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Endocrinology Vol. 140, No. 8 3581-3586
Copyright © 1999 by The Endocrine Society

ARTICLES

Growth Hormone (GH)-Releasing Hormone (GHRH) and the GH Secretagogue (GHS), L692,585, Differentially Modulate Rat Pituitary GHS Receptor and GHRH Receptor Messenger Ribonucleic Acid Levels1

Rhonda D. Kineman, Jun Kamegai2 and Lawrence A. Frohman

Department of Medicine, Section of Endocrinology and Metabolism, University of Illinois at Chicago, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Lawrence A. Frohman, M.D., Department of Medicine (M/C 787), University of Illinois at Chicago, 840 South Wood, Chicago, Illinois 60612. E-mail: frohman{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ability of synthetic GH secretagogues (GHSs) to elicit a maximal release of GH in vivo is dependent on an intact GH-releasing hormone (GHRH) signaling system. The role of GHRH in GHS-induced GH release has been attributed primarily to the ability of GHS to release GHRH from hypothalamic neurons. However, GHS also releases GH directly at the pituitary level. Several lines of evidence suggest that GHRH is necessary to maintain pituitary responsiveness to GHS by stimulating GHS receptor (GHS-R) synthesis. To test this hypothesis, male rats (250–290 g) were anesthetized with ketamine/xylazine (which does not alter pulsatile GH secretion) and infused iv with a GHRH analog ([des-NH2Tyr1,D-Ala15]hGRF-(1–29)-NH2; 10 µg/h) or saline for 4 h. Serum was analyzed for GH, pituitaries were collected, and GHS-R and GHRH receptor (GHRH-R) messenger RNA (mRNA) levels were determined by RT-PCR. GHRH infusion resulted in a 10-fold increase in circulating GH concentrations that were accompanied by an increase in GHS-R mRNA levels to 200% of those in saline-treated controls (P < 0.01). In contrast, GHRH reduced GHRH-R mRNA levels slightly, but not significantly (P < 0.07). The stimulatory effect of GHRH on GHS-R mRNA levels was independent of somatostatin tone, as pretreatment with somatostatin antiserum did not alter the effectiveness of GHRH infusion. In contrast, blockade of somatostatin actions up-regulated GHRH-R mRNA levels under basal conditions and unmasked the inhibitory effects GHRH on its own receptor mRNA. These observations suggest GHRH-R mRNA is tonically suppressed by somatostatin. The stimulatory effect of GHRH on GHS-R mRNA levels was independent of circulating GH, as GHRH infusion in spontaneous dwarf rats, which do not have immunodetectable GH, increased GHS-R mRNA levels to 150% of those in saline-treated controls (P < 0.05). To determine whether this effect occurred by a direct action on the pituitary, primary cell cultures from normal rat pituitaries were incubated with GHRH (0.01–10 nM) or forskolin (10 µM) for 4 h. These GH secretagogues did not alter GHS-R mRNA levels in vitro. However, GHRH and forskolin reduced GHRH-R mRNA levels by 40% (P < 0.05). To determine whether the synthesis of the GHS-R, like that of the GHRH-R, is negatively mediated by its own ligand, anesthetized rats were infused with the nonpeptidyl secretagogue, L-692,585 (100 µg/h) for 4 h. Neither circulating GH (at 4 h) nor GHRH-R mRNA levels were significantly altered by L-692,585, whereas GHS-R mRNA levels were reduced by 50% (P < 0.05). Taken together, these results indicate that GHRH-induced up-regulation of pituitary GHS-R synthesis in vivo is indirect and independent of both somatostatin and GH. They also demonstrate that GHS-R synthesis, like that of GHRH-R, can be rapidly down-regulated by its own ligand.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL lines of evidence indicate that in vivo responsiveness to the synthetic GH secretagogues (GHSs) is dependent on an intact GH-releasing hormone (GHRH) signaling system. First, hypothalamic-pituitary stalk disconnection reduces or completely blocks the response to peptidyl and nonpeptidyl GHSs in both humans and rats (1, 2, 3). Second, pretreatment with GHRH antiserum (4) or a GHRH antagonist (5) substantially reduces the response to a subsequent GHS challenge. Finally, lit/lit mice, which do not respond to GHRH (6) due to a point mutation in the ligand-binding region of the GHRH receptor (GHRH-R) gene (7), are equally unresponsive to GH-releasing peptide-6 (GHRP-6) in vivo (8), and their heterozygous littermates show an intermediate response (9). One proposed link between the GHS and GHRH signaling systems is the ability of GHSs to stimulate hypothalamic GHRH release. In the rat, systemic administration of GHS stimulates neuronal activity within the arcuate nucleus (10, 11) and increases c-fos expression in GHRH-containing neurons (12). Two groups (13, 14) have reported that systemic administration of GHRP-6 (hexarelin) to sheep increased GHRH levels in the portal vascular system. More recently, Tannebaum et al. (15) demonstrated that GHRH neurons express the GHS receptor (GHS-R). Taken together, these observations have led to the conclusion that the primary mechanism by which GHS stimulates GH release is by directly stimulating the release of GHRH from hypothalamic arcuate neurons.

It is also well documented that GHSs release GH by a direct action within the pituitary. GHSs bind to somatotropes by a cell surface receptor that is distinct from the GHRH-R. The GHS-R belongs to the G protein-coupled receptor superfamily and specifically interacts with Gq11 (16). Upon ligand binding, the phospholipase C signaling pathway is activated (17, 18), intracellular Ca2+ stores are released (19), plasma membrane conductance increases, and GH is secreted. Several lines of evidence suggest that GHRH is required to maintain pituitary responsiveness to GHSs. First, pituitary cell cultures that are deprived of hypothalamic influence are less responsive to acute GHS stimulation (alone or in combination with a maximum dose of GHRH) compared with the response observed in vivo (20, 21). Second, hypophysectomized rats with pituitaries transplanted under the kidney capsule maintain responsiveness to GHRP-6 by prior GHRH priming (22). Finally, we have observed a positive association between hypothalamic GHRH messenger RNA (mRNA) and pituitary GHS-R mRNA levels in the spontaneous dwarf rat (23, 24), which has no immunodetectable GH due to a splice site mutation in the GH gene (25). Under basal conditions, spontaneous dwarf rat (SDR) hypothalamic GHRH mRNA and pituitary GHS-R mRNA levels are greater than those in normal controls. Suppression of SDR GHRH mRNA levels by GH infusion (72 h) results in a concomitant suppression of pituitary GHS-R mRNA levels. Taken together, these observations suggest that GHRH can act to augment pituitary responsiveness to GHSs by increasing GHS-R expression. The present experiments were designed to directly test this hypothesis by examining the acute effect of GHRH on GHS-R mRNA levels in vivo and in vitro in the presence or absence of endogenous somatostatin and GH. As GHRH acutely suppresses GHRH-R mRNA levels in vitro (26), we also examined whether GHRH would effectively down-regulate GHRH-R mRNA levels in vivo. In addition, we examined whether the GHS, L-692,585, could modulate GHRH-R and/or GHS-R synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Intravenous infusion of GHRH and L-692,585
Male rats (250–290 g; Harlan Sprague Dawley, Inc., Indianapolis, IN) were anesthetized with ketamine (100 mg/kg)/xylazine (6 mg/kg) anesthesia, and an iv cannula was inserted into the right jugular vein to approximately 30 mm rostral to the right atrium. The anesthetic was chosen because it does not alter pulsatile GH secretion (27). To test the effect of GHRH on pituitary GHS-R and GHRH-R mRNA levels in the absence or presence of endogenous somatostatin, animals were infused for 4 h with either saline (vehicle; n = 10) or the GHRH analog [(des-NH2Tyr1,D-Ala2, Ala15]hGRF-(1–29)-NH2; Dr. R. M. Campbell, Hoffmann-La Roche, Inc., Nutley, NJ; 10 µg/h; n = 10). Half the animals in each group received an iv injection of somatostatin antiserum (0.5 ml/rat; Dr. A. Arimura, Tulane University, Belle Chase, LA) or normal sheep serum (NSS) 5 min before the start of the GHRH infusion.

To test the effects of GHRH infusion on receptor mRNA levels in the absence of GH, male SDR rats (4–5 months; 100–150 g) were anesthetized, cannulated, and infused with either the GHRH agonist (n = 5) or saline (n = 5) as described above.

To determine whether GHS can alter GHRH-R or GHS-R mRNA levels, anesthetized rats were infused with saline or L-692,585 (100 µg/h; Dr. R. G. Smith, Merck & Co., Inc., Rahway, NJ) for 4 h. Immediately after the infusions, animals were killed by decapitation, trunk blood was collected, and serum was stored for analysis of GH by RIA (28). In addition, anterior pituitaries were collected and frozen for analysis of GHS-R and GHRH-R mRNA by RT-PCR (see below for details). All procedures were conducted according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the University of Illinois at Chicago Animal Care Research Committee.

Treatment of primary pituitary cell cultures with GHRH
Anterior pituitaries were enzymatically and mechanically dissociated into single cells and cultured for 3 days before experimental treatment, as previously described (26). Culture medium was then replaced with serum-free medium (0.1% BSA in {alpha}MEM), and rat GHRH-(1–44)NH2 (0.01–10 nM; Peninsula Laboratories, Inc., Belmont, CA) or forskolin (10 µM; Sigma Chemical Co., St. Louis, MO) was added (n = 4 wells/treatment group). After a 4-h incubation, cells were recovered, and total RNA was extracted as previously described (26).

RT-PCR of GHRH-R and GHS-R mRNA
Total RNA (1 µg) from whole pituitaries or primary cell cultures was used as a template to generate complementary DNA (cDNA) by RT with random hexamer priming. A constant amount of synthetic RNA generated from a rat GHRH-R subclone modified by excision of an internal 235-bp fragment (rat pituitary standard-1) was added to each RNA sample to correct for variability in the RT reaction. RT products were amplified by PCR in separate reactions using primers for the rat GHRH-R cDNA (GenBank no. L01407), rat GHS-R cDNA (GenBank no. U94321), or rat glyceraldehyde 3'-phosphate dehydrogenase (GAPDH) cDNA (GenBank no. X02231). PCR products were gel electrophoresed, transferred to nylon membranes, and hybridized to specific radiolabeled cDNA probes generated by random oligonucleotide labeling. Membranes were washed at high stringency conditions and exposed to a phosphorscreen for 1.5 h. Hybridization signals were detected by phosphorimager, and band intensity was evaluated by image analysis (Molecular Dynamics, Inc., Sunnyvale, CA). The GHRH-R signal was adjusted by rat pituitary standard-1 and GAPDH, whereas the GHS-R signal was adjusted by GAPDH. Details of the procedure and validation of the quantitative RT-PCR for the GHS-R and GHRH-R mRNA levels have been previously described (24, 26).

Data analysis
All comparisons were made between samples electrophoresed on the same gel. The effects of GHRH on serum GH levels and pituitary receptor mRNA levels in the absence or presence of somatostatin antiserum were evaluated by two-way ANOVA, whereas the in vitro effects of GHRH were determined by one-way ANOVA. Group comparisons were performed using Duncan’s new multiple range test. Student’s t test was used to compare the effects of GHRH and L-692,585 infusion on receptor mRNA levels in SDR and normal rats, respectively. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A 4-h infusion of the GHRH agonist resulted in a greater than 10-fold increase in circulating GH levels (Fig. 1Go, upper panel), which was associated with a 2-fold increase in pituitary GHS-R mRNA (P < 0.01; Fig. 1Go, middle panel). In contrast, GHRH-R mRNA levels tended to be reduced after the infusion of the agonist, but this decline was not statistically significant (P < 0.07; Fig. 1Go, lower panel). Blockade of endogenous somatostatin action by somatostatin antiserum (SS AS) increased circulating GH levels above NSS-treated control values (P < 0.05), but did not affect basal or stimulated GHS-R mRNA levels. However, in the absence of somatostatin tone (SS AS), GHRH-R mRNA levels were increased above NSS-treated control values (P < 0.05), and the GHRH agonist significantly suppressed these elevated levels to values comparable to those observed after GHRH administration in the presence of NSS.



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  		Figure 1. Effect of GHRH infusion on serum GH levels and pituitary GHS-R and GHRH-R mRNA levels in the presence and absence of somatostatin effects. Blood and pituitaries were collected from anesthetized male Sprague Dawley rats infused for 4 h with either a GHRH analog (10 µg/h) or vehicle. Five minutes before the start of the GHRH infusion, half of the animals received an iv injection of SS AS (0.5 ml/rat) or NSS. Serum GH levels were determined by RIA, and GHS-R and GHRH-R mRNA levels were quantified by RT-PCR. Data are expressed as a percentage of the vehicle-treated control value and represent the mean ± SEM (n = 5 animals/group). Values with different letters (a, b, and c) are statistically significant (P < 0.05).

 
The effect of GHRH agonist infusion on pituitary GHS-R and GHRH-R mRNA levels in the SDR, an animal model with no immunodetectable GH, is presented in Fig. 2Go. The GHRH agonist increased GHS-R mRNA levels in the SDR, as in normal rats. However, this increase was only half that observed in normal animals. The GHRH agonist did not significantly alter SDR GHRH-R mRNA.



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  		Figure 2. Effect of GHRH infusion on pituitary GHS-R and GHRH-R mRNA levels in the absence of endogenous GH. SDRs, lacking immunodetectable GH, were infused (4 h) with a GHRH analog or vehicle, and pituitary GHS-R and GHRH-R mRNA levels were assessed by RT-PCR. Data are expressed as a percentage of the vehicle-treated control value and represent the mean ± SEM (n = 5 animals/group). **, P < 0.01.

 
The in vitro effects of GHRH on pituitary GHS-R and GHRH-R mRNA levels are shown in Fig. 3Go (representative of three separate experiments). Incubation of primary pituitary cell cultures with GHRH did not affect GHS-R mRNA levels at any dose tested. In addition, forskolin did not significantly alter GHS-R mRNA levels. GHRH did suppress its own receptor mRNA level in a dose-dependent manner, and this effect was also observed with forskolin.



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  		Figure 3. Effect of GHRH in vitro on pituitary GHRH-R and GHS-R mRNA levels. Normal rat pituitaries were enzymatically dispersed and plated at 1 x 106 cells/well in {alpha}MEM-10% horse serum. After 3 days of culture, cells were washed in serum-free medium and incubated with GHRH (0.01–10 nM) or forskolin (FSK; 10 µM) for 4 h. Total cellular RNA was extracted, and GHS-R and GHRH-R mRNA levels were determined. Data are expressed as a percentage of the basal value and represent the mean ± SEM (n = 3–4 wells/treatment group). *, P < 0.05. These results are representative of three separate experiments.

 
The effects of a 4-h infusion of the GHS-R ligand, L-692,585, on circulating GH levels and pituitary GHS-R and GHRH-R mRNA levels are presented in Fig. 4Go. Although L-692,585 did not significantly increase circulating GH levels at the single time point measured, it did decrease pituitary GHS-R mRNA values to 50% of those in vehicle-treated controls (P < 0.05), whereas GHRH-R mRNA levels were not significantly altered.



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  		Figure 4. Effect of GHS infusion on serum GH levels and pituitary GHS-R and GHRH-R mRNA levels in normal rats. Blood and pituitaries were collected from anesthetized male Sprague Dawley rats infused for 4 h with either the nonpeptidyl GHS, L-692,585 (100 µg/h), or vehicle. Serum GH levels were determined by RIA, whereas pituitary GHS-R and GHRH-R mRNA levels were assessed by RT-PCR. Data are expressed as a percentage of the vehicle-treated control value and represent the mean ± SEM (n = 5 animals/group). *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study demonstrate that an iv GHRH infusion rapidly increases pituitary GHS-R mRNA levels. These observations provide evidence for the hypothesis that GHRH can augment pituitary responsiveness to GHSs by stimulating GHS-R synthesis. Despite the robust increase in GHS-R mRNA after GHRH infusion in vivo, GHRH did not alter GHS-R mRNA levels in vitro. These observations indicate that additional factors, either central or systemic, are required for the stimulatory actions of GHRH on GHS-R synthesis. In this report, GHRH infusion resulted in a 10-fold increase in circulating GH concentrations. As elevation of serum GH levels initiates a feedback loop that ultimately leads to a decrease in GHRH synthesis and an increase in somatostatin release (29, 30, 31, 32), the possibility exists that the GHRH-induced changes in GHS-R synthesis could occur by GH-mediated changes in somatostatin tone. However, pretreatment with somatostatin antiserum, which resulted in a characteristic rise in basal GH levels (33, 34), did not inhibit the GHRH-induced increase in pituitary GHS-R mRNA levels. These results indicate that the mechanism by which GHRH stimulates GHS-R mRNA levels is independent of somatostatin’s actions at the pituitary level. However, these results do not exclude the possibility that GH activates somatostatinergic neurons that do not terminate at the median eminence (inaccessible to SS AS), but serve as interneurons that modulate the release of other factors critical for pituitary GHS-R regulation. As these proposed pathways would be initiated by an increase in circulating GH concentrations, it was of interest to determine whether GHRH could stimulate GHS-R mRNA levels in the absence of GH. The effect of a GHRH infusion was, therefore, assessed in SDRs, an animal model with no immunodetectable GH (35). GHRH evoked a 50% increase in SDR GHS-R mRNA levels. The reduced effectiveness of GHRH in SDRs compared with that in normal rats (50% vs. 100% of respective vehicle-treated control values) may be explained by the fact that basal levels of GHS-R mRNA in this dwarf model are nearly 4 times normal values (24). Despite the variable sensitivity, our results demonstrate that the acute stimulatory effect of GHRH on GHS-R synthesis is independent of the central and peripheral actions of GH.

Although the exact mechanism by which GHRH mediates the up-regulation of GHS-R synthesis remains to be determined, these observations emphasize the close interaction of GHRH and GHS on the stimulation of GH release from pituitary somatotropes. To determine whether GHSs have a complimentary effect on the GHRH-R, we examined the effects of L-692,585, a nonpeptidyl GHS, on GHRH-R mRNA levels in vivo and found that an infusion of L585,692 did not significantly alter GHRH-R mRNA levels. However, GHS-R mRNA levels were significantly reduced after infusion of the ligand, providing a mechanism by which repetitive or constant GHS treatment decreases the response to subsequent GHS challenge both in vivo and in vitro (20, 36, 37, 38, 39). Ligand-induced desensitization, also known as homologous down-regulation, is commonly observed with many G protein-coupled receptors. Ligand-mediated desensitization can occur by multiple pathways, including 1) a phosphorylation-dependent decrease in receptor affinity, 2) removal of the receptor from the cell surface by internalization, and 3) reduction in receptor synthesis by decreasing receptor gene transcription and/or decreasing receptor mRNA stability (40). The results of the present study indicate that GHS-mediated desensitization is at least in part due to a decrease in receptor synthesis. However, it remains to be determined whether the ligand-mediated reduction in GHS-R mRNA is due to a transcriptional or a posttranscriptional mechanism.

The GHRH/GHRH-R signaling system, like the GHS/GHS-R signaling system, undergoes homologous desensitization in vivo and in vitro (41, 42, 43, 44). The present report confirms our previous demonstration that GHRH-induced desensitization in vitro is associated with a rapid (within 4 h) decline in GHRH-R mRNA levels (26). As the inhibitory effect of GHRH on its own receptor synthesis can be duplicated by forskolin, a receptor-independent activator of adenylate cyclase, it appears that the ligand-initiated reduction in GHRH-R mRNA levels is a cAMP-dependent process. In the present study we have extended these observations by demonstrating that acute GHRH treatment also decreases GHRH-R mRNA levels in vivo. However, the inhibitory actions of GHRH are not fully recognized unless the effects of somatostatin are eliminated. In fact, blockade of somatostatin’s actions raises basal GHRH-R mRNA levels, indicating that GHRH-R synthesis is tonically inhibited by this neurohormone. It is unlikely that the increase in GHRH-R mRNA levels after somatostatin immunoneutralization is due to the enhancement of adenylate cyclase activity because acute activation of adenylate cyclase decreases GHRH-R mRNA levels (26). Therefore, somatostatin appears to act through a cAMP-independent process to suppress GHRH-R synthesis. This hypothesis is consistent with the fact that activation of somatostatin receptors not only regulates cAMP-mediated intracellular events (45), but also affects multiple intracellular signaling systems, including phospholipase A, serine/threonine phosphatases, and tyrosine phosphatases (46).

In summary, the present results indicate that the interdependency of the GHS and GHRH signaling pathways is at least in part due to the ability of GHRH to up-regulate GHS-R mRNA levels in vivo. The fact that GHRH does not induce GHS-R mRNA levels in vitro suggests that other systemic or central factors are necessary for GHRH-initiated augmentation of GHS-R synthesis. These studies also indicate that GHS, like GHRH, can rapidly down-regulate its own receptor message. Both observations emphasize the important role of receptor synthesis in dynamic modulation of pituitary sensitivity to both GHS and GHRH.


   Acknowledgments
 
We thank Dr. Akira Arimura (Tulane University, Hebert Center, Belle Chase, LA) for the somatostatin antiserum, Dr. Roy G. Smith (Merck Research Laboratories, Rahway, NJ) for the L585,692, Dr. Robert M. Campbell (Hoffmann-La Roche, Inc., Milford, NJ) for the GHRH analog, and Dr. Terry G. Unterman (Chicago Veterans Administration Health Care System, West Side Division, Chicago, IL) for the spontaneous dwarf rats.


   Footnotes
 
1 This work was supported by NIH Grant DK-30667 and the Bane Scholar Fund (to L.A.F.). Back

2 Visiting scientist from the Department of Medicine, Nippon Medical School, 1–1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan. Recipient of the Japan Private School Promotion Foundation Award for Overseas Training. Back

Received January 14, 1999.


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 Discussion
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