Luce Boulanger

DIFFERENTIAL IN VIVO REGULATION OF THE PITUITARY GROWTH HORMONE-RELEASING HORMONE (GHRH) RECEPTOR BY GHRH IN YOUNG AND AGED RATS

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 s.c. 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.

Catabolism of rat growth hormone-releasing factor(1–29) amide in rat serum and liver

Clinical and veterinary uses of growth hormone-releasing factor [GRF(1- 29)NH2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1-29)NH2 processing in serum and liver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1-29)NH2 (30 microM) was incubated (0-120 min, 37 degrees C) in serum (49 +/- 8 mg prot./ml). The rGRF(1-29)NH2 (10 microM) was also incubated (0-120 min, 37 degrees C) with liver homogenate (200 +/- 6 micrograms prot./ml). Time course studies of rGRF(1-29)NH2 disappearance showed apparent half-lives of 18 +/- 4 min and 13 +/- 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1-20)OH and (3-20)OH. A small amount of rGRF(12-29)NH2, coeluting with rGRF(3-20)OH, was also found by sequencing. In the liver, rGRF(1-18)OH, (3-18)OH, and (1-10)OH were identified. The peptide bond Ala2-Asp3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg11-Arg12 and Arg20-Lys21 (trypsin-like cleavage site) in the serum, and Tyr10-Arg11 and Tyr18-Ala19 (chymotrypsin-like cleavage site) in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Restricted Presence of the Growth Hormone-Releasing Hormone Receptor to Somatotropes in Rat and Human Pituitaries

Specific binding of growth hormone-releasing hormone (GHRH) to its plasma membrane receptor represents the first step of cellular signals leading to exocytotic GH secretion in the anterior pituitary. The GHRH receptor (GHRH-R) has been cloned and belongs to the secretin/glucagon/vasoactive intestinal peptide subfamilly of G-protein-coupled receptors. To study its characteristics in rat and human pituitaries and examine its cellular and subcellular localization, a site-directed polyclonal antibody recognizing the C-terminal portion 392–404 of the rat and human GHRH-R was used. Immunohistochemistry was performed on paraffin-embedded pituitary sections while ultrastructural immunocytology was done on frozen and Lowicryl-resin-embedded ultrathin sections. GHRH-R-like immunoreactivity was restricted to somatotropes and colocalized with GH in both rat and human tissues. No signal was detected in gonadotropes, lactotropes, corticotropes and thyrotropes. At the subcellular level, gold particles were associated with the plasma membrane (observed on ultrathin frozen sections), secretory granule membrane, cytoplasmic matrix, nuclear membrane and nuclear matrix. In the nucleus, gold particles were mainly observed at the junction between eu- and heterochromatin. The highest density of labeling was observed in the cytoplasm (55 vs. 45% in the nucleus), mainly in secretory granules (59% of cytoplasmic labeling) and the plasma membrane. These results support the hypothesis that GHRH-mediated actions in the pituitary are specific to somatotropes and that GHRH-R isoforms and/or ligand-receptor complexes are involved in intracellular trafficking, recycling processes and nuclear functions.

Characterization of [125I-Tyr10]human growth hormone-releasing factor(1–44) amide binding to rat pituitary: evidence for high and low affinity classes of sites

A sensitive binding assay was developed to determine binding characteristics of commercially available [125I-Tyr10]human growth hormone-releasing factor (hGRF) (1-44)NH2 in rat pituitary using 0.1 gland homogenate (70-75 micrograms protein) per incubation tube. Under standard assay conditions, addition of 5 mM EDTA efficiently prevented the degradation of both human and rat GRF for at least 3 h. Association of the ligand was time-dependent: equilibrium was reached within 30 min of incubation at 23 degrees C and remained stable for an additional 150 min (K1 = 5.01 +/- 0.86 nM-1.min-1). Specific binding increased linearly with the amount of protein present in the assay, from 15 to 170 micrograms per incubation tube. This binding was reversible, dissociation occurring almost completely after a 120-min period (K-1 = 8.13 +/- 0.29 x 10(-3) min-1). A concentration of 5-10 mM Mg2+ was required for optimal specific binding whereas 50 mM Mg2+ or monovalent cations such as Na+, K+, Li+ decreased it. Scatchard analysis of cold saturation studies by the Ligand program statistically revealed the presence of two distinct classes of binding sites; the first was of high affinity (0.68 +/- 0.11 nM) and low capacity (140 +/- 22 fmol/pituitary), the second was of lower affinity (590 +/- 347 nM) and higher capacity (38.7 +/- 18.7 pmol/pituitary). Similar values were obtained with various bovine serum albumin (BSA) concentrations and when using crude or washed pituitary homogenates, suggesting that the second low affinity site was not BSA or a soluble protein from the homogenate.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a Site-Directed Polyclonal Antibody against the Pituitary Growth Hormone-Releasing Hormone Receptor and Its Use to Estimate GHRH Receptor Concentration in Normal and Hypothyroid Rats

A site-directed polyclonal antipeptide antibody was generated in rabbits against segment 392–404 of the rat pituitary growth hormone-releasing hormone receptor (GHRH-R), using a multiple antigenic peptide system strategy of immunization. This C-terminal intracellular region of the rat GHRH-R exhibits 85% sequence identity with the human GHRH-R. The purified anti-GHRH-R(392–404) IgGs were characterized in cell lines expressing the human GHRH-R and in rat and human anterior pituitary, using immunoblotting. The polyclonal antibody recognized a 45-kD protein in human GHRH-R-transfected BHK 570 cell membrane preparations but not in wild-type cells. A 45-kD Nα-tagged human GHRH-R was immunodetected with both antitag and anti-GHRH-R antibodies in human GHRH-R-transfected HEK 293 cells. Cross-linking of [125I-Tyr10]hGHRH(1–44)NH2 to GHRH-R-transfected BHK cells led to the detection of a major and specific 45-kD radioactive complex. Its probing with the anti-GHRH-R(392–404) IgGs led also to the detection of a 45-kD entity. In rat anterior pituitary homogenates or membrane preparations, immunoblotting led to the detection of 44-, 47- and 65-kD proteins. In human anterior pituitary membrane preparations, immunoblotting led to the detection of 52- and 55-kD proteins. No immunoreactive signal was observed in the rat liver. Cross-linking of [125I-Tyr10]hGHRH(1–44)NH2 to rat anterior pituitary homogenates revealed the presence of specific 28-, 47- and 65-kD radioactive complexes. Probing of these radioactive complexes with the anti-GHRH-R(392–404) IgGs resulted in the visualization of 28-, 47- and 65-kD entities and of an additional immunoreactive 44-kD protein. To assess the usefulness of this GHRH-R antibody, estimation of changes in the concentration of rat anterior pituitary GHRH-R was performed by immunoblotting and compared to binding data after a 3-week antithyroid treatment. The treatment known to depress the 2.5- and 4-kb GHRH-R mRNA transcripts by at least 1.7-fold decreased the apparent maximal concentration of high (Bmax1) and low (Bmax2) affinity binding sites by 4.6- and 15.2-fold, respectively, and the 47- and 65-kD GHRH-R proteins by 3.5- and 1.25-fold, respectively. Altogether, the characteristics of the anti-GHRH-R(392–404) polyclonal antibody indicate that it specifically recognizes the human and rat GHRH-R. It also represents an additional valuable tool to estimate variations of GHRH-Rs in physiopathological conditions known to affect GHRH-R mRNA and/or GHRH binding site concentrations.

Long-Term Calorie Restriction Protects Rat Pituitary Growth Hormone-Releasing Hormone Binding Sites from Age-Related Alterations

In mammals, middle age and late adulthood is characterized by a decrease of growth hormone (GH) secretion and insulin-like growth factor 1 (IGF-1) serum levels, contributing to tissue and organ atrophy. This condition is related, at least in part, to alterations of pituitary GH-releasing hormone (GHRH) receptor-binding sites. Prevention of age-related deterioration of tissues and organs, retardation of the onset or progression of a wide range of age-related diseases and extension of both mean and maximum life span can be achieved through life-long moderate calorie restriction (CR). Because CR has been reported to positively modulate the somatotropic axis resulting in the maintenance of a youthful GH secretory pattern in aged rats, we investigated whether or not benefits of a long-term (10 months) 40% CR, started in 8-month-old male Sprague-Dawley rats, was accomplished by preventing age-related alterations of pituitary GHRH receptor binding sites. We also studied whether or not a short-term (50 days) 40% CR, started in 16-month-old rats, could revert them. Potential hormonal and metabolic modulators of the GHRH receptors were investigated as well. GHRH binding parameters were derived from saturation studies performed in pituitary homogenates with [125I-Tyr10]hGHRH (1-44)NH2. As previously reported, the high affinity GHRH receptor-binding sites were blunted in 18-month-old ad libitum-fed rats and the apparent concentration of total binding sites was reduced. Short-term CR neither restored high affinity GHRH binding sites nor increased the apparent concentration of total binding sites. On the contrary, long-term calorie-restricted 18-month-old rats exhibited high and low affinity GHRH binding sites (Kd1: 1.73 ± 0.35 nM; Kd2: 310 ± 41 nM; Bmax1: 183 ± 55 fmol/mg protein; Bmax2: 30 ± 3 pmol/mg protein) as found in 2-month-old rats (Kd1: 0.68 ± 0.15 nM; Kd2: 350 ± 47 nM; Bmax1: 219 ± 53 fmol/mg protein; Bmax2: 84 ± 9 pmol/mg protein). Our results imply that CR must be implemented before age-related alterations of GHRH receptor-binding sites become too severe or that CR has to be carried out for a long period of time, independently from the age at which it begins. Protection of pituitary GHRH binding sites from age-related alterations could not be attributed to changes in circulating levels of total or free T4 or free fatty acids. Finally, the anti-aging effect of a long-term CR observed at the level of pituitary GHRH receptors does not result in a significant increase of total IGF-1 circulating levels. Identification of molecular and cellular mechanisms responsible for these actions will deserve attention in order to identify centrally and/or peripherally active classes of molecules that could preserve, in aging mammals, the functionality of the somatotropic axis through selective regulation of pituitary GHRH receptors.

Characterization of a growth hormone-releasing hormone binding site in the rat renal medulla.

Receptor binding analysis was performed in the renal medulla from 2-month-old rats, an extrapituitary tissue containing the highest level of GHRH receptor mRNA. At 4 degrees C, in the presence of a cocktail of protease inhibitors, binding of [125I-Tyr(10)]hGHRH (1-44)NH(2) to medullary homogenates was specific, time-dependent, reversible and saturable (K(d): 28 nM; B(max): 30 fmol/mgprot.). In these experimental conditions, no change of binding parameters could be detected in the course of aging. The structure-affinity profile was different in the two tissues and chemical cross-linking revealed the presence of 65-, 55- and 38-kDa 125I-GHRH-labeled complexes in the renal medulla compared to 65-, 47- and 28-kDa radioactive complexes in the anterior pituitary. It is suggested that GHRH binding sites, and possibly the receptor, may be different in the two tissues.

Pheochromocytomas are diagnosed incidentally and at older age in neurofibromatosis type 1

Guidelines do not currently recommend routine systematic hormonal screening for pheochromocytoma (PHEO) in all/normotensive patients with neurofibromatosis type 1 (NF1), in contrast to other PHEO‐predisposing genetic syndromes such as Von Hippel–Lindau syndrome and multiple endocrine neoplasia type 2.

Proteolytic degradation of rat growth hormone-releasing factor(1-29) amide in rat pituitary and hypothalamus.

The identification of peptide bonds vulnerable to tissue peptidases is a valuable approach to design peptide agonists which exhibit a longer duration of action than the native molecules. Therefore, the kinetic of disappearance of rat growth hormone-releasing factor (rGRF(1-29)NH2) and the identification of its metabolites were studied in rat pituitary and hypothalamus. Synthetic rGRF(1-29)NH2 (10 microM) was incubated (0-120 min, 37 degrees C) in the presence of a pituitary (237 +/- 51 micrograms protein/ml) or hypothalamus homogenate (576 +/- 27 micrograms protein/ml). Using analytical high pressure liquid chromatography (HPLC), apparent half-lives of 22 +/- 3 min and 25 +/- 4 min were found in pituitary and hypothalamus, respectively. In both tissues, three degradation products, all less hydrophobic than the native peptide, were detected and isolated by preparative HPLC. The identification of the purified metabolites was ascertained by amino acid analysis, sequencing and chromatography with synthetic homologs. These results indicate that the main sites of cleavage in the pituitary and hypothalamus are Lys21-Leu22 (trypsin-like cleavage site), Leu14-Gly15 and Tyr10-Arg11 (chymotrypsin-like cleavage sites). TLCK and leupeptin did not affect the formation of fragment (1-21)OH while TPCK blocked the cleavage of Leu14-Gly15. The low affinity of fragment (1-21)NH2 for pituitary GRF binding sites suggests that hydrolysis of the Lys21-Leu22 bond inactivates rGRF(1-29)NH2 in this target tissue.