| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ARTICLES |
Division of Neurophysiology, National Institute for Medical Research, The Ridgeway, London, United Kingdom NW7 IAA
Address all correspondence and requests for reprints to: Dr. I. C. A. F. Robinson, Division of Neurophysiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom NW7 IAA. E-mail: irobins{at}nimr.mrc.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although the endocrine effects of GHSs are relatively specific for GH release, this specificity is not absolute. Most of the GHSs tested also release small but significant amounts of ACTH and PRL (4, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Earlier studies have suggested that these secretagogues do not act directly on the corticotrophs (21), but release ACTH indirectly, again via a hypothalamic action (22), possibly involving CRH or AVP (16). GHSs also stimulate small but significant amounts of PRL release in human subjects (12, 13, 15, 20, 23, 24), but the mechanisms involved are unknown, largely because the PRL responses are minimal or even absent in conscious experimental animals (14, 25) and thus difficult to study.
We wished to examine the PRL-releasing activity of GHRP-6 in the rat. In addition to normal male and female rats, we chose to study GH-deficient dwarf (dw/dw) rats (26), because these show increased hypothalamic GHS-R expression (9), respond robustly to GHSs (27), and maintain high pituitary PRL stores despite profound GH deficiency (26, 28). Whereas GHRP-6 had little effect on PRL release in normal rats of either sex, more consistent PRL responses were observed in dw/dw females. Furthermore, PRL responses to GHRP-6 were abolished in dw/dw females by ovariectomy and were induced in dw/dw males by treatment with estrogen (E2), which also increased their hypothalamic GHS-R expression. Thus, GHRP-6 does release PRL in the rat, but this response varies markedly with GH status and the gonadal steroid milieu.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Hormone release studies
Rats were anesthetized with sodium pentobarbitone (60 mg/kg, ip;
Sagatal, May & Baker, Dagenham, Essex, UK) and were maintained under
anesthesia throughout the experiments. Jugular catheters were inserted
for iv injections, and manual blood sampling was begun 1 h later,
as previously described (30). TRH (Cambridge Laboratories, Newcastle,
UK), GH-releasing hormone [human GHRH-(1–29)NH2], and
GHRP-6 (Ferring, Malmö, Sweden) were dissolved in saline
containing 0.05% BSA, diluted in heparinized saline, and injected in
100 µl, flushed in with 100 µl saline. Peptide doses and group
sizes are detailed in the text or figure legends. For each peptide
injection, samples of blood were withdrawn before and 5 and 15 min
after injection. A period of 3 h was allowed between injections,
which were given consecutively to the same animals, as detailed in the
figure legends. Blood samples were centrifuged, and the plasma was
separated and frozen for hormone measurements. RIAs for GH, PRL, and
TSH were performed on 25-µl aliquots as previously described (28, 30), using reagents provided by the NIDDK, and the results were
expressed as nanograms per ml in terms of the NIDDK standards: GH RP-2,
PRL RP-3, and TSH RP-3.
In situ hybridization for GHS-R
Rats were killed by an ip overdose of Sagatal, and the brains
were removed, frozen on dry ice, and stored at -70 C. Sections were
cut at -16 C, thaw-mounted onto gelatin- and chrome alum-coated
slides, and stored at -70 C until use. In situ
hybridization for GHS-R was performed on 12-µm coronal brain sections
through the ARC and ventromedial nuclei (VMN) using full-length
35S-labeled GHS-R antisense or sense control riboprobes,
and the autoradiographic images were analyzed densitometrically using
the program Image exactly as previously described (9). Comparisons were
made on sections from all animals exposed concurrently.
Statistical comparisons
Unless otherwise stated, results are expressed as the mean
± SEM. Groups were compared by Student’s t
test or ANOVA followed by Student-Newman-Keuls test for multiple
comparisons, as appropriate. Differences with P < 0.05
were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
shows the PRL and GH
responses in groups of six prepubertal female rats of both normal (AS)
and dw/dw strains given three different doses (0.1–2.5
µg) of GHRP-6. GHRP-6 had no effect on PRL release in normal AS
females, whereas dw/dw females showed a greater PRL response
to GHRP-6, which was significant at the lowest dose tested (Fig. 1A).
This was not seen in GH responses to GHRP-6, which were lower in
dwarves but increased dose dependently in both strains (Fig. 1B).
Similar results were obtained when GHRP-6 was given to groups of adult
female rats (not shown), but no PRL responses to GHRP-6 were observed
in male rats of either strain despite their marked GH responses to
GHRP-6 (see below; Fig. 2).
|
|
Effects of E2 treatment on GH and PRL
release
The effects of E2 treatment on PRL and GH
responses to GHRP-6 and TRH were compared in groups of young male
normal and dw/dw rats (n = 6), and the results are
shown in Fig. 2
. PRL release was completely refractory to GHRP-6 and
TRH in untreated males of either strain (Fig. 2, A and B).
E2 treatment raised baseline PRL levels in all groups. PRL
release in normal males remained unresponsive to GHRP-6 after
E2 treatment, but showed small responses to TRH (Fig. 2A),
whereas dw/dw males treated with E2 showed very
large PRL responses to both GHRP-6 and TRH (Fig. 2B). In the same
samples, GH release was stimulated by GHRP-6 in both normal and
dw/dw rats (Fig. 2, C and D). E2 treatment had
no effect on GHRP-6-induced GH release in normal rats, but reduced the
GH response in dw/dw rats. TRH did not affect GH release in
dw/dw males with or without E2 treatment (Fig. 2D).
Similar results were obtained when the same experiment was repeated in adult rats (n = 6/group). Adult dw/dw rats treated with E2 showed reduced GH responses to GHRP-6, but dramatically increased PRL responses to both GHRP-6 and TRH [from 94.0 ± 32 to 389 ± 67 ng/ml (P < 0.005) and from 48.9 ± 7.5 to 570 ± 48 ng/ml (P < 0.0001)]. E2 treatment did not induce PRL responses to GHRP-6 in adult normal males (from 36.3 ± 2.9 to 38.5 ± 4.8 ng/ml); their PRL responses to TRH increased, but this difference just failed to reach statistical significance (from 21.6 ± 3.9 to 124 ± 48.8; P = 0.06).
Effects of ovariectomy on GH and PRL release
As the PRL responses to GHRP-6 were present in dw/dw
females and could be induced in males by E2, the effects of
GHRP-6 and TRH were also tested in Ovx dw/dw female rats and
compared with those in sham-operated dw/dw controls (Fig. 3). The effects of ovariectomy on PRL
responses were dramatic; basal PRL levels were reduced, and the
responses to both GHRP-6 and TRH were abolished (Fig. 3A). The effects
on GH, measured in the same samples, were in the same direction but
were much less marked. Basal GH levels were the same in all groups, but
ovariectomy significantly reduced the GH responses to GHRP-6 and TRH
compared with those in the sham-operated dw/dw rats (Fig. 3B).
|
). GHRP-6
had no effect on TSH release in any group, whereas TRH increased plasma
TSH in all groups, with higher responses in the normal rats than in the
dw/dw animals regardless of whether they were treated with
E2. The lack of TSH response to GHRP-6 but not TRH, and the
lack of an induction of TSH responses to GHRP-6 after E2
suggest that the effects of GHRP-6 on PRL in dw/dw rats are
not mediated via TRH release.
|
, and the results
of quantitation in VMN and ARC of all animals are shown in Fig. 5B. The
overall hypothalamic distribution of specific GHS-R mRNA expression was
unaffected by E2 treatment, and no new sites of expression
were noted compared with those in sham-treated controls. However, the
abundance of GHS-R mRNA was significantly increased in both ARC and VMN
of E2-treated animals compared with that in untreated
controls. Similar increases in ARC GHS-R expression were observed in 12
female rats given the same E2 treatment for 14 days (not
shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PRL responses to GH secretagogues are also observed in human subjects (12, 13, 15, 18, 20, 23, 24), but there is no information about the mechanisms involved. PRL responses are much less prominent in conscious experimental animals (1, 14, 25) and thus are difficult to study. As GH responses to GHRP-6 are greater in young female rats than in older males (32, 33) and are more reproducible in anesthetized than in conscious rats, we compared the PRL and GH responses to GHRP-6 and TRH in both young and adult male and female animals under anesthesia. PRL responses were also studied in dw/dw rats, as these animals are fully responsive to the hypothalamic effects of GHRP-6 (27) and show increased hypothalamic expression of the newly identified GHS-R (9). Furthermore, unlike other dwarf rodent models, dw/dw rats maintain high PRL production and lactotroph numbers in the face of profound GH deficiency and somatotroph hypoplasia. In our initial description of this dw/dw rat (26), we found pituitary PRL concentrations unchanged although total PRL contents were reduced compared with those in heterozygous animals. Our more extensive studies suggested that PRL concentrations are actually increased in these rats (28).
We show here for the first time that female dw/dw rats release PRL at doses of GHRP-6 that produce no significant PRL release in male normal or dw/dw rats. Furthermore, PRL responses to GHRP-6 are sensitive to E2. They were eliminated in dw/dw females by ovariectomy and were induced in dw/dw males by E2 treatment. This study is also the first to examine PRL responses to TRH in the dw/dw strain, which were enhanced compared with those in normal females of the same strain. As in normal males, PRL release in dw/dw male rats was relatively unresponsive to TRH, but a strong PRL response to both TRH and GHRP-6 was induced in dw/dw males by treating them with E2.
The effectiveness of GHRP-6-induced GH release in rats is affected by the gonadal steroid environment (4, 32, 33). Mallo et al. (34) reported that the GH responses to GHRP-6 were reduced by gonadectomy in adult males, but could be increased by testosterone or E2 treatment. In intact adult males, E2 increased the GH response to GHRP-6 (34), whereas it decreased the response to GHRH. We found no change in GH release after GHRP-6 treatment in normal adult rats and a small diminution in dw/dw rats after E2 treatment. Studies in adult human subjects show no major sex differences in GH responses to GHRP-6 (35), which are similar across the menstrual cycle (36) and are unaltered by E2 treatment in postmenopausal women (37). On the other hand, GH responses to the GHRP-6 analog, hexarelin, are greater in girls than in boys at puberty (38).
In contrast to their effects on GH, the effects of gonadal steroids on PRL responses to GHRP-6 have not been investigated. Although there was little effect of E2 on PRL responses to GHRP-6 in normal rats, the PRL response in dw/dw rats was clearly dependent on E2 and could be differentiated from the effects on GH. Ovariectomy drastically reduced their PRL responses while leaving their GH responses intact, whereas E2 treatment induced a robust PRL response to GHRP-6 in dw/dw males while inhibiting their GH responses to GHRP-6.
How might the PRL-releasing effect of GHRP-6 be mediated? For GH release, GHRP-6 acts in part by releasing GHRH, with which it synergizes (39, 40). However, GHRH is unlikely to mediate GHRP-6-induced PRL release, as GHRH administration produced robust GH release without affecting PRL in either normal or dw/dw strains, and GHRH does not synergize with hexarelin on PRL release as it does for GH (18, 40).
An analogous possibility was that GHRP-6-induced PRL release could be mediated via a stimulation of TRH release, especially as the PRL responses to TRH were affected by E2 treatment and ovariectomy in exactly the same fashion as those to GHRP-6. However, TRH and GHRP-6 do not interact on each other’s receptors (7, 41), no increase in TSH was observed after GHRP-6 administration in rats that showed strong TSH responses to TRH, nor did E2 treatment induce a TSH response to GHRP-6. Although the GHS hexarelin has similar ED50 values for inducing PRL and GH release in man (18), this secretagogue produces a much smaller PRL response than does TRH (42). Furthermore, TSH is either unaffected or decreased by GHS administration in human subjects (43, 44). We thus believe it unlikely that GHRP-6 releases PRL in the rat simply by releasing TRH.
We cannot rule out the possibility that lactotrophs and thyrotrophs in dw/dw rats are differentially sensitive to TRH, or that GHRP-6 might release another factor, such as somatostatin, that could block the effects of TRH on TSH but leave PRL responses unaffected. Again, this is unlikely, as the sensitivity of PRL to somatostatin inhibition in the rat is increased, not decreased, by E2 treatment (45).
GHRP-6 could also stimulate PRL release by inhibiting the output of dopamine (DA) from tuberoinfundibular dopaminergic (TIDA) neurons in the ARC or by functionally antagonizing DA at the pituitary. Enhanced PRL responses in E2-treated animals might then be explained by their increased TIDA DA synthesis and metabolism (46). Although many ARC cells are stimulated by GHSs (10, 47), other cells are inhibited (10), although these may not project to the median eminence (48). Few of the cells that respond to GHRP-6 with increased c-fos expression contain tyrosine hydroxylase (47), although c-fos expression may not be a good marker for revealing cells inhibited by GHRP-6. TIDA cell immunofluorescence is unchanged in dw/dw rats despite their high pituitary PRL stores (49), and GHRP-6-induced PRL release in anesthetized dw/dw females is not affected by pretreatment with DA antagonists (Carmignac, D. F., unpublished data), so an inhibition of TIDA cell activity in dw/dw rats by GHRP-6 is unlikely to explain an acute PRL response.
The simplest explanation of our results is that GHRP-6-induced PRL release in anesthetized rats is a direct pituitary effect, insignificant in males, more pronounced in females or after E2 treatment in males, and greatest in dw/dw rats, which have increased pituitary PRL cells relative to GH cells (49). As the PRL responses to TRH and GHRP-6 were affected by E2 and ovariectomy in the same way, both peptides may act on the same population of E2-sensitive PRL-producing cells, which is prominent in dw/dw rats.
It is well established that E2 increases lactotroph TRH receptor expression, PRL responsiveness to TRH, and PRL synthesis (50, 51). The latter effect would increase the amount of PRL available for release by GHRP-6. E2 can also affect PRL responses to TRH indirectly, as TRH is degraded by a pituitary enzyme activity that is reduced by E2 treatment (52). However, unless this enzyme also degrades GHRP-6, this would not explain the heightened PRL response to this secretagogue after E2 treatment.
Our study cannot distinguish an effect of E2 on lactotrophs (to render their PRL release GHRP-6 responsive), from an effect of E2 on somatomammotrophs (to secrete both GH and PRL in response to GHRP-6 and TRH). We favor the latter hypothesis, because the animals that showed the most pronounced PRL responses to GHRP-6 (female dw/dw rats or male dw/dw rats treated with E2) also showed small GH responses to TRH. Furthermore, the PRL responses to TRH and GHRP-6 were both abolished in Ovx dw/dw females, whereas the GH responses to GHRP-6 (most of which we assume derive from somatotrophs) were unchanged.
Although all pituitary cells exhibiting GHS binding apparently contain some GH (4), not all GH-containing cells respond to GHSs, and subpopulations of GH-containing cells have been described that differ in their responses to GHRP-6 or GHRH (53). Other data also suggest that PRL release induced by GHRP-6 is direct and derives mainly from somatomammotrophs. PRL responses to GHSs can be observed in vitro and are more pronounced in pituitary cells from lactating rats (4), which have an increased proportion of somatomammotrophs (54). Furthermore, E2 treatment increases somatomammotroph cell numbers (45, 54, 55).
The small GH response to TRH we observed in some animals accords with the observation that 7% of rat pituitary cells responding to GHRP-6 also respond to TRH (4). Paradoxical GH responses to TRH are also observed in acromegaly and in critical illness, conditions in which PRL responses to GHSs may also be demonstrated (15, 24, 56), although PRL was not responsive to hexarelin in patients with idiopathic hyperprolactinemia (24).
We also examined the effects of E2 on GHS-R expression because this treatment had opposite effects on GH and PRL responses to GHRP-6. We have previously found it difficult to reveal specific GHS-R expression in the pituitary gland using in situ hybridization (9), whereas GHS-R expression is readily demonstrable in the hypothalamus under the same conditions. In that study (9), a sex difference in GHS-R expression was found in VMN but not in ARC. In the present study, no additional sites of hypothalamic GHS-R expression were revealed after E2 administration, but we show here that the levels of GHS-R mRNA in both ARC and VMN were strongly up-regulated in E2-treated rats.
An increased GH response to GHS in short normal children given priming doses of testosterone or E2 was not seen when a nonaromatizable androgen was used (57), implicating a specific estrogenic effect on the GH response to GHSs, and the researchers speculated that E2 could increase hypothalamic GHS-R expression. Our results provide direct evidence in favor of this and might also explain why GHRP-6 responses are more pronounced in female rats (32, 33). However, in our study, it was the PRL, rather than GH, responses to GHRP-6 that were increased by E2. As GHSs also have other nonendocrine effects (such as stimulation of food intake) that are also sensitive to E2, it is unwise to interpret changes in hypothalamic GHS-R expression induced by E2 solely in terms of effects on the GH axis.
These studies were largely focused on anesthetized female dw/dw rats, simply because they exhibit the most robust PRL responses to GHRP-6. However, PRL responses to GHRP-6 are demonstrable in conscious female rats and also in conscious male dw/dw rats treated with E2 (Thomas, G. B., D. F. Carmignac, and I. C. A. F. Robinson, unpublished observations). Thus, although the PRL responses to GHRP-6 depend heavily on the gender, GH status, and gonadal steroid milieu in the rat, they are not simply a consequence of anesthesia. In human subjects, PRL responses to GHSs are usually observed, but the plasma concentrations generally remain within the normal physiological range (12), so the physiological or clinical significance of this response may be questioned. Nevertheless, most clinical studies with these agents have been performed in male subjects; our results suggest that PRL responses to GH secretagogues in GH-deficient female subjects should be given further attention.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received January 8, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  I. Garcia-Tornadu, M. Rubinstein, B. D Gaylinn, D. Hill, E. Arany, M. J Low, G. Diaz-Torga, and D. Becu-Villalobos GH in the dwarf dopaminergic D2 receptor knockout mouse: somatotrope population, GH release, and responsiveness to GH-releasing factors and somatostatin. J. Endocrinol., September 1, 2006; 190(3): 611 - 619. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Huerta-Ocampo, H. C. Christian, N. M. Thompson, M. M. El-Kasti, and T. Wells The Intermediate Lactotroph: A Morphologically Distinct, Ghrelin-Responsive Pituitary Cell in the Dwarf (dw/dw) Rat Endocrinology, November 1, 2005; 146(11): 5012 - 5023. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Chandrashekar, D. Zaczek, and A. Bartke The Consequences of Altered Somatotropic System on Reproduction Biol Reprod, July 1, 2004; 71(1): 17 - 27. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. van der Lely, M. Tschop, M. L. Heiman, and E. Ghigo Biological, Physiological, Pathophysiological, and Pharmacological Aspects of Ghrelin Endocr. Rev., June 1, 2004; 25(3): 426 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. M. Thompson, D. A. S. Gill, R. Davies, N. Loveridge, P. A. Houston, I. C. A. F. Robinson, and T. Wells Ghrelin and Des-Octanoyl Ghrelin Promote Adipogenesis Directly in Vivo by a Mechanism Independent of the Type 1a Growth Hormone Secretagogue Receptor Endocrinology, January 1, 2004; 145(1): 234 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Hurley, D. V. Birch, M. C. Almond, I. J. Estrada, and C. J. Phelps Reduced Hypothalamic Neuropeptide Y Expression in Growth Hormone- and Prolactin-Deficient Ames and Snell Dwarf Mice Endocrinology, November 1, 2003; 144(11): 4783 - 4789. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Broglio, A. Benso, C. Castiglioni, C. Gottero, F. Prodam, S. Destefanis, C. Gauna, A. J. van der Lely, R. Deghenghi, M. Bo, et al. The Endocrine Response to Ghrelin as a Function of Gender in Humans in Young and Elderly Subjects J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1537 - 1542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Weekers, E. Van Herck, W. Coopmans, M. Michalaki, C. Y. Bowers, J. D. Veldhuis, and G. Van den Berghe A Novel in Vivo Rabbit Model of Hypercatabolic Critical Illness Reveals a Biphasic Neuroendocrine Stress Response Endocrinology, March 1, 2002; 143(3): 764 - 774. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. C. Palyha, S. D. Feighner, C. P. Tan, K. K. McKee, D. L. Hreniuk, Y.-D. Gao, K. D. Schleim, L. Yang, G. J. Morriello, R. Nargund, et al. Ligand Activation Domain of Human Orphan Growth Hormone (GH) Secretagogue Receptor (GHS-R) Conserved from Pufferfish to Humans Mol. Endocrinol., January 1, 2000; 14(1): 160 - 169. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology |
) or
dw/dw (•) rats were given consecutive iv injections
(arrows) of three doses of GHRP-6 (0.1, 0.5, or 2.5
µg) at intervals of 3 h. Blood samples were withdrawn before and
5 and 15 min after each injection and assayed for PRL (A, upper
panel) or GH (B, lower panel). Data shown are
the mean ± SEM. **, P < 0.01,
normal vs. dw/dw.
and •), whereas the other six received sham
implants (
). Blood samples were withdrawn and assayed for
PRL (A and B) or GH (C and D) as before. The data shown are the
mean ± SEM. **, P < 0.01; ***,
P < 0.001 (E2 treated
vs. sham-treated rats).
,
P < 0.05, E2-treated
vs. nontreated normal rats.