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Growth hormone secretion from chicken adenohypophyseal cells in primary culture: Effects of human pancreatic growth hormone-releasing factor, thyrotropin-releasing hormone, and somatostatin on growth hormone release
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
65, 408-414 (1987)
Growth
Hormone Secretion from Chicken Adenohypophyseal Cells in Primary Culture: Effects of Human Pancreatic Growth Hormone-Releasing Factor, Thyrotropin-Releasing Hormone, and Somatostatin on Growth Hormone Release FRANK M. PEREZ.*,? SASHA MALAMED,*
*Department of Anatomy, UMDNJ-Robert and tDepartment of Animal Sciences,
Wood Rutgers-The
ANDCOLIN G. SCANES~
Johnsorl Medical School. Piscatutiaay. State University, New Brunswick.
NeM, Jersey Ne,lx Jersey
08854. 08903
Accepted October 10, 1986 A primary culture of chicken adenohypophyseal cells has been developed to study the regulation of growth hormone (GH) secretion. Following collagenase dispersion, cells were exposed for 2 hr to vehicle (control) or test agents. Human pancreatic (tumor) growth hormone-releasing factor (hpGRF) and rat hypothalamic growth hormone-releasing factor stimulated GH release to similar levels. GH release was increased by the presence of dibutyryl cyclic AMP. Thyrotropin-releasing hormone (TRH) alone did not influence GH release: however, TRH plus hpGRF together exerted a synergistic (greater than additive) effect. increasing GH release by 100 to 300% over the sum of the values for each secretagogue acting alone. These relationships between TRH and hpGRF were further examined in cultured cells exposed to secretagogues for two consecutive 2-hr incubations. TRH pretreatment enhanced subsequent hpGRF-stimulated GH release by about 80% over that obtained if no secretagogue was present during the first incubation. In other experiments, somatostatin (SRIF) alone did not alter GH secretion. However, SRIF reduced hpGRF-stimulated GH release to levels found in controls. Furthermore, GH release stimulated by the presence of both TRH and hpGRF was lowered to control values by SRIF. The results of these studies demonstrate that a primary culture of chicken adenohypophyseal cells is a useful model for the study of GH secretion. Indeed, these results suggest that TRH and hpGRF regulate GH secretion by mechanisms which are not identical. ?a 1987 Academic Pres. Inc.
In mammals growth hormone (GH) secretion is regulated by stimulatory and inhibitory factors derived from the hypothalamus (Martin, 1976). A recently isolated rat hypothalamic growth hormone-releasing factor (rhGRF) is a potent and specific stimulator of GH secretion (Spiess et al., 1983). In addition, human pancreatic (tumor) growth hormone-releasing factor (hpGRF), chemically similar to rhGRF, also stimulates GH secretion (Guillemin et al., 1982; Rivier et al., 1982). Somatostatin (SRIF) has been isolated from the mammalian hypothalamus (Brazeau ef al., 1973) and counteracts GRF-induced GH release (Vale ef al., 1983; Law et al., 1984). In birds also, evidence indicates that GH secretion is controlled by hypothalamo-hy-
pophysiotrophic factors (Scanes et al., 1984a). Thyrotropin-releasing hormone (TRH) and hpGRF stimulate GH secretion (Harvey et al., 1978; Scanes et al., 1984b) and SRIF inhibits GH release (Hall and Chadwick, 1976). Much information concerning the regulation of GH secretion in mammals has been obtained through the use of in vitro systems of pituitary cells which are easily varied under controlled conditions. However, less is known about avain GH secretion and few studies of the bird pituitary have used in vitro systems. This report presents a method for preparing primary cultures of chicken adenohypophyseal cells to study the regulation of GH secretion. In addition, the effects of hpGRF, rhGRF, di408
0016-6480187 $1.50 Copyright 10 1987 by Academic Prey. Inc. All rights of reproduction in any form rexwed.
CHICKEN
butyryl cyclic AMP, TRH, GH release are examined. MATERIALS
GH SECRETION
and SRIF on
AND METHODS
Cell processing. Male domestic fowl (White Leghorn strain) were obtained commercially (Moyers Hatcheries, PA) at 1 day old and reared under a daily photoperiod (16 hr light:8 hr dark) with food and water available ad libitum. Sterile conditions were maintained during cell processing and culture. Anterior pituitaries from immature cockerels (6-8 weeks old) were removed immediately following decapitation and placed in a petri dish containing Hanks’ balanced salt solution, Ca2+ and Mg2+ free (HBSS-CMF, GIBCO, Grand Island, NY). Glands were diced into approximately 2-mm cubes and transferred to a conical test tube (15 x 75 mm) containing 0.35% collagenase (Worthington Enzymes, Freehold, NJ) (Vale et ul.. 1972) in HBSS-CMF. Adenohypophyseal cells were dispersed in this medium for 60 min at 37” in a water bath with agitation; dispersion was facilitated by periodic mechanical trituration with a flame-tapered Pasteur pipet. Following dispersion, cells were washed twice with HBSS-CMF and either incubated (2 hr) in medium 199 (Ml99, GIBCO) or placed in culture medium: Ml99 containing 20% fetal bovine serum (GIBCO), 2.0 mM glutamine (GIBCO). 100 U/ml penicillin, 100 &ml streptomycin, and 2.5 p&ml fungizone (GIBCO). Aliquots of 1 ml (3.0-3.5 x lo5 cells) were placed in a 24-well culture plate (Falcon No. 3847) and cultured for 48 hr in a humidified chamber under 5% CO, and air. Secretagogue studies. Following culture. the media were removed and the attached cells were washed once with phosphate-buffered saline prior to incubation (2 hr) with fresh Ml99 containing the test agents. Following exposure to agents, the media excluding attached cells were collected and centrifuged, and the supernatant solutions were stored at -20” until assay. Media from cells incubated for 2 hr immediately after dispersion were treated similarly. Concentrations of GH were determined in duplicate by a homologous radioimmunoassay (Harvey and Scanes, 1977). The antibody used in this assay is specific for chicken GH; binding with chicken PRL is less than 1% (Harvey and Scanes. 1977). Thyrotropin-releasing hormone and dibutyryl cyclic AMP were purchased from Sigma (St. Louis, MO); rhGRF was generously donated by Dr. W. Vale (Salk Institute, La Jolla, CA), and hpGRF (l-44 NH,) was supplied by Dr. T. Mowles (Hoffman La Roche, Nutley. NJ). Sfarisfical analysis. Data were analyzed by analysis of variance (ANOVA) and Fisher Least significant difference. Each value (represented as mean 2 SEM) is from at least 18 replicates (three separate experiments
409
in Vim
with 6 replicates each). All data having a P value of less than 0.05 were considered statistically different. Somatotroph ultrastructure. Electron microscopy was used to evaluate the morphology of somatotrophs. The attached cells in primary culture were removed with a rubber policeman and processed for routine transmission electron microscopy. Cells were first fixed (1 hr at 5”) in 2.5% glutaraldehyde and then postfixed (30 min at 5”) in 1.0% 0~0, (Karnovsky, 1965), dehydrated in ethanol, and embedded in Epon 812. Ultrathin sections were cut on a Porter-Blum Ultramicrotome (MT-2), stained with uranyl acetate and lead citrate, and examined with a Philips 300 electron microscope.
RESULTS
Morphological Study After 48 hr in culture the ultrastructure of somatotrophs (Fig. 1) was similar to that of cells in situ (Malamed et al., 1984). Functional Studies Values obtained in the absence of test agents were taken as control values. The following results pertain to cells treated for 2 hr after 48 hr in culture. Other results (not shown) with cell incubated for 2 hr immediately following enzymatic dispersion showed an insignificant response to GH secretagogues. hpGRF, rhGRF, and dbcAMP The effects of hpGRF and rhGRF on GH release are compared in Table I. GH release was unaltered by either hpGRF or rhGRF at the lowest concentration (0.05 rig/ml). However, higher concentrations (0.5 and 5.0 rig/ml) significantly stimulated GH release. At the highest concentration (5.0 rig/ml), hpGRF elevated GH release by 133 * 41% (mean 2 SEM) over that of controls and rhGRF stimulated GH release by 168 + 40%. The preparations of hpGRF and rhGRF produced similar increases in GH release; the potency estimate (Bliss, 1952) for hpGRF was 1.43 (95% confidence limits, 0.33-6.0 ng rhGRF/ng hpGRF). Dibutyryl cyclic AMP stimulated GH release in a dose-dependent manner (Table 2).
PEREZ, MALAMED,
AND SCANES
FIG. I. Section through a chicken somatotroph fixed following 48 hr in culture. Secretory granules are abundant in the cytoplasm. x 12,740.
The lowest concentration (0.02 mM) had no effect on GH release; a higher concentration (0.2 mM) stimulated GH release to a value 30 ?z 10% over that of controls. Although a further increase (93 ? 17%) in GH release was induced by dbcAMP at the highest concentration (2.0 mM), this eleva-
tion in GH release was significantly less than that evoked (171 2 21%) by hpGRF (5.0 rig/ml). TRH and ‘pGRF zn2eractions Cultured cells were exposed to TRH in the presence or the absence of hpGRF
CHICKEN
GH SECRETION
in Vitro
411
agents (same concentrations) elevated GH release by 161 & 46%. When cells were exposed to both TRH and hpGRF at higher Growth hormone concentrations (5.0 rig/ml each), GH re(rig/ml; Treatment means 2 SEM) lease was increased by 335 + 61% above control values. Ml99 only 166 k 14a The effects of preexposure to either TRH 235 2 19” hpGRF (0.05 ngiml) hpGRF (0.5 rig/ml) or hpGRF on GH release were examined. 371 f 586 hpGRF (5.0 rig/ml) 387 2 68b Cultured cells were exposed to these secrerhGRF (0.05 ngiml) 224 f 21” tagogues for two consecutive 2-hr incubarhGRF (0.5 rig/ml) 3.54 f 41b tions (Fig. 3). During the first incubation 444 ” 66b rhGRF (5.0 rig/ml) (pretreatment), GH release was increased Note. Cells were exposed (2 hr) to secretagogues (183 + 26% above that of controls) by following 48 hr in culture. Each value is a mean of 32 replicates (four separate experiments). Values with no hpGRF (5.0 rig/ml), but unaffected by TRH with TRH (5.0 letters in common differ from each other with P < (5.0 rig/ml). Pretreatment rig/ml) had no effect on GH release in TRH 0.05. (5.0 rig/ml)-treated cells during the second incubation. However, TRH pretreatment (Fig. 2). The effects of these secretagogues potentiated hpGRF (5.0 rig/ml)-induced GH on GH release were synergistic (more than release during the second incubation. In additive) at several concentrations. For exTRH pretreated cells, hpGRF stimulated ample, TRH (0.5 &ml) alone did not sigGH release by 189 +- 31% over control nificantly increase GH release; hpGRF (5.0 values of the second incubation as comrig/ml)-induced GH release was 71 5 26% pared with an increase of 106 f 11% in over that of controls. The presence of both cells receiving no pretreatment. Other reagents at these concentrations markedly sults showed that in cells treated with stimulated GH release to a value 183 ? hpGRF (5.0 rig/ml) during the first incuba39% above that of controls. When the concentrations of secretagogues were re- tion, subsequent hpGRF (5.0 ng/ml)-induced GH release was reduced to a value versed, synergy was again demonstrated. Thus, TRH (5.0 rig/ml) alone had no effect lower than that of the Iirst incubation. on GH release; hpGRF (0.5 rig/ml) stimuSRIF, hpGRF, and TRH lated GH release by 52 ?z 14% over that of controls. However, the addition of both Table 3 presents the effects of SRIF on GH release in the presence and absence of hpGRF, TRH, or both hpGRF and TRH. TABLE 2 GH release was unaltered by SRIF (0.5 and EFFECTS OF CAMP ON GH RELEASE 5.0 &ml) or TRH (5.0 rig/ml). The pres% of control ence of hpGRF (5.0 rig/ml) stimulated GH Treatment (means c SEM) release to a value 65 + 13% above conMl99 only 1W trols; this induction was reduced to control dbcAMP (0.02 m&f) 122 2 9” values by SRIF (either 0.5 or 5.0 &ml). dbcAMP (0.2 mM) 130 k lob The synergistic (124 t 21% over controls) dbcAMP (2.0 r&f) 193 * 17’ hpGRF (5 .O &ml) effects of TRH and hpGRF (5 .O rig/ml each) 271 2 21d on GH release were diminished by the Note. After 48 hr in culture, cells were treated (2 hr) lower concentration (0.5 rig/ml) of SRIF with test agents. Each value is a mean of 40 replicates and reduced to control values by the higher (five separate experiments). Values with no letters in common differ from each other with P c 0.05. concentration (5.0 &ml). EFFECTS
OF
TABLE 1 hpGRF AND rhGRF ON GH SECRETION
412
PEREZ, MALAMED,
AND SCANES
cd
de
7
11 bed
0
0 ICI
OS0.5 so TAH
05 05 50 hp G
RF
05 05 50
05 05 50
hbGRF
-hpGRF
.05+mi
0.5;Wi
05 05 50 hp
RF
5.0TTRH
Fig. 2. The effects of TRH in the presence of hpGRF on GH release. Following culture (48 hr), cells were exposed (2 hr) to secretagogues. Each value is a mean of values from three separate experiments (24 replicates). Bars indicate SEM. Values with no letters in common differ from each other with P C 0.05.
DISCUSSION
We have developed an in vitro system of chicken adenohypophyseal cells to study GH secretion in birds. These studies demonstrate that the control of GH release in vitro in birds shares some features with those of mammals. For example, rhGRF and hpGRF stimulated GH release (Table 1); these secretagogues stimulate GH release in rat pituitary cells (Guillemin et al., 1982; Rivier et al., 1982; Spiess et al., 1983). The inhibitory effect of SRIF on hpGRF-induced GH release (Table 3) is consistent with a previous report in birds (Leung and Taylor, 1983) and with its effects on mammalian GH secretion in vitro (Brazeau et al., 1973). Furthermore, SRIF inhibition of GH release stimulated by the presence of both TRH and hpGRF (Table 3) is a novel finding and lends further support
for an inhibitory role of SRIF in the control of avian GH secretion. Other results from these studies have provided some insight into the mode of action of hpGRF. Although the isolation of an avian GRF or its receptor has not been reported, the stimulatory effects of rhGRF and hpGRF on GH secretion (Table 1) suggests the existence of an avian GRF and the presence of a GRF receptor on the somatotroph cell. The ability of dbcAMP to stimulate GH release (Table 2) suggests a role for CAMP as a “second messenger” in avian GH secretion. In mammals, CAMP mediates GRF-induced GH release (Brazeau et al., 1982). Another mode of GH regulation involves TRH. In mammals, TRH is not a consistent GH secretagogue. However, TRH does stimulate GH release during neonatal development in rats (Szabo et al., 1985) and
CHICKEN 600
r
GH SECRETION
ef PRE- TREATMENT
413
in Vitro
TABLE 3 GH RELEASEINTHE TRH AND hpGRF
EFFECTSOF SRIF ON PRESENCEANDTHEABSENCEOF
Treatment Ml99 only SRIF (0.5 ngiml) SRIF (5.0 rig/ml) TRH (5.0 ngiml) No SRIF SRIF (0.5 &ml) SRIF (5.0 rig/ml) hpGRF (5.0 r&ml) No SRIF SRIF (0.5 rig/ml) SRIF (5.0 nglml) TRH + hpGRF (5.0 @ml) No SRIF SRIF (0.5 ngiml) SRIF (5.0 ngiml)
TREATMENT =
f 600 J
I
$ 400 H
a 200
[ k
C 0
TRH hV3f?F 5.0 5.0
C 0
TRli hpGRF
C
5.0
0
5.0
TRH hpOFzF
5.0 5.0 ngllnl
FIG. 3. The effects of TRH and hpGRF preexposure on subsequent treatment with TRH and hpGRF. Following culture (48 hr), cells were exposed to secretagogues for two consecutive 2-hr incubations. The upper panel represents GH release after the first incubation; the lower panel shows GH release after the second incubation. Each value is a mean of values from 24 replicates (three experiments). Bars indicate SEM. Values with no letters in common differ from each other with P c 0.05.
in hypothyroid adult rats (Szabo et al., 1984). In birds TRH evokes a greater GH release in viva than does hpGRF (Harvey and Scanes, 1984). In the present studies the lack of effect of TRH on GH release (Table 3, Figs. 1 and 2) may be due to the absence of a hypothalamic component in the in vitro system. In avian species the regulation of GH secretion may involve two stimulatory factors, TRH and GRF. Thus, the possibility exists that the presence of one agent may influence the actions of another. Indeed, results of studies in domestic fowl indicate that GRF has a potentiating effect on TRH-induced GH release in vivo (Harvey
GH release (&ml; means 2 SEM) 394 ? 27” 397 2 28” 398 2 34a 444 2 32”tb 431 k 37”,b 465 2 38”,b 654 f 52’ 432 2 37”,b 373 k 19” 886 t 83d 539 -+ 36b 418 f 28”
Note. Cultured (48 hr) cells were exposed to test agents for 2 hr. Each value is a mean of values from three separate experiments having six replicates each. Values with no letters in common differ from each other with P c 0.05.
and Scanes, 1984). In the present experiments, TRH and GRF exerted synergistic effects on GH release (Fig. 2). Furthermore, preexposure to TRH potentiated subsequent hpGRF-induced GH release (Fig. 3). These results indicate two useful models for investigating cell surface receptor regulation and signal transduction mechanisms. Among the possibilities to be examined are TRH regulation of GRF receptors and post-receptor events involving Ca*+, CAMP, and phosphatidylinositol (Berridge, 1984). In conclusion, the primary culture system of chicken adenohypophyseal cells described here is a useful model for studies of the regulation of GH secretions. In this in vitro system GH release is increased by secretagogues such as hpGRF, rhGRF, and dbcAMP; GH release is inhibited by SRIF. Furthermore, the synergistic effects of TRH and hpGRF on GH release provide an opportunity to investigate various aspects
414
PEREZ,
MALAMED,
of GH secretion involving receptor regulation and “second messenger” mediation. ACKNOWLEDGMENTS This is a paper of the Journal Sciences, New Jersey Agriculture Experimental Station (Project No. 06141). supported by the State and Hatch Act Funds. and by grants from the National Science Foundation (PMC-8022727) and the Minority Access to Research Careers Predoctoral Fellowship Program (No. GM08741 ).
REFERENCES Berridge, M. J. (1984). Inositol trisphosphate and diacylglycerol as second messengers. Biocllenl. J. 220, 34.5-360. Bliss, C. 1. (1952). “The Statistics of Bioassay.” pp. 483-523. Academic Press, New York. Brazeau, P.. Ling, N.. Esch, F., Bohlen, P.. Mougin, C.. and Guillemin, R. (1982). Somatocrinin (growth hormone releasing factor) in vitro bioactivity; Ca+ + involvement, CAMP mediated action and additivity of effect with PGE,. Biochem. Biophys. Res. Commun. 109, 588-594. Brazeau. P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier. C.. and Guillemin. R. (1973). Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179, 77-79. Guillemin, R., Brazeau, P., Bohlen, P., Esch, F., Ling, N.. and Wehrenberg. W. B. (1982). Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218, 585-587. Hall, T. R., and Chadwick, A. (1976). Effects of growth hormone inhibiting factor (somatostatin) on the release of growth hormone and prolactin from pituitaries of the domestic fowl in vitro. J. Endocrinol. 68, 163- 164. Harvey, S., and Scanes, C. G. (1984). Interactions of human pancreatic growth hormone releasing factor and thyrotrophin releasing hormone on in vivo growth hormone secretion in the domestic fowl. Horm. Metab. Res. 17, 113-l 19. Harvey. S.. and Scanes, C. G. (1977). The purification and radioimmunoassay of chicken growth hormone. .I. Endocrinol. 13, 321-329. Harvey, S., Scanes, C. G., Chadwick, A., and Bolton, N. J. (1978). The effect of thyrotropin releasing hormone (TRH) and somatostatin (GHRIH) on growth hormone and prolactin secretion in vitro and in vivo in the domestic fowl (Gallus domesticus). Neuroendocrinology 26, 249-260. Karnovsky, M. J. (1965). A formaldehyde-glutaralde-
AND SCANES hyde fixative of high osmolality for use in electron microscopy. .I. Cell Biol. 27, 137A. Law, G. J., Ray. K. P., and Wallis, M. (1984). Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells. FEBS 116, 189-193. Leung, F. C., and Taylor. J. E. (1983). In viva and in vitro stimulation of growth hormone release in chickens by synthetic human pancreatic growth hormone releasing factor. (hpGRFs). Endocrinology 113, 1913-1915. Malamed. S.. Gibney, J. A., Loesser, K. E., and Scanes, C. G. (1984). Age-related changes of the somatotrophs of the domestic fowl Gallus domesticus. Cell Tissue Res. 239, 87-91. Martin, J. B. (1976). Brain regulation of growth hormone secretion. In “Frontiers in Neuroendocrinology” (L. Martin and W. F. Ganong. Eds.). Vol. 4, pp. 129-147. Raven Press, New York. Rivier. J.. Spiess. J.. Thorner, M., and Vale. W. (1982). Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nurure (London) 300, 276-278. Scanes. C. G.. Hall. T. R., and Harvey, S. (1984a). The physiology of growth hormone in poultry. Domes?. Anim. Endocrinol. 1, 201-215. Scanes, C. G.. Carsia. R. V., Lauterio, T. J.. Huybrechts. L. Rivier, J., and Vale, W. (1984b). Synthetic human pancreatic growth hormone releasing factor (GRF) stimulates growth hormone secretion in the domestic fowl (Gallus domesticus). L$e Sci. 34, 1127- 1134. Spiess, J.. Rivier, J.. and Vale. W. (1983). Characterization of rat hypothalamic growth hormone-releasing factor. Nuture (London) 303, 532-535. Szabo, M.. Stachura. M. E.. Paleologos, N.. Bybee, D. E., and Frohman. L. A. (1984). Thyrotropinreleasing hormone stimulates growth hormone release from the anterior pituitary of hypothyroid rats in virro. Endocrinology 114, 1344- 135 1. Szabo. M.. Welsh, J.. and Cuttler, L. (1985). TRH is a potent GH secretagogue during fetal/neonatal development in the rat. Program of the 67th Annual Meeting of fire Endoc :ke Sociery. Baltimore MD, p. 86 (Abstract) Vale, W., Grant. G.. Amoss. M., Blackwell. R.. and Guillemin. R. (1972). Culture of enzymatically dispersed anterior pituitary cells: Functional validation of a method. Endocrinology 91, 562-572. Vale, W.. Vaughan, J., Yamamoto. G.. Speiss. J.. and Rivier, J. (1983). Effects of synthetic human pancreatic (tumor) GH releasing factor and somatostatin. triiodothyronine and dexamethasone on GH secretion in vifro. Endocrinology 112, 1553-1.555.
AND
COMPARATIVE
ENDOCRINOLOGY
65, 408-414 (1987)
Growth
Hormone Secretion from Chicken Adenohypophyseal Cells in Primary Culture: Effects of Human Pancreatic Growth Hormone-Releasing Factor, Thyrotropin-Releasing Hormone, and Somatostatin on Growth Hormone Release FRANK M. PEREZ.*,? SASHA MALAMED,*
*Department of Anatomy, UMDNJ-Robert and tDepartment of Animal Sciences,
Wood Rutgers-The
ANDCOLIN G. SCANES~
Johnsorl Medical School. Piscatutiaay. State University, New Brunswick.
NeM, Jersey Ne,lx Jersey
08854. 08903
Accepted October 10, 1986 A primary culture of chicken adenohypophyseal cells has been developed to study the regulation of growth hormone (GH) secretion. Following collagenase dispersion, cells were exposed for 2 hr to vehicle (control) or test agents. Human pancreatic (tumor) growth hormone-releasing factor (hpGRF) and rat hypothalamic growth hormone-releasing factor stimulated GH release to similar levels. GH release was increased by the presence of dibutyryl cyclic AMP. Thyrotropin-releasing hormone (TRH) alone did not influence GH release: however, TRH plus hpGRF together exerted a synergistic (greater than additive) effect. increasing GH release by 100 to 300% over the sum of the values for each secretagogue acting alone. These relationships between TRH and hpGRF were further examined in cultured cells exposed to secretagogues for two consecutive 2-hr incubations. TRH pretreatment enhanced subsequent hpGRF-stimulated GH release by about 80% over that obtained if no secretagogue was present during the first incubation. In other experiments, somatostatin (SRIF) alone did not alter GH secretion. However, SRIF reduced hpGRF-stimulated GH release to levels found in controls. Furthermore, GH release stimulated by the presence of both TRH and hpGRF was lowered to control values by SRIF. The results of these studies demonstrate that a primary culture of chicken adenohypophyseal cells is a useful model for the study of GH secretion. Indeed, these results suggest that TRH and hpGRF regulate GH secretion by mechanisms which are not identical. ?a 1987 Academic Pres. Inc.
In mammals growth hormone (GH) secretion is regulated by stimulatory and inhibitory factors derived from the hypothalamus (Martin, 1976). A recently isolated rat hypothalamic growth hormone-releasing factor (rhGRF) is a potent and specific stimulator of GH secretion (Spiess et al., 1983). In addition, human pancreatic (tumor) growth hormone-releasing factor (hpGRF), chemically similar to rhGRF, also stimulates GH secretion (Guillemin et al., 1982; Rivier et al., 1982). Somatostatin (SRIF) has been isolated from the mammalian hypothalamus (Brazeau ef al., 1973) and counteracts GRF-induced GH release (Vale ef al., 1983; Law et al., 1984). In birds also, evidence indicates that GH secretion is controlled by hypothalamo-hy-
pophysiotrophic factors (Scanes et al., 1984a). Thyrotropin-releasing hormone (TRH) and hpGRF stimulate GH secretion (Harvey et al., 1978; Scanes et al., 1984b) and SRIF inhibits GH release (Hall and Chadwick, 1976). Much information concerning the regulation of GH secretion in mammals has been obtained through the use of in vitro systems of pituitary cells which are easily varied under controlled conditions. However, less is known about avain GH secretion and few studies of the bird pituitary have used in vitro systems. This report presents a method for preparing primary cultures of chicken adenohypophyseal cells to study the regulation of GH secretion. In addition, the effects of hpGRF, rhGRF, di408
0016-6480187 $1.50 Copyright 10 1987 by Academic Prey. Inc. All rights of reproduction in any form rexwed.
CHICKEN
butyryl cyclic AMP, TRH, GH release are examined. MATERIALS
GH SECRETION
and SRIF on
AND METHODS
Cell processing. Male domestic fowl (White Leghorn strain) were obtained commercially (Moyers Hatcheries, PA) at 1 day old and reared under a daily photoperiod (16 hr light:8 hr dark) with food and water available ad libitum. Sterile conditions were maintained during cell processing and culture. Anterior pituitaries from immature cockerels (6-8 weeks old) were removed immediately following decapitation and placed in a petri dish containing Hanks’ balanced salt solution, Ca2+ and Mg2+ free (HBSS-CMF, GIBCO, Grand Island, NY). Glands were diced into approximately 2-mm cubes and transferred to a conical test tube (15 x 75 mm) containing 0.35% collagenase (Worthington Enzymes, Freehold, NJ) (Vale et ul.. 1972) in HBSS-CMF. Adenohypophyseal cells were dispersed in this medium for 60 min at 37” in a water bath with agitation; dispersion was facilitated by periodic mechanical trituration with a flame-tapered Pasteur pipet. Following dispersion, cells were washed twice with HBSS-CMF and either incubated (2 hr) in medium 199 (Ml99, GIBCO) or placed in culture medium: Ml99 containing 20% fetal bovine serum (GIBCO), 2.0 mM glutamine (GIBCO). 100 U/ml penicillin, 100 &ml streptomycin, and 2.5 p&ml fungizone (GIBCO). Aliquots of 1 ml (3.0-3.5 x lo5 cells) were placed in a 24-well culture plate (Falcon No. 3847) and cultured for 48 hr in a humidified chamber under 5% CO, and air. Secretagogue studies. Following culture. the media were removed and the attached cells were washed once with phosphate-buffered saline prior to incubation (2 hr) with fresh Ml99 containing the test agents. Following exposure to agents, the media excluding attached cells were collected and centrifuged, and the supernatant solutions were stored at -20” until assay. Media from cells incubated for 2 hr immediately after dispersion were treated similarly. Concentrations of GH were determined in duplicate by a homologous radioimmunoassay (Harvey and Scanes, 1977). The antibody used in this assay is specific for chicken GH; binding with chicken PRL is less than 1% (Harvey and Scanes. 1977). Thyrotropin-releasing hormone and dibutyryl cyclic AMP were purchased from Sigma (St. Louis, MO); rhGRF was generously donated by Dr. W. Vale (Salk Institute, La Jolla, CA), and hpGRF (l-44 NH,) was supplied by Dr. T. Mowles (Hoffman La Roche, Nutley. NJ). Sfarisfical analysis. Data were analyzed by analysis of variance (ANOVA) and Fisher Least significant difference. Each value (represented as mean 2 SEM) is from at least 18 replicates (three separate experiments
409
in Vim
with 6 replicates each). All data having a P value of less than 0.05 were considered statistically different. Somatotroph ultrastructure. Electron microscopy was used to evaluate the morphology of somatotrophs. The attached cells in primary culture were removed with a rubber policeman and processed for routine transmission electron microscopy. Cells were first fixed (1 hr at 5”) in 2.5% glutaraldehyde and then postfixed (30 min at 5”) in 1.0% 0~0, (Karnovsky, 1965), dehydrated in ethanol, and embedded in Epon 812. Ultrathin sections were cut on a Porter-Blum Ultramicrotome (MT-2), stained with uranyl acetate and lead citrate, and examined with a Philips 300 electron microscope.
RESULTS
Morphological Study After 48 hr in culture the ultrastructure of somatotrophs (Fig. 1) was similar to that of cells in situ (Malamed et al., 1984). Functional Studies Values obtained in the absence of test agents were taken as control values. The following results pertain to cells treated for 2 hr after 48 hr in culture. Other results (not shown) with cell incubated for 2 hr immediately following enzymatic dispersion showed an insignificant response to GH secretagogues. hpGRF, rhGRF, and dbcAMP The effects of hpGRF and rhGRF on GH release are compared in Table I. GH release was unaltered by either hpGRF or rhGRF at the lowest concentration (0.05 rig/ml). However, higher concentrations (0.5 and 5.0 rig/ml) significantly stimulated GH release. At the highest concentration (5.0 rig/ml), hpGRF elevated GH release by 133 * 41% (mean 2 SEM) over that of controls and rhGRF stimulated GH release by 168 + 40%. The preparations of hpGRF and rhGRF produced similar increases in GH release; the potency estimate (Bliss, 1952) for hpGRF was 1.43 (95% confidence limits, 0.33-6.0 ng rhGRF/ng hpGRF). Dibutyryl cyclic AMP stimulated GH release in a dose-dependent manner (Table 2).
PEREZ, MALAMED,
AND SCANES
FIG. I. Section through a chicken somatotroph fixed following 48 hr in culture. Secretory granules are abundant in the cytoplasm. x 12,740.
The lowest concentration (0.02 mM) had no effect on GH release; a higher concentration (0.2 mM) stimulated GH release to a value 30 ?z 10% over that of controls. Although a further increase (93 ? 17%) in GH release was induced by dbcAMP at the highest concentration (2.0 mM), this eleva-
tion in GH release was significantly less than that evoked (171 2 21%) by hpGRF (5.0 rig/ml). TRH and ‘pGRF zn2eractions Cultured cells were exposed to TRH in the presence or the absence of hpGRF
CHICKEN
GH SECRETION
in Vitro
411
agents (same concentrations) elevated GH release by 161 & 46%. When cells were exposed to both TRH and hpGRF at higher Growth hormone concentrations (5.0 rig/ml each), GH re(rig/ml; Treatment means 2 SEM) lease was increased by 335 + 61% above control values. Ml99 only 166 k 14a The effects of preexposure to either TRH 235 2 19” hpGRF (0.05 ngiml) hpGRF (0.5 rig/ml) or hpGRF on GH release were examined. 371 f 586 hpGRF (5.0 rig/ml) 387 2 68b Cultured cells were exposed to these secrerhGRF (0.05 ngiml) 224 f 21” tagogues for two consecutive 2-hr incubarhGRF (0.5 rig/ml) 3.54 f 41b tions (Fig. 3). During the first incubation 444 ” 66b rhGRF (5.0 rig/ml) (pretreatment), GH release was increased Note. Cells were exposed (2 hr) to secretagogues (183 + 26% above that of controls) by following 48 hr in culture. Each value is a mean of 32 replicates (four separate experiments). Values with no hpGRF (5.0 rig/ml), but unaffected by TRH with TRH (5.0 letters in common differ from each other with P < (5.0 rig/ml). Pretreatment rig/ml) had no effect on GH release in TRH 0.05. (5.0 rig/ml)-treated cells during the second incubation. However, TRH pretreatment (Fig. 2). The effects of these secretagogues potentiated hpGRF (5.0 rig/ml)-induced GH on GH release were synergistic (more than release during the second incubation. In additive) at several concentrations. For exTRH pretreated cells, hpGRF stimulated ample, TRH (0.5 &ml) alone did not sigGH release by 189 +- 31% over control nificantly increase GH release; hpGRF (5.0 values of the second incubation as comrig/ml)-induced GH release was 71 5 26% pared with an increase of 106 f 11% in over that of controls. The presence of both cells receiving no pretreatment. Other reagents at these concentrations markedly sults showed that in cells treated with stimulated GH release to a value 183 ? hpGRF (5.0 rig/ml) during the first incuba39% above that of controls. When the concentrations of secretagogues were re- tion, subsequent hpGRF (5.0 ng/ml)-induced GH release was reduced to a value versed, synergy was again demonstrated. Thus, TRH (5.0 rig/ml) alone had no effect lower than that of the Iirst incubation. on GH release; hpGRF (0.5 rig/ml) stimuSRIF, hpGRF, and TRH lated GH release by 52 ?z 14% over that of controls. However, the addition of both Table 3 presents the effects of SRIF on GH release in the presence and absence of hpGRF, TRH, or both hpGRF and TRH. TABLE 2 GH release was unaltered by SRIF (0.5 and EFFECTS OF CAMP ON GH RELEASE 5.0 &ml) or TRH (5.0 rig/ml). The pres% of control ence of hpGRF (5.0 rig/ml) stimulated GH Treatment (means c SEM) release to a value 65 + 13% above conMl99 only 1W trols; this induction was reduced to control dbcAMP (0.02 m&f) 122 2 9” values by SRIF (either 0.5 or 5.0 &ml). dbcAMP (0.2 mM) 130 k lob The synergistic (124 t 21% over controls) dbcAMP (2.0 r&f) 193 * 17’ hpGRF (5 .O &ml) effects of TRH and hpGRF (5 .O rig/ml each) 271 2 21d on GH release were diminished by the Note. After 48 hr in culture, cells were treated (2 hr) lower concentration (0.5 rig/ml) of SRIF with test agents. Each value is a mean of 40 replicates and reduced to control values by the higher (five separate experiments). Values with no letters in common differ from each other with P c 0.05. concentration (5.0 &ml). EFFECTS
OF
TABLE 1 hpGRF AND rhGRF ON GH SECRETION
412
PEREZ, MALAMED,
AND SCANES
cd
de
7
11 bed
0
0 ICI
OS0.5 so TAH
05 05 50 hp G
RF
05 05 50
05 05 50
hbGRF
-hpGRF
.05+mi
0.5;Wi
05 05 50 hp
RF
5.0TTRH
Fig. 2. The effects of TRH in the presence of hpGRF on GH release. Following culture (48 hr), cells were exposed (2 hr) to secretagogues. Each value is a mean of values from three separate experiments (24 replicates). Bars indicate SEM. Values with no letters in common differ from each other with P C 0.05.
DISCUSSION
We have developed an in vitro system of chicken adenohypophyseal cells to study GH secretion in birds. These studies demonstrate that the control of GH release in vitro in birds shares some features with those of mammals. For example, rhGRF and hpGRF stimulated GH release (Table 1); these secretagogues stimulate GH release in rat pituitary cells (Guillemin et al., 1982; Rivier et al., 1982; Spiess et al., 1983). The inhibitory effect of SRIF on hpGRF-induced GH release (Table 3) is consistent with a previous report in birds (Leung and Taylor, 1983) and with its effects on mammalian GH secretion in vitro (Brazeau et al., 1973). Furthermore, SRIF inhibition of GH release stimulated by the presence of both TRH and hpGRF (Table 3) is a novel finding and lends further support
for an inhibitory role of SRIF in the control of avian GH secretion. Other results from these studies have provided some insight into the mode of action of hpGRF. Although the isolation of an avian GRF or its receptor has not been reported, the stimulatory effects of rhGRF and hpGRF on GH secretion (Table 1) suggests the existence of an avian GRF and the presence of a GRF receptor on the somatotroph cell. The ability of dbcAMP to stimulate GH release (Table 2) suggests a role for CAMP as a “second messenger” in avian GH secretion. In mammals, CAMP mediates GRF-induced GH release (Brazeau et al., 1982). Another mode of GH regulation involves TRH. In mammals, TRH is not a consistent GH secretagogue. However, TRH does stimulate GH release during neonatal development in rats (Szabo et al., 1985) and
CHICKEN 600
r
GH SECRETION
ef PRE- TREATMENT
413
in Vitro
TABLE 3 GH RELEASEINTHE TRH AND hpGRF
EFFECTSOF SRIF ON PRESENCEANDTHEABSENCEOF
Treatment Ml99 only SRIF (0.5 ngiml) SRIF (5.0 rig/ml) TRH (5.0 ngiml) No SRIF SRIF (0.5 &ml) SRIF (5.0 rig/ml) hpGRF (5.0 r&ml) No SRIF SRIF (0.5 rig/ml) SRIF (5.0 nglml) TRH + hpGRF (5.0 @ml) No SRIF SRIF (0.5 ngiml) SRIF (5.0 ngiml)
TREATMENT =
f 600 J
I
$ 400 H
a 200
[ k
C 0
TRH hV3f?F 5.0 5.0
C 0
TRli hpGRF
C
5.0
0
5.0
TRH hpOFzF
5.0 5.0 ngllnl
FIG. 3. The effects of TRH and hpGRF preexposure on subsequent treatment with TRH and hpGRF. Following culture (48 hr), cells were exposed to secretagogues for two consecutive 2-hr incubations. The upper panel represents GH release after the first incubation; the lower panel shows GH release after the second incubation. Each value is a mean of values from 24 replicates (three experiments). Bars indicate SEM. Values with no letters in common differ from each other with P c 0.05.
in hypothyroid adult rats (Szabo et al., 1984). In birds TRH evokes a greater GH release in viva than does hpGRF (Harvey and Scanes, 1984). In the present studies the lack of effect of TRH on GH release (Table 3, Figs. 1 and 2) may be due to the absence of a hypothalamic component in the in vitro system. In avian species the regulation of GH secretion may involve two stimulatory factors, TRH and GRF. Thus, the possibility exists that the presence of one agent may influence the actions of another. Indeed, results of studies in domestic fowl indicate that GRF has a potentiating effect on TRH-induced GH release in vivo (Harvey
GH release (&ml; means 2 SEM) 394 ? 27” 397 2 28” 398 2 34a 444 2 32”tb 431 k 37”,b 465 2 38”,b 654 f 52’ 432 2 37”,b 373 k 19” 886 t 83d 539 -+ 36b 418 f 28”
Note. Cultured (48 hr) cells were exposed to test agents for 2 hr. Each value is a mean of values from three separate experiments having six replicates each. Values with no letters in common differ from each other with P c 0.05.
and Scanes, 1984). In the present experiments, TRH and GRF exerted synergistic effects on GH release (Fig. 2). Furthermore, preexposure to TRH potentiated subsequent hpGRF-induced GH release (Fig. 3). These results indicate two useful models for investigating cell surface receptor regulation and signal transduction mechanisms. Among the possibilities to be examined are TRH regulation of GRF receptors and post-receptor events involving Ca*+, CAMP, and phosphatidylinositol (Berridge, 1984). In conclusion, the primary culture system of chicken adenohypophyseal cells described here is a useful model for studies of the regulation of GH secretions. In this in vitro system GH release is increased by secretagogues such as hpGRF, rhGRF, and dbcAMP; GH release is inhibited by SRIF. Furthermore, the synergistic effects of TRH and hpGRF on GH release provide an opportunity to investigate various aspects
414
PEREZ,
MALAMED,
of GH secretion involving receptor regulation and “second messenger” mediation. ACKNOWLEDGMENTS This is a paper of the Journal Sciences, New Jersey Agriculture Experimental Station (Project No. 06141). supported by the State and Hatch Act Funds. and by grants from the National Science Foundation (PMC-8022727) and the Minority Access to Research Careers Predoctoral Fellowship Program (No. GM08741 ).
REFERENCES Berridge, M. J. (1984). Inositol trisphosphate and diacylglycerol as second messengers. Biocllenl. J. 220, 34.5-360. Bliss, C. 1. (1952). “The Statistics of Bioassay.” pp. 483-523. Academic Press, New York. Brazeau, P.. Ling, N.. Esch, F., Bohlen, P.. Mougin, C.. and Guillemin, R. (1982). Somatocrinin (growth hormone releasing factor) in vitro bioactivity; Ca+ + involvement, CAMP mediated action and additivity of effect with PGE,. Biochem. Biophys. Res. Commun. 109, 588-594. Brazeau. P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier. C.. and Guillemin. R. (1973). Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179, 77-79. Guillemin, R., Brazeau, P., Bohlen, P., Esch, F., Ling, N.. and Wehrenberg. W. B. (1982). Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218, 585-587. Hall, T. R., and Chadwick, A. (1976). Effects of growth hormone inhibiting factor (somatostatin) on the release of growth hormone and prolactin from pituitaries of the domestic fowl in vitro. J. Endocrinol. 68, 163- 164. Harvey, S., and Scanes, C. G. (1984). Interactions of human pancreatic growth hormone releasing factor and thyrotrophin releasing hormone on in vivo growth hormone secretion in the domestic fowl. Horm. Metab. Res. 17, 113-l 19. Harvey. S.. and Scanes, C. G. (1977). The purification and radioimmunoassay of chicken growth hormone. .I. Endocrinol. 13, 321-329. Harvey, S., Scanes, C. G., Chadwick, A., and Bolton, N. J. (1978). The effect of thyrotropin releasing hormone (TRH) and somatostatin (GHRIH) on growth hormone and prolactin secretion in vitro and in vivo in the domestic fowl (Gallus domesticus). Neuroendocrinology 26, 249-260. Karnovsky, M. J. (1965). A formaldehyde-glutaralde-
AND SCANES hyde fixative of high osmolality for use in electron microscopy. .I. Cell Biol. 27, 137A. Law, G. J., Ray. K. P., and Wallis, M. (1984). Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells. FEBS 116, 189-193. Leung, F. C., and Taylor. J. E. (1983). In viva and in vitro stimulation of growth hormone release in chickens by synthetic human pancreatic growth hormone releasing factor. (hpGRFs). Endocrinology 113, 1913-1915. Malamed. S.. Gibney, J. A., Loesser, K. E., and Scanes, C. G. (1984). Age-related changes of the somatotrophs of the domestic fowl Gallus domesticus. Cell Tissue Res. 239, 87-91. Martin, J. B. (1976). Brain regulation of growth hormone secretion. In “Frontiers in Neuroendocrinology” (L. Martin and W. F. Ganong. Eds.). Vol. 4, pp. 129-147. Raven Press, New York. Rivier. J.. Spiess. J.. Thorner, M., and Vale. W. (1982). Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nurure (London) 300, 276-278. Scanes. C. G.. Hall. T. R., and Harvey, S. (1984a). The physiology of growth hormone in poultry. Domes?. Anim. Endocrinol. 1, 201-215. Scanes, C. G.. Carsia. R. V., Lauterio, T. J.. Huybrechts. L. Rivier, J., and Vale, W. (1984b). Synthetic human pancreatic growth hormone releasing factor (GRF) stimulates growth hormone secretion in the domestic fowl (Gallus domesticus). L$e Sci. 34, 1127- 1134. Spiess, J.. Rivier, J.. and Vale. W. (1983). Characterization of rat hypothalamic growth hormone-releasing factor. Nuture (London) 303, 532-535. Szabo, M.. Stachura. M. E.. Paleologos, N.. Bybee, D. E., and Frohman. L. A. (1984). Thyrotropinreleasing hormone stimulates growth hormone release from the anterior pituitary of hypothyroid rats in virro. Endocrinology 114, 1344- 135 1. Szabo. M.. Welsh, J.. and Cuttler, L. (1985). TRH is a potent GH secretagogue during fetal/neonatal development in the rat. Program of the 67th Annual Meeting of fire Endoc :ke Sociery. Baltimore MD, p. 86 (Abstract) Vale, W., Grant. G.. Amoss. M., Blackwell. R.. and Guillemin. R. (1972). Culture of enzymatically dispersed anterior pituitary cells: Functional validation of a method. Endocrinology 91, 562-572. Vale, W.. Vaughan, J., Yamamoto. G.. Speiss. J.. and Rivier, J. (1983). Effects of synthetic human pancreatic (tumor) GH releasing factor and somatostatin. triiodothyronine and dexamethasone on GH secretion in vifro. Endocrinology 112, 1553-1.555.