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Other Original Studies |
Institute of Endocrinology (T.R., I.S.) and Departments of Neurosurgery (M.H.) and Human Genetics (G.B.), Chaim Sheba Medical Center, Tel-Hashomer, Israel 52621; and Cedars-Sinai Research Institute (S.M.), Los Angeles, California 90048
Address correspondence and requests for reprints to: Ilan Shimon, M.D., Institute of Endocrinology, Chaim Sheba Medical Center, Tel-Hashomer, 52621 Israel. E-mail: i_shimon{at}netvision.net.il
Abstract
The hypothalamic peptide PRL-releasing peptide (PrRP) has recently been cloned and identified as a ligand of an orphan pituitary receptor that stimulates in vitro PRL secretion. PrRP also induces PRL release in rats in vivo, especially in normal cycling females. However, no information on the effects of PrRP in the human is available. To elucidate the role of PrRP in regulating human anterior pituitary hormones, we used human PrRP-31 in primary cultures of human pituitary tissues, including fetal (20–27 weeks gestation) and normal adult pituitaries, as well as PRL- and GH-secreting adenomas. PrRP increased PRL secretion from human fetal pituitary cultures in a dose-dependent manner by up to 35% (maximal effect achieved with 10 nM), whereas TRH was slightly more potent for PRL release. Coincubation with estradiol resulted in enhanced fetal PRL response to PrRP, and GH release was only increased in the presence of estradiol. Although PRL secretion from PRL-cell adenomas was not affected by PrRP, PrRP induced PRL release from cultures of a GH-cell adenoma that cosecreted PRL. PrRP enhanced GH release in several GH-secreting adenomas studied by 25–27%, including GH stimulation in a mixed PRL-GH-cell tumor. These results show for the first time direct in vitro effects of PrRP-31 on human pituitary cells. PrRP is less potent than TRH in releasing PRL from human fetal lactotrophs and is unable to release PRL from PRL-cell adenomas in culture, but stimulated GH from several somatotroph adenomas. Thus, PrRP may participate in regulating GH, in addition to PRL, in the human pituitary.
SPECIFIC HYPOTHALAMIC RELEASING factors regulate each of the anterior pituitary hormones, including GH, gonadotropins, ACTH, and thyroid-stimulating hormone. Until recently, no PRL-releasing peptide was identified, and the major mechanism known to regulate PRL secretion was tonic dopaminergic inhibition (1). Recently, Hinuma et al. (2) identified a novel "orphan" G protein-coupled receptor (hGR3) in the human pituitary, and its putative ligand in the hypothalamus. This novel hypothalamic peptide was found to be a PRL-releasing factor for rat anterior pituitary cells and was, thus, named PRL-releasing peptide (PrRP) (2). The peptide possesses two molecular forms, a 31-amino acid peptide (PrRP-31) and the C-terminal 20-residue peptide (PrRP-20). Their sequences are highly conserved among several species, including bovine, rat, and human (2), suggesting an important role in mammals. Zhang et al. (3) have shown that normal human pituitary glands express messenger RNA (mRNA) for both PrRP and its receptor. They also identified the receptor’s mRNA in all PRL- and GH-secreting adenomas studied using RT-PCR, as well as in nonfunctional pituitary tumors (3). Thus, expression of PrRP-receptor mRNA in human pituitary tissues, in both normal and adenomatous tissues, seems to be ubiquitous. Interestingly, the PrRP was detected in half of these adenomas (3).
Since its identification, contradictory data on PrRP-stimulatory effects have been reported. On one hand, in vivo studies showed that PrRP enhances PRL release in female rats during the estrus stage (4). This effect was also seen in male rats administered supraphysiological PrRP concentrations (5). On the other hand, Jarry et al. (6) could not detect an effect of PrRP in female lactating rats. Hinuma et al. (2) found that PrRP affects in vitro PRL release specifically, with no secretory effect on other pituitary hormones. In contrast, others could not detect a physiological effect of PrRP on PRL release from pituitary cells (7), and in vivo studies showed an effect of this peptide on ACTH secretion (8) and a possible role in regulating LH and FSH release (9).
The effects of PrRP on human anterior pituitary hormone secretion, both in vitro and in vivo, are unknown. In this study, we used primary cultures of human fetal pituitaries as well as PRL- and GH-secreting pituitary adenomas to investigate the regulation of human PRL and GH by PrRP-31. We demonstrate a modest in vitro stimulation of PRL secretion in the human fetal pituitary by PrRP, whereas in secreting pituitary adenomas PRL was not affected and GH release was mildly enhanced.
Materials and Methods
Peptides
Human PRL-releasing peptide (PrRP-31) was purchased from Phoenix Pharmaceuticals, Inc. (Mountain View, CA). TRH, GHRH (1–40), and 17ß-estradiol were all obtained from Sigma (St. Louis, MO).
Human pituitary tissues
Human fetal pituitary tissues of 20–27 weeks gestation (both males and females) were obtained after therapeutic pregnancy terminations. Studies of human fetal pituitaries followed guidelines of the National Advisory Board on Ethics in Reproduction (10). Written informed consent was obtained from pregnant subjects. Specimens of PRL- and GH-secreting pituitary adenomas were obtained at the time of transsphenoidal surgery. Normal human pituitary tissue was found attached to a specimen of nonfunctioning pituitary adenoma after surgical resection and was used for in vitro studies. Both GH and PRL were secreted from cultures of this unique tissue, and GH release was stimulated by GHRH, as expected.
Primary human fetal pituitary and adenoma cell culture
Fetal specimens were harvested from pathologic specimens within
0.5–2 h of the termination procedure. Pituitary adenoma specimens were
collected during transsphenoidal procedures. Fetal pituitary and tumor
specimens were treated similarly. Tissues were washed in low-glucose
DMEM supplemented with 0.3% BSA, 2 mM glutamine,
and antibiotics, then minced and enzymatically dissociated using 0.35%
collagenase and 0.1% hyaluronidase (both from Sigma) for
45–60 min. Cell suspensions were filtered through 80 µM
naylon mash (Millipore Corp., Bedford, MA) and resuspended
in low-glucose DMEM supplemented with 10% FBS, 2 mM
glutamine, and antibiotics. For primary cultures, 
5 x
104 cells were seeded in 48-well tissue culture
plates (Costar, Cambridge, MA) in 0.5 mL medium and
incubated for 72–96 h in a humidified atmosphere of 95% air/5%
CO2, at 37 C. Medium was then changed to
serum-free defined low-glucose DMEM containing 0.2% BSA, 120
nM transferrin, 100 nM hydrocortisone, 0.6
nM triiodothyronine, 5 U/L insulin, 3 nM
glucagon, 50 nM PTH, 2 mM glutamine, 15
nM epidermal growth factor, and antibiotic, and
cells were treated for 4 h with 1–100 nM PrRP with or
without a 16-h preincubation with 17ß-estradiol (10 nM).
A single pituitary (either fetal or adenoma) was divided and plated
into 60–80 wells, depending on the age and size of the specimen. In
each experiment six wells served as controls (treated with vehicle
solution), and groups of six wells were treated with PrRP (as
specified), TRH, estradiol, or GHRH (10 nM for all). Medium
was then collected and stored at -20 C for later hormone
measurements.
Hormone assays
Human PRL levels were measured by immunoradiometric assay and GH by RIA (both obtained from Diagnostic Products, Los Angeles, CA), after appropriate sample dilutions. Because absolute hormonal levels differ from one specimen to the other, both in fetal tissues and adenoma cells, we expressed all our data as percentage of control.
Statistical analysis
Results are expressed as mean ± SD. Data were analyzed by t test and by one-way ANOVA, as appropriate, and P values less than 0.05 were considered significant.
Results
Effects of PrRP on human fetal and adult PRL and GH
To examine the effect of PrRP on PRL secretion from human fetal
pituitary, primary cultures of human fetal pituitaries of 20–27 weeks
gestation were incubated with human PrRP-31, and PRL release was
stimulated in a dose-dependent manner (Fig. 1
). Maximal effect (35% increase; from
3.9 ± 0.5 µg/L at baseline to 5.3 ± 1.2 µg/L,
P < 0.05) was achieved at concentrations of 10
nM, after 4-h incubations, in tissues derived
from both male and female fetuses. However, this stimulatory effect of
PrRP was somehow variable, resulting usually in 25–35% increase in
PRL release. As previous studies showed that PrRP stimulated in
vitro PRL release mainly in pituitaries derived from lactating or
cycling female rats, we studied the effects of PrRP in cultures of
fetal pituitaries preincubated with estradiol (Fig. 2). Pretreatment of fetal pituitary
cultures with estradiol (10 nM) for 16 h,
and then coincubating the cells with both PrRP and estradiol, resulted
in a 33% elevation of PRL levels, whereas estradiol alone had a mild
effect (13%) on PRL release (Fig. 2A). Based on several experiments in
fetal pituitary tissues, TRH was slightly more potent (5–10%) than
PrRP (without estradiol pretreatment) in stimulating PRL secretion from
fetal lactotrophs.
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emphasizing the role of estrogen in modulating both PRL and GH response
to PrRP. PRL and GH secretion from cell cultures of adult human pituitary was not affected by PrRP-31 (data not shown). However, these experiments were performed without estrogen pretreatment.
PrRP effects in PRL-secreting adenomas
We studied three PRL-secreting adenomas obtained during
transsphenoidal procedures from patients with dopamine agonist
resistance, dopamine agonist intolerance, and from a patient with
invasive adenoma and apoplexy. PrRP failed to stimulate in
vitro PRL secretion in all three adenomas studied, even after
overnight preincubation with estradiol. One of these cultured adenomas
(immunostained for both PRL and GH) also secreted GH to the culture
medium. Interestingly, both PrRP and estradiol
(10-nM concentrations) increased GH secretion by
35–41% (Fig. 3), without affecting PRL
release.
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Four tissue specimens obtained from patients with GH-secreting
adenomas were cultured and then incubated with PrRP. In two of these
tumors PrRP (10 nM) stimulated GH secretion to the culture
medium by 25–27% (Fig. 4) compared with
no effect on GH elicited by GHRH. No effect on GH was found in the two
other somatotroph adenomas. However, one of these adenomas secreted PRL
in addition to GH. Preoperative hormonal evaluation revealed elevated
PRL level (80 µg/L) in addition to high-serum GH. PRL release from
this tumor (in vitro baseline, 9.6 µg/L; expressed as
100%) was stimulated by PrRP (100 nM) in the
presence of estradiol (Fig. 5), with no
concomitant GH stimulation (in vitro, 33.3 µg/L).
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This study shows that PrRP stimulates PRL secretion from human fetal pituitary cell cultures. In contrast, the peptide does not release PRL from cultured human lactotroph adenomas, but can regulate GH secretion in a subgroup of human GH-secreting adenomas. Since the identification of PrRP by Hinuma et al. (2) as a specific stimulator of PRL secretion in rat pituitary cell cultures, this is the first report on the hormonal effects of PrRP in human pituitary cells.
In PRL-secreting adenomas PrRP had no effect on PRL secretion. It was previously shown that all normal pituitaries and pituitary adenoma samples studied, including PRL-cell adenomas, express PrRP receptor mRNA (3). However, it is not surprising that cultured prolactinoma samples did not respond to PrRP, because prolactinomas may express very low levels of PrRP receptors, detected only by the sensitive method of RT-PCR. Similarly, GH-secreting adenomas frequently do not respond in vitro to stimulation with GHRH by GH release like the GH response of normal pituitaries, although these tumors specifically express normal GHRH receptors, similarly to normal somatotrophs (11). This phenomenon may also be explained by receptor or postreceptor signal transudation defects [i.e. the PrRP receptor, although expressed, is not functional or the signaling pathway it triggers (cAMP, extracellular signal-regulated kinase, and c-jun-NH2-kinase) (12) is altered]. It may also reflect a constitutive activation of the hormonal secretion mechanism, where the known physiological stimulators (PrRP in this case) would not provide additive releasing effects to an already over-saturated regulating mechanism.
In our studies with human fetal pituitary cultures PrRP alone was
slightly less potent than TRH in stimulating PRL release. However, when
estradiol and PrRP were added together, the effect on PRL release was
additive and similar to the effect obtained by TRH (Fig. 2). This
hormonal effect was specific and consistent in fetal pituitaries
derived from different gestational stages, although the magnitude was
somewhat variable. In their original description of PrRP, Hinuma
et al. (2) found that PrRP-31 was comparable
with TRH in its potency to release PRL from anterior pituitary cells
derived from lactating female rats. However, these pituitary cells, in
contrast to the cell cultures we used, are originally derived from a
hormonal milieu where the pituicytes are already exposed to estradiol,
resembling the cultures used by us pretreated with estradiol. In rats
estrogens modulate expression of Pit-1 in the anterior
pituitary and, thus, stimulate PRL expression (13). This
offers a possible explanation for the additive effect of estrogen and
PrRP on PRL secretion from fetal cultures. Our results in human fetal
pituitary cultures are supported by in vitro
(7) and in vivo studies in rats (4, 5) where the effect of PrRP on PRL release is influenced
considerably by the estrous cycle and sex; thus, female rats,
especially those at proestrus, are more sensitive to PrRP-induced PRL
secretion than male rats. Moreover, in ovariectomized rats treated with
estrogens, a dose-dependent increase of PRL secretion in response to
PrRP was observed (4). Thus, it is suggested that PrRP
plays an important role in the neuroendocrine regulation of PRL
secretion in lactating and cycling female rats. However, our in
vitro reported results are the only data available for PrRP in
humans. Importantly, we did not find differences in the effects of PrRP
on PRL secretion in human fetal female and male cultures. This may be
associated with a recent report on PRL concentrations in human fetal
serum from midpregnancy to term pregnancy, where no difference between
PRL levels in females and males was observed (14).
Here, we show that in the human the secretory effect of PrRP is not PRL
specific and the peptide can modulate GH secretion. Several cultures of
GH-secreting adenomas responded to PrRP-31 by a modest elevation of GH
release to the medium, including one mixed PRL/GH-cell adenoma where
PrRP stimulated GH but not PRL secretion (Fig. 3). Moreover, PrRP
induces GH secretion from fetal cultures in combination with estradiol,
providing a synergistic effect. The mechanism for GH modulation by this
specific PRL-releasing peptide is unclear. Human fetal somatotrophs and
lactotrophs are derived from mammosomatotroph cells, bihormonal
primitive stem cells that secrete both PRL and GH (15).
These pituitary stem cells secrete GH and PRL during the second
trimester of human fetal life and may explain the GH response of human
fetal cultures to PrRP stimulation observed by us. This resembles the
phenomenon of PRL release from cultures of human fetal pituitaries that
respond to GHRH stimulation in parallel with GH (15, 16),
in contrast to adult pituitary PRL that is not affected by GHRH. More
puzzling is the PrRP-mediated GH secretion from GH- secreting
adenomas. Indeed, GH-secreting adenomas express PrRP receptor
(3), which may mediate this GH stimulatory effect through
ligand binding.
PrRP and its receptor were originally identified in the hypothalamus and the pituitary, respectively (2), and these characteristics suggested that PrRP is a novel hypophysiotropic peptide that specifically stimulates PRL production and secretion. Recently, immunocytochemical studies of rat brain located PrRP in several hypothalamic and thalamic nuclei (17, 18), but not in the external region of the median eminence, which is the release site of several hypophysiotropic hormones, including GHRH and somatostatin, into the hypophyseal portal system. In situ hybridization histochemistry performed for PrRP mRNA (19, 20) supports these results and suggests a novel route of the hypophysiotropic action of PrRP, or an alternative physiological function for PrRP in the central nervous system other than the classical hypophysiotropic role. Different groups reported recently that intracerebroventricular administration of PrRP-31 to the rat brain resulted in LH and FSH stimulation (9) and CRH-mediated increase in ACTH (8). In addition, PrRP released LH-releasing hormone from hypothalamic explants (9), and also galanin and vasoactive intestinal peptide (9), both known stimulators of PRL secretion (21). Thus, the PRL-releasing potential of PrRP may be mediated through a direct effect on pituitary lactotrophs, but also via an indirect hypothalamic action.
Our results support the hypothesis that in the human pituitary PrRP has a direct stimulatory action on PRL release. However, this effect on PRL regulation is probably less important compared with the tonic inhibition that dopamine exerts on PRL secretion. PrRP may be more significant during pregnancy and lactation, whereas in males and postmenopausal females it may lose its PRL-releasing ability.
Footnotes
1 Supported by the Israel Ministry of Health, the Chief Scientist
(4529; to I.S.), and the Doris Factor Molecular Endocrinology
Laboratory. 
Received November 28, 2000.
Revised February 5, 2001.
Accepted February 14, 2001.
References
-adrenergic and opioidergic mechanism. Brain Res. 507:321–324.[CrossRef][Medline]
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) and GH (B,
) secretion from human fetal pituitary cell culture (21 weeks
gestation). Each bar represents mean (±SD)
of hormone secretion in six wells. C, Control. *,
P < 0.05 vs. untreated wells.
