Skip to main content
PMC home page
Endocrinology logoLink to Endocrinology
. 2009 Apr 2;150(7):3177–3185. doi: 10.1210/en.2008-1482

Use of the Metallothionein Promoter-Human Growth Hormone-Releasing Hormone (GHRH) Mouse to Identify Regulatory Pathways that Suppress Pituitary Somatotrope Hyperplasia and Adenoma Formation due to GHRH-Receptor Hyperactivation

Raul M Luque 1, Beatriz S Soares 1, Xiao-ding Peng 1, Sonia Krishnan 1, Jose Cordoba-Chacon 1, Lawrence A Frohman 1, Rhonda D Kineman 1
PMCID: PMC2703537  PMID: 19342460

Abstract

Hyperactivation of the GHRH receptor or downstream signaling components is associated with hyperplasia of the pituitary somatotrope population, in which adenomas form relatively late in life, with less than 100% penetrance. Hyperplastic and adenomatous pituitaries of metallothionein promoter-human GHRH transgenic (Tg) mice (4 and > 10 months, respectively) were used to identify mechanisms that may prevent or delay adenoma formation in the presence of excess GHRH. In hyperplastic pituitaries, expression of the late G1/G2 marker Ki67 increased, whereas the proportion of 5-bromo-2′-deoxyuridine-labeled cells (S phase marker) did not differ from age-matched controls. These results indicate cell cycle progression is blocked, with further evidence suggesting that enhanced p27 activity may contribute to this process. For adenomas, formation was associated with loss of p27 activity (nuclear localization and mRNA). Increased endogenous somatostatin (SST) tone may also slow the conversion from hyperplastic to adenomatous state because mRNA levels for SST receptors, sst2 and sst5, were elevated in hyperplastic pituitaries, whereas adenomas were associated with a decline in sst1 and sst5 mRNA. Also, SST-knockout Tg pituitaries were larger and adenomas formed earlier compared with those of SST-intact Tg mice. Unexpectedly, these changes were independent of changes in proliferation rate within the hyperplastic tissue, suggesting that endogenous SST controls GHRH-induced adenoma formation primarily via modulation of apoptotic and/or cellular senescence pathways, consistent with the predicted function of some of the most differentially expressed genes (Casp1, MAP2K1, TNFR2) identified by membrane arrays and confirmed by quantitative real-time RT-PCR.

Both cell cycle modulators and endogenous somatostatin tone exert restraining influences on the progression from GHRH-induced hyperplastic to adenomatous somatotropes and decreases in their activity over time removes this restraint, allowing for adenoma development.

GHRH is a hypothalamic neuropeptide that stimulates the release and synthesis of GH from the anterior pituitary (1,2). In addition, GHRH directly stimulates proliferation of GH-producing cells, as assessed by tritiated thymidine incorporation of primary pituitary cell cultures from adult rats (3). This action is mimicked by forskolin, a receptor-independent stimulator of adenylyl cyclase (AC), suggesting that the proliferative action of GHRH is transduced by activation of a 3′, 5′-cAMP-dependent signal transduction pathway. The proliferative action of GHRH is clinically relevant in that patients with tumors that ectopically express GHRH have elevated circulating GH levels associated with an enlargement of the pituitary gland due to somatotrope hyperplasia, and in some rare cases of hypothalamic GHRH-producing gangliocytomas, GH adenomas form (4,5,6,7). In addition, pituitary expression of GHRH, which is low or undetectable in normal tissue, is highly expressed in a large cohort of GH-producing tumors (8,9), suggesting that locally produced GHRH may enhance somatotrope proliferation.

Independent of GHRH stimulation, approximately 40% of all sporadic GH-producing pituitary adenomas in humans express a constitutively active mutant form of Gsα (gsp), which activates AC and raises intracellular cAMP levels (10). Approximately 20% of patients with McCune-Albright syndrome, a developmental disease with a mosaic tissue distribution of the gsp mutation, have elevated circulating GH levels associated with somatotrope hyperplasia, and GH-producing tumors form in some subjects (10). In addition to genetic abnormalities in Gsα, a subset of gsp-negative, GH-producing adenomas show elevated expression of wild-type Gsα (11). Another disease associated with somatotrope hyperplasia and GH-producing pituitary tumors is the Carney complex (CNC) (10,12). As in McCune-Albright syndrome, pituitary adenomas form in only a subset of CNC patients (∼10%). Despite the low incidence of GH-producing tumors, up to 80% of individuals with CNC show paradoxical GH responses to various stimuli (such as to TRH) or have elevated IGF-I, indicative of somatotrope hyperactivity (10,12). In some families with CNC, the disease segregates with an inactivating mutation of the protein kinase A (PKA) regulatory subunit Iα (PKARIA). It has been postulated that in the absence of functional PKARIA, the catabolic subunit of PKA is constitutively active (i.e. does not require cAMP activation), resulting in chronic phosphorylation of nuclear targets. Whereas global knockout of PKARIA in mice is embryonic lethal and heterozygotes do not show abnormalities in somatotrope growth or function, the ability of a mutant PKARIA to predispose pituitaries to adenoma formation was recently suggested by the observation that somatotrope-directed inactivation of PKARIA results in an increase in the number of GH-producing microadenomas late in life [18 months (13)].

In the spontaneous and experimental disease states outlined above, aberrant activation of the GHRH receptor (GHRH-R)/cAMP/PKA pathway results in somatotrope hyperplasia and subsequent adenoma formation. In that pituitary tumors form relatively late in life with less than 100% penetrance, it can be assumed that hyperactivation of the GHRH-R/cAMP/PKA pathway predisposes the pituitary somatotrope to tumor development but cannot independently transform the somatotrope to the neoplastic state. Therefore, secondary events are required. Although a plethora of information has accumulated regarding the differences between adenomatous and normal pituitary tissue in humans, it is difficult to obtain tissue in the hyperplastic stage, and due to this limitation, important events may be missed that are key to understanding somatotropinoma initiation and/or progression, associated with hyperactivation of the GHRH-R/cAMP/PKA pathway. To circumvent this problem, we used the metallothionein promoter-human GHRH transgenic (MT-hGHRH, subsequently referred to as Tg) mouse, as a model of ectopic GHRH-mediated pituitary tumorigenesis (14,15,16,17,18,19). We previously reported that the appearance of pituitary tumors in Tg mice is preceded by a prolonged static pituitary growth phase that is histologically characterized by hyperplastic/hypertrophic GH-producing cells (18). When tumors do form, they appear relatively late in life (>8 months) with incomplete penetrance. These experimental findings, coupled with clinical observations, suggest that counterregulatory mechanisms quell aberrant GHRH-R/cAMP/PKA-stimulated somatotrope proliferation, and these mechanisms may converge to generate a balance between cell proliferation and programmed cell death (apoptosis). Therefore, in the current report, we sought to identify the counterregulatory mechanism(s) that may slow adenoma development in Tg mice by assessing anterior pituitary cell proliferation rate by 5-bromo-2′-deoxyuridine (BrdU) incorporation and assessing mRNA expression levels of putative tumor suppressors, by quantitative real-time RT-PCR (qrtRT-PCR), with a particular emphasis on the role that endogenous somatostatin (SST) signaling may play in this process. In addition, membrane gene arrays were used to identify novel genes that are dramatically up-regulated or down-regulated in the hyperplastic and adenomatous state, as targets for future studies.

Materials and Methods

Animals

All experimental procedures were approved by the animal care and use committees of the University of Illinois at Chicago. Tg mice were originally obtained from Dr. Ralph L. Brinster (14,15) and maintained as heterozygotes on a C57BL/6J background. Offspring positive for Tg were identified by PCR genotyping at weaning and the genotype confirmed by an increase in body weight relative to wild-type (WT) littermates, as previously described (19). Four months represents an age at which the pituitaries of Tg mice display clear signs of somatotrope hyperplasia and hypertrophy without macroscopic or microscopic signs of adenoma; therefore, a subset of mice were used at 4 months to assess anterior pituitary cell proliferation rate by in vivo BrdU incorporation or pituitaries were frozen for subsequent qrtRT-PCR of mRNA for a proliferation marker (Ki67), SST receptors, and putative tumor suppressor genes (men1 and p27). The remaining mice were allowed to age and monitored closely for early signs of pituitary tumor enlargement (rapid weight loss, ataxia, lethargy, rough hair coat, and/or deformation of the skull). Some Tg mice began to show signs of morbidity after 10 months of age and were killed and their pituitaries examined for macroscopic signs of adenomas. Pituitaries from aged Tg mice with adenomas and age/sex-matched WT controls were frozen and used for mRNA expression analysis.

Mice carrying the MT-hGHRH transgene, but lacking the endogenous SST allele, were generated by cross-breeding heterozygote Tg mice to SST−/− mice, originally obtained from Dr. Ute Hochgeschwender (20) and maintained on a C57BL/6 background. Genotype of the offspring was assessed by PCR (19,21). Tg+/−,SST+/− mice were then cross-bred to SST−/− or SST+/− mice, and the pituitaries of the offspring were collected at 4 months of age and fixed, weighed, and processed for general histology, in which a subset of male mice were infused with BrdU to assess pituitary cell proliferation rate.

BrdU incorporation

To mark cells undergoing DNA synthesis, mice were implanted, sc, with osmotic minipumps (Alzet osmotic pumps; Durect Corp., Cupertino, CA) containing BrdU (Sigma-Aldrich, St. Louis, MO; 20 mg/ml PBS, release rate of 20 μg/h) and killed 72 h later. Pituitaries were fixed in methacarn (22), paraffin embedded, sectioned (5 μm), and immunostained for BrdU using the Dako ARK peroxidase kit (Dako North America Inc., Carpinteria, CA) in conjunction with an anti-BrdU monoclonal antibody (Becton Dickinson, Franklin Lakes, NJ), and the sections were counterstained with hematoxylin. The proportion of BrdU-positive cells was determined by counting greater than 2000 cells/section, two sections per pituitary. As a positive control, a portion of the liver was also collected and similarly processed for BrdU incorporation.

p27 immunocytochemistry

p27 immunocytochemistry was performed after antigen retrieval using 10 mm sodium citrate and heat (85 C, 10 min). Pituitary sections were then sequentially treated with normal goat serum, mouse monoclonal antibody to Kip1/p27 (1:500, 4 C overnight; BD Bioscience, San Jose, CA), and biotinylated secondary antibody (Vectastain ABC reagent; Vector Laboratories, Burlingame, CA) and diaminobenzidine (Sigma).

Pituitary cell cultures

Pituitaries of male Tg or WT mice (4 months of age) were dispersed into single cells and cultured (50,000 cells/well, 24-well plates) in serum containing medium, as previously described (23). After 24 h of culture, medium was removed and wells were washed in serum-free medium, and WT and Tg pituitary cells were subsequently treated with hGHRH (10 nm; Sigma) for 4 h to evaluate GH release into the medium or 15 min for intracellular cAMP determinations. GH and cAMP determinations were performed as previously described (24). In addition, primary pituitary cell cultures from WT mice (100,000 cells/well, 24-well plates) were treated for 24 h with hGHRH (10 nm), SST,1–14 (10 nm; Phoenix Pharmaceuticals, Burlingame, CA) or IGF-I (10 nm; Sigma), and total cellular RNA was extracted for determination of SST receptor expression by qrtRT-PCR.

Quantitative assessment of mRNA levels

Whole pituitaries or cultured cells were processed for recovery of total RNA, reversed transcribed, and select cDNA transcripts amplified by qrtRT-PCR as previously described (21,23). Specific primer sequences, GenBank accession numbers, and product sizes are provided as supplemental information (Table S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

To control for variations in the amount of RNA used in the reverse transcription reaction and the efficiency of the reverse transcription reaction, the expression levels (copy number) of five commonly used housekeeping genes (HKGs), glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine ribosyltransferase (HPRT), β-actin, cyclophilin A, and 18S, were determined for each sample. The absolute mRNA copy number for each HKG tested in male pituitary samples is provided as supplemental Table S2. ANOVA, using Newman-Keuls for post hoc comparisons demonstrated that glyceraldehyde-3-phosphate dehydrogenase, β-actin, and cyclophilin were significantly reduced in Tg pituitaries compared with controls, whereas the mean levels of HPRT and 18S did not significantly differ between experimental groups (refer to supplemental Table S2). Similar results were observed in females (data not shown). Therefore, only HPRT and 18S copy numbers were used to calculate a normalization factor using the GeNorm 3.3 visual basic application for Microsoft Excel (http://medgen.ugent.be/∼jvdesomp/genorm) [Vandesompele et al. (25)]. Within each sample, the copy number for the gene of interest was divided by the normalization factor and the data presented as the mean ± sem of the adjusted values, within group.

Membrane gene arrays

Membrane arrays were purchased from CLONTECH Laboratories (Mountain View, CA; atlas array count no. 7741-1). In each experiment two blots were separately hybridized overnight with radiolabeled cDNA generated from either three WT pituitaries and two hyperplastic pituitaries or two hyperplastic pituitaries and one adenomatous (>100 mg) pituitary. Radiolabeled cDNA was generated by reverse transcription of 6 μg of total RNA (for each sample) in the presence of [α-33P]dATP and the cDNA products column purified according to the manufacturer’s instructions. The membranes were then washed and exposed to a phosphorImage screen (72 h) and the signal intensity determined using the ImageQuant software (Molecular Dynamics Inc., Sunnyvale, CA). The experiment comparing hyperplastic vs. adenomatous tissue was repeated using RNA from different pituitary extracts. Signals within each blot were corrected by subtracting local background. Only signals that exceeded 500 pixel density above background were considered for further analysis and these signals represented 192 of the 588 genes spotted on the array. Signals were then normalized using two separate methods: 1) dividing each by the combined signal of ubiquitin and S29 [HKGs] and 2) dividing by the total (cumulative) signal of the blot (a method used in many microarray analysis systems). After correction within each blot, the relative expression level of each gene in the hyperplastic sample was compared with that of the WT sample or the relative expression level of each gene in the tumor sample was compared with that of the hyperplastic sample. Signals that were reduced to less than one third or increased by more than 3-fold using both correction methods are listed in Table 1. For those genes with the most dramatic change, the results were confirmed by qrtRT-PCR as described above.

Table 1.

Screening for differential gene expression by membrane arrays

Gene name
Array position
Genbank/gene ID
Primary function
Δa
Relative changes in gene expression in hyperplastic vs. WT pituitaries
Caspase 1 (Casp1) F7a L28095/12362 Initiates apoptosis 6.08b
Slc6a11 solute carrier family 6 member 11 E5g L04662/243616 GABA transporter 6.80b
Relative changes in gene expression in adenomatous vs. hyperplastic pituitaries
Mitogen-activated protein kinase kinase1 (MAP2K1) B6a L02526/26395 Signal transduction, associated with cell survival 0.11b
Lysosomal-associated membrane protein (LAPTM4a) B2d U34259/17775 Function not yet defined 0.15b
Nuclear factor erythroid derived 2, like 2 (NRF2) D5 h U20532/18024 Transcription factor activated by antioxidants (carcinogens) 0.19
Ras-related protein (RAB2A) B7b X95403/59021 Vesicular trafficking 0.19
Adenomatosis polyposis coli binding protein (Mapre1) A1e U51196/13589 Micotubule stability 0.19
Tight junction protein 1 (TJP1) A2d D14340/21872 Cell-cell interaction 0.20
Heat shock protein 84 (Hsp90ab1) B1c M36829/15516 Stress defense, protein folding aggregation and trafficking 0.21
Glutathione S-transferease, mu2 (GSTM2) C2b J04696/14863 Activated by antioxidants (carcinogens) 0.22
Heat shock protein 86 (Hsp90aa1) B1d M36830/15519 Stress defense, protein folding aggregation and trafficking 0.27
Proliferating cell nuclear antigen (PCNA) C7b X53068/18103 DNA replication and processing 3.72
Expressed in non-metastatic cells 2 (NME2) C4c X68193/18103 Nucleoside diphosphate kinase 4.29
Zyxin (ZYX) B7n X99063/22793 Actin filament assembly 5.98
Tumor necrosis factor receptor 1b (TNFR2) C5d M59378/21938 Expressed largely in cells of the vascular compartment, initiates apoptosis 7.55b
Open in a new tab

GABA, γ -Aminobutyric acid. 

a

The values represent the average fold changes of the two separate experiments for hyperplastic vs. adenomatous pituitaries and the changes observed in a single experiment comparing hyperplastic vs. WT pituitaries. 

b

Differential gene expression confirmed by qrtRT-PCR (see supplemental Fig. S2B). 

Statistical analysis

Raw data were evaluated for heterogeneity of variance and where found, values were log transformed. Comparison of the effect of genotype and age on pituitary mRNA expression levels, BrdU labeling, and pituitary weight, and the impact of in vitro GHRH treatment on GH release and cAMP accumulation, was assessed by ANOVA, followed by Newman-Keuls post hoc test. In the case of the impact of in vitro treatment of WT pituitary cell cultures on SST receptor mRNA levels, differences between vehicle- and hormone-treated groups were assessed by unpaired Student’s t test. All values are expressed as mean ± sem. All statistical analyses were performed using the GB-STAT software package (Dynamic Microsystems, Inc., Silver Spring, MD).

Results and Discussion

The proliferation rate of hyperplastic Tg pituitaries does not differ from WT controls

Consistent with the predicted proliferative actions of GHRH, pituitaries of 2-month-old Tg mice are 2–3 times the size of WT controls due to a generalized expansion of the number and size of somatotropes (14,15,16,17,18). However, our laboratory previously reported pituitary weights of Tg mice do not appreciably change between 2 and 8 months of age, whereas pituitary weight dramatically increases as tumors form between 10 and 12 months of age in most (but not all) mice (18). If GHRH stimulates somatotrope proliferation, why do pituitary weights in adult Tg mice remain relatively constant until older than 8 months of age? Several possibilities could explain these seemingly incongruent observations. First, expression of the hGHRH transgene during early pituitary development could result in expansion of the somatotrope stem cell population, but the adult somatotropes may become desensitized to the proliferative actions of excess GHRH. Second, the normal somatotrope may be capable of only a finite number of divisions and suffers replicative senescence in an environment of chronic GHRH stimulation. Alternatively, GHRH hyperstimulation could result in an increase in somatotrope proliferation independent of age, but the increase in the rate of proliferation is counterbalanced by an increase in the rate of cell death in the adult gland.

To begin to distinguish among these possibilities, in the current study, we assessed the proliferation rate of anterior pituitary cells of WT and Tg mice at 4 months of age by determining the level of Ki67 mRNA, which is expressed in late G1 to G2, as well as determining the proportion of cells labeled with BrdU, as a direct marker of DNA synthesis. Ki67 expression declined with age in normal pituitaries in both male and female mice, but these differences reached statistical significance only in females (Fig. 1A). Compared with age-matched WT controls, Ki67 mRNA levels in 4-month-old Tg mice were increased to 150% in males and 187% in females (P < 0.05, as assessed by Student’s t test), consistent with a previous report showing pituitaries of 2-month-old Tg mice have more Ki67 immunopositive cells compared with WT controls (26). As would be predicted, Ki67 mRNA levels were greater in adenomatous pituitaries taken from mice older than 10 months of age, compared with WT and hyperplastic pituitaries. Despite these observations, there was no significant difference in the proportion of BrdU-labeled pituitary cells in WT and Tg pituitaries from 4-month-old male mice (Fig. 1, B and C). However, we did observe a dramatic increase in BrdU labeling of hepatocytes (Fig. 1, B and C), consistent with the increase in the number of mitotic figures previously observed in the livers of Tg mice (17), thereby confirming the technique used is capable of detecting an increase in cells that have undergone DNA synthesis. Taken together these observations suggest hyper-GHRH stimulation in young adult mice may promote entry of pituitary cells into G1 (as indicated by enhanced Ki67 expression), but progression to S phase may be delayed or blocked.

Figure 1.

Figure 1

Open in a new tab

A, Ki67 expression in the pituitaries of male and female Tg mice at 4 (hyperplastic) and older than 10 (adenomatous) months of age and age-matched WT controls (n = 5–10 mice/group). B, Percent BrdU-labeled cells in 4-month-old WT pituitaries (n = 7) and livers (n = 3) and 4-month-old Tg pituitaries (n = 10) and livers (n = 3). C, Representative micrographs of BrdU immunostaining in 4-month-old WT and Tg pituitaries and livers. @, BrdU immunopositive pituicyte; †, BrdU immunopositive hepatocyte; #, BrdU immunopositive lymphocyte. D, men1 and p27 mRNA levels in pituitaries of WT and Tg male mice at 4 and older than 10 months of age (n = 5–10 mice/group). E, Representative examples of pituitary p27 immunostaining in WT and Tg male mice. The dotted line shown in the lower panel demarcates the transition area between hypertrophic/hyperplastic somatotropes (right) and adenomatous cells (left). a, Values that differ from age-matched WT controls (P < 0.05) and asterisks (*, P < 0.05; **, P < 0.01) indicate values that significantly differ between age within genotype.

mRNA levels of the putative pituitary tumor suppressors, menin, and p27, are reduced in adenoma- bearing pituitaries

men1

A subset of patients with multiple endocrine neoplasia type I (MENI) develop pituitary adenomas, in which about 9% are GH producing (10). It is now appreciated that the primary defect in MEN1 is inactivating mutations in the MEN1 gene, which encodes for a putative tumor suppressor protein referred to as menin. It is possible that a rise in menin expression may serve to slow tumor progression based on the observation that MEN1 mRNA levels were reported to be increased in GH-producing sporadic human adenomas, using conventional RT-PCR techniques (27). However, in the current study, evaluation of menin expression by qrtRT-PCR revealed men1 mRNA levels did not differ between WT and hyperplastic pituitaries but were significantly reduced in pituitaries bearing adenomas (Fig. 1D, top panel). Therefore, we might speculate that suppression of menin expression may contribute to adenoma formation and/or progression in this model system. However, the low incidence of GH-producing tumors in MEN1 patients suggests additional factors are involved.

p27

The exact mechanism by which menin might serve as a pituitary tumor suppressor remains to be clarified, but there is evidence that it may be linked to regulation of the cell cycle inhibitor, p27. As previously reviewed (28), MEN1 tumors show decreased p27 expression compared with normal endocrine cells, the expression of p27 protein is down-regulated in human pituitary adenomas, and knockout of p27 accelerates somatotropinoma formation in Tg mice (19). In the current study, the expression of p27 followed the same pattern as that for menin (Fig. 1D, lower panel), in which p27 mRNA levels did not differ between WT and hyperplastic pituitaries but were significantly reduced in adenomatous pituitaries, thereby supporting a link between menin and p27.

It is interesting to note that p27 nuclear staining was reduced in adenomatous cells, compared with the surrounding hyperplastic/hypertrophic cells in Tg pituitaries (Fig. 1E), consistent with the suppression of p27 mRNA levels, between the hyperplastic (4 months) and the neoplastic state (>10 months). However, nuclear immunostaining for p27 was more intense in hyperplastic 4-month-old, Tg pituitaries compared with age-matched WT controls. Given these early changes occurred independent of changes in p27 mRNA levels suggests that posttranscriptional activation of p27 might help to maintain pituitary mass in the hyperplastic state.

Alteration in GHRH sensitivity does not appear to be involved in regulating somatotrope hyperplasia in Tg mice

Our laboratory previously reported that pituitary GHRH-R mRNA levels do not differ between WT and Tg mice at 2 months of age (29), and this observation was confirmed in 4-month-old mice (data not shown). The integrity of GHRH signaling was supported in the current study showing primary pituitary cell cultures from WT and Tg mice were equally responsive to the stimulatory actions of GHRH, as demonstrated by the amount of GH released (4 h) and intracellular cAMP accumulated (15 min) after GHRH treatment (Fig. 2). Consistent with these observations, GHRH-R mRNA and GHRH-R protein levels in human GH-producing adenomas do not significantly differ from that observed in normal pituitary tissue (30,31). Therefore, it is unlikely that suppression of pituitary GHRH-R synthesis or sensitivity contributes to the static pituitary growth observed between 2 and 8 months of age in the Tg mouse. However, it should be noted that basal cAMP levels of Tg pituitary cell cultures were only half of that observed in WT cultures (Fig. 2, lower panel). The reduced levels of intracellular cAMP could be the result of a decrease in the rate of cAMP synthesis due to a reduction in AC activity and/or the consequence of an increase in the rate of cAMP degradation brought about by an increase in phosphodiesterase activity, as observed in gsp-positive tumors (32). One or both of these mechanisms may act to reduce the chronic effects of GHRH, thereby slowing pituitary growth.

Figure 2.

Figure 2

Open in a new tab

Basal and GHRH-stimulated GH release (4 h) and intracellular cAMP generation (15 min) in primary pituitary cell cultures from 4-month-old WT and Tg (hyperplastic) male mice (n = 3 separate experiments). Data are adjusted to account for an increase in the proportion of somatotropes, 55% (WT) and 75% (Tg) as previously reported (18). a, Values that differ from vehicle-treated controls (P < 0.01), within genotype, and asterisks (**, P < 0.01) indicate values that significantly differ between WT and Tg cultures within treatment.

Alterations in SST synthesis and signaling are associated with somatotrope hyperplasia and adenoma formation

SST receptor expression

A role for endogenous SST signaling in suppressing hyper-GHRH-mediated somatotrope expansion is supported by our previous observations that hypothalamic SST and pituitary SST receptors, sst2 and sst5, are increased in the 2-month-old Tg male mice, compared with WT controls (29). Such a rise in SST tone could serve to antagonize the actions of GHRH because SST or its synthetic analog, octreotide, activates Giα to inhibit AC activity, reduces intracellular cAMP signaling (33), and regulates cAMP-independent pathways (34). Indeed, SST has been shown to inhibit GHRH-mediated stimulation of somatotrope proliferation in primary rat pituitary cell cultures (35) and cause a transient GO/G1 block in the rat GH-producing cell line, GH3 (36). Also, long-acting SST analogs have been shown to decrease the size of GH-secreting tumors in humans (37,38).

In the current study, we extended our previous observations and assessed the mRNA levels of pituitary SST receptor subtypes (sst1, sst2, sst3, sst4, sst5) of Tg male and female mice at 4 months (without adenoma) and older than 10 months (with adenomas) of age and compared them with that of age-matched WT controls. As shown in Fig. 3, both sst2 and sst5 mRNA levels were elevated in Tg pituitaries at 4 months of age, consistent with previous results (29). At older than 10 months, sst5 expression declined to WT levels in both genders, whereas sst2 expression was reduced, only in pituitaries of female Tg mice. In contrast, expression of sst1 was suppressed in Tg pituitaries at 4 months and declined further in adenomatous pituitaries. Expression of sst3 did not change with genotype or age and sst4 mRNA levels were below the sensitivity of the assay in all samples tested (data not shown). The current observations, taken together with previous reports, suggest that an increase in SST synthesis and signaling may help contribute to the maintenance of pituitary mass in Tg mice between 2 and 8 months of age, whereas a reduction in SST signaling may favor the transition from the hyperplastic to the adenomatous state. This hypothesis is supported by the recent findings that in a heterologous in vitro cell system, sst2 agonists are more efficacious in suppressing AC activity and inducing p27 in cells that express both sst2 and sst5 (39).

Figure 3.

Figure 3

Open in a new tab

SST receptor subtype (sst1, sst2, and sst5) mRNA levels in Tg male and female mice at 4 (hyperplastic) and older than 10 (adenomatous) months of age and age-matched WT controls (n = 5–10 mice/group). a, Values that differ from age-matched WT controls (P < 0.01) and asterisks (*, P < 0.05; **, P < 0.01) indicate values that significantly differ between age within genotype. It should be indicated the expression of sst3 did not significantly differ across age or genotype (data not shown), and sst4 expression levels were below the sensitivity of the assay (<10 copies).

Because it has been previously reported that somatotropes preferentially express sst2 and sst5 (40), the rise in sst2 and sst5 expression in 4-month-old Tg pituitaries, compared with age-matched WT controls (Fig. 3), could be due in part to the relative increase in the contribution of somatotropes, which represent about 75% of the pituitary cell types in Tg male pituitaries compared with about 55% in WT pituitaries (18). However, the magnitude of change in sst2 and sst5 expression in 4-month-old Tg pituitaries, compared with WT controls, cannot solely be accounted for by an increase in somatotrope numbers but also to an increase in sst2 and sst5 mRNA on a per-cell basis. To determine whether hyper-GHRH stimulation can directly contribute to these changes or whether GHRH indirectly impacts SST receptor expression by GH-mediated increases in SST or IGF-I input (18), pituitary cell cultures from WT male mice were treated with GHRH, SST, or IGF-I for 24 h and sst1, sst2, and sst5 mRNA levels were assessed by qrtRT-PCR. As shown in Fig. 4, GHRH increased sst1 and sst2 mRNA levels 2- and 4-fold, respectively, but suppressed sst5 expression, compared with vehicle-treated controls (shown by the dotted line set at 100%). IGF-I also enhanced the expression of sst1 and sst2 as well as sst5. In contrast, SST treatment had no effect in this regard.

Figure 4.

Figure 4

Open in a new tab

In vitro regulation of sst1, sst2, and sst5 by GHRH, IGF-I, and SST (24 h cultures of WT pituitaries, three to four wells/treatment; n = 3–4 separate experiments). Asterisks (**, P < 0.01) indicate values that significantly differ from controls (shown by the dotted line set at 100%).

These in vitro findings, coupled with the changes in SST receptor expression profile in hyperplastic and adenomatous Tg pituitaries, implicate a direct action of GHRH excess in promoting sst2 expression, whereas IGF-I may serve to augment this effect. However, in vivo changes in pituitary sst1 and sst5 expression cannot be simply explained by the direct actions of GHRH, IGF-I, or SST.

Endogenous SST

The question remains, does elevated SST tone play a significant role in slowing adenoma formation in the presence of hyper-GHRH stimulation? To directly answer this question, we examined pituitary size and BrdU uptake in 4-month-old, SST knockout mice with and without the MT-hGHRH Tg. As shown in Fig. 5A, loss of SST did not alter pituitary weight in the absence of Tg, consistent with our recent observations that pituitary cell numbers and the proportion of GH immunopositive cells do not differ between SST−/− and WT controls (21). Expression of Tg more than doubled pituitary weight, in which the pituitaries of both female and male SST−/−, Tg mice were about 25% larger than those of SST+/+, Tg mice. In addition, pituitaries of female SST+/−, Tg mice were larger than that of SST+/+, Tg mice. Histological evaluation revealed multiple adenomas in one of three pituitaries from male SST−/−, Tg mice (refer to supplemental Fig. S1), whereas pituitaries from female SST−/−, Tg mice showed somatotrope hyperplasia but remained adenoma free (data not shown). A subset of male mice were infused with BrdU before the animals were killed and the pituitaries were processed for BrdU immunostaining. As shown in supplemental Fig. S1, there were multiple BrdU-labeled cells within the adenomas of one male SST−/−, Tg mouse. However, examination of the proportion of BrdU-labeled cells in the surrounding hyperplastic tissue of this pituitary, as well as those without adenomas, showed that the presence or absence of SST did not alter the proportion of cells that underwent S phase (Fig. 5B). Taken together, these observations suggest endogenous SST does play a role in suppressing the expansion of the somatotrope population and slowing adenoma formation in response to ectopic GHRH excess. However, the mechanism by which SST acts, at least in the adult gland, appears to be proliferation independent and therefore may be related to alterations in cell senescence and apoptotic pathways.

Figure 5.

Figure 5

Open in a new tab

Impact of SST knockout on pituitary weight of male and female mice (A) and percent BrdU-labeled cells in male mice (B) in the presence or absence of Tg (excluding adenomas that formed in one SST−/−,Tg mice (refer to supplemental Fig. S1). Asterisks (*, P < 0.05) indicate values that significantly differ between SST+/+ mice in the absence (WT) or presence (Tg) of the MT-GHRH transgene.

Use of membrane arrays to screen for changes in gene expression that may contribute to GHRH-mediated somatotrope hyperplasia and adenoma formation

Summarized in Table 1 are the genes that were shown by membrane array to be at least 3-fold reduced, or 3-fold elevated, in hyperplastic Tg pituitaries compared with age-matched (4 months) WT pituitaries and adenomatous compared with hyperplastic Tg pituitaries. Supplemental Fig. S2A shows an enlargement of comparable areas in membrane arrays hybridized with radiolabeled cDNA from hyperplastic or adenomatous pituitary extracts, which includes two genes (TNFR1b, NME2) that were up-regulated and one gene (GSTM2) that was down-regulated in tumor-bearing Tg pituitaries. It should be noted that the sensitivity of the membrane array is limited, and therefore only those genes that are expressed at high levels are detected, in which about 30% of those represented were above background levels and only a small subset of these showed differential regulation based on our conservative criteria. To confirm these findings, qrtRT-PCR was used to screen for those genes showing the most dramatic changes (CASP1, Slc6a11, MAP2K1, LAPTM4a, and TNFR2), using the same cDNA generated from male pituitaries shown in Figs. 1 and 3, and the results are shown in supplemental Fig. S2B. It should be noted that adenomatous pituitaries used for qrtRT-PCR expression analysis were far smaller (20–40 mg), compared with those samples used for the membrane array (>100 mg), and therefore represent an earlier stage in adenoma progression. Nonetheless, using this more sensitive and quantitative methodology, we confirmed the directional changes revealed by the membrane array and extended these findings by simultaneously comparing gene expression between genotypes and across age groups. These findings are preliminary and require detailed followed-up studies to establish their physiological significance; however, it is noteworthy that none of the differentially regulated genes would be predicted to promote adenoma formation and/or progression, and in fact, many of the down-regulated genes are associated with cell survival, whereas many of the up-regulated genes are associated with apoptosis, consistent with delayed appearance and slow progression of pituitary adenomas.

Summary and conclusions

Constitutive activation of the GHRH-R or its downstream signaling components is associated with hyperplasia of the pituitary somatotrope population, in which adenomas form relatively late in life, with less than 100% penetrance. The term hyperplasia indicates that abnormal multiplication or increase in the number of normal cells has occurred, but normal arrangement of the tissue is maintained. The results of the current study showing that the proliferation rate did not differ between hyperplastic Tg and WT pituitaries, coupled with previous findings demonstrating pituitary weights of Tg mice are maintained between 2 and 8 months of age (18), indicates that the proliferative actions of excess GHRH is limited to the expansion of the somatotrope population during early pituitary development, whereas the adult somatotrope becomes desensitized to this process. Evidence is presented that p27, a cell cycle inhibitor, may be important in this process. By extension, these results suggest that the adenomas that initiate from a background of excess GHRH signaling are not simply due to an increase in the number of genetic mutations that would be predicted to occur if proliferation rate was chronically enhanced. However, once a transforming mutation does occur, an environment of excess GHRH may still serve to promote tumor progression.

Desensitization of the somatotrope to the proliferative action of excess GHRH could not be attributed to loss of GHRH-R expression or immediate downstream signaling. However, our previous observations of enhanced hypothalamic expression of SST (18), coupled with an increase in sst2 and sst5 expression in hyperplastic pituitaries of Tg mice suggested an increase in endogenous SST tone may play a role in slowing the proliferative process. Unexpectedly, hyperplastic pituitary tissue from Tg mice lacking endogenous SST did not show an increase in the rate of cellular proliferation compared with SST-intact Tg mice, despite the fact that pituitaries were larger and adenomas formed earlier in the absence of SST. These results indicate that endogenous SST controls GHRH-induced adenoma formation primarily via modulation of apoptotic and/or cellular senescence pathways, consistent with the predicted function of some of the most differentially expressed genes (Casp1, MAP2K1, TNFR2) identified by membrane arrays and confirmed by qrtRT-PCR.

Supplementary Material

[Supplemental Data]
en.2008-1482_index.html (2.3KB, html)

Acknowledgments

The authors thank Dr. Ralph L. Brinster (University of Pennsylvania, Philadelphia, PA) for the original MT-hGHRH Tg mice and Dr. Ute Hochgeschwender (Oklahoma Medical Research Foundation, Oklahoma City, OK) for the SST knockout mice. We would also like to thank Dr. Steven Swanson (University of Illinois at Chicago) for advice on fixation and processing for optimal BrdU immunostaining.

Footnotes

This work was supported by Grant RYC-2007-00186 from the Programa Ramon y Cajal del Ministerio de Educación y Ciencia and Grant BFU2008-01136/BFI, Spain (to R.M.L.); a grant from the Kabbes Foundation for undergraduate research (to S.K.); Grant FI06/00804Ayudas Predoctorales de Formacion en Investigacion en Salud del Fondo de Investigación Sanitaria, Spain (to J.C.-C.); National Institute of Diabetes and Digestive and Kidney Diseases Grant 30677 and the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development Merit Award (to R.D.K.).

Current address for B.S.S.: Department of Pharmacology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

Current address for X.P.: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60612.

Disclosure Summary: The authors have nothing to disclose.

First Published Online April 2, 2009

Abbreviations: AC, Adenylyl cyclase; BrdU, 5-bromo-2′-deoxyuridine; CNC, Carney complex; GHRH-R, GHRH receptor; HKG, housekeeping gene; HPRT, hypoxanthine ribosyltransferase; MENI, multiple endocrine neoplasia type I; MT-hGHRH, metallothionein promoter-human GHRH transgenic; PKA, protein kinase A; PKARIA, PKA regulatory subunit Iα; qrtRT-PCR, quantitative real-time RT-PCR; SST, somatostatin; Tg, transgenic; WT, wild type.

References

  1. Mayo KE, Miller TL, DeAlmeida V, Zheng J, Godfrey PA 1996 The growth-hormone-releasing hormone receptor: signal transduction, gene expression and physiological function in growth regulation. Ann NY Acad Sci 805:184–203 [DOI] [PubMed] [Google Scholar]
  2. Frohman LA, Kineman RD 1999 Growth hormone-releasing hormone: discovery, regulation, and actions. In: Kostyo J, ed. Handbook of physiology: hormonal control of growth. New York: Oxford University Press; 189–221 [Google Scholar]
  3. Billestrup N, Swanson LW, Vale W 1986 Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA 83:6854–6857 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Thorner MO, Frohman LA, Leong DA, Thominet J, Downs T, Hellman P, Chitwood J, Vaughan JM, Vale W 1984 Extra-hypothalamic growth hormone-releasing factor (GRF) secretion is a rare cause of acromegaly: plasma GRF levels in l77 acromegalic patients. J Clin Endocrinol Metab 59:846–849 [DOI] [PubMed] [Google Scholar]
  5. Sano T, Asa SL, Kovacs K 1988 Growth hormone-releasing hormone-producing tumors: clinical, biochemical, and morphological manifestations. Endocr Rev 9:357–373 [DOI] [PubMed] [Google Scholar]
  6. Ezzat S, Asa SL, Stefaneanu L, Whittom R, Smyth HS, Horvath E, Kovacs K, Frohman LA 1994 Somatotroph hyperplasia without pituitary adenoma associated with a longstanding GHRH-producing bronchial carcinoid tumor. J Clin Endocrinol Metab 78:555–560 [DOI] [PubMed] [Google Scholar]
  7. Saeger W, Puchner MJ, Lüdecke DK 1994 Combined sellar gangliocytoma and pituitary adenoma in acromegaly or Cushing’s disease. A report of 3 cases. Virchows Arch 425:93–99 [DOI] [PubMed] [Google Scholar]
  8. Thapar K, Kovacs K, Stefaneanu L, Scheithauer B, Killinger DW, Lloyd RV, Smyth HS, Barr A, Thorner MO, Gaylinn B, Laws Jr ER 1997 Overexpression of the growth-hormone-releasing hormone gene in acromegaly-associated pituitary tumors. An event associated with neoplastic progression and aggressive behavior. Am J Pathol 151:769–784 [PMC free article] [PubMed] [Google Scholar]
  9. Wakabayashi I, Inokuchi K, Hasegawa O, Sugihara H, Minami S 1992 Expression of growth hormone (GH)-releasing factor gene in GH-producing pituitary adenoma. J Clin Endocrinol Metab 74:357–361 [DOI] [PubMed] [Google Scholar]
  10. Horvath A, Stratakis CA 2008 Clinical and molecular genetics of acromegaly: MEN1, Carney complex, McCune-Albright syndrome, familial acromegaly and genetic defects in sporadic tumors. Rev Endocr Metab Disord 9:1–11 [DOI] [PubMed] [Google Scholar]
  11. Bertherat J, Chanson P, Montminy M 1995 The cyclic adenosine 3′,5′-monophosphate-responsive factor CREB is constitutively activated in human somatotroph adenomas. Mol Endocrinol 9:777–783 [DOI] [PubMed] [Google Scholar]
  12. Pack SD, Kirschner LS, Pak E, Zhuang Z, Carney JA, Stratakis CA 2000 Genetic and histologic studies of somatomammotropic pituitary tumors in patients with the “complex of spotty skin pigmentation, myxomas, endocrine overactivity and Schwannomas” (Carney Complex). J Clin Endocrinol Metab 85:3860–3865 [DOI] [PubMed] [Google Scholar]
  13. Yin Z, Williams-Simons L, Parlow AF, Asa S, Kirschner LS 2008 Pituitary-specific knockout of the Carney Complex gene Prkar1a leads to pituitary tumorigenesis. Mol Endocrinol 22:380–387 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hammer RE, Brinster RL, Rosenfeld MG, Evans RM, Mayo KE 1985 Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth. Nature 315:413–416 [DOI] [PubMed] [Google Scholar]
  15. Mayo KE, Hammer RE, Swanson LW, Brinster RL, Rosenfeld MG, Evans RM 1988 Dramatic pituitary hyperplasia in transgenic mice expressing a human growth hormone-releasing factor gene. Mol Endocrinol 2:606–612 [DOI] [PubMed] [Google Scholar]
  16. Stefaneanu L, Kovacs K, Horvath E, Asa SL, Losinski NE, Billestrup N, Price J, Vale W 1989 Adenohypophysial changes in mice transgenic for human growth hormone-releasing factor: a histological, immunocytochemical, and electron microscopic investigation. Endocrinology 125:2710–2718 [DOI] [PubMed] [Google Scholar]
  17. Lloyd RV, Jin L, Chang A, Kulig E, Camper SA, Ross BD, Downs TR, Frohman LA 1992 Morphologic effects of hGRH gene expression on the pituitary, liver, and pancreas of MT-hGRH transgenic mice. An in situ hybridization analysis. Am J Pathol 141:895–906 [PMC free article] [PubMed] [Google Scholar]
  18. Kineman RD, Teixeira LT, Amargo GV, Coschigano KT, Kopchick JJ, Frohman LA 2001 The effect of GHRH on somatotrope hyperplasia and tumor formation in the presence and absence of GH signaling. Endocrinology 142:3764–3773 [DOI] [PubMed] [Google Scholar]
  19. Teixeira LT, Kiyokawa H, Peng X-D, Christov KT, Frohman LA, Kineman RD 2000 p27(Kip1)-deficient mice exhibit accelerated growth hormone-releasing hormone-induced somatotrope proliferation and adenoma formation. Oncogene 19:1875–1884 [DOI] [PubMed] [Google Scholar]
  20. Zeyda T, Diehl N, Paylor R, Brennan MB, Hochgeschwender U 2001 Impairment in motor learning of somatostatin null mutant mice. Brain Res 906:107–117 [DOI] [PubMed] [Google Scholar]
  21. Luque RM, Kineman RD 2007 Gender-dependent role of endogenous somatostatin in regulating growth hormone (GH)-axis function in mice. Endocrinology 148:5998–6006 [DOI] [PubMed] [Google Scholar]
  22. McGinley JN, Knott KK, Thompson HJ 2000 Effect of fixation and epitope retrieval on BrdU indices in mammary carcinomas. J Histochem Cytochem 48:355–362 [DOI] [PubMed] [Google Scholar]
  23. Luque RM, Gahete MD, Hochgeschwender U, Kineman RD 2006 Evidence that endogenous somatostatin (SST) inhibits adrenocorticotropin (ACTH) and ghrelin expression by independent pathways. Am J Physiol Endocrinol Metab 291:E395–E403 [DOI] [PubMed] [Google Scholar]
  24. Kineman RD, Kamegai J, Frohman LA 1999 Growth hormone-releasing hormone (GHRH) and the growth hormone secretagogue (GHS), L692,585, differentially modulate rat pituitary GHS receptor (GHS-R) and GHRH receptor (GHRH-R) mRNA levels. Endocrinology 140:3581–3586 [DOI] [PubMed] [Google Scholar]
  25. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3:research0034 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jirawatnotai S, Aziyu A, Osmundson EC, Moons DS, Zou X, Kineman RD, Kiyokawa H 2004 Cdk4 is indispensable for postnatal proliferation of the anterior pituitary. J Biol Chem 279:51100–51106 [DOI] [PubMed] [Google Scholar]
  27. McCabe CJ, Gittoes NJL, Sheppard MC, Franklyn JA 1999 Increased MEN1 mRNA expression in sporadic pituitary tumors. Clin Endocrinol (Oxf) 50:727–733 [DOI] [PubMed] [Google Scholar]
  28. Lytras A, Tolis G 2006 Growth hormone-secreting tumors: genetic aspects and data from animal models. Neuroendocrinology 83:166–178 [DOI] [PubMed] [Google Scholar]
  29. Peng X-D, Park S, Gadelha MR, Coschigano KT, Kopchick JJ, Frohman LA, Kineman RD 2001 The growth hormone (GH)-axis of GH receptor/binding protein gene-disrupted and metallothionein-human GH-releasing hormone transgenic mice: hypothalamic neuropeptide and pituitary receptor expression in the absence and presence of GH feedback. Endocrinology 142:1117–1123 [DOI] [PubMed] [Google Scholar]
  30. Lopes MB, Gaylinn BD, Thorner MO, Stoler MH 1997 Growth hormone-releasing hormone receptor mRNA in acromegalic pituitary tumors. Am J Pathol 150:1885–1891 [PMC free article] [PubMed] [Google Scholar]
  31. Oka H, Kameya T, Sato Y, Naritaka H, Kawano N 1999 Significance of growth hormone-releasing hormone receptor mRNA in non-neoplastic pituitary and pituitary adenomas: a study by RT-PCR and in situ hybridization. J Neurooncol 41:197–204 [DOI] [PubMed] [Google Scholar]
  32. Lania A, Persani L, Ballaré E, Mantovani S, Losa M, Spada A 1998 Constitutively active Gsα is associated with an increased phosphodiesterase activity in human growth hormone-secreting adenomas. J Clin Endocrinol Metab 83:1624–1628 [DOI] [PubMed] [Google Scholar]
  33. Tentler JJ, Hadcock JR, Gutierrez-Hartmann A 1998 Somatostatin acts by inhibiting the cyclic 3′, 5′-adenosine monophosphate (cAMP)/protein kinase A pathway, cAMP response element-binding protein (CREB) phosphorylation, and CREB transcription potency. Mol Endocrinol 11:859–866 [DOI] [PubMed] [Google Scholar]
  34. Ferjoux G, Bousquet C, Cordelier P, Benali N, Lopez F, Rochaix P, Buscail L, Susini C 2000 Signal transduction of somatostatin receptors negatively controlling cell proliferation. J Physiol 94:205–210 [DOI] [PubMed] [Google Scholar]
  35. Bilezikjian LM, Vale WW 1983 Stimulation of adenosine 3′,5′-monophosphate production by growth hormone-releasing factor and its inhibition by somatostatin in anterior pituitary cells in vitro. Endocrinology 113:1726–1731 [DOI] [PubMed] [Google Scholar]
  36. Cheung NW, Boyages SC 1995 Somatostatin-14 and its analog octreotide exert a cytostatic effect on GH3 rat pituitary tumor proliferation via a transient G0/G1 cell cycle block. Endocrinology 136:4174–4181 [DOI] [PubMed] [Google Scholar]
  37. Bevan JS, Melmed S 2005 The antitumoral effects of somatostatin analog therapy in acromegaly. J Clin Endocrinol Metab 90:1856–1863 [DOI] [PubMed] [Google Scholar]
  38. Melmed S, Sternberg R, Cook D, Kilbanski A, Chanson P, Bonert V, Vance ML, Rhew D, Kleinberg D, Barkan A 2005 A critical analysis of pituitary tumor shrinkage during primary medical therapy in acromegaly. J Clin Endocrinol Metab 90:4405–4410 [DOI] [PubMed] [Google Scholar]
  39. Grant M, Alturaihi H, Jaquet P, Collier B, Kumar U 2008 Cell growth inhibition and functioning of human somatostatin receptor type 2 are modulated by receptor heterodimerization. Mol Endocrinol 22:2278–2292 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Day R, Dong W, Panetta R, Kracier J, Greenwood MT, Patel YC 1995 Expression of mRNA for somatostatin receptor (sstr) types 2 and 5 in individual rat pituitary cells. A double labeling in situ hybridization analysis. Endocrinology 136:5232–5235 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental Data]
en.2008-1482_index.html (2.3KB, html)
en.2008-1482_1.pdf (92.7KB, pdf)
en.2008-1482_2.pdf (42.2KB, pdf)
en.2008-1482_3.pdf (15.4KB, pdf)
en.2008-1482_4.pdf (19.9KB, pdf)

Articles from Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES