GRF stimulatory effects were present at 10~12 M, and maximal stimulation represented a 7-fold increase over basal release. GRF did not stimulate PRL release in PPC. Effects of TRH and VIP on GH and PRL release GH secretion by P o cells was stimulated by TRH at concentrations of 10"9 to 10~7 M (Fig. 2). Maximal stimulation of GH was 75% above basal release. A comparable stimulation of PRL release (69% above basal) was seen at similar concentrations. VIP also stimulated GH release at concentrations of 10"8 to 10~6 M with a maximal increase of 48% above basal release. PRL release was increased at comparable concentrations with a maximal increase of 28% above basal release. In PPC, TRH did not stimulate GH release, although PRL release was enhanced at concentrations of 10~8 M and greater. Similarly, VIP was ineffective in increasing GH release, although it also stimulated PRL release at concentrations of 10~8 M and greater. Effect of SRIF on GH and PRL release GH secretion by P o cells was inhibited by SRIF at concentrations of 10~8 M and greater (Fig. 3). At a concentration of 10~6 M, GH release was inhibited by 60%. There was a parallel inhibition of PRL secretion in P o cells beginning at 10~9 M SRIF, and PRL release was inhibited by 53% at a concentration of 10~6 M. GH secretion by PPC was inhibited by SRIF at concentrations of 10~n M and greater, and at a concentration of 10~7 M GH release was inhibited by 80%. In contrast to P o cells, PRL secretion by PPC was not significantly inhibited by SRIF.
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SOMATOMAMMOTROPH GH AND PRL SECRETION 40
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FIG. 1. Left, Effect of rGRF on GH and PRL release in rat somatomammotroph (Po) cells determined during a 4-h incubation. Shown are the mean ±SE of quadruplicate incubations in one representative experiment. Right, Effect of rGRF on GH and PRL release in primary cultures of dispersed rat pituitary cells during a 4-h incubation. Each point represents the mean ±SE of quadruplicate incubations from one of two separate experiments. **, P < 0.01 vs. control incubation.
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FIG. 2. Left, Effects of TRH (top) and VIP (bottom) on GH and PRL release in rat somatomammotroph (Po) cells during a 4-h incubation. Shown are the mean ± SE of quadruplicate incubations from one of three experiments with comparable results. Right, Effects of TRH and VIP on GH and PRL release in primary cultures of dispersed rat pituitary cells during a 4-h incubation. *, P < 0.05. **, P < 0.01 vs. control incubation.
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Effect of (Bu)2cAMP and forskolin on GH and PRL release
(Bu)2cAMP exhibited comparable stimulatory effects on GH and PRL secretion in P o cells (Fig. 4). Stimulation was observed at concentrations of 10~5 M and greater. At a concentration of 10"3 M, the increase in GH and PRL release was 3.4-fold and 2.9-fold, respectively, over basal release. The stimulatory effects of forskolin on GH and PRL secretion were also comparable. Stimulation was observed at 10~8 M and greater with maximal increases of GH and PRL secretion of 3.2-fold and 2.6-fold, respectively, over basal secretion. (Bu)2cAMP stimulated GH secretion in PPC to a
comparable degree as in P o cells. The increase in release at a concentration of 10~3 M was 5 times that of basal release. The effects on PRL release were much less pronounced with a maximal response of only 68% above basal release. Similarly, in PPC, forskolin stimulated GH and PRL secretion at concentrations from 10~8 to 10~5 M, with maximal increases of 6.4-fold for GH as compared with only 96% for PRL. Effects of phorbol ester and DAG on GH and PRL release
PMA stimulated the release of GH from P o cells at concentrations of 10~8 to 10~7 M and of PRL at 10~9 to
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SOMATOMAMMOTROPH GH AND PRL SECRETION
Endo • 1990 Voll27-No3 -10.6
FIG. 3. Left, Effect of SRIF on GH and PRL release in rat somatomammotroph (Po) cells during a 4-h incubation. Right, Effect of SRIF on GH and PRL release in primary cultures of dispersed rat pituitary cells. *, P < 0.05. **, P < 0.01 vs. control incubation.
FIG. 4. Left, Effects of (Bu)2cAMP (top) and forskolin (bottom) on GH and PRL release in rat somatomammotroph (Po) cells during a 4-h incubation. Right, Effects of (Bu)2cAMP and forskolin on GH and PRL release in primary cultures of dispersed rat pituitary cells during a 4-h incubation. *, P < 0.05. **, P < 0.01 vs. control incubation.
10 7 Forskolin, M
10 7 M (Fig. 5). The maximal increases in GH and PRL release were each 2.1 times that of basal release. DAG also stimulated both GH and PRL release from P o cells at concentrations of 10~6 to 10~3 M. The maximal increases over basal release were 118% for GH and 72% for PRL. In PPC, PMA increased the secretion of GH at concentrations of 10~10 to 1CT7 M and of PRL at 10~9 to 10"7 M. Maximal increases over basal release were 4.8-fold for GH but only 51% for PRL. DAG stimulated GH release at a concentration of 10~3 M with a maximal increase of 2.9 times basal release. PRL secretion was not stimulated by DAG in PPC cells.
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Discussion Several studies have shown that the pituitary contains a population of cells in which both GH and PRL are present, called somatomammotrophs, and it has been proposed that these cells represent progenitor cells for both somatotrophs and lactotrophs (1-3). The mitogenic potential of this cell type is most likely responsible for its ability to survive in continuous cell culture, which, in our hands, has now persisted for more than 2 yr (1). P o cells contain only 1 to 2% of the GH content of freshly dispersed pituitary cells (1, 17). Under the conditions used in the present study, the basal 4-h GH secretion rate in P o cells constitutes approximately 30% of cell
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SOMATOMAMMOTROPH GH AND PRL SECRETION
1133
FIG. 5. Left, Effects of phorbol myristate acetate (top) and diacylglycerol (bottom) on GH and PRL release in rat somatomammotroph (Po) cells during a 4-h incubation. Right, Effects of phorbol myristate acetate and diacylglycerol on GH and PRL release in primary cultures of dispersed rat pituitary cells during a 4-h incubation. *, P < 0.05. **, P < 0.01 us. control incubation.
Diacylglycerol, M
content as compared with 2% in freshly dispersed cells. The majority of cells exhibited immunofluorescent staining for both GH and PRL, and each of 9 cell lines cloned by limiting dilution secreted both GH and PRL (although in varying proportions), suggesting that each of the cells is truly bihormonal. Although it is not known whether GH and PRL are colocalized in the same secretory granules in the P o cells, the results of the present study indicate that there is a common mechanism of release of the two hormones. Four major second messenger systems have been implicated in hypophysiotropic hormone-mediated secretion of anterior pituitary hormones: the adenylate cyclase-cAMP system, the phosphatidyl inositol-DAG-protein kinase C pathway, the arachadonic acid-eicosanoid pathways, and the Ca++-calmodulin system (18). The adenylate cyclase system is believed to be the major pathway involved in GRF- and SRIF-mediated GH secretion, although Ca++ translocation is also considered important. In contrast, TRH stimulation of PRL secretion in the lactotroph is primarily under the control of the phosphatidyl inositol-protein kinase C pathway. It was therefore of importance to compare the effects of stimuli for each of these systems on GH and PRL secretion by somatomammotroph cells with those in mature somatotrophs and lactotrophs. Initial experiments with GRF and TRH revealed that each releasing hormone stimulated both GH and PRL secretion from P o cells, in contrast to mature somatotrophs and lactotrophs where their effects were hormone specific: GRF stimulated only GH release and TRH stimulated only PRL. VIP, which stimulates PRL release
10 s 10 Diacylglycerol, M
in mature lactotrophs by an adenylate cyclase-mediated mechanism (9, 19), stimulated the release of GH as well as PRL in P o cells. All three of these natural secretogogues stimulated the secretion of the two hormones in P o cells with a similar minimal effective dose. However, although the effects of TRH and VIP in P o cells were comparable to those in mature lactotrophs with respect to both the minimal effective dose and the percentage increase over basal secretion, those of GRF were less potent and seen in the majority of experiments although not in every one. The reason for the less pronounced and less consistent effect of GRF is not entirely clear, although preliminary experiments suggest an impairment in the activation of adenylate cyclase (data not shown). SRIF, the primary inhibitor of GH secretion (20), did not affect PRL secretion in mature lactotrophs. However, in P o cells, SRIF inhibited both GH and PRL secretion. As with GRF, though, the effects of SRIF on GH release were less potent in P o cells compared with mature somatotrophs by a factor of at least 103, with respect to the minimal effective dose. We evaluated the function of the adenylate cyclase system in P o cells by the use of (Bu)2cAMP and forskolin, a plant diterpine that directly stimulates the catalytic subunit of adenylate cyclase (21). (Bu)2cAMP stimulated GH and PRL to a comparable degree in P o cells and to a level that was almost as great, in relation to basal hormone release, as that observed for GH release in mature somatotrophs (4- to 6-fold). These effects were not mirrored in mature lactotrophs where maximal stimulation of PRL was less than twice basal hormone release. Comparable effects were seen with forskolin and
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SOMATOMAMMOTROPH GH AND PRL SECRETION
also with cholera toxin, another stimulator of adenylate cyclase (22) (data not shown), where the stimulation of GH and PRL release in P o cells were similar to that of GH in mature somatotrophs but considerably greater than that on PRL release from mature lactotrophs. Thus, the secretory response of somatomammotrophs to activation of the adenylate cyclase system appears to resemble somatotrophs more than lactotrophs. Enhancement of inositol phospholipid turnover results in stimulation by 1,2-DAG of protein kinase C, another important intracellular mediator of hormone release (23, 24). We therefore investigated the effects of a synthetic DAG and PMA, a tumor-promoting phorbol ester that activates protein kinase C (25), on hormone secretion in somatomammotroph cells. These agents have previously been reported to stimulate GH (26-28) and in some reports (29) PRL release from freshly dispersed anterior pituitary cells. In the present studies, PMA stimulated GH and PRL release in both somatomammotroph cells and dispersed anterior pituitary cells. It is important to note, however, that although the extent of stimulation of GH release in mature somatotrophs was similar to that in somatomammotroph cells, PRL release by mature lactotrophs in response to PMA was relatively modest and less pronounced than in somatomammotrophs. DAG also stimulated both GH and PRL release in somatomammotroph cells. However, in mature pituitary cells, only GH release was stimulated and at a 103-fold greater concentration than in somatomammotroph cells. Thus, the secretory responses to activators of protein kinase C in somatomammotroph cells also tend to resemble those of somatotrophs more than of lactotrophs. The overall results of our studies demonstrate several features of somatomammotrophs that may in part explain some of the discordant literature reports relating to specificity of hormonal responses to various stimuli. The present findings indicate the existence of functional receptors on P o cells for each of the hypophysiotropic peptides that are linked with several second messenger systems. The secretory response to virtually every stimulus evaluated was not only bihormonal, but the least effective dose and the relative increase above basal secretion tended to be similar. Although we do not at present have morphological evidence to support the colocalization of GH and PRL in individual secretory granules, the secretory responses are very suggestive of this possibility. Alternatively, it is possible that individual hormone-containing secretory granules could be similarly affected by each of the intracellular secondary signaling mechanisms. In either case, the results may explain some of the reports demonstrating concomitant GH and PRL responses to secretogogues such as GRF, TRH, VIP, and SRIF. TRH has been reported to stimulate both GH and PRL release in normal pituitaries
E n d o • 1990 Vol 127 • No 3
(30-32) and particularly in hypothyroidism (31, 32). In superfusion experiments using pituitary cell aggregates, the release of both GH and PRL was increased by VIP (33). Although GH release was not stimulated by VIP in our static incubation system of dispersed adult pituitaries, it did occur in somatomammotrophs. SRIF has also been reported to decrease PRL as well as GH secretion in dispersed rat pituitaries previously exposed to estradiol (34). The extent to which somatomammotrophs may contribute to these bihormonal responses in a mixed population of cells is difficult to determine, but their observed frequency, approximately one third of the total GH- and/or PRL-secreting cells in adult male rat pituitaries (3), could explain the results. However, another important variable in such experiments is the hormonal environment during the culture period. For example, our preincubation media for P o cells included 50 nM hydrocortisone, which was essential for demonstrating the GH response to GRF. However, this concentration of glucocorticoid significantly inhibited basal but not stimulated PRL secretion (data not shown). Overall, the spectrum of responses to the various stimuli tested indicate that somatomammotrophs possess the secretory characteristics of both somatotrophs and lactotrophs, although where differences exist between the mature cell types, there is more resemblance to somatotrophs. In the absence of any specific markers for somatotrophs or lactotrophs, with the exception of GH and PRL and of membrane receptors for the hypophysiotropic hormones, it is not possible to conclude from the present study whether somatomammotrophs are the precursors of mature somatotrophs and lactotrophs. This has been suggested by others based on the detection of somatomammotrophs early in development (35) but is not consistent with a recent report in which the cell type apparently responsible for repopulating both somatotrophs and lactotrophs after transgene-induced cytotoxicity is a "stem" somatotroph that does not express the PRL gene (36). A better understanding of the factors necessary for the terminal differentiation of somatomammotrophs will most likely be required to resolve this question. In summary, our studies have shown that the rat pituitary somatomammotroph cell line possesses secretory characteristics of both somatotrophs and lactotrophs, as shown by their responsiveness to GRF, SRIF, TRH, and VIP. The responses to each of the agents are bihormonal and appear to be regulated by a common mechanism. Acknowledgments The authors acknowledge with appreciation the excellent technical assistance of Jane Withrow. Reagents for the rat GH and PRL RIAs were provided by the NIH National Hormone and Pituitary Program.
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SOMATOMAMMOTROPH GH AND PRL SECRETION
References 1. Chomczynski P, Brar A, Frohman LA 1988 Growth hormone synthesis and secretion by a somatomammotroph cell line derived from normal adult pituitary of the rat. Endocrinology 123:22762283 2. Nikitovitch-Winer MB, Atkin J, Maley BE 1987 Colocalization of prolactin and growth hormone within specific adenohypophyseal cells in male, female and lactating female rats. Endocrinology 121:625-630 3. Frawley LS, Boockfor FR, Hoeffer JP 1985 Identification by plaque assays of a pituitary cell type that secretes both growth hormone and prolactin. Endocrinology 116:734-737 4. Brazeau P, Ling N, Bohlen P, Esch F, Ying S, Guillemin R 1982 Growth hormone releasing factor, somatocrinin, releases pituitary growth hormone in vitro. Proc Natl Acad Sci USA 79:7909-7913 5. Bilezikjian LM, Vale W 1983 Stimulation of adenosine 3',5'monophosphate by growth hormone-releasing factor and its inhibition by somatostatin in anterior pituitary cells in vitro. Endocrinology 113:1726-1731 6. Law GJ, Ray KP, Wallis M 1985 Effects of growth hormonereleasing factor and somatostatin on growth hormone secretion and cellular cyclic AMP levels. FEBS Lett 179:12-16 7. Gourdji D, Bataille D, Vauclin N, Grouselle D, Rosselin G, TixierVidal A 1979 Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) release and cAMP production in a rat pituitary cell line (GH3/B6). Additive effects of VIP and TRH on PRL release. FEBS Lett 104:165-168 8. Robberecht P, Deschodt-Lanckman M, Camus J-C, De Neef P, Lambert M, Christophe J 1979 VIP activation of rat anterior pituitary adenylate cyclase. FEBS Lett 103:229-233 9. Rotsztejn WH, Dussaillant M, Nobou F, Rosselin G 1981 Rapid glucocorticoid inhibition of vasoactive intestinal peptide-induced cyclic AMP accumulation and prolactin release in rat pituitary cells in culture. Proc Natl Acad Sci USA 78:7584-7588 10. Sutton CA, Martin TFJ 1982 Thyrotropin-releasing hormone (TRH) selectively and rapidly stimulates phophatidylinositol turnover in GH pituitary cells: a possible second step of TRH action. Endocrinology 110:1273-1280 11. Rebecchi MJ, Gershengorn MC 1983 Thyroliberin stimulates rapid hydrolysis of phophatidylinositol 4,5-bisphosphate by a phosphodiesterase in rat mammotrophic pituitary cells. Biochem J 216:287294 12. Rebecchi M, Kolesnick RM, Gershengorn MC 1983 Thyrotropinreleasing hormone stimulates rapid loss of phosphatidylinositol and its conversion to 1,2-diacylglycerol and phophatidic acid in rat mammotrophic pituitary cells. J Biol Chem 258:227-234 13. Wilfinger WW, Larsen WJ, Downs TR, Wilbur DL 1984 An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells. Tissue Cell 16:483-497 14. Frohman LA, Downs TR 1986 Measurement of growth hormonereleasing factor. Methods Enzymol 124:371-389 15. Frohman LA, Bernardis LL 1968 Growth hormone and insulin levels in weanling rats with ventromedial hypothalamic lesions. Endocrinology 82:1125-1132 16. Szabo M, Frohman LA 1976 Dissociation of prolactin-releasing activity from thyrotropin-releasing hormone in porcine stalk median eminence. Endocrinology 98:1451-1459 17. Katakami H, Downs TR, Frohman LA 1986 Decreased hypothalamic growth hormone-releasing hormone content and pituitary responsiveness in hypothyroidism. J Clin Invest 77:1704-1711 18. Denef C 1988 Mechanism of action of pituitary hormone releasing and inhibiting factors. In: Cooke BA, King RJB, van der Molen HJ (eds) Hormones and Their Actions, Part II. Elsevier Science Publishers, Amsterdam, pp 113-134
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19. Enjalbert A, Arancibia S, Ruberg M, Priam M, Bluet-Pajot MT, Rotsztejn WH, Kordon C 1980 Stimulation of in vitro prolactin release by vasoactive intestinal peptide. Neuroendocrinology 31:200-204 20. Brazeau P, Vale W, Burgus R, Ling N, Butcher M, Rivier J, Guillemin R 1973 Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:77-79 21. Seamon K, Daly JW 1981 Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. J Biol Chem 256:9799-9801 22. Cassel D, Selinger Z 1977 Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Proc Natl Acad Sci USA 74:3307-3311 23. Takai Y, Kishimoto A, Kikkawa U, Mori T, Nishizuka Y 1979 Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system. Biochem Biophys Res Commun 91:1218-1224 24. Kishimoto A, Takai Y, Mori T, Kikkawa U, Nishizuka Y 1980 Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J Biol Chem 255:2273-2276 25. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y 1982 Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 257:7847-7851 26. Smith MA, Vale WW 1980 Superfusion of rat anterior pituitary cells attached to cytodex beads: validation of a technique. Endocrinology 107:1425-1431 27. Ohmura E, Friesen HG 1985 12-0-tetradecanoyl phorbol-13-acetate stimulates rat growth hormone (GH) release through different pathways from that of human pancreatic GH-releasing factor. Endocrinology 116:728-737 28. Judd AM, Koike K, Yasumoto T, MacLeod RM 1986 Protein kinase C activators and calcium-mobilizing agents synergistically increase GH, LH and TSH secretion from anterior pituitary cells. Neuroendocrinology 42:197-202 29. Ohmura E, Tsushima T, Murakami H, Wakai K, Shizume K 1984 Effect of phorbol esters on the release of growth hormone and prolactin from rat pituitary cells cultured in monolayer. Acta Endocrinol (Copenh) 107:185-191 30. Carlson HE, Mariz IK, Daughaday WH 1974 Thyrotropin-releasing hormone stimulation and somatostatin inhibition of growth hormone secretion from perfused rat adenohypophyses. Endocrinology 94:1709-1713 31. Chihara K, Kato Y, Ohgo S, Iwasaki Y, Maeda K, Miyamoto Y, Imura H 1976 Effects of hyperthyroidism and hypothyroidism on rat growth hormone release induced by thyrotropin-releasing hormone. Endocrinology 98:1396-1400 32. Szabo M, Stachura ME, Paleologos N, Bybee DE, Frohman LA 1984 Thyrotropin-releasing hormone stimulates growth hormone release from the anterior pituitary of hypothyroid rats in vitro. Endocrinology 114:1344-1351 33. Denef C, Schramme C, Baes M 1985 Stimulation of growth hormone release by vasoactive intestinal peptide and peptide PHI in rat anterior pituitary reaggregates. Neuroendocrinology 40:88-91 34. 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 35. Hoeffler JP, Boockfor FR, Frawley LS 1985 Ontogeny of prolactin cells in neonatal rats: initial prolactin secretors also release growth hormone. Endocrinology 117:187-195 36. Borelli E, Heyman RA, Arias C, Sawchenko PE, Evans RM 1989 Transgenic mice with inducible dwarfism. Nature 339:538-541
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Vol. 127, No. 3 Printed in U.S.A.
Growth Hormone and Prolactin Secretion in Cultured Somatomammotroph Cells* YOICHI KASHIO, PIOTR CHOMCZYNSKI, THOMAS R. DOWNS, AND LAWRENCE A. FROHMAN Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
cells by adenylate cyclase-related agents ((Bu)2 cAMP and forskolin) was comparable to that for GH secretion in mature somatotrophs but much greater than that of PRL release in mature lactotrophs. Stimulation of GH and PRL release in Po cells by protein kinase C-related agents (diacylglycerol and phorbol ester) was also similar to that observed for GH release from mature pituitary cells, whereas minimal or undetectable effects were observed on PRL release from mature cells. The results indicate that the Po somatomammotropic cell line possesses receptors, second messenger systems, and secretory characteristics of both somatotrophs and lactotrophs, although where differences exist, there is more resemblance to somatotrophs. They also demonstrate that the responses to each of the agents studied are bihormonal and appear to be regulated by a common mechanism. (Endocrinology 127: 1129-1135, 1990)
ABSTRACT. A somatomammotropic cell line (Po) derived from adult rat pituitaries has been maintained in culture for 2 yr. Secretion of GH and PRL by this cell line has been studied in response to hypophysiotropic peptides known to affect the release of both hormones as well as agents that affect second messenger systems in an attempt to characterize the stimulussecretion mechanisms used by these cells. GH and PRL release during short term (4 h) incubations of Po cells and primary cultures of dispersed rat pituitary cells was initially measured in response to GRF, TRH, vasoactive intestinal peptide (VIP), and SRIF. In Po cells, the minimal effective dose of each of the hypophysiotropic peptides was comparable with respect to GH and PRL secretion. The effects of TRH and VIP were similar to those in freshly dispersed cells with respect to PRL release, whereas those of GRF and SRIF were less potent with respect to GH release. The stimulation of GH and PRL release in P o
W
E HAVE recently described a pituitary cell line derived from primary cultures of dispersed adult male rat anterior pituitary cells by repeated and selective subculturing (1). Immunofluorescent staining revealed that virtually all of the cells contained GH, whereas 72% also contained PRL, and nine separate clones, obtained by limiting dilution of the cell line, each secreted GH and PRL, although in markedly varying ratios. These somatomammotroph cells differ from the pituitary tumor-derived cell lines GC, GH3, and GH4 in that they do not grow to confluence in culture, do not produce tumors after syngeneic transplantation, and maintain responsiveness to GRF. We have proposed that these cells may represent progenitor cells that can differentiate into somatotrophs and lactotrophs, although such transformations have yet to be demonstrated. Similar suggestions have been made by others on the basis of electron microscopic findings in somatomammotrophs (2) and bihormonal secretory patterns using the hemolytic plaque
assay (3). In an attempt to characterize the control of hormone secretion in somatomammotroph cells, we have studied the role of the hypothalamic hormones [GRF, TRH, vasoactive intestinal peptide (VIP), and somatostatin (SRIF)] known to affect secretion of GH and PRL in adult somatotrophs or lactotrophs. GRF and VIP exert their actions primarily through the activation of a cAMP-dependent protein kinase (4-9), whereas SRIF acts, at least in part, by inhibiting cAMP production (5, 6). In contrast, TRH action is mediated primarily by a protein kinase C-dependent pathway (10-12). We therefore also examined the effects of dibutyryl (Bu)2cAMP and forskolin to assess the adenylate cyclase system and those of phorbol myristate acetate (PMA) and diacylglycerol (DAG), activators of protein kinase C. The same agents were evaluated in primary cultures of dispersed anterior pituitary cells (from which the somatomammotrophs were derived) to compare the responses in developmental and mature cells.
Received March 23, 1990. Address all correspondence and requests for reprints to: Dr. Lawrence Frohman, Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, 231 Bethesda Avenue, M.L. #547, Cincinnati, Ohio 45267. * These studies were supported in part by USPHS Grant DK-30667.
Materials and Methods Pituitary cell line culture The origin of the pituitary cell line (herein designated as Po) has been previously described (1). Cells were maintained in a-
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SOMATOMAMMOTROPH GH AND PRL SECRETION
minimum essential medium (aMEM, Gibco, Grand Island, NY) supplemented with 10% horse serum (Sigma, St. Louis, MO), gentamicin (25 Mg/ml, Gibco) and nystatin (25 U/ml, Gibco) at 37 C in a water-saturated atmosphere containing 5% CO2. The culture medium was changed every 2 days. For experiments performed to study acute hormone release, cells were plated at a density of 1 to 2 x 105 cells/16-mm well and grown as monolayer cultures in aMEM supplemented with 10% horse serum, 50 nM hydrocortisone, and 50 fig/m\ gentamicin for 48 h. The cells were rinsed and then incubated for 4 h in 1 ml aMEM containing 0.1% BSA (Metrix, Chicago, IL) and specific test substances. The medium was then aspirated, and any floating cells were removed by centrifugation at 100 X g for 10 min at 4 C. The cell count was repeated at the end of the incubation (1). Each experimental protocol was performed from 2 to 5 times. Hormone secretion, expressed as the quantity of GH or PRL released during the 4-h incubation per 105 cells, is presented as the mean ± SE of quadruplicate incubations from a single representative experiment. Rat pituitary cell primary culture Primary monolayer cultures of dispersed pituitary cells were prepared as previously described (13, 14). After a 3-day culture period in aMEM containing 10% horse serum and 25 fig/ml gentamicin, the medium was removed and the cells washed twice with aMEM containing 0.1% BSA. The incubation medium and duration of the experimental period were identical to that described previously for the Po cells. Hormone measurements and data analysis GH and PRL were measured by RIA in duplicate as previously described (15, 16). Rat GH Reference Preparation (RP)1 and rat PRL RP-1 served as reference standards. Samples from each experiment were measured in a single assay with intra- and interassay coefficients of variation of 8.6% and 14.4% (GH) and 9.0% and 11.4% (PRL), respectively. The difference between group means was determined by analysis of variance using Dunnet's modified method for multiple groups compared with a single control after logarithmic transformation of the raw data to normalize variance. Drugs Rat (r) GRF (Peninsula Laboratories, Inc., Belmont, CA) was initially dissolved in 5 mM acetic acid containing 0.1% BSA. Porcine VIP(1o-28) (Peninsula), TRH (Abbott Laboratories, Chicago, IL), SRIF (Sigma), and (Bu)2cAMP (SchwartzMann, Orangeburg, NY) were dissolved in 0.05 M PBS containing 1% BSA and forskolin (Calbiochem, La Jolla, CA) in absolute ethanol. The latter was subsequently diluted so that the final concentration of ethanol in the incubation medium was less than 0.1%. PMA (Sigma) and l-oleoyl-2-acetyl-snglycerol (a synthetic DAG; Sigma) were initially dissolved and stored in dimethylsulfoxide (DMSO) and diluted so that the final concentration of DMSO in the incubation medium was less than 0.1%.
Results Effect of rGRF on GH and PRL release rGRF stimulated both GH and PRL release in pituitary somatomammotroph (Po) cells (Fig. 1). A significant
E n d o • 1990 Vol 127 • No 3
increase in GH release occurred at a concentration of 10~9 M and a maximal response was observed at 10~8 M to 10~7 M in several experiments. No stimulation of GH release was detected at 10"10 M rGRF in three separate incubations (data not shown). The maximum response constituted a 96% increase over basal hormone release. Basal GH release during a 4-h incubation period represented 33% of initial cell content (six experiments). rGRF also stimulated PRL release in P o cells at similar concentrations with a maximal increase of 198% over basal levels. Basal PRL release during the 4-h incubation period represented 109% of initial cell content. Twelve separate incubations were performed in which GRF was used as a stimulus. GH secretion was significantly increased in eight experiments but not in the other four. No explanation is presently available for the lack of consistency of the response to GRF, which stands in contrast to that of other stimuli, the effects of which were always reproducible. In primary pituitary cell cultures (PPC), in contrast, basal GH release represented 2% of cell content. GRF stimulatory effects were present at 10~12 M, and maximal stimulation represented a 7-fold increase over basal release. GRF did not stimulate PRL release in PPC. Effects of TRH and VIP on GH and PRL release GH secretion by P o cells was stimulated by TRH at concentrations of 10"9 to 10~7 M (Fig. 2). Maximal stimulation of GH was 75% above basal release. A comparable stimulation of PRL release (69% above basal) was seen at similar concentrations. VIP also stimulated GH release at concentrations of 10"8 to 10~6 M with a maximal increase of 48% above basal release. PRL release was increased at comparable concentrations with a maximal increase of 28% above basal release. In PPC, TRH did not stimulate GH release, although PRL release was enhanced at concentrations of 10~8 M and greater. Similarly, VIP was ineffective in increasing GH release, although it also stimulated PRL release at concentrations of 10~8 M and greater. Effect of SRIF on GH and PRL release GH secretion by P o cells was inhibited by SRIF at concentrations of 10~8 M and greater (Fig. 3). At a concentration of 10~6 M, GH release was inhibited by 60%. There was a parallel inhibition of PRL secretion in P o cells beginning at 10~9 M SRIF, and PRL release was inhibited by 53% at a concentration of 10~6 M. GH secretion by PPC was inhibited by SRIF at concentrations of 10~n M and greater, and at a concentration of 10~7 M GH release was inhibited by 80%. In contrast to P o cells, PRL secretion by PPC was not significantly inhibited by SRIF.
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SOMATOMAMMOTROPH GH AND PRL SECRETION 40
1131
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Po Cells
FIG. 1. Left, Effect of rGRF on GH and PRL release in rat somatomammotroph (Po) cells determined during a 4-h incubation. Shown are the mean ±SE of quadruplicate incubations in one representative experiment. Right, Effect of rGRF on GH and PRL release in primary cultures of dispersed rat pituitary cells during a 4-h incubation. Each point represents the mean ±SE of quadruplicate incubations from one of two separate experiments. **, P < 0.01 vs. control incubation.
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FIG. 2. Left, Effects of TRH (top) and VIP (bottom) on GH and PRL release in rat somatomammotroph (Po) cells during a 4-h incubation. Shown are the mean ± SE of quadruplicate incubations from one of three experiments with comparable results. Right, Effects of TRH and VIP on GH and PRL release in primary cultures of dispersed rat pituitary cells during a 4-h incubation. *, P < 0.05. **, P < 0.01 vs. control incubation.
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Effect of (Bu)2cAMP and forskolin on GH and PRL release
(Bu)2cAMP exhibited comparable stimulatory effects on GH and PRL secretion in P o cells (Fig. 4). Stimulation was observed at concentrations of 10~5 M and greater. At a concentration of 10"3 M, the increase in GH and PRL release was 3.4-fold and 2.9-fold, respectively, over basal release. The stimulatory effects of forskolin on GH and PRL secretion were also comparable. Stimulation was observed at 10~8 M and greater with maximal increases of GH and PRL secretion of 3.2-fold and 2.6-fold, respectively, over basal secretion. (Bu)2cAMP stimulated GH secretion in PPC to a
comparable degree as in P o cells. The increase in release at a concentration of 10~3 M was 5 times that of basal release. The effects on PRL release were much less pronounced with a maximal response of only 68% above basal release. Similarly, in PPC, forskolin stimulated GH and PRL secretion at concentrations from 10~8 to 10~5 M, with maximal increases of 6.4-fold for GH as compared with only 96% for PRL. Effects of phorbol ester and DAG on GH and PRL release
PMA stimulated the release of GH from P o cells at concentrations of 10~8 to 10~7 M and of PRL at 10~9 to
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SOMATOMAMMOTROPH GH AND PRL SECRETION
Endo • 1990 Voll27-No3 -10.6
FIG. 3. Left, Effect of SRIF on GH and PRL release in rat somatomammotroph (Po) cells during a 4-h incubation. Right, Effect of SRIF on GH and PRL release in primary cultures of dispersed rat pituitary cells. *, P < 0.05. **, P < 0.01 vs. control incubation.
FIG. 4. Left, Effects of (Bu)2cAMP (top) and forskolin (bottom) on GH and PRL release in rat somatomammotroph (Po) cells during a 4-h incubation. Right, Effects of (Bu)2cAMP and forskolin on GH and PRL release in primary cultures of dispersed rat pituitary cells during a 4-h incubation. *, P < 0.05. **, P < 0.01 vs. control incubation.
10 7 Forskolin, M
10 7 M (Fig. 5). The maximal increases in GH and PRL release were each 2.1 times that of basal release. DAG also stimulated both GH and PRL release from P o cells at concentrations of 10~6 to 10~3 M. The maximal increases over basal release were 118% for GH and 72% for PRL. In PPC, PMA increased the secretion of GH at concentrations of 10~10 to 1CT7 M and of PRL at 10~9 to 10"7 M. Maximal increases over basal release were 4.8-fold for GH but only 51% for PRL. DAG stimulated GH release at a concentration of 10~3 M with a maximal increase of 2.9 times basal release. PRL secretion was not stimulated by DAG in PPC cells.
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Discussion Several studies have shown that the pituitary contains a population of cells in which both GH and PRL are present, called somatomammotrophs, and it has been proposed that these cells represent progenitor cells for both somatotrophs and lactotrophs (1-3). The mitogenic potential of this cell type is most likely responsible for its ability to survive in continuous cell culture, which, in our hands, has now persisted for more than 2 yr (1). P o cells contain only 1 to 2% of the GH content of freshly dispersed pituitary cells (1, 17). Under the conditions used in the present study, the basal 4-h GH secretion rate in P o cells constitutes approximately 30% of cell
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SOMATOMAMMOTROPH GH AND PRL SECRETION
1133
FIG. 5. Left, Effects of phorbol myristate acetate (top) and diacylglycerol (bottom) on GH and PRL release in rat somatomammotroph (Po) cells during a 4-h incubation. Right, Effects of phorbol myristate acetate and diacylglycerol on GH and PRL release in primary cultures of dispersed rat pituitary cells during a 4-h incubation. *, P < 0.05. **, P < 0.01 us. control incubation.
Diacylglycerol, M
content as compared with 2% in freshly dispersed cells. The majority of cells exhibited immunofluorescent staining for both GH and PRL, and each of 9 cell lines cloned by limiting dilution secreted both GH and PRL (although in varying proportions), suggesting that each of the cells is truly bihormonal. Although it is not known whether GH and PRL are colocalized in the same secretory granules in the P o cells, the results of the present study indicate that there is a common mechanism of release of the two hormones. Four major second messenger systems have been implicated in hypophysiotropic hormone-mediated secretion of anterior pituitary hormones: the adenylate cyclase-cAMP system, the phosphatidyl inositol-DAG-protein kinase C pathway, the arachadonic acid-eicosanoid pathways, and the Ca++-calmodulin system (18). The adenylate cyclase system is believed to be the major pathway involved in GRF- and SRIF-mediated GH secretion, although Ca++ translocation is also considered important. In contrast, TRH stimulation of PRL secretion in the lactotroph is primarily under the control of the phosphatidyl inositol-protein kinase C pathway. It was therefore of importance to compare the effects of stimuli for each of these systems on GH and PRL secretion by somatomammotroph cells with those in mature somatotrophs and lactotrophs. Initial experiments with GRF and TRH revealed that each releasing hormone stimulated both GH and PRL secretion from P o cells, in contrast to mature somatotrophs and lactotrophs where their effects were hormone specific: GRF stimulated only GH release and TRH stimulated only PRL. VIP, which stimulates PRL release
10 s 10 Diacylglycerol, M
in mature lactotrophs by an adenylate cyclase-mediated mechanism (9, 19), stimulated the release of GH as well as PRL in P o cells. All three of these natural secretogogues stimulated the secretion of the two hormones in P o cells with a similar minimal effective dose. However, although the effects of TRH and VIP in P o cells were comparable to those in mature lactotrophs with respect to both the minimal effective dose and the percentage increase over basal secretion, those of GRF were less potent and seen in the majority of experiments although not in every one. The reason for the less pronounced and less consistent effect of GRF is not entirely clear, although preliminary experiments suggest an impairment in the activation of adenylate cyclase (data not shown). SRIF, the primary inhibitor of GH secretion (20), did not affect PRL secretion in mature lactotrophs. However, in P o cells, SRIF inhibited both GH and PRL secretion. As with GRF, though, the effects of SRIF on GH release were less potent in P o cells compared with mature somatotrophs by a factor of at least 103, with respect to the minimal effective dose. We evaluated the function of the adenylate cyclase system in P o cells by the use of (Bu)2cAMP and forskolin, a plant diterpine that directly stimulates the catalytic subunit of adenylate cyclase (21). (Bu)2cAMP stimulated GH and PRL to a comparable degree in P o cells and to a level that was almost as great, in relation to basal hormone release, as that observed for GH release in mature somatotrophs (4- to 6-fold). These effects were not mirrored in mature lactotrophs where maximal stimulation of PRL was less than twice basal hormone release. Comparable effects were seen with forskolin and
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SOMATOMAMMOTROPH GH AND PRL SECRETION
also with cholera toxin, another stimulator of adenylate cyclase (22) (data not shown), where the stimulation of GH and PRL release in P o cells were similar to that of GH in mature somatotrophs but considerably greater than that on PRL release from mature lactotrophs. Thus, the secretory response of somatomammotrophs to activation of the adenylate cyclase system appears to resemble somatotrophs more than lactotrophs. Enhancement of inositol phospholipid turnover results in stimulation by 1,2-DAG of protein kinase C, another important intracellular mediator of hormone release (23, 24). We therefore investigated the effects of a synthetic DAG and PMA, a tumor-promoting phorbol ester that activates protein kinase C (25), on hormone secretion in somatomammotroph cells. These agents have previously been reported to stimulate GH (26-28) and in some reports (29) PRL release from freshly dispersed anterior pituitary cells. In the present studies, PMA stimulated GH and PRL release in both somatomammotroph cells and dispersed anterior pituitary cells. It is important to note, however, that although the extent of stimulation of GH release in mature somatotrophs was similar to that in somatomammotroph cells, PRL release by mature lactotrophs in response to PMA was relatively modest and less pronounced than in somatomammotrophs. DAG also stimulated both GH and PRL release in somatomammotroph cells. However, in mature pituitary cells, only GH release was stimulated and at a 103-fold greater concentration than in somatomammotroph cells. Thus, the secretory responses to activators of protein kinase C in somatomammotroph cells also tend to resemble those of somatotrophs more than of lactotrophs. The overall results of our studies demonstrate several features of somatomammotrophs that may in part explain some of the discordant literature reports relating to specificity of hormonal responses to various stimuli. The present findings indicate the existence of functional receptors on P o cells for each of the hypophysiotropic peptides that are linked with several second messenger systems. The secretory response to virtually every stimulus evaluated was not only bihormonal, but the least effective dose and the relative increase above basal secretion tended to be similar. Although we do not at present have morphological evidence to support the colocalization of GH and PRL in individual secretory granules, the secretory responses are very suggestive of this possibility. Alternatively, it is possible that individual hormone-containing secretory granules could be similarly affected by each of the intracellular secondary signaling mechanisms. In either case, the results may explain some of the reports demonstrating concomitant GH and PRL responses to secretogogues such as GRF, TRH, VIP, and SRIF. TRH has been reported to stimulate both GH and PRL release in normal pituitaries
E n d o • 1990 Vol 127 • No 3
(30-32) and particularly in hypothyroidism (31, 32). In superfusion experiments using pituitary cell aggregates, the release of both GH and PRL was increased by VIP (33). Although GH release was not stimulated by VIP in our static incubation system of dispersed adult pituitaries, it did occur in somatomammotrophs. SRIF has also been reported to decrease PRL as well as GH secretion in dispersed rat pituitaries previously exposed to estradiol (34). The extent to which somatomammotrophs may contribute to these bihormonal responses in a mixed population of cells is difficult to determine, but their observed frequency, approximately one third of the total GH- and/or PRL-secreting cells in adult male rat pituitaries (3), could explain the results. However, another important variable in such experiments is the hormonal environment during the culture period. For example, our preincubation media for P o cells included 50 nM hydrocortisone, which was essential for demonstrating the GH response to GRF. However, this concentration of glucocorticoid significantly inhibited basal but not stimulated PRL secretion (data not shown). Overall, the spectrum of responses to the various stimuli tested indicate that somatomammotrophs possess the secretory characteristics of both somatotrophs and lactotrophs, although where differences exist between the mature cell types, there is more resemblance to somatotrophs. In the absence of any specific markers for somatotrophs or lactotrophs, with the exception of GH and PRL and of membrane receptors for the hypophysiotropic hormones, it is not possible to conclude from the present study whether somatomammotrophs are the precursors of mature somatotrophs and lactotrophs. This has been suggested by others based on the detection of somatomammotrophs early in development (35) but is not consistent with a recent report in which the cell type apparently responsible for repopulating both somatotrophs and lactotrophs after transgene-induced cytotoxicity is a "stem" somatotroph that does not express the PRL gene (36). A better understanding of the factors necessary for the terminal differentiation of somatomammotrophs will most likely be required to resolve this question. In summary, our studies have shown that the rat pituitary somatomammotroph cell line possesses secretory characteristics of both somatotrophs and lactotrophs, as shown by their responsiveness to GRF, SRIF, TRH, and VIP. The responses to each of the agents are bihormonal and appear to be regulated by a common mechanism. Acknowledgments The authors acknowledge with appreciation the excellent technical assistance of Jane Withrow. Reagents for the rat GH and PRL RIAs were provided by the NIH National Hormone and Pituitary Program.
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SOMATOMAMMOTROPH GH AND PRL SECRETION
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