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Max-Planck-Institute of Psychiatry, Clinical Institute, Department of Endocrinology (U.R., U.P., G.K.S.), Munich; Department of Neurology, Technical University of Munich (T.A., S.L., A.W.); Departments of Neurosurgery of the University of Munich (A.M., E.U.) and the Clinic of Mannheim (M.L.), Germany
Address all correspondence and requests for reprints to: Ulrich Renner, Max Planck Institute of Psychiatry, Clinical Institute, Department of Endocrinology, Kraepelinstr. 10, D-80804 Munich, Germany. E-mail: renner{at}mpipsykl.mpg.de
Abstract
Little is known about differences in the expression, localization, and function of the two dopamine D2 receptor subtypes, D2short and D2long (D2s and D2l), in either normal or adenomatous pituitary. We investigated the messenger RNA (mRNA) expression of the D2 receptor (D2R) subtypes in clinically nonfunctioning pituitary adenomas by in situ hybridization using subtype-specific oligonucleotides. The five normal pituitaries studied expressed similar ratios of D2R subtypes mRNA with a predominant expression of the D2l isoform. In 2 of 18 clinically inactive adenomas no D2R mRNA was found, whereas in 16 a heterogenous expression of D2R isoforms was observed. Six adenomas expressed only the D2l and 2 adenomas only the D2s subtype mRNA; the remaining 8 expressed extremely varying proportions of the two subtypes. The D2R was found only in a subset of the nonfunctioning adenoma cells. In gonadotropin-immunopositive adenomas, the D2R was mainly localized in LH- and FSH-immunopositive cells. Probably because of the heterogenous D2R subtype expression, suppression of cell proliferation was observed in only 3 of 9 adenoma cell cultures in which the growth inhibitory effect of bromocriptine was studied. Although there is some evidence that the presence of the D2s receptor subtype favors the growth inhibitory response to bromocriptine, further studies with a larger number of inactive adenomas are needed to confirm this speculation.
THE five types of dopamine receptors (DRs) can be divided into two groups by their molecular, biochemical, and pharmacological differences (1, 2). The dopamine D1R family consists of the D1Rs and D5Rs, whereas D2Rs, D3Rs, and D4Rs represent the D2R family. Two D2R subtypes exist, which are generated by alternative splicing. The human D2R isoforms consist of 414 (D2short, D2s) or 443 (D2long, D2l) amino acids, and the mouse and rat isoforms contain 415 and 444 amino acids (3, 4, 5, 6). The D2Rl subtype differs from the D2Rs subtype by an insertion of 29 amino acids in the third intracytoplasmatic protein loop. This polypeptide region is involved in G protein coupling of the D2R, and therefore, the two D2R subtypes may be associated with different G proteins or may induce different effects when coupled to the same G protein (1, 2).
In the normal pituitary, the D2R mediates the tonic inhibitory control of PRL secretion (7) by dopamine. In prolactinomas, the size of the tumor and the excessive PRL secretion can usually be reduced and even normalized by long-acting and potent dopamine agonists such as bromocriptine (BC) (reviewed in Ref.8). This indicates that in these tumors functional DRs are still expressed (9). Only in prolactinomas that are not responsive to dopamine agonist treatment are DR either absent or expressed in reduced amounts or are functionally inactive (10, 11, 12).
Ligand binding studies and scintigraphic evaluations have shown the presence of dopamine binding sites (most likely D2R) in other pituitary tumor types (13, 14), and therefore the effects of dopamine agonists in these tumors were investigated both in vivo and in vitro (8). Suppressive effects of dopamine agonists on GH and ACTH secretion have been reported in some somatotropinomas (15, 16) and Nelson tumors (17), respectively. In some somatotroph and inactive adenomas an antiproliferative effect of dopamine agonists has also been observed (16, 18, 19, 20, 21), but in others this effect has not been seen (13, 22).
No genomic mutations in the coding regions of the D2R gene have been detected in pituitary adenomas (23). Thus, posttranscriptional or posttranslational events seem to be responsible for the missing response of D2R-positive pituitary adenomas to dopamine agonist treatment. We investigated the expression of dopamine D2R subtype messenger RNA (mRNA) in nonfunctioning pituitary tumors at the cellular level by in situ hybridization (ISH). In addition, we tried to correlate the growth inhibitory effects of BC on clinically inactive adenomas in vitro with the predominant mRNA expression of a particular D2R subtype.
Materials and Methods
Materials
Except where stated, materials and methods were obtained from Gibco (Karlsruhe, Germany), Falcon (Heidelberg, Germany), Nunc (Wiesbaden, Germany), Seromed (Berlin, Germany), Flow (Meckenheim, Germany), and Sigma Chemical Co. (St. Louis, MO). Monoclonal antibodies against human FSH and LH for immunohistochemistry (IHC) were purchased from DAKO (Hamburg, Germany), BC from Novartis (Nuremburg, Germany), and haloperidol from RBI (Natick, MA).
Pituitary adenomas and normal pituitaries
Tumor specimens were obtained from 18 patients (see Table 1
)
with clinically nonfunctioning adenomas. Four tumors were recurrences
that were detected during the follow-up of 4 patients who had already
had surgery for an inactive adenoma. None of the 4 patients had had any
medical treatment before the second operation. The remaining 14 cases
were newly diagnosed nonfunctioning adenomas. Visual field loss and
headache were the presenting symptoms in all patients. All tumors were
macroadenomas with para- or suprasellar extension, as documented by
computerized tomography or magnetic resonance imaging. No clinical
symptoms because of tumor-derived hormone excess could be observed. The
results of the routine immunohistological examinations of the adenoma
tissues performed in the neuropathology department are listed in Table 1.
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Tissue preparation and adenoma cell culture
Tissue fragments of the adenomas and normal pituitaries were shock-frozen on dry ice for ISH and subsequent IHC. To establish primary cell cultures, nine adenomas were dispersed as previously described (21). The adenoma cells were washed and seeded (100,000 cells per well in 48-well plates) in D-valine minimal essential medium (D-Val-MEM, pH 7.3) supplemented with 10% FCS, 2.2 g/L NaHCO3, 10 mmol/L HEPES, 2 mmol/L glutamine, 10 mL/L nonessential amino acids, 10 mL/L MEM vitamins, 5 mg/L insulin, 5 mg/L transferrin, 2.5 mg/L amphotericin B, 105 U/L penicillin/streptomycin, 20 µg/L sodium selenite, and 30 pmol/L T3. D-Val-MEM was used to suppress the growth of contaminating fibroblasts. After 48 h, when the cells had attached to the culture dishes, they were used for growth experiments. Immunocytochemical investigations of the cultured adenoma cells showed a similar pattern of hormone expression similar to that observed in the adenoma tissue.
In situ hybridization
ISH was performed as previously described with minor
modifications (24, 25). In brief, 8-µm tissue sections of the
shock-frozen tissue were cut in a cryostat, thaw-mounted onto sterile,
poly-L-lysine-coated slides, fixed in 4%
phosphate-buffered paraformaldehyde, and stored in 96% ethanol until
use. An oligonucleotide corresponding to position 1022–1063 of the
coding sequence of the long human D2R isoform was used to detect the
mRNA of both receptor isoforms (D2total). Oligonucleotides
corresponding to position 724–765 of the long D2R isoform or to
position 704–743 of the short D2R isoform were used to recognize mRNA
expression of the D2l and D2s receptor
isoforms, respectively. The oligonucleotides were 3'-end labeled with
[
-33P]deoxycytidine ATP (Dupont, Bad Homburg, Germany)
by terminal transferase (Boehringer Mannheim, Mannheim, Germany). After
pretreatment in 0.25% acetic anhydride in 0.1 mol/L
triethanolamine-HCl/0.9% NaCl for 10 min, sections were hybridized
with the labeled probe (the SA ranged from 2.5–3.5 x
105 cpm/µL) diluted 1:50 in hybridization buffer (50%
formamide, 4 x SSC, 10% dextran sulfate, 100 µg/mL
polyadenylic acid, 5 x Denhardt’s solution, 25 mmol/L sodium
phosphate, 1 mmol/L sodium pyrophosphate) at 42 C for 20 h.
Consecutive washes were performed in 1 x SSC for 10 min at room
temperature, 1 x SSC for 30 min at 55 C, and 1 x SSC and
0.1 x SSC for 1 min each at room temperature. After dehydration
in ethanol, air-dried sections were dipped in Ilford K5 photoemulsion
(Ilford Photo Corp., Paramus, NJ). Slides were developed after 28 days
and counterstained with toluidine blue.
The specificity of our probes was confirmed by a specific signal in the normal human anterior lobe as previously described (3). For negative control, a 100-fold excess of nonlabeled oligonucleotides was added to the radioactive probe.
Colocalization of D2total mRNA with LH or FSH
To examine the colocalization of D2R mRNA with normal and adenomatous LH and FSH immunopositive cells, IHC for LH and FSH was performed after ISH for D2total mRNA. The slides were incubated with monoclonal antibodies against FSH (diluted 1:200) and LH (1:500) overnight at 4 C. Subsequently, the indirect streptavidin-biotin-peroxidase-method (DAKO) was performed according to the manufacturer’s instructions to detect immunopositive cells. For the final enzyme reaction (5 min at room temperature), 0.01% H2O2 was used as the substrate and 0.1% diaminobenzidine as the chromogen.
Growth experiments
Proliferation studies ([3H]thymidine incorporation) were performed in serum-free D-Val-MEM culture medium after an initial 24 h serum washout period (26). BC (1, 10, and 100 nM) with and without haloperidol (1 µM) was added to the cell cultures as well as [3H]thymidine (0.5 µCi/mL). After an incubation period of 48 h, the supernatants were removed. The cells were precipitated with 10% ice-cold trichloroacetic acid and washed with PBS. The DNA was hydrolized using 0.5 mol/L NaOH and 0.1% Triton X-100. Incorporated [3H]thymidine was determined with a liquid scintillation counter.
Statistical analyses
Growth experiments were performed at least in quadruplicates. Values are expressed as mean ± SD, and statistics were performed by ANOVA in combination with Scheffe’s test (27).
Results
D2 receptor subtype mRNA expression in normal and adenomatous pituitary
In all 5 normal pituitaries, a similar, strong signal for the
D2total mRNA was found by ISH. In all 5, the
D2l subtype mRNA was predominantly expressed (Table 1
). In
2 of 18 nonfunctioning adenomas, no D2total mRNA was found.
In 3 of 16 D2R-positive adenomas, the intensity of the
D2total signal was similar to that of normal pituitaries,
whereas it was lower in all the others. In contrast to normal
pituitaries, in the D2total-positive adenomas a
heterogenous ratio of D2l and D2s mRNA
expression was found (Table 1). Six adenomas expressed only
D2l mRNA and 2 tumors only the D2s mRNA. Of the
8 adenomas that expressed mRNA of both D2R isoforms, 4 showed a
predominant expression of D2l mRNA (see example shown in
Fig. 1), 2 showed an equal expression of
the two isoforms, and 2 showed a stronger expression of D2s
mRNA (Fig. 2). It should be noted that in
D2R-positive tumors, not all adenoma cells expressed D2R transcripts
(Table 1).
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Nonfunctioning adenoma cells are very often positive for
gonadotropins (28). By combining ISH for D2total mRNA
expression and IHC for LH and FSH, the presence of D2total
mRNA in normal and adenomatous gonadotropes was studied. Only a few
single LH- or FSH-immunopositive cells coexpressed D2total
mRNA in the normal pituitary (Fig. 3). In
contrast, nearly all gonadotropin-immunopositive adenoma cells
expressed D2total mRNA (Fig. 3). In adenomas that were both
LH- and FSH-immunopositive, no evidence of a predominant expression of
D2total mRNA in one of the two gonadotroph cell types was
found.
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In 9 adenomas, enough tissue was available to establish
primary cell cultures and to study [3H]thymidine
incorporation as a parameter of cell proliferation. One of the adenomas
studied was negative for mRNA of both D2R subtypes, 3 expressed only
D2l receptor subtype mRNA, and 1 expressed only
D2s; in 4 tumors both receptor subtypes were found. In all
adenoma cultures, the proliferation rate of the adenoma cells under
serum-free conditions was very low (Table 2). BC suppressed the
[3H]thymidine incorporation in 3 of 9 adenoma cell
cultures (Fig. 4 and Table 2). The
dopamine D2R antagonist haloperidol reversed the inhibitory action of
BC in all three cases, indicating the specificity of the growth
suppressive effect (Fig. 4 and Table 2). Growth inhibition was observed
in the adenoma that expressed only the D2s receptor subtype
mRNA and in 2 tumors that were positive for both receptor subtypes,
with a predominant expression of the D2s isoform in one of
these 2 tumors. In the adenomas that expressed only D2l
receptor subtype mRNA, no effect of BC on growth was observed.
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We studied the expression and localization of D2total receptor mRNA as well as D2l and D2s receptor subtype mRNA in inactive pituitary adenomas by ISH. This technique allows examination of the expression of D2l and D2s receptor subtypes at the cellular level and discrimination between subtype positive and negative cells within normal and adenomatous pituitary tissues. This is not possible with other methods such as scintigraphic examinations of D2R in whole adenomas, ligand binding studies with membrane preparations, and D2R mRNA measurements in adenoma extracts by RT-PCR. However, because the medical treatment of nonfunctioning adenomas with dopamine agonists is still under discussion (19), it is essential to know how many cells of an inactive pituitary adenoma express D2R and thus represent a possible target for dopamine agonists.
In normal human pituitaries, D2total receptor mRNA was found in more than 75% of all cells, indicating that D2R are not expressed in lactotropes only, which represent only 10–30% of all pituitary cells (7). Both receptor subtypes were found, and the predominant expression of D2l in normal pituitary, already described by others, could be confirmed (3, 4, 29, 30). In inactive adenomas, the D2total mRNA expression was lower than in normal pituitary in most cases, and the ratio of D2l to D2s subtype expression was extremely heterogenous. Two of the tumors investigated were completely D2R mRNA negative, in 8 tumors mRNA of both D2R subtypes could be detected, and in 6 tumors only the long isoform and in 2 tumors only the short isoform could be detected. In all D2R mRNA positive adenomas only a part of the adenoma cells expressed D2R transcripts.
By combining ISH with IHC we found that in the LH- and FSH-immunopositive adenomas, D2R mRNA could be detected in nearly all gonadotropin-positive cells, whereas in normal human pituitary it could be detected in only a few scattered gonadotropes. Our finding of D2R expression in the majority of LH- and FSH-immunopositive cells would explain the results of previous studies in which BC suppressed the release of LH and FSH from inactive adenomas in vitro (31).
Clinically inactive adenomas represent a heterogenous tumor group (28), which is also reflected by the variable mRNA expression of the two D2R subtypes. Moreover, our observation that in many nonsecreting adenomas only one particular D2R subtype mRNA is expressed indicates that changes in the mechanism of alternative D2R subtype splicing may occur during tumorigenesis of inactive adenomas. The nature of these changes remains unclear, because very little information is available about the mechanism of alternative splicing. The D2R splicing process is affected by sex steroid hormones that induce alterations in the D2s/D2l ratio in lactotropes (30, 32). Disturbances of D2R mRNA splicing have also been found in dopamine-responsive lactotroph MMQ rat pituitary tumor cells (33), which express only the D2s subtype (34), and in BC-resistant mammotroph adenomas (35).
The affinity of dopamine and dopamine agonists for the D2R
isoforms is nearly identical (6, 36), but it seems that after binding,
the subsequent signaling is different. The two D2R subtypes are coupled
differently to G proteins such as Gs,
Gi2, and Gi3, which regulate adenylate
cyclase as well as K+ or Ca2+ fluxes in
different ways. Thus, the heterogenous G protein coupling would allow a
variable second-messenger activation by the two D2R subtypes (36, 37, 38, 39, 40, 41, 42).
In D2R-positive but BC-resistant prolactinomas, a decreased
Gi2 expression was found (43), suggesting that the
corresponding Gi2-coupled D2R subtype was functionally
less active. A heterogenous dopamine signal transduction was also
observed in D2R-positive nonfunctioning pituitary adenomas in
vitro (44).
Conflicting results have been reported about the effect of dopamine agonists on the growth of nonfunctioning adenomas. In some clinical studies no effect on the size of the tumor could be found (13, 22), whereas in others tumor shrinkage was reported in up to 20% of the patients (reviewed in Ref.19). To investigate whether the different growth inhibitory response to dopamine agonist treatment is because of the heterogenous pattern of D2 receptor subtype mRNA expression in nonfunctioning adenomas, we performed growth studies with nine such tumors. In three tumors a dose-dependent and specific, haloperidol-reversible suppression of [3H]thymidine incorporation was observed in vitro. This growth inhibitory action was found in one adenoma in which only the mRNA of the D2s isoform was expressed and in two adenomas with a predominant or an equal expression of D2s vs. D2l receptor subtype. In adenomas in which only or predominantly the D2l isoform was found, no growth suppression after application of BC was observed. Our results suggest that the presence of the D2s isoform in the adenomas favors the growth suppressive response to BC. However, up until now only a few nonfunctioning pituitary adenomas have been investigated. Further studies including a larger number of inactive tumors are needed to confirm our speculation that the different expression of D2R isoforms plays a role in the different growth response of nonfunctioning adenomas to dopamine agonist treatment.
Footnotes
1 This work was supported by a grant from Novartis Pharma GmbH,
Nuremburg, Germany. 
2 U.R. and T.A. are joint first authors.
Received July 7, 1997.
Revised December 16, 1997.
Accepted December 22, 1997.
References
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bromocriptine resistant prolactinomas. J Neuroendocrinol. 8:737–746.[CrossRef][Medline]
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