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Inhibition of growth hormone and thyrotropin release by growth hormone-release inhibiting hormone
Molecular and Cellular Endocrinology
1 (1974) 329-339. 0 North-Holland
Pub]. Comp.
INHIBITION OF GROWTH HORMONE AND THYROTROPIN RELEASE BY GROWTH HORMONE-RELEASE INHIBITING HORMONE
Alain BELANGER,
MRC
Fernand LABRIE, Pierre BORGEAT, Jean COTE and Jacques DROUIN
Group in Molecular
Endocrinology,
Centre
Hospitalier
Muriel SAVARY,
de I’UniversitP
Lava& Quebec,
Quebec Gl V 4G2
Andrew Veterans
V. SCHALLY,
Administration
Hospital
Hans IMMER,
H. COY and Esther J. COY
and Tulane
University,
Louisiana
70146. U.S.A.
K. SESTANJ,
Ayerst
Received 13 February
David
Research
School
V. NELSON
Laboratories,
of Medicine,
and Manfred
Montreal,
New
Orleans,
GOTZ
Canada
1974
Accepted 8 March 1974
Addition of increasing doses of synthetic growth hormone-release inhibiting hormone (GH-RIH) leads to a progressive decrease of the basal and N6-monobutyryl cyclic AMP-, theophylline- and prostaglandin E2-induced release of immunoreactive growth hormone (GH) and thyrotropin (TSH) release from rat anterior pituitary cells in monolayer culture. A halfmaximal effect is measured at 3 x 10m9M GH-RIH while a maximal inhibition to 10-20°/0 of the control level is found at 1 x lo-’ M. Using rat hemipituitaries and measurement of GH release by both polyacrylamide gel electrophoresis and radioimmunoassay, a maximal effect of GH-RIH was found in the first 5 min of incubation. The inhibitory effect of GH-RIH on GH release remained constant for at least 3 h. GH-RIH does not affect the basal or induced release of prolactin and luteinizing hormone nor the high K+-induced release of GH and TSH. Keywords:
anterior pituitary;
The hypothalamus secretion of growth stimulatory and one 1965; Krulich et al.,
somatostatin;
hypothalamic
secretes neurohormones which hormone (GH) by the anterior being inhibitory (Deuben and 1968; Schally et al., 1968, 1973;
hormone.
exert a dual control pituitary gland, one Meites, 1964; Pecile McCann and Porter,
on the being et al., 1969).
330
A. Belanger
The neurohormone
which stimulates
et al.
GH release has been named GH-releasing
hormone (Schally et al., 1968) and has been purified from porcine, sheep and rat hypothalami (Schally et al., 1969; Malacara and Reichlin, 1971; Wilber et al., 1971). Such GH-releasing activity has been found in hypothalamic extracts of many mammals, including man, as well as in some birds and amphibia (Schally et al., 1968; Schally and Kastin, 1971). The existence in ovine hypothalamic extracts of a substance(s) inhibiting GH release was first reported by Krulich et al. (1968). Recently, the tetradecapeptide H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH (somatostatin or GH-RIH, GH-release inhibiting hormone) has been isolated and characterized from ovine hypothalami (Vale et al., 1972; Brazeau et al., 1973 ; Burgus et al., 1973 ; Rivier et al., 1973) and found to inhibit immunoreactive GH release both in vitro and in vivo (Vale et al., 1972; Brazeau et al., 1973; Burgus et al., 1973; Coy et al., 1973; Rivier et al., 1973). As a preliminary step to a study of the mechanism of action of this peptide, we have investigated the specificity of its action on adenohypophyseal hormone release and its interaction with the known stimulatory effects of theophylline, cyclic AMP, prostaglandins and high K+ (Vale et al., 1972; Labrie et al., 1973a).
MATERIALS Preparation
AND
METHODS
and incubation of cells
Monolayer cultures of enzymatically dispersed cells from adult male rat anterior pituitaries were prepared as described (Vale et al., 1972; Labrie et al., 1973a). After 4 days in culture, the cells were washed and incubated for 5 h at 37 “C in Dulbecco modified Eagle’s medium (DMEM) in the presence or absence of the indicated concentrations of GH-RIH, theophylline, N6-monobutyryl adenosine 3’,5’-monophosphate (mbcAMP) or prostaglandin E,
Preparation
and incubation of hemipituitaries
Hemipituitaries (8 per group) from adult male Sprague-Dawley rats (225-250 g) were prepared and incubated as described (Labrie et al., 1973b) and indicated in the legends to tables and figs. When a high K+ (25 mM) medium was used, the concentration of Na+ was decreased accordingly. Abbreviations: GH, growth hormone; TSH, thyrotropin; PRL, prolactin; LH, luteinizing hormone; GH-RIH, growth hormone-release inhibiting hormone (also called somatostatin); mbcAMP, N6-monobutyryl adenosine 3’,5’-monophosphate; PGE,, prostaglandin E2; KRBG, Krebs-Ringer bicarbonate buffer containing 11 mM D-glucose.
Znhibition of growth hormone and thyrotropin release
331
measurements of GH, PRL, TSH and LH The release of GH, PRL, TSH and LH was measured by double-antibody radioimmunoassay (Birge et al., 1967; Ode11 et al., 1967) using hormones and antisera provided by the Rat Pituitary Hormone Program. Radioimmunoassay data were analysed with an IBM-340-APL computer essentiahy as described (Robard, 1971). In some experiments performed with hemipituitaries, GH and PRL release were also determined by densitometry of stained proteins and counting of incorporated radioactivity in the corresponding bands after separation of the components of the incubation medium by polyacrylamide gel etectrophoresis (Lemay, 1972; Labrie et al., 1973b). Materials GH-RIH (AY 24, 910) was prepared by classical fragment synthesis (Immer et al., 1974). PGE, was generously provided by Dr. John E. Pike, Upjohn Co., Kalamazoo. Antisera to rat GH, PRL, LH and TSH (Anti GH-S-2, anti TSHS-l, anti LH-S-l and anti PRL-S-2) and rat hormones used for radioiodination (Rat GH-I-2, TSH-I-I, LH-E-3 and PRL-I-l) and standards (Rat GH-RP-I, TSH-RP-1, LH-RP-1 and PRL-RP-1) were kindly supplied by the National Institute of Arthritis and Metabolic Diseases, Rat Pituitary Hormone Program.
RESULTS Eflect of GH-RIH on the basal release of GH and TSH Addition of increasing doses of synthetic GH-RIH leads to a progressive decrease of GH release from rat anterior pituitary cells in monolayer culture (fig. I). A near-maximal inhibition of GH release to 35% of the control rate is found at 1 x IOV M GH-RIH while hormone release is inhibited to 20% of the control rate at 1 x 10m6M GH-RIH. Fig. 2 shows that 1 x 10e7 M GH-RIH reduces the basal release of newly-synthesized GH from rat hemipituitaries to approximately 20% of the control rate during six consecutive 30-min incubation periods. In similar experiments, it was found that the release of immunoreactive GH and TSH was inhibited to approximately 50% of the control rate as early as 2 min after addition of GH-RIH (data not shown).
A.
SOMATOSTATIN
CONC
MINUTES
(M)
Fig. 1. Effect of increasing concentrations
of synthetic GH-RIH
OF
Belangerrr at.
I~CUBATIO~
on the basal release of GH
from rat anterior pituitary cells in monolayer culture. GH was measured by radioimmunoassay Results are presented as mean & SD of data obtained from triplicate dishes. Fig. 2. Effect of GH-RIH
on the basal release of GH from male rat hemipituitaries in vitro. After a preliminary incubation of 90 min at 37 “C with 60 @i/ml of (%)L-leucine, and a 90-min chase with 2 mM unlabeled L-leucine, half-pituitaries (8 per group) were incubated for 6 successive periods of 30 min in the presence or absence of 1 x 10d7 M GH-RIH. GH release was measured by counting of radioactivity associated with the GH band after separation of the components of the incubation medium by polyacrylamide get ektrophoresis (Labrie et al. 1973a, b). Data are presented as mean & SEM.
EApect of GH-RIH on the N”-monobutyqd cyclic AMP-induced release of GH md TSH In the presence of a concentration of mbcAMP leading alone to a 4.5fold stimulation of GH release from pituitary cells in monolayer culture, increasing concentrations of GH-RIH lead to a progressive lowering of GH release to approximately 15% of the control level at 1 x lop7 M GH-RIH (fig. 3a). Half-maximal inhibition is measured at 3 x 10Wg M GH-RIH.
Inhibition of growth hormone and thyrotropin release
a
10-9
10”
to-’
333
04.
IO* 0 SOMATOSTATIN CONC
*
13-*
10-8
10-T
lo4
(Ml
Fig. 3. Effect of increasing concentrations of synthetic GH-RIH on the NC-monobutyryl cyclic AMP-stimulated release of GH (a) and TSH (b) from rat anterior pituitary cells in monolayer culture. Experimental conditions were as in fig. 1 except for the presence of 2.5 mM mbcAMP. TSH was measured by radioimmunoassay.
Fig. 3b shows that the 2-fold mbcAMP-induced release of TSH is reduced to approximately 20% of the control at concentrations of GH-RIH higher than 3 x lop9 M. Efleect ofGH-RIH
on the th~~~hyllin~-induced release of GH and TSH
Table 1 shows that reduced to respectively incubation with 1 x on both GH and TSH Effect of GH-RIH
the theophylline-induced release of GH and TSH is 12 and 16% of the control rate during the first 5 min of lo-’ M GH-RIH. This inhibitory effect of GH-RIH release remains significant up to 2 h of incubation..
on the PGEpstimulated
release of GH and TSH
In the presence of 2 x 10U6 M PGE2, a concentration leading to a 2.2-fold stimulation of GH release when added alone to pituitary cells in monolayer culture, increasing doses of GH-RIH led to a maximal inhibition of GH release to 8% of the control at 1 x IO-’ M (data not shown). A marked inhibition of PGE,-induced TSH release was also observed at concentrations of GH-RIH above 3 x 1O-1o M.
334
A. Eelanger
et al.
Table 1 Time course of the effect of GH-RIH on the theophylIine-induced release of GH and TSH. After a preliminary incubation of 2 h at 37 “C, male rat hemipituitaries (3 per group) were incubated with 10 mM theophylline in the presence or absence of 1 x 10e7 M GH-RIH for the indicated time periods. GH and TSH were measured by radioimmunoassay. Data are presented as mean & SEM. Minutes of incubation
GH release (ug GH-RP-l/ml) Control
GH-RIH
_
Control -
5
15 30 60 120 150
21.3 17.5 32.9 47.8 84.2 68.3
$r & + * rt +
0.5 4.7 2.9 8.5 5.4 4.7
2.6 7.4 23.4 21.0 48.0 61.5
f f & f & i
TSH release (ug TSH-RP-I /ml)
0.1 0.6 3.2 3.4 6.5 9.9
Control
GH-RIH
% 12 42 71 44 57 90
Control %
39.5 26.8 89.9 138 272 243
& i + + & &
5.3 6.5 4.9 17.4 IO.3 47.5 6.1 78.4 22 202 16.3 262
f f :t rt :k +
1.6 3.2 6.4 8.6 13.1 12.7
16 65 53 57 74 108
on basal and cyclic ASP-induced release of PRL Table 2 shows that the presence of 2 x lo-’ or 2 x 10e6 M GH-RIH during a 60-min incubation period of rat hemipituitaries has no effect on either the mbcAMP - or theophylline-induced release of PRL measured by densitometry of total hormone released or counting of radioactivity associated with the newly-synthesized hormone. In the same experiment (table 2), the release of GH is markedly inhibited. In fact, total GH release measured by densitometry is inhibited to 69 to 53% while the release of the newly-synthesized hormone is inhibited to 4 to 15 % of the control rate. Basal and mbcAMP-induced release of LH was not affected by GH-RIH.
Absente of eflect of Gff-RIH
on the high K~-ir~d~ced release if GH and Ts;i-I Table 3 shows that the presence of 1 x: 10e7 M GH-RlH during a 90-min incubation of rat hemipituitaries has no inhibitory effect on the high K+induced release of GH and TSH. That this absence of effect of GH-RIH measured after a 90-min incubation period is not due to the insensivity of our experimental design to detect a transient effect of GH-RIH was confirmed by an absence of inhibitory effect of the neurohormone on GH release during six consecutive 30-min incubations in high K+ (data not shown).
Absence of effect ~~~~-RI~
-.
140 sr 7.5
66 i 4.0
Theophylline
Theophylline C IS.5 x 1W7 M CH-RIH
75 + 3.5
162 sr 8.6
Control
Theophylline
47 + 4.2
20 & 2.1
mbcAMP + 2 x lo-’ M GH-RIH
mbcAMP + 2 x 1O-6 M GH-RIH
mbcAMP
55 i 0.7
17 * 0.4
mbcAMP
16 ” 1.4
48 i 2.1
Control
~~.
Q-Q?)
__
mbcAMP
Addition
47
216
44
31
303
% of control
CPM
260
.-
1820
40
1250
205
10535
2700 ;cl
19280 &
460
400
17820 i_ 3085
5060 *
2180 &
21015 $
1040 *
27845 i
27880 & 4210
4310 i
-_.____.
GH release
14
352
IO
37
647
..-.-
% of control
i
1.1
& 1.4
* 2.8
+ 2.8 26.5 f 2.1
30
32
13.5 f 2.1
9.5 & 0.7
11
9.7 + 1.8
9.2 * 0.4
10
5.2 i4
o-ig)
.--
88
237
86
105
195
% of control
115
390
190
1925
535
125
745
1525
8000 * 1440 7745 f
-
97
197
110
98
187
__-
% of control
--.__I_
6.515 & 1010
3305 f
13785 &
12470 i
12805 &
13015 i
13340 i
7140 f
CPM
PRL release
Effect of GH-RIH on monobutyryl cyclic AMP- and theophylline-stimulated release of GI-I and PRL from paired rat hemipituitaries. After a preincubation of 2.5 h with (3H)leucine (7 pCi/ml) and GH-RIH for 20 min in the appropriate groups, paired hemipituitaries (4 per group) were incubated for 60 min in the presence or absence of 4 mM mbcAMP, 10 mM theophylline, 2 x 10e6 or 2 x lo-’ M GH-RIH. GH and PRL release were measured in duplicate by densitometry and counting of the corresponding protein bands after polyacrylamide gel electrophoresis of the proteins of the incubation medium (Labrie et al. 1973a, b). Data are presented as mean & SD.
Table 2
336
A. Belanger
et al.
DISCUSSION Growth hormone exerts metabolic effects at the different stages of development of probably all mammalian tissues. The recent availability of a synthetic tetradecapeptide (Vale et al., 1972, 1973; Brazeau et al., 1973; Burgus et al., 1973; Coy et al., 1973; Rivier et al., 1973; Yamashiro and Li, 1973; Immer et al., 1974), inhibiting GH release from the anterior pituitary gland offers the possibility of further studying the mechanism controlling the secretion of this hormone. The present data (figs. l-3, table 1) show clearly that GH-RIH inhibits not only the basal release of both GH and TSH but that the increased hormonal secretion induced by mbcAMP, theophylline and PGE, is also markedly inhibited by the tetradecapeptide. Such inhibition is observed both with pituitary cells in monolayer culture and with hemipituitaries. Table 1 shows that the inhibitory effect of GH-RIH on both GH and TSH release is rapid, a maximal effect being detected in the first 5 min of incubation. Fig. 2 indicates clearly that the inhibitory effect remains constant for at least six successive 30-min incubations. In contrast with the marked inhibitory effect of GH-RIH on the mbcAMP-, theophyllineand PGE,-induced release of GH and TSH, the neurohormone does not affect the high K+--stimulated release of these hormones (table 3). Although the high K.-induced release of GH (Parsons, 1970) and TSH is well known, the present findings of a marked inhibitory effect of GH-RiH on the cyclic AMP-, theophyllineand PGE,-induced release of these two hormones and an absence of effect of the same agent on the I(+ -induced hormonal release suggest strongly that hormone release induced by high K+ occurs independently of the cyclic AMP pathway likely to be the physiological stimulus of the release of these two hormones (Labrie et al., 1973a, b, 1974; Borgeat et al., 1973). The data of table 2 also confirm our previous findings of a preferential release of newly-synthesized GH under the stimulatory influence of high K+ (Labrie et al., 1973a). In fact, high K-1 leads to a 3.4-fold stimulation of total GH release measured by densitometry of the protein band on polyacrylamide gels while a IO-fold stimulatory effect is found when the release of the newlysynthesized hormone is measured. Table 2 shows also that not only the release of newly-synthetized proteins is preferentially stimulated but that the inhibitory effect of GH-RIH is also preferentially exerted on the newly-synthesized hormone. In fact, while GH-RIH leads to a 53 to 69 % inhibition of total GH release, the inhibitory effect of the neurohormone on newly-synthesized GH is 86 to 96%.
Inhibition
of growth
hormone and thyrotropin
release
33i
338
A. Eelanger et al.
Although no explanation for this phenomenon is yet available, it seems to indicate that the newly-synthesized granules are more sensitive to the intracellular signals for rapid changes of the rates of hormone release. These observations strongly suggest the presence of more than one pool of GH in the adenohypophysis. Data obtained by radioimmunoassay more closely resemble those of total hormone release measured by densitometry. As a prerequisite to a detailed study of the mechanism of action of GH-RIH, these studies indicate clearly the temporal and dose characteristics of the action of this neurohormone on pituitary cells in monolayer culture and in intact hemipituitaries. They also indicate the specificity of action of GH-RIH on both GH and TSH release. Such dual action of GH-RIH could well offer an explanation for the inhibitory effect of stress on both GH and TSH release in the rat (Brown-Grant et al., 1954; Fortier et al., 1970; Howard and Martin, 1971).
REFERENCES Birge, C. A., Peake, G. T., Marix, 1. K. and Daughaday, W. H. (1967) Endocrinology 81,195. Borgeat, P., Labrie, F., Poirier, G., Chavancy, G. and Schally, A. V. (1973) Trans. Am. Phys. Ass., in press. Brazeau, P., Vale, W., Burgus, R., Ling, V., Butcher, M., Rivier, J. and Guillemin, R. (1973) Science 119, 77. Brown-Grant, K., Harris, G. W. and Reichlin, S. (1954) J. Physiol. (London) 126,29. Burgus, R., Ling, N., Butcher, M. and Guillemin, R. (1973) Proc. Nat!. Acad. Sci. U.S. 70,684. Coy, D. H., Coy, E. J. and Schally, A. V. (1973) Biochem. Biophys. Res. Commun. 54, 1267. Deuben, R. and Meites, J. (1964) Endocrinology 74, 408. Fortier, C., Delgado, A., Ducommun, P., Ducommun, S., Dupont, A., Jobin, M., Kraicer, J., Macintosh-Hardt, B., Marceau, I-I., Mialhe, P., Mialhe-Voloss, C., Rerup, C. and Van Rees, P. P. (1970) Can. Med. Ass. J. 103, 864. Howard, N. and Martin, .I. M. (1971) End~rinology 88, 497. Immer, H., Sestanj, K., Nelson, V. and Gotx, M. (Manuscript in preparation). Krulich, L., Dhariwal, A. P. S. and McCann, S. M. (1968) Endocrinology 83, 783. Labrie, F., Pelletier, G., Lemay, A., Borgeat, P., Barden, N., DuPont, A., Savary, M., C&B, J. and Boucher, R. (1973a) In: Sixth Karolinska Symposia on Research Methods in Reproductive Endocrinol, Ed. : W. Diczfalusy, pp. 301. Labrie, F., Gauthier, M., Pelletier, G., Borgeat, P., Lemay, A. and Gouge, J. J. (1973b) Endocrinology 93, 903. Labrie, F., Pelletier, G., Barden, N., Lemay, A., Lemaire, S. and Borgeat, P. (1974) In: Advances in Sex Hormone Research, Eds.: J. A. Thomas and R. Singhal (Academic Press) in press. Lemay, A. and Labrie, F. (1972) FEBS Letters 20, 7. Malacara, J. M. and Reichlin, S. (1971) Fed. Proc. 30, 198 Abstract. McCann, S. M. and Porter, J. C. (1969) Physiol. Rev. 49, 240.
Inhibition
of growth
hormone and thyrotropin
release
339
Midgley, A. R. Jr. (1967) J. Clin. Endocr. Metab. 27, 295. Odell, W. D., Rayford, P. L. and Ross, G. T. (1967) J. Lab. Clin. Med. 70,973. Parsons, J. A. (1970) J. Physiol. (London) 217, 1599. Pecile, A., Mullet-, E. E., Falconi, G. and Martini, L. (1965) Endocrinology 77, 241. Rivier, R., Brazeau, P., Vale, W., Ling, N., Burgus, R., Gilin, C., Yardley, J. and Guillemin, R. (1973) Compt. Rend. Acad. Sci. Paris, Ser. D, 276, 2737. Rodbard, D. (1971) In: Principles of Competitive Protein-Binding Assays, Eds.: W. D. Ode11 and W. H. Daughaday (The J. B. Lippincott Co.) p. 204. Schally, A. V. and Kastin, A. J. (1971) Acta Physiol. Polonica 22, 721. Schally, A. V., Arimura, A., Bowers, C. Y., Kastin, A. J., Sawano, S. and Redding, T. W. (1968) Recent Progr. Horm. Res. 24, 497. Schally, A. V., Sawano, S., Arimura, A., Barret, J. F., Wakabayashi, I. and Bowers, C. Y. (1969) Endocrinology 84, 1493. Schally, A. V., Arimura, A. and Kastin, A. J. (1973) Science 179, 341. Vale, W., Burgus, R. and Guillemin, R. (1967) Experientia 23, 855. Vale, W., Brazeau, P., Grant, G., Nussey, A., Burgus, R., Rivier, J., Ling, N. and Guillemin, R. (1972) Compt. Rend. Acad. Sci. Paris, Serie D. 275, 2913. Vale, W., Grant, G., Amoss, M., Blackwell, R. and Guillemin, R. (1972) Endocrinology 91, 562. Vale, W., Brazeau, P., Rivier, C. J., Grant, G., Burgus, R. and Guillemin, R. (1973) Fed. Proc. 32, 211 (Abstract). Wilber, J., Nagel, T. and White, W. F. (1971) Endocrinology 89, 1419. Yamashiro, D. and Li, C. H. (1973) Biochem. Biophys. Res. Commun. 54,882.
1 (1974) 329-339. 0 North-Holland
Pub]. Comp.
INHIBITION OF GROWTH HORMONE AND THYROTROPIN RELEASE BY GROWTH HORMONE-RELEASE INHIBITING HORMONE
Alain BELANGER,
MRC
Fernand LABRIE, Pierre BORGEAT, Jean COTE and Jacques DROUIN
Group in Molecular
Endocrinology,
Centre
Hospitalier
Muriel SAVARY,
de I’UniversitP
Lava& Quebec,
Quebec Gl V 4G2
Andrew Veterans
V. SCHALLY,
Administration
Hospital
Hans IMMER,
H. COY and Esther J. COY
and Tulane
University,
Louisiana
70146. U.S.A.
K. SESTANJ,
Ayerst
Received 13 February
David
Research
School
V. NELSON
Laboratories,
of Medicine,
and Manfred
Montreal,
New
Orleans,
GOTZ
Canada
1974
Accepted 8 March 1974
Addition of increasing doses of synthetic growth hormone-release inhibiting hormone (GH-RIH) leads to a progressive decrease of the basal and N6-monobutyryl cyclic AMP-, theophylline- and prostaglandin E2-induced release of immunoreactive growth hormone (GH) and thyrotropin (TSH) release from rat anterior pituitary cells in monolayer culture. A halfmaximal effect is measured at 3 x 10m9M GH-RIH while a maximal inhibition to 10-20°/0 of the control level is found at 1 x lo-’ M. Using rat hemipituitaries and measurement of GH release by both polyacrylamide gel electrophoresis and radioimmunoassay, a maximal effect of GH-RIH was found in the first 5 min of incubation. The inhibitory effect of GH-RIH on GH release remained constant for at least 3 h. GH-RIH does not affect the basal or induced release of prolactin and luteinizing hormone nor the high K+-induced release of GH and TSH. Keywords:
anterior pituitary;
The hypothalamus secretion of growth stimulatory and one 1965; Krulich et al.,
somatostatin;
hypothalamic
secretes neurohormones which hormone (GH) by the anterior being inhibitory (Deuben and 1968; Schally et al., 1968, 1973;
hormone.
exert a dual control pituitary gland, one Meites, 1964; Pecile McCann and Porter,
on the being et al., 1969).
330
A. Belanger
The neurohormone
which stimulates
et al.
GH release has been named GH-releasing
hormone (Schally et al., 1968) and has been purified from porcine, sheep and rat hypothalami (Schally et al., 1969; Malacara and Reichlin, 1971; Wilber et al., 1971). Such GH-releasing activity has been found in hypothalamic extracts of many mammals, including man, as well as in some birds and amphibia (Schally et al., 1968; Schally and Kastin, 1971). The existence in ovine hypothalamic extracts of a substance(s) inhibiting GH release was first reported by Krulich et al. (1968). Recently, the tetradecapeptide H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH (somatostatin or GH-RIH, GH-release inhibiting hormone) has been isolated and characterized from ovine hypothalami (Vale et al., 1972; Brazeau et al., 1973 ; Burgus et al., 1973 ; Rivier et al., 1973) and found to inhibit immunoreactive GH release both in vitro and in vivo (Vale et al., 1972; Brazeau et al., 1973; Burgus et al., 1973; Coy et al., 1973; Rivier et al., 1973). As a preliminary step to a study of the mechanism of action of this peptide, we have investigated the specificity of its action on adenohypophyseal hormone release and its interaction with the known stimulatory effects of theophylline, cyclic AMP, prostaglandins and high K+ (Vale et al., 1972; Labrie et al., 1973a).
MATERIALS Preparation
AND
METHODS
and incubation of cells
Monolayer cultures of enzymatically dispersed cells from adult male rat anterior pituitaries were prepared as described (Vale et al., 1972; Labrie et al., 1973a). After 4 days in culture, the cells were washed and incubated for 5 h at 37 “C in Dulbecco modified Eagle’s medium (DMEM) in the presence or absence of the indicated concentrations of GH-RIH, theophylline, N6-monobutyryl adenosine 3’,5’-monophosphate (mbcAMP) or prostaglandin E,
Preparation
and incubation of hemipituitaries
Hemipituitaries (8 per group) from adult male Sprague-Dawley rats (225-250 g) were prepared and incubated as described (Labrie et al., 1973b) and indicated in the legends to tables and figs. When a high K+ (25 mM) medium was used, the concentration of Na+ was decreased accordingly. Abbreviations: GH, growth hormone; TSH, thyrotropin; PRL, prolactin; LH, luteinizing hormone; GH-RIH, growth hormone-release inhibiting hormone (also called somatostatin); mbcAMP, N6-monobutyryl adenosine 3’,5’-monophosphate; PGE,, prostaglandin E2; KRBG, Krebs-Ringer bicarbonate buffer containing 11 mM D-glucose.
Znhibition of growth hormone and thyrotropin release
331
measurements of GH, PRL, TSH and LH The release of GH, PRL, TSH and LH was measured by double-antibody radioimmunoassay (Birge et al., 1967; Ode11 et al., 1967) using hormones and antisera provided by the Rat Pituitary Hormone Program. Radioimmunoassay data were analysed with an IBM-340-APL computer essentiahy as described (Robard, 1971). In some experiments performed with hemipituitaries, GH and PRL release were also determined by densitometry of stained proteins and counting of incorporated radioactivity in the corresponding bands after separation of the components of the incubation medium by polyacrylamide gel etectrophoresis (Lemay, 1972; Labrie et al., 1973b). Materials GH-RIH (AY 24, 910) was prepared by classical fragment synthesis (Immer et al., 1974). PGE, was generously provided by Dr. John E. Pike, Upjohn Co., Kalamazoo. Antisera to rat GH, PRL, LH and TSH (Anti GH-S-2, anti TSHS-l, anti LH-S-l and anti PRL-S-2) and rat hormones used for radioiodination (Rat GH-I-2, TSH-I-I, LH-E-3 and PRL-I-l) and standards (Rat GH-RP-I, TSH-RP-1, LH-RP-1 and PRL-RP-1) were kindly supplied by the National Institute of Arthritis and Metabolic Diseases, Rat Pituitary Hormone Program.
RESULTS Eflect of GH-RIH on the basal release of GH and TSH Addition of increasing doses of synthetic GH-RIH leads to a progressive decrease of GH release from rat anterior pituitary cells in monolayer culture (fig. I). A near-maximal inhibition of GH release to 35% of the control rate is found at 1 x IOV M GH-RIH while hormone release is inhibited to 20% of the control rate at 1 x 10m6M GH-RIH. Fig. 2 shows that 1 x 10e7 M GH-RIH reduces the basal release of newly-synthesized GH from rat hemipituitaries to approximately 20% of the control rate during six consecutive 30-min incubation periods. In similar experiments, it was found that the release of immunoreactive GH and TSH was inhibited to approximately 50% of the control rate as early as 2 min after addition of GH-RIH (data not shown).
A.
SOMATOSTATIN
CONC
MINUTES
(M)
Fig. 1. Effect of increasing concentrations
of synthetic GH-RIH
OF
Belangerrr at.
I~CUBATIO~
on the basal release of GH
from rat anterior pituitary cells in monolayer culture. GH was measured by radioimmunoassay Results are presented as mean & SD of data obtained from triplicate dishes. Fig. 2. Effect of GH-RIH
on the basal release of GH from male rat hemipituitaries in vitro. After a preliminary incubation of 90 min at 37 “C with 60 @i/ml of (%)L-leucine, and a 90-min chase with 2 mM unlabeled L-leucine, half-pituitaries (8 per group) were incubated for 6 successive periods of 30 min in the presence or absence of 1 x 10d7 M GH-RIH. GH release was measured by counting of radioactivity associated with the GH band after separation of the components of the incubation medium by polyacrylamide get ektrophoresis (Labrie et al. 1973a, b). Data are presented as mean & SEM.
EApect of GH-RIH on the N”-monobutyqd cyclic AMP-induced release of GH md TSH In the presence of a concentration of mbcAMP leading alone to a 4.5fold stimulation of GH release from pituitary cells in monolayer culture, increasing concentrations of GH-RIH lead to a progressive lowering of GH release to approximately 15% of the control level at 1 x lop7 M GH-RIH (fig. 3a). Half-maximal inhibition is measured at 3 x 10Wg M GH-RIH.
Inhibition of growth hormone and thyrotropin release
a
10-9
10”
to-’
333
04.
IO* 0 SOMATOSTATIN CONC
*
13-*
10-8
10-T
lo4
(Ml
Fig. 3. Effect of increasing concentrations of synthetic GH-RIH on the NC-monobutyryl cyclic AMP-stimulated release of GH (a) and TSH (b) from rat anterior pituitary cells in monolayer culture. Experimental conditions were as in fig. 1 except for the presence of 2.5 mM mbcAMP. TSH was measured by radioimmunoassay.
Fig. 3b shows that the 2-fold mbcAMP-induced release of TSH is reduced to approximately 20% of the control at concentrations of GH-RIH higher than 3 x lop9 M. Efleect ofGH-RIH
on the th~~~hyllin~-induced release of GH and TSH
Table 1 shows that reduced to respectively incubation with 1 x on both GH and TSH Effect of GH-RIH
the theophylline-induced release of GH and TSH is 12 and 16% of the control rate during the first 5 min of lo-’ M GH-RIH. This inhibitory effect of GH-RIH release remains significant up to 2 h of incubation..
on the PGEpstimulated
release of GH and TSH
In the presence of 2 x 10U6 M PGE2, a concentration leading to a 2.2-fold stimulation of GH release when added alone to pituitary cells in monolayer culture, increasing doses of GH-RIH led to a maximal inhibition of GH release to 8% of the control at 1 x IO-’ M (data not shown). A marked inhibition of PGE,-induced TSH release was also observed at concentrations of GH-RIH above 3 x 1O-1o M.
334
A. Eelanger
et al.
Table 1 Time course of the effect of GH-RIH on the theophylIine-induced release of GH and TSH. After a preliminary incubation of 2 h at 37 “C, male rat hemipituitaries (3 per group) were incubated with 10 mM theophylline in the presence or absence of 1 x 10e7 M GH-RIH for the indicated time periods. GH and TSH were measured by radioimmunoassay. Data are presented as mean & SEM. Minutes of incubation
GH release (ug GH-RP-l/ml) Control
GH-RIH
_
Control -
5
15 30 60 120 150
21.3 17.5 32.9 47.8 84.2 68.3
$r & + * rt +
0.5 4.7 2.9 8.5 5.4 4.7
2.6 7.4 23.4 21.0 48.0 61.5
f f & f & i
TSH release (ug TSH-RP-I /ml)
0.1 0.6 3.2 3.4 6.5 9.9
Control
GH-RIH
% 12 42 71 44 57 90
Control %
39.5 26.8 89.9 138 272 243
& i + + & &
5.3 6.5 4.9 17.4 IO.3 47.5 6.1 78.4 22 202 16.3 262
f f :t rt :k +
1.6 3.2 6.4 8.6 13.1 12.7
16 65 53 57 74 108
on basal and cyclic ASP-induced release of PRL Table 2 shows that the presence of 2 x lo-’ or 2 x 10e6 M GH-RIH during a 60-min incubation period of rat hemipituitaries has no effect on either the mbcAMP - or theophylline-induced release of PRL measured by densitometry of total hormone released or counting of radioactivity associated with the newly-synthesized hormone. In the same experiment (table 2), the release of GH is markedly inhibited. In fact, total GH release measured by densitometry is inhibited to 69 to 53% while the release of the newly-synthesized hormone is inhibited to 4 to 15 % of the control rate. Basal and mbcAMP-induced release of LH was not affected by GH-RIH.
Absente of eflect of Gff-RIH
on the high K~-ir~d~ced release if GH and Ts;i-I Table 3 shows that the presence of 1 x: 10e7 M GH-RlH during a 90-min incubation of rat hemipituitaries has no inhibitory effect on the high K+induced release of GH and TSH. That this absence of effect of GH-RIH measured after a 90-min incubation period is not due to the insensivity of our experimental design to detect a transient effect of GH-RIH was confirmed by an absence of inhibitory effect of the neurohormone on GH release during six consecutive 30-min incubations in high K+ (data not shown).
Absence of effect ~~~~-RI~
-.
140 sr 7.5
66 i 4.0
Theophylline
Theophylline C IS.5 x 1W7 M CH-RIH
75 + 3.5
162 sr 8.6
Control
Theophylline
47 + 4.2
20 & 2.1
mbcAMP + 2 x lo-’ M GH-RIH
mbcAMP + 2 x 1O-6 M GH-RIH
mbcAMP
55 i 0.7
17 * 0.4
mbcAMP
16 ” 1.4
48 i 2.1
Control
~~.
Q-Q?)
__
mbcAMP
Addition
47
216
44
31
303
% of control
CPM
260
.-
1820
40
1250
205
10535
2700 ;cl
19280 &
460
400
17820 i_ 3085
5060 *
2180 &
21015 $
1040 *
27845 i
27880 & 4210
4310 i
-_.____.
GH release
14
352
IO
37
647
..-.-
% of control
i
1.1
& 1.4
* 2.8
+ 2.8 26.5 f 2.1
30
32
13.5 f 2.1
9.5 & 0.7
11
9.7 + 1.8
9.2 * 0.4
10
5.2 i4
o-ig)
.--
88
237
86
105
195
% of control
115
390
190
1925
535
125
745
1525
8000 * 1440 7745 f
-
97
197
110
98
187
__-
% of control
--.__I_
6.515 & 1010
3305 f
13785 &
12470 i
12805 &
13015 i
13340 i
7140 f
CPM
PRL release
Effect of GH-RIH on monobutyryl cyclic AMP- and theophylline-stimulated release of GI-I and PRL from paired rat hemipituitaries. After a preincubation of 2.5 h with (3H)leucine (7 pCi/ml) and GH-RIH for 20 min in the appropriate groups, paired hemipituitaries (4 per group) were incubated for 60 min in the presence or absence of 4 mM mbcAMP, 10 mM theophylline, 2 x 10e6 or 2 x lo-’ M GH-RIH. GH and PRL release were measured in duplicate by densitometry and counting of the corresponding protein bands after polyacrylamide gel electrophoresis of the proteins of the incubation medium (Labrie et al. 1973a, b). Data are presented as mean & SD.
Table 2
336
A. Belanger
et al.
DISCUSSION Growth hormone exerts metabolic effects at the different stages of development of probably all mammalian tissues. The recent availability of a synthetic tetradecapeptide (Vale et al., 1972, 1973; Brazeau et al., 1973; Burgus et al., 1973; Coy et al., 1973; Rivier et al., 1973; Yamashiro and Li, 1973; Immer et al., 1974), inhibiting GH release from the anterior pituitary gland offers the possibility of further studying the mechanism controlling the secretion of this hormone. The present data (figs. l-3, table 1) show clearly that GH-RIH inhibits not only the basal release of both GH and TSH but that the increased hormonal secretion induced by mbcAMP, theophylline and PGE, is also markedly inhibited by the tetradecapeptide. Such inhibition is observed both with pituitary cells in monolayer culture and with hemipituitaries. Table 1 shows that the inhibitory effect of GH-RIH on both GH and TSH release is rapid, a maximal effect being detected in the first 5 min of incubation. Fig. 2 indicates clearly that the inhibitory effect remains constant for at least six successive 30-min incubations. In contrast with the marked inhibitory effect of GH-RIH on the mbcAMP-, theophyllineand PGE,-induced release of GH and TSH, the neurohormone does not affect the high K+--stimulated release of these hormones (table 3). Although the high K.-induced release of GH (Parsons, 1970) and TSH is well known, the present findings of a marked inhibitory effect of GH-RiH on the cyclic AMP-, theophyllineand PGE,-induced release of these two hormones and an absence of effect of the same agent on the I(+ -induced hormonal release suggest strongly that hormone release induced by high K+ occurs independently of the cyclic AMP pathway likely to be the physiological stimulus of the release of these two hormones (Labrie et al., 1973a, b, 1974; Borgeat et al., 1973). The data of table 2 also confirm our previous findings of a preferential release of newly-synthesized GH under the stimulatory influence of high K+ (Labrie et al., 1973a). In fact, high K-1 leads to a 3.4-fold stimulation of total GH release measured by densitometry of the protein band on polyacrylamide gels while a IO-fold stimulatory effect is found when the release of the newlysynthesized hormone is measured. Table 2 shows also that not only the release of newly-synthetized proteins is preferentially stimulated but that the inhibitory effect of GH-RIH is also preferentially exerted on the newly-synthesized hormone. In fact, while GH-RIH leads to a 53 to 69 % inhibition of total GH release, the inhibitory effect of the neurohormone on newly-synthesized GH is 86 to 96%.
Inhibition
of growth
hormone and thyrotropin
release
33i
338
A. Eelanger et al.
Although no explanation for this phenomenon is yet available, it seems to indicate that the newly-synthesized granules are more sensitive to the intracellular signals for rapid changes of the rates of hormone release. These observations strongly suggest the presence of more than one pool of GH in the adenohypophysis. Data obtained by radioimmunoassay more closely resemble those of total hormone release measured by densitometry. As a prerequisite to a detailed study of the mechanism of action of GH-RIH, these studies indicate clearly the temporal and dose characteristics of the action of this neurohormone on pituitary cells in monolayer culture and in intact hemipituitaries. They also indicate the specificity of action of GH-RIH on both GH and TSH release. Such dual action of GH-RIH could well offer an explanation for the inhibitory effect of stress on both GH and TSH release in the rat (Brown-Grant et al., 1954; Fortier et al., 1970; Howard and Martin, 1971).
REFERENCES Birge, C. A., Peake, G. T., Marix, 1. K. and Daughaday, W. H. (1967) Endocrinology 81,195. Borgeat, P., Labrie, F., Poirier, G., Chavancy, G. and Schally, A. V. (1973) Trans. Am. Phys. Ass., in press. Brazeau, P., Vale, W., Burgus, R., Ling, V., Butcher, M., Rivier, J. and Guillemin, R. (1973) Science 119, 77. Brown-Grant, K., Harris, G. W. and Reichlin, S. (1954) J. Physiol. (London) 126,29. Burgus, R., Ling, N., Butcher, M. and Guillemin, R. (1973) Proc. Nat!. Acad. Sci. U.S. 70,684. Coy, D. H., Coy, E. J. and Schally, A. V. (1973) Biochem. Biophys. Res. Commun. 54, 1267. Deuben, R. and Meites, J. (1964) Endocrinology 74, 408. Fortier, C., Delgado, A., Ducommun, P., Ducommun, S., Dupont, A., Jobin, M., Kraicer, J., Macintosh-Hardt, B., Marceau, I-I., Mialhe, P., Mialhe-Voloss, C., Rerup, C. and Van Rees, P. P. (1970) Can. Med. Ass. J. 103, 864. Howard, N. and Martin, .I. M. (1971) End~rinology 88, 497. Immer, H., Sestanj, K., Nelson, V. and Gotx, M. (Manuscript in preparation). Krulich, L., Dhariwal, A. P. S. and McCann, S. M. (1968) Endocrinology 83, 783. Labrie, F., Pelletier, G., Lemay, A., Borgeat, P., Barden, N., DuPont, A., Savary, M., C&B, J. and Boucher, R. (1973a) In: Sixth Karolinska Symposia on Research Methods in Reproductive Endocrinol, Ed. : W. Diczfalusy, pp. 301. Labrie, F., Gauthier, M., Pelletier, G., Borgeat, P., Lemay, A. and Gouge, J. J. (1973b) Endocrinology 93, 903. Labrie, F., Pelletier, G., Barden, N., Lemay, A., Lemaire, S. and Borgeat, P. (1974) In: Advances in Sex Hormone Research, Eds.: J. A. Thomas and R. Singhal (Academic Press) in press. Lemay, A. and Labrie, F. (1972) FEBS Letters 20, 7. Malacara, J. M. and Reichlin, S. (1971) Fed. Proc. 30, 198 Abstract. McCann, S. M. and Porter, J. C. (1969) Physiol. Rev. 49, 240.
Inhibition
of growth
hormone and thyrotropin
release
339
Midgley, A. R. Jr. (1967) J. Clin. Endocr. Metab. 27, 295. Odell, W. D., Rayford, P. L. and Ross, G. T. (1967) J. Lab. Clin. Med. 70,973. Parsons, J. A. (1970) J. Physiol. (London) 217, 1599. Pecile, A., Mullet-, E. E., Falconi, G. and Martini, L. (1965) Endocrinology 77, 241. Rivier, R., Brazeau, P., Vale, W., Ling, N., Burgus, R., Gilin, C., Yardley, J. and Guillemin, R. (1973) Compt. Rend. Acad. Sci. Paris, Ser. D, 276, 2737. Rodbard, D. (1971) In: Principles of Competitive Protein-Binding Assays, Eds.: W. D. Ode11 and W. H. Daughaday (The J. B. Lippincott Co.) p. 204. Schally, A. V. and Kastin, A. J. (1971) Acta Physiol. Polonica 22, 721. Schally, A. V., Arimura, A., Bowers, C. Y., Kastin, A. J., Sawano, S. and Redding, T. W. (1968) Recent Progr. Horm. Res. 24, 497. Schally, A. V., Sawano, S., Arimura, A., Barret, J. F., Wakabayashi, I. and Bowers, C. Y. (1969) Endocrinology 84, 1493. Schally, A. V., Arimura, A. and Kastin, A. J. (1973) Science 179, 341. Vale, W., Burgus, R. and Guillemin, R. (1967) Experientia 23, 855. Vale, W., Brazeau, P., Grant, G., Nussey, A., Burgus, R., Rivier, J., Ling, N. and Guillemin, R. (1972) Compt. Rend. Acad. Sci. Paris, Serie D. 275, 2913. Vale, W., Grant, G., Amoss, M., Blackwell, R. and Guillemin, R. (1972) Endocrinology 91, 562. Vale, W., Brazeau, P., Rivier, C. J., Grant, G., Burgus, R. and Guillemin, R. (1973) Fed. Proc. 32, 211 (Abstract). Wilber, J., Nagel, T. and White, W. F. (1971) Endocrinology 89, 1419. Yamashiro, D. and Li, C. H. (1973) Biochem. Biophys. Res. Commun. 54,882.