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Original Studies |
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 |
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| Introduction |
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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 Additionally, the rat pituitary tumor cell line GH3 does not respond to GHRPs (Adams, E. F., unpublished observations).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 |
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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 manufacturers instructions. The extracted RNA (15 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. Bartholomews 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 Students t test.
| Results |
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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
genes 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. 1
). 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. 2
). 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. 3
). 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. 4
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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 25 fold increase found
with the responsive tumors (data not shown). Despite the lack of
response of tumors 3 and 6 to GHRP-2, Fig. 1
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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. 5
), 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. 6
), 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 (3050% increases).
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| Discussion |
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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 (
3050% 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 |
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Received February 28, 1997.
Revised July 31, 1997.
Accepted October 21, 1997.
| References |
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chain
of Gs and stimulate adenylyl cyclase in human pituitary
tumours. Nature. 340:692696.[CrossRef][Medline]
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