0013-7227/90/1265-2369$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 5 Printed in U.S.A.
Activin-A Modulates Growth Hormone Secretion from Cultures of Rat Anterior Pituitary Cells* LOUISE M. BILEZIKJIAN, ANNE Z. CORRIGAN, AND WYLIE VALEt The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037
ABSTRACT. Activins, initially identified as FSH-releasing proteins, have now been shown to exert effects on other cell types of the anterior pituitary, including the somatotrophs. In the present study the inhibitory action of activin-A (/?A/?A) o n GH secretion was characterized using primary cultures of rat anterior pituitary cells. Activin-A suppressed basal GH secretion for up to 72 h (the longest time tested). Immediately after the treatment period with activin-A, when the cells were thoroughly washed and further incubated with or without rat GH-releasing factor (rGRF), basal and stimulated GH secretion were partially inhibited as well. In parallel, activin-A pretreatment diminished rGRF-stimulated cAMP accumulation. The effects of activin-A were time- and concentration-dependent, with half-maximal inhibition occurring in the range of 20-30 pM activin-A. A minimum pretreatment time of 3 h was required for maximal effect, and when rGRF and activin-A were added simultaneously, no inhibition was evident. Secretory responses of activin-Apretreated cells to rGRF were influenced by glucocorticoids. When cells were cultured in the presence of the synthetic glucocorticoid dexamethasone, pretreatment (72 h) with activin-A attenuated rGRF-stimulated GH secretion only during short (1h), but not longer (3-h), exposure periods to the neuropeptide. In the absence of dexamethasone, rGRF-stimulated GH secre-
tion was inhibited at all incubation times tested (up to 3 h). A 3-h exposure to the protein factor did not alter total (cellular plus secreted) immunoreactive GH levels, suggesting that the inhibition of secretion with the shorter treatment was not secondary to attenuated GH biosynthesis. However, longer (72-h) treatment with activin-A decreased total GH levels, indicating lower GH biosynthetic rates, as previously shown. Somatostatin is recognized as the primary negative modulator of GH secretion. Activin-A and SRIF inhibited GH secretion additively, suggesting distinct mechanisms of action for each. GH secretion in response to other secretagogues, such as 12-O-tetradecanoylphorbol-13-acetate, forskolin, cholera toxin, and 8-bromocAMP, was also suppressed after activin-A pretreatment. The presence of the RNA synthesis inhibitor actinomycin-D completely blocked the inhibitory effect of a 3-h activin-A pretreatment on subsequent rGRF-stimulated GH secretion. Pertussis toxin was only partially effective in preventing the inhibition by activin-A. The results of this study indicate that activin-A plays a crucial role as a modulator of somatotropic function, inhibiting GH secretion at the level of the secretory process and secondary to the inhibition of GH biosynthesis. {Endocrinology 126: 23692376, 1990)
S
ECRETION of GH by the somatotrophs of the anterior pituitary is known to be inversely regulated by GH-releasing factor (GRF) and somatostatin (SRIF) (1-4). GRF enhances intracellular cAMP levels (5) and activates the cAMP-dependent protein kinases (6), suggesting that the adenylate cyclase-activated signaling pathway mediates the actions of GRF. Calcium, however, is also required for GH secretion, and GRF has been reported to elevate intracellular calcium concentrations (7). Moreover, the activation of other second messenger systems, such as protein kinase-C, also promotes GH secretion (8), indicating the existence of multiple mechanisms that can potentially regulate the secretory process. The transcriptional effects of GRF on the GH gene, Received December 11,1989. Address requests for reprints to: Dr. Louise M. Bilezikjian, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, P.O. Box 85800, San Diego, California 92138. * This work was supported by NIH Grant DK-26741 and was conducted in part by The Clayton Foundation for Research, California Division. t Senior Clayton Foundation Investigator.
on the other hand, appear to be primarily mediated by cAMP (9, 10). Glucocorticoids and thyroid hormones also exert a positive control on the GH gene (10, 11) and augment the response to GRF (3), while other factors, such as insulin-like growth factor-I, inhibit its expression (12). SRIF acutely inhibits the secretion of GH in response to any of the known GH secretagogues (8, 13), but does not acutely attenuate the stimulation of transcription of the GH gene (9). The mechanism of action of SRIF is not well understood and may involve inhibition of calcium fluxes (14) and/or the inhibitory Gprotein (G;) of adenylate cyclase (15). In addition to SRIF, recent evidence indicates that a newly characterized protein, referred to as activin (16, 17), also exerts negative effects on GH secretion (16, 18) and biosynthesis (19). Activins and inhibins, initially isolated and characterized based on their ability to, respectively, enhance and inhibit FSH secretion from cultured rat anterior pituitary cells (16, 17, 20-23), are members of a functionally
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diverse but structurally related family of proteins known as transforming growth factor-/? (TGF0) (24-26). This family consists of various proteins, including the multiple forms of TGFjS, Mullerian inhibiting substance, cartilage-inducing factors, and decapentaplegic gene complex polypeptide, and is identical to erythroid differentiation factor (25, 26). Activins and inhibins are disulfide-linked dimeric proteins; inhibins are heterodimers comprised of an a-chain and either a j8A- or a /3B-chain, and activins are homodimers of any combination of the inhibin (3chains. The cDNAs encoding the three known subunits (a, /3A, and j8B) have been cloned and sequenced (24, 2730). Inhibin a and ft mRNAs have been detected in a number of tissues, including the ovary, testis, placenta, brain, bone marrow, and pituitary (31). Moreover, receptors for activin are expressed on a number of different cell types (32-35), consistent with the varied effects of these proteins that have thus far been reported (16, 18, 19, 35-40). The first recognized actions of the activins and the inhibins at the anterior pituitary level were the stimulation and the inhibition, respectively, of basal FSH (but not basal LH) secretion (16, 17, 20-23). In contrast to GnRH, the effects of these proteins on FSH secretion are slower in onset. Further examination, however, indicated that activin, and possibly inhibin, modulates the activity of other cell types of the anterior pituitary as well. Long term treatment of cultured rat anterior pituitary cells with activin-A inhibits basal GH and ACTH secretion (16, 18, 19) as well as PRL secretion (18). Additionally, activin blocks the proliferative effects of GRF on somatotrophs (19). The present study was undertaken to further examine the inhibitory effects of activin-A (J8A/?A) on GH secretion from cultures of rat anterior pituitary cells and define some of the characteristics of this action. The results indicate that activin-A has dual actions on somatotrophs: inhibition of basal and GRF-stimulated GH secretion as well as GH biosynthesis.
Materials and Methods Anterior pituitaries from male Sprague-Dawley rats (200220 g) were dissociated by collagenase and plated (0.15 X 106 cells/0.5 ml-well in 48-well multiwelled dishes) in medium containing 2% fetal bovine serum, 30 pM T3, and, unless stated otherwise, dexamethasone (DEX; 5 or 10 nM), as described previously (41). The cells were allowed to recover for 3 days before initiating the 72-h pretreatments with activin-A or for up to 5 days for shorter pretreatment times. The cells were washed twice and placed in fresh medium containing either 2% fetal bovine serum or 0.1% BSA for the 72-h or the 3-h pretreatments, respectively. For experiments with protein synthesis inhibitors, the agent was added 30 min before initiating the 3-h treatment with activin-A. At the end of the preincu-
Endo • 1990 Vol 126 • No 5
bation period, the media were collected for determination of basal GH secretion. The cells were rewashed three times with medium and treated with various GH secretagogues for 1 or 3 h in the same medium that was used during the pretreatment period. Activin-A and the protein synthesis inhibitors were not reintroduced if the stimulation period was 1 h, but were added back for longer stimulation periods. Medium was collected for the analysis of secreted GH levels, and the cells were extracted for the determination of either GH content or intracellular cAMP levels. The cells were lysed with 0.5% Nonidet P-40 for GH analysis and with 95% ethanol-0.1 N HC1 for the determination of cAMP levels, as previously described (42). GH was measured using a RIA kit provided by the National Hormone and Pituitary Program of the NIDDK. Intracellular cAMP levels were quantified using a commercially available RIA kit (Biomedical Technologies, Inc., Cambridge, MA). The activin-A (/3A/3A) used in these experiments was either a highly purified protein isolated from porcine follicular fluid (16) or recombinantly expressed human activin-A (43) (generous gift of Genentech, San Francisco, CA). Equivalent responses were obtained with the protein from either source. All data were subjected to analysis of variance, and differences between groups were assessed using the multiple range test of Duncan. Reported values represent the mean ± SEM of triplicate wells for representative experiments or for normalized means from three or more independent experiments, each performed in triplicate.
Results The time-course of activin-A action on GH secretion was determined using rat anterior pituitary cells cultured in the presence of 5 nM DEX. Secretion in response to a 1-h challenge with 10 nM rGRF was attenuated as a function of the length of preincubation with the protein (Fig. IB). These results indicated that preexposure to activin-A was necessary for its inhibitory action on subsequent stimulated secretion; when activin-A and rat GRF (rGRF) were added simultaneously, no inhibition was observed for up to 5 h (the longest time tested). As short as a 3-h preexposure to a maximal (0.7 nM) concentration of activin-A was sufficient to obtain the full effect; the inhibitions of basal and stimulated GH secretion after 3 h of activin-A pretreatment were 25 ± 4% and 44 ± 2% (n = 15), respectively. The inhibition of rGRF-stimulated GH secretion by activin-A pretreatment was accompanied by a parallel attenuation of rGRF-stimulated cAMP generation (Fig. 1A). Activin-A by itself had no significant effect on basal cAMP levels during or after the treatment periods. Basal secretion during the pretreatment period with activin-A was also inhibited in a time-dependent manner, but was not statistically significant during a period of less than 3 h. Incubation of the rat anterior pituitary cells with a maximal concentration of activin-A for 3 h inhibited basal GH secretion by 21 ± 9% (n = 7). After 72 h, maximal inhibition was 31 ± 4% (n = 5), with an
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ACTIVIN AND GH SECRETION 4.0
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11 23
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FIG. 2. Concentration-dependent inhibition of basal GH secretion during a 72-h treatment with activin-A from cells (0.3 x 106/ml) cultured without DEX. The data are the mean ± SEM of triplicate wells of a representative experiment.
1.0 -
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Secret ion |
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0.6-
0.4-
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Activin A Pretreatment (h) FlG. 1. The time course of the inhibition of basal (•) and rGRFstimulated (H) cAMP accumulation (A) and GH secretion (B) during a 1-h incubation after pretreatment with 0.7 nM activin-A for the indicated lengths of time. The cells (0.15 X 106/0.5 ml) were cultured in the presence of 5 nM DEX for 3 days before initiating the treatments with activin-A in the presence of DEX. The data are the mean ± SEM of triplicate wells of a representative experiment.
IC50 of approximately 20-30 pM (Fig. 2). A complex interaction between glucocorticoids and activin-A was noted. Basal GH secretion was inhibited to approximately the same extent regardless of whether the cells had been cultured with or without the synthetic glucocorticoid dexamethasone (DEX) during the 72-h pretreatment period with a maximally effective concentration of activin-A (0.7 nM; 39 ± 2% and 31 ± 4% inhibition in DEX-treated and untreated cells, respectively; n = 5). The activin-A effect on the subsequent
rGRF
-
+
-
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FIG. 3. The effect of DEX on basal (•) and rGRF-stimulated (M) GH secretion after a 72-h pretreatment with activin-A (0.7 nM). Rat anterior pituitary cells (0.3 x 106 cells/ml) were cultured in the presence or absence of 5 nM DEX for 3 days, washed, and treated or not treated with 0.7 nM activin-A for an additional 72 h with or without DEX. The cells were rewashed, and GH secretion was measured during a 3-h incubation in the presence or absence of 10 nM rGRF. The data are the mean ± SEM of triplicate wells of a representative experiment.
stimulation period with rGRF, however, was influenced by DEX. GH secretion in response to a 1-h challenge with rGRF was inhibited to the same extent regardless of whether the cells had been cultured with or without DEX, equivalent to the data in Fig. 1. In contrast, when GH secretion was measured in response to a 3-h challenge with rGRF after a 72-h activin-A pretreatment, inhibition was observed only in cells cultured without DEX (Fig. 3). In the absence of DEX, the half-maximal concentration of activin-A required during a 72-h pretreatment period to inhibit subsequent GH secretion in response to a 3-h challenge with rGRF was in the range of 20-30 pM (Fig. 4A). This value was equivalent to that
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ACTIVIN AND GH SECRETION
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1.0
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Endo • 1990 Vol 126 • No 5
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Activin A (M) FlG. 4. Concentration-dependence of the inhibition of basal (A) or 10 nM rGRF-stimulated (•) GH secretion A) during a 3-h incubation period from cells (0.3 X 106 cells/ml) cultured in the absence of DEX and pretreated for 72 h with activin-A, and B) during a 1-h incubation from cells (0.15 X 106 cells/0.5 ml) cultured with 5 nM DEX and pretreated for 3 h with the indicated concentrations of activin-A. The data are the mean ± SEM of triplicate wells of a representative experiment.
required during a 3-h pretreatment period for the inhibition of GH secretion in response to a 1-h challenge with rGRF, using cells cultured in DEX (Fig. 4B). After either the 3-h treatment with activin-A in DEXtreated cells (Fig. 5A) or the 72-h treatment period without DEX (Fig. 5B), GH secretion was attenuated at all doses of rGRF. Total GH levels (cellular plus secreted) after treatment with 0.7 nM activin-A for 3 h were not significantly different from control values, but were 83%
rGRF (M) FIG. 5. The inhibition of rGRF-stimulated GH secretion from cells pretreated with activin-A (A) for 3 h in the presence of 5 nM DEX 0.15 x IO6 cells/0.5 ml) or (B) for 72 h in the absence of DEX (0.3 x 106 cells/ml). • , 0.7 nM activin-A-pretreated cells; • , untreated cells. The data are the mean ± SEM of triplicate wells of a representative experiment.
of control values after treatment for 72 h (Table 1). The inhibition of GH secretion by activin-A was reversible. After either a 3- or 24-h treatment with a maximal concentration of activin-A, rGRF-stimulated GH secretion was restored to control levels within 24 h (the first time point checked) by washing the cells thoroughly (data not shown). The interaction of activin-A with SRIF was also investigated. In cells pretreated with activin-A for 3 h, GH secretion was attenuated, as discussed above. SRIF by
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ACTIVIN AND GH SECRETION
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TABLE 1. The effect of eithera 3- or 72-h pretreatment with activinA on basal GH secretion and cellular GH content
Activin-A
Control 3-h preincubation 1-h basal secretion Cellular Total
0.28 ± 0.15 ± 2.79 ± 3.22 ±
0.01 0.01 0.04 0.04
0.18 0.08 2.96 3.22
72-h preincubation 3-h basal secretion Cellular Total
16.3 ± 0.65 ± 4.64 ± 21.6 ±
0.66 0.03 0.32 0.90
10.6 ± 0.68 ± 6.72 ± 18.0 ±
± ± ± ±
0.03 0.01 0.08 0.05 0.51° 0.03 0.35" 0.42°
The data are from representative experiments, showing the mean ± SEM of triplicate wells. The effects of a 3-h (upper panel) and a 72-h (lower panel) activin-A pretreatment on GH levels in cells (0.15 x 106/ 0.5 ml) cultured with 5 nM DEX are shown. 0 P < 0.05 compared to the corresponding control values. TABLE 2. The interaction of activin-A and SRIF on GH secretion. Hg GH secreted/ml • h Control
-rGRF
+rGRF
Activin-A
-rGRF
+rGRF
£* E O)
zL C O
ecr
ng GH/well
(/}
X
o
Basal
Basal 0.17 ± 0.01 0.88 + 0.06 0.13 + 0.01 0.48 + 0.01° 0.5 nM SRIF 0.11 ± 0.03 0.32 ± 0.01" 0.06 ± 0.01 0.15 ± 0.010* 10 nM SRIF 0.11 ± 0.04 0.18 ± 0.026 0.07 ± 0.01 0.10 ± O.OOl^
itself inhibited both basal and rGRF-stimulated GH secretion in a concentration-dependent manner (Table 2). The percent inhibition of rGRF-stimulated GH secretion by SRIF was comparable in activin-A pretreated and untreated cells, suggesting that these factors exert their actions by different mechanisms (Table 2). The inhibition of basal GH secretion was more variable, and a statistically significant effect was not observed. When GH secretion was stimulated with various GH secretagogues other than GRF, a 3-h activin-A pretreatment suppressed GH secretion in all cases (Fig. 6). Activin-A inhibited secretion in response to cholera toxin and forskolin, two activators of the adenylate cyclase system (44, 45), by 37 ± 4% and 45 ± 6% (n = 6), respectively. Secretion in response to 0.3 mM 8-bromocAMP was also attenuated by 43 ± 6% (n = 3). When the cells were challenged with 12-O-tetradecanoylphor-
TPA
1.4 -i
ion (\i tg/ml/
1.2
o
GH Sect
The cells (0.15 X 106/0.5 ml) were cultured with 5 nM DEX and pretreated or not with 0.7 nM activin-A for 3 h. After washing the cells, they were either treated or not treated with 0.5 nM rGRF in the presence or absence of the indicated concentrations of SRIF for an additional hour. The data are the mean ± SEM of triplicate wells from a representative experiment. "P < 0.002 compared to the corresponding nonactivin-A-treated values. 6 P < 0.005 compared to rGRF only values of activin-A-treated and untreated groups. C P < 0.05 compared to the corresponding nonactivin-A-treated values.
FORS
rGRF
FIG. 6. The inhibition of GH secretion in response to a 1-h challenge with the indicated secretagogues after pretreatment with 0.7 nM activin-A for 3 h from cells (0.15 x 106/0.5 ml) cultured with 5 nM DEX. The concentrations of the agents were: rGRF, 10 nM; cholera toxin (CT), 20 ng/ml; forskolin (FORS), 10 nM; and TPA, 10 nM. • , ActivinA pretreated; M, untreated. The data are the mean ± SEM of triplicate wells of a representative experiment.
1.0 0.8 0.6 0.4 0.2
Control
PT
Activin A
Activin A PT
FIG. 7. Partial reversal by pertussis toxin (PT; 100 ng/ml) of the inhibitory effect of a 3-h pretreatment with activin-A (0.7 nM) of basal (•) and rGRF-stimulated (M) GH secretion. PT was added to the cells (0.15 x 106/0.5 ml) 30 min before activin-A. The data are the mean ± SEM of triplicate wells of a representative experiment.
bol-13-acetate (TPA), an activator of protein kinase-C (46), secretion was inhibited by 31 ± 6% (n = 6). The addition of pertussis toxin 30 min before initiating the 3-h treatment with activin-A partially reversed the inhibitory effect of the protein on rGRF-stimulated GH secretion, suggesting the involvement of the inhibitory G-protein (G;) of the adenylate cyclase (Fig. 7); inhibitions without and with the toxin were 50 ± 2% and 19 ±
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ACTIVIN AND GH SECRETION
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8% (n = 4), respectively. Pertussis toxin by itself slightly, but not significantly, enhanced rGRF-stimulated GH secretion (122 ± 11% of control; P > 0.1; n = 4). The inhibition of RNA synthesis by the introduction of actinomycin-D (2.5 Mg/ml) completely prevented the inhibitory effect of activin-A on rGRF-stimulated GH secretion (Fig. 8). Actinomycin-D by itself had a modest and statistically significant stimulatory effect on rGRFstimulated secretion (118 ± 8% of control; P < 0.05; n = 7). After 3 h of activin-A treatment, rGRF-stimulated secretion was 55 ± 2% of the control value (rGRF only), and actinomycin-D restored this value to 110 ± 10% of control (n = 7). In parallel, the inclusion of actinomycinD blocked the inhibitory action of activin-A on rGRFstimulated cAMP accumulation (data not shown). Equivalent results were obtained regardless of whether the cells had been cultured in the presence or absence of DEX.
Discussion The results presented in this study provide evidence for a dual role of activin in the regulation of GH secretion and biosynthesis in vitro. Prolonged (72-h) treatment of cultured rat anterior pituitary cells with activin-A resulted not only in the inhibition of basal GH secretion, but also in decreased total (secreted and cellular) GH levels. The decrease in total GH probably reflects attenuated biosynthesis, consistent with our recent study in which GH biosynthesis was monitored directly (19). The 1.4 1.2
1
1.0
(
o> secret ion
0.8
vs
X
o
0.6 0.4 0.2
Control
Act D
Activin A
Activin A
ActD FlG. 8. The effect of actinomycin-D (Act D; 2.5 Mg/ml) on the inhibition of basal (•) and rGRF-stimulated (M) GH secretion. The cells (0.15 X 106/0.5 ml) were pretreated with actinomycin-D for 30 min before the addition of activin-A (0.7 nM) and incubated for a further 3 h. The cells were subsequently washed and incubated with and without 10 nM rGRF for 1 h. The data are the mean ± SEM of triplicate wells of a representative experiment.
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inhibition of basal secretion in long term treated cells, therefore, may in part reflect the inhibitory effects of activin on synthesis. Shorter (3-h) exposure to the protein also attenuated basal secretory rates, but in this case, total GH levels were not altered, indicating that activin can modulate the secretory process without affecting GH biosynthesis. The primary regulators of GH secretion and biosynthesis are recognized to be GRF and SRIF, both of hypothalamic origin (1-4). GRF stimulates the secretion of GH, expression of the GH gene, and proliferation of somatotrophs (9, 47). SRIF generally opposes the actions of GRF (4). Both neuropeptides exert their effects acutely. GRF stimulates the cAMP/adenylate cyclase pathway (5, 6), and cAMP is probably the mediator of the actions of GRF. SRIF partially inhibits GRF-stimulated cAMP accumulation (5), although whether this action is necessary or sufficient for its ability to inhibit GH secretion is unclear. The present data suggest that activin is another factor that may play a role in regulating somatotroph function. Unlike GRF and SRIF, the exact source of activin has not been identified. It is known that the gonads express measurable amounts of inhibin /3subunits and secrete activin-like biological activity (48, 49), but whether activin circulates has not been established. Inhibin a and /3B mRNAs are present within the gonadotrophs of the anterior pituitary (50), but intrapituitary levels of the dimeric forms of the proteins, inhibins and activins, have not been quantified. The presence of their mRNAs, though, suggests a possible paracrine role. The inhibitory latency of action of activin on GH secretion differs from that of SRIF in that, unlike the latter, pretreatment with activin is required to observe its inhibitory effects on basal or stimulated GH secretion. Moreover, at least during the first hour of stimulated GH secretion, subsequent to the pretreatment period the continuous presence of activin is not required to obtain its full effect. The latter, of course, may be due to residual activin-A remaining associated with the cells even after thorough washing. SRIF effects, in contrast to those of activin, are observed acutely and only while the peptide is present. In fact, removal of the peptide results in the well described rebound of basal GH secretion (51), which is not observed after removal of activin. The actions of these two agents are similar, in that both agents only partially inhibit basal and stimulated GH secretion. They also partially attenuate GRF-stimulated cAMP accumulation (5). The two agents, though, probably exert their effects via distinct mechanisms of action, based on the observation that SRIF and activin-A exhibit effect additivity in inhibiting GH secretion. Both agents also appear to be capable of inhibiting GH secretion in response not only to GRF but also to other activators of
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ACTIVIN AND GH SECRETION
the adenylate cyclase pathway and the protein kinase-C pathway. Glucocorticoids are well known to modulate somatotropic functions. They stimulate GH gene expression (11) and up-regulate the number of GRF-binding sites (52), thus making the cells more responsive to GRF. The results of this study indicate that glucocorticoids also affect the response of these cells to activin. The continued (3-h) presence of GRF enabled the cells to overcome the inhibitory effects of activin-A on GH secretion if they had been cultured in the presence of glucocorticoids, but not in their absence. The higher rate of GH synthesis or the higher density of GRF receptors due to the presence of the synthetic glucocorticoid DEX may account for this observation. Alternatively, the steroid may influence the synthesis or the stability of a putative intermediate factor/protein required for the action of activin; the experiments with actinomycin-D are indicative of the requirement for protein synthesis. The mechanism of action of activin is not known. The protein had no effect on basal levels of intracellular cAMP, but was able to attenuate GRF-stimulated cAMP accumulation. Cotreatment with pertussis toxin, partially reversed the effect of activin-A on GRF-stimulated GH secretion, suggesting that G; may be a target for the protein factor. In a recent report activin was shown to stimulate inositol trisphosphate production and increase intracellular calcium concentrations in cultured hepatocytes (39). However, in hepatocytes the actions of activin were stimulatory, increasing glucose production, rather than inhibitory and, therefore, were difficult to extrapolate to its actions on somatotrophs. Furthermore, due to the heterogeneity of the anterior pituitary cell population and the ability of activin to modulate not only somatotrophs, but also gonadotrophs, corticotrophs, and possibly lactotrophs (16, 18, 19), intracellular events are difficult to assign to a particular cell type. In preliminary experiments using mixed populations of rat anterior pituitary cells activin-A did not alter phosphatidylinositol turnover (our personal observation). There currently is no information regarding the effects of activin on plasma GH levels in vivo. The results of this study and two previous reports (16, 19) clearly suggest that activin can modulate somatotropic functions. Acknowledgments We thank Cindy Donaldson, Diane Jolley, and Amy Blount for their technical assistance and Sandra Guerra for assistance with manuscript preparation. We also thank Dr. Jean Rivier for providing synthetic SRIF and GRF.
References 1. Rivier J, Spiess J, Thorner M, Vale W 1982 Characterization of a growth hormone-releasing factor from a human pancreatic islet
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tumour. Nature 300:276 2. Guillemin R, Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg WB 1982 Growth hormones-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585 3. Vale W, Vaughan J, Yamamoto G, Spiess J, Rivier J 1983 Effects of synthetic human pancreatic (tumor) GH releasing factor and somatostatin, triiodothyronine and dexamethasone on GH secretion in vitro. Endocrinology 112:1553 4. Brazeau P, Vale W, Burgus R, Ling N, Batcher M, Rivier J, Guillemin J 1973 Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:177 5. Bilezikjian LM, Vale WW 1983 Stimulation of adenosine 3',5'monophosphate production by growth hormone-releasing factor and its inhibition by somatostatin in anterior pituitary cells in vitro. Endocrinology 113:1726 6. Bilezikjian LM, Erlichman J, Fleischer N, Vale WW 1987 Differential activation of type I and type II 3',5'-cyclic adenosine monophosphate-dependent protein kinases by growth hormone-releasing factor. Mol Endocrinol 1:137 7. Holl RW, Thorner MO, Leong DA 1988 Intracellular calcium concentration and growth hormone secretion in individual somatotropes: effects of growth hormone-releasing factor and somatostatin. Endocrinology 122:2927 8. Summers ST, Canonico PL, MacLeod RM, Rogol AD, Cronin MJ 1985 Phorbol esters affect pituitary growth hormone (GH) and prolactin release: the interaction with GH releasing factor, somatostatin and bromocriptine. Eur J Pharmacol 111:371 9. Barinaga M, Bilezikjian LM, Vale WW, Rosenfeld MG, Evans RM 1985 Independent effects of growth hormone releasing factor on growth hormone release and gene transcription. Nature 314:279 10. Copp RP, Samuels HH 1989 Identification of an adenosine 3',5'monophosphate (cAMP)-responsive region in the rat growth hormone gene: evidence for independent and synergistic effects of cAMP and thyroid hormone on gene expression. Mol Endocrinol 3:790 11. Evans RM, Birnberg NC, Rosenfeld MG 1982 Glucocorticoids and thyroid hormones transcriptionally regulate growth hormone gene expression. Proc Natl Acad Sci USA 79:7659 12. Yamashita S, Melmed S 1986 Insulin-like growth factor I action on rat anterior pituitary cells: suppression of growth hormone secretion and messenger ribonucleic acid levels. Endocrinology 118:176 13. Sheppard MS, Eatock BA, Bala RM 1987 Characteristics of phorbol ester stimulated growth hormone release: inhibition by insulinlike growth factor I, somatostatin, and low calcium medium and comparison with growth hormone releasing factor. Can J Physiol Pharmacol 65:2302 14. Koch BD, Blalock JB, Schonbrunn A 1988 Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. I. An increase in potassium conductance is responsible for both the hyperpolarization and the decrease in intracellular free calcium produced by somatostatin. J Biol Chem 263:216 15. Jakobs KH, Aktories K, Schultz G 1983 A nucleotide regulatory site for somatostatin inhibition of adenylate cyclase in S49 lymphoma cells. Nature 303:177 16. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321:776 17. Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R 1986 Pituitary FSH is released by a heterodimer of the betasubunits from the two forms of inhibin. Nature 321:779 18. Kitaoka M, Kojima I, Ogata E 1988 Activin-A: a modulator of multiple types of anterior pituitary cells. Biochem Biophys Res Comraun 157:48 19. Billestrup N, Gonzalez-Manchon C, Potter E, Vale W 1990 Inhibition of somatotroph growth and GH biosynthesis by activin-A in vitro. Mol Endocrinol 4:356 20. Robertson DM, Foulds LM, Leversha L, Morgan FJ, Hearn MT, Burger HG, Wettenhall RE, de Kretser DM 1985 Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun
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ACTIVIN AND GH SECRETION
126:220 21. Miyamoto K, Hasegawa Y, Fukuda M, Nomura M, Igarashi M, Kangawa K, Matsuo H 1985 Isolation of porcine follicular fluid inhibin of 32K daltons. Biochem Biophys Res Commun 129:396 22. Rivier J, Spiess J, McClintock R, Vaughan J, Vale W 1985 Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun 133:120 23. Ling N, Ying SY, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 82:7217 24. Mason AJ, Hayflick JS, Ling N, Esch F, Ueno N, Ying SY, Guillemin R, Niall H, Seeburg PH 1985 Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-beta. Nature 318:659 25. Massague J 1987 The TGF-/3 family of growth and differentiation factors. Cell 49:437 26. Vale W, Hsueh A, Rivier C, Yu J, The inhibin/activin family of hormones and growth factor. In: Sporn MA, Roberts AB (eds) Peptide Growth Factors and Their Receptors, Handbook of Experimental Pharmacology. Springer-Verlag, New York, in press 27. Mayo KE, Cerelli GM, Spiess J, Rivier J, Rosenfeld MG, Evans RM, Vale W 1986 Inhibin A-subunit cDNAs from porcine ovary and human placenta. Proc Natl Acad Sci USA 83:5849 28. Forage RG, Ring JM, Brown RW, Mclnerney BV, Cobon GS, Gregson RP, Robertson DM, Morgan FJ, Hearn MT, Findlay JK, Wettenhall REH, Burger HG, de Kretser DM 1986 Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc Natl Acad Sci USA 83:3091 29. Esch FS, Shimasaki S, Cooksey K, Mercado M, Mason AJ, Ying S-Y, Ueno N, Ling N 1987 Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol Endocrinol 5:388 30. Woodruff TK, Meunier H, Jones PBC, Hsueh AJW, Mayo KE 1987 Rat inhibin: molecular clong of a- and /3-subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol 8:561 31. Meunier H, Rivier C, Evans RM, Vale W 1988 Gonadal and extragonadal expression of inhibin alpha, beta A, and beta B subunits in various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247 32. Campen CA, Vale W 1988 Characterization of activin-A binding sites on the human leukemia cell line K562. Biochem Biophys Res Commun 157:844 33. Cheifetz S, Ling N, Guillemin R, Massague J 1988 A surface component on GH3 pituitary cells that recognizes transforming growth factor-beta, activin, and inhibin. J Biol Chem 263:17225 34. Sugino H, Nakamura T, Hasegawa Y, Miyamoto K, Igarashi M, Eto Y, Shibai H, Titani K 1988 Identification of a specific receptor for erythroid differentiation factor on follicular granulosa cell. J Biol Chem 263:15249
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35. LaPolt PS, Soto D, Su J-G, Campen CA, Vaughn J, Vale W, Hsueh AJW 1989 Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells. Mol Endocrinol 3:1666 36. Yu J, Shao LE, Lemas V, Yu AL, Vaughan J, Rivier J, Vale W 1987 Importance of FSH-releasing protein and inhibin in erythrodifferentiation. Nature 330:765 37. Gonzalez-Manchon C, Vale W 1989 Activin-A, inhibin and TGF/3 modulate growth of two gonadal cell lines. Endocrinology 125:1666 38. Bilezikjian LM, Activin-A modulates POMC mRNA levels and ACTH secretion in a clonal AtT20 cell line. 71st Annual Meeting of The Endocrine Society, Seattle WA, 1989, p 953 (Abstract) 39. Mine T, Kojima I, Ogata E 1989 Stimulation of glucose production by activin-A in isolated rat hepatocytes. Endocrinology 125:586 40. Hedger MP, Drummond AE, Robertson DM, Risbridger GP, de Kretser DM 1989 Inhibin and activin regulate [3H]thymidine uptake by rat thymocytes and 3T3 cells in vitro. Mol Cell Endocrinol 61:133 41. Vale W, Vaughan J, Yamamoto G, Bruhn T, Douglas C, Dalton C, Rivier C, Rivier J 1983 Assay of corticotropin releasing factor. In: Conn PM (ed) Methods of Enzymology. Academic Press, New York, p 565 42. Bilezikjian LM, Vale WW 1983 Glucocorticoids inhibit corticotropin-releasing factor-induced production of adenosine 3',5'-monophosphate in cultured anterior pituitary cells. Endocrinology 113:657 43. Schwall RH, Nikolics K, Szonyi E, Gorman C, Mason AJ 1988 Recombinant expression and characterization of human activin-A. Mol Endocrinol 2:1237 44. Spiegel AM 1987 Signal transduction by guanine nucleotide binding proteins. Mol Cell Endocrinol 49:1 45. Laurenza A, Khandelwal Y, DeSouza NJ, Rupp RH, Metzger H, Seamon KB 1987 Stimulation of adenylate cyclase by water-soluble analogues of forskolin. Mol Pharmacol 32:133 46. Nishizuka Y 1984 The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308:693 47. Billestrup N, Swanson LW, Vale W 1986 Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA 83:6854 48. Steinberger A, Steinberger E 1976 Secretion of an FSH-inhibiting factor by cultured Sertoli cells. Endocrinology 99:918 49. Bicsak TA, Tucker EM, Cappel S, Vaughan J, Rivier J, Vale W, Hsueh AJ 1986 Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 119:2711 50. Roberts V, Meunier H, Vaughan J, Rivier J, Rivier C, Vale W, Sawchenko P 1989 Production and regulation of inhibin subunits in pituitary gonadotropes. Endocrinology 124:552 51. Cowan JS, Chow MA, Kraicer J 1983 Characteristics of the postsomatostatin rebound in growth hormone secretion from perifused somatotrophs. Endocrinology 113:1056 52. Seifert H, Perrin M, Rivier J, Vale W 1985 Growth hormonereleasing factor binding sites in rat anterior pituitary membrane homogenates: modulation by glucocorticoids. Endocrinology 117:424
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Vol. 126, No. 5 Printed in U.S.A.
Activin-A Modulates Growth Hormone Secretion from Cultures of Rat Anterior Pituitary Cells* LOUISE M. BILEZIKJIAN, ANNE Z. CORRIGAN, AND WYLIE VALEt The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037
ABSTRACT. Activins, initially identified as FSH-releasing proteins, have now been shown to exert effects on other cell types of the anterior pituitary, including the somatotrophs. In the present study the inhibitory action of activin-A (/?A/?A) o n GH secretion was characterized using primary cultures of rat anterior pituitary cells. Activin-A suppressed basal GH secretion for up to 72 h (the longest time tested). Immediately after the treatment period with activin-A, when the cells were thoroughly washed and further incubated with or without rat GH-releasing factor (rGRF), basal and stimulated GH secretion were partially inhibited as well. In parallel, activin-A pretreatment diminished rGRF-stimulated cAMP accumulation. The effects of activin-A were time- and concentration-dependent, with half-maximal inhibition occurring in the range of 20-30 pM activin-A. A minimum pretreatment time of 3 h was required for maximal effect, and when rGRF and activin-A were added simultaneously, no inhibition was evident. Secretory responses of activin-Apretreated cells to rGRF were influenced by glucocorticoids. When cells were cultured in the presence of the synthetic glucocorticoid dexamethasone, pretreatment (72 h) with activin-A attenuated rGRF-stimulated GH secretion only during short (1h), but not longer (3-h), exposure periods to the neuropeptide. In the absence of dexamethasone, rGRF-stimulated GH secre-
tion was inhibited at all incubation times tested (up to 3 h). A 3-h exposure to the protein factor did not alter total (cellular plus secreted) immunoreactive GH levels, suggesting that the inhibition of secretion with the shorter treatment was not secondary to attenuated GH biosynthesis. However, longer (72-h) treatment with activin-A decreased total GH levels, indicating lower GH biosynthetic rates, as previously shown. Somatostatin is recognized as the primary negative modulator of GH secretion. Activin-A and SRIF inhibited GH secretion additively, suggesting distinct mechanisms of action for each. GH secretion in response to other secretagogues, such as 12-O-tetradecanoylphorbol-13-acetate, forskolin, cholera toxin, and 8-bromocAMP, was also suppressed after activin-A pretreatment. The presence of the RNA synthesis inhibitor actinomycin-D completely blocked the inhibitory effect of a 3-h activin-A pretreatment on subsequent rGRF-stimulated GH secretion. Pertussis toxin was only partially effective in preventing the inhibition by activin-A. The results of this study indicate that activin-A plays a crucial role as a modulator of somatotropic function, inhibiting GH secretion at the level of the secretory process and secondary to the inhibition of GH biosynthesis. {Endocrinology 126: 23692376, 1990)
S
ECRETION of GH by the somatotrophs of the anterior pituitary is known to be inversely regulated by GH-releasing factor (GRF) and somatostatin (SRIF) (1-4). GRF enhances intracellular cAMP levels (5) and activates the cAMP-dependent protein kinases (6), suggesting that the adenylate cyclase-activated signaling pathway mediates the actions of GRF. Calcium, however, is also required for GH secretion, and GRF has been reported to elevate intracellular calcium concentrations (7). Moreover, the activation of other second messenger systems, such as protein kinase-C, also promotes GH secretion (8), indicating the existence of multiple mechanisms that can potentially regulate the secretory process. The transcriptional effects of GRF on the GH gene, Received December 11,1989. Address requests for reprints to: Dr. Louise M. Bilezikjian, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, P.O. Box 85800, San Diego, California 92138. * This work was supported by NIH Grant DK-26741 and was conducted in part by The Clayton Foundation for Research, California Division. t Senior Clayton Foundation Investigator.
on the other hand, appear to be primarily mediated by cAMP (9, 10). Glucocorticoids and thyroid hormones also exert a positive control on the GH gene (10, 11) and augment the response to GRF (3), while other factors, such as insulin-like growth factor-I, inhibit its expression (12). SRIF acutely inhibits the secretion of GH in response to any of the known GH secretagogues (8, 13), but does not acutely attenuate the stimulation of transcription of the GH gene (9). The mechanism of action of SRIF is not well understood and may involve inhibition of calcium fluxes (14) and/or the inhibitory Gprotein (G;) of adenylate cyclase (15). In addition to SRIF, recent evidence indicates that a newly characterized protein, referred to as activin (16, 17), also exerts negative effects on GH secretion (16, 18) and biosynthesis (19). Activins and inhibins, initially isolated and characterized based on their ability to, respectively, enhance and inhibit FSH secretion from cultured rat anterior pituitary cells (16, 17, 20-23), are members of a functionally
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diverse but structurally related family of proteins known as transforming growth factor-/? (TGF0) (24-26). This family consists of various proteins, including the multiple forms of TGFjS, Mullerian inhibiting substance, cartilage-inducing factors, and decapentaplegic gene complex polypeptide, and is identical to erythroid differentiation factor (25, 26). Activins and inhibins are disulfide-linked dimeric proteins; inhibins are heterodimers comprised of an a-chain and either a j8A- or a /3B-chain, and activins are homodimers of any combination of the inhibin (3chains. The cDNAs encoding the three known subunits (a, /3A, and j8B) have been cloned and sequenced (24, 2730). Inhibin a and ft mRNAs have been detected in a number of tissues, including the ovary, testis, placenta, brain, bone marrow, and pituitary (31). Moreover, receptors for activin are expressed on a number of different cell types (32-35), consistent with the varied effects of these proteins that have thus far been reported (16, 18, 19, 35-40). The first recognized actions of the activins and the inhibins at the anterior pituitary level were the stimulation and the inhibition, respectively, of basal FSH (but not basal LH) secretion (16, 17, 20-23). In contrast to GnRH, the effects of these proteins on FSH secretion are slower in onset. Further examination, however, indicated that activin, and possibly inhibin, modulates the activity of other cell types of the anterior pituitary as well. Long term treatment of cultured rat anterior pituitary cells with activin-A inhibits basal GH and ACTH secretion (16, 18, 19) as well as PRL secretion (18). Additionally, activin blocks the proliferative effects of GRF on somatotrophs (19). The present study was undertaken to further examine the inhibitory effects of activin-A (J8A/?A) on GH secretion from cultures of rat anterior pituitary cells and define some of the characteristics of this action. The results indicate that activin-A has dual actions on somatotrophs: inhibition of basal and GRF-stimulated GH secretion as well as GH biosynthesis.
Materials and Methods Anterior pituitaries from male Sprague-Dawley rats (200220 g) were dissociated by collagenase and plated (0.15 X 106 cells/0.5 ml-well in 48-well multiwelled dishes) in medium containing 2% fetal bovine serum, 30 pM T3, and, unless stated otherwise, dexamethasone (DEX; 5 or 10 nM), as described previously (41). The cells were allowed to recover for 3 days before initiating the 72-h pretreatments with activin-A or for up to 5 days for shorter pretreatment times. The cells were washed twice and placed in fresh medium containing either 2% fetal bovine serum or 0.1% BSA for the 72-h or the 3-h pretreatments, respectively. For experiments with protein synthesis inhibitors, the agent was added 30 min before initiating the 3-h treatment with activin-A. At the end of the preincu-
Endo • 1990 Vol 126 • No 5
bation period, the media were collected for determination of basal GH secretion. The cells were rewashed three times with medium and treated with various GH secretagogues for 1 or 3 h in the same medium that was used during the pretreatment period. Activin-A and the protein synthesis inhibitors were not reintroduced if the stimulation period was 1 h, but were added back for longer stimulation periods. Medium was collected for the analysis of secreted GH levels, and the cells were extracted for the determination of either GH content or intracellular cAMP levels. The cells were lysed with 0.5% Nonidet P-40 for GH analysis and with 95% ethanol-0.1 N HC1 for the determination of cAMP levels, as previously described (42). GH was measured using a RIA kit provided by the National Hormone and Pituitary Program of the NIDDK. Intracellular cAMP levels were quantified using a commercially available RIA kit (Biomedical Technologies, Inc., Cambridge, MA). The activin-A (/3A/3A) used in these experiments was either a highly purified protein isolated from porcine follicular fluid (16) or recombinantly expressed human activin-A (43) (generous gift of Genentech, San Francisco, CA). Equivalent responses were obtained with the protein from either source. All data were subjected to analysis of variance, and differences between groups were assessed using the multiple range test of Duncan. Reported values represent the mean ± SEM of triplicate wells for representative experiments or for normalized means from three or more independent experiments, each performed in triplicate.
Results The time-course of activin-A action on GH secretion was determined using rat anterior pituitary cells cultured in the presence of 5 nM DEX. Secretion in response to a 1-h challenge with 10 nM rGRF was attenuated as a function of the length of preincubation with the protein (Fig. IB). These results indicated that preexposure to activin-A was necessary for its inhibitory action on subsequent stimulated secretion; when activin-A and rat GRF (rGRF) were added simultaneously, no inhibition was observed for up to 5 h (the longest time tested). As short as a 3-h preexposure to a maximal (0.7 nM) concentration of activin-A was sufficient to obtain the full effect; the inhibitions of basal and stimulated GH secretion after 3 h of activin-A pretreatment were 25 ± 4% and 44 ± 2% (n = 15), respectively. The inhibition of rGRF-stimulated GH secretion by activin-A pretreatment was accompanied by a parallel attenuation of rGRF-stimulated cAMP generation (Fig. 1A). Activin-A by itself had no significant effect on basal cAMP levels during or after the treatment periods. Basal secretion during the pretreatment period with activin-A was also inhibited in a time-dependent manner, but was not statistically significant during a period of less than 3 h. Incubation of the rat anterior pituitary cells with a maximal concentration of activin-A for 3 h inhibited basal GH secretion by 21 ± 9% (n = 7). After 72 h, maximal inhibition was 31 ± 4% (n = 5), with an
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ACTIVIN AND GH SECRETION 4.0
25 T
2371
i
CM
20 -
u ^ «- o
15
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•
2.0 -
11 23
10 •
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0 -I
1.2 -
1012
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B
FIG. 2. Concentration-dependent inhibition of basal GH secretion during a 72-h treatment with activin-A from cells (0.3 x 106/ml) cultured without DEX. The data are the mean ± SEM of triplicate wells of a representative experiment.
1.0 -
E 0.8 -
Secret ion |
1010
DEX Activin A
0.6-
0.4-
X
o
0.2-
Control
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72
Activin A Pretreatment (h) FlG. 1. The time course of the inhibition of basal (•) and rGRFstimulated (H) cAMP accumulation (A) and GH secretion (B) during a 1-h incubation after pretreatment with 0.7 nM activin-A for the indicated lengths of time. The cells (0.15 X 106/0.5 ml) were cultured in the presence of 5 nM DEX for 3 days before initiating the treatments with activin-A in the presence of DEX. The data are the mean ± SEM of triplicate wells of a representative experiment.
IC50 of approximately 20-30 pM (Fig. 2). A complex interaction between glucocorticoids and activin-A was noted. Basal GH secretion was inhibited to approximately the same extent regardless of whether the cells had been cultured with or without the synthetic glucocorticoid dexamethasone (DEX) during the 72-h pretreatment period with a maximally effective concentration of activin-A (0.7 nM; 39 ± 2% and 31 ± 4% inhibition in DEX-treated and untreated cells, respectively; n = 5). The activin-A effect on the subsequent
rGRF
-
+
-
+
-
•
-
FIG. 3. The effect of DEX on basal (•) and rGRF-stimulated (M) GH secretion after a 72-h pretreatment with activin-A (0.7 nM). Rat anterior pituitary cells (0.3 x 106 cells/ml) were cultured in the presence or absence of 5 nM DEX for 3 days, washed, and treated or not treated with 0.7 nM activin-A for an additional 72 h with or without DEX. The cells were rewashed, and GH secretion was measured during a 3-h incubation in the presence or absence of 10 nM rGRF. The data are the mean ± SEM of triplicate wells of a representative experiment.
stimulation period with rGRF, however, was influenced by DEX. GH secretion in response to a 1-h challenge with rGRF was inhibited to the same extent regardless of whether the cells had been cultured with or without DEX, equivalent to the data in Fig. 1. In contrast, when GH secretion was measured in response to a 3-h challenge with rGRF after a 72-h activin-A pretreatment, inhibition was observed only in cells cultured without DEX (Fig. 3). In the absence of DEX, the half-maximal concentration of activin-A required during a 72-h pretreatment period to inhibit subsequent GH secretion in response to a 3-h challenge with rGRF was in the range of 20-30 pM (Fig. 4A). This value was equivalent to that
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ACTIVIN AND GH SECRETION
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1.0
10 -,
E
X CD
0.8 -
1
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Endo • 1990 Vol 126 • No 5
0.6 H
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Activin A (M) FlG. 4. Concentration-dependence of the inhibition of basal (A) or 10 nM rGRF-stimulated (•) GH secretion A) during a 3-h incubation period from cells (0.3 X 106 cells/ml) cultured in the absence of DEX and pretreated for 72 h with activin-A, and B) during a 1-h incubation from cells (0.15 X 106 cells/0.5 ml) cultured with 5 nM DEX and pretreated for 3 h with the indicated concentrations of activin-A. The data are the mean ± SEM of triplicate wells of a representative experiment.
required during a 3-h pretreatment period for the inhibition of GH secretion in response to a 1-h challenge with rGRF, using cells cultured in DEX (Fig. 4B). After either the 3-h treatment with activin-A in DEXtreated cells (Fig. 5A) or the 72-h treatment period without DEX (Fig. 5B), GH secretion was attenuated at all doses of rGRF. Total GH levels (cellular plus secreted) after treatment with 0.7 nM activin-A for 3 h were not significantly different from control values, but were 83%
rGRF (M) FIG. 5. The inhibition of rGRF-stimulated GH secretion from cells pretreated with activin-A (A) for 3 h in the presence of 5 nM DEX 0.15 x IO6 cells/0.5 ml) or (B) for 72 h in the absence of DEX (0.3 x 106 cells/ml). • , 0.7 nM activin-A-pretreated cells; • , untreated cells. The data are the mean ± SEM of triplicate wells of a representative experiment.
of control values after treatment for 72 h (Table 1). The inhibition of GH secretion by activin-A was reversible. After either a 3- or 24-h treatment with a maximal concentration of activin-A, rGRF-stimulated GH secretion was restored to control levels within 24 h (the first time point checked) by washing the cells thoroughly (data not shown). The interaction of activin-A with SRIF was also investigated. In cells pretreated with activin-A for 3 h, GH secretion was attenuated, as discussed above. SRIF by
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ACTIVIN AND GH SECRETION
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TABLE 1. The effect of eithera 3- or 72-h pretreatment with activinA on basal GH secretion and cellular GH content
Activin-A
Control 3-h preincubation 1-h basal secretion Cellular Total
0.28 ± 0.15 ± 2.79 ± 3.22 ±
0.01 0.01 0.04 0.04
0.18 0.08 2.96 3.22
72-h preincubation 3-h basal secretion Cellular Total
16.3 ± 0.65 ± 4.64 ± 21.6 ±
0.66 0.03 0.32 0.90
10.6 ± 0.68 ± 6.72 ± 18.0 ±
± ± ± ±
0.03 0.01 0.08 0.05 0.51° 0.03 0.35" 0.42°
The data are from representative experiments, showing the mean ± SEM of triplicate wells. The effects of a 3-h (upper panel) and a 72-h (lower panel) activin-A pretreatment on GH levels in cells (0.15 x 106/ 0.5 ml) cultured with 5 nM DEX are shown. 0 P < 0.05 compared to the corresponding control values. TABLE 2. The interaction of activin-A and SRIF on GH secretion. Hg GH secreted/ml • h Control
-rGRF
+rGRF
Activin-A
-rGRF
+rGRF
£* E O)
zL C O
ecr
ng GH/well
(/}
X
o
Basal
Basal 0.17 ± 0.01 0.88 + 0.06 0.13 + 0.01 0.48 + 0.01° 0.5 nM SRIF 0.11 ± 0.03 0.32 ± 0.01" 0.06 ± 0.01 0.15 ± 0.010* 10 nM SRIF 0.11 ± 0.04 0.18 ± 0.026 0.07 ± 0.01 0.10 ± O.OOl^
itself inhibited both basal and rGRF-stimulated GH secretion in a concentration-dependent manner (Table 2). The percent inhibition of rGRF-stimulated GH secretion by SRIF was comparable in activin-A pretreated and untreated cells, suggesting that these factors exert their actions by different mechanisms (Table 2). The inhibition of basal GH secretion was more variable, and a statistically significant effect was not observed. When GH secretion was stimulated with various GH secretagogues other than GRF, a 3-h activin-A pretreatment suppressed GH secretion in all cases (Fig. 6). Activin-A inhibited secretion in response to cholera toxin and forskolin, two activators of the adenylate cyclase system (44, 45), by 37 ± 4% and 45 ± 6% (n = 6), respectively. Secretion in response to 0.3 mM 8-bromocAMP was also attenuated by 43 ± 6% (n = 3). When the cells were challenged with 12-O-tetradecanoylphor-
TPA
1.4 -i
ion (\i tg/ml/
1.2
o
GH Sect
The cells (0.15 X 106/0.5 ml) were cultured with 5 nM DEX and pretreated or not with 0.7 nM activin-A for 3 h. After washing the cells, they were either treated or not treated with 0.5 nM rGRF in the presence or absence of the indicated concentrations of SRIF for an additional hour. The data are the mean ± SEM of triplicate wells from a representative experiment. "P < 0.002 compared to the corresponding nonactivin-A-treated values. 6 P < 0.005 compared to rGRF only values of activin-A-treated and untreated groups. C P < 0.05 compared to the corresponding nonactivin-A-treated values.
FORS
rGRF
FIG. 6. The inhibition of GH secretion in response to a 1-h challenge with the indicated secretagogues after pretreatment with 0.7 nM activin-A for 3 h from cells (0.15 x 106/0.5 ml) cultured with 5 nM DEX. The concentrations of the agents were: rGRF, 10 nM; cholera toxin (CT), 20 ng/ml; forskolin (FORS), 10 nM; and TPA, 10 nM. • , ActivinA pretreated; M, untreated. The data are the mean ± SEM of triplicate wells of a representative experiment.
1.0 0.8 0.6 0.4 0.2
Control
PT
Activin A
Activin A PT
FIG. 7. Partial reversal by pertussis toxin (PT; 100 ng/ml) of the inhibitory effect of a 3-h pretreatment with activin-A (0.7 nM) of basal (•) and rGRF-stimulated (M) GH secretion. PT was added to the cells (0.15 x 106/0.5 ml) 30 min before activin-A. The data are the mean ± SEM of triplicate wells of a representative experiment.
bol-13-acetate (TPA), an activator of protein kinase-C (46), secretion was inhibited by 31 ± 6% (n = 6). The addition of pertussis toxin 30 min before initiating the 3-h treatment with activin-A partially reversed the inhibitory effect of the protein on rGRF-stimulated GH secretion, suggesting the involvement of the inhibitory G-protein (G;) of the adenylate cyclase (Fig. 7); inhibitions without and with the toxin were 50 ± 2% and 19 ±
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ACTIVIN AND GH SECRETION
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8% (n = 4), respectively. Pertussis toxin by itself slightly, but not significantly, enhanced rGRF-stimulated GH secretion (122 ± 11% of control; P > 0.1; n = 4). The inhibition of RNA synthesis by the introduction of actinomycin-D (2.5 Mg/ml) completely prevented the inhibitory effect of activin-A on rGRF-stimulated GH secretion (Fig. 8). Actinomycin-D by itself had a modest and statistically significant stimulatory effect on rGRFstimulated secretion (118 ± 8% of control; P < 0.05; n = 7). After 3 h of activin-A treatment, rGRF-stimulated secretion was 55 ± 2% of the control value (rGRF only), and actinomycin-D restored this value to 110 ± 10% of control (n = 7). In parallel, the inclusion of actinomycinD blocked the inhibitory action of activin-A on rGRFstimulated cAMP accumulation (data not shown). Equivalent results were obtained regardless of whether the cells had been cultured in the presence or absence of DEX.
Discussion The results presented in this study provide evidence for a dual role of activin in the regulation of GH secretion and biosynthesis in vitro. Prolonged (72-h) treatment of cultured rat anterior pituitary cells with activin-A resulted not only in the inhibition of basal GH secretion, but also in decreased total (secreted and cellular) GH levels. The decrease in total GH probably reflects attenuated biosynthesis, consistent with our recent study in which GH biosynthesis was monitored directly (19). The 1.4 1.2
1
1.0
(
o> secret ion
0.8
vs
X
o
0.6 0.4 0.2
Control
Act D
Activin A
Activin A
ActD FlG. 8. The effect of actinomycin-D (Act D; 2.5 Mg/ml) on the inhibition of basal (•) and rGRF-stimulated (M) GH secretion. The cells (0.15 X 106/0.5 ml) were pretreated with actinomycin-D for 30 min before the addition of activin-A (0.7 nM) and incubated for a further 3 h. The cells were subsequently washed and incubated with and without 10 nM rGRF for 1 h. The data are the mean ± SEM of triplicate wells of a representative experiment.
Endo • 1990 Vol 126 • No 5
inhibition of basal secretion in long term treated cells, therefore, may in part reflect the inhibitory effects of activin on synthesis. Shorter (3-h) exposure to the protein also attenuated basal secretory rates, but in this case, total GH levels were not altered, indicating that activin can modulate the secretory process without affecting GH biosynthesis. The primary regulators of GH secretion and biosynthesis are recognized to be GRF and SRIF, both of hypothalamic origin (1-4). GRF stimulates the secretion of GH, expression of the GH gene, and proliferation of somatotrophs (9, 47). SRIF generally opposes the actions of GRF (4). Both neuropeptides exert their effects acutely. GRF stimulates the cAMP/adenylate cyclase pathway (5, 6), and cAMP is probably the mediator of the actions of GRF. SRIF partially inhibits GRF-stimulated cAMP accumulation (5), although whether this action is necessary or sufficient for its ability to inhibit GH secretion is unclear. The present data suggest that activin is another factor that may play a role in regulating somatotroph function. Unlike GRF and SRIF, the exact source of activin has not been identified. It is known that the gonads express measurable amounts of inhibin /3subunits and secrete activin-like biological activity (48, 49), but whether activin circulates has not been established. Inhibin a and /3B mRNAs are present within the gonadotrophs of the anterior pituitary (50), but intrapituitary levels of the dimeric forms of the proteins, inhibins and activins, have not been quantified. The presence of their mRNAs, though, suggests a possible paracrine role. The inhibitory latency of action of activin on GH secretion differs from that of SRIF in that, unlike the latter, pretreatment with activin is required to observe its inhibitory effects on basal or stimulated GH secretion. Moreover, at least during the first hour of stimulated GH secretion, subsequent to the pretreatment period the continuous presence of activin is not required to obtain its full effect. The latter, of course, may be due to residual activin-A remaining associated with the cells even after thorough washing. SRIF effects, in contrast to those of activin, are observed acutely and only while the peptide is present. In fact, removal of the peptide results in the well described rebound of basal GH secretion (51), which is not observed after removal of activin. The actions of these two agents are similar, in that both agents only partially inhibit basal and stimulated GH secretion. They also partially attenuate GRF-stimulated cAMP accumulation (5). The two agents, though, probably exert their effects via distinct mechanisms of action, based on the observation that SRIF and activin-A exhibit effect additivity in inhibiting GH secretion. Both agents also appear to be capable of inhibiting GH secretion in response not only to GRF but also to other activators of
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ACTIVIN AND GH SECRETION
the adenylate cyclase pathway and the protein kinase-C pathway. Glucocorticoids are well known to modulate somatotropic functions. They stimulate GH gene expression (11) and up-regulate the number of GRF-binding sites (52), thus making the cells more responsive to GRF. The results of this study indicate that glucocorticoids also affect the response of these cells to activin. The continued (3-h) presence of GRF enabled the cells to overcome the inhibitory effects of activin-A on GH secretion if they had been cultured in the presence of glucocorticoids, but not in their absence. The higher rate of GH synthesis or the higher density of GRF receptors due to the presence of the synthetic glucocorticoid DEX may account for this observation. Alternatively, the steroid may influence the synthesis or the stability of a putative intermediate factor/protein required for the action of activin; the experiments with actinomycin-D are indicative of the requirement for protein synthesis. The mechanism of action of activin is not known. The protein had no effect on basal levels of intracellular cAMP, but was able to attenuate GRF-stimulated cAMP accumulation. Cotreatment with pertussis toxin, partially reversed the effect of activin-A on GRF-stimulated GH secretion, suggesting that G; may be a target for the protein factor. In a recent report activin was shown to stimulate inositol trisphosphate production and increase intracellular calcium concentrations in cultured hepatocytes (39). However, in hepatocytes the actions of activin were stimulatory, increasing glucose production, rather than inhibitory and, therefore, were difficult to extrapolate to its actions on somatotrophs. Furthermore, due to the heterogeneity of the anterior pituitary cell population and the ability of activin to modulate not only somatotrophs, but also gonadotrophs, corticotrophs, and possibly lactotrophs (16, 18, 19), intracellular events are difficult to assign to a particular cell type. In preliminary experiments using mixed populations of rat anterior pituitary cells activin-A did not alter phosphatidylinositol turnover (our personal observation). There currently is no information regarding the effects of activin on plasma GH levels in vivo. The results of this study and two previous reports (16, 19) clearly suggest that activin can modulate somatotropic functions. Acknowledgments We thank Cindy Donaldson, Diane Jolley, and Amy Blount for their technical assistance and Sandra Guerra for assistance with manuscript preparation. We also thank Dr. Jean Rivier for providing synthetic SRIF and GRF.
References 1. Rivier J, Spiess J, Thorner M, Vale W 1982 Characterization of a growth hormone-releasing factor from a human pancreatic islet
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tumour. Nature 300:276 2. Guillemin R, Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg WB 1982 Growth hormones-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585 3. Vale W, Vaughan J, Yamamoto G, Spiess J, Rivier J 1983 Effects of synthetic human pancreatic (tumor) GH releasing factor and somatostatin, triiodothyronine and dexamethasone on GH secretion in vitro. Endocrinology 112:1553 4. Brazeau P, Vale W, Burgus R, Ling N, Batcher M, Rivier J, Guillemin J 1973 Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:177 5. Bilezikjian LM, Vale WW 1983 Stimulation of adenosine 3',5'monophosphate production by growth hormone-releasing factor and its inhibition by somatostatin in anterior pituitary cells in vitro. Endocrinology 113:1726 6. Bilezikjian LM, Erlichman J, Fleischer N, Vale WW 1987 Differential activation of type I and type II 3',5'-cyclic adenosine monophosphate-dependent protein kinases by growth hormone-releasing factor. Mol Endocrinol 1:137 7. Holl RW, Thorner MO, Leong DA 1988 Intracellular calcium concentration and growth hormone secretion in individual somatotropes: effects of growth hormone-releasing factor and somatostatin. Endocrinology 122:2927 8. Summers ST, Canonico PL, MacLeod RM, Rogol AD, Cronin MJ 1985 Phorbol esters affect pituitary growth hormone (GH) and prolactin release: the interaction with GH releasing factor, somatostatin and bromocriptine. Eur J Pharmacol 111:371 9. Barinaga M, Bilezikjian LM, Vale WW, Rosenfeld MG, Evans RM 1985 Independent effects of growth hormone releasing factor on growth hormone release and gene transcription. Nature 314:279 10. Copp RP, Samuels HH 1989 Identification of an adenosine 3',5'monophosphate (cAMP)-responsive region in the rat growth hormone gene: evidence for independent and synergistic effects of cAMP and thyroid hormone on gene expression. Mol Endocrinol 3:790 11. Evans RM, Birnberg NC, Rosenfeld MG 1982 Glucocorticoids and thyroid hormones transcriptionally regulate growth hormone gene expression. Proc Natl Acad Sci USA 79:7659 12. Yamashita S, Melmed S 1986 Insulin-like growth factor I action on rat anterior pituitary cells: suppression of growth hormone secretion and messenger ribonucleic acid levels. Endocrinology 118:176 13. Sheppard MS, Eatock BA, Bala RM 1987 Characteristics of phorbol ester stimulated growth hormone release: inhibition by insulinlike growth factor I, somatostatin, and low calcium medium and comparison with growth hormone releasing factor. Can J Physiol Pharmacol 65:2302 14. Koch BD, Blalock JB, Schonbrunn A 1988 Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. I. An increase in potassium conductance is responsible for both the hyperpolarization and the decrease in intracellular free calcium produced by somatostatin. J Biol Chem 263:216 15. Jakobs KH, Aktories K, Schultz G 1983 A nucleotide regulatory site for somatostatin inhibition of adenylate cyclase in S49 lymphoma cells. Nature 303:177 16. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321:776 17. Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R 1986 Pituitary FSH is released by a heterodimer of the betasubunits from the two forms of inhibin. Nature 321:779 18. Kitaoka M, Kojima I, Ogata E 1988 Activin-A: a modulator of multiple types of anterior pituitary cells. Biochem Biophys Res Comraun 157:48 19. Billestrup N, Gonzalez-Manchon C, Potter E, Vale W 1990 Inhibition of somatotroph growth and GH biosynthesis by activin-A in vitro. Mol Endocrinol 4:356 20. Robertson DM, Foulds LM, Leversha L, Morgan FJ, Hearn MT, Burger HG, Wettenhall RE, de Kretser DM 1985 Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun
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126:220 21. Miyamoto K, Hasegawa Y, Fukuda M, Nomura M, Igarashi M, Kangawa K, Matsuo H 1985 Isolation of porcine follicular fluid inhibin of 32K daltons. Biochem Biophys Res Commun 129:396 22. Rivier J, Spiess J, McClintock R, Vaughan J, Vale W 1985 Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun 133:120 23. Ling N, Ying SY, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 82:7217 24. Mason AJ, Hayflick JS, Ling N, Esch F, Ueno N, Ying SY, Guillemin R, Niall H, Seeburg PH 1985 Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-beta. Nature 318:659 25. Massague J 1987 The TGF-/3 family of growth and differentiation factors. Cell 49:437 26. Vale W, Hsueh A, Rivier C, Yu J, The inhibin/activin family of hormones and growth factor. In: Sporn MA, Roberts AB (eds) Peptide Growth Factors and Their Receptors, Handbook of Experimental Pharmacology. Springer-Verlag, New York, in press 27. Mayo KE, Cerelli GM, Spiess J, Rivier J, Rosenfeld MG, Evans RM, Vale W 1986 Inhibin A-subunit cDNAs from porcine ovary and human placenta. Proc Natl Acad Sci USA 83:5849 28. Forage RG, Ring JM, Brown RW, Mclnerney BV, Cobon GS, Gregson RP, Robertson DM, Morgan FJ, Hearn MT, Findlay JK, Wettenhall REH, Burger HG, de Kretser DM 1986 Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc Natl Acad Sci USA 83:3091 29. Esch FS, Shimasaki S, Cooksey K, Mercado M, Mason AJ, Ying S-Y, Ueno N, Ling N 1987 Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol Endocrinol 5:388 30. Woodruff TK, Meunier H, Jones PBC, Hsueh AJW, Mayo KE 1987 Rat inhibin: molecular clong of a- and /3-subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol 8:561 31. Meunier H, Rivier C, Evans RM, Vale W 1988 Gonadal and extragonadal expression of inhibin alpha, beta A, and beta B subunits in various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247 32. Campen CA, Vale W 1988 Characterization of activin-A binding sites on the human leukemia cell line K562. Biochem Biophys Res Commun 157:844 33. Cheifetz S, Ling N, Guillemin R, Massague J 1988 A surface component on GH3 pituitary cells that recognizes transforming growth factor-beta, activin, and inhibin. J Biol Chem 263:17225 34. Sugino H, Nakamura T, Hasegawa Y, Miyamoto K, Igarashi M, Eto Y, Shibai H, Titani K 1988 Identification of a specific receptor for erythroid differentiation factor on follicular granulosa cell. J Biol Chem 263:15249
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35. LaPolt PS, Soto D, Su J-G, Campen CA, Vaughn J, Vale W, Hsueh AJW 1989 Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells. Mol Endocrinol 3:1666 36. Yu J, Shao LE, Lemas V, Yu AL, Vaughan J, Rivier J, Vale W 1987 Importance of FSH-releasing protein and inhibin in erythrodifferentiation. Nature 330:765 37. Gonzalez-Manchon C, Vale W 1989 Activin-A, inhibin and TGF/3 modulate growth of two gonadal cell lines. Endocrinology 125:1666 38. Bilezikjian LM, Activin-A modulates POMC mRNA levels and ACTH secretion in a clonal AtT20 cell line. 71st Annual Meeting of The Endocrine Society, Seattle WA, 1989, p 953 (Abstract) 39. Mine T, Kojima I, Ogata E 1989 Stimulation of glucose production by activin-A in isolated rat hepatocytes. Endocrinology 125:586 40. Hedger MP, Drummond AE, Robertson DM, Risbridger GP, de Kretser DM 1989 Inhibin and activin regulate [3H]thymidine uptake by rat thymocytes and 3T3 cells in vitro. Mol Cell Endocrinol 61:133 41. Vale W, Vaughan J, Yamamoto G, Bruhn T, Douglas C, Dalton C, Rivier C, Rivier J 1983 Assay of corticotropin releasing factor. In: Conn PM (ed) Methods of Enzymology. Academic Press, New York, p 565 42. Bilezikjian LM, Vale WW 1983 Glucocorticoids inhibit corticotropin-releasing factor-induced production of adenosine 3',5'-monophosphate in cultured anterior pituitary cells. Endocrinology 113:657 43. Schwall RH, Nikolics K, Szonyi E, Gorman C, Mason AJ 1988 Recombinant expression and characterization of human activin-A. Mol Endocrinol 2:1237 44. Spiegel AM 1987 Signal transduction by guanine nucleotide binding proteins. Mol Cell Endocrinol 49:1 45. Laurenza A, Khandelwal Y, DeSouza NJ, Rupp RH, Metzger H, Seamon KB 1987 Stimulation of adenylate cyclase by water-soluble analogues of forskolin. Mol Pharmacol 32:133 46. Nishizuka Y 1984 The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308:693 47. Billestrup N, Swanson LW, Vale W 1986 Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA 83:6854 48. Steinberger A, Steinberger E 1976 Secretion of an FSH-inhibiting factor by cultured Sertoli cells. Endocrinology 99:918 49. Bicsak TA, Tucker EM, Cappel S, Vaughan J, Rivier J, Vale W, Hsueh AJ 1986 Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 119:2711 50. Roberts V, Meunier H, Vaughan J, Rivier J, Rivier C, Vale W, Sawchenko P 1989 Production and regulation of inhibin subunits in pituitary gonadotropes. Endocrinology 124:552 51. Cowan JS, Chow MA, Kraicer J 1983 Characteristics of the postsomatostatin rebound in growth hormone secretion from perifused somatotrophs. Endocrinology 113:1056 52. Seifert H, Perrin M, Rivier J, Vale W 1985 Growth hormonereleasing factor binding sites in rat anterior pituitary membrane homogenates: modulation by glucocorticoids. Endocrinology 117:424
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