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Endocrinology Vol. 143, No. 4 1475-1484
Copyright © 2002 by The Endocrine Society

GRH-SOMATOSTATIN-GH

Localization and Regulation of a Functional GHRH Receptor in the Rat Renal Medulla

Chantale Boisvert1, Caroline Paré1, Christelle Veyrat-Durebex, Alma Robert, Sophie Dubuisson, Gérard Morel and Pierrette Gaudreau

Laboratory of Neuroendocrinology of Aging, Centre Hospitalier de l’Université Montréal Research Center, Notre Dame Hospital and Department of Medicine, University of Montréal, Montréal, Québec, Canada H2L 4M1; and Centre National de la Recherche Scientifique, UMR 5578, Université Claude Bernard Lyon I (G.M.), Villeurbanne 69622, France

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To provide information about the kidney GHRH receptor (GHRH-R), we assessed its tissue and cellular localization, defined its pattern of expression in developing and aging rats, and studied the effects of GHRH on the regulation of GHRH-R mRNA levels and receptor internalization. In situ hybridization and ribonuclease protection assay demonstrated that GHRH-R mRNA is restricted to the Henle’s loop (HL). GHRH-R mRNA levels were low in the medulla from 3- and 12-d-old male rats, increased significantly in that from 30- to 70-d-old rats, and decreased in that from 12- and 18-month-old animals. Compared with the GHRH-R mRNA profile obtained in the pituitary, these data support the concept of a tissue-specific regulation of GHRH-R. In HL cell cultures from 70-d-old rats, a 4-h incubation with 1–100 nM rat GHRH-(1–29)NH2 reduced GHRH-R mRNA levels significantly. As anti-GHRH-R- (392–404) immunoreactivity was demonstrated in HL cells, internalization of [N{alpha}-5-carboxyfluoresceinyl-D-Ala2,Ala8, Ala15,Lys22]hGHRH-(1–29)NH2 in a time- and temperature-dependent manner and inhibition of this process by phenyl arsine oxide indicate that desensitization to GHRH involves both GHRH-R internalization and down-regulation of GHRH-R mRNA levels. Localization of a functional GHRH-R in HL and its regulation during development and aging suggest roles associated with cellular proliferation, differentiation, and/or water/electrolyte transport.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH PLAYS A crucial role in promoting growth and development in young mammals and maintaining tissue integrity and functionality in adults. GH is also an important regulator of protein, lipid, and carbohydrate metabolism. Its effects either result from a direct action on target tissues or are mediated by IGF-I (1). In the anterior pituitary, GH pulsatile release is under the dual control of two hypothalamic peptides, GHRH and somatostatin (2). Activation of specific GHRH and somatostatin plasma membrane receptors on somatotroph cells (3, 4) leads to the stimulation or inhibition of cAMP production (5) and the regulation of GH secretion and synthesis (6, 7, 8). GHRH is also involved in somatotroph proliferation and differentiation (9, 10, 11, 12, 13).

The GHRH receptor (GHRH-R) has been cloned in rat (3, 14), mouse (14), porcine (15), bovine (16), ovine (16), and human (3, 17, 18) pituitary and in human pituitary adenomas (17, 18, 19). It belongs to the secretin-glucagon-VIP subfamily of G protein-coupled receptors (3). In mouse and rat pituitaries, the presence of 2- to 2.5-kb and 3.1- to 4-kb GHRH-R mRNA transcripts has been reported (3, 14). Although the 2- to 2.5-kb transcript probably generates the 423-amino acid functional GHRH-R (20), the structure and role of the 4-kb transcript is currently unknown. In human pituitary adenomas, 2-, 2.8-, and 4.5-kb transcripts were identified (18). The higher mol wt transcripts encode C-terminal-modified forms of the GHRH-R (17, 18) that may interfere with the functional GHRH-R to decrease GHRH-induced cAMP signaling (21). In rat pituitary, GHRH-R variants, with either a longer third intracytoplasmic loop than the 423-amino acid receptor (3) or a modified C terminus (22), retain a similar affinity to GHRH, but may exhibit a different ability to mediate GHRH-induced cAMP production. GHRH-R mRNA levels are subjected to a number of changes in the pituitary of developing (23) and aging (24) rats. Moreover, GHRH, glucocorticoids, sex steroids, and thyroid hormones participate in a complex regulation of GHRH-R levels (for review, see Ref. 25).

Low concentrations of GHRH and GHRH-R mRNA have been detected, by RT-PCR, in the rat brain, heart, lung, duodenum, small intestine, spleen, kidney, epididymis, and skeletal muscle, but not in the liver (26). Interestingly, the kidney represents the sole extrapituitary tissue containing a sufficient concentration of GHRH-R mRNA to allow its detection without amplification (26). As no data exist on its precise localization in this organ, the regulation of its expression, or its functionality, the specific aims of the present study were 1) to localize the GHRH-R mRNA at the tissue and cellular levels, 2) to define the pattern of GHRH-R mRNA expression in the developing and aging rat kidney and compare it with that of the anterior pituitary, and 3) to investigate the effect of an in vitro stimulation to GHRH on the regulation of GHRH-R mRNA levels and GHRH-R internalization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal and tissue preparations
Three-, 12-, 30-, 45-, and 70-d-old male; 30-, 45-, and 70-d-old female (Charles River Laboratories, Inc., St. Constant, Canada); and 2-, 12-, 18-, and 22-month-old male Sprague Dawley rats (obtained from our aging rat colony, bought at 2 months of age from Charles River Laboratories, Inc.) were used. They were kept in temperature (22 C)-, humidity (65%)-, and lighting- (12-h cycles; lights on at 0700 h)-controlled rooms and had free access to food and water. After 3–4 d of acclimatization to the animal facilities, they were killed in a block design fashion between 0900 and 1200 h by rapid decapitation. Pituitaries, kidneys, and livers were excised immediately, and anterior pituitary, renal cortex, medulla, and pelvis dissected out. All tissues were snap-frozen in liquid nitrogen and stored at -80 C until RNA extraction. For in situ hybridization, tissues from 2-month-old male rats were fixed 2 h in 4% paraformaldehyde/100 mM mono-di-PBS (300 mM NaCl) before freezing in liquid nitrogen. For isolation of collecting duct (CD) and Henle’s loop (HL) cells, medullas were washed and minced in ice-cold HEPES-Ringer buffer (118 mM NaCl, 16 mM H-HEPES, 16 mM Na-HEPES, 14 mM glucose, 3.2 mM KCl, 2.5 mM CaCl2, 1.8 mM MgSO4, 1.8 mM KH2PO4, and 290 mosmol, pH 7.4) (27). For primary culture of semipurified HL cells, kidney excision and renal medulla dissection and mincing were performed aseptically, using HEPES-Ringer containing 250 U/ml penicillin/250 µg/ml streptomycin (Life Technologies, Inc., Burlington, Canada) and 1.25 µg/ml amphotericin (Wisent Canada Laboratory, St. Bruno, Canada). The animal protocol was approved by the animal care committee of our institution in compliance with the guidelines of the Canadian Council on Animal Care.

Isolation of medullary CD and HL cells
Cell dispersion was performed using 12 70-d-old male rats, according to a published protocol with some modification (27). Minced medullas were incubated at 37 C in HEPES-Ringer collecting buffer containing 0.2% (wt/vol) collagenase II (Life Technologies, Inc.) and 0.2% hyaluronidase (ICN Pharmaceuticals, Inc., Montréal, Canada) for 75 min. After 30 min, 0.001% deoxyribonuclease (Roche Molecular Biochemicals Canada, Laval, Canada) was added to dissociate cell aggregates. Cells were also mechanically dispersed every 30 min by gentle pipetting with a 5-ml plastic pipette. Microscopic evaluation of the final suspension, using a Neubauer chamber, showed a majority of single cells and few aggregates (<=20 cells). The cell suspension was centrifuged at 28 x g for 2 min at 16 C, and the pellet was resuspended in 15 ml HEPES-Ringer collecting buffer. This step was repeated twice for enrichment of CD cells in the pellet. The first two supernatants were kept for purification of thin limb HL cells. The pellet was resuspended in 1–2 ml HEPES-Ringer buffer, and CD cells were purified by affinity chromatography using Dolichos biflorus agglutinin (DBA)-coated magnetic beads (28). Tosyl-activated beads (4 x 108 beads/ml; Dynal, Lake Success, NY) were prepared by incubation with 0.1 mM avidin DX (Vector Laboratories, Inc., Burlingame, CA) in 150 mM NaCl and 50 mM NaHCO3 (pH 8.5) at 37 C for 16 h. After washing in PBS (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, and 1.8 KH2PO4), beads were incubated with 0.05 mM biotinylated DBA in 150 mM NaCl, 0.1 mM CaCl2, and 10 mM HEPES (pH 7.4) at 37 C for 2 h. Unbound biotinylated DBA was removed by additional washing. CD cells were incubated with DBA-coated beads (bead/cell ratio, 20:1) at 37 C for 10 min with gentle agitation, pelleted in a magnetic field, and resuspended in HEPES-Ringer buffer. Elution of bound cells was performed by agitation with 300 mM N-acetyl-D-galactosamine (Sigma-Aldrich Corp., Oakville, Canada) at room temperature for 2 h. Cells were pelleted (1000 x g, 4 C, 5 min) and washed twice with HEPES-Ringer buffer.

The HL cell suspension (first two supernatants) was centrifuged at 150 x g for 10 min (16 C) and washed twice in HEPES-Ringer buffer. This pellet was resuspended in 2 ml HEPES-Ringer buffer and treated with DBA-coated beads to isolate the residual medullary CD cells (<=20% in our experimental conditions). Unbound material was used to purify thin limb HL cells by differential centrifugation, using a continuous gradient of Nycodenz. The gradient was prepared by solubilizing 28% (wt/vol) Nycodenz [5-(N-2,3-dihydroxypropylacetamido)-2,4,6-tri-iodo-N,N'-bis (2,3-dihydroxypropyl)-isophthalamide (Life Technologies, Inc.) in 5 mM Tris-HCl buffer containing 0.3 mM CaNa2-EDTA and 3 mM KCl, pH 7.4. Twenty and 8% solutions of Nycodenz were prepared by appropriate dilutions with 7.45% (wt/vol) sucrose solubilized in the Tris-HCl buffer. The gradient was obtained according to the protocol of Grupp et al. (27). Thin limb HL cells were recovered in fraction I of the gradient after centrifugation at 1500 x g (16 C, 45 min) and washed twice in HEPES-Ringer buffer (430 x g, 16 C, 10 min).

Immunological characterization of medullary CD and HL cells
Equivalent amounts of CD and HL cells (~2 x 105) were spun on each glass slide by cytocentrifugation (32 x g, room temperature, 2 min; Cytospin 3 centrifuge, Shandon, Pittsburgh, PA) and fixed in acetone-methanol (50:50, -20 C, 15 min) and ethanol (70%, room temperature, 1 min), respectively. They were incubated in a 5% (wt/vol) BSA (Sigma-Aldrich Corp.)-PBS blocking solution (room temperature, 30 min). CD cells were labeled using fluorescein isothiocyanate (FITC)-labeled DBA (1:1000 diluted in PBS, room temperature, 10 min; Sigma-Aldrich Corp.), thin limb HL cells, using a rabbit polyclonal antiaquaporin-1 antibody (1:200 diluted in PBS, 37 C, 60 min; Alamone Laboratories, Jerusalem, Israel) and interstitial/vascular cells, using a mouse monoclonal anti-vimentin antibody (1:10 diluted in PBS, 37 C, 60 min; Amersham Pharmacia Biotech, Baie d’Urfé, Canada). Slide-mounted cells were washed three times with PBS and HL, and interstitial/vascular cells were visualized with a fluorescein-conjugated goat antirabbit antibody (1:5000 diluted in PBS, room temperature, 60 min; Molecular Probes, Inc., Eugene, OR) and a Texas Red-X-conjugated goat antimouse antibody (1:10,000 diluted in PBS, room temperature, 60 min; Molecular Probes, Inc.), respectively. The specificity of labeling was assessed by substituting primary antibodies with normal IgGs. All procedures with fluorescent probes were performed in the dark. Cells were visualized using a Nikon Eclipse TE600 (x20 objective), equipped with a coolsnap camera, a Nikon super high pressure mercury lamp and a filter for excitation/emission of fluorescein (485/520 nm) and Texas Red (595/660) (Nikon Canada, Montréal, Canada).

Primary culture of semipurified HL cells
Medullas from 70-d-old male rats were enzymatically and mechanically dissociated in HEPES-Ringer containing antibiotics, as described above. Dispersed cells were centrifuged at 28 x g (2 min, 4 C), and the pellet was resuspended in HEPES-Ringer buffer containing antibiotics and recentrifuged. The two supernatants were pooled and centrifuged at 150 x g (10 min, 4 C). The pellet was washed three times with DMEM/F-12 (Life Technologies, Inc.) containing 250 U/ml penicillin/250 µg/ml streptomycin and 1.25 µg/ml amphotericin and once with the culture medium containing 50 U/ml penicillin/50 µg/ml streptomycin and 0.25 µg/ml amphotericin. At this step, approximately 40 x 106 cells were recovered from each medulla, with a viability consistently between 95–98%, as assessed by trypan blue exclusion. After being cultured in 100-mm (id) petri dishes overnight at 37 C in a humidified atmosphere containing 95% air and 5% CO2, cells were collected and rinsed twice with serum-free culture medium containing 0.1% BSA and were preincubated for 1 h at 37 C in this medium. Cells were subsequently exposed for 4 h to 1, 10, and 100 nM rat (r) GHRH-(1–29)NH2 [synthesized in our laboratory (29)] or the rGHRH vehicle (culture medium). At the end of the incubation period, cells were collected on ice, centrifuged (3000 x g, 10 min, 4 C), and washed in serum-free medium. Total RNA was extracted and analyzed by ribonuclease (RNase) protection assay (RPA).

Northern blot hybridization
Total RNA was isolated from anterior pituitary, renal medulla, and liver from 2-month-old male rats using a single step, acid guanidinium-phenol/chloroform method with TRIzol (Life Technologies, Inc.). Pituitary and medullary polyadenylated (poly A) RNA was isolated by chromatography using oligo(deoxythymidine)-cellulose (Ambion, Inc., Austin, TX). Northern blot hybridization was performed as previously described with minor modifications (24). Aliquots of 1.5 µg poly A 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 and subjected to electrophoresis on 1.2% agarose/17.5% formaldehyde gels, using 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 Life Science Products, Boston, MA) and covalently attached by UV cross-linking and heating (80 C, 2 h). Blots were hybridized with the RPR64 probe corresponding to the 3'-end of the rat GHRH-R cDNA (nucleotide position 1044–1611) (3). The probe was labeled with [32P]deoxy-CTP (3000 Ci/mmol; Amersham Pharmacia Biotech), using random hexamer primers and the Klenow fragment of Escherichia coli DNA polymerase (Life Technologies, Inc.) and purified by chromatography using a G-50 column (Amersham Pharmacia Biotech). Hybridization was performed at 42 C for 16 h in 50% formamide, 5x SSC (1x SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.0), 10% dextran sulfate, 1x Denhardt’s solution (50x = 1% BSA, 1% Ficoll 400, and 1% polyvinylpyrrolidone), 20 mM Tris (pH 7.5), 0.1% SDS, and 100 µg/ml DNA salmon sperm. Membranes were subsequently washed in 2x SSC/0.1% SDS at room temperature, in 1x SSC/0.1% SDS at 65 C, and in 0.5x SSC/0.1% SDS at 65 C for 30 min each time and exposed to XAR-5 films (Eastman Kodak Co., Rochester, NY) at -80 C, for 7 d with an intensifying screen. Membranes were stripped in a boiling aqueous solution of 0.1% SDS and rehybridized with a 1.2-kb rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA to assess the amount of RNA present in each lane. Quantification of each GHRH-R mRNA transcript was performed using an IS1000 Digital imaging system (Alpha Innotech Corp./Canberra Packard, Missisauga, Canada). The specificity of the [32P]RPR64 probe was assessed in each experiment using 5 µg liver total RNA. The linearity of protected signals was verified routinely using 0.5–5 µg poly A RNA.

RPA
Renal medullas and anterior pituitaries from 70-d-old or younger male or female rats and cortex and pelvis from 70-d-old male rats were pooled to maximize RNA yields. Total RNA from these tissues and from semipurified HL cells was isolated with TRIzol. The antisense 32P- labeled RPR64 riboprobe was generated by in vitro transcription of BamHI-linearized RPR64-pGEM3z plasmid, using T7 RNA polymerase (MAXIscript transcription kit, Ambion, Inc.) in the presence of [{alpha}-32P]UTP (800 Ci/mmol; Amersham Pharmacia Biotech). GAPDH mRNA levels were assessed using a probe derived from exons 5–8 of the rat GAPDH cDNA (nucleotide position 369–685). The antisense-labeled probe was generated by in vitro transcription of the XbaI-linearized p-TRI-GAPDH plasmid (Ambion, Inc.) using T7 RNA polymerase. The specific activity of the [32P]RPR64 riboprobe, determined after trichloroacetic acid precipitation onto GF/B Whatman filters (Fisher Scientific, Montreal, Canada), was 275–325 x 106 cpm/µg RNA, and the percentage of [{alpha}-32P]UTP incorporation was 67–75%. RPA was performed according to published protocols with some modifications (23, 30, 31), using the RPA II kit (Ambion, Inc.). Twenty micrograms of total RNA from renal cortex, medulla, and pelvis; 5 µg total RNA from anterior pituitary or semipurified HL cells; and 5 or 20 µg total RNA from liver (pituitary or kidney studies, respectively) were hybridized overnight at 50 C with the 4 x 106 cpm/ml [32P]riboprobe. To normalize GHRH-R mRNA levels for experimental variations, 40 pg of the RPR64 MscI cRNA standard (corresponding to nucleotide position of RPR64 cDNA 1044–1203) were added to RNA samples before hybridization. A second step of normalization, with GAPDH as internal standard, was used in semipurified HL cell preparations to maintain the intraassay coefficient of variation to 10% or less. Nonannealed nucleic acids were digested with RNase A (1 U/ml) and RNase T (40 U/ml) at 37 C for 30 min. Stable hybrids were resolved on 1.5 mM 5% polyacrylamide-8 M urea denaturing gels. Autoradiography was performed at -80 C using XAR-5 or Biomax Ms-1 films (Kodak) with an intensifying screen. Tissue GHRH-R, GAPDH mRNA, and cRNA standard levels were quantified by densitometry using a IS1000 Digital imaging system. The intraassay coefficient of variation of normalized GHRH-R mRNA levels was 10% or less in all experiments. The specificity of the [32P]RPR64 riboprobe was assessed in each experiment using 5 µg pituitary total RNA and 5 or 20 µg liver total RNA. The linearity of protected signals was verified routinely using 10–30 µg medulla total RNA or 1–10 µg anterior pituitary or semipurified HL cell total RNA. Results were expressed as the percentage of relative density to a control group (70-d-old or 2-month-old rats) or condition (purified cell populations, incubation with rGHRH vehicle), using a fixed amount of total RNA. Results were also expressed as GHRH-R mRNA relative densities per anterior pituitary or renal medulla total RNA content.

In situ hybridization
In situ hybridization was performed according to published protocols with some modifications (3, 32). Ten-micron cryosections of kidney, liver, and pituitary were incubated in 20 mM Tris-2 mM CaCl2 buffer with 1 µg/ml proteinase K (Roche, Meylan, France) for 15 min at 37 C and postfixed for 5 min in 4% paraformaldehyde and for 1 min in 100 mM triethanolamine (pH 8.0)/0.25% anhydrous acetic acid. Slides were dehydrated using ethanol series and were air-dried. GHRH-R mRNA was detected using the RPR64 riboprobe. Antisense and sense 35S-labeled RPR64 riboprobes were generated by in vitro transcription as described above, using T7 and SP6 RNA polymerase (MAXIscript transcription kit, Ambion, Inc.), respectively, in the presence of [{alpha}-35S]UTP (1000 Ci/mmol; Amersham Pharmacia Biotech). Riboprobes were purified on 5% denaturing polyacrylamide gel and eluted in 0.5 M ammonium acetate, 1 nM EDTA, and 0.2% SDS. Slide-mounted tissue sections were incubated in hybridization buffer containing 50% deionized formamide, 10% dextran sulfate, 4x SSC, 2x Denhardt’s solution, 100 µg/ml yeast transfer RNA, 10 mM dithiothreitol, 100 µg/ml DNA salmon sperm, and [35S]GHRH-R riboprobe (~6 x 106 cpm/ml hybridization buffer). In situ hybridization was performed overnight at 55 C. Sections were washed sequentially in 2x SSC for 1 h at room temperature, 2x SSC for 1 h at 60 C, and 1x SSC at room temperature. Sections were then treated with RNase A (100 µg/ml) for 45 min at 37 C, washed in 0.5x SSC for 1 h and 0.1x SSC for 45 min each at room temperature, and dehydrated in ethanol. Slides were dipped in NTB2 nuclear emulsion (Kodak), exposed at 4 C for 5–15 d, developed in D19 (Kodak), and stained with 1% toluidine blue. Slides were examined using fluorescence light microscopy by epipolarization (Olympus Corp. Provis, Rungis, Cedex, France). The specificity of the reaction was assessed in the kidney by hybridization with the sense [35S]GHRH-R riboprobe, and tissue specificity was assessed in the pituitary (positive control) and liver (negative control) with the antisense [35S]riboprobe.

Immunocytochemical localization of GHRH-R
Semipurified HL cells (~2 x 105) were spun on each glass slide by cytocentrifugation (32 x g, room temperature, 2 min) and fixed in 4% paraformaldehyde-PBS (20 min, room temperature), washed twice in PBS (2 x 10 min), and permeabilized in 0.2% Triton X-100 (Sigma-Aldrich Corp.; 15 min, room temperature). Slides were then washed in PBS (four times, 5 min each time, room temperature), blocked with 5% BSA/PBS (30 min, room temperature), washed in PBS (three times, 5 min each time), and incubated with 0.5 µg purified anti-GHRH-R-(392–404) polyclonal antibody (33) in 100 µl PBS containing 1% BSA overnight at 4 C in a humid atmosphere. Cells were rinsed in PBS (twice, 10 min each time), incubated for 1 h at room temperature in the presence of Alexa-conjugated goat antirabbit IgGs (Molecular Probes; 1:15,000 in PBS-BSA buffer), and washed in PBS (twice, 10 min each time). All steps in the presence of the fluorescent secondary antibody were performed in the dark. Slides were kept in a humid atmosphere for fluorescence microscopy examination. Analysis was performed using a Nikon Eclipse TE600 microscope (x20 objective).

Internalization of [N{alpha}-5-carboxyfluoresceinyl-D-Ala2,Ala8,Ala15,Lys22]hGHRH-(1–29)NH2 (Fluo-GHRH)
Fluo-GHRH was synthesized by solid phase methodology (34). Phenyl arsine oxide (PAO, Sigma-Aldrich Corp.) was solubilized in 50% dimethylsulfoxide (DMSO)/50% picopure H2O to a final volume of 0.1% DMSO at 10 µM PAO. Semipurified HL cells were incubated at 4 C for 45 min in the presence of 1 nM Fluo-GHRH to study temperature dependency and kinetics of internalization. After centrifugation (2000 x g, 5 min, 4 C), supernatant was removed, and cells were washed once in cold HEPES-Ringer buffer. Cells were then either suspended in cold buffer and spun (~2 x 105) on glass slides by cytocentrifugation (32 x g, room temperature, 2 min) for immediate visualization or warmed to 37 C for 30, 60, 90, and 120 min to allow internalization. Inhibition of internalization at 37 C was performed by preincubating cells for 10 min with 10 µM PAO before the addition of Fluo-GHRH and including PAO during the 45-min incubation at 4 C and the 90-min incubation at 37 C. The effect of 0.1% DMSO alone was tested on the internalization of Fluo-GHRH. Nonspecific binding was determined in the presence of 1 µM rGHRH-(1–29)NH2. The reaction was stopped by placing tubes on ice and cytocentrifugation. Slides were kept on ice for immediate fluorescence microscopy examination. Analysis was performed using a Nikon Eclipse TE600 microscope (x20 objective).

Statistical analysis
Results were expressed as the mean ± SEM. Comparisons of GHRH-R mRNA levels among groups or conditions were performed by ANOVA, followed by Tukey’s or Dunnett’s multiple range tests or t test. Statistical significance of differences was established at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of renal medulla GHRH-R mRNA transcripts
As shown in Fig. 1Go by Northern blotting, two GHRH-R poly A mRNA transcripts of 3.7 and 2.3 kb (doublets) were detected in the renal medulla, as in the anterior pituitary, using the RPR64 riboprobe. The total relative level of mRNA transcripts was 1.6 times lower in the medulla compared with that in the pituitary; the relative intensities of 3.7- and 2.3-kb transcripts were 1.3 and 2.3 lower than those in the pituitary, respectively. Similar results were obtained using RPR13, a riboprobe corresponding to nucleotide position 265-1134 of the rat GHRH-R cDNA (data not shown). No signal was detected in the liver with these probes (data not shown).



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  		Figure 1. Northern blot autoradiographic representation of pituitary (P) and renal medulla (M) GHRH-R mRNA transcripts. An average of four pituitaries and medullas from 2-month-old male rats were pooled, and 1.5 µg poly A RNA were analyzed by Northern blotting. Results are representative of two independent experiments.

 
Localization of GHRH-R mRNA in the kidney by in situ hybridization
High density labeling was restricted to HL cells of the renal medulla (Fig. 2Go, A and B), whereas low to very low intensity of labeling was observed in CD (Fig. 2BGo) and cortex (Fig. 2CGo) with the antisense RPR64 riboprobe or in the renal medulla when tissue sections were hybridized with the sense riboprobe (Fig. 2DGo). In these hybridization conditions, positive and negative signals were obtained in pituitary and liver tissue sections, respectively (data not shown).



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  		Figure 2. In situ localization of GHRH-R mRNA in the rat kidney. Kidneys from 2-month-old male rats were used. Labeling of GHRH-R mRNA with the antisense RPR64 riboprobe in HL cells of the medulla (A), CD and HL cells of the medulla (B), and glomerular cell of the cortex (C) or in the medulla (D) with the sense RPR64 riboprobe. Bar, 50 µM. Results are representative of three independent experiments.

 
Quantification of protected GHRH-R mRNA fragments in RPA
Densitometric analysis of GHRH-R mRNA protected fragments from renal medulla, semipurified HL cells, and anterior pituitary showed a linear relationship with the amount of RNA loaded, in the range of 1–30 µg total RNA for renal tissue and in the range of 1–10 µg total RNA for pituitary (data not shown). The mean r2 ± SEM was 0.97 ± 0.01. Routinely, 20 µg medulla, cortex, or pelvis and 5 µg semipurified HL cells or pituitary total RNA were used. As previously reported, two distinct bands were detected using the RPR64 probe (23). The sum of both signals was considered the total density of GHRH-R mRNA. It was 2–3 times lower in the medulla than in the pituitary (data not shown). In the kidney of 70-d-old male rats (Fig. 3Go), the medulla contained the highest GHRH-R mRNA levels. Signals were 13 and 7 times lower in the cortex and pelvis than in the medulla, respectively (P < 0.01). Similar results were obtained in the kidney from 45-d-old rats, with signals 24 and 5 times lower in the cortex and pelvis than in the medulla, respectively.



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  		Figure 3. Quantification of GHRH-R mRNA levels in the rat kidney. An average of eight cortexes, medullas, and pelvises from 70-d-old male rats were pooled. Twenty micrograms of total RNA were analyzed by RPA. Results were expressed as the percentage of relative density to that obtained in the medulla and are the mean ± SEM of four independent experiments performed in duplicate. **, P < 0.01 (compared with GHRH-R mRNA levels in the medulla, by Dunnett’s test).

 
Cellular distribution of GHRH-R mRNA in cell populations of the renal medulla
Nycodenz-purified thin limb HL and DBA-purified CD cells were used to determine the cellular localization of GHRH-R mRNA in the medulla. Immunocytochemical characterization indicated that 53 ± 3% of purified HL cells were specifically labeled with the antiaquaporin-1 antibody, a specific marker of descending thin limb HL cells, whereas DBA-FITC- and antivimentin-labeled cells represented approximately 2% and 26 ± 3% of purified cells, respectively. Seventy-six ± 7% of DBA-purified cells were labeled with DBA-FITC, whereas antiaquaporin-1 and antivimentin labeling accounted for about 1% and 12 ± 3% of cells, respectively. As shown in Fig. 4Go, GHRH-R mRNA levels were 17.3 and 3.4 times higher in purified HL cells than in CD cells (P < 0.01) and total medulla (P < 0.05), respectively.



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  		Figure 4. 4.GHRH-R mRNA levels in purified CD and HL cells and in total medulla. Twenty-four medullas from 70-d-old male rats were pooled for each purification. Twenty micrograms of total RNA were analyzed by RPA. Results were expressed as a percentage of relative density to that obtained in the medulla and are the mean ± SEM of two independent experiments performed in duplicate and using two independent groups of rats. *, P < 0.05; **, P < 0.01 (compared with GHRH-R mRNA levels in HL cells, by Dunnett’s test).

 
Regulation of GHRH-R mRNA levels in male and female renal medulla during development and comparison with GHRH-R mRNA levels in male anterior pituitary
In the developing male rat, GHRH-R mRNA signals were very low in the medulla from 3- and 12-d-old rats and increased significantly between 30 and 70 d of age (Fig. 5AGo). When expressed per 20 µg total RNA (Fig. 5BGo), these levels were 15.3 and 6.0 times lower compared with those in 70-d-old rats, respectively (P < 0.001). They were also significantly lower compared with those in 30- and 45-d-old rats (P < 0.001). Moreover, GHRH-R mRNA levels were 1.4 times higher in the medulla from 45-d-old rats compared with that of 70-d-old rats (P < 0.05). No significant difference was observed between GHRH-R mRNA levels of 30- and 70-d-old rats. When GHRH-R mRNA levels were analyzed per medulla total RNA content (Fig. 5C), they were 213 and 30 times (P < 0.001) lower in the medulla from 3- and 12-d-old rats compared with that of 70-d-old rats, respectively, and 1.6 times higher in the medulla from 45-d-old rats compared with that of 70-d-old rats (P < 0.01).



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  		Figure 5. GHRH-R mRNA levels in the renal medulla of the developing male rat. An average of 20 and 6 medullas from 3- to 45-d-old and 70-d-old rats were pooled, respectively. Twenty micrograms of total RNA were analyzed by RPA for each age group. A, Autoradiographic representation of GHRH-R mRNA and RPR-64 MscI RNA standard (40 pg) signals in various age groups. B and C, GHRH-R mRNA densities expressed per 20 µg total RNA (B) or per medulla total RNA content (C) as a percentage of relative density to that obtained in the medulla from 70-d-old rats. Results are the mean ± SEM of nine experiments performed in triplicate, using three independent pools. *, P < 0.05 (compared with GHRH-R mRNA levels in the medulla from 70-d-old rats, by t test). **, P < 0.01; ***, P < 0.001 (compared with GHRH-R mRNA levels in the medulla from 70-d-old rats, by Tukey’s test).

 
In the developing female rat, GHRH-R mRNA signal was 1.6 times higher in the medulla from 45-d-old female rats compared with that of 70-d-old female rats (P < 0.01) when expressed per 20 µg total RNA (Fig. 6AGo). No significant difference was observed between GHRH-R mRNA levels from 30- and 70-d-old female rats. However, the GHRH-R mRNA level was 1.4 times higher in the medulla from 70-d-old male rats compared with that of 70-d-old female rats (P < 0.05). When GHRH-R mRNA levels were analyzed per total RNA content of medulla (Fig. 6BGo), they were 2.2 times higher in the medulla from 45-d-old than in that of 70-d-old female rats (P < 0.001) and 1.4 times higher in the medulla of 70-d-old male rats compared with that of 70-d-old female rats (P < 0.001).



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  		Figure 6. GHRH-R mRNA levels in the renal medulla of the developing female rat. An average of six medullas from 30- to 70-d-old rats were pooled. Twenty micrograms of total RNA were analyzed by RPA for each age group. Results were expressed per 20 µg total RNA (A) or per medulla total RNA content (B) as a percentage of relative density to that obtained in the medulla from 70-d-old female rats. Data are the mean ± SEM of four independent experiments performed in triplicate. F, Female; M, male. *, P < 0.05 (compared with GHRH-R mRNA levels in the medulla from 70-d-old female rats, by t test). **, P < 0.01 (compared with GHRH-R mRNA levels in the medulla from 70-d-old female rats, by Tukey’s test). ***, P < 0.001 (GHRH-R mRNA levels from 45-d-old female compared with those of 70-d-old female rats, by Tukey’s test). ***, P < 0.001 (GHRH-R mRNA levels from 70-d-old male compared with those of 70-d-old female rats, by t test).

 
A study of the developmental regulation of GHRH-R mRNA levels was also performed in the anterior pituitary of male rats, allowing a tissue-specific comparison of the process. Levels of pituitary GHRH-R mRNA varied with age. When expressed per 5 µg total RNA, GHRH-R mRNA levels were 1.8 (P < 0.05) and 2.2 times higher (P < 0.01) in the pituitary from 3- and 12-d-old rats compared with that of 70-d-old rats and decreased thereafter (Fig. 7AGo). When GHRH-R mRNA levels were analyzed per anterior pituitary total RNA content (Fig. 7BGo), they were 13.1 and 5.5 times (P < 0.01) lower in the pituitary from 3- and 12-d-old rats compared with that of 70-d-old rats due to the rapid growth of the gland.



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  		Figure 7. GHRH-R mRNA levels in the anterior pituitary of the developing male rat. An average of 10 and 3 anterior pituitaries from 3- to 45-d-old and 70-d-old rats were pooled, respectively. Five micrograms of total RNA were analyzed by RPA for each age group. Results were expressed per 5 µg total RNA (A) or per anterior pituitary total RNA content (B) as a percentage of relative density to that obtained in the pituitary from 70-d-old rats. Data are the mean ± SEM of four independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01 (compared with GHRH-R mRNA levels in the anterior pituitary from 70-d-old rats, by Dunnett’s test).

 
Regulation of GHRH-R mRNA levels in male renal medulla during aging
GHRH-R mRNA levels decreased significantly in the medulla from 12- and 18-month-old male rats, but not in that of 22-month-old rats (Fig. 8AGo). When expressed per 20 µg total RNA (Fig. 8BGo), GHRH-R mRNA signals were 1.5 times lower in the medulla from 12- and 18-month-old rats compared with those of 2-month-old rats (P < 0.01). When GHRH-R mRNA levels were analyzed per medulla total RNA content (Fig. 8C), no significant difference was observed between groups.



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  		Figure 8. GHRH-R mRNA levels in the renal medulla of aging male rat. Medullas from each rat were pooled. At least three rats were used in each age group. Twenty micrograms of total RNA from each rat were analyzed by RPA. A, Autoradiographic representation of GHRH-R mRNA and RPR-64 MscI RNA (40 pg) standard signals in various age groups. B and C, GHRH-R mRNA densities expressed per 20 µg total RNA (B) or per medulla total RNA content (C) as a percentage of relative density to that obtained in the medulla from 2-month-old rats. Results are the mean ± SEM of 2 independent experiments performed in triplicate. **, P < 0.01 (compared with GHRH-R mRNA levels in the medulla from 2-month-old rats, by Tukey’s test).

 
In vitro regulation of GHRH-R mRNA levels by GHRH in semipurified HL cells
The effect of GHRH on the regulation of GHRH-R mRNA levels was investigated in semipurified HL cells. A 4-h stimulation with 1, 10, and 100 nM rGHRH-(1–29)NH2 induced a 3.2-, 3.7-, and 2.0-fold decrease in GHRH-R mRNA levels compared with a 4-h incubation with the rGHRH vehicle (P < 0.01; Fig. 9Go, A and B).



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  		Figure 9. GHRH-R mRNA levels in semipurified HL cells treated with GHRH. Semipurified HL cells from 2-month-old male rats (two medulla equivalents) were incubated for 4 h in the presence of 1, 10, or 100 nM rGHRH-(1–29)NH2 or the vehicle in serum-free medium. A, Five micrograms of total RNA were analyzed by RPA. Autoradiographic representation of GHRH-R mRNA, GAPDH mRNA, and RPR-64 MscI cRNA (40 pg) signals of control and GHRH-treated cells. B, GHRH-R mRNA densities expressed per 5 µg total RNA. Results were expressed as a percentage of relative density to that obtained in control HL cells and are the mean ± SEM of two (1–10 nM) or three independent experiments performed in duplicate or triplicate. **, P < 0.01 (compared with GHRH-R mRNA levels in control cells, by Tukey’s test).

 
Visualization and internalization of GHRH-R in semipurified HL cells
Immunocytochemical localization of GHRH-R with the anti-GHRH-R-(392–404) primary antibody revealed its presence in semipurified HL (Fig. 10aGo). No signal was observed when primary antibodies were substituted with normal goat antirabbit IgGs (Fig. 10bGo).



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  		Figure 10. Visualization of GHRH-R in semipurified HL cells with the anti-GHRH-R-(392–404) antibody and Fluo-GHRH. a, GHRH-R was visualized in semipurified HL cells from 2-month-old male rats with the anti-GHRH-R-(392–404) antibody and Alexa-conjugated goat antirabbit IgGs. b, Specificity of labeling was assessed by substituting the primary antibody with normal goat antirabbit IgGs. Bar, 10 µm. Results are representative of three independent experiments. Fluorescence imaging of Fluo-GHRH in semipurified HL cells. Cells were incubated at 4 C for 45 min in the presence of 1 nM Fluo-GHRH and in the absence (A) or presence (B) of 1 µM rGHRH-(1–29)NH2 or were incubated at 4 C for 45 min and warmed at 37 C for 30 (C), 90 (D), or 120 min (E) or warmed at 37 C for 90 min (F) in the presence of 10 µM PAO. Bar, 10 µm. Results are representative of three independent experiments.

 
Internalization of the GHRH-R was assessed using Fluo-GHRH, a high affinity ligand for the human and rat pituitary GHRH-R (34). At 4 C, maximal removal of fluorescence was obtained in presence of 1 µM rGHRH-(1–29)NH2 (Fig. 10AGo, total binding; Fig. 10BGo, nonspecific binding). The distribution of GHRH fluorescence varied as a function of incubation temperature. After a 45-min incubation at 4 C, low to moderate levels of fluorescence were diffusely distributed over the cell surface (Fig. 10AGo). When cells were warmed at 37 C for 90 min, a punctate pattern of fluorescence appeared. This labeling was distributed throughout the cytoplasm, sparing the nucleus (Fig. 10DGo). Internalization of Fluo-GHRH was time dependent. After 30-min incubation at 37 C, cells exhibited a more intense labeling than at 4 C. However, this labeling remained largely at the cell surface after 30 and 60 min (Fig. 10CGo). Fluo-GHRH continued to internalize progressively at 37 C (Fig. 10DGo, 90-min incubation) and appeared to be completely internalized after a 120-min incubation at 37 C (Fig. 10EGo). After a 90-min incubation in the presence of 10 µM PAO, labeling was less intense, and Fluo-GHRH remained diffuse at the cell surface (Fig. 10FGo). This effect was specific to PAO, as incubation in the presence of 0.1% DMSO alone did not block internalization of Fluo-GHRH (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report for the first time that high levels of renal GHRH-R mRNA are localized in HL of the renal medulla by RPA and in situ hybridization. The procedure used to purify thin descending and ascending HL cell has been reported to give highly purified cells (27); however, we cannot exclude the potential presence of thick ascending limb cells in our preparation. Moreover, in situ hybridization on cryosections allowed discrimination of CD from HL cells, but not subpopulations of HL cells.

When these GHRH-R mRNA signals were analyzed by Northern blotting, it was shown that the approximately 2.5- and 4-kb transcripts initially described in the anterior pituitary by Mayo (3) are both present in the renal medulla. Their total abundance is almost 2 times lower in the medulla than in the pituitary, with the concentration of the 4-kb transcript being 1.3 times lower and that of the 2.5-kb transcript being 2.3 times lower. As data from partial sequencing of renal GHRH-R cDNA revealed a complete homology with that of the pituitary receptor (26), it might be suggested that the same GHRH-R isoforms are generated in both tissues, but in different concentrations. Elucidation of complete cDNA/mRNA structures of medullary GHRH-R will be needed to further substantiate this hypothesis. The present results are at variance with those reported by Matsubara et al. (26), showing a sole 4-kb transcript in rat medulla, which could be attributed to a lower sensitivity of their assay. Based on immunological data revealing the presence of a 52- and 50-kDa proteins in human kidney and pituitary membrane preparations, respectively (35), a tissue-specific GHRH-R heterogeneity resulting from posttranslational modifications should also be considered.

RPA was found to be a sensitive and reliable method to perform a valid quantification of GHRH-R mRNA levels in rat medulla. As for Northern blotting, a single pattern was identified in medulla and pituitary, and the relative abundance of protected fragments was 2–3 times lower in the medulla. To help understand some of the regulatory mechanisms by which GHRH-R mRNA levels are affected in the renal medulla, studies were performed in developing male and female rats and aging male rats, with pituitary comparison. In the course of development, GHRH-R mRNA levels were low in the medulla during the perinatal period and became elevated around 30–70 d, being maximal at 45 d. GHRH-R mRNA patterns were concordant using a fixed amount of RNA or medulla total RNA content. The pattern of GHRH-R mRNA expression observed between 30- and 70-d-old animals in the medulla of male rats was also present in females. However, when GHRH-R mRNA levels were compared in the medulla of 70-d-old males and females, they were significantly higher in males. This observation suggests the existence of a sexual dimorphism in the medulla as proposed in the pituitary. In the pituitary of 3-month-old female Sprague Dawley rats, GHRH-R mRNA levels were 15% of those found in males (36). In 2- and 4-month-old female LOU/C rats, levels of 2.5- and 4-kb GHRH-R mRNA transcripts were 2–3 times lower in females than in males (37). However, the absence of difference in GHRH-R mRNA levels has also been reported in neonate and adult rats (38, 39). These discrepancies might be due to differences in age groups, animals strains, estrous cycles, or RNA analysis techniques. Studies in gonadectomized male and female rats submitted, or not, to hormonal replacement therapy will be required to determine the tissue-specific impact of gonadal steroids and to assess a sexual dimorphic expression of GHRH-R in the renal medulla.

In the present study the highest levels of GHRH-R mRNA were seen in the pituitary during the postnatal period between 3 and 12 d of age, with a subsequent decline, reaching a nadir at 45–70 d. Previous studies performed by RPA (23) or RT-PCR (39) showed the highest levels of GHRH-R mRNA during the perinatal period (embryonic d 19.5–2) and a subsequent decline between 10 and 75 d of age (23, 39). At variance with Kamegai et al. (39), Korytko et al. (23) reported a drastic decrease of GHRH-R mRNA levels in the pituitary of 12-d-old rats, followed by a significant increase in that of 30-d-old rats. This latter observation could not be confirmed in the present study. In contrast to that in the medulla, a different pattern of GHRH-R mRNA levels was observed in the anterior pituitary when using a fixed amount of RNA or the total tissue content. In total pituitary, GHRH-R mRNA levels increased drastically between 3 and 30 d of age and stabilized thereafter due to its rapid growth. These results suggest an important contribution of GHRH-R in somatotroph proliferation (9, 10, 11, 12). Such a role has been further supported by studies in the lit/lit dwarf mouse, showing that a mutated and functionally defective somatotroph GHRH-R prevents both proliferation and terminal differentiation in the mature anterior pituitary (11, 12). In addition, data in the chick suggest that GHRH could also act during embryonic development to regulate somatotroph differentiation (13). Together, these results demonstrated a tissue-specific regulation of GHRH-R mRNA levels during development. Moreover, the presence of low levels of GHRH-R mRNA in the medulla of newborn rats added to the fact that the rat kidney is fully developed at 20 d of age but reaches its maximal capacity to concentrate urine around 45 d (40) may suggest a preferential role in proliferation of mature cells or in cellular differentiation.

In the renal medulla of aging rats, levels of GHRH-R mRNA diminished, but this was compensated by an increase in kidney size, which doubled in weight between 2 and 12 months. The greater variability observed in GHRH-R mRNA levels from 22-month-old rats may be related to the smaller number of animals compared with other age groups and/or to the fact that some survivors are physiologically younger and maintain higher levels of GHRH-R mRNA. Whether a decrease of GHRH-R mRNA concentrations correlates with an age-dependent decreased sensitivity to GHRH or a possible defect in urinary concentrating capacity remains to be explored. Interestingly, in the medullary thick ascending limb of HL from 20- to 24-month-old mice, the appearance of a defect in the urinary concentrating capacity was associated with a decreased sensitivity to vasopressin (41). In the pituitary of aging male rats, Northern blot analysis showed that the 4-kb GHRH-R mRNA transcript increased significantly between 2 and 18 months of age, whereas the 2.5-kb transcript increased between 2 and 8 months of age and decreased thereafter (42). A similar pattern was observed in 16- and 24-month-old male F344 rats compared with 6-month-old rats (43). As Northern blotting cannot be used for a precise quantification of GHRH-R mRNA levels in the renal tissue, a differential regulation of GHRH-R transcripts, impacting on physiological processes, cannot be excluded in the aging medulla. However, as the total density of transcripts remains unchanged in the pituitary from 2- and 18-month-old rats (24) as well as that of protected GHRH-R mRNA fragments in the pituitary from 70-d-old and 12-month-old rats (23), these results instead suggest that tissue-specific regulation of GHRH-R mRNA levels also exists in this physiological situation.

To help establish the functionality of GHRH-R in LH cells, the effect of a stimulation to GHRH on the regulation of GHRH-R mRNA levels was investigated. A 4-h exposure to 1, 10, or 100 nM rGHRH-(1–29)NH2 induced a 50–70% decrease in GHRH-R mRNA content, consistent with results obtained in anterior pituitary cell cultures (44, 45), showing that a 4-h exposure to 0.1 and 1 nM rGHRH-(1–44)NH2 induced an approximately 50% decrease in GHRH-R mRNA. In somatotrophs, this down-regulation of GHRH-R mRNA levels was mediated via a cAMP-dependant mechanism (44). Preliminary results obtained in our laboratory indicate that GHRH induces a concentration-dependent increase in cAMP production in semipurified HL cells (data not shown), suggesting that the phenomenon could be mediated at least in part via a cAMP pathway. To further document the receptor-mediated action of GHRH in semipurified HL cells, its presence was assessed by immunocytochemistry, using a specific anti-GHRH-R antibody recognizing the portion 392–404 of rat and human pituitary (33). The result suggests that GHRH-R species found in the rat medulla share at least partial sequence identity with the pituitary receptor, as proposed for the kidney and pituitary human receptor (35). Finally, assessment of GHRH-R functionality, using a fluorescent GHRH agonist (46), indicated that GHRH is internalized in a specific temperature- and time-dependent manner in HL cells. As PAO, a general inhibitor of endocytosis, blocked internalization of Fluo-GHRH, GHRH-mediated internalization of GHRH-R may be considered a part of the GHRH desensitization process together with down-regulation of GHRH-R mRNA. In the anterior pituitary internalization is dependent upon fatty acid acylation of GHRH-R (47), but such a mechanism has not yet been documented in HL cells.

The presence in the kidney of a functional GHRH-R that is regulated in physiological conditions such as development suggests a physiological relevance. GHRH-R could play a role in water and/or electrolyte transport directly or by regulating the expression or function of renal proteins involved in concentrating processes, some of which increase in the rat kidney between 10 and 40 d of age (40, 48). GHRH-R could also mediate, as in somatotrophs (9, 10, 11, 12, 13), cell proliferation and/or differentiation postnephrogenesis. As various components of the somatotroph axis are found in HL cells, GHRH could act directly or via a GH/IGF-I system (49).


   Acknowledgments
 
We are grateful to Dr. Kelly E. Mayo (Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanstown, IL) for providing us with the RPR64 rGHRH-R cDNA, to Dr. N. E. Petersen (Department of Biochemistry and Clinical Genetics, Odense University, Odense, Denmark) for supplying us with very helpful documentation on RPA, and to Dr. P. Chartrand (CHUM Research Center, University of Montréal) for giving us a GAPDH cDNA.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research. The colony of aged rats was supported by a grant from the Dairy Farmers of Canada. A France Québec exchange program supported the collaboration with G.M. C.B. received studentships from Fonds de la Recherche en Santé du Québec and the Faculty of Graduate Studies, University of Montréal. C.P. received a studentship from the Faculty of Graduate Studies, University of Montréal. C.V.D. and P.G. received a postdoctoral fellowship and a scholarship chercheur-boursier national from Fonds de la Recherche en Santé du Québec, respectively. S.D. received a summer studentship from Fondation Rx&D des Compagnies de Recherche Pharmaceutique du Canada and the Faculty of Medicine, University of Montréal.

1 C.B. and C.P. contributed equally to this work. Back

Abbreviations: CD, Collecting duct; DBA, Dolichos biflorus agglutinin; DMSO, dimethylsulfoxide; FITC, fluorescein isothiocyanate; Fluo-GHRH, [N{alpha}-5-carboxyfluoresceinyl-D-Ala2,Ala8,Ala15,Lys22]hGHRH-(1–29)NH2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GHRH-R, GHRH receptor; PAO, Phenyl arsine oxide; poly A, polyadenylated; r, rat; RNase, ribonuclease; RPA, ribonuclease protection assay.

Received October 30, 2001.

Accepted for publication December 18, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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