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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-2148
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 6 2257-2263
Copyright © 2006 by The Endocrine Society

Cortistatin Inhibits Growth Hormone Release from Human Fetal and Adenoma Pituitary Cells and Prolactin Secretion from Cultured Prolactinomas

Hadara Rubinfeld, Moshe Hadani, Gad Barkai, John E. Taylor, Michael D. Culler and Ilan Shimon

Institute of Endocrinology and Metabolism and Felsenstein Medical Research Center (H.R., I.S.), Rabin Medical Center, Petach Tikva 49100, Israel; Departments of Neurosurgery (M.H.) and Human Genetics (G.B.), Chaim Sheba Medical Center, Tel Hashomer 52621, Israel; Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; and IPSEN Group (J.E.T., M.D.C.), Milford, Massachusetts 01757

Address all correspondence and requests for reprints to: Ilan Shimon, M.D., Institute of Endocrinology and Metabolism, Rabin Medical Center, Beilinson Campus, Petach Tikva 49100, Israel. E-mail: ilanshi{at}clalit.org.il.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Cortistatin (CST) is a neuropeptide that shares high homology with somatostatin and binds with high affinity to all somatostatin receptor (SSTR) subtypes. Many of its endocrine and biological activities overlap with those of somatostatin.

Objective/Design: The objective of the study was to assess the direct in vitro effects of CST on human pituitary hormone secretion.

Setting: This study was performed in the endocrine laboratory of a tertiary academic medical center.

Materials: Primary cell cultures of human fetal (21–25 wk gestation) pituitary tissues and cultured hormone-secreting adenoma cells were used in this study.

Interventions: Cell cultures were incubated with CST-14 or CST-17, somatostatin, GHRH, SSTR analogs, and ghrelin analogs, and hormone secretion was analyzed.

Outcome Measures: GH and prolactin (PRL) medium concentrations were tested by hormone assay, and SSTR mRNA was tested by RT-PCR.

Results: CST-14 (10 nM) inhibited GH secretion by up to 65% in all fetal pituitary specimens after 4-h incubation (P < 0.05). CST-14 or CST-17 (10 nM) inhibited basal GH secretion in six of the 13 GH-cell adenomas and two of the three GH-PRL mixed adenomas. CST-17 (100 nM) suppressed the GH response to GHRH and ghrelin analog (10 nM each) by 30–50% in adenomas (P < 0.05). Three PRL-adenomas treated with CST-17 (10 nM) showed a 20–40% inhibition of PRL release (P < 0.05), whereas in three others no suppression or mild response was achieved at this concentration. A comparable inhibition of PRL secretion was obtained with SSTR5-selective analog but significantly less with SSTR2-preferential compounds. RT-PCR revealed the expression of both SSTR2 and SSTR5 in all GH-cell and mixed adenomas studied and all PRL-secreting adenomas studied, except for two of the CST-resistant prolactinomas, in which SSTR5 was absent.

Conclusions: This is the first report of in vitro CST suppression of human GH and PRL in cultured pituitary tissues. The regulation of PRL release from cultured adenomas appears to be primarily mediated by SSTR5.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
GH RELEASE FROM the pituitary is tonically suppressed by the hypothalamic hormone somatostatin, which acts through ligand binding to a family of five distinct G protein-coupled somatostatin receptors (SSTR-1–5) (1). Cortistatin (CST) is a neuropeptide with high homology to somatostatin. It shares many pharmacological and functional properties with somatostatin (2), although it is encoded by a different gene (2, 3). Prepro-CST mRNA is expressed mainly in the brain cortex and hippocampus as well as the adenohypophysis (4, 5) but also in peripheral tissues including kidney, stomach, testes, and the immune system (3, 4, 5). It is produced by the cleavage of its precursor prepro-CST into two mature products, CST-14 and CST-29 in rats (2, 3, 6) and CST-17 and CST-29 in humans (3, 7). CST-14 binds to SSTRs on GH4 pituitary cells and inhibits the accumulation of cAMP (2). Likewise, the human homolog, CST-17, binds to SSTRs overexpressed in Chinese hamster ovary cells and inhibits forskolin-stimulated cAMP production (7). In stably transfected cells, none of the SSTRs showed marked selectivity for CST or somatostatin (8). Unlike somatostatin, however, CST binds the GH secretagogue (GHS) receptor (GHS-R) of the endogenous ligand, ghrelin (9). Furthermore, an orphan G protein-coupled receptor, MrgX2, was found to be specific for CST but unable to bind somatostatin (10). These unique characteristics may explain why not all CST actions overlap with those of somatostatin, including the induction of slow-wave sleep and the reduction of locomotor activity and the possible role of CST in the human immune system (11, 12, 13, 14).

Several in vivo studies support the notion that CST is a potential GH suppressor: rat CST effectively inhibited GH release (15), and both rat CST-14 and human CST-17 exerted the same endocrine effects of somatostatin-14 (16, 17), namely inhibition of basal GH secretion as well as GH response to GHRH and ghrelin. None of these peptides affected basal prolactin (PRL) or ACTH release.

The aim of the present study was to investigate the direct effects of CST on human GH and PRL secretion using dispersed human fetal pituitaries and cultured hormone-secreting pituitary adenomas. We also used receptor-specific analogs of SSTRs to identify the mechanisms involved in hormone regulation by CST.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Peptides

Somatostatin-14, octreotide, and GHRH were purchased from Sigma Chemicals Co. (St. Louis, MO). Cortistatin-14 [rat; IC50 of 0.1–0.3 nM for the human SSTR2–5 (7)] was first available and purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA) for the initial experiments, and cortistatin-17 [human; IC50 of 0.4–0.6 nM (7)] was available from Bachem AG (Bubendorf, Switzerland) only later and thus used for later experiments. BIM-23120 and BIM-23206, somatostatin analogs selective for SSTR2 and SSTR5, respectively (18), and BIM-28152, selective analog for ghrelin (19), were obtained from IPSEN Group (Milford, MA). The specific binding affinities of these compounds (BIM-23120 and BIM-23206) for the different human SSTRs or for GHS-R1a (BIM-28152) were determined by radioligand membrane receptor binding assays, as previously described (20).

Human pituitary tissues

Human pituitary tissues from male and female fetuses of 21–25 wk gestation were obtained within 0.5–2 h of therapeutic pregnancy terminations from pathological specimens. The studies of human fetal pituitaries were performed in accordance with the guidelines of the National Advisory Board on Ethics in Reproduction (21), and written informed consent was obtained from the pregnant subjects. Specimens of pituitary adenoma (GH, PRL, and mixed GH-PRL tumors) were obtained during transsphenoidal surgical resections, after informed consent was provided by the patients. The clinical characteristics of the patients are presented in Table 1Go. All tissues were placed in culture medium for cell culture studies. Adenomatous pituitary tissues were also snap frozen for RNA assays.


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  		TABLE 1. Clinical characteristics of patients with secreting pituitary adenomas

 
Human fetal pituitary and pituitary adenoma primary cell cultures

The fetal pituitary and tumor specimens were treated similarly. The specimens were washed in low-glucose DMEM supplemented with 0.3% BSA, 2 mM glutamine, and antibiotics and 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 nylon mesh (Millipore, Bedford, MA) and resuspended in low-glucose DMEM supplemented with 10% fetal bovine serum, 2 mM 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 and incubated for 48–72 h in a humidified atmosphere of 95% air-5% CO2 at 37 C. The medium was then changed to serum-free defined (SFD) low-glucose DMEM containing 0.2% BSA, 120 nM transferrin, 100 nM hydrocortisone, 0.6 nM T3, 5 U/liter insulin, 3 nM glucagon, 50 nM PTH, 2 mM glutamine, 15 nM epidermal growth factor, and antibiotics. Fetal pituitary cells were treated for 4 h with CST-14, somatostatin-14, or BIM-23120 (1–10 nM each). GH-cell and mixed GH-PRL adenomas cultures were treated with CST-14 (for M-1 and GH-1–6) or CST-17 (for M-2, M-3, GH-7–13) at concentrations of 10–1000 nM. Cultures of GH-adenomas GH-7, GH-12, and GH-13 were also treated with 10 nM of octreotide, BIM-23120, BIM-23206 (GH-12), and somatostatin-14 (GH-7). Cultures of GH-adenoma GH-8 and mixed GH-PRL adenoma M-2 were treated with 10 nM of GHRH or BIM-28152, either alone or in combination with CST-17 (100 nM). PRL-adenoma cultures A-F were treated with CST-17, BIM-23206, BIM-23120, somatostatin-14, and octreotide at 10 nM. A single pituitary specimen (either fetal or adenoma) was divided and plated into 60–80 wells, depending on its size. In each experiment 6–8 wells served as controls (treated with vehicle solution), and groups of 6–8 wells were treated as indicated. The medium was then collected and stored at –20 C for later hormone measurements.

Hormone assays

Human GH was measured by RIA (Diagnostic Products Corp., Los Angeles, CA). Intra- and interassay precisions were 1.5 and 3.4%, respectively. Human PRL was measured by immunoradiometric assay (Diagnostic Products). Intra- and interassay precisions were 2.0 and 3.6%, respectively.

Pituitary RNA extraction

Pituitary adenomas were harvested and kept at –70 C for RNA extraction. After homogenization, total RNA was extracted using guanidium isothiocyanate-phenol-chloroform (TRizol; Invitrogen Inc., Carlsbad, CA), and aliquots of RNA were electrophoresed through Tris-borate EDTA gel to confirm RNA integrity.

RT-PCR

Reverse transcription (RT) followed by PCR amplification was performed to detect human SSTR2 and SSTR5 mRNA expression in adenomatous pituitary tissues. RNA was treated with deoxyribonuclease (DNA free; Ambion, Inc., Austin, TX) before the RT reaction to eliminate contaminating genomic DNA. RNA was then used in a 20-µl RT reaction containing Oligo(dT)16 as a primer and SuperScript II (Invitrogen). Samples were also incubated without RT as negative controls. RT reactions were incubated at 42 C for 50 min and then at 70 C for 15 min. The resulting cDNA and negative controls were used for subsequent PCR amplification of SSTR2, SSTR5, and ß-actin in the presence of 2 mM MgCl2 and 5 U Taq DNA polymerase (Ex Taq, Hot Start version; TaKaRa Shuzo Co., Ltd., Otsu, Japan). Amplifications were carried out for 35 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 54 C, and elongation at 72 C; each step lasted 1 min. The following primer sets were used: human SSTR2, 5'-GACAAGCAATGCAGTCCTCA (480–499) and 3'-CCATCCACAGTCATGACCAC (709–728; GenBank accession no. BC019610); human SSTR5, 5'-AACACGCTG GTCATCTACGTGGT (172–194) and 3'-AGACACTGGTGAACTGGTTGAC (361–382; GenBank accession no. NM_001053); and ß-actin, 5'-ATCTGGCACCACACC TTCTACAATGAGCTGCG (234–265, exon 3) and 3'-CGTCATACTCCTG CTTGCTGATCCACATCTGC (1040–1071, exon 5; GenBank accession no. BC008633). This process generated PCR products of 249 bp for SSTR2, 210 bp for SSTR5, and 837 bp for ß-actin. The products were visualized with ethidium bromide after electrophoresis on 2% agarose gel.

Statistical analysis

Results are presented as mean ± SD. Because absolute hormonal levels differed among fetal specimens and among adenoma specimens, the hormonal data were expressed as percentage of control (100%). Data were analyzed by one-way ANOVA, and P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Effect of CST on human fetal GH secretion

The incubation of human fetal pituitary cells (21 and 25 wk gestation) with CST-14 (10 nM) for 4 h significantly inhibited GH secretion by up to 65% (Fig. 1Go), which was greater than the suppression induced by somatostatin-14 (Fig. 1AGo). This inhibitory effect was consistent in four different fetal specimens studied (data not shown). Comparison of the action of CST and SSTR2-specific analog, BIM-23120, revealed the same magnitude of GH inhibition for both at a concentration of 10 nM (65%; Fig. 1BGo) but a higher potency of somatostatin analog at 1 nM.


Figure 1
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  		FIG. 1. Human fetal GH (hGH) inhibition by CST, somatostatin, and BIM-23120. Human fetal pituitaries (21 and 25 wk gestation) were harvested, and cells were cultured at 5 x 104 cells/well. Forty-eight hours later, cultures were treated with CST-14, somatostatin-14 (SRIF), or BIM-23120 (120) at 10 nM or as indicated, in SFD medium for 4 h. Control cells (C) were treated with vehicle solution. Medium was then collected for later GH measurements. Each of the bars represents the mean ± SD of GH levels in six wells, compared with control wells (100%). *, P < 0.05.

 
Effect of CST on GH release from human GH-secreting pituitary adenomas

In contrast to the consistent inhibitory effect of CST in all human fetal tissues studied, CST (-14 or -17, as indicated; 10–100 nM) significantly inhibited GH release (20–30%) in six of the 13 GH-secreting adenomas tested as well as in two of the three mixed GH-PRL adenomas (Fig. 2AGo). A significant inhibition of GH release from cultures of adenoma GH-11 (30%) was achieved already at CST-17 concentration of 1 nM (Fig. 2BGo). Interestingly, in cell cultures of adenoma GH-7, GH secretion was suppressed by 24–36% by somatostatin-14, octreotide, and BIM-23120 at concentrations of 10 nM (Fig. 2CGo), but CST-17 had a significant effect on GH secretion only at a higher concentration of 100 nM (Fig. 2AGo). In adenoma GH-12, however, GH was significantly suppressed by octreotide, BIM-23120, and BIM-23206 as well as CST-17 (all 10 nM; Fig 2CGo). In contrast, adenoma GH-13 did not respond to CST-17 (10 nM; Fig. 2AGo) or any of the somatostatin analogs (data not shown).


Figure 2
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  		FIG. 2. CST effects on cultured GH-cell and mixed GH-PRL adenomas. A, Human GH-adenomas (GH-1–13) and mixed GH-PRL adenomas (M-1–3) were harvested during transsphenoidal procedures, and then cells were cultured at 5 x 104 cells/well. Forty-eight hours later, cultures were treated with CST-14 (for M-1 and GH-1–6) or CST-17 (for M-2, M-3, GH-7–13), at the indicated concentrations, in SFD medium for 4 h. Control cells (white bars) were treated with vehicle solution. B, CST dose-dependent effects on GH-cell adenoma. Cultures of GH-adenoma (GH-11) were treated with 0.1–100 nM of CST-17 in SFD medium for 4 h. Control cells (C) were treated with vehicle solution. C, Treatment of cultured GH-cell adenomas with CST and somatostatin analogs. Cultures of GH-adenomas (GH-7 and GH-12) were treated with 10 nM of CST-17, octreotide (OCT), BIM-23120 (120), BIM-23206 (206; GH-12), and somatostatin-14 (SRIF; GH-7). D, SSTR mRNA expression in GH-cell adenomas. Human SSTR2, SSTR5, and ß-actin mRNA expression was studied by RT-PCR in GH-cell adenomas 9–13. The expected PCR products of SSTR2 (249 bp) and SSTR5 (210 bp) as well as the PCR product of ß-actin (837 bp) and control for RT-PCR are depicted. E, CST effects on stimulated GH secretion. Cultures of GH-adenoma (GH-8; left panel) and mixed GH-PRL adenoma (M-2; right panel) were treated with 10 nM of GHRH or BIM-28152 (152), either alone or in combination with CST-17 (100 nM), in SFD medium for 4 h. M-2 adenoma was treated also with CST alone (10 nM). Control cells (C) were treated with vehicle solution. Each of the bars represents the mean ± SD of GH levels in six wells, compared with control wells (100%). *, P < 0.05, vs. control cells. **, P < 0.05, vs. cells untreated with CST. hGH, Human GH.

 
Whether the lack of CST efficacy in several adenomas reflects expression profile of SSTRs was tested in adenomas 9–13, for which RNA was available. As shown in Fig. 2DGo, all five of these adenomas were found to express both SSTR2 and SSTR5.

We next examined whether CST affects stimulated GH secretion. The addition of CST-17 (100 nM) to cultures of GH-secreting adenoma cells (GH-8) or mixed GH-PRL adenoma cells (M-2) suppressed the induction of GH secretion by GHRH (10 nM) by 33–50% (vs. GHRH) (Fig. 2EGo). Similarly, CST-17 (100 nM) inhibited the GH response to ghrelin analog (BIM-28152; 10 nM) by 30–56% (vs. BIM-28152; Fig. 2EGo).

Effect of CST on PRL-secreting adenomas

The effect of CST on PRL secretion was studied in PRL-secreting and mixed GH-PRL adenomas. CST-17 (10–100 nM) suppressed PRL release from cultured PRL-cell adenomas (prolactinomas A, B, C) in the range of 20–40% (P < 0.05; Fig. 3AGo), gained also by rat CST (CST-14) in prolactinoma C. A similar rate of PRL suppression was observed for BIM-23206 (10 nM), the SSTR5-specific agonist (Fig. 3AGo). By contrast, the action of the SSTR2-specific agonist, BIM-23120, was not consistent, exerting no effect on PRL release in prolactinoma A and only a minor, nonsignificant effect in prolactinoma B but inducing 24% PRL inhibition in prolactinoma C (Fig. 3AGo).


Figure 3
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  		FIG. 3. A, PRL inhibition by CST and somatostatin analogs in cultured PRL-secreting adenomas. Human PRL-adenomas harvested during transsphenoidal procedures were then cultured at 5 x 04 cells/well. Forty-eight hours later cultures were treated with CST-17, CST-14 (prolactinoma C), BIM-23206 (206), and BIM-23120 (120) at the indicated concentrations for 4 h in SFD medium. Medium was then collected for later PRL measurements. Each of the bars represents the mean ± SD of PRL levels in six wells, compared with control wells (C; 100%). B, CST effects in cultured mixed GH-PRL adenomas. Human mixed GH-PRL adenomas (M-1–3) harvested during transsphenoidal procedures were cultured and treated with CST-17 (10 nM); M-1 and M-3 cultures were also treated with BIM-23206 (206) and BIM-23120 (120), and M-3 also was treated with octreotide (OCT) at 10 nM for 4 h in SFD medium. Medium was then collected for later GH and PRL measurements. Each of the bars represents the mean ± SD of GH or PRL levels in six wells, compared with control wells (C; 100%). *, P < 0.05.

 
In the mixed GH-PRL adenomas, M-1, M-2, and M-3, a significant inhibition of PRL by CST-17 was observed (Fig. 3BGo). A comparable inhibition of PRL was achieved by SSTR5- and SSTR2-selective agonists in adenoma M-3, whereas SSTR5 but not SSTR2 agonist significantly suppressed PRL in adenoma M-1 (Fig. 3BGo). Interestingly, the same pattern of suppression was obtained for GH in the mixed GH-PRL adenomas, M-1 and M-3, although no significant effect on GH was observed in adenoma M-2 (Fig. 3BGo).

In contrast to the high potency of CST in prolactinomas A, B, and C and the mixed adenomas, its effect on cells derived from prolactinomas D and E was minor, achieving significant inhibition of PRL secretion only at a concentration of 1000 nM (Fig. 4AGo). In addition, treatment of prolactinomas D and E with 10 nM of CST-17, SSTR2-selective analog, or octreotide (in prolactinoma D only) resulted in a mild and nonsignificant suppression of PRL secretion. The same effect was achieved by SSTR5-selective analog and somatostatin-14 in prolactinoma E, yet, no effect was detected for SSTR5-selective analog or somatostatin-14 in prolactinoma D (Fig. 4AGo). Prolactinoma F also did not respond to CST-17 or SSTR5-selective analog (10 nM; Fig. 4AGo).


Figure 4
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  		FIG. 4. A, PRL inhibition by CST and somatostatin analogs in prolactinomas D, E, and F. Human PRL-adenoma cells were cultured at 5 x 104 cells/well. Forty-eight hours later, all cultures were treated with CST-17 and BIM-23206 (206), and prolactinomas D and E were also treated with BIM-23120 (120), somatostatin-14 (SRIF), and octreotide (OCT; prolactinoma D) at 10 nM (right) or CST-17 at the indicated concentrations (left) or for 4 h in SFD medium. Medium was then collected for later PRL measurements. Each of the bars represents the mean ± SD. PRL levels in six wells, compared with control wells (C; 100%), are shown. B, SSTR mRNA expression in prolactinomas and mixed GH-PRL adenomas. Human SSTR2, SSTR5, and ß-actin mRNA expression was studied by RT-PCR in PRL-cell adenomas B-F and in mixed adenomas M-2 and M-3. Left panel, The expected PCR products of SSTR2 (249 bp) and SSTR5 (210 bp). Right panel, ß-actin (PCR product of 837 bp), control for RT-PCR. C, PRL inhibition in cultured PRL-secreting adenoma cotreated with CST and an SSTR5-specific analog. Cell cultures of human PRL-adenoma (prolactinoma A) were treated with CST-17 (20 nM), BIM-23206 (206; 20 nM), and a combination of both (10 nM each) for 4 h in SFD medium. Medium was then collected for later PRL measurements. Each of the bars represents the mean ± SD of PRL levels in six wells, compared with control wells (C, 100%). *, P < 0.05.

 
The resistance of prolactinomas D and F to an SSTR5-selective compound and their mild/no response to CST and compounds with enhanced binding affinity for SSTR2 could reflect a different expression pattern of SSTR2 and SSTR5 in these cells. To test this assumption, RNA was extracted from the prolactinomas and mixed adenomas and subjected to RT followed by PCR amplification of human SSTR2 and SSTR5 mRNAs, in addition to ß-actin as the RT-PCR control. We found that both SSTR2 and SSTR5 were expressed in prolactinomas B, C, and E and the mixed GH-PRL adenomas, M-2 and M-3, whereas only SSTR2 was expressed in prolactinomas D and F (Fig. 4BGo). RNA from prolactinoma A and adenoma M-1 was not available for RT-PCR studies.

The specific involvement of SSTR5 in CST regulation of PRL in adenoma cells was further evaluated by the administration of CST-17 with the SSTR5-selective analog. The PRL suppression induced by the combination of these two peptides (10 nM each; 26%, P < 0.05; Fig. 4CGo) was comparable with that achieved by CST or BIM-23206 individually (20 nM each; 30 and 23%, respectively; P < 0.05), providing further support to the notion that SSTR5 is an important mediator of the effect of CST on PRL.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The present study demonstrates for the first time that CST is a potent suppressor of human GH secretion in fetal pituitary cells and several GH-cell adenomas, suggesting impaired signaling integrity in nonresponding adenomas. CST significantly inhibited PRL secretion from PRL-secreting adenomas and mixed GH-PRL tumors.

Although CST has been found to bind SSTRs in pharmacological assays, whether these interactions take place in vivo, or have functional implications, remains unclear. Membrane-radioligand binding studies reported that CST-17 can displace [125I-Tyr3]octreotide binding to its receptor (probably SSTR2) on tissue sections of human thymus (14). In wild-type and SSTR2 knockout mice, CST-14 interacted with somatostatin receptors in the brain (22), but the functional significance of these findings requires further investigation. In addition, CST-14 decreased glutamate-induced responses in mouse hypothalamic neuronal primary cultures, similar to somatostatin-14 and octreotide, suggesting the involvement of SSTR2 (12).

The present finding of an inhibitory effect of CST on GH secretion in fetal pituitary cultures is in accordance with previous reports of an in vivo CST effect in normal human adults (16, 17) and patients harboring GH-secreting adenomas (23). Our use of isolated cultured fetal human pituitary cells points to a direct effect of CST at the pituitary level. This was not evident, however, in half of the GH-secreting adenomas studied here. Although we addressed the pattern of expression of the SSTRs in only five adenomas, we found expression of both SSTR2 and SSTR5 in all adenomas tested, compatible with an earlier quantitative analysis of SSTRs expression in cells derived from acromegalic tumors (24) showing a consistent expression of both SSTR2 and SSTR5. Despite the higher level of SSTR5 mRNA, GH inhibition by somatostatin and its analogs correlated significantly with the level of expression of SSTR2; SSTR5-selective analog inhibited GH release in only half of these tumors (24). In our panel of cultured GH-cell adenomas, six of 13 tumors responded to CST. In the subgroup of adenomas proved for both SSTR2 and -5 expression, there were tumors responding and not responding to CST. Moreover, in adenoma GH-12, GH was suppressed by all peptide treatments, whereas in adenoma GH-13, GH did not respond to CST or any somatostatin analog. Therefore, it is impossible to conclude now that the inhibitory effects of CST on GH secretion are SSTR dependent. However, using SSTR-selective antagonists in the future may add more information to our understanding of this inhibitory mechanism.

Recently Grottoli et al. (23) demonstrated an in vivo inhibitory effect on PRL release in patients with prolactinomas. The present findings in PRL-secreting adenomas reveal a significant effect of CST and SSTR5 agonist in most prolactinomas studied, whereas inhibition of PRL by SSTR2 agonist was observed in only one prolactinoma. These results confirm previous observations that SSTR5 primarily regulates PRL suppression by somatostatin in prolactinoma cells (25). Similarly, PRL regulation by CST appears to be mediated mainly by SSTR5 because the two PRL adenomas that did not express SSTR5 did not respond to CST. However, a mild effect of CST at high concentration was observed in prolactinoma D, which lacks the SSTR5, suggesting contribution of other receptors, i.e. SSTR1, which was previously shown to be involved in PRL regulation in pituitary adenomas (26). Moreover, SSTR5 expression does not necessarily ensure efficient inhibition by CST (prolactinoma E), supporting the involvement of a specific receptor for CST or postreceptor signal transduction defects. However, MrgX2, the specific CST receptor, usually is not expressed in human neuroendocrine tissues, including pituitary tumors (5). Interestingly, the inhibitory effect of CST on PRL secretion in cultured prolactinomas was not apparent in normal human adults (16, 17). Similarly, somatostatin has been shown to suppress PRL secretion in cultured prolactinomas (25, 27) but not in normal adults (16, 17) or patients with prolactinomas (28, 29).

Some of the actions of CST are distinct from those of somatostatin, such that CST may exert them independently of the SSTRs. Accordingly, one study (30) noted that the interaction of CST with GHS-R probably mediates CST’s antiproliferative effect on thyroid cancer cells. It is possible that the CST-GHS-R interaction displaces GHS binding from the receptor (9, 31). Therefore, the inhibition of GHS-induced GH secretion by CST may be a reflection of this interaction, in concert with the activation of SSTRs, leading to an enhanced inhibitory effect. Others have suggested that ghrelin antagonizes somatostatin by counteracting its hyperpolarizing effect on the cell membrane (32, 33). This may be true also for CST. Our results indicate a similar inhibitory effect of CST on the GH response to ghrelin analog or GHRH. Moreover, previous studies in humans showed that CST inhibits ghrelin-induced GH release to the same extent as somatostatin (16, 17). The functional ability of CST to bind GHS-R in vivo is not yet evident, and the endocrine interactions of CST with GHS-R require further study.

In summary, we have shown that human GH and PRL secretion can be suppressed in vitro by CST directly at the pituitary level, using normal fetal and adenoma cells. The use of receptor-specific analogs of the human SSTRs implies that SSTR5 may be an important contributor to CST regulation of PRL release from cultured pituitary adenomas, similar to somatostatin inhibition of PRL in cultured prolactinomas.


   Acknowledgments
 
The authors thank Gloria Ginzach for her editorial assistance.


   Footnotes
 
First Published Online April 4, 2006

Abbreviations: CST, Cortistatin; GHS, GH secretagogue; GHS-R, GHS receptor; PRL, prolactin; RT, reverse transcription; SFD, serum-free defined; SSTR, somatostatin receptor.

Received September 27, 2005.

Accepted March 24, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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R. M. Luque and R. D. Kineman
Gender-Dependent Role of Endogenous Somatostatin in Regulating Growth Hormone-Axis Function in Mice
Endocrinology, December 1, 2007; 148(12): 5998 - 6006.
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