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Original Studies |
Department of Medicine, Cedars-Sinai Research Institute, University of California School of Medicine, Los Angeles, California 90048
Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, B-131, Los Angeles, California 90048. E-mail: Melmed{at}CSMC.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo administration of both GHRH and GHRP-6 to normal male subjects results in a powerful synergistic GH discharge (12). However, GHRP probably exerts its main effect at the hypothalamic level, as its GH stimulation requires intact hypothalamo-pituitary function (13). Moreover, functional GHRH receptors are required for GHRP action, as exemplified by the GHRH receptor mutation (lit/lit mouse) impeding the expected in vitro and in vivo responses to GHRP (14). In addition, cAMP increases before GH induction by GHRH, whereas the action of GHRP is probably mediated via protein kinase C and not cAMP (15). However, combinations of GHRH and GHRP in vitro do, in fact, increase cAMP levels synergistically, and both signaling pathways are associated with an increase in intracellular calcium within somatotrophs (14). Thus, the hypothalamus is a major target for GHRP in vivo in addition to the direct effect of these peptides on the pituitary.
In vivo the actions of the GHRPs have been systematically studied in normal subjects (12, 16, 17) and in short-statured children (17, 18), whereas all in vitro studies have employed rodent (19, 20), bovine (21), or ovine pituitary cells (22). The in vitro effects of GHRPs on normal human pituitary cells have not yet been reported. In this study we thus assessed direct effects of GHRPs on hormone regulation in primary cultures derived from human fetal pituitary cells and also studied expression of the GHRP receptor in these cells.
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Materials and Methods |
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GHRP-6 (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) and human GHRH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) amide were obtained from Peninsula Laboratories (Belmont, CA). GHRP-2 (D-Ala-D-bNal-Ala-Trp-D-Phe-Lys-NH2) was provided by Dr. Cyril Y. Bowers (Tulane University Medical Center, New Orleans, LA). Human recombinant IGF-I was purchased from R&D Systems (Minneapolis, MN). Somatostatin analog (BIM-23190) (23) was obtained from Biomeasure (Milford, MA).
Human fetal pituitary tissue
Human fetal pituitary glands (18–31 weeks gestation) were obtained from an independent facility, with no direct or indirect involvement of our investigators in the third party pregnancy termination referral. Studies of human tissues followed the guidelines of the National Advisory Board on Ethics in Reproduction (24), and written informed consent was obtained for anonymous distribution of aseptic tissue specimens.
Human GHRP receptor expression
Normal adult human pituitary glands (obtained from Zion Diagnostics, New York, NY) and fetal pituitaries (18 and 31-week gestation) were harvested and kept at -70 C for ribonucleic acid (RNA) extraction. Tissues were homogenized, and total RNA was extracted using TRIzol (Life Technologies, Grand Island, NY). Reverse transcriptase (RT) followed by PCR amplification to detect human GHRP receptor was performed as previously described (23). Briefly, 1 µg of each RNA sample was treated with deoxyribonuclease 1 (amplification grade; Life Technologies) to eliminate contaminating genomic DNA. RNA then was used in a 20-µL RT reaction containing oligo(deoxythymidine)16 as a primer and SuperScript II (Life Technologies). RT incubation was performed for 50 min at 42 C. Samples were also incubated without RT enzyme for negative controls. Aliquots (1 µL) from the generated complementary DNA and the negative control reactions were used for subsequent PCR amplification in the presence of 1.5 mmol/L MgCl2 and 5 U Taq DNA polymerase (Life Technologies). Amplifications were carried out for 40 cycles, with an initial denaturation step at 95 C for 5 min and a final 7-min extension step at 72 C. Each cycle consisted of denaturation at 94 C, annealing at 58 C, and elongation at 72 C; each step lasted 1 min. The following primer sets were used: 5'-TTCTGTCTCACGGTCCTCTACAGT (nucleotides 676–699) and 3'-CCAGAAGTCTGAACACTGCCAC [nucleotides 994-1015; GenBank accession no. U60179 for the human GHRP receptor type 1a messenger RNA (mRNA)]. The PCR product thus generated was 340 bp, and it was digested by Sau3AI (Life Technologies) and visualized with ethidium bromide after electrophoresis on 2% agarose gel.
Primary fetal pituitary cell culture
Specimens were harvested within 1–2 h, and pituitary tissues were washed in low glucose DMEM supplemented with 0.3% BSA, 2 mmol/L glutamine, and penicillin/streptomycin, then minced and enzymatically dissociated using 0.35% collagenase and 0.1% hyaluronidase (both from Sigma Chemical Co., St. Louis, MO) for 45–60 min. Cell suspensions were filtered and resuspended in low glucose DMEM supplemented with 10% FBS, 2 mmol/L glutamine, and antibiotics. For primary cultures, approximately 5 x 104 cells were seeded in 48-well tissue culture plates (Costar, Cambridge, MA) in 0.5 mL medium. A single pituitary was divided and plated into 60–80 wells, depending upon the age and size of the specimen, 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 nmol/L transferrin, 100 nmol/L hydrocortisone, 0.6 nmol/L T3, 5 U/L insulin, 3 nmol/L glucagon, 50 nmol/L PTH, 2 mmol/L glutamine, 15 nmol/L epidermal growth factor, and antibiotics, and cells were treated for 2 h with GHRP-6 (or GHRP-2; 100 nmol/L), GHRH (10 nmol/L), and GHRP with GHRH (6–8 wells for each treatment group, including vehicle-treated control wells). Other doses of GHRP (1–1000 nmol/L) and GHRH (1 nmol/L) and different incubation intervals (30 min and up to 6 h) were also used. In addition, GHRP-6 was used to treat cells in the presence of either IGF-I or somatostatin analog. After treatment, medium was collected and stored at -20 for later hormone measurements.
Hormone assays
For human GH measurements we used a RIA kit from Diagnostics Products Corp. (Los Angeles, CA) after appropriate dilutions (1:3 to 1:10) of conditioned medium. Human PRL was measured by immunoradiometric assay, and ACTH was measured by RIA (both purchased from Diagnostics Products Corp.).
Statistical analysis
Results are expressed as the mean ± SEM. Differences were assessed by the unpaired t test or one-way ANOVA where appropriate. For both statistical tests, P < 0.05 was considered significant.
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Results |
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RNA extracted from adult human pituitary and fetal pituitaries of
18 and 31 weeks gestation was subjected to RT followed by PCR
amplification. PCR reaction revealed the presence of human GHRP
receptor isoform 1a (340-bp PCR product) in both adult and fetal
pituitaries (Fig. 1
). The specificity of
the expressed bands was confirmed by incubation with Sau3A1
restriction enzyme, appropriately digesting the 340-bp bands
representing GHRP receptor to the expected 221- and 119-bp restriction
products (Fig. 1). All sample products were negative when reactions
without RT were amplified by PCR, thus confirming that the positive PCR
products represent the presence of pituitary GHRP receptor mRNA
transcripts.
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To determine the effect of GHRP on GH secretion from normal human
pituitary cells, primary cultures of human fetal pituitary cells
(20–25 weeks gestation) were incubated for 2 h with GHRP-6 (100
nmol/L), human GHRH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) (10 nmol/L), and GHRP-6 together with GHRH.
GHRP-6 alone usually induced GH secretion by 40–80%
(P < 0.001), GHRH stimulated GH release by 60–120%
(P < 0.001), and GHRP together with GHRH had no more
than an additive effect, inducing GH from fetal somatotrophs up to
2.8-fold (P < 0.0005; Fig. 2). Even when lower doses of GHRP (10
nmol/L) and GHRH (1 nmol/L) were combined to treat human pituitary
cultures, only additive effects on GH release were demonstrated.
Consistently, the in vitro stimulatory effect of GHRP-6 on
human GH was only 65% of the GHRH effect. Interestingly, PRL secretion
was not affected by GHRP, whereas GHRH modestly stimulated PRL
secretion from cells derived from 24-week gestation pituitary tissue
(P < 0.05; Fig. 2). In addition, ACTH levels were not
altered after incubations with GHRP, but were mildly suppressed by GHRH
(P < 0.05; Fig. 2).
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). The stimulation of GH secretion in
these fetal pituitary cultures by either GHRP-6 or GHRH was time
dependent, with a maximal effect seen after 2 h (Fig. 4). Shorter incubation intervals of
1 h (Fig. 4) and 30 min (data not shown) resulted in decreased
secretagogue induction of GH, especially by GHRH.
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In vitro and in vivo GH expression and
secretion are regulated by IGF-I and somatostatin in addition to GHRH.
We studied the effects of these peptides, when combined with GHRP, on
human fetal pituitary GH secretion. Both IGF-I (10 nmol/L; 4-h
incubation) and somatostatin analog (10 nmol/L; 2-h incubation)
suppressed GH release (Fig. 5;
P < 0.05), as expected. Addition of GHRP (100 nmol/L)
to pituitary cell cultures cotreated with either IGF-I or somatostatin
enhanced GH (Fig. 5; P < 0.0001 for IGF-I and
P < 0.05 for somatostatin) over hormone levels
measured in cultures with no added GHRP.
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There was no significant difference between GHRP-6 and GHRP-2 (both at 100 nmol/L) in the stimulation of GH from human fetal pituitaries in cultures after 2-h treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For our studies, we used primary cultures of human fetal pituitaries at 18–31 weeks gestation. At this age, all hormone-producing cells, including somatotrophs and corticotrophs, are well differentiated and functional (25, 26). GH immunoreactivity is detected in differentiated human fetal somatotrophs by 8 weeks (25). We have previously shown expression of human GHRH, IGF-I, and somatostatin receptors, respectively, in fetal cells derived from second trimester pituitaries (23). Human fetal pituitary cells are responsive to hypothalamic releasing and inhibitory hormones, including GHRH (26, 27), and somatostatin (23, 26, 27) as early as 10–14 weeks gestation. In the present study we show the expression and functional maturity of the GHRP receptor, another important element in pituitary GH regulation, as early as 18 weeks gestation. The PCR primers used for human GHRP receptor expression studies were designed to identify the biologically active 366-amino acid type 1a GHRP receptor isoform that contains 7 transmembrane domains, rather than the shorter nonactive 289-amino acid 1b isoform containing only 5 transmembrane domains (9). Thus, the complex regulation of human GH expression and secretion appears functional early in human gestation.
We have used a unique model of human pituitary cells in primary culture to show, for the first time, direct effects of GHRP on human pituitary GH secretion. It has been clearly demonstrated that GHRP synergizes with GHRH in stimulating in vivo GH release (12, 28). However, whether the synergy occurs primarily in the hypothalamus or at the level of the pituitary is still controversial. Studies using primary cultures of rat (29, 30), bovine (21), and ovine pituitary cells (22) showed that GHRP was only additive with GHRH to induce GH release from cultured cells. In contrast, others reported that GHRP-6 synergizes with GHRH in rat pituitary cell cultures (31). We now show in human fetal pituitary cultures only an additive effect of GHRP-6 with GHRH on GH secretion. These observations support the idea that in man, the hypothalamic level of the GH axis is responsible for the synergistic interaction between these two important modulators of GH. Moreover, as in many previous reports using nonhuman pituitary cultures (21, 29), the in vitro effects we demonstrate in human fetal pituitaries are much less prominent than those shown in vivo (12, 14). Thus, this differential in vivo and in vitro stimulation may be explained by the induction of endogenous hypothalamic GHRH or another unknown factor (U factor) by GHRP. In sheep, hexarelin, a GHRP analog, enhanced GHRH levels in hypophyseal portal blood (32), supporting this hypothesis. In rats, GHRH antiserum attenuated the GHRP-induced GH release (33). However, studies in humans do not support the role of hypothalamic GHRH in GH release induced by GHRP (12, 14).
Remarkably, the in vitro effect of GHRP (10 and 100 nmol/L) in human fetal pituitary cultures was less pronounced than that of GHRH (10 nmol/L). This is in contrast to the GH levels achieved after GHRP administration in vivo, which are higher than those seen after maximal doses of GHRH (13, 28). In addition, each of these peptides has relatively moderate stimulatory effects on GH (usually <2-fold) compared with their actions in adult rat somatotrophs, where GH levels may be enhanced up to 4-fold (19, 20). Interestingly, for different classes of peptide GHRPs, in vivo and in vitro potencies in rats may diverge widely; some are more potent in vivo, whereas others are more effective in vitro (19). This may result from different structural characteristics required for activation of the pituitary and hypothalamic GHRP receptor, although the same receptor has recently been cloned in both tissues (9). In addition, the maximal effect of GHRP-6 (and GHRH) on GH was only seen after 2 h. This contrasts with other studies showing maximal in vitro (19, 20) and in vivo GH stimulatory effects occurring after 15–30 min (6, 13, 18). This different time course of GHRP action observed in the human fetal pituitary cannot be explained by the method used for primary pituitary cell cultures, which does not differ from that used in other reports on rodents (19, 20). More modest effects of GHRP and GHRH on GH release may be operative in human fetal somatotrophs, thus confirming previous results showing that during the second trimester of fetal life there is a gradual increase in fetal somatotroph activity (27).
All classes of GHRPs (34, 35), including nonpeptidyl secretagogues (10), release small, but significant, amounts of ACTH and cortisol when administered in vivo. The effect on ACTH is probably not mediated by direct stimulation of pituitary corticotrophs, but reflects other hypothalamic actions of GHRPs. Our study confirms that GHRP-6 does not induce ACTH when incubated with human corticotrophs in vitro. In contrast, we show that GHRH modestly, but significantly, suppresses human ACTH secretion from fetal pituitary cell cultures. Interestingly, PRL levels were mildly enhanced by GHRH, but not by GHRP. This differential effect of the two peptides on PRL secretion, which before 24 weeks gestation is derived solely from bihormonal primitive mammosomatotroph stem cells secreting both GH and PRL, is important and may suggest that the GHRP receptor, unlike the GHRH receptor, is not completely active before human somatotroph maturation and differentiation occur. Alternatively, these two receptors may mediate hormone release via different intracellular signaling cascades.
The results shown here indicate that both GHRH and GHRP integrate with known physiological suppressors of GH, IGF-I and somatostatin, in regulating GH secretion from human somatotrophs. Thus, by simulating an endogenous inhibitory tone on in vitro pituitary GH expression, we show that GHRP participates in the complex mechanism responsible for determining intrauterine circulating fetal GH levels.
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Acknowledgments |
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Footnotes |
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Received July 17, 1997.
Accepted September 18, 1997.
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