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High Plasma Growth Hormone (GH) Levels Inhibit Expression of GH Secretagogue Receptor Messenger Ribonucleic Acid Levels in the Rat Pituitary¹

Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia, Charlottesville, Virginia 22908; and Department of Statistics, Ohio State University (M.P.), Columbus, Ohio 43210

Address all correspondence and requests for reprints to: Dr. Michael O. Thorner, Department of Medicine, University of Virginia, Box 800466, Charlottesville, Virginia 22908-0466. E-mail: mot{at}virginia.edu

	Abstract

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Synthetic GH secretagogues (GHSs) act via a receptor (GHS-R) distinct from that of GH-releasing hormone. The GHS-R has been cloned from the pituitary and is expressed not only in the pituitary but also in specific areas of the brain, including the hypothalamus. Recent studies suggest that hypothalamic GHS-R expression is regulated by GH. This study was designed to investigate whether pituitary GHS-R expression is modulated by GH. Female Wistar-Furth rats were injected sc with either saline (control) or GC tumor cells (GC) that secrete rat GH. The tumors were allowed to develop for 1–4 weeks. At weeks 1–4, control (n = 4–8) and GC rats (n = 3–8) were killed. Pituitary GHS-R messenger RNA (mRNA) was measured by a quantitative competitive PCR assay. The endogenous GHS-R mRNA levels were measured by determining the amount of competitive template RNA required to produce equimolar amounts of native and competitive template PCR products. The mean log plasma GH levels were significantly greater in the GC rat group than in the control group at weeks 2, 3, and 4. At these times, the mean log pituitary GHS-R mRNA contents were significantly lower in the GC rat group than in the control group. No relationship could be established between log estradiol levels and GHS-R levels. These data indicate that pituitary GHS-R expression is modulated by GH.

	Introduction

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GH SECRETION is controlled by at least two hypothalamic hormones, GH-releasing hormone (GHRH) which stimulates GH release from the somatotrophs, and somatostatin (SS), which inhibits GH release (1). The synthetic peptide GH secretagogue (GHS), GH-releasing peptide-6, can stimulate GH release by a pathway distinct from that of GHRH, implying the presence of a third potential modulator of GH secretion (2). A natural ligand for the GHS receptor (GHS-R) has recently been discovered (3). Like GHRH and somatostatin, GHS acts via specific cell surface receptors. Howard et al. (4) identified, cloned, and sequenced a GHS-R from human, pig, and, more recently, rat (5). The GHS-R is a G protein-coupled receptor that has its closest identity to the neurotensin and TRH receptors (6). Recent data suggest that the GHS system is complex and appears to involve multiple receptor subtypes. There are at least two GHS-R subtypes expressed in the pituitary. GHS-R type 1a and type 1b result from alternative messenger RNA (mRNA) splicing. Type 1a message encodes a biologically active receptor consisting of a 336-amino acid polypeptide possessing 7 transmembrane domains. Type 1b message encodes a 289-amino acid polypeptide with only 5 transmembrane domains and does not appear to result in a functional receptor (4).

Although the exact physiological role of GHS-R has not been fully established, probable sites of action include both the pituitary and the hypothalamus. The ligands for the GHS-R act directly on somatotrophs to cause GH secretion, amplify the effects of GHRH, and behave as functional antagonists of somatostatin (7). Recent clinical studies suggest that the natural ligand of this receptor has an impact on pulsatility of GH secretion and increases pulse height (8). Studies reported by Bennett et al. (9) suggest that hypothalamic GHS-Rs may be involved in the feedback regulation of GH. It is not known, however, whether regulation of GHS-R at the pituitary is involved in the negative feedback of GH. The objective of the present study was to investigate the expression of pituitary GHS-R mRNA in response to altered GH levels. Using the transplantation of GH-secreting tumor cells to female Wistar-Furth rats, we examined the impact of elevated serum GH levels on GHS-R mRNA expression. To investigate a potential impact of estradiol on the GHS-R mRNA expression, estradiol levels were measured and related to GHS-R mRNA levels in the rat pituitary. To measure GHS-R mRNA expression in the pituitary, we developed a quantitative PCR-based assay.

	Materials and Methods

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Cell culture, tumor growth, and animal model
The GC cell subclone of the rat GH₃ pituitary tumor cell line was grown as a monolayer in culture according to the method described by Laverriere et al. (10). The cells were harvested by treatment with 0.05% trypsin-0.53 mM EDTA, washed twice by centrifugation with Dulbecco’s PBS, and resuspended at approximately 2 x 10⁷ cells/ml in Dulbecco’s PBS.

Female Wistar-Furth rats were maintained in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Rats were injected sc with either saline or the resuspended GC pituitary tumor cells (1 x 10⁷ cells suspended in saline). Only female animals were used, because the GC cells do not develop in male animals. Eight animals from each group were killed weekly. Tumors became palpable between 2 and 3 weeks after injection. Before death, the animals were weighed, then decapitated, and trunk blood was collected. Pituitaries were rapidly excised and immediately frozen in liquid nitrogen. RNA was extracted using a phenol/guanidine thiocyanate single step extraction (11).

Measurement of pituitary GHS-R mRNA
As pituitary GHS-R mRNA is present in low abundance, the solution hybridization method used to quantitate message levels for other receptors lacked the required level of sensitivity. Therefore, we developed a quantitative RT-PCR assay using an established method (12) and demonstrated that it had characteristics (quantitative, sensitive, and specific) that allowed us to measure GHS-R in pituitary. The GHS-R complementary DNA (cDNA) was subcloned using EcoRI-NotI sites into the pcDNA I/Amp-vector (Promega Corp., Madison, WI). An EcoRI/XbaI fragment was then inserted into the HindIII-/XbaI sites of the pSP64 (polyadenylase) vector (Promega Corp.) with the EcoRI site filled and blunt ended into the blunted HindIII site. This cDNA was then cut by restriction enzymes PstI and HindIII to remove a 516-bp segment, and then a 783-bp fragment of unrelated DNA (pBR322) was inserted to create a size-altered, competitive template (CT) cDNA, 267 bp longer than the native GHS-R cDNA. The identity of the CT construct was verified by restriction enzyme digest and partial nucleotide sequencing. As the next step, in vitro sense strand synthesis of polyadenylated RNA was performed. For the assay, quadruplicate samples of CT (1, 10, 50, and 100 fg) and native total RNA (200 ng) were reverse transcribed [50 mM KCl, 10 mM Tris (pH 8.3), 5 mM MgCL2, 5 mM dithiothreitol, 2.5 µM oligo(deoxythymidine) primer, and deoxynucleotide triphosphates (1 mM dATP, dTTP, dGTP, and dCTP)] at 42 C for 45 min (Moloney murine leukemia virus, Life Technologies, Inc.). The reaction was stopped by heating to 99 C for 15 min. To each sample 20 µl of a solution containing 50 mM KCl, 10 mM Tris (pH 8.3), 3.3 mM MgCl₂, and 0.17 µM oligonucleotide primers were added along with 2.5 U/sample Taq polymerase (Promega Corp.; total volume per tube, 30 µl). This method allows for use of the same oligonucleotide primers for amplification of both native and competitive template cDNAs. The sequences of the sense (5') and reverse (3') primers, which hybridize to regions flanking the unrelated DNA insert, are CGT GAA GCT GGT CAT CCT TGT (473–494 bp) and GAA CTC TCA TCC TTC AGA GTG (3096–3074 bp) (4), respectively. Optimal amplification of the 855-bp CT and 588-bp native segments was produced over 35 cycles containing a 94 C denaturation phase (30 sec), a 60 C annealing phase (30 sec), and a 72 C extension phase (60 sec), followed by a final 72 C phase for 5 min. Thirty microliters of product from each tube were loaded onto a 1% agarose gel and electrophoresed for 1 h.

The gels were stained with ethidium bromide and quantitated with the Fluoroimager 595 using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). After establishing the appropriate conditions and linear range, experimental samples were spiked with varying amounts of CT mRNA. The amount of CT mRNA required to give a 1:1 molar ratio of native and CT PCR products was then determined graphically. This gives a measure of the endogenous level of the native GHS-R mRNA. The differences in length of the two templates required a correction factor of 0.69 to convert fluorescence ratios to molar ratios. Assays of in vitro synthesized native template of known concentration gave concentrations in the expected range. The inter- and intraassay coefficients of variation of this assay are 24% and 7%, respectively.

The region of mRNA selected for amplification flanks an intron, so that any amplification of contaminating genomic DNA would result in larger products that would not be confused with those derived from the mRNA. As an additional control, RNA from each sample was amplified in the absence of reverse transcriptase, and no products were detected.

Measurement of plasma GH
GH was measured in each sample in duplicate using a standard RIA with the rat GH reference preparation NIDDK rGH RP-2. Reagents were supplied by the NIDDK. The sensitivity of the assay was 3.5 ng/ml. The interassay coefficient of variation was 14.7% at 1.9 ± 0.3 ng/ml. The intraassay coefficient of variation was 6.6% at 2.9 ± 0.19 ng/ml.

Measurement of plasma estradiol
Estradiol was measured in each sample in duplicate using a commercial RIA kit (Diagnostic Products, Los Angeles, CA). The sensitivity of the assay was 8 pg/ml. The interassay coefficient of variation was 8.1% at 48 pg/ml and 4.2% at 1025 pg/ml. The intraassay coefficient of variation was 7% at 50 pg/ml and 4% at 1082 pg/ml.

Statistical analysis
Four parameters were measured: weight change, plasma GH, pituitary GHS-R mRNA, and plasma estradiol. Forty-eight rats yielded GH measurements that could be analyzed. Due to the early (ribonuclease protection) assay requirements, the pituitary glands of some of these rats had to be pooled to obtain adequate tissue samples.

A preliminary examination of plots of the GH, GHS-R mRNA, and estradiol measurements indicated that their variability was not constant over time and across the two treatment groups. Thus, a logarithmic transformation of the measurements for these three variables was performed. This preliminary transformation of the data is essential. Fitting a linear model to the data on the original scale would violate the basic assumption that the measurements have homogeneous variance and would cause the reported levels of significance to be inaccurate.

For the weight measurements and each of the three transformed responses, statistical analysis was conducted according to the following steps. 1) Compute least squares estimates of the differences in the mean measured levels between the GC-injected and the control rats at weeks 1–4. 2) Compute the SEs of the differences in the mean levels under the assumption of homogeneous variance. 3) Construct 95% simultaneous confidence intervals for the differences in the mean levels using Bonferroni adjustment (13). The weekly differences (GC rat group minus control group) in mean levels and the 95% confidence intervals are displayed in Fig. 7. For each of the three sets of comparisons, the adjustment adequately inflates the width of the intervals to account for the fact that simultaneous inferences were made.

View larger version (29K): [in this window] [in a new window]   Figure 7. Summary of the comparisons made for the weight change, GH, and GHS-R mRNA measurements. Weekly differences (weeks 1–4 after injection of tumor cells or saline) between mean levels in the GC rat group and in the control group, and the Bonferroni-adjusted, 95% simultaneous confidence intervals for the differences in mean levels are shown. Intervals excluding zero indicate a significant difference in mean level between the GC and control groups.

	Results

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View larger version (11K): [in this window] [in a new window]   Figure 1. Plots of the weight changes compared with baseline (in grams) in adult female control (circles) and GC (diamonds) rats at weeks 1–4 after injection of tumor cells. Each horizontal line denotes the mean of the corresponding group of measurements. The greater increase in weight seen in the GC tumor-bearing rats demonstrates the effect of their increased GH levels.

View larger version (10K): [in this window] [in a new window]   Figure 2. Plots of the peripheral GH concentrations (nanograms per ml) in adult female control (circles) and GC (diamonds) rats at weeks 1–4 after injection of tumor cells. Each horizontal line denotes the geometric mean of the corresponding group of measurements.

GHS-R RT-PCR assay characterization
As shown in Fig. 3, we established the linear range of the standard curve by using serial dilutions of competitive template (range, 0.5–1000 fg), with a constant amount of pituitary RNA (200 ng). The PCR reaction was linear between 1 and 100 fg, which we used as the range for the standard curve (Fig. 4). Using this range of CT, we next determined whether linear changes in the concentration of pituitary RNA resulted in linear changes in measured GHS-R mRNA. The GHS-R mRNA measurement was linear from 50–400 ng total pituitary RNA; therefore, we used 200 ng total RNA in subsequent assays (Fig. 5).

View larger version (32K): [in this window] [in a new window]   Figure 3. Ethidium bromide-stained gel after RT-PCR of a representative pituitary mRNA. To determine the linear range of the standard curve we decreased the CT (upper band; 855 bp) in a range from 1000 to 0.5 fg. The native product (NT) is shown as the lower band (588 bp). The amount of CT per reaction is listed at the top of each lane. As the PCR reaction showed the best linearity between 1 and 100 fg, we used this range for the standard curve. (Native T/total T) = molar fraction of native template/total template; total template = CT x 0.69 + NT.

View larger version (26K): [in this window] [in a new window]   Figure 4. RT-PCR of pituitary GHS-R mRNA. A, Ethidium bromide-stained gel after RT-PCR of pituitary mRNA from a control (week 4) and a GC tumor rat (week 4). The CT (upper band) and native product (lower band) sizes are displayed on the left. The amounts of CT per PCR reaction were 100, 50, 10, and 1 fg. B, The semilog plot of the standard curves is shown on the right (control rat, r = 0.99; GC tumor, r = 0.96). The calculated quantity of GHS-R mRNA in the control animal was 17.3 fg, and that in the GC tumor rat was 5.8 fg. (Native T/total T) = molar fraction of native template/total template; total template = CT x 0.69 + NT.

View larger version (13K): [in this window] [in a new window]   Figure 5. RT-PCR GHS-R mRNA quantitation showed linearity from 50–400 ng total pituitary RNA. We used 200 ng total pituitary RNA for subsequent measurements.

View larger version (11K): [in this window] [in a new window]   Figure 6. Plot of the pituitary GHS-R mRNA concentrations (attograms per ng total RNA) in adult female control (circles) and GC (diamonds) rats at weeks 1–4 after injection of tumor cells. Each horizontal line denotes the geometric mean of the corresponding group of measurements.

	Discussion

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We used GC tumor cell transplants to produce constant rat GH hypersecretion. Mounier and colleagues demonstrated that the GH secretion in this animal model is derived entirely from the GC cell implants (17); this is a GH-secreting cell line that does not secrete PRL. Specifically, passive immunization with either SS or GHRH antiserum did not alter plasma GH levels in rats bearing GC tumors, in contrast to the observations in normal rats (17).

We demonstrated that 2 weeks after GC cell transplantation, plasma GH levels were markedly increased and remained greater than control levels through the conclusion of the experiment at 4 weeks. Similarly, IGF-I levels (data not shown) showed a tendency to increase after 2 weeks of transplantation of the GC cells. The in vivo responses to increased GH included an increase in the animal’s body weight, with GC animals gaining more weight than controls at weeks 3 and 4 of treatment.

To measure GHS-R mRNA expression, a quantitative RT-PCR assay was developed. This method has been used to successfully measure changes in other mRNA of low abundance and provides a sensitive method to study physiological variations in gene expression (18). After exposure to high circulating GH levels, pituitary GHS-R mRNA expression was significantly decreased (3.3-fold) at week 2 in animals with GC cell implants. This difference persisted through the 4 weeks of the study.

We could not determine whether the observed changes in pituitary GHS-R mRNA levels in the tumor rats are related to direct GH effects at the pituitary or at the hypothalamus, i.e. whether they are a result of changed concentrations of the natural ligand for this receptor at the hypothalamus. However, Szabo and co-workers (19) showed in transgenic mice that regulation of the pituitary GHRH receptor mRNA through changes in hypothalamic GHRH activity occurs, and this concept could also apply to the GHS system.

In addition, chronic GH hypersecretion induces a decrease in hypothalamic GHRH mRNA content and an increase in hypothalamic SS mRNA content (20), supporting the theory that GH exerts feedback at the hypothalamus. Further kinetic studies are needed to address the question of whether changes at the hypothalamic level, i.e. changes in SS or GHRH, precede the GHS-R mRNA changes observed at the pituitary level in our study.

Yamashita et al. (21) found that IGF-I exerts negative feedback on GH secretion from the pituitary. It is not clear, however, whether IGF-I feeds back on the GHS-R system. Notably, the decline in pituitary GHS-R mRNA expression in our experiment occurred concomitantly with a significant increase in GH and a trend for IGF-I levels to increase (data not shown); due to the variation in IGF-I levels in both the control and GC-implanted animals, this did not reach statistical significance. Whether the inhibition of GHS-R gene expression is a direct result of GH itself or is secondary to increases in IGF-I remains to be determined. However, the results reported by Kamegai and colleagues (14) suggest that the feedback effect of GH at the hypothalamus is more likely. In addition, it has to be considered that the GC tumor cells may produce tumor-specific factors, which could have an impact on the change in GHS-R mRNA expression.

The findings published by Bennett et al. (9) suggest that GHS-R is transcribed differently in the hypothalamus of GH-deficient male and female rats. Data collected in rats show that the 24-h GH profile is significantly different in male and female rats, suggesting a role for estrogen in regulating GH pulsatility (22). In our experiment there was no relation between the measured estrogen levels and GHS-R mRNA expression in either group.

We developed a quantitative RT-PCR method for measuring GHS-R mRNA in the pituitary. We demonstrated that at the level of the pituitary, GHS-R mRNA expression is regulated by GH. These data extend the previous observation of GH regulation of GHS-R in the hypothalamus. Whether this is directly mediated at the pituitary or indirectly through the hypothalamus, i.e. via inhibition of the release of the natural ligand for the GHS-R from the hypothalamus, has yet to be determined. Our findings further support the concept that in addition to the hypothalamus, the pituitary itself is directly involved in the regulation of GH release through the pituitary GHS-R.

	Acknowledgments

 
We thank Dr. A. Howard, Merck Research Laboratories (Rahway, NJ) for providing the GHS-R cDNA. We thank Eric Bissonette for valuable advice about the statistical analysis.

	Footnotes

 
¹ This work was supported in part by NIH Grants U54-HD-28934, RO1-DK-32632, and RO1-DK-45350 and grants from the Deutsche Forschungsgemeinschaft (Na 317/1–1, Na 317/1–2).

Received August 17, 1999.

	References

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nass, R. Articles by Thorner, M. O. Search for Related Content PubMed PubMed Citation Articles by Nass, R. Articles by Thorner, M. O.