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Institut National de la Santé et de la Recherche Médicale U 413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23) (F.S., V.C., H.L., D.A.-A., M.G., D.C., A.D., N.C., Y.A., H.V., C.D.), University of Rouen, 76821 Mont-Saint-Aignan, France; and Department of Endocrinology and Metabolic Diseases (H.L.), Centre Hospitalier Universitaire of Rouen, 76031 Rouen, France
Address all correspondence and requests for reprints to: Dr. Catherine Delarue, European Institute for Peptide Research (Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U 413, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: catherine.delarue{at}univ-rouen.fr.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: The objective of this study was to investigate the possible involvement of NT in the control of cortisol secretion in the human adrenal gland.
Design: In vitro studies were conducted on cultured human adrenocortical cells.
Setting: This study was conducted in a university research laboratory.
Patients: Adrenal explants from patients undergoing expanded nephrectomy for kidney cancer were studied.
Main Outcome Measure: Cortisol secretion from cultured adrenocortical cells was measured.
Results: NT1–11, the N-terminal fragment of NT, dose-dependently inhibited basal and ACTH-stimulated cortisol production by human adrenocortical cells in primary culture. In contrast, NT had no influence on cortisol output at concentrations up to 10–6 M. HPLC and RT-PCR analyses failed to detect any significant amounts of NT and NT mRNA, respectively, in adrenal extracts. Molecular and pharmacological studies were performed to determine the type of NT receptor involved in the corticostatic effect of NT1–11. RT-PCR analysis revealed the expression of NT receptor type (NTR) 3 mRNA but not NTR1 and NTR2 mRNAs in the human adrenal tissue. However, the pharmacological profile of the adrenal NT1–11 receptor was different from that of NTR3, indicating that this receptor type is not involved in the action of NT1–11 on corticosteroidogenesis.
Conclusion: Our results indicate that NT1–11 may act as an endocrine factor to inhibit cortisol secretion through activation of a receptor distinct from the classical NTR1, NTR2, and NTR3.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In mammals, NT and related peptides exert their biological effects through three distinct types of receptors termed NT receptor type (NTR) 1, NTR2, and NTR3. NTR1 and NTR2 belong to the family of seven-transmembrane domain G protein-coupled receptors, whereas NTR3 is a single-transmembrane domain non-G protein-coupled receptor (1). All types of NT receptors are able to bind the C-terminal region of NT, NT8–13, which is the shortest biological active fragment of the peptide (1). The three NT receptors are expressed in the central nervous system and in peripheral organs (1). For instance, NTR1 mRNA is expressed in the gastrointestinal tract (12), NTR2 mRNA is present in the uterus (13) and gastric fundus (14), and NTR3 mRNA is found in the spinal cord, heart, thyroid, placenta, and testis (15). However, the occurrence of NT receptors has never been reported in the adrenal gland in mammals.
In the present study, we have investigated in vitro the involvement of NT in the regulation of glucocorticoid secretion from the human adrenal gland. The presence of NT and NT precursor mRNA in the adrenal tissue has been explored by HPLC analysis and RT-PCR experiments, respectively. The effects of NT and NT analogs on basal and ACTH-stimulated cortisol secretion have been determined using cultured adrenocortical cells. Finally, the human adrenocortical NT receptor has been characterized by pharmacological and molecular approaches.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Normal human adrenal explants were obtained at surgery, after informed consent, from patients undergoing expanded nephrectomy for kidney cancer. The protocol of collection of the tissue and the experimental procedures were approved by the regional ethics committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Haute-Normandie, Loi Huriet, January 1990). Tissues were either collected and frozen at –80 C until biochemical analysis and RNA extraction or immersed in DMEM supplemented with 0.5% antibiotic-antimycotic solution and rapidly transported to the laboratory for cell cultures.
Reagents
Ham-F12 culture medium, insulin, collagenase type I, deoxyribonuclease I, apotransferrin, ascorbic acid, human ACTH, NT, NT8–13, NT1–11, NT1–8, and porcine NMN (pNMN) were purchased from Sigma (St. Quentin Fallavier, France). The antibiotic-antimycotic solution, DMEM, and fetal bovine serum were from Invitrogen (Cergy-Pontoise, France). [1,2,6,7-3H]cortisol (78 Ci/mmol) and [125I]NT(2000 Ci/mmol) were obtained from Amersham International (Les Ulis, France). Levocabastine was a generous gift from Janssen Pharmaceuticals (Beerse, Belgium). SR48692 (2-[(1-[7-chloro-4-quinolinyl]-5-[2,6-dimethoxyphenyl]pyrazol-3-yl) carbonylamino]tricyclo-(3.3.1.1)decan-2-carboxylic acid) (SR 48692) and SR 142948A (2-[(5-[2,6-dimethoxyphenyl]-1-[4-(N-[3-dimethylaminopropyl]-N-methylcarbamoyl)-2-isopropylphenyl]-1H-pyrazole-3-carbonyl) amino]adamantane-2-carboxylic acid hydrochloride) were kindly provided by Sanofi-Recherche (Montpellier, France). The antiserum to bovine NT was raised in rabbit, and the antibodies were directed against the conserved C-terminal region of the peptide (16).
Characterization of NT-like immunoreactivity in human adrenal extracts
The tissues were boiled in 0.5 M acetic acid for 15 min, homogenized in a glass Potter, and centrifuged (4000 x g; 30 min; 4 C). The supernatant was prepurified on Sep-Pak C18 cartridges (Waters Associates, Milford, MA) as previously described (17). Bound material was eluted from the cartridges with 60% (vol/vol) acetonitrile/water and evaporated in a Speed-Vac concentrator (Savant Instruments, Hickville, NY). The dry adrenal extract was redissolved in 0.12% trifluoroacetic acid/water (900 µl), centrifuged three times (13,000 x g; 5 min), and filtered through a 0.22-µm sieve. The filtrate was injected directly onto a Vydac 218TP1010 C18 reversed-phase HPLC column equilibrated with acetonitrile/water/trifluoroacetic acid (7.0:92.88:0.12, vol/vol) at a flow rate of 1 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 50% (vol/vol) over 60 min. One-minute fractions were collected and the content of NT-like immunoreactivity was determined by RIA. Synthetic NT, used as a reference peptide, was chromatographed under the same conditions as the human adrenal extract.
The concentration of NT in HPLC fractions was measured by RIA using [125I]NT as a radioligand and the NT antiserum at the dilution of 1:3000. The EC50 of the assay was 430 pg/tube, and the minimum detectable amount of peptide was 10 pg/tube.
Cell culture
Adrenocortical explants, free of fat and medullary tissues, were diced into small pieces and enzymatically dispersed as previously described (18). Briefly, the adrenal tissue was mechanically stirred for 45 min in DMEM containing collagenase (2 mg/ml) and DNAse (0.1 mg/ml). After digestion, the tissue was disaggregated by gentle aspiration through a 10-ml pipette. The suspension containing both dissociated cells and tissue fragments was filtered, and the fragments were resubmitted to the dispersion procedure. Dispersed cells were centrifuged (100 x g; 24 C; 20 min), and the pellet was suspended in DMEM-Ham-F12 (1:1) supplemented with ascorbic acid (0.2 µg/ml), apo-transferrin (10 µg/ml), insulin (5 µg/ml), 0.5% antibiotic-antimycotic solution, and 5% fetal bovine serum. Adrenocortical cells were seeded on 24-well plates at the density of 2 x 105 cells per well. The cells were kept at 37 C in an incubator with a humidified atmosphere (95% O2/5% CO2). The culture medium was changed 24 h after plating. After 2 or 3 d in culture, cells were incubated for 24 h with DMEM (control) or DMEM containing different test substances. Aliquots of culture medium were collected and kept at –20 C until assay. Cortisol concentration was determined as previously described (19).
RNA extraction and RT-PCR analysis
Total RNAs from adrenocortical explants derived from 10 different glands were extracted according to the method of Chomczynski and Sacchi (20) using Tri-Reagent (Sigma). The concentration of total RNA was determined by spectrophotometry analysis (UV-1605; Shimadzu, Kyoto, Japan). Total RNA (2 µg) from normal adrenal gland was converted into single-stranded cDNA using either Superscript II (Life Technologies, Eragny, France) with the oligo (deoxythymidine)12–18 primer (500 µg/ml) for NT and NT receptors or the ImProm-II Reverse Transcription System (Promega, Charbonnières, France) for neprilysin (NEP). Amplification of the cDNAs encoding NT, NTR1, NTR2, NTR3, and NEP was performed by PCR using specific primers (Table 1
). Two other primers of the cloned glyceraldehyde-3-phosphate dehydrogenase sequence were used for semiquantitation of reverse-transcribed mRNAs (Table 1). PCR amplification for NT and NT receptors was performed in a final volume of 50 µl containing 1/10 RT reaction, 3 U DNA Taq polymerase (Life Technologies), PCR buffer (Life Technologies), 1.5 mM MgCl2, 0.4 mM dNTP, and 20 pM of each primer. The PCRs were performed for 30 cycles (94 C, 30 sec; 45 C, 60 sec; 72 C, 60 sec) in a DNA Thermal Cycler (Perkin-Elmer, Courtaboeuf, France). The PCR products were separated by electrophoresis on a 1.5% agarose gel and visualized by UV illumination in the presence of ethidium bromide. Individual bands were excised, purified using the MinElute gel extraction kit (QIAGEN, Courtaboeuf, France), and ligated into pGEM-T vector (Promega). Each band was characterized by restriction mapping and subsequently sequenced using the Thermosequenase kit (Amersham, Orsay, France) on a Li-Cor 4200L DNA sequencer (ScienceTec, Les Ulis, France). PCR amplification for NEP was performed in a final volume of 50 µl containing first strand cDNA (10% of RT reaction), PCR Master Mix version ReddyMix (1.25 U Thermoprime Plus DNA Polymerase, 1.5 mM MgCl2), Abgene, and 20 pM of each primer. The PCRs were performed for 35 cycles (94 C, 30 sec; 60 C, 30 sec; 72 C, 30 sec). The amplified products were separated on 1.5% agarose gels, blotted on a nylon membrane, and hybridized with a [32P]ATP-labeled internal gene-specific oligonucleotide (5'-TTC ACG TTC TGT TCG TCT GC-3') corresponding to bases 2024–2043 of the NEP cDNA (GenBank accession no. AF336981.1).
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Each response curve was established as the mean profile of cortisol secretion (±SEM) calculated from at least three independent experiments. The concentration-response curves were fitted using the Prism 4.00 software (GraphPad Software, San Diego, CA), and maximum response and IC50 (the agonist concentration eliciting half-maximum effect) were derived from this analysis. To determine the affinities of the receptor antagonists, adrenocortical cells were incubated with NT1–11 in the absence or presence of 10–6 M NTR1 or NTR2 antagonists. For each antagonist, the dissociation constant (KB) was determined according to the equation: KB = [B]/(dose ratio – 1), where [B] is the concentration of the antagonist, and the dose ratio is the quotient of the EC50 of the agonist in the presence or absence of antagonist. The results were expressed as the negative logarithm of KB (pKB) (21). Statistical analysis was performed by Dunnett’s Multiple Comparison test after one-way ANOVA.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The effects of NT and its N-terminal fragment NT1–11 on cortisol secretion by human adrenocortical cells in primary culture are shown in Fig. 1A. NT1–11 induced a dose-dependent inhibition of cortisol secretion. The half-maximum effective dose of NT1–11 (IC50) was 3.3 x 10–10 M, and maximum response (–23.4 ± 0.9%) was obtained with a concentration of 10–7 M (P < 0.01). In contrast, NT had no influence on cortisol output at concentrations up to 10–6 M (Fig. 1A). Similarly, pNMN, NT8–13, and NT1–8 (10–10 to 10–6 M) did not affect cortisol production (data not shown). The effect of NT1–11 was then studied on the cortisol response evoked by ACTH (10–9 M) in cultured adrenocortical cells. Graded doses of NT1–11 (10–9 to 10–6 M) caused a concentration-dependent inhibition of ACTH-induced cortisol secretion (Fig. 1B).
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Biochemical characterization of NT in Sep-Pack-prepurified adrenocortical extracts was carried out by combining reversed-phase HPLC analysis with RIA detection (Fig. 2A). No immunoreactive material coeluted with synthetic NT. In addition, RT-PCR analysis showed the absence of NT mRNA in six human adrenal extracts (Fig. 2A, inset). As a positive control, a band of the expected size for the NT PCR product was observed in a human liver extract (Fig. 2A, inset). The expression of the mRNA encoding NEP, an endopeptidase involved in the breakdown of NT into N-terminal products, was also detected in four human adrenocortical extracts by RT-PCR (Fig. 2B).
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Coincubation of adrenocortical cells with NT and pNMN (10–6 M) suppressed the inhibitory effect of NT1–11 on cortisol production with a pKB more than 10 (Fig. 3, A and B). NT8–13 and NT1–8 also inhibited NT1–11-induced inhibition of cortisol secretion with pKB of 9.68 and 8.73, respectively (Fig. 3, C and D).
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In an attempt to determine the pharmacological profile of the receptor involved in the inhibitory effect of NT on corticosteroidogenesis, we have investigated the action of nonpeptide ligands of NTR1 and NTR2 on the cortisol response to NT1–11 in cultured adrenocortical cells. The NTR1 antagonists SR 48692 (1 µM) and SR 142948A (1 µM) both antagonized the inhibitory effect of NT1–11 on cortisol secretion (Fig. 4A). SR 48692 exhibited higher potency (pKB > 10) than SR 142948A (pKB = 8.06). In contrast, the specific NTR2 antagonist levocabastine (1 µM) did not affect NT1–11-evoked inhibition of cortisol output (Fig. 4B).
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). Oligonucleotides were designed to amplify cDNA fragments of 292, 430, and 547 bp corresponding, respectively, to NTR1, NTR2, and NTR3 mRNAs. A band of the predicted size of the NTR3 PCR product was observed in the six adrenal extracts examined. In contrast, no PCR products corresponding to the NTR1 and NTR2 mRNAs were detected in the adrenal extracts, whereas NTR1 and NTR2 transcripts were found in a human cerebellum extract (positive control). |
Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Several regulatory peptides are synthesized within the adrenal gland by chromaffin cells or released by nerve terminals (29). Thus, it was conceivable that NT could occur in the human adrenal gland and exert a paracrine inhibitory action on corticosteroid secretion after local conversion into NT1–11. Therefore, we have looked for the presence of NT in adrenal extracts by combining HPLC purification with RIA quantification, and we have searched for the occurrence of NT mRNA by RT-PCR analysis. We did not detect any significant amounts of immunoreactive NT nor NT mRNA amplification product, indicating that the peptide is neither synthesized in the human adrenal gland nor contained in nerve fibers. Indeed, several observations suggest that NT1–11 may rather act as an endocrine factor to inhibit glucocorticoid production: 1) NT1–11 has been detected in the plasma of rat and human (30, 31), and the circulating levels of immunoreactive N-terminal NT fragments are higher than those of intact NT in human (31); 2) NT1–11 is cleared more slowly than NT in rat plasma (30); 3) the fairly low IC50 of NT1–11 (3.3 x 10–10 M) is consistent with an endocrine mode of action; and 4) adrenocortical capillaries are largely fenestrated (32), indicating that plasma NT1–11, like ACTH, can easily reach adrenocortical cells. It is also conceivable that NT1–11 could be formed from circulating NT within the adrenocortical tissue itself since we have observed the presence of NEP (EC 3.4.24.11), an endopeptidase involved in the breakdown of NT into N-terminal products, in human adrenal extracts. Unfortunately, we have not been able to characterize NT1–11 in adrenal extracts since we did not have access to antibodies against the N-terminal region of NT.
The functional significance of NT1–11-induced cortisol inhibition remains to be elucidated. However, it is possible that NT fragments may be involved in the postprandial regulation of plasma cortisol concentration. For instance, several studies conducted in humans have reported an increase in plasma NT and NT fragment levels after a fat-rich meal (33, 34) and a positive correlation between the concentration of circulating NT and meal energy (34, 35). In particular, the postprandial concentration of N1–11 has been found to peak between 30 and 120 min (34), whereas serum cortisol concentrations are significantly reduced 1 h after a fat-rich meal (36). These observations, together with the present data, suggest that NT fragments might be responsible for the postprandial inhibition of cortisol concentration and may thus participate in the regulation of the energy homeostasis. They also imply that NT1–11 may be regarded as an endocrine link between the gastrointestinal tract and the adrenal gland, probably aimed at favoring the positive action of insulin on the storage of ingested nutrients through inhibition of cortisol, which rather promotes catabolic events. The fact that supraphysiological concentrations (10–6 M) of NT1–11 are required for inhibition of the stimulatory effect of ACTH on cortisol secretion indicates that the adrenal response to stress is preserved during the postprandial periods.
To investigate the type(s) of receptor mediating the inhibitory effect of NT1–11 on cortisol secretion, adrenocortical cells were exposed to various NT short-chain analogs and available NT receptor ligands. Our data show that neither NT nor NT8–13 had any effect on basal cortisol output, but NT, pNMN, NT8–13, and NT1–8 blocked the inhibitory action of NT1–11 at supraphysiological concentrations. Similarly, the NTR1 antagonists SR 48692 and SR 142948A markedly attenuated the effect of NT1–11 on cortisol secretion, suggesting that NTR1 could have been involved in the secretory response to NT1–11. However, this hypothesis could be refuted on the basis of molecular studies that failed to detect NTR1 mRNA in adrenal tissues. Concurrently, the lack of effect of the NTR2 antagonist levocabastine on NT1–11-evoked inhibition of cortisol production and the absence of NTR2 mRNA in the human adrenal gland indicated that the effect of NT1–11 on adrenocortical cells cannot be mediated through NTR2. Although RT-PCR amplification studies revealed the expression of NTR3 mRNA in the human adrenal tissue, the pharmacological profile of the receptor clearly showed that the action of NT1–11 is not mediated through the NTR3 type. In particular, NT and pNMN, which bind NTR3 with high affinity (37), did not affect cortisol secretion. However, it is conceivable that the adrenal NTR3 may mediate a possible mitogenic effect of NT on human adrenocortical cells as previously shown in cancer cell lines (38). Altogether, our data indicate that NT1–11 inhibits cortisol production by interacting with a receptor that exhibits pharmacological properties obviously distinct from those of the cloned mammalian NT receptors, i.e. NTR1, NTR2, and NTR3. Consequently, these results suggest that the inhibitory action of NT1–11 on glucocorticoid secretion is mediated by a novel NT receptor that remains to be identified. Consistent with this hypothesis, previous reports have shown that, in the gastrointestinal tract, N-terminal fragments of NT, i.e. NT1–8, NT1–10, and NT1–11, but not the C-terminal fragments NT8–13 and NT 9–13, exert their biological effects through activation of a receptor different from the classical NT receptors (39, 40).
In conclusion, our results show that NT1–11 may act as an endocrine factor to inhibit cortisol secretion. Although RT-PCR analysis demonstrated the occurrence of NTR3 in human adrenal tissue, pharmacological studies revealed that the cortisol response to NT1–11 is mediated through a novel type of NT receptor that remains to be identified. These data suggest that N-terminal fragments of NT may play a role in the postprandial decrease of plasma cortisol levels.
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Acknowledgments |
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Footnotes |
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First Published Online May 16, 2006
Abbreviations: NEP, Neprilysin; NMN, neuromedin N; NT, neurotensin; NTR, NT receptor type; pNMN, porcine NMN.
Received January 17, 2006.
Accepted May 5, 2006.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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) and NT1–11 (•) from 10–11 to 10–6 M on cortisol production. The results are expressed as a percentage of the basal secretory rate. B, Effects of graded concentrations of NT1–11 (10–9 to 10–6 M) on the cortisol response evoked by ACTH (10–9 M). The results are expressed as a percentage of the response induced by ACTH in the absence of NT1–11. Each curve represents the mean ± SEM of two to 15 independent experiments. The mean basal levels of cortisol secretion in these experiments were: A, 223.6 ± 38.0 ng/24 h per 2 x 105 cells; and B, 212.0 ± 14.3 ng/24 h per 2 x 105 cells. *, P < 0.05; **, P < 0.01 vs. control.
) of 1 µM NT (A), pNMN (B), NT8–13 (C), and NT1–8 (D). The results are expressed as a percentage of the basal secretory rate. Each curve represents the mean ± SEM of two to 15 independent experiments. The mean basal level of cortisol secretion in these experiments was 228.1 ± 50.1 ng/24 h per 2 x 105 cells. *, P < 0.05; **, P < 0.01 vs. NT1–11 alone.
). B, Concentration-response curves of NT1–11 on cortisol secretion in the absence (•, control) or presence of the NTR2 antagonist levocabastine (
, P < 0.05; **, P < 0.01 vs. NT1–11 alone. C, RT-PCR analysis of mRNA encoding NTR1, NTR2, and NTR3 in six normal adrenal glands (ag1 to ag6) and fetal human cerebellum. Specific primers for NTR1, NTR2, and NTR3 were used to amplify fragments of 292, 430, and 547 bp, respectively.