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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 2 638-642
Copyright © 1998 by The Endocrine Society

Original Studies

Presence of Growth Hormone Secretagogue Receptor Messenger Ribonucleic Acid in Human Pituitary Tumors and Rat GH3 Cells1

Eric F. Adams, Bei Huang, Michael Buchfelder, Andrew Howard, Roy G. Smith, Scott D. Feighner, Lex H. T. van der Ploeg, Cyril Y. Bowers and Rudolf Fahlbusch

Department of Neurosurgery, University of Erlangen-Nuremberg, 91054 Erlangen, Germany; Merck Research Laboratories (A.H., R.G.S., S.D.F., L.H.T.v.d.P.), Rahway, New Jersey 07065; and the Department of Medicine, Tulane University Medical Center (C.Y.B.), New Orleans, Louisiana 70112-2699

Address all correspondence and requests for reprints to: Dr. Eric Adams, Neuroendokrinologisches Labor, Neurochirurgische Klinik der Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A novel G11-protein-coupled receptor specific for synthetic GH-releasing peptides (GHRPs) has recently been cloned and sequenced. Two forms exist, types 1a and 1b, the latter of which is biologically inactive. Using RT-PCR, we looked for the presence in tumorous pituitary cells of messenger ribonucleic acid (mRNA) for this novel GH secretagogue receptor (GHS-R). Both subtypes of GHS-R mRNA were detected in all six human pituitary somatotropinomas removed from patients with acromegaly. In culture, four of the tumors exhibited strong responses to GHRP-2 in terms of both phosphatidylinositol (PI) hydrolysis and GH secretion, but two were resistant. There was no apparent difference in the type 1a and type 1b expression pattern, as judged by RT-PCR, between responsive and nonresponsive tumors. Similarly, the rat pituitary tumor cell line, GH3, was found to express GHS-R mRNA, although these cells also did not respond to GHRPs. RT-PCR failed to detect GHS-R mRNA in eight functionless human pituitary tumors. In contrast, prolactinomas were found to express the receptor and, in culture, significant stimulation of PRL secretion and PI hydrolysis occurred in two of three tumors tested. These results demonstrate that tumorous somatotrophs express the GHS-R gene and that the occasionally observed nonresponsiveness of somatotropinomas to GHRPs is not due to the absence of the biologically active type 1a receptor. Additionally, human pituitary prolactinomas also express GHS-R and are able to respond to GHRPs in terms of PI hydrolysis and PRL secretion. In contrast, GHS-R gene expression does not appear to be associated with human functionless pituitary tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE EARLY 1980s, Bowers and co-workers developed short synthetic peptides that possess GH-releasing activity (1, 2). These GH-releasing peptides (GHRPs) have a dual site of action, exerting effects on both hypothalamus and pituitary (3). Unlike the cAMP dependence of GHRH, GHRPs exert their effects via protein kinase C (PKC), probably via hydrolysis of membrane phosphatidylinositol (PI) (4, 5, 6, 7, 8). In recent years, the potential clinical and physiological significance of GHRPs has become increasingly apparent. Thus, nonpeptidyl analogs of GHRPs have been developed that possess oral activity and could prove to be the foundation for designing alternative therapies for GH deficiency syndromes (9, 10, 11). Additionally, it is almost certain that there exists a natural counterpart to GHRPs that plays a pivotal role in the physiological control of GH secretion (11, 12). In complete support of this latter concept, the specific cell surface receptor able to bind GHRPs [GH secretagogue receptor (GHS-R)] has now been identified in hypothalamic and pituitary tissues (12). Cloning and sequence analysis revealed that the GHS-R is unique, with little homology to other types of pituitary and hypothalamic cell surface receptors. Interestingly, at least 2 full-length GHS-R subtypes are expressed by human pituitary cells. These have been termed types 1a and 1b and are probably the result of alternative messenger ribonucleic acid (mRNA) splicing. The type 1a receptor is biologically active and consists of a 366-amino acid polypetide possessing 7 transmembrane domains together with 3 intracellular and 3 extracellular loops, typical of G protein-coupled receptors. Indeed, biochemical studies show that this GHS-R is coupled to G11, which activates phospholipase C, thus supporting the evidence that GHRPs mediate their effects through PI hydrolysis (5, 6, 7, 8, 12). In contrast, the type 1b mRNA diverges significantly from the type 1a sequence beyond codon 265, resulting in a 289-amino acid polypeptide with a completely different carboxyl-terminal sequence of 24 residues. Consequently, type 1b GHS-R contains only 5 transmembrane domains rather than 7 as in other G protein-coupled receptors. Moreover, this GHS-R subtype does not transduce GHRP signals (12).

To date, the GHS-R status of pituitary tumor cells has not been examined. However, as well as effects on normal somatotrophs, it is well established that GHRPs stimulate GH secretion and PI hydrolysis by human pituitary somatotropinomas (5, 6, 13). Nevertheless, a subgroup of somatotropinomas exhibits relatively high basal PI hydrolysis, and some of these are resistant to GHRPs (14, 15). Additionally, the rat pituitary tumor cell line GH3 does not respond to GHRPs (Adams, E. F., unpublished observations). The reasons for this nonresponsiveness to GHRPs remains unknown, but may be related to absent GHS-R expression or alterations in relative expression of the two receptor subtypes, particularly as type 1b is biologically inactive. Therefore, in the present study, we used RT-PCR to investigate GHS-R gene expression in human pituitary somatotropinomas and rat GH3 cells and correlated the findings to the in vitro effect of GHRP-2, the most potent GHRP, on PI hydrolysis. Additionally, we examined human prolactinomas and functionless pituitary tumors, because the GHS-R status and effects of GHRPs on these types of tumor have not yet been fully established.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RT-PCR for GHS-R

Experiments were performed on 6 somatotropinomas, 3 prolactinomas, 8 functionless pituitary tumors, and rat GH3 cells. The somatotropinomas were a selected group to include 3 tumors exhibiting relatively high basal rates of PI hydrolysis, 2 of which shown to be nonresponsive to GHRP in culture. Total RNA was extracted from a portion of human tissue and confluent GH3 cell cultures using Ultraspec (Biotecx, Wak Chemie, Bad Homburg, Germany) according to the manufacturer’s instructions. The extracted RNA (1–5 µg) was denatured by incubation for 2 min at 95 C, followed by rapid cooling on ice. Single stranded complementary DNA (cDNA) was synthesized by mixing the RNA with ribonuclease inhibitor (50 U), reaction buffer (10 mmol Tris/L and 50 mmol KCl/L, pH 8.3), MgCl2 (5 mmol/L), deoxynucleotide triphosphate mix [1 mmol/L each of deoxy (d)-ATP, dCTP, dTTP, and dGTP], oligo-(deoxythymidine)15 primer (1.6 µg), and 20 U avian myeloblastosis virus reverse transcriptase (total volume, 20 µL; all ingredients from Boehringer Mannheim, Mannheim, Germany) followed by sequential incubation for 10 min at room temperature, for 1 h at 42 C, and for 5 min at 95 C. PCR for the type 1a GHS-R was performed on the cDNA product under the following conditions. cDNA (20 µL) was mixed with 5'- and 3'-amplimers (1 µmol/L each; 5'-amplimer sequence, 5'-TTCTGTCTCACGGTCCTCTACAGT-3'; 3'-amplimer sequence, 5'-GGACACGAGGTTGCAGTACTGGCT-3'), deoxynucleotide triphosphate mix (dGTP, dCTP, dATP, and dTTP; 200 mmol/L each), Tris (10 mmol/L), KCl (50 mmol/L), MgCl2 (1.5 mmol/L), and 2.5 U Taq DNA polymerase (Perkin-Elmer, Ueberlingen, Germany) in a total volume of 100 µL and overlayed with 100 µL light mineral oil (Sigma Chemie, Deisenhofen, Germany). The reaction was carried through 35 cycles of 95 C (1 min), 53 C (2 min), and 72 C (3 min). Samples of the reaction products (10 µL each) were electrophoresed through 1% agarose gels (6 x 6 cm) and visualized with a UV transilluminator. The remaining PCR products were salt-ethanol precipitated, dissolved in 15 µL water, electrophoresed through agarose from which the GHS-R bands were excised, and purified with Quiaex (Diagen, Duesseldorf, Germany). The PCR DNAs were then directly sequenced by the dideoxy method and using conditions previously described for the primer annealing reaction (16), gel electrophoresis and autoradiography (17). To assess type 1b GHS-R mRNA expression, identical protocols were used, except that the 3'-amplimer sequence was 5'-TCAGAGAGAAGGGAGAAGGCACAGG-3'. This sequence is specific for the 3'-terminus of type 1b cDNA (GenBank accession no. U60181) and differs completely from the type 1a receptor.

Cell culture and PI hydrolysis

A portion of freshly resected human pituitary tumor tissue was dispersed with collagenase and placed into cell culture as previously described in detail (15), followed by assessment of the effect of GHRP-2 (100 nmol/L) on the rate of PI hydrolysis in vitro. Additionally, experiments were performed on confluent rat GH3 cells. The methods used have been fully described previously (7). In brief, cultured pituitary cells were prelabeled with [3H]inositol, washed, and then incubated for 2 h in medium containing LiCl (10 mmol/L) without (controls) and with GHRP-2 (100 nmol/L). After incubation, media were collected for hormone assay, the cells were extracted with perchloric acid (3.3%, vol/vol), and the cell membranes were dissolved in NaOH (1 mol/L). Inositol phosphates were removed from the extracts by anionic exchange chromatography, using Dowex columns (AG 1-X8, Bio-Rad, Munich, Germany). Results are expressed as the amount of radioactivity in the free inositol phosphate fractions as a percentage of total radioactivity (membranes plus free) and are representative of the PI hydrolysis rate. Hormone concentrations in the collected media were determined by ELISA using kits obtained from NETRIA (St. Bartholomew’s Hospital, London, UK).

Gsp oncogenes

Gsp oncogenes cause constitutive adenylyl cyclase activity and are found in about 40% of pituitary somatotropinomas (18). The gsp oncogene status of each tumor was therefore determined by direct sequence analysis of PCR-generated DNA as previously described (15, 17).

Statistical analyses

Statistical significance was determined by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human pituitary somatotropinomas

The GHS-R gene consists of two exons separated by a single intron (18). For the type 1a receptor, the PCR amplimers used corresponded to mRNA (cDNA) coding sequences within the two exons, either side of the gene’s intronic region, and were designed to yield RT-PCR DNA of 261 bp in length. Bands estimated to be of this size were observed after RT-PCR of somatotropinoma RNA isolated from all six tumors (Fig. 1Go). Conclusive proof that the RT-PCR DNAs were truly representative of type 1a GHS-R cDNA (i.e. mRNA) was obtained by direct sequencing, which revealed the coding sequences without the intervening intronic region (Fig. 2Go). The sequence found in all six cases proved to be identical to that published (GenBank accession no. U60179), at least within the readable regions between the two amplimers used for these studies. PCR performed directly on genomic DNA and RNA preparations, without prior reverse transcription, failed to yield visible amplified bands, excluding the possibility of the presence of related intronless genes (data not shown). All six somatotropinomas, however, also expressed the type 1b GHS-R mRNA as revealed by RT-PCR, using a specific 3'-amplimer designed to yield a band size of 194 bp (Fig. 3Go). Direct sequencing of these bands confirmed that they were representative of the type 1b receptor. A specific portion of the sequence is shown in Fig. 4Go.



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  		Figure 1. RT-PCR for type 1a GHS-R cDNA using RNA derived from six human pituitary somatotropinomas (lanes 1–6). Single bands were observable for all tumors, running just behind the 244-bp marker. M, Markers; bp, number of base pairs in marker bands (arrowed).

 


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  		Figure 2. Partial sequence of RT-PCR DNA bands depicted in Fig. 1Go. The sequence reads 3'-CCGTTTGTGGTGATGTCGGACGTAAA-5', is identical to type 1a GHS-R cDNA (mRNA), and is without the genes’s intervening intronic region situated, as arrowed, between the two underlined G residues (GenBank accession no. U60179) (12).

 


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  		Figure 3. RT-PCR for type 1b GHS-R cDNA using RNA derived from the same six human pituitary somatotropinomas (lanes 1–6) as those used in Fig. 1Go. Single bands were observable for all tumors, running just in front of the 244-bp marker. M, Markers; bp, number of base pairs in marker bands (arrowed).

 


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  		Figure 4. Partial sequence of RT-PCR DNA bands depicted in Fig. 3Go. The sequence reads 3'-GACTCTGGGTGGGTC-5' and is identical to a specific 3'-terminal region of the type 1b GHS-R cDNA (mRNA; GenBank accession no. U60181) (12).

 
Table 1Go summarizes the clinical and biochemical details of the six human pituitary somatotropinomas, including the effect of GHRP-2 on the rate of PI hydrolysis in vitro. This selected patient group consisted of three men and three women, aged 25–56 yr, with preoperative serum GH levels of 8.7–188 ng/mL. Three of the tumors were gsp positive (tumors 1–3). Thus, the series fairly well represents the spectrum of acromegalic patients. The basal rate of PI hydrolysis in vitro were relatively high (>=9.9%/2 h) in three tumors (no. 3, 4, and 6) and was not significantly stimulated by GHRP-2 in two of these (no. 3 and 6), although tumor 4 exhibited a response similar to those of the other somatotropinomas that had lower basal PI hydrolysis levels (tumors 1, 2, and 5). GH secretion by tumors 3 and 6 was also not stimulated by GHRP-2, in contrast to the 2–5 fold increase found with the responsive tumors (data not shown). Despite the lack of response of tumors 3 and 6 to GHRP-2, Fig. 1Go (lanes 3 and 6) clearly shows that the GHS-R mRNA for biologically active type 1a receptor was produced in these.


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  		Table 1. Characteristics of six human pituitary somatotrophinomas and in vitro effect of GHRP-2 (100 nmol/L) on PI hydrolysis

 
Human prolactinomas, functionless tumors, and rat GH3 cells

Eight tumors, clinically diagnosed as functionless, were studied. Although not associated with endocrine dysfunction, five of the tumors secreted small amounts of LH and FSH in culture, indicating the presence of gonadotrophs. RT-PCR failed to detect type 1a GHS-R mRNA in all eight tumors, and there was no effect of GHRP-2 on PI hydrolysis in cell culture (data not shown). In contrast, type 1a GHS-R mRNA was detected in human prolactinomas and rat GH3 cells (examples shown in Fig. 5Go), the identity of which was confirmed by sequence analysis. In culture, GH3 cells were not responsive to GHRP-2 (rate of PI hydrolysis per 2 h in controls and GHRP-2-treated cultures, 0.9 ± 0.07% and 1.1 ± 0.06%, respectively). However, GHRP-2 significantly (P < 0.05) and quite strongly stimulated PI hydrolysis by cell cultures of two human prolactinomas (Fig. 6Go), but was without effect on a third tumor tested (data not shown). In parallel, PRL secretion was also significantly (P < 0.05) increased in the responsive tumors, although the magnitude of stimulation was low compared with the effect on PI hydrolysis (30–50% increases).



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  		Figure 5. RT-PCR for the GHS-R cDNA using RNA derived from rat GH3 cells (lane 1) and a human pituitary prolactinoma (lane 2). M, Markers; bp, number of base pairs in marker bands (arrowed).

 


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  		Figure 6. Stimulatory effect of GHRP-2 (100 nmol/L) on PI hydrolysis (left panels) and PRL secretion (right panels) by cell cultures of two human pituitary prolactinomas (tumors A and B). Cells were incubated in the absence (control) or presence of GHRP-2 for 2 h. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The discovery of GHRPs and their mechanism of action has introduced new concepts into models of GH secretory control. Specifically, although it is well established that hypothalamic control of GH secretion involves adenylyl cyclase through the combined influence of GHRH and somatostatin (19), studies with GHRPs have shown clearly that receptor-mediated activation of PI, PKC, and intracellular Ca2+ and K+ channels also plays a significant role (4, 5, 6, 7, 8, 12). It is thus of considerable importance that the GHS-R has been identified and sequenced, further supporting the concept of additional physiological pathways influencing pituitary GH secretion (12). Such findings raise the possibility that abnormalities in hormone secretion by pituitary tumors may be at least in part due to defects within the presumed GHS-R/PI/PKC pathway. It is, therefore, relevant to characterize the pattern of GHS-R expression in relation to biochemical parameters such as response to GHRPs and rate of PI hydrolysis. Previous studies have shown that about 25% of human pituitary somatotropinomas exhibit relatively high basal rates of PI hydrolysis (15), and we have demonstrated that some of these fail to respond to GHRPs (Adams E. F., Buchfelder M., Fahlbusch R., unpublished observations). Thus, the somatotropinomas for the present investigation were selected to include tumors with high and low basal rates of PI hydrolysis and two tumors nonresponsive to GHRP. Despite this variability, our results demonstrate consistent expression of the GHS-R gene by different human pituitary somatotropinomas, even in those tumors resistant to GHRP-2. Additionally, GH3 cells, which also do not respond to GHRPs, nevertheless expressed the type 1a GHS-R mRNA. Hence, the observation of an absent effect of GHRP-2 on rat GH3 cells and some human somatotropinomas is not due to lack of GHS-R expression per se. Further work will be required to resolve this problem, but one possibility is that resistance is related to altered expression of GHS-R subtypes, as type 1b does not transduce GHRP signals (12). The present results show that both GHS-R subtypes are expressed by human pituitary somatotropinomas, and we have no evidence of complete failure to express the biologically active type 1a form. It should be emphasized, however, that RT-PCR is nonquantitative, and as type 1b receptors do not elicit a response to GHRPs (12), it will be important to use fully quantitative procedures to determine whether nonresponsive human pituitary somatotropinomas and GH3 cells express predominantly this form of GHS-R. Alternatively, a distinct possibility is that relatively high basal PI hydrolysis and nonresponsiveness to GHRP are due to defects in components downstream of the GHS-R, such as a mutated G11 protein. Such a situation would be analogous to that for gsp oncogenes, in which a mutated Gs{alpha} subunit results in excessive adenylyl cyclase activity and some degree of resistance to GHRH (19).

The absence of GHS-R gene expression in functionless pituitary tumors is consistent with the observation of no effect of GHRP-2 on PI hydrolysis. As functionless human pituitary tumors are often associated with gonadotrophs (20), as were five of the tumors in this study, these results further support the findings that GHRPs do not modulate LH and FSH secretion (21). In earlier unpublished studies, we failed to detect effects of GHRP-6 on PI hydrolysis in functionless tumors regardless of the presence or absence of gonadotropin secretion in vitro. In contrast to these findings, GHS-R mRNA was detected in prolactinomas, and a significant effect of GHRP-2 on PI hydrolysis and PRL secretion occurred in at least two of the three tumors studied. These results are in agreement with in vivo studies in normal humans and acromegalic subjects, which showed elevated serum PRL levels after iv administration of GHRPs or nonpeptidyl analogs (21, 22, 23). Moreover, PRL secretion and PI hydrolysis by mixed somatotropic-lactotropic pituitary tumors are markedly increased by the nonpeptidyl GHRP analog, L-692,429, in vitro (24). The present results are at variance, however, with the findings of Ciccarelli et al. (23), who reported no effect of hexarelin, a methylated derivative of GHRP-6, on serum PRL levels in five patients with prolactinomas, whereas stimulation of PRL secretion occurred in normal subjects and acromegalic patients. The reasons for this discrepancy are not clear, but may be related to the fact that in vivo, GHRPs also exert effects via the hypothalamus (3). As pointed out by Cicarelli et al. (23), there is evidence for hypothalamic alterations in patients with prolactinomas, which may explain the absent effect of GHRPs on serum PRL levels. Alternatively, as one of the three PRL-secreting tumors used in the present study also did not respond to GHRP-2, it is possible that a variable effect of GHRPs on prolactinomas will be found in a larger series, perhaps reflecting differing intracellular dysfunctions or variable receptor status and responsiveness. This latter concept merits consideration in view of analogous systems with respect to other hypothalamic ligands, as shown by paradoxical responses of PRL and GH secretion to GHRH and TRH, respectively, in acromegaly (24, 25, 26). It is noteworthy that although PI hydrolysis was increased to a similar degree as found with somatotropinomas and mixed somatotropic-lactotropic tumors, the effect on PRL secretion (~30–50% stimulation) was low compared to that found with the mixed tumors, in which up to 2.5-fold stimulation of PRL secretion occurred (24). As PI hydrolysis was, nevertheless, strongly stimulated in vitro, these findings may be indicative of some degree of decoupling of the GHS-R/PI/PKC transduction from PRL secretion in pure prolactinomas, as has been suggested to occur with the dopamine receptor (27). Comparative studies using normal pituitary cells will be required to further investigate this possibility.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (Ref: Ad 100/2–2). Back

Received February 28, 1997.

Revised July 31, 1997.

Accepted October 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  5. Lei T, Buchfelder M, Fahlbusch R, Adams EF. 1995 Growth hormone releasing peptide (GHRP-6) stimulates phosphatidylinositol (PI) turnover in human pituitary somatotroph cells. J Mol Endocrinol. 14:135–138.[Abstract/Free Full Text]
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S. Petersenn, A. C. Rasch, M. Penshorn, F. U. Beil, and H. M. Schulte
Genomic Structure and Transcriptional Regulation of the Human Growth Hormone Secretagogue Receptor
Endocrinology, June 1, 2001; 142(6): 2649 - 2659.
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M. Korbonits, S. A. Bustin, M. Kojima, S. Jordan, E. F. Adams, D. G. Lowe, K. Kangawa, and A. B. Grossman
The Expression of the Growth Hormone Secretagogue Receptor Ligand Ghrelin in Normal and Abnormal Human Pituitary and Other Neuroendocrine Tumors
J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 881 - 887.
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H. Kaji, S. Tai, Y. Okimura, G. Iguchi, Y. Takahashi, H. Abe, and K. Chihara
Cloning and Characterization of the 5'-Flanking Region of the Human Growth Hormone Secretagogue Receptor Gene
J. Biol. Chem., December 18, 1998; 273(51): 33885 - 33888.
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A. Giustina and J. D. Veldhuis
Pathophysiology of the Neuroregulation of Growth Hormone Secretion in Experimental Animals and the Human
Endocr. Rev., December 1, 1998; 19(6): 717 - 797.
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M. M. Skinner, R. Nass, B. Lopes, E. R. Laws, and M. O. Thorner
Growth Hormone Secretagogue Receptor Expression in Human Pituitary Tumors
J. Clin. Endocrinol. Metab., December 1, 1998; 83(12): 4314 - 4320.
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M. Korbonits, R. A. Jacobs, S. J. B. Aylwin, J. M. Burrin, P. L. M. Dahia, J. P. Monson, J. Honegger, R. Fahlbush, P. J. Trainer, S. L. Chew, et al.
Expression of the Growth Hormone Secretagogue Receptor in Pituitary Adenomas and Other Neuroendocrine Tumors
J. Clin. Endocrinol. Metab., October 1, 1998; 83(10): 3624 - 3630.
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