0013-7227/98/$03.00/0
Endocrinology
Copyright © 1998 by The Endocrine Society
Vol. 139, No. 8
Printed in U.S.A.
Effects of Growth Hormone Secretagogues on Prolactin
Release in Anesthetized Dwarf (dw/dw) Rats*
DANIELLE F. CARMIGNAC, PAMELA A. BENNETT,
AND
IAIN C. A. F. ROBINSON
Division of Neurophysiology, National Institute for Medical Research, The Ridgeway, London, United
Kingdom NW7 IAA
ABSTRACT
In addition to stimulating GH release, GH secretagogues such as
GH-releasing peptide-6 (GHRP-6) stimulate small amounts of ACTH
and PRL release. Although the effects on ACTH have recently been
studied, there is little information about the effects of GHRP-6 on
PRL. We have now studied GHRP-6-induced GH and PRL release and
their regulation by estrogen (E2) in anesthetized male and female rats
and in GH-deficient dwarf (dw/dw) rats that maintain high pituitary
PRL stores and show elevated hypothalamic GH secretagogue receptor expression. Whereas GHRP-6 (0.1–2.5 mg, iv) did not induce PRL
release in normal male or female rats, significant PRL responses were
observed in dw/dw females. These responses were abolished by ovariectomy and could be strongly induced in male dw/dw rats by E2
treatment. These effects could be dissociated from GHRP-6-induced
G
GH release in the same animals, but not from PRL release induced
by TRH, which was also abolished by ovariectomy and induced in
males by E2 treatment. However, the effects of GHRP-6 on PRL were
unlikely to be mediated by TRH because in the same animals, TSH
levels were unaffected by GHRP-6 whereas they were increased by
TRH. The increased PRL response could reflect an increase in GH
secretagogue receptor expression that was observed in the arcuate
and ventromedial nuclei of E2-treated rats. Our results suggest that
the minimal PRL-releasing activity of GHRP-6 in normal rats becomes prominent in GH-deficient female dw/dw rats and is probably
exerted directly at the pituitary; these GHRP-6 actions may be modulated by E2 at both hypothalamic and pituitary sites. (Endocrinology
139: 3590 –3596, 1998)
H secretagogues (GHSs), developed from the prototype
hexapeptide GH-releasing peptide-6 (GHRP-6), are
potent releasers of GH in animals and man (1– 4) and act both
at the pituitary and in the hypothalamus via a novel receptor
that has recently been cloned and sequenced (5–7). The distribution (8) and regulation by GH (9) of this GHS receptor
(GHS-R) in the rat hypothalamic arcuate nucleus (ARC) support earlier evidence suggesting that GHSs release GH in
part via the activation of ARC neurons (10). However, GHS-R
expression is seen in several other central nervous system
sites not obviously involved in the regulation of GH (8),
suggesting that GHSs may have other functions.
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, 11–20). 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.
Received January 8, 1998.
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: [email protected].
* This work was supported by the United Kingdom Medical Research
Council.
Hormone release studies
Materials and Methods
Animals and treatments
All experiments were carried out under our institutional and national
ethical guidelines. Normal and dwarf (dw/dw) rats (26) from the same
(NIMR:AS) strain were housed under controlled conditions (22 C; 12-h
light, 12-h dark cycle) with food and water ad libitum. Animals were
studied at 35– 40 days of age or at 10 weeks of age in different experiments. Some dw/dw rats were ovariectomized (Ovx) under halothane
anesthesia at 8 weeks of age and studied 2 weeks later, whereas controls
were sham operated (29). Other groups of normal or dw/dw male rats
were implanted sc with controlled release pellets (Innovative Research
of America, Toledo, OH) delivering estradiol (E2) at 25 mg/day for 2
weeks, as previously described (29). Controls for these experiments
underwent a sham implant procedure.
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, New-
3590
PRL RELEASE BY GHRP-6
3591
castle, 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 ml, flushed
in with 100 ml 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-ml 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 270 C. Sections were cut at
216 C, thaw-mounted onto gelatin- and chrome alum-coated slides, and
stored at 270 C until use. In situ hybridization for GHS-R was performed
on 12-mm 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 6 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
Figure 1 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 mg) 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).
In another experiment, GH and PRL responses to GHRH
were examined in groups (n 5 5– 6) of anesthetized normal
and dw/dw prepubertal female rats. GHRH (100 ng, iv) stimulated a large release of GH in both strains (from 7.1 6 1.1
to 345 6 26 ng/ml and from 9.1 6 1.7 to 57 6 7.7 ng/ml; P ,
0.005 in AS vs. dw/dw animals, respectively) without affecting
PRL release in either strain (from 2.3 6 0.6 to 1.5 6 0.5, and
from 2.1 6 0.4 to 2.6 6 0.5 ng/ml PRL; not significant). Thus,
the PRL response to GHRP-6 is not simply a nonspecific
response accompanying a large GH release in dw/dw rats.
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 5 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
FIG. 1. Effects of GHRP-6 on PRL and GH release in anesthetized
female rats. Groups of six prepubertal normal (M) or dw/dw (F) rats
were given consecutive iv injections (arrows) of three doses of GHRP-6
(0.1, 0.5, or 2.5 mg) 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 6 SEM.
**, P , 0.01, normal vs. dw/dw.
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 5 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 6 32 to 389 6 67 ng/ml (P , 0.005) and from 48.9 6
7.5 to 570 6 48 ng/ml (P , 0.0001)]. E2 treatment did not induce
PRL responses to GHRP-6 in adult normal males (from 36.3 6
2.9 to 38.5 6 4.8 ng/ml); their PRL responses to TRH increased,
but this difference just failed to reach statistical significance
(from 21.6 6 3.9 to 124 6 48.8; P 5 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
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PRL RELEASE BY GHRP-6
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Vol 139 • No 8
FIG. 2. The effects of E2 treatment on PRL and GH responses to GHRP-6 and TRH. Groups (n 5 12) of young normal (left panels) or dwarf
(right panels) male rats were anesthetized and given iv injections of GHRP-6 (0.5 mg) followed 3 h later by TRH (1 mg). Six animals from each
strain received E2 treatment from sc pellets implanted 14 days previously (f and F), whereas the other six received sham implants (M and
E). Blood samples were withdrawn and assayed for PRL (A and B) or GH (C and D) as before. The data shown are the mean 6 SEM. **, P ,
0.01; ***, P , 0.001 (E2 treated vs. sham-treated 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).
Effects of GHRP-6 on TSH release
An obvious candidate mechanism for PRL release by
GHRP-6 is a stimulation of hypothalamic TRH release. As
this would also be expected to stimulate TSH, plasma TSH
responses to GHRP-6 and TRH were also measured in young
normal and dw/dw males, with and without E2 treatment (Fig.
4). 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.
Effects of E2 treatment on hypothalamic GHS-R expression
As the effects of GHRP-6 on PRL release were increased by
E2 treatment, an additional group of eight adult normal male
rats was treated with E2 (25 mg/day for 14 days), and their
hypothalamic GHS-R messenger RNA (mRNA) expression
was evaluated by in situ hybridization. Individual examples
are illustrated in Fig. 5A, 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 shamtreated controls. However, the abundance of GHS-R mRNA
was significantly increased in both ARC and VMN of E2treated animals compared with that in untreated controls.
Similar increases in ARC GHS-R expression were observed
PRL RELEASE BY GHRP-6
FIG. 3. Effects of ovariectomy on PRL and GH responses to GHRP-6
and TRH in female dw/dw rats. Groups of six sham-operated (F) and
five Ovx (E) dw/dw female rats were anesthetized and given iv injections of GHRP-6 (0.5 mg) followed 3 h later by TRH (1 mg) as before.
Blood samples were withdrawn and assayed for PRL (A) or GH (B).
The data shown are the mean 6 SEM. *, P , 0.05; **, P , 0.01; ***,
P , 0.001 (Ovx vs. sham-operated controls).
in 12 female rats given the same E2 treatment for 14 days (not
shown).
Discussion
Although early studies with GHRP-6 showed that this
secretagogue was relatively specific for GH release in rats,
subsequent investigations in humans revealed slight but significant stimulation of ACTH and PRL by a variety of GH
secretagogues (see Refs. 4 and 35 for review). The mechanisms involved in GHS-stimulated GH release in vivo are
complex, but are much better understood than the mechanisms mediating other endocrine effects of these compounds.
We and others have recently investigated ACTH stimulation
by GHSs and have suggested that, as for GH release, the
major site of action is hypothalamic (22, 31), probably involving endogenous ACTH secretagogues (16), although this
may differ between species (19).
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
3593
FIG. 4. The effects of E2 treatment on TSH responses to GHRP-6 and
TRH in normal and dw/dw male rats (same experiment as that in Fig.
2). Blood samples were assayed for TSH after iv administration of
GHRP-6 (0.5 mg) followed 3 h later by TRH (1 mg). Groups (n 5 12)
were either normal (M and f) or dw/dw (E and F) young male rats;
six animals in each group (f and F) had received E2 treatment for 14
days, and the others (M and E) were given sham implants. The data
shown are the mean 6 SEM. **, P , 0.01, AS vs. dw/dw; †, P , 0.05,
E2-treated vs. nontreated normal rats.
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
3594
PRL RELEASE BY GHRP-6
FIG. 5. Effects of E2 treatment on hypothalamic GHS-R expression in
normal male rats. Groups of eight adult male normal rats were given
E2 (25 mg/day for 14 days), and their hypothalamic GHS-R mRNA
expression was evaluated by in situ hybridization and compared with
that in untreated male rats. The upper panel (A) shows individual
sections from control and E2-treated animals, showing expression in
ARC and VMN. The lower panel (B) shows the results of quantification of similar sections from all animals in the group. The data shown
are the mean 6 SEM. **, P , 0.01; ***, P , 0.001 (E2-treated vs.
untreated controls).
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
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Vol 139 • No 8
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-6induced 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 pitu-
PRL RELEASE BY GHRP-6
itary 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.
3595
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
We are grateful to Dave Flavell for the neuropeptide Y clone, Andrew
Howard, and Roy Smith at Merck Research Laboratories for the rat
GHS-R complementary DNA, and Dr. Jerzy Trojnar, Ferring, for the GH
secretagogues. The provision of RIA reagents for GH, PRL, and TSH by
the NIDDK is also gratefully acknowledged.
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