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Endocrinology Vol. 140, No. 6 2836-2842
Copyright © 1999 by The Endocrine Society

ARTICLES

Differential in Vivo Regulation of the Pituitary Growth Hormone-Releasing Hormone (GHRH) Receptor by GHRH in Young and Aged Rats1

Nathalie Girard, Luce Boulanger2, Stéphan Denis and Pierrette Gaudreau3

Laboratory of Neuroendocrinology of Aging, Notré-Dame Hospital Research Center, and the Department of Medicine, University of Montréal, Montréal, Québec, Canada H2L 4M1

Address all correspondence and requests for reprints to: Dr. Pierrette Gaudreau, Laboratory of Neuroendocrinology of Aging, Notre Dame Hospital Research Center, Room M-5226, 1560 East Sherbrooke Street, Montréal, Québec, Canada H2L 4M1. E-mail: gaudreap{at}ere.umontreal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In aging, alterations of pituitary GH-releasing hormone (GHRH) receptor (GHRH-R)-binding sites have been proposed as one of the initiating factors contributing to the loss of somatotroph responsiveness to GHRH. Changes in the characteristics and/or concentration of the functional GHRH-R could take place in the course of aging and reduce the sensitivity of the somatotroph axis to GHRH. Because chronic exposure to GHRH has been proposed to resensitize aged somatotroph cells, better knowledge of its effects on the regulation of the somatotroph axis is required, particularly at the level of GHRH-R. Two- and 18-month-old male Sprague Dawley rats were treated for 14 days with a daily sc injection of 0.5 or 1.0 mg/kg BW human GHRH-(1–29)NH2 or saline. In 2-month-old rats, treatment with 0.5 mg/kg GHRH increased the number of high affinity pituitary GHRH-R-binding sites by 2-fold (P < 0.05) and hypothalamic somatostatin (SRIF) content by 45% (P < 0.05). It did not affect hypothalamic GHRH content, serum total insulin-like growth factor I (IGF-I), or body weight gain. Treatment with 1.0 mg/kg GHRH decreased the number of high affinity pituitary GHRH-R-binding sites by 2.4-fold compared with that in rats treated with 0.5 mg/kg BW (P < 0.05) and increased hypothalamic SRIF content by 45% (P < 0.05), but did not affect GHRH content. It also decreased circulating levels of IGF-I by 13% (P < 0.05) and slowed the growth rate by 17% (P < 0.05). In 18-month-old rats, treatment with 0.5 mg/kg GHRH for 14 days was not sufficient to rejuvenate pituitary GHRH binding parameters. However, treatment with 1.0 mg/kg GHRH restored the affinities of high and low affinity classes of GHRH-binding sites to values similar to those found in 2-month-old rats. Binding capacities of the high and low affinity classes of sites were increased by 1.8- and 3-fold, respectively, although significance was only reached for the low affinity site (P < 0.05). These changes were associated with a normalization of the level of 2.5-kb GHRH-R messenger RNA transcript, which was decreased by 31% in aging rats (P < 0.05), and by a trend for an increase in the 4-kb GHRH-R messenger RNA transcript, which was already increased by 49% in 18-month-old rats (P < 0.05). A normalization of serum IGF-I levels, which were decreased by 11% in 18-month-old control rats (P < 0.01), was also observed. No treatment effect was detected on body weight or hypothalamic SRIF and GHRH contents. We conclude that a 14-day administration of GHRH induces a differential GHRH-R-mediated regulation at the level of the pituitary and probably the hypothalamus as a function of age.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE anterior pituitary, GH pulsatile release is under the dual control of two hypothalamic peptides: GH-releasing hormone (GHRH) and somatostatin (SRIF) (1). These peptides stimulate specific Gs and Gi protein-coupled plasma membrane receptors on somatotroph cells (2, 3). The resulting activation of adenylate cyclase and production of cAMP constitute the primary events leading to GH secretion and synthesis (4, 5).

In mammals, middle and late adulthood is characterized by a decrease in spontaneous and stimulated GH secretion and circulating levels of insulin-like growth factor I (IGF-I). It is thought that these endocrine changes are responsible for a diminution of muscle mass, an increase in adipose tissue mass, and a deterioration of several tissue and organ functions (6, 7, 8, 9). It has been hypothesized that the age-related decline of GH secretion, and consequently IGF-I, results more from a decreased responsiveness of somatotrophs to GHRH (6, 10, 11, 12, 13) than from a defect in the mechanisms of GH release (14). In 24-month-old rats, it has been partly related to a decrease in both GHRH hypothalamic content (15, 16) and gene expression (16), but not to a loss of GHRH-containing neurons (15). The contribution of hypothalamic SRIF to decreased GH secretion remains controversial, as divergent results were reported on levels of messenger RNA (mRNA) expression, peptide content, and release (10, 11, 12, 15, 17, 18, 19). We have shown that a significant diminution of the maximal GHRH-induced GH response appears in vitro and in vivo in male rats around 12 months of age (10). This is accompanied by an approximately 15% decrease in pituitary GH mRNA levels (17), which is not translated to a decrease in the immunoreactive content (10). In 20- to 24-month-old rats, the GHRH-induced GH response is severely depressed (10), and GH mRNA levels and immunoreactive content are diminished by 50% (17) and 30% (10), respectively. Alterations in pituitary GHRH receptor (GHRH-R)-binding sites, starting around 8 months of age in male rats, have been proposed as one of the initiating factors contributing to the loss of somatotroph responsiveness to GHRH in aging (20, 21). In 18-month-old rats, this leads to a blunting of the high affinity GHRH-R binding sites and a reduction of the total number of binding sites (20, 21). In addition, a 45% reduction of the maximal GHRH-induced cAMP production is seen in 20- to 24-month-old rats (22). Changes in the characteristics and/or concentrations of functional GHRH-R could therefore take place in the course of aging and reduce the sensitivity of the somatotroph axis to GHRH.

One strategy to resensitize aged somatotroph cells could be the stimulation of de novo synthesis of pituitary GHRH-R by chronic in vivo administration of GHRH. In aged men, the effects of a chronic treatment with GHRH include an increase in GHRH-stimulated GH release and mean 24-h GH levels (23, 24, 25). In young and aged rats, variable results for GH mRNA levels and GH secretion have been reported after GHRH treatment depending upon sex, dosage, and route, frequency, and duration of administration (23, 26, 27, 28, 29).

Because new therapeutic applications may be developed in the near future for GHRH, a better knowledge of its chronic effects on the regulation of the somatotroph axis is required. To understand the desensitization/resensitization mechanisms of the somatotroph axis to GHRH, particularly through GHRH-R regulation, 2- and 18-month-old male rats were treated with either 0.5 or 1.0 mg/kg BW human (h) GHRH-(1–29)NH2, sc, for 14 days. Pituitary GHRH-R binding parameters and mRNA levels were examined, and immunoreactive hypothalamic GHRH and SRIF contents and serum total IGF-I concentrations were measured.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental procedures
Two- and 18-month-old male Sprague Dawley rats (Camm Research Laboratory Animals, Wayne, NJ) were housed in Plexiglass cages in groups of four and two, respectively, under controlled temperature (22 C), humidity (65%), and lighting (cycles of 12 h; lights on at 0700 h) conditions. Chow and water were available ad libitum. After 2–3 days of acclimatization to our animal facilities, the rats were weighed to determine hGHRH-(1–29)NH2 dosage and were reweighed after 7 days of treatment to readjust it. In the first experiment, 2-month-old rats received in the back a daily sc injection of 0.5 or 1.0 mg/kg BW hGHRH-(1–29)NH2 (GHRH) for 14 days between 0815–0900 h. In the second experiment, 18-month-old rats received a daily sc injection of 0.5 or 1.0 mg/kg BW of the peptide. hGHRH-(1–29)NH2 (synthesized in our laboratory) (30) was solubilized in physiological saline each morning and kept on ice until administration. Control 2- and 18-month-old rats received an isovolumetric amount of saline. The doses of GHRH were selected on the basis of our previous data indicating that sc administration of 1.0 mg/kg BW hGHRH-(1–29)NH2 to 2-month-old male Sprague Dawley rats induces a GH peak equivalent to that obtained with the iv GHRH dose of 3 µg/kg BW (approximate ED50). Rats were killed by decapitation, in a block design fashion, 24 h after the last injection. The animal protocol was approved by the animal care committee of Notre-Dame Hospital Research Center in compliance with the guidelines of the Canadian Council on Animal Care.

Tissue handling
For binding assays, the anterior pituitaries were collected in ice-cold 50 mM Tris-acetate buffer, pH 7.4, containing 5 mM MgCl2 and 5 mM EDTA and used within 15 min. For determination of pituitary GHRH-R mRNA levels and hypothalamic GHRH and SRIF immunoreactive contents, tissues were snap-frozen in liquid nitrogen and kept at -80 C until use. For determination of total IGF-I immunoreactive concentrations, trunk blood was collected, and serum was kept at -80 C.

Binding assay
Tissue preparation and cold saturation studies were performed as previously reported, using [125I-Tyr10]hGHRH-(1–44)NH2 ([125I]GHRH; 2000 Ci/mmol, purchased from Amersham Canada, Oakville, Canada) as radioligand (20, 21). For each saturation curve, two anterior pituitaries from 2-month-old rats and one anterior pituitary from an 18-month-old rat were homogenized for 8–10 sec, with a microultrasonic cell disrupter (Kontes, Vineland, NJ), in 0.5 ml ice-cold 50 mM Tris-acetate buffer, pH 7.4, containing 5 mM MgCl2 and 5 mM EDTA. The homogenates were further diluted to 1.3 ml, and 50 µl of this preparation were used in each assay tube [51–85 µg protein, as determined by the Lowry method (31)] with 35–50 pM [125I]GHRH and increasing concentrations of [127I-Tyr10]hGHRH-(1–44)NH2 (0.1–1000 nM) in a total volume of 300 µl Tris-acetate, pH 7.4, containing 5 mM MgCl2, 5 mM EDTA, and 0.42% BSA. Incubations were carried out at 23 C for 60 min and were stopped by centrifugation (12,000 x g, 5 min, 4 C). Nonspecific binding was determined in presence of 1.0 µM rat (r) GHRH-(1–29)NH2. [127I-Tyr10]hGHRH-(1–44)NH2 and rGHRH-(1–29)NH2 were synthesized in our laboratory (30). In the first experiment, percentages of specific binding were 76 ± 1%, 76 ± 2%, and 76 ± 1% for 2-month-old control rats, 2-month-old rats treated with 0.5 mg/kg BW GHRH, and 2-month-old rats treated with 1.0 mg/kg BW GHRH, respectively. In the second experiment, they were 70 ± 1%, 66 ± 1%, 68 ± 1%, and 69 ± 1% for 2-month-old control rats, 18-month-old control rats, 18-month-old rats treated with 0.5 mg/kg BW GHRH, and 18-month-old rats treated with 1.0 mg/kg BW GHRH, respectively. Control pituitaries from 2-month-old rats were always assayed along with those of 2-month-old treated rats, whereas control pituitaries from 2- and 18-month-old rats were assayed with those of 18-month-old treated rats.

RIAs
Each hypothalamus was homogenized in 0.1 N acetic acid, boiled 10 min, and centrifuged (12,000 x g, 30 min, 4 C). Immunoreactive contents of GHRH and SRIF were determined in supernatants using commercial RIA kits (Advanced ChemTech, Inc., Louisville, KY) and were expressed per mg protein (31). The sensitivities of the GHRH and SRIF assays were 2.5 and 4.0 pg/tube, respectively. Serum total IGF-I concentrations were measured after acid-ethanol treatment, using a commercial RIA kit (Diagnostics Systems Laboratories, Inc., Webster, TX). The sensitivity of the assay was 21 ng/ml. For each RIA, all samples from each experiment were determined in duplicate in a single assay. Intraassay coefficients of variation were 4.4%, 4.4%, and 6.1% for GHRH, SRIF, and total IGF-I, respectively.

Northern blot hybridization
Total RNA was isolated from each anterior pituitary using a single step acid guanidinium-phenol/chloroform procedure with Trizol (Life Technologies, Gaithersburg, MD). Aliquots of 18 µg total RNA were denatured by heating (65 C, 10 min) in a 50% formamide-17.5% formaldehyde-15 mM MOPS [3-(N-morpholino)propanesulfonic acid] solution. They were subjected to electrophoresis on 1.2% agarose gels containing 17.5% formaldehyde, using a 33 mM MOPS buffer, pH 7.0, containing 5 mM sodium acetate and 1 mM EDTA (pH 8.0). RNA was transferred by capillary elution to a nylon membrane (GeneScreen, NEN Research Products, Boston, MA) and covalently attached by UV cross-linking (Stratagene, La Jolla, CA) and heating (80 C, 2 h). Blots were hybridized with the RPR-64 complementary DNA (cDNA) specific for the rat GHRH-R (2). The probe was labeled with [32P]deoxy-CTP (Amersham Canada), using random hexamer primers and the Klenow fragment of Escherichia coli DNA polymerase (Life Technologies, Burlington, Canada) and purified by chromatography, using a G-50 column (Pharmacia Biotech, Baie d’Urfé, Quebec). Hybridization was performed overnight at 42 C in 50% formamide, 5 x SSC (1 x SSC = 150 mM NaCl and 15 mM Na citrate, pH 7.0), 10% dextran sulfate, 1 x Denhart’s reagent, 20 mM Tris (pH 7.5), 1% SDS, and 100 µg/ml DNA salmon sperm. Membranes were subsequently washed (2 x SSC-1% SDS, room temperature; 1 x SSC-1% SDS, 65 C; 0.5 x SSC-1% SDS, 65 C; 30 min each time) and exposed to Kodak XAR film (Eastman Kodak Co., Rochester, NY) at -80 C, for 24 h with an intensifying screen. The membranes were then stripped in a boiling aqueous solution of 0.1% SDS and sequentially rehybridized with 1.2 kb rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 28S ribosomal RNA probes (American Type Culture Collection, Manassas, VA). Amounts of GHRH-R mRNA were normalized in each lane with GAPDH. Compared with the expression levels of 28S ribosomal RNA, GAPDH mRNA levels were constant in all of our experimental conditions.

Data analysis
The Ligand program was used to analyze cold saturation studies (32). The F test was used to compare the goodness of the fit between the one-site model, corrected or not for nonspecific binding, and the two-site model. If the F test indicated P > 0.05, the less complex model was accepted as the model that better fit the experimental data. The sum of the square error values for the three models along with their corresponding degrees of freedom were used to define the F value.

As quantification of each GHRH-R mRNA transcript, visible in the anterior pituitary as a doublet at about 2.5 and 4 kb, could not be performed independently with accuracy even after overnight gel migration, the total densities of each doublet at 2.5 and 4 kb were determined using an IS1000 digital imaging system (Alpha Innotech Corp./Canberra Packard, Montréal, Québec, Canada). Results were expressed as the mean ± SEM. The statistical significance of differences was determined by unpaired Student’s t test and was established at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight
In the first experiment, 2-month-old control rats increased their body weight (BW) by 40% (initial BW, 245 ± 3 g; final BW, 342 ± 7 g; P < 0.005) during the 14-day treatment with GHRH, whereas 2-month-old rats treated with 0.5 or 1.0 mg/kg GHRH increased their BW by 35% and 33%, respectively (initial BW, 251 ± 2 and 250 ± 3 g; final BW, 339 ± 6 and 333 ± 6 g, respectively). The rats treated with the higher dose of GHRH had a 17% slower growth rate than controls (P < 0.05).

In the second experiment, 2-month-old control rats increased their BW by 45% (initial BW, 222 ± 1 g; final BW, 321 ± 4 g; P < 0.05) over the 14-day treatment with GHRH, whereas 18-month-old control rats or rats treated with 0.5 or 1.0 mg/kg GHRH exhibited only a slight weight gain. Their respective BW increased by 4%, 7%, and 6% (initial BW, 689 ± 11, 702 ± 22, and 679 ± 6 g; final BW, 718 ± 14, 752 ± 23, and 717 ± 10 g). No significant difference was observed between 18-month-old control and treated rats.

Anterior pituitary GHRH binding parameters
The effect of 14-day daily sc administration of GHRH on pituitary binding parameters in 2-month-old rats is shown in Table 1Go. As previously reported (20, 21), coanalysis of binding data derived from cold saturation studies revealed the presence of high and low affinity classes of binding sites in this tissue. Administration of 0.5 or 1.0 mg/kg GHRH did not alter the preference of the Ligand program for the two-site model (P < 0.05) and did not significantly affect Kd1, Kd2, or Bmax2 values. However, it differentially regulated the binding capacity (Bmax) of the high affinity class of sites depending upon the dose of GHRH. Treatment with 0.5 mg/kg GHRH increased the Bmax1 by 2-fold compared with that in control rats (P < 0.05), whereas treatment with 1.0 mg/kg GHRH decreased the Bmax1 by 2.4-fold compared with that of rats treated with 0.5 mg/kg GHRH (P < 0.05).


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  		Table 1. Estimate of GHRH binding parameters in pituitary homogenates from 2-month-old rats: effect of a daily sc administration of hGHRH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 )NH2 for 14 days

 
The effect of 14-day daily sc administration of GHRH on pituitary binding parameters in 18-month-old rats is summarized in Table 2Go and Fig. 1Go. As previously reported (20, 21), the two-site model was no longer preferred in 18-month-old control rats. In this group, the high affinity site was blunted, and the Kd was 159 times lower than the Kd1 and 11 times higher than the Kd2 in pituitaries from 2-month-old control rats (Table 2Go; P < 0.01). Moreover, the total number of binding sites was 10 times lower than that in 2-month-old control rats (Table 2Go; P < 0.01). As observed in 2-month-old rats, [125I]GHRH binding parameters were differentially regulated by GHRH treatment depending upon dosage. Pituitaries from 18-month-old rats treated with 0.5 mg/kg GHRH exhibited one class of binding sites, similarly to their age-matched controls. As in 18-month-old control rats, the high affinity site was blunted. The Kd was 103 times lower than the Kd1 and 17 times higher than the Kd2 of pituitaries from 2-month-old control rats (Table 2Go; P < 0.01). The total number of binding sites was 22 times lower than that in 2-month-old control rats (Table 2Go; P < 0.01). In addition, the Kd and Bmax appeared diminished compared with those for pituitaries from 18-month-old control rats (P < 0.05). In pituitaries from 18-month-old rats treated with 1.0 mg/kg GHRH, analysis of the data showed a significant preference for the two-site model (P < 0.05). Kd1 and Kd2 values reverted to those observed in pituitaries from 2-month-old control rats and were different from the Kd of 18-month-old control rats or rats treated with 0.5 mg/kg GHRH (P < 0.01). In addition, the Bmax1 and Bmax2 were increased by 1.8- and 3-fold, respectively, compared with those of pituitaries from 2-month-old control rats. However, only the Bmax2 value reached significance (P < 0.01).


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  		Table 2. Estimate of GHRH binding parameters in pituitary homogenates from 2- and 18-month-old rats: effect of a daily sc administration of hGHRH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 )NH2 for 14 days in 18-month-old rats

 


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  		Figure 1. Representative [125I]GHRH binding profile in anterior pituitary homogenates from 2-month-old control rats ({blacksquare}; 2C), 18-month-old control rats ({square}; 18C), and 18-month-old rats treated with 1.0 mg/kg GHRH (•; 18T). Data were expressed as the ratio of specific binding, corrected for 75 µg protein, over the total amount of radioligand added (B/T).

 
Anterior pituitary GHRH receptor mRNA levels
Quantification of mRNA levels performed by the IS1000 digital imaging system showed a linear increase in signals with the amount of RNA loaded, within the range of 5–35 µg total RNA, for the 2.5- and 4-kb GHRH-R mRNA doublets, GAPDH mRNA and 28S ribosomal RNA (data not shown). As shown in Fig. 2Go, the presence of approximately 2.5- and 4-kb GHRH-R mRNA transcripts (doublets) was detected in the anterior pituitaries of 2- and 18-month-old rats. In pituitaries from 18-month-old control rats, the relative intensity of the 2.5-kb doublet decreased by 31% (P < 0.05), and that of the 4-kb doublet increased by 49% compared with those of 2-month-old control rats (P < 0.05; Figs. 2Go and 3Go). Combined levels of 2.5- and 4-kb transcripts were not significantly different in these two groups.



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  		Figure 2. Northern blot autoradiographic analysis of pituitary GHRH-R mRNA levels in 2-month-old control rats (2 C), 18-month-old control rats (18 C), and 18-month-old rats treated with 1.0 mg/kg GHRH (18 T).

 


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  		Figure 3. Densities of pituitary GHRH-R mRNA transcripts in 2-month-old control rats (2 C), 18-month-old control rats (18 C) and 18-month-old rats treated with 1.0 mg/kg GHRH (18 T). Data were expressed as a percentage of the 2-month-old control values in arbitrary density units and represent the mean ± SEM of four individual samples performed in duplicate for each group. *, P < 0.05 when 2.5- or 4-kb transcripts from 18-month-old control rats were compared with those from 2-month-old control rats or when combined densities of 2.5- and 4-kb transcripts from 18-month-old treated rats were compared with those of 18-month-old control rats. **, P < 0.02 when the 4-kb transcript from 18-month-old treated rats was compared with that from 2-month-old control rats.

 
In 18-month-old rats, the effect of a 14-day daily sc administration of 1.0 mg/kg GHRH on levels of 2.5- and 4-kb GHRH-R mRNA transcripts is shown in Figs. 2Go and 3Go. The intensity of the 2.5-kb doublet was normalized to that of pituitaries from 2-month-old control rats and increased by 35% (P < 0.05) compared with pituitaries from 18-month-old controls. The intensity of the 4-kb doublet was increased by 126% compared with that in pituitaries from 2-month-old control rats (P < 0.02) and by 51% compared with that in pituitaries from 18-month-old control rats, although this increase did not reach significance. Combined levels of 2.5- and 4-kb transcripts were increased by 36% in 18-month-old treated rats compared with those in 18-month-old controls (P < 0.05).

Immunoreactive hypothalamic GHRH and SRIF contents and serum total IGF-I concentration
As shown in Table 3Go, a 14-day daily sc administration of 0.5 and 1.0 mg/kg GHRH had no significant effect on hypothalamic GHRH content of 2-month-old rats. However, it increased the hypothalamic SRIF content by 45% (P < 0.05). The serum total IGF-I concentration was not significantly altered by a 14-day daily sc administration of 0.5 mg/kg GHRH, but was decreased 13% by a 1.0 mg/kg GHRH treatment (P < 0.05). As also shown in Table 4Go, no significant changes in hypothalamic GHRH and SRIF contents were observed between 2- and 18-month-old control and 18-month-old treated rats. However, the serum total IGF-I concentration was decreased by 12% in 18-month-old control rats compared with that in 2-month-old controls (P < 0.01) and was normalized by a 14-day daily sc administration of 1.0 mg/kg GHRH.


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  		Table 3. Immunoreactive hypothalamic GHRH and SRIF contents and serum total IGF-I concentration in 2-month-old rats: effect of a daily sc administration of hGHRH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 )NH2 for 14 days

 

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  		Table 4. Immunoreactive hypothalamic GHRH and SRIF contents and serum total IGF-I concentration in 2- and 18-month-old rats: effect of a daily sc administration of hGHRH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 )NH2 for 14 days in 18-month-old rats

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In aged men, chronic administration of GHRH has been shown to increase GH secretion and circulating levels of IGF-I (23, 24, 25). In 14-month-old female, but not male, Sprague Dawley rats, 3-day treatment with rGHRH-(1–43)OH (5 µg/rat·day, subdivided in three iv injections delivered at 0800, 1400, and 2000 h) elicited a slight, but unequivocal, priming effect on the in vitro subsequent GHRH-induced cAMP maximal response (28). However, this priming effect was not sufficient to impact on GHRH-induced GH secretion in cultured anterior pituitary cells (28). In 2-month-old male and female Sprague Dawley rats, the same GHRH treatment was shown to significantly decrease the subsequent in vitro response to GHRH (28). In the present study we report the description of a model of hGHRH-(1–29)NH2 administration in 18-month-old male Sprague Dawley rats that successfully mimics GHRH treatment in aged men, as serum total IGF-I levels were normalized to those in 2-month-old rats.

Analysis of the regulation of the somatotroph axis by 14-day administration of GHRH revealed striking differences between young and aged male rats. In 2-month-old rats, administration of 0.5 mg/kg GHRH significantly increased the number of high affinity pituitary GHRH-R-binding sites. Treatment with 1.0 mg/kg GHRH probably also induced a short period of up-regulation, followed by an observable process of down-regulation at the end of the treatment. These results indicate that depending upon its circulating level, GHRH is capable of up- or down-regulating its own receptor. Horikawa et al. (33) and Miki et al. (34) reported modulatory effects of GHRH on pituitary GHRH-R expression in neonatal or 3-month-old rats. The first group demonstrated that passive immunization with GHRH decreased GHRH-R mRNA levels. The second group showed that a pharmacologically induced GHRH deficiency led to an increase in GHRH-R mRNA levels, whereas replacement therapy normalized GHRH-R mRNA levels to those in control rats. In addition, Bilezikijan et al. reported that incubation of 2-month-old rat primary pituitary cells with GHRH induced a 50% reduction of GHRH-binding sites (35), whereas Wehrenberg et al. showed down-regulation of pituitary GHRH-R-binding sites by iv infusions of GHRH (60–900 µg/kg BW) (36). Aleppo et al. showed that in pituitary cell cultures from 2-month-old rats, GHRH inhibits the production of its own receptor by a receptor-mediated, cAMP-dependent reduction of GHRH-R mRNA accumulation (37). As studies from Mayo indicate that the cloned GHRH-R, originating from a 2.5-kb mRNA transcript, exhibits high affinity binding and functionality (2), our data suggest that in 2-month-old rats, the GHRH-R high affinity binding site is more sensitive to up- and down-regulation by circulating levels of GHRH than the low affinity site. The mechanisms of action of GHRH on rat GHRH-R gene expression or GHRH-R mRNA stability, however, remain to be investigated.

Treatment with 0.5 mg/kg also induced a 45% increase in hypothalamic SRIF content, but had no effect on GHRH content. This negative feedback counterbalanced a potential increase in GH secretion resulting from up-regulated GHRH-R high affinity binding sites and did not lead to a significant reduction of IGF-I circulating levels and BW gain. Interestingly, treatment with 1 mg/kg GHRH induced a 45% increase in hypothalamic SRIF content and down-regulation of pituitary GHRH-R high affinity binding sites, leading to a significant reduction of IGF-I circulating levels and BW gain. The resulting peripheral endocrine and metabolic effects that we observed after chronic treatment with 1 mg/kg BW GHRH were similar to those reported by Kovacs et al. (38), using treatment with a GHRH-R antagonist. Young rats, treated for 2 weeks with [Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1–28)Agm exhibited a 21% reduction of body weight gain, a 15% decrease in serum IGF-I levels, and a 48% diminution of pituitary GHRH-R concentration (38). Together, these data reinforce the important sensor role of the pituitary GHRH-R in the maintenance of a functional somatotroph axis. At the central level, our results suggest the existence of functional GHRH-R on hypothalamic SRIF neurons that would be activated by exogenous GHRH or one of its bioactive metabolites and involved in a negative feedback mechanism of GH secretion. Although the presence of GHRH-R mRNA has been reported in the periventricular, arcuate, and ventromedial nuclei of the hypothalamus (39), its precise localization on SRIF neurons will have to be established.

In 18-month-old rats, administration of 0.5 mg/kg GHRH for 14 days was not sufficient to rejuvenate GHRH binding parameters. Interestingly, treatment with 1.0 mg/kg GHRH restored the high and low affinity class of GHRH binding sites, leading to binding affinities similar to those observed in 2-month-old rats. This GHRH treatment also increased the binding capacity of the high and low affinity classes of sites; however, significance was only reached for the low affinity site. These changes were associated with a normalization of the 2.5-kb GHRH-R mRNA transcript, which is decreased in aging rats, and by a trend for an increase in the 4-kb pituitary GHRH-R mRNA transcript, which is already increased in aged rats, thus resulting in an increase in combined levels of transcripts. This suggests that GHRH can stimulate de novo synthesis of high and low affinity GHRH-R-binding sites in aged rats. Whether they represent the 2.5- and 4.0-kb GHRH-R mRNA transcripts, respectively, requires further investigation. These sites, however, seem to exhibit a different level of sensitivity to regulation by circulating levels of GHRH compared with those of 2-month-old pituitaries. Elucidation of transcriptional and/or posttranscriptional mechanisms regulating the expression of high and low affinity pituitary GHRH-R-binding sites in aging and by GHRH administration in aged rats will now be required. Among the numerous transcription factors regulating GHRH-R expression, the pituitary-specific factor Pit-1 has been shown to be essential (40). Although, no difference in Pit-1 mRNA levels has been reported in the pituitaries of 70-day-old and 12-month-old rat (41), it will be necessary to determine Pit-1 protein and mRNA concentrations in older rats treated, or not, with GHRH.

At the hypothalamic level, the sensitive negative feedback GHRH mechanism seen in 2-month-old rats did not seem to operate in 18-month-old rats. Thus, 14-day administration of 1.0 mg/kg BW GHRH was not sufficient to increase SRIF hypothalamic content and counteract the effects of GHRH. The resulting peripheral effect of this GHRH treatment was therefore normalization of circulating IGF-I levels, with no effect on BW gain. Similarly, De Geranno Colonna et al. reported the presence of GH negative feedback effects on hypothalamic GHRH and SRIF mRNA levels of 8-month-old, but not 20-month-old, rats, submitted to 4-day treatment with hGH (125 µg, ip, twice daily) (42). These results were interpreted as diminished sensitivity of the GHRH and SRIF neurons to GH, related to either altered GH receptor signaling processes or peptide gene expression (42). Whether hypothalamic GHRH-R characteristics/concentrations and/or signaling pathways are altered in aging rats remains to be investigated.

Together, our results indicate that a 14-day sc administration of GHRH differentially regulates the high and low affinity classes of pituitary GHRH-R-binding sites and GHRH-R mRNA transcripts in 2- and 18-month-old male rats depending upon GHRH dosage. It might be hypothesized that different cellular mechanisms are triggered in the somatotroph depending upon the abundance and functional state of plasma membrane GHRH-R and the intensity and duration of GHRH-R stimulation. For example, a high level of GHRH-R stimulation could induce massive internalization of GHRH-R, leading to decreased levels of intracellular cAMP, phosphorylated CREB, and GHRH-R gene expression (43), whereas a moderate level of GHRH-R stimulation may not affect normal GHRH-R internalization processes, thus leading to increased levels of the intracellular messengers responsible for GHRH-R gene expression.

Chronic administration of GHRH to rejuvenate the somatotroph axis in aging mammals therefore appears to be an attractive replacement therapy, as appropriate dosage and duration of treatment should normalize the pituitary high affinity GHRH-R-binding site concentration and serum IGF-I levels without affecting hypothalamic GHRH and SRIF functions. However, optimal conditions of long term replacement therapy will have to be developed as a function of age and based upon residual somatotroph function.


   Acknowledgments
 
We thank Dr. Kelly E. Mayo (Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanstown, IL) for providing the RPR-64 rGHRH-R cDNA, and Dr. Pierre Chartrand (University of Montréal, Montréal, Québec, Canada) for the supply of GAPDH cDNA.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada. Back

2 Recipient of a studentship from the Faculty of Graduate Studies of the University of Montreal. Back

3 Recipient of a scholarship from the Fonds de la Recherche en Santé du Québec. Back

Received August 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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